Gene Duplication

GENE DUPLICATION
Edited by Felix Friedberg

Gene Duplication
Edited by Felix Friedberg
Published by InTech
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Gene Duplication, Edited by Felix Friedberg
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Contents

Preface IX
Part 1 General Aspects 1
Chapter 1 A Theoretical Scheme of the
Large-Scale Evolution by Generating
New Genes from Gene Duplication 3
Jinya Otsuka
Chapter 2 Duplicated Gene Evolution Following
Whole-Genome Duplication in Teleost Fish 27
Baocheng Guo, Andreas Wagner and Shunping He
Chapter 3 Detection and Analysis of Functional
Specialization in Duplicated Genes 37
Owen Z. Woody and Brendan J. McConkey
Chapter 4 Predicting Tandemly Arrayed
Gene Duplicates with WebScipio 59
Klas Hatje and Martin Kollmar
Chapter 5 The LRR and TM Containing
Multi-Domain Proteins in Arabidopsis 77
Felix Friedberg
Chapter 6 Partial Gene Duplication
and the Formation of Novel Genes 95
Macarena Toll-Riera, Steve Laurie,
Nria Rad-Trilla and M.Mar Alb
Part 2 A Look at Some Gene Families 111
Chapter 7 Immunoglobulin Polygeny:
An Evolutionary Perspective 113
J. E. Butler, Xiu-Zhu Sun and Nancy Wertz
VI Contents

Chapter 8 Gene Duplication in Insecticide Resistance 141
Si Hyeock Lee and Deok Ho Kwon
Chapter 9 Gene Duplication and the
Origin of Translation Factors 151
Galina Zhouravleva and Stanislav Bondarev
Chapter 10 Analysis of Duplicate Gene Families in Microbial
Genomes and Application to the Study of Gene
Duplication in M. tuberculosis 173
Venu Vuppu and Nicola Mulder
Chapter 11 The Evolutionary History of CBF
Transcription Factors: Gene Duplication of
CCAAT Binding Factors NF-Y in Plants 197
Alexandro Cagliari, Andreia Carina Turchetto-Zolet,
Felipe dos Santos Maraschin, Guilherme Loss,
Rogrio Margis and Marcia Margis-Pinheiro
Part 3 Examining Bundles of Genes 223
Chapter 12 L- Myo-Inositol 1-Phosphate Synthase (MIPS) in
Chickpea: Gene Duplication and Functional Divergence 225
Manoj Majee and Harmeet Kaur
Chapter 13 On the Specialization History of
the ADP-Dependent Sugar Kinase Family 237
Felipe Merino and Victoria Guix
Chapter 14 Duplication of Coagulation Factor Genes and
Evolution of Snake Venom Prothrombin Activators 257
Shiyang Kwong and R. Manjunatha Kini
Chapter 15 A Puroindoline Mutigene Family
Exhibits Sequence Diversity in Wheat
and is Associated with Yield-Related Traits 279
Feng Chen, Fuyan Zhang,
Craig F. Morris and Dangqun Cui
Chapter 16 Evolution of GPI-Aspartyl
Proteinases (Yapsines) of Candida spp 289
Berenice Parra-Ortega, Lourdes Villa-Tanaca
and Csar Hernndez-Rodrguez
Chapter 17 Clues to Evolution of the SERA
Multigene Family in the Genus Plasmodium 315
Nobuko Arisue, Nirianne M. Q. Palacpac,
Kazuyuki Tanabe and Toshihiro Horii
Contents VII

Chapter 18 Molecular Evolution of Juvenile Hormone Signaling 333
Aaron A. Baumann and Thomas G. Wilson
Chapter 19 Gene Duplication and Subsequent Differentiation
of Esterases in Cactophilic Drosophila Species 353
Rogrio P. Mateus, Luciana P. B. Machado and Carlos R. Ceron
Chapter 20 SNCA Gene Multiplication:
A Model Mechanism of Parkinson Disease 373
Kenya Nishioka, Owen A. Ross and Nobutaka Hattori
Chapter 21 Bucentaur (Bcnt) Gene Family:
Gene Duplication and Retrotransposon Insertion 383
Shintaro Iwashita and Naoki Osada

Preface

The book Gene Duplication consists of 21 chapters divided in 3 parts: General Aspects,
A Look at Some Gene Families and Examining Bundles of Genes.
The importance of the study of Gene Duplication stems from the realization that the
dynamic process of duplication is the sine qua non underlying the evolution of all
living matter. Genes may be altered before or after the duplication process thereby
undergoing neofunctionalization, thus creating in time new organisms which populate
the Earth.
Osaka (Chapter I) suggests that similarities in amino acid sequences exhibited by
paralogous proteins prove that evolution proceeds via in toto gene duplication. If the
ancestral and the newly created gene perform the same function, the new gene would
be labeled a subfunctional gene. It should be added that such a duplicated gene
encoding an identical product might also be engaged by different cellular regulatory
signals (e.g. methylation of nucleotide sites) which in turn, could hamper the
expression of such a duplicated gene. (See e.g. Woody et al. Chapter 3). If this
duplicated gene subsequently undergoes mutations that allow a function for the new
gene that is different from the parent gene (neofunctionalization) that would represent
a far more positive evolutionary event. The first three chapters in this book focus on
such in toto gene duplications whereby in evolutionary time neofunctionalization
could have taken hold. There are also several specific cicumscribed examples given in
this book. (See e.g. Majee&Kaur, Chapter 12). Undoubtedly, duplication contributes
substantially to the formation of new genes. But there is a caveat: In time, the majority
of duplicated genes mutates into oblivion.
In recent years, however, attention has been paid to another possible path for creating
a new gene: The formation of the chimeric gene, a gene immediately ready for a new
function. Such a gene might result from altering the position of spliced introns, or
more likely from retropositioning of a new encoding domain into the gene: I.e. partial
gene duplications and combination. It is obvious that such processes are particularly
suited for the creation of genes encoding multi-domain proteins and that they may
accelerate considerably the natural process of neofunctionalization. (See Hatje
et al.Chapter 4; Friedberg, Chapter 5; Toll-Riera et al. Chapter 6 and Iwashita et al.
Chapter 21). Retrotransposons are capable of promoting such segmental duplications.
X Preface

Retroduplication contributes significantly to the formation of new genes. These
genes, in turn may also be duplicated and eventually be erased into oblivion by
mutations.

Prof. Felix Friedberg
Howard University Medical School,
Washington DC,
USA

Part 1
General Aspects

1
A Theoretical Scheme of the Large-Scale
Evolution by Generating New Genes
from Gene Duplication
Jinya Otsuka
JO Institute of Biophysics, Tokyo,
Japan
1. Introduction
In the famous book The Origin of Species by Darwin (1859), the gradual accumulation of
selectively advantageous variants has been proposed qualitatively by obtaining a hint from
the artificial selection of domestic animals and plants as well as from the observation of
unique species in a geographically isolated region. The core of this proposal has become
evident, after the re-discovery of Mendelian heredity, by the detection of hereditary
variants, i. e., mutants, and extensive investigations have been carried out for the behavior
of mutants especially in the Drosophila population (Dobzhansky, 1941; Mayer, 1942; Huxley,
1943; Simpson, 1944). In parallel, Darwinian evolution is mathematically formulated in
population genetics to estimate the probability that a spontaneously generated mutant is
fixed in, or eliminated from, the population according to the positive or negative value of a
selective parameter (Fisher, 1930; Wright, 1949). Although the accumulation of such mutants
as those found in the Drosophila was supposed to explain the whole process of evolution, the
mutants detected at that time were mainly due to the point mutations in established genes,
and most of them were defective. Thus, doubts remain about whether the gradual
accumulation of such mutants gives rise to radically new organs such as wings and eyes.
Another criticism against the survival of the fittest in Darwinian evolution is also raised by
the ecological fact of diversity that different styles of organisms coexist in the same area
(Nowak et al., 1994).
The gene and genome sequencing, which started in the latter half of the last century, has
brought new information about the evolution of organisms. First, the amino acid sequence
similarities of paralogous proteins strongly suggest that the repertoire of protein functions
has been expanded by gene duplication, succeeding nucleotide base substitutions, partial
insertion and deletion, and further by domain shuffling in some cases (Ingram, 1963; Gilbert,
1978; Ferris & White, 1979). Such examples are now increasing, proposing many protein
families and superfamilies. Second, the clustering analysis of proteomes reveals a
characteristic feature that the proteins functioning in the core part are essentially common to
both prokaryotes and eukaryotes, and that the decisive difference in gene repertoire
between the organisms is observed in the peripheral parts displaying different living styles
(Kojima & Otsuka, 2000 a, b, c; Kojima & Otsuka, 2002). These sequence data are now
compiled into databases (e. g., Wheeler et al., 2004; Birney et al., 2006).

Gene Duplication

4
Although the importance of gene duplication in evolution was already indicated in the
last century (Ohno, 1970), this indication still remained describing the circumstantial
evidence of gene duplication and the fossil record of vertebrate organs in a qualitative
way. Theoretically, some new concept is needed to formulate the evolution by gene
duplication, going beyond the narrow view of population genetics which only focuses on
a mutated gene. For this purpose, the author has recently proposed the new concept of
biological activity, which is determined by a whole genome, and explained the
divergence of the original style of organisms and the new style of organisms having a new
gene generated from the counterpart of duplicated genes (Otsuka, 2005; 2008). This
evolution by gene duplication will be called the large-scale evolution, being distinguished
from Darwinian evolution.
In this chapter, the explanatory remarks are first given for the concept of biological activity
and the large-scale of evolution will be then investigated in detail on the three types of
organisms, which are different in their genome constitution and transmission. The genome
is a single DNA molecule in most prokaryotes and it is a set of chromosomes in lower
eukaryotes. These organisms will be tentatively called the monoploid organisms as the first
type of organisms. Some lower eukaryotes exchange homologous chromosomes through
the process of conjugation. These lower eukaryotes are treated as the second type of
organisms. In higher animals and plants, each of the cells constituting the adult form carries
the genome consisting of the plural number of homologous chromosome pairs, and the
monoploid state only appears in the gametes (egg and sperm). These higher eukaryotes will
be treated as the third type, being called the diploid organisms in the sense that the present
study focuses on the evolution of the characters expressed in their diploid state. The main
purpose of the present study is to elucidate the difference between the three types of
organisms, especially in the probabilities that two or more kinds of new genes are generated
from different origins of gene duplication. This study reveals that the second type organism
is most suitable to generate many kinds of new genes and the third type organism is next in
line. The cell differentiation is a representative character, which requires many kinds of
genes for its expression, and the present result provides an explanation for the fact that the
cell differentiation has started in the second type of organisms and then evolved to the
higher hierarchy in the third type of organisms.
2. The concept of biological activity
Although the biological activity is a macroscopic quantity generally characterizing various
biological systems such as an ecological system, an organism, an individual cell of a
multicelluar organism etc. (Otsuka, 2004, 2005, 2008), it will be explained focusing on an
organism for the present purpose of considering the large-scale evolution of organisms by
gene duplication. In general, an organism may be characterized by a set of two macro-
variables, the genome size N and its systematization - S
N
of genes and their products. The
systematization corresponds to the negentropy, which should be measured for the specific
arrangement of nucleotides in individual genes, the degree of accuracy in transmitting the
genetic information to the amino acid sequences of proteins, the formation of metabolic
pathways by enzyme protein functions, the regulation and control at various levels of
biological processes, the cell structure constructed by the interaction of metabolic products,
and for furthering the communication between differentiated cells in the case of
multicellular organisms. The energy acquired by an organism depends not only on the
A Theoretical Scheme
of the Large-Scale Evolution by Generating New Genes from Gene Duplication

5
genome size N and systematization - S
N
but also on the material and energy source M
available from the environment. Thus, the energy acquired by the organism during its
lifetime is expressed as E
a
(M; N, S
N
), which may be an increasing function of N and S
N
as
well as of M. On the other hand, the organism utilizes the acquired energy and materials to
construct the biomolecules for its growth and self-reproduction. The energy E
s
(N, S
N
) stored
in the form of biomolecules is also another increasing function of N and S
N
. The difference
between the acquired energy and the stored energy, E
a
(M; N, S
N
) E
s
(N, S
N
), is lost as heat.
According to the second law of thermodynamics, the entropy production by the heat must
compensate for the entropy reduction, i. e., - S
N
, by the systematization. Thus, the following
inequality must hold:
( ; , ) ( , ) 0
a N s N N
E M N S E N S TS > (1)
where T is the temperature. In other words, this indicates the upper boundary of
systematization (negentropy) by entropy production (Otsuka & Nozawa, 1998). However,
organisms must have developed the systematization to increase the acquired energy
through the evolutionary process of gene and genome duplication, nucleotide base
substitutions and selection, and this is the main problem in the present study. The larger
value of E
a
(M; N, S
N
) E
s
(N, S
N
) TS
N
gives a measure for the biological processes to
proceed more smoothly. In this sense, the quantity of E
a
(M; N, S
N
) E
s
(N, S
N
) TS
N
, which
an organism produces during one generation, will be called the biological activity of the
organism. The biological activity has thermodynamic connotation as a departure from
equilibrium, but this is in a reverse relation to the free energy in thermodynamics, which
decreases upon any change in a given system by the decrease in internal energy and/or by
the increase in entropy. In an organism, the acquired energy is stored in ATP and NADH
molecules as chemical energy, and it is gradually consumed in the syntheses of
biomolecules under the guidance of the enzymes, without drastically raising the
temperature. In such moderate reactions, the temperature is almost constant, and the
quantity obtained from the biological activity divided by the product of the Boltzmann
constant k and temperature T is considered to be approximately proportional to the self-
reproducing rate of an organism, which will be denoted by R(M; N, S
N
) hereafter.
This concept of biological activity or self-reproducing rate is useful to formulate the large-
scale of evolution arising from the gene duplication and succeeding generation of new
genes. The essence of the present theory considers the following process of evolution in
terms of biological activity. First, the enlarged genome size N + N due to gene duplication
makes the stored energy E
s
(N+N, S
N
) larger than E
s
(N, S
N
), while the acquired energy E
a
(M;
N+N, S
N
) remains almost equal to E
a
(M; N, S
N
). Thus, the biological activity of a variant
bearing duplicated genes becomes lower than that of the original style organism. Moreover,
the biological activity of the variant further decreases by the increase in systematization
from S
N
to S
N+N
, as a new gene generated from the counterpart of duplicated genes is
incorporated into an extended system of regulation and control. However, such a variant
with the lower activity is not necessarily extinct but has a chance to recover as a new style of
organisms, if the new gene begins expressing a new biological function to raise the acquired
energy from E
a
(M, N+N, S
N
) to E
a
(M, N+N, S
N+N
) by utilizing the new material and
energy source M other than M, or by moving to a new living area or by utilizing M more
efficiently in the case of M = M. This process of the large-scale evolution will be
mathematically formulated to estimate the probabilities of generating new genes, for the

Gene Duplication

6
first type of organisms in section 3, for the second type in section 4 and for the third type in
section 5.
3. Prokaryotes and lower eukaryotes in the monoploid state
For the mathematical description, the set of variables (N
i
, S
Ni
) characterizing a variant i will
be simply denoted as a single variable x
i
, unless the description of changes in its content is
necessary. In the population of monoploid organisms taking a common material and energy
source M, the number n(x
i
;t) of variants, each characterized by the monoploid genome x
i
,
obeys the following time-change equation.

,
( )
( ; ) { ( ) ( ; ) ( )} ( ; ) ( ) ( ; ) ( ; )
i xi i i i xi xj j j
j i
d
n x t Q t R M x D x n x t q t R M x n x t
dt

= +

(2)
where the self-reproducing rate and death rate of the variant x
i
are denoted by R(M;x
i
) and
D(x
i
), respectively. The apparent decrease factor Q
xi
(t) in the self-reproducing rate of the
variant x
i
is related with the mutation term q
xj,xi
(t) from the variant x
i
to other kinds of
variants x
j
s in the following way.

,
( )
( ) 1 ( )
xi xj xi
j i
Q t q t
=

(3)
If the quantity q
xi,xi
(t) defined by Q
xi
(t) -1 is introduced, the restriction j i can be removed
from the summation of the second term on the right side of Eq. (2). For investigating the
population behavior, Eq. (2) is transformed into the following two types of equations; one
concerning the total number of all kinds of variants defined by B(t) =
i
n(x
i
;t) and another
concerning the fraction f(x
i
;t) of variants x
i
defined by n(x
i
;t)/B(t).
( ) ( ; ) ( )
av
d
B t W M t B t
dt
= (4)

,
( ; ) { ( ; ) ( ; )} ( ; ) ( ) ( ; ) ( ; )
i i av i xi xj j j
j
d
f x t W M x W M t f x t q t R M x f x t
dt
= +
(5)
where the increase rate W(M; x
i
) of variant x
i
and the average increase rate W
av
(M;t) of
organisms in the population are defined by the following forms, respectively.

( ; ) ( ; ) ( )
i i i
W M x R M x D x
(6)
( ; ) ( ; ) ( ; )
av i i
i
W M t W M x f x t
(7)
Strictly, the nucleotide base change occurs due to the miss in repairing damaged bases,
while the gene duplication occurs by the illegitimate crossing over of DNA strands upon
replication. Although they are simply represented by the mutation term q
xi,xj
(t) in the above
mathematical formulation, the point mutation due to nucleotide base change and the gene
duplication are distinguished from each other in the following mathematical treatment.

7
Darwinian evolution corresponds to the evaluation of the time-change of variant fractions
mainly by the first term on the right side of Eq. (5), as discussed by Eigen (1971). If the
increase rate W(M;x
i
) of an occasionally generated mutant x
i
is greater than the average
increase rate, that is, W(M;x
i
) - W
av
(M;t) > 0, the fraction f(x
i
;t) increases with time according
to the first term on the right side of Eq. (5). The increase in the fraction of such variants x
i

gradually raises the average increase rate W
av
(M;t), resulting in the increase in the total
number B(t) of organisms according to Eq. (4), although this increase is ultimately stopped
by the decrease in available material M. On the other hand, the fraction f(x
i
;t) decreases
when W(M;x
i
) W
av
(M;t) < 0. Thus, the organisms taking a common material and energy
source M are elaborated by mutation and selection, and most of them finally reach the ones
with the optimum increase rate, each characterized by x
opt
. However, such Darwinian
evolution may only hold for the point mutations in existing genes.
The large-scale evolutionary process of generating new gene(s) from gene duplication is
obtained by evaluating the fraction of variants up to the first and higher orders of the
mutation term. For this illustration, Eq. (5) will be formally integrated with respect to
time t:

,
0 0
0
( ; ) exp[ { ( ; ) ( ; )} ][ ( ) ( ; ) ( ; )
[ { ( ; ) ( ; ')} '] ( ; 0)]
t t
i i av xi xj j j
j
i av i
f x t W M x W M d q R M x f x
W M x W M d d f x

=
+
(8)
After the organisms x
opt
have become dominant in the population, W
av
(M;t) is approximately
equal to W(M; x
opt
), the fractions of variants except for x
opt
are neglected on the right side of
Eq. (8), and the mutation term q
xi,xopt
(t) is replaced by the mutation rate q
xi,xopt
defined as an
average of mutation terms during a sufficiently long time t, i. e.,

, ,
0
1
( )
t
xi xopt xi xopt
q q d
t

(9)
Then, the fraction f(x
i
) of variants x
i
is finally related with the fraction f(x
opt
) of dominant
organisms x
opt
in the following form.

,
( ; )
( ) ( )
( ; ) ( ; )
xi xopt opt
i opt
opt i
q R M x
f x f x
W M x W M x
=
(10)
Among such satellite variants, the variant arising from the gene duplication is especially
notable in the sense that it has the potential to generate a new gene from the counterpart of
duplicated genes. If the probability of generating a new gene I from the duplicated part in x
i

is denoted by q
xI
,
xi
, a new style of the organism carrying the new gene I is generated from
the original style of an organism with the following probability P
m1
(x
I
x
i
x
o
).

, ,
1
( ; )
( )
( ; ) ( ; )
xI xi xi xo o
m I i o
o i
q q R M x
P x x x
W M x W M x
=
(11)
where x
opt
is rewritten into x
o
with the meaning of the original style of an organism. Here, x
i

and x
I
correspond to (N+N, S
N
) and (N+N,S
N+N
), respectively, in terms of the set of
variables characterizing an organism in section 2.

Gene Duplication

8
When a biologically meaningful character is newly exhibited by two new genes generated
from different origins of gene duplication, the variant, which experienced gene duplication
i, must successively experience further gene duplication j in the other part of the genome to
exhibit such a new character. The fraction f(x
ij
; t) of such variants x
ij
obeys the following
equation as a special case of Eq. (5).

,
( ; ) { ( ; ) ( ; )} ( ; ) ( ) ( ; ) ( ; )
ij ij av ij xij xi i i
d
f x t W M x W M t f x t q t R M x f x t
dt
= + (12)
where q
xij,xi
(t) represents the mutation term from the variant x
i
to the variant x
ij
and the
smaller terms including the mutation from the variant x
ij
to other variants are neglected. By
formally integrating Eq. (12), the fraction f(x
ij
) of variants x
ij
is finally expressed as

,
( ; )
( ) ( )
( ; ) ( ; )
xij xi i
ij i
opt ij
q R M x
f x f x
W M x W M x
=
(13)
where W
av
(M;t) is approximated to be W(M;x
opt
) and the mutation term q
xij,xi
(t) is replaced
by the mutation rate q
xij,xi
, i. e.,

, ,
0
1
( )
t
xij xi xij xi
q q d
t

(14)
By inserting the expression (10) of fraction f(x
i
) into the right side of Eq. (13), the fraction
f(x
ij
) of variants x
ij
is related with the fraction f(x
opt
) of dominant organisms x
opt
by the second
order of mutation rates in the following form.

, ,
( ; ) ( ; )
( ) ( )
( ; ) ( ; ) ( ; ) ( ; )
xij xi i xi xopt opt
ij opt
opt ij opt i
q R M x q R M x
f x f x
W M x W M x W M x W M x
=

(15)
Thus, a new style of the organism x
IJ
carrying new genes I and J is generated from the
original style of an organism x
o
with the following probability P
m2
(x
IJ
x
ij
x
o
).

, , ,
,
2
( ; )
( ; )
( )
( ; ) ( ; ) ( ; ) ( ; )
xIJ xIj xI xi xij xi i
xi xo o
m IJ ij o
o ij o i
q q q R M x
q R M x
P x x x
W M x W M x W M x W M x
=

(16)
where q
xIJ,xIj
is the probability of generating the new gene J from the duplicated part in j. This
procedure can be easily extended to the general case of successively generating three or
more new genes.
Before describing the result of the general case, the expression of probabilities (11) and (16)
will be simplified by assuming that the gene duplication only reduces the self-reproducing
rate of the variant without any influence on the death rate. When the self-reproducing rate
of the original style organism is simply denoted by R and that of the variant x
i
is expressed
as R(1-s
1
) with the reduction factor satisfying 0 < s
1
< 1, the probability (11) is simply
expressed as

1
1
1
( )
m I i o
Q
P x x x
s
= (17)

9
where q
xI,xi
q
xi,xo
is denoted by Q
1
. In the same way, the self-reproducing rate of the variant x
ij
is
denoted as R(1- s
1
- s
2
) with the additional reduction factor s
2
under the condition of 0 < s
1
+ s
2
<
1 and q
xIJ,xIj
q
xI,xi
q
xij,xi
q
xi,xo
is denoted by Q
2
. The expression of the probability (16) then becomes

0
5
10
15
20
25
1 3 5 7 9 11
12s
P
m
n
/
Q
n
Pm1/Q1
Pm2/Q2
Pm3/Q3
Pm4/Q4
Fig. 1. The probabilities of generating new genes from gene duplication in the monoploid
organism. On the basis of Eq. (20), the values of P
mn
/Q
n
are plotted against the twelve-fold
reduction factor 12s for n = 1, 2, 3 and 4. Although the value of Q
n
becomes smaller for a
larger value of n, the plotting of the probability P
mn
in the unit of Q
n
makes the figure
compact. The probability P
m1
is present in a whole range of reduction factor 0 < s < 1. As the
number of n increases, however, the range of reduction factor s, where the probability P
mn
is
present, is narrowed to 0 < s < 1/n.

1
2 2
1 1 2
(1 )
( )
( )
m IJ ij o
s
P x x x Q
s s s
=
+
(18)
This expression of probabilities (17) and (18) is easily extended to express the probability of
successively generating n kinds of new genes in the following way.

1 1 2 1 2 3 1
1 1 2 1 2 3
(1 )(1 ) (1 )
( ) ( )
n
mn n
n
s s s s s s s
P Q
s s s s s s s

=
+ + + + +
(19)
The reduction factors s
i
s in Eq. (19) are in the relations of 0 < s
1
+ s
2
+
.
+ s
n
< 1 and 0 < s
1
,
s
2
,
, s
n
< 1. Strictly, the values of s
i
s are different depending on the length of duplicated
sequences and on the order of gene duplication events. For the simple investigation of the n
dependence of P
mn
, however, these reduction factors are assumed to be commonly equal to
one variable s. Then, the first relation becomes 0 < s < 1/n, and Eq. (19) is reduced to

(1 )(1 2 )(1 3 ) {1 ( 1) }
!
mn n
n
s s s n s
P Q
n s

= (20)

Gene Duplication

10
On the basis of this expression (20), the probabilities P
mn
s for several values of n are plotted
against the reduction factor s in Fig. 1. In the case of n = 1, the reduction factor s is permitted
in a whole range of 0 < s < 1 and the probability P
m1
of generating a new gene is present in
this range. This means that the monoploid organism is suitable to create a new gene step by
step, testing the biological function of the new gene product, even if the gene size is large.
As the value of n increases, however, the reduction factor s is restricted to the narrower
range of 0 < s < 1/n. When the monoploid organism creates simultaneously multiple kinds of
new genes from different origins of gene duplication, therefore, these genes are obliged to
be of a smaller size. Moreover, the probability P
mn
is also decreased as the value of n
increases. This is because Q
n
becomes smaller for the larger value of n. Thus, it is difficult for
the monoploid organism to evolve a new character which requires the expression of many
kinds of new and large genes. This result is common to the prokaryote with a single DNA
molecule and the lower eukaryote with the plural number of chromosomes, if the latter does
not conjugate to exchange homologous chromosomes.
4. The monoploid eukaryotes that exchange homologous chromosomes
through conjugation
Some monoploid eukaryotes with the plural number of chromosomes conjugate to form a
zygote during their life cycle, and the zygote produces monoploid descendants by
exchanging homologous chromosomes upon the meiosis. Although the conjugation also
occurs in prokaryotes, it only takes place to exchange plasmids and partial genes. Originally,
the conjugation would have evolved to avoid the accumulation of disadvantageous
mutations in a special lineage and to maintain the stability of a population by weakening the
influence of such mutations. However, the conjugation in the eukaryote with the plural
number of chromosomes makes it possible to produce the descendant receiving two or more
new genes, even if these new genes are relatively large. Thus, the conjugation of such
eukaryotes is considered to be the strategy to overcome the difficulty of generating many
and large new genes from the successive gene duplication in a single lineage of monoploid
organisms. For this illustration, several examples will be first listed in the following
subsections 4.1 to 4.3, and they are used to estimate the probabilities of producing the
descendant received more new genes by the conjugation of variants, each carrying a smaller
number of new genes.
4.1 The probability of producing the descendant received two new genes
Such a descendant is produced from the conjugation of two types of variants, one carrying a
new gene I on a chromosome C
1
and another carrying a new gene J on another kind of
chromosome C
2
. The genome of the variant carrying the new gene I is denoted by (C
1I
, C
20
)
and the genome of another variant carrying the new gene J is by (C
10
, C
2J
). The conjugation
of these two types of variants yields the zygote, whose genome constitution is represented
by (C
1I
, C
10
; C
2J
, C
20
). If the homologous chromosomes are randomly partitioned into two
daughter cells, the probability P
c2
of producing the new monoploid descendant received the
genome (C
1I
, C
2J
) is calculated to be P
m1
2
/2.
4.2 The probability of producing the descendant received three new genes
The descendant received three new genes I, J and K can be produced from the conjugation of
variants, one carrying one new gene I and another carrying two new genes J and K. Two
cases are considerable for this production.

11
One is the case that the new gene I is encoded on the chromosome C
1
and both new genes J
and K are encoded on another kind of chromosome C
2
. Then, the genome of the variant
carrying the new gene I is denoted by (C
1I
, C
20
) and the genome of another variant carrying
the new genes J and K is denoted by (C
10
, C
2JK
). The conjugation of these two variants forms
the zygote (C
1I
, C
10
; C
2JK
, C
20
), which can produce four types of monoploid descendants, (C
1I
,
C
2JK
), (C
1I
, C
20
), (C
10
, C
2JK
) and (C
10
, C
20
). If the homologous chromosomes are equivalently
partitioned into two daughter cells, regardless of carrying new genes or not, the new
monoploid descendant (C
1I
, C
2JK
) is produced with the probability of P
m1
P
m2
/2.
In the second case, the new genes J and K are encoded on separate chromosomes. If the
chromosome carrying the new gene K is denoted by C
3
, the genome of the variant carrying
new genes J and K is represented by (C
10
, C
2J
, C
3K
). The conjugation of this variant and the
variant (C
1I
, C
20
, C
30
) forms the zygote (C
1I
, C
10
; C
2J
, C
20
; C
3K
, C
30
). Under the random partition
of homologous chromosomes, this zygote yields a new monoploid descendant (C
1I
, C
2J
, C
3K
)
with the probability of P
m1
P
m2
/4 .
As a whole, 3P
m1
P
m2
/4 is obtained for the probability P
c3
of producing a new monoploid
organism received three new genes by conjugation.
4.3 The probability of producing the descendant received four new genes
The highest probability of producing the descendant received four new genes is obtained
by the conjugation of two variants, one carrying two new genes I and J, and another
carrying other two new genes K and L. The following three cases (i) ~ (iii) are
considerable. (i) The new genes I and J are encoded on the chromosome C
1
in one variant,
while the new genes K and L are encoded on the chromosome C
2
in another variant. The
conjugation of these two variants forms the zygote (C
1IJ
, C
10
; C
2KL
, C
20
), which yields four
types of monoploid descendants, (C
1IJ
, C
2KL
), (C
1IJ
, C
20
), (C
10
, C
2KL
) and (C
10
, C
20
). If the
homologous chromosomes are randomly partitioned into two descendants, the
probability of producing the monoploid descendant (C
1IJ
, C
2KL
m2
2
/2.
(ii) The new genes I and J are encoded on the chromosome C
1
in one variant but the new
genes K and L are encoded on the chromosomes C
2
and C
3
, respectively, in another
variant. The conjugation of these two variants forms the zygote (C
1IJ
, C
10
; C
2K
, C
20
; C
3L
,
C
30
). If the homologous chromosomes in each kind of 1, 2 and 3 are randomly partitioned
into two daughter cells, the probability of producing the monoploid descendant (C
1IJ
, C
2K
,
C
3L
m2
2
/4. (iii) The new genes I and J are encoded on the chromosomes
C
1
and C
2
, respectively, in one variant, while the new genes K and L are encoded on the
chromosomes C
3
and C
4,
respectively, in another variant. The conjugation of these two
variants forms the zygote (C
1I
, C
10
; C
2J
, C
20
; C
3K
, C
30
; C
4L
, C
40
), and yields the monoploid
descendant (C
1I
, C
2J
, C
3k
, C
4L
) with the probability P
m2
2
/8.
The monoploid organism receiving four new genes can be also produced by the conjugation
of a variant with one new gene I on the chromosome C
1
and another variant with three new
genes J, K and L. The following three cases (iv) ~ (vi) are considerable for the location of the
three new genes J, K and L. (iv) The three new genes are encoded on the same chromosome
C
2
. In this case, the conjugation of the two variants forms the zygote (C
1I
, C
10
; C
2JKL
, C
20
) and
yields the monoploid descendant (C
1I
, C
2JKL
) with the probability of P
m1
P
m3
/2. (v) The new
gene J is encoded on the chromosome C
2
and the other two new genes K and L are encoded
on the chromosome C
3
. The conjugation of these variants forms the zygote (C
1I
, C
10
; C
2J
, C
20
;
C
3KL
, C
30
) and yields the descendant monoploid (C
1I
, C
2J
, C
3KL
) with the probability of

Gene Duplication

12
P
m1
P
m3
/4. (vi) The new genes J, K and L are encoded on the chromosomes C
2
, C
3
and C
4
,
respectively. In this case, the probability of producing the monoploid descendant (C
1I
, C
2J
,
C
3K
, C
4L
) is further decreased to be P
m1
P
m3
/8.
As illustrated in the above examples in subsections 4.1 to 4.3, the probability P
c2n
of
producing the monoploid descendant received the even number 2n of new genes through
one time of conjugation is generally expressed as

0
10
20
30
40
1 2 3 4 5 6 7 8 9 10 11 12
12s
P
c
2
n
/
Q
2
n
,
P
c
2
n
+
1
/
Q
2
n
+
1
Pc2/Q2
Pc3/Q3
Pc4/Q4
Pc5/Q5
Fig. 2. The probabilities of producing the descendants received multiple kinds of new genes
by the conjugation of monoploid organisms. The probability P
c2n
of producing the
descendant received 2n kinds of new genes is simply expressed as the square of the
probability P
mn
, i. e., P
c2n
= P
mn
2
. In the same way, the probability P
c2n+1
of producing the
descendant received (2n+1) kinds of new genes is expressed as the product of the
probabilities P
mn+1
and P
mn
, i. e., P
c2n+1
= P
mn+1
P
mn
. Using the relations of Q
2n
= Q
n
2
and Q
2n+1

= Q
n
Q
n+1
, P
c2n
/Q
2n
and P
c2n+1
/Q
2n+1
are plotted against the twelve- fold reduction factor 12s for
n = 1 and 2. It should be noted that the probabilities P
c2n
and P
c2n+1
are present in the wider
range of reduction factor than the probabilities P
m2n
and P
m2n+1
shown in Fig. 1, respectively.

2
2 , 1, 1 1 1
................
c n n n mn n n mn mn
P a P b P P
+ +
= + +
(21)
and the probability P
c2n+1
of producing the monoploid descendant received the odd number
(2n+1) of new genes is expressed as

2 1 , 1 1 2, 1 2 1
...........
c n n n mn mn n n mn mn
P a P P b P P
+ + + + +
= + +
(22)
Although the coefficients a
n,n
, a
n,n+1
, b
n+1,n-1
, b
n+2, n-1
etc. depend not only on the number of
new genes but also on the distribution of new genes over chromosomes in a complex way,
the first terms are most important on the right sides of Eqs. (21) and (22), respectively. This
is because the probabilities P
mn
and P
mn+1
in these terms are present in the wider range of
reduction factor than those in other terms, as indicated in the preceding section. Thus, P
c2
~
P
mn
2
and P
c2n+1
~ P
mn
P
mn+1
, without the coefficients a
n,n
and a
n,n+1
, are plotted against the

13
reduction factor s for some values of n in Fig. 2. The probability P
c2n
is present in the same
range of reduction factor as the probability P
mn
is present and the probability P
c2n+1
is
present in the same range of reduction factor as the probability P
mn+1
is. This indicates that
the larger size of new genes not generated from the successive gene duplication in a single
lineage of monoploid organisms can be assembled into an organism through conjugation.
Although the values of P
c2n
and P
c2n+1
are smaller than those of P
mn
and P
mn+1
, respectively,
due to the relations of Q
2n
< Q
n
and Q
2n+1
< Q
n+1
, the smaller value of the probability only
means the longer time for the monoploid organism to receive 2n or (2n+1) new genes
through the conjugation of variants than the time for a single lineage of monoploid
organisms to generate n or (n+1) new genes from gene duplication. If these larger new genes
assembled by conjugation endow the descendant with a superior new character, such
descendants increase their fraction as a new style of organisms. In this sense, it should be
also noted that the descendant receiving n( = 3, 4, 5
,..
) kinds of new genes can be
produced with the lower probability of (1/2)
n(n-1)/2
P
m1
n
by the successive conjugation of
variants having experienced gene duplication on different kinds of chromosomes. Such
successive hybridization of different variants, each of them carrying one new gene, may
become the main course to yield a new style of organisms carrying three or more new genes,
if the homologous chromosomes different in carrying two or more new genes, such as those
appeared in the first case of subsection 4.2 and in (i), (ii), (iv) and (v) of subsection 4.3, are
severely incompatible upon the meiosis in the zygote.
At any rate, the eukaryote with the plural number of chromosomes is suitable to create new
characters each expressed by many kinds of new genes, through the conjugation exchanging
homologous chromosomes. This explains the diversity of various living styles of
eukaryotes, ranging from the unicellular organisms called the Protoctista evolving various
intracellular organs to the multicellular organisms evolving cell differentiation. As will be
discussed in the last section, it is evident from the phylogeny of eukaryotes that the
multicellularity and cell differentiation have also started in the monoploid eukaryotes,
although the higher hierarchy of cell differentiation has developed in the diploid
eukaryotes.
5. Higher eukaryotes in the diploid state
The higher eukaryote in the diploid state is characterized by the pairs of homologous
chromosomes, and its large-scale evolution contains the process to establish the homozygote
of new genes as well as their generation from gene duplication. Although the number of
homologous chromosome pairs is different depending on the species of diploid organisms, a
specific pair of homologous chromosomes (x
i
, x
k
) will be first focused for simplicity, where
the suffixes i and k denote different mutations on the respective chromosomes. The number
n(x
i
,x
k
;t

) of variants carrying such a pair (x
i
,x
k
) obeys the following time-change equation in
the population of organisms exchanging the homologous chromosomes upon reproduction.

,
' ' ' ' ' ' ' ' ' '
', '( , ) ,
( , ; ) ( , ; ) ( ; , ) ( , ; ) ( , ; ) ( , ) ( , ; )
( , , ; ) ( ; , ) ( , ; ) ( , ; )
i k i k ijxkl i k ijxkl i j k l i k i k
j l
i k i k i j k l i k i j k l i j k l
i k i k j l
d
n x x t Q x x t R M x x n x x t n x x t D x x n x x t
dt
q x x x x t R M x x n x x t n x x t

=
+

(23)

Gene Duplication

14
where R(M; x
i
, x
k
)
ijxkl
is the rate of producing the children (x
i
, x
k
) from the mating of a variant
(x
i
, x
j
) with another variant (x
k
, x
l
) under a common material and energy source M, and D(x
i
,
x
k
) is the death rate of the organism (x
i
, x
k
). The apparent decrease factor Q(x
i
, x
k
; t)
ijxkl
is
related with the mutation term q(x
i
,x
k
x
i
,x
k
; t)
ijxkl
in the following way.

' '
', '( , )
( , ; ) 1 ( , , ; )
i k ij kl i k i k ij kl
i k i k
Q x x t q x x x x t

(24)
Although Eq. (23) makes no distinction between the male and the female for simplicity, this
distinction does not essentially alter the following process of evolution.
In the same way as for monoploid organisms, the population behavior of diploid organisms
becomes transparent by transforming Eq. (23) into the equation concerning the total number
of organisms given by B(t) =ikn(x
i
, x
k
; t) and that concerning the fraction of variants (x
i
, x
k
)
defined by f(x
i
, x
k
; t) = n(x
i
, x
k
; t)/B(t). These equations are expressed in the following forms,
respectively.

( ) ( ) ( )
d
B t W t B t
dt
=
(25)

', ' ' ' ' ' ' ' ' '
', ' ,
( , ; ) { ( , ; ) ( )} ( , ; )
( , ; ) ( ; , ) ( , ; ) ( , ; ) ( )
i k i k i k
i k i k i jxk l i k i j k l i j k l
i k j l
d
f x x t W x x t W t f x x t
dt
q x x x x t R M x x f x x t f x x t B t
=
+
(26)
where the increase rate W(x
i
, x
k
; t) of the variant (x
i
, x
k
) is defined by

( , ; ) ( ; , ) ( , ; ) ( , ; ) ( ) / ( , ; ) ( , )
i k i k ij kl i j k l i k i k
j l
W x x t R M x x f x x t f x x t B t f x x t D x x
(27)
the average increase rate W (t) is by

( ) ( , ; ) ( , ; )
i k i k
i k
W t W x x t f x x t
(28)
and q(x
i
,x
k
x
i
,x
k
; t)
ijxkl
is defined by Q(x
i
, x
k
; t)
ijxkl
1. If the suffixes i, j, k and l denote the
point mutations in existing genes, Eq. (26) represents Darwinian evolution gradually leading
to the organisms with an optimal increase rate, each characterized by (x
opt
, x
opt
).
Because the gene duplication occurs only rarely, it is natural to consider that the large-scale
evolution due to gene duplication starts after the organisms (x
opt
, x
opt
) have been dominant in
the population. If the chromosome having experienced gene duplication is newly denoted
by x
i
and the point mutation is neglected, the fraction f(x
i
, x
opt
; t) of variants (x
i
, x
opt
) obeys
the following equation as a special case of Eq. (26).

2
( , ; ) { ( , ; ) ( )} ( , ; )
( , , ; ) ( ; , ) ( , ; ) ( )
i opt i opt i opt
i opt opt opt optoptxoptopt opt opt optoptxoptopt opt opt
d
f x x t W x x t W t f x x t
dt
q x x x x t R M x x f x x t B t
=
+
(29)
where the increase rate W(x
i
,x
opt
;t) of the variant (x
i
, x
opt
) is given by
( , ; ) ( ; , ) ( , ; ) ( ) ( , )
i opt i opt iopt optopt opt opt i opt
W x x t R M x x f x x t B t D x x
= (30)

15
and the average increase rate ( ) W t is by
( ) ( , ; ) ( , ; ) ( , ; ) ( , ; )
i opt i opt opt opt opt opt
W t W x x t f x x t W x x t f x x t = + (31)
The probability of generating a new style of organisms carrying a new gene is derived from
Eq. (29). In the population where the organisms (x
opt
, x
opt
) are dominant, W (t) is
approximately equal to W(x
opt
, x
opt
), and both f(x
opt
, x
opt
; t) and B(t) are hardly dependent on
time. Eq. (29) is then integrated to give the following relation between the fraction of
variants f(x
i
, x
opt
) and that of dominant organisms f(x
opt
, x
opt
).

2
,
( , , ) ( ; , )
( , ) ( , )
( ) ( , )
i opt opt opt optoptxoptopt opt opt optopt optopt
i opt opt opt
opt opt i opt
q x x x x R M x x
f x x f x x B
W x x W x x

(32)
where the rate of generating the gene duplication i is defined for a sufficiently long time t by

0
1
( , , ) ( , , ; )
t
i opt opt opt optoptxoptopt i opt opt opt optoptxoptopt
q x x x x q x x x x d
t

(33)
Although this relation (32) seems to be different in including the population size B from Eq.
(10) of monoploid organisms at first glance, the denominator on the right side of Eq. (32)
also contains the population size B as seen in Eqs. (27) and (30). If the population size is
large enough to neglect the difference in death rate between the variant (x
i
, x
opt
) and the
dominant organism (x
opt
, x
opt
), therefore, the difference in the increase rate W(x
opt
, x
opt
) - W(x
i
,
x
opt
) is approximately equal to {R(M; x
opt
, x
opt
)
optoptxoptopt
- R(M; x
i
, x
opt
)
ioptxoptopt
}Bf(x
opt
, x
opt
), and
Eq. (32) is reduced to be

( , , ) ( ; , )
( , ) ( , )
( ; , ) ( ; , )
i opt opt opt optoptxoptopt opt opt optopt optopt
i opt opt opt
opt opt optopt optopt i opt iopt optopt
q x x x x R M x x
f x x f x x
R M x x R M x x

(34)
This is essentially the same form as Eq. (10) of the monoploid organism in the case when the
gene duplication hardly changes the death rate, i. e., D(x
i
) D(x
opt
). Denoting the probability
of generating a new gene I from the gene duplicated part i by q(x
I
x
i
), the probability
P
d1
(x
I
,x
o
x
o
,x
o
) that a new style of the organism (x
I
, x
o
) carrying the new gene I
heterogeneously is generated from the original style of an organism (x
o
, x
o
) is expressed as

1
( ) ( , , ) ( ; , )
( , , )
( ; , ) ( ; , )
I i i o o o ooxoo o o oo oo
d I o i o
o o oo oo i o io oo
q x x q x x x x R M x x
P x x x x
R M x x R M x x

=
(35)
where x
opt
in Eq. (34) is rewritten into x
o
with the meaning of the original type chromosome.
Thus, a new style diploid organism also arises from the minor members in the population
just like the case of monoploid organisms.
However, the content of the above probability in diploid organisms is different from the
case of monoploid organisms in the following points. First of all, the reproducing rate R(M;
x
i
, x
o
)
ioxoo
is only the half of R(M; x
o
, x
o
)
ooxoo
even in the random partition of homologous
chromosomes, and the former may be further decreased by the lowering of the biological
activity of the variant (x
i
, x
o
). Second, the further gene duplication to produce two or more
new genes is hardly expected in the homologous chromosomes (x
i
, x
o
), because the fraction

Gene Duplication

16
of such variants experienced successive gene duplication becomes much lower, not only due
to the severer lowering of biological activity but also by the severer incompatibility of
homologous chromosomes or by the separation of the chromosomes carrying different
origins of duplicated genes in the descendants. That is, if the further gene duplication j
occurs on the chromosome x
i
to yield x
ij
, for example, the incompatibility of chromosomes x
ij
and x
o
becomes severer upon the mitosis and/or the meiosis. If the gene duplication j occurs
on the chromosome x
o
to yield x
j
, on the contrary, the chromosome x
j
is separated from the
chromosome x
i
in the descendants.
In spite of such conservative property, the diploid organism with the plural number of
homologous chromosome pairs can give rise to a new style of an organism getting together
two or more new genes, through the successive hybridization among the satellite variants
having experienced gene duplication on different kinds of chromosomes. As the first
example, the appearance of a new style organism received two kinds of new genes I and J
will be considered by this mechanism of hybridization. In this case, two pairs of
homologous chromosomes (x
0
, x
0
; y
0
, y
0
) are focused, and the probability of generating the
heterozygote (x
I
,x
o
;y
J
,y
o
) from the original style of organisms (x
o
,x
o
;y
o
,y
o
) is considered
through the hybridization of two types of variants (x
i
,x
o
;y
o
,y
o
) and (x
o
,x
o
; y
j
, y
o
). According to
Eq. (35), this probability P
d2
(x
I
,x
o
;y
J
,y
o
x
o
,x
o
;y
o
,y
o
) is given by

2 ,
0
( ; , , ; , )
( ) ( , , ) ( ; , ; , )
( ; , ; , ) ( ; , ; , )
( ) ( , , ) ( ; , ; , )
( ; ,
d I o J o o o o o
I i i o o o ooooxoooo o o o o oooo oooo
o o o o oooo oooo i o o iooo oooo
J j j o o o ooooxoooo o o o o oooo oooo
o
P x x y y x x y y
q x x q x x x x R M x x y y
R M x x y y R M x x y y
q y y q y y y y R M x x y y
R M x
2
; , ) ( ; , ; , )
o o o oooo oooo o o j o oojo oooo
r
x y y R M x x y y

(36)
where r
2
is the ratio of the children received two kinds of new genes I and J, taking the value
of (1/2)
2
in the case of random partition of homologous chromosomes. In order to show the
result of further hybridization process, Eqs. (35) and (36) will be simplified in their
expression at this stage. The probabilities q(x
I
x
i
) and q(x
J
x
j
) of generating new genes I
and J from duplicated parts i and j in Eqs. (35) and (36) may be equal to the corresponding
probabilities q
xI,xi
and q
xIJ,xIj
in Eqs. (11) and (16), respectively, because the nucleotide base
substitution rate is almost common to both eukaryotes and prokaryotes (Kimura, 1980;
Otsuka et al., 1997). Although it is still difficult to estimate the occurrence frequency of gene
duplication, this frequency is also assumed to be common to both monoploid and diploid
organisms, i. e., q
xi,xo
~ q(x
i
,x
o
x
o
,x
o
)
ooooxooo
and q
xij,xi
~ q(y
j
,y
o
y
o
,y
o
)
ooooxoooo
, for simplicity.
The reproducing rates R(M; x
o
,x
o
; y
o
,y
o
)
ooooxoooo
, R(M; x
i
,x
o
; y
o
,y
o
)
ioooxoooo
and R(M;x
o
,x
o
;y
j
,y
o
)
oojoxoooo
are simply denoted by R, R(1 - S
1
) and R(1 - S
2
), respectively, with the reduction
factors S
1
and S
2
, where both S
1
and S
2
satisfy the relation 1/2 < S
1
, S
2
< 1 as noted already.
Eqs. (35) and (36) are then rewritten into

1
1
1
( , , )
d I o o o
Q
P x x x x
S
= (37)
and

2
2 2
1 2
( , ; , , ; , )
d I o J o o o o o
Q
P x x y y x x y y r
S S
= (38)

17
respectively. Here, Q
1
and Q
2
represent the terms q(x
I
x
i
)q(x
i
,x
o
x
o
,x
o
)
ooxoo
and
q(x
I
x
i
)q(x
i
,x
o
x
o
,x
o
)
ooooxoooo
q(y
J
y
j
)q(y
j
,y
o
y
o
,y
o
)
ooooxoooo
, respectively. As the extension,
the probability P
dn
, with which a new style diploid organism carrying n kinds of new genes
heterogeneously is generated from the successive hybridization of variants, is expressed in
the following form.

1 2
n
dn n
n
Q
P r
S S S
=

(39)
where S
i
(i=1, 2,
,n) is the reduction factor in the producing rate of the variant carrying
duplicated genes on the chromosome i, Q
n
is the product of the probabilities of generating n
kinds of new genes from gene duplication on the respective chromosomes and r
n
is the ratio
of the children received these new genes. Although reduction factors S
i
s in Eq. (39)
independently take values in the range of 1/2 < S
i
<1, they are tentatively represented by a
common variable S for a simple illustration of n dependence of P
dn
in a figure. Then, the
probability P
dn
is simply expressed by

n
dn n
n
Q
P r
S
= (40)
These probabilities P
dn
s in Eq. (40) are plotted against the reduction factor S in Fig. 3 for
several values of n. As noted already, the reduction factor S is restricted to the

0
5
10
15
20
25
30
35
1 2 3 4 5 6 7 8 9 10 11 12
12S
P
d
n
/
Q
n
r
n
Pd1/Q1
Pd2/Q2r2
Pd3/Q3r3
Pd4/Q4r4
Pd5/Q5r5

Fig. 3. The probabilities of generating new genes from gene duplication and successive
hybridization in diploid organisms. On the basis of Eq. (40), the values of P
dn
/Q
n
r
n
are
plotted against the twelve-fold reduction factor 12S for n = 1, 2, 3, 4 and 5. The value of r
1
is
equal to one, and the curve of P
d1
vs S is consistent with the curve of P
m1
vs s in Fig. 1, but the
range of reduction factor S is restricted to the range of 1/2 < S < 1. For a larger value of n,
however, the probability P
dn
is still present in the range of 1/2 < S < 1. Although
P
dn+1
/Q
n+1
r
n+1
is larger than P
dn
/Q
n
r
n
in the figure, P
dn+1
is smaller than P
dn
. This is because
Q
n+1
r
n+1
is smaller than Q
n
r
n
, as discussed in the text.

Gene Duplication

18
range of 1/2 < S < 1, and the probability P
dn
is within the range of 2
n
Q
n
r
n
> P
dn
> Q
n
r
n
. If the
homologous chromosomes are randomly partitioned into the children regardless of carrying
a new gene or not, r
n
takes the value of (1/2)
n
and the above relation of P
dn
becomes Q
n
> P
dn

> Q
n
/2
n
. Moreover, the value of Q
n
becomes smaller for the larger value of n, and the
probability P
dn
becomes lower as the number of new genes assembled by hybridization is
increased. The lower probability means the longer time or more generations for a new style
organism carrying more kinds of new genes to appear. Thus, the diploid organism has a
chance to acquire many kinds of new genes by hybridization, but it takes a longer time to
realize this chance.
Moreover, the process to establish the homozygote is further continued after the new style
organism carrying n kinds of new genes heterogeneously is generated with the probability
P
dn
. Although it is laborious to follow this process completely, the essence of this process can
be elucidated by investigating the ratio of children that receive these new genes
homogeneously and heterogeneously from the mating between the organisms each carrying
n kinds of new genes heterogeneously. If the chromosomes in each homologous pair are
randomly partitioned into the children regardless of carrying a new gene or not, the ratio of
children receiving (n-k) kinds of new genes is calculated to be
n
C
k
3
n-k
/4
n
with the
normalization factor 4
n
, where k takes a value ranging from zero to n. This indicates that
more than half of the children receive all new genes (k = 0) for n = 1, 2. If the one or two new
genes exhibit an excellent character, therefore, the descendants increase their fraction
monotonously as a new style of organisms. However, the ratio of children receiving a full
set of new genes becomes smaller for a larger value of n. In the case of n = 5, for example,
the ratio of the children that receive five kinds of new genes (k = 0) decreases to (3/4)
5
, while
other five types of children each appear with the ratio of (3/4)
4
/4 by receiving four kinds of
new genes (k = 1) in different ways. When a biologically meaningful character is expressed
by five kinds of new genes, therefore, only (3/4)
5
of the children succeed in expressing this
character but other five types of children are reserved as those carrying hidden genes for
producing other characters by further hybridization with other types of variants. Such
divergence of characters becomes more outstanding when a larger number of new genes are
required for the expression of a character. This divergent property in the process to establish
many kinds of new genes as the homozygote explains the explosive divergence of body
plans that has occasionally occurred in diploid organisms, because the cell differentiation is
a representative character expressed by many kinds of genes and its hierarchical evolution
constructs body plans, as will be discussed in the next section. Until the new style organisms
are established as the homozygote, the mating between the variants of heterozygote also
regenerates the original style of organisms. The phenomenon called the reversion or
atavism in classical biology may be the vestige of this evolutionary process to establish the
homozygote.
If the influence of transposons is explicitly considered, it makes the above process more
complicated in such a way that duplicated genes are separately transferred to different
kinds of chromosomes. When various origins of duplicated genes or new genes are
concentrated on one chromosome, however, the descendants received such a chromosome
may be extinct due to the incompatibility of this chromosome with its partner chromosome
not carrying any new gene. Thus, many kinds of new genes for expressing a new character
may be scattered over different kinds of chromosomes in survivors just like the result of the
present model scheme.

19
6. Conclusions and discussion
The variants, which experienced gene duplication, first decline to be minor members in a
population by the load of carrying extra gene(s), but some of them revives as a new style of
organisms by the generation of new gene(s) from the counterpart of duplicated genes. After
the new gene(s) appear, the new style organisms increase their fraction being further
elaborated by Darwinian evolution. This course of the large-scale evolution is essentially the
same in any type of organisms, and this is a necessary condition for the new style of
organisms and the original style of organisms to be able to coexist utilizing different
material and energy sources or to live in separate areas, showing a striking contrast to the
survival of the fittest in Darwinian evolution. This evolutionary pattern also gives an
explanation to the punctuated mode of evolution, which has been proposed from
paleontology against the gradual accumulation of variants in Darwinian evolution
(Eldredge & Gould, 1972).
However, the detailed processes of this large-scale evolution are different depending on the
types of genome constitution and transmission. The monoploid organism is suitable to
generate one new gene step by step testing its biological function, but hardly generates
many kinds of new genes simultaneously. The lower eukaryote, whose genome consists of
the plural number of chromosomes, resolves this difficulty to produce a new style of
organisms receiving many kinds of new genes by the conjugation of variants carrying
different origins of new genes. The diploid organism can also produce a new character
responsible for multiple kinds of new genes by the successive hybridization of different
variants but its conservative property requires the succeeding process to establish the
homozygote of these genes. This process becomes longer for a larger number of new genes
to be established. During this long process, the further hybridization with other variants
also occurs, occasionally yielding the explosive divergence of new characters depending on
the combinatorial sets of new genes. This conclusion of the present study explains the
recently revealed evolutionary patterns of prokaryotes and eukaryotes to a great extent,
getting an insight into the problems how and why the monoploid eukaryotes have evolved
to the diploid eukaryotes.
According to the analyses of base-pair changes in ribosomal RNAs, the main lineages of
present-day prokaryotes diverged 3.0x10
9
years ago, developing various chemical syntheses,
O
2
-releasing photosynthesis and O
2
respiration, respectively (Otsuka et al., 1999), after the
earlier divergence of archaebacteria, eubacteria and eukaryotes (Sugaya & Otsuka, 2002).
Several stages from simple electron transport pathways to O
2
respiration and O
2
-releasing
photosynthesis are still observed in the present-day eubacteria and the elongation of the
pathways has taken place stepwise by gene duplication, as can be traced from the amino
acid sequence similarities between their component proteins and the ubiquitous permeases
(Otsuka, 2002; Otsuka & Kawai, 2006), although such similarity search of amino acid
sequences is not systematically carried out yet for chemical syntheses. However, the
excellent abilities of O
2
respiration and O
2
-releasing photosynthesis cannot be fully exhibited
in the simple cell structure of prokaryotes (Otsuka, 2005), and the genome size of the
eubacteria having these abilities is also limited to the order of 10
6
bp compactly encoding
3,000 ~ 4,000 genes like the other prokaryotes (Wheeler et al., 2004).
On the other hand, the eukaryotes have experienced much more evolutionary events until
some of them establish the diploid state. The ancestral eukaryote probably became the
predator of eubacteria by developing the intracellular structure, endocytosis and exocytosis

Gene Duplication

20
as well as the signal transduction network. Such cell structure would have been suitable to
acquire the mitochondria as the endosymbionts of O
2
-respiratory eubacteria, which is
estimated to have occurred 2.0x10
9
years ago (Margulis, 1981; Yang et al., 1985; Otsuka et al.,
1999). Under the supply of abundant ATP molecules efficiently synthesized by the
mitochondria, the yeast Sacchromyces serevisiae, which appeared 1.8 x10
9
years ago (Otsuka et
al., 1997), has expanded its genome to 1.2x10
7
bp encoding 6, 300 genes (Wheeler et al., 2004)
and can take the diploid state under nutrient conditions, although it usually takes the
monoploid state. The enlarged genome consisting of the plural number of chromosomes
also requires the special apparatus for the faithful segregation of sister DNAs upon cell
division, in contrast to the prokaryotes where the membrane attachment mechanism of
DNA only operates (Jacob et al., 1963; Ogden et al., 1988; de Boer, 1993). Multiple kinds of
gene products such as the primitive spindle pole and kinetochore and polar microtubules
are already present in the yeast (Alberts et al., 1994) while several bundles of microtubules
only pass through tunnels in the typical Dinoflagellates (Kubai, 1975; Hearth, 1980; Wise,
1988). Thus, the components of this auxiliary apparatus for cell division may have evolved
step by step at the stage of unicellular eukaryotes. Although the molecular mechanism
underlying the switching from the monoploid to diploid states and vice versa is not fully
clarified yet, the example of yeast indicates that this mechanism itself has also evolved at the
stage of unicellular eukaryotes.
However, the evolution from the monoploid eukaryote to the diploid eukaryote has taken
place considerably gradually via several stages. This is reasonable because the diploid state
is an extreme case of gene duplication. If the genome size jumps from N to 2N, this means
the increase in the stored energy and systematization from E
s
(N,S
N
) and S
N
to E
s
(2N, S
2N
)
and S
2N
. Thus, the acquired energy must be also increased to maintain the biological activity.
As indicated already (Otsuka, 2008), this increase in acquired energy is possibly attained by
the cooperative action of differentiated cells. However, the evolution of cell differentiation
cannot occur suddenly. On this problem, the present result throws light, in the point that the
conjugation of lower eukaryotes with the plural number of chromosomes is suitable to
assemble many kinds of new genes necessary for cell differentiation. In fact, the recently
revealed phylogeny of eukaryotes strongly suggests at least the following five stages in the
evolution from the monoploid to diploid eukaryotes. (a) First, the monoploid eukaryote
evolves the conjugation to exchange the homologous chromosomes. (b) Second, this
eukaryote then develops multicellularity and cell differentiation in the monoploid state by
assembling many kinds of new genes. (c) Third, the cell differentiation also advances to the
cells in the diploid state. (d) Fourth, the eukaryote evolves to alternate the monoploid
generation and the diploid generation. (e) Finally, the eukaryote evolves to the diploid
organism with the higher hierarchy of cell differentiation.
As far as the present knowledge of the phylogeny of eukaryotes and their genome
constitution (Otsuka et al., 1997) is concerned, the first lineages having evolved
multicellularity and cell differentiation are some of the fungi that appear after yeast and the
sea algae, which have further acquired photosynthetic eubacteria as the endosymbionts in
the lineage of fungi (Van den Eynde et al., 1988). However, the most advanced one of them
still remains at the stage (d), alternating the monoploid generation and the diploid
generation. Apart from the lineages of fungi and algae, the evolution of advancing the cell
differentiation to the diploid state has taken place in the animals and the green plants,
whose divergence is estimated to have occurred 1.2x10
9
years ago (Dickerson, 1971). Among

21
them, the green plants, which have also acquired the chloroplasts as the endosymbionts of
photosynthetic eubacteria independently of sea algae, provide a representative example of
the above five stages of evolution from the monoploid organisms to the diploid organisms.
The Cojugatae such as Roya and Spirogyra are at the stage (a), the Chara of Charophyta is at the
stage (b), the Bryophyta is at the stage (c), where the fertilized egg on female gametophyte
grows into sporangium, the Pterophyta is at the stage (d), and the seed plants are at the stage
(e). According to the recent analysis of neutral nucleotide base substitutions in rbcL genes on
the chloroplast genomes (Kawai & Otsuka, 2004), the divergence of Charophyta and
Bryophyta occurred more than 10
9
years ago, the divergence of Bryophyta and Pterophyta
occurred around 4.7x10
8
years ago, and the divergence of Pterophyta and seed plants
occurred about 3.8x10
8
years ago.
The molecular mechanism underlying the cell differentiation is not fully clarified yet, but it
is probably based on a set of receptors, the corresponding ligands, signal transduction
proteins, transcriptional regulators as well as the proteins exhibiting the respective cell-type
specific functions. Moreover, the amino acid sequences of these proteins under the control of
signal transduction network become longer by the attachment of special amino acid residue
repeats such as serines and threonines. Thus, the assembly of so many kinds of large genes
into a genome must have first progressed under the scheme of the conjugation of monoploid
eukaryotes with the plural number of chromosomes. After a set of genes responsible for cell
differentiation are established in the monoploid state, the increase in the repertoire of the
respective members would have occurred relatively easily. In particular, a small number of
nucleotide base substitutions could bring about the expansion of such protein families as
transcriptional regulators, receptors and kinases associated with the signal transduction
network, although these kinds of proteins have their origins at the stage of unicellular
eukaryotes. The increase in acquired energy by the cell differentiation in the monoploid
state makes it possible to realize the cell differentiation in the diploid state. The example of
green plants suggests that the cell differentiation in the diploid state has started from the
zygote and gradually spread to form other organs of diploid cells, resulting in the
alternation of the monoploid generation and the diploid generation. The diploid state is
suitable to protect the differentiated cells from the point mutations, as will be discussed in
the last part of this section, but it takes a longer time or many generations to establish a set
of many genes for advancing the further cell differentiation in the diploid state as the
homozygote. Although this is the barrier lying between the stage (d) and the stage (e), the
diploid organisms having gone over this barrier receive a good chance to produce various
combinatorial sets of new genes leading to the explosive divergence of morphological
characters. Such explosive divergence has the merit of testing simultaneously various
characters for survival.
Although any example of animals at the stages (b) and (c) is hardly found at the present
time, the Cnidaria still alternates the monoploid generation and the diploid one. The
divergence of Cnidaria and the common ancestor of other animals occurred immediately
after the animal-plant divergence (Otsuka & Sugaya, 2003). The famous explosion of body
plans giving rise to Annelida, Mollusca, Arthropoda, Echinodermata and Chordata, which is first
found by the fossil record of Ediacara and Avalon faunas (Mathews & Missarzhersky, 1975;
Rozanov & Zhuravlev, 1992) and of Cambrian Burgess Shale (Gould, 1989) and then
estimated to have occurred successively during the period of 9~6x10
8
years ago by the
analysis of neutral nucleotide base substitutions (Otsuka & Sugaya, 2003), is probably based
on the evolutionary scheme of diploid organisms described in sections 5, because these

Gene Duplication

22
animals show the living style defined as the diploid organism in the present chapter. Such
divergence of body plans occasionally occurred afterwards in each of the above phyla. The
examples well investigated in paleontology are the divergence of Placodermi, cartilaginous
fish and bony fish, the divergence of amphibians, reptiles and mammals, and the divergence
of dinosaurs and birds, which occurred in the Chordata within the recent 4x10
8
years
(Carroll, 1988). The seed plants also show the similar tendency in the successive divergence
of Coniferophyta, Anthophyta and their relatives (Fairon-Demaret & Scheckler, 1987; Rothwell
et al., 1989; Rowe, 1992; Stewart & Rothwell, 1993; Kawai & Otsuka, 2004), although many of
these seed plants can also self-reproduce by the parthenogenesis and their explosive feature
seems mild. Although the explosive divergence of body plans can be also explained by the
biological activity expressed in terms of the interaction between differentiated cells (Otsuka,
2008), the present study derives this divergence from the aspect of the generation of new
genes from gene duplication in diploid organisms.
The fossil record of these examples indicates that the original style of organisms prospered
over a wide region when new styles of organisms diverged, being consistent with the
present theory. The prosperity of the original style of organisms means that their biological
activity is high, and this is necessary to permit the existence of variants carrying duplicated
genes in the population and further to enhance the chance of assembling many kinds of new
genes into a genome by hybridization. This is in contrast with Darwinian evolution
generating new species adapted to the special environment of a geographically isolated
district by accumulating point mutations.
Finally, some discussions will be given to the problem why the cell differentiation has
been shifted from the monoploid state to the diploid state. This problem arises from the
present result that the diploid organism is not necessarily superior to the monoploid
organism with the ability of exchanging homologous chromosomes in assembling many
kinds of new genes for cell differentiation. The main reason of this shifting may be the
protection of differentiated cells from the point mutations due to the miss in repairing
damaged nucleotide bases. First of all, many more genes are needed to develop the higher
hierarchy of cell differentiation. In fact, the genome size of higher eukaryotes is expanded
to the order of 10
8
~ 10
9
bp, e. g., 1.2x10
8
bp encoding 24,000 genes in Arabidopsis thaliana,
1.4x10
8
bp encoding 13,000 genes in Drosophila melanogaster and 3.1x10
9
bp encoding 30,000
genes in Homo sapiens (Wheeler et al., 2004). Second, it takes a longer time, one or more
years, to develop the higher hierarchy of cell differentiation to form an adult form in the
higher eukaryotes, although the growth rate and the lifetime seem to be further regulated
differently depending on species. On the other hand, the mutation rate due to the miss in
repair is 10
-9
per site per year in eukaryotes as well as in prokaryotes (Kimura, 1980;
Otsuka et al., 1997). As the evidence for the above discussion, the males of some species of
ants and bees are born by the haploid parthenogenesis, showing that the monoploid state is
sufficient for the high hierarchy of cell differentiation during their short lifetime.
Although the accuracy in repairing damaged DNAs can be raised by the additional
energy for proofreading (Hopfield, 1974), the evolution of organisms has not been
directed to use such additional energy. On the contrary, the nucleotide base substitution
rate becomes about tenfold faster in animal mitochondrial genome than in the host cell
genome, as is used to resolve the phylogeny of recently diverged animals (Hasegawa et
al., 1985; Pesole et al., 1999; Otsuka et al., 2001). This faster mutation rate strongly
suggests that the energy to proofread the small genome of mitochondria is diminished
and instead the saved energy is used to raise the biological activity of the host cell. For the

23
same sequence length of gene duplication, therefore, the reduction factor may take a
smaller value in animals than in lower eukaryotes and prokaryotes. Thus, the fraction of
variants carrying the hidden genes generated from gene duplication may be high
enough to hybridize between them in higher eukaryotes, especially in animals. Such
hidden genes belong to the category of genetic polymorphism, which has been first
proposed by Ford (1965) and is subsequently disclosed by electrophoretic studies,
although the genetic polymorphism was only regarded as the result of random fixation
of selectively neutral or nearly neutral mutations by the neutralist (Kimura, 1977).
It is still somewhat mysterious that the introns and spacers are more expanded in animal
genomes than in the genomes of other eukaryotes. Such expansion can be seen from the
ratio of the genome size to the number of encoded genes described above. It is conceivable
that the introns are necessary for messenger RNAs to pass through the nuclear membrane
and the spacers enhance the crossing over of homologous chromosomes without injuring
established genes, but the expansion of introns and spacers in the higher eukaryotes might
imply any other biological role of their nucleotide sequences.
7. References
Alberts, B.; Bray, D.; Lewis, J.; Raff, M.; Roberts, K. & Watson, J. D. (1994). Molecular Biology
of the Cell. 3
rd
Ed. Garland Publishing Inc., New York & London. pp. 941-943
Birney, E. & Other fifty persons (Ensembl 2006). Database Issue. Nucleic Acids Res. Vol. 1,
D556-D561
Carroll, R. L. (1988). Vertebrate Paleontology and Evolution. W. H. Freeman, New York
Darwin, C. (1859). The Origin of Species. John Murry, London
de Boer, R. A. J. ( 1993). Chromosome Segregation and Cytokinesis in Bacteria. Curr. Opin.
Cell Biol., Vol 5, pp. 232-237
Dickerson, R. E. (1971). The Structure of Cytochrome c and the Rate of Evolution. J. Mol.
Evol., Vol. 1, pp. 26-45
Dobzhansky, T. (1941). Genetics and the Origin of Species. 2
nd
Ed. Columbia University Press,
New York
Eigen, E. (1971). Selforganization of Matter and Evolution of Biological Macromolecules. Die
Natrwissenshaften, Vol. 58, pp. 465-523
Eldredge, N. & Gould, S. J. (1972). Punctuated Equilibria: An Alternative to Phyletic
Gradualism. In: Models in Paleobiology, T. J. M. Schopf, (Ed.). p. 82, Freeman and
Cooper, San Francisco
Fairon-Demaret, M. & Scheckler, E. S. (1987). Typification and Redescription of Moresnetia
zalesskyi Stockmans, 1948, an Early Seed Plant from the Upper Famennian of
Belgium. Bull Inst Roy Sci Nat Belg Sci Terre, Vol. 57, pp. 193-199
Ferris, S. D. & Whitt, G. S. (1979). Evolution of the Differential Regulation of Duplicated
Gene after Polyploidization. J. Mol. Evol., Vol. 12, pp. 267-317
Fisher, R. A. (1930). The General Theory of Natural Selection. Oxford Univ. Press, London and
New York
Ford, E. B. (1965). Genetic Polymorphism. Faber & Faber, London
Gilbert, W. (1978). Why Genes in Pieces ? Nature, Vol. 271, p. 501
Gould, S. J. ( 1989). Wonderful Life. The Burgess Shale and the Nature of History. W. W. Norton
& Company Inc., New York

Gene Duplication

24
Hasegawa, M.; Kishino, H. & Yano, T. (1985). Dating of the Human-Ape Splitting by a
Molecular Clock of Mitochondrial DNA. J. Mol. Evol., Vol. 22, pp. 160-174
Hearth, I. B. (1980). Variant Mitosis in Lower Eukaryotes: Indicators of the Evolution of
Mitosis? Int. Rev. Cytol., Vol. 64, pp. 1-80
Hopfield, J. J. (1974). Kinetic Proofreading: A New Mechanism for Reducing Errors in
Biosynthetic Processes Requiring High Specificity. Proc. Nat. Acad. Sci. USA, Vol. 71,
pp. 4135-4139
Huxley, J. (1943). Evolution: The Modern Synthesis. The Grandson of T. H. Huxley Explores
Mendelism, Evolutionary Trends, and Genetic Systems. Harper & Row, New York
Ingram, V. M. (1963). The Hemoglobin in Genetics and Evolution. Columbia Press, New York
Jacob, F.; Brenner, S. & Cuzin, F. (1963). On the Regulation of DNA Replication in Bacteria.
Cold Spr. Harb. Symp. Quant. Biol., Vol. 28, pp. 329-348
Kawai, Y. & Otsuka, J. (2004). The Deep Phylogeny of Land Plants Inferred from a Full
Analysis of Nucleotide Base Changes in Terms of Mutation and Selection. J. Mol.
Evol., Vol. 58, pp. 479-489
Kimura, M. (1977). Causes of Evolution and Polymorphism at the Molecular Level.
Proceedings of the Second Taniguchi International Symposium on Biophysics, pp. 1-28,
Mishima, Japan
Kimura, M. (1980). A Simple Method for Estimating Evolutionary Rate of Base Substitutions
through Comparative Studies of Nucleotide Sequences. J. Mol. Evol., Vol. 16, pp.
111-120
Kojima, S. & Otsuka, J. (2000a). Characterization of Organisms by the Paralogous
Relationships of Proteins. Part I. Escherichia coli. Res. Commun. in Biochemi Cell &
Molec. Biology, Vol. 4, pp. 59-82
Kojima, S. & Otsuka, J. (2000b). Characterization of Organisms by the Paralogous
Relationships of Proteins. Part II. Methanococcus jannaschii. Res. Commun. in
Biochemi. Cell & Molec. Biology, Vol. 4, pp. 83-100
Kojima, S. & Otsuka, J. (2000c). Characterization of Organisms by the Paralogous
Relationships of Proteins. Part III. Saccharomyces cerevisiae. Res. Commun. in Biochemi.
Cell & Molec. Biology, Vol. 4, pp. 101-138
Kojima, S. & Otsuka, J. (2002). Characterization of Proteome by Similarity Linkages of
Paralogous Functional Domains and Special Amino Acid-Rich Regions. Part IV.
Drosophila melanogaster. Res. Commun. in Biochemi. Cell & Molec. Biology, Vol. 6, pp.
72-102
Kubai, D. F. (1975). The Evolution of the Mitotic Spindle. Int. Rev. Cytol., Vol. 43, pp. 167-227
Margulis, L. (1981). Symbiosis in Cell Evolution: Life and its Environment on the Early Earth. W.
H. Freeman, San Francisco
Mathews, S. C. & Missarzhevsky, V. (1975). Small Shelly Fossils of Late Precambrian and
Early Cambrian Age: A Review of Recent Work. Q. J. Geol. Soc. London, Vol. 131, pp.
289-304
Mayer, E. (1942). Systematics and the Origin of Species. A Correlation of the Evidence and Points of
View of Systematics WithThose of Other Biological Disciplines, Particularly Genetics and
Ecology. Columbia University Press, New York
Nowak, M. A.; Bonhoeffer, S. & May, R. M. (1994). Spacial Games and the Maintenance of
Cooperation. Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 4877-4881

25
Ogden, G. B.; Pratt, M. J. & Schaechter, M. (1988). The Replication Origin of the Escherichia
coli Chromosome Binds to Cell Membrane only When Hemimethylated. Cell, Vol.
54, pp. 127-135
Ohno, S. (1970). Evolution by Gene Duplication. Spring-Verlag, Berlin.
Otsuka, J.; Nakano, T. & Terai, G. (1997). A Theoretical Study on the Nucleotide Changes
under a Definite Functional Constraint of Forming Stable Base-Pairs in the Stem
Regions of Ribosomal RNAs; Its Application to the Phylogeny of Eukaryotes. J.
Theor. Biol., Vol. 184, pp. 171-186
Otsuka, J. & Nozawa, Y. (1998). Self-Reproducing System can Behave as Maxwells Demon:
Theoretical Illustration under Prebiotic Conditions. J. Theor. Biol., Vol. 194, pp.
205-221
Otsuka, J.; Terai, G. & Nakano, T. (1999). Phylogeny of Organisms Investigated by the Base-
Pair Changes in the Stem Regions of Small and Large Ribosomal Subunit RNAs. J.
Mol. Evol., Vol. 48, pp. 218-235
Otsuka, J.; Kawai, Y. & Sugaya, N. (2001). The Influence of Selection on the Evolutionary
Distance Estimated from the Base Changes between Homologous Nucleotide
Sequences. J. Theor. Biol., Vol. 213, pp. 129-144
Otsuka, J. (2002). An Inquiry into the Evolutionary History of Photosynthetic and
Respiratory Systems from the Similarity Relationships of Member Proteins. In:
Recent Research Developments in Proteins, Transworld Research Network, (Ed.), Vol.
1, pp. 229-256, Kerala, India
Otsuka, J. & Sugaya, N. (2003). Advanced Formulation of Base Pair Changes in the Stem
Regions of Ribosomal RNAs; Its Application to Mitochondrial rRNAs for Resolving
the Phylogeny of Animals. J. Theor. Biol., Vol. 222, pp. 447-460
Otsuka, J. (2004). A Theoretical Characterization of Ecological System by Circular Flow of
Materials. Ecological Complexity, Vol. 1, pp. 237-252
Otsuka, J. (2005). A Theoretical Scheme for the Large-Scale Evolution of Organisms towards
a Higher Order of Organization and Diversity. In: Recent Research Developments in
Experimental & Theoretical Biology, Transworld Research Network, (Ed.), Vol. 1, pp.
93-122, Kerala, India
Otsuka, J. & Kawai, Y. (2006). Phylogenetical Relationships among Permeases and the
Membrane Proteins in Photosynthetic and Respiratory Systems. Trends in
Photochemistry and Photobiology, Vol. 11, pp. 1-22
Otsuka, J. (2008). A Theoretical Approach to the Large-Scale Evolution of Multicellularity
and Cell Differentiation. J. Theor. Biol., Vol. 255, pp.129-136
Pesole, G.; Gissi, C.; Chirico, A. D. & Saccone, C. (1999). Nucleotide Substitution Rate of
Mammalian Mitochondrial Genomes. J. Mol. Evol., Vol. 44, pp. 427-434
Rothwell, G. W.; Scheckler, S. E. & Gillespie, W. H. (1989). Elkinsia gen. nov., a Late
Devonian Gymnosperm with Cupulate Ovules. Bot Gazette, Vol. 150, pp. 170-189
Rowe, N. P. (1992). Winged Late Devonian Seeds. Nature, Vol. 359, p. 682
Rozanov, A. Y. & Zhuravlev, A. Y. (1992). The Lower Cambrian Fossil Record of the Soviet
Union, In: Origin and Early Evolution of the Metazoa. J. H. Lipps & P. W. Signor,
(Eds.), pp. 205-282, Plenum Press, New York and London
Simpson, G. G. (1944). Tempo and Mode in Evolution: A Synthesis of Paleontology and Genetics,
Columbia University Press, New York

Gene Duplication

26
Stewart, N. S. & Rothwell, G. W. (1993). Paleobotany and the Evolution of Plants. pp. 438-467,
Cambridge University Press, Cambridge
Sugaya, N. & Otsuka, J. (2002). The Lineage-Specific Base-Pair Contents in the Stem Regions
of Ribosomal RNAs and Their Influence on the Estimation of Evolutionary
Distances. J. Mol. Evol., Vol. 55, pp. 584-594
Van den Eynde, H.; De Baere, R.; De Roeck, E.; Van de Peer, Y.; Vandenberghe, A.;
Willekens, P. & De Wachter, R. (1988). The 5S Ribosomal RNA Sequences of a Red
Algal Rhodoplast and Gymnosperm Chloroplast: Implication for the Evolution of
Plastids and Cyanobacteria. J. Mol. Evol., Vol. 27, pp. 126-132
Wheeler, D. L.; Church, D. M.; Edgar, R.; Federhen, S.; Helmberg, W.; Madden, T. L.;
Pontius, J. U.; Schuler, G. D.; Schriml, L. M.; Sequeira, E.; Suzek, T. O.; Tatusova, T.
A. & Wagner, L. (2004). National Center of Biotechnology Information. Nucl. Acid
Res., Vol. 32, Database Issue D35
Wise, D. M. (1988). The Diversity of Mitosis: the Value of Evolutionary Experiments.
Biochem. Cell Biol., Vol. 66, pp. 515-529.
Wright, S. (1949). Adaptation and Selection. In: Genetics, Paleontology and Evolution. G. L.
Jepson; G. G. Simpson & E. Mayer, (Eds.), pp. 365-389, Princeton Univ. Press,
Princeton, New Jersey
Yang, D.; Oyaizu, Y.; Oyaizu, H.; Olsen, G. J. & Woese, C. R. (1985). Mitochondrial Origin.
Proc. Natl. Acad. Sci. USA, Vol. 82, pp. 4443-4447
2
Duplicated Gene Evolution Following
Whole-Genome Duplication in
Teleost Fish
Baocheng Guo
1,2,3
, Andreas Wagner
1,2
and Shunping He
3*

1
Institute of Evolutionary Biology and Environmental Studies,
University of Zurich, Zurich
2
The Swiss Institute of Bioinformatics, Quartier Sorge-Batiment Genopode, Lausanne
3
Fish Phylogenetics and Biogeography Group, Key Laboratory of Aquatic Biodiversity and
Conservation, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan
1,2
Switzerland
3
PR China
1. Introduction
Gene and genome duplication have been thought to play an import part during evolution
since the 1930s (Bridges 1936; Stephens 1951; Ohno 1970) . Ohno (1970) proposed that the
increased complexity and genome size of vertebrates has resulted from two rounds (2R) of
whole genome duplication (WGD) in early vertebrate evolution, which provided raw
materials for the evolutionary diversification of vertebrates. Recent genomic sequence data
provide substantial evidence for the abundance of duplicated genes in many organisms.
Extensive comparative genomics studies have demonstrated that teleost fish experienced
another round of genome duplication, the so-called fish-specific genome duplication (FSGD)
(Amores et al. 1998; Taylor et al. 2003; Meyer and Van de Peer 2005). Because the timing of this
WGD and the radiation of teleost species approximately coincided, it has been suggested that
the large number (about 27,000 speciesmore than half of all vertebrate species (Nelson,
2006)) of teleosts and their tremendous morphological diversity might be causally related to
the FSGD event (Amores et al. 1998; Taylor et al. 2001; Taylor et al. 2003; Christoffels et al.
2004; Hoegg et al. 2004; Vandepoele et al. 2004). Semon and Wolfe (2007) showed thousands of
genes that remained duplicated When Tetraodon and zebrafish diverged underwent
reciprocal loss subsequently in these two species may have been associated with
reproductive isolation between teleosts and eventually contributed to teleost diversification. A
study in yeast demonstrated that speciation of polyploid yeasts may be associated with
reciprocal gene loss at duplicated loci (Scannell et al. 2006). Thus, speciation accompanied by
differential retention and loss of duplicated genes after genome duplication may be a powerful
lineage-splitting force (Lynch and Conery 2000).
For two reasons, teleost fish represent an excellent model system to study the retention and
loss of duplicated genes as well as their evolutionary trajectory following whole-genome

*
Corresponding author

Gene Duplication

28
duplication. First, many duplicated genes that resulted from the FSGD event were preserved
in teleost genomes. Second, five teleost genomes have been sequenced and more teleost
genomes are being sequenced. Here, we investigate retention, loss, and molecular evolution
of duplicate genes after the FSGD in five available teleost geomes that include the genomes
of zebrafish Danio rerio, stickleback Gasterosteus aculeatus, medaka Oryzias latipes, Takifugu
Takifugu rubripes, and Tetraodon Tetraodon nigroviridis.
2. Identifying duplicated genes that resulted from the FSGD event throughout
the teleost genomes
We obtained 23,155 gene families from the database HOMOLENS version 4
(ftp://pbil.univ-lyon1.fr/databases/homolens4.php) (Penel et al. 2009), which is based on
the Ensembl release 49. We chose HOMOLENS, because it allowed us to reliably retrieve
sets of orthologous genes for our evolutionary analysis. HOMOLENS is devoted to
metazoan genomes from Ensembl and contains gene families from complete animal
genomes found in Ensembl. HOMOLENS has the same architecture as HOVERGEN (Duret
et al. 1994), in which genes are organized in families and include precalculated alignments
and phylogenies. In HOMOLENS 4, alignments are computed using MUSCLE (Edgar 2004)
with default parameters; phylogenetic trees are computed with PHYML, using the JTT
amino acid substitution model (Jones et al. 1992). Phylogenies are computed based on
conserved blocks of the alignments selected with Gblocks (Castresana 2000). Each
phylogenetic tree is reconciled with a species tree using the program RAP (Dufayard et al.
2005), which, combined with the tree pattern search functionality, allows detection of
ancient gene duplications or selection of orthologous genes (Penel et al. 2009). Several
studies on duplicated gene evolution have been performed with data retrieved from
HOMOLENS (Brunet et al. 2006; Studer et al. 2008).
We employed a topology-based method to identify duplicated genes that resulted from the
FSGD event in the five teleost genomes we study. Briefly, if two teleosts have been subject to
the same whole genome duplication event, a gene X that has been duplicated in this event
and retained in both genomes, should form two gene lineages Xa and Xb (Figure 1A).
We identified gene trees with the topology shown in Figure 1A using the TreePattern
functionality (Dufayard et al. 2005) of the FamFetch client for HOMOLENS. We required
duplicated genes to exist in at least two species to increase the likelihood that they result
from the FSGD event (Figure 1B). In total, we identified 1,500 gene families with duplicated
genes in this way.
3. Differential retention and loss of duplicated genes during teleost
diversification
The most common fate of a duplicated gene is nonfunctionalization (pseudogenization). After
a whole genome duplication event, many genes share this fate, so that a genomes gene
content may only appear be slightly increased long after the duplication (Wolfe and Shields
1997; Jaillon et al. 2004). Our data suggest that only 3.3 percent (zebrafish) to 7.2 percent
(Takifugu) of genes in current teleost genomes result from the FSGD event (Table 1). These
percentages are lower than the 13 percent of retained duplicates in yeast (Wolfe and Shields
1997). One possible reason for this difference might lie in our topology-based method to
identify likely FSGD duplicates (Figure 1), which enforces duplicated genes to exist in at least

Duplicated Gene Evolution Following Whole-Genome Duplication in Teleost Fish

29
two teleost genomes. Thus, our method would overlook duplicated genes that result from the
FSGD and that are retained in only one teleost genome. While we cannot exclude this
possibility, we note that our observations are consistent with a genome-wide study of
Tetraodon, in which Jaillon et al. (2004) showed that up to 3 percent of duplicated genes may
have been retained since the FSGD event. One plausible explanation of the difference in
duplicated gene retention between teleost and yeast may come from the different ages of the
genome duplication event. In addition, Kassahn et al. (2009) suggested that a minimum of 3 to
4 percent of protein-coding loci have been retained in two copies in each of the five model

(A)
Species A
Species B
Species A
Species B
Gene_Xa
Gene_Xb
Duplication
Speciation
(B)
Homo sapiens
FSGD
Gene_Xa
Gene_Xb
Gene_X
N2
N2
(A)
Species A
Species B
Species A
Species B
Gene_Xa
Gene_Xb
Duplication
Speciation
(A)
Species A
Species B
Species A
Species B
Gene_Xa
Gene_Xb
Duplication
Speciation
Species A
Species B
Species A
Species B
Gene_Xa
Gene_Xb
Duplication
Speciation
(B)
Homo sapiens
FSGD
Gene_Xa
Gene_Xb
Gene_X
N2
N2

Fig. 1. (A) Expected phylogenetic relationship of duplicated gene Xa and Xb in two related
species A and B when speciation occurred after the duplication event; (B) Tree topology we
used for duplicated gene identification in the database HOMLENS 4. N 2 means that
duplicated gene pairs must exist at least in two species to increase the likelihood that the
duplicated genes actually resulted from the FSGD event.

Gene Duplication

30

Number
of genes

*
Gene families with likely FSGD duplicates
FSGD Duplicates Singleton Double loss
D. rerio 21,420 731 541 228
G. aculeatus 20,839 681 669 150
O.latipes 19,687 1,162 311 27
T. rubripes 18,709 1,340 148 12
Te. nigroviridis 27,991 1,047 397 56
*

Total gene number in each genome, data based on the Ensembl release 49.
Table 1. Summary of different gene retention and loss in the 1,500 duplicated gene families
we identified.
fish genomes. The FSGD occurred between 253 and 404 Million years ago (MYA) (Hoegg et
al. 2004; Vandepoele et al. 2004), whereas the yeast whole genome duplication may have
occurred more recently, between 100 and 150 MYA (Sugino and Innan 2005). More time has
elapsed since the FSGD, allowing more duplicate genes to be lost.
Differential retention and loss of duplicated genes is a common phenomenon during
speciation after genome duplication. It has been observed in yeast (Scannell et al. 2006) as
well as in teleosts (Semon and Wolfe 2007), and is believed to lead to speciation. We thus
expected that our dataset would contain many gene families with differential gene retention
and loss, as well as fewer families where both copies are retained in all five teleost genomes.
Indeed, when we consider all five species together, we observed that 90.4 percent of the
1,500 gene families we identified show differential retention and loss of duplicated genes,
and in only 9.6 percent (144 gene families) are both copies retained in all five teleost
genomes. Figure 2 and Table 1 show relevant data, broken down by study species. In 45.4
percent to 89.3 percent (depending on the species) of the 1,500 gene families we identified,
both duplicates were retained. In 9.9 percent to 44.6 percent of the duplicates (depending on
the species), one copy was lost. Our data also indicate that differences in differential gene
retention are associated with the phylogenetic position and the relatedness between two
teleost species (Figure 2). Taken together, these observations indicate that differential
duplicated gene retention and loss are pervasive in teleosts, that the loss of duplicated genes
is an ongoing process that has continued for hundreds of million years after the FSGD event,
and that this process may be associated with teleost diversification.
We next discuss an illustrative example of differential duplicate gene retention and loss. It
involves Hox genes, which encode a subclass of homeodomain transcription factors that help
determine the anteriorposterior axis of bilaterian animals (McGinnis and Krumlauf 1992). In
vertebrates, Hox genes have evolved a highly compact organization, where genes are arranged
in clusters on chromosomes. Hox gene clusters are one of the best-studied systems for
assessing gene retention and loss after the FSGD event (Amores et al. 1998; Prohaska and
Stadler 2004; Hoegg et al. 2007; Guo et al. 2010), due to their genomic architecture and gene
complement variation in teleosts. Seven or eight Hox clusters with different complements of


31
Hox genes exist in extant diploid teleosts. They are a result of the FSGD event, which was
followed by loss of some Hox gene duplicates. The putative Hox cluster complement of the
teleost ancestor and the Hox clusters of several model teleost species are shown in Figure 3.
Hox clusters exhibit remarkably different gene complements in different teleost lineages after
the FSGD event. Theoretically, 8 Hox clusters containing at least 80 Hox genes genes may have
existed in the ancestor of teleosts after the FSGD event. Up to now, 66 of these Hox genes have
been found in different teleost species and extant evolutionary diploid teleost usually have 45
to 49 Hox genes in their genome (Figure 3). According to the summary of Hoegg et al (2007)
(Figure 3), the Ostariophysii have lost seven Hox genes since their hypothetical common
ancestor with the Neoteleosts; during the evolution of the Neoteleosts eight Hox genes were
lost; and the pufferfish lineage lost three genes in the common lineage leading to Takifugu
and Tetraodon. Some Hox genes are specifically preserved in different teleosts, for example,
HoxA1b has been identified thus far only in the Japanese eel (Guo et al. 2010). At the cluster
level, eight Hox clusters were retained in basal species such as the Japanese eel (Guo et al.
2010) and the goldeye (Chambers et al. 2009), whereas one Hox cluster (C or D) was lost
respectively in the Otocephala (Amores et al. 1998) and Euteleostei (Kurosawa et al. 2006).
Based on the phylogeny of teleosts, Guo et al. (2010) proposed that the HoxDb cluster

Homo sapiens
Danio rerio
Gasterosteus aculeatus
Oryzias latipes
Tetraodon nigroviridis
Takifugu rubripes
FSGD
731* / 541** / 228***
681 / 669 / 150
1,162 / 311 / 27
1,340 / 148 / 12
1,047 / 397 / 56
1500
Homo sapiens
Danio rerio
Oryzias latipes
Takifugu rubripes
FSGD
731* / 541** / 228***
681 / 669 / 150
1,162 / 311 / 27
1,340 / 148 / 12
1,047 / 397 / 56
1500

Fig. 2. Differential retention and loss of duplicated genes during teleost diversification. The
topology is adopted from (Negrisolo et al. 2010). *: retention of both copies; **: retention of
one copy; ***: loss of both copies.

Gene Duplication

32

Hypothetical Hox cluster complement of teleost ancestor
Teleost ancestor
Danio rerio
Takifugu rubripes
Oryzias latipes
Astatotilapia burtoni
Oreochromis niloticus
Hypothetical Hox cluster complement of teleost ancestor
Teleost ancestor
Hypothetical Hox cluster complement of teleost ancestor Hypothetical Hox cluster complement of teleost ancestor
Teleost ancestor
Danio rerio
Takifugu rubripes
Oryzias latipes
Astatotilapia burtoni
Oreochromis niloticus

Fig. 3. Hox gene clusters, the best-studied examples of differential duplicate gene retention
and loss in teleosts. Hypothetical Hox clusters of the teleost ancestor (modified from Guo et
al. 2010), and Hox clusters of teleost model fish species, together with specific gene loss
events shown on a phylogenetic tree of select fish species (adapted form Hoegg et al. 2007).
was lost independently in the Otocephala and Euteleostei after the FSGD event. The
ongoing process of Hox gene loss and retention in teleosts illustrates again that degeneration
of functionally important duplicated genes can last for hundreds of millions of years after
the FSGD event.
4. Molecular evolution of duplicated genes
We next wished to study patterns of sequence evolution in the 1,500 duplicate gene families
we had identified. To this end, we downloaded both nucleic acid and amino acid sequences
for genes in these families. For each species, we retained only one gene copy in each
duplicated clade (Figure 1B) for further analysis, and discarded all other copies in those
gene families where additional duplications have occurred after the FSGD event. We
then aligned the amino acid sequences within each gene family with MUSCLE (Edgar


33
2004), and calculated DNA alignments from protein alignments with RevTrans
(Wernersson and Pedersen 2003). The following computations were then done on the
new DNA alignments. We estimated the nucleic acid evolutionary distance between fish
genes and their human orthologs using the LogDet nucleotide substitution model
(Tamura and Kumar 2002) in PHYLIP-3.6b (Felsenstein 2004).
Previous studies show that duplicated genes in yeast often diverge asymmetrically (Kellis et al.
2004), meaning that one copy evolves significantly faster than the other. We asked whether
this is also the case for teleost duplicates. To this end, we compared evolutionary distances of
duplicated genes with their human orthologs within the 1,500 gene families we had identified.
There is indeed evidence for asymmetric evolution between duplicated gene pairs from the
FSGD event (Table 2). Average evolutionary distances to the human homologue between
members of duplicated gene pairs are significantly different for each of our five teleost species
(paired t-test: P < 4.8 10
95
). As all duplicated gene pairs stemming from the FSGD diverged
at the same time from their human orthologs, we can directly convert differences between
evolutionary distances into differences between evolutionary rates. Taken together, our
observations suggest that duplicate genes tend not to accumulate sequence change at the same
rate. Our results are consistent with previous works in teleosts (Brunet et al. 2006; Steinke et al.
2006) and yeast (Kellis et al. 2004), and confirm that asymmetric sequence evolution between
duplicated genes is a frequent pattern of duplicated gene evolution after a genome duplication
event.

D. rerio G. aculeatus O.latipes T. rubripes Te. nigroviridis
Duplicate_L 0.613 0.243 0.607 0.229 0.621 0.230 0.623 0.229 0.614 0.224
Duplicate_S 0.529 0.213 0.526 0.200 0.536 0.195 0.535 0.195 0.505 0.182
P-value* 4.1 10
105
4.8 10
95
1.9 10
165
7.3 10
175
8.9 10
133

Duplicate_L: duplicated gene in each duplicate pair that has the larger distance to the human
orthologue (distances averaged over all duplicate gene families); Duplicate_S: duplicated gene in each
duplicate pair that has the smaller distance to the human orthologue (distances averaged over all
duplicate gene families). All means are one standard deviation.
* paired t-test
Table 2. Average evolutionary distances of duplicated genes in five teleost species to their
human orthologs.
5. Conclusion
In summary, we used a phylogenetic method to identify 1,500 duplicated gene families in
five teleost species that are likely to have resulted from the FSGD event. Only a small
fraction of genes in extant teleost genomes have been retained in the FSGD event.
Differential retention and loss of duplicated gene is pervasive in the five species we
studied, as is illustrated by genes in the teleost Hox gene clusters. Sequence analysis
suggests that some duplicated genes pairs may evolve asymmetrically. Our work
provides a framework for future studies of the evolutionary trajectory of duplicated genes
in the teleost genome.

Gene Duplication

34
6. Acknowledgement
Support was provided by the National Natural Science Foundation of China (NSFC) to
Shunping He.
7. References
Amores A, Force A, Yan YL, et al. 1998. Zebrafish hox clusters and vertebrate genome
evolution. Science 282:1711-1714.
Bridges CB. 1936. The bar gene a duplication. Science 83:210-211.
Brunet FG, Crollius HR, Paris M, Aury JM, Gibert P, Jaillon O, Laudet V, Robinson-Rechavi
M. 2006. Gene loss and evolutionary rates following whole-genome duplication in
teleost fishes. Mol Biol Evol 23:1808-1816.
Castresana J. 2000. Selection of conserved blocks from multiple alignments for their use in
phylogenetic analysis. Mol Biol Evol 17:540-552.
Chambers KE, McDaniell R, Raincrow JD, Deshmukh M, Stadler PF, Chiu CH. 2009. Hox
cluster duplication in the basal teleost Hiodon alosoides (Osteoglossomorpha).
Theory Biosci 128:109-120.
Christoffels A, Koh EG, Chia JM, Brenner S, Aparicio S, Venkatesh B. 2004. Fugu genome
analysis provides evidence for a whole-genome duplication early during the
evolution of ray-finned fishes. Mol Biol Evol 21:1146-1151.
Dufayard JF, Duret L, Penel S, Gouy M, Rechenmann F, Perriere G. 2005. Tree
pattern matching in phylogenetic trees: automatic search for orthologs or
paralogs in homologous gene sequence databases. Bioinformatics 21:
2596-2603.
Duret L, Mouchiroud D, Gouy M. 1994. HOVERGEN: a database of homologous vertebrate
genes. Nucleic Acids Res 22:2360-2365.
Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accuracy and high
throughput. Nucleic Acids Res 32:1792-1797.
Felsenstein J. 2004. PHYLIP (Phylogeny Inference Package) version 3.6. Distributed
by the author. Department of Genome Sciences, University of Washington,
Seattle.
Guo B, Gan X, He S. 2010. Hox genes of the Japanese eel Anguilla japonica and Hox cluster
evolution in teleosts. J Exp Zool B Mol Dev Evol 314:135-147.
Hoegg S, Boore JL, Kuehl JV, Meyer A. 2007. Comparative phylogenomic analyses of teleost
fish Hox gene clusters: lessons from the cichlid fish Astatotilapia burtoni. BMC
Genomics 8:317.
Hoegg S, Brinkmann H, Taylor JS, Meyer A. 2004. Phylogenetic timing of the fish-specific
genome duplication correlates with the diversification of teleost fish. J Mol Evol
59:190-203.
Jaillon O, Aury JM, Brunet F, et al. 2004. Genome duplication in the teleost fish
Tetraodon nigroviridis reveals the early vertebrate proto-karyotype. Nature
431:946-957.
Jones DT, Taylor WR, Thornton JM. 1992. The rapid generation of mutation data matrices
from protein sequences. Comput Appl Biosci 8:275-282.


35
Kassahn KS, Dang VT, Wilkins SJ, Perkins AC, Ragan MA. 2009. Evolution of gene function
and regulatory control after whole-genome duplication: comparative analyses in
vertebrates. Genome Res 19:1404-1418.
Kellis M, Birren BW, Lander ES. 2004. Proof and evolutionary analysis of ancient genome
duplication in the yeast Saccharomyces cerevisiae. Nature 428:617-624.
Kurosawa G, Takamatsu N, Takahashi M, et al. 2006. Organization and structure of hox
gene loci in medaka genome and comparison with those of pufferfish and zebrafish
genomes. Gene 370:75-82.
Lynch M, Conery JS. 2000. The evolutionary fate and consequences of duplicate genes.
Science 290:1151-1155.
McGinnis W, Krumlauf R. 1992. Homeobox genes and axial patterning. Cell 68:283-302.
Meyer A, Van de Peer Y. 2005. From 2R to 3R: evidence for a fish-specific genome
duplication (FSGD). Bioessays 27:937-945.
Negrisolo E, Kuhl H, Forcato C, Vitulo N, Reinhardt R, Patarnello T, Bargelloni L.
2010. Different Phylogenomic Approaches to Resolve the Evolutionary
Relationships among Model Fish Species. Mol Biol Evol 27:2757-2774.
Ohno S. 1970. Evolution by gene duplication. Springer-Verlag, New York.
Penel S, Arigon AM, Dufayard JF, Sertier AS, Daubin V, Duret L, Gouy M, Perriere G. 2009.
Databases of homologous gene families for comparative genomics. BMC
Bioinformatics 10 Suppl 6:S3.
Prohaska SJ, Stadler PF. 2004. The duplication of the Hox gene clusters in teleost fishes.
Theory Biosci 123:89-110.
Scannell DR, Byrne KP, Gordon JL, Wong S, Wolfe KH. 2006. Multiple rounds of
speciation associated with reciprocal gene loss in polyploid yeasts. Nature 440:
341-345.
Semon M, Wolfe KH. 2007. Reciprocal gene loss between Tetraodon and zebrafish after
whole genome duplication in their ancestor. Trends Genet 23:108-112.
Stephens SG. 1951. Possible Significance of Duplication in Evolution. In: M Demerec, editor.
Advances in Genetics: Academic Press. p. 247-265.
Studer R, Duret L, Penel S, Robinson-Rechavi M. 2008. Pervasive positive selection on
duplicated and non duplicated vertebrate protein coding genes. Genome Res
18:1393-1402.
Sugino RP, Innan H. 2005. Estimating the time to the whole-genome duplication and
the duration of concerted evolution via gene conversion in yeast. Genetics 171:
63-69.
Tamura K, Kumar S. 2002. Evolutionary distance estimation under heterogeneous
substitution pattern among lineages. Mol Biol Evol 19:1727-1736.
Taylor JS, Braasch I, Frickey T, Meyer A, Van de Peer Y. 2003. Genome duplication, a trait
shared by 22000 species of ray-finned fish. Genome Res 13:382-390.
Taylor JS, Van de Peer Y, Braasch I, Meyer A. 2001. Comparative genomics provides
evidence for an ancient genome duplication event in fish. Philos Trans R Soc Lond B
Biol Sci 356:1661-1679.
Vandepoele K, De Vos W, Taylor JS, Meyer A, Van de Peer Y. 2004. Major events in the
genome evolution of vertebrates: paranome age and size differ considerably

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36
between ray-finned fishes and land vertebrates. Proc Natl Acad Sci U S A 101:
1638-1643.
Wernersson R, Pedersen AG. 2003. RevTrans: Multiple alignment of coding DNA from
aligned amino acid sequences. Nucleic Acids Res 31:3537-3539.
Wolfe KH, Shields DC. 1997. Molecular evidence for an ancient duplication of the entire
yeast genome. Nature 387:708-713.
3
Detection and Analysis of Functional
Specialization in Duplicated Genes
Owen Z. Woody and Brendan J. McConkey
University of Waterloo
Canada
1. Introduction
Gene duplication has long been recognized as a powerful mechanism facilitating
development and evolution in genomes. Duplication events produce additional copies of
genomic information, perhaps including one or more genes. While in some cases these
duplicated elements may be of immediate benefit (i.e. increasing availability and effective
dosage of a desired gene product), often they are initially at least somewhat redundant,
and either neutral or mildly detrimental to the fitness of the organism. It is perhaps no
surprise, then, that the majority of duplicated genes are quickly deactivated by mutations
abolishing transcription or translation. Some duplicated genes, however, survive and
persist, suggesting that their retention has some benefit. Many of these genes seem to
have acquired properties that distinguish them from their progenitors they may be
expressed in a novel tissue type, for example, or differ in their functional specificity. In
these cases, it appears as though duplication has facilitated evolution, either by allowing
specialization and refinement or, perhaps most intriguingly, generating genes free to
mutate and acquire novel functions. These retained duplicates form a family of genes
related through common ancestry. As a result of their common origin, gene sequences
within gene families are often quite similar, complicating the task of assigning them
unique and specific functions. As such, there has been a significant effort to study and
characterize the evolution of function in the aftermath of a duplication event. This chapter
will briefly cover the various modes of gene duplication, and then will focus on the
various functional outcomes of duplication. The theoretical models for functional
specialization following a duplication event are discussed, as are practical techniques for
applying these models to observed gene duplicates.
2. Mechanisms of duplication
There are several different mutational mechanisms through which gene duplicates can be
produced. Depending on the type of event, the nature and scale of what is duplicated can
differ significantly; single genes may be copied, with or without their peripheral regulatory
elements, or entire genome can be duplicated. While each mechanism ultimately results in
the duplication of one or more genes, the mechanisms differ in three key respects; how
much regulatory information the duplicated genes retain, where the duplicates are
integrated into the genome, and how many interaction partners are duplicated. Duplication

Gene Duplication

38
mechanisms can be broadly categorized into three groups DNA/RNA-mediated
transposition, unequal recombination, and genome doubling/hybridization. These
mechanisms all produce paralogs -- homologous genes that are both present in and native to
the same genome (in contrast to orthologs, where speciation acts as a 'duplication event' and
the homologous genes are components of different genomes). Figure 1 provides a diagram
depicting various modes of duplication.
2.1 DNA/RNA transposition
DNA/RNA transposition refer to mechanisms by which a specific short nucleotide
sequence, either mRNA (as in retrotransposition) or DNA (e.g., transposon-mediated
duplication) is copied from one location in the genome to another. The insertion location is
essentially random; any compatible destination locus will do, and thus the produced
duplicate need not necessarily be located near its progenitor template. RNA-mediated
retrotransposition is unique in that it uses post-transcription sequence as a template for the
nascent duplicate. Hence, upstream and downstream regulatory sequences lying outside the
transcribed gene sequence are not preserved, and the newly produced gene will have most
or all introns (and possibly some exons) spliced out. The new gene may also possess a
genetically encoded poly-A tail. Since RNA-mediated retrotransposition does not preserve
most non-coding elements, the duplicate gene must depend on the a-priori availability or
acquisition of promoter/regulatory sequences in order to be transcribed. Absence of these
elements effectively means the new gene duplicate is a pseudogene.
DNA-mediated duplications, as mediated by transposons, for example, often retain
regulatory information and intron/exon structure. Nonetheless, they still operate on a very
specific subsequence of DNA, and elements relocated by DNA-mediated transposition can
be inserted in any eligible location in the genome.
2.2 Segmental duplication/unequal cross-over
Errors during homologous recombination can produce serial duplications of genetic
sequence. Unequal crossing-over is an error stemming from the mis-alignment of
homologous chromosomes during mitosis/meiosis. Ordinarily, homologous sequences are
aligned and cross-over events result in balanced exchanges of sequence information across
chromosomes. An abundance of repetitive sequences can, however, cause chromosomes to
misalign, in which case a segment of one chromosome is inserted into its sister chromatid
(thus producing a duplication and a reciprocal deletion).
Since multiple rounds of unequal crossing over tend to gradually inflate the number of
candidate repeat regions, some genomic regions are hotbeds for sequence duplication and
can give rise to a large number of duplicate genes in series. These serially arranged
duplicates are referred to as tandem duplicates. These tandem gene arrays are highly
localized in the genome, and tandem duplicates retain most or all of their intron/exon
structure and peripheral non-coding elements. Unequal crossing-over also plays a role in the
generation of copy number variations (Redon et al., 2006).
2.3 Whole genome duplication/allopolyploids
In some circumstances, errors during segregation can produce diploid gametes, and the
fusion of these diploid gametes can result in a complete doubling of genomic content (all
chromosomes present in duplicate). While very rare, these whole genome duplication

Detection and Analysis of Functional Specialization in Duplicated Genes

39

Fig. 1. Modes of gene duplication. Upper left: transposition mediated by either a DNA or
RNA intermediate can produce gene locations at distant locations in the genome. RNA
intermediates retain little of the regulatory sequence surrounding the parent gene. Upper
right: Errors during homologous recombination can produce tandem arrays of genes,
situated in series. Bottom Left: Doubling of all chromosomes will produce duplicates of all
genes in the genome. Bottom Right: Allopolyploids contain genomes from two compatible
species, with duplicate gene pairs from formerly orthologous genes.

Gene Duplication

40
(WGD) events have a dramatic impact on the content of the genome, and a number of WGD
events have been hypothesized in the history of various lineages (Van de Peer et al., 2009).
By their nature, WGD events result in the duplication of all loci, preserving non-coding
elements, intron/exon structure, and even overall stoichiometry within gene/protein
interaction networks. Interestingly, it has been observed that lineages that undergo separate,
distinct WGD events (in this case, Xenopus tropicalis and zebrafish) often ultimately retain
similar (i.e. orthologous) duplicates that is to say, WGD duplicates that becomes fixed in
one lineage, were also often fixed in the other (Semon & Wolfe, 2008). Pairs of duplicate
genes that arose through WGD are sometimes referred to as Ohnologs (Turunen et al., 2009).
WGD events are relatively common in plant lineages, which may have interesting
implications for the evolution of gene regulation (Lockton & Gaut, 2005).
Allopolyploids are a variant of whole genome duplications in which the diploid gametes
come from two different species. These genomic hybrids contain two formerly independent
complete genomes. The most commonly studied allopolyploids are plants, though a number
of examples have been documented elsewhere in the animal kingdom (including the model
organism Xenopus Laevis). Duplicates produced through allopolyploidy (i.e. formerly
orthologous genes now present in the same organism) are often referred to as
homeologs(Flagel et al., 2008).
3. Defining gene function
One significant hurdle to the discussion of duplicate functional specialization is defining
gene function. Gene function may be broken into two broad categories regulation and
gene product (MacCarthy & Bergman, 2007). Regulation encompasses the when, where,
why, and how much aspects of a genes transcription non-coding elements around a gene
(such as enhancers and signaling sequences) can direct when a gene should be expressed,
and in what quantity. These non-coding elements are responsive to various cellular and
environmental triggers. Changes to regulation alone may be sufficient to bring about the
specialization of a new duplicate.
Gene products, on the other hand, primarily dictate the how in a genes function (along
with some regulatory and subcellular localization information present in the 5' and 3'
untranslated regions (UTRs)). Studies of duplicated genes have focused on changes to
various coding sequence properties, such as binding sites, eligible cofactors, indels, and
catalytic residues (Turunen et al., 2009). Its theoretically possible for a duplicate gene to
become functionally specialized without any change to its regulation (Des Marais &
Rausher, 2008).
It is worth noting that changes falling into these two categories can occur serially or in
concert. For example, a change in tissue localization may precede structural mutations
adapting a protein to a new environment.
4. Theoretical models for duplicate retention and functional specialization
A number of theoretical models have been proposed to describe how a parental genes
functions can be partitioned between offspring, and how this partitioning affects the chances
of these genes to avoid pseudogenization and eventual deletion.
Three archetypal outcomes specifically, nonfunctonalization, subfunctionalization, and
neofunctionalization, are based on concepts typically attributed to Ohno (1970).


41
Nonfunctionalization describes the situation where one duplicates expression is abolished,
making it invisible to natural selection and thus free to accumulate mutations. While it is
technically possible for a nonfunctionalized gene to have its function restored, the vast
majority become relics progressively crippled by the accumulation of disabling and
deleterious mutations. There has been some interest in studying the impact losing a
duplicate via nonfunctionalization has on sibling genes for example, whole genome
duplication events can lead to cases of ohnologs gone missing, where a WGD duplicate
has been lost (Canestro et al., 2009). Reciprocal duplicate loss has been hypothesized as one
means of speciation. Figure 2 depicts two hypothetical duplications and their respective
functional specializations.
4.1 Neofunctionalization
Neofunctionalization refers to the scenario where one duplicate gene acquires mutations
that allow it to acquire previously unexplored functions, either through changes in
regulation (e.g. tissue localization) or coding sequence. Claims of neofunctionalization tend
to focus on the generation of new functions, though it should be noted that these
developments may also result in the loss of ancestral function(s) (Turunen et al., 2009).
A specific example of neofunctionalization can be found in a recent study of the MADS-box
gene family in angiosperms. MADS-box genes are well-known for their role in
developmental processes, but the functions of some gene family members have been
difficult to determine. Viaene et al. (2010) provide evidence that a group of these genes, the
AGL6 subfamily, can be neatly divided into two groups based on duplication history. One
of these groups retains the ancestral function of guiding reproductive development, while
the other seems to have acquired a novel role in regulating the growth of vegetative tissues.
A second example, describing the functional differentiation of two paralogs in maize, shows
that ancestral functions can still be retained even when one duplicate acquires a novel
function (Goettel & Messing, 2010). Two paralogs, named p1 and p2, both drive the
synthesis of maysin, which in turn contributes to resistance against earworm. In addition,
the p1 gene also has a secondary role in controlling the accumulation of red pigments. The
authors propose of a series of recombination events that describe how these genes acquired
their distinct characters.
4.2 Subfunctionalization
Subfunctionalization involves each gene taking upon a complementary subset of the
parental genes functions, such that neither is independently capable of fulfilling all the
parental genes roles. Subfunctionalization is conceptually synonymous with the
Duplication, Degeneration, and Complementation (DDC) model. Regulatory
subfunctionalization could result in non-overlapping tissue distributions for the nascent
duplicates, with the union of the expression profiles matching the parental gene's range.
Jarinova et al. (2008) describe an instance of subfunctionalization the Hox genes of zebrafish.
Through a careful analysis of peripheral non-coding elements, the authors show how the
two hoxb complexes in zebrafish, hoxb5a and hoxb5b, acquired non-overlapping expression
profiles. In particular, the experimental removal of one regulatory element unique to hoxb5a
resulted in the two paralogs (re)acquiring a similar expression profile.
The idea of structural subfunctionalization is perhaps best captured in the "Escape from
Adaptive Conflict" (EAC) hypothesis. Consider a hypothetical gene product with multiple

Gene Duplication

42

Fig. 2. Possible functional specializations following duplication. Two hypothetical examples
showing how retention models can apply either to regulatory regions or gene products. A)
Duplicated genes subfunctionalize at the regulatory level, partitioning their parental
regulatory domains and suggesting subdivided roles. The gene product, however, has
acquired a novel element (i.e. new exon), suggesting neofunctionalization at the coding
sequence level. B) Following duplication, one gene loses its regulatory domains and is
interrupted by an early stop codon, reflecting nonfunctionalization both at the regulatory
and gene product levels.
interaction partners (e.g. an enzyme with two possible substrates). Selection for
bifunctionality in this enzyme may limit the binding/catalytic efficiency of either specific
reaction -- mutations that improve one may inhibit the other, hence the "adaptive conflict".


43
Should this gene be duplicated, however, each offspring gene could be free to acquire
mutations that optimize binding to one specific substrate, thus escaping the conflict without
a loss of functionality. The EAC model essentially describes this process, where a single
enzyme with multiple interaction partners gives rise to duplicate genes with more specific
but enhanced functionality.
EAC is interesting in that it lies somewhere on the boundary between subfunctionalization
and neofunctionalization. Three claims are required to invoke the model: that i) both
duplicates accumulate adaptive changes post mutation, that ii) these mutations enhance
ancestral functions, and lastly that iii) the ancestral gene was constrained from improving
functions (Barkman & Zhang, 2009). The key difference (and challenge) lies in proving the
ancestral form was bi-functional. Studies demonstrating the EAC model in action are still
relatively uncommon. An early attempt to apply the model to the genes from the
anthocyanin biosynthetic pathway of morning glories has come under criticism for not
clearly providing these three veins of supporting evidence (Barkman & Zhang, 2009; Des
Marais & Rausher, 2008).
The EAC process has also been invoked to describe the evolution of a novel anti-freeze
protein in an Antarctic zoarcid fish (Deng et al., 2010). The authors demonstrate that an
ancestral gene had a rudimentary ice-binding affinity in addition to its primary catalytic
function (a sialic acid synthase), and that a duplication event allowed one copy of this gene
to abolish this ancestral function and refine its ice-binding capability. The discussion
includes a careful analysis of the three EAC criteria listed above.
While duplicated genes are generally relegated to one of the fates listed above, a number of
case studies have shown that recent duplicates can maintain identical functional profiles.
One possible explanation for this is that the duplicates have acquired mutations that have
restored the status quo that was present prior to duplication. If mutations cause the sum
of the duplicate genes' expression levels to equal the expression level of their ancestor, both
genes could experience some level of selective pressure to maintain expression despite being
fully redundant. Ganko et al. (2007) observe that a vast majority of duplicates, regardless of
duplication mechanism, showed asymmetric expression, with one gene consistently
showing higher levels of expression than its sibling across all tissues. This suggests that a
limited form of subfunctionalization may play an initial role in the retention of duplicates.
Asymmetrical expression divergence was also observed in a study of duplicated genes in
the fly, with a tendency for the parent gene of the duplicate to have high expression levels
(Langille & Clark, 2007). Interestingly, Qian et al. (2010) point out that many gene duplicates
are synthetically lethal or deleterious, and they suggest that expression load may being
shared by both genes after duplication.
4.3 Alternative models and odd cases
A number of other subtle variations have been proposed to augment these three primary fates.
Subneofunctionalization, for example, is a model that argues that subfunctionalization
followed by neofunctionalization is a common and sequential process. Subfunctionalization
permits a relaxation of selection on various subregions of the gene, which in turn allows the
(eventual) evolution of novel functions, suggesting that subfunctionalization is more of a
midstep than an endpoint (Johnson & Thomas, 2007).
In addition, while subfunctionalization does not make any a priori claims about the
proportion of functions lost by each duplicate, it appears that in some cases the losses are

Gene Duplication

44
highly asymmetrical. Panchin et al. (2010) show that in human duplicate genes one
duplicate appears to remain totally unchanged, while its sibling accumulates the majority of
functional (in this case, amino acid) mutations.
Contrary to expectations, many gene duplicate pairs appear to be retained despite total
apparent functional redundancy. A relatively recent model has been proposed to explain
this phenomenon. The theory, coined "originalization", uses arguments based on
purifying selection and recombination to support the preservation of both duplicate
copies (i.e. prevent non-functionalization) for an extended period of time (Xue & Fu, 2009;
Xue et al., 2010).
It has also been suggested that models of duplicate retention are focusing on too small a
unit, and that protein interaction networks (themselves composed of a number of co-
expressed and functionally related proteins) provide a more coherent perspective on the size
of perturbation required to have a phenotypically relevant effect (MacCarthy & Bergman,
2007). The authors argue that cases of regulatory subfunctionalization and
neofunctionalization often have no phenotypic consequence on the output of a protein
network, and are thus effectively neutral for longer than duplicate-oriented models would
suggest. A number of studies have reported an unexplained level and duration of retention
for redundant duplicates (Skamnioti et al., 2008).
The relative importance of these models to the retention of duplicates is a subject of
continued interest. Whole genome duplication events, which effectively introduce a
paralogous copy of every gene in the genome, present an opportunity to tally the cases for
which each model applies best. For a review of the relative importance of these various
retention models specifically as they pertain to duplicates produced in plant WGD events,
see (Edger & Pires, 2009).
5. Factors affecting the rate and trajectory of duplicate gene divergence
5.1 Gene conversion
Gene conversion describes the process by which the sequence content of one genetic locus
is used as a template to alter and "paste over" the genetic sequence at a distal location.
Gene conversion has the potential to enforce similarity across duplicate loci, both in terms
of regulation and structure. A recent study on duplicated segments in a pair of Drosophila
species made noted several anomalies that were suggestive of gene conversion.
Interestingly, the edges of duplicated regions accumulated distinguishing mutations
faster than more central regions, suggesting that these regions were being maintained by
gene conversion and that the size of the region being converted was gradually being
reduced by sequence mutations near the borders. Furthermore, paralogs near the
boundaries of duplicated segments showed more divergence than those located near the
centre (Osada & Innan, 2008). The authors note that this phenomenon could result in
misleading estimates of synonymous divergence, as the conversion process would
periodically homogenize the two sequences.
The requirements for a neofunctionalized gene to escape gene conversion and achieve
fixation have been studied from a population genetics perspective (Teshima & Innan,
2008). The fit of the model is tested on a pair of human opsins, which differ in their light
sensitivity.
Additional evidence that gene conversion may play a role in duplicate divergence was
found in a study of WGD duplicates in rice. Duplicates that contained subsequences of


45
particularly high sequence similarity also showed a tendency towards retaining similar
regulation profiles. The authors suggest that this was a consequence of the promoter regions
being propagated via gene conversion activity acting on the locally similar subsequences
(Yim et al., 2009).
5.2 Alternative splicing
In general, multiple splice forms (and the potential for these splice variants to have distinct
functions) have not received much attention in studies of gene duplication and functional
divergence. In a first step towards addressing this oversight, Zhan et al. (2011) studied the
potential for alternative splicing in Drosophila duplicates. New genes tended to show lower
levels of alternative splicing, and the subset of duplicates that retained the potential for
multiple spliceforms were expressed in fewer tissues, at lower levels, and had had their
expression breadth shifted towards preferential expression in testes. The authors also noted
that a duplicates alternative splicing potential depended on duplication mode, with
retrotransposed genes being copied with a specific and frozen configuration of
exons/introns.
5.3 Properties of genes that influence duplicate specialization
The rate at which duplicated genes acquire novel functions is of great interest. Studies have
been done to compare the standard metrics of gene evolution (synonymous distance, non-
synonymous distance) to measures of functional differentiation across duplicate genes.
While initial studies demonstrated only a weak correlation between expression divergence
and sequence divergence, subsequent studies have drawn attention to a number of gene
parameters that strongly influence the rate and extent of functional differentiation across
duplicates.
The mode of duplication has been cited in multiple instances as an important determinant of
eventually retention/functional specialization. In a study comparing the functional
evolution of genes duplicated through different mechanisms, Ganko et al. (2007) found that
WGD-derived duplicates tended to be expressed at higher levels and were more broadly
expressed (in contrast to duplicates derived in smaller scale duplications). Wang et al. (2010)
found that tandem gene duplicates tended to have conserved function, whereas
retrotransposed genes were more likely to have undergone EAC.
Duplicate genes can differ in their tissue distribution, and certain tissues seem to have a
greater propensity to adopt genes with novel function than others. In particular, novel
duplicates show a tendency towards expression in the testes. Langille and Clark (2007)
found that retrotransposed duplicates in particular showed testis-biased expression.
Mikhaylova et al. (2008) also found that duplicated genes expressed in the testes tended to
show particularly divergent expression across species. Gallach (2010) illustrate a trend for
mitochondrial-associated proteins to preferentially fixate in autosomes (i.e. avoiding the X
chromosome), and to have a strong tissue bias towards testes expression.
Han et al. (2009) revealed an interesting trend for duplicate genes that had been relocated
(i.e. created by transposition). In instances where these duplicated genes showed
asymmetric sequence evolution, in more than 80% of cases it was the relocated gene that
showed stronger support for positive selection. This suggests an important role for
chromosomal distribution in the evolution of gene function and duplicate divergence. A
study by Tsankov et al. (2010) also showed that local chromatin organization (i.e.

Gene Duplication

46
nucleosome positions) has a strong effect on gene expression, which suggests that
translocated duplicates may show expression divergence by virtue of chromosomal position
alone. In addition, Ren et al. (2005) found that tandem duplicates that shared expression
domains tended to have dissimilar sequence-based functions. Shoja et al. (2007) noted that
tandem gene duplicates tended to show a relationship between expression divergence and
chromosomal distance.
In their work on the possible action of gene conversion on the evolution of duplicated
segments in Drosophila, Osada and Innan (2008) noted that duplications lying near the edges
of duplicated segments showed more sequence divergence, suggesting that sublocation
within a duplicated segment is an additional factor to consider in studies of duplicate
divergence.
The broad functional category to which a gene belongs can also influence its freedom to
explore divergent functions. In an analysis of genes in the rice genome produced through
a specific WGD, Yim et al. (2009) found that duplicate genes with divergent functions
showed a significant enrichment towards metabolism-related activity. Langille and Clark
(2007) showed that cell physiological process genes were particularly amenable to
duplication via transposition. Perhaps reflecting similar functional pressures, Li et al.
(2010) found that subcellular localization also influenced the divergence of expression
between duplicate genes.
The mode of retention may also depend on the amount of selective pressure acting on its
coding sequence. Semon and Wolfe (2008) showed that duplicates undergoing slow rates of
sequence evolution seemed particularly prone to regulatory subfunctionalization. This
observation is echoed in Arnaiz et al. (2010), who find that duplicate pairs in Tetraurelia with
divergent expression profiles were unlikely to undergo sequence subfunctionalization. Li et
al. (2009) found that the mode of duplication had a substantial effect on the degree of
expression divergence between duplicates, based on microarray expression profiles of rice
tissues.
Nielsen et al. (2010) suggest that genes under strong selective pressure produce duplicates
that are quickly nonfunctionalized, suggesting low tolerance for (poisonous) isoforms of
essential products. Thus, a genes essentiality and, by consequence, age, may both determine
the extent to which gene duplicates may be retained.
6. Tools for measuring gene regulation
While a genes regulatory control is partly controlled by its complement of non-coding
elements (as well as its genomic location, e.g., proximity to histones/heterochromatin),
efforts to predict regulation from sequence alone have met with limited success, owing to
non-linear interactions between various regulatory domains (Jarinova et al., 2008). A
separate study found that transcription factor binding site turnover was insufficient to
explain cis-regulatory evolution across orthologs (Venkataram & Fay, 2010).
Since accurate predictions of gene regulation based on genomic context and peripheral
regulatory elements remain elusive, most studies of gene regulation depend on empirical
measurements of gene products (i.e. mRNA or protein) as evidence for a genes
expression under given conditions. Tools for quantifying the abundance of specific
mRNA and/or protein species, such as PCR and Western blots, are standard laboratory
techniques.


47
Within the past decade, however, a number of high-throughput technologies have become
available that allow the localization and abundance of gene products to measured
empirically on a genomic/proteomic scale. At present, the most widely used platform is the
microarray, an assay with a very large number of transcript-specific probes. Each probe is
specific to a known transcript, allowing the potential for complete coverage of all known
and predicted genes in a known genome sequence. Custom arrays can also be built from
cDNA libraries when working with non-model organisms. Databases replete with
microarray data are now publicly available for data mining, allowing a genes expression (or
lack thereof) to be profiled across tissues, timepoints, and stimuli. This aggregate gene
behaviour is referred to as an expression profile, and can serve as an empirical proxy of
overall gene function. As more microarray data becomes available, the quality of this proxy
will improve.
Expression measurement technologies measure gene activation directly, and are agnostic to
the regulatory inputs/mechanisms that lead to transcription. In some cases, cis-regulatory
regions can undergo substantial changes/shuffling without having much effect on the
ultimate transcription behaviour of a gene -- transcription measurement technologies can
help distinguish these cases from those that have actually changed a gene's expression
phenotype (Comelli & Gonzalez, 2009).
In addition to general purpose (i.e. gene, exon) microarrays, several arrays have been
designed to be maximally sensitive to differences between closely related genes.
Microarrays use probes that measure targets by hybridizing to nucleotides directly via base
complementation. Studies have previously demonstrated that the nucleotides at the center
of the probe have the most influence on binding strength. In order to minimize the potential
for cross-hybridization, some researchers have designed microarrays for comparing closely
related genes (e.g. homeologs) by using probes that feature a known distinguishing SNP at
the central position in a probe (Chaudhary et al., 2009; Flagel & Wendel., 2010; Flagel et al.,
2008; Udall et al., 2006). This design should minimize cross-hybridization, though it should
be noted that previous studies have found that cross-hybridization is only of concern when
target sequences are >90-95% identical (Rajashekar et al., 2007). For duplicate genes that
have highly similar sequences, alternative measurement technologies like deep sequencing
can be used to obtain unbiased paralog-specific expression profiles.
Quantitative proteomics techniques such as iTRAQ (Burkhart et al., 2011) or 2D
differential in-gel electrophoresis provide a similarly high-throughput platform for the
quantitation of protein abundance. The data differs from microarray data in two respects
the identities of quantified proteins are often not known in advance, and the coverage of
the proteome is not complete and is sensitive to experimental parameters. However,
protein abundances may be a more accurate reflection of gene action, as proteins are the
active products of genes in most cases and mRNA abundance doesnt always correlate
with protein abundance.
Gibson and Goldberg (2009) conducted a study on yeast duplicates using a novel metric of
functional differentiation -- number and type of protein interactions. The authors used both
affinity-precipitation mass spectometry and yeast-2-hybrid assays to construct networks of
protein interactions, and then sought to test whether the patterns of functional
differentiation better fit models of subfunctionalization or neofunctionalization. Their work
expands on previous studies that describe the functional evolution of the genome/proteome
in terms of the growth of (novel) protein interactions. They illustrate how existing methods

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48
overlook self-self interactions in the parents/progeny, and propose means of avoiding this
bias. In general, they found that subfunctionalization was the prevalent driver of protein
interaction network evolution.
Recently, sequencing and mass spectrometry have both achieved levels of throughput that
make it possible to survey the transcriptome or proteome directly. While these
technologies have considerable promise as a source for expression data, at present there are
less data available from these platforms (but see Harhay et al., 2010). However, the essential
idea of the expression profile holds constant, irrespective of the specific sort (and indeed,
mixture) of data that is mined.
7. Models of parental gene function
Since available sequence data is generally restricted to present-day organisms, it is not
directly possible to measure a genes function pre- and post-duplication. As such, when
presented with a pair of paralogs, it is not often clear which genes have retained the
ancestral function, if any. In this section, a number of proposed techniques for
estimating pre-duplication function are described. These techniques can be broadly
broken into two categories techniques which seek to find an appropriate reference
organism elsewhere in nature (i.e. a sister species with a somewhat divergent duplication
history), and techniques which attempt to estimate/reconstruct ancestral function from
those observed in extant species.
If gene information is available for two closely related species, it is often possible to find a
number of examples where a gene duplication event has only occurred in one of the two
species. In these cases, paralogs in one genome will correspond to a single ortholog in
another. By comparing the functions of paralogs to an unduplicated ortholog, it may be
possible to infer which of the two paralogs has undergone more dramatic functional
changes. Unfortunately, this approach is restricted to genes present in a 2-to-1 fashion, and
even in this cases caution must be taken to ensure the duplication event truly post-dates the
speciation event.
One interesting variant of this strategy is to use distantly related members of the same
gene family from within the same species (Panchin et al., 2010). Since most genes belong
to families with several members, recent duplicates can take advantage of ancient and
highly diverged gene family members to serve as a proxy for an orthologous outgroup.
This process is useful for calculating rates of sequence evolution between recent
duplicates.
Comparisons between lineages which have and have not undergone WGD events can also
shed light on the evolution of function post-duplication. For example, Kassahn et al. (2009)
used mouse orthologs as reference points for evaluating the post-WGD expression
divergence in duplicate genes from five teleost fish species, suggesting that this approach is
viable even when the organisms being compared are distant relatives.
Allopolyploids present a unique opportunity for studying gene evolution in the aftermath
of widespread duplication. Allopolyploids are hybrids of distinct species, and in many cases
the unhybridized lineages have persisted alongside their allopolyploid cousins to the
present day. In these cases, the history of gene functional evolution can be inferred by
examining gene expression behaviour at four stages: pre-hybridization (the two present day
diploid parental strains), day zero hybridization (a cross of the two modern day parentals to


49
create an F1 allopolyploid), and post-hybridization (the present day allopolyploid). Thus,
the functions of both parental genes can be compared to novel and mature hybrids,
revealing the immediate effects upon and eventual trajectory of functional evolution.
The utility of this approach can be seen in Chaudhary et al. (2009), where the functional
profiles of homeologous genes could be succinctly depicted as two-component pie charts.
The dominance of one genome's homelog over another can be visualized as an unequal
partitioning in the pie, and changes to this partitioning following the transition from
diploidy to allopolyploidy mark possible instances of functional specialization.
However, in many cases there are no suitable extant orthologs available to serve as models
for ancestral gene function. In these cases, there are a number of algorithms for estimating
ancestral gene function based directly on the functions of descendent (and other related)
genes. Estimation methods can try to infer both gene regulation and gene
sequence/structure from present day data.
Microarray-based gene expression profiles have been used in several efforts to estimate
ancestral gene function. In a study of stress response genes in Arabidopsis, estimates of
ancestral gene function were constructed using BayesTraits (Pagel & Meade, 2006), with the
present day response profiles used as primary data. For each extant stress response gene,
responses to various stresses were coded based on expression level changes (up-regulation,
down-regulation, no response). By adjusting the parameters of the Bayestraits program, the
authors were able to select a model for gain/loss of response behaviour. This information,
when combined with phylogenetic trees mapping out the sequence relationships for each
gene family, allowed estimates of the stress response behaviour of ancestral genes (internal
nodes on the tree) (Zou et al., 2009).
Another microarray-based approach was explored in Doxey et al. (2007). The study
examined the beta-(1,3)-glucanase gene family in Arabidopsis, using expression profiles
constructed from microarray measurements on tissue and stress response patterns. The
expression data for all genes in the family were grouped using hierarchical clustering,
such that genes with similar (correlated) expression profiles were grouped together. Based
on this clustering, genes were assigned labels according to their functional groups, and
these labels were then used as primary data for the reconstruction of ancestral states on
the gene family phylogenetic tree via parsimony. Using this approach, the expression
profile of ancestral, pre-duplication sequences could be estimated from on the values
reconstructed on the tree.
This approach of reconstructing gene functions as characters on a gene phylogenetic tree has
a lot of potential, as it allows all members of a gene family to contribute information about
the functional breadth explored in a gene family. The exact quantity reconstructed on the
tree can vary from simple binary tissue presence/absence (Karanth et al., 2009) to the exact
expression abundance as measured by a high-throughput assay (Guo et al., 2007; Li et al.,
2005; Oakley et al., 2005).
There have also been efforts to reconstruct ancestral gene sequences, with the hope of
reconstructing gene function. Working from the extant variety of fluorescent proteins,
Field and Matz (2010) modeled the evolution of fluorescence color in the family by
estimating and producing gene sequences at the internal nodes of the fluorescent protein
family phylogenetic tree. By producing proteins based on the estimated ancestral
sequences, the authors were able to estimate the fluorescence colors of evolutionary
intermediates in the family.

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50
8. Making a case for duplicate specialization
The most important techniques for studying functional specialization focus on different
aspects of gene function, but are all generally associated with the task of distinguishing the
roles and fates of duplicate genes. Figure 3 provides a diagram summarizing the various
aspects of gene function that are amenable to these techniques.
8.1 Biochemical function and analysis
Unequivocal evidence for functional specialization can be drawn from studies of
enzyme kinetics. By measuring the substrate affinity and catalytic rates of enzymes, for
example, it is possible to quantitatively measure differences in performance between
duplicate genes. Biochemical approaches are very labor intensive and only readily
applicable to certain classes of genes, but the evidence they provide is direct and readily
interpreted.
In a study highlighting the potential importance of the EAC model, Des Marais and
Rauscher (2008) used enzyme affinity assays to demonstrate the enzymatic function of
paralogous anthocyanin biosynthetic pathway genes in morning glories. Enzyme
kinetics were compared across different species that differed by a duplication of a
specific enzyme, with the unduplicated ortholog acting as a proxy for the ancestral
function.
A recent innovative approach to studying gene function used directed (in lab) evolution to
try and encourage a derived gene to revert to an hypothesized ancestral function (Bershtein
& Tawfik, 2008). The authors studied the rate of reversion and how this rate varied when
various degrees of selective pressure (selecting for the ancestral function, the current
function, or both) were applied. By studying the transitional states the gene underwent as its
function shifted, the authors found evidence that best fit the subfunctionalization model of
duplicate gene evolution.
8.2 Expression profiling and reconstruction
Expression profiles (mined from expression assays like high-throughput sequencing) can
provide immediate evidence of functional differentiation between duplicated genes
(based on divergent, non-overlapping expression behaviour). For example, Yasukawa et
al. (2010) use RNA in-situ and reporter analysis to determine precise expression
localization and timing of duplicates in Drosophila. Rajashekar (2007) used microarray
expression profiles to analyze the similarity of duplicates in the hydrophobin gene family
in a fungus, Paxillus involutus.
By augmenting comparisons of gene expression profiles with reconstructed estimates of
parental gene expression (via ancestral character estimation projected onto phylogenetic
trees, see section 7), it is further possible to estimate how each gene progeny specialized
from its parent following duplication. Case examples are the studies by Doxey et al. (2007)
and Zou et al. (2009), both mentioned previously. Zou et al. (2009) reconstructed the
expression behaviour in stress response genes in Arabidopsis, and with the additional
information made available in the estimates of ancestral behaviour, the authors were able
to infer patterns of subfunctionalization and neofunctionalization leading to the
expression behaviour in extant duplicates. Karanth et al. (2009) reconstructed the
ancestral tissue expression patterns of fatty acid binding proteins in zebrafish, revealing


51
an apparent neofunctionalization event followed by subfunctionalization in a subsequent
duplication.
8.3 Comparing with a non-duplicated ortholog
As discussed earlier, one effective technique for estimating the function of the ancestor of a
pair of duplicate genes is to refer to a related species where the locus is unduplicated. In this
case, the assumption is that the orthologous gene is behaving in the related genome as the
parental gene was behaving prior to the duplication event. This point of reference makes it
possible to distinguish between models of duplicate retention, lending to support towards
subfunctionalization versus neofunctionalization, for example.
In a study of zebrafish-specific WGD-produced duplicates, Kassahn et al. (2009) use
unduplicated mouse orthologs as a reference, despite the considerable distance separating
these two organisms. Multiple gene properties were compared between paralogs and their
mouse ortholog, including sequence, structure, and expression information. The authors
found support for neofunctionalization in a number of duplicates, and that regulatory
changes were far more common than changes to gene products.
In a study of human genes, Panchin et al. (2010) chose to use distantly related gene family
members as proxies for ancestors of recent paralogs. They demonstrated that, in many cases,
the recent duplicates are evolving asymmetrically, with one duplicate accumulating
sequence mutations much faster than its sibling.
Semon and Wolfe (2008) conducted a study comparing the fate of WGD duplicates in
X.laevis, an allopolyploid, to X. tropicalis, a related species that did not undergo any WGD.
Expression patterns were compared across 11 tissue types, and related losses of tissue
breadth to possible subfunctionalization. In addition to this, the authors also compared the
fate of duplicated genes produced through two different large-scale duplication
mechanisms by comparing X.laevis to zebrafish, a species with a well studied WGD that did
not stem from allopolyploidy. They find that duplicates retained in the X.laevis duplication
were also frequently retained in duplicate in zebrafish, suggesting common influences on
the duplicability of these gene varieties.
Another example of a well-studied allopolyploid, cotton, has been discussed in previous
sections (Chaudhary et al., 2009; Flagel et al., 2008; Flagel & Wendel, 2010). One unique
observation made possible in this system is the phenomenon of transgressive segregation,
where the expression profiles of homeologous genes eventually evolve to resemble neither
of the parental strains, suggesting a unique adaptation to the presence of two essentially
complete genomes within a single cell (Flagel & Wendel, 2010).
8.4 Comparing gene product properties
While not as easily assayed as gene expression, the transcribed content of genes (i.e.
proteins) can also suggest the gain and loss of functions. As a simple example, the rate of
protein sequence evolution can be compared between duplicates by comparing their
respective rates of synonymous and non-synonymous mutation. While not necessarily
illustrative of the nature of the difference, this method can provide evidence for
asymmetrical selection, suggesting one duplicate is acquiring amino acid altering mutations
faster than the other (Ganko et al., 2007). Working from a list of 15 of the most
asymmetrically diverged WGD-derived protein sequences in S. cerevisiae, Turunen et al.
(2009) noted substantial indels in addition to changes in important catalytic residues and

Gene Duplication

52

Fig. 3. Gene properties that can be examined for evidence of functional specialization. The
top set (orange) are approaches that check for differences in gene regulation; expression
levels reflect measurements of transcription in tissues in response to a series of stresses (e.g.
as obtained from microarrays). The bottom set (blue) are aspects of the gene product that
may differ between duplicates. Sequence logos may be generated using the WebLogo
software (Crooks et al., 2004).


53
active/cofactor binding sites. A literature search seemed to support that many of these
highly modified duplicates had acquired novel functions.
Other aspects of gene function, such as the position and number of introns or methylation
sites, have also been used to characterize divergence between duplicated genes. For
example, Xiong et al. (2010) include intron position in a study of the expansion of the ABC
transporter gene family in the ciliate Tetrahymena thermophila. In addition to comparing the
expression profiles constructed by clustering gene expression data, the authors also compare
intron positions to group the family members into functional subcategories (Xiong et al.,
2010). A similar study has examined differential splicing forms in duplicate genes of
Drosophila (Zhan et al., 2011).
Yang et al. (2006) compared the DNA-binding with one finger (DOF) gene family across
three plant species rice, Arabidopsis thaliana, and poplar. Their multifaceted approach to
describing gene function included an analysis of protein motif gain/loss and changes to
methylation patterns. Combined with information about gene regulation drawn from
microarrays, PCR and massively parallel signature sequencing, the authors compared the
relative applicability of various duplicate retention models to the DOF family.
When the information is available, the protein-protein interaction partners of duplicates can
also be compared to study duplicate specialization. Nielsen et al. (2010) compared a set of
residues in the tail ends of tubulin genes in fruit flies, noting divergence in these regions
which may reflect changes in protein-protein interaction partners. Studying the applicability
of models like subfunctionalization and neofunctionalization at the level of gene networks
has helped integrate duplicate specialization into a broader systems biology context (Gibson
& Goldberg, 2009; MacCarthy & Bergman, 2007).
9. Conclusion
Studies of the evolution of duplicate genes are pushing the field towards more exacting
standards and definitions for gene function. Since the rate and extent of duplicate gene
specialization is dependent on so many factors, and since novel functions can emerge in so
many different ways, integrative approaches will be of paramount importance to
understanding this key aspect of genomic evolution. Future studies can benefit in particular
from the inclusion of data from gene families as a whole, as this additional information
helps both with estimating ancestral gene functions and with evaluating the breadth of
function previously covered by related genes.
While empirical evidence of differential catalytic function remains the gold standard for
proving functional specialization of duplicated genes, high-throughput studies exploiting
the vast quantities of minable expression data provide a cheap and effective means for
studying functional specialization at the level of whole gene families. Genomes with
annotations beyond expression profiles (such as gene-by-gene interaction profiles and
essentiality data) should be helpful for determining the extent to which functional changes
at the regulatory level actually impact phenotype.
10. References
Arnaiz, O., Gout, J. F., Betermier, M., Bouhouche, K., Cohen, J., Duret, L., Kapusta, A.,
Meyer, E. & Sperling, L. (2010). Gene expression in a paleopolyploid: a

Gene Duplication

54
transcriptome resource for the ciliate Paramecium tetraurelia. BMC Genomics, Vol. 11,
No. 547
Barkman, T. & Zhang, J. (2009). Evidence for escape from adaptive conflict? Nature, Vol. 462,
No. 7274
Bershtein, S. & Tawfik, D. S. (2008). Ohno's model revisited: measuring the frequency of
potentially adaptive mutations under various mutational drifts. Mol.Biol.Evol., Vol.
25, No. 11, pp. 2311-2318
Burkhart, J. M., Vaudel, M., Zahedi, R. P., Martens, L. & Sickmann, A. (2011). iTRAQ protein
quantification: A quality-controlled workflow. Proteomics, Vol. 11, No. 6, pp. 1125-
1134
Canestro, C., Catchen, J. M., Rodriguez-Mari, A., Yokoi, H. & Postlethwait, J. H. (2009).
Consequences of lineage-specific gene loss on functional evolution of surviving
paralogs: ALDH1A and retinoic acid signaling in vertebrate genomes. PLoS Genet.,
Vol. 5, No. 5
Chaudhary, B., Flagel, L., Stupar, R. M., Udall, J. A., Verma, N., Springer, N. M. & Wendel, J.
F. (2009). Reciprocal silencing, transcriptional bias and functional divergence of
homeologs in polyploid cotton (gossypium). Genetics, Vol. 182, No. 2, pp.
503-517
Comelli, R. N. & Gonzalez, D. H. (2009). Divergent regulatory mechanisms in the response
of respiratory chain component genes to carbohydrates suggests a model
for gene evolution after duplication. Plant. Signal. Behav., Vol. 4, No. 12, pp.
1179-1181
Crooks, G. E., Hon, G., Chandonia, J. M. & Brenner, S. E. (2004). WebLogo: a sequence logo
generator. Genome Res., Vol. 14, No. 6, pp. 1188-1190
Deng, C., Cheng, C. H., Ye, H., He, X. & Chen, L. (2010). Evolution of an antifreeze protein
by neofunctionalization under escape from adaptive conflict. Proc. Natl Acad. Sci.
U.S.A., Vol. 107, No. 50, pp. 21593-21598
Des Marais, D. L. & Rausher, M. D. (2008). Escape from adaptive conflict after duplication in
an anthocyanin pathway gene. Nature, Vol. 454, No. 7205, pp. 762-765
Doxey, A. C., Yaish, M. W., Moffatt, B. A., Griffith, M. & McConkey, B. J. (2007). Functional
divergence in the Arabidopsis beta-1,3-glucanase gene family inferred by
phylogenetic reconstruction of expression states. Mol. Biol. Evol., Vol. 24, No. 4, pp.
1045-1055
Edger, P. P. & Pires, J. C. (2009). Gene and genome duplications: the impact of dosage-
sensitivity on the fate of nuclear genes. Chromosome Res., Vol. 17, No. 5, pp.
699-717
Field, S. F. & Matz, M. V. (2010). Retracing evolution of red fluorescence in GFP-like proteins
from Faviina corals. Mol. Biol. Evol., Vol. 27, No. 2, pp. 225-233
Flagel, L., Udall, J., Nettleton, D. & Wendel, J. (2008). Duplicate gene expression in
allopolyploid Gossypium reveals two temporally distinct phases of expression
evolution. BMC Biol., Vol. 6, No. 16
Flagel, L. E. & Wendel, J. F. (2010). Evolutionary rate variation, genomic dominance and
duplicate gene expression evolution during allotetraploid cotton speciation. New
Phytol., Vol. 186, No. 1, pp. 184-193


55
Gallach, M., Chandrasekaran, C. & Betran, E. (2010). Analyses of nuclearly encoded
mitochondrial genes suggest gene duplication as a mechanism for resolving
intralocus sexually antagonistic conflict in Drosophila. Genome Biol. Evol., Vol. 2, pp.
835-850
Ganko, E. W., Meyers, B. C. & Vision, T. J. (2007). Divergence in expression
between duplicated genes in Arabidopsis. Mol. Biol. Evol., Vol. 24, No. 10, pp.
2298-2309
Gibson, T. A. & Goldberg, D. S. (2009). Questioning the ubiquity of neofunctionalization.
PLoS Comput. Biol., Vol. 5, No. 1
Goettel, W. & Messing, J. (2010). Divergence of gene regulation through chromosomal
rearrangements. BMC Genomics, Vol. 11, No. 678
Guo, H., Weiss, R. E., Gu, X. & Suchard, M. A. (2007). Time squared: repeated measures on
phylogenies. Mol. Biol. Evol., Vol. 24, No. 2, pp. 352-362
Han, M. V., Demuth, J. P., McGrath, C. L., Casola, C. & Hahn, M. W. (2009). Adaptive
evolution of young gene duplicates in mammals. Genome Res., Vol. 19, No. 5, pp.
859-867
Harhay, G. P., Smith, T. P., Alexander, L. J., Haudenschild, C. D., Keele, J. W., Matukumalli,
L. K., Schroeder, S. G., Van Tassell, C. P., Gresham, C. R., Bridges, S. M., Burgess, S.
C. & Sonstegard, T. S. (2010). An atlas of bovine gene expression reveals novel
distinctive tissue characteristics and evidence for improving genome annotation.
Genome Biol., Vol. 11, No. 10
Jarinova, O., Hatch, G., Poitras, L., Prudhomme, C., Grzyb, M., Aubin, J., Berube-Simard, F.
A., Jeannotte, L. & Ekker, M. (2008). Functional resolution of duplicated hoxb5
genes in teleosts. Development, Vol. 135, No. 21, pp. 3543-3553
Johnson, D. A. & Thomas, M. A. (2007). The monosaccharide transporter gene family in
Arabidopsis and rice: a history of duplications, adaptive evolution, and functional
divergence. Mol. Biol. Evol., Vol. 24, No. 11, pp. 2412-2423
Karanth, S., Denovan-Wright, E. M., Thisse, C., Thisse, B. & Wright, J. M. (2009). Tandem
duplication of the fabp1b gene and subsequent divergence of the tissue-specific
distribution of fabp1b.1 and fabp1b.2 transcripts in zebrafish (Danio rerio). Genome,
Vol. 52, No. 12, pp. 985-992
Kassahn, K. S., Dang, V. T., Wilkins, S. J., Perkins, A. C. & Ragan, M. A. (2009). Evolution of
gene function and regulatory control after whole-genome duplication: comparative
analyses in vertebrates. Genome Res., Vol. 19, No. 8, pp. 1404-1418
Langille, M. G. & Clark, D. V. (2007). Parent genes of retrotransposition-generated gene
duplicates in Drosophila melanogaster have distinct expression profiles. Genomics,
Vol. 90, No. 3, pp. 334-343
Li, Q., Liu, X., He, Q., Hu, L., Ling, Y., Wu, Y., Yang, X. & Yu, L. (2011). Systematic analysis
of gene expression level with tissue-specificity, function and protein subcellular
localization in human transcriptome. Mol. Biol. Rep., Vol. 38, No. 4, pp. 2597-2602.
Li, Z., Zhang, H., Ge, S., Gu, X., Gao, G. & Luo, J. (2009). Expression pattern divergence of
duplicated genes in rice. BMC Bioinformatics, Vol. 10 Suppl 6
Li, Z., Liu, Q., Song, M., Zheng, Y., Nan, P., Cao, Y., Chen, G., Li, Y. & Zhong,
Y. (2005). Detecting correlation between sequence and expression divergences

Gene Duplication

56
in a comparative analysis of human serpin genes. BioSystems, Vol. 82, No. 3, pp.
226-234
Lockton, S. & Gaut, B. S. (2005). Plant conserved non-coding sequences and paralogue
evolution. Trends Genet., Vol. 21, No. 1, pp. 60-65
MacCarthy, T. & Bergman, A. (2007). The limits of subfunctionalization. BMC Evol.Biol., Vol.
7, No. 213
Mikhaylova, L. M., Nguyen, K. & Nurminsky, D. I. (2008). Analysis of the Drosophila
melanogaster testes transcriptome reveals coordinate regulation of paralogous genes.
Genetics, Vol. 179, No. 1, pp. 305-315
Nielsen, M. G., Gadagkar, S. R. & Gutzwiller, L. (2010). Tubulin evolution in insects: gene
duplication and subfunctionalization provide specialized isoforms in a functionally
constrained gene family. BMC Evol.Biol., Vol. 10, No. 113
Oakley, T. H., Gu, Z., Abouheif, E., Patel, N. H. & Li, W. H. (2005). Comparative methods for
the analysis of gene-expression evolution: an example using yeast functional
genomic data. Mol.Biol.Evol., Vol. 22, No. 1, pp. 40-50
Ohno, S. (1970). Evolution by Gene Duplication, Springer-Verlag, 0-04-575015-7, New York
Osada, N. & Innan, H. (2008). Duplication and gene conversion in the Drosophila melanogaster
genome. PLoS Genet., Vol. 4, No. 12
Pagel, M. & Meade, A. (2006). Bayesian Analysis of Correlated Evolution of Discrete
Characters by Reversible-Jump Markov Chain Monte Carlo. Am. Nat., Vol. 167, No.
6
Panchin, A. Y., Gelfand, M. S., Ramensky, V. E. & Artamonova, I. I. (2010). Asymmetric
and non-uniform evolution of recently duplicated human genes. Biol. Direct,
Vol. 5
Qian, W., Liao, B. Y., Chang, A. Y. & Zhang, J. (2010). Maintenance of duplicate genes and
their functional redundancy by reduced expression. Trends Genet., Vol. 26, No. 10,
pp. 425-430
Rajashekar, B., Samson, P., Johansson, T. & Tunlid, A. (2007). Evolution of nucleotide
sequences and expression patterns of hydrophobin genes in the ectomycorrhizal
fungus Paxillus involutus. New Phytol., Vol. 174, No. 2, pp. 399-411
Redon, R., Ishikawa, S., Fitch, K. R., Feuk, L., Perry, G. H., Andrews, T. D., et al. (2006).
Global variation in copy number in the human genome. Nature, Vol. 444, No. 7118,
pp. 444-454
Ren, X. Y., Fiers, M. W., Stiekema, W. J. & Nap, J. P. (2005). Local coexpression domains of
two to four genes in the genome of Arabidopsis. Plant Physiol., Vol. 138, No. 2, pp.
923-934
Semon, M. & Wolfe, K. H. (2008). Preferential subfunctionalization of slow-evolving genes
after allopolyploidization in Xenopus laevis. Proc. Natl Acad. Sci. U.S.A., Vol. 105, No.
24, pp. 8333-8338
Shoja, V., Murali, T. M. & Zhang, L. (2007). Expression divergence of tandemly arrayed
genes in human and mouse. Comp. Funct. Genomics, 60964
Skamnioti, P., Furlong, R. F. & Gurr, S. J. (2008). The fate of gene duplicates in the genomes
of fungal pathogens. Commun. Integr. Biol., Vol. 1, No. 2, pp. 196-198


57
Teshima, K. M. & Innan, H. (2008). Neofunctionalization of duplicated genes under the
pressure of gene conversion. Genetics, Vol. 178, No. 3, pp. 1385-1398
Tsankov, A. M., Thompson, D. A., Socha, A., Regev, A. & Rando, O. J. (2010). The
role of nucleosome positioning in the evolution of gene regulation. PLoS Biol.,
Vol. 8, No. 7
Turunen, O., Seelke, R. & Macosko, J. (2009). In silico evidence for functional specialization
after genome duplication in yeast. FEMS Yeast Res., Vol. 9, No. 1, pp. 16-31
Udall, J. A., Swanson, J. M., Nettleton, D., Percifield, R. J. & Wendel, J. F. (2006). A novel
approach for characterizing expression levels of genes duplicated by polyploidy.
Genetics, Vol. 173, No. 3, pp. 1823-1827
Van de Peer, Y., Maere, S. & Meyer, A. (2009). The evolutionary significance of ancient
genome duplications. Nat. Rev. Genet., Vol. 10, No. 10, pp. 725-732
Venkataram, S. & Fay, J. C. (2010). Is transcription factor binding site turnover a sufficient
explanation for cis-regulatory sequence divergence? Genome Biol. Evol., Vol. 2, pp.
851-858
Viaene, T., Vekemans, D., Becker, A., Melzer, S. & Geuten, K. (2010). Expression divergence
of the AGL6 MADS domain transcription factor lineage after a core
eudicot duplication suggests functional diversification. BMC Plant. Biol., Vol. 10,
No. 148
Wang, Z., Dong, X., Ding, G. & Li, Y. (2010). Comparing the retention mechanisms of
tandem duplicates and retrogenes in human and mouse genomes. Genet. Sel. Evol.,
Vol. 42, pp. 24
Xiong, J., Feng, L., Yuan, D., Fu, C. & Miao, W. (2010). Genome-wide identification and
evolution of ATP-binding cassette transporters in the ciliate Tetrahymena
thermophila: A case of functional divergence in a multigene family. BMC Evol.Biol.,
Vol. 10, No. 330
Xue, C., Huang, R., Liu, S. Q. & Fu, Y. X. (2010). Recombination facilitates
neofunctionalization of duplicate genes via originalization. BMC Genet., Vol. 11,
No. 46
Xue, C. & Fu, Y. (2009). Preservation of duplicate genes by originalization. Genetica, Vol. 136,
No. 1, pp. 69-78
Yang, X., Tuskan, G. A. & Cheng, M. Z. (2006). Divergence of the Dof gene families in
poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after
duplication. Plant Physiol., Vol. 142, No. 3, pp. 820-830
Yasukawa, J., Tomioka, S., Aigaki, T. & Matsuo, T. (2010). Evolution of expression patterns
of two odorant-binding protein genes, Obp57d and Obp57e, in Drosophila. Gene,
Vol. 467, No. 1-2, pp. 25-34
Yim, W. C., Lee, B. M. & Jang, C. S. (2009). Expression diversity and evolutionary dynamics
of rice duplicate genes. Mol. Genet. Genomics, Vol. 281, No. 5, pp. 483-493,
1617-4623
Zhan, Z., Ren, J., Zhang, Y., Zhao, R., Yang, S. & Wang, W. (2011). Evolution of alternative
splicing in newly evolved genes of Drosophila. Gene, Vol. 470, No. 1-2, pp. 1-6

Gene Duplication

58
Zou, C., Lehti-Shiu, M. D., Thomashow, M. & Shiu, S. H. (2009). Evolution of stress-
regulated gene expression in duplicate genes of Arabidopsis thaliana. PLoS Genet.,
Vol. 5, No. 7, e1000581
4
Predicting Tandemly Arrayed Gene
Duplicates with WebScipio
Klas Hatje and Martin Kollmar
Abteilung NMR basierte Strukturbiologie, Max-Planck-Institut fr
Biophysikalische Chemie, Am Fassberg 11, Gttingen
Germany
1. Introduction
Since the first high-quality eukaryotic genome assemblies became available the large scale
analysis of the origin of new genes came into the focus of many studies (Shoja & Zhang, 2006;
Zhou et al., 2008). New genes can originate through multiple mechanisms including gene
duplication, gene fusion/fission, exon shuffling, retroposition, horizontal gene transfer, and de
novo from noncoding sequences (Long et al., 2003). Although initial models proposed that
new copies of genes soon become nonfuntional (Nei & Roychoudhury, 1973; Ohno, 1970) it has
since been shown for numerous genes that they retain function through creating redundancy,
subfunctionalization, and neofunctionalization (Hahn, 2009; Li et al., 2005; Massingham et al.,
2001). While de novo origination from noncoding sequence has been shown to play an
unexpectedly important role (Zhou et al., 2008) most of the new genes are derived through
duplications. Gene duplicates are normally classified into dispersed and tandem duplicates.
Tandem duplications of clusters of genes, single genes, groups of exons, or single exons are
thought to be formed by unequal crossing-over events, or misaligned homologous
recombinational repair (Babushok et al., 2007; Zhang, 2003). A comparative analysis of the
human, mouse, and rat genome has shown that about 15 % of all genes represent tandemly
arrayed genes (Shoja & Zhang, 2006). A similar number of about 20 % has been found for the
fruit fly Drosophila melanogaster (Quijano et al., 2008). All these analyses rely on the particular
dataset of annotated genes used and the specific methods for defining genes as tandem genes.
However, first annotations of genomes are in most cases done by automatic gene prediction
programs, nowadays often supported by incorporating additional EST data, and therefore
miss many genes, include artificially fused neighbouring genes, and contain mis-predicted
exons and introns. Although these errors seem small, in the case of distinguishing tandem
gene duplicates from genomic region duplication and trans-spliced genes they are essential. In
addition, defining tandem genes by a certain number of nucleotides appearing in-between
cannot separate tandem gene duplicates from duplications of small genomic regions.
Tandemly arrayed gene duplicates are often conserved between species. Examples are the
olfactory receptor genes that constitute a very large gene family of several hundred genes per
species in vertebrates (Aloni et al., 2006) and the HOX genes (Garcia-Fernandez, 2005; Zhang &
Nei, 1996). While algorithms have been developed to reconstruct the history and evolution of
tandemly arrayed genes (Bertrand et al., 2008; Elemento et al., 2002) specific programs are not
available for the prediction and local reconstruction of these gene arrays.

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WebScipio is a web application to reconstruct genes based on a given protein query sequence
and a genomic DNA target sequence (Odronitz et al., 2008). The reconstruction is done with
Scipio (Keller et al., 2008), a post-processing script for the output of a BLAT run (Kent, 2002).
BLAT is a very fast tool for the alignment of protein or DNA sequences if these sequences are
almost identical. However, BLAT is not able to reconstruct intron and exon borders, it does not
identify very short exons and very divergent exons, and it is not able to reconstruct genes
spread on several pieces of contiguous DNA (contigs), which is very common in low-coverage
genome assemblies. Furthermore, BLAT is not able to identify sequencing and assembly errors
like additional or missing bases in exon regions or base substitutions leading to in-frame stop
codons. Scipio is able to correct all these errors and extend the BLAT output for the missing
sequences of short or divergent exons and of exon borders. In addition, Scipio assembles genes
spread on several contigs. WebScipio has been developed as a web interface to Scipio so that
the user does not have to install scripts and libraries. Moreover, WebScipio offers access to
about 2300 genome assembly files of more than 650 sequenced eukaryotes (July 2011), and
provides graphical and human-readable analyses of the results.
Here, we present an extension to the WebScipio web application to search for and predict
tandemly arrayed gene duplicates for a given query sequence. This extension is not
available via the Scipio command-line script. The user can search for gene duplicates in
hundreds of species for which reliable annotations are not available yet, because WebScipio
provides access to thousands of genome files.
2. Implementation
The new algorithm to predict tandemly arrayed gene duplicates is fully integrated into the web
application WebScipio to make it usable for the inexperienced user and to visualize the results for
immediate analysis. It was implemented in the Ruby programming language (Ruby
Programming Language, 2011) using the BioRuby library (Goto et al., 2010) to handle sequences.
WebScipio is based on the web framework Ruby on Rails (Ruby on Rails, 2011), which includes
the Javascript libraries Prototype (Prototype JavaScript framework: Easy Ajax and DOM
manipulation for dynamic web applications, 2011) and Scriptaculous (script.aculo.us - web 2.0
javascript, 2011). To keep the web application responsive, the search algorithm runs in the
background with the help of the Ruby on Rails plug-ins Workling (purzelrakete's workling at
master - GitHub, 2011) and Spawn (tra's spawn at master - GitHub, 2011). To store the user
session data, the database backend Tokyo Tyrant is used in combination with Tokyo Cabinet
(Tokyo Cabinet: a modern implementation of DBM, 2011). The results of the search are presented
as SVG pictures (W3C SVG Working Group, 2011) and several human-readable representations,
most notably a detailed alignment of protein query, target DNA sequence, and target translation.
The raw results can be downloaded as General Feature Format (GFF) files or as YAML files (The
Official YAML Web Site, 2011) for future upload and analysis. Specific results are available in
various formats for further inspection, like the human-readable log-files, or publication quality
figures, like the SVGs.
2.1 Search algorithm
The overall workflow of the search algorithm is shown in Fig. 1. The search for tandem gene
duplications is based on the exon-intron structure of a gene generated by Scipio. Thus the
first step of the algorithm includes a WebScipio run generating a new gene structure or the
upload of an existing Scipio result.

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61

Fig. 1. Activity flow diagram of the search for tandem gene duplications: The activity
diagram shows the processing steps of the search algorithm and the influence of the
parameters on each step. The run starts with an exon-intron gene structure determined by
Scipio. Based on the chosen parameters the exons and up- and downstream regions are
selected and searched for candidate exons of gene duplicates. The candidates are processed
and filtered. These steps are repeated for exons that have not been found. Those exons are
splitted and the search is repeated with fragments. In the end, the algorithm outputs the
exon-intron structure of the original gene and all gene duplicates.

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2.1.1 Query and target selection
The next steps are the selection of the query and the target for the search. All exons, which
are longer than a minimal length, are selected as query. The minimal length can be adjusted
by the minimal exon length parameter, which is given in number of amino acids coded by the
exon. In addition, the algorithm is able to generate exon tuples by the fusion of
neighbouring exons to one exon. This means that all pairs (2-tuples) of consecutive exons,
triplets (3-tuples), 4-tuples, 5-tuples, up to all exons are concatenated and used as query
exons. This option can be enabled by the search for concatenated exons parameter. The
nucleotide sequences of the up- and downstream regions of the gene are used as target
sequences. The lengths of these sequences are determined by the Scipio parameter region size
in number of nucleotides. The up- and downstream sequences are scanned in forward and
reverse direction. For the reverse strand the reverse complements of the given target
sequences are created.
2.1.2 Candidate identification
The query and target selection steps are followed by the search for exon candidates in the
target sequences. The search algorithm assumes that exons of gene duplications have a
similar length, share sequence similarity, are translated in the same reading frame and have
conserved splice sites. Candidate exons are determined in the target sequences for each exon
of the original gene and each exon tuple. The target nucleotide sequences are scanned for
sequence sections, which do not differ more than a maximal number of nucleotides from the
original exon length. This maximal difference is given by the allowed length difference for exons
parameter in number of amino acids. In addition, the sequence section, which determines an
exon candidate, must be flanked by a splice site pattern that corresponds to the introns
surrounding the original exon or exon tuple. Allowed splice site patterns for the first two
and last two nucleotides of these introns are GT---AG, GC---AG, GG---AG, and AT---AC.
The first exon of a gene must start with the start codon ATG and the last exon must be
followed by one of the stop codons TAG, TAA, or TGA. To allow searches for partial genes,
the algorithm is able to find candidates corresponding to the first and last exon of the gene
fragment that share splice site patterns instead of having a start codon or stop codon. This
behaviour can be adjusted by the search with start codon for first exon and search with stop codon
for last exon parameters.
2.1.3 Candidate translation and alignment
Candidate sequences are translated to amino acids in the same reading frame as the original
exon. If a candidate sequence includes a stop codon, the candidate is rejected immediately.
The translations of the candidate exons are aligned to the original exon translations by a
global alignment algorithm. The pair_align tool of the SeqAn package (Doring et al., 2008) is
used for this task. The resulting alignment score is divided by the score resulting from the
alignment of the original exon translation to itself. This normalised score makes exons of
different lengths and amino acid compositions comparable. Finally, exon candidates having
a score lower than the score given by the minimal score for exons parameter are rejected.
2.1.4 Hit filtering
The resulting candidate hits are filtered. If candidate sequences are overlapping, the lower
scoring candidates are rejected. Neighbouring candidate exons are combined to genes if they


63
are in the same order as the original exons. For each identified tandem gene a score is
calculated that reveals how many residues of the original gene were found in the tandem gene
duplication. The score is calculated as the number of residues of the original gene that are
aligned to residues in the tandem gene duplicate (and not to gaps) divided by the number of
all residues of the original gene. The tandem gene duplications that have a low score are
rejected. This behaviour can be adjusted by the minimal tandem gene score parameter.
2.1.5 Exon split run
If exons of a duplicated gene are missing, either in between two neighbouring exons, at the
start of the gene or at the end, the search is repeated for these exons by splitting the missing
original exons into pieces. The original exon sequences are split in two parts at each
nucleotide as long as the smaller part is longer than the minimum exon length. The
algorithm scans the intron regions of the duplicated genes that miss exons for candidates
corresponding to these exon splits, each composed of two parts. Thus, exons, which are split
by an intron in the duplicated gene, are found too. This option can be enabled by the search
for splitted exons parameter.
2.1.6 Results output
The output of the search algorithm is the exon-intron structure of all identified tandem gene
duplications combined in one result, and the exon-intron structure of each duplicated gene
alone. For every result a gene structure drawing is shown, as well as several options to
further examine gene details like the alignment of the query sequence to the translation of
the hit and the hit itself (Fig. 2).
2.2 WebScipio integration
The search algorithm is fully integrated into the web interface of WebScipio. The search for
tandem gene duplications can be enabled in the Advanced Options section. WebScipio
provides an interface to easily set the parameters, suggests default parameters, which will
be suitable for most cases, and offers documentation at several help pages and examples.
The raw results for the gene cluster can be downloaded all together in one YAML file or the
result for each gene of the cluster in a separate file. In addition to the raw data, the SVG
figures of the gene structures and FASTA files of the sequences (cDNA, genomic DNA,
exons, introns, target translation) are available for download. WebScipio provides an upload
option for downloaded YAML files to let the user analyse his results at a later date.
3. Results and discussion
WebScipio uses the command-line tool Scipio to reconstruct the gene structures of given
protein sequences based on the available eukaryotic genome assemblies. Scipio has been
developed for the case that protein sequences and target genome sequence are from the same
organism. Nevertheless, Scipio allows several mismatches that might result from sequencing
and assembly errors like missing or additional bases, which lead to frame-shifts, or in-frame
stop codons that would lead to premature gene stops. Mismatches might also be the result
from differences in the source of the protein sequence, which might have been obtained from
cDNA libraries of a certain strain, and the specific sequenced strain of the species. To
accomplish this task, Scipio relies on BLAT, which is one of the fastest tools available for

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Fig. 2. WebScipio result view of the search for tandem gene duplications of the Drosophila
melanogaster CG14502 gene (Flybase sequence accession FBpp0085935): Exons are illustrated
as coloured rectangles, introns as grey narrow rectangles, and gaps as red narrow
rectangles. Gaps indicate missing exons of the tandem gene duplicates. For the search the
default parameters were used except for the minimal score for exons parameter that was set to
5 % to find some exon duplicates of the first exon.
the alignment of almost similar protein or DNA sequences. As Scipio tolerates a certain
amount of mismatches between query and target sequence it can also successfully be used
for cross-species gene reconstructions and predictions (Odronitz et al., 2008). Because Scipio
relies on BLAT the success of the cross-species search depends on the difference between the


65
query and target gene. If genes are highly conserved in evolution Scipio is able to correctly
reconstruct genes in species that diverged hundreds of million years ago. If genes evolve
fast Scipio can predict genes only in very related organisms. This behaviour can also be used
to predict gene duplicates in the same organism, and is implemented as multiple results
parameter in the Scipio options. Again, because Scipio relies on BLAT, only those duplicates
will be identified that are very similar. An advantage of this option is that Scipio is able to
find dispersed as well as tandem duplicates.
In an analysis of the origin of new genes in the Drosophila species complex (Zhou et al., 2008)
it has been shown that the majority of the constrained functional new genes are dispersed
duplicates. In contrast, tandem duplications were found to be young events and to lead to
lower survival rates. Thus, tandem duplicates are often pseudogenes most probably because
the introduction of frame shifts and in-frame stop codons does not demand too many
mutations to destroy the transcription and expression of the new gene. If duplicates are kept
in the genome they acquire new functions through neofunctionalization and
subfunctionalization by accumulation of many substitutions (Ohno, 1970). Those genes are
too divergent to be identified by the multiple results option of Scipio. However, although
accumulating many substitutions tandem duplicates very often retain the gene structure of
the original gene including intron splice sites and reading frames of exons. Occasionally
further introns might be introduced or prior existing introns lost because these changes
would not destroy transcription and translation. To use this knowledge in tandem gene
duplicate identification we developed an algorithm that searches for duplicates of a query
sequence based on the restrictions imposed by its gene structure. Every piece of DNA in the
up- and downstream region of the original exon that has the same splice sites and shares
sequence homology to the original exon, when translated in the same reading frame, is
thought to be a candidate for an exon of a duplicated gene. In the case that introns have
been lost or gained in the duplicated genes the splice site restrictions apply to the outer
borders of the fused or split exons. WebScipio is able to correctly reconstruct the gene
structure for a given protein sequence and is thus very suited as starting point for searches
for candidate exons of duplicated genes.
To search for tandem gene duplicates an extension to WebScipio was implemented
providing several parameters to adjust the search according to users or genome-specific
needs. In most cases, however, the standard parameters will provide reasonable and
interpretable results. As soon as the search is done, WebScipio shows an overview of the
results as small gene structure pictures (Quick View), which reveal the exon regions of the
found tandem genes (Fig. 2). For convenient analysis the genomic region comprising the
gene structure of the query sequence and the exons of the predicted tandem genes is shown
in a combined graph and provided as one YAML file. The exons of the original gene are
dark coloured and the corresponding predicted exons have the same but lighter colour. The
darkness of the colour relates to the similarity of the predicted exon to the original one. The
same colour scheme is used to highlight the various exons in the Alignment view of the
genomic regions (Fig. 3). The Alignment view shows the nucleotide sequence of the gene
ordered in exons and introns. For every exon the genomic DNA and the corresponding
translation are shown, as well as the alignment of the query sequence to the translation.
To demonstrate the application, quality, and limitations of the new algorithm we provide
some example searches in the following sections. Tandemly arrayed gene duplicates have
several characteristics that need to be considered. Gene duplications can be found on both
the forward and the reverse strand. The duplicated genes might contain fused exons or

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Fig. 3. Alignment view of the first two exons of the third gene duplicate of the
Drosophila melanogaster CG14502 gene (see also Fig. 2): Each exon is named by the
tandem gene number and the exon number. In addition, the tandem gene score and the
exon score is given for each exon in percentage. The alignment indicates the positions of
the sequences in the genome and the protein. The first line of the alignment represents
the nucleotide sequence of the gene and the second line the translation of this sequence.
The third line shows how the amino acids of this translation match the amino acids
coded by the original exon, which are shown in the forth line. Mismatches are
represented by an X in dark red or, if amino acids are chemically similar, in light red.
Gaps in the alignment are shown as hyphens in green. The Duplicated Exon 3.3
alignment has been closed for representation purposes.
might contain additional introns. In the case of retroposed genes, which are derived from
the reverse transcription and insertion of processed genes, gene duplications do not have


67
any introns. Although gene duplications are more often found for small genes consisting
of one or only a few exons, gene duplicates can also be identified for genes consisting of
dozens of exons spanning large genomic regions. Because tandem gene duplicates are
defined by being located next to each other in the genome, intergenic regions are expected
to be short. This is also the reason why the parameter for bordering the search in up- and
downstream regions of the original gene limits this region to 300,000 nucleotides.
However, WebScipio cannot exclude that there may be additional genes in-between gene
duplicates. An example for such a scenario would be the duplication or multiple
duplications of small genomic regions that encode several genes. In most cases we
considered examples from the fruit fly Drosophila melanogaster and sequences from Flybase
(Tweedie et al., 2009), because the corresponding genome is of high quality and the
annotation of the genome is already at a very advanced stage. Fragmented genomes, like
draft genomes for which only short contigs are available, or chromosome assemblies
containing many gaps, are useful to screen for interesting candidates but do not provide
the reliability needed for tests of the algorithms quality and limitations. An advanced
annotation provides the advantage that genomic locations of most genes have already
been identified. Thus the gene order is already established although there might still be
errors in the annotation of single exons.
3.1 Examples of tandemly arrayed gene duplicates
3.1.1 Gene duplicates on both the forward and the reverse strand
The WebScipio tandem gene duplication extension has been developed to find tandem
gene duplications on the forward as well as on the reverse strand in relation to the query
gene. The example in Fig. 4 shows five gene duplicates of the Drosophila melanogaster heat
shock protein 23 gene (Hsp23), which consists of one exon. The first duplicate (Hsp67Bc)
and the forth duplicate (Hsp26) in the genomic region are on the reverse strand, the other
duplications Hsp22, CG4461, and Hsp27 are in the same reading direction as Hsp23. This
search was performed with default parameters except increasing the allowed length
difference for exons parameter to 30 amino acids. The most divergent gene duplication
Hsp67Ba (Table 1), which is encoded in the genomic region between Hsp26 and Hsp23,
was not found. This example shows that although the sequence identity is very low
between the duplicates and the Hsp23 search sequence (Table 1), five duplicates could be
identified. The length difference between Hsp23 and Hsp67Ba was too large so that
candidates of the length of Hsp67Ba were not included in the search with the given search
parameters.

Hsp67Bc Hsp22 CG4461 Hsp26 Hsp67Ba Hsp23 Hsp27
Length [aa] 199 174 200 208 445 186 213
Identity to
Hsp23
0.29 0.31 0.26 0.49 0.15 1.00 0.41
Strand rev for for rev rev for for
Table 1. Comparison of the length, similarity, and reading direction of the genes of the
Drosophila melanogaster heat shock protein cluster.

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Fig. 4. Drosophila melanogaster heat shock protein gene duplicates: The figure shows the
duplications found by the algorithm with Hsp23 as query. The genomic region contains,
from the left to the right side in the drawing, the identified genes Hsp67Bc, Hsp22, CG4461,
Hsp26, the query gene Hsp23, and another gene duplicate Hsp27. Gene duplications on the
reverse strand are marked by an arrow in reverse direction.
3.1.2 Duplicated exons in six tandemly arrayed genes including a lost intron and a
pseudogene
The new algorithm is able to reconstruct tandemly arrayed gene duplications containing
many exons and gene duplicates. The Drosophila melanogaster CG30047 gene includes 12
exons. Five duplicates of this gene could be identified with the algorithm (Fig. 5, top). In the
second duplicated gene an intron loss could be identified. The exons 11 and 12 of CG30047
are translated as one exon in this duplicate (Fig. 5, bottom). To find such lost introns the
option to search for concatenated exons has been enabled. The third duplicate most probably
represents a pseudogene, because exon 11 contains a frame shift and could thus not be
found. Other reasons for the frame shift could be sequencing and assembly errors. However,
the Drosophila melanogaster genome (Adams et al., 2000) is one of the best available and a lot
of effort has been spent in the finishing process. Thus, it is more probable that the third
duplicate is a pseudogene. Exon 1, which codes for seven amino acids, has low complexity
and could therefore only be identified in the second gene duplication by setting the minimal
exon length parameter to 7 aa.
3.1.3 Myosin heavy chain gene duplicates
Mammals encode two clusters of muscle myosin heavy chain genes, one cluster containing
the - and -cardiac muscle myosin heavy chain genes (Saez et al., 1987; Weydert et al.,
1985), and one cluster containing six skeletal muscle myosin heavy chain genes in the order
embryonic, 2a, 2x, 2b, perinatal, and extraocular (Sun et al., 2003; Weydert et al., 1985). These
myosin genes consist of 38 exons each. Based on their gene size and number of exons the
genes of the muscle myosin gene cluster should be on the upper limit of the complexity of a
search for tandem gene duplicates. With the new WebScipio extension all genes of the
muscle myosin cluster in Homo sapiens could be identified (Fig. 6). For the search the region
size parameter was set to 300,000 nucleotides and the minimal score for exons to 50 %. This
example also shows the advantage of the new WebScipio extension compared to the multiple
results option in Scipio. When searching with the multiple results option of Scipio and the 2a
gene as starting sequence, mixed genes are found for every additional gene candidate
(Fig. 6). Scipio does not know about gene borders and analyses all BLAT hits according to
their score. Therefore, Scipio combines the highest scoring hits to gene candidate one (2a),
the next highest scoring hits to gene candidate two (2x), and so on. The third gene


69

Fig. 5. Drosophila melanogaster CG30047: Five gene duplications were found for the CG30047
gene. In the second duplication the intron between exon 11 and 12 was lost as shown in the
alignment. The alignment of exon 11 (CG30047) and the alignment of the corresponding
region in the duplicated gene were shortend by amino acids 682 to 801 for representation
purposes.

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Fig. 6. Homo sapiens muscle myosin heavy chain gene cluster: The skeletal muscle myosin
heavy chain cluster consists of the genes embryonic, 2a, 2x, 2b, perinatal, and extraocular,
from left (5' end) to the right (3' end). The WebScipio search for tandem gene duplicates
based on the 2a gene identifies all other genes of the cluster. The Scipio search with the
parameter multiple results also identifies six gene candidates but only the search sequence
(the 2a gene) is found correctly while the other gene candidates consist of fusions of
different parts of the other muscle myosin heavy chain genes.
candidate, for example, mainly consists of the exons of the perinatal muscle myosin heavy
chain gene, but the N-terminus of the 2b gene has a higher homology to the 2a gene than the
N-terminus of the perinatal gene and therefore the 2b N-terminus is combined with the C-
terminus of perinatal.
The nile tilapia Oreochromis niloticus contains another type of a muscle myosin heavy chain
gene cluster (Fig. 7). Here, two genes (Mhc6 and Mhc13) are encoded on the forward strand,
and Mhc7 is encoded on the reverse strand. Nevertheless, WebScipio correctly reconstructed
the complete cluster when searching with the Mhc13 gene. When searching with Mhc6 or
Mhc7, the small C-terminal exons of the respective other genes could not be identified.
These examples demonstrate that WebScipio with the new extension is able to correctly
identify arrays of very large and complex genes. For this search the minimal score for exons
parameter was set to 30 % and the region size parameter to 50,000 nucleotides.


71

Fig. 7. Oreochromis niloticus muscle myosin heavy chain gene cluster: The nile tilapia
contains a cluster of three muscle myosin heavy chain genes (Mhc13, Mhc6, and Mhc7) of
which Mhc7 is encoded in the opposite direction. The last exon is too divergent to be
identified in most cases. Only when searching with the Mhc13 gene, the tandem genes Mhc6
and Mhc7 are reconstructed completely.
3.1.4 Revealing a pseudogene
For the Drosophila melanogaster CG3397 gene the first exon is splitted into two exons in the
prediction of the gene duplication. For this search the default parameters were used and the
option to search for splitted exons was enabled. The predicted gene is most probably a
pseudogene, because either the predicted intron between the two splitted exons is too short
to be spliced, or the exon translation results in a frame shift if both parts are considered as
one potential exon. The details are shown in the alignment (Fig. 8).
3.2 Examples of non-tandemly arrayed gene duplicates
3.2.1 Duplicated gene regions
Tandemly arrayed genes evolve through unequal recombination. In this process not only
single genes might be duplicated but small genomic regions containing several genes. The
result would be a tandemly arrayed group of genes. Because WebScipio is searching for
each gene separately it cannot separate a group of duplicated genes from a tandem array of
single genes. An example for duplicated genomic regions is the region in Drosophila
melanogaster containing genes coding for histones (Fig. 9). The new algorithm identified
many duplicates for each of the His1, His2A, His2B, His3, and His4 genes in the Drosophila
genome. As query the genes CG33825 (His1), CG33826 (His2A), CG33894 (His2B), CG33827
(His3), and CG33893 (His4) were used. The His2B and His4 genes are on the reverse strand
in comparison to the other genes. The genes are very similar (some code for the same
protein sequence) resulting in alignment scores between 99 % and 100 %. Only two more
divergent gene duplicates were found for the His2A gene. The first two gene duplicates of
His2A have alignment scores of 79 %.
3.2.2 Trans-spliced genes
Tandem gene duplicates and trans-spliced genes could evolve through the same gene
duplication process during evolution, except that only part of the gene is duplicated instead
of the complete gene. The exon-intron structure of tandem gene duplicates and trans-spliced
genes look very similar, which complicates their differentiation during the process of gene
identification. If, for example, the constitutive part of the trans-spliced gene consists of only

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Fig. 8. Drosophila melanogaster CG3397 gene: A gene duplication could be identified
downstream of the CG3397 gene, which, however, most probably is a pseudogene.
one exon while the trans-spliced part consists of groups of similar alternative exons the
correct reconstruction of the trans-spliced gene would not look different compared to a
partial reconstruction of a cluster of duplicated genes for which the first (or last) exons were
not found because of low similarity. The gene CG1637 of Drosphila is a trans-spliced gene
(McManus et al., 2010). The WebScipio algorithm predicts tandemly arrayed genes for
isoform A and B of CG1637, although the first exons of the potential tandem gene
candidates were not found (Fig. 10). The close inspection of the three isoforms shows that
the predicted exons do not belong to duplicated genes, but to trans-spliced variants of the
same gene. Another type of problem is demonstrated by the dynein intermediate chain gene
of Drosophila melanogaster. Here, the dynein intermediate chain gene is annotated as four
separate genes (Sdic1, Sdic2, Sdic3 and Sdic4) in Flybase (version of June 24
th
, 2011). The
problem is, however, that the real first two exons of the gene are not annotated in Flybase.


73

Fig. 9. Drosophila melanogaster histones: The results for the separate searches for gene
duplicates of the histones His1, His2A, His2B, His3, and His4 are shown. Based on the
results of the search for each single gene it is not possible to distinguish between a gene and
a genomic region duplication. The results of all searches at the same scale shows that not
single genes but a genomic region containing all five histone genes has been duplicated
several times.

Fig. 10. Drosophila melanogaster CG1737 gene and Drosophila melanogaster dynein
intermediate chain: The algorithm identified duplicated exons in the trans-spliced CG1737
and dynein intermediate chain genes. The search was done with default parameters and the
search for concatenated exons and search for splitted exons options were enabled. To reveal the
last and most divergent exon the region size parameter was set to 35,000 nucleotides and the
allowed length difference parameter to 30 amino acids for the dynein intermediate chain gene.
The sequence encoded by the true first exons is conserved throughout all major branches of
the eukaryotic tree of life that express a cytoplasmic dynein, in chromalveolates, Excavata,
and Opisthokonta. In addition, this N-terminal part of the dynein intermediate chain is of
high functional importance because it connects dynein to dynactin by interacting with the

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dynactin p150 gene. Based on these facts and the found exon order of the genomic region,
we expect the gene to be trans-spliced (Fig. 10, bottom).
4. Conclusion
Our algorithm provides a method to consistently predict and reconstruct tandemly arrayed
gene duplicates. It has been integrated into the web interface of WebScipio allowing the
search for gene duplicates of a given query protein sequence in the respective genome
assemblies. WebScipio provides access to more than 2300 genome assembly files from more
than 650 eukaryotes (July 2011) and is updated as soon as further genome assemblies
become available whether from newer versions of already sequenced species or from newly
sequenced genomes. The search results are presented in drawings coloured according to the
sequence similarity of the gene duplicate to the search sequence, and in several human-
readable formats like detailed alignments of the found exons to the genomic DNA.
Sequences and figures can be downloaded, as well as the complete raw data for later upload
or further computational analysis. The new algorithm is based on the precondition that gene
duplicates rather retain the gene structure of the original gene than the sequence. We could
show that the new extension to WebScipio is able to correctly predict and reconstruct gene
duplicates on both the forward and the reverse strand. Also, the new algorithm is able to
correctly reconstruct complicated gene structures spread over hundreds of thousands of
nucleotides like the skeletal muscle myosin heavy chain gene cluster in mammals. Gene
duplications often accumulate gene function destroying mutations that lead to frame shifts
and in-frame stop codons. Those potential pseudogenes are identified by WebScipio but the
user has to carefully inspect the results to distinguish between sequencing errors and real
pseudogenes. WebScipio cannot distinguish between gene duplicates and duplications of
small genomic regions that might encode several genes. Here, WebScipio can identify and
reconstruct the duplicates of one gene but does not provide any hints about other genes in
the intergenic regions. Trans-spliced genes often contain clusters of alternative exons. Those
clusters will be identified by WebScipio, but again the user needs to evaluate the results to
distinguish between cases of trans-spliced genes, where the constitutive part is encoded by
just a few exons, or real gene duplications, for which some terminal exons could not be
identified because of very low sequence similarity or even assembly gaps. Altogether,
WebScipio provides an easy to use way to analyse the genomic region of every gene of
interest for the very common event of tandem gene duplication.
5. Acknowledgments
MK has been funded by grant KO 2251/6-1 of the Deutsche Forschungsgemeinschaft. We
thank Bjrn Hammesfahr for fruitful discussions.
6. References
Adams, M. D., Celniker, S. E., Holt, R. A., Evans, C. A., Gocayne, J. D., Amanatides, P. G.,
Scherer, S. E., Li, P. W., Hoskins, R. A., Galle, R. F. et al. (2000). The genome
sequence of Drosophila melanogaster, Science, Vol.287, No.5461, pp. 2185-2195
Aloni, R., Olender, T. & Lancet, D. (2006). Ancient genomic architecture for mammalian
olfactory receptor clusters, Genome Biol, Vol.7, No.10, pp. R88


75
Babushok, D. V., Ostertag, E. M. & Kazazian, H. H., Jr. (2007). Current topics in genome
evolution: molecular mechanisms of new gene formation, Cell Mol Life Sci, Vol.64,
No.5, pp. 542-554
Bertrand, D., Lajoie, M. & El-Mabrouk, N. (2008). Inferring ancestral gene orders for a family
of tandemly arrayed genes, J Comput Biol, Vol.15, No.8, pp. 1063-1077
Doring, A., Weese, D., Rausch, T. & Reinert, K. (2008). SeqAn an efficient, generic C++
library for sequence analysis, BMC Bioinformatics, Vol.9, pp. 11
Elemento, O., Gascuel, O. & Lefranc, M. P. (2002). Reconstructing the duplication history of
tandemly repeated genes, Mol Biol Evol, Vol.19, No.3, pp. 278-288
Garcia-Fernandez, J. (2005). The genesis and evolution of homeobox gene clusters, Nat Rev
Genet, Vol.6, No.12, pp. 881-892
Goto, N., Prins, P., Nakao, M., Bonnal, R., Aerts, J. & Katayama, T. (2010). BioRuby:
Bioinformatics software for the Ruby programming language, Bioinformatics
Hahn, M. W. (2009). Distinguishing among evolutionary models for the maintenance of gene
duplicates, J Hered, Vol.100, No.5, pp. 605-617
Keller, O., Odronitz, F., Stanke, M., Kollmar, M. & Waack, S. (2008). Scipio: using protein
sequences to determine the precise exon/intron structures of genes and their
orthologs in closely related species, BMC Bioinformatics, Vol.9, pp. 278
Kent, W. J. (2002). BLAT--the BLAST-like alignment tool, Genome Res, Vol.12, No.4, pp.
656-664
Li, W. H., Yang, J. & Gu, X. (2005). Expression divergence between duplicate genes, Trends
Genet, Vol.21, No.11, pp. 602-607
Long, M., Betran, E., Thornton, K. & Wang, W. (2003). The origin of new genes: glimpses
from the young and old, Nat Rev Genet, Vol.4, No.11, pp. 865-875
Massingham, T., Davies, L. J. & Lio, P. (2001). Analysing gene function after duplication,
Bioessays, Vol.23, No.10, pp. 873-876
McManus, C. J., Duff, M. O., Eipper-Mains, J. & Graveley, B. R. (2010). Global analysis of
trans-splicing in Drosophila, Proc Natl Acad Sci U S A, Vol.107, No.29, pp. 12975-
12979
Nei, M. & Roychoudhury, A. K. (1973). Probability of fixation and mean fixation time of an
overdominant mutation, Genetics, Vol.74, No.2, pp. 371-380
Odronitz, F., Pillmann, H., Keller, O., Waack, S. & Kollmar, M. (2008). WebScipio: an online
tool for the determination of gene structures using protein sequences, BMC
Genomics, Vol.9, pp. 422
The Official YAML Web Site, (2011). Available from http://www.yaml.org/
Ohno, S. (1970). Evolution by Gene Duplication, Berlin, Springer
Prototype JavaScript framework: Easy Ajax and DOM manipulation for dynamic web
applications, (2011). Available from http://www.prototypejs.org
purzelrakete's workling at master - GitHub, (2011). Available from
http://github.com/purzelrakete/workling
Quijano, C., Tomancak, P., Lopez-Marti, J., Suyama, M., Bork, P., Milan, M., Torrents, D. &
Manzanares, M. (2008). Selective maintenance of Drosophila tandemly arranged
duplicated genes during evolution, Genome Biol, Vol.9, No.12, pp. R176
Ruby on Rails, (2011). Available from http://rubyonrails.org
Ruby Programming Language, (2011). Available from http://www.ruby-lang.org/

Gene Duplication

76
Saez, L. J., Gianola, K. M., McNally, E. M., Feghali, R., Eddy, R., Shows, T. B. & Leinwand, L.
A. (1987). Human cardiac myosin heavy chain genes and their linkage in the
genome, Nucleic Acids Res, Vol.15, No.13, pp. 5443-5459
script.aculo.us - web 2.0 javascript, (2011). Available from http://script.aculo.us
Shoja, V. & Zhang, L. (2006). A roadmap of tandemly arrayed genes in the genomes of
human, mouse, and rat, Mol Biol Evol, Vol.23, No.11, pp. 2134-2141
Sun, Y. M., Da Costa, N. & Chang, K. C. (2003). Cluster characterisation and temporal
expression of porcine sarcomeric myosin heavy chain genes, J Muscle Res Cell Motil,
Vol.24, No.8, pp. 561-570
Tokyo Cabinet: a modern implementation of DBM, (2011). Available from
http://fallabs.com/tokyocabinet/
tra's spawn at master - GitHub, (2011). Available from http://github.com/tra/spawn
Tweedie, S., Ashburner, M., Falls, K., Leyland, P., McQuilton, P., Marygold, S., Millburn, G.,
Osumi-Sutherland, D., Schroeder, A., Seal, R. et al. (2009). FlyBase: enhancing
Drosophila Gene Ontology annotations, Nucleic Acids Res, Vol.37, Database issue,
pp. D555-559
W3C SVG Working Group, (2011). Available from http://www.w3.org/Graphics/SVG/
Weydert, A., Daubas, P., Lazaridis, I., Barton, P., Garner, I., Leader, D. P., Bonhomme, F.,
Catalan, J., Simon, D., Guenet, J. L. et al. (1985). Genes for skeletal muscle myosin
heavy chains are clustered and are not located on the same mouse chromosome as a
cardiac myosin heavy chain gene, Proc Natl Acad Sci U S A, Vol.82, No.21, pp.
7183-7187
Zhang, J. (2003). Evolution by gene duplication: an update, Trends Ecol Evol, Vol.18, pp.
292-298
Zhang, J. & Nei, M. (1996). Evolution of Antennapedia-class homeobox genes, Genetics,
Vol.142, No.1, pp. 295-303
Zhou, Q., Zhang, G., Zhang, Y., Xu, S., Zhao, R., Zhan, Z., Li, X., Ding, Y., Yang, S. & Wang,
W. (2008). On the origin of new genes in Drosophila, Genome Res, Vol.18, No.9, pp.
1446-1455
5
The LRR and TM Containing
Multi-Domain Proteins in Arabidopsis
Felix Friedberg
Howard University Medical School Washington, DC
USA
1. Introduction
Thousands of different multi-domain proteins, each the product of one separate specific
gene, which exhibit one or multiple transmembrane (TM) domains, are expressed in
Arabidopsis as well as in Homo sapiens (Sallman-Almen et al.,2009). These molecules may
carry one or multiple TM domains very close to the amino or carboxyl terminus or even
dispersed throughout the molecule Thus one may conclude that the TM domain existed
prior to the branching of plants and metazoa. In both organisms, a very small percent of the
TM domain containing proteins additionally possess multiple leucine rich repeats (LRR)
domains. These domains seem to transmit ligand perception. So one should presume that
such domains also existed prior to the branching of these two forms of life. (A caveat,
however, is in order: Many of the combinations of the various domains which one finds in
plants are not present in the same combination in animals and vice versa.). As demonstrated
in this paper, subsequently, during evolution, on several occasions, the genes for these
multi-domain proteins duplicated and during this process they were often altered slightly to
allow generation of proteins that could provide new specific functioning (i.e.they
underwent neofunctionalization).
Unlike the adaptive immune system which exists in animals but not in plants, an innate
immune system is present in all multicellular organisms (animals and plants ). This latter
system operates by way of receptors: the Toll-like receptors (TLRs) (first identified in
Drosophila ) which bind lipopolysaccharides (endotoxins). In Drosophila, these receptors
not only activate innate immunity; they also act in dorsal-ventral specifications. When one
compares these molecules as they are encoded e.g. .in Arabidopsis (Dangl & Jones, 2001)
with those present in Homo Sapiens, one finds that in animals but not in plants these
receptor proteins possess a cysteine rich domain just prior to entering the membrane and
also a signaling domain (which is not a protein kinase but which acts as a docking site) on
the backside of the TM domain. TLRs target pathogen associated molecular patterns
(PAMPs) by way of the LRRs domains.
There are, however, besides of TLRs, many other multi-domain proteins that contain both TM
and LRR domains, encoded in Arabidopsis and also in Homo sapiens. Many exist in both of
these two species but some of them are present only in one or in the other. Below we list more
than a hundred of these various proteins (each expressed from an individual gene) present in
Arabidopsis. The TM domain is an about 22 AA residue domain and the LRR is an about 20-29
residue domain (which contains about 6 Leu ). Both domains are present in proteins that

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78
participate in protein-protein interactions but which have different functions and cellular
locations. In each case, the protein is presented below, in an order guided by a Clustal X
arrangement (http:www.clustal.org/) and is labeled by its NCBI (http://www.ncbi.nlmn.nih.
gov/protein) protein identification number, followed by its chromosomal locus tag, in diagram
form as given by SMART (http://smart.embl-heidelberg.de).
1.1 Proteins with extracellular multiple LRR domains, a single TM domain (blue
rectangle) positioned close to the carboxyl terminus and a short cytoplasmic tail

NP_199740;AT5G49290; Protein Binding

NP_177558;AT1G74180;Receptor Like Protein 14; Protein Binding

NP_180117;AT2G25470;Receptor Like Protein 21; Protein Binding
(NP_199740, NP_177558 and NP_180117 display 60% AA identity.)

NP_177559;AT1G74190; Receptor Like Protein 15; Protein Binding


NP_190892;AT3G53240; Receptor Like Protein 45, Protein Binding

The LRR and TM Containing Multi-Domain Proteins in Arabidopsis

79

NP_176115;AT1G58190; Receptor Like Protein 9, Protein Binding

NP_178125:AT1G80080; TMM (Too Many Mouths); Protein Binding/Receptor. (Note: This
protein promotes cell fate progression in stomatal development of stems (Bhave et al.,2009)).

P_176717;AT1G65380; Clavata 2; Protein Binding /Receptor Signaling Protein. (This protein
forms a distinct CLE binding receptor complex regulating stem cell specification (Guo et
al.,2010)).

NP_188941; AT3G23010; Receptor Like Protein 36 (Disease Resistance Protein)

NP_187188; AT3G05370; Receptor Like protein 31; Protein Binding

NP_177296; AT1G71400; Receptor Like Protein 12; Protein Binding


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80

NP_197963; AT5G25910; Receptor Like Protein 52; Protein Binding (Note: It is a putative
disease resistance protein induced e.g. by chitin oligomers (Ramonell et al., 2005)).





Note: It is possible that the primary function of most if not of all of the proteins listed above is to
provide disease resistance.


81
1.2 Proteins with one single TM domain positioned at the amino terminus and another
one close to the carboxyl terminus

NP_198058:AT5G27060; Receptor Like Protein 53; protein binding

NP_187216;AT3G05650: Receptor Like Protein 32; protein binding

NP_175225;AT1G47890; Receptor Like Protein 7; protein binding. Note: NP_175225
possesses two TM domains at the AA terminus. ( NP_198058, NP_187216 and NP_175225
exhibit 44 % AA identity.)

NP_188953;AT3G23120; Receptor Like Protein 38; protein binding:

NP_188952;AT3G23110; Receptor Like Protein 37; protein binding



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82

NP_180864;AT2G33050; Receptor Like Protein 26; protein binding . Notice loss of carboxyl
terminal TM.


2. Receptor Like Kinases (RLKs)
2.1 Proteins with extracellular N-terminal LRR domains, a TM domain and an
intracellular STYKc (tyrosine kinase catalytic) domain which is close to the C-
terminus
These proteins are expected to play roles in intercellular communication during tissue
identity maintenance and regulation of development. Still some of them have been shown to
play a role in innate immunity. Interaction with the extracellular LRR domains results in
activation of the intracellular STYK catalytic domain. Then, the activated receptor, in
response, catalyses the phosphorylation of tyrosine residues in intracellular proteins.

NP_191196;AT3G56370; Leucine rich repeat transmembrane protein kinase. (Inflorescence
and root apices receptor-like kinase (IRK). (Hattan et al., 2004)

NP_195809;AT5G01890; Leucine rich repeat transmembrane protein kinase


83



NP_193747;AT4G20140; Leucine rich repeat transmembrane protein kinase.
(GASSHO 1).

NP_199283;AT5G44700; Leucine rich repeat transmembrane protein kinase.
(GASSSHO 2). (GASSHO 1 and 2 show 76% AA homology. They are required for the
formation of normal epidermal surface during embryogenesis (Tsuwamoto et al., 2008).

NP_201371;AT5G65700; Leucine rich repeat transmembrane protein kinase. (BAM1/ Barely
any meristem 1). Bam receptors regulate stem cell specification and organ development
through interactions with Clavata signaling (DeYoung & Clark,2008).

NP_190536;AT3G49670; Leucine rich repeat transmembrane protein kinase. (Bam2)

NP_193760;AT4G20270; Leucine rich repeat transmembrane protein kinase (Bam3). BAM 1,2
and 3 exhibit 49% AA identity). Notice the presence of a second TM at the amino terminus
of Bam 3 which evolution diluted for Bam 1 and 2.

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84


NP_174166;AT1G28440; ATP binding/protein serine/threonine kinase (HAESA-Like1)

NP_194578;AT4G28490; ATP binding/protein serine/threonine kinase (HAESA)

NP_201372;AT5G65710; ATP binding /protein serine/threonine kinase (HAESA-Like2) The
three HAESA proteins function in developmentally regulated floral organ abscision (Jinn et
al., 2000). (NP_174166, NP_194578 and NP_201372 exhibit 37% AA identity.)

NP_197965;AT5G25930; Leucine rich repeat family protein kinase

NP-199777;AT5G49660; Leucine rich repeat transmembrane protein kinase

NP_188604;AT3G19700; ATP binding / protein kinase (Haiku 2) Haiku 2 may play a role in
the determination of seed size by endosperm (Garcia et al.,2003).


85

NP_178330;AT2G02220; ATP binding / protein kinase (Phytosulfokine (PSK) Receptor
1).PSK may play a role in pathogen or herbivore interactions (Loivamaki et al.,(2010)
(NP_188604 and NP_178330 exhibit 29.3% AA identity.)

NP_196311;AT5G06940; Leucine rich repeat family protein

NP_190342; AT3G47580; Leucine rich repeat transmembrane protein kinase




NP_197548; AT5G20480; ATP binding / protein kinase (EF-TU receptor) It recognizes
bacterial PAMP EF Tu. (Zipfel, et al., 2006).

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86

NP_199445;AT5G46330; ATP binding / protein kinase (Flagellin sensitive 2 (FLS) .It
recognizes bacterial flagellin and evokes plant innate immunity (Lu, et al., 2010)

NP_180150;AT2G25790: Leucine rich repeat / transmembrane protein kinase

NP_178304;AT2G01950; BRI1-like2 ATP binding / protein serine-threonine kinase. This
kinase interacts with vascular-specific adaptor proteins to influence leaf venation. (Ceserani
et al.,2009.) Note absence of amino terminal TM.

NP_187946;AT3G13380; BRI1-like3 ATP binding / protein serine-threonine kinase. Note
absence of amino terminal TM.

NP_195650;AT4G39400; Brassinosteroid insensitive 1 (BRI1) ATP binding / protein serine-
threonine kinase. (Brassinosteroids regulate plant development by way of a signal
transduction pathway involving BRI1 and BAK1 transmembrane receptor kinases (Wang et
al., 2008).

NP_175957;AT1G55610; BRI1-like1 ATP binding / protein serine threonine kinase.

NP_196345;AT5G07280; EMS1 (Excess Microsporocytes 1) Transmembrane receptor protein
kinase. (This protein controls somatic and reproductive cell fates in the anther of
Arabidopsis.) (Zhao et al., 2002) Note absence of the amino terminal TM.


87

NP_178330;AT2G02220; Phytosulfokine receptor (PSK R1) ATP binding / protein serine-
threonine kinase. (PSK represents a class of hormones that affects cellular longevity and
growth ( Matsubayashi et al., 2006).

NP_200200;AT5G53890; Leucine-rich repeat transmembrane protein kinase

NP_181713;AT2G41820: Leucine-rich repeat transmembrane protein kinase



NP_193826;AT4G20940, Leucine rich repeat transmembrane protein kinase
Note: For molecules such as NP_172468 SMART does not recognize any LRR regions. Hence, these
molecules are not listed here.

Gene Duplication

88
2.2 The members below possess a TM domain at the amino terminus and a second
one in front of the STYKc domain:

NP_201029;AT5G62230; Erecta like 1 (ERL1); kinase. Mediates morphological alterations.
(Uchida et al.;2011).

NP_196335;AT5G07180; Erecta like 2 (ERL 2); kinase

NP_173217;AT1G17750;Leucine rich repeat / transmembrane protein kinase. PEP receptor 2
(PEPR2). The amino terminal TM islost (overly mutated).

NP_177451; AT1G73080; ATP binding / protein serine/ threonine kinase, PEP1 receptor
(PEPR1). (PEP1and 2 are implicated in Arabidopsis development and immunity) ( Postel , et
al.;2010). (PEPR1 and PEPR2, both perceive the existence of an endogenous danger signal
Peptide 1 when such a peptide is present.) (Krol et al.;2010). (NP_173217 and NP_177451
exhibit 70% AA identity.)

NP_199705;AT5G48940 Leucine rich repeat transmembrane protein kinase




89


NP_174809:AT1G35710; Leucine rich repeat transmembrane protein kinase

NP_192625, AT4G08850; Leucine rich repeat transmembrane protein kinase

NP_200956;AT5G61480; Leucine rich repeat transmembrane protein kinase..PXY receptor.
(This receptor like-kinase is essential for polarity during plant vascular tissue development.
(Fisher &Turner,2007)



NP_177374;AT1G72300; Leucine rich repeat transmembrane protein kinase, (This protein
perceives phytosulfokines (Amano et al., 2007)).

NP_190742;AT3G51740; Inflorescence Meristem Receptor-Like Kinase (IMK2)

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90

NP_201529;AT5G67280; Receptor-Like Kinase (RLK)
The Strubbelig Receptor Family Kinases:
;
NP_196300;AT5G06820; (SRF2) Strubbelig receptor family 2; kinase. An amino terminal TM
is lost. SRFs affect the formation and shape of several organs by influencing cell
morphogenesis, the orientation of the division plane and cell proliferation. (Chevalier et al.,
2005).

NP_566444;AT3G13065; (SRF4) Strubbelig receptor family 4; kinase

NP_178019;AT1G78980; (SRF5) Strubbelig receptor family 5, kinase . The amino terminal
TM is lost.

NP_175777;AT1G53730; (SRF6) Strubbelig receptor family 6; kinase

NP_188052;AT3G14350; (SRF7) Strubbelig receptor family 7; kinase.. The amino terminal
TM is lost.


91
Not shown:
NP_565489;AT2G20850; (SRF1) Strubbelig receptor family 1; kinase. All LRR domains are
lost. (SMART).
NP_192248;AT4G03390; (SRF3) Strubbelig receptor family 3; kinase. The amino terminal TM
is duplicated. All LRR domains are lost. (SMART).
NP_193944;AT4G22130; (SRF8) Strubbelig receptor family 8, kinase. All LRR domains are
lost.(SMART).
Note: For molecules such as NP_177363 SMART does not recognize any LRR regions. Hence, these
molecules are not listed here.
3. Proteins that contain an S_TKc domain: (AA homology is 44% for S_TKc
vs. STYKc.)

NP_173166;AT1G17230; Leucine rich repeat / transmembrane protein kinase




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92
4. Conclusion
Gene duplication is rampant. It should be noted that the products depicted above show
considerable variation both in number of, and distances between, the LRR domains.
Where, when and how do the genes listed here function? For most of them there is no
evidence that they deliver innate immunity in the plant. Of course, some of them do! For
many the function is not known. Gene duplications, carrying amino acid changes
resulting from mutations, often end in neofunctionalization even though duplicate genes
may also merely provide tissue specific expression for the original ancestral gene.
Subsequent alternate splicing of genes, in turn, might also give new roles to the genes. But
if domains are the units that built proteins, then domain shuffling provides a more
efficient source for expressed gene versatility: (Thereby, nature promotes evolution of
disparate proteins for novel functions.) Most of the genes listed in this chapter certainly
exist because of duplication. These genes could be grouped further, however, because
another domain had first been added for the projected protein molecules, long before the
gene duplications occurred. Possibly, domains represent the evolutionary building blocks
for all proteins. At present we can only speculate as to the mechanism of such random
multi-domain protein formation. Were transposons involved? (Retroprocession, the
process that is responsible for pseudogene formation, possibly could have also facilitated
the creation of new disparate proteins!). Specific domain combinations might have been
built randomly maybe sometimes just once during the evolution of an organism and
then sometimes only to be rearranged during duplication or even to loose domains by
mutating them away thereafter. (.See e.g..the Strubbelig family members 1-8.)
5. References
Amano Y, Tsubouchi H, Shinohara H. et al. Tyrosine-sulfated glycopeptides involved in
cellular proliferation and expansion in Arabidopsis, PNAS (2007) 104: 18333-8.
Bhave NS, Veley KM, Nadeau JA, et al. Mutations of TMM causes stomatal patterning
defects in leaves and eliminates stomata formation in stems, Planta (2009) 229: 357-
67.
Ceserani T, Trofka A, Gandora N, et al. VH1/BRL2 receptor-like kinase interacts with
vascular-specific adaptor proteins VIT and VIK to influence leaf venation, Plant J
(2009) 57: 1000-14.
Chevalier D, Batoux M, Fulton L, et al. Strubbelig defines a receptor kinase mediating
signaling pathway regulating organ development in Arabidopsis, PNAS (2005) 102:
9074-79.
Clark SE, Williams RW, Myerowitz EM, The Clavata 1 gene encodes a putative receptor
kinase that controls shoot and floral meristem size in Arabidopsis, Cell (1997)
89:575-85.
Dangl JL, Jones JDG, Plant pathogens and integrated defence responses to infection, Nature
(2001) 411: 826-33.
DeYoung BJ, Clark SE, Signalling through the Clavata 1 Receptor complex, Plant Mol Biol
(2001) 46:505-13.


93
Fisher K, Turner S, PXY a receptor like kinase essential for maintaining polarity during
plant vascular-tissue development,
Curr Biol (2007) 17: 1061-6
Garcia D, Saingery V,Chambrier P, et al., Arabidopsis haiku mutants reveal new controls of
seed size by endosperm, Plant Physiol. (2003) 131: 1661-70.
Gomez-Gomez L, and Boller T, FLS2: A LRR receptor like kinase involved in the perception
of the bacterial elicitor Flagellin in Arabidopsis, Mol Cell (2000) 5, 1003-11.
Guo Y, Han L, Hymes L,et al., Clavata 2 forms a distinct CLE binding receptor complex
regulating Arabidopsis stem cell specification, The Plant Journal (2010) 63: 889-
900.
Hattan J, Kanamoto H,Takemura M, et al., Molecular characterization of the cytoplasmic
interacting protein of the receptor kinase IRK expressed in the inflorescence and
root apices of Arabidopsis, Biosci. Biotechnol.Biochem. (2004) 68:2598-606.
Jinn T-L, Stone JM, and Walker JC, HAESA, An Arabidopsis leucine-rich repeat receptor
kinase controls floral organ abscission, Genes, Dev (2000) 14:108-17.
Krol E, Mentzel T, Chincilla D, et al.., Perception of the Arabidopsis danger signal peptide 1
involves the pattern recognition receptor AtPEPR1 and its close homologue
AtPEPR2, J Biol Chem (2010) 285:13471-9.
Loivamaki M, Stuhrwohldt N, Deeken R, et al. A role for PSK signaling in wounding and
microbial interactions in Arabidopsis. Physiol Plant (2010) 139:348-57.
Lu D, Wu S, Gao X, Zhang Y, et al., A receptorlike cytoplasmic kinase, BIK1, associates
with a flagellin receptor complex to initiate plant innate immunity, PNAS (2010)
107:496-501.
Matsubayashi Y, Shinohara H, and Ogawa M, Identification and functional
characterization of phytosulfokine receptor using a ligand-based approach, Chem
Rec (2006) 6:356-64.
Postel S, Kufner I,Beuter C, et al.. The multifunctional leucine-rich repeat receptor kinase
BAK1 is implicated in Arabidopsis development and immunity, European J Cell
Biol (2010) 89: 169-.74.
Ramonell K, Berrocal-Lobo M, Koh S, et al. Loss-offunction mutations in chitin responsive
genes show increased susceptibility to the powdery mildew pathogen Ersiphe
chicoracecearum. Plant Physiol. (2005) 138: 1027-36.
Sallman-Almen M, Nordstrom KJ, Fredriksson R, et al.. Mapping the human membrane
proteome. BMC Biology 2009
Tori KU, Mitsukawa N, Oosumi T, et al. The Arabidopsis Erecta gene encodes a
putative receptor kinase with extracellular leucine rich repeats, Plant cell (1996)
8: 735-46.
Tsuwamoto R, Fukuoka H,Takahata Y, GASSHO1 and GASSHO2 encoding a putative LRR
repeat TM-type receptor kinase are essential for the normal development of the
epidermal surface of Arabidopsis embryos, The Plant J. (2008) 54: 32-42.
Uchida N, Igan K, Bogenschutz NL, et al. Arabidopsis ERECTA-family receptor kinases
mediate morphological alterations stimulated by activation of NB-LRR type UNI
proteins, Plant Cell Physiol. (2011) in Press

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Zhao DZ, Wang GF, Speal B, Ma H, The excess microsporocytes 1 gene encodes a putative
leucine rich repeat receptor protein kinase that controls somatic and reproductive
cell fates in the Arabidopsis anther, Genes Dev. (2002) 16:2021-31.
Zipfel C, Kunze G, Cinchilla D, et al. Perception of the bacterial PAMP EF-TU by the
receptor EFR restricts Agrobacterium mediated transformation, Cell (2006) 125:749-
60.
6
Partial Gene Duplication and the
Formation of Novel Genes

Macarena Toll-Riera
1
, Steve Laurie
1
, Nria Rad-Trilla
1
and M.Mar Alb
1,2

1
Evolutionary Genomics Group, Biomedical Informatics Programme, Universitat Pompeu
Fabra (UPF) - Institut Municipal dInvestigaci Mdica (IMIM)

2
Catalan Institution for Research and Advanced Studies (ICREA), Barcelona
Spain
1. Introduction
The publication of the first fully sequenced genomes represented a landmark in the
biological sciences. The comparison of genomes from different organisms provides us with
unprecedented opportunities to address many long-standing evolutionary questions in a
more comprehensive way.
1.1 Lineage-specific genes
The availability of several genomes from related organisms permits the identification of
newly evolved genes in different lineages or species, the study of their mechanisms of
formation and the investigation of their role in adapting to new environments or
physiological conditions (Domazet-Loso & Tautz, 2003; Guo et al., 2007; Khalturin et al.,
2009; Kuo & Kissinger, 2008; Siepel, 2009; Toll-Riera et al., 2009a; Zhou et al., 2008). Recently
formed genes give us the opportunity to study the action of natural selection in recent times
and to investigate the processes associated with gene creation (Zhou & Wang, 2008).
The number of species-specific genes, or orphan genes, is not insignificant. They represent
around 14% of the genes in 60 fully sequenced microbial genomes (Siew & Fischer, 2003)
and between 20-30% in Drosophila species (Domazet-Loso & Tautz, 2003; Drosophila 12
Genomes Consortium, 2007). Genes restricted to particular lineages include vomeronasal
receptors and casein milk proteins in mammals, which are known to be involved in specific
physiological adaptations in this lineage (International Chicken Genome Sequencing
Consortium, 2004). Additionally, several lineage-specific genes have been found to be
involved in defence against pathogens, such as dermcidin in primates (Toll-Riera et al.,
2009a) and surface antigens in apicomplexan parasites (Kuo & Kissinger, 2008).
Interestingly, it has been noticed that rice orphan genes are more often expressed under
environmental pressure (injury and hormone treatment) than non-orphan genes, indicating
that novel genes help in adaptation to changing conditions (Guo et al., 2007).
Many newly evolved genes are derived from partial or complete gene duplication of pre-
existing genes (Long et al., 2003; Marques et al., 2005; Toll-Riera et al., 2009a; Zhou et al.,
2008). Alternative processes of gene formation include exaptation from mobile elements

Gene Duplication 96
(Nekrutenko & Li, 2001; Toll-Riera et al., 2009b), gene fusion or fission (Parra et al., 2006)
and de novo gene formation from non-coding sequences (Cai et al., 2008; Heinen et al., 2009;
Knowles & McLysaght, 2009; Levine et al., 2006; Toll-Riera et al., 2009a). A genome-wide
study in Drosophila melanogaster has reported that gene duplication is the most common
mechanism for the formation of novel genes in this species (Zhou et al., 2008).
1.2 Gene duplication
In the early thirties Haldane (Haldane, 1932) and Muller (Muller, 1935) were the first to
propose gene duplication as a mechanism for the generation of new genes. Later, in the
seventies, Ohno published an influential book about the role of gene duplication in
evolution (Ohno, 1970), in which he emphasised the importance of gene duplication in
generating protein functional diversity. With the availability of complete genome sequences
it has become possible to estimate genomic rates of gene duplication (Lynch & Conery,
2000), analyse the pattern of evolution of the two duplicated copies, and identify lineage-
specific gene family expansions. Expanded gene families that have been analysed in detail
include olfactory receptors in mouse (Mouse Genome Sequencing Consortium 2002) and
human (Gilad et al., 2005), and KRAB-associated zinc-finger in primates (Castresana et al.,
2004). Genomic studies have shown that gene duplication is associated with increased
coding sequence evolutionary rates (Lynch and Conery 2000; Scannell and Wolfe 2008),
higher tissue expression divergence (Gu et al., 2002; Makova & Li, 2003), and higher
regulatory sequence divergence (Farre & Alba, 2010).
The molecular mechanisms that have been proposed to be involved in duplication are non-
allelic homologous recombination, transposon-mediated transposition and illegitimate
recombination. The first two mechanisms imply the presence of sequence homology (Zhou
et al., 2008). Yang and colleagues (Yang et al., 2008) found an excess of repetitive sequences
at the breakpoints of the duplicated regions of a group of Drosophila lineage-specific young
duplicates, suggesting the action of non-allelic homologous recombination. Another study
in Drosophila found that dispersed duplicates have mainly arisen through non-allelic
homologous recombination, while tandem duplicates most often arose through illegitimate
recombination (Zhou et al., 2008). It has also been hypothesized that segmental duplications
may arise from the recombination of Alu repeat sequences (Bailey et al., 2003).
Duplicated genes appear at a very high rate. It has been estimated that, on average, 0.01
duplicates arise per gene per million years (Lynch & Conery, 2000). The most frequent fate
following gene duplication is believed to be the silencing of one of the duplicated copies due
to the accumulation of degenerative mutations, a process that may take approximately 4
million years to complete (Lynch & Conery, 2000). However, sometimes both copies survive.
The duplicated copy can acquire beneficial mutations and consequently gain a novel
function with respect to the parental gene (neofunctionalisation), while the parental copy
preserves its original function (Ohno, 1970). The duplicated copy may also be retained due
to the split of the original function between the two gene copies (subfunctionalisation)
(Hughes, 1994). Finally, if an increase in dosage of a particular gene is beneficial, the new
copy may become fixed by positive selection maintaining the same gene structure and
function as the parental gene (Kondrashov & Koonin, 2004).
Duplicated genes may confer adaptive advantages. For example, trichromatic colour vision
in Old World Monkeys is associated with a pigment gene duplication that occurred after the

Partial Gene Duplication and the Formation of Novel Genes 97
separation of New World Monkeys, and which gave rise to differentiated red and green
pigments (Nathans et al., 1986). Zhang and colleagues (Zhang et al., 1998) reported on
another example of the action of positive selection after gene duplication. The eosinophil
cationic protein (ECP) and eosinophil-derived neurotoxin (EDN) genes are present in Old
World Monkeys and hominoids, and probably originated by tandem gene duplication after
the divergence of New World Monkeys. EDN is an antiviral agent (Domachowske &
Rosenberg, 1997) and ECP is a potent toxin for bacteria and parasites (Rosenberg & Dyer,
1995). The authors detected a non-random accumulation of arginine substitutions in ECP,
which may contribute to the generation of pores in pathogens membranes. Another
example refers to pancreatic ribonuclease 1B (RNASE1B), which originated through gene
duplication of RNASE1, an enzyme used to digest bacteria in the small intestine, in the douc
langur (Pygathrix nemaeus) around 2-4 million years ago (Zhang et al., 2002). Douc langurs
are folivorous monkeys, in which leaves are digested through fermentation by symbiotic
bacteria residing in the foregut. The newly duplicated copy, RNASE1B has evolved very
rapidly (non-synonymous to synonymous nucleotide substitution rate of 4.03), contrary to
the paralogous copy, RNASE1, which has not undergone change. These results indicate a
burst of positive selection acting on the duplicated copy. Moreover, most of the substitutions
imply the gain of negatively charged residues, lowering the optimal pH for RNASE1B,
which could be related to an increase in digestive efficiency, given the lower pH found in
the small intestine of douc langurs.
1.3 Partial gene duplication
Not all duplicated proteins are identical to their parental copies at birth. In fact, it has
been reported that in C. elegans only about 40% of the new duplicates are borne out of
complete gene duplications, the remainder representing cases of partial gene duplication
(Katju & Lynch, 2003). These partially duplicated genes may recruit sequences from their
genomic neighbourhood or from other genes (Katju & Lynch, 2006). In the first case,
adjacent non-coding sequences are co-opted for a coding function. Katju and Lynch (Katju
& Lynch, 2006) found that about half of the partially duplicated genes did not recruit any
surrounding sequences but accumulated mutations, for example in initiation or
termination codons, that altered the coding sequence. In Drosophila melanogaster, around
30% of the newly formed genes recruited various genomic sequences or formed chimeric
gene structures (Zhou et al., 2008). Partially duplicated and chimeric genes are expected to
adopt new functions immediately, which may increase their probability of being retained
(Patthy, 1999; Zhou et al., 2008). An example of a gene that has arisen by partial
duplication is the Hun gene in Drosophila, located on the X-chromosome. Hun arose from a
partial duplication of the Bllchen gene, which is on chromosome 3R. Hun lacks 3 coding
sequence with respect to Bllchen, but has gained 33 amino acids from a nearby intergenic
sequence. Further, while Bllchen is expressed ubiquitously, Hun shows testes-specific
expression (Arguello et al., 2006).
The sequence similarity that exists between completely duplicated gene copies and parental
gene copies is often sufficient to detect homologues in a whole range of organisms.
However, this is often not the case for partially duplicated genes, especially if the sequence
common to both duplicates is short and the rate of divergence of the novel gene duplicate is
abnormally high. As a result, many partially duplicated genes are identified as orphan or
lineage-specific genes, that is, genes that do not yield any significant hits in database protein

Gene Duplication 98
searches of more distant organisms (Chen et al., 2010; Domazet-Loso & Tautz, 2003; Toll-
Riera et al., 2009a). In a recent study that showed that newly formed genes in Drosophila
melanogaster are as likely to perform essential functions as older genes, it was found that 28
out of the 50 new genes that had arisen through gene duplication corresponded to partial
duplications (Chen et al., 2010). These young genes were found to evolve very rapidly,
showing a median of 47.3% divergence, at the amino-acid level, from their parents. In an
analysis of the mechanisms of formation of primate-specific genes, we observed that about
24% of the newly formed genes had originated through gene duplication, frequently
involving partial gene duplication and the recruitment of additional sequences (Toll-Riera et
al., 2009a). One example is human XAGE-1, a cancer/testis-associated gene that has partial
homology to human XAGE-2, a gene that is well conserved in other mammals. The
similarity is limited to the C-terminal half of the orphan XAGE-1 protein. We showed that,
in the conserved region, the rate of amino acid sequence evolution of XAGE-1 was double
that of XAGE-2, suggesting that the recruitment of additional sequences in XAGE-1 resulted
in a marked asymmetry in the evolutionary rates of the two copies.
Partial gene duplication is likely to be very important for the formation of novel gene
structures and the evolution of new protein functions, but studies focusing on this type of
gene duplication are still scarce. To shed new light on this issue, we decided to analyse the
evolutionary patterns of several primate-specific genes (orphan genes) formed, at least
partially, by gene duplication. The results show that increased evolutionary rates in the
partially duplicated copy are the norm, reinforcing the role of partial gene duplication in the
formation of novel genes with distinct functions.
2. Results
Here we use a similar approach to that employed in Toll-Riera et al. (Toll-Riera et al., 2009a)
to identify a set of primate-specific genes that show significant similarity to human genes
(parental genes) that are well conserved in non-primate species. We investigate the
differences in the rate of evolution of the novel and parental genes and discuss the role of
partial duplication in increasing the protein functional repertoire.
2.1 Identification of primate lineage-specific genes formed by gene duplication
We identified a set of genes present in human and macaque but absent in 13 non-primate
genomes (Mus musculus, Rattus norvegicus, Bos Taurus, Canis familiaris, Gallus gallus,
Xenopus tropicalis, Danio rerio, Takifugu rubripes, Caenorhabditis elegans, Drosophila
melanogaster, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Arabidopsis
thaliana). The existence of a homologue in a specific genome was determined by the
presence of a BLASTP (Altschul et al., 1997) hit with an expectation value (E-value) smaller
than 10
-4
, as previously described (Alba and Castresana 2005). Orphan genes were defined
as those for which we could not detect any homologues in any of the species mentioned
above. As they were, by definition, present in human and macaque, our collection of orphan
genes corresponded to primate-specific genes, presumably formed after the split of the
rodent and primate branches and before the speciation of the human and macaque lineages.
Once we had this set of orphan genes, we investigated which ones could have arisen
through gene duplication by performing BLASTP searches against all human proteins, using
a relaxed E-value (E<0.5). We kept those cases for which we could identify human

paralogues that were not primate-specific. In such cases, the closest hit in human was
considered the putative human parental gene, and the closest non-primate orthologue of the
parental gene was taken as the outgroup gene (Figure 1). Protein sequences were aligned
with T-Coffee (Notredame et al., 2000), and the alignments between primate-specific genes
and parental genes were carefully examined to discard any spurious associations. We also
removed any regions that were completely divergent (non-alignable) between the orphan
and parental genes.

Fig. 1. Tree topology corresponding to gene families containing duplicated orphan genes.
The orphan and parental genes are from human, the outgroup gene from a non-primate
species.
The final set consisted of 14 orphan genes. Table 1 shows the orphan, parental and outgroup
gene names, protein identifiers, and the percent of parental protein that could be reliably
aligned with the orphan protein, corresponding to the portion of the protein that had
duplicated. Of the 14 orphan genes, 4 represented single copies and the rest belonged to
orphan gene families. In only one case, dermcidin, sequence similarity supported a complete
gene duplication event.
We used the protein multiple alignments to estimate the number of amino acid substitutions
per site (K) in the orphan, parental and outgroup branches. We used PROML, a maximum
likelihood based method in the Phylip package for this purpose (Felsenstein, 2005). The
results of these computations are discussed below.
2.2 Dermcidin and lacritin
The first example of an orphan gene that arose through gene duplication is dermcidin. This
gene encodes a short protein of 110 amino acids in length. The corresponding parental gene
is lacritin, which has orthologues in other mammals, and is located on chromosome 12
adjacent to the dermcidin gene. The two genes have a similar exonic structure, and although
they are highly divergent, sequence similarity between the two is still detectable (Wang et
al., 2006). Dermcidin is secreted in sweat glands, having an antimicrobial activity (Schittek et
al., 2001), and may also be involved in neural survival and cancer (Porter et al., 2003),
whereas lacritin is expressed in the lacrimal glands (Ma et al., 2008).
Figure 2 shows the alignment of the complete protein sequences of human dermcidin,
human lacritin and cat lacritin. The number of amino acid substitutions per site in the
orphan branch was 1.026, about double the number of amino acid substitutions per site in
the parental and outgroup branches (0.434 and 0.505, respectively).

Gene Duplication 100
Orphan
Name
Parental name
Parental
protein
Outgroup %
Dermcidin Lacritin
ENSP00000
257867
ENSFCAP0000000931
7 (cat)
100%
FAM9A
Synaptonemal
complex protein 3
ENSP00000
266743
ENSMUSP0000002025
2 (mouse)
87.29%
FAM9B idem idem idem idem
FAM9C idem idem idem idem
AL023807.2 AL365202.1
ENSP00000
382846
ENSCINP00000011125
(vase tunicate)
64.42%
XAGE-1A XAGE-2
ENSP00000
333775
XP_001249434.1 (cow) 36.03%
XAGE-1B idem idem idem idem
XAGE-1C idem idem idem idem
XAGE-1D idem idem idem idem
XAGE-1E idem idem idem idem
NPIP-like 1
Acyl-CoA
synthetase
medium-chain
family member 1
ENSP00000
428098
ENSMUSP0000003614
0 (mouse)
18,07%
C2orf27A
Ral guanine
nucleotide
dissociation
stimulator like-4
ENSP00000
290691
ENSCAFP0000003110
2 (dog)
12.05%
C2orf27B idem idem idem idem
AL133216.1
Arsenite-resistance
protein 2
ENSP00000
314491
ENSMUSP0000004312
3 (mouse)
9.36%

Table 1. List of primate-specific genes that have arisen by gene duplication. Protein
identifiers are from Ensembl (ENSP) or Genbank (XP). % refers to the percentage of the
parental protein that showed homology to the orphan protein.
2.3 Partially duplicated orphan genes
The remaining primate-specific genes that have arisen through gene duplication
corresponded to partial duplications of the parental gene (Table 1). They included 3
individual genes (AL023807.2, NPIP-like 1 and AL133216.1) and 3 gene families (FAM9,
XAGE-1 and C2orf27). The percentage of protein sequence from the parental protein that
could by identified as homologous in the orphan protein ranged from 9.4 to 87.3% (Table 1).
With the exception of NPIP-like 1, the orphan gene is located on a different chromosome
from the parental gene, although the presence of introns in all orphan genes suggests that
they were not retrotransposed copies. We aligned the conserved regions of orphan, parental
and outgroup proteins (Figure 3). These alignments were used for the estimation of the

number of amino acid substitutions per site in the orphan, parental and outgroup branches.
We also investigated the presence of any known protein domains in the region conserved
between parental and orphan proteins, using the Pfam web server (Finn et al., 2010).

Fig. 2. Alignment of dermcidin (orphan), human lacritin (parental) and cat lacritin
(outgroup) proteins. Identical residues are in green, similar residues in yellow.
The FAM9 family (family with sequence similarity 9) is composed of three genes: FAM9A,
FAM9B and FAM9C. They are all predicted to have a Cor1/Xlr/Xmr domain in the region
of similarity to the parental gene (E-values ranging from 0.049 to 4.4e
-13
), related to meiotic
prophase chromosomes. The parental gene, synaptonemal complex protein 3 (SYCP3) is
involved in the assembly of the synaptonemal complex during meiosis (Martinez-Garay et
al., 2002), but the exact physiological functions of the FAM9 proteins remain unknown.
The largest orphan gene family is XAGE-1, which has 5 members with identical amino acid
sequences that are contiguous on the X chromosome. The region conserved between the
XAGE-1s and XAGE-2 includes the GAGE domain. The function of GAGE (G antigen) and
XAGE (X antigen) domains is unknown, but XAGE and GAGE proteins have been
implicated in several human cancers (Zendman et al., 2002).
The two genes belonging to the C2orf27 family are contiguous in the genome, though
C2orf27A is located on the forward strand of chromosome 2 whereas C2orf27B is located on
the reverse strand. Their function is unknown, but they derive from a protein annotated as
Ral guanine nucleotide dissociation stimulator-like 4. The parental protein contains the
RasGEF domain, which is a guanine nucleotide exchange factor for Ras-like small GTPases.
The duplicated region overlaps minimally with this domain (14 amino acids).
NPIP-like 1 belongs to the nuclear pore complex-interacting protein (NPIP) family. The
parental protein contains two AMP-binding domains that are at the N-terminal region of the
protein, not the area conserved in the orphan protein, which is the C-terminal part. The
NPIP family (Nuclear Pore Interacting Protein), also named morpheus, is located on a
duplicated segment of chromosome 16. It has been suggested to have experienced a burst of
positive selection during the emergence of Homininae (Johnson et al., 2001).
Finally, AL133216.1 and AL023807.2 are two primate-specific genes of unknown function
containing putative coding sequences of length 151 and 121 amino acids respectively. The
parental copy of AL133216.1 modulates arsenic sensitivity, is involved in cell cycle
progression, and in RNA-mediated gene silencing by microRNA (Gruber et al., 2009). It also
contains an arsenite-resistance protein 2 domain (Pfam hit E-value = 3.1e
-18
). The orphan

copy does not contain this domain even though it is located in the conserved region,
suggesting that this region has lost its ancestral function in the orphan protein.
Table 2 shows the estimated amino acid substitution rates in the orphan, parental and
outgroup branches. In the case of identical copies (for example C2orf27A and C2orf27B)
only one is taken as representative. In the case of divergent copies (the FAM9 family) the
amino acid substitution rates are summed up for all branches from the ancestor to the
derived node (see Figure 4). In all cases the duplicated protein is evolving much faster than
the parental gene, and in some cases, such as the FAM9 and NPIP-like 1 proteins, more than
six times faster. These results indicate that orphan proteins are evolving under much more
relaxed constraints, and/or adapting to a new function with respect to their parental copies.

Orphan Name Orphan protein Orphan Parental Outgroup
Dermcidin ENSP00000293371 1.02595 0.43458 0.5055
FAM9A ENSP00000370391 1.28971 0.17014 0.15423
FAM9B ENSP00000318716 1.13565 0.17014 0.15423
FAM9C ENSP00000369999 1.15328 0.17014 0.15423
AL023807.2 ENSP00000381423 0.19840 0.12203 0.23096
XAGE-1A ENSP00000382698 0.52961 0.17820 0.94188
NPIP-like 1 ENSP00000350444 0.40089 0.02020 0.11929
C2orf27B ENSP00000304065 0.55865 0.21080 0.68342
AL133216.1 ENSP00000382606 1.37580 0.00010 0.00010
Table 2. Estimated number of amino acid substitutions per site (K) for orphan, parental and
outgroup branches. Orphan protein identifiers are from Ensembl. See Table 1 for more
details.
2.4 Role of low-complexity sequences
Low complexity regions (LCRs) are sequences in which one or a few residues are highly
overrepresented. Several studies have shown that duplicated gene copies can gain new
functions through the acquisition of LCRs (Fondon & Garner, 2004; Salichs et al., 2009). It
has also been shown that young proteins contain more LCRs than old proteins (Alba &
Castresana, 2005). Therefore, we inspected the presence of LCRs in our set of orphan
proteins using the SEG algorithm with default parameters (Wootton & Federhen, 1996).
We found that the FAM9A protein contained a very conspicuous low-complexity
sequence. Figure 4 shows the detailed phylogenetic tree of the FAM9 gene family
(including the parental and outgroup SYCP3 genes). The ancestral FAM9 evolved very
rapidly and eventually underwent two duplication events, leading to FAM9A, FAM9B
and FAM9C. The multiple alignment of the region surrounding the LCR in FAM9A shows
how, from a small region containing several acidic residues in SYCP3, a larger acidic
region was formed in the common FAM9 ancestor, which finally expanded to a 75 amino
acid stretch in FAM9A containing a long glutamic acid repeat, as well as poly-alanine and
poly-glycine repeats.
As is the case for the SYCP3 proteins, all three human FAM9 proteins show testis-specific
expression. However, the cellular localization is different depending on the protein studied:
FAM9B and FAM9C are localized in the nucleus with low protein levels being detectable in
the cytoplasm, whereas FAM9A is present at high levels in the nucleolus (Martinez-Garay et
al., 2002). The distinct location of FAM9A may be due to the long glutamic acid repeat, as


Fig. 3. Multiple alignments of the conserved regions between orphan, parental and outgroup
proteins. For the XAGE-1 family only XAGE-1A is shown, as the other orphan sequences
were identical at the amino acid level. The same is true for the C2orf27 family. Identical
residues are in green, similar residues in yellow. See Table 1 for more details.


Fig. 4. Phylogenetic tree of the FAM9 gene family. Branch lengths correspond to the
estimated number of amino acid substitutions per site, using the alignment in Fig 3. The
protein alignment shown corresponds to exon 5 in FAM9B and FAM9C and to exon 6 in
FAM9A, human SYCP3 and mouse SYCP3. The expanded low-complexity region in FAM9A
is depicted above the alignment.
acidic clusters have been shown to mediate protein nucleolar retention (Ochs et al., 1996;
Shu-Nu et al., 2000; Ueki et al., 1998).In FAM9A, the low complexity sequence is located
within the Cor1/Xlr/Xmr conserved region, perhaps interfering with its function. In fact,
FAM9A shows higher sequence divergence from the common ancestor than FAM9B.
3. Discussion
The role of partial gene duplication in the formation of novel genes is still poorly
understood, although recent reports in Drosophila (Chen et al., 2010; Zhou et al., 2008) and
C.elegans (Katju & Lynch, 2006; 2003) indicate that partially duplicated gene copies are very
frequent. The present study analyses a set of primate-specific genes formed by partial gene
duplication. We find that the rate of divergence of the partially duplicated copy is, in all
cases, higher that the rate of divergence of the parental copy, generalizing previous
observations for XAGE1-A (Toll-Riera et al., 2009a). This, together with the fact that most
partially duplicated genes recruit additional sequences, strengthens the notion that partial
duplication is a major process for the formation of genes with novel structures and
functions. In these genes, any remaining similarity to the homologous proteins is being
quickly erased by high sequence turnover. As a consequence, distant homologues are
difficult to identify and these proteins end up being classified as orphans. This fits the
model of Domazet-Loso and Tautz in explaining the high number of orphan genes in
Drosophila: orphan genes are created by gene duplication followed by a period of rapid
sequence divergence that erases the similarity with its homologues (Domazet-Loso & Tautz,
2003). Although we now have evidence that not all orphan genes are generated in this
manner (Toll-Riera et al., 2009a; Toll-Riera et al., 2009b; Zhou et al., 2008), a significant
portion is.
A large fraction of the duplicated gene copies that become fixed in a population are
subsequently lost, presumably because the new copy is completely redundant and thus
dispensable. However, the formation of chimeric gene structures, encoding part of an
existing protein together with additional sequences, could in principle favour their
retention, as these genes are not going to be functionally equivalent to the ancestral gene
(Patthy, 1999; Zhou et al., 2008). In support of this, in Drosophila it was found that the
proportion of novel genes corresponding to complete gene duplications decreased with
gene age, suggesting that complete gene duplications had a shorter lifespan than partial
gene duplications (Zhou et al., 2008).

Orphan genes are in general poorly annotated and their function is unknown in most cases
(Kuo & Kissinger, 2008). The fact that organisms had lived perfectly well without them until
recent times when they made their appearance, has led scientists to think that orphan genes
were, for the most part, dispensable. However, a recent study by Chen and colleagues (Chen
et al., 2010) has challenged this viewpoint. In their study, the authors identified new young
genes in Drosophila melanogaster (around 34 million years old) and designed RNA
interference lines to knoch each of them out (KO). Surprisingly, they found that 30% of these
young genes KOs were lethal, as Drosophila could not survive without them. These young
genes had mainly arisen through duplication and they showed higher evolutionary rates
than the parental gene, indicating the action of positive selection, or relaxation of functional
constraints. They hypothesized that new genes are quickly integrated into existing
pathways, and hence many of them soon become essential for the viability of the organism.
Capra and colleagues (Capra et al., 2010) compared the evolutionary patterns of genes that
arose by duplication with those that did not (named novel genes). They argued that the
evolutionary pressures should be different in each case as, contrary to novel genes,
duplicated genes were functionally and structurally well formed from birth. They showed
that although duplicated genes are initially more integrated into cellular networks, both
types of new genes gain functions and interactions with time, though novel genes do it more
rapidly than duplicated genes. Additionally, novel genes also increase in length through the
incorporation of transposable elements or surrounding sequences. This increase in length
could be related with the rapid gain of function and interactions experienced by novel
genes. They also found that genes tended to interact with genes similar in age and mode of
origin. Thus, the mechanism by which a gene originates seems to significantly impact on its
subsequent evolution.
Several studies have demonstrated that duplicated genes show increased protein
evolutionary rates with respect to non-duplicated genes in the same lineage (Castillo-Davis
et al., 2004; Cusack & Wolfe, 2007; Kondrashov et al., 2002; Lynch & Conery, 2000;
Nembaware et al., 2002; Scannell & Wolfe, 2008; Van de Peer et al., 2001). Here we identified
a very strong asymmetry in the rates of evolution of the newly evolved copy (orphan) and
the well-conserved copy (parental), the former evolving much faster than the latter.
Surprisingly, the parental protein copy did not evolve consistently faster than the outgroup
protein (not duplicated), highlighting the fact that we are dealing with a special type of gene
duplication in which the copy containing the partially duplicated segment rapidly departs
from the ancestral family, which remains essentially unaffected.
Increased evolutionary rates may reflect either relaxation of purifying selection, positive
selection, or the combined effects of both these forces. The orphan genes under study
predated the split of the human and macaque lineages, which occurred approximately 25
million years ago so, if relaxed selection was the only factor for their increased rates, the
genes should by now have become pseudogenes and not be expressed. However, all genes
were expressed at the RNA level in one or several tissues. Therefore we must hypothesize
that, at least to some extent, positive selection has influenced the evolution of these genes.
We compared the rates of evolution of the protein regions that were conserved between
orphan and parental proteins, but what about the unique sequences contained in the orphan
proteins? These sequences lacked any similarity to other protein-coding genes, so they may
be ancestral non-coding sequences that have been co-opted for a coding function (Long et
al., 2003). Genes generated de novo from non-coding sequences are among the fastest
evolving genes (Levine et al., 2006), and there is no reason to believe that unique sequences

in orphan proteins will evolve slower than the conserved protein regions, rather the
contrary would seem more logical. In a previous study we showed that the non-
synonymous to synonymous nucleotide substitution rates of primate-specific genes,
measured for human and macaque orthologues, were, on average, twice as high as those of
mammalian-specific genes and five times higher than those of deeply conserved eukaryotic
proteins (Toll-Riera et al., 2009a). The differences in amino acid substitution rates between
orphan and parental genes described here reinforce the idea that the evolution of a new
gene is strongly associated with very rapid sequence change.
4. Concluding remarks and future research
We have examined the evolutionary dynamics of a group of novel primate-specific genes
(orphan genes) that have arisen by gene duplication. These genes typically form new
structures in which only part of the protein sequence is shared with the parental copy,
presumably because of partial gene duplication, and the rest of the protein sequence is
unique. The orphan proteins accumulate a much larger number of amino acid substitutions
per site than the parental proteins, denoting rapid functional diversification. The parental
gene copies appear to act as donors of sequence but do not experience any obvious
sequence evolution alterations, thus they probably preserve their ancestral functions. Future
research in this area, using computational as well as experimental studies, should help
clarify how frequent is partial gene duplication with respect to complete gene duplication,
the differences in gene copy survival in both cases, and how partial and complete gene
duplication contribute to the generation of evolutionary novelties.
5. Acknowledgments
We received financial support from Ministerio de Educacin, Gobierno de Espaa (FPU to
M.T-R, BIO2009-08160), Generalitat de Catalunya (FI to S.L.), Fundaci Javier Lamas
Miralles (Ajut Predoctoral Javier Lamas Miralles to N.R-T) and Instituci Catalana de
Recerca i Estudis Avanats (M.M.A).
6. References
Alba, M.M. & Castresana, J. 2005. Inverse relationship between evolutionary rate and age of
mammalian genes. Mol Biol Evol 22(3): 598-606.
Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J.
1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search
programs. Nucleic Acids Res 25(17): 3389-3402.
Arguello, J.R., Chen, Y., Yang, S., Wang, W. & Long, M. 2006. Origination of an X-linked
testes chimeric gene by illegitimate recombination in Drosophila. PLoS Genet 2(5):
e77.
Bailey, J.A., Liu, G. & Eichler, E.E. 2003. An Alu transposition model for the origin and
expansion of human segmental duplications. Am J Hum Genet 73(4): 823-834.
Cai, J., Zhao, R., Jiang, H. & Wang, W. 2008. De novo origination of a new protein-coding
gene in Saccharomyces cerevisiae. Genetics 179(1): 487-496.
Capra, J.A., Pollard, K.S. & Singh, M. 2010. Novel genes exhibit distinct patterns of function
acquisition and network integration. Genome Biol 11(12): R127.

Castillo-Davis, C.I., Hartl, D.L. & Achaz, G. 2004. cis-Regulatory and protein evolution in
orthologous and duplicate genes. Genome Res 14(8): 1530-1536.
Castresana, J., Guigo, R. & Alba, M.M. 2004. Clustering of genes coding for DNA binding
proteins in a region of atypical evolution of the human genome. J Mol Evol 59(1):
72-79.
Chen, S., Zhang, Y.E. & Long, M. 2010. New genes in Drosophila quickly become essential.
Science 330(6011): 1682-1685.
Cusack, B.P. & Wolfe, K.H. 2007. Not born equal: increased rate asymmetry in relocated and
retrotransposed rodent gene duplicates. Mol Biol Evol 24(3): 679-686.
Domachowske, J.B. & Rosenberg, H.F. 1997. Eosinophils inhibit retroviral transduction of
human target cells by a ribonuclease-dependent mechanism. J Leukoc Biol 62(3):
363-368.
Domazet-Loso, T. & Tautz, D. 2003. An evolutionary analysis of orphan genes in
Drosophila. Genome Res 13(10): 2213-2219.
Drosophila 12 Genomes Consortium. 2007. Evolution of genes and genomes on the
Drosophila phylogeny. Nature 450(7167): 203-218.
Farre, D. & Alba, M.M. 2010. Heterogeneous patterns of gene-expression diversification in
mammalian gene duplicates. Mol Biol Evol 27(2): 325-335.
Felsenstein, J. 2005. PHYLIP (Phylogeny Inference Package) version 3.6. Distributed by the
author. Department of Genome Sciences, University of Washington, Seattle.
Finn, R.D., Mistry, J., Tate, J., Coggill, P., Heger, A., Pollington, J.E., Gavin, O.L.,
Gunasekaran, P., Ceric, G., Forslund, K., Holm, L., Sonnhammer, E.L., Eddy, S.R. &
Bateman, A. 2010. The Pfam protein families database. Nucleic Acids Res
38(Database issue): D211-222.
Fondon, J.W., 3rd & Garner, H.R. 2004. Molecular origins of rapid and continuous
morphological evolution. Proc Natl Acad Sci U S A 101(52): 18058-18063.
Gilad, Y., Man, O. & Glusman, G. 2005. A comparison of the human and chimpanzee
olfactory receptor gene repertoires. Genome Res 15(2): 224-230.
Gruber, J.J., Zatechka, D.S., Sabin, L.R., Yong, J., Lum, J.J., Kong, M., Zong, W.X., Zhang, Z.,
Lau, C.K., Rawlings, J., Cherry, S., Ihle, J.N., Dreyfuss, G. & Thompson, C.B. 2009.
Ars2 links the nuclear cap-binding complex to RNA interference and cell
proliferation. Cell 138(2): 328-339.
Gu, Z., Nicolae, D., Lu, H.H. & Li, W.H. 2002. Rapid divergence in expression between
duplicate genes inferred from microarray data. Trends Genet 18(12): 609-613.
Guo, W.J., Li, P., Ling, J. & Ye, S.P. 2007. Significant comparative characteristics between
orphan and nonorphan genes in the rice (Oryza sativa L.) genome. Comp Funct
Genomics: 21676.
Haldane, J.B.S. 1932. The causes of evolution. London: Longmans and Green.
Heinen, T.J., Staubach, F., Haming, D. & Tautz, D. 2009. Emergence of a new gene from an
intergenic region. Curr Biol 19(18): 1527-1531.
Hughes, A.L. 1994. The evolution of functionally novel proteins after gene duplication. Proc
Biol Sci 256(1346): 119-124.
International Chicken Genome Sequencing Consortium. 2004. Sequence and comparative
analysis of the chicken genome provide unique perspectives on vertebrate
evolution. Nature 432(7018): 695-716.

Johnson, M.E., Viggiano, L., Bailey, J.A., Abdul-Rauf, M., Goodwin, G., Rocchi, M. & Eichler,
E.E. 2001. Positive selection of a gene family during the emergence of humans and
African apes. Nature 413(6855): 514-519.
Katju, V. & Lynch, M. 2003. The structure and early evolution of recently arisen gene
duplicates in the Caenorhabditis elegans genome. Genetics 165(4): 1793-1803.
Katju, V. & Lynch, M. 2006. On the formation of novel genes by duplication in the
Caenorhabditis elegans genome. Mol Biol Evol 23(5): 1056-1067.
Khalturin, K., Hemmrich, G., Fraune, S., Augustin, R. & Bosch, T.C. 2009. More than just
orphans: are taxonomically-restricted genes important in evolution? Trends Genet
25(9): 404-413.
Knowles, D.G. & McLysaght, A. 2009. Recent de novo origin of human protein-coding
genes. Genome Res 19(10): 1752-1759.
Kondrashov, F.A. & Koonin, E.V. 2004. A common framework for understanding the origin
of genetic dominance and evolutionary fates of gene duplications. Trends Genet
20(7): 287-290.
Kondrashov, F.A., Rogozin, I.B., Wolf, Y.I. & Koonin, E.V. 2002. Selection in the evolution of
gene duplications. Genome Biol 3(2): RESEARCH0008.
Kuo, C.H. & Kissinger, J.C. 2008. Consistent and contrasting properties of lineage-specific
genes in the apicomplexan parasites Plasmodium and Theileria. BMC Evol Biol
8: 108.
Levine, M.T., Jones, C.D., Kern, A.D., Lindfors, H.A. & Begun, D.J. 2006. Novel genes
derived from noncoding DNA in Drosophila melanogaster are frequently X-linked
and exhibit testis-biased expression. Proc Natl Acad Sci U S A 103(26): 9935-9939.
Long, M., Betran, E., Thornton, K. & Wang, W. 2003. The origin of new genes: glimpses from
the young and old. Nat Rev Genet 4(11): 865-875.
Lynch, M. & Conery, J.S. 2000. The evolutionary fate and consequences of duplicate genes.
Science 290(5494): 1151-1155.
Ma, P., Wang, N., McKown, R.L., Raab, R.W. & Laurie, G.W. 2008. Focus on molecules:
lacritin. Exp Eye Res 86(3): 457-458.
Makova, K.D. & Li, W.H. 2003. Divergence in the spatial pattern of gene expression between
human duplicate genes. Genome Res 13(7): 1638-1645.
Marques, A.C., Dupanloup, I., Vinckenbosch, N., Reymond, A. & Kaessmann, H. 2005.
Emergence of young human genes after a burst of retroposition in primates. PLoS
Biol 3(11): e357.
Martinez-Garay, I., Jablonka, S., Sutajova, M., Steuernagel, P., Gal, A. & Kutsche, K. 2002. A
new gene family (FAM9) of low-copy repeats in Xp22.3 expressed exclusively in
testis: implications for recombinations in this region. Genomics 80(3): 259-267.
Mouse Genome Sequencing Consortium. 2002. Initial sequencing and comparative analysis
of the mouse genome. Nature 420(6915): 520-562.
Muller, H.J. 1935. The origination of chromatin deficiencies as minute deletions subject to
insertion elsewhere. Genetica 17: 237-252.
Nathans, J., Thomas, D. & Hogness, D.S. 1986. Molecular genetics of human color vision: the
genes encoding blue, green, and red pigments. Science 232(4747): 193-202.
Nekrutenko, A. & Li, W.H. 2001. Transposable elements are found in a large number of
human protein-coding genes. Trends Genet 17(11): 619-621.

Nembaware, V., Crum, K., Kelso, J. & Seoighe, C. 2002. Impact of the presence of paralogs
on sequence divergence in a set of mouse-human orthologs. Genome Res 12(9):
1370-1376.
Notredame, C., Higgins, D.G. & Heringa, J. 2000. T-Coffee: A novel method for fast and
accurate multiple sequence alignment. J Mol Biol 302(1): 205-217.
Ochs, R.L., Stein, T.W., Jr., Chan, E.K., Ruutu, M. & Tan, E.M. 1996. cDNA cloning and
characterization of a novel nucleolar protein. Mol Biol Cell 7(7): 1015-1024.
Ohno, S. 1970. Evolution by gene duplication. New York: Springer-Verlag.
Parra, G., Reymond, A., Dabbouseh, N., Dermitzakis, E.T., Castelo, R., Thomson, T.M.,
Antonarakis, S.E. & Guigo, R. 2006. Tandem chimerism as a means to increase
protein complexity in the human genome. Genome Res 16(1): 37-44.
Patthy, L. 1999. Genome evolution and the evolution of exon-shuffling--a review. Gene
238(1): 103-114.
Porter, D., Weremowicz, S., Chin, K., Seth, P., Keshaviah, A., Lahti-Domenici, J., Bae, Y.K.,
Monitto, C.L., Merlos-Suarez, A., Chan, J., Hulette, C.M., Richardson, A., Morton,
C.C., Marks, J., Duyao, M., Hruban, R., Gabrielson, E., Gelman, R. & Polyak, K.
2003. A neural survival factor is a candidate oncogene in breast cancer. Proc Natl
Acad Sci U S A 100(19): 10931-10936.
Rosenberg, H.F. & Dyer, K.D. 1995. Eosinophil cationic protein and eosinophil-derived
neurotoxin. Evolution of novel function in a primate ribonuclease gene family. J
Biol Chem 270(37): 21539-21544.
Salichs, E., Ledda, A., Mularoni, L., Alba, M.M. & de la Luna, S. 2009. Genome-wide analysis
of histidine repeats reveals their role in the localization of human proteins to the
nuclear speckles compartment. PLoS Genet 5(3): e1000397.
Scannell, D.R. & Wolfe, K.H. 2008. A burst of protein sequence evolution and a prolonged
period of asymmetric evolution follow gene duplication in yeast. Genome Res 18(1):
137-147.
Schittek, B., Hipfel, R., Sauer, B., Bauer, J., Kalbacher, H., Stevanovic, S., Schirle, M.,
Schroeder, K., Blin, N., Meier, F., Rassner, G. & Garbe, C. 2001. Dermcidin: a novel
human antibiotic peptide secreted by sweat glands. Nat Immunol 2(12): 1133-1137.
Shu-Nu, C., Lin, C.H. & Lin, A. 2000. An acidic amino acid cluster regulates the nucleolar
localization and ribosome assembly of human ribosomal protein L22. FEBS Lett
484(1): 22-28.
Siepel, A. 2009. Darwinian alchemy: Human genes from noncoding DNA. Genome Res
19(10): 1693-1695.
Siew, N. & Fischer, D. 2003. Analysis of singleton ORFans in fully sequenced microbial
genomes. Proteins 53(2): 241-251.
Toll-Riera, M., Bosch, N., Bellora, N., Castelo, R., Armengol, L., Estivill, X. & Alba, M.M.
2009a. Origin of primate orphan genes: a comparative genomics approach. Mol Biol
Evol 26(3): 603-612.
Toll-Riera, M., Castelo, R., Bellora, N. & Alba, M.M. 2009b. Evolution of primate orphan
proteins. Biochem Soc Trans 37(Pt 4): 778-782.
Ueki, N., Kondo, M., Seki, N., Yano, K., Oda, T., Masuho, Y. & Muramatsu, M. 1998. NOLP:
identification of a novel human nucleolar protein and determination of sequence
requirements for its nucleolar localization. Biochem Biophys Res Commun 252(1):
97-102.

Van de Peer, Y., Taylor, J.S., Braasch, I. & Meyer, A. 2001. The ghost of selection past: rates of
evolution and functional divergence of anciently duplicated genes. J Mol Evol 53(4-
5): 436-446.
Wang, J., Wang, N., Xie, J., Walton, S.C., McKown, R.L., Raab, R.W., Ma, P., Beck, S.L.,
Coffman, G.L., Hussaini, I.M. & Laurie, G.W. 2006. Restricted epithelial
proliferation by lacritin via PKCalpha-dependent NFAT and mTOR pathways. J
Cell Biol 174(5): 689-700.
Wootton, J.C. & Federhen, S. 1996. Analysis of compositionally biased regions in sequence
databases. Methods Enzymol 266: 554-571.
Yang, S., Arguello, J.R., Li, X., Ding, Y., Zhou, Q., Chen, Y., Zhang, Y., Zhao, R., Brunet, F.,
Peng, L., Long, M. & Wang, W. 2008. Repetitive element-mediated recombination
as a mechanism for new gene origination in Drosophila. PLoS Genet 4(1): e3.
Zendman, A.J., Van Kraats, A.A., Weidle, U.H., Ruiter, D.J. & Van Muijen, G.N. 2002. The
XAGE family of cancer/testis-associated genes: alignment and expression profile in
normal tissues, melanoma lesions and Ewing's sarcoma. Int J Cancer 99(3): 361-369.
Zhang, J., Rosenberg, H.F. & Nei, M. 1998. Positive Darwinian selection after gene
duplication in primate ribonuclease genes. Proc Natl Acad Sci U S A 95(7):
3708-3713.
Zhang, J., Zhang, Y.P. & Rosenberg, H.F. 2002. Adaptive evolution of a duplicated
pancreatic ribonuclease gene in a leaf-eating monkey. Nat Genet 30(4): 411-415.
Zhou, Q. & Wang, W. 2008. On the origin and evolution of new genes--a genomic and
experimental perspective. J Genet Genomics 35(11): 639-648.
Zhou, Q., Zhang, G., Zhang, Y., Xu, S., Zhao, R., Zhan, Z., Li, X., Ding, Y., Yang, S. & Wang,
W. 2008. On the origin of new genes in Drosophila. Genome Res 18(9): 1446-1455.

Part 2
A Look at Some Gene Families

7
Immunoglobulin Polygeny:
An Evolutionary Perspective
J. E. Butler, Xiu-Zhu Sun and Nancy Wertz
Department of Microbiology & Interdisciplinary Immunology Program
Carver College of Medicine, University of Iowa, Iowa City,
USA
1. Introduction
The immune system of vertebrates is characterized by genes of the Ig-superfamily (IGSF)
that encode the immunoglobulin (Ig) genes, genes that encode the T cell receptor (TCR), a
portion of the structure of the genes encoding the major histocompatibility molecules
(MHC), Ig cell surface and transport receptors, some families of cytokines and chemokines
as well as numerous other proteins important to the immune system. IGSF genes also
encode proteins in sponges, coelenterates and flatworms (Blumbach et al., 1998; Miller &
Steele, 2000; Ogawa et al., 1998). While not a topic for this chapter, we acknowledge that the
IGSF genes are not the only family of genes used to generate an antibody repertoire in
vertebrates. The VLR-based receptors of jawless fishes that belong to the LRR family of
receptors, have had a parallel evolution (Herrin & Cooper, 2010).
Figure 1 illustrates the signature features of proteins encoded by the IGSF genes. Highly
diagnostic is the so-called |-barrel or Ig fold. Anti-parallel |-pleated sheets form the
staves of the barrel that are joined at each end by flexible polypeptide chains. These flexible
polypeptides on the face of a heavy chain variable region domain (VH; Fig. 1A) contain
three combinatorial determining regions (CDRs). The variable light chain domain (VL; not
shown) also contributes three CDRs. CDRs from both VH and VL domains coalesce to form
the antibody binding site (Fig. 1B). A striking feature of IGSF genes that encode the variable
region domain of Igs is the degree of polygeny such that duplicated VH genes alone can
occupy > three megabases (Matsuda et al., 1990). There are three such variable region loci in
mammals: VH, V and Vk. The former encodes the variable heavy chain domain (Fig. 1A)
while V and Vk encode the light chains variable region domains. All three loci are
independent (non-linked) although a few orphan human VH genes can be found in other
linkage groups (Matsuda et al., 1990). Popular textbooks suggest that this polygeny explains
why antibodies can recognize >10
10
different antigens. It is argued that if each specific
antibody required a completely separate gene, more DNA would be needed than exists in
the mammalian genome. To reduce the need for so many different germline encoded
antibody binding sites, a system of somatic gene segment recombinations and later, somatic
hypermutation (SHM) or somatic gene conversion (SCG), evolved.
The complete antibody molecule (and other proteins encoded by IGSF genes) is often
composed of a tandem series of |-barrel domains as illustrated in Fig. 1B. Each domain in
such multi-domain molecules differs slightly in structure and correspondingly, in function.

Gene Duplication

114
In this article we will focus on the genes encoding the VH domain of Ig (Fig. 1A) as well as
those encoding the so-called constant regions (C) of IgG, the mammalian flagship
antibody isotype. We use as examples the sequences of duplicated VH genes in swine and
bats (opposite extremes) and the duplicated C genes encoding the subclasses of swine IgG,
as evidence to suggest how this polygeny occurred.

A
B
Ig Domain
icon
Multidomain
Ig
VH
VH
VL
VL

Fig. 1. Duplication/diversification of Ig genes resulted in macromolecules with repeating
units. A. The variable heavy chain domain (VH) with its characteristic |-barrel or Ig fold.
The dark polypeptides connecting the barrel staves contain the CDR regions. B.
Complete Igs are multidomain molecules comprised of many Ig fold domains. The CDR-
containing peptides occur only in the VH and VL domains. The remaining C-domains
comprise the constant region of the Ig. The monomeric Ig shown in Fig. 1B is bivalent,
with two identical VH/VL pairs that contain the antigen binding sites.
In the interests of those who are not immunologists, we describe the different Ig-loci, how
they vary among vertebrates and the processes involved in the generation of the antibody
repertoire (Section 2). Section 3 discusses the gene duplication phenomenon which resulted
in the polygeny that characterizes the vertebrate Ig genome, while Section 4 reviews the
somatic processes that lead to the synthesis and secretion of antibodies in higher vertebrates.
Section 5 discusses the selection processes involved in gene usage. Finally, we provide data
from studies in fetal/neonatal piglets, newborn rabbits and the chicken, to support the view
that only a small number of the many duplicated V-region Ig genes are actually used. We
provide examples in which only one or a few VH genes are needed to generate the antibody
repertoire so long as the machinery for somatic recombination and somatic mutation is in
place. Based on these examples and comparing them to antibody repertoire development in
lower vertebrates, we hypothesize that the extensive polygeny in the Ig loci of higher
vertebrates exists as an evolutionary vestige but is retained because of its redundancy value.
While the recent duplication/diversification of C allows for specialized effector function,
IgG in rabbits did not diversify, yet few would argue against the success of this mammalian
order. Thus, many of the C duplicons in other mammals may also have been retained for

Immunoglobulin Polygeny: An Evolutionary Perspective

115
their redundancy value. This could explain why individuals with major deletions of certain
C genes remain healthy (see Section 6.3).
2. Organization of the Ig Loci
2.1 Translocon organization of gene segments characterizes higher vertebrates
Figure 2A shows the organization of the light and heavy chain loci. Each locus can be
divided into subloci that, from 5 to 3, are known as the V, D, J and C regions. The light
chain loci are similar but lack D subloci. As discussed above, the V, D, and J regions encode
the antibody binding site for the heavy and light chain, and are comprised of a large number
of duplicated gene segments that vary among species (Table 1). The VH and VL gene
segments are the largest (~ 300 nucleotides) and encode both framework regions (FR) and
CDR1 and CDR2. The FR regions encode the |-pleated sequences of the |-barrel (Fig. 1A).
Displayed in linear fashion FR1, CDR1, FR2, CDR2 and FR3 comprise a VH (or VL) gene
(Fig. 4 and 5). The 3 portion of the JH segment (after the tryptophan codon; Fig. 8) encodes
FR4 while CDR3 results from the recombination of V-D-J or V-J (see Section 4).

Remainder of C-region
see Part B, below
B
Human
Switch C Co C3 C1 Cc2 Co1 C C2 C4 Cc1 Co
Duplicon 1 Duplicon 2
Rabbit
Switch C C1 Cc Co1 Co13
n=13
A
N~100 n=30 n=9
Heavy V
H
D
H
J
H
Ck
n=66 n= 5
Kappa Vk Jk
n=70 n=7
Lambda V JC
Switch C Exons
Remainder of C-region
see Part B, below
B
Human
Switch C Co C3 C1 Cc2 Co1 C C2 C4 Cc1 Co
Duplicon 1 Duplicon 2
Rabbit
Switch C C1 Cc Co1 Co13
n=13
A
N~100 n=30 n=9
Heavy V
H
D
H
J
H
Ck
n=66 n= 5
Kappa Vk Jk
n=70 n=7
Lambda V JC
Switch C Exons

Fig. 2. The translocon organization of Ig genes of mammals. A. Organization of the variable
region gene segments of the human heavy chain (V
H
), kappa (k) and lambda () loci. Brackets
indicate the number (n) of gene segments of a particular type. Switch regions are depicted with
diagonal strips. B. Organization of the constant region of the heavy chain locus of human and
rabbit. The site of intralocus segment duplication in humans is indicated.

Gene Duplication

116
The C- region Sublocus is composed of exons of the genes that encode the constant
domains of the antibody molecule (Fig. 1B). Fig. 2A illustrates the four exons that encode
IgM (C). Each exon encodes one of the constant region domains illustrated in Fig. 1B. Each
domain possesses the |barrel structures that characterizes the minimal structure of
proteins encoded by IGSF genes (Fig. 1B). Within the C-region sublocus, sets of exons
encode different antibody isotypes: e.g. IgM, IgD, IgG, IgE IgA (Fig. 2B). Two variations of
the C-region sublocus are illustrated by the human and rabbit. The distribution of the
encoded isotypes among common vertebrates is summarized in Table 2. Of interest to the
theme of this review, the C-region sublocus contains a region that contains stretches of
exons of duplicated C genes encoding IgG subclasses (Fig. 2B; most mammals) or multiple
IgA subclasses (Fig. 2B; in rabbit).

Species V
H
(F*) D
H
J
H
V
(F*) J
V
k
(F*) J
k
C
k

Human 87 (7) 30 9 70 (7) 7 7** 66 (7) 5 1
Mouse >100 (14) 11 4 3 (3) 4 4** 140 (4) 4 1
Rat >100 (11) ? 5 15 (4) 1 1 18 (?) 6 ?
Rabbit >100 (1) 12 6 ? (?) 2 2 >36 (?) 5 2
Swine > 20 (1) 2 1 ? (4) >3 >3 60 (2) 5 1
Horse >10 (2) >7 >5 25 (3) 4 4 >20 (?) 5 1
Cattle >15 (2) 3 5 30 (?) >2 4 ? (?) ? 1
Sheep >10 (1) ? 6 >100 (3) 2 2 10 (4) 3 1
VHH 42 (1)
Camelid
VH 50 (1) 10 6 ? (?) ? 2 ? (?) ? ?
Bat >250 (5) ? 13 ? (?) ? ? ? (?) ? ?
Oppossum 12 ? ? 30 (3) 6 6 35 (4) >2 1
Platypus 25 (1) >5 7 15-25 (2) 6 4 ? (4) ? ?
* Number of families (F) of variable region genes.
** J
-C
occurs as duplicons (see Fig. 2A).

Table 1. Variable region gene duplication among mammalian antibody genes

Species
IgM (C) IgD (Co) IgG (C) IgE (Cc) IgA (Co) C
C
k

Human 1 1 >4 1* >1 1* 2 >4 3* 1
Mouse 1 1 4 1 1 >3 1* 1
Rat 1 1 4 1 1 1 ?
Rabbit 1 0 1 1 13 8 2
Swine 1 1 6 1 1 >3 1
Horse 1 1 7 1 1 4 1
Cattle 1 1 3 1 1 4 1
Sheep 1 1 >2 1 1 >1 1
Camel 1 ? >3 4* ? ? 2 1
Cat 1 ? >2 1 ? >1 1
Dog 1 1 4 1 1 >1 1
Bat 1 1** 1 5** 1 1 ? ?
Oppossum 1 0 1 1 1 6 1
Platypus 1 0 2 1 2 4 ?
* Additional pseudogenes
** Varies with species

Table 2. Constant region gene duplication among mammalian antibody genes


117
2.2 The Ig loci in sharks and the chicken are differently organized
Figure 3 illustrates examples of the organization of V-D-J-C segments in three different
shark species which are organized as repeating cassettes rather than in translocon fashion.
Interestingly, Figure 2A also shows that an apparent evolutionary remnant of this form of
organization is still found in the lambda light chain locus of mammals. In the shark, an
entire cassette is used for encoding an antibody; recombination among cassettes is unusual.
Furthermore, segments within the cassettes of certain sharks are fused in the genome so
recombination (Section 4) does not occur. It is believed that the tandem repeat system of
sharks later evolved into the translocon system (Marchalonis et al., 1998). In the translocon
system, recombination among the various V, D, and J segments can occur and the
rearranged VDJ is later spliced to a C region exon (Fig. 4A).

V
H
D
H
D
H
J
H
C
H
n=200
Shark
Chicken
V
H
V
H
1 D
H
J
H
C
H
n=25 n=14 n=1
V
H
D
H
D
H
J
H
C
H
V
H
D
H
D
H
J
H
C
H
V
H
D
H
D
H
J
H
C
H
n=200
Shark
Chicken
V
H
V
H
1 D
H
J
H
C
H
n=25 n=14 n=1
V
H
D
H
D
H
J
H
C
H
V
H
D
H
D
H
J
H
C
H

Fig. 3. Organization of heavy chain loci in sharks and chicken. Three different types of
clusters are shown for sharks, some in which VDJs are fused in the genome; n= number of
repeating clusters. Modified from Dooley & Flajnik, 2006. In the diagram for chicken, the
number (n) of gene segments of each type is indicated. Only VH1 of chicken is a functional
VH gene.
The chicken also displays a translocon system but there is only one functional VH (and one
V; not shown), multiple highly similar DH segments and only one JH. All VH genes
upstream of VH1 in the chicken are pseudogenes (Fig. 3; Ratcliffe, 2006). These pseudo VH
genes are used in SGC to create the chicken antibody repertoire (Reynaud et al., 1987;
Ratcliffe, 2006).
3. Duplication and diversification of Ig genes
3.1 VH genes display evidence of duplication and genomic gene conversion
Genomic gene conversion was originally described in yeast (Meselson & Radding, 1975;
Szostak et al., 1983) and is a form of non-homologous recombination in which the end result

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118
is that a segment of one gene is translocated to another gene. When this process is
combined with gene duplication, an array of modified duplicons results. Figure 4 shows the
VH gene sequences for swine and Figure 5 the VH3 genes of the little brown bat (Myotis
lucifugus). We have used color-coding to show that in swine, there are only four different
FR1 sequences among the 24 known VH genes, only two FR2 sequences but a larger number
of different FR3 sequences, although five are often shared (Fig. 4). Also shown is that CDR
regions are often shared. Assuming these genes are the result of a combination of
duplication and genomic gene conversion, VHT could be derived from VHE with CDR2 and
FR3 translocated from VHF. A similar pattern of shared gene segments is seen among the
VH3 germline gene repertoire of the little brown bat (Fig. 5). As shown, many of these share
common FR1 sequences, a smaller number share CDR1, FR2 and CDR2 while the greatest
diversity is seen in FR3 (Bratsch et al., 2011). This pattern of similarity among duplicated Ig
genes is also seen in human and mouse, suggesting that after duplication and genomic gene
conversion, the 3 segment was subjected to a higher rate of germline mutation and
selection. We believe these examples support the hypothesis that the polygeny of VH took
place by a combination of gene duplication and genomic gene conversion. It is well-
documented that within a sublocus, intralocus duplication of segments containing several
genes also occurs. This is illustrated for the C-region sublocus for humans and rabbits (Fig.
2B). The same phenomenon occurs in the VH sublocus of mice (Retter et al., 2007; Johnston
et al., 2006) humans (Matsuda et al., 1990) and in swine (Eguchi-Ogawa et al., 2010). For
example, the genomic segment in swine that contains VHA, VHB and VHE has been
duplicated to yield VHA*, VHB* and VHF.

Genebank V
H
# gene
AF064686 V
H
A
AB513624 V
H
A*
AF064687 V
H
B
AB513624 V
H
B*
AF064688 V
H
C
V
H
D
AF064689 V
H
E
AF064690 V
H
F
DQ886395 V
H
G
DQ886392 V
H
H
AY911501 V
H
J
AF064692 V
H
K
AY911500 V
H
L
AF321841 V
H
N
AF321842 V
H
O
AF321844 V
H
Q
AF321845 V
H
R
AF321846 V
H
S
AF321847 V
H
T
AF321848 V
H
U
AF321849 V
H
V
AY911502 V
H
X
AY911504 V
H
ZZ
DQ886393 V
H
Y
EEKLVESGGGLVQPGGSLRLSCVGSGFTFS
EEKLVESGGGLVQPGGSLRLSCVGSGFDFS
EEKLVESGGGLVQPGGSLRLSCVGSGYTFS
EEKLVESGGGLVQPGGSLRLSCVGSGITFS
EVKLVESGGGLVQPGGSLRLSCVGSVFDFS
EVKLVESGGGLVQPGGSLRLSCVGSGYTFS
FR1
STYIN
STYIN
DNAFS
DYAFS
SYEIS
SYEIS
SYAVS
SYGVG
SYGMS
SYPIG
SYAVE
SSPIG
SYAVS
SYSMS
SYPIG
SYEIS
SYPIG
SYNMI
SYAVS
SYEIS
STYIN
SYGIG
SYSMS
SYEIS
CDR1
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWLA
WVRQAPGKGLEWVA
WVRQAPGKGLEWLA
WVRQAPGKGLESLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLESLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWVA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
FR2
AISTSGG
AISTSGG
AIASSDYDG
AIASSDYDG
GIYSSGS
DICSGG
GIDSGSYSG
SIGSGSYIG
GIDSGSYSG
SIGRGRYRG
SIGSGSYIG
SIGSGSYSG
AIYSGGS
GIYSSGS
AISTSGS
AISTSGA
CIYSSGS
YITSSGG
SIGSGSYIG
AIGCGSYSG
AIASSDYDG
GIYSGG
CIYSSGS
AISTSGG
CDR2
STYYADSVKGRFTISRDNSQNTAYLQMNSLRTEDTARYYCAT
STYYADSVKGRFTISRDDSQNTAYLQMNSLRTEDTARYYCAT
STYYADSVKGRFTISRDNSQNTVYLQMNSLRTEDTARYYCAI
STYYADSVKGRFTISRDNSQNTAYLQMNSLRTEDTARYYCAR
STDYADSVKGRFTISSDNSQNTAYLQMNSLRTEDTARYYCAR
STYYADYVKGRFTISRDNSQNTAYLQMNSLRTEDTARYYCAR
STDYADSVKGRFTISSDDSQNTVYLQMNSLRTEDTARYYCAR
STYYADSVNGRFTISRDNSQNTAYLQMNSLRTEDTARYYCAR
STYYADSVKGRFTISKDNSQNTAYLQMNSLRTEDTARYYCAT
STYYADSVKGRFTISSDNSQNTAYLQMNSLRTEDTARYYCAR
STVYADSVKDRFIYSRDNSQNTAYLQMNRNTAYLQMTYYCAR
STYYADSVKGRFTISKDNSQNTAYLQMNSLRTEDTARYYCAR
STYYADSVKGRFTISSDNSQNTAYLQMTRNTAYLQMTYYCAR
STDYADSVKGRFTISSDDSQNTVYLQMNRNTAYLQMTYYCAR
FR3
Genebank V
H
# gene
AF064686 V
H
A
AB513624 V
H
A*
AF064687 V
H
B
AB513624 V
H
B*
AF064688 V
H
C
V
H
D
AF064689 V
H
E
AF064690 V
H
F
DQ886395 V
H
G
DQ886392 V
H
H
AY911501 V
H
J
AF064692 V
H
K
AY911500 V
H
L
AF321841 V
H
N
AF321842 V
H
O
AF321844 V
H
Q
AF321845 V
H
R
AF321846 V
H
S
AF321847 V
H
T
AF321848 V
H
U
AF321849 V
H
V
AY911502 V
H
X
AY911504 V
H
ZZ
DQ886393 V
H
Y
EVKLVESGGGLVQPGGSLRLSCVGSVFDFS
EVKLVESGGGLVQPGGSLRLSCVGSGYTFS
FR1
STYIN
STYIN
DNAFS
DYAFS
SYEIS
SYEIS
SYAVS
SYGVG
SYGMS
SYPIG
SYAVE
SSPIG
SYAVS
SYSMS
SYPIG
SYEIS
SYPIG
SYNMI
SYAVS
SYEIS
STYIN
SYGIG
SYSMS
SYEIS
CDR1
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWVA
WVRQAPGKGLEWVA
WVRQAPGKGLEWLA
WVRQAPGKGLEWVA
WVRQAPGKGLEWLA
WVRQAPGKGLESLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLESLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWVA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
WVRQAPGKGLEWLA
FR2
AISTSGG
AISTSGG
AIASSDYDG
AIASSDYDG
GIYSSGS
DICSGG
GIDSGSYSG
SIGSGSYIG
GIDSGSYSG
SIGRGRYRG
SIGSGSYIG
SIGSGSYSG
AIYSGGS
GIYSSGS
AISTSGS
AISTSGA
CIYSSGS
YITSSGG
SIGSGSYIG
AIGCGSYSG
AIASSDYDG
GIYSGG
CIYSSGS
AISTSGG
CDR2
STYYADSVKGRFTISRDDSQNTAYLQMNSLRTEDTARYYCAT
STDYADSVKGRFTISSDNSQNTAYLQMNSLRTEDTARYYCAR
STYYADYVKGRFTISRDNSQNTAYLQMNSLRTEDTARYYCAR
STDYADSVKGRFTISSDDSQNTVYLQMNSLRTEDTARYYCAR
STYYADSVNGRFTISRDNSQNTAYLQMNSLRTEDTARYYCAR
STYYADSVKGRFTISKDNSQNTAYLQMNSLRTEDTARYYCAT
STYYADSVKGRFTISSDNSQNTAYLQMNSLRTEDTARYYCAR
STVYADSVKDRFIYSRDNSQNTAYLQMNRNTAYLQMTYYCAR
STDYADSVKGRFTISSDDSQNTVYLQMNRNTAYLQMTYYCAR
FR3

Fig. 4. Deduced amino acid sequences for the framework (FR) and combinatorial determining
regions (CDR) of germline porcine VH genes. Regions of FR and CDR regions that are shared
among genes are color-coded. Those sequences that are not colored indicate segments with
sequences that differ by one or a few changes that are not shared by other sequences.


119

Genebank # group FR1 CDR1 FR2 CDR2 FR3
GQ923685 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS DYSMN WVRQAPGKGLEWVA YTSYGSGNPI YYADSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923622 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS EYGMN WVRQAPGKGPEWVS YISSPSGSNI YYAASVKGRFTISRENAKNSLYLQMSSLRAEDTAVYYCAR
GQ923683 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS EYGMN WVRQAPGKGLEWVS YISGDSSSDI YYAASVKGRFTISRDNAKNMVYLQMNSLRAEDTALCYCVR
GQ923616 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS EYGMN WVRQAPGKGLEWVS YISSPSGSNI YYAASVKGRFTISRENAKNSLYLQMSSLRAEDTAVYYCAR
GQ923644 LVESGGGLVQPGGSLRLSCAASGFTFS NYDMH WIRQAPGKELEWVA HIWTDGSQK YYAESVKGRFTISRDNTKNMAYLQMNSLRVKDTALYYCAR
GQ923675 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS NYGMN WVRQAPGKGLEWIA YTSSGDGNPI YYADSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923628 LVESGGGLVQPGGSLRLSCAASGFTFS NYYMN WVRQAPGKGLEWVA SISDGSSYI YYGEAVKGRFTISRDNTKNMLYLQMNSLRAEDSAVYYCAR
GQ923647 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS NYYMN WVRQAPGKGLEWVA YISSDGSSYI NYANAVKGRFTISRDNAKNMVYLQMSSLRAEDTAMYYCAR
GQ923618 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SNSMN WVRQAPGKGLEWVA LISSGGGST YYAASVKGRFTISRDNAKNSLYLQMNSLRAEDMALYYCAR
GQ923650 LVESGGGLVQPGGSLRLSCAASGFTFS SNWMS WVRQAPGKGLEWVG IISTDGGTT NYADSVKGRFTISRDNAKNTLYLQMNSLTAKDTAVYYCAK
GQ923681 3-1 LVESGGGLVQPGGSLRLSCAASGFTFS SSWMV GVRQAPGKGLEWVS LINPDGSIT NYANSVKGRFTISSDNAKNMLYLQMNSLRAEETAMYYCAR
GQ923662 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYDMN WVRQAPGKGLEWVA LISTDGGST YYANSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCAR
GQ923669 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYDMN WVRQAPGKGLEWVS LISPSGGST YYADSVKGRFTISRDNAKNMVYLQMSSLKAEDKAVYFCAR
GQ923625 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYDMN WVRQAPGKGLEWVS LISPSGGST YYADSVKGRFTISRDNAKNMVSLQMSSLRAEDTAVYYCAR
GQ923612 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYDMN WVRQAPGKGLEWVA YISSASNTI YYANSVKGRFTISIDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923621 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYDMS WVRQAPGKGLEWVS AISNGGGST YYAASVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
GQ923679 LVESGGGLVQPGGSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA YQYISSDGRNYI NYAASVKGRFTISRDNAKNTAYLQMNSLKAEDTAVYYCAR
GQ923617 LVESGGGLVQPGGSLRLSCAASGFTFS SYGMH WVRQAPGKGLEWVA YQYISSDGRNYI NYAASVKGRFTISRDNAKNMLYLQMSSLRAEDMAVYYCAR
GQ923658 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS SYGMH WIRQAPGKGLEWVS RIGSDGRSYI HYADSVKSRFTISRDNAKNMLYLQMSSLRSEDTALYYCAR
GQ923672 3-4 LVESGGGLVQPGGSLRLSCAASGFTFS SYGMN WVHQVLGKGLECVS GVSSIGGTT YYADSVKSRFTVSRDNTTSMLYLQMNSLRTEDMAVYYCAR
GQ923613 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS SYGMY WVGQVPGKGLEWVS LISSDGSSTI YYANSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR
GQ923657 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYHMN WVRQAPGKGLEWVA FISNGGSTI YYAASVKGRFTISRDNAKNMLYLQMNSLRAEDTALYYCAR
GQ923638 LVESGGGLVQPGGSLRLSCAASGFTFS SYQMH WVRQAPGKGLEWVE LISSSGGTI YYADSVKGRFTISRDNAKNTLFLQMSSLRADDTAMYYCAR
GQ923629 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYSMD WVRQAPGKGLEWVA YISSASSTI YYANSVKGRFTISRDNAKNTLYLQMSSLRAEDTAMYYCAR
GQ923642 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYSMN WVRQAPGKGLEWVA VISSSGGTI YYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAR
GQ923684 3-1 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMD WVRQAPGKGLEWLC RMNPDGSTT HYANSVKGRFTISRDNAKNMLYLQMNSLRAEETAMYYCAR
GQ923619 3-3 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGLEWVS RISSSGSTI SYAASVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAR
GQ923632 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGLEWVS LISSDGSSTI YYANSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923668 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGLEWVS LISSDGSSYI YYAASVKGRFTISRDNAKNTLYLQMSSLKAEDTAVYYCAR
GQ923640 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMN WVRQAPGKRLEWVS AISSSGGST YYADSVKGRFTISRDNAKNTLFLQMSSLRVEDTAVYYCAK
GQ923627 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMS WVRQAPGKGLEWVA HINSGGST YYADSVKGRFTISRHNAKNSLYLQMSSLRAEDTVGYYCVR
GQ923663 3-1 LVESGGGLVQPGGSLRLSCAASGFTFS SYWMY WVRQAPGKGLEWLC RMNPDGSTT NYANSVKGRFTISRDNAKNTLYLQMNSLSTQDTAMYYCST
GQ923623 3-2 LVESGGGLVQPGGSLRLSCAASGFTFS SYYMN WVRQAPGEGLEWVA SISSGGGSYI YYAASVKGRFTISRDNTKNTLYLQMNSLRAEDTAVYYCAR
GQ923680 LVESGGGLVLPGGSLRLSCAASGFTFS DYWVH WVCQAPWKGLEWVS DFRGDGGTT YYADSVKGRSTLSRDNVKNSLYLQMSSLRAKDTAIYYCAR
GQ923635 LVESGGGLVLPGGSLRLSCAASGFTFS GYWIS WARQAPGKKLEWVS DISGDSSIT YYAASVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCAR
GQ923636 LVESGGGLVLPGGSLRLSCAASGFTFS NYDMH WIRQAPGKGLEWVA HIWTDGSQK YYAESVKGRFTISRDNAKNSLYLQMNSLKAEDSALYYCAR
GQ923673 3-1 LVESGGGLVLPGGSLRLSCAASGFTFS SNWMH WVRQAPGKGLEWLC RMNPDGSTT NYANSVKGRFTISRDSAKNMLYLQMNSLRAEETAMYYCTT
GQ923655 3-1 LVESGGGLVLPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGPEWLC QINSDGNTI YYANSVKGRFTISRDNAKNMLHLQMNSLRAEESALYYCAR
GQ923615 3-1 LVESGGGLVLPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGLEWLC QINSDGNTI YYANSVKGRFTISRDNAKNMLHLQMNSLRAEESALYYCAR
GQ923676 3-3 LVESGGGLVQPGGSLRISCAASGFTFS SYWMS WVRQAPGKGPEWVS HISDGGGST YYANSVKGRFTNSRDNAKNSLSLQMNSLKPEDTALYYCAR
GQ923631 LVESGGGLVQPGGSLRLSCAASGFIFS SYGMS WVRQAPGNGLEWVS GVSSIGGTTG YYADSVKGRFTVSRDNGKNMLFLQMNSLRAKDTAVYYCAR
GQ923611 3-2 LVESGGGLVQPGGSLRLSCAASGFSFS SYWMG WVRQAPGKGLEWVA LISTGGGGNT YYATSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAR
GQ923667 3-3 LVESGGGLVQPGGSLRLSCAASGFSFS IYGMN WVRQAPGKGLEWVS GISTGGGST YYAASVKGRFTISRDNAKNSLYLQMNSLKAEDTAVYYCAR
GQ923654 3-2 LVESGGGLVQPGGSLRLSCAATGFTFS SYWMH WVRQAPGKGLEWVS LISSDGSSYI YYAASVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923645 3-3 LVESGGGLVQPGGSLRLSCSGSGFTFS SYSMD WVRQAPGKGLEWVA YISSASNTI YYANSVKGRFTISIDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923660 3-3 LVESGGGLVQPGGSLRLSCSGSGFTFS SYSMD WVRQTPGKGLEWVA YISSASNTI YYANSVKGRFTISIDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923677 3-6 LVESGGGVVPPGGSLSLSCKASGFTFT NYSMD WVSQAPGKGLQWVT RVSKPKGMTQ WYAPAVQGRFTIFRDNPMSTASLEITKLTSEDMAMYYCAR
GQ923648 3-6 LVESGGGVVPPGGSLSLSCKASGFTFT NYSMD WVSQAPGKGLQWVT RVSKPKGMTQ WYAPAVQGRFTIFRDNPMSTVSLEITKLTSEDMAMYYCTR
GQ923671 3-6 LVESGGGVVPPGGSLSLSCKASGFTFT NYSMD WVRQAPGKGLQWVA RVSQPKGTTQ WYAPAVRGRFTISRDNPTSTVSLKMTKLTSEDTAVYYCTR
GQ923626 LVESGGDLVQPGGSLRLSCAASGFTFS SYWMH WVRQAPGKGLEWVS LVNPAGSST YYANSVKGRFTISRDNAKNTLYLQMSSLRAEDTGVYYCAR
GQ923653 LVESGGDLVQPGGSLRLSCAASGFTFS SYWMH WVRQAPGEGLEWVS LINPAGSST YYANSVKGRFTISRDNAKNMLYLQMNSLRAEDTALYYCSR
GQ923674 3-1 LVESGGGFVQPGGSLRLSCAASGFTFS SSSMD WVRQAPGKGLEWLC RINPDGSTT YYANSVKGKFTTSRDNAKNLLYLQMNSLRAQDMAVYYCVT
GQ923652 LVESGGGLGKPEGSLRLSCAASGFASS SYYMN WIRQTPGKGLEWMA VISYNGNNT YYADSVKGRFTISRDNAKNMVYLQMSSLRAEDTALYYCVR
GQ923661 3-5 LVESGGGLGKPEGSLRLSCAASGYASS SYYMN WVRQTPGKGLEWIC AITANGDST YYADSVKGRFTISRDNAKNTIYLQMSSLKSEDTAVYYCST
GQ923624 3-5 LVESGGGLGKPEGTLRLSCAASGYASS SYYMN WVRQTPGKGLEWIC AITGNSDST YYADSVKGRFTISRDNAKNTIYLQMNSLRAEDMTVYYCAT
GQ923633 3-5 LVESGGGLGKPEGTLRLSCAASGYASS SYYMN WVRQTPGKGLEWIC AITGNSDST YYADSVKGRFTISRDNAKNTIYLQMNSLRAEDTGVYYCAK
GQ923637 3-3 LVESGGGLVKPGGSLRLSCAASGFTFS SYSMD WVCQAPGKGLEWVA YISSASSTI YYANSVKGRFTISIDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923614 3-2 LVESGGGLVKPGGSLRLSCAASGFTFS EYGMN WVRQAPGKGLEWVS YISGDSSSDI YYAASVKGRFTISRDNAKNMVYLQMNSLRAEDTAVYYCAR
GQ923651 LVESGGGLVPHGGSLRLSCAASGFTFN DYEMN WVRQAPGKGLEWVS RITSTGGST HYAASVKGRFTISRDNAKNTLFLQMNSLRAEDQAMYYCTA
GQ923659 3-1 LVESGGGLVPPGGSLRLSCAASGFTFS SYWNT WVRQAPGKGLEWLG EINPDGSTT NYANAVKGRFTISRDNAKNTLYLQMNSLSAQDMAVYYCVR
GQ923646 LVESGGGLVPPGGSLRLSCAASGFTFS SYWMG WVRQAPGKGLEWVS FVTDYGGSI YYADSVKGRFSISRDNAKNTLYLQMTSLRATDTAVYYCAR
GQ923630 LVESGGGLVPPGGSLRLSCATSGFTFS GYWIS WARQAPGKKLEWVS DINGDSSTT YYAASVKGRFTTSRDNAKNMLFLQMSSLRAEDTAVYYCAR
GQ923682 LVESGGGLVPPGGSLRLSCATSGFTFS GYWIS WARQAPGKKLEWVS DINGDSSTT YYAASVKGRFTISRDNAKNTLYLQMNSLRAEDTALYYCAR
GQ923649 3-2 LVESGGGLVQPGGSLRLSCAASGFSFW SYPMN WVRQAPGKGLEWVA LISSGRDGNT YYATSVKGRFTISRDNAKNSLYLQMSSLRSDDTALYYCAR
GQ923634 LVESGGGLVQPGGSLRLSCAASGFTFD DYYMH WVRQAPGKGLEWVT SISEGGSYI YYANAVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
GQ923641 3-2 LVESGGGLVQPGGSLRLSCAASGFTFG SYWMH WVRQAPGKGLEWVS RIGSDGSSYI YYADSVKGHFTISRDNAKNMLYLQMSSLRSEDTAVYYCAR
GQ923665 3-3 LVESGGGLVQPGGSLRLSCAASGSTFS SYSMD WVRQAPGKGLEWVA YISSASSTI YYANSVKGRFTISRDNAKNSLYLQMSSLRAEDTAVYYCAR
GQ923670 3-3 LVESGGGLVQPGGSLRLSCTGSGFSFS SYSMD WVRQAPGKGLEWVA YISSASNTI YYANSVKGRFTISIDNAKNTLYLQMSSLRAEDTAVYYCAR
GQ923639 3-4 LVESGGGLVQPGGSLRLSFAASGFTFS SYGMN WAHQFPGKGLEWVS GVSSIGGTT YYADSVKGRFTVSRDNAKNTLYLQMNSLRTEDMAVYYCAR
GQ923656 3-4 LVESGGGLVQPGGSLRLSFAASGFTFS SYGMN WAHQFPGKGLEWVS GVSSIGGTT YYADSVKGRFTVSRDNAKNTLYLQMNSLRTEDMAVYYCAR
GQ923620 LVESGGGLVQPWGSLRLSCKGSGFTFS DYYMN WIRQTPGKGLEWVG LIRNKANGHTT EYAASVKGRVAISRDDSKSTADLQMSSLIAEDTAMYNYAR
GQ923664 LVESGGGLVQTGGSLRLSCAASGFTFS SYWMN WVRQAPGKGLEWVS TITHTDGST YYPDSVKGRFTISRDNTKNMLYLQMNSLRSEDTAVYYCAR
GQ923643 3-6 LVESGGGMVPPGGSLSLSCKASGFTFT NYSMY WVRQAPGKGLQWVA GVSKPTGKNQ WYAPAVQGRFMISRDNPTSTVSLEITKLTSEDTALYYCSR
GQ923678 LVESGGGLVQPGGSLRLSCAASGYTFS SYWMG WVCQAPCKGLEWVS LITGNGGST YYANTAKVRFTISRDNTKNMLYLQMNSLTAEDTAVYYCAR
GQ923666 3-5 LVESGGSLVQPEGSLRLTCAASGFTSN SYYMN WFHQTPGKGLEWIC AITANGDST YYSDSVKGSFTISRDTAKNPIYLQMSSLRSKDTAVYYCAR

Fig. 5. The deduced amino acid sequences for the framework (FR) and combinatorial
determining regions (CDR) for 75 members of the VH3 family from the little brown bat (M.
lucifugus). Shared sequences are color-coded as in Fig. 4. From Bratsch et al., 2011.

Gene Duplication

120
3.2 Duplication and deletion of Ig genes in the C-region sublocus
Figure 2B illustrates that in humans, a major segment of the C- region sublocus has been
duplicated resulting in two IgEs, two IgAs and two pair of IgG genes (Lefranc et al., 1982;
Flanagan & Rabbitts, 1982). A C pseudogenes separates them. In mammals, the target of
duplications in the C-region has been those genes encoding IgG (or IgA in rabbits; Fig. 2B;
Table 2). The duplication process in the CH region suggests it also occurred together with
genomic gene conversions to produce an array of modified C genes (Fig. 6). In the example
provided the IgG1 and IgG2 alleles share a common CH1 domain that is also found in the
IgG4 alleles. The allelic variants of IgG1 and IgG4 differ only in their hinge exons. However,
IgG1
a
and IgG4
a
have the same hinge as do IgG1
b
and IgG4
b
. The difference between IgG1
and IgG4 is in the CH3 domain which is not shared with any other C subclass gene.
Another example is IgG5
a
and IgG6 which share a common CH1 and hinge exons. IgG5
a

also has the same CH2 exon as IgG6
b
, but the difference is in CH3. Thus Fig. 6 also shows
that apparent genomic gene conversion events also involve allelic variants as well as entire
genes. The pattern shows that the CH1, hinge and CH2 of
IgG1
a
and IgG4
a
were derived from the same ancestral C gene but the IgG1
b
and IgG4
b

were derived from another ancestral gene. The reverse effects of genomic gene conversion
may explain certain heterozygous C deletions (Migone et al., 1984; Keyeux et al., 1989).
Some swine lack certain C genes (Butler et al., 2009a) and deletions in the human C
sublocus are well documented (LeFranc et al., 1983a; Rabbani et al., 1995).

Fig. 6. Alignment of the constant region domains of the porcine C genes. Regions of >95%
homology are designated with the same texture. Superscripts in the gene designation denote
allotypic variants. From Butler et al., 2009a.
As we have shown elsewhere, porcine IgG3 has a gene structure which is most similar to the
consensus C genes of other mammals and therefore is closest to the ancestral C gene of all
mammals (Butler et al., 2009a). IgG3 in humans, mice andswine all occupy the same 5
position which is immediately downstream of Co (Eguchi-Ogawa-et al., 2010). Our studies
indicate that the remainder of the porcine C genes were derived from an ancestral C that


121
diverged early from IgG3 (Butler et al., 2009a). We hypothesize that the
duplication/diversification of C subclass genes in mice and humans followed the same
pattern.
4. Mammalian antibody repertoires result from somatic events
4.1 Somatic gene segment recombination characterizes B cell lymphogenesis
B lymphocytes, named because they form in the Bone marrow or the chicken Bursa of
Fabricius, are the cells that synthesize and secrete antibodies. This developmental process
occurs in what are called primary lymphoid tissues. These include the bone marrow, the
chicken bursa, fetal liver, yolk sac and according to some, certain hindgut lymphoid tissues
of artiodactyls. Among lower vertebrates, other tissues like the head kidney(pronephros),
epigonal organ and Leydig organ are involved in this process (Solem & Stenvik, 2006;
Rumfelt et al., 2002; Dooley & Flajnik, 2006) .
Somatic recombination is illustrated in Figure 7A. This process is mediated by Recombinase
Activation Genes (RAGs) as well as a variety of DNA repair and ligation enzymes. In the
heavy chain locus this process first involves recombination of one J region gene segment and
one D region gene segment. This event also produces a circular DNA product containing the
intervening DNA sequence that is excised and is known as a signal joint circle (Fig. 7B).
Single joint circles are diagnostic evidence that B cell lymphogenesis has recently occurred
since this nuclear product is rapidly degraded. The rearrangement process then proceeds to
the rearrangement of the DJ unit with some V gene segment and generation of another
signal joint circle (Fig. 7A). The selection of the J, D, and V gene segments is poorly
understood and will be discussed in Section 5. A similar series of events occurs among
segments in the light chain loci except that there are no D segments involved.

Fig. 7. The somatic rearrangement process among the gene segments of the variable heavy
chain locus of mammals. A. Sequential rearrangement of D to J, then DJ to V and finally
splicing of the primary transcript for VDJ to the exons encoding the C-region of IgM. B.
Generation of a signal joint circle during the excision of intervening DNA during
recombination of D and J.

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The rearranged VDJ (heavy chain) and VJ (light chains) rearrangements are then transcribed
and the primary transcript spliced to some set of C-region exons in the heavy and light
chain loci respectively. For the heavy chain this is initially IgM in all higher vertebrates (Fig.
7A). The resulting VDJ-C and VJ-C transcripts are then translated into the light and heavy
polypeptide chains that combine to form the complete antibody molecule (Fig. 1B). As
shown in Fig. 1A and discussed above, the antigen-binding site is located in the peptide
loops from the VH and VL domains that coalesce at the end of theseV-domain and that
contain the CDRs; three from the VDJ and three from the light chain VJ rearrangements.
CDR1 and CDR2 of the VH and VL are encoded within the germline genes whereas the
CDR3 region is the result of the combining ofV-D-J segments (heavy chain) and V-J (light
chains; Fig. 8). The CDR3 region of the heavy chain, hereafter designated HCDR3, is
considered most important to the specificity of the binding site (Amit et al., 1986; Padlan,
1996; Xu & Davis2000; Mageed et al., 2001). In fact the same set of V-genes encoding CDR1
and CDR2 can theoretically and actuallycontribute the binding site for antibodies of quite
different specificity in the context of different HCDR3 regions (Thomson et al., 2011;
Ichiyoshi & Casali, 1994) as might be envisioned from a comparison of the CDR3 sequences
shown in Fig. 8.

FR3 5' N-add D
H
A 3' N-add J
H
FR4
TGNGCNAGN GAATTGCTATAGCTATGGTGCTAGTTGCTATATGATGAC ATTACTATGTTATGGATCTCTGGGGCCCA
TGTGCAAGT TGCTATAGCTATGGTGCTAGTTGC TTTTGGACAAGATCA TACTATGCTATGGATCTCTGGGGCCCA
TGTGCAAGA GGCTGTTTTC GCTATAGCTATGGTGCTAGTTGCTAT GATGTCG ACTATGCTATGGATCTCTGGGGCCCA
TGTGCAA CAGGCGAT TGCTATAGCTA GGTGCTAGTTGCACCGGGATG GCTATGGATCTCTGGGGCCCA
TGTGCAA TT GCTATAGCTATGGTGCTAGTTG T TATGGATCTCTGGGGCCCA
TGTGCAA CAGAG TGCTATAGCTATGGTGCTAGTTGCTATAT GTATGC TATGGATCTCTGGGGCCCA
TGTGCAAGA G ATAGCTATGGTGCTAGTT ACCCCTC TATGGATCTCTGGGGCCCA
TGTGC CCAG GCTATAGCTATGGTGCTAGT CCAGGATG TGGATCTCTGGGGCCCA
TGTGCAA CAGGC ATAGCTATGGTGCTAGTTGCTAT GAAGA TGGATCTCTGGGGCCCA
TGTGCAAG GTCC AATTGCTATAGC TCCGGTGGTGAGTGCTATGGTTACCCTTGGGGTTATGTTGCTG TGGATCTCTGGGGCCCA
TGTGCAA TT GCTATAGCTATGGTGCTAGTT AGATC GGATCTCTGGGGCCCA

Fig. 8. The diversity of HCDR3 sequences resulting from the recombination of the same VH,
DH and JH segments. Remnants of the DH germline segments are underlined. The 5 and 3
nucleotide additions are indicated. TGG is the codon for the invariant tryptophan found in
the JH gene segments of all mammals while TGT is the codon for C that is nearly invariant
in the FR3 of all VH3 family genes.
4.2 Maturation of the antibody repertoire involves class switch and somatic
hypermutation
All immunologists, immunopathologist and physicians in specialties such as rheumatology
know that most Igs are IgG (serum) or IgA (secretions). This means that the rearrangements
involved in B cell lymphogenesis that initially favors the expression of IgM (Fig. 7A) switch
to these isotypes. After environmental exposure, the concentration of the major Igs in serum
is elevated 100-300 fold compared to newborn piglets or those reared in germfree isolators
(Fig. 9A). The transition from newborn to conventionally-reared young adults favors IgG in
serum (Fig. 9A) and IgA in secretions (Butler et al., 2011a). This change involves class switch
recombination (CSR) which is mediated by activation-induced cytidine deaminase (AID) of
the APOBEC family which facilitates the splicing of RNA encoding the rearranged VDJ to
transcripts encoding IgG and IgA rather than IgM. This maturation process typically occurs
in tandem with somatic hypermutation (SHM) of the rearranged VDJs or VJs prior to their
transcription. SHM is another mechanism for repertoire diversification and is triggered


123
when the neonate encounters environmental antigen (Fig. 9B). Since both CSR and SHM
occur simultaneously, it is not surprising that both are mediated by AID and that AID is also
correlated with SGC (Withers et al., 2005; Arakawa et al., 1996). These events occur in
germinal centers (GCs) of secondary lymphoid tissues after exposure to environmental
antigen. GCs are found only in mammals and birds (Yasuda et al., 2003; Vigliano et al., 2006;
Du Pasquier et al., 2000). Although lacking GCs, there is SHM and CSR in Xenopus (Marr et
al., 2007) although it may be less efficient. However, for these events to occur, the nave
immune system must first or simultaneously be exposed to Pathogen Associated Molecular
Patterns (PAMPs) that are recognized by a variety of innate immune system receptors. This
dependence was demonstrated using the isolator piglet model (Butler et al., 2002; 2005;
2009b; Butler & Sinkora 2007).
SHM is not random across the entire rearranged VDJ-C transcript. Rather it is largely
concentrated in the CDR regions of the rearranged VDJ or VJ segments (Fig. 9C). This is
generally believed to result from selection of B centrocytes in GCs rather than specific
targeting. Although Fig. 9C only shows the accumulation of somatic mutations in CDR1 and
CDR2, the same occurs in CDR3. As discussed previously, the CDRs are those segments of
the encoded protein that coalesce to form the antibody binding site (Fig. 1A; Fig 8). There is
little evidence to suggest that SHM proceeds downstream from segments of transcript that
begins with the codon for the invariant tryptophan in FR4 (Fig. 8) or to sequences further
downstream in the C-sublocus.
4.3 The association of VH genes and VH- VL pairing in generation of specific
antibodies
Much of the early studies on antibody specificity that appeared when VH or VL polygeny
became known, attempted to correlate particular response to the use of certain VH or VL
genes. We do not review that literature here but do provide a few examples. Cerato et al.,
(1997) studied hybridomas to show a lack of correlation between VH usage and specificity
while Mo and Holmdahl (1996) show that mAbs to different epitopes used the same VH/Vk
combinations. Boffey et al., (3004) showed that only 6/15 anti-LPS mAbs used the same VH
gene (VH7183.3b). These observations should not be surprising considering the importance
of HCDR3 in the specificity of antibodies (see Sectiion 4.3; 6.2). Lavoie et al., (1997) showed
that nearly all mAbs to HEL use VH36-60 but differ in affinity because of SHM or HCDR3
differences. The antibody binding site involves CDRs (including CDR3) of both H and L
chains (Fig 1B); this has been shown by separation and reassociation experiments. These
experiments show that binding site specificity depends on both H and L chains even for
antibodies specific for the same hapten since heterologous light chains seldom restore the
full binding site (Kranz & Voss, 1981). This mutual dependence is also demonstrated by the
non-random pairing found in antibodies of certain specificity such as to the capsular
polysaccharides of S. pneumoniae (Thomson et al., 2011). Further evidence for the effect of H-
L pairing comes from studies of autoantibodies in a phenomenon called receptor editing
(deWildt et al., 1999). This in vivo phenomenon involves reactivation of recombinase activity
in lymph nodes resulting in the replacement of the light chain with a new one. In this way, B
cells expressing autoreactive BCRs acquire a new light chain which alters their specificity
and removes or diminishes their autoreactivity apoptotic elimination (Tiegs et al., 1993; Gay
et al., 1993).
While L-H pairing is important for binding site specificity, there are situations in which light
chains are not needed to form an antibody binding site. The best known examples are the

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124
naturally occurring single chain antibodies of the camelid group and some sharks (Hamers-
Casterman et al., 1993; De Genst et al., 2006; Dooley et al., 2003; Diaz et al., 2002; Nguyen et
al., 2002). Based on the convenience of producing single chain antibodies from these species
for therapy and the evidence that the HCDR3 domainplays the major role in forming the
antibody binding site (see Section 6.2) there have also been various attempts to
developsynthetic single chain antibodies or camelized antibodies (Janssens et al., 2006;
Reiter et al., 1999; Davies & Riechmann,1995). Among the camelids, single chain antibodies
use a separate set of VH genes (called VHH) that encode a much largerportion of the
binding sites than the conventional VH genes which compensates for the lack of a light
chain. This topic has been recently reviewed (Muyldermanns et al., 2009). We mention these
single chain antibodies here because we believe they further support the role played by
HCDR3 in binding antigen and diminishes the value of polygeny of conventional VH genes
(Section 6.2). It also shows that the extensive and universal Vk and V polygeny among
mammals (Table 1) is unnecessary.

Fetal GF PIC
0
10
20
30
40
50
IgM
IgG
IgA
51
2051
4051
6051
6052
16052
26052
g

/

m
l
Fetal GF PIC
0
20
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M
u
t
a
t
i
o
n
/
K
i
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o
b
a
s
e
FR1 CDR1 FR2 CDR2 FR3
0
50
100
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Fetal
GF
PIC
M
u
t
a
t
i
o
n

/

K
i
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o
b
a
s
e
A B
C
Fetal GF PIC
0
10
20
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40
50
IgM
IgG
IgA
51
2051
4051
6051
6052
16052
26052
g

/

m
l
Fetal GF PIC
0
20
40
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M
u
t
a
t
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/
K
i
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o
b
a
s
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FR1 CDR1 FR2 CDR2 FR3
0
50
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150
200
250
Fetal
GF
PIC
M
u
t
a
t
i
o
n

/

K
i
l
o
b
a
s
e
A B
C

Fig. 9. The effect of antigen exposure on: A. Serum Ig levels; B. Frequency of SHM and C.
Accumulation of somatic mutations in various segments of the VH genes. Germfree piglets
are reared in isolators for 5 weeks and their only contact with potentially foreign antigen is
food protein. PIC=conventionally-reared young pigs that are heavily antigenized through
colonization and also infected with nematodes. The horizontal line (9B) in the scattergram is
the mean frequency of SHM. SHM is significantly greater in PIC piglets than in fetal and
germfree piglets. In 9C SHM accumulates in CDR regions as opposed to FR regions that
encode the |-pleated staves of the |-barrel (Fig. 1A).


125
5. Patterns of V, D and J gene segment usage
5.1 VH usage is biased to favor certain VH genes
Table 1 shows that higher vertebrates have many duplicated V-region gene segments
available for use in the formation of their antibody repertoire using the recombinatorial
process illustrated in Figure 7. Humans have available ~ 100 VH segments, ~30 DH
segments and 9 JH segments (Fig. 2A). By contrast, swine have <30 VH genes belonging to a
single family (Fig. 4), only two functional DH segments and like the chicken (Fig. 3) one
functional JH segment (Sun et al., 1994; Butler et al., 1996; Eguchi-Ogawa et al., 2010). While
the ancestral VH3 family (Schroeder et al., 1990) dominates the V-region loci of many
species, the ~100 VH genes of mice and human belong to 14 and 7 different families
respectively (Table 1).
Usage of VH genes in rabbit is biased to the most 3 VH gene, which accounts for 90% of VH
usage in the pre-immune repertoire although there are >100 VH genes in the rabbit
repertoire (Currier et al., 1988; Table 2). In humans there is bias for V3-23,V3-30,V3-33 and
V4-34 (Glas et al., 2000). While some suggest that VH usage is random in mice (Dildrop et
al., 1985) studies on J558 usage (one-half of the mouse genome) indicates that usage in
unequal and rather scattered across the entire J558 genome even in the pre-immune
repertoire (Gu et al., 1991) and that usage is not affected by SHM or CSR. Foster et al., (1997)
showed that while most Vk genes were used, usage was non-random and the same was true
for Jk. Sheehan et al., (1993) showed that fetal VH usage can differ from 0.1 to 1.0 but that
most 5 VH genes are underrepresented. In swine VHA (IGHV4) and its near duplicate
(IGHV10; see Figs. 4 & 10) account for one-third to one-half of the pre-immune repertoire
(Butler et al., 2006; Eguchi-Ogawa et al., 2010; Butler et al., 2011b). Interestingly, the majority
of these preferred genes in all these species belong to the ancestral VH3 family (Schroder et
al., 1990; Brezinschek et al., 1997).
5.2 Variable region gene segment usage is not position dependent
Early studies suggested that VH usage was biased during early stages of B cell
lymphogenesis to favor the most JH proximal DH segments and the most 3 VH genes
(Schroeder et al., 1987; Yancopoulos et al., 1984) but that this pattern became normalized
in adults (Malynn et al., 1987). This concept gained support when it was found that young
rabbits use their 3 most VH gene > 90% of the time and then further diversfied their
repertoire using upstream VH genes and SGC; perhaps a type of developmental
normalization (Knight 1992; Becker & Knight 1990). However, additional studies in humans
neither substantiated the positional 3 bias (Matsuda et al., 1993) nor have our studies in
swine (Eguchi-Ogawa et al., 2010; Fig. 10). The most 3 functional VH in swine (IGHV2) is
almost never used while upstream VH15 (IGHV15) can account for ~13% of VH usage (Fig.
10). Thus, the position hypothesis to explain VH usage has not been universally fulfilled.
5.3 VH gene usage remains constant in fetal and young pigs
Vertical studies on VH usage in especially humans and mice are difficult because: (a) the V-
D-J repertoire of these species is complex (Table 2) and could require up to 56 primer sets to
recover all VDJ rearrangements in mouse and 42 sets for human (b) maternal regulatory
factors transmitted in utero or via colostrum/milk can influence pre-natal and postnatal
development (Wikler et al., 1980; Rodkey & Adler, 1983; Klobasa et al., 1981; Wang &
Shlomchik 1998; Yamaguchi et al., 1983) and (c) control of environmental and maternal

Gene Duplication

126
effects is difficult or impossible in species with altricial offspring. Therefore we addressed
this issue using a piglet model in which there is no transfer of maternal factors in utero and
the influence of environmental factors postnatally on their precosial offspring can be
controlled by the experimenter (Butler & Sinkora 2007; Butler et al., 2009b). Use of this
model revealed that VH usage was constant during fetal life and that seven major genes
accounted for >90% of the repertoire (Fig. 10; Butler et al. 2011b) while four can explain
>80% of the repertoire Interestingly in piglets exposed to viral infection, gut colonization or
nematode parasites after birth, ~ 75% of the mutated genes used were the same seven (Fig.
10; extreme right). Furthermore, proportional usage of these genes was similar to what was
seen in the pre-immune repertoire, albeit somatically mutated. Some modest changes were
observed such as an increase in VHY and decreases in VHA* and VHN. In other words,
swine seldom select other genes from their repertoire after exposure to environmental
antigen, but continue to use the same VH genes that comprise ~93% of the pre-immune
repertoire.

0
10
20
30
40
50
60
70
80
90
100
VHG VHA VHB VHE VHA* VHF VHY VHN/VHZZ VHC VHZ % Major
P
r
o
p
o
r
t
i
o
n
a
l

o
f

U
s
a
g
e
20-50DG (2108)
95DG (3357)
Mutated in antigen-exposed piglets(136)
(VH2) (VH4) (VH6/ VH12) (VH8) (VH10) (VH11) (VH14) (VH15) (VH?) (VH?) (seven)

Fig. 10. VH gene usage in fetal piglets and among neonates that are antigen-exposed
remains relatively constant. DG= days of gestation. The number of VH gene clones tested is
given in the legend. The mutated VH genes are no longer recognized because they do not
hybridize with VH gene-specific probes. Their identity must then be determined by
sequencing. The bar graph on the extreme right gives the proportion of all mutated VH
genes that are accounted for by the major seven genes used in the pre-immune repertoire by
the fetus.
These observations should not be surprising considering that the specificity of binding site is
heavily dependent on HCDR3 (Section 6.2). The HCDR3 repertoire in swine is diverse when
only one VH, one DH and one JH segment are used (Fig. 8). Furthermore, the limited use of
VH genes should not limit specificity, since their CDR regions can also be somatically
mutated (Fig. 9B, C). As discussed in 4.1, the variants from a single VH (Fig. 8) can pair
with different light chains, which can further reduce the need for large numbers of different
VH genes for repertoire formation.
5.4 Why is the germline VH repertoire large but usage of the repertoire limited?
Use of the piglet model demonstrates that most of the piglet VH repertoire (Fig. 4 versus
Fig. 10) is seldom used to form the antibody repertoire. While corresponding vertical
studied are lacking in mice and human because of the logistic and experimental reasons


127
discussed in the last section, the bias usage of certain VH genes reported for these species
(Section 5.1) suggests the outcome might be similar if such studies could be efficiently
performed. The answer to the question may reside in understanding the evolution of the
genes encoding antibody specificity that generated the vast array of Ig variable region genes
first seen in more primitive vertebrates and the later evolution of somatic events that would
appear to have made the original polygeny unnecessary.
6. The value of gene duplication to adaptive immunity
6.1 Adaptive versus innate immunity
The innate immune system is the first responder element of immune protection for higher
vertebrates and may be the sole system for most invertebrate. Innate immunity works
through phagocytic and epithelial cells that bear so-called innate immune receptors, e.g.
Toll-like (TLR) that recognize bacterial and viral entities that are not produced by eucaryotic
cells of the host and are therefore considered foreign and dangerous. For vertebrates these
include lipopolysaccharide, flagellin, bacterial DNA (non-methylated) and double-strained
RNA. These are referred to as PAMPs (Pathogen Associated Molecular Patterns). Products
liberated from cells stimulated when their innate receptors recognize these PAMPs, then
stimulate lymphocytes that leads to development of the adaptive immune system. This is
demonstrated in studies using the isolator piglet model in which colonization or purified
PAMPs are required for an adaptive immune response (Butler et al., 2002; 2005) and by
infection with influenza virus which generates double-stranded RNA during replication
(Butler, Lager, Vincent, Sun unpublished).
Unlike antibodies, the ligand binding sites of innate immune receptors do not change their
specificity by any somatic process when they encounter a PAMP. The capacity for receptor
modification after antigen encounter is the property of the adaptive immune system, as
implied by the name. This adaptive capacity is illustrated in Figure 9 by showing there is a
profound increase in Ig secretion and a shift in isotype usage (Fig. 9A) and an increase in
SHM of the adaptive immune system antigen receptors after antigen exposure (Fig. 9B). As
illustrated, these somatic hypermutations accumulate in those regions of the VH genes that
encode the antibody binding site, i.e. the CDRs (Fig. 9C; Fig. 1A).
6.2 Are multiple V
H
genes required for host immune protection?
Since swine use a very small number of VH genes to generate a VDJ repertoire capable of
protecting the host at all ages (Sun et al., 1998; Butler et al., 2006), we did a statistical
itineration to suggest that >95% of the adaptive VDJ repertoire was the result of diversity
within HCDR3 (Butler et al., 2000). HCDR3 is not encoded by any particular V-region
gene segment but rather by the recombination of VH-DH-JH (Fig. 7 & 8). Joining of VH-
DH-JH involves exonuclease removal of nucleotides from the gene segments involved as
well as nucleotide additions using deoxynucleotide transferase. The types of variations
generated are illustrated in Fig. 8 which shows different HCDR3 sequences found among
the recombinants that use just one VH, DH

and JH segment. The importance of the
diversity generated in HCDR3 was empirically shown by Xu and Davis (2000) to be
sufficient to allow most adaptive immune response using a transgenic mouse given only
on VH gene but an intact DH and JH genome. Studies in rabbit also show that a single VH
gene is primarily used in the establishment of the antibody repertoire (Knight 1992) but
following antigen encounter this VH gene can be modified by SHM as well as by SGC

Gene Duplication

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(Winstead et al., 1999; Schiaffella et al., 1999). We show that in swine seven VH genes can
account for 93% of the entire pre-repertoire and that the two duplicated VHA genes
(which have idetntical CDR1 and CDR regions; Fig. 4) can alone account for 30-50% (Fig.
10). Collectively these studies raise the question as to why the VH repertoire has been so
heavily duplicated while so few of these duplicons are used. The answer may be found
among bats, or at least in the insectivorous microbats. Myotis lucifugus has >250 VH3
family genes (Fig. 5) and probably 350 total VH genes, including all families (Bratsch et
al., 2011). However, SHM occurs at the frequency seen in fetal piglets (Fig. 9B). Perhaps
some older mammalian orders like the Chiroptera, rely more heavily on VH polygeny
than somatic modification.
6.3 How does duplication/ diversification in the C-region effect protective immunity
The survival value of C-region polygeny can be appreciated because antibody isotypes
encoded by the exons for IgM, IgG etc have distinct biological functions (Janeway et al.,
2005). Additional duplication of C and Co genes has generated modified duplicons
encoding Ig subclasses also with different important biological functions. In the case of IgG
subclasses, these involve features like the ability to be recognized by different Fc receptors
on phagocytic cells, transport across epithelial barriers, serum half-life differences involving
FcRn and an association with antibodies of certain specificities. Duplication of C genes in
cattle (and other domestic ruminants) has lead to subclass IgG1 that is selectively
transported by acinar epithelial cells of the mammary gland to provide essential antibodies
for the survival of the newborn (Butler 1974; 1998). Neither IgG2 nor IgG3 function in this
capacity. While there have been no functional studies on the many IgG subclass antibodies
in horse or swine, it is possible that each of these subclass antibodies are best suited for
particular activities in the same manner as described for human IgG subclasses.
The duplicated human Co genes also differ in a number of features. Most striking is the
susceptibility of IgA1 to IgA proteases produced by many common Gram positive bacteria
while IgA2 is resistance due to the lack of the 13 amino acid hinge which is the substrate for
these proteases in IgA1 (Plaut et al., 1974). Differential tissue expression of rabbit IgA
subclasses also suggests a division of labor ( Spieker-Polet et al., 1993). IgA in swine lack the
long hinge of human IgA1 and is therefore not susceptible to the classical bacterial IgA1
proteases although a protease from H. suis can cleave the porcine o-chain (M. Mullins, K.
Register, D.O. Bayles, J.E. Butler, unpub).
The experiment of nature is whether mammals with a deficiency in their C sublocus are
immunologically impaired. There are no known ruminates that like lack ruminat IgG1, so
there are no data; perhaps such a deficiency would be a developmental lethal. The
mammals best-studied for C subclass deficiencies are humans. For example, humans
lacking IgG2 have a deficiency in their response to bacterial polysaccharide antigens
(Hammarstrom et al., 1986). Additional deletions of C genes have been described,
including individuals lacking C1, C2, C4 and Co1 (the 5duplicon shown in Figure 2B).
However, such individuals remain healthy and asymptomatic (Lefranc et al., 1983b;
Notoaramgelo et al., 2009). Selective IgG1 deficiciency which accounts for the major portion
of serum IgG, is not correlated with lower serum igG levels (Olsson et al., 1993) while some
appear to be (Rabbani et al., 1995). In swine, the IgA
b
allotypic variant lacks a major portion
of its hinge (Brown et al., 1995), yet this defect has not been correlated with any risk of
disease; (Navarro et al., 2000).


129
6.4 Polygeny is widespread in other loci important to immunity
There are various examples of polygeny in the immune system but in these loci SHM is not
important in repertoire formation. The loci encoding the various T cell receptors are similar
to those encoding the Igs. Recombination of gene segments occurs in the same manner as for
Ig genes, i.e. recombination requires RAGs, DNA excision, and splicing and DNA repair
enzymes(see Fig. 7). However there is no convincing evidence for SHM after segment
recombination so polygeny in the TCR is theoretically more important in repertoire
generation than in the Ig loci where SHM can further diversify the repertoire.
The genes encoding the major histocompatibility genes (MHC) are partially encoded by
IGSF genes and determine: (a) individualism and self recognition as demonstrated in
tissue typing and (b) recognition of peptides generated and presented by antigen-presenting
cells. The MHC gene system is highly conserved among mammals with three genes
encoding MHC I and 6-8 encoding MHC II. The enormous diversity of MHC is not achieved
by polygeny or somatic processes but rather by an enormous degree of polymorphism of the
MHC genes within the population (Janeway et al., 2005). There can be as many as 300 allelic
variants of any one MHC gene.
The selective advantage of polygeny for genes encoding the MHC, innate immune receptors,
the CH and C subclasses and even the TCR is easy to appreciate. However, such polygeny
in loci encoding the variable Ig genes in higher vertebrates is more difficult to explain if
these species can generate an effective antibody response using a single VH gene (Section
6.2). This creates an enigma for VH and VL polygeny that we shall attempt to explain from
an evolutionary perspective.
7. Conclusions: Ig polygeny and redundancy in higher vertebrates is an
evolutionary vestige
7.1 The case of mammalian IgD
Vestiges of genes are not unusual and mammalian IgD is an example. IgD was discovered as
a myeloma protein nearly 50 years ago and its function has remained an enigma since that
time (Rowe & Fahey, 1965). The considerable research funding invested to determine the
function of IgD has largely generated only hypotheses. IgD is the least homologous isotype
among mammals, e.g. <40%,(in part the reason it was overlooked in some mammals; Butler
et al., 1996) whereas most other isotypes share 70-90% homology (Butler, 2006). IgD is even
missing from the genome of some mammals (Table 2; Fig. 2B) and perhaps all birds. In mice
and humans, IgD and IgM occur as dual B cell receptors but IgD-deficient mice have normal
immune responses (Nitschke et al., 1993) although IgD can compensate for the loss of
functional IgM (Lutz et al., 1998). While numerous studies have attempted to define a
unique role for mammalian IgD, most of these have not been very convincing (Monroe et al.,
1983; Liu et al., 1996; Roes et al., 1993; Vitetta et al., 1977).
Comparative immunologists have put the role of IgD into perspective beginning with the
observation in catfish of a seven domain Ig with distant homology to mammalian IgD
(Wilson et al., 1997; Bengten et al., 2002). This was followed by the discovery of a similar
multi-domain IgD in Xenopus (Zhao et al., 2006) and in other teleosts (Hordvik et al., 1999;
Srisapoome et al., 2004; Stenvik & Jorgensen, 2000). IgD has subsequently been found in the
genome of many other lower vertebrates and in protherian mammals (reviewed by Edholm
et al., 2010). Collectively these studies would morph into the realization that IgD and IgM
are the primordial vertebrates Ig isotypes (Ohta & Flajnik, 2006; Bengten et al., 2006).

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Interesting among the studies in catfish and swine, is that IgD can be produced as a chimeric
Ig using the C1 domain of IgM and the various domain exons of Co (Zhao et al., 2002;
2003). Thus, the expression of IgD in mice and humans by RNA splicing rather than classical
CSR (Maki et al., 1981) has a primitive history.
Compared to the IgD of fish and Xenopus, the exons encoding for mammalian IgD appear to
be relics. Depending on the mammal selected, the number of hinge and domain exons is
highly variable (Butler et al., 2010). While switch regions are typically >3kb in length,
mammalian So (when present) exists as a short remnant of <0.5 kb (Zhao et al., 2003).
Nevertheless it appears to function in some cases in CSR in humans and swine (Zhao et al.,
2002; 2003; Arpin et al., 1998; Koelsch et al., 2007; Zheng et al., 2004).
Recent findings show that basophils have abundant membrane IgD but not T cells, NK cells,
dendritic cells or monocytes (Chen et al., 2009; Dawichi & Marshall, 2007) although this
observation was not supported by the recent report by Karasuyami et al., (2009). Assuming
the case for adventious IgD on basophils is true, it agrees with studies in catfish showing
surface IgD on granulocytes (Edholm et al., 2010). These observations would support a
unique role of this ancestral Ig. In spite of the observation of IgD on human basophils, the
experiment of nature that IgD deficiency does not impair mammalian adaptive immunity
(Nitschke et al., 1993) has lead us to conclude that IgD is an evolutionary relic for mammals
but persists because of its redundant value including its role as a BCR and its presence as an
adventious Ig on basophils (Lutz et al., 1998; Chen et al., 2009). Thus, the relic remains
because there has been no negative selection to completely remove IgD from most
mammalian genomes.
7.2 CH duplication/diversification provides antibodies with specialized effector
function but also redundancy
Isotype diversity in sharks and bony fish is limited to IgM and IgD (IgW in sharks). In
tetrapods the CH repertoire diversified; 3 in birds and reptiles, 5-6 in amphibians and 5 in
mammals (Table 2). In mammals this includes subclasses of IgG and IgA. Each major
isotypes in mammals, perhaps with the exception of IgD, has some specialized effector
function (Janeway et al., 2005). Perhaps the terrestrial environment offered a new challenge
to survival and with the addition of homeothermia, the need for a more specialized adaptive
immune system that could respond more quickly and lead to the evolution of GCs (Sections
3.2; 6.3). As discussed in Section 3-2, there is also evidence that the polygeny of C genes
resulted from a combination of gene duplication and genomic gene conversion. This
duplication event was restricted to mammals that appeared in the last minute before
12PM on the evolutionary clock, appearing after mammalian speciation (Butler et al.,
2009a). The subclass duplication/diversification in mammals resembles the pattern that
produced V region polygeny. In sharks and bony fishes, isotype diversity is limited to IgM
and IgD, while in higher vertebrates duplication/ diversification extends downstream into
the CH sublocus, and in mammals, especially to the C sublocus. However, IgG subclass
deficiencies are not lethal defects, suggesting that even in late-evolving sites of Ig gene
duplication, such duplication seem unnecessary.
7.3 Somatic mechanisms render much of the polygeny in VH, V and Vk loci to relics
As we show in the piglet model, very few VH genes are needed and after antigen encounter,
further repertoire diversification is by SHM of the same genes (Fig. 10). Furthermore, the


131
fact that >90% of the repertoire is generated by junctional diversity in HCDR3 (Section 6.2;
Fig. 8) and that a transgenic mouse with only one VH is fully immune competent,
strengthens the argument. Studies in rabbit also support this view in which one VH gene
account for 90% of the early repertoire which can be diversified SHM and SGC after antigen
encounter (Winstead et al., 1999; Schiaffella et al., 1999). Detractors from this view may
argue that while few (or only one) VH gene are needed, polygeny in the DH and J
H
regions
is still necessary (Table 1). Again studies in swine counter this argument since they have
only two functional DH segments and one J
H
(Butler et al., 1996; Eguchi-Ogawa et al., 2010).
In addition, the chicken has only a single JH, DH segments that are nearly identical and only
one functional VH and one function V gene (Fig.3). This species uses the relics of
upstream pseudogenes for use in repertoire diversification by SGC (Reynaud et al., 1987;
Ratcliffe 2006), a mechanism also available to rabbits (Becker & Knight, 1990). Thus we
propose that the evolution of gene segment recombination and SHM rendered polygeny in
the VH and VL loci of higher vertebrates unnecessary. We believe this polygeny is derived
from the tandem array of V-D-J-C clusters in sharks that do not require somatic
recombination (Fig. 3). Sharks lack germinal centers as do bony fished and amphibians
which makes SHM a less efficient process (DuPasquir et al., 2000). While SHM had been
described in these vertebrate classes, we cannot cite head-to-head studies on the frequency
of SHM like that presented in Fig. 9B.
We believes that like IgD, the VH, V and Vk polygeny of the most evolved mammals and
birds, remain in the genome largely as relics and because of the lack of negative selection.
Early vertebrates have as many as four light chain loci but with evolution the number
diminished and birds have lost all but lambda. That different Ig isotypes and certain IgG
subclass have specific biological functions suggests a selective advantage for polygeny
which we believe was true at the dawn of VH and VL duplicatation that lead to the
polygeny in V-region loci. The alternative explanation for especially VH and VL polygeny is
that this polygeny occurs in a hot spot of RAG-dependent gene segment recombination.
For such recombination events to occur, opening of the chromatin structure to provide
access to nuclear enzymes, is considered necessary. Perhaps such exposure to repeated
recombination activity explains the instability of the locus (Lefranc et al 1983a; Matsuda et al
1990) which renders genes in the locus vulnerable to the molecular machinery involved in
gene duplication and genomic gene conversion.
Mainstream immunology has invested almost entirely in studies of mouse and human
immune systems and therefore seems to have missed the evolutionary significance of Ig
polygeny. To avoid a similar criticism our analysis of polygeny reviewed the major elements
of the Ig genes of all vertebrates that have been seriously studied and the mechanisms they
use to generate their antibody repertoire. From these comparisons we have offered a
hypothesis to explain the polygeny of the major Ig loci in mammals and the reason why at
the highest levels of vertebrate development, this polygeny appears to be an evolutionary
vestige.
8. Conclusions
Gene duplication is a common feature of eucaryotic genomes although the degree varies
among gene families and loci. It is estimated that ~5% of the human genome is comprised of
duplicated genes (Lewin, 2004). Among these are genes of the immunoglobulin supergene
family (IGSF). This polygeny is widespread in loci important to the immune system as well as

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132
encoding proteins with only indirect roles in immunity. The |-barrel encoded by genes of the
IGSF has obviously been a successful structural motif which can explain its conservation
during evolution and diversification into many variants. While IGSF polygeny is widespread,
the degree of polygeny is especially pronounced among those that encode the variable heavy
and light chain genes of antibodies and the T cell receptor (TCR). Early estimates suggested
there were as many as 1000 variable heavy (VH) genes in mice and hundreds in humans.
While subsequently studies, including genome projects, have lowered the number of VH
genes to ~100-150 in these species, this is still a very large number of homologous genes to
occupy a single locus. Similar duplication is seen among genes encoding the variable light
chain genes, i.e. V and Vk. However, there are large variations in the numbers and features of
duplicated VH and VL genes among mammals and other vertebrates.
This article surveys the duplicated Ig genes in a number of species and uses examples
indicating that Ig polygeny resulted from a combination of duplication and genomic gene
conversion. Since understanding the evolutionary forces at work in this process requires
some understanding of the role played by these duplicated genes in humoral immunity, we
review the processes involved in the generation of the antibody repertoire such as Ig gene
segment recombination, junctional diversity, somatic hypermutation (SHM) and somatic
gene conversion (SGC). We review these processes in various vertebrates but focus on data
obtained using the neonatal and newborn piglet model to suggest that evolutionary
improvements in somatic processes have reduced the need for the Ig polygeny that evolved
among lower vertebrates. We also describe the more recent duplication of the C genes of
mammals that indicates the process was similar. C genes encode the subclasses of
mammalian IgG, the flagship mammalian antibody that is unique to this vertebrate class.
Since this duplication event occurred more recently, we thought it could provide insight
into the advantages conferred by gene duplication.
We propose that the extensive polygeny of VH, V and Vk genes among vertebrates gave
adaptive advantage to the earliest vertebrates for generating a diverse repertoire of antibody
specificities much as the more recent evolutionary diversification of C genes resulted in
IgG subclass antibodies with different effector functions. We suggest that the evolutionary
appearance of mechanisms to somatically alter V-region genes reduced the importance of
polygeny in V-region loci for certain mammals and birds. In higher mammals these
mechanisms make it possible for a complete functional repertoire to be generated using just
one or a few VH genes. This hypothesis can explain why so many of the V-region genes of
higher mammals are seldom used, and why deletions of VH genes and C genes have no
effect. We propose that these genes remain as evolutionary vestiges or redundant back-ups
in the genome in a manner that parallels the retention of IgD in most mammals. An
alternative hypothesis is that the extensive somatic recombination which characterizes the
variable region loci (Section 4) creates instability that promotes duplication and genomic
gene conversion. In any case, these hypotheses challenge the existing paradigm that random
VH, DH and JH recombination among the many gene segments is necessary for survival
(presented in immunology textbooks) by placing Ig polygeny into evolutionary perspective.
9. Acknowledgement
The authors acknowledge the Molecular Cell Biology Program of the National Science
Foundation (USA) and Biological Mimetics of Fredrick, Md for their support of the studies
described.


133
10. References
Amit., A.G., R. A. Mariuzza, S. E. Phillips, & R. J. Pliak. (1986). Three dimensional structure
of an antigen-antibody complex at 2.8 A resolution. Science 233 pp. 747-753.
Arakawa, H., Furusawa S., Ekino, S., & Yamaguchi, H. (1996). Immunoglobulin gene
hyperconversion ongoing in chicken splenic germinal centers. EMBO J. 15 pp. 2540-
2546.
Arpin, C., de Bouteillier, O., Razanajaona, D., Briere, F., Banchereau, J., Lecque, S. & Liu, Y. J.
(1997). Human peripheral B- cell development. sIgM-IgD + CD38+ hypermutatied
germinal center centroblasts preferentially express lambda light chains and have
undergone mu-to-delta switch. Ann. N.Y. Acad. Sci. 815 pp. 193-198.
Becker, R.S. & Knight, K.L. (1990). Somatic diversification of immunoglobulin heavy chain
VDJ genes: evidence for somatic gene conversion in rabbit. Cell 63 pp. 987-997.
Bengten, E., Quiniou, S., Hikima, J., Waldbleser, G., Warr, G.W., Miller, N.W. & Wilson, M.
(2006). Structure of the catfish IGH locus: analysis of the region including the single
functional IGHM gene. Immunogenetics 58 pp. 831-844.
Bengten, E., S.M. Quiniou, T.B. Stuge, T. Katagirai, N.W. Miller, L.W. Clem, G.W. Warr and
M. Wilson. (2002). The IgH locus of the channel cafish, Ictalurus punctatus,contains
multiple constant region gene sequences: different genes encode heavy chains of
membrane and secreted IgD, J. Immunol. 169: 2488-2497.
Blumbach, B., Diehl-Seifer, B., Seack, J., Steffen,R., Muller, I.M. & Muller, W.E.G. (1999).
Cloning and expression of new receptors belonging to the immunoglobulin
superfamily from the marine sponge Geodia cydonium. Immunogenetics 49 pp. 751-
763.
Boffey, J., Nicholl, D., Wagner, E.R., Townson, K., Goodyear, C., Furukawa, K., Furukawa,
K., Conner, J. & Willison, H.J. (2004). Innate murine B cells produce anti-disialosyl
antibodies reactive with Camylobacter jejuni LPS and gangliosides that are
polyreactive and encoded by a restricted set of unmutated V genes. J.
Neuroimmunol. 152 pp. 98-111.
Bratsch, S., Wertz, N., Chaloner, K., Kunz, T. H. & Butler, J. E. (2011). The little brown bat
displays a highly diverse V
H
, D
H
and J
H
repertoire but little evidence of somatic
hypermutation. Develop. Comp. Immunol. 35 pp. 421-430.
Brezinschek, H-P, Foster, S. J., Brezinschek, R. I., Doerner, T., Domiati-Saad, R. & Lipsky P.
E. (1997). Analysis of the human VH gene repertoire. J. Clin. Invest. 99 pp. 2488-
2501.
Brown, W. R., Kacskovics, I., Amendt, B. Shinde, R., Blackmore, N., Rothschild, M. & Butler,
J.E. (1995). The hinge deletion variant of porcine IgA results from a mutation at
the splice acceptor site in the first Co intron. J. Immunol. 154 pp. 3836-3842.
Butler, J. E. (1998). Immunoglobulins and immune cells in animal milks. In: Mucosal
Immunology, Ogra, P. L., Mestecky, J., Lamm, M. E., Strober, W., McGhee, J. R.&
Bienenstock, J. (Eds.), , Chapter 98. Academic Press, New York pp. 1531-1554.
Butler, J.E., Santiago-K. Mateo, K., Sun, X-Z, Wertz, N., Sinkora, M., Harvey, R. & Francis,
D.L. (2011a). Antibody repertoire development in fetal and neonatal piglets. XX.
The ileal Peyers patches are not a site of B cell lymphogenesis and are not required
for systemic B cell proliferation and Ig synthesis.

Gene Duplication

134
Butler, J. E., (1974). Immunoglobulins of the mammary secretions. In: Lactation, a
Comprehensive Treatise, (Larson, B. L. & Smith, V., eds.), Vol. III, Chapter V, pp. 217-
255. Academic Press, New York.
Butler, J. E., & Kehrle Jr., M. E. (2005). Immunocytes and immunoglobulins in milk. In:
Mucosal Immunology, (J. Mestecky, M. E. Lamm, W. Strober, J. R. McGhee, L. Mayer
and J. Bienenstock, eds.), 3rd Edition, Academic Press, NY. pp. 1763-1793.
Butler, J. E., Francis, D., Freeling, J., Weber, P., Sun, J. & Krieg, A. M. (2005). Antibody
repertoire development in fetal and neonatal piglets. IX. Three PAMPs act
synergistically to allow germfree piglets to respond to TI-2 and TD antigens. J.
Immunol. 175 pp. 6772-6785.
Butler, J. E., Sun, J. & Navarro, P. (1996). The swine immunoglobulin heavy chain locus has a
single J
H
and no identifiable IgD. International Immunology 8 pp. 1897-1904.
Butler, J. E., Weber, P., Sinkora, M., Baker, D., Schoenherr, A., Mayer, B. & Francis, D.
(2002). Antibody repertoire development in fetal and neonatal piglets. VIII.
Colonization is required for newborn piglets to make serum antibodies to T-
dependent and type 2 T-independent antigens. J. Immunol. 169 pp. 6822-6830.
Butler, J. E., Weber, P., Sinkora, M., Sun, J., Ford, S. J. & Christenson, R. (2000). Antibody
repertoire development in fetal and neonatal piglets. II. Characterization of heavy
chain CDR3 diversity in the developing fetus. J. Immunol. 165 pp. 6999-7011.
Butler, J. E. & Sinkora, M. (2007). The isolator piglet: A model for studying the development
of adaptive immunity. Immunol. Res. 39 pp. 33-51.
Butler, J. E., Sun, X-Z , Wertz, N., Lager, K. M., Urban Jr., J. , Nara, P. & Tobin, G. (2011b).
Antibody repertoire development in fetal and neonatal piglets. XXI. VH usage
remains constant during development in fetal piglets and postnatally in pigs
exposed to environmental antigen.
Butler, J. E., Zhao, Y., Sinkora, M., Wertz, N. & Kacskovics, I. (2009a). Immunoglobulins,
antibody repertoire and B cell development. Devel. Comp. Immunol. 33 pp. 321-333
Butler, J. E., Lager, K. M., Splichal, I., Francis, D., Kacskovics, I., Sinkora, M., Wertz, N., Sun,
J., Zhao, Y., Brown, W. R., DeWald, R., Dierks, S., Muyldermanns, S., Lunney, J.K.,
McCray, P. B., Rogers, C. S., Welsh, M. J., Navarro, P., Klobasa, F., Habe, F. &
Ramsoondar, J. (2009b).

The Piglet as a Model for B cell and Immune System
Development. Vet. Immunol. Immunopath. 128 pp. 147-170.
Butler, J. E., Wertz, N., Zhao, Y., Kunz, T. H., Bratsch, S., Whitaker, J. & Schountz, T. (2010).
Two suborders of bats have the canonical isotypes repertoire of other eutherian
mammals. Devel. Com. Immunol. 35 pp. 272-284.
Butler, J. E., Weber, P. & Wertz, N. (2006). Antibody repertoire development in fetal and
neonatal pigs. XIII. Hybrid VH genes and the pre-immune repertoire revisited. J.
Immunol.177 pp. 5459-5470.
Cerato, E., Birkle, S., Portoukalian, J., Mezazigh, A., Chatal, J.F. & Aubry, J. (1997). Variable
region gene segments of nine monoclonal antibodies specific to disialgangliosides
(GD2, GD3) and their O-acetylated derivatives. Hybridoma 16 pp. 307-316.
Chen, K., Xu, W., Wilson, M., He, B., Miller, N. W., Begnten, E., Edholm, E-S, Santini, P.A.,
Rath, R., Chiu, A., Cattalinei, M., Litzman, J., Busseel, J., Huang, B., Meini, A.,
Riesbaeck, K., Cunningham-Rudles, C., Plebani, A. & Cerutti, A. (2009).
Immunoglobulin D enhances immune surveillance by activating antimicrobioal,
proinflammatory and B cell-stimulating programs in basophils. Nat. Immunol. 10
pp. 889-898.


135
Currier, S. J., Gallara, J. L. & Knight K. L. (1998). Partial genetic map of the rabbit VH
chromosomal region. J. Immunol. 140 pp. 1651-1659.
Davies, J. & Riechmannn, L. (1995). Antibody VH domains as small recognitions units.
Biotechnol. 13 pp. 475-479.
Dawichki, W. & Marshall, J.S. (2007). New and emerging roles for mast cells in host defense.
Curr. Opin. Immunol. 19 pp. 31-18.
De Genst, E., Saerens,D., Muyldermans, S. & Conrath, K. (2006). Antibody repertoire
development in camelids. Dev. Comp. Immunol. 30 pp. 187-198.
deWildt, R. M., Hoet, R. M., van Venrooig, W. J., Tomlinson, I. M. & Winter, G. (1999).
Analysis of heavy and light chain pairing indicates that receptor editing shapes the
human antibody repertoire. J. Mol. Bio. 285 pp. 895-901.
Diaz, M., Stanfield, R.L., Greenberg, S. & Flajnik, M. F. (2002). Structural analysis, selection
and ontogeny of the shark new antigen receptor (IgNAR): identification of a new
locus preferentially expressed in early development. Immunogenetics 54: 501-512.
Dildrop, R, Krawinkel, U., Winter, E. & Rajewsky, K. (1985). VH gene expression in murine
lipopolysaccharide blasts distribute over the nine known VH-groups and may be
random. Eur. J. Immunology 15 pp. 1154-1156.
Dooley, H., Flajnik, M.F. & Porter, J. (2003). Selection and characterization of naturally
occurring single domain (IgNAR) antibody fragments from immunized sharks by
phage display. Mol. Immunol. 40 pp. 25-33.
Dooley, H. & Flajnik, M.F. (2006). Antibody repertoire development in cartilagenous fish.
Devel. Comp. Immunol. 30 pp. 43-56.
Du Pasquier, L., Robert, J., Courtet, M., & Mussmann, R. (2000). B cell development in the
amphibian Xenopus. Immunol. Rev. 175 pp. 201-213.
Edholm, E-S, Bengton, E., Staffor, J. L., Sahoo, M., Taylor, E. R. , Miller, N. W. & Wilson, M.
(2010). Identification of two IgD+ B cell populations in channel catfish, Ictalurus
punctatus. J. Immunol. 185 pp. 4082-4094.
Eguchi-Ogawa, T, Sun, X-Z., Wertz, N., Uenishi, H., Puimi, F., Chardon, P., Wells, K., Tobin,
G. J. & Butler, J. E. (2010). Antibody repertoire development in fetal and neonatal
piglets. XI. The relationship of VDJ usage and the genomic organization of the
variable heavy chain locus. J. Immunol. 184 pp. 3734-3742.
Flanagan, J.G. & Rabbitts, T. H. (1982) Arrangement of human immunoglobulin heavy
chain constant region genes implies evolutionary duplication of a segment
containing , c, and o genes. Nature 300 pp. 709-713.
Foster, S. J., Brezinschek, H. P., Brezinschek, R. I. & Lipsky, P. E. (1997). Molecular
mechansism and selective influences that shape the kappa gene repertoire of IgM+
B cells. J. Clin. Invest. 99 pp. 1614-1627.
Gay, D., Saunders, T., Camper, S. & Weigert, M. (1993). Receptor editing: an approach by
autoreactive B cells to escape tolerance. J. Exp. Med. 177 pp. 999-1008.
Glas, A. M., van Monfort, E. H. N. & Milner, E. C. B. (2000). The human antibody repertoire:
Old notions, current realities and VH gene-dependent biases. In: The antibodies,
(Zanetti, M. &Capra, J.D., eds). Vol 6 , pp 63-79. Harwood Academic Publishers ,
Amsterdam.
Gu, H., Tarlinton, D., Muller, W., Rajewsky, K. & Forster, I. (1991). Most peripheral B cells in
mice are ligand restricted. J. Exp. Med. 173 pp. 1357-1371

Gene Duplication

136
Hamers-Casterman, C., Atarhouch, T., Muylermans, S., Robinson, G., Hamers, C., Songa, E.
B., Bendahman, N. & Hammers, R. (1993). Naturally occurring antibodies devoid of
light chains. Nature 363 pp. 446-448.
Hammarstrom L., Lefranc G., Lefranc M-P, Persson, M.A.A. & Smith, C.I.E. (1986). Monogr.
Allergy 20 pp. 18-25.
Herrin, B. R. & Cooper, M. D. (2010). Alternative adaptive immunity in jawless vertebrates.
J. Immunol. 185 pp. 1367-1374.
Hordvik, I., Thevarajan, J., Sandal, I., Bastani, N. & Krossoy, B. (1999). Molecular cloning
and phylogenetics analysis of the Atlantic salmon immunoglobulin D gene. Scand.
J. Immunol. 50 pp. 202-210.
Ichiyoshi, Y. & Casali, P. (1994). Analysis of the structural correlates for antibody
polyreactivity by multiple reassortments of chimeric human immunoglobulin
heavy and light chain V segments J. Exp. Med. 180 pp. 885-895.
Janeway, C.A., Travers, P., Walport, M., & Shlomchik, M.J. (2005). Immunobiology, 6
th

Edition, Garland Science, New York.
Janssens, R., Dekker, S., Hendriks, R. W., Panayotou, G., van Remoortere, A., San, J. K.,
Groveld, F. & Drabek, D. (2006). Generation of heavy-chain-only antibodies in
mice. Proc. Natl. Acad. Sci. USA 103 pp. 15130-15135.
Johnston, C. M., Wood, A. L., Bolland, D. J. & Corcoran, A. E. (2006). Complete sequence
assembly and characterization of the C57BL/6 mouse heavy chain V region. J.
Immunol. 176 pp. 4221-4234.
Karasuyama, H., Mukai, K., Tsujimura, Y., & Obata, K. (2009). Newly discovered roles for
basophils : a neglected minority gains new respect. Nature Rev. Immunol. 9 pp. 9-13.
Keyeux, G, Lefranc, G. & Lefranc, M. P. (1989). A multigene deletion in the human IGH
constant region locus involves highly homologous hot spots of recombination.
Genomics 5 pp. 432-441.
Klobasa, F., Werhahn, E., & Butler, J.E. (1981). Regulation of humoral immunity in the
piglet by immunoglobulins of maternal origin. Res. Vet. Sci. 31 pp. 195-206.
Knight, K. L. (1992). Restricted VH usage and generation of antibody diversity in rabbit.
Ann. Rev. Immunol. 10 pp. 593-616.
Koelsch, K., Zheng, N. Y., Zhang, Q., Duty, A., Helms, C., Mathias, M. D., Jared, M., Smith,
K., Capra, J. D., & Wilson, P. C. (2007). Mature B cells class switch to IgD are
autoreactive in healthy individuals. J. Clin Invest. 117 pp. 1558-1565.
Kranz, D. M. & Voss, Jr., E. W. (1981). Restricted reassociation of heavy and light chains
from hapten-specific monoclonal antibodies. Proc. Natl Acad. Sci. 78 pp. 5807-5811.
Lavoie, T. B., Mohan, S., Lipschults, C. A., Grivel, J-C., Li, Y. l., Mainhart, C. R., Kam-
Morgan, L. N. W., Drohan, W. N. & Smith-Gill, S. J. (1999). Structural differences
among monoclonal antibodies with distinct fine specificities and kinetic properties.
Mol. Immunol. 36 pp. 1189-1205.
Lefranc, M. P., Lefranc, G., de Lange, G., Out ,T. A., van den Broek, P. J., van Nieuwkoop, J.,
Radl, J., Hela, A. N., Chaabani, H., van Loghem, E. & Rabbitts, T. H. (1983a)
Instability of the human immunoglobulin heavy chain constant region locus
indicated by different inherited chromosomal deletions. Mol. Biol Med. 1 pp. 207-
217.
Lefranc, G., Chaabani, H., van Loghem, E., Lefranc, M. P., de Lange, G. & Helal, A. N.
(1983b). Simultaneous absence of the human IgG1, IgG2, IgG4 and IgA1


137
subclasses: immunological and immunogenetical considerations. Eur. J. Immunol 13
pp. 240-244.
Lefranc, M. P., Lefranc, G. & Rabbitts, T. H. (1982). Inherited deletion of immunoglobulin
heavy chain constant region genes in normal individuals. Nature 300 pp. 760-762.
Lewin, B., (2004). Genes VIII. Pearson-Prentice Hall, Upper Saddle River, N.J. p. 87.
Liu, Y.J., de Bouteiller, O., Arpin, C., Briere, F., Gailbert, L., Ho, S. , Martinez-Valdez, H.,
Banchereau, J. & Lebecque, S. (1990). Normal IgD+IgM- germinal center B cells can
express up to 80 mutations in the variable region of their IgD transcripts. Immunity
4 pp. 603-613.
Lutz, C., Ledermann, B., Kosco-Vilbois, M. H., Ochsenbein, A. F., Zingernagel, R. M., Kohler,
G. & Brombacher, F. (1998). IgD can largely substitute for loss of IgM function in B
cells. Nature 393 pp. 797-801.
Mageed, R. A., Marmer, I. J. , Wynn, S. L., Moyes, S. P, Maziak, B. B., Bruggemann, M. &
MacKworth-Young, C. G. (2001). Rearrangement of the human heavy chain
variable region gene V3-23 in transgenic mice generates antibodies reactive with a
range of antigens on the basis of VHCDR3 and residues intrinsic to the heavy chain
variable region. Clin. Exp. Immunol. 123 pp. 1-8.
Maki, R., Roeder W., Trawnecker, A., Sidman, C., Wabl, M., Raschke, W. & Tonegawa, S.
(1981). The role of DNA rearrangement and alternative RNA processing in the
expression of immunoglobulin delta. Cell. 24 pp. 353-365
Malynn, B. A., Berman, J. E., Yancopous, G. D., Bona. C. A. & Alt, F. W. (1987). Expression of
the immunoglobulin heavy-chain variable gene repertoire. Curr. Top. Microbiol.
Immunol. pp. 135: 75-94.
Marchalonis, J. J., Schluter, S. F., Bernstein, R. M. & Edmundson, A. B. (1998). Phylogenetic
emergence and molecular evolution of the immunoglobulin family. Adv. Immunol.
70 pp. 417-506.
Marr, S., Morales, H., Bottaro, A., Cooper, M., Flajnik, M. & Robert, J. (2007). Localization
and differential expression of activation-induced cytidine deaminase in the
amphibian Xenopus upon antigen stimulation and during early development. J.
Immunol. 179 pp. 6783-6789.
Matsuda, F., Shin, E. K., Nagaoka, H., Matsumura, R., Haino, M., Fukita, Y., Taka-ishi, S.,
Imai, T., Riley, J. H., Amand, R., Soeda, E. & Honjo, T. (1993). Structure and
physical map of 64 variable segments in 3 0.8-megabase region of the human
immunoglobulin heavy-chain locus. Nature Genet. 3 pp. 88-94.
Matsuda, F., Ishii, K., Bourvagnet, P., Kuma, K., Hayashida, H., Miyata, T. & Honjo, T.
(1998). The complete nucleotide sequence of the human immunoglobulin heavy
chain region locus J. Expt. Med. 188 pp. 2151-2161.
Matsuda, F., Sin, E. K., Hirabayashi, Y., Nagaoka, H., Yoshida, M. C., Zong, S. Q. & Honjo, T.
(1990). Organization of variable region segments of the human immunoglobulin
heavy chain: duplication of the D5 cluster within the locus and interchromosomal
translocation of variable region segments. EMBO J. 9: pp. 2501-2506.
Meselson, M. S. & Radding, C. M. (1975). A general model for genetic recombination. Proc.
Natl Acad. Sci. USA 72 pp. 358-361.
Migone, N., Oliviero, S., de Lange, G., Delacroix, D. L., Boschis, D., Altruda, F., Silengo, L.,
Demarchi, M., & Carbonaro, A. O. (1984). Multiple gene deletions within the
human heavy-chain cluster. Proc. Natl Acad. Sci. USA 81 pp. 5811-5815.

Gene Duplication

138
Miller, M. A., & Steele, R.E. (2000). Lemon encodes an unusual receptor protein-tyrosine
kinase expressed during gametogenesis in Hydra. Dev. Biol. 224:286-298.
Mo, J.A., & Holmdahl, R. (1996). The B cell response to autologous type II collagen: biased V
gene repertoire with V gene sharing and epitope shift. J. Immunol. 157 pp. 2440-2448.
Monroe, J. G., Havran, W. L., & Cambier, J. C. (1983). B lymphocyte activation entry into
cell cycle is accompanied by decrease expression of IgD but not IgM. Eur. J.
Immunol. 13 pp. 208-213.
Muyldermans, S., Ghassabeh, G.H., & Saerens D. (2009). Single-domain antibodies. In :
Recombinant antibodies for immunotherapy, Little, M. (ed.), Cambridge University
Press. Chapter 16, pp. 216-230.
Navarro, P., Christenson, R. Ekhardt, G, Lunney, J.K., Rothschild,M., Bosworth, B, Lemke, J.
& Butler, J.E. (2000). Genetic differences in the frequency of the hinge variants of
porcine IgA is breed dependent. Vet. Immunol. Immunopath. 73 pp. 287-295.
Nguyen, V., Su, C., Muyldermans, S. van der Loo, W. (2002). Heavy chain antibodies in
Camelidea; a case of evolutionary innovation. Immunogenetics 54 pp. 39-47.
Nitschke, L., Kosco, M. L., Kohler, G., & Lamers, M. C. (1993). Immunoglobulin D deficient
mice can mount normal immune responses to thymus independent and
dependent antigens. Proc. Natl Acad Sci. USA 90 pp.1887-1891.
Notarangelo, L. D., Fischer, A.,Geha, R.S., Casanova, J-L., Chapel, H, Conley, M.E.,
Cunningham-Rundles, C., Etzioni, A., Hammarstrom, L., Nonoyama, S., Ochs,
H.D., Puck, J., Roifman, C., Seger, R., & Wedgwood, J. (2009). Primary
immunodeficiencies: 2009 update. J. Allergy Clin. Immunol. 124: 1161-1178.
Ogawa, K., Wakayama, A., Kunsisada, T., Oril, H., Watanabe, K. & Agata, K. (1998).
Identification of a receptor tyrosine kinase involved in germ cell differentiation in
planarians. Biochem. Biophys Res. Commun. 248 pp. 204-209.
Ohta, Y. & Flajnik, M. (2006). IgD, like IgM , is a primordial immunoglobulin class
perpetuated in most jawed vertebrates. Proc. Natl. Acad. Sci. USA 103 pp. 10723-
10728.
Olsson, P.G., Rabbani, H., Hammarstromn, L. & Smith, C.I.E. (1993). Novel human
immunoglobulin heavy chain constant region gene deletion haplotypes
characterized by pulsed -field electrophoresis. Clin. Exp. Immunol. 94: pp. 84-90.
Padlan, E. A. (1994). Anatomy of the antibody molecule. Mol. Immunol. 31 pp. 169-217.
Plaut, A.G., Wustar, R. Jr. & Capra, J.D. (1974). Differential susceptibility of human IgA
immunoglobulins to streptococal IgA proteases. J. Clin. Invest. 54 pp. 1295-1300.
Rabbani, H., Kondo, N., Smith, C.I., Hammarstrom, L. (1995). The influence of gene deletion
and duplication within the IGHC locus on serum immunoglobulin subclass levels.
Clin. Immunl. Immunopathol. 76:S pp. 214-218.
Ratcliffe, M. J. H. (2006). Antibodies, immunoglobulin genes and the bursa of Fabricius in
chicken. B cell development. Devel. Comp. Immunol. 30 pp. 101-118.
Reiter, Y., Schuck, P., Boyd, L. F. & Plaksin, D. (1999). An antibody single domain phage
display library of a native heavy chain variable region: Isolation of functional
single-domain VH molecules with a unique interface. J. Mol. Bio. 290 pp. 685-698.
Retter, I., Chevillard, C., Scharfe, M., Conrad, A., Hafner, M., Im, T-H , Ludewig, M.,
Nordsied,G., Severitt, S., Thies, S., Mauhar, A., Bloecker, H., Mueller, W. & Riblet,
R. (2007). Sequence and characterization of the Ig heavy chain constant and partial
variable region of the mouse strain 129S1. J. Immunol. 179 pp. 2419-2427.


139
Reynaud, C.A., Anquez, V., Daher,A. & Weill, J. (1987). A hyperconversion mechanism
generates the chichen pre-immune light chain repertoire. Cell. 48 pp. 379-388.
Rodkey, L. S. & Adler, F. L. (1983). Regulation of natural anti- allotypic antibody responses
by network induced auto-anti-idiotypic responsiveness of their offspring. J. Exp.
Med. 152 pp. 1024-1035.
Roes, J. & Rajewsky, K. (1993). Immunoglobulin D (IgD)-deficient mice reveal an auxiliary
receptor function for IgD in antigen-mediated recruitment of B cells. J. Exp. Med.
177 pp. 45-55.
Rowe, D. S. & Fahey, J. L. (1965). A new class of human immunoglobulins. J. Expt. Med. 121
pp. 171-199.
Rumfelt, L. L., McKinney, E. C., Taylor, E. & Flajnik, M. F. 2002). The development of
primary and secondary lymphoid tissues In the nurse shark Ginglymostoma
cirratum. B cell zones precede dendritic cell immigration and T cell cell zones
formation during ontogeny of the spleen. Scand. J. Immunol. 56 pp. 130-148.
Schiaffella, E., Sehgal, D., Anderson, A. O. & Mage,R. G. (1999). Gene conversion and
hypermutation during diversification of VH sequences in developing germinal
centers of immunized rabbits. J. Immunol. 162 pp. 3984-3995.
Schroeder, H. W. Jr., Hillson, H. L. & Perlmutter, R. M. (1987). Early restriction of human
antibody repertoire. Science 238 pp. 791-793.
Schroeder. H.W. Jr, Hillson, H.L. & Perlmutter, R.M. (1990). Structure and evolution of
mammalian VH families. Intl Immunol 2 pp. 41-45.
Sheehan, K. M., Mainville, C. A., Willert, S. & Brodeur, P. H. (1993). The utilization of
individual V
H
exons in the primary repertoire of adult BALB/c mice. J. Immunol.
151 pp. 5363-5375.
Solem, S.T. & Stenvik, J. (2006). Antibody repertoire development in teleosts--a review with
emphasis on salmonids and Gadus morrhua L. Develop. Comp. Immunology 30 pp. 57-
76.
Spieker-Polet, H., Yam, P-C., & Knight K. L. (1993). Differential espression of 13 IgA-heavy
chain genes in rabbit lymphoid tissues. J. Immunol. 150 pp. 5457-5465.
Srisapoome, P., Ohira, T., Hirona, I. & Aoki, T. (2004). Genes of the constant regions of
functional heavy chain of Japanese flounder. Parealichthys olivaceus. Immunogenetics
56 pp. 292-300.
Stenvik, J. & Jorgensen, T. O. (2000). Immunoglobulin D (IgD) of Atlantic cod has a unique
structure. Immunogenetics 51 pp. 452-461.
Sun, J., Hayward, C., Shinde, R., Christenson, R., Ford, S.P. & Butler, J.E. (1998). Antibody
repertoire development in fetal and neonatal piglets. I. Four V
H
genes account for
80% of V
H
usage during 84 days of fetal life. J. Immunol. 161 pp. 5070-5078
Sun, J., Kacskovics, I., Brown, W. R. & Butler, J. E. (1994). Expressed swine V
H
genes belong to
a small V
H
gene family homologous to human V
H
III. J. Immunol. 153 pp. 5618-5627.
Szostak, J. W., Orr-Weaver, T. L. & Rothstein, R. J. (1983). The double-strand break repair
model for recombination. Cell. 33 pp. 25-35.
Thomson, C. A., Little, K. Q., Reason, D. C. &Schrader, J. W. (2011). Somatic diversity in
CDR3 loops allows single V-genes to encode innate immunological memories for
multiple pathogens. J. Immumnol. 186: pp. 2291-2298.
Tiegs, S. L., Russell, D. M. & Nemazee, D. (1993). Receptor editing in self-reactive bone
marrow B cells. J. Exp. Med. 177 pp. 1009-1020.

Gene Duplication

140
Vigliano, F. A., Bemudez, R., Quiroga, M. I. & Nieto, J. M. (2006). Evidence for melamno-
macrophage centers of telosts as evolutionary precursors of germinal centers of
higher vertebrates: An immunohistochemical study. Fish and Shellfish Immunology
21 pp. 467-471.
Vitetta, E. S., Cambier, J. C., Ligler, F. S., Kettman, J. R. & Uhr, J. W. (1977). B cell tolerance.
IV. Differential role of surface IgM and IgD in determining tolerance susceptibility
of murine B cells. J. Exp. Med. 146 pp. 1804-1808.
Wang, H. & Shlomchik., M. J. (1998). Maternal Ig mediates neonatal tolerance in
rheumatoid factor transgenic mice but tolerance breaks down in adult mice. J.
Immunol. 160 pp. 2263-2271.
Wikler, M., Demeur, C., Dewasne, G. & Urbain, J. (1980). Immunoregulatory role of
maternal idiotypes. Ontogeny of immune networks. J. Exp. Med. 152 pp. 1024-1035.
Wilson, M., Bengten, E., Miller, N. W., Chen, L. W., Du Pasquier, L. & Warr, G. W. (1997). A
novel chimeric Ig heavy chain from a teleost fish shares similarities to IgD. Proc.
Natl. Acad. Sci. USA 94 pp. 4593-4597.
Winstead, C. R, Zhai, S. K., Sethupathi, P. & Knight, K. L. (1999). Antigen-induced somatic
diversification of rabbit IgA genes: gene conversion and point mutation. J. Immunol
162 pp. 6602-6612.
Withers, D. R., Davison, T. F. & Young, J. R. (2005). Developmentally programmed
expression of AID in chicken B cells. Devel. Comp. Immunol. 29 pp. 651-662.
Xu, J. L. & Davis., M. M. (2000). Diversity in the CDR3 region of V
H
is sufficient for most
antibody specificities. Immunity 13 pp. 37-45.
Yamaguchi, N., Shimizu, S., Hara, T. & Saito, T. (1983). The effector maternal antigenic
stimulation upon the active immune responsiveness of their offspring. Immunology
50 pp. 229-238.
Yancopoulos, G. D., Desiderio, S. V., Paskind, M., Kearney, J. F., Baltimore, D. V. & Alt, F.
W. (1984). Preferential usage of the most JH proximal VH gene segments in pre-B
cell lines. Nature 311 pp. 717-733.
Yasuda, M., Kajiwara, E., Ekino, S., Taura, Y., Hirota, Y., Horiuchi, H., Matsuda, H.,
Furusawa, S. (2003). Immunobiology of chicken germinal center: I. Changes in
surface Ig class expression in the chicken splenic germinal center after antigenic
stimulation. Develp. Comp. Immunol. 27 pp. 159-166.
Zhao, Y, Kacskovics, I., Pan-Hammarstrom, Q, Liberies, D. A., Geli, J., Davis, S. K., Rabbani,
H. & Hammarstrom, L. (2002). Artiodactyl IgD: the missing link J. Immunol. 169 pp.
4408-4416.
Zhao, Y., Pan-Hammarstrom, Q., Kacskovics, I. & Hammarstrom, L. (2003). The porcine Ig
delta gene: unique chimeric splicing of the first constant region domain in its heavy
chain transcripts. J. Immunol. 171: pp. 1312-1318.
Zhao, Y., Pan-Hammarstrom, Q., Yu, S., Wertz, N., Zhang, X., Li , N., Butler, J.E. &.
Hammarstrom, L. (2006). Identification of IgF, a hinge-region containing Ig class
and IgD in Xenopus tropicalis. Proc. Natl. Acad. Sci (USA) 103 pp. 12087-12092.
Zheng, N. Y., Wilson, K., Wang, X., Boston, A., Kolar, G., Jackson, S. M, Liu, Y. I., Pascual,
V., Capra, J.D. & Wilson, P.C. (2004). Human immunoglobulin selection associated
with class switch and possible tolerogenic origin for C delta class-switched B cells.
J. Clin Invest. 113 pp. 1188-1201.
8
Gene Duplication in Insecticide Resistance
Si Hyeock Lee and Deok Ho Kwon
Department of Agricultural Biotechnology, Seoul National University, Seoul
Republic of Korea
1. Introduction
Gene duplication is a widely observed phenomenon in all three kingdoms of life and is
considered to be a major driving force in the evolution of genomes and organisms. Gene
duplication refers to any duplication event of a region of DNA that contains genes,
eventually giving rise to gene families. In a classical sense, gene duplication is considered to
predate functional diversification. When a duplicated copy is generated, the surplus copy is
released from the selective pressure that is posed by random mutations, which allows the
rapid accumulation of mutations without deleterious consequences to the organism (Zhang,
2003). The accumulated mutations can increase the fitness of the organism or create a novel
function, thereby playing a major role in evolution through functional divergence (Ohno,
1970; Taylor and Raes, 2004). Paralogous gene family members that share a common
ancestor gene are generated from a duplication event, which is distinguished from the
orthologous genes in different genomes that share a common ancestor as a result of a
speciation event (Hurles, 2004). In another theory, instead one copy retains the original
function after gene duplication, both of the two copies become to undergo complementary
functional diversification, allowing the evolution of an organism over generations (Force et
al., 1999). Whole genome duplication events are also common particularly in plant species
having polyploidy genomes. Whole genome duplication has influenced the evolutionary
path in many species.
One example of extensive gene duplication is the gene amplification. Contrary to gene
duplication, which is a doubling mechanism of one gene, gene amplification refers to the
process by which the copy number of a particular gene is specifically increased to a greater
extent compared to those of other genes, resulting in a dramatic increase in gene dosage.
Gene amplification generally results from the repeated replication of a stretch of DNA in a
specific region of a genome. Because gene amplification increases the copy number of a gene
relatively quickly, it is commonly involved in gene expression control during the
development of an organism. Increased copy numbers of a particular gene enables rapid
production of a large amount of protein within a short period.
The most common mechanism of gene duplication is homologous recombination by
unequal crossing-over between short repeated sequences on homologous segments of
chromosomes during meiosis. The replication slippage is also responsible for the
duplication of small contiguous repeats of DNA. The possibility and frequency of gene
duplication depend on the degree of repetitive sequence distribution between two
homologous chromosomes. Detailed information on gene duplication mechanisms can be
found elsewhere in this book.

Gene duplication has been implicated as one of insecticide resistance mechanisms.
Amplification of detoxification genes such as esterase in the Culex mosquito and green
peach aphid, Myzus persicae, is the first example of a gene duplication (amplification) that is
associated with insecticide resistance. These aspects of gene amplification were previously
reviewed by Devonshire and Field (Devonshire and Field, 1991). Although the degree is not
as extensive as the gene amplification, another example of detoxification gene duplication is
found in organophosphate (OP)-resistant sheep blow flies, in which E7 gene is duplicated.
In this case, the duplication of E7 is considered as a way to simultaneously maintain two
different resistance alleles. Tandem duplication of two cytochrome P450 (Cyp450) genes was
found in a pyrethroid-resistant strain of a major malaria vector mosquito species, suggesting
that Cyp450 gene duplication may contribute to pyrethroid resistance by enhancing
monooxygenase activity.
Insecticide target gene duplication is involved in insecticide resistance as well. Duplication
of the Rdl -amino butyric acid (GABA) receptor subunit gene is a possible cause for the
resistance to cyclodiene insecticides in M. persicae. Dual copies of acetylcholinesterase
(AChE) play a role in resistance and adaptation in Culex mosquitoes. AChE duplications
reduce the fitness cost associated with the mutant ace allele in Culex mosquitoes. Recently,
multiple copies of AChE were confirmed to confer resistance to an OP insecticide in the two-
spotted spider mite. Such extensive duplication of AChE provides adaptive advantages in
fitness compensation and resistance. Unlike the duplication of detoxification genes, gene
dosage control may become a more important issue in the duplication of insecticide target
genes because the encoded gene products are directly involved in the transmission of nerve
impulses and the maintenance of nervous system homeostasis. Therefore, adaptive
compensation for target gene duplication is necessary, particularly when the extent of
duplication is severe and linked to a fitness disadvantage.
In this chapter, we reviewed various reported cases of gene duplication involved in
insecticide resistance, and we discussed its roles in the resistance evolution and fitness
compensation.
2. Amplification and duplication of detoxification genes
2.1 Esterase gene in Culex mosquitoes
The overexpression of two types of esterases, coded at two loci, Est-3 (A esterase) and Est-2
(B esterase), increases esterase activity to confer resistance to OP insecticides in Culex
mosquito species (Mouches et al., 1986; Mouches et al., 1990; Guillemaud et al., 1997).
Multiple overexpressed allozymes have been described as follows: six at the B esterase locus
(B1, B2, B4, B5, B6 and B7) and four (A1, A2, A4 and A5) at the A esterase locus (Raymond et
al., 1998). In Culex pipiens quinquefasciatus, an 800-fold OP resistance is caused by ca. 250-fold
amplification of the B1 esterase (Est-2 locus) (Mouches et al., 1986). Sequence analysis and
the characterization of the amplified gene structure has revealed that the amplicon covers at
least 30 kb and contains a highly conserved 25-kb "core" carrying a single copy of the
esterase gene (2.8 kb) (Mouches et al., 1990). The core is enclosed by two repetitive DNA
sequences in other parts of the genome, but not in proximity to the B1 esterase gene,
suggesting that the repetitive sequences have a role in the amplification process (Mouches et
al., 1990). Analysis of the genomic structure of the Est genes in different OP-resistant strains
revealed that two types of genetic alteration mechanisms (transcriptional regulation and
gene amplification) are involved in the development of resistance. Overexpression of one A1

Gene Duplication in Insecticide Resistance 143
esterase reported in the southern French mosquito strain was due to a transcriptional
regulatory mechanism, whereas in other cases, the coamplification of the A and B esterase
loci or an amplification of the B esterase locus alone resulted in overproduction of esterase
(Guillemaud et al., 1997; Raymond et al., 1998). The level of gene amplification varies
depending on different esterase alleles. For example, the copy number of B1 esterase
reached 250 copies (Mouches et al., 1986), whereas that of B4 esterase did not exceed a few
copies (Guillemaud et al., 1997). The level of gene amplification is also different within and
between populations as in the case of the coamplified A2-B2 esterases (Callaghan et al.,
1998). The number of independent amplification events at the loci of A and B esterases
cannot be precisely estimated. Considering the protein quantification profiles and the
molecular data published to date, however, the number of independent amplification events
may range from five to ten events as a minimum figure (Raymond et al., 1998). This
relatively low frequency of independent amplification events on a global scale suggests that
the fitness advantage of esterase gene amplification may be restrictive (Raymond et al.,
1998).
2.2 Esterase gene in the green peach aphid, Muzus persicae, and other aphids
Overexpression of the esterase (E4) responsible for broad insecticide resistance in the green
peach aphid, M. persicae Sulz, was also associated with amplification of the structural E4
gene or its closely related variant (FE4). The extent of amplification was well correlated with
the activity of the esterase and the level of resistance (Field et al., 1988). Molecular studies
revealed that the presence of the amplified E4 gene is correlated with an autosomal 1,3
translocation, whereas amplified FE4 genes are found in insecticide-resistant aphids with
normal untranslocated chromosomes (Field et al., 1988). Subsequent in situ hybridization
assays revealed that a single amplified site is located on autosome 3 near the breakpoint of
the autosomal 1,3 translocation in all of the E4-producing aphid clones except one having
two other E4 loci. In the most resistant aphid clone producing FE4, however, the
amplification sites were widely distributed around the genome (from three to eight sites)
(Blackman et al., 1995; Blackman et al., 1999). The relative esterase gene copy numbers in
aphid clones with different levels of insecticide resistance (R1, R2 and R3) were determined
to increase ca. 4-fold between susceptible, R1, R2 and R3 aphids, reaching a maximum
increase of approximately 80-fold amplification in R3 aphids. This proportionate correlation
between amplification and resistance further suggested that transcriptional upregulation of
amplified genes may not be involved in resistance (Field et al., 1999). The amplified esterase
genes are arranged as tandem repeats at a single locus in some aphid clones, whereas
amplicons are dispersed throughout the genome in other clones. The amplified E4 and FE4
genes are methylated at the CpG repeats within the gene (Hick et al., 1996). However, the
methylation is absent from upstream regions, including the 5 CpG-rich region around the
regulatory region, and from 3 flanking DNA. Contrary to the common belief that
methylation suppresses gene expression, methylated E4 genes are expressed and loss of the
5-methylcytosine is correlated with transcription suppression, suggesting that the
methylation of E4 has a positive role in expression (Field, 2000).
2.3 Esterase gene in the sheep blow fly, Lucilia cuprina
Resistance to diazinon and malathion is primarily due to two point mutations (Gly137Asp
and Trp251Leu) in a carboxylesterase gene (LcE7), encoding both OP hydrolase and
malathion carboxylesterase (MCE) activities (Campbell et al., 1997; Campbell et al., 1998).

The OP hydrolase activity, which is responsible for the resistance to diazinon and the
majority of other OPs, is conferred by Gly137Asp. In contrast, high MCE activity, which is
associated with resistance that is limited to a type of OPs with carboxyl ester bonds such as
malathion, is conferred by Trp251Leu mutation. Flies showing double resistance to both
malathion and diazinon were also found though they are not common. Since the allele
containing double mutations of Gly137Asp and Trp251Leu cannot confer resistance to both
malathion and diazinon (Heidari et al., 2004), it was proposed that a duplication of the
region containing the LcE7 gene generated two loci, each carrying different mutation
(Newcomb et al., 2005). As in the case of LcE7, when two (or multiple) mutations cannot
exit together in a single locus, duplication may be the most useful option to acquire the
benefits of both resistance mutations. In this scenario, the LcE7 duplication events most
likely postdate the origin of the two resistance alleles, representing a case of gene that share
preceding gene duplications (Newcomb et al., 2005). The relatively low frequencies of cases
indicating double resistance suggests restrictions on the rate of recombination or fitness
costs associated with the duplications.
2.4 Cyp450 gene in M. persicae
Cyp450-mediated detoxification acts as a primary mechanism in neonicotinoid resistance in
M. persicae (Puinean et al., 2010). Microarray analysis of all known detoxification genes in M.
persicae revealed constitutive over-expression (22-fold) of a single Cyp450 gene (Cyp6CY3).
The overexpression of Cyp450 is due, at least in part, to an approximately 9-fold gene
amplification, as quantitative PCR of genomic DNA showed that the diploid genome of a
susceptible aphid clone carries two copies of the Cyp6CY3, whereas the neonicotinoid-
resistant clone has 18 copies (Puinean et al., 2010). Transcriptional upregulation based on
mutations in cis-acting and/or trans-acting regulatory loci has been reported to be mainly
responsible for the overexpression of Cyp450 genes in insecticide-resistant insects (Li et al.,
2007). Therefore, this may be the first case of Cyp450 amplification that is associated with
insecticide resistance in an agriculturally important insect pest but the Cyp6CY3
amplification mechanism remains to be elucidated.
3. Duplication of insecticide-target site genes
3.1 Duplication of -aminobutyric acid (GABA) receptor
A point mutation (Ala302Ser or Ala302Gly) in the Resistance to dieldrin (Rdl) gene
encoding a GABA receptor subunit is known to confer resistance to cyclodiene insecticides
in Drosophila and other insects (ffrench-Constant et al., 1993; ffrench-Constant et al., 2000).
Because Rdl is a single copy gene in most insects, individuals can carry only two different
alleles. In contrast, M. persicae is reported to have up to four different Rdl-like alleles
(Anthony et al., 1998). Along with the wild-type allele (encoding Ala302 or allele A), three
other alleles encoding Gly302 (allele G), Ser302 (encoded from TCG codon; allele S) and
Ser302 (encoded from AGT codon; allele S) were found in M. persicae (Anthony et al.,
1998). Three of these alleles (A, G and S) were common in individual aphids or aphid clones.
The presence of two independent Rdl loci in M. persicae has been confirmed by Southern
analysis in conjunction with sequencing downstream of the exon containing the mutation.
Sequence comparison between two loci has suggested that the loci may have been generated
through a recent gene duplication event. Interestingly, only one locus carrying the opposite

alleles of the alanine vs. glycine was responsible for resistance, whereas the other locus
carrying the two serine-containing alleles (S or S) was not associated with resistance
(Anthony et al., 1998). Taken together, it appears that, after gene duplication, one locus
(S/S) of Rdl likely had been fixed in aphids regardless of their resistance status, whereas the
other (A/G) locus had been specialized as a mechanism for GABA receptor insensitivity to
dieldrin. This is a typical example of the duplication of a gene encoding an insecticide target
site followed by the functional diversification with respect to insecticide resistance.
3.2 Duplication of acetylcholinesterase (AChE) gene in Culex mosquitoes
In C. pipiens mosquitoes, the ace1 locus encodes acetylcholinesterase 1 (AChE1), which is the
target of OP and carbamate insecticides. Most OP-resistant populations showed insensitive
AChE1, whereas, in two Caribbean populations, individual mosquito displayed a mixture of
sensitive and insensitive AChE1. The parsimonious explanation for these phenomenon is
the existence of two ace1 loci encoding both resistant and susceptible AChE1 (phenotype
RS), which were most likely generated by gene duplication (Bourguet et al., 1996). The OP-
resistant allele, ace1R, has been determined to be due to a single amino acid substitution,
Gly119Ser (Weill et al., 2003; Weill et al., 2004), which causes not only reduced sensitivity to
OP insecticide but also high fitness cost by modifying the catalytic properties of AChE1
(Weill et al., 2003). Later on, a similar duplication event was suggested for C. pipiens
populations from Southern France, where an excess of the [RS] phenotype was observed in
natural populations (Lenormand et al., 1998). The duplication event in Southern France was
dated back to 1993, 15 years after ace1R was first detected in the area, and then has gradually
replaced the ace1R allele in treated areas (Lenormand et al., 1998).
Advantages and costs of ace1 duplications in relation to OP resistance have previously been
described from the perspective of gene dosage (Labb et al., 2007). Gene dosage is very
important to maintain essential cellular processes, and increase in gene dosage by
duplication likely disturb this balance (Kondrashov et al., 2002; Papp et al., 2003; Veitia,
2005). Duplication of ace1 produces higher levels of AChE1, resulting in hyperactivity. In the
resistant form of AChE1 (AChE1R), however, the catalytic activity is less than 60% of the
susceptible form of AChE1 (AChE1S) (Bourguet et al., 1996; Bourguet et al., 1997). Therefore,
ace1 duplication may restore its normal gene dosage, otherwise phenotypically reduced by
the resistance mutation (Labb et al., 2007). A duplication of two ace1R copies may partly
restore normal AChE1 activity to a level that is comparable to that of a single copy of ace1S,
serving as a transitional step to ace1D haplotypes (having both ace1S and ace1R). AChE1
activity in ace1D homozygotes is similar to or greater than those in susceptible ace1S
homozygotes (Bourguet et al., 1996). The slightly higher AChE1 activity generated by ace1D
may result in another type of fitness cost by rapidly degrading the neurotransmitter
acetylcholine. In summary, ace1 duplication generating both resistant and susceptible copies
may be selected as a compensatory mechanism for the fitness cost by the homozygous ace1R
allele (Labb et al., 2007). The generation of persistent heterozygosis, in which the
susceptible ace1 allele is always expressed, likely reduces the fitness cost associated with the
resistant allele. The duplication event was proposed to occur relatively more frequently than
generally conceived and very recently (i.e., within less than past 40 years), demonstrating
that the gene duplication associated with insecticide resistance is a typical example of
rapidly developing evolutionary events (Labb et al., 2007).

3.3 Extensive duplication of AChE gene in the two-spotted spider mite (TSSM)
Three point mutations (Gly228Ser, Ala391Thr and Phe439W) have been identified using
extensive sequence comparisons of the AChE gene from the TSSM (Tuace) between OP-
resistant and susceptible strains. In addition, their functional roles have been assessed by
analyzing the correlation between mutation frequencies and actual resistance levels of
several field populations (Kwon et al., 2010b). The frequencies of the Gly228Ser and
Phe439Trp mutations in resistant strains never reached 100% even after extensive selection
with monocrotophos (Kwon et al., 2010b). To determine whether the lack of saturation for
these mutation frequencies is due to the heterozygosity of the Tuace allele in individual
mites, the frequencies of the three mutations in an individual diploid virgin female and her
parthenogenetic haploid male progenies were have been determined using quantitative
sequencing (Kwon et al., 2010a). The actual frequencies of the G228S, A391T and F439W
mutations in a female mite and its haploid male progenies have been estimated as
approximately 50%, 100% and 75%, respectively. These findings clearly suggested the
presence of multiple copies of Tuace. Determination of Tuace copy number in three mite
strains (highly resistant AD, moderately resistant PyriF and susceptible UD strains) using
quantitative PCR has revealed that resistant strains have relatively more Tuace copies than
the susceptible strain and that the levels of transcript were directly proportional to copy
numbers (Kwon et al., 2010a). AChEs from the AD and PyriF strains have shown reduced
catalytic efficiencies based on lower k
cat
values, suggesting that the resistant form of AChE is
likely accompanied by fitness cost. Relative copy numbers of Tuace in field populations of
TSSM ranged from 2.4 to 6.1 and are highly correlated with the respective resistance level,
suggesting that Tuace duplication itself contributes to resistance (Kwon et al., 2010a).
Western blot analysis using AChE-specific antibodies has been conducted to determine
whether Tuace duplication results in TuAChE overproduction. The protein quantities of
TuAChE in seven field-collected mite populations precisely correlated with the copy
numbers (Lee and Kwon, unpublished data). To investigate the effects of each mutation on
AChE insensitivity and possible fitness costs, eight variants of TuAChE were expressed in
vitro using the baculovirus expression system. Kinetic analysis revealed that the Ala391Thr
mutation did not alter the kinetic properties of AChE, whereas the Gly228Ser and
Phe439Trp mutations significantly increased the insensitivity to monocrotophos. Moreover,
when the Gly228Ser and Phe439Trp mutations were co-expressed, insensitivity increased
over 1000-fold. These results show that both mutations confer resistance in a synergistic
manner. However, the presence of the mutations considerably reduced the catalytic
efficiency of AChE, suggesting an apparent fitness cost in monocrotophos-resistant mites.
Reconstitution of the multiple copies of AChE with different compositions of the mutations
revealed that the catalytic efficiencies of the six-copy and two-copy AChEs (resembling the
AD and PyriF strains of mite, respectively) were lower but comparable to that of wildtype
AChE. These finding clearly suggest that multiple rounds of Tuace duplication is needed to
compensate the reduced catalytic activity of AChE caused by mutations. Whether mutation
or gene duplication occurs first is unknown. However, the introduction of a single mutation
(Gly228Ser or Phe439Trp) or a double mutation in a single copy of Tuace is unlikely because
the fitness cost is severe based on the dramatic reductions in the catalytic efficiency.
Therefore, at least a single event of Tuace duplication predates the introduction of mutations.
A single Gly228Ser mutation likely occurs first in one of the duplicates as seen in the PyriF
strain, in which the Gly228Ser mutation has been identified in one of the Tuace duplicates.
Under continuous selection pressure by OPs, further duplication of mutations might have

been required to compensate for the further reduction of catalytic efficiency that is
attributed to an additional Phe439Trp mutation. In summary, monocrotophos resistance in
TSSM may have evolved through a combination of phased gene duplication and mutation
accumulation.
4. Conclusions
Duplications (or amplifications) of resistance-related genes are frequent mechanisms in
insecticide resistance. Extensive forms of gene duplication (i.e., amplification) are commonly
found in metabolic genes that are involved in insecticide detoxification, including esterase
and Cyp450. In these cases, even with dramatically increased gene dosage, the apparent
fitness cost is not severe. In other words, low fitness costs that are associated with high
dosages of metabolic genes allow the amplification of genes. Gene duplication events have
been found in insecticide target genes, including ace and Rdl, which play crucial functions in
nerve impulse transmission. Unlike the amplification of metabolic genes, the level of
duplication of these genes is precisely regulated due to the necessity to maintain the normal
gene dosage. Mutations conferring target site insensitivity are always accompanied with
duplication events. Because mutations in the insecticide target sites frequently alter catalytic
or functional properties of target proteins, which usually increase fitness costs, duplication
may act as a compensatory mechanism to restore the normal activity of the target protein,
which is otherwise detrimental to maintaining the nervous system homeostasis. Conversely,
if the increase of the target protein dose due to an incidental gene duplication event has
different fitness consequences, the introduction and selection of any target site mutations
conferring insecticide resistance is facilitated following duplication because the mutations
that are associated with resistance usually reduce the normal function of target proteins.
Taken together, it is difficult to determine whether duplication or mutation occurs first.
However, these evolutionary events to acquire insecticide resistance may interact each other
to balance the level of resistance and accompanied fitness costs. Genetic introgression
between different populations plays a crucial role in spreading and formulating the degree
of gene duplication and mutation. In Anopheles gambiae, for example, the genetic traits of the
ace1R mutation and the ace1 duplication are shared between populations through
introgression (Djogbnou et al., 2008).
5. References
Anthony, N., Unruh, T., Ganser, D. & ffrench-Constant, R. (1998). Duplication of the Rdl
GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae.
Molecular and General Genetics, Vol.260, No.2-3, (November 1998), pp. 165-75, ISSN
0026-8925
Blackman, R. L., Spence, J. M., Field, L. M. & Devonshire, A. L. (1995). Chromosomal
location of the amplified esterase genes conferring resistance to insecticides in
Myzus persicae (Homoptera: Aphididae). Heredity, Vol.75, No.3, (September 1995),
pp. 297-302, ISSN 0018-067X
Blackman, R. L., Spence, J. M., Field, L. M. & Devonshire, A. L. (1999). Variation in the
chromosomal distribution of amplified esterase (FE4) genes in Greek field
populations of Myzus persicae (Sulzer). Heredity, Vol.82, No.2, (February 1999), pp.
180-186, ISSN 0018-067X

Bourguet, D., Lenormand, T., Guillemaud, T., Marcel, V., Fournier, D. & Raymond, M.
(1997). Variation of dominance of newly arisen adaptive genes. Genetics, Vol.147,
No.3, (November 1997), pp. 1225-1234, ISSN 0016-6731
Bourguet, D., Raymond, M., Bisset, J., Pasteur, N. & Arpagaus, M. (1996). Duplication of the
Ace.1 locus in Culex pipiens mosquitoes from the Caribbean. Biochemical Genetics,
Vol.34, No.9-10, (October 1996), pp. 351-62, ISSN 0006-2928
Callaghan, A., Guillemaud, T., Makate, N. & Raymond, M. (1998). Polymorphisms and
fluctuations in copy number of amplified esterase genes in Culex pipiens
mosquitoes. Insect Molecular Biology, Vol.7, No.3, (August 1998), pp. 295-300, ISSN
1365-2583
Campbell, P. M., Newcomb, R. D., Russell, R. J. & Oakeshott, J. G. (1998). Two different
amino acid substitutions in the ali-esterase, E3, confer alternative types of
organophosphorus insecticide resistance in the sheep blowfly, Lucilia cuprina. Insect
Biochemistry and Molecular Biology, Vol.28, No.3, (March 1998), pp. 139-150, ISSN
0965-1748
Campbell, P. M., Trott, J. F., Claudianos, C., Smyth, K. A., Russell, R. J. & Oakeshott, J. G.
(1997). Biochemistry of esterases associated with organophosphate resistance in
Lucilia cuprina with comparisons to putative orthologues in other Diptera.
Biochemical Genetics, Vol.35, No.1-2, (February 1997), pp. 17-40, ISSN 0006-2928
Devonshire, A. L. & Field, L. M. (1991). Gene Amplification and Insecticide Resistance.
Annual Review Entomology, Vol.36, No.1, (January 1991), pp. 1-21, ISSN 0066-4170
Djogbnou, L., Chandre, F., Berthomieu, A., Dabir, R., Koffi, A., Alout, H. & Weill, M.
(2008). Evidence of introgression of the ace-1
R
mutation and of the ace-1 duplication
in west african Anopheles gambiae s. s. PLoS ONE, Vol.3, No.5, (May 2008), pp. e2172,
ISSN 1932-6203
ffrench-Constant, R. H., Anthony, N., Aronstein, K., Rocheleau, T. & Stilwell, G. (2000).
Cyclodiene insecticide resistance: from molecular to population genetics. Annual
Review Entomology, Vol.45, No.1, (January 2000), pp. 449-466, ISSN 0066-4170
ffrench-Constant, R. H., Rocheleau, T. A., Steichen, J. C. & Chalmers, A. E. (1993). A point
mutation in a Drosophila GABA receptor confers insecticide resistance. Nature,
Vol.363, No.6428, (June 1993), pp. 449-451, ISSN 1476-4687
Field, L. M. (2000). Methylation and expression of amplified esterase genes in the aphid
Myzus persicae (Sulzer). Biochemical Journal, Vol.349 Pt 3, (August 2000), pp. 863-8,
ISSN 0264-6021
Field, L. M., Blackman, R. L., Tyler-Smith, C. & Devonshire, A. L. (1999). Relationship
between amount of esterase and gene copy number in insecticide-resistant Myzus
persicae (Sulzer). Biochemical Journal, Vol.339, No.3, (May 1999), pp. 737-742, ISSN
0264-6021
Field, L. M., Devonshire, A. L. & Forde, B. G. (1988). Molecular evidence that insecticide
resistance in peach-potato aphids (Myzus persicae Sulz.) results from amplification
of an esterase gene. Biochemical Journal, Vol.251, No.1, (April 1988), pp. 309-312,
ISSN 0264-6021
Force, A., Lynch, M., Pickett, F. B., Amores, A., Yan, Y. L. & Postlethwait, J. (1999).
Preservation of duplicate genes by complementary, degenerative mutations.
Genetics, Vol.151, No.4, (April 1999), pp. 1531-45, ISSN 0016-6731

Guillemaud, T., Makate, N., Raymond, M., Hirst, B. & Callaghan, A. (1997). Esterase gene
amplification in Culex pipiens. Insect Molecular Biology, Vol.6, No.4, (November
1997), pp. 319-327, ISSN 1365-2583
Heidari, R., Devonshire, A. L., Campbell, B. E., Bell, K. L., Dorrian, S. J., Oakeshott, J. G. &
Russell, R. J. (2004). Hydrolysis of organophosphorus insecticides by in vitro
modified carboxylesterase E3 from Lucilia cuprina. Insect Biochemistry and Molecular
Biology, Vol.34, No.4, (April 2004), pp. 353-63, ISSN 0965-1748
Hick, C. A., Field, L. M. & Devonshire, A. L. (1996). Changes in the methylation of amplified
esterase DNA during loss and reselection of insecticide resistance in peach-potato
aphids, Myzus persicae. Insect Biochemistry and Molecular Biology, Vol.26, No.1,
(January 1996), pp. 41-47, ISSN 0965-1748
Hurles, M. (2004). Gene duplication: the genomic trade in spare parts. PLoS Biology, Vol.2,
No.7, (July 2004), pp. E206, ISSN 1545-7885
Kondrashov, F. A., Rogozin, I. B., Wolf, Y. I. & Koonin, E. V. (2002). Selection in the
evolution of gene duplications. Genome Biology, Vol.3, No.2, (January 2002), pp.
RESEARCH0008, ISSN 1465-6914
Kwon, D. H., Clark, J. M. & Lee, S. H. (2010a). Extensive gene duplication of
acetylcholinesterase associated with organophosphate resistance in the two-spotted
spider mite. Insect Molecular Biology, Vol.19, No.2, (April 2010), pp. 195-204, ISSN
1365-2583
Kwon, D. H., Im, J. S., Ahn, J. J., Lee, J.-H., Marshall Clark, J. & Lee, S. H. (2010b).
Acetylcholinesterase point mutations putatively associated with monocrotophos
resistance in the two-spotted spider mite. Pesticide Biochemistry and Physiology,
Vol.96, No.1, (January 2010), pp. 36-42, ISSN 0048-3575
Labb, P., Berticat, C., Berthomieu, A., Unal, S., Bernard, C., Weill, M. & Lenormand, T.
(2007). Forty years of erratic insecticide resistance evolution in the mosquito Culex
pipiens. PLoS Genetics, Vol.3, No.11, (November 2007), pp. e205, ISSN 1553-7404
Lenormand, T., Guillemaud, T., Bourguet, D. & Raymond, M. (1998). Evaluating gene flow
using selected markers: a case study. Genetics, Vol.149, No.3, (July 1998), pp. 1383-
1392, ISSN 0016-6731
Li, X., Schuler, M. A. & Berenbaum, M. R. (2007). Molecular mechanisms of metabolic
resistance to synthetic and natural xenobiotics. Annual Review Entomology, Vol.52,
(August 2006), pp. 231-53, ISSN 0066-4170
Mouches, C., Pasteur, N., Berge, J., Hyrien, O., Raymond, M., de Saint Vincent, B., de
Silvestri, M. & Georghiou, G. (1986). Amplification of an esterase gene is
responsible for insecticide resistance in a California Culex mosquito. Science,
Vol.233, No.4765, (August 1986), pp. 778-780, ISSN 0036-8075
Mouches, C., Pauplin, Y., Agarwal, M., Lemieux, L., Herzog, M., Abadon, M., Beyssat-
Arnaouty, V., Hyrien, O., de Saint Vincent, B. R., Georghiou, G. P. & Pasteur, N.
(1990). Characterization of amplification core and esterase B1 gene responsible for
insecticide resistance in Culex. Proceedings of the National Academy of Sciences of the
United States of America, Vol.87, No.7, (April 1990), pp. 2574-8, ISSN 0027-8424
Newcomb, R. D., Gleeson, D. M., Yong, C. G., Russell, R. J. & Oakeshott, J. G. (2005).
Multiple mutations and gene duplications conferring organophosphorus
insecticide resistance have been selected at the Rop-1 locus of the sheep blowfly,

Lucilia cuprina. Journal of Molecular Evolution, Vol.60, No.2, (February 2005), pp. 207-
220, ISSN 0022-2844
Ohno, S. (1970). Evolution by gene duplication, Springer-Verlag, ISBN 0-04-575015-7, New
York, USA
Papp, B., Pal, C. & Hurst, L. D. (2003). Dosage sensitivity and the evolution of gene families
in yeast. Nature, Vol.424, No.6945, (July 2003), pp. 194-7, ISSN 1476-4687
Puinean, A. M., Foster, S. P., Oliphant, L., Denholm, I., Field, L. M., Millar, N. S.,
Williamson, M. S. & Bass, C. (2010). Amplification of a cytochrome P450 gene is
associated with resistance to neonicotinoid insecticides in the aphid Myzus persicae.
PLoS Genetics, Vol.6, No.6, (June 2010), pp. e1000999, ISSN 1553-7404
Raymond, M., Chevillon, C., Guillemaud, T., Lenormand, T. & Pasteur, N. (1998). An
overview of the evolution of overproduced esterases in the mosquito Culex pipiens.
Philosophical Transactions of the Royal Society B: Biological Sciences, Vol.353, No.1376,
(October 1998), pp. 1707-11, ISSN 0962-8436
Taylor, J. S. & Raes, J. (2004). Duplication and divergence: the evolution of new genes and
old ideas. Annual Review Genetics, Vol.38, No.1, (June 2004), pp. 615-643, ISSN 0066-
4197
Veitia, R. A. (2005). Gene dosage balance: deletions, duplications and dominance. Trends in
Genetics, Vol.21, No.1, (January 2005), pp. 33-35, ISSN 0168-9525
Weill, M., Lutfalla, G., Mogensen, K., Chandre, F., Berthomieu, A., Berticat, C., Pasteur, N.,
Philips, A., Fort, P. & Raymond, M. (2003). Comparative genomics: insecticide
resistance in mosquito vectors. Nature, Vol.423, No.6936, (May 2003), pp. 136-7,
ISSN 1476-4687
Weill, M., Malcolm, C., Chandre, F., Mogensen, K., Berthomieu, A., Marquine, M. &
Raymond, M. (2004). The unique mutation in ace-1 giving high insecticide
resistance is easily detectable in mosquito vectors. Insect Molecular Biology, Vol.13,
No.1, (February 2004), pp. 1-7, ISSN 1365-2583
Zhang, J. (2003). Evolution by gene duplication: an update. Trends in Ecology and Evolution,
Vol.18, No.6, (June 2003), pp. 292-298, ISSN 0169-5347
9
Gene Duplication and the Origin
of Translation Factors
Galina Zhouravleva and Stanislav Bondarev
Department of Genetics and Breeding, St Petersburg State University
Russia
1. Introduction
Charles Darwin is famous for his contribution to the development of evolutionary theory.
Less commonly known is that Darwin was a good botanist. He wrote several books devoted
to flowering plants. Being an honest scientist, he did not conceal the inability of his theory of
evolution to explain the sudden appearance and rapid spread of angiosperms, calling this
phenomenon an abominable mystery. One possible solution to the puzzle that agitated
Darwin may be the several successive duplications of the ancient ancestral genome at the
beginning of the divergence of angiosperms that gave them the ability to rapidly accumulate
changes (Cui et al., 2006). Speculation about the possible role of gene duplication in
evolution began in the middle of the last century (Sturtevant, 1925; Haldane, 1932; Muller,
1936; Lewis, 1951), but only the later rapid development of molecular biology allowed the
identification of numerous repeated sequences that revealed a high frequency of gene
duplication in evolution. Based on this information, S. Ohno (Ohno, 1970) suggested that
gene duplication was the only way new genes could emerge.
2. Types of duplications
Duplication of DNA can occur in many ways: (1) partial duplication of a gene (or an internal
duplication), (2) duplication of a single gene, (3) partial duplication of a chromosome, (4)
duplication of an entire chromosome, and (5) genome duplication, or polyploidy. The first
four types of duplication are sometimes combined under the term SSD (smaller scale
duplication) (Davis & Petrov 2005). Other authors prefer the terms "paralogon" (derived
from paralog), for extended duplicated regions containing paralogs, and SGD (single gene
duplication), for duplications of individual genes (Durand & Hoberman, 2006). Duplication
of the entire genome is designated as WGD (whole genome duplication) (Davis & Petrov,
2005). According to Ohno, duplication of the genome rather than its individual parts is more
important for evolution, because the partial duplication of regulatory genes or other
restricted elements of the genome may lead to regulatory imbalances (Ohno 1970).
2.1 Whole genome duplications
Ancient polyploidizations of the genome have been identified in all four eukaryotic
kingdoms: plants, animals, fungi and protists. In all cases, the proportion of genes in the

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152
form of duplicated copies ranges from 10 to 50% and often correlates with the time elapsed
since duplication (Scannell et al., 2006).
WGD is widespread in plants (Vision et al., 2000; Adams & Wendel, 2005). Estimates of the
incidence of polyploidy in angiosperms vary from 30 to 80%, and about 3% of speciation
events are explained by genome duplications (Otto & Whitton, 2000). Many, if not all,
species of plants may thus have at least one polyploid ancestor. Most eudicots are assumed
to have an ancient hexaploid ancestor, with subsequent tetraploidization in some taxa
(Jaillon et al., 2007).
Duplication of the entire genome in the yeast Saccharomyces cerevisiae led to an initial
increase in the number of genes from 5000 to 10 000, but the subsequent loss of paralogs has
led to the preservation in modern Saccharomyces of about 5500 protein-coding genes, of
which 1102 form 551 paralogous pairs (Byrne & Wolfe, 2005). A special term, ohnologs,
dedicated to S. Ohno, was proposed for paralogs resulting from WGD (Wolfe, 2000).
Detection of natural polyploidy is a difficult task, especially for ancient events. Recent
duplications can be detected by comparing closely related species, one of which underwent
diploidization and therefore contains twice as many chromosomes as species that did not
undergo WGD. For example, a comparison of the genomes of Ashbya gossypii and S.
cerevisiae revealed that both species evolved from a single ancestor that had seven or eight
chromosomes (Dietrich et al., 2004). Changes in chromosome number due to mutations (in
particular translocations) led to the ancestors of A. gossypii and S. cerevisiae. WGD in S.
cerevisiae has provided this species with new opportunities for functional divergence absent
in A. gossypii. A similar comparative analysis was also carried out for S. cerevisiae and its
closest non-WGD relative, Kluyveromyces waltii (Kellis et al., 2004).
The older the duplication, the harder the analysis, because a period of diploidization often
follows polyploidization, which "transforms" the polyploid genome to the diploid state.
Diploidization is achieved by an intensive loss of genes, rearrangements of the genome and
the divergence of duplicated genes. Recent analyses have also shown that the duplication of
individual genes in evolution has occurred much more frequently than was previously
thought (Lynch & Conery, 2000; Lynch et al., 2001). Diploidization has been studied in many
genomes including those of plants (Chapman et al., 2006; Jaillon et al., 2007; Tuskan et al.,
2006), bony fishes (Brunet et al., 2006), yeasts (Piskur, 2001; Kellis et al., 2004; Scannell et al.,
2006; Scannell et al., 2007), Paramecium (Aury et al., 2006) and vertebrata (Blomme
et al., 2006).
Plants have repeatedly undergone polyploidization during evolution, presumably aided by
their ability to propagate vegetatively and by the existence of specific regulatory
mechanisms in plant cells. In particular, model polyploids have been characterized by a
rapid loss of some genes and the specific inactivation of others by methylation (Kashkush et
al., 2002; Comai et al., 2000; Lee & Chen, 2001). Epigenetic silencing may protect the
duplicated copies from pseudogenization, thus facilitating the acquisition of new functions
(Rodin & Riggs, 2003).
Vertebrate genomes contain many families of genes that are not found in invertebrates, and
many gene duplications apparently occurred early in the evolution of the chordates (Taylor
& Raes, 2004). Ohno suggested that the complex genome of vertebrates arose as a result of
two rounds (2R) of WGD (Ohno, 1970). This view was once supported by the belief that the
human genome contained about 100 000 genes, which was four times more than the
estimated number of genes in the genomes of invertebrates. Sequencing of the human
genome has since reduced the estimate of the number of genes to 20 000-25 000 but has not

Gene Duplication and the Origin of Translation Factors

153
yet answered the question of the number of duplications of the ancestral genome. Some
authors continue to support the 2R hypothesis (Larhammar et al., 2002; Spring, 1997; Meyer
& Schartl, 1999; Wang & Gu, 2000; Dehal & Boore, 2005), others find evidence of only one
round of WGD (X.Gu et al., 2002; Guigo et al., 1996; McLysaght et al., 2002), while others
disclaim the possibility of WGD entirely and discuss only duplications of a limited number
of segments (Friedman & Hughes, 2001; 2003).
2.2 Smaller scale duplication
Ohno (1970) argued that duplication of the genome rather than its individual parts is more
important for evolution, because partial duplications can lead to regulatory imbalances.
Nevertheless, partial and complete duplications of genes also play very important roles in
evolution. WGDs have occurred several times during the evolutionary history of organisms,
while SSDs arise continuously through multiple mechanisms. Several mechanisms have
been suggested for the improvement in function of existing proteins and for the creation of
new functions. One such mechanism is the internal (partial) duplication of genes, which is
important for increasing the functional complexity of genes in evolution (Li, 1997). Such
duplications are believed to have played a key role in the emergence of complex genes.
Many proteins of modern organisms contain internal repeats of amino acids, and these
repeats often correspond to functional or structural domains of proteins. These data suggest
that the genes encoding these proteins were formed by internal duplications (Lavorgna et
al., 2001). Internal duplication provides the possibility of improving protein function by
increasing the number of active sites. Internal duplications can also lead to the acquisition of
new functions by the modification of duplicated regions or the reorganization of modules.
Numerous data on the role of intragenic duplications in the early stages of evolution of
proteins were obtained by comparative analyses of sequenced genomes (Marcotte et al.,
1999; Lavorgna et al., 2001; Conant & Wagner 2005; Chen et al. 2007). Duplicated regions can
accumulate mutations that contribute to the divergence of the repeated fragments, which
can then become fixed. Often, only traces of duplications in the form of imperfect repeats
can be detected in contemporary amino acid sequences (Li, 1997). Eukaryotic proteins have
more repeats than do prokaryotic proteins (Marcotte et al., 1999; Chen et al., 2007).
3. The fate of duplicated genes
Tens of millions of years after WGD in Arabidopsis thaliana and S. cerevisiae, only about 30% and
10%, respectively, of the genes are preserved in the form of duplicated copies (Seoighe &
Wolfe, 1999; Wong et al., 2002; Blanc et al., 2003). Preservation of duplicated copies in
evolution can be achieved by one of three processes: (1) conservation, in which the copies are
stored in an unaltered state (Hahn 2009); (2) subfunctionalization, in which both paralogs are
necessary for performing the functions previously provided by the ancestral gene (both terms
were offered by Force (Force et al., 1999)); and (3) neofunctionalization, in which one of the
paralogs acquires a new function and the other preserves the old function. Characteristically,
in (2) and (3), the regulatory and/or structural parts of the gene may be changed (Figure 1).
3.1 Conservation of duplicated copies
Duplicated genes are retained unchanged in cases where the normal development of the
organism needs many copies of genes with similar function, which allows the synthesis of a

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larger amount of specific RNA or protein (Ohno, 1970). An increase in the number of copies
of these genes correlates with the increasing complexity of the organism (Chen et al., 2007).
Amplification of genes in microorganisms leads to resistance to antibiotics and heavy
metals, increased virulence and other adaptive properties (Romero & Palacios, 1997; Reams
& Neidle, 2004; Andersson & Hughes, 2009). In plants, amplification of genes provides
resistance to herbicides (Harms et al., 1992; Shyr et al., 1992). The best known examples of
conservation of duplicated copies in various organisms are genes for rRNA, tRNA and
histones, many of which are organized in tandem repeats, which allows the maintenance of
homogeneity by unequal crossing over or gene conversion (Hurles, 2004).

Fig. 1. Possible consequences of gene duplication (modified from (Hahn, 2009)). A and C -
regulatory sequence changes; B and D coding sequence changes. Since variant 3
(conservation) does not change the duplicated copies, it is not represented in the diagram.
OF (grey) - old function, NF (black) new function, LF (white) lost function (attributed to
both regulatory and structural sequences).
One of the most interesting questions related to the preservation of duplicated copies of
genes is whether the loss of genes is an occasional event or is subjected to natural selection.
Which duplicates are lost, and which persist after polyploidization? About 10% of yeast
genes are preserved in the form of duplicated copies, and most are not needed for viability
(Z.Gu et al., 2002). The most frequently duplicated genes encode cyclins, components of the
signal transduction pathway, and cytoplasmic (but not mitochondrial) ribosomal proteins.
Most are characterized by high levels of expression. Perhaps selection for increasing the
level of expression was the major factor for the preservation of duplicated genes (Seoighe &
Wolfe, 1999).
Analysis of the most recent WGD in Arabidopsis showed a preferential retention of genes
involved in transcription and signal transduction, whereas genes involved in DNA repair or
encoding proteins of organelles were characterized by more frequent loss (Blanc & Wolfe,
2004). Interestingly, genes preserved as paralogs after duplication have a high probability of
remaining duplicated after the next round of duplication (Seoighe & Gehring, 2004). Loss of
duplicates is thus not a random process.


155
3.2 Subfunctionalization
This hypothesis is to some extent the opposite to Ohnos hypothesis of evolution, because it
assumes the existence of both functions before the duplication (Figure 1). The first evidence
for it was the discovery of the phenomenon of gene sharing (Piatigorsky et al., 1988;
Piatigorsky, 2003).
This model explains the emergence of new genes by the duplication of multifunctional
genes. Such genes encode proteins that already perform different functions. Gene sharing
was discovered in crystallins, proteins found in the lens of the eye. Crystallins make up 70%
of the contents of cells, but remain in soluble form without forming aggregates (formation of
aggregates leads to cataracts). Under such conditions, a majority of proteins would form
insoluble aggregates within seconds. Another feature of these proteins is a record longevity
(equal to the lifetime of an individual, for example 80 years); most proteins last for only
minutes or hours. The eyes of all vertebrates have a standard set of crystallins (, , ), and
additional species-specific crystallins are encoded by genes that in other tissues encode
enzymes. In most cases, this double life is ensured not by duplications but by a "division of
functions": enzyme and crystallin are encoded by the same gene, but the protein can
perform additional functions without changing its amino acid sequence. This phenomenon
was thus called gene sharing (Piatigorsky et al., 1988; Piatigorsky, 2003). In gene sharing, a
gene acquires a second function, without duplication and without loss of its primary
function. A change in tissue specificity or regulation during development, however, may
occur. Acquisition of a new function without duplication was first detected in crystallin in
birds and crocodiles (up to 23% of the total protein of the lens). The amino acid sequence of
crystallin was identical to lactate dehydrogenase B (LDH), and the protein had an activity
similar to LDH. Subsequent work showed that both proteins were encoded by the same
gene. Similarly, crystallin in lampreys, bony fishes, reptiles and birds is identical to and
encoded by the same gene as enolase. Zeta-crystallin is identical to quinone reductase.
Crystallins , and thus illustrate examples of "division of functions", when a gene has
acquired additional functions, without duplication. Multifunctional genes are characterized
by significant limitations in the capabilities of any adaptive changes, since mutation that
improves one function may disturb another. Duplication could provide a possible resolution
of this "adaptive conflict". The molecular mechanisms leading to subfunctionalization have
not been studied in detail until recently. Such analyses only became possible with the
comparative analysis of genes in closely related species, for example in genes involved in
galactose utilization in S. cerevisiae and K. lactis (Hittinger & Carroll, 2007). Divergence in the
expression of duplicated genes over long periods of time attracted the interest of scientists
as an important stage in the emergence of a new gene by duplication (Ohno, 1970; Ferris &
Whitt, 1979). Thus in some cases, duplicates may have identical coding sequences but
different regulatory sequences (Figure 1). Some pairs of duplicated genes can diverge in
concert, forming two groups that are expressed in different tissues or under different
conditions (Blanc & Wolfe, 2004). This process, which explains the divergence of metabolic
pathways, is called "concerted divergence.
3.3 Neofunctionalization
The stable maintenance of duplicated copies in the genome requires functional
divergence. From Ohnos (1970) position, functional divergence is achieved by ensuring
that one copy of the gene retains the old function, while other copies acquire new

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156
functions. An inevitable intermediate stage in this process would be the emergence of a
pseudogene, as most mutations will disrupt or inactivate a gene rather than giving rise to
new functions. Because this event is considered extremely unlikely, an extended
hypothesis of neofunctionalization (NF) has been proposed, which includes the following
possibilities: (1) a new gene acquires a new function but keeps the old function (NF-I), (2)
a new gene completely looses the old function (NF-II), or (3) a new gene retains part of the
old function (NF-III) (He & Zhang, 2005). Many examples of neofunctionalization have
been described in recent years (see (Hahn, 2009)), although distinguishing
neofunctionalization from subfunctionalization is sometimes difficult and has led to the
creation of a "subneofunctionalization" model (He & Zhang, 2005).
3.4 Exon shuffling as a mechanism of neofunctionalization
One of the options for neofunctionalization is the formation of "chimeric" or fusion genes
(Long, 2000). This phenomenon is possible due to the duplication of a gene or part of a gene,
because only then can the original gene remain functional. After gene duplication, one of the
copies can capture an exon(s) from an unrelated adjacent gene. Another possibility is the
addition of flanking non-coding DNA as an additional open reading frame. The model,
known as exon shuffling (Gilbert, 1978), suggests that recombination in introns can
provide a mechanism for exchanging exon sequences between genes. However, the event
will be evolutionarily significant only if it involves a structural or functional domain.
Moreover, the shuffling of domains can occur without the involvement of introns (Doolittle,
1995). We are thus more correct to discuss the shuffling of domains rather than exons.
Introns do not occur in prokaryotic genes, but many cases of domain shuffling have been
described. The presence of introns, though, greatly facilitates the shuffling of domains,
especially in vertebrates. In the 30 years since the discovery of introns, many examples of
exon shuffling in a variety of organisms (vertebrates, invertebrates, plants) have been found.
Only relatively recently have retrotransposition and illegal recombination been shown to be
responsible for these phenomena (Long et al., 2003; van Rijk & Bloemendal, 2003).
4. Translation factors as examples of subneofunctionalization
4.1 The main stages of translation
In the process of protein synthesis, or translation, four distinct phases are usually
distinguished: initiation, elongation, termination and recycling (Figure 2).
During initiation, the ribosome is assembled at the initiation codon of the mRNA, and the
initiating methionyl-tRNA is attached to the peptidyl (P) center of the ribosome. The main
objectives of the initiation of translation are identical in bacteria and eukaryotes, but
initiation is much more complex in eukaryotes than in bacteria (Kapp & Lorsch, 2004). Three
initiation factors occur in bacteria, but eukaryotes have at least 12, which contain about 23
different proteins (Sonenberg & Dever, 2003). Interestingly, the initiation of translation in
archaea is intermediate in complexity between bacterial and eukaryotic translation.
During elongation, the aminoacyl-tRNA binds to the aminoacyl center (or A-site) of the
ribosome, where the information recorded on the mRNA is translated into the language of
proteins. This process involves elongation factor eEF1A (EF-Tu in bacteria) in complex with
GTP. The ribosomes catalyze the formation of peptide bonds when the anticodons of tRNAs
correspond to the codons of the mRNA. After translocation of the mRNA in the P-center,
with the help of eEF2 (EF-G in bacteria), a next codon arrives in the A-center, and the


157
process repeats. In contrast to initiation, the main components involved in elongation are
highly conserved in all three domains. For example, the human elongation factor eEF1A and
EF-Tu of Escherichia coli are 33% identical along their entire length, exhibiting a higher
degree of similarity in the GTP-binding domains (Cavallius et al. 1993). The proteins
a/eEF1A and a/eEF2 reveal significant structural similarities, both in the free state and in
complex with the ribosome (Andersen et al., 2001; Stark et al., 2002; Valle et al., 2002;
Jorgensen et al., 2003). The similarity of elongation factors in bacteria, archaea and
eukaryotes suggests that the mechanisms of elongation in eukaryotes in many respects
correspond to those in bacteria and archaea (Ramakrishnan, 2002).
Termination of translation begins when the stop codon (UAA, UAG or UGA) enters the A-
site of the ribosome. As the result of this process, the newly synthesized polypeptide chain
is released. The stop codon is recognized by a release factor (RF1/RF2 in prokaryotes and
eRF1 in eukaryotes) that triggers release of the nascent peptide from the ribosome. The
efficiency of termination is enhanced by the GTPase release factor, RF3 in prokaryotes and
eRF3 in eukaryotes (Kisselev et al., 2003). At least some stages of the termination of
translation, such as recognition of the stop codon and hydrolysis of peptidyl-tRNAs, are
assumed to be similar in archaea and eukaryotes. This hypothesis is based on data of the
homology of aRF1 and eRF1 and the finding that aRF1 of Methanococcus jannaschii is able to
function in an in vitro system containing mammalian ribosomes (Dontsova et al., 2000).
Archaea, however, do not have homologs of RF3 and eRF3, which does not necessarily
mean the absence of proteins with similar functions. Alternatively, these proteins may be
absent due to a reduction of the apparatus of translation during the evolution of archaea
(Lecompte et al., 2002).
During the final stage of translation, recycling, the dissociation of the ribosome occurs
together with the release of the mRNA and deacylated tRNAs. An essential feature of this
stage is the preparation of a new round of initiation. The details of this process are known
only for bacteria.
4.2 Termination factors have arisen by the duplication of genes encoding elongation
factors
Comparison of amino acid sequences in the family of elongation factors raised speculation
that the progenitors of EF-G and EF-Tu arose as a result of duplication and subsequent
divergence of a gene encoding an ancient GTPase, and further duplications led to the
emergence of modern elongation and termination factors (Nakamura & Ito, 1998; Inagaki &
Doolittle, 2000) (Figure 3). RF1, RF2 and RF3, as well as eRF1 and elongation factor eEF-2,
are assumed to have been derived from the bacterial elongation factor EF-G (Nakamura &
Ito 1998), while eRF3 arose from the duplication of the gene encoding eukaryotic elongation
factor eEF1-A (Inagaki & Doolittle 2000).
The amino acid sequences of RF1 and RF2 are 36% identical, suggesting that the genes prfA
and prfB arose from a common precursor by duplication (Craigen et al., 1990). Homologs of
eRF1 are found in different species, and the eRF1 protein from different species is able to
replace eRF1 of S. cerevisiae, indicating a high degree of functional conservation (Urbero et
al., 1997). An almost complete lack of similarity in the sequences of bacterial and eukaryotic
termination factors probably indicates their independent origin (Kisselev et al., 2003). On the
other hand, the first class factors (RF1, RF2, aRF1 and eRF1) could be so divergent that they
have lost any resemblance, with the exception of the GGQ motif (Frolova et al., 1999;
Lecompte et al., 2002; Seit-Nebi et al., 2001). The lack of homology between the amino acid

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158

Fig. 2. Evolutionarily related proteins perform similar functions and interact with the same
sites of the ribosome during translation. The most significant participants are shown. The
arrows indicate the sequence of events. IF - initiation factor; EF - elongation factor; RF
release, or termination, factor; e eukaryotic.


159

Fig. 3. The origin of the proteins involved in elongation, termination and mRNA quality
control. The genes duplicated only in certain taxa are marked with asterisks: * - duplication
unique to mammals (Hoshino et al., 1998; Jakobsen et al., 2001); ** - duplication described
only from Saccharomyces (Atkinson et al., 2008); *** - duplication specific to several species of
ciliates (Liang et al., 2001; Atkinson et al., 2008) and A. thaliana (Chapman & Brown, 2004).
Branch lengths are not to scale. The progenitors of prokaryotic EF-G and EF-Tu were
proposed to have first diverged from a common ancestral GTPase, and then each gave rise
to two protein families corresponding to the elongation and termination factors (Nakamura
& Ito, 1998; Inagaki & Doolittle, 2000; Atkinson et al., 2008). EF elongation factor, RF -
release factor, e eukaryotic, a archaeal.
sequences of bacterial and eukaryotic termination factors does not mean that these proteins
lack similarity at other levels of the organization of protein molecules. Indeed, the spatial
structure of many translation factors are characterized by a number of common features that
fit the hypothesis of "molecular mimicry" (Nissen et al., 2000; Nakamura & Ito, 2003).
In contrast to eRF1, eRF3 is a much less conserved protein, especially in its N-terminal
domain, which can either be completely absent, as in the case of Giardia lamblia (Inagaki &
Doolittle, 2000), or demonstrate species-specific differences in length (maximum length is
321 amino acids in Leishmania major (Atkinson et al., 2008)) and amino acid sequence. This
lack of conservation may underlie species-specific regulation of the activity of this protein
(Kodama et al., 2007). In some species of yeast, the N-terminus is enriched in QN residues

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160
and provides prionogenic properties to the protein (Kushnirov & Ter Avanesyan, 1998).
The same amino acid composition is also detected in the N-terminal domains of eRF3 in
the kinetoplastid protists L. major and Trypanosoma cruzi, but this similarity is unlikely to
be homologous (Atkinson et al., 2008). For termination of translation and maintenance of
viability, only the C-terminal domain of eRF3 (homologous to elongation factor eEF1A) is
necessary. eRF3 may have arisen in the early stages of eukaryotic evolution, since neither
bacterial nor archaeal genomes contain homologues of eRF3 (Inagaki & Doolittle, 2000).
Recent studies have shown that the functions of eRF3 can be performed in archaea by
aEF1A (Saito et al., 2010).
The termination factor eRF3, preserving the functions typical of elongation factors (GTP-ase
activity and interaction with the A-site of the ribosome), lost the capacity to bind tRNA but
acquired the capacity to interact with eRF1 (Table 1). From this standpoint, elongation factor
EF1A of archaea is functionally intermediate between elongation and termination factors: it
acquired the ability to stimulate aRF1 while maintaining all the properties of an elongation
factor (Saito et al., 2010). Termination factor eRF1 is a striking example of
neofunctionalization, because it has acquired a variety of functions absent in elongation
factors, including the ability to decode stop signals and to catalyze the release of nascent
peptides from eukaryotic ribosomes in response to stop codons.

GTP-
binding
tRNA binding and
delivering to the
A-site of ribosome
Recognition of
stop-signal in
A-site of
ribosome
Function of acessory
protein
Archaea aEF1A aEF1A aRF1
aEF1A (for aRF1),
aEF1B
(for aEF1A)
Eubacteria
EF-Tu,
RF3,
EF-G
EF-Tu RF1, RF2
RF3
(for RF1 or RF2),
EF-Ts
(for EF-Tu)
Eucarya
eEF1A,
eRF3,
eEF-2
eEF1A eRF1
eRF3 (for eRF1),
eEF1B
(for eEF1A)
Table 1. Functional homology between elongation and termination factors in Archaea,
Bacteria and Eukaryota
4.3 Additional paralogs of termination factors in several species
Additional duplication of genes encoding termination factors have been found in several
species (Figure 3). For example, an additional copy of eRF1 is present in some lineages of
ciliates (Liang et al., 2001; Atkinson et al., 2008). These organisms differ from most
eukaryotes by their reassignment of one or two stop codons to encode amino acids
(Lozupone et al., 2001). UGA, for instance, encodes cysteine in Euplotes (Meyer et al., 1991).
The presence of two copies of eRF1 in Euplotes octocarinatus may be associated with a
different codon specificity of eRF1 proteins for UAA and UAG codons (Liang et al., 2001).
Later studies showed that both eRF1a and eRF1b recognized UAA and UAG as stop codons


161
(Wang et al., 2010). The precise functions of each protein thus remain to be discovered. The
plant A. thaliana has three paralogs of eRF1, all of which are able to rescue the sup45-2(ts)
mutation in SUP45 (encoding eRF1) in S. cerevisiae (Chapman & Brown, 2004).
Another example of duplication, found only in some taxonomic groups, is the presence of
two paralogous genes encoding eRF3 in mammals. In mammals, proteins homologous to
eRF3 can be divided into two subfamilies based on the sequence of their N-termini. The first
subfamily includes human hGSPT1 (or eRF3a) and mouse mGSPT1 (Hoshino et al., 1989;
Hoshino et al., 1998; Jean-Jean et al., 1996), while the second subfamily includes human
hGSPT2 (eRF3b) and mouse mGSPT2 (Hoshino et al., 1998; Jakobsen et al., 2001).
Complementation experiments have shown that only mGSPT2 is able to complement the
SUP35 gene (encoding eRF3) mutation (Le Goff et al., 2002). GSPT2 is a paralog of GSPT1
that has perhaps arisen as a result of retrotransposition of the GSPT1 transcript into the
genome of the common ancestor of mouse and human. GSPT2 may thus be a functional
retrogene (Zhouravleva et al., 2006). Both eRF3a and eRF3b are able to serve as termination
factors in mammalian cells and interact with eRF1 (Chauvin et al., 2005). However, eRF3a is
considered the main factor (Chauvin et al., 2005) that is expressed in all tissues, while eRF3b
is detected only in the brain (Hoshino et al., 1998; Chauvin et al., 2005). This duplication
event may not have led to the emergence of a new gene function but may have contributed
to the complexity of regulatory processes by tissue-specific expression of these genes.
4.4 Subneofunctionalization in a family of termination factors gave rise to proteins
participating in mRNA quality control
A necessary condition of protein synthesis is to obtain functionally active proteins, so the
control of accuracy of protein synthesis occurs at each stage of translation (Valente & Kinzy,
2003). The accuracy of initiation is achieved by proper identification of the start codon by a
multifactorial initiation complex (Asano et al., 2001). Elongation requires the control of
various events, including maintenance of the correct reading frame. Shifts in the reading
frame occur at a frequency near 3 x 10
-5
(Atkins et al., 1991) and may lead to the synthesis of
non-functional products because shifts in the reading frame will often create a premature
termination codon (PTC).
Eukaryotic cells possess a mechanism known as nonsense-mediated mRNA decay (NMD)
that recognizes and degrades mRNA molecules containing premature termination codons
(Amrani et al., 2006) (Figure 4). NMD is mediated by the trans-acting factors Upf1, Upf2 and
Upf3, all of which directly interact with eRF3; only Upf1 interacts with eRF1 (Czaplinski et
al., 1998; Wang et al., 2001). In addition to NMD, eukaryotic cells contain two additional
mechanisms of mRNA quality control. No-go decay (NGD) releases ribosomes that are
stalled on the mRNA (Doma & Parker, 2006). In yeast, NGD involves the proteins Hbs1 and
Dom34 (Pelota in mammals). Another mechanism, non-stop decay (NSD), leads to the
release of ribosomes that have read through the stop codon instead of terminating
(Vasudevan et al., 2002). NSD has only been found in S. cerevisiae and involves the Ski7
protein (van Hoof et al., 2002). A common feature of these processes is that all involve the
termination factors eRF1 and eRF3 (NMD) or their paralogs (Dom34/eRF1 and Hbs1/eRF3
in NGD; Ski7/eRF3 in NSD).
Hbs1 is a paralog of eEF1A and eRF3 (Wallrapp et al., 1998; Inagaki & Doolittle, 2000), while
Dom34 is a paralog of eRF1 (Koonin et al., 1994; Davis & Engebrecht, 1998) (Figure 3). The
C-terminus of Hbs1, homologous to that of eRF3, is sufficient to interact with Dom34, which
assumes the same structure of the complex of two pairs of proteins (Hbs1-Dom34 and eRF3-

Gene Duplication

162
eRF1) (Carr-Schmid et al., 2002). Indeed, Hbs1 forms a complex with Dom34 and GTP
(Dom34-Hbs1-GTP), similar to that of eRF1-eRF3-GTP (Hauryliuk et al., 2006; Graille et al.,
2008; Chen et al., 2010; Shoemaker et al., 2010; van den Elzen et al., 2010). The central event
of NGD is mRNA cleavage, and Dom34 has the necessary RNase activity (Lee et al., 2007;
Graille et al., 2008), although the proposed endonuclease activity of Dom34 is not required
for mRNA cleavage in NGD (Passos et al., 2009). Dom34 of S. cerevisiae consists of three
domains, two of which are homologous to the corresponding domains in eRF1, while the N-
terminal domain of Dom34 is different from that of eRF1 and is probably necessary for the

Fig. 4. Neofunctionalization of termination factors in mRNAs quality control systems. Three
systems described for S. cerevisiae are shown. NSD (Non-stop decay) is responsible for the
degradation of transcripts lacking stop codons. NGD (No-go decay) removes mRNA
secondary structures that prevent translation. NMD (Nonsense-mediated decay) destroys
transcripts containing nonsense mutations. See text for details.


163
recognition of the mRNA stem (Graille et al., 2008). Lack of the Hbs1 protein in archaea is
apparently compensated by its homolog aEF1A (Kobayashi et al,. 2010), which also
performs the functions of eRF3 in archaeal termination of translation (Saito et al., 2010).
In one more pathway of mRNA degradation, non-stop decay (NSD), participates In one
more pathway of mRNA degradation, non-stop decay (NSD), participates Ski7 protein
that is paralog of Hbs1 and eRF3 (Benard et al., 1999). This mechanism is necessary to
destroy mRNAs lacking all termination codons (Frischmeyer et al., 2002; van Hoof et al.,
2002). Ski7 protein that is paralog of Hbs1 and eRF3 (Benard et al., 1999). This mechanism
is necessary to destroy mRNAs lacking all termination codons (Frischmeyer et al., 2002;
van Hoof et al., 2002) Ski7, involved in NSD, arose from duplication of Hbs1 by WGD
(Kellis et al., 2004) or by an independent duplication of Hbs1 before WGD and the
subsequent loss in several species (Atkinson et al., 2008) (Figure 3). An interesting
hypothesis links the appearance of Ski7 with the existence of the prion [PSI
+
] (Atkinson et
al., 2008). [PSI
+
] is the aggregated (prion) form of the yeast protein Sup35 (eRF3)
(Kushnirov & Ter Avanesyan, 1998). Formation of [PSI
+
] decreases the amount of
functional Sup35, leading to the efficient read-through of nonsense mutations in ORFs
(and possibly at the normal terminator codons) (Serio & Lindquist, 1999). The emergence
of Ski7 in such organisms would thus create an additional system of mRNA quality
control. However, [PSI
+
] formation has not been detected in the natural, industrial and
clinical isolates of Saccharomyces. In addition, the prionic properties of Sup35 are
conserved in various species of Saccharomyces as well as in Candida albicans and Pichia
methanolica (Inge-Vechtomov et al., 2003), species in which Ski7 has not been found
(Atkinson et al., 2008).
5. Conclusion
Successive duplications of genes encoding elongation factors for translation led to the
emergence of several protein complexes with different properties. The eRF1-eRF3 complex
terminates translation, and the Dom34-Hbs1 complex is involved in the quality control of
mRNA. Both eRF1 and eRF3 interact not only with each other but also with additional
proteins. Some of these interactions are possibly mutually exclusive, and some of the
proteins interacting with eRF1/eRF3 can be components of the complex terminating
translation. Possible candidates for involvement in termination are poly(A) binding protein
(PABP) and Upf proteins (Upf1, Upf2 and Upf3). Interaction of eRF3 with PABP links
termination of translation with initiation (Hoshino et al., 1999), while interaction with Upf
involves eRF proteins in nonsense-mediated decay (Amrani et al., 2006). The genetic data,
derived mostly from S. cerevisiae, strongly suggest that the functions of eRF1 and eRF3 are
not restricted to termination of translation (Inge-Vechtomov et al., 2003). Further studies are
needed to characterize other non-translational functions of both proteins, as was shown for
eEF1A (Mateyak & Kinzy, 2010).
6. Acknowledgments
This work was supported by the Russian Foundation for Basic Research (10-04-00237) and
the Program of the Presidium of the Russian Academy of Sciences, The Origin and the
Evolution of the Biosphere.

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7. References
Adams, K.L. & Wendel, J.F. (2005) Polyploidy and genome evolution in plants. Curr
Opin.Plant Biol., Vol. 8, pp. 135-141.
Amrani, N., Dong, S., He, F., Ganesan, R., Ghosh, S., Kervestin, S., Li, C., Mangus, D.A.,
Spatrick, P., & Jacobson, A. (2006) Aberrant termination triggers nonsense-
mediated mRNA decay. Biochem.Soc.Trans., Vol. 34, pp. 39-42.
Andersen, G.R., Valente, L., Pedersen, L., Kinzy, T.G., & Nyborg, J. (2001) Crystal structures
of nucleotide exchange intermediates in the eEF1A-eEF1Balpha complex.
Nat.Struct.Biol., Vol.8, pp. 531-534.
Andersson, D.I. & Hughes, D. (2009) Gene Amplification and Adaptive Evolution in
Bacteria. Annu.Rev.Genet., Vol.43, pp. 167-95.
Asano, K., Phan, L., Valasek, L., Schoenfeld, L.W., Shalev, A., Clayton, J., Nielsen, K.,
Donahue, T.F., & Hinnebusch, A.G. (2001) A multifactor complex of eIF1, eIF2,
eIF3, eIF5, and tRNA(i)Met promotes initiation complex assembly and couples GTP
hydrolysis to AUG recognition. Cold Spring Harb.Symp.Quant.Biol., Vol.66, pp.
403-415.
Atkins, J.F., Weiss, R.B., Thompson, S., & Gesteland, R.F. (1991) Towards a genetic dissection
of the basis of triplet decoding, and its natural subversion: programmed reading
frame shifts and hops. Annu.Rev.Genet., Vol.25, pp. 201-228.
Atkinson, G.C., Baldauf, S.L., & Hauryliuk, V. (2008) Evolution of nonstop, no-go and
nonsense-mediated mRNA decay and their termination factor-derived
components. BMC.Evol.Biol., Vol.8, pp. 290.
Aury, J.M., Jaillon, O., Duret, L., Noel, B., Jubin, C., Porcel, B.M., Segurens, B., Daubin, V.,
Anthouard, V., Aiach, N., Arnaiz, O., Billaut, A., Beisson, J., Blanc, I., Bouhouche,
K., Camara, F., Duharcourt, S., Guigo, R., Gogendeau, D., Katinka, M., Keller, A.M.,
Kissmehl, R., Klotz, C., Koll, F., Le Mouel, A., Lepere, G., Malinsky, S., Nowacki,
M., Nowak, J.K., Plattner, H., Poulain, J., Ruiz, F., Serrano, V., Zagulski, M., Dessen,
P., Betermier, M., Weissenbach, J., Scarpelli, C., Schachter, V., Sperling, L., Meyer,
E., Cohen, J., & Wincker, P. (2006) Global trends of whole-genome duplications
revealed by the ciliate Paramecium tetraurelia. Nature, Vol.444, pp. 171-178.
Benard, L., Carroll, K., Valle, R.C., Masison, D.C., & Wickner, R.B. (1999) The Ski7 antiviral
protein is an EF1-alpha homolog that blocks expression of non-Poly(A) mRNA in
Saccharomyces cerevisiae. J.Virol., Vol.73, pp. 2893-2900.
Blanc, G., Hokamp, K., & Wolfe, K.H. (2003) A recent polyploidy superimposed on older
large-scale duplications in the Arabidopsis genome. Genome Res., Vol.13, pp.
137-144.
Blanc, G. & Wolfe, K.H. (2004) Widespread paleopolyploidy in model plant species inferred
from age distributions of duplicate genes. Plant Cell, Vol.16, pp. 1667-1678.
Blomme, T., Vandepoele, K., De Bodt, S., Simillion, C., Maere, S., & Van de, P.Y. (2006) The
gain and loss of genes during 600 million years of vertebrate evolution. Genome
Biol., Vol.7, R43.
Brunet, F.G., Crollius, H.R., Paris, M., Aury, J.M., Gibert, P., Jaillon, O., Laudet, V., &
Robinson-Rechavi, M. (2006) Gene loss and evolutionary rates following whole-
genome duplication in teleost fishes. Mol.Biol.Evol., Vol.23, pp. 1808-1816.


165
Byrne, K.P. & Wolfe, K.H. (2005) The Yeast Gene Order Browser: combining curated
homology and syntenic context reveals gene fate in polyploid species. Genome Res.,
Vol.15, pp. 1456-1461.
Carr-Schmid, A., Pfund, C., Craig, E.A., & Kinzy, T.G. (2002) Novel G-protein complex
whose requirement is linked to the translational status of the cell. Mol.Cell Biol.,
Vol.22, pp. 2564-2574.
Cavallius, J., Zoll, W., Chakraburtty, K., & Merrick, W.C. (1993) Characterization of yeast
EF-1 alpha: non-conservation of post-translational modifications.
Biochim.Biophys.Acta, Vol.1163, pp. 75-80.
Chapman, B. & Brown, C. (2004) Translation termination in Arabidopsis thaliana:
characterisation of three versions of release factor 1. Gene, Vol.341, pp. 219-225.
Chapman, B.A., Bowers, J.E., Feltus, F.A., & Paterson, A.H. (2006) Buffering of crucial
functions by paleologous duplicated genes may contribute cyclicality to
angiosperm genome duplication. Proc.Natl.Acad.Sci.U.S.A., Vol.103, pp.2730-2735.
Chauvin, C., Salhi, S., Le Goff, C., Viranaicken, W., Diop, D., & Jean-Jean, O. (2005)
Involvement of human release factors eRF3a and eRF3b in translation termination
and regulation of the termination complex formation. Mol.Cell Biol., Vol.25, pp.
5801-5811.
Chen, C.C., Li, W.H., & Sung, H.M. (2007) Patterns of internal gene duplication in the course
of metazoan evolution. Gene, Vol.396, pp. 59-65.
Chen, L., Muhlrad, D., Hauryliuk, V., Cheng, Z., Lim, M.K., Shyp, V., Parker, R., & Song, H.
(2010) Structure of the Dom34-Hbs1 complex and implications for no-go decay.
Nat.Struct.Mol.Biol. 17, 1233-1240.
Comai, L., Tyagi, A.P., Winter, K., Holmes-Davis, R., Reynolds, S.H., Stevens, Y., & Byers, B.
(2000) Phenotypic instability and rapid gene silencing in newly formed arabidopsis
allotetraploids. Plant Cell, Vol.12, pp. 1551-1568.
Conant, G.C. & Wagner, A. (2005) The rarity of gene shuffling in conserved genes. Genome
Biol., Vol.6, R50.
Craigen, W.J., Lee, C.C., & Caskey, C.T. (1990) Recent advances in peptide chain
termination. Mol.Microbiol., Vol.4, pp. 861-865.
Cui, L., Wall, P.K., Leebens-Mack, J.H., Lindsay, B.G., Soltis, D.E., Doyle, J.J., Soltis, P.S.,
Carlson, J.E., Arumuganathan, K., Barakat, A., Albert, V.A., Ma, H., & dePamphilis,
C.W. (2006) Widespread genome duplications throughout the history of flowering
plants. Genome Res., Vol.16, pp. 738-749.
Czaplinski, K., Ruiz-Echevarria, M.J., Paushkin, S.V., Han, X., Weng, Y., Perlick, H.A., Dietz,
H.C., Ter Avanesyan, M.D., & Peltz, S.W. (1998) The surveillance complex interacts
with the translation release factors to enhance termination and degrade aberrant
mRNAs. Genes Dev., Vol.12, pp. 1665-1677.
Davis, J.C. & Petrov, D.A. (2005) Do disparate mechanisms of duplication add similar genes
to the genome? Trends Genet, Vol.21, pp. 548-551.
Davis, L. & Engebrecht, J. (1998) Yeast dom34 mutants are defective in multiple
developmental pathways and exhibit decreased levels of polyribosomes. Genetics,
Vol.149, pp. 45-56.

Gene Duplication

166
Dehal, P. & Boore, J.L. (2005) Two rounds of whole genome duplication in the ancestral
vertebrate. PloS.Biol., Vol.3, e314.
Dietrich, F.S., Voegeli, S., Brachat, S., Lerch, A., Gates, K., Steiner, S., Mohr, C., Pohlmann,
R., Luedi, P., Choi, S., Wing, R.A., Flavier, A., Gaffney, T.D., & Philippsen, P. (2004)
The Ashbya gossypii genome as a tool for mapping the ancient Saccharomyces
cerevisiae genome. Science, Vol.304, pp. 304-307.
Doma, M.K. & Parker, R. (2006) Endonucleolytic cleavage of eukaryotic mRNAs with stalls
in translation elongation. Nature, Vol.440, pp. 561-564.
Dontsova, M., Frolova, L., Vassilieva, J., Piendl, W., Kisselev, L., & Garber, M. (2000)
Translation termination factor aRF1 from the archaeon Methanococcus jannaschii is
active with eukaryotic ribosomes. FEBS Lett., Vol.472, pp. 213-216.
Doolittle, R.F. (1995) The multiplicity of domains in proteins. Annu.Rev.Biochem., Vol.64, pp.
287-314.
Durand, D. & Hoberman, R. (2006) Diagnosing duplications-can it be done? Trends Genet,
Vol.22, pp. 156-164.
Ferris, S.D. & Whitt, G.S. (1979) Evolution of the differential regulation of duplicate genes
after polyploidization. J.Mol.Evol., Vol.12, pp. 267-317.
Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.L., & Postlethwait, J. (1999)
Genetics, Vol.151, pp. 1531-1545.
Friedman, R. & Hughes, A.L. (2001) Pattern and timing of gene duplication in animal
genomes. Genome Res., Vol.11, pp. 1842-1847.
Friedman, R. & Hughes, A.L. (2003) The temporal distribution of gene duplication events in
a set of highly conserved human gene families. Mol.Biol.Evol., Vol.20, pp.
154-161.
Frischmeyer, P.A., van Hoof, A., O'Donnell, K., Guerrerio, A.L., Parker, R., & Dietz, H.C.
(2002) An mRNA surveillance mechanism that eliminates transcripts lacking
termination codons. Science, Vol.295, pp. 2258-2261.
Frolova, L.Y., Tsivkovskii, R.Y., Sivolobova, G.F., Oparina, N.Y., Serpinsky, O.I., Blinov,
V.M., Tatkov, S.I., & Kisselev, L.L. (1999) Mutations in the highly conserved GGQ
motif of class 1 polypeptide release factors abolish ability of human eRF1 to trigger
peptidyl-tRNA hydrolysis. RNA., Vol.5, pp. 1014-1020.
Gilbert, W. (1978) Why genes in pieces? Nature, Vol.271, pp. 501.
Graille, M., Chaillet, M., & van Tilbeurgh, H. (2008) Structure of yeast Dom34: a protein
related to translation termination factor eRF1 and involved in No-Go decay.
J.Biol.Chem., Vol.283, No.11, pp. 7145-7153.
Gu, X., Wang, Y., & Gu, J. (2002) Age distribution of human gene families shows significant
roles of both large- and small-scale duplications in vertebrate evolution. Nat.Genet.,
Vol.31, pp. 205-209.
Gu, Z., Cavalcanti, A., Chen, F.C., Bouman, P., & Li, W.H. (2002) Extent of gene duplication
in the genomes of Drosophila, nematode, and yeast. Mol.Biol.Evol., Vol.19, pp.
256-262.
Guigo, R., Muchnik, I., & Smith, T.F. (1996) Reconstruction of ancient molecular phylogeny.
Mol.Phylogenet.Evol., Vol.6, pp. 189-213.


167
Hahn, M.W. (2009) Distinguishing among evolutionary models for the maintenance of gene
duplicates. J.Hered. Vol.100, pp. 605-617.
Haldane, J. (1932) The causes of evolution, NY: Longmans, Green.
Harms, C.T., Armour, S.L., DiMaio, J.J., Middlesteadt, L.A., Murray, D., Negrotto, D.V.,
Thompson-Taylor, H., Weymann, K., Montoya, A.L., Shillito, R.D., et al. (1992)
Herbicide resistance due to amplification of a mutant acetohydroxyacid synthase
gene. Mol.Gen.Genet., Vol.233, pp. 427-435.
Hauryliuk, V., Zavialov, A., Kisselev, L., & Ehrenberg, M. (2006) Class-1 release factor eRF1
promotes GTP binding by class-2 release factor eRF3. Biochimie., Vol.88, pp.
747-757.
He, X. & Zhang, J. (2005) Rapid subfunctionalization accompanied by prolonged and
substantial neofunctionalization in duplicate gene evolution. Genetics, Vol.169, pp.
1157-1164.
Hittinger, C.T. & Carroll, S.B. (2007) Gene duplication and the adaptive evolution of a classic
genetic switch. Nature, Vol.449, pp. 677-681.
Hoshino, S., Miyazawa, H., Enomoto, T., Hanaoka, F., Kikuchi, Y., Kikuchi, A., & Ui, M.
(1989) A human homologue of the yeast GST1 gene codes for a GTP-binding
protein and is expressed in a proliferation-dependent manner in mammalian cells.
EMBO J., Vol.8, pp. 3807-3814.
Hoshino, S., Imai, M., Mizutani, M., Kikuchi, Y., Hanaoka, F., Ui, M., & Katada, T. (1998)
Molecular cloning of a novel member of the eukaryotic polypeptide chain-releasing
factors (eRF). Its identification as eRF3 interacting with eRF1. J.Biol.Chem., Vol.273,
pp. 22254-22259.
Hoshino, S., Imai, M., Kobayashi, T., Uchida, N., & Katada, T. (1999) The eukaryotic
polypeptide chain releasing factor (eRF3/GSPT) carrying the translation
termination signal to the 3'-Poly(A) tail of mRNA. Direct association of eRF3/GSPT
with polyadenylate-binding protein. J.Biol.Chem., Vol.274, pp. 16677-16680.
Hurles, M. (2004) Gene duplication: the genomic trade in spare parts. PloS.Biol., Vol.2,
E206.
Inagaki, Y. & Doolittle, F.W. (2000) Evolution of the eukaryotic translation termination
system: origins of release factors. Mol.Biol.Evol., Vol.17, pp. 882-889.
Inge-Vechtomov, S., Zhouravleva, G., & Philippe, M. (2003) Eukaryotic release factors (eRFs)
history. Biol.Cell, Vol.95, pp. 195-209.
Jaillon, O., Aury, J.M., Noel, B., Policriti, A., Clepet, C., Casagrande, A., Choisne, N.,
Aubourg, S., Vitulo, N., Jubin, C., Vezzi, A., Legeai, F., Hugueney, P., Dasilva, C.,
Horner, D., Mica, E., Jublot, D., Poulain, J., Bruyere, C., Billault, A., Segurens, B.,
Gouyvenoux, M., Ugarte, E., Cattonaro, F., Anthouard, V., Vico, V., Del Fabbro, C.,
Alaux, M., Di Gaspero, G., Dumas, V., Felice, N., Paillard, S., Juman, I., Moroldo,
M., Scalabrin, S., Canaguier, A., Le, C., I, Malacrida, G., Durand, E., Pesole, G.,
Laucou, V., Chatelet, P., Merdinoglu, D., Delledonne, M., Pezzotti, M., Lecharny,
A., Scarpelli, C., Artiguenave, F., Pe, M.E., Valle, G., Morgante, M., Caboche, M.,
Adam-Blondon, A.F., Weissenbach, J., Quetier, F., & Wincker, P. (2007) The
grapevine genome sequence suggests ancestral hexaploidization in major
angiosperm phyla. Nature, Vol.449, pp. 463-467.

Gene Duplication

168
Jakobsen, C.G., Segaard, T.M., Jean-Jean, O., Frolova, L., & Justesen, J. (2001) Identification
of a novel termination release factor eRF3b expressing the eRF3 activity in vitro and
in vivo. Mol.Biol.(Mosk), Vol.35, pp. 672-681.
Jean-Jean, O., Le Goff, X., & Philippe, M. (1996) Is there a human [psi]? C.R.Acad.Sci.III.,
Vol.319, pp. 487-492.
Jorgensen, R., Ortiz, P.A., Carr-Schmid, A., Nissen, P., Kinzy, T.G., & Andersen, G.R. (2003)
Two crystal structures demonstrate large conformational changes in the eukaryotic
ribosomal translocase. Nat.Struct.Biol., Vol. 10, pp. 379-385.
Kapp, L.D. & Lorsch, J.R. (2004) The molecular mechanics of eukaryotic translation.
Annu.Rev.Biochem., Vol.73, pp. 657-704.
Kashkush, K., Feldman, M., & Levy, A.A. (2002) Gene loss, silencing and activation in a
newly synthesized wheat allotetraploid. Genetics., Vol.160, pp. 1651-1659.
Kellis, M., Birren, B.W., & Lander, E.S. (2004) Proof and evolutionary analysis of ancient
genome duplication in the yeast Saccharomyces cerevisiae. Nature, Vol.428, pp.
617-624.
Kisselev, L., Ehrenberg, M., & Frolova, L. (2003) Termination of translation: interplay of
mRNA, rRNAs and release factors? EMBO J., Vol.22, pp. 175-182.
Kobayashi, K., Kikuno, I., Kuroha, K., Saito, K., Ito, K., Ishitani, R., Inada, T., & Nureki, O.
(2010) Structural basis for mRNA surveillance by archaeal Pelota and GTP-bound
EF1alpha complex. Proc.Natl.Acad.Sci.U.S.A., Vol.107, pp. 17575-17579.
Kodama, H., Ito, K., & Nakamura, Y. (2007) The role of N-terminal domain of translational
release factor eRF3 for the control of functionality and stability in S. cerevisiae. Genes
Cells, Vol.12, pp. 639-650.
Koonin, E.V., Bork, P., & Sander, C. (1994) A novel RNA-binding motif in omnipotent
suppressors of translation termination, ribosomal proteins and a ribosome
modification enzyme? Nucleic Acids Res., Vol.22, pp. 2166-2167.
Kushnirov, V.V. & Ter Avanesyan, M.D. (1998) Structure and replication of yeast prions.
Cell, Vol. 94, pp. 13-16.
Larhammar, D., Lundin, L.G., & Hallbook, F. (2002) The human Hox-bearing chromosome
regions did arise by block or chromosome (or even genome) duplications. Genome
Res., Vol.12, pp. 1910-1920.
Lavorgna, G., Patthy, L., & Boncinelli, E. (2001) Were protein internal repeats formed by
"bricolage"? Trends Genet, Vol.17, pp. 120-123.
Le Goff, C., Zemlyanko, O., Moskalenko, S., Berkova, N., Inge-Vechtomov, S., Philippe, M.,
& Zhouravleva, G. (2002) Mouse GSPT2, but not GSPT1, can substitute for yeast
eRF3 in vivo. Genes Cells, Vol.7, pp. 1043-1057.
Lecompte, O., Ripp, R., Thierry, J.C., Moras, D., & Poch, O. (2002) Comparative analysis of
ribosomal proteins in complete genomes: an example of reductive evolution at the
domain scale. Nucleic Acids Res., Vol.30, pp. 5382-5390.
Lee, H.H., Kim, Y.S., Kim, K.H., Heo, I., Kim, S.K., Kim, O., Kim, H.K., Yoon, J.Y., Kim, H.S.,
Kim, d.J., Lee, S.J., Yoon, H.J., Kim, S.J., Lee, B.G., Song, H.K., Kim, V.N., Park,
C.M., & Suh, S.W. (2007) Structural and functional insights into Dom34, a key
component of no-go mRNA decay. Mol.Cell., Vol.27, pp. 938-950.


169
Lee, H.S. & Chen, Z.J. (2001) Protein-coding genes are epigenetically regulated in Arabidopsis
polyploids. Proc.Natl.Acad.Sci.U.S.A., Vol.98, pp. 6753-6758.
Lewis, E.B. (1951) Pseudoallelism and gene evolution. Cold Spring Harb.Symp.Quant.Biol.,
Vol.16, pp. 159-174.
Li, W.H. (1997) Molecular evolution Sunderland, Mass: Sinauer Associates.
Liang, A., Brunen-Nieweler, C., Muramatsu, T., Kuchino, Y., Beier, H., & Heckmann, K.
(2001) The ciliate Euplotes octocarinatus expresses two polypeptide release factors of
the type eRF1. Gene, Vol.262, pp. 161-168.
Long, M. (2000) A new function evolved from gene fusion. Genome Res., Vol.10, pp.
1655-1657.
Long, M., Betran, E., Thornton, K., & Wang, W. (2003) The origin of new genes: glimpses
from the young and old. Nat.Rev.Genet., Vol.4, pp. 865-875.
Lozupone, C.A., Knight, R.D., & Landweber, L.F. (2001) The molecular basis of nuclear
genetic code change in ciliates. Curr.Biol., Vol.11, pp. 65-74.
Lynch, M. & Conery, J.S. (2000) The evolutionary fate and consequences of duplicate genes.
Science, Vol.290, pp. 1151-1155.
Lynch, M., O'Hely, M., Walsh, B., & Force, A. (2001) The probability of preservation of a
newly arisen gene duplicate. Genetics, Vol.159, pp. 1789-1804.
Marcotte, E.M., Pellegrini, M., Yeates, T.O., & Eisenberg, D. (1999) A census of protein
repeats. J.Mol.Biol., Vol.293, pp. 151-160.
Mateyak, M.K. & Kinzy, T.G. (2010) eEF1A: thinking outside the ribosome. J.Biol.Chem.,
Vol.285, pp. 21209-21213.
McLysaght, A., Hokamp, K., & Wolfe, K.H. (2002) Extensive genomic duplication during
early chordate evolution. Nat.Genet., Vol.31, pp. 200-204.
Meyer, A. & Schartl, M. (1999) Gene and genome duplications in vertebrates: the one-to-four
(-to-eight in fish) rule and the evolution of novel gene functions. Curr Opin.Cell
Biol., Vol.11, pp. 699-704.
Meyer, F., Schmidt, H.J., Plumper, E., Hasilik, A., Mersmann, G., Meyer, H.E., Engstrom, A.,
& Heckmann, K. (1991) UGA is translated as cysteine in pheromone 3 of Euplotes
octocarinatus. Proc.Natl.Acad.Sci.U.S.A., Vol.88, pp. 3758-3761.
Muller, H.J. (1936) Bar duplication. Science, Vol.83, pp. 528-530.
Nakamura, Y. & Ito, K. (1998) How protein reads the stop codon and terminates translation.
Genes Cells, Vol.3, pp. 265-278.
Nakamura, Y. & Ito, K. (2003) Making sense of mimic in translation termination. Trends
Biochem.Sci., Vol.28, pp. 99-105.
Nissen, P., Kjeldgaard, M., & Nyborg, J. (2000) Macromolecular mimicry. EMBO J., Vol.19,
pp. 489-495.
Ohno, S. (1970). Evolution by Gene Duplication. NY: Springer Verlag.
Otto, S.P. & Whitton, J. (2000) Polyploid incidence and evolution. Annu.Rev.Genet., Vol.34,
pp. 401-437.
Passos, D.O., Doma, M.K., Shoemaker, C.J., Muhlrad, D., Green, R., Weissman, J., Hollien, J.,
& Parker, R. (2009) Analysis of Dom34 and its function in no-go decay.
Mol.Biol.Cell., Vol.20, pp. 3025-3032.

Gene Duplication

170
Piatigorsky, J. (2003) Crystallin genes: specialization by changes in gene regulation may
precede gene duplication. J.Struct.Funct.Genomics, Vol.3, pp. 131-137.
Piatigorsky, J., O'Brien, W.E., Norman, B.L., Kalumuck, K., Wistow, G.J., Borras, T.,
Nickerson, J.M., & Wawrousek, E.F. (1988) Gene sharing by delta-crystallin and
argininosuccinate lyase. Proc.Natl.Acad.Sci.U.S.A., Vol.85, pp. 3479-3483.
Piskur, J. (2001) Origin of the duplicated regions in the yeast genomes. Trends Genet., Vol.17,
pp. 302-303.
Ramakrishnan, V. (2002) Ribosome structure and the mechanism of translation. Cell, Vol.108,
pp. 557-572.
Reams, A.B. & Neidle, E.L. (2004) Selection for gene clustering by tandem duplication.
Annu.Rev.Microbiol., Vol.58, pp. 119-142.
Rodin, S.N. & Riggs, A.D. (2003) Epigenetic silencing may aid evolution by gene
duplication. J.Mol.Evol., Vol.56, pp. 718-729.
Romero, D. & Palacios, R. (1997) Gene amplification and genomic plasticity in prokaryotes.
Annu.Rev.Genet., Vol.31, pp. 91-111.
Saito, K., Kobayashi, K., Wada, M., Kikuno, I., Takusagawa, A., Mochizuki, M., Uchiumi, T.,
Ishitani, R., Nureki, O., & Ito, K. (2010) Omnipotent role of archaeal elongation
factor 1 alpha (EF1alpha) in translational elongation and termination, and quality
control of protein synthesis. Proc.Natl.Acad.Sci.U.S.A. Vol.107, pp. 19242-19247.
Scannell, D.R., Butler, G., & Wolfe, K.H. (2007) Yeast genome evolution--the origin of the
species. Yeast, Vol.24, pp. 929-942.
Scannell, D.R., Byrne, K.P., Gordon, J.L., Wong, S., & Wolfe, K.H. (2006) Multiple rounds of
speciation associated with reciprocal gene loss in polyploid yeasts. Nature, Vol.440,
pp. 341-345.
Seit-Nebi, A., Frolova, L., Justesen, J., & Kisselev, L. (2001) Class-1 translation termination
factors: invariant GGQ minidomain is essential for release activity and ribosome
binding but not for stop codon recognition. Nucleic Acids Res., Vol.29, pp.
3982-3987.
Seoighe, C. & Gehring, C. (2004) Genome duplication led to highly selective expansion of
the Arabidopsis thaliana proteome. Trends Genet., Vol.20, pp. 461-464.
Seoighe, C. & Wolfe, K.H. (1999) Yeast genome evolution in the post-genome era. Curr
Opin.Microbiol., Vol.2, pp. 548-554.
Serio, T.R. & Lindquist, S.L. (1999) [PSI+]: an epigenetic modulator of translation
termination efficiency. Annu.Rev.Cell Dev.Biol., Vol.15, pp. 661-703.
Shoemaker, C.J., Eyler, D.E., & Green, R. (2010) Dom34:Hbs1 promotes subunit dissociation
and peptidyl-tRNA drop-off to initiate no-go decay. Science, Vol.330, pp.
369-372.
Shyr, Y.Y., Hepburn, A.G., & Widholm, J.M. (1992) Glyphosate selected amplification of the
5-enolpyruvylshikimate-3-phosphate synthase gene in cultured carrot cells.
Mol.Gen.Genet., Vol.232, pp. 377-382.
Sonenberg, N. & Dever, T.E. (2003) Eukaryotic translation initiation factors and regulators.
Curr Opin.Struct.Biol., Vol.13, pp. 56-63.
Spring, J. (1997) Vertebrate evolution by interspecific hybridisation are we polyploid?
FEBS Lett., Vol.400, pp. 2-8.


171
Stark, H., Rodnina, M.V., Wieden, H.J., Zemlin, F., Wintermeyer, W., & van Heel, M. (2002)
Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-
recognition complex. Nat.Struct.Biol., Vol.9, pp. 849-854.
Sturtevant, A.H. (1925) The Effects of Unequal Crossing over at the Bar Locus in Drosophila.
Genetics, Vol.10, pp. 117-147.
Taylor, J.S. & Raes, J. (2004) Duplication and divergence: the evolution of new genes and old
ideas. Annu.Rev.Genet., Vol.38, pp. 615-643.
Tuskan, G.A., Difazio, S., Jansson, S., Bohlmann, J., Grigoriev, I., Hellsten, U., Putnam, N.,
Ralph, S., Rombauts, S., Salamov, A., Schein, J., Sterck, L., Aerts, A., Bhalerao, R.R.,
Bhalerao, R.P., Blaudez, D., Boerjan, W., Brun, A., Brunner, A., Busov, V., Campbell,
M., Carlson, J., Chalot, M., Chapman, J., Chen, G.L., Cooper, D., Coutinho, P.M.,
Couturier, J., Covert, S., Cronk, Q., Cunningham, R., Davis, J., Degroeve, S., Dejardin,
A., Depamphilis, C., Detter, J., Dirks, B., Dubchak, I., Duplessis, S., Ehlting, J., Ellis, B.,
Gendler, K., Goodstein, D., Gribskov, M., Grimwood, J., Groover, A., Gunter, L.,
Hamberger, B., Heinze, B., Helariutta, Y., Henrissat, B., Holligan, D., Holt, R., Huang,
W., Islam-Faridi, N., Jones, S., Jones-Rhoades, M., Jorgensen, R., Joshi, C., Kangasjarvi,
J., Karlsson, J., Kelleher, C., Kirkpatrick, R., Kirst, M., Kohler, A., Kalluri, U., Larimer,
F., Leebens-Mack, J., Leple, J.C., Locascio, P., Lou, Y., Lucas, S., Martin, F., Montanini,
B., Napoli, C., Nelson, D.R., Nelson, C., Nieminen, K., Nilsson, O., Pereda, V., Peter,
G., Philippe, R., Pilate, G., Poliakov, A., Razumovskaya, J., Richardson, P., Rinaldi, C.,
Ritland, K., Rouze, P., Ryaboy, D., Schmutz, J., Schrader, J., Segerman, B., Shin, H.,
Siddiqui, A., Sterky, F., Terry, A., Tsai, C.J., Uberbacher, E., Unneberg, P., Vahala, J.,
Wall, K., Wessler, S., Yang, G., Yin, T., Douglas, C., Marra, M., Sandberg, G., Van de,
P.Y., & Rokhsar, D. (2006) The genome of black cottonwood, Populus trichocarpa (Torr.
& Gray). Science, Vol. 313, pp. 1596-1604.
Urbero, B., Eurwilaichitr, L., Stansfield, I., Tassan, J.P., Le Goff, X., Kress, M., & Tuite, M.F.
(1997) Expression of the release factor eRF1 (Sup45p) gene of higher eukaryotes in
yeast and mammalian tissues. Biochimie, Vol.79, pp. 27-36.
Valente, L. & Kinzy, T.G. (2003) Yeast as a sensor of factors affecting the accuracy of protein
synthesis. Cell Mol.Life Sci., Vol.60, pp. 2115-2130.
Valle, M., Sengupta, J., Swami, N.K., Grassucci, R.A., Burkhardt, N., Nierhaus, K.H.,
Agrawal, R.K., & Frank, J. (2002) Cryo-EM reveals an active role for aminoacyl-
tRNA in the accommodation process. EMBO J., Vol. 21, pp. 3557-3567.
van den Elzen, A.M., Henri, J., Lazar, N., Gas, M.E., Durand, D., Lacroute, F., Nicaise, M.,
van Tilbeurgh, H., Seraphin, B., & Graille, M. (2010) Dissection of Dom34-Hbs1
reveals independent functions in two RNA quality control pathways.
Nat.Struct.Mol.Biol. Vol.17, pp. 1446-1452.
van Hoof, A., Frischmeyer, P.A., Dietz, H.C., & Parker, R. (2002) Exosome-mediated
recognition and degradation of mRNAs lacking a termination codon. Science,
Vol.295, pp. 2262-2264.
van Rijk, A. & Bloemendal, H. (2003) Molecular mechanisms of exon shuffling: illegitimate
recombination. Genetica, Vol.18, pp. 245-249.
Vasudevan, S., Peltz, S.W., & Wilusz, C.J. (2002) Non-stop decay-a new mRNA surveillance
pathway. Bioessays, Vol.24, pp. 785-788.

Gene Duplication

172
Vision, T.J., Brown, D.G., & Tanksley, S.D. (2000) The origins of genomic duplications in
Arabidopsis. Science, Vol.290, pp. 2114-2117.
Wallrapp, C., Verrier, S.B., Zhouravleva, G., Philippe, H., Philippe, M., Gress, T.M., & Jean-
Jean, O. (1998) The product of the mammalian orthologue of the Saccharomyces
cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3)
but does not carry eRF3-like activity. FEBS Lett., Vol.440, pp. 387-392.
Wang, W., Czaplinski, K., Rao, Y., & Peltz, S.W. (2001) The role of Upf proteins in
modulating the translation read-through of nonsense-containing transcripts. EMBO
J., Vol.20, pp. 880-890.
Wang, Y., Chai, B., Wang, W., & Liang, A. (2010) Functional characterization of polypeptide
release factor 1b in the ciliate Euplotes. Biosci.Rep., Vol.30, pp. 425-431.
Wang, Y. & Gu, X. (2000) Evolutionary patterns of gene families generated in the early stage
of vertebrates. J.Mol.Evol., Vol.51, pp. 88-96.
Wolfe, K. (2000) Robustness-it's not where you think it is. Nat.Genet., Vol.25, pp. 3-4.
Wong, S., Butler, G., & Wolfe, K.H. (2002) Gene order evolution and paleopolyploidy in
hemiascomycete yeasts. Proc.Natl.Acad.Sci.U.S.A., Vol.99, pp. 9272-9277.
Zhouravleva, G., Schepachev, V., Petrova, A., Tarasov, O., & Inge-Vechtomov, S. (2006)
Evolution of translation termination factor eRF3: Is GSPT2 generated by
retrotransposition of GSPT1's mRNA? IUBMB.Life., Vol.58, pp. 199-202.
10
Analysis of Duplicate Gene Families in Microbial
Genomes and Application to the Study of Gene
Duplication in M. tuberculosis
Venu Vuppu and Nicola Mulder
Computational Biology Group, Department of Clinical Laboratory Sciences
Institute of Infectious Diseases and Molecular Medicine, Health Science Faculty
University of Cape Town
South Africa
1. Introduction
Though considerable sequence information from different organisms was available prior
to the recent advances in genome sequencing technology, the foundation for our current
understanding of the mechanisms of bacterial pathogenesis was laid by the release of the
first complete genome sequence of Heamophilus influenza in 1995 (Fraser-Liggett, 2005).
Ever since, significant progress in the availability of data for different genomes has been
possible due to the contribution of various genome sequencing projects (Koonin & Wolf,
2008). Despite the complete genome sequences of many pathogenic organisms being
available, the mortality rates due to these infectious agents still remains a problem,
highlighting the need to decipher the complex molecular mechanisms responsible for
survival of the bacteria. The wealth of complete genome information for pathogens can be
effectively explored using comparative genomic tools for the identification of common
and unique sets of genes involved in the propagation of virulence. Sequence comparison
tools have been developed to identify homologous genes from the complete genomes of
microorganisms. Homologous genes which arise from speciation tend to maintain
functions similar to that of their ancestral molecule and are known as orthologs, while the
genes originating from duplication events often evolve new functions and are defined as
paralogs (Tatusov et al., 1997).
The world of microbes is highly diverse with genome complexity differing across a wide
range of microorganisms. In general, the difference in the complexity of genomes is dictated
by the life style and environment of the organism (Cordero & Hogeweg, 2009). Life style
plays an important role in regulating the genome dynamics of an organism, and functional
novelty provided by gene duplication is thought to enhance the adaptation capability of the
organism. In addition, horizontal transfer of operons or functional units of genes from
external sources may provide an immediate functional benefit to the organism, thereby
adding to the functional complexity of the genomes. The availability of complete genome
sequences of important mycobacteria such as Mycobacterium tuberculosis, Mycobacterium
ulcerans, Mycobacterium bovis, Mycobacterium leprae, Mycobacterium paratuberculosis,
Mycobacterium avium and others, can be used to gain deeper insights into possible

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mechanisms of prokaryotic gene innovation. Genome data has been used in recent studies to
compare different species of mycobacteria, as well as different strains, to understand the
evolution and pathogenesis of M. tuberculosis (Marri et al., 2006). In our study, duplicate
gene sets from different mycobacteria were investigated to identify the distribution of
important functional classes of protein families, and the evolution of these functional classes
was further analyzed by comparing their genetic divergence following duplication.
The importance of gene duplication in prokaryotic gene innovation is well established and
comparative analysis of duplicate genes with basic characteristic features of genomes like
GC content and genome size may aid in deciphering their contributions. In contrast to
eukaryotes, GC content varies widely across different bacterial genomes (Mann & Chen,
2010), and analysis of GC variations between related bacteria could be useful in establishing
evolutionary relationships (Mann et al., 2010). The focus of the majority of earlier studies
was on deciphering the role of GC composition in HGT (Nelson et al., 1999; Hamady et al.,
2006), transcription start and stop sites (Zhang et al., 2004), nucleotide substitution rates
(DeRose-Wilson & Gaut, 2007), optimal growth temperature (Basak & Ghosh, 2005; Musto,
2006) and metabolic characteristics (Naya et al., 2002). Furthermore, genome size has been
reported to increase with an increase in number of genes in duplicate gene families (Snel et
al., 2002; Pushker et al., 2004). In this study we analyzed the genomes of 56 pathogenic and
20 non-pathogenic microorganisms to identify and characterize the expanded gene families
across these organisms. In addition to the GC content, we investigated the relationship
between genome size and duplicate gene percentage. On finding sufficient evidence for a
correlation between genome size and extent of gene duplication, we further investigated the
significance of duplicate genes in enhancing genome complexity. He and Zhang (2005)
previously reported the importance of gene duplication in enhancing genome and organism
complexity in eukaryotes. However, due to the difference in the selective pressures
operating on prokaryotic and eukaryotic genomes, we used the duplicate and single copy
genes to investigate the influence of protein lengths on genome and organism complexity of
prokaryotic organisms, with a specific focus on investigating the role of duplicate genes in
enhancing the genome complexity of M. tuberculosis.
2. Materials and methods
2.1 Data selection and identification of homologous sequences
Comparative sequence analysis of different genomes is the most common approach for
identifying orthologs and paralogs. However, here we used both the sequence and protein
signature data as the latter could substantiate the former, and enables identification of more
distantly related members of a protein family. We collected non-redundant protein sets for 76
microorganisms, including pathogens and non-pathogens, to identify expanded gene families in
these organisms. The selection of the non-pathogenic bacteria in this study is of value, since
many of these may also contain virulent genes which could act as barriers conferring protection
against the defense mechanisms of the host, thus enhancing the survival capabilities and
adapting the organism to intracellular conditions. In addition, acquisition of specific virulent
gene clusters can transform these non-pathogenic agents to pathogenic microorganisms.
For the selected organisms, approximately 1,91,497 protein signatures, 2,47,858 protein
sequences, Genome size and G+C composition data were retrieved from the InterPro
(http://www.ebi.ac.uk/interpro) (Apweiler et al., 2001; Mulder et al., 2007) and Integr8
(http://www.ebi.ac.uk/integr8) (Kersey et al., 2005) databases respectively. The protein
Analysis of Duplicate Gene Families
in Microbial Genomes and Application to the Study of Gene Duplication in M. tuberculosis

175
signature data from InterPro enabled the identification of approximately 27,827 proteins
which exhibited complete domain identity (same InterPro matches) over their entire length
to one or more proteins in M. tuberculosis strain H37Rv. Within each organism and across all
organisms, the proteins showing complete domain identity were grouped together as
duplicate gene sets or ortholog and paralog sets, respectively, and those with no common
signature matches were considered to be single copies. In addition to the identification of
expanded families using InterPro data, homologous sequences were clustered using
BlastClust in two separate clustering procedures:
a. Independent Genome Clustering: This involves within genome clustering to generate
clusters of paralogs or protein families for each genome. BlastClust was executed at a
wide range of percentage identities over varying lengths of the sequence to select the
optimum parameters. Amongst the tested parameters, a 30% similarity over 60%
sequence length cut-off was chosen, as it generated a suitable number of clusters (in line
with previously reported numbers of duplicated families for M. tuberculosis).
b. Multiple Genome Clustering: In this, all of the 76 genomes were appended together
for the clustering of related proteins (orthologs and paralogs). In addition, the
clustering of six of the mycobacterial species was performed separately for the
evolutionary analysis of expanded gene families in M. tuberculosis.
2.2 Evolutionary analysis
For evolutionary studies, in addition to 66 paralogous gene clusters, 116 multiple genome
clusters from the phylogenetic matrix of six of the closely related mycobacterial genomes
that showed gene family expansions in both M. tuberculosis and M. leprae, as well as other
mycobacteria, were selected. The proteins in each of the clusters were aligned with T-coffee
(Notredame et al., 2000), and poorly aligned regions were edited using the Gblocks program
(Castresana, 2000) with adjustments in the default settings for the generation of optimal
sequence alignments. For each of these protein alignments, selection of the best-fitting
amino acid substitution model was performed according to the Akaike informational
criterion, and the gamma correction factor (alpha), the proportion of invariable sites (I), and
observed amino acid frequencies (F) were estimated and selected for subsequent
phylogenetic analysis using ProtTest (Abascal et al., 2005). Since, PhyML is a maximum
likelihood method with the ability to incorporate the estimated values of alpha, proportion
of invariable sites, and observed frequencies, the tree topologies for the gene sets in the
identified clusters were constructed using this program (Guindon & Gascuel, 2003). The
genetic distance measures from each of the estimated tree topologies were used to compute
average and maximum genetic distance using Perl scripts.
3. Results and discussion
3.1 Identification of expanded gene families and relation to GC content and genome
size
We used sequence clustering and protein signature data to identify expanded genes families
within and across several different microbial genomes. The across-genome clustering of
protein sequence data yielded 1,984 expanded genes in 441 clusters for M. tuberculosis
H37Rv. The protein signature method allowed us to group 30,885 proteins into 2238 clusters
from all the organisms. InterPro signatures usually match between 50% and 80% of a
genome, so data is not available for every protein. Since signature data enables identification

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of more distantly related members of a cluster, but loses data where proteins do not match
InterPro, the sequence and signature-based cluster data was merged, and used to generate a
phylogenetic profile, which reflected the number of copies in each expanded family for each
organism. From this, we identified 2011 duplicate/expanded genes in 461 clusters for M.
tuberculosis, confirming previous reports that the duplicate genes make up approximately
half of the M. tuberculosis genome (Tekaia et al., 1999). The percentages derived from the 2
methods are shown in Table 1 for the 6 mycobacteria studied. The 461 clusters in the merged
data include gene families that are also expanded in different organisms.

S.No Organism Sequence Signature Union
1 M. tuberculosis 31.47% 38% 50.96%
2 M. bovis 30.28% 42% 48.69%
3 M. paratuberculosis 39.75% 49% 56.46%
4 M. avium 42.06% 49% 55.19%
5 M. ulcerans 36.82% 46% 53.51%
6 M. leprae 12.03% 20% 30.44%
Table 1. Percentage of the genome belonging to expanded gene families for the
mycobacteria. Data was generated using sequence clustering, protein signatures and a
combination of the two (union).
Next, we investigated the GC composition of different bacteria in relation to the duplicate
gene percentages (Figure 1) to understand the characteristic features of genomes
maintaining high percentages of duplicate genes. A statistical analysis of the data using
Pearsons correlation, revealed a moderate correlation between the GC content and
estimated duplicate gene percentages. However, the analysis of the trend lines of the scatter
plot in figure 1 reveals the presence of three different kinds of relationships in the data: i) an
initial increase of the trend line, ii) the initial increase is followed by a phase of neutrality,
iii) and a steady increase of the trend line follows the phase of neutrality. We analyzed
histograms of GC content and duplicate gene percentages and observed differences in the
modality of the data distribution; GC percentages followed a trimodal distribution, while
the duplicate gene percentages followed a unimodal distribution. Thus, although a positive
correlation could be inferred from the analysis of the scatter plot, the correlation coefficient
could be subdued by the differences in the modality of the data distributions. Hence, based
on the analysis of the scatter plot and trimodal distribution of the GC percentage histogram,
the organisms were grouped into three categories based on their GC compositions:
Group 1: Organisms having GC content greater than 54 percent.
Group 2: Organisms having GC content greater than 44 percent and less than 54 percent.
Group 3: Organisms having GC content less than 44 percent.
We then performed a one-way ANOVA on the data (Table 2). Taking into consideration the
mean square values (Mean Sq) and the calculated p-value of 2 x 10
-16
, we predicted that the
mean variance between groups is significant compared to the within sample variance. These
results indicate the existence of differences in the means of the three groups of organisms,
and hence, we reject the null hypothesis and accept the alternative. Further, to estimate how
significantly different the means of each group are compared to one another, a Tukeys
Honest Significant Difference (Tukeys HSD) test was performed, and significant differences
in the mean values of group2 and group1, group3 and group1, and group3 and group2 were
found. From the results table of the Tukeys multiple comparison test (Table 3), it can be

177

Fig. 1. Scatter plot analysis of the relationship between GC content and duplicate gene
percentages of the selected organisms. The percent GC content is plotted on the X-axis and
duplicate gene percentage on the Y-axis. The graph suggests that a positive correlation
between GC content and duplicate gene percentages exists for the majority of the
investigated organisms.
Df Sum Sq Mean Sq F value Pr(>F)
Groups 2 13174.8 6587.4 417.28 <2.2e-16***
Residuals 73
Signif Codes 0 *** 0.001 ** 0.01 * 0.05 .0.1 1
Table 2. One-Way ANOVA Results. The columns of the table display the degrees of freedom
(df), sum square values (Sum Sq), Mean square values (Mean Sq), F value and p-value
(Pr(>F)) reported by One-Way ANOVA for the data.

Tukeys Multiple Comparison of Means
Groups Diff Lwr Upr
G2-G1 -16.36 -19.08 -13.64
G3-G1 -31.54 -34.15 -28.92
G3-G2 -15.18 -17.88 -12.48
Table 3. The table displays the differences between the mean values of the groups. The Groups
column represents the investigated groups: group2 and group1 (G2-G1), group3-group1 (G3-
G1), and group3-group2 (G3-G2). The differences in the means of the groups are given by the
difference (diff) column, and the lower (lwr) and upper (upr) columns represent the lower and
upper boundaries for the estimated mean difference between the groups.

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inferred that both groups, G2-G1 and G3-G2 exhibit similar mean differences (-16.36 and -
15.18). However, the mean differences of both these groups are higher than the mean
difference (-31.54) of group G3-G1. Therefore, group2 organisms, which have higher mean
differences compared with group1 and group3, could be responsible for the reduced
correlation coefficient values. Hence, their elimination from the list of investigated
organisms could result in the prediction of strong positive correlation between the GC
composition and duplicate gene percentages of group1 and group3 organisms. Thus, we
suggest that gene duplication events may be a characteristic feature of GC rich bacterial
genomes. Since all of the selected mycobacterial species in the present study are
representatives of group1, the phenomenon of gene duplication in this genus could be
attributed to its high GC content.
In addition to GC compositions, we analyzed the influence of duplicate genes on the
physical expansion of the genomes (Figure 2). An observed correlation coefficient value of
0.84 at a p-value of 2 x 10
-16
between the genome size and duplicate genes provides sufficient
evidence to prove the contribution of duplicate genes to genome expansion of these
organisms. This is not surprising, since the addition of genes through gene duplication will
obviously increase genome size unless some genes are lost in the process.

Fig. 2. The graph displays the relationship between duplicate gene percentage and genome
size for the selected organisms. The identified duplicate gene percentages are plotted on the
X-axis and genome size on the Y-axis. From the graph, a positive correlation can be
observed between duplicate gene percentages and genome size.

179
3.2 Investigation of functional complexity of the duplicate and single copy genes
In eukaryotes, single copy genes were reported to be shorter and to contain fewer domains
than duplicate genes (He & Zhang, 2005). Here, we investigated the gene lengths and
complexity (using domain number) of both pathogenic and non-pathogenic bacteria to
determine the role of gene duplication in enhancing genome complexity in prokaryotes. A
preliminary determination of the functional complexity of the expanded genes in the
selected organisms was graphically analyzed by plotting the mean gene lengths of both the
duplicate and single copy genes. Figure 3 shows that the average gene length in the majority
of the organisms is comparatively higher for duplicate genes than for single copy genes.
The difference in the mean gene lengths of duplicate and single copy genes was statistically
analyzed using the Mann-Whitney U test. An observed W value of 4880 at a p-value of 2 x
10
-13
estimated from the Mann-Whitney U test confirms that the mean gene lengths of the
duplicate genes are significantly higher than that of the single copy genes.

Fig. 3. Comparison of Functional Complexity of the Expanded and Single Copy Gene
Families Based on Nucleotide Sequence Data. The graph displays the mean gene lengths of
duplicate and single copy genes in the investigated organisms. The organisms are plotted on
the X-axis and the corresponding mean gene length on the Y-axis.
We went on to investigate the domain complexity of single and duplicate copy genes to
further enhance our understanding of the functional complexity of these organisms. As a
measure of domain complexity, the number of domains present in each of the

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180
corresponding proteins of the genes was computed from InterPro data, and the mean for the
total number of domains was estimated for the duplicate and single copy genes using Perl
scripts. From figure 4, we can see that the mean number of domains per duplicate gene is
lower than that of the single copy genes. This suggests that single copy genes should be
more complex due to the presence of more domains. We further analyzed the results, by
statistically comparing the difference in the mean domain numbers of duplicate and single
copy genes using the Mann-Whitney U test. From the resulting W value of 5717 at a p-value
of 2 x 10
-16
, we inferred that the mean number of domains per single copy genes is
significantly higher than that of duplicate genes. Thus, these studies suggest that the single
copy genes are functionally more complex than duplicate genes. This was a surprising
result, given that the mean length of duplicate genes was found to be higher than that of the
single copy genes. Therefore, we specifically investigated the influence of gene lengths on
the domain complexity of M. tuberculosis, and compared this statistic in two other
organisms, Leptospira interrogans and the model organism, Escherichia coli.

Fig. 4. Comparison of Functional Complexity of the Expanded and Single Copy Genes Based
on InterPro Signature Data in Selected organisms. The graph displays the mean number of
domains per protein in the duplicate and single copy genes of each organism. The
organisms investigated are plotted on the X-axis and the corresponding mean number of
domains per protein for each organism on the Y-axis.

181
For each of these three organisms, the total number of genes in the genome was retrieved,
and for every gene, we estimated the total number of domains to determine the relationship
between gene length and domain number (Figure 5 -Whole Genome Analysis). In addition,
the number of domains for each of the duplicate (Figure 6) and single copy genes (Figure 7)
were also estimated. The preliminary analysis of the relationships using scatter plots
suggested that the number of domains per gene does not necessarily increase with an
increase in the gene length. Further, correlation coefficient values estimated from the
Pearson moment correlation were used for statistical confirmation of the relationships.

Fig. 5. Investigation of number of domains per gene in the M. tuberculosis H37Rv, E. coli and
L. interrogans genomes (Whole Genome Analysis). The graph displays the relationships
between gene length and number of domains. The sequence lengths of each gene are plotted
on the X-axis and the corresponding number of domains per gene on the Y-axis. From the
graph, it can be inferred that the gene length is not necessarily dependent on domain
number.

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Fig. 6. Investigation of number of domains per duplicate gene in the M. tuberculosis H37Rv,
E. coli and L. interrogans genomes (Duplicate Gene Analysis). The graph displays the
relationship between sequence length (X-axis) and number of domains (Y-axis) of the
duplicate genes.
The correlation coefficient values for the whole genome, duplicate gene and single copy
gene analysis in E. coli were 0.48, 0.47, and 0.49, respectively, while the values of 0.39, 0.25,
and 0.53 were reported for L. interrogans (Table 4). The results from these two organisms
suggest that the number of domains does not increase significantly with the increase in gene
length and hence, domain complexity may be independent of the gene length or vice versa.
Although the reported correlation coefficient values of 0.58, 0.62 and 0.59 corresponding to
the whole genome, duplicate and single copy gene analyses, respectively, in M. tuberculosis
are higher than those for E. coli and L. interrogans, these correlation coefficient values still do
not suggest significant positive correlation between domain complexity and gene lengths.
Thus, the specific protein complexity studies of these three genomes show that it is not
necessarily surprising that while the duplicate genes are generally longer than single copy
genes, they tend to contain fewer domains.

183

Fig. 7. Investigation of number of domains per single copy gene in the M. tuberculosis
H37Rv, E. coli and L. interrogans genomes (Single Copy Gene Analysis). The graph displays
the relationship between sequence length and number of domains of the single copy genes
in these genomes.

Pearsons product-moment correlation
Organism
and P-value
E. coli P-value H37Rv P-Value L. interrogans P-Value
All Proteins 0.48 2.2e-16 0.58 2.2e-16 0.39 2.2e-16
Duplicate 0.47 2.2e-16 0.62 2.2e-16 0.25 2.64e-10
Single 0.49 2.2e-16 0.59 2.2e-16 0.53 2.2e-16
Table 4. Correlation Coefficient and P-values of the whole genome, duplicate and single
copy gene analysis in E. coli, M. tuberculosis H37Rv and L. interrogans. The table displays the
results of Pearsons product-moment correlation.

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3.3 Functional and evolutionary analysis of expanded genes in M. tuberculosis
The protein sequence and signature data for the 76 genomes were clustered into related sets
of duplicate genes for the study of relationships between percentage duplication and the GC
content, genome size and gene complexity described above. However, since the comparison
of closely related organisms is better for inferring evolutionary relationships, we separately
clustered six of the closely related mycobacterial genomes and identified 390 duplicate gene
clusters in M. tuberculosis. The results were represented as a phylogenetic profile and a
summary is shown in Table 5.

Organism Total
Genes
TGC SCG DGC Total
duplicate
Genes
Estimated
Duplicate Gene
Percentages
M. tuberculosis 3947 2815 2425 390 1521 38.53
M. bovis 3910 2817 2439 378 1471 37.62
M. paratuberculosis 4316 2807 2343 464 1973 45.71
M. avium 5040 3199 2679 520 2361 46.84
M. ulcerans 4206 2755 2359 396 1847 43.91
M. leprae 1036 1603 1261 119 342 21.33
Table 5. Protein sequence clustering of the mycobacterial group. The columns of the table
represent the selected organisms, total number of genes in the genome, total number of
identified gene clusters (TGC), total number of single copy genes (SCG), total number of
duplicate gene clusters (DGC) in the organism, total number of duplicate genes in the
duplicate gene clusters identified, and the percentage of duplicate genes estimated for each
organism.
The biggest expanded family in M. tuberculosis was the PE/PPE/PGRS family with 164
members, followed by a family of alcohol dehydrogenases and oxidoreductases with 44
members, the fatty-acid-CoA ligase family with 33 members, then acyl-CoA dehydrogenase
with 27 members. A manual assignment of high-level functional classes was done
previously in the laboratory for all M. tuberculosis proteins. This was used here to determine
the functional distribution of all 390 expanded families in this organism. Figure 8 shows the
number of families and number of proteins belonging to each of the functional classes. The
biggest class is made up of enzymes or proteins involved in metabolism, followed by
proteins of unknown function. From the data, we selected 116 gene clusters which showed
gene family expansions in M. tuberculosis and M. leprae, as well as other mycobacteria. We
are interested in expansion in M. leprae, as this is a highly reduced mycobacterial genome, so
expanded genes that have been maintained are likely to be important. When considering
only the 116 families that are also expanded in M. leprae, the distribution of functional
classes is similar, except for a large reduction in the number of unknown protein families.
For each of the 116 clusters of interest, we calculated the genetic distance between family
members and investigated the relationship between genetic distance and gene family size.
Our results suggest that the genetic distance between two of the most distant proteins in the
clusters increases with an increase in cluster size. In addition, the correlation coefficient
value of 0.87 at a p-value of 2.2 x 10
-16
is indicative of strong positive correlation between
these factors. These sets of duplicate copies of proteins are clustered from different
mycobacterial genomes, and since the estimated maximum genetic distance between the 2

185

Fig. 8. Distribution of functions in M. tuberculosis (all 390 clusters) and M. leprae-shared (116
clusters) expanded families.
most distant proteins in each of these sets increases with an increase in cluster size, it was
inferred that some of the duplicate copies show a tendency to diverge from the original
ancestral functions after multiple duplication events in bigger families. To investigate the
average divergence of proteins in these clusters, the relationship between average genetic
distance and cluster size was determined. The results suggest that the average genetic
distance between the gene families does not increase with the cluster size, except perhaps
for the few larger families. In order to statistically verify the results, correlation coefficient
values were estimated using the Pearson's product-moment correlation. The correlation
coefficient value of 0.43 at a p-value of 1.17 x 10
-6
indicates the presence of moderate

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correlation between the average genetic distance and cluster size. It suggests that the
majority of homologous gene families identified from these mycobacterial species have not
undergone significant functional divergence and still show close evolutionary relatedness,
but these results may be skewed by the fact that the clusters contain orthologs and paralogs.
Orthologs are generally predicted to maintain similar functions, while paralogs are known
to have diverged functions.
The relationship between cluster size and genetic distance was also studied for 66
paralogous families only (within genome clustering). Within each of the selected
mycobacterial species, the estimated tree topologies were used to investigate the genetic
divergence of the identified paralogous gene families by computing the maximum genetic
distance between two of the most distant paralogs in each of the clusters. A scatter plot
analysis of the computed maximum genetic distances and cluster sizes was performed
(Figure 9), and correlation coefficient values were estimated for studying the genetic
divergence of these paralog gene families. From the analysis of the scatter plot (Figure 9)
and correlation coefficient values (Table 6), we inferred that the genetic distance between the
two most distant proteins increases with the cluster size.

Organism df Pearsons
correlation
P-value
M. tuberculosis 64 0.88 2.20e-16
M. bovis 62 0.81 4.4e-16
M. paratuberculosis 59 0.93 2.20e-16
M. avium 56 0.95 2.20e-16
M. ulcerans 59 0.93 2.20e-16
M. leprae 36 0.66 5.94e-06
Table 6. Results of the correlation calculations for maximum genetic distance versus cluster
size, including degrees of freedom (df), Pearsons correlation coefficient values, and the
corresponding p-values.
In addition to maximum genetic distance, the average genetic distance for each of the
paralog gene families was computed to investigate the evolutionary relationships between
the members within the selected mycobacterial genomes. To provide statistical significance
for the scatter plot observations, correlation coefficient values were estimated using the
Pearson's product-moment correlation (Table 7). The scatter plot (Figure 10) and correlation
coefficient values (Table 7), suggest a moderate negative correlation between the average
genetic distance and cluster size of the paralogous gene families.
3.4 Further analysis of one example expanded gene family in M. tuberculosis
While we have evolutionary data for all the orthologous and paralogous families of M.
tuberculosis, we cannot show all the results, so we have selected an important class of
regulatory proteins as an example. The adaptability of M. tuberculosis to enable successful
survival of the stressful conditions in the host during infection is attributed to the existence
of a diverse class of sigma factors in the organism (Fontan, 2009). The organism is suggested
to contain numerous sigma factors that bind to the core subunit of RNA polymerase to
provide promoter specificity (Fontan, 2008). To investigate the phylogenetic diversification
of sigma factors in M. tuberculosis and other mycobacteria, we studied the sigma factors

187
Organism df Pearsons
correlation
P-value
M. tuberculosis 64 -0.44 0.0001966
M. bovis 62 -0.5 2.38e-05
M. paratuberculosis 59 -0.41 0.0007649
M. avium 56 -0.43 0.0007819
M. ulcerans 59 -0.43 0.000633
M. leprae 36 -0.44 0.005978
Table 7. Pearsons correlation coefficient results for the relationship between the average
genetic distance and cluster size. The columns include degrees of freedom (df), Pearsons
correlation coefficient values, and the corresponding P-values.
Fig. 9. Relationship between maximum genetic distance and cluster size for families of M.
tuberculosis H37Rv, M. bovis, M. paratuberculosis, M. avium, M. ulcerans and M. leprae. The X-
axis represents the cluster size (total proteins in each cluster) and Y-axis shows the genetic
distance between the two most distant proteins in the clusters of each organism. The genetic
distance appears to increase with the cluster size, suggesting a correlation between them.
identified by our ortholog and paralog clustering methods. From the analysis of the sigma
factor phylogenetic trees of M. tuberculosis (Figure 11), we infer that gene duplication events
followed by divergence could have resulted in the bifurcation of the sigma factor class of
proteins into two subfamilies (marked as A and B in the Figure).

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Fig. 10. Relationship between average genetic distance and cluster size for duplicate gene
families of M. tuberculosis H37Rv, M. bovis, M. paratuberculosis, M. avium, M. ulcerans and M.
leprae. The X-axis represents the cluster size (total proteins in each cluster) and Y-axis shows
the average genetic distance between the identified gene families of each organism. The
average genetic distance appears to decrease with the cluster size, suggesting a negative
correlation between these two factors.
3.4.1 Analysis of sigma factor proteins in subfamily A
Following duplication, the proteins of this subfamily have diverged into 2 groups: SigE and
SigM (Figure 11). The 2 proteins in the SigE group have further diverged following
duplication and divergence. However, one of the proteins in the sigE group was identified
to have no orthologs in M. leprae (Figure 12), and loss of various sigma factors is suggested
to be the reason for M. leprae reductive genome evolution (Babu, 2003). Interestingly, all the
paralogs of M. tuberculosis appear to have orthologs in M. bovis, but the absence of sigM
proteins in M. bovis, and the large divergence of this protein group in M. tuberculosis
compared to other mycobacteria enables us to speculate on its significance in M. tuberculosis
evolution. Though an error in available M. bovis sequences could have resulted in incorrect
annotation of the sigM locus as a psuedogene (Manganelli et al., 2004), the extent of
divergence of this protein in M tuberculosis compared to other mycobacteria prompts further
investigation into its possible paths of pseudogenization or neofunctionalization.

189

Fig. 11. Phylogenetic tree of the Sigma factor paralog cluster inferred by the maximum
likelihood method. The duplication events are marked by As and Bs. The figure displays the
phylogenetic diversification of sigma factors (sigE, sigM, sigL, sigK, sigC and sigD).
3.4.2 Analysis of the sigma factor proteins in subfamily B
From the analysis of the sigma factor class of paralogs in GroupB (Figure 11), we infer that
duplication events followed by divergence resulted in the 2 groups of sigma factor
subfamilies (marked as B1 and B2 in Figure 11). The sigma factor proteins in each of the
subfamilies have significantly diverged after gene duplication. For the two sigma proteins
(sigK and sigL) in one of the subfamilies (Figure 12), sigK was identified to have no
orthologs in M, avium, M paratuberculosis or M. leprae, and sigL was noted to have no
orthologs in M. leprae. These results are inconsistent with the published reports of
Manganelli et al, 2003. For the other subfamily of sigma proteins (sigC and sigD) on the
phylogenetic tree (Figure 12), we did not identify orthologs for sigC in M. paratuberculosis or
sigD in M. leprae.
4. Discussion
The availability of complete genome sequences of many bacteria and significant progress in
the development of modern computational biology methods has resulted in the evolution of
a powerful platform for the comparative investigation of genome diversity across different
organisms. Here, we make use of the wealth of genome information and bioinformatics
tools to understand the significance of gene duplication in M. tuberculosis evolution. The
investigation of relationships between the GC composition and duplicate gene percentages
identified from the sequence and InterPro domain data provides sufficient evidence to
suggest a positive correlation between them for group1 and group3 organisms. Here, the
mycobacterial species are part of the group1 organisms, so the maintenance of

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Fig. 12. Phylogenetic tree of the Sigma factor ortholog and paralog cluster inferred by the
maximum likelihood method. The duplication events are marked by As and Bs. The labels
Mtu, Mbo, Mav, Mul, Mpa and Mle represent proteins from M. tuberculosis, M. bovis, M.
avium, M. ulcerans, M. paratuberculosis and M. leprae respectively.
high duplicate gene percentages in these species, with the exception of M. leprae, could be
attributed to the high GC composition of their genomes. Unsurprisingly, the study has also
shown a correlation between duplicate gene percentage and genome size, suggesting that
gene duplication increases genome size. Further, our investigations on protein complexity
provide deeper insights into the general trend in gene length and domain number in
duplicate genes in these organisms.
He and Zhang (2005), investigating Saccharomyces cerevisiae, showed duplicate genes to be
complex molecules with longer sequences containing more functional domains. From the
investigations of the mean gene lengths of 76 pathogenic and non-pathogenic organisms, it
is evident that the average length of the duplicate genes is comparatively higher than that of
single copy genes. However, the analysis of mean number of domains in the duplicate and
single copy genes reveals the presence of a higher number of domains in the single copy
genes compared to the duplicate genes. According to Stoltfus (1999), partial loss-of-function

191
mutations lead to the preservation of duplicate genes with single functions (Stoltfus, 1999;
Lynch & Force, 2000). Moreover, it has been suggested that duplicate genes lose one of the
domains that were originally present in the ancestral molecule, and by complementation of
the lost domains, both the daughter copies are reported to reflect the original ancestral
function. Thus, gene complexity is suggested to be reduced after subfunctionalization of
duplicate genes (He & Zhang, 2005). Further, the complementary loss of subfunctions is
considered to facilitate the preservation of duplicate gene pairs, and due to relaxed
evolutionary constraints following subfunctionalization, the chances of long-term evolution
of new functions is enhanced (Force et al., 1999). However, since deleterious mutations are
more common than beneficial mutations (Cun, 2010), evolution of new and essential protein
functions is considered to be a rare event (Nadeau & Sankoff, 1997; Force et al., 1999).
According to the predominant argument, the evolution of new domains would be favored
only if they can perform a function different to that of preexisting domains or domain
combinations (Lagomarsino et al., 2009). Further, the majority of duplicate genes are
predicted to develop new functions from the already existing ancestral gene functions, and
if new functions evolve by mutation from prior domains, it is less likely that all of the
domains would evolve into new domains due to the mutational bridge for new domain
evolution being too far from the ancestral molecule (Lagomarsino et al., 2009). Hence,
evolution of new functions following subfunctionalization could be a rare event. Therefore,
the presence of fewer domains in the duplicate genes compared to the single copy genes
could be due to evolution of duplicate genes by subfunctionalization, where complementary
loss of subfunctions is viewed to primarily facilitate preservation of the duplicate gene.
Alternatively, the addition of new domains into a bacterial genome could be due to
acquisition by HGT. Indeed, acquisition of one or more domains by HGT in 30 to 50 percent
of bacteria has been reported (Choi & Kim, 2007). The acquired gene or gene segment is
known to be beneficial only if it has some properties different to that of recipient genome
(Kinsella et al., 2003). Since the selection of a new domain would depend upon its ability to
perform a biological function that is not covered by pre-existing domains, addition of such
rare domains by HGT could be an uncommon phenomenon (Lagomarsino et al., 2009).
Adaptation of bacteria to new environments requires evolution of new functions (Hooper &
Berg, 2003), and gene duplication is viewed to be the general mechanism of adaptation to
different environmental conditions (Kondrashov, 2002). However, a recent study suggests
HGT to be a far more important route to adaptation compared to gene duplication (Koonin &
Wolf, 2009). Further, duplication of horizontally transferred genes with weak or no functions is
suggested to accelerate the evolutionary process of gene innovation. Since both gene
duplication and HGT are considered to be important routes of bacterial adaptation to
changing environments, and amplification of weak ancillary functions is considered to be the
easiest route to gene innovation, quick adaptation of bacteria to changing environments could
be due to amplification of weak ancillary functions. Thus, reduced functional complexity of the
investigated duplicate genes compared to single copy genes could be due to preservation of
the majority of the paralogs by subfunctionalization, and the rare event of neofunctionalization
could have been either due to divergence of subfunctions over an evolutionary period of time
following preservation of subfunctionalized paralogs, or mostly due to rapid amplification of
weak ancillary functions after gene duplication. To gain deeper insights into the functional
complexity of duplicate genes in M. tuberculosis, we focussed on the evolutionary analysis of
the duplicate genes in six of the closely related mycobacterial species.

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From the analysis of maximum genetic distance between the two most distant proteins of the
mycobacterial multiple genome clusters, we suggest that the divergence of at least one of the
duplicate gene copies from the ancestral gene increases with the increase in cluster size. These
homologous gene families consist of orthologs and paralogs. The lack of a strong correlation
between the average genetic distance and cluster size of the duplicate gene copies in the
multiple genome clusters indicates that the homologous gene families including proteins from
different mycobacterial species have not undergone complete functional divergence. This is to
be expected for orthologs, which tend to maintain their functions.
The average genetic distance estimated for single genome paralogous gene clusters, on the
other hand, decreases with the increase in cluster size, suggesting that, on average, smaller
families tend to diverge more rapidly than the larger families. This is apart from some
members of the larger families, which have obviously diverged further as they are
contributing to the increased maximum genetic distance with cluster size. Though gene
duplication is considered to be an important mechanism for acquiring new genes, and
creating evolutionary novelty (Torgerson and Singh, 2004), horizontal gene transfer (HGT)
is also known to be a wide spread phenomenon, and a significant proportion of genes in
bacteria are accepted to have been acquired by HGT (Price et al., 2007). The genome of M.
tuberculosis is known to contain 19 genes of eukaryotic origin, and it is speculated that the
organism may have also acquired genes from other prokaryotes by HGT (Kinsella et al.,
2003). In addition, the occurrence of many intraspecies HGT events in the progenitor of M.
tuberculosis has been reported (Rosas-Magallanes et al., 2006). The ability of HGT to
incorporate a new gene which is homologous to an existing gene family member is well
recognized (Ochman, 2001; Kinsella et al., 2003; Krzywinska, 2004), and in comparison to its
gene family members, the newly introduced gene may be more divergent in sequence and
function (Pushker et al., 2004). Following duplication, such laterally transferred genes with
already divergent functions may further diversify in the process of evolving new functions,
and this could result in an increase in genetic distance between the laterally transferred
duplicate gene and its paralog gene family members. The chance of this should increase
with the number of members.
Phylogenetic analysis of the sigma factors in M. tuberculosis suggested that most of the sigma
factors have orthologs in other mycobacteria. However, we could not observe orthologs in
M. leprae for a few of the subfamilies, and this could have been due to the extensive loss of
sigma factors during its reductive genome evolution. Agarwal et al., 2007 reports that sigM
proteins control only a small subset of genes, and their loss would not influence M.
tuberculosis virulence (Agarwal et al., 2007). The difference in the divergence of sigM in M.
tuberculosis compared to other mycobacteria, and absence of its ortholog in M. bovis should
be considered further to study the importance of the sigM factor in M. tuberculosis virulence.
5. Conclusions and future work
The estimated duplicate gene percentages for M. tuberculosis from independent genome
clustering (31%), InterPro signature methods (38%), across genome clustering (49%) and a
union of the methods (51%) were all relatively high, showing the significance of gene
duplication in M. tuberculosis genome evolution. The investigation of relationships between
the GC composition and duplicate gene percentages identified from the sequence and
InterPro domain data provides sufficient evidence to suggest that for the mycobacterial
species, with the exception of M. leprae, the maintenance of high duplicate gene percentages

193
could be attributed to the high GC composition of their genomes. The study has also shown
a correlation between duplicate gene percentage and genome size, suggesting that gene
duplication increases genome size, which is a logical result. Interestingly, our functional
complexity results were in contrast to recent finding in eukaryotes, and we show that, on
average, duplicate genes have longer sequences but fewer domains than single copy genes
in the investigated organisms. The reduced functional complexities of duplicate genes could
be due to their evolution by subfunctionalization following duplication.
We also show that duplicate gene families of mycobacterial multiple genome clusters have
not undergone complete functional divergence following gene duplication and still tend to
maintain their functions. Our maximum genetic distance results suggest that multiple
duplication events in a few of the duplicate copies of bigger families may result in their
functional divergence from the original ancestral functions. Our paralog maximum genetic
distance results suggest that the increase in genetic distance between the two most distant
proteins with the size of the gene family may be due to duplication followed by paralog
evolution of some of the distant genes that already have divergent functions compared to its
paralog members. From the study of average genetic distance of paralogs, we suggest that
slow evolution of paralogs of large families in M. tuberculosis could be due to preservation of
original ancestral functions by the mechanism of subfunctionalization.
For future studies, we are investigating selection pressure and comparison between smaller
and larger gene family evolution to shed light on the evolutionary fate of duplicate genes
and functional innovation in M. tuberculosis. In addition, since the functional constraints on
amino acid residues are known to differ due to the potential changes in protein function, we
have studied site specific rate differences between the amino acids of closely related
mycobacterial species to aid in deciphering specific subfamily evolutionary divergence
following gene duplication. Such predicted critical amino acids when mapped on to protein
secondary structure could help in evaluation of important structural locations in functional
diversification. In addition to functional divergence, gene expression data from different
experimental conditions is of use to understand the degree of expression divergence of the
genes following duplication events. Overall, we are working on further investigation of M
tuberculosis duplicate genes with the integration of phylogeny-sequence-structure-function-
expression information, which will be valuable for understanding the functional and
evolutionary fate of genes following gene duplication in M. tuberculosis.
6. Acknowledgement
We thank the National Bioinformatics Network and Computational Biology Group,
University of Cape Town, South Africa for supporting this work.
7. References
Abascal, F.; Zardoya, R. & Posada, D. (2005). ProtTest: selection of best-fit models of protein
evolution. Bioinformatics Applications Note, Vol. 21, No. 9, pp. 21042105.
Agarwal, N.; Woolwine SC, Tyagi S, Bishai WR. (2007). Characterization of the
Mycobacterium tuberculosis Sigma Factor SigM by Assessment of Virulence and
Identification of SigM-Dependent Genes. Infection and Immunity, Vol. 75, No 1, pp.
452461.

Gene Duplication

194
Apweiler, R.; Attwood TK, Bairoch A, Bateman A, Birney E, Biswas M, Bucher P, Cerutti L,
Corpet F, Croning MD, Durbin R, Falquet L, Fleischmann W, Gouzy J, Hermjakob
H, Hulo N, Jonassen I, Kahn D, Kanapin A, Karavidopoulou Y, Lopez R, Marx B,
Mulder NJ, Oinn TM, Pagni M, Servant F, Sigrist CJ, Zdobnov EM. (2001). The
InterPro database, an integrated documentation resource for protein families,
domains and functional sites. Nucleic Acids Research, Vol. 29, No. 1, pp. 37-40.
Babu, MM. (2003). Did the loss of sigma factors initiate pseudogene accumulation in M.
leprae? Trends in Microbiology, Volume 11, No. 2, pp. 5961.
Basak, S. & Ghosh,

TC. (2005). On the origin of genomic adaptation at high temperature for
prokaryotic organisms. Biochemical and Biophysical Research Communications, Vol.
330, No. 3, pp. 629-632.
Castresana, J. (2000). Selection of Conserved Blocks from Multiple Alignments for Their Use
in Phylogenetic Analysis. Molecular Biology and Evolution, Vol. 17, Vol. 4, pp.
540552.
Choi, I. & Kim, S. (2007). Global extent of horizontal gene transfer. Proceedings of National
Academy of Science, Vol. 104, No. 11, pp. 44894494.
Cordero, OX. & Hogeweg, P. (2009). The impact of long-distance horizontal gene transfer on
prokaryotic genome size. Proceedings of National Academy of Science, Vol. 106, No.
51, pp. 2174821753.
Cun Y. (2010). The Evolutionary Dynamics of Mutant Allele at Duplicate Loci
arXiv:1007.0333v1.
DeRose-Wilson, LJ. & Gaut, BS. (2007). Transcription-related mtations and GC content drive
variation in nucleotide substitution rates across the genomes of Arabidopsis thaliana
and Arabidopsis lyrata. BMC Evolutionary Biology, Vol. 7, No. 66.
Fontan, PA.; Voskuil MI, Gomez M, Tan D, Pardini M, Manganelli R, Fattorini L, Schoolnik
GK, Smith I. (2009). The Mycobacterium tuberculosis Sigma Factor B Is Required
for Full Response to Cell Envelope Stress and Hypoxia In Vitro, but It Is
Dispensable for In Vivo Growth. Journal of Bacteriology, Volume 191, No. 18, pp.
56285633.
Fontan, PA.; Aris V, Alvarez ME, Ghanny S, Cheng J, Soteropoulos P, Trevani A, Pine R, and
Smith I. (2008 ). Mycobacterium tuberculosis Sigma Factor E Regulon Modulates
the Host Inflammatory Response. Vol. 198, pp. 877-85.
Force, A.; Lynch M, Pickett FB, Amores A, Yan YL, Postlethwait J. (1999). Preservation of
duplicate genes by complementary, degenerative mutations. Genetics, Vol. 151, No.
4, pp. 1531-1545.
Fraser-Liggett, CM. (2005). Insights on biology and evolution from microbial genome
sequencing. Genome Research, Vol. 15, No. 12, pp.16031610.
Guindon, S. & Gascuel, O. (2003). A Simple, Fast, and Accurate Algorithm to Estimate Large
Phylogenies by Maximum Likelihood. Systematic Biology, Vol. 52, No. 5, pp.
696704.
Hamady, M; Betterton, MD., & Knight, R. (2006). Using the nucleotide substitution rate
matrix to detect horizontal gene transfer. BMC Bioinformatics, Vol. 7, No. 476.
He, X. & Zhang, J. (2005). Gene Complexity and Gene Duplicability. Current Biology. Vol. 15,
No. 11, pp. 1016-1021.
Hooper, DS. & Berg, GO. (2003). On the Nature of Gene Innovation: Duplication patterns in
Microbial Genomes. Molecular Biology and Evolution, Volume 20, No. 6, pp. 945-954.

195
Kersey, P.; Bower L, Morris L, Horne A, Petryszak R, Kanz C, Kanapin A, Das U, Michoud
K, Phan I, Gattiker A, Kulikova T, Faruque N, Duggan K, Mclaren P, Reimholz B,
Duret L, Penel S, Reuter I and Apweiler R. (2005). Integr8 and Genome Reviews:
integrated views of complete genomes and proteomes. Nucleic Acids Research, Vol.
33, Database issue, D297D302.
Kinsella, RJ.; Fitzpatrick DA, Creevey CJ, McInerney JO. (2003). Fatty acid biosynthesis in
Mycobacterium tuberculosis: Lateral gene transfer, adaptive evolution, and gene
duplication. Proceedings of National Academy of Science, Vol. 100, No. 18, pp. 10320
10325.
Kondrashov, FA.; Rogozin IB, Wolf YI, Koonin EV. (2002). Selection in the evolution of gene
duplications. Genome Biology, Vol. 3, No. 2.
Koonin, EV. & Wolf YI. (2008). Genomics of bacteria and archaea: the emerging dynamic
view of the prokaryotic world. Nucleic Acids Research, Vol. 36, No. 21.
Koonin, EV. & Wolf, YI. (2009). Is evolution Darwinian or/and Lamarckian? Biology Direct, Vol.
4, No. 42.
Krzywinska, E.; Krzywinski J, & Schorey JS. 2004. Naturally occurring horizontal gene
transfer and homologous recombination in Mycobacterium. Microbiology, Vol. 150,
No. Pt 6, pp. 1707-1712.
Lagomarsino, MC. (2009). Universal features in the genome-level evolution of protein domains.
Genome Biology, Vol. 10, No. 1.
Manganelli, R.; Provvedi R, Rodrigue S, Beaucher J, Gaudreau L, Smith I. (2004). Factors
and Global Gene Regulation in Mycobacterium tuberculosis. Journal of Bacteriology,
Volume 186, No. 4, pp. 895902.
Marri, PR.; Bannantine, JP. & Golding, GB. (2006). Comparative genomics of metabolic
pathways in mycobacterium species: gene duplication, gene decay and lateral gene
transfer FEMS. Microbiology Review, Vol. 30, No. 6, pp. 906-925.
Mulder, NJ; Apweiler R, Attwood TK, Bairoch A, Bateman A, Binns D, Bork P, Buillard V,
Cerutti L, Copley R, Courcelle E, Das U, Daugherty L, Dibley M, Finn R,
Fleischmann W, Gough J, Haft D, Hulo N, Hunter S, Kahn D, Kanapin A, Kejariwal
A, Labarga A, Langendijk-Genevaux PS, Lonsdale D, Lopez R, Letunic I, Madera
M, Maslen J, McAnulla C, McDowall J, Mistry J, Mitchell A, Nikolskaya AN,
Orchard S, Orengo C, Petryszak R, Selengut JD, Sigrist CJ, Thomas PD, Valentin F,
Wilson D, Wu CH, Yeats C. (2007). New developments in the InterPro database.
Nucleic Acids Research. Vol. 35, No. D224-D228.
Musto, H.; Naya H, Zavala A, Romero H, Alvarez-Valn F,

and Bernardi

G. (2006). Genomic GC
level, optimal growth temperature, and genome size in prokaryotes. Biochemical and
Biophysical Research Communications, Vol. 347, No. 1, pp. 1-3.
Mann, S. & Chen, YP. (2010). Bacterial genomic G + C composition-eliciting environmental
adaptation. Genomics, Vol. 95, No. 1, pp. 7-15.
Mann, S.; Li, J. & Chen YP. (2010). Insights into Bacterial Genome Composition through
Variable Target GC Content Profiling. Journal of Computational Biology, Vol. 17, No.
1, Pages 79-96.
Nadeau, JH. & Sankoff, D. (1997). Comparable Rates of Gene LOSS and Functional
Divergence After Genome Duplications Early in Vertebrate Evolution. Genetics, Vol.
147, No. 3, pp. 1259-1266.

Gene Duplication

196
Naya, H.; Romero H, Zavala A, Alvarez B, Musto H. (2002). Aerobiosis Increases the
Genomic Guanine Plus Cytosine Content (GC%) in Prokaryotes. Journal of Molecular
Evolution, Vol. 55, No. 3, pp. 260-264.
Nelson, KE.; Clayton RA, Gill SR, Gwinn ML, Dodson RJ, Haft DH, Hickey EK, Peterson JD,
Nelson WC, Ketchum KA, McDonald L, Utterback TR, Malek JA, Linher KD,
Garrett MM, Stewart AM, Cotton MD, Pratt MS, Phillips CA, Richardson D,
Heidelberg J, Sutton GG, Fleischmann RD, Eisen JA, White O, Salzberg SL, Smith
HO, Venter JC, Fraser CM. (1999). Evidence for lateral gene transfer between
Archaea and Bacteria from genome sequence of Thermotoga maritime. Nature, Vol.
399, pp. 323-329.
Notredame, C.; Higgins, DG. & Heringa J. (2000). T-Coffee: A novel method for fast and
accurate multiple sequence alignment. Journal of Molecular Biology, Vol. 302, No.
1, pp. 205-217.
Ochman, H. 2001. Lateral and oblique gene transfer. Current Opinion in Genetics &
Development, Vol. 11, No. 6, pp. 616619.
Price, MN.; Dehal PS, & Arkin AP. 2007. Orthologous transcription factors in bacteria have
different functions and regulate different genes. PLoS Computational Biology, Vol. 3,
No. 9, pp. 1739-1750.
Pushker, R.; Mira A, & Rodriguez-Valera F. (2004). Comparative genomics of gene-family
size in closely related bacteria. Genome Biology, Vol. 5, No. 4.
Rosas-Magallanes, V.; Deschavanne P, Quintana-Murci L, Brosch R, Gicquel B, Neyrolles O.
2006. Horizontal Transfer of a Virulence Operon to the Ancestor of Mycobacterium
tuberculosis. Molecular Biology and Evolution, Vol. 23, No. 6, pp. 11291135.
Snel. B.; Bork, P. & Huynen MA. (2001). Genomes in Flux: The Evolution of Archaeal and
Proteobacterial. Gene Content Genome Research, Vol 12, No. 1, pp.1725.
Stoltzfus, A. (1999). On the possibility of constructive neutral evolution. Journal of Molecular
Biology, Vol 49, No. 2, pp. 169181.
Tatusov, RL.; Koonin, EV. & Lipman DJ. (1997). A Genomic Perspective on Protein Families.
Science, Vol. 278, No. 631.
Tekaia, F.; Gordon SV, Garnier T, Brosch R, Barrell BG, Cole ST. (1999). Analysis of
proteome of mycobacterium tuberculosis in silico. Tubercle and Lung Diseases, Vol.
79, No. 6, pp. 329-342.
Torgerson, DG. & Singh RS. 2004. Rapid Evolution Through Gene Duplication and
Subfunctionalization of the Testes-Specific 4 Proteasome Subunits in Drosophila.
Genetics, Vol. 168, No. 3, pp. 14211432.
Zhang, L.; Kasif S, Cantor CR, Broude NE. (2004). GC/AT-content spikes as genomic
punctuation marks. Proceedings of National Academy of Science, Vol. 101, No. 48, pp.
1685516860.
11
The Evolutionary History of CBF Transcription
Factors: Gene Duplication of CCAAT
Binding Factors NF-Y in Plants
Alexandro Cagliari, Andreia Carina Turchetto-Zolet, Felipe dos Santos
Maraschin, Guilherme Loss, Rogrio Margis and Marcia Margis-Pinheiro
Universidade Federal do Rio Grande do Sul/UFRGS
Brazil
1. Introduction
Eukaryotic gene expression is often controlled by complex and refined combinatorial
transcription factor networks composed of multiprotein complexes that derive their gene
regulatory capacity from both intrinsic properties and from their trans-acting partners
(Singh, 1998; Wolberger, 1998; Remenyi et al., 2004). Participation in such higher complex
order allows an organism to use single transcription factors to control multiple genes with
different temporal and spatial expression patterns (Siefers et al., 2009).
In this chapter, we provide a synopsis of the genetic and genomic mechanisms that might be
responsible for the gene copy diversification observed in the eukaryotic NF-Y transcription
factor family. We identify the genes coding for NF-Y transcription factors in eukaryotes with
an emphasis on the duplication of the NF-Y family in the plant lineage and discuss the
important consequences of its gene diversification.
2. The CCAAT cis-element promoter
Eukaryotic genes contain numerous cis-regulatory elements that mediate their induction,
repression or basal transcription (Dynan and Tjian, 1985; Myers et al., 1986; Maity and de
Crombrugghe, 1998). These regulatory elements can be found in the proximity of
transcribed genes, such as the promoter region and/or in distant regions of the genes where
they may act as enhancers (de Silvio et al., 1999).
The transcriptional regulation of several eukaryotic genes is coordinated through sequence-
specific binding of proteins to the promoter region located upstream of the gene. During
evolution, many of these protein-binding sequences, which are found in a wide variety of
organisms, have shown a high degree of conservation (Edwards et al., 1998).
The CCAAT box is one of the most common upstream elements, found in approximately
2530% of eukaryotic promoters (Bucher, 1990; Mantovani, 1998). It is typically located
between 60100 bp upstream of the transcription start site and it can function in direct or in
inverted orientations (Dorn et al., 1987b; Bucher, 1990; Edwards et al., 1998; Mantovani, 1998;
Stephenson et al., 2007) with possible cooperative interactions between multiple boxes
(Tasanen et al., 1992) or other conserved motifs (Muro et al., 1992; Rieping and Schoffl, 1992;

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198
Edwards et al., 1998). CCAAT boxes are highly conserved within homologous genes across
species in terms of position, orientation, and flanking nucleotides (Mantovani, 1998). In
addition, the spacing between the CCAAT box and other promoter-specific cis-elements is
also conserved among species (Dorn et al., 1987a; Chodosh et al., 1988; Maity and de
Crombrugghe, 1998).The expression of genes under the control of promoters that contain
CCAAT boxes may be ubiquitous or tissue/stage specific, suggesting that the gene
expression pattern is also determined by other cis and trans elements (Stephenson et al.,
2007).
In Sacharomyces cerevisiae, CCAAT boxes are found in the promoters of cytochrome genes, in
genes coding for proteins that are activated by non-fermentable carbon sources (McNabb et
al., 1995) and in genes involved in nitrogen metabolism (Dang et al., 1996). In the
filamentous fungus Aspergillus nidulans, CCAAT boxes are present in genes involved with
penicillin biosynthesis (Steidl et al., 1999). In higher eukaryotes, a multitude of promoters
contain CCAAT boxes, including those of developmentally controlled and tissue-specific
genes (Berry et al., 1992), housekeeping and inducible genes (Roy and Lee, 1995) and cell-
cycle regulated genes (Mantovani, 1998). In addition, many cell-cycle regulated promoters
lack a recognizable TATA-box, but contain more than one CCAAT box in a position close to
and sometimes overlapping with the start site of transcription (Zwicker and Muller, 1997).
3. The CBF/NF-Y transcription factor
Several CCAAT-binding proteins have been isolated and described, including CBF/NF-Y
(CCAAT Binding Factor/Nuclear Factor of the Y box), CTF/NF1 (CCAAT Transcription
Factor/Nuclear Factor 1), C/EBP (CCAAT/Enhancer Binding Protein) and CDP (CCAAT
Displacement Protein) (Mantovani, 1999). Among them, NF-Y is the most ubiquitous and
specific one acting as a key proximal promoter factor in the transcriptional regulation of an
array of different eukaryotic genes. Unlike other CCAAT-binding proteins, NF-Y requires a
high degree of conservation of the CCAAT pentanucletide sequence and shows strong
preference for specific flanking sequences (Dorn et al., 1987a; Stephenson et al., 2007).
Therefore, the NF-YC transcription factor can be distinguished from the other CCAAT-
binding proteins based on its DNA sequence requirements (Maity and de Crombrugghe,
1998).
The CBF/NF-Y transcription factor, which will be referenced in this chapter as NF-Y, is a
conserved oligomeric transcription factor found in all eukaryotes that is involved in the
regulation of diverse genes (Maity et al., 1992; McNabb et al., 1995; Edwards et al., 1998;
Mantovani, 1998; Siefers et al., 2009). NF-Y typically acts in concert with other regulatory
factors to modulate gene expression in a highly controlled manner (Nelson et al., 2007). In
many eukaryotic promoters, the functional NF-Y-binding sites are relatively close to the
TATA motif (Bucher, 1990) and are invariably flanked by at least one additional functionally
important cis-element. Several reports have shown that various factors, including
transcription factors, co-activators, and TATA-binding proteins, interact with NF-Y or its
subunits in promoting transcriptional regulation (Mantovani, 1999; Yazawa and Kamada,
2007). NF-Y was originally identified as the protein that recognizes the MHC class II
conserved Y box in Ea promoters (Dorn et al., 1987a; Matuoka and Chen, 2002). It specifically
recognizes the consensus sequence 5-CTGATTGGYYRR-3 or 5-YYRRCCAATCAG-3(Y is
5 pyrimidines and R is 5 purines) present in the promoter region of eukaryotic genes.
Bioinformatic analyses indicate that about 30% of mammalian promoters have predicted
The Evolutionary History of CBF Transcription Factors:
Gene Duplication of CCAAT Binding Factors NF-Y in Plants

199
NF-Y binding sites (Bucher, 1990; Testa et al., 2005), and chromatin immunoprecipitation
data have demonstrated additional widespread NF-Y binding in nonpromoter sites.
Suggesting the importance of binding context, NF-Y-regulated gene expression can be tissue
specific, developmentally regulated, or constitutive (Maity and de Crombrugghe, 1998;
Siefers et al., 2009).The transcriptional activity of NF-Y can be regulated by differential
expression, alternative splicing, proteinprotein interactions, and cellular redox potential
(Matuoka and Yu Chen, 1999).
NF-Y has been shown to be involved in the regulation of some G1/S genes whose
expressions are attenuated during the senescence process (Matuoka and Yu Chen, 1999).
NF-Y plays a pivotal role in the cell cycle regulation of the mammalian cyclin A, cdc25C, and
cdc2 genes, in the S-phase of the cell cycle (Currie, 1998). Additionally, there are a number of
genes involved in the cellular response to damage and stress, including the phospholipid
hydroperoxide glutathione peroxidase genes (Huang et al., 1999), which are regulated by
NF-Y, indicating its pivotal role in the removal of damaging agents from cells (Matuoka and
Chen, 2002). Although NF-Y functions basically as a transactivator of gene expression, it is
also involved, directly or indirectly, in the downregulation of transcription. For instance,
NF-Y binds to the mouse CCAAT box renin enhancer and blocks the binding of positive
regulatory elements (Shi et al., 2001). In this case, NF-Y dysfunction would lead to the
damage of systems that control blood pressure (Matuoka and Chen, 2002).
NF-Y is composed of three different subunits named NF-YA (also known as HAP-2 or CBF-
B), NF-YB (HAP3 or CBF-A), and NF-YC (HAP5 or CBF-C) that interact to form a complex
that can bind CCAAT DNA motifs and control the expression of target genes (Figure 1).
Each subunit is required for DNA binding, subunit association and transcriptional
regulation in both vertebrates and plants (Sinha et al., 1995; Stephenson et al., 2007). Yeast
possesses a fourth subunit, called HAP4, which provides a transcriptional activation domain
to the complex (Forsburg and Guarente, 1989; Lee et al., 2003). The yeast HAP4 protein is not
needed for DNA-binding but contains an acidic domain that is essential to promote
transactivation when associated with the HAP2/HAP3/HAP5 complex (Olesen and
Guarente, 1990; Serra et al., 1998). In vertebrates, the function of this fourth domain was
incorporated into other subunits (Forsburg and Guarente, 1989; Yazawa and Kamada, 2007).
Despite the wide cellular distribution and functional variability of NF-Y-regulated genes,
most eukaryotic genomes have only one or two genes encoding each NF-Y subunit (Maity
and de Crombrugghe, 1998; Riechmann and Ratcliffe, 2000). Fungi and animals, for
example, present single genes encoding each protein subunit. Thus, there is minimal
combinatorial diversity in the subunit composition of the heterotrimeric NF-Y in these
organisms (Siefers et al., 2009). In contrast, the NF-Y complex in vascular plants is generally
encoded by gene families (Riechmann and Ratcliffe, 2000).
3.1 NF-Y subunits
NF-Y is the only transcription factor thus far identified for which the interaction of three
heterologous subunits creates the DNA binding domain (Maity and de Crombrugghe, 1992;
McNabb et al., 1995; Sinha et al., 1995). All three NF-Y subunits are essential for the DNA
binding activity and one molecule of each subunit forms the NF-Y-DNA complex (Maity
and De Crombrugghe, 1996). Each NF-Y subunit contains a conserved domain with
identities greater than 70% across species. This highly conserved domain is located at the C-
terminus of NF-YA; in the central part of NF-YB; and at the N-terminus of NF-YC (Li et al.,
1992).

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The NF-YA conserved domain can be divided in two functionally distinct regions: an N-
terminal region that is required for NF-YB and NF-YC association and a C-terminal region
required for DNA-binding (Maity and de Crombrugghe, 1992). Additionally, NF-YA
usually contains a glutamine (Q)-rich and a serine/threonine (S/T)-rich regions. There are
numerous variants of NF-YA due to alternative splicing at the Q-S/T domains (Li et al.,
1992) and, although the expression of these isoforms is variable depending of tissue and cell
types, they all seem intact in terms of transcriptional function (Matuoka and Chen, 2002).
Both NF-YB and NF-YC subunits possess the highly conserved histone-fold motif (HFM)
and are structurally similar to core histone subunits H2B and H2A, respectively, and to the
archaebacterial histone-like protein Hmf-2 (Arents and Moudrianakis, 1995; Baxevanis et al.,
1995; Mantovani, 1998). In terms of identity, NF-YB is 30% identical to H2B, 14% to H2A,
17% to H4 and 18% to H3; NF-YC is 21% identical to H2A, 15% to H4 and H3 and 20% to
H2B (Liberati et al., 1999). Other proteins showing a remarkable identity (25-30%) to both
NF-YB and NF-YC are present in Archaea. These proteins homodimerize and associate with
DNA, forming nucleosome-like structures (Sandman et al., 1990). The NF-YB and NF-YC
subunits also contain residues that are important for their contact with DNA (Romier et al.,
2003; Stephenson et al., 2007). In contrast, the conserved segment of NF-YA has no homology
with the histone-fold motif, or with any of the known dimerization motifs present in other
heteromeric DNA-binding proteins (Maity and de Crombrugghe, 1992).
Some portions of NF-YA, NF-YB and NF-YC present a high degree of identity with yeast
HAP3, HAP2 and HAP5, respectively. These HAP genes, which are components of the yeast
CCAAT-binding protein, are necessary for the expression of genes encoding components of
the electron transport chain. Yeast strains mutated for either of the three genes failed to
grow on media containing a nonfermentable carbon source such as lactate or glycerol, a
characteristic respiratory-defect phenotype (McNabb et al., 1995).
Assembly of the NF-Y heterotrimer in mammals (where this complex is better studied)
follows a strict, stepwise pattern (Sinha et al., 1995; Sinha et al., 1996) (Figure 1). Initially, the
NF-YB and NF-YC subunits form a tight heterodimer (Figure 1a) similar to those of the
HFM, a conserved proteinprotein and DNA-binding interaction module (Luger et al., 1997)
composed by 65 amino acid stretch common to all histones that is required for nucleosome
formation (Baxevanis et al., 1995; Luger et al., 1997; de Silvio et al., 1999). This dimer then
moves to the nucleus, where the third subunit (NF-YA, Figure 1b) is recruited to generate
the complete, heterotrimeric NF-Y (Figure 1c). Interestingly, NF-YA is unable to interact
with the NF-YB or NF-YC alone, interacting only with the NF-YB-NF-YC heterodimer (Serra
et al., 1998). The complete NF-Y is able to bind promoters containing the core pentamer
nucleotide sequence CCAAT (Figure 1d) with high specificity and affinity resulting in either
positive or negative transcriptional regulation (Figure 1e) (Peng and Jahroudi, 2002; 2003;
Ceribelli et al., 2008; Siefers et al., 2009).
Because the NF-Y transcription factor contains H2B-like and H2A-like molecules (NF-YB
and NF-YC, respectively), the complex presents all the core histone components and could
mimic the interaction of the nucleosome core with genomic DNA (Struhl and Moqtaderi,
1998). In this scenario, it has been demonstrated that the NF-YA/NF-YB/NF-YC trimer or
the NF-YB/NF-YC dimer can bind to H3/H4 tetramer during nucleosome assembly (Caretti
et al., 1999). In addition, the NF-Y complex also can bind to the chromatin even after
nucleosome formation, indicating the ability of NF-Y to interact with genomic DNA
assembled in the nucleosome. The interaction between the NF-Y transcription factor and the
DNA molecule causes local disruption of the nucleosomal architecture (Coustry et al., 2001).

201
This disruption results in a partial dissociation of DNA from the histone core, which might
enable the access of the general transcription machinery to initiate the transcription process.

Fig. 1. Assembly of NF-Y subunits and its binding to DNA. Initially, the NF-YB and NF-YC
subunits form a tight heterodimer via proteinprotein interactions (a). The dimer then
moves to the nucleus, where is recruited the third subunit (NF-YA) (b) to generate the
complete, heterotrimeric NF-Y (c) that is able to bind promoters containing the core
pentamer nucleotide sequence CCAAT (d) resulting in either positive or negative
transcriptional regulation (e). Adapted from Mantovani (1999). White circles and oblong
black circles into each NF-Y subunit represent the DNA-binding domain and the NF-Y
interaction domain of NF-Y subunits, respectively.
4. Gene duplication and evolution
DNA duplication act as one of the main forces driving the evolution of organisms by
creating the raw genetic material that natural selection can subsequently modify. Gene
duplications arise in eukaryotes at a rate of 0.01 paralogs per gene per million years (Lynch
and Conery, 2000), the same order of magnitude of the mutation rate per nucleotide per year
(De Grassi et al., 2008). Duplication of individual genes, chromosomal segments, or entire
genomes represent the primary source for the origin of evolutionary novelties, including
new gene functions and expression patterns (Holland et al., 1994; Sidow, 1996; Lynch and
Conery, 2000). However, how duplicated genes successfully evolve from an initial state of
complete redundancy, wherein one copy is likely to be expendable, to a stable situation in
which both copies are maintained by natural selection, is unclear (Sidow, 1996; Lynch and
Conery, 2000; Ober, 2010).
In the evolutionary history of plants, genome duplications have been relatively common,
leading to the hypothesis that most angiosperms are to some extent polyploidal (Soltis,
2005). The genome of Arabidopsis, for example, possesses traces of at least three polyploidy
events (Vision et al., 2000; Simillion et al., 2002), followed by subsequent gene loss (Bowers
et al., 2003; Ober, 2010).
Similar to a point mutation, a duplication that occurs in an individual can be fixed or lost in
the population. Compared with pre-existing alleles, if a new allele of the duplicate gene is
selectively neutral, it has a small probability (1/2N) to be fixed in a diploid population
(where N is the effective population size). This suggests that the majority of duplicated
genes will be lost. For those duplicated genes that do become fixed, the fixation time
averages is 4N generations (Kimura, 1989; Zhang, 2003).

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202
On an evolutionary scale, gene duplication may result in new functions via different
scenarios. Although the most likely outcome is a loss of function in one of the two gene
copies (nonfunctionalization, Figure 2a), in rare instances one copy may acquire a novel
evolutionarily advantageous function and become preserved by natural selection
(neofunctionalization, Figure 2b), while the other copy retains the original function.
Alternatively, after duplication, mutations may occur in both genes leading to specialization
to perform complementary functions (subfunctionalization, Figure 2c) (Lynch and Conery,
2000; Lynch and Force, 2000). This process produces novel genetic variants that drive
genetic innovation (Lynch and Conery, 2000; Conrad and Antonarakis, 2007). Because gene
duplication generates functional redundancy, it is often not advantageous to the organism
to possess two identical genes. In nonfunctionalization (Figure 2a), the accumulation of
deleterious mutations might lead to the loss of the original function of one paralogue.
Alternatively, instead of being completely lost, many duplicated genes are silenced or
become pseudogenes and are thus either unexpressed or functionless (Gallagher et al., 2004;
Nicole et al., 2006; Yang et al., 2006; Beisswanger and Stephan, 2008; Xiong et al., 2009).
Pseudogenization is the most frequent fate of duplicated genes. In Caenorhabditis elegans,
for example, genomic analyses have identified 2168 pseudogenes or approximately one
pseudogene for every eight functional genes (Harrison et al., 2001). In humans, one
pseudogene was identified for approximately every two functional genes (Harrison et al.,
2002). As pseudogenes generally do not confer a selective advantage, they have a low
probability of being fixed in large populations (Ober, 2010).
Unless the presence of an extra amount of gene product is advantageous, it is unlikely that
two genes with the same function will be stably maintained in the genome of the organism
(Nowak et al., 1997). In subfunctionalization (Figure 2c), both duplicated copies may
become, by accumulation of mutations, partially compromised to the point at which their
total capacity is reduced to the level of the single-copy ancestral gene (Force et al., 1999;
Stoltzfus, 1999; Lynch and Force, 2000). Subfunctionalization can occur through the
modification of the regulatory elements by mutations (Force et al., 1999; Hinman and
Davidson, 2007) or by epigenetic silencing (Rodin and Riggs, 2003). In an evolutionary scale,
one of the most important forms of subfunctionalization is the division of gene expression
after duplication (Force et al., 1999). For example, zebrafish ENGRAILED 1 and
ENGRAILED 1-B, generated by a chromosomal segmental duplication, are a pair of
transcription factors that occurred in the lineage of ray-finned fish. While ENGRAILED-1 is
expressed in the pectoral appendage bud, ENGRAILED 1-B is expressed in a specific set of
neurons in the hindbrain/spinal cord (Force et al., 1999). In yeast, more than 40% of gene
pairs exhibit significant expression divergence (Gu et al., 2002). Also, the comparison of 17
fungal genomes revealed that duplicated genes rarely diverge with respect to biochemical
function, but typically diverge with respect to regulatory control (Wapinski et al., 2007). On
the other hand, if two redundant gene copies were retained without significant functional
divergence in the genome, the organism may acquire increased genetic robustness against
harmful mutations (Figure 1d) (Conrad and Antonarakis, 2007).
In neofunctionalization (Figure 2b), the ancestral gene keeps its ancestral function, while the
duplicated gene gains a new function under positive selection for advantageous mutations
(De Grassi et al., 2008). However, in many cases, rather than an entirely new function, a
related function evolves after gene duplication. For example, the red and green-sensitive
opsin genes of humans where the result of a gene duplication that occurred in hominoids
and Old World monkeys (Yokoyama and Yokoyama, 1989). After the duplication process,

203
functional divergence of the two opsins resulted in a 30-nanometer difference in their
maximum absorption wavelength. This difference conferred a sensitivity to a wide range of
colors for humans and related primates (Zhang, 2003).

Fig. 2. Evolutionary fate of duplicated genes. Gene duplication may result in new functions
via different scenarios. (a) nonfunctionalization; (b) neofunctionalization;
(c) subfunctionalization; (d) genetic robustness and (e) gene conversion.
Adapted from Conrad and Antonarakis (2007).
The fate of a gene that suffers duplication seems to be the result of diverse and, in some cases,
interdependent factors (Taylor et al., 2001). These variables include its functional category
(Papp et al., 2003; Kondrashov and Koonin, 2004; Marland et al., 2004), degree of conservation
(Conant and Wagner, 2002; Davis and Petrov, 2004; Jordan et al., 2004; Braybrook and Harada,
2008), sensitivity to dosage effects (Kondrashov and Koonin, 2004), as well as its regulatory
and architectural complexity (He and Zhang, 2005). Some observations indicate that natural
selection created a preferential association of duplications with certain gene categories. For
example, genes encoding proteins that interact with the environment are more frequently
retained after the duplication process than genes which interact at intracellular compartments

Gene Duplication

204
(Li et al., 2003; Marland et al., 2004). In addition, genomes tend to retain duplicated genes
involved in signal transduction and transcription, but to lose duplicated DNA repair genes
(Blanc and Wolfe, 2004; Maere et al., 2005; Paterson et al., 2010).

Fig. 3. Functional divergence of duplicated genes. (a) The independent evolution of cis-
regulatory and the protein-coding regions. (b1) protein sequence divergence after
duplication; (b2) protein network divergence with increase or loss of partners and (c) DNA
sequence regulatory divergence after duplication (certain regulatory motifs are lost in one
copy of the duplicated gene sequence). t; evolutionary time. Adapted from Conrad and
Antonarakis (2007).

205
It has been shown that shortly after duplication the protein-coding sequence and cis-
regulatory regions of some duplicated genes can evolve independently (Figure 3a) (Wagner,
2000). This independent evolution can generate protein sequence divergence of duplicated
genes (Figure 3b1) or protein network divergence (Figure 3b2), where the protein interaction
domains (cis-regulatory elements) of the original sequence evolve by maintenance, gain, or
loss of interacting partners. Alternatively, the divergence of cis-regulatory motifs in the
promoter-proximal region (Figure 3c) can generate expression divergence between the
duplicated genes (Conrad and Antonarakis, 2007).
5. Gene duplication of NF-Y in plants
While duplication of NF-Y genes is poorly understood in the plant lineage, many of the
functional mechanistic details are likely conserved across plant, animal and fungal lineages.
This inference comes from strong cross-kingdom conservation of functional important
amino acid residues in mammalian and yeast NF-Ys (Maity and de Crombrugghe, 1992;
Maity et al., 1992; Sinha et al., 1995; Coustry et al., 1996; Kim et al., 1996; Sinha et al., 1996;
Mantovani, 1998; Romier et al., 2003). CCAAT-like motifs are found in several plant
promoters, and binding activity to CCAAT sequences has been identified in plant nuclear
extracts (Yazawa and Kamada, 2007). Besides, at least some plant NF-YA and NF-YB
subunits have been shown to complement yeast mutant strains lacking the corresponding
NF-Y subunit. Additionally, several groups have demonstrated that each of the three plant
NF-Y proteins can substitute their yeast counterparts in gene expression assays (Edwards et
al., 1998; Masiero et al., 2002; Ben-Naim et al., 2006; Siefers et al., 2009). These observations
indicate that plant NF-Y subunits might act as general transcription factors, as in mammals
(Yamamoto et al., 2009).
Although a complete functional plant NF-Y complex has not yet been described, the
individual subunits are known to be involved in a number of important physiological
processes, such as specific developmental processes and response to environmental stimuli
(Lotan et al., 1998; Kusnetsov et al., 1999; Miyoshi et al., 2003; Ben-Naim et al., 2006; Combier
et al., 2006; Wenkel et al., 2006; Cai et al., 2007; Nelson et al., 2007; Warpeha et al., 2007; Siefers
et al., 2009). A well-established example is the NF-YB subunit gene called LEAFY
COTYLEDON-1 (LEC1), which specifically controls embryo development, especially the
maturation phase. LEC1 plays specialized roles not only because of its developmentally
regulated expression but also due to its distinct molecular activity, as the in vivo function of
LEC1 cannot be replaced by other NF-YB subunits, except for the most closely related Leafy
Cotyledon 1 Like (L1L) (Kwong et al., 2003; Lee et al., 2003; Yamamoto et al., 2009). In
Arabidopsis, many NF-Y subunit genes are expressed ubiquitously, although some are
differentially expressed. For example, while the AtNF-YC-4 transcript accumulates in seeds
7 days after germination, AtNF-YB-9 is only expressed in green siliques (Gusmaroli et al.,
2001).
Plant NF-Y function also appears to be important for responses to drought stress. Although
a specific mechanism of action remains unclear, overexpression of the AtNF-YB1 subunit
and its orthologue in maize (Zea mays), ZmNF-YB2, leads to enhanced drought resistance
(Nelson et al., 2007). Another study showed that overexpression of maize NF-YA5 reduced
drought susceptibility, anthocyanin production and stomatal aperture, while nf-ya5 mutants
had the expected opposite phenotype in each situation (Li et al., 2008). In addition, several

Gene Duplication

206
publications strongly suggest that NF-Y transcription factors are also involved in
photoperiod-regulated flowering (Ben-Naim et al., 2006; Wenkel et al., 2006; Siefers et al.,
2009).
We adopted a high throughput comparative genomic approach to conduct a broad survey of
fully sequenced genomes, including representatives of amoebozoa, yeasts, fungi, algae,
mosses, plants, vertebrate and invertebrate species to identify the presence of homologous
genes coding for each of the three subunits that form the NF-Y transcription factor (Table 1).
NF-Y gene and protein sequences were obtained through blast searches (blastp, blastx and
tblastx) against the Protein and Genome databases with the default parameters at the NCBI
(National Center for Biotechnology Information - http://www.ncbi.nlm.nih.gov) and
against completed genome projects database at the JGI (Joint Genome Institute -
http://www.jgi.doe.gov).
The results point to a scenario where all fungi and the majority of metazoa possess single
genes coding for each of the NF-Y subunits (Table 1). The metazoa exceptions include the
amphioxus Branchiostoma floridae, the nematode Caenorhabditis elegans and the gastropod
Lottia gigantea, all of each present a proportional duplication of the three subunits,
possessing two genes for each subunit (Table 1).
In contrast, plants possess gene families coding for each NF-Y subunit (Table 1). For
instance, in the model plant Arabidopsis thaliana 10 genes coding for NF-YA, 13 for NFY-B,
and 13 for NF-YC were identified. Because of the heterotrimeric composition, the 36
Arabidopsis NF-Y subunits could theoretically combine to generate 1.690 unique
transcription factors (Siefers et al., 2009). This Arabidopsis NF-Y expansion is a general
feature of the plant lineage, including monocots and eudicots. In rice (Oryza sativa), for
example, 11 genes were identified coding for the NF-YA subunit, 10 for NF-YB and 8 for
NFY-C. Four of the rice NF-YB subunits have been characterized and at least one of these
genes is involved in chloroplast development (Miyoshi et al., 2003; Yazawa and Kamada,
2007). Interestingly, the moss Physcomitrella patens and the lycophyte Selaginella mollendorffii
possess single genes coding for NF-YA subunits whereas the other subunits are encoded by
multiple genes (Table 1).
Since the evolutionary rates can be species dependent, the difference observed in the
number of genes of NF-Y subunits in eukaryotic class (Table 1), especially in vascular plants,
can be result of recent duplication process that contribute to the establishment of genes
families coding each NF-Y subunit. However, some duplicated genes might have suffered
high level of diversification what could be responsible to prevent their identification in our
analyses.
Representative plants genes (monocot and eudicot) were selected to perform phylogenetic
analyses of the NF-Y subunits. The phylogenetic analysis was reconstructed after protein
sequence alignments using a Bayesian approach in MrBayes 3.1.2 (Ronquist and
Huelsenbeck, 2003). The mixed amino acid substitution model plus gamma and invariant
sites was used in two independent runs of 5,000,000 generations each with two Metropolis-
coupled Monte Carlo Markov chains (MCMCMC) that were run in parallel (starting each
from a random tree). Markov chains were sampled every 100 generations, and the first 25%
of the trees were discarded as burn-in. The remaining ones were used to compute the
majority rule consensus tree, the posterior probability of clades and branch lengths (Figure 4
to 6).

207
Phylo Specie Code
Subunit A
Genes
Subunit B Genes Subunit C Genes
Metazoa Homo sapiens Hsa 1 1 1
Mus musculus Mmu 1 1 1
Rattus norvegicus Rno 1 1 1
Canis familaris Cfa 1 1 1
Monodelphis domestica Mdo 1 1 1
Gallus gallus Gga 1 1 1
Xenopus tropicalis Xtr 1 1 1
Gasterosteus aculeatus Gac 1 1 1
Oryzias latipes Ola 1 1 1
Takifugu rubripes Tru 1 1 1
Danio rerio Dre 1 1 1
Ciona savignyi Csa 1 1 1
Branchiostoma floridae Bfl 2 2 2

Strongylocentrotus
purpuratus
Spu
1 1 1

Drosophila
melanogaster
Dme
1 1 1
Anopheles gambiae Aga 1 1 1
Tribolium castaneum Tca 1 1 1
Caenorhabditis elegans Cel 2 2 2
Lottia gigantea Lgi 2 2 2
Nematostella vectensis Nve 1 1 1
Fungi Neurospora crassa Ncr 1 1 1
Candida tropicalis Ctr 1 1 1
Tuber melanosporum Tme 1 1 1
Pyrenophora teres Pte 1 1 1
Aspergillus nidulans Ani 1 1 1
Chaetomium globosum Cgl 1 1 1
Penicillium marneffei Pma 1 1 1
Talaromyces stipitatus Tst 1 1 1
Sordaria macrospora Sma 1 1 1
Heterolobosea Naegleria gruberi Ngr 1 1 1
Metaphyta Manihot esculenta Mes 12 15 9
Ricinus communis Rco 6 12 7
Populus tricocharpa Ptr 8 17 9
Medicago truncatula Mtr 5 10 5
Glycine max Gma 21 25 11
Cucumis sativus Csa 6 11 3
Prunus persica Ppe 6 13 6
Arabidopsis thaliana Ath 10 13 13
Carica papaya Cpa 5 9 3
Vitis vinifera Vvi 7 12 5
Sorghum bicolor Sbi 9 10 7
Zea mays Zma 10 20 14
Oryza sativa Osa 11 10 8
Brachipodyum Bdi 7 13 10

Gene Duplication

208
distachyon
Selaginella mollendorffii Smo 1 5 3
Physcomitrella patens Ppa 1 6 6
Heterokonta
Phaeodactylum
tricornutum
Ptri
1 1 1
Table 1. NF-Y genes identified in the fully eukaryotic sequenced genomes.
10.0
Gma6
Sbi6
Ath7
Mes1
Ptr7
Gma13
Ath2
Mes7
Gma12
Ath6
Gma15
Ptr8
Gma19
Ptr3
Gma17
Osa3
Gma9
Ath5
Osa6
Mes5
Mtr4
Bdi7
Ath9
Ptr2
Sbi2
Vvi4
Rco5
Mes11
Bdi1
Ath4
Gma8
Gma10
Mes3
Mes2
Gma4
Gma16
Osa8
Vvi2
Rco2
Osa1
Bdi5
Gma1
Vvi1
Vvi5
Mes9
Ptr1
Mes10
Vvi6
Osa10
Mtr3
Rco6
Mes12
Vvi7
Gma5
Gma3
Ptr5
Bdi4
Sbi3
Ptr6
Gma2
Gma7
Gma20
Ath3
Rco3
Osa4
Bdi3
Mtr2
Gma14
Sbi1
Osa9
Ath10
Mes4
Vvi3
Bdi6
Mes8
Osa2
Ath8
Rco1
Bdi2
Sbi4
Sbi9
Gma18
Osa7
Mes6
Sbi5
Sbi8
Ath1
Gma21
Sbi7
Ptr4
Mtr5
Mtr1
Gma11
Osa5
Rco4
1
0,95
1
0,99
0,97
1
1
1
0,99
0,95
1
1
1
1
0,98
0,63
0,6
0,77
1
0,95
1
1
0,57
1
0,92
0,98
0,77
1
1
1
0,76
1
1
1
1
0,95
0,51
1
1
0,98
1
1
1
0,87
1
1
0,95
0,99
1
0,85
1
0,76
1
0,98
1
1
1
0,96
0,85
1
0,95
0,95
0,99
1
1
1
1
1
0,99
0,81
0,76
1
1
1
1
1
1
1
0,73
1
0,94
0,99
0,99
0,95
1
0,99
1
1
1
D
D
D
D
M
M
M
M
I
II
III
IV

Fig. 4. Phylogenetic tree of monocot and eudicot representatives of NF-YA subunit.M:
monocots; D: eudicots; Rco: Ricinus communis; Mes: Manihot esculenta; Ptr: Populus
tricocharpa; Gma: Glycine max; Mtr: Medicago truncatula; Vvi: Vitis vinifera; Ath: Arabidopsis
thaliana; Sbi: Sorghum bicolor; Bdi: Brachipodyum distachyon; Osa: Oryza sativa; red square:
event of duplication inside the specie; green square: event of duplication inside the same
plant family; black arrows: genes that possess an unresolved position in the phylogenetic
tree; I to IV: independent phylogenetic gene clusters.

209
20.0
Mes14
Bdi12
Ptr11
Bdi11
Vvi1
Sbi3
Mes6
Ptr9
Vvi2
Vvi7
Sbi8
Ath8
Gma10
Gma21
Mes9
Ptr10
Sbi10
Ptr1
Mes15
Gma15
Bdi1
Sbi1
Gma5
Ath4
Gma23
Mtr7
Osa4
Rco12
Gma19
Mes13
Mtr8
Gma9
Bdi7
Rco11
Bdi3
Mes3
Osa2
Ptr7
Mes11
Osa5
Rco6
Gma22
Vvi9
Mtr6
Rco5
Mes4
Ptr13
Bdi8
Ptr16
Mes8
Ptr14
Ath10
Bdi2
Sbi9
Mtr4
Gma2
Ptr6
Vvi12
Vvi4
Bdi10
Gma25
Gma16
Bdi9
Gma13
Vvi8
Ath6
Mes7
Bdi13
Ptr8
Rco2
Osa9
Vvi6
Gma18
Ath3
Sbi4
Osa7
Bdi6
Ptr12
Osa3
Bdi5
Rco8
Mes12
Gma6
Sbi2
Mtr1
Ath5
Sbi7
Gma14
Ath1
Ath2
Mtr2
Vvi5
Rco9
Ptr15
Gma1
Osa6
Ptr2
Sbi6
Gma3
Rco10
Ptr3
Gma24
Sbi5
Vvi3
Ptr4
Gma8
Osa1
Vvi10
Osa8
Mes10
Gma17
Mes5
Gma20
Gma4
Ath7
Vvi11
Rco1
Rco4
Mes1
Mtr5
Mes2
Mtr10
Ath9
Rco3
Gma7
Ptr5
Gma12
Rco7
Bdi4
Mtr9
Mtr3
Ptr17
Osa10
Gma11
0,63
0,99
0,72
0,81
0,99
0,68
0,9
0,66
0,98
1
0,63
1
1
1
1
1
0,97
1
0,8
0,83
1
0,81
0,98
0,98
0,71
0,71
1
0,95
0,83
1
1
1
0,94
0,59
0,74
0,62
0,92
0,99
0,95
0,93
0,97
1
0,78
0,63
1
0,71
0,99
0,55
0,9
1
0,99
0,71
0,97
1
0,94
0,92
0,51
0,63
0,73
0,99
1
0,53
1
1
0,98
0,71
0,53
1
1
0,7
0,7
0,64
0,99
1
0,91
1
0,89
0,97
0,96
0,67
0,99
1
0,55
0,92
0,71
0,93
1
0,91
0,98
1
0,92
0,94
0,7
1
0,94
0,9
0,77
0,98
0,79
0,59
0,51
0,68
0,95
0,89
0,97
0,72
1
0,89
0,99
0,98
0,98
0,61
1
1
1
0,6
1
0,97
0,99
0,83
1
1
0,95
0,83
1
D
D
D
D
D
M
D
M
M
M
M
D
I
II
III

Fig. 5. Phylogenetic tree of monocot and eudicot representatives of NF-YB subunit.
For details see legend of Figure 4.

Gene Duplication

210
9.0
Ptr7
Gma6
Gma4
Mtr2
ATh9
Ptr2
Gma9
Bdi10
Bdi6
Vvi2
ATh8
Gma11
Osa8
Rco5
ATh1
Bdi7
Osa5
Osa2
Mtr1
Ptr4
Rco4
Osa3
ATh6
Gma5
Ptr5
Bdi5
ATh13
Rco6
ATh3
Ptr6
Mes3
Rco3
Ath4
Sbi5
Vvi3
Vvi5
Bdi3
Osa7
Bdi9
Mtr5
Mes2
Mes9
Osa4
Sbi6
Gma2
Gma8
Mtr3
Bdi8
Rco2
Gma1
ATh2
Gma10
Sbi3
Gma7
Rco7
Ath12
Osa6
Ptr8
Ptr3
Mes7
Mtr4
Bdi4
Mes5
Vvi1
Mes8
Sbi1
Sbi2
Ptr9
Sbi4
ATh7
Rco1
Bdi1
Ptr1
Gma3
ATh5
Mes1
Vvi4
ATh10
ATh11
Mes6
Bdi2
Osa1
Sbi7
Mes4
1
0,81
1
1
0,99
1
0,8
0,96
1
0,76
0,99
1
1
1
1
1
0,72
0,8
0,99
0,86
1
0,6
1
1
1
0,62
1
0,93
1
1
0,9
0,87
1
0,65
0,92
1
0,97
0,71
0,94
0,91
1
0,99
0,75
1
0,68 1
0,55
0,78
0,61
0,71
0,72
1
1
1
0,87
1
0,97
0,71
1
1
1
1
1
0,91
0,95
1
0,89
1
1
1
1
0,94
1
0,62
0,99
M
M
M
D
D
D
D
D
V
IV
III
II
I

Fig. 6. Phylogenetic tree of monocot and eudicot representatives of NF-YC subunit.
For details see legend of Figure 4.

211
Phylogenetic analysis showed that the gene diversification of all NF-Y subunits likely
resulted from several duplication events along evolution and diversification of plants
(Figure 4 to 6). It was possible to observe the formation of four independent highly
supported clusters for the NF-YA subunit (I to IV, Figure 4), three for NF-YB (I to III, Figure
5) and five for NF-YC (I to V, Figure 6). Based on these results, we suggest that each cluster
might possess an independent ancestral subunit that the duplicated members of each group
originated from. However, independent duplication events have occurred in many species
after the divergence of monocots and eudicots. For example, the soybean and Arabidopsis
genomes have experienced a series of recent duplication events (red squares in figures 4 to
6) that could be the result of chromosome duplication or could be derived from
polyploidization events (soybean is a good example of a recent polyploidization). These
duplications can help us to explain the differences observed in the number of genes coding
for the NF-Y subunits in plants (Table 1). Additionally, these duplications seem to be
relatively recent and can provide the raw material for neofunctionalization (Figure 2b) and
functional divergence of duplicated genes (Figure 3). With few exceptions (genes that
possess an unresolved position in the phylogenetic tree are plotted with black arrows,
Figures 4 to 6), all clusters of a specific NF-Y subunit are formed by well-defined sub-
clusters of monocot and eudicot representatives (Figure 4 to 6). Events of duplication inside
a specific plant family were also observed between the two fabaceae species Glycine max and
Medicago truncatula (green square, Figure 4), which could indicate concerted evolution of
duplicated genes between these related species (Figure 2e). This is similar to the clade-
specific shifts in selective constraint following concerted duplication events observed for
MADS box transcription factors in angiosperms (Shan et al., 2009).
The duplication process is a prominent feature of plant genomic architecture (Figure 1). This
has led many researchers to speculate that gene duplication may have played an important
role in the evolution of phenotypic novelty within the plant lineage (Flagel and Wendel,
2009). As a result of pervasive and recurring small-scale duplications, which may be
followed by functional divergence, many nuclear genes in plants are members of gene
families and may exhibit copy number variation lineages (Blanc and Wolfe, 2004; Schlueter
et al., 2004), as can be observed in table 1.
Evidence for frequent gene duplication has also been observed in the evolutionary history of
numerous gene families that have expanded during the diversification of the angiosperms
(De Bodt et al., 2005; Zahn et al., 2005; Duarte et al., 2010). In multigene families descended
from a common ancestor, individual genes in the group exert similar functions and have
similar DNA sequences (Conrad and Antonarakis, 2007). One concept, concerted evolution,
applies particularly to localized and typically tandem copies of a gene. The concept posits
that all genes in a given group evolve coordinately, and that homogenization is the result of
gene conversion (Figure 2e) (Conrad and Antonarakis, 2007).
The emerging picture points to plant NF-Y complexes acting as essential regulatory hubs for
many processes. Multiple NF-Y subunits in vascular plants may associate with each other in
various combinations that regulate the expression of specific gene sets and might provide
similar levels of combinatorial diversity for transcriptional fine-tuning (Siefers et al.,
2009).The amplification observed in the plant lineage (Table 1) raises the possibility that new
and divergent functions of heterotrimeric complexes have evolved in plants (Nelson et al.,
2007) indicating a more complex regulatory role for the various NF-Y proteins in plants than
in other organisms (Stephenson et al., 2007).

Gene Duplication

212
The existence of multiple genes for each subunit in the plant genome indicates that the
specificity of subunit interaction may be determined by preferential proteinprotein
interaction, tissue or cell-specific expression of each gene or a combination of both (Yazawa
and Kamada, 2007). The large number of possible combinations has hindered the analysis of
plant NF-Y complexes and suggests that they might act in a more intricate system than in
vertebrates and yeast, which have only one gene that encodes each HAP subunit (Yazawa
and Kamada, 2007). Additionally, the multiple copies for each NF-Y subunit raises a
question if a specific NF-Y subunit interacts with any other two NF-Y subunits or if the NF-
Y subunit interacts with only specific member(s) of the other two subunits (Thirumurugan et
al., 2008).
Although the presence of many genes encoding NF-Y subunits suggests a high degree of
genetic redundancy in plants, the analysis of mutants in single NF-Y genes in Arabidopsis
has been associated with defects in development and enhanced stress sensitivity, suggesting
a specialized function for each member (Lotan et al., 1998; Kwong et al., 2003; Lee et al., 2003;
Zanetti et al., 2010). This could indicate that duplicated genes have passed through a
neofunctionalization process (Figure 2b).
Some proteins may require several key substitutions before acquiring a new function, while
others may be more mutationally labile. An example includes the terpene synthase gene
family in Norway spruce (Picea abies). These genes appear to have undergone repeated
rounds of neofunctionalization (Figure 2b) (Keeling et al., 2008) and a small number of key
amino acid substitutions among paralogs was sufficient to alter the substrate specificity and
terpenoid product profiles (Flagel and Wendel, 2009). Another example of
neofunctionalization (Figure 2b) in plants is observed in Arabidopsis, where a specific
amino acid residue identified in LEC1 and LEC1-LIKE (L1L) is responsible for
differentiating their functions (seed development) from those of other NF-YB members
(Kwong et al., 2003; Lee et al., 2003; Yamamoto et al., 2009). In addition, the analysis of amino
acid substitution rates in plants has been appointed for the asymmetric evolution of certain
duplicates of NF-YB and NF-YC subunits, which appears to be coupled with the asymmetric
divergence in gene function (Yang et al., 2005; Yamamoto et al., 2009).
With respect to expression patterns, the Arabidopsis NF-Y gene family presents some
members that are ubiquitously expressed and others that are tissue specific or induced only
after the switch to reproductive growth in flowers and siliques (Gusmaroli et al., 2001; 2002;
Yazawa and Kamada, 2007). The difference observed in the expression pattern of these
genes could represent an example of cis-regulatory divergence (Figure 3c), where the cis-
element of gene evolves independently from the other members of gene family, and
becomes regulated by different stimuli and/or trans-activators.
Because genes that harbor NF-Y binding domains include genes that are constitutive,
inducible, and cell-cycle-dependent, the regulation of the expression of these genes cannot
be exclusively due to NF-Y binding to DNA. In this scenario, the interaction with other
transcription factors, either functionally or physically, will contribute to the NF-Y action
(Matuoka and Chen, 2002). In addition, the independent evolution of protein-binding
domains present in duplicated gene architecture can contribute to protein network
divergence (Figure 3b2), increasing the numbers of possible interacting partners of NF-Y
genes.
When compared with other forms of mutation, a notable feature of duplication is that it
creates genetic redundancy. This redundancy fosters evolutionary innovation, creating the
opportunity for duplicates to explore new evolutionary terrain (Flagel and Wendel, 2009).

213
The most important contribution of gene duplication to evolution is to supply new genetic
material for mutation, drift and selection to act upon. This leads to the creation of new genes
and new gene functions (Hurley et al., 2005; Woollard, 2005; Schmidt and Davies, 2007), two
important factors in the origin of genomic and organismal complexity (Gu et al., 2002; Taylor
and Raes, 2004; Sterck et al., 2007).
The plasticity of a genome or species in adapting to environmental changes would be
severely limited without gene duplication, because no more than two variants (alleles) exist
at any locus within a diploid individual. A good example is the dozens of duplicated
immunoglobulin genes that constitute the vertebrate adaptive immune system. It seems
difficult to imagine how this system could have acquired this high complexity level without
gene duplication (Zhang, 2003).
Plant gene families are largely conserved even over evolutionary time scales that encompass
the diversification of all angiosperms and nonflowering plants (Rensing et al., 2008). This
property of plant genomes indicates that plants have not created new gene families, but
have been endowed with a basic genetic toolkit of ancient origin. Despite the evolutionary
conservation of gene families, lineage-specific fluctuations in gene family size are frequently
observed among taxa (Velasco et al., 2007; Ming et al., 2008; Rensing et al., 2008), which
suggests that the diversity and lineage-specific phenotypic variation observed in land plants
may not be explained by an equally diverse set of entirely novel genes. Indeed, much of
plant diversity may have arisen from the duplication and adaptive specialization processes
of pre-existing genes (neofunctionalization and subfunctionalization, Figure 2b and c,
respectively). This perspective assigns gene duplication a central role in plant
diversification, being a key process that generates the raw material necessary for adaptive
evolution (Flagel and Wendel, 2009).
6. Conclusions
Whereas various classes of structural and metabolic genes preferentially return to a single
copy state following whole-genome duplication (Paterson et al., 2010), transcription factors
tend to be preferentially retained among the duplicated genes in A. thaliana (Flagel and
Wendel, 2009). Our findings support the hypotheses that this preference seems to be true for
all plant species, based on the number of genes identified for each NF-Y subunit. Certainly,
further studies encompassing functional assays are required to ascertain the role of these
genes in plant metabolism.
The number of interacting partners in a molecular network (connectivity) of a particular
gene also influences the probability of duplication gene retention (Flagel and Wendel, 2009).
In this scenario, the high number of genes coding for the three subunits of NF-Y
transcription factor in higher plants leads to numerous interaction possibilities among
different genes of each subunit and among these genes and other transcription factors what
could contribute to gene retention of the NF-Y transcription factor family in plants.
7. Acknowledgement
This work was supported by a CNPq (Conselho Nacional de Desenvolvimento Cientfico e
Tecnolgico), CAPES (Coordenao de Aperfeioamento de Pessoal de Nvel Superior),
FAPERGS (Fundao de Amparo a Pesquisa do Estado do Rio Grande do Sul), FINEP
(Financiadora de Projetos) and MCT (Ministrio de Cincia e Tecnologia). A. Cagliari

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214
received a Ph.D. fellowship from CNPq. and G. Loss a Ph.D. fellowship from CAPES. A.
Turchetto-Zolet and F. Maraschin have a PNPD-CAPES fellowship and M. Margis-Pinheiro
and R. Margis are recipients of CNPq research fellowships number 308708/2006-7 and
303967/2008-0, respectively.
8. References
Arents, G. and Moudrianakis, E.N. (1995) The histone fold: a ubiquitous architectural motif
utilized in DNA compaction and protein dimerization. Proc Natl Acad Sci U S A, 92,
11170-11174.
Baxevanis, A.D., Arents, G., Moudrianakis, E.N. and Landsman, D. (1995) A variety of
DNA-binding and multimeric proteins contain the histone fold motif. Nucleic Acids
Res, 23, 2685-2691.
Beisswanger, S. and Stephan, W. (2008) Evidence that strong positive selection drives
neofunctionalization in the tandemly duplicated polyhomeotic genes in
Drosophila. Proc Natl Acad Sci U S A, 105, 5447-5452.
Ben-Naim, O., Eshed, R., Parnis, A., Teper-Bamnolker, P., Shalit, A., Coupland, G., Samach,
A. and Lifschitz, E. (2006) The CCAAT binding factor can mediate interactions
between CONSTANS-like proteins and DNA. Plant J, 46, 462-476.
Berry, M., Grosveld, F. and Dillon, N. (1992) A single point mutation is the cause of the
Greek form of hereditary persistence of fetal haemoglobin. Nature, 358, 499-502.
Blanc, G. and Wolfe, K.H. (2004) Widespread paleopolyploidy in model plant species
inferred from age distributions of duplicate genes. Plant Cell, 16, 1667-1678.
Bowers, J.E., Chapman, B.A., Rong, J. and Paterson, A.H. (2003) Unravelling angiosperm
genome evolution by phylogenetic analysis of chromosomal duplication events.
Nature, 422, 433-438.
Braybrook, S.A. and Harada, J.J. (2008) LECs go crazy in embryo development. Trends Plant
Sci, 13, 624-630.
Bucher, P. (1990) Weight matrix descriptions of four eukaryotic RNA polymerase II
promoter elements derived from 502 unrelated promoter sequences. J Mol Biol, 212,
563-578.
Cai, X., Ballif, J., Endo, S., Davis, E., Liang, M., Chen, D., DeWald, D., Kreps, J., Zhu, T. and
Wu, Y. (2007) A putative CCAAT-binding transcription factor is a regulator of
flowering timing in Arabidopsis. Plant Physiol, 145, 98-105.
Caretti, G., Motta, M.C. and Mantovani, R. (1999) NF-Y associates with H3-H4 tetramers and
octamers by multiple mechanisms. Mol Cell Biol, 19, 8591-8603.
Ceribelli, M., Dolfini, D., Merico, D., Gatta, R., Vigano, A.M., Pavesi, G. and Mantovani, R.
(2008) The histone-like NF-Y is a bifunctional transcription factor. Mol Cell Biol, 28,
2047-2058.
Chodosh, L.A., Baldwin, A.S., Carthew, R.W. and Sharp, P.A. (1988) Human CCAAT-
binding proteins have heterologous subunits. Cell, 53, 11-24.
Combier, J.P., Frugier, F., de Billy, F., Boualem, A., El-Yahyaoui, F., Moreau, S., Vernie, T.,
Ott, T., Gamas, P., Crespi, M. and Niebel, A. (2006) MtHAP2-1 is a key
transcriptional regulator of symbiotic nodule development regulated by
microRNA169 in Medicago truncatula. Genes Dev, 20, 3084-3088.

215
Conant, G.C. and Wagner, A. (2002) GenomeHistory: a software tool and its application to
fully sequenced genomes. Nucleic Acids Res, 30, 3378-3386.
Conrad, B. and Antonarakis, S.E. (2007) Gene duplication: a drive for phenotypic diversity
and cause of human disease. Annu Rev Genomics Hum Genet, 8, 17-35.
Coustry, F., Maity, S.N., Sinha, S. and de Crombrugghe, B. (1996) The transcriptional activity
of the CCAAT-binding factor CBF is mediated by two distinct activation domains,
one in the CBF-B subunit and the other in the CBF-C subunit. J Biol Chem, 271,
14485-14491.
Coustry, F., Hu, Q., de Crombrugghe, B. and Maity, S.N. (2001) CBF/NF-Y functions both in
nucleosomal disruption and transcription activation of the chromatin-assembled
topoisomerase IIalpha promoter. Transcription activation by CBF/NF-Y in
chromatin is dependent on the promoter structure. J Biol Chem, 276, 40621-40630.
Currie, R.A. (1998) NF-Y is associated with the histone acetyltransferases GCN5 and P/CAF.
J Biol Chem, 273, 1430-1434.
Dang, V.D., Bohn, C., Bolotin-Fukuhara, M. and Daignan-Fornier, B. (1996) The CCAAT
box-binding factor stimulates ammonium assimilation in Saccharomyces cerevisiae,
defining a new cross-pathway regulation between nitrogen and carbon
metabolisms. J Bacteriol, 178, 1842-1849.
Davis, J.C. and Petrov, D.A. (2004) Preferential duplication of conserved proteins in
eukaryotic genomes. PLoS Biol, 2, E55.
De Bodt, S., Maere, S. and Van de Peer, Y. (2005) Genome duplication and the origin of
angiosperms. Trends Ecol Evol, 20, 591-597.
De Grassi, A., Lanave, C. and Saccone, C. (2008) Genome duplication and gene-family
evolution: the case of three OXPHOS gene families. Gene, 421, 1-6.
de Silvio, A., Imbriano, C. and Mantovani, R. (1999) Dissection of the NF-Y transcriptional
activation potential. Nucleic Acids Res, 27, 2578-2584.
Dorn, A., Bollekens, J., Staub, A., Benoist, C. and Mathis, D. (1987a) A multiplicity of
CCAAT box-binding proteins. Cell, 50, 863-872.
Dorn, A., Durand, B., Marfing, C., Le Meur, M., Benoist, C. and Mathis, D. (1987b)
Conserved major histocompatibility complex class II boxes--X and Y--are
transcriptional control elements and specifically bind nuclear proteins. Proc Natl
Acad Sci U S A, 84, 6249-6253.
Duarte, J.M., Wall, P.K., Edger, P.P., Landherr, L.L., Ma, H., Pires, J.C., Leebens-Mack, J. and
dePamphilis, C.W. (2010) Identification of shared single copy nuclear genes in
Arabidopsis, Populus, Vitis and Oryza and their phylogenetic utility across various
taxonomic levels. BMC Evol Biol, 10, 61.
Dynan, W.S. and Tjian, R. (1985) Control of eukaryotic messenger RNA synthesis by
sequence-specific DNA-binding proteins. Nature, 316, 774-778.
Edwards, D., Murray, J.A. and Smith, A.G. (1998) Multiple genes encoding the conserved
CCAAT-box transcription factor complex are expressed in Arabidopsis. Plant
Physiol, 117, 1015-1022.
Flagel, L.E. and Wendel, J.F. (2009) Gene duplication and evolutionary novelty in plants.
New Phytol, 183, 557-564.

Gene Duplication

216
Force, A., Lynch, M., Pickett, F.B., Amores, A., Yan, Y.L. and Postlethwait, J. (1999)
Genetics, 151, 1531-1545.
Forsburg, S.L. and Guarente, L. (1989) Identification and characterization of HAP4: a third
component of the CCAAT-bound HAP2/HAP3 heteromer. Genes Dev, 3, 1166-1178.
Gallagher, C.E., Matthews, P.D., Li, F. and Wurtzel, E.T. (2004) Gene duplication in the
carotenoid biosynthetic pathway preceded evolution of the grasses. Plant Physiol,
135, 1776-1783.
Gu, Z., Nicolae, D., Lu, H.H. and Li, W.H. (2002) Rapid divergence in expression between
duplicate genes inferred from microarray data. Trends Genet, 18, 609-613.
Gusmaroli, G., Tonelli, C. and Mantovani, R. (2001) Regulation of the CCAAT-Binding NF-Y
subunits in Arabidopsis thaliana. Gene, 264, 173-185.
Gusmaroli, G., Tonelli, C. and Mantovani, R. (2002) Regulation of novel members of the
Arabidopsis thaliana CCAAT-binding nuclear factor Y subunits. Gene, 283, 41-48.
Harrison, P.M., Echols, N. and Gerstein, M.B. (2001) Digging for dead genes: an analysis of
the characteristics of the pseudogene population in the Caenorhabditis elegans
genome. Nucleic Acids Res, 29, 818-830.
Harrison, P.M., Hegyi, H., Balasubramanian, S., Luscombe, N.M., Bertone, P., Echols, N.,
Johnson, T. and Gerstein, M. (2002) Molecular fossils in the human genome:
identification and analysis of the pseudogenes in chromosomes 21 and 22. Genome
Res, 12, 272-280.
He, X.L. and Zhang, J.Z. (2005) Gene complexity and gene duplicability. Current Biology, 15,
1016-1021.
Hinman, V.F. and Davidson, E.H. (2007) Evolutionary plasticity of developmental gene
regulatory network architecture. Proc Natl Acad Sci U S A, 104, 19404-19409.
Holland, P.W., Garcia-Fernandez, J., Williams, N.A. and Sidow, A. (1994) Gene duplications
and the origins of vertebrate development. Dev Suppl, 125-133.
Huang, H.S., Chen, C.J. and Chang, W.C. (1999) The CCAAT-box binding factor NF-Y is
required for the expression of phospholipid hydroperoxide glutathione peroxidase
in human epidermoid carcinoma A431 cells. FEBS Lett, 455, 111-116.
Hurley, I., Hale, M.E. and Prince, V.E. (2005) Duplication events and the evolution of
segmental identity. Evol Dev, 7, 556-567.
Jordan, I.K., Wolf, Y.I. and Koonin, E.V. (2004) Duplicated genes evolve slower than
singletons despite the initial rate increase. BMC Evol Biol, 4, 22.
Keeling, C.I., Weisshaar, S., Lin, R.P. and Bohlmann, J. (2008) Functional plasticity of
paralogous diterpene synthases involved in conifer defense. Proc Natl Acad Sci U S
A, 105, 1085-1090.
Kim, I.S., Sinha, S., de Crombrugghe, B. and Maity, S.N. (1996) Determination of functional
domains in the C subunit of the CCAAT-binding factor (CBF) necessary for
formation of a CBF-DNA complex: CBF-B interacts simultaneously with both the
CBF-A and CBF-C subunits to form a heterotrimeric CBF molecule. Mol Cell Biol, 16,
4003-4013.
Kimura, M. (1989) The neutral theory of molecular evolution and the world view of the
neutralists. Genome, 31, 24-31.

217
Kondrashov, F.A. and Koonin, E.V. (2004) A common framework for understanding the
origin of genetic dominance and evolutionary fates of gene duplications. Trends
Genet, 20, 287-290.
Kusnetsov, V., Landsberger, M., Meurer, J. and Oelmuller, R. (1999) The assembly of the
CAAT-box binding complex at a photosynthesis gene promoter is regulated by
light, cytokinin, and the stage of the plastids. J Biol Chem, 274, 36009-36014.
Kwong, R.W., Bui, A.Q., Lee, H., Kwong, L.W., Fischer, R.L., Goldberg, R.B. and Harada, J.J.
(2003) LEAFY COTYLEDON1-LIKE defines a class of regulators essential for
embryo development. Plant Cell, 15, 5-18.
Lee, H., Fischer, R.L., Goldberg, R.B. and Harada, J.J. (2003) Arabidopsis LEAFY
COTYLEDON1 represents a functionally specialized subunit of the CCAAT
binding transcription factor. Proc Natl Acad Sci U S A, 100, 2152-2156.
Li, W.H., Gu, Z., Cavalcanti, A.R. and Nekrutenko, A. (2003) Detection of gene duplications
and block duplications in eukaryotic genomes. J Struct Funct Genomics, 3, 27-34.
Li, W.X., Oono, Y., Zhu, J., He, X.J., Wu, J.M., Iida, K., Lu, X.Y., Cui, X., Jin, H. and Zhu, J.K.
(2008) The Arabidopsis NFYA5 transcription factor is regulated transcriptionally
and posttranscriptionally to promote drought resistance. Plant Cell, 20, 2238-2251.
Li, X.Y., Mantovani, R., Hooft van Huijsduijnen, R., Andre, I., Benoist, C. and Mathis, D.
(1992) Evolutionary variation of the CCAAT-binding transcription factor NF-Y.
Nucleic Acids Res, 20, 1087-1091.
Liberati, C., di Silvio, A., Ottolenghi, S. and Mantovani, R. (1999) NF-Y binding to twin
CCAAT boxes: role of Q-rich domains and histone fold helices. J Mol Biol, 285, 1441-
1455.
Lotan, T., Ohto, M., Yee, K.M., West, M.A., Lo, R., Kwong, R.W., Yamagishi, K., Fischer,
R.L., Goldberg, R.B. and Harada, J.J. (1998) Arabidopsis LEAFY COTYLEDON1 is
sufficient to induce embryo development in vegetative cells. Cell, 93, 1195-1205.
Luger, K., Mader, A.W., Richmond, R.K., Sargent, D.F. and Richmond, T.J. (1997) Crystal
structure of the nucleosome core particle at 2.8 A resolution. Nature, 389, 251-260.
Lynch, M. and Conery, J.S. (2000) The evolutionary fate and consequences of duplicate
genes. Science, 290, 1151-1155.
Lynch, M. and Force, A. (2000) The probability of duplicate gene preservation by
subfunctionalization. Genetics, 154, 459-473.
Maere, S., De Bodt, S., Raes, J., Casneuf, T., Van Montagu, M., Kuiper, M. and Van de Peer,
Y. (2005) Modeling gene and genome duplications in eukaryotes. Proc Natl Acad Sci
U S A, 102, 5454-5459.
Maity, S.N. and de Crombrugghe, B. (1992) Biochemical analysis of the B subunit of the
heteromeric CCAAT-binding factor. A DNA-binding domain and a subunit
interaction domain are specified by two separate segments. J Biol Chem, 267, 8286-
8292.
Maity, S.N., Sinha, S., Ruteshouser, E.C. and de Crombrugghe, B. (1992) Three different
polypeptides are necessary for DNA binding of the mammalian heteromeric
CCAAT binding factor. J Biol Chem, 267, 16574-16580.
Maity, S.N. and De Crombrugghe, B. (1996) Purification, characterization, and role of
CCAAT-binding factor in transcription. Methods Enzymol, 273, 217-232.

Gene Duplication

218
Maity, S.N. and de Crombrugghe, B. (1998) Role of the CCAAT-binding protein CBF/NF-Y
in transcription. Trends Biochem Sci, 23, 174-178.
Mantovani, R. (1998) A survey of 178 NF-Y binding CCAAT boxes. Nucleic Acids Res, 26,
1135-1143.
Mantovani, R. (1999) The molecular biology of the CCAAT-binding factor NF-Y. Gene, 239,
15-27.
Marland, E., Prachumwat, A., Maltsev, N., Gu, Z. and Li, W.H. (2004) Higher gene
duplicabilities for metabolic proteins than for nonmetabolic proteins in yeast and E.
coli. J Mol Evol, 59, 806-814.
Masiero, S., Imbriano, C., Ravasio, F., Favaro, R., Pelucchi, N., Gorla, M.S., Mantovani, R.,
Colombo, L. and Kater, M.M. (2002) Ternary complex formation between MADS-
box transcription factors and the histone fold protein NF-YB. J Biol Chem, 277,
26429-26435.
Matuoka, K. and Yu Chen, K. (1999) Nuclear factor Y (NF-Y) and cellular senescence. Exp
Cell Res, 253, 365-371.
Matuoka, K. and Chen, K.Y. (2002) Transcriptional regulation of cellular ageing by the
CCAAT box-binding factor CBF/NF-Y. Ageing Res Rev, 1, 639-651.
McNabb, D.S., Xing, Y. and Guarente, L. (1995) Cloning of yeast HAP5: a novel subunit of a
heterotrimeric complex required for CCAAT binding. Genes Dev, 9, 47-58.
Miyoshi, K., Ito, Y., Serizawa, A. and Kurata, N. (2003) OsHAP3 genes regulate chloroplast
biogenesis in rice. Plant J, 36, 532-540.
Muro, A.F., Bernath, V.A. and Kornblihtt, A.R. (1992) Interaction of the -170 cyclic AMP
response element with the adjacent CCAAT box in the human fibronectin gene
promoter. J Biol Chem, 267, 12767-12774.
Myers, R.M., Tilly, K. and Maniatis, T. (1986) Fine structure genetic analysis of a beta-globin
promoter. Science, 232, 613-618.
Nelson, D.E., Repetti, P.P., Adams, T.R., Creelman, R.A., Wu, J., Warner, D.C., Anstrom,
D.C., Bensen, R.J., Castiglioni, P.P., Donnarummo, M.G., Hinchey, B.S., Kumimoto,
R.W., Maszle, D.R., Canales, R.D., Krolikowski, K.A., Dotson, S.B., Gutterson, N.,
Ratcliffe, O.J. and Heard, J.E. (2007) Plant nuclear factor Y (NF-Y) B subunits confer
drought tolerance and lead to improved corn yields on water-limited acres. Proc
Natl Acad Sci U S A, 104, 16450-16455.
Nicole, M.C., Hamel, L.P., Morency, M.J., Beaudoin, N., Ellis, B.E. and Seguin, A. (2006)
MAP-ping genomic organization and organ-specific expression profiles of poplar
MAP kinases and MAP kinase kinases. BMC Genomics, 7, 223.
Nowak, M.A., Boerlijst, M.C., Cooke, J. and Smith, J.M. (1997) Evolution of genetic
redundancy. Nature, 388, 167-171.
Ober, D. (2010) Gene duplications and the time thereafter - examples from plant secondary
metabolism. Plant Biol (Stuttg), 12, 570-577.
Olesen, J.T. and Guarente, L. (1990) The HAP2 subunit of yeast CCAAT transcriptional
activator contains adjacent domains for subunit association and DNA recognition:
model for the HAP2/3/4 complex. Genes Dev, 4, 1714-1729.
Papp, B., Pal, C. and Hurst, L.D. (2003) Dosage sensitivity and the evolution of gene families
in yeast. Nature, 424, 194-197.

219
Paterson, A.H., Freeling, M., Tang, H. and Wang, X. (2010) Insights from the comparison of
plant genome sequences. Annu Rev Plant Biol, 61, 349-372.
Peng, Y. and Jahroudi, N. (2002) The NFY transcription factor functions as a repressor and
activator of the von Willebrand factor promoter. Blood, 99, 2408-2417.
Peng, Y. and Jahroudi, N. (2003) The NFY transcription factor inhibits von Willebrand factor
promoter activation in non-endothelial cells through recruitment of histone
deacetylases. J Biol Chem, 278, 8385-8394.
Remenyi, A., Scholer, H.R. and Wilmanns, M. (2004) Combinatorial control of gene
expression. Nat Struct Mol Biol, 11, 812-815.
Riechmann, J.L. and Ratcliffe, O.J. (2000) A genomic perspective on plant transcription
factors. Curr Opin Plant Biol, 3, 423-434.
Rieping, M. and Schoffl, F. (1992) Synergistic effect of upstream sequences, CCAAT box
elements, and HSE sequences for enhanced expression of chimaeric heat shock
genes in transgenic tobacco. Mol Gen Genet, 231, 226-232.
Rodin, S.N. and Riggs, A.D. (2003) Epigenetic silencing may aid evolution by gene
duplication. J Mol Evol, 56, 718-729.
Romier, C., Cocchiarella, F., Mantovani, R. and Moras, D. (2003) The NF-YB/NF-YC
structure gives insight into DNA binding and transcription regulation by CCAAT
factor NF-Y. J Biol Chem, 278, 1336-1345.
Ronquist, F. and Huelsenbeck, J.P. (2003) MrBayes 3: Bayesian phylogenetic inference under
mixed models. Bioinformatics, 19, 1572-1574.
Roy, B. and Lee, A.S. (1995) Transduction of calcium stress through interaction of the human
transcription factor CBF with the proximal CCAAT regulatory element of the
grp78/BiP promoter. Mol Cell Biol, 15, 2263-2274.
Sandman, K., Krzycki, J.A., Dobrinski, B., Lurz, R. and Reeve, J.N. (1990) HMf, a DNA-
binding protein isolated from the hyperthermophilic archaeon Methanothermus
fervidus, is most closely related to histones. Proc Natl Acad Sci U S A, 87, 5788-5791.
Schlueter, J.A., Dixon, P., Granger, C., Grant, D., Clark, L., Doyle, J.J. and Shoemaker, R.C.
(2004) Mining EST databases to resolve evolutionary events in major crop species.
Genome, 47, 868-876.
Schmidt, E.E. and Davies, C.J. (2007) The origins of polypeptide domains. Bioessays, 29, 262-
270.
Serra, E., Zemzoumi, K., di Silvio, A., Mantovani, R., Lardans, V. and Dissous, C. (1998)
Conservation and divergence of NF-Y transcriptional activation function. Nucleic
Acids Res, 26, 3800-3805.
Shan, H., Zahn, L., Guindon, S., Wall, P.K., Kong, H., Ma, H., DePamphilis, C.W. and
Leebens-Mack, J. (2009) Evolution of plant MADS box transcription factors:
evidence for shifts in selection associated with early angiosperm diversification and
concerted gene duplications. Mol Biol Evol, 26, 2229-2244.
Shi, Q., Gross, K.W. and Sigmund, C.D. (2001) NF-Y antagonizes renin enhancer function by
blocking stimulatory transcription factors. Hypertension, 38, 332-336.
Sidow, A. (1996) Gen(om)e duplications in the evolution of early vertebrates. Curr Opin
Genet Dev, 6, 715-722.

Gene Duplication

220
Siefers, N., Dang, K.K., Kumimoto, R.W., Bynum, W.E.t., Tayrose, G. and Holt, B.F., 3rd
(2009) Tissue-specific expression patterns of Arabidopsis NF-Y transcription factors
suggest potential for extensive combinatorial complexity. Plant Physiol, 149, 625-
641.
Simillion, C., Vandepoele, K., Van Montagu, M.C., Zabeau, M. and Van de Peer, Y. (2002)
The hidden duplication past of Arabidopsis thaliana. Proc Natl Acad Sci U S A, 99,
13627-13632.
Singh, K.B. (1998) Transcriptional regulation in plants: the importance of combinatorial
control. Plant Physiol, 118, 1111-1120.
Sinha, S., Maity, S.N., Lu, J. and de Crombrugghe, B. (1995) Recombinant rat CBF-C, the
third subunit of CBF/NFY, allows formation of a protein-DNA complex with CBF-
A and CBF-B and with yeast HAP2 and HAP3. Proc Natl Acad Sci U S A, 92, 1624-
1628.
Sinha, S., Kim, I.S., Sohn, K.Y., de Crombrugghe, B. and Maity, S.N. (1996) Three classes of
mutations in the A subunit of the CCAAT-binding factor CBF delineate functional
domains involved in the three-step assembly of the CBF-DNA complex. Mol Cell
Biol, 16, 328-337.
Soltis, P.S. (2005) Ancient and recent polyploidy in angiosperms. New Phytol, 166, 5-8.
Steidl, S., Papagiannopoulos, P., Litzka, O., Andrianopoulos, A., Davis, M.A., Brakhage,
A.A. and Hynes, M.J. (1999) AnCF, the CCAAT binding complex of Aspergillus
nidulans, contains products of the hapB, hapC, and hapE genes and is required for
activation by the pathway-specific regulatory gene amdR. Mol Cell Biol, 19, 99-106.
Stephenson, T.J., McIntyre, C.L., Collet, C. and Xue, G.P. (2007) Genome-wide identification
and expression analysis of the NF-Y family of transcription factors in Triticum
aestivum. Plant Mol Biol, 65, 77-92.
Sterck, L., Rombauts, S., Vandepoele, K., Rouze, P. and Van de Peer, Y. (2007) How many
genes are there in plants (... and why are they there)? Curr Opin Plant Biol, 10, 199-
203.
Stoltzfus, A. (1999) On the possibility of constructive neutral evolution. J Mol Evol, 49, 169-
181.
Struhl, K. and Moqtaderi, Z. (1998) The TAFs in the HAT. Cell, 94, 1-4.
Tasanen, K., Oikarinen, J., Kivirikko, K.I. and Pihlajaniemi, T. (1992) Promoter of the gene
for the multifunctional protein disulfide isomerase polypeptide. Functional
significance of the six CCAAT boxes and other promoter elements. J Biol Chem, 267,
11513-11519.
Taylor, J.S., Van de Peer, Y. and Meyer, A. (2001) Genome duplication, divergent resolution
and speciation. Trends Genet, 17, 299-301.
Taylor, J.S. and Raes, J. (2004) Duplication and divergence: the evolution of new genes and
old ideas. Annu Rev Genet, 38, 615-643.
Testa, A., Donati, G., Yan, P., Romani, F., Huang, T.H., Vigano, M.A. and Mantovani, R.
(2005) Chromatin immunoprecipitation (ChIP) on chip experiments uncover a
widespread distribution of NF-Y binding CCAAT sites outside of core promoters. J
Biol Chem, 280, 13606-13615.

221
Thirumurugan, T., Ito, Y., Kubo, T., Serizawa, A. and Kurata, N. (2008) Identification,
characterization and interaction of HAP family genes in rice. Mol Genet Genomics,
279, 279-289.
Vision, T.J., Brown, D.G. and Tanksley, S.D. (2000) The origins of genomic duplications in
Arabidopsis. Science, 290, 2114-2117.
Wagner, A. (2000) Decoupled evolution of coding region and mRNA expression patterns
after gene duplication: implications for the neutralist-selectionist debate. Proc Natl
Acad Sci U S A, 97, 6579-6584.
Wapinski, I., Pfeffer, A., Friedman, N. and Regev, A. (2007) Natural history and
evolutionary principles of gene duplication in fungi. Nature, 449, 54-61.
Warpeha, K.M., Upadhyay, S., Yeh, J., Adamiak, J., Hawkins, S.I., Lapik, Y.R., Anderson,
M.B. and Kaufman, L.S. (2007) The GCR1, GPA1, PRN1, NF-Y signal chain
mediates both blue light and abscisic acid responses in Arabidopsis. Plant Physiol,
143, 1590-1600.
Wenkel, S., Turck, F., Singer, K., Gissot, L., Le Gourrierec, J., Samach, A. and Coupland, G.
(2006) CONSTANS and the CCAAT box binding complex share a functionally
important domain and interact to regulate flowering of Arabidopsis. Plant Cell, 18,
2971-2984.
Wolberger, C. (1998) Combinatorial transcription factors. Curr Opin Genet Dev, 8, 552-559.
Woollard, A. (2005) Gene duplications and genetic redundancy in C. elegans. WormBook, 1-6.
Xiong, A.S., Peng, R.H., Zhuang, J., Gao, F., Zhu, B., Fu, X.Y., Xue, Y., Jin, X.F., Tian, Y.S.,
Zhao, W. and Yao, Q.H. (2009) Gene duplication, transfer, and evolution in the
chloroplast genome. Biotechnol Adv, 27, 340-347.
Yamamoto, A., Kagaya, Y., Toyoshima, R., Kagaya, M., Takeda, S. and Hattori, T. (2009)
Arabidopsis NF-YB subunits LEC1 and LEC1-LIKE activate transcription by
interacting with seed-specific ABRE-binding factors. Plant J, 58, 843-856.
Yang, J., Xie, Z. and Glover, B.J. (2005) Asymmetric evolution of duplicate genes encoding
the CCAAT-binding factor NF-Y in plant genomes. New Phytol, 165, 623-631.
Yang, X., Tuskan, G.A. and Cheng, M.Z. (2006) Divergence of the Dof gene families in
poplar, Arabidopsis, and rice suggests multiple modes of gene evolution after
duplication. Plant Physiol, 142, 820-830.
Yazawa, K. and Kamada, H. (2007) Identification and characterization of carrot HAP factors
that form a complex with the embryo-specific transcription factor C-LEC1. J Exp
Bot, 58, 3819-3828.
Yokoyama, S. and Yokoyama, R. (1989) Molecular evolution of human visual pigment
genes. Mol Biol Evol, 6, 186-197.
Zahn, L.M., Leebens-Mack, J., DePamphilis, C.W., Ma, H. and Theissen, G. (2005) To B or
Not to B a flower: the role of DEFICIENS and GLOBOSA orthologs in the evolution
of the angiosperms. J Hered, 96, 225-240.
Zanetti, M.E., Blanco, F.A., Beker, M.P., Battaglia, M. and Aguilar, O.M. (2010) A C subunit
of the plant nuclear factor NF-Y required for rhizobial infection and nodule
development affects partner selection in the common bean-Rhizobium etli
symbiosis. Plant Cell, 22, 4142-4157.

Gene Duplication

222
Zhang, J.Z. (2003) Evolution by gene duplication: an update. Trends in Ecology & Evolution,
18, 292-298.
Zwicker, J. and Muller, R. (1997) Cell-cycle regulation of gene expression by transcriptional
repression. Trends Genet, 13, 3-6.
Part 3
Examining Bundles of Genes
12
L- Myo-Inositol 1-Phosphate
Synthase (MIPS) in Chickpea:
Gene Duplication and Functional Divergence
Manoj Majee and Harmeet Kaur
National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi
India
1. Introduction
Gene duplication is one of the key driving forces in the evolution of genes and important
features of genomic architecture of living organisms including plants. Moreover, much of
the plant diversity may have arisen largely due to duplication, followed by divergence and
adaptive specialization of the pre existing genes (Ohno,1970; Zhang, 2003; Flagel &
Wendel,2009). Current impetus on genomic sequence data provides substantial evidence for
the profusion of duplicated genes in all organisms surveyed. Functional divergence after
gene duplication can possibly result in two alternative evolutionary fates: i)
neofunctionalization where one copy acquires an entirely new function whereas the other
copy maintains the original function. ii) Subfunctionalization, in which each copy adopts
part of the task of their parental gene (Ohno,1970; Nowak et al., 1997; Jenesen,1976;
Orgel,1977;Hughes,1994). However, subfunctionalization is reported as a more prevalent
outcome than neofunctionalization in nature. In any case, functional divergence of such
paralogous proteins is found to be the key force shaping molecular network in organisms
(Ohno, 1970). Recent studies also suggest that duplicate genes diverge mostly through the
partitioning of gene expression as in subfunctionalization (Force et al.,1999; Wagner,2000;
Gu et al.,2002). In addition, subfunctionalization can also take place at the protein function
level leading to functional specialization, when one of the duplicated genes becomes better
at performing one of the original functions of the progenitor gene (Hughes, 1994; Gu et
al.,2002; Conant &Wolfe, 2008; Hughes, 1999; Zhang et al., 2002).
Myo-inositol-1-phosphate synthase (MIPS;EC5.5.1.4) is an evolutionary conserved enzyme
which catalyzes the rate limiting step in well conserved inositol biosynthetic pathway and is
extremely widespread in living organisms including plants (Loewus & Murthy, 2000;
Majumder et al., 2003). The evolution of MIPS gene/ protein among the prokaryotes seems
to be more divergent and complex than amongst the eukaryotes, however they preserve a
conserve core catalytic domain among the MIPS proteins (Majumder et al., 2003).
Many of the plant species are known to contain more than one copy of gene encoding MIPS
and are hypothesized to arise through gene duplication. Expression studies of multiple gene
encoding MIPS have revealed the possibility of specialized role for individual enzyme
isoforms. Previously, two genes encoding MIPS have been identified and characterized from
chickpea by Kaur et al. A comparative study of two divergent genes (CaMIPS1 & CaMIPS2)

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reveals features of both functional redundancy and diversification (Kaur et al., 2008). This
chapter explores how a possible gene duplication of MIPS gene in chickpea lead to a
functional diversification that perhaps contributed adaptive evolution to the plant.
2. Gene duplication and functional divergence of MIPS in chickpea
2.1 Evolution and diversification of MIPS
The inositols are the nine isomeric forms of cyclohexane hexitols and myo-inositol is the
most abundant and physiologically favored molecule in the biological system.
The biosynthesis of myo-inositol has been acknowledged as an evolutionary conserved
pathway and its importance across biological organisms from different domains of life has
been recognized for long time. The first and rate limiting step of this pathway is catalyzed
by an evolutionary conserved enzyme named as MIPS.
MIPS particularly catalyzes the conversion of glucose 6- phosphate (Glc6P) to myo inositol 1-
phosphate (Ins1P) through an internal oxidoreduction reaction involving NAD
+
.
Subsequently inositol 1- phosphate is dephosphorylated to produce free inositol by Mg
2+

dependent myo- inositol 1- phosphate phosphatase (IMP: EC 3.1.3.25) (Loewus &
Loewus,1983;Loewus,1990). This free myo inositol occupies the central position in inositol
metabolism since this free inositol can be chanellized to various metabolic routes and
produce different inositol derivatives (Fig-1) (Loewus & Murthy, 2000; Loewus,1990).

MIPS (rate limiting enzyme)
Glc6P
Ins1P
Inositol
Signaling
pathway
Conjugation
pathway
Methylation
pathway
Oxidation
pathway
IMP
MIPS (rate limiting enzyme)
Glc6P
Ins1P
Inositol
Signaling
pathway
Conjugation
pathway
Methylation
pathway
Oxidation
pathway
IMP
Glc6P
Ins1P
Inositol
Signaling
pathway
Conjugation
pathway
Methylation
pathway
Oxidation
pathway
IMP

Fig. 1. Inositol biosynthesis and its consumption in other pathway.
This free inositol and its derivatives have acquired diverse functions over the course of
evolution. As for example, inositol containing phospholipids are the important constituents
of many archaea. Few thermophilic archaea also use inositol phosphodiester as thermo
protective solutes. Then with the emergence and diversification of eukaryotes, function of
L- Myo-Inositol 1-Phosphate Synthase
(MIPS) in Chickpea: Gene Duplication and Functional Divergence

227
inositol and its derivatives proliferated dramatically. So far, inositol and its derivatives have
been shown to be involved in growth regulation, membrane biogenesis, hormone
regulation, signal transduction, pathogen resistance and stress adaptation in higher plants
(Loewus & Murthy, 2000; Stevenson et al., 2000; Michell,2008).
Since the usage and distribution of inositol and inositol derivatives are reported in all
domains of life, it is imperative to contain MIPS enzyme in diverse organisms such as
archaea, eubacteria, parasites, animals, higher plants and many others. Few higher plants
and algae are reported to have both cytosolic and chloroplastic isoforms of MIPS. However,
the biochemical and enzymatic properties of these two forms do not differ significantly
between each other. Recent studies suggest that rice chloroplastic MIPS is coded by OsINO1-
1 gene located on chromosome 3 (RayChaudhuri et al.1997;Ray et al., 2010).
The structural gene coding (INO1) for this ancient enzyme was first identified and cloned in
Saccharomyces cerevisiae (Donahue & Henry,1981). Subsequently more than 80 INO1 genes
were reported from various sources including both prokaryotes and eukaryotes.
Evolution and diversification of MIPS has been highlighted by Majumder et al. (2003) and a
clear difference between prokaryotic and eukaryotic MIPS protein sequences was observed
when compared among each other. The MIPS protein sequences of prokaryotes are quite
divergent among themselves and significantly distinct than any other known eukaryotic
sequences. In contrast, the eukaryotic MIPS sequences show remarkable similarities among
each other. A phylogenetic tree constructed to include few representative MIPS sequences
from diverse organisms present an overall evolutionary divergence of this enzyme in the
biological kingdom. The higher plants constitute one close subgroup, while the higher
animals, protozoa, fungi form the other subgroups in the eukaryotic cluster (Fig-2). In
Archeoglobus, MIPS shows more sequences similarity to the eukaryotic MIPS than the other
known prokaryotic ones and thereby all eukaryotic MIPS seems to have evolved from one
common stock, probably from the fusion of an archaebacterial and eubacterial MIPS genes
(Fig-2 & 3).
Four stretches of amino acid residues (GWGGNG, LWTANTERY, NGSPQNTFVPGL and
SYNHLGNNDG) are found to be conserved in MIPS proteins of all eukaryotes and among
them; SYNHLGNNDG is identified as highly conserved. Interestingly among higher plants,
MIPS enzyme shows greater conservation in addition to these four domains (Fig-3). Many of
the plant species also possess multiple genes encoding MIPS and are thought to arise
through gene duplication in course of time.
Subsequent analysis of crystal structures of various MIPS proteins provide ample evidence
towards the presence of conserved core structure in all MIPS proteins throughout
evolution. Moreover, some of the important amino acid residues are identified in the active
site of the yeast MIPS and are shown to be highly conserved in all eukaryotic MIPS. These
amino acids are considered to be the part of a eukaryotic core structure which has
remained largely the same during evolution, despite the divergence in rest of the sequences
over time (Fig-3) (Stein & Geiger, 2002; Norman et al.,2002).
Crystal structure analysis of MIPS from Saccharomyces cerevisiae also revealed that each
monomer of the homo-tetrameric MIPS has three functionally important structural domains
namely the NAD binding Rossman fold, the catalytic binding site and the core domain. This
study also exemplifies a case of induced fit model for binding of the substrate with the
catalytic domain of the enzyme. (Stein & Geiger, 2002)

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Fig. 2. A phylogenetic tree of few representative MIPS amino acid sequences from various
domains of living organisms. Neighbour-Joining algorithm was used to construct tree from
the distance matrix using Clustal X. Thousand rounds of bootstrapping were performed to
ensure the validity of the tree.

229
CaMIPS1 ------MFIENFKVDSPNVKYTETEIQSVYNYETTELVHENRNGTYQWIVKPKTVKYEFK
CaMIPS2 ------MFIESFKVESPNVKYTDTEIQSVYSYETTELVHENRNNTYQWVVKPKTIKYEFK
OsMIPS ------MFIESFRVESPHVRYGAAEIESDYQYDTTELVHESHDGASRWIVRPKSVRYNFR
HsMIPS -----MEAAAQFFVESPDVVYGPEAIEAQYEYRTTRVSREG----GVLKVHPTSTRFTFR
ScMIPS MTEDNIAPITSVKVVTDKCTYKDNELLTKYSYENAVVTKTAS---GRFDVTPTVQDYVFK
PfMIPS ------------------------------------------------------------
MtMIPS ------------------------------------------------------MSEHQS
AfMIPS ------------------------------------------------------------

CaMIPS1 TDTHVP-KLGVMLVGWGGNNGSTLTGGVIANREGISWATKDNIQQANYFGSLTQASATRV
CaMIPS2 TQTHVP-KLGVMLVGWGGNNGSTLTGGVIANREGISWATKDKIQQSNYFGSLTQASAIRV
OsMIPS TTTTVP-KLGVMLVGWGGNNGSTLTAGVIANREGISWATKDKVQQANYYGSLTQASTIRV
HsMIPS TARQVP-RLGVMLVGWGGNNGSTLTAAVLANRLRLSWPTRSGRKEANYYGSLTQAGTVSL
ScMIPS LDLKKPEKLGIMLIGLGGNNGSTLVASVLANKHNVEFQTKEGVKQPNYFGSMTQCSTLKL
PfMIPS --------MVRVAIIGQGYVASIFAVGLERIKE----------GELGYYG----------
MtMIPS LPAPEASTEVRVAIVGVGNCASSLVQGVEYYYN--------ADDTSTVPG----------
AfMIPS --------MKVWLVGAYGIVSTTAMVGARAIERGIAPKIGLVSELPHFEG----------
: . *

CaMIPS1 GSFQ-GEEIYAPFKSLLPMVNPDDIVFGGWDISDMNLADAMARA-RVFDIDLQKQLRPYM
CaMIPS2 GSFQ-GEEIYAPFKSLLPMVNPEDIVWGGWDINNMNLADAMGRA-RVFDIDLQKQLRPYM
OsMIPS GSYN-GEEIYAPFKSLLPMVNPDDLVFGGWDISNMNLADAMTRA-KVLDIDLQKQLRPYM
HsMIPS GLDAEGQEVFVPFSAVLPMVAPNDLVFDGWDISSLNLAEAMRRA-KVLDWGLQEQLWPHM
ScMIPS GIDAEGNDVYAPFNSLLPMVSPNDFVVSGWDINNADLYEAMQRS-QVLEYDLQQRLKAKM
PfMIPS ----------IPLANELPIKVEDIKIVASYDVDKTKIGLPLSEI-VQRYWKGNVPESLQE
MtMIPS ----------LMHVRFGPYHVRDVKFVAAFDVDAKKVGFDLSDA-IFASENNTIKIADVA
AfMIPS ------------IEKYAPFSFEFGGHEIRLLSNAYEAAKEHWELNRHFDREILEAVKSDL
* . .

CaMIPS1 ESMVPLPGIYDPDFIAANQGDRANNVIKGTKR---------EQINQIIKDIKEFKEANKV
CaMIPS2 ESMVPLPGIYDPDFIAANQGDRANNVINGTKK---------EQLQQIIKDIKEFKEASKI
OsMIPS ESMVPLPGIYDPDVIAANQGSRANNVIKGTKK---------EQMEQIIKDIREFKEKSKV
HsMIPS EALRPRPSVYIPEFIAANQSARADNLIPGSRA---------QQLEQIRRDIRDFRSSAGL
ScMIPS SLVKPLPSIYYPDFIAANQDERANNCINLDEKGNVTTRGKWTHLQRIRRDIQNFKEENAL
PfMIPS VFVRKGIHLGSLRNLPIEATGLEDEMT---------------LKEAIERLVEEWKEKKVD
MtMIPS PTNVIVQRGPTLDGIGK-----------------------------YYADTIELSDAEPV
AfMIPS EGIVARKGTALNCGSGIKELGDIKTLEGEGLS-----------LAEMVSRIEEDIKSFAD
: .

CaMIPS1 DRVVVLWTANTERYSNLVVGLNDTMENLFAAVDRNE-SEISPSTLFAIACVTENVPFING
CaMIPS2 DKVVVLWTANTERYSNVVVGLNDTMENLLASVDKNE-AEISPSTLYALACVLENVPFING
OsMIPS DKVVVLWTANTERYSNVCVGLNDTMENLLASVDKNE-AEISPSTLYAIACVMEGIPFING
HsMIPS DKVIVLWTANTERFCEVIPGLNDTAENLLRTIELG--LEVSPSTLFAVASILEGCAFLNG
ScMIPS DKVIVLWTANTERYVEVSPGVNDTMENLLQSIKNDH-EEIAPSTIFAAASILEGVPYING
PfMIPS VIINVPTTEAFTPFGKLEELEKAIKDNNKERLTATQ-AYAYAAAQYAKE--VGGAAFVNA
MtMIPS DVVQALKEAKVDVLVSYLPVGSEE-----------------ADKFYAQCAIDAGVAFVNA
AfMIPS DETVVINVASTEPLPNYSEEYHGSLEGFERMIDEDRKEYASASMLYAYAALKLGLPYANF
. . . :* . .: *

CaMIPS1 SP-QNTFVPGLIDLAIKRNTLIGGDDFK--SGQTKMKSVLVDFLVGAGIKPTSIVSYNHL
CaMIPS2 SP-QNTFVPGLIDLAIQRNSLIGGDDFK--SGQTKMKSVLVDFLVGAGIKPTSIVSYNHL
OsMIPS SP-QNTFVPGLIDLAIKNNCLIGGDDFK--SGQTKMKSVLVDFLVGAGIKPTSIVSYNHL
HsMIPS SP-QNTLVPGALELAWQHRVFVGGDDFK--SGQTKVKSVLVDFLIGSGLKTMSIVSYNHL
ScMIPS SP-QNTFVPGLVQLAEHEGTFIAGDDLK--SGQTKLKSVLAQFLVDAGIKPVSIASYNHL
PfMIPS IPTLIANDPAFVELAKESNLVIFGDDGA--TGATPLTADILGHLAQRNRHVLDIVQFNIG
MtMIPS LPVFIASDPVWAKKFTDAGVPIVGDDIKSQVGATITHRVLAKLFEDRGVQLDRTMQLNVG

Gene Duplication

230
AfMIPS TPSPGSAIPALKELAEKKGVPHAGNDGK--TGETLVKTTLAPMFAYRNMEVVGWMSYNIL
* : * . *:* * * : : . . *

CaMIPS1 GNNDGMNLSAPQTFRSKEISKSNVVDDMVNSNGILY--APGEHPDHVVVIKYVPYVGDSK
CaMIPS2 GNNDGMNLSAPQTFRSKEISKSNVVDDMVNSNAILY--QPGEHPDHVVVIKYVPYVADSK
OsMIPS GNNDGMNLSAPQTFRSKEISKSNVVDDMVSSNAILY--ELGEHPDHVVVIKYVPYVGDSK
HsMIPS GNNDGENLSAPLQFRSKEVSKSNVVDDMVQSNPVLY--TPGEEPDHCVVIKYVPYVGDSK
ScMIPS GNNDGYNLSAPKQFRSKEISKSSVIDDIIASNDILYNDKLGKKVDHCIVIKYMKPVGDSK
PfMIPS GNTDFLALTDKERNKSKEYTKSSVVEDILG----------YDAPHFIKPTGYLEPLGDKK
MtMIPS GNMDFLNMLERERLESKKISKTQAVTSNLKR-------EFKTKDVHIGPSDHVGWLDDRK
AfMIPS GDYDGKVLSARDNKESKVLSKDKVLEKMLG-----------YSPYSITEIQYFPSLVDNK
*: * : .** :* . : . : :. : * *

CaMIPS1 RAMDEYTSEIFMGGKSTIVLHNTCEDSLLAAPIILDLVLLAELSTRIQFKSEA-----EN
CaMIPS2 RAMDEYISEIFMGGKNTIVLHNTCEDSLLAAPIILDLVLLAELSTRIQFKSQH-----ED
OsMIPS RAMDEYTSEIFMGGKSTIVLHNTCEDSLLAAPIILDLVLLAELSTRIQLKAEG-----EE
HsMIPS RALDEYTSELMLGGTNTLVLHNTCEDSLLAAPIMLDLALLTELCQRVSFCTDM-----DP
ScMIPS VAMDEYYSELMLGGHNRISIHNVCEDSLLATPLIIDLLVMTEFCTRVSYKKVDPVKEDAG
PfMIPS FIAMHIEYISFNGARDELIIAGRINDSPALAGLLVDLARLGKIAVDK------------K
MtMIPS WAYVRLEGRAFGDVPLNLEYKLEVWDSPNSAGVIIDAVRAAKIAKDRGIG----------
AfMIPS TAFDFVHFKGFLGKLMKFYFIWDAIDAIVAAPLILDIARFLLFAKKKGVKG
: . : *: :::* :.

CaMIPS1 KFHTFHPVATILSYLTKAPLVPPGTPVVNALSKQRAMLENIMRACVGLAPENNMILEYK-
CaMIPS2 KFHSFHPVATILSYLTKAPLVPPGTPVVNALSKQRAMLENILRACVGLAPENNMILEYK-
OsMIPS KFHSFHPVATILSYLTKAPLVPPGTPVVNALAKQRAMLENIMRACVGLAPENNMILEYK-
HsMIPS EPQTFHPVLSLLSFLFKAPLVPPGSPVVNALFRQRSCIENILRACVGLPPQNHMLLEHKM
ScMIPS KFENFYPVLTFLSYWLKAPLTRPGFHPVNGLNKQRTALENFLRLLIGLPSQNELRFEERL
PfMIPS ---EFGTVYPVNAFYMKNPGPKEAKNIPRIIAYEKLRQWAGLPPRYL-------------
MtMIPS -----GPVIPASAYLMKSPPEQLPDDIARAQLEEFIIG----------------------
AfMIPS -------VVKEMAFFFKSPMDTNVINTHEQFVVLKEWYSNLK------------------

CaMIPS1 --------------------------------------------------
CaMIPS2 --------------------------------------------------
OsMIPS --------------------------------------------------
HsMIPS ERPGPSLKRVGPVAATYPMLNKKGPVPAATNGCTGDANGHLQEEPPMPTT
ScMIPS L-------------------------------------------------
PfMIPS --------------------------------------------------
MtMIPS --------------------------------------------------
AfMIPS --------------------------------------------------

Fig. 3. Multiple sequence alignment of MIPS from prokaryotes and eukaryotes. Proposed
common active site amino acid residues for the Mycobacterium tuberculosis and
Saccharomyces cerevisiae MIPS sequence are highlighted in green color and four conserved
domains of eukaryotes (GWGGNG, LWTANTERY, NGSPQNTFVPGL and
SYNHLGNNDG) are highlighted in yellow color. 43 variant positions between CaMIPS1
and 2 have been highlighted in blue color.
2.2 MIPS from chickpea: A case of functional divergence
Chickpea is an annual self- pollinated diploid legume crop which is mostly grown in the
arid and semi arid regions of the world. Long term evolution and adaptation to harsh
conditions make chickpea rich in resistant genes for environmental stresses including
drought and cold. Several classes of genes controlling potential resistance have been

231
identified through genomic and proteomic studies (Ahmaed et al., 2005; Mantri ,2007;
Bhusan et al., 2007).
In this particular plant, inositol seems to play an important role in drought tolerance
besides growth and development, since inositol content and MIPS transcript was found to
be significantly increased under dehydration condition (Boominathan et al.,2004).
Subsequently, chickpea is reported to have two MIPS coding genes (CaMIPS1 and CaMIPS2)
(Kaur et al. 2008) and both genes are revealed to have overall similar structure consisting of
9 introns and 10 exons (Fig-4). Sequence analysis of these two genes show high similarity
(>85%) in their coding regions but their non-coding or 5' and 3' flanking regions are
extremely divergent. Moreover length of each exon is similar between these two genes while
the size of introns varies. Such findings suggest that these two MIPS genes most likely arose
by ancestral gene duplication and have undergone considerable sequence divergence.
In spite of the remarkable resemblance in their coding sequences, some base substitutions
occurs in exons leading to changes in 43 amino acids in protein sequences, however,
maintaining four highly conserved functional domains and known active site amino acids
of MIPS (Fig-3) (Majumder et al. 2003). Among these 43 amino acids, 19 amino acids differ
considerably between CaMIPS1 and CaMIPS2 while rest of the amino acid substitutions are
relatively insignificant, i.e. substitution between amino acids having similar physico
chemical properties (Kaur et al., 2008).

188 69 136 248 227 116 177 189 63 120
(2979 bp)
(4107 bp)
Exon
CaMIPS1
CaMIPS2
Intron
188 69 136 248 227 116 177 189 63 120
96 111 325 143 91 250 89 88 253
109 316 1345 141 156 103 134 103 167
188 69 136 248 227 116 177 189 63 120
(2979 bp)
(4107 bp)
Exon
CaMIPS1
CaMIPS2
Intron
188 69 136 248 227 116 177 189 63 120
96 111 325 143 91 250 89 88 253
109 316 1345 141 156 103 134 103 167

Fig. 4. Diagrammatic representation of CaMIPS1 and CaMIPS2 genomic structure. Length of
exon and intron indicated in bp. [Modified from Kaur et al., 2008]
Functional divergence after gene duplication can result in following alternative fates: One
copy acquires a novel function (neofunctionalization) or one copy loses its function
completely or each copy adopts part of the task of their parental gene (subfunctionalization)
(Ohno, 1970; Nowak et al., 1997; Jenesen, 1976; Orgel,1977;Hughes,1994).
Functional complementation and in-vitro enzymatic properties were analyzed to check the
fate of these two genes. First to check the functional identity of these two divergent genes, a
complementation experiment was carried out in natural inositol auxotroph
Schizosaccharomyces pombe PR109 which clearly demonstrates that both CaMIPS1 and

Gene Duplication

232
CaMIPS2 indeed encode functional MIPS enzymes. Subsequently, the enzymatic properties
of these two enzymes were examined since CaMIPS1 and CaMIPS2 polypeptides are
reported to have some differences in their amino acid sequences.
Both enzymes showed nearly same Km values for Glc6P suggesting the similar substrate
specificity. For both proteins, the optimum temperature for enzyme activity is at 35C and
optimum pH is 7.0 suggesting the similar biochemical characteristics (table1).
Further the enzymatic activities of each protein under stress environment in invitro
conditions were examined and the activities of these two enzyme proteins were shown to
differ significantly in response to high temperature and salt concentration (Kaur et al., 2008).
CaMIPS1 activity is considerably affected at high temperature or in presence of increasing
sodium chloride concentration while the CaMIPS2 activity is less affected in similar
conditions and thereby retaining higher activity than CaMIPS1 (Fig-5).
The amino acid substitutions in protein sequence as analyzed by sequence comparison and the
higher enzyme activity in CaMIPS2 under stress condition also indicates that it might be
evolved during the course of time to function better under stress conditions. This differential
activity towards high temperature and salt of these two enzymes could be supported by the
bioinformatics analysis in respect to the available yeast MIPS crystal structure and salt tolerant
PcINO1 (MIPS coding gene from Salt tolerant Porteresia coarctata) protein sequence (Majee et
al., 2004). Based on the bioinformatics study, CaMIPS2 appears to be more stable towards
destabilizing factors such as high temperature, salt, etc, thereby retains better functionality
under such conditions (Kaur et al., 2008). Subsequently, growth pattern of CaMIPS1 &
CaMIPS2 transformed Schizosaccharomyces pombe under stress conditions were analyzed and
CaMIPS2 transformed S. pombe cells were reported to grow or survive better than CaMIPS1
transformants both at high temperature and salt environment (Fig-6) suggesting CaMIPS2
gene product functions more efficiently under stress conditions due to its stress tolerant
property and hence provide sufficient inositol to grow as compared to CaMIPS1.

CaMIPS1
CaMIPS2
E
n
z
y
m
e

a
c
t
i
v
i
t
y

(
%
)
0
20
40
60
80
100
0 100 200 300 400
NaCl (mM)
35C 45C 55C
E
n
z
y
m
e

a
c
t
i
v
i
t
y

(
%
)
0
20
40
60
80
100
Temperature
A B
CaMIPS1
CaMIPS2
CaMIPS1
CaMIPS2
E
n
z
y
m
e

a
c
t
i
v
i
t
y

(
%
)
0
20
40
60
80
100
0 100 200 300 400
NaCl (mM)
35C 45C 55C
E
n
z
y
m
e

a
c
t
i
v
i
t
y

(
%
)
0
20
40
60
80
100
Temperature
A B

Fig. 5. Effect of salt (A) & temperature (B) on CaMIPS1 and CaMIPS2 enzyme activity.
[Modified from Kaur et al., 2008]

233
Characters CaMIPS1 CaMIPS2
Km
Gluc 6-P
NAD
+

Vmax
Gluc 6-P
NAD
+

pH optima
Temp. optima

2.63 mM
0.181 mM

0.074 mole min
-1

0.069 mole min
-1

7.5
35C

2.70 mM
0.192 mM

0.075 mole min
-1

0.070 mole min
-1

7.5
35C

[Modified from Kaur et al., 2008]
Table 1. Biochemical characterization of recombinant CaMIPS1 and CaMIPS2 enzymes.

E
m
p
t
y

v
e
c
t
o
r
C
a
M
I
P
S
1
C
a
M
I
P
S
2
Control
40C
250mM
200mM
NaCl
E
m
p
t
y

v
e
c
t
o
r
C
a
M
I
P
S
1
C
a
M
I
P
S
2
Control
40C
250mM
200mM
NaCl
Control
40C
250mM
200mM
NaCl

Fig. 6. Growth pattern of Schizosaccharomyces pombe transformed with CaMIPS1 and
CaMIPS 2 at high temperature and salt environment. [Modified from Kaur et al., 2008]
Recent studies suggest that duplicate genes diverge mostly through the partitioning of gene
expression as in subfunctionalization and thereby being expressed in a differential manner;
redundant genes may acquire functional divergence (Force et al., 1999; Wagner, 2000; Gu et
al., 2002). This hypothesis was examined on CaMIPS1 and 2.
CaMIPS1 gene was shown to express in root, shoot, leaves, and flower in fairly equal
abundance but no transcript was observed in seed, while CaMIPS2 transcript was observed

Gene Duplication

234
in all examined tissues including seed. This result proposes that CaMIPS1 and CaMIPS2
genes are indeed differentially regulated in different organs to coordinate inositol
metabolism with cellular growth as hypothesized previously (Loweus & Murthy,2000).
Subsequently, expression pattern of these two genes are examined in various environmental
stresses. Interestingly, CaMIPS2 was shown to be induced at different level in various
environmental stresses while level of CaMIPS1 transcript was found to be unaltered by such
stresses (Fig-7). This differential expression is also supported by the divergence of their
upstream regulatory sequences.

d
e
h
y
d
r
a
t
i
o
n
CaMIPS1
CaMIPS2
0
5
10
15
200
R
e
l
a
t
i
v
e

q
u
a
n
t
i
f
i
c
a
t
i
o
n

n
o
r
m
a
l
h
e
a
t
s
a
l
t
c
o
l
d
d
e
h
y
d
r
a
t
i
o
n
CaMIPS1
CaMIPS2
0
5
10
15
200
R
e
l
a
t
i
v
e

q
u
a
n
t
i
f
i
c
a
t
i
o
n

n
o
r
m
a
l
h
e
a
t
s
a
l
t
c
o
l
d

Fig. 7. Expression analysis of CaMIPS1 and CaMIPS2 through real time PCR analysis under
various stresses. [Modified from Kaur et al., 2008]
3. Conclusion
Gene duplication, followed by sequence divergence leads to functional divergence of the
paralogous proteins, is a major force for adaptation of living organisms. Without gene
duplication, the plasticity of genome or organism in adapting to changing environment
would be very limited. Chickpea plants are know to be evolved and diversified considerably
over time and acquired subsequently various potential genes for their adaptation to
environmental stresses. It seems that this drought tolerant legume plant requires more
inositol for their adaptation particularly under drought condition and hence acquired
CaMIPS2 over time. Collectively, our results exemplified that CaMIPS1 and CaMIPS2 are
differentially expressed in chickpea to play discrete though overlapping roles in plant;

235
however CaMIPS2 is likely to be evolved through gene duplication, followed by adaptive
changes in its sequences to function better under environmental stresses and thereby play a
key role in environmental stress adaptation along with other aspects of inositol metabolism
in chickpea.
4. Acknowledgement
This work was supported by a grant from Department of Biotechnology (Next Generation
Challenge Programme on Chickpea Genomics), Department of Science and Technology
(Fast Track Scheme) , Government of India.
We also like to acknowledge the Research support from National Institute of Plant Genome
Research, New Delhi. H.K. thanks the Council of Scientific and Industrial Research,
Government of India, for Senior Research Fellowship.
5. References
Ahmad, F.; Gaur, P. M. & Croser, J. (2005) Chickpea (Cicer arietinum L.) In genetic resources,
Chromosome Engineering and Crop Improvement. Grain legumes Edited by Singh
R, Jauhar P. vol1,pp 187-217, CRC Press. USA
Bhusan, D.; Pandey, A.; Choudhary, M.K.; Datta, A.; Chakraborty, S. & Chakraborty, N.
(2007) Comparative proteomics analysis of differentially expressed proteins in
chickpea extracellular matrix during dehydration stress. Molecular & Cellular
Proteomics,vol 6:1868-1884.
Boominathan, P.; Shukla, R.; Kumar, A.; Manna, D.; Negi, D.; Verma, P.K. &
Chattopadhyay, D. (2004) Long term transcript accumulation during the
development of dehydration adaptation in Cicer arietinum. Plant Physiology, vol
135: 1608-1620
Conant, G.C. & Wolfe, K.H. (2008) Turning a hobby into a job: How duplicated genes find
new functions. Nature Review Genetics, vol 9: 938-950
Donahue, T.F. & Henry, S.A. (1981) Myo-inositol 1- phosphate synthase. Characteristics of
the enzyme and identification of its structural gene in yeast. Journal of Biological
Chemistry,vol 256: 7077-7085
Flagel, L.E. & Wendel, J.F. (2009) Gene duplication and evolutionary novelty in plants. New
Phytologist, vol 183: 557-564.
Force, A.; Lynch, M.; Pickett,F.B.; Amroes A.; Yan,Y-L. & Postlethwait, J. (1999) Preservation
of duplicate genes by complementary, degenerative mutations. Genetics, vol 151,
1531-1545
Gu, Z.; Nicolae, Lu, H-S. & Li, H.W. (2002) Rapid divergence in expression between
duplicate genes inferred from microarray data. Trends in Genetics, vol 18,609-613
Hughes, A.L. (1994) The evolution of functionally novel proteins after gene duplication.
Proceedings of the Royal Society, Lond Ser. B 256: 119- 124
Hughes, A.L. (1999) Adaptive Evolution of genes and genomes, Oxford University press.
Jensen, R.A. (1976) Enzyme recruitment in the evolution of new function. Annual Review of
Microbiology, vol 30, 409-425
Kaur, H.; Shukla, R.; Yadav, G.; Chattopadhyay, D. & Majee, M. (2008) Two divergent genes
encoding L -myo inositol 1 phosphate synthase1 (CaMIPS1) and 2 (CaMIPS2) are
differentially expressed in chickpea. Plant, Cell &Environment, vol31, 1701-1716

Gene Duplication

236
Loewus, F.A. & Loewus, M.W. (1983) Myo- inositol: its biosynthesis and metabolism. Annual
Review of Plant Physiology, vol 34,137-161
Loewus, F.A. & Murthy, P.N. (2000) Myo- inositol metabolism in plants. Plant Science, vol
150, 1-19
Loewus, F.A. (1990) Inositol biosynthesis. In Inositol metabolism in plants (Morre D.J., Boss W.F.
& Loewus F.A .eds). pp13-19, New York, Wiley-Liss., USA
Majee, M.; Maitra, S.; Dastidar, K.G.; Pattnaik, S.; Chatterjee, A.; Hait, N.C.; Das, K.P. &
Majumder, A.L. ( 2004) A novel salt tolerant L myo- inositol -1-phosphate synthase
from Porteresia coarctata (Roxb) Tateoka, a halophytic wild rice. Journal of Biological
Chemistry, vol 279, 28539-28552
Majumder, A.L.; Chatterjee, A.; Dastidar, K.G. & Majee, M. (2003) Diversification and
evolution of L- myo-inositol 1 phosphate synthase. FEBS Letter ,vol 533, 3-10.
Mantri, N.L.; Ford, R.; Coram, T.E. & Pang, E.C. (2007) Transcriptional profiling of chickpea
genes differentially regulated in response to high salinity, cold, and drought. BMC
genomics, vol 8:303
Michell, R.H. (2008) Inositol derivatives: evolution and functions. Nature reviews Molecular
Cell Biology ,vol9, 151-161
Norman, R.A.; McAlister, M.S.B.; Murray Rust, J.; Movahedzadeh, F.; Stoker, N.G. &
McDonald, N.Q. (2002) Crystal structure of inositol 1 phosphate synthase from
Mycobacterium tuberculosis, a key enzyme in phosphotidyl inositol synthesis.
Structure ,vol10: 393-402
Nowak, M.A.; Boerlijst, M.C.; Cooke, J, Smith, M.J. (1997) Evolution by genetic redundancy.
Nature, vol 388,167-171
Ohno, S. (1970) Evolution by gene duplication. Springer Verlag. New York,, USA
Orgel, L.E. (1977) Gene duplication and the origin of proteins with novel functions. Journal of
Theoretical Biology, vol 67,773
Ray, S.; Patra, B.; Das Chaterjee, A.;, Ganguli, A. & Majumder, A.L. (2010) Identification and
organization of chloroplastic and cytosolic L-myo-inositol 1- phosphate synthase
coding gene (s) in Oryza sativa: comparison with the wild halophytic rice, Porteresia
coarctata. Planta 231:1211-1227
RayChaudhuri, A.; Hait, N.C.; DasGupta, S.; Bhaduri, T.J.; Deb, R. & Majumder, A.L. (1997)
L myo- inositol 1-phosphate synthase from plant sources (characteristics of the
chloroplastic and cytosolic enzymes). Plant Physiology 115: 727-736
Stein, A.J. & Geiger, J.H. (2002) The crystal structure and mechanism of L myo inositol 1
phosphate synthase. Journal of Biological Chemistry 277: 9484-9491.
Stevenson, J.M.; Perera, I.Y.; Heilmann, I.; Persson, S. & Boss, W.F. (2000) Inositol signaling
and Plant Growth. Trends in Plant Science 5: 252-258.
Wagner, A. (2000) Decoupled evolution of coding region and mRNA expression patterns
after gene duplication: Implication for the neutralist selectionist debate. Proceedings
of National Academy of Sciences USA. vol 97: 6579-6584
Zhang, J. (2003) Evolution by gene duplication; an update: Trends in Ecology and evolution,
vol18, 292-298.
Zhang, J.; Zhang, Y.P. & Rosenberg, H.F. (2002) Adaptive evolution of duplicated pancreatic
ribonuclease gene in a leaf eating monkey. Nature Genetics, vol 30, 411-415
0
On the Specialization History of the
ADP-Dependent Sugar Kinase Family
Felipe Merino and Victoria Guix
Laboratorio de Bioqmica y Biologa Molecular
Facultad de Ciencias
Universidad de Chile
Chile
1. Introduction
Sugars are one of the most common carbon sources used by heterotrophic organisms. Indeed,
sugar phosphorylation is thought to be a key step in the cellular metabolism since, just
after transport into the cell, these molecules are phosphorylated to trap them for further
metabolic processing. There are several known pathways used to produce pyruvate from
the incoming sugar (like glucose or galactose) which is accompanied by the synthesis of ATP
and the production of reductive power. Amongst them, the Embden-Meyerhof pathway,
or glycolysis, seems to be the most commonly used. Some microorganisms can also use
the Entner-Doudoroff pathway. Also, although the pentose phosphate pathway is generally
associated with nucleotide synthesis and reductive power in the form of NADPH it also
can be linked to the ux from glucose to pyruvate as this pathway has fructose-6-phosphate
and glyceraldehyde-3-phosphate as intermediates. Some microorganisms, such as Lactococcus
lactis, use a pathway very similar to glycolysis, but instead of start from glucose they use
galactose as main carbon source. In this fashion glucokinases are replaced by galactokinases
and phosphosfructokinases by tagatose-6-phosphate kinases (van Rooijen et al., 1991).
Interestingly, all the above mentioned pathways ultimately converge through
glyceraldehyde-3-phosphate. In this way, the main difference between them is what happens
with the hexoses. Here, one of the most important reactions are the initial phosphorylations,
e.g. phosphorylation of glucose, fructose-6-phosphate, galactose, tagatose-6-phosphate.
Early on the 90s it was already recognized that the transfer of the -phosphate of ATP to
several sugars was catalyzed by at least three different non-homologous protein families: the
hexokinase family, the ribokinase family, and the galactokinase family (Bork et al., 1993).
The hexokinase family contains enzymes with wide specicities including glucokinases,
ribulokinases, gluconokinases, xylulokinases, glycerokinases, fructokinases, rhamnokinases,
and fucokinases (Bork et al., 1993). The galactokinase family contains enzymes that catalyze
the phosphorylation of galactose, mevalonate, P-mevalonate, and homoserine (Bork et al.,
1993). The ribokinase family on the other hand is very interesting since its members catalyze
the transfer of the terminal phosphate of ATP to sugars like ribose, fructose, sugar containing
molecules such as nucleosides, and sugar phosphate molecules like fructose-6-phosphate,
fructose-1-phosphate, and tagatose-6-phosphate (Bork et al., 1993). This makes the ribokinase
family the group with the broadest specicity amongst the above mentioned. It is clear
that while the three groups share some similar substrates and hence are a great example of
13
2 Will-be-set-by-IN-TECH
convergent evolution the ribokinase family is the only one that contains enzymes able to
phosphorylate sugar phosphates.
In particular, glucokinases have been extensively studied since they are in the top on many
metabolic pathways, and hence some sort of metabolic hub, and also they are responsible
for most of the ux control in glycolysis (Torres et al., 1988). On the other hand, while in
normal conditions the phosphofructokinase from rat liver shows almost no control over the
glycolytic ux, in starving conditions it becomes almost as important as glucokinase (Torres
et al., 1988) which suggests that they become key in gluconeogenic conditions. Moreover,
phosphofructokinases are extensively studied because they are highly regulated enzymes.
In this light, phosphofructokinases have also been recognized as one of the key enzymes of
glycolysis.
From the ribokinase family, one of the most studied enzyme is the phosphofructokinase-2
from Escherichia coli which is often referred to as a member of the PfkB subfamily
(Cabrera et al., 2010). It is possible to nd a second phosphofructokinase, called
phosphofructokinase-1
1
, in the genome of E. coli which belongs to another family called PfkA.
In this family, the most extensively studied members are the phosphofructokinase-1 from E.
coli and the phosphofructokinase from Bacillus stearothermophilus (Evans et al., 1981; Schirmer
& Evans, 1990). Initially it was thought that both PfkB and PfkA groups had a common origin
(Wu et al., 1991), but now we know that they are two non-homologous families. Interestingly,
while not phylogenetically related, both phosphofructokinase-1 and phosphofructokinase-2
from E. coli show strong inhibition at high concentrations of their substrate MgATP (Atkinson
& Walton, 1965; Kotlarz & Buc, 1981), which suggests that this is a key requirement of this
metabolic step. This reinforces the idea that these enzymes are strongly related to the balance
between glycolysis and gluconeogenesis.
Indeed, it has been already demonstrated that the substrate inhibition is needed
for the avoidance of a futile cycle of phosphorylation/dephosphorylation of
fructose-6-phosphate/fructose-1,6-bisP which will ultimately lead to a net hydrolysis of
ATP (Torres et al., 1997). Interestingly, some microorganisms present phosphofructokinases
(also members of the PfkA family) which use polyphosphates as a source of phosphate and
hence they do not appear to be regulated (Peng & Mansour, 1992).
2. Glucose degradation in the members of the Archaea domain
Nowadays, organisms can be classied in three principal domains of life: Bacteria, Eukarya,
and Archaea (Woese & Fox, 1977; Woese et al., 1990). Interestingly, although there are some
known archaea that growth in mesophilic conditions, most of them are extremophiles. Two
main phylogenetic groups can be found inside Archaea: Euryarchaeota and Crenarchaeota
(Allers & Mevarech, 2005). Also, recently based in environmental samples, two more groups
called Korarchaeota and Nanoarchaeota has been proposed (Allers & Mevarech, 2005).
Considering the potential for technological applications, most of the attention has been
directed to study those archaea able to grow in extreme temperature conditions (known
as thermophiles or hyperthermophiles), extremely high salinities (known as halophiles),
extremely low pH (known as acidophiles), and most commonly a combination of them. From
the Crenarchaeota, the Sulfolubus and Aeropyrum genera receive lots of attention since both
are aerobic thermophilic organisms. In the Euryarchaeota, the methanogenic organisms are
1
Sometimes phosphofructokinase-1 and phosphofructokinase-2 from E. coli are called the major and
minor enzyme respectively.
238 Gene Duplication
On the Specialization History of the ADP-Dependent Sugar Kinase Family 3
intensively studied. One of the most studied organism here is Methanocaldococcus jannaschii
2
(Jones et al., 1983) since it is one of the few organisms known to produce methane at extreme
temperatures. Besides it, the Halobacterium and Haloferax genera are used as models for
halophilic organisms while organisms from the Thermococcus and Pyrococcus genera are used
as models of hyperthermophilic organisms. Here, by far, the most studied organism is
Pyrococcus furiosus.
In these organisms, sugar degradation proceeds either through the Entner-Doudoroff or
the Embden-Meyerhof pathway (Verhees et al., 2003). For instance, members of the
Thermoproteus, Thermoplasma, and Sulfolobus genera degrade glucose through a modied
version of the Entner-Doudoroff pathway where sugars are phosphorylated only at the
2-keto-3-deoxygluconate or glycerate level. While the former version is still able to produce
one ATP molecule per glucose the later does not produce any ATP (for a review see Verhees
et al. (2003)). On the other hand, up until the early 90s it was thought that some archaea of the
Euryarchaeota used a modied unphosphorylated version of the Entner-Doudoroff pathway
to degrade glucose (Mukund & Adams, 1991) which was called pyroglycolysis. However,
in 1994 it was possible to demonstrate that, in fact, the ux to pyruvate proceeds through
a highly modied version of the Embden-Meyerhof pathway (Kengen et al., 1994).Here,
although all the intermediates are present, only four of the ten textbook enzymes are
conserved (Verhees et al., 2003). In this pathway, the redox reactions are carried out by
ferredoxin containing enzymes which latter use the electrons to reduce protons (producing
hydrogen) to couple the proton motive force to ATP synthesis by means of a membrane
bound hydrogenase enzyme (Sapra et al., 2003). Between the oxido-reductases present in these
organisms, perhaps the most interesting is the glyceraldehyde-3-phosphate oxido-reductase.
This enzyme is responsible for the single-step conversion of glyceraldehyde-3-phosphate
to 3-phosphoglycerate in a phosphate independent manner (Mukund & Adams, 1995).
Besides redox reactions, one of the most striking modications seen in this version of the
Embden-Meyerhof pathway is that the phosphorylation of glucose and fructose-6-phosphate
is carried out by enzymes that use ADP and not ATP or polyphosphates as the phosphoryl
donor (Kengen et al., 1994). These ADP-dependent enzymes are, in fact, homologous to
each other and they show no sequence identity over the noise level with any of the hitherto
known ATP, or polyphosphate dependent kinases (Tuininga et al., 1999). For this reason it
was initially proposed that they belong to a new protein family called PfkC.
Given that these ADP-dependent enzymes were initially discovered in the hyperthermophilic
archaeon P. furiosus (Kengen et al., 1994), it has been argued in the literature that the main
reason for this ADP-dependence is the fact that ADP has a higher thermostability than
ATP and also that both nucleotides are essentially equivalent since both have a similar
standard G of hydrolysis. However, these arguments are highly misleading since, (i) as
metabolism is a non-equilibrium process the free energy change upon phosphoryl transfers
depends on the concentration of the metabolites, (ii) several ATP-dependent enzymes can be
found in hyperthermophilic organisms, (iii) the ADP-dependent enzymes are also present in
mesophilic organisms (see below), and (iv) the half life of ATP at high temperatures is higher
than some other metabolic intermediates present in the Embden-Meyerhof pathway (Drr
et al., 2003).
The adaptive value of the appearance of the ADP-dependent enzymes has been a matter of
great debate. As we have argued before (Guix & Merino, 2009), it is most likely unrelated
2
This organism was initially named Methanococcus jannaschii and was later renamed as
Methanocaldococcus jannaschii to aknowledge the fact that those organisms from the Methanococcus
genus are not thermophilic.
239 On the Specialization History of the ADP-Dependent Sugar Kinase Family
to the temperature at which most of the thermococcales grow. The most intriguing question
arising here is what happens with the adenylate charge inside these archaea. As they present
a glyceraldehyde-3-phosphate ferredoxin oxidoreductase (Mukund & Adams, 1995) which
produces 3-phosphoglycerate in a single step that does not produce ATP and also considering
both glucose and fructose-6-phosphate are phosphorylated using ADP as phosphoryl donor,
it was though that this modied glycolysis had a net ATP production of zero. However, it has
been demonstrated by Sakuraba et al. (2004) that the pyruvate kinase from P. furiosus catalyze
the synthesis of ATP from AMP, phosphoenolpyruvate, and Pi. In this way, the pathway from
glucose to pyruvate produces two ATP molecules from every glucose molecule degraded.
Up until now, we have three protein families that contain phosphofructokinases: PfkA, the
ribokinase family (which contains the PfkB-like kinases), and PfkC. While the rst PfkA
crystal structure (The phosphofructokinase from B. stearothermophilus) was solved in the 80s
(Evans et al., 1981), the rst PfkB-like crystal structure (the ribokinase from E. coli) (Sigrell
et al., 1998) was solved in the late 90s, and the rst PfkCcrystal structure (The ADP-dependent
glucokinase from Thermococcus litoralis) just in 2001 (Ito et al., 2001). As all of them were
discovered before the middle 90s most of the phylogenetic analysis were performed only on
the basis of sequence data. Quite surprisingly, despite the extremely low sequence identity,
the PfkC family can be structurally classied as another member of the ribokinase group (Ito
et al., 2001) which is now known as the ribokinase superfamily.
3. The ribokinase superfamily
Structurally, the PfkC and PfkB-like groups contain enzymes that present two domains. The
large domain, which contains the core ribokinase-like fold, is an structure where a central
-sheet mainly composed of parallel strands is anked by -helices on both sides. Also, they
present a smaller domain which in general is used as a scaffold for dimerization (Sigrell
et al., 1998). However, some of the enzymes are monomers. In this case, the hydrophobic core
of the small domain is formed by the insertion of some -helices (Ito et al., 2001; Mathews
et al., 1998). While not all the known PfkC enzymes are monomers (Jeong et al., 2003; Koga
et al., 2000; Tuininga et al., 1999) all of them present those -helices in the small domain.
Interestingly, the way in which many of them form multimers is not known, but seems to be
highly enzyme specic.
The active site of these enzymes is located in a cleft between both domains (Ito et al., 2001;
Sigrell et al., 1998). For some members of the ribokinase family, it has been shown by means
of x-ray crystallography that the relative orientation of the domains can be modied by the
binding of the phosphoryl acceptor ligand (Schumacher et al., 2000; Sigrell et al., 1999) which
has been suggested as a key step in the catalytic mechanism of these enzymes. In the PfkC
case, a similar scenario has been suggested (Ito et al., 2003; Tsuge et al., 2002). Here, although
the evidence is also crystallographic, it is indirect because the only enzyme crystallized in
the apo form and complexed with a substrate is the ADP-dependent phosphofructokinase
from Pyrococcus horikoshii (Currie et al., 2009) which does not show any domain movement.
However, it was not possible to obtain a crystalline form of the enzyme in the presence of
fructose-6-phosphate which could be the key component to induce the domain closing. In fact,
it has been previously shown by us based on molecular modeling that the open conformation
of these enzymes is most likely inactive (Merino & Guix, 2008).
Table 1 shows most of the members of the ribokinase superfamily with known
crystallographic structures. Based on this structural data it is possible to add other specicities
PDB Code Organism Function
P
f
k
C
l
i
k
e
1UA4 Pyrococcus furiosus Glucokinase
1GC5 Thermococcus litoralis Glucokinase
1L2L Pyrococcus horikoshii Glucokinase
1U2X Pyrococcus horikoshii Fructose-6-phosphate kinase
V
i
t
a
m
i
n
k
i
n
a
s
e
l
i
k
e
1JXH Salmonella typhimurium 4-amino-5-hydroxymethyl-2-methylpyrimidine
phosphate kinase
1EKQ Bacillus subtilis Hydroxyethylthiazole kinase
1V8A Pyrococcus horikoshii Hydroxyethylthiazole kinase
1UB0 Thermus thermophilus Phosphomethylpyrimidine kinase
1LHP Ovis Aries Pyridoxal kinase
1TD2 Escherichia coli Pyridoxal kinase (PdxY)
2DDM Escherichia coli Pyridoxal kinase (PdxK)
2F7K Homo sapiens Pyridoxal kinase
2I5B Bacillus subtilis Pyridoxal kinase
1KYH Bacillus subtilis Unknown function
2AX3 Thermotoga maritima Unknown function
2R3B Enterococcus faecalis Unknown function
P
f
k
B
l
i
k
e
2AFB Thermotoga maritima 2-keto-3-deoxygluconate kinase
2VAR Sulfolobus solfataricus 2-keto-3-deoxygluconate kinase
2DCN Sulfolobus tokodaii 2-keto-3-deoxygluconate kinase
1V1A Thermus thermophilus 2-keto-3-deoxygluconate kinase
2QCV Bacillus halodurans 5-dehydro-2-deoxygluconate kinase
1TZ6 Salmonella enterica Aminoimidazol riboside kinase
1BX4 Homo sapiens Adenosine kinase
1LII Toxoplasma gondii Adenosine kinase
2PKN Mycobacterium
tuberculosis
Adenosine kinase
2C49 Methanocaldococcus
jannaschii
Nucleoside kinase
1RKD Escherichia coli Ribokinase
1VM7 Thermotoga maritima Ribokinase
2FV7 Homo sapiens Ribokinase
2QHP Bacteroides
thetaiotaomicron
Fructokinase
2HW1 Homo sapiens Ketohexokinase
2ABQ Bacillus halodurans Fructose-1-phosphate kinase
2F02 Enterococcus Faecalis Tagatose-6-phosphate kinase
2JG1 Staphylococcus aureus Tagatose-6-phosphate kinase
3CQD Escherichia coli Fructose-6-phosphate kinase
3BF5 Thermoplasma
acidophilum
Unknown function
1VK4 Thermotoga maritima Unknown function
2NWH Agrobacterium
tumefaciens
Unknown function
2RBC Agrobacterium
tumefaciens
Unknown function
2AJR Thermotoga maritima Unknown function
2JG5 Staphylococcus aureus Unknown function
Table 1. Crystal structures of the ribokinase superfamily found in the PDB database.
Fig. 1. Schematic representation of the three branches of the ribokinase superfamily. For the
vitamin kinase like branch the pyridoxal kinase (pdxK) from E. coli (PDBID 2DDM) is used
as example, for the PfkB like branch the ribokinase from E. coli (PDBID 1RKD) is used, and
for the ADP-dependent branch the glucokinase from T. litoralis (PDBID 1GC5) is shown.
to the superfamily, such as adenosine kinase
3
(Mathews et al., 1998), 2-keto-3-deoxygluconate
kinase (Ohshima et al., 2004), and aminoimidazole riboside kinase (Zhang et al., 2004). Beyond
the sugar containing molecules, three dimensional structure comparison showed that kinases
like 4-methyl-5--hydroxyethylthiazole kinase (Campobasso et al., 2000), pyridoxal kinase
(Li et al., 2002), and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase (Cheng
et al., 2002) are also members of the ribokinase superfamily. Interestingly, these enzymes lack
the small domain.
Already based on substrate specicities three major branches can be recognized (Figure 1).
One of them contains those enzymes that catalyze the transfer of the -phosphate of ATP to
molecules such as pyridoxal, or pyrimidine derivatives which we know as vitamin kinase like
branch. The second contains all the enzymes that catalyze the transfer of the -phosphate of
ATP to sugar containing molecules, such as fructose-6-phosphate, adenosine, aminoimidazole
riboside, etc. We knowthis as the PfkB like branch. The last of themcontains the enzymes that
catalyze the transfer of the -phosphate of ADP to glucose and fructose-6-phosphate which,
as was mentioned before is known as PfkC family or ADP-dependent sugar kinase family.
Based mainly on the presence of the small domain and the monomer complexity Zhang et al.
(2004) proposed that the most ancient activity of the superfamily should be that catalyzed by
the simplest enzyme which is 4-methyl-5--hydroxyethylthiazole kinase. In that way, they
propose that the increase of complexity in the monomers fold indicates a newer enzyme. By
this hypothesis, the ADP-dependent enzymes and the monomeric adenosine kinases should
be the newest acquisitions of the superfamily. However, this hypothesis was never tested.
Nevertheless, although it could capture the essence of the evolutionary history of this group,
considering the linearity of the hypothesis, it is rather unlikely that the true history of the
group is entirely represented by it.
As it can be inferred from Figure 1 and Table 1 the ribokinase superfamily is an excellent
example of how gene duplication has been used several times by nature to produce new
specicities. This process has been recognized before as one of the most important steps in the
creation of new protein functions (Chothia et al., 2003). Indeed, most of the proteins present
inside a genome belong to a few protein families or a combination of them (see for example
Chothia et al. (2003)). This degeneracy causes that the number of protein families represented
in a genome are much smaller that the number of genes there.
Just as an example, a simple PSI-BLAST search on the genome of E. coli using
the phosphofructokinase-2 as query nds 28 non-redundant proteins including:
6-phosphofructokinase, 1-phosphofructokinase, ribokinase, 2-keto-3-deoxygluconate
kinase, and several proteins of unknown function. All of them present the PfkB-like fold (see
Figure 1) which shows that this family is a very interesting example of gene duplications.
However, the study of this feature is complicated by the lack of information on the function
of several of the PfkB-like proteins.
4. Structural evolution of the substrate specicity in the ADP-dependent sugar
kinase family
The ADP-dependent sugar kinases have been found in several members of the
Pyrococcus, Thermococcus, Methanosarcina, Methanosaeta, Methanococcoides, Methanococcus,
Methanocaldococcus, and Archaeoglobus genera (Hansen & Schnheit, 2004; Kengen et al., 1994;
Koga et al., 2000; Tuininga et al., 1999; Verhees et al., 2001). Also, it has been possible to
3
These enzymes have a slightly different fold compared with the other nucleoside kinases (such as
inosine-guanosine kinases) from the superfamily mentioned by Bork et al. (1993)
identify a distant homolog of these enzymes in the genome of higher eukaryotes, which has
been proven to be an ADP-dependent glucokinase (Ronimus & Morgan, 2004). The metabolic
role of the eukaryotic ADP-dependent glucokinases is unclear, but they have been suggested
to be used in ischemic conditions (Ronimus & Morgan, 2004).
To date, the crystallographic structures of the ADP-dependent glucokinases from
Thermococcus litoralis (Ito et al., 2001), Pyrococcus horikoshii (Tsuge et al., 2002), Pyrococcus
furiosus (Ito et al., 2003), and the ADP-dependent phosphofructokinase from Pyrococcus
horikoshii (Currie et al., 2009) have been solved. As opposed to the vitamin kinase or the
PfkB-like branches of the ribokinase superfamily, to date just two specicities have been
observed in the ADP-dependent branch (see Table 1). Considering that the ribokinase
superfamily contains enzymes that catalyze the transfer the terminal phosphate of a
nucleotide phosphate to the methyl alcohol end of a big number of small molecules which
includes pyridoxal, pyrimidine derivatives, nucleosides, and several sugars, the PfkC family
seems to be the one with the smallest substrate specicity in this group.
While there are many phosphoryl acceptor substrates in this superfamily, just two nucleotides,
ADP and ATP, are described as the primary phosphoryl donors. Given the metabolic
importance of the phosphoryl donor this specicity problem has received more attention than
the acceptor problem in the literature. Of course, the specicity is not strict, and some other
nucleotides can replace them. For instance, it has been shown that several ADP-dependent
enzymes can use other purines (such as GDP) or even pyrimidines (such as UDP, (Currie
et al., 2009)) as phosphoryl donors (Guix & Merino, 2009). Also, GTP can be used by
the phosphofructokinase-2 from E. coli and even produce substrate inhibition (unpublished
results). Yet, it is important to remember that only those nucleotides with the right number of
phosphates (either two for the ADP dependent enzymes or three for the ATP dependent) can
be used, as it has been reviewed by us elsewhere (Guix & Merino, 2009). This shows that any
hint for the transition between nucleotide specicities has been obscured by evolution and
specialization.
From an evolutionary perspective, while Zhangs hypothesis (Zhang et al., 2004) can be
oversimplifying the problem, it captures the most common trend in the evolution of protein
families: newer versions within the group tend to increase their structural complexity (Fong
et al., 2007). Through their reasoning, ADP-dependent kinases should be closely related to the
monomeric adenosine kinases. What has not been properly mentioned in the literature before
is the fact that while the tertiary structure of the PfkC enzymes is almost equivalent to that
of the PfkB enzymes, the topology of the C-terminal region is completely different (Figure 2).
Indeed, this is the reason why it was not possible to group the ADP-dependent enzymes with
the other members of the ribokinase superfamily just based on sequence comparison.
A BLAST search on the genome sequence of the archaeon P. furiosus reveals three PfkB-like
enzymes of unknown function. As it is also possible to nd vitamin kinase like enzymes in
the genome of the thermococcales (see for instance Table 1), then it is possible to deduce that all
three modern branches of the ribokinase superfamily have been originated by ancient gene
duplication events followed by extensive topological modications. While the addition of
the small domain can be viewed as a trivial modication since it only involves the insertion
of sequence, the C-terminal topological reordering involves a non-cyclic permutation. Now,
considering that the ADP-dependent enzymes should be the modern ones, in order to be
compatible with the topological reordering an ATP-dependent enzyme should present an
extra strand in the C-terminal end of the protein extending the central -sheet. Figure 2 shows
that indeed, this requirement is fullled by some PfkB-like enzymes. Quite surprisingly, the
sugar-phosphate kinases and not adenosine kinases are those who showthe extra strand. This
Fig. 2. -meander region of several members of the ribokinase superfamily such as Pfk-2
from E. coli (A), a fructose-1-phosphate kinase from B. halodurans (B), a putative
phosphofructokinase from S. aureus (C), and the ADP-dependent glucokinase from P. furiosus
(D). In red is shown the C-terminal extension thought to be needed for the circular
permutation.
suggests that the ADP-dependent enzymes are most closely related to the other glycolytic
enzymes present in the superfamily, which seems to be reasonable given the similarity of
their substrates.
Indeed, this C-terminal reordering is quite interesting since this same region constitutes
almost all the nucleotide binding site. However, while this permutation almost certainly alters
the dynamics of the binding pocket, we are not sure if it will alter the specicity of the enzyme.
Nevertheless, it requires empirical testing which is now being performed in our laboratory.
Interestingly, the and phosphates of ADP are accommodated in the binding site of the PfkC
enzymes almost in the same way as the and phosphates of ATP in the remaining members
of the superfamily. This led Ito et al. (2001) to suggest that the bulky side chain of Y357 in
the ADP-dependent glucokinase from T. litoralis which is located below the ribose moiety of
ADP was pushing the nucleotide forward and then rendering an enzyme unable to use ATP.
However, we indirectly demonstrated that this is not the case since for the ADP-dependent
phosphofructokinase from P. horikoshii the presence of a signicantly less bulky side chain
(I340) does not produce an enzyme with ATP-dependent activity (Currie et al., 2009).
While in most of the members of the Euryarchaeota there are two ADP-dependent enzymes
coded in their genomes, the archaeon M. jannaschii presents just one copy of these genes.
Surprisingly, the enzyme is able to catalyze the transfer of the -phosphate of ADP to either
glucose or fructose-6-phosphate (Sakuraba et al., 2002). Based on this feature, it was proposed
that this enzyme represent an ancestral state of the family, which later gave rise to the separate
specicities through a gene duplication event (Sakuraba et al., 2002). However, this hypothesis
had to wait six years to be tested (Merino & Guix, 2008).
To test this hypothesis we used the Bayesian method of phylogenetic inference implemented
in the MrBayes 3.1 software (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck,
2003). Initially, a structural based sequence prole was built by means of a structural
alignment of the ADP-dependent glucokinases from T. litoralis, P. horikoshii, and P. furiosus
and the ADP-dependent phosphofructokinase from P. horikoshii. Later all the ADP-dependent
kinases from archaeal source were aligned to this prole. After several rounds of alignment
renement the eukaryotic ADP-dependent enzymes were added. As they share only about 15
to 20% sequence identity with the archaeal versions the alignment was guided by secondary
structure predictions.
Fig. 3. Phylogenetic tree of the archaeal part of the ADP-dependent sugar kinase family. The
eukaryotic sequences were used as outgroup. For displaying clarity just the posterior
probabilities of the most important nodes are shown in the gure. The node shown in bold
letters correspond to the bifunctional enzyme from M. jannaschii. Modied from (Merino &
Guix, 2008).
Considering that in the Archaea most organisms present ADP-dependent glucokinases and
phosphofructokinases while in the Eukarya just glucokinases can be found we thought that
the divergence between both domains happened before the gene duplication event. In this
light, the eukaryotic enzymes seem to be a reasonable choice for the outgroup. Nevertheless,
for consistency, other members of the ribokinase superfamily were tested as outgroups as
well.
Figure 3 shows the resulting evolutionary tree. It clearly shows four main clades: a group
containing the glucokinases from the methanosarcinales, a group containing the glucokinases
from the thermococcales, a group containing the phosphofructokinases from the thermococcales,
and a group containing the phosphofructokinases from methanococcales and methanosarcinales.
Surprisingly, irrespective of the outgroup used, the root of the tree appears between both
glucokinases groups and not dividing both specicities as should be expected from the
bifunctional ancestor hypothesis. This demonstrates that the bifunctional enzyme from M.
jannaschii does not represent an ancestral state of the family. However, given its basal position
inside the phosphofructokinases it still could represent a transitional form between both
specicities.
Given the tree topology, it is possible to infer that the rst separation between thermococcales
and methanosarcinales, i.e. the glucokinases separation close to the root, was produced by a
speciation event. Most likely, the gene duplication event is located close to the last common
ancestor between both specicities. As this node is located after the speciation event, it is
necessary to have an extra horizontal gene transfer event in order to explain the presence of
ADP-dependent glucokinases and phosphofructokinases in the genomes of thermococcales and
Fig. 4. Dendrogram grouping the archaeal ADP-dependent genes according to their average
difference in relative synonymous codon usage. Modied from (Merino & Guix, 2008).
methanosarcinales. Indeed a similar scenario for the generation of paralogous genes has been
proposed before (Gogarten et al., 2002).
To test this hypothesis we analyzed the relative synonymous codon usage of the archaeal
PfkC genes (McInerney, 1998). By this methodology, the frequency of any given codon in a
gene is calculated relative to the frequency expected for an unbiased codon usage. Figure 4
shows that, in general, genes are grouped very close to their paralogous. If this is not the case,
they are at least inside a group that contains closely related species. The only exception is
the glucokinase from Methanosaeta thermophila which is located inside the thermococcales group
(Figure 4). Indeed, when the codon usage of this gene is compared with the codon usage of
the archaeal genomes, it seems to be more related to the genome of T. litoralis than to its own
genome (not shown).
While the data present above are not enough to prove the horizontal transfer hypothesis it still
strongly suggests that this process has been involved in the evolution of the ADP-dependent
sugar kinase family. It is important to stress out that if the event of horizontal gene transfer
is ancient enough, then the accumulation of a sufcient number of mutations should have
masked it. If this is our case then, to our knowledge, there is no sequence based technique to
prove the hypothesis.
Sakuraba et al. (2002) demonstrated that when the bifunctional enzyme was using
fructose-6-phosphate as substrate glucose can act as a competitive inhibitor. They proposed
that this was produced because both sugars bind to the same site. It is important to mention
that competitive inhibition does not necessarily indicates that substrate and inhibitor have the
same site, but in this case it is certainly the case. To take advantage of this fact we modeled
the bifunctional enzyme and its interaction with both sugars. In this way, it is possible to gain
as much information as possible about the structural determinants of the sugar specicity.
Figure 5 shows the predicted interaction geometries for both substrates. For clarity just the
residues in a 5 radius are shown. As it was inferred by Sakuraba et al. (2002) the interaction
between the protein and both substrates are very similar. Indeed, just three of the residues
seems to differ signicantly in the way they interact with the sugars. For instance, while
Fig. 5. Left. Glucose docked to the bifunctional enzyme. Right. Fructose-6-phosphate
docked to the bifunctional enzyme. Bottom. Results of the real value evolutionary trace
analysis for all the residues within 5 from the ligands. The results for the glucokinase
specicity are shown in black, those for the phosphofructokinase specicity in red, and those
for the whole family in green. Modied from (Merino & Guix, 2008).
E82 makes a hydrogen bond with the hydroxyl located at C2 in glucose it does not seem
to interact in any specic way with fructose-6-phosphate. Indeed, this side chain has been
proposed by other authors as key for the glucokinase specicity (Ito et al., 2003; Sakuraba
et al., 2002). On the other hand, R203 is making a close salt bridge with the phosphate moiety
of fructose-6-phosphate while it does not interact with glucose. Although K170 is not in the 5
radius we had strong evidence that, as in the R203 case, this side chain was also involved in
the phosphate binding (see below).
To quantify the conservation degree of the residues inside the sugar binding site we used a
tree-based residue ranking system called real value evolutionary trace (Mihalek et al., 2004).
Briey, the method ranks the residues as follows:
First, let us consider a rooted evolutionary tree with N leaves (sequences). If we number the
nodes in the tree starting with the root being 1 then, using as example Figure 3, the node
number 2 should be the one with 0.98 posterior probability, and so on. Using this method
it is possible to number N 1 nodes. Each node denes some groups g of sequences. The
root node of course creates a group with all of them. Node number 2 creates a group that
contains the ADP-dependent glucokinases from methanosarcinales and other with the rest of
the sequences. By this nomenclature one can dene a measurement of the conservation of
each position in the alignment i where
r
i
= 1 +
N1
n=1
0 if position i conserved within each group g

1 otherwise
(1)
It is clear that if a residue is conserved from the root of the tree, then it will have a r
i
of 1. As it
gets less and less conserved r
i
will be higher. To account for the sequence conservation within
each group, the r
i
value was weighted by sequence entropy given the expression
i
= 1 +
N1
n=1
1
n
n
g=1
20
aa=1
f
g
ia
log f
g
ia
(2)
where f
g
ia
stands for the frequency of appearance of amino acid a inside the group g.
Figure 5 (bottom) shows the result of the ranking applied to the whole PfkC family, and both
separated specicities. It is clear from the gure that most of the residues are conserved
in the whole family. Interestingly, E82 is only conserved inside the glucokinase specicity,
which is in good agreement with the role proposed above. Also, K170 and R203 are only
conserved inside the phosphofructokinase specicity which makes them the inverse case of
the E82 residue.
Interestingly, N172 is conserved inside both specicities, but it is not in the whole family. The
reason for this is that within phosphofructokinases this residue is strictly an asparagine while
inside the glucokinases is always a histidine. This suggests that this residue is also related
with sugar specicity, but the reason is not as clear as the above examples.
Recently, we used a more elegant method known as explicit likelihood of subset covariation
(ELCS) (Dekker et al., 2004) to explore the correlation between mutations to search for the
structural specicity determinants.
Figure 6 shows the group of side chains with the highest ELCS score. Surprisingly, the
group contain a side chain that belongs to a highly conserved motif called NXXE which
has been related with metal binding to the enzymes of the superfamily (Maj et al., 2002;
Parducci et al., 2006; Rivas-Pardo et al., 2011). In fact, we have demonstrated that this motif
is related to the binding of the catalytic and regulatory metals in the ADP-dependent sugar
kinase family (To be published). Also, the rst group found by the ELCS method contains
some residues that we proposed before as specicity related. The role of the R48/D65
4
,
R65/S76, P73/F90 mutations is not clear, but seem to be related to the dynamics of the small
domain. K158/C174 (equivalent to K170 in the bifunctional enzyme), N160/H176 (equivalent
to N172 in the bifunctional enzyme), and R191/D203 (equivalent to R203 in the bifunctional
enzyme) are clearly interacting with the sugars. Interestingly, when the position R191/D203
presents an arginine, this positive side chain coordinates the phosphate group present in the
fructose-6-phosphate molecule. On the other hand, when it presents an aspartic acid, this
side chain interacts with the histidine in the N160/H176 position, allowing the histidine to be
correctly positioned to make an h-bond with the O2 hydroxyl group of glucose. Curiously,
the position equivalent to E82 from the bifunctional enzyme does not appear to be correlated
with other positions by the ELCS method. However this could be due to the small amount of
sequence information used for the analysis.
4
We use the numbering of PhPFK/Pf GK for the correlated mutations. See Figure 6 for clarity.
Fig. 6. First cluster of correlated mutations in the PfkC family identied by the ELCS method.
A. Crystal structure of the glucokinase from P. furiosus. Glucose and AMP are shown. B.
Structural model for the ternary complex between the phosphofructokinase from P. horikoshii,
ADP and fructose-6-phosphate. The coordinates were derived from the molecular dynamics
simulation performed in (Currie et al., 2009)
We have tested the predictions made by the evolutionary trace and the ELCS methods by
means of mutagenesis using the ADP-dependent phosphofructokinase from horikoshii (Currie
et al., 2009) as a model.
Table 2 shows the effect of each mutation on the kinetic parameters of fructose-6-phosphate
and MgADP. As it can be predicted, either the mutations R191A, R191E, or K158A produce a
high increase in the K
M
value for fructose-6-phosphate with a little effect on k
cat
or K
M
from
MgADP.
On the other hand, the N160A increase three-fold k
cat
with a concomitant high increase in
the K
M
value for fructose-6-phosphate. The reason for the increase in activity is not clear,
but it suggests that while this interaction increases the afnity of the protein for the sugar it
Enzyme k
cat
K
M
F6P k
cat
/K
M
F6P
s
1
M M
1
s
1
Wild Type 45.5 4.0 15.2 2.5 2.98 10
6
A71E 39.3 9.9 22.2 2.6 1.77 10
6
K158A 41.0 6.7 6500 1300 6.30 10
3
N160A 151 16 415 13 3.65 10
5
N160Q 14.7 0.6 6300 720 2.33 10
3
R191A 27.4 1.5 254.4 26.1 1.07 10
5
R191E 42.5 1 4870 170 8.73 10
3
Table 2. Kinetic parameters of wild type and mutant versions of the ADP-dependent
phosphofructokinase from P. horikoshii. Modied from (Currie et al., 2009). All experiments
were performed at 50 C.
imposes a strain in the transition state, which results in a decrease of k
cat
. However, none of
these mutations produce an enzyme with glucokinase activity.
The A71E mutation does not affect the catalytic constants nor K
M
for fructose-6-phosphate.
Surprisingly, it produces an enzymes that now can catalyze the transfer of the -phosphate of
ADP to glucose with a k
cat
of 2.7 0.05 s
1
and a K
M
of 3.95 0.2 mM
5
. Also we recently have
produced a N160H mutant. It dramatically increases the K
M
value for fructose-6-phosphate
to 6.3 0.72 mM and decreases the k
cat
almost four-fold (Table 2). As in the A71E case, it
also produces a bifunctional enzyme that can use glucose as substrate. However, for this
mutant no clear saturation is seen even for 25 mM glucose. Based on a Lineweaver-Burk plot,
it is possible to estimate a k
cat
of 2.42 s
1
and a K
M
value of 25.3 mM. Clearly, this mutation
produces a much stronger effect than A71E.
The last two mutations are key to understanding the specialization problem since they
not only enable the phosphofructokinase from P. horikoshii to use glucose as substrate.
Competition experiments with this enzyme have shown that glucose does not bind to the wild
type version, which demonstrates that the mutations somehow unblock the binding site for
the binding of glucose. Curiously, both mutations points to the interaction between the protein
and the hydroxyl group at C2 of glucose. This suggests that the specicity determinants are
not evenly distributed amongst the binding site, but rather concentrated in hot-spots. In this
light, in order to revert the specicities, just a couple of mutations are needed.
5. Are two ADP-dependent kinases better than one?
Considering that the glycolysis of M. jannaschii is functional with just one enzyme in charge
of the phosphorylation of glucose and fructose-6-phosphate it is not clear, at a rst glance,
why two genes were select by nature in the other members of the Euryarchaeota. As it was
mentioned above, glucokinases are on the top of several pathways and hence the modication
of their activity affects a big part of the metabolism. Indeed, this enzyme generally have a
great control of the carbon ux. On the other hand, phosphofructokinases seem to be closely
related with the balance between glycolysis and gluconeogenesis. In the archaeon P. furiosus
it has been shown that the switching between these two metabolic pathways is controlled
at the expression level (Schut et al., 2003). When the ADP-dependent phosphofructokinase
is expressed the fructose-1,6-bisPase is repressed and vice versa. Of course, just shutting the
5
Glucokinase experiments were performed at 40 Cgiven the instability of the auxiliar enzyme used.
bifunctional enzyme down in M. jannaschii will not only decrease the phosphofructokinase
activity, but it will have the undesirable side effect of decreasing the glucokinase activity. In
this light, the use of one enzyme for each specicity has a great impact in how the cell can
regulate the carbon ux. Indeed, the fact the sugar specicity residues are correlated with
some others related with regulation (such as the mutation in the NXXE motif) strongly favors
our explanation.
What kind of process produce this? It is now a generally accepted hypothesis that less
important genes or parts of a gene tend to change or evolve faster than less important ones,
which is known as the Kimura-Ohta principle (for a review see Camps et al. (2007)). It is
clear that the upon a gene duplication, the phenotypic effect of a mutation in any copy of
these genes should be fairly null with the only exception of those that produces specialized
genes. It can be argued that even those mutations that produce inactive proteins which should
be deleterious and removed by purifying selection under normal conditions are now nearly
neutral since the only extra cost is to produce a non-functional protein.
It has been shown recently that upon a change in the tness optimum (either produced by
an environmental change or an internal redistribution of uxes) most mutations are xed by
natural selection up until the genes reach a nearly optimal sequence. Then they accumulated
mutations according to a neutral model (Razeto-Barry et al., 2011). From the arguments
given above, it is clear that the only way in which a duplicated gene can break the neutral
regime is when a rare specializing mutation is xed. In this case, the organism must adapt
to the new distribution of internal uxes. Ohta (1987) reached a similar conclusion based
on simulations. He stated: Positive natural selection favors those chromosomes with more
benecial mutations in redundant copies than others in the population, but accumulation of
deteriorating mutations (pseudo genes) have no effect on tness so long as there remains a
functional gene. The results imply the following: Positive natural selection is needed in order
to acquire gene families with new functions. Without it, too many pseudo genes accumulate
before attaining a functional gene family.
As we have shown here, for our protein family, this would imply just one or two mutations
since for instance, just the change of a single interaction can change the balance between
both specicities. Of course, upon specialization, mutations that modulate regulation (such
as those related with the NXXE motif) increases the exibility of the metabolism.
Interestingly, although most of the Euryarchaeota that present the ADP-dependent kinases
have two separated specicities the glucokinase frompsychrophilic archaeon Methanococcoides
burtonii have a big C-terminal deletion that should make it non-functional (Merino & Guix,
2008). The fact that it is still possible to know that it was a glucokinase suggests that this
deletion was recent. The phosphofructokinase gene in this archaeon present a glutamic acid
in the position equivalent to E82 in the bifunctional enzyme, which suggests that this could
be a bifunctional enzyme too. In this way, it appears that until the phosphofructokinase gene
is entirely specialized it still exist the possibility of loosing the specic glucokinase gene.
6. Concluding remarks
Our studies about the evolution of the ADP-dependent sugar kinase family showed that
the root of the family is located inside the glucokinase group, demonstrating that the
bifunctional (glucokinase/phosphofructokinase) enzyme is not an ancestral form, but could
be a transitional form from glucokinase to phosphofructokinase. Unfortunately, to date it has
not been possible to obtain the crystal structure of any ADP-dependent phosphofructokinase
in the presence of fructose-6-phosphate. However, based on structural modeling we have
been able to understand partially the structure/specicity relation up to the point where we
can produce bifunctional enzymes from specic ones. Strikingly, the sugar discrimination is
somehow concentrated in very few hot-spots in the structure. Indeed, the introduction of
just one hydrogen bond or some salt bridges seems to modulate the afnity for glucose or
fructose-6-phosphate respectively. Unfortunately, to date, we have been unable to absolutely
switch the specicity of these enzymes.
The gene duplication event itself seems to be related with the separated regulation of the
glucokinase and phosphofructokinase activity, along with the balance between glycolysis and
gluconeogenesis. Indeed, with two different enzymes a nest tuning of the carbon ux inside
the cell can be achieved.
7. Acknowledgements
We are very grateful to Dr. Ricardo Cabrera for his help in the creation of this chapter by
means of enlightening discussions about the evolutionary implications of the gene duplication
in the modied Embden-Meyerhof pathway. This work was supported by Fondo Nacional de
Desarrollo Cientco y Tecnolgico (Fondecyt) through the grant 1110137.
8. References
Allers, T. &Mevarech, M. (2005). Archaeal genetics - the third way., Nat. Rev. Genet. 6(1): 5873.
Atkinson, D. E. & Walton, G. M. (1965). Kinetics of regulatory enzymes. Escherichia coli
phosphofructokinase., J. Biol. Chem. 240: 757763.
Bork, P., Sander, C. & Valencia, A. (1993). Convergent evolution of similar enzymatic function
on different protein folds: the hexokinase, ribokinase, and galactokinase families of
sugar kinases., Protein Sci. 2(1): 3140.
Cabrera, R., Babul, J. &Guix, V. (2010). Ribokinase family evolution and the role of conserved
residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia
coli., Arch. Biochem. Biophys. 502(1): 2330.
Campobasso, N., Mathews, I. I., Begley, T. P. & Ealick, S. E. (2000). Crystal structure
of 4-methyl-5-beta-hydroxyethylthiazole kinase from Bacillus subtilis at 1.5
resolution., Biochemistry 39(27): 78687877.
Camps, M., Herman, A., Loh, E. & Loeb, L. A. (2007). Genetic constraints on protein
evolution., Crit. Rev. Biochem. Mol. Biol. 42(5): 313326.
Cheng, G., Bennett, E. M., Begley, T. P. & Ealick, S. E. (2002). Crystal structure of
4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase from Salmonella
typhimurium at 2.3 resolution., Structure 10(2): 225235.
Chothia, C., Gough, J., Vogel, C. &Teichmann, S. A. (2003). Evolution of the protein repertoire.,
Science 300(5626): 17011703.
Currie, M. A., Merino, F., Skarina, T., Wong, A. H. Y., Singer, A., Brown, G., Savchenko,
A., Caniuguir, A., Guix, V., Yakunin, A. F. & Jia, Z. (2009). ADP-dependent
6-phosphofructokinase from Pyrococcus horikoshii OT3: structure determination and
biochemical characterization of PH1645., J. Biol. Chem. 284(34): 2266422671.
Dekker, J. P., Fodor, A., Aldrich, R. W. & Yellen, G. (2004). A perturbation-based method
for calculating explicit likelihood of evolutionary co-variance in multiple sequence
alignments., Bioinformatics 20(10): 15651572.
Drr, C., Zaparty, M., Tjaden, B., Brinkmann, H. & Siebers, B. (2003). The hexokinase
of the hyperthermophile Thermoproteus tenax. ATP-dependent hexokinases and
ADP-dependent glucokinases, two alternatives for glucose phosphorylation in
Archaea., J. Biol. Chem. 278(21): 1874418753.
Evans, P. R., Farrants, G. W. & Hudson, P. J. (1981). Phosphofructokinase: structure and
control., Philos. Trans. R Soc. Lond. B Biol. Sci. 293(1063): 5362.
Fong, J. H., Geer, L. Y., Panchenko, A. R. & Bryant, S. H. (2007). Modeling the
evolution of protein domain architectures using maximum parsimony., J. Mol. Biol.
366(1): 307315.
Gogarten, J. P., Doolittle, W. F. & Lawrence, J. G. (2002). Prokaryotic evolution in light of gene
transfer., Mol. Biol. Evol. 19(12): 22262238.
Guix, V. & Merino, F. (2009). The ADP-dependent sugar kinase family: kinetic and
evolutionary aspects., IUBMB Life 61(7): 753761.
Hansen, T. & Schnheit, P. (2004). ADP-dependent 6-phosphofructokinase, an extremely
thermophilic, non-allosteric enzyme from the hyperthermophilic, sulfate-reducing
archaeon Archaeoglobus fulgidus strain 7324., Extremophiles 8(1): 2935.
Huelsenbeck, J. P. & Ronquist, F. (2001). MrBayes: Bayesian inference of phylogenetic trees.,
Bioinformatics 17(8): 754755.
Ito, S., Fushinobu, S., Jeong, J.-J., Yoshioka, I., Koga, S., Shoun, H. & Wakagi, T. (2003). Crystal
structure of an ADP-dependent glucokinase from Pyrococcus furiosus: implications
for a sugar-induced conformational change in ADP-dependent kinase., J. Mol. Biol.
331(4): 871883.
Ito, S., Fushinobu, S., Yoshioka, I., Koga, S., Matsuzawa, H. & Wakagi, T. (2001). Structural
basis for the ADP-specicity of a novel glucokinase from a hyperthermophilic
archaeon., Structure 9(3): 205214.
Jeong, J.-J., Fushinobu, S., Ito, S., Shoun, H. & Wakagi, T. (2003). Archaeal ADP-dependent
phosphofructokinase: expression, purication, crystallization and preliminary
crystallographic analysis., Acta Crystallogr. D Biol. Crystallogr. 59(Pt 7): 13271329.
Jones, W., Leigh, J., Mayer, F., Woese, C. & Wolfe, R. (1983). Methanococcus jannaschii sp. nov.,
an extremely thermophilic methanogen from a submarine hydrothermal vent, Arch.
Microbiol. 136(4): 254261.
Kengen, S. W., de Bok, F. A., van Loo, N. D., Dijkema, C., Stams, A. J. & de Vos, W. M. (1994).
Evidence for the operation of a novel embden-meyerhof pathway that involves
ADP-dependent kinases during sugar fermentation by Pyrococcus furiosus., J Biol
Chem 269(26): 1753717541.
Koga, S., Yoshioka, I., Sakuraba, H., Takahashi, M., Sakasegawa, S., Shimizu, S. & Ohshima,
T. (2000). Biochemical characterization, cloning, and sequencing of ADP-dependent
(AMP-forming) glucokinase fromtwo hyperthermophilic archaea, Pyrococcus furiosus
and Thermococcus litoralis., J. Biochem. 128(6): 10791085.
Kotlarz, D. & Buc, H. (1981). Regulatory properties of phosphofructokinase 2 from Escherichia
coli., Eur. J. Biochem. 117(3): 569574.
Li, M., Kwok, F., Chang, W., Lau, C., Zhang, J., Lo, S. C. L., Jiang, T. & Liang, D. (2002). Crystal
structure of brain pyridoxal kinase, a novel member of the ribokinase superfamily., J.
Biol. Chem. 277(48): 4638546390.
Maj, M. C., Singh, B. & Gupta, R. S. (2002). Pentavalent ions dependency is a conserved
property of adenosine kinase from diverse sources: identication of a novel motif
implicated in phosphate and magnesium ion binding and substrate inhibition.,
Biochemistry 41(12): 40594069.
Mathews, I. I., Erion, M. D. & Ealick, S. E. (1998). Structure of human adenosine kinase at 1.5
resolution., Biochemistry 37(45): 1560715620.
McInerney, J. O. (1998). GCUA: general codon usage analysis., Bioinformatics 14(4): 372373.
Merino, F. &Guix, V. (2008). Specicity evolution of the ADP-dependent sugar kinase family:
in silico studies of the glucokinase/phosphofructokinase bifunctional enzyme from
Methanocaldococcus jannaschii., FEBS J. 275(16): 40334044.
Mihalek, I., Res, I. & Lichtarge, O. (2004). A family of evolution-entropy hybrid methods for
ranking protein residues by importance., J. Mol. Biol. 336(5): 12651282.
Mukund, S. & Adams, M. W. (1991). The novel tungsten-iron-sulfur protein of the
hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin
oxidoreductase. evidence for its participation in a unique glycolytic pathway., J Biol
Chem 266(22): 1420814216.
Mukund, S. & Adams, M. W. (1995). Glyceraldehyde-3-phosphate ferredoxin oxidoreductase,
a novel tungsten-containing enzyme with a potential glycolytic role in the
hyperthermophilic archaeon Pyrococcus furiosus., J. Biol. Chem. 270(15): 83898392.
Ohshima, N., Inagaki, E., Yasuike, K., Takio, K. & Tahirov, T. H. (2004). Structure of Thermus
thermophilus 2-keto-3-deoxygluconate kinase: evidence for recognition of an open
chain substrate., J. Mol. Biol. 340(3): 477489.
Ohta, T. (1987). Simulating evolution by gene duplication., Genetics 115(1): 207213.
Parducci, R. E., Cabrera, R., Baez, M. & Guix, V. (2006). Evidence for a catalytic Mg
2+
ion and effect of phosphate on the activity of Escherichia coli phosphofructokinase-2:
regulatory properties of a ribokinase family member., Biochemistry 45(30): 92919299.
Peng, Z. Y. &Mansour, T. E. (1992). Purication and properties of a pyrophosphate-dependent
phosphofructokinase from Toxoplasma gondii., Mol. Biochem. Parasitol. 54(2): 223230.
Razeto-Barry, P., Daz, J., Cotoras, D. & Vsquez, R. A. (2011). Molecular evolution, mutation
size and gene pleiotropy: a geometric reexamination., Genetics 187(3): 877885.
Rivas-Pardo, J. A., Caniuguir, A., Wilson, C. A. M., Babul, J. & Guix, V. (2011). Divalent
metal cation requirements of phosphofructokinase-2 from E. coli. evidence for a high
afnity binding site for Mn
2+
., Arch. Biochem. Biophys. 505(1): 6066.
Ronimus, R. S. & Morgan, H. W. (2004). Cloning and biochemical characterization of a novel
mouse ADP-dependent glucokinase., Biochem. Biophys. Res. Commun. 315(3): 652658.
Ronquist, F. & Huelsenbeck, J. P. (2003). MrBayes 3: Bayesian phylogenetic inference under
mixed models., Bioinformatics 19(12): 15721574.
Sakuraba, H., Goda, S. & Ohshima, T. (2004). Unique sugar metabolism and novel enzymes
of hyperthermophilic archaea., Chem. Rec. 3(5): 281287.
Sakuraba, H., Yoshioka, I., Koga, S., Takahashi, M., Kitahama, Y., Satomura, T., Kawakami, R.
& Ohshima, T. (2002). ADP-dependent glucokinase/phosphofructokinase, a novel
bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii.,
J. Biol. Chem. 277(15): 1249512498.
Sapra, R., Bagramyan, K. & Adams, M. W. W. (2003). A simple energy-conserving
system: proton reduction coupled to proton translocation., Proc. Natl. Acad. Sci. USA
100(13): 75457550.
Schirmer, T. & Evans, P. (1990). Structural basis of the allosteric behaviour of
phosphofructokinase, Nature 343: 140145.
Schumacher, M. A., Scott, D. M., Mathews, I. I., Ealick, S. E., Roos, D. S., Ullman, B. &Brennan,
R. G. (2000). Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel
catalytic mechanism and prodrug binding., J. Mol. Biol. 298(5): 875893.
Schut, G. J., Brehm, S. D., Datta, S. & Adams, M. W. W. (2003). Whole-genome dna microarray
analysis of a hyperthermophile and an archaeon: Pyrococcus furiosus grown on
carbohydrates or peptides., J. Bacteriol. 185(13): 39353947.
Sigrell, J. A., Cameron, A. D., Jones, T. A. & Mowbray, S. L. (1998). Structure of Escherichia coli
ribokinase in complex with ribose and dinucleotide determined to 1.8 resolution:
insights into a new family of kinase structures., Structure 6(2): 183193.
Sigrell, J. A., Cameron, A. D. & Mowbray, S. L. (1999). Induced t on sugar binding activates
ribokinase., J. Mol. Biol. 290(5): 10091018.
Torres, J. C., Guix, V. & Babul, J. (1997). A mutant phosphofructokinase produces a futile
cycle during gluconeogenesis in Escherichia coli., Biochem. J. 327: 675684.
Torres, N. V., Mateo, F. & Melndez-Hevia, E. (1988). Shift in rat liver glycolysis
control from fed to starved conditions. ux control coefcients of glucokinase and
phosphofructokinase., FEBS Lett. 233(1): 8386.
Tsuge, H., Sakuraba, H., Kobe, T., Kujime, A., Katunuma, N. & Ohshima, T. (2002). Crystal
structure of the ADP-dependent glucokinase from Pyrococcus horikoshii at 2.0-
resolution: a large conformational change in ADP-dependent glucokinase., Protein
Sci. 11(10): 24562463.
Tuininga, J. E., Verhees, C. H., van der Oost, J., Kengen, S. W., Stams, A. J. & de Vos,
W. M. (1999). Molecular and biochemical characterization of the ADP-dependent
phosphofructokinase from the hyperthermophilic archaeon Pyrococcus furiosus., J.
Biol. Chem. 274(30): 2102321028.
van Rooijen, R. J., van Schalkwijk, S. & de Vos, W. M. (1991). Molecular cloning,
characterization, and nucleotide sequence of the tagatose 6-phosphate pathway gene
cluster of the lactose operon of Lactococcus lactis., J. Biol. Chem. 266(11): 71767181.
Verhees, C. H., Kengen, S. W. M., Tuininga, J. E., Schut, G. J., Adams, M. W. W., De Vos, W. M.
& Van Der Oost, J. (2003). The unique features of glycolytic pathways in Archaea.,
Biochem. J. 375(2): 231246.
Verhees, C. H., Tuininga, J. E., Kengen, S. W., Stams, A. J., van der Oost, J. & de Vos,
W. M. (2001). ADP-dependent phosphofructokinases in mesophilic and thermophilic
methanogenic archaea., J. Bacteriol. 183(24): 71457153.
Woese, C. R. & Fox, G. E. (1977). Phylogenetic structure of the prokaryotic domain: the
primary kingdoms., Proc. Natl. Acad. Sci. USA 74(11): 50885090.
Woese, C. R., Kandler, O. & Wheelis, M. L. (1990). Towards a natural system of organisms:
proposal for the domains Archaea, Bacteria, and Eucarya., Proc. Natl. Acad. Sci. USA
87(12): 45764579.
Wu, L. F., Reizer, A., Reizer, J., Cai, B., Tomich, J. M. &Saier, M. H. (1991). Nucleotide sequence
of the Rhodobacter capsulatus fruK gene, which encodes fructose-1-phosphate kinase:
evidence for a kinase superfamily including both phosphofructokinases of Escherichia
coli., J. Bacteriol. 173(10): 31173127.
Zhang, Y., Dougherty, M., Downs, D. M. & Ealick, S. E. (2004). Crystal structure of
an aminoimidazole riboside kinase from Salmonella enterica: implications for the
evolution of the ribokinase superfamily., Structure 12(10): 18091821.
14
Duplication of Coagulation Factor
Genes and Evolution of Snake
Venom Prothrombin Activators
Shiyang Kwong
1
and R. Manjunatha Kini
1,2

1
Department of Biological Sciences, Faculty of Science
National University of Singapore, Singapore
2
Department of Biochemistry, Medical College of Virginia
Virginia Commonwealth University, Richmond,Virginia
1
Singapore
2
USA
1. Introduction
Snake venom is a complex mixture of pharmacologically active molecules which are
responsible for immobilization, paralysis, death and digestion of prey organisms. This
armory of toxins has evolved to target two key systems, namely the neuromuscular and
circulatory systems, in order to induce rapid immobilization and death. So far, several
hundreds of protein toxins from snake venoms have been purified and characterized. Most
of these toxins have been documented to be structurally, and at times functionally, similar to
proteins expressed in different tissues of the body. For example, elapid phospholipase A
2

toxins are structurally and catalytically similar to mammalian pancreatic phospholipase A
2

enzymes (Robin Doley et al. 2009). Similarly, sarafotoxins are structurally and functionally
similar to endothelins produced primarily in endothelium (Landan et al. 1991a). Based on
such structural and functional similarities, it is hypothesized that toxin proteins are
recruited from body proteins by gene duplication (Fry 2005). Accordingly, the genes of
body proteins are duplicated and modified to have differential and specific expression in
venom glands. This phenomenon is broadly termed as recruitment. This recruitment
process of body proteins has not only been observed in snakes but also in various other
venomous animals, such as cone snails, spiders, scorpions and sea anemones as well as
hematophagous animals (Fry et al. 2009). Although this overarching concept existed in the
field of snake venom toxins for decades, there is not much direct molecular evidence for this
process of recruitment.
Our laboratory has extensively characterized prothrombin activators from Australian elapid
snake venoms and documented their structural and functional similarity with mammalian
plasma coagulation factors. Through systematic, detailed studies, we provided the
molecular details of the recruitment of venom prothrombin activators from plasma
coagulation factors after gene duplication. We also identified several key structural changes
that make these prothrombin activators better toxins. In this chapter, we will describe the
first molecular evidence for the recruitment process and the evolution of prothrombin
activators in venoms of Australian elapid snakes.

2. Snake venom prothrombin activators
As mentioned above, the circulatory system is one of the main targets of snake venom
toxins. These toxins affect heart function (e.g., cardiotoxins), vasculature and blood pressure
(e.g., sarafotoxins, natriuretic peptides and hemorrhagic toxins), blood coagulation (e.g.,
procoagulant and anticoagulant proteins), platelet aggregation (e.g., agonists and
antagonists) and fibrinolysis (e.g., direct and indirect fibrinolytic proteinases) (Braud et al.
2000; Chow and Kini 2001; Hutton and Warrell 1993; Kini 2004; Kini and Chow 2001; Kini
and Evans 1990; Markland 1997; Markland 1998; Morita 2004). All procoagulant proteins are
generally proteinases that promote blood coagulation by activating zymogens of specific
plasma coagulation factors (Davie 2003). Prothrombin activators are a group of
procoagulant proteins that specifically activate prothrombin to thrombin, which then
induces blood coagulation through fibrin clot formation.
Several prothrombin activators have been identified and characterized from snake venoms
(Gao et al. 2002; Hasson et al. 2003; Joseph and Kini 2001; Kornalik and Blomback 1975;
Morita and Iwanaga 1978; Rosing and Tans 1991; Rosing and Tans 1992; Schieck et al. 1972;
Silva et al. 2003; Speijer et al. 1986; St Pierre et al. 2005; Yamada and Morita 1997), and based
on their properties (structure, cofactor requirements and end-products formed), these snake
venom prothrombin activators have been classified into four groups (Kini et al. 2001) (Table
1). Group A and B prothrombin activators, such as ecarin from Echis carinatus venom
(Kornalik and Blomback 1975; Morita and Iwanaga 1978; Nishida et al. 1995; Schieck et al.
1972) and multactivase from E. multisquamatus venom (Yamada and Morita 1997), are
metalloproteinases which induce coagulation by converting prothrombin to meizothrombin.
These proteins are structurally distinct from blood coagulation factors. Group C and D
prothrombin activators, such as oscutarin from Oxyranus scutellatus venom (Owen and
Jackson 1973; Speijer et al. 1986; Walker et al. 1980; Welton and Burnell 2005) and notecarin
from Notechis scutatus scutatus venom (Tans et al. 1985), are serine proteinases that induce
coagulation by converting prothrombin to thrombin. These proteins exhibit functional
similarity to mammalian plasma coagulation factors. However, no detailed structural
information of these proteins was available. To fill this void, we initiated structural studies
of these prothrombin activators. We characterized one representative each of group C
(pseutarin C) and group D (trocarin D) prothrombin activators. Our results show that snake
venom prothrombin activators are structurally and functionally similar to mammalian
plasma coagulation factors.

Group
Cofactor
Requirements
Type of
proteinase
Product
formed
Estimated Size
Similar to Plasma
Coagulation
Factors
Examples
A None
Metalloproteinase Meizothrombin
~47 kDa
None
Ecarin
B Ca
2+

Two subunits of
~25 and ~60 kDa
Carinactivase,
multactivase
C
Ca
2+
plus
phospholipids
Serine proteinase Thrombin
Two subunits of
~60 and ~220
kDa
FXa-FVa
complex
Pseutarin C
oscutarin,
D
Ca
2+
plus
phospholipids
plus factor Va
~60 kDa FXa
Trocarin D,
hopsarin D,
notanarin D
Table 1. Classification of snake venom prothrombin activators

Duplication of Coagulation Factor Genes and Evolution of Snake Venom Prothrombin Activators 259
2.1 Group D prothrombin activators
Group D prothrombin activators are found exclusively in the venom of Australian elapid
snakes (Rosing and Tans 1991). Notecarin from Notechis scutatus scutatus venom was the first
member of this group to be isolated and characterized (Tans et al. 1985). Since then, similar
prothrombin activators have been characterized from several other snake venoms. They are
glycoproteins with a molecular weight of ~50 kDa (Table 1). As a group of proteins, they
share striking resemblances and requirements for optimal activity with activated
mammalian plasma coagulation factor X (FXa) (Table 1) (Joseph et al. 1999; Marsh et al.
1997; Rao and Kini 2002; Stocker et al. 1994; Tans et al. 1985).
Venom of the Australian elapid Tropidechis carinatus (rough-scaled snake) was documented
to have procoagulant properties 29 years ago (Chester and Crawford 1982). A prothrombin
activator was isolated using gel filtration and benzamidine-based affinity chromatography
and was partially characterized (Marsh et al. 1997). Our laboratory purified a prothrombin
activator, trocarin D, from T. carinatus venom to homogeneity using a series of high
performance liquid chromatography techniques including gel filtration, ion-exchange and
reverse-phase chromatographies (Joseph et al. 1999). This purification procedure was
refined to a single-step reverse-phase chromatographic method and was subsequently used
for the purification of several other group D prothrombin activators such as notanarin D
from N. ater niger venom, notecarin D from N. scutatus venom and hopsarin D from
Hoplocephalus stephensi venom (Rao et al. 2003a). Our laboratory characterized trocarin D for
its functional and structural properties in detail as a representative of group D prothrombin
activators.
Functionally, trocarin D has properties which are similar to mammalian plasma coagulation
FXa. They both promote blood coagulation by activating prothrombin to thrombin (Joseph
et al. 1999). Trocarin D and FXa achieve activation of prothrombin by cleaving the same
peptide bonds (Arg
274
-Thr
275
and Arg
323
-Ile
324
). Both proteins have identical co-factor
requirements of Ca
2+
ions, phospholipids and activated factor V (FVa) for their optimal
activities (Joseph et al. 1999). We determined the amino acid sequence of trocarin D and its
precursor using Edman degradation (Joseph et al. 1999) and cDNA sequencing (Reza et al.
2005a), respectively. Trocarin D and mammalian FXa share significant sequence identity
(~53-60%) and exhibit identical domain architecture (Joseph et al. 1999; Rao et al. 2003a)
(Figure 1). Both proteins comprise two chains: a heavy chain, which has a serine proteinase
with the characteristic catalytic triad (His
42
, Asp
88
and Ser
185
), and a light chain, which has a
Gla domain followed by two epidermal-growth factor-like domains (EGF-I and EGF-II).
These two chains are held together by a single inter-chain disulfide bond (Joseph et al. 1999)
(Figure 1). The differences between trocarin D and mammalian FXa reside in an insertion in
the heavy chain, the size of the activation peptide and post-translational modifications.
Firstly, there is a 12-residue insert in the heavy chain of trocarin D (Reza et al. 2005a).
However, the functional importance of this insertion is not clear. Secondly, the activation
peptide of trocarin D precursor is only 27 residues long (Reza et al. 2005a) compared to the
activation peptides of mammalian FXs which ranges from 48 to 52 residues (Figure 1).
Lastly, post-translational modifications show that trocarin D is glycosylated, but
mammalian FXa is not. In addition, trocarin D also contains a O-linked carbohydrate at Ser
52

of the light chain and a N-linked carbohydrate at Asn
45
of the heavy chain (Joseph et al.
1999) (Figure 1). Interestingly, the O-linked carbohydrate moiety has a N-acetylglucosamine
moiety, which is found commonly in nuclear and cytoplasmic proteins but rarely in secreted
proteins (Hanover et al. 1987; Holt et al. 1987; Holt and Hart 1986; Snow et al. 1987). The

function of glycosylation is hypothesized to protect trocarin D from inactivation by
proteolysis and confer it with thermal stability (Rao et al. 2003a; Wang et al. 1996). Although
human FX contains two O-glycosylation and two N-glycosylation moieties (Inoue and
Morita 1993), all the carbohydrate moieties are exclusively found on the activation peptide,
which is removed when FX is activated. Therefore, activated mammalian FXa is not
glycosylated. In contrast, the short activation peptide of trocarin D does not have any
glycosylation sites. The last difference in post-translational modification is that the Asp
63
of
FX light chain EGF-I domain is -hydroxylated (McMullen et al. 1983; Stenflo et al. 1987),
but the corresponding residue in trocarin D is not (Joseph et al. 1999) (Figure 1).

Fig. 1. Domain architecture of mammalian FX, and precursors of trocarin D and PCCS. The
Ys represent Gla residues; open triangles represent the O-linked carbohydrate at Ser
52
on
trocarin D and PCCS; solid triangles represent the N-linked carbohydrate at Asn
45
on
trocarin D and PCCS; the solid diamond represents -hydroxylation at Asp
62
on mammalian
FXa.
2.2 Group C prothrombin activators
The group C snake venom prothrombin activators are also found exclusively in the venoms
of Australian elapid snakes (Rosing and Tans 1991). Oscutarin from Oxyuranus scutellatus
venom was the first member of the group C prothrombin activators to be isolated and
characterized (Owen and Jackson 1973; Speijer et al. 1986; Walker et al. 1980; Welton and
Burnell 2005). This group of proteins is generally ~300 kDa in size and comprises two
subunits (~60 and ~220 kDa) (Table 1). The smaller enzymatic subunit is a serine proteinase
and has characteristics of FXa, whereas the nonenzymatic subunit resembles activated
mammalian plasma coagulation factor V (FVa). Overall, group C prothrombin activators
have striking resemblances to and similar co-factor requirements as the mammalian plasma
coagulant FXa-FVa complex (Filippovich et al. 2005; Masci et al. 1988; Rao and Kini 2002;
Speijer et al. 1986; Walker et al. 1980) (Table 1).

Pseutarin C was purified from P. textilis venom, and it activates prothrombin to thrombin.
For its optimal activity, pseutarin C requires only Ca
2+
ions and phospholipids (Rao and
Kini 2002). These functional characteristics are similar to that of the mammalian FXa-FVa
complex. As with other group C prothrombin activators, pseutarin C comprises two
subunits of ~60 kDa and ~220 kDa (Rao and Kini 2002) (Table 1). The smaller subunit, with
serine proteinase activity, was termed the pseutarin C catalytic subunit (PCCS) and the
larger subunit, which has no enzymatic activity, was termed the pseutarin C nonenzymatic
subunit (PCNS) (Rao and Kini 2002). A comparison of the protein quantities in the venom
and plasma revealed that pseutarin C is expressed ~4,200 times higher in the venom than
the amount of FV and FX in the plasma (Rao and Kini 2002). We purified pseutarin C and its
subunits and characterized them both functionally and structurally as representatives of the
group C prothrombin activators.
1. Pseutarin C catalytic subunit (PCCS) Functionally, PCCS is similar to mammalian FXa and
group D prothrombin activators (Rao and Kini 2002). They have the same co-factor
requirements, including Ca
2+
ions, phospholipids and FVa, for their optimal activity and
activate prothrombin by cleaving the same two peptide bonds (Arg
274
-Thr
275
and Arg
323
-
Ile
324
). The enzymatic activity (Vmax) of PCCS is enhanced by the presence of FVa (Rao and
Kini 2002). The amino acid sequence of PCCS and its precursor was determined using both
Edman degradation and cDNA sequencing (Rao et al. 2004; Rao and Kini 2002). Structurally,
PCCS is also similar to mammalian FXa and group D prothrombin activators (Figure 1). Its
sequence shows ~42% identity to mammalian FXa and 74-83% identity to group D
prothrombin activators (Rao et al. 2004). Like mammalian FXa and group D prothrombin
activators (Rao et al. 2004), the domain architecture of PCCS consists of a light and a heavy
chain that are linked by a single disulfide bond (Rao et al. 2004). The light chain has a Gla
domain followed by two EGF-like domains, and the heavy chain contains a serine
proteinase domain (Figure 1). Despite such functional and structural similarities, the
differences between PCCS and mammalian FXa reside in the size of the activation peptide
and post-translational modifications (Rao et al. 2004). Similar to trocarin D precursor, the
activation peptide of PCCS precursor is 27 residues long and is significantly shorter than
those of mammalian FXs (Figure 1). Like trocarin D, it has an insertion in its heavy chain.
Interestingly, the PCCS insert is 13 residues long and is distinctly different from the 12-
residue insert in trocarin D (Rao et al. 2004; Reza et al. 2005b). This strongly indicates that
the evolution of groups C and D prothrombin activators are independent. Like trocarin D,
the Ser
52
and Asn
45
residues of the light and heavy chains of PCCS are O- and N-
glycosylated, respectively (Figure 1). However, as mentioned previously, these two residues
have no post-translational modifications in mammalian FX/FXa. These functional and
structural characteristics suggest that PCCS is a homologue of mammalian FXa and group D
prothrombin activators.
2. Pseutarin C nonenzymatic subunit (PCNS) Structurally, PCNS is similar to mammalian FV.
The amino acid sequence of PCNS and its precursor was determined using Edman
degradation and cDNA sequencing (Rao et al. 2003b; Rao and Kini 2002). PCNS shares ~50%
identity with the mammalian FV and has identical domain architecture with mammalian FV
(Rao et al. 2003b). Both PCNS and mammalian FV have six domains: A1, A2, B, A3, C1 and
C2 (Figure 2). Domains A and C are functionally important, and these domains are highly
conserved in PCNS and FVs of other species (Rao et al. 2003b). These structural similarities
suggest that PCNS is a homologue of mammalian FV (Rao et al. 2003b).


Fig. 2. Domain architecture of bovine FV, PCNS precursor and PFV. N-glycosylation sites
are shown as colored knobs. Black knobs are conserved, while red knobs are found only in
PCNS and PFV, and blue knobs are found only in PFV.
Despite being a FV homologue, PCNS shows several differences with FV from other species.
Firstly, the domain B size of PCNS is significantly smaller (127 residues) than that of fishes
(fugu: 530 residues and zebrafish: 756 residues) and mammals (murine: 843 residues,
bovine: 869 residues, and human: 882 residues) (Rao et al. 2003b) (Figure 2). During FV
activation, domain B is removed by thrombin or FXa by cleavage at three activation sites:
Arg
709
, Arg
1018
and Arg
1545
(bovine FV numbering) (Foster et al. 1983; Nesheim et al. 1979;
Suzuki et al. 1982) (Figure 3B). Although only two of these sites (Arg709 and Arg1545) are
conserved in PCNS (Figure 3B), the complete domain B can still be cleaved off during the
activation of PCNS (Rao et al. 2003b). Thus, the difference in domain B size should not have
any effect on the function of PCNS. Secondly, PCNS and FV of other species have different
post-translation modifications. While bovine FV has 29 glycosylation sites, PCNS has only
11 potential N-glycosylation sites (Rao et al. 2003b) (Figure 2). Mammalian FV is
phosphorylated at the Ser692 residue, but PCNS is not (Rao et al. 2003b). In addition, there
are six sulfation sites in human FV that are absent in PCNS (Rao et al. 2003b). This difference
in post-translation modifications is interesting, as sulfation and phosphorylation are
important for regulating the activation of human FV by thrombin (Kalafatis et al. 1994;
Pittman et al. 1994).
Aside from these differences, it is noted that PCNS possesses certain modifications and
associations that allow it to function efficiently as a toxin (Bos et al. 2009). Firstly, PCNS has
evolved a way to evade inactivation by protein C. In the human coagulation system, protein
C is activated by thrombin in the presence of thrombomodulin, and activated protein C
(APC) subsequently inactivates FVa in a negative feedback loop (Esmon 2001). This
inactivation occurs by proteolytic cleavage at three sites on the FVa heavy chain: Arg
306
,
Arg
506
and Arg
662
(Kalafatis et al. 1994; Mann et al. 1997) in bovine FVa (Figure 3B). PCNS is
able to evade APC inactivation, as it does not have any of the three APC cleavage sites (Rao
et al. 2003b). Even an alternate less efficient cleavage site at Arg
316
(van der Neut et al. 2004b)

is not conserved in PCNS (Rao et al. 2003b). Secondly, FVa is inactivated by phosphorylation
at Ser
692
(Kalafatis et al. 1994). This phosphorylation site is not present in PCNS (Rao et al.
2003b). Lastly, PCNS is shielded from APC inactivation (Nesheim et al. 1982; Rao et al.
2003b) and is kept activated through its constant and stable association with FXa-like PCCS
(Rao et al. 2003a; Rao et al. 2004; Thorelli et al. 1998) in the venom. We have shown
experimentally that pseutarin C is unaffected by APC, while bovine FXa-FVa complex is
completely inactivated (Bos et al. 2009; Rao et al. 2003b) (Figure 4). Overall, PCNS is a good
example of how a toxin gene is duplicated from an ancestral gene and undergoes
modifications to gain unique characteristics that allow it to function efficiently as a toxin.

Fig. 3. Functionally important proteolytic sites in PCNS and FV. (A) Activation sites.
Thrombin and FXa cleavage sites of PCNS, PFV and bovine FV are shown in blue, green and
red, respectively. Critical activation sites are conserved in PCNS and PFV. (B) Inactivation
sites. Activated protein C (APC) cleavage sites of PFV and bovine FV are shown in green
and red, respectively. In addition to Arg
505
, PFV has a primitive inactive site at Arg
316
shown
in purple arrow. Inactivation sites are missing in PCNS.


Fig. 4. APC resistance assay (Rao et al. 2003b). Varying concentrations of APC were added
either to pseutarin C (E; 8 nM) or bovine FXa-FVa (F; FXa 42 nM, FVa 2 nM) complex which
was diluted in 50 mM Tris-HCl buffer (pH 7.5) containing 100 mM NaCl, 5 mM CaCl2, and
0.5 mg/mL BSA. The reaction mixture was incubated for 30 minutes at room temperature.
Prothrombin was added to a final concentration of 2.8 M and thrombin formed was
assayed using thrombin-specific chromogenic substrate S-2238. Each point represents an
average of 2 independent experiments each carried out in triplicates.
3. Parallel prothrombin activator system in Australian elapid snakes
As described above, groups C and D snake venom prothrombin activators are functional
and structural homologues of mammalian blood coagulation factors. As snakes are
vertebrates, their hemostatic system should contain plasma coagulation factors. Thus,
Australian elapid snakes should possess parallel prothrombin activating systems: one in
their venom, which is used as an offensive weapon to attack the hemostatic system of the
prey, and the other in their plasma, which is used for their own hemostatic purpose. We
examined the presence of plasma coagulation factors in the snakes hemostatic system and
determined the relationship between the snake venom and plasma coagulation factors.
3.1 Trocarin D and FX from Tropidechis carinatus (TrFX)
Since the liver mainly expresses plasma coagulation factors, the cDNA encoding T. carinatus
FX (TrFX) was sequenced from liver tissue (Reza et al. 2005a). The deduced amino acid
sequence of TrFX is similar to mammalian FX (~50%) and trocarin D (~80%) (Figure 5).
Structurally, TrFX is similar to trocarin D. They both have conserved cysteine residues and
identical domain architecture. However, there are some differences between TrFX and
trocarin D. The activation peptide of TrFX is similar to the mammalian FXs and not to that of
0
20
40
60
80
100
120
0 100 200 300 400 500
F
V
a

A
c
t
i
v
i
t
y

(
%
)
nM APC
BFXa-FVa
Pseutarin C

the venom prothrombin activators (Reza et al. 2005b). It is 57 residues long compared to 27
residues in trocarin D (Figure 5). In addition, there is no 12-residue insert in the heavy chain
of TrFX as was observed to be present in the trocarin D precursor (Reza et al. 2005b). These
differences in amino acid sequences, and the lengths of activation peptides and insertion in
the heavy chain, suggest that TrFX and trocarin D are encoded by two independent genes.
Hence, this confirms the presence of a parallel prothrombin activator system. TrFX is more
similar to trocarin than to mammalian FX in terms of post-translational modifications (Reza
et al. 2005a). TrFX and trocarin D both have N- and O-glycosylation modifications that are
not found in mammalian FXs (as described previously).

Fig. 5. Alignment of deduced amino acid sequences of FX-like proteins from T. carinatus
(TrFX and trocarin D) and P. textilis (PCCS, PFX1 and PFX2) snakes.
Trocarin D and TrFX differ in their physiological roles. Trocarin D plays an offensive role as
a toxin in the venom that is used for killing prey. Upon envenomation, like other
prothrombin activators (Masci et al. 1988; Rao et al. 2003a), it induces cyanation and death in
experimental animals (Joseph et al. 1999) through disseminated intravascular coagulopathy.
On the other hand, TrFX plays a crucial role in the coagulation cascade and prevents
excessive blood loss by promoting blood coagulation when there is a vascular injury.
Trocarin D is an active enzyme and is found in large quantities in the venom. In contrast,
TrFX is found as a zymogen, which gets activated only when required and is found in much
smaller concentrations in the plasma. Real-time polymerase chain reaction (RT-PCR) was
used to determine the amount of expression of these two closely related proteins in the liver
and venom gland. The results indicate that trocarin D is expressed in the venom gland but
90
90 iver_type_1
90 iver_type_2
90 Cat
90
MAPQLLLCLILTFLWSLSEAESNVFLKSKVANRFLQRTKRANSLFEEFKAGNIERECIEERCSKEEAREAFEDNEKTETFWNVYVDGDQC
MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRANSLFEEFKSGNIERECIEERCSKEEAREAFEDDEKTETFWNVYVDGDQC
MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRANSLVEEFKSGNIERECIEERCSKEEAREAFEDDEKTETFWNVYVDGDQC
MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRANSLVEEFKSGNIERECIEERCSKEEAREAFEDDEKTETFWNVYVDGDQC
MAPQLLLCLILTFLWSLPEAESNVFLKSKVANRFLQRTKRSNSLFEEIRPGNIERECIEEKCSKEEAREVFEDNEKTETFWNVYVDGDQC
M
M
M
M
M
A
A
A
A
A
P
P
P
P
P
Q
Q
Q
Q
Q
L
L
L
L
L
L
L
L
L
L
L
L
L
L
L
C
C
C
C
C
L
L
L
L
L
I
I
I
I
I
L
L
L
L
L
T
T
T
T
T
F
F
F
F
F
L
L
L
L
L
W
W
W
W
W
S
S
S
S
S
L
L
L
L
L
P
P
P
P
E
E
E
E
E
A
A
A
A
A
E
E
E
E
E
S
S
S
S
S
N
N
N
N
N
V
V
V
V
V
F
F
F
F
F
L
L
L
L
L
K
K
K
K
K
S
S
S
S
S
K
K
K
K
K
V
V
V
V
V
A
A
A
A
A
N
N
N
N
N
R
R
R
R
R
F
F
F
F
F
L
L
L
L
L
Q
Q
Q
Q
Q
R
R
R
R
R
T
T
T
T
T
K
K
K
K
K
R
R
R
R
R
A
A
A
A
N
N
N
N
N
S
S
S
S
S
L
L
L
L
L
F
F
F
E
E
E
E
E
E
E
E
E
E
F
F
F
F
I
K
K
K
K
R
S
S
S
G
G
G
G
G
N
N
N
N
N
I
I
I
I
I
E
E
E
E
E
R
R
R
R
R
E
E
E
E
E
C
C
C
C
C
I
I
I
I
I
E
E
E
E
E
E
E
E
E
E
R
R
R
R
K
C
C
C
C
C
S
S
S
S
S
K
K
K
K
K
E
E
E
E
E
E
E
E
E
E
A
A
A
A
A
R
R
R
R
R
E
E
E
E
E
A
A
A
A
F
F
F
F
F
E
E
E
E
E
D
D
D
D
D
N
D
D
D
N
E
E
E
E
E
K
K
K
K
K
T
T
T
T
T
E
E
E
E
E
T
T
T
T
T
F
F
F
F
F
W
W
W
W
W
N
N
N
N
N
V
V
V
V
V
Y
Y
Y
Y
Y
V
V
V
V
V
D
D
D
D
D
G
G
G
G
G
D
D
D
D
D
Q
Q
Q
Q
Q
C
C
C
C
C
180
180 iver_type_1
180 iver_type_2
180 Cat
180
SSNPCHYGGTCKDGIGSYTCTCLAGYEGKNCQYVLYQSCRVDNGNCWHFCKPVQNEIQCSCAESYLLGDDGYSCVAGGDFSCGRNIKARN
SSNPCHYGGTCKDGIGSYTCTCLSGYEGKNCEYVLYKSCRVDNGDCWHFCKPVQNGIQCSCAESYLLGEDGHSCVAGGDFSCGRNIKTRN
SSNPCHYRGICKDGIGSYTCTCLSGYEGKNCERVLYKSCRVDNGNCWHFCKHVQNDIQCSCAEGYLLGEDGHSCVAGGNFSCGRNIKTRN
SSNPCHYRGICKDGIGSYTCTCWSGYEGKNCERVLYKSCRVDNGNCWHFCKSVQNDIQCSCAEGYLLGEDGHSCVAGGNFSCGRNIKTRN
SSNPCHYRGTCKDGIGSYTCTCLPNYEGKNCEKVLYQSCRVDNGNCWHFCKRVQSETQCSCAESYRLGVDGHSCVAEGDFSCGRNIKARN
S
S
S
S
S
S
S
S
S
S
N
N
N
N
N
P
P
P
P
P
C
C
C
C
C
H
H
H
H
H
Y
Y
Y
Y
Y
R
R
R
G
G
G
G
G
T
T
T
C
C
C
C
C
K
K
K
K
K
D
D
D
D
D
G
G
G
G
G
I
I
I
I
I
G
G
G
G
G
S
S
S
S
S
Y
Y
Y
Y
Y
T
T
T
T
T
C
C
C
C
C
T
T
T
T
T
C
C
C
C
C
L
L
L
W
L
S
S
S
G
G
G
G
Y
Y
Y
Y
Y
E
E
E
E
E
G
G
G
G
G
K
K
K
K
K
N
N
N
N
N
C
C
C
C
C
Q
E
E
E
E
V
V
V
V
V
L
L
L
L
L
Y
Y
Y
Y
Y
K
K
K
S
S
S
S
S
C
C
C
C
C
R
R
R
R
R
V
V
V
V
V
D
D
D
D
D
N
N
N
N
N
G
G
G
G
G
N
D
N
N
N
C
C
C
C
C
W
W
W
W
W
H
H
H
H
H
F
F
F
F
F
C
C
C
C
C
K
K
K
K
K
V
V
V
V
V
Q
Q
Q
Q
Q
N
N
N
N
I
I
I
I
Q
Q
Q
Q
Q
C
C
C
C
C
S
S
S
S
S
C
C
C
C
C
A
A
A
A
A
E
E
E
E
E
S
S
G
G
S
Y
Y
Y
Y
Y
L
L
L
L
L
L
L
L
L
G
G
G
G
G
D
E
E
E
D
D
D
D
D
G
G
G
G
G
H
H
H
H
S
S
S
S
S
C
C
C
C
C
V
V
V
V
V
A
A
A
A
A
G
G
G
G
E
G
G
G
G
G
D
D
N
N
D
F
F
F
F
F
S
S
S
S
S
C
C
C
C
C
G
G
G
G
G
R
R
R
R
R
N
N
N
N
N
I
I
I
I
I
K
K
K
K
K
T
T
T
R
R
R
R
R
N
N
N
N
N
270
270 iver_type_1
241 iver_type_2
241 Cat
241
KREASLPDFQTDFSDDYDAIDENNFVETPTNFSGLVPTVQSQNATLLKKSDNPSPDIRVVNGTDCKLGECPWQALLINDQGDGFCGGTIL
KREANLPDFQTDFSDDYDEIDENNFVETPTNFSGLVLTVQSQNATLLKKSDNPSPDIRVVNGTDCKLGECPWQALLLNDEGDGFCGGTIL
KREANLPDF.............................VQSQNATLLKKSDNPSPDIRIVNGMDCKLGECPWQAALVDEKEGVFCGGTIL
KREASLPDF.............................VQSQNAPLLKISDNPSPDIRIVNGMDCKLGECPWQAALVDDKKGVFCGGTIL
KREASLPDF.............................VQSQKATLLKKSDNPSPDIRIVNGMDCKLGECPWQAVLINEKGEVFCGGTIL
K
K
K
K
K
R
R
R
R
R
E
E
E
E
E
A
A
A
A
A
S
S
S
L
L
L
L
L
P
P
P
P
P
D
D
D
D
D
F
F
F
F
F
V
V
V
V
V
Q
Q
Q
Q
Q
S
S
S
S
S
Q
Q
Q
Q
Q
N
N
N
N
A
A
A
A
A
T
T
T
T
L
L
L
L
L
L
L
L
L
L
K
K
K
K
K
K
K
K
K
S
S
S
S
S
D
D
D
D
D
N
N
N
N
N
P
P
P
P
P
S
S
S
S
S
P
P
P
P
P
D
D
D
D
D
I
I
I
I
I
R
R
R
R
R
V
V
I
I
I
V
V
V
V
V
N
N
N
N
N
G
G
G
G
G
M
M
M
D
D
D
D
D
C
C
C
C
C
K
K
K
K
K
L
L
L
L
L
G
G
G
G
G
E
E
E
E
E
C
C
C
C
C
P
P
P
P
P
W
W
W
W
W
Q
Q
Q
Q
Q
A
A
A
A
A
L
L
L
L
L
N
N
D
D
N
D
D
E
D
E
K
K
K
G
G
E
G
V
V
V
F
F
F
F
F
C
C
C
C
C
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
I
I
I
I
I
L
L
L
L
L
347
347 iver_type_1
327 iver_type_2
331 Cat
330
SPIYVLTAAHCINQTKYIRVVVGEIDISRKKTGRLLSVDKIYVHQKFVP.............STYDYDIALIQMKTPIQFSENVVPACLP
SPIYVLTAAHCINQTKYITVVVGEIDISSKKTGRLHSVDKIYVHQKFVP.............ATYDYDIAIIQLKTPIQFSENVVPACLP
SPIYVLTAAHCINETETISVVVGEIDKSRIETGPLLSVDKIYVHKKFVPPQKAY....KFDLAAYDYDIAIIQMKTPIQFSENVVPACLP
SPIYVLTAAHCINETETISVVVGEIDRSRAETGPLLSVDKVYVHKKFVPPKKSQEFYEKFDLVSYDYDIAIIQMKTPIQFSENVVPACLP
SPIHVLTAAHCINQTKSVSVIVGEIDISRKETRRLLSVDKIYVHTKFVPPN.YYYVHQNFDRVAYDYDIAIIRMKTPIQFSENVVPACLP
S
S
S
S
S
P
P
P
P
P
I
I
I
I
I
Y
Y
Y
Y
V
V
V
V
V
L
L
L
L
L
T
T
T
T
T
A
A
A
A
A
A
A
A
A
A
H
H
H
H
H
C
C
C
C
C
I
I
I
I
I
N
N
N
N
N
Q
Q
E
E
Q
T
T
T
T
T
K
K
K
I
I
I
I
V
S
S
S
V
V
V
V
V
V
V
V
V
I
V
V
V
V
V
G
G
G
G
G
E
E
E
E
E
I
I
I
I
I
D
D
D
D
D
I
I
I
S
S
S
S
S
R
R
R
R
K
K
K
E
E
E
T
T
T
T
T
G
G
G
G
R
R
R
L
L
L
L
L
L
L
L
L
S
S
S
S
S
V
V
V
V
V
D
D
D
D
D
K
K
K
K
K
I
I
I
V
I
Y
Y
Y
Y
Y
V
V
V
V
V
H
H
H
H
H
K
K
K
K
K
F
F
F
F
F
V
V
V
V
V
P
P
P
P
P
P
P
P
F
F
F
D
D
D
Y
Y
Y
Y
Y
D
D
D
D
D
Y
Y
Y
Y
Y
D
D
D
D
D
I
I
I
I
I
A
A
A
A
A
L
I
I
I
I
I
I
I
I
I
Q
Q
Q
Q
M
L
M
M
M
K
K
K
K
K
T
T
T
T
T
P
P
P
P
P
I
I
I
I
I
Q
Q
Q
Q
Q
F
F
F
F
F
S
S
S
S
S
E
E
E
E
E
N
N
N
N
N
V
V
V
V
V
V
V
V
V
V
P
P
P
P
P
A
A
A
A
A
C
C
C
C
C
L
L
L
L
L
P
P
P
P
P
437
437 iver_type_1
417 iver_type_2
421 Cat
420
TADFANQVLMKQDFGIVSGFGRTRERGQTSNTLKVVTLPYVDRHTCMLSSNFPITQNMFCAGYNTLPQDACQGDSGGPHITAYRDTHFIT
TADFANQVLMKQNFGIVSGFGRTRERGKTSNTLKVVTLPYVDRHTCMLSSNFPITQNMFCAGYDTLPQDACQGDSGGPHITAYRDTHFIT
TADFANQVLMKQDFGIVSGFGRIFEKGPKSKTLKVLKVPYVDRHTCMVSSETPITPNMFCAGYDTLPRDACQGDSGGPHTTVYRDTHFIT
TADFANQVLMKQDFGIVSGFGGIFGRGPNSKTLKVLKVPYVDRHTCMLSSNFPITPTMFCAGYDTLPQDACQGDSGGPHITAYRDTHFIT
TADFANEVLMKQDSGIVSGFGRIQFKQPTSNTLKVITVPYVDRHTCMLSSDFRITQNMFCAGYDTLPQDACQGDSGGPHITAYRDTHFIT
T
T
T
T
T
A
A
A
A
A
D
D
D
D
D
F
F
F
F
F
A
A
A
A
A
N
N
N
N
N
Q
Q
Q
Q
E
V
V
V
V
V
L
L
L
L
L
M
M
M
M
M
K
K
K
K
K
Q
Q
Q
Q
Q
D
N
D
D
D
F
F
F
F
G
G
G
G
G
I
I
I
I
I
V
V
V
V
V
S
S
S
S
S
G
G
G
G
G
F
F
F
F
F
G
G
G
G
G
R
R
R
R
I
I
I
E
E
E
G
R
R
K
R
K
G
G
G
G
P
P
P
T
T
T
S
S
S
S
S
N
N
N
T
T
T
T
T
L
L
L
L
L
K
K
K
K
K
V
V
V
V
V
T
T
T
L
L
V
V
V
P
P
P
P
P
Y
Y
Y
Y
Y
V
V
V
V
V
D
D
D
D
D
R
R
R
R
R
H
H
H
H
H
T
T
T
T
T
C
C
C
C
C
M
M
M
M
M
L
L
V
L
L
S
S
S
S
S
S
S
S
S
S
N
N
E
N
D
F
F
F
F
P
P
P
P
I
I
I
I
I
T
T
T
T
T
Q
Q
Q
N
N
N
N
M
M
M
M
M
F
F
F
F
F
C
C
C
C
C
A
A
A
A
A
G
G
G
G
G
Y
Y
Y
Y
Y
N
D
D
D
D
T
T
T
T
T
L
L
L
L
L
P
P
P
P
P
Q
Q
Q
Q
D
D
D
D
D
A
A
A
A
A
C
C
C
C
C
Q
Q
Q
Q
Q
G
G
G
G
G
D
D
D
D
D
S
S
S
S
S
G
G
G
G
G
G
G
G
G
G
P
P
P
P
P
H
H
H
H
H
I
I
I
I
T
T
T
T
T
A
A
A
A
Y
Y
Y
Y
Y
R
R
R
R
R
D
D
D
D
D
T
T
T
T
T
H
H
H
H
H
F
F
F
F
F
I
I
I
I
I
T
T
T
T
T
483
483 iver_type_1
463 iver_type_2
449 Cat
455
GIISWGEGCAQTGKYGAYTKVSRFILWIKRIMRLKLPSTESSTGRL...
GIVSWGEGCAQTGKYGVYTKVSKFILWIKRIIRQKQPSTESSTGRL...
GIVSSGEGCARNGKYGIYTKLSKFIPWIKRIMRQKLPSTESSTGRL...
GIVSWGEGCARKGRYGIYTKLSKFIPWI.....................
GIISWGEGCARKGKYGVYTKVSKFIPWIKKIMSLK..............
G
G
G
G
G
I
I
I
I
I
I
V
V
V
I
S
S
S
S
S
W
W
W
W
G
G
G
G
G
E
E
E
E
E
G
G
G
G
G
C
C
C
C
C
A
A
A
A
A
R
R
R
G
G
G
G
G
K
K
K
R
K
Y
Y
Y
Y
Y
G
G
G
G
G
Y
Y
Y
Y
Y
T
T
T
T
T
K
K
K
K
K
V
V
L
L
V
S
S
S
S
S
R
K
K
K
K
F
F
F
F
F
I
I
I
I
I
P
P
P
W
W
W
W
W
I
I
I
I
I
K
K
K
K
R
R
R
K
I
I
I
I
M
I
M
M
R
R
R
K
K
K
K
P
P
P
S
S
S
T
T
T
E
E
E
S
S
S
S
S
S
T
T
T
G
G
G
R
R
R
L
L
L
TrFX
PFX1
PFX2
PCCS
Trocarin D
TrFX
PFX1
PFX2
PCCS
Trocarin D
TrFX
PFX1
PFX2
PCCS
Trocarin D
TrFX
PFX1
PFX2
PCCS
Trocarin D
TrFX
PFX1
PFX2
PCCS
Trocarin D
TrFX
PFX1
PFX2
PCCS
Trocarin D
Signal peptide
Activation peptide
Propeptide

not in the liver, while TrFX is expressed in the liver but not in the venom gland. Further, the
expression of trocarin D is ~30 times higher in the venom gland than TrFX in the liver (Reza
et al. 2007). Such differential expression patterns of trocarin D and TrFX strongly support
the distinct physiological roles of these two proteins.
3.2 PCCS and FX from Pseudonaja textilis (PFX)
To understand the evolution of group C prothrombin activators, we also determined the
cDNA sequence of the P. textilis FX (PFX) from the liver. Interestingly, two PFX isoforms
(PFX1 and PFX2) were detected in the liver, and their cDNA sequences are ~85% similar
(Reza et al. 2006). The domain architecture and cysteine residues of these two isoforms are
also conserved compared to group D prothrombin activators. Amino acid sequence
comparison shows that PFX1 is more similar to TrFX (~94%), while PFX2 is more similar
PCCS and trocarin D (~90%) (Figure 5). Further, PFX1 has a longer activation peptide,
similar to plasma FXs, whereas PFX2 has a shorter activation peptide, similar to PCCS and
trocarin D. Also, PFX2 has a 9-residue insert, which is not present in PFX1. These structural
differences suggest that PFX1, PFX2 and PCCS are encoded by three independent genes and
that PFX2 is an evolutionary intermediate between PFX1 and PCCS (Reza et al. 2006) (Figure
5). This similarly confirms the presence of a parallel prothrombin activator system. The
expression profiles of PFX1, PFX2 and PCCS were determined in liver and venom gland
tissues by RT-PCR (Reza et al. 2006). The results show that PFX1 and PFX2 are expressed only
in the liver, while PCCS is expressed only in the venom gland. PFX1 is also found to be
expressed ~55,000 times higher than PFX2 in the liver, and PCCS is expressed ~80 times higher
in the venom gland than is PFX1 in the liver (Reza et al. 2006). In summary, the sequence
comparisons and expression profiles indicate that PCCS has evolved from PFX1 by gene
duplication and PFX2 is an intermediary product of this recruitment process (Figure 6).

Fig. 6. Schematic diagram showing the probable evolutionary path in the recruitment of FX
protein as toxin in the venom (Reza et al. 2006).

3.3 PCNS and FV from Pseudonaja textilis (PFV)
The cDNA sequence of P. textilis FV (PFV) was determined from its liver (Minh et al. 2005).
The deduced amino acid sequence of PFV shows similarities to other mammalian and non-
mammalian FVs (~50%) and PCNS (~96%) and shares identical domain architecture (Minh
et al. 2005). Like the FVs of other species, PFV and PCNS comprise A1, A2, B, A3, C1 and C2
domains (Figure 3A). Functionally important domains A and C are highly conserved in both
PFV and PCNS, whereas domain B is the most variable (Minh et al. 2005). The domain B
(126 residues) of PFV is one residue shorter than that of PCNS (127 residues), and is much
shorter than that of mammalian and non-mammalian FVs. A more detailed comparison
shows that all the FXa and thrombin proteolytic cleavage sites (which are important for
activation of these nonenzymatic proteins) are conserved in PFV and PCNS (Figure 3A).
However, PFV has an additional FXa proteolytic cleavage site at Arg
1765
(Minh et al. 2005;
Rao et al. 2003b). This cleavage site also exists in mammalian FV but not in FVs of teleosts
(Minh et al. 2005). This is evolutionarily interesting as this additional cleavage site may be a
characteristic found only in tetrapod FVs. However, the functional implication of this
cleavage with regards to procoagulant activity of FV is not yet known.
As mentioned previously, PCNS has evolved to be resistant to inactivation by activated
protein C (APC), which is crucial to its function as a toxin. On the other hand, PFV is similar
to other FV, as it can still be inactivated by APC. PFV can be inactivated by APC by cleavage
at Arg
316
, a primitive inactive site (van der Neut et al. 2004a), and at Arg
506
(Minh et al. 2005)
(Figure 3B). The expression profiles of PFV and PCNS in the liver and the venom gland were
determined using RT-PCR. As with other venom prothrombin activator genes, PCNS is
expressed only in the venom gland, while PFV is expressed only in the liver. It was found
that PCNS is expressed ~280 times higher in the venom gland than is PFV in the liver (Minh
et al. 2005). Thus, PCNS and PFV are have differential expressions (Minh et al. 2005).
Based on sequence comparisons, we confirmed the presence of parallel prothrombin
activator systems in Australian elapid snakes and showed for the first time that groups C
and D prothrombin activators in snake venom and their plasma coagulation factor
counterparts are closely related. We also proposed that these venom prothrombin activators
evolved from their plasma coagulation factor counterparts by gene duplication and were
subsequently modified to function efficiently as toxins.
4. Phylogenetic relationship between snake venom and plasma prothrombin
activators
A phylogenetic tree of the snake venom and plasma prothrombin activators with other
known FX sequences was constructed to understand their evolutionary relationships using
zebrafish FX as the out group (Reza et al. 2006; St Pierre et al. 2005). All the reptilian
sequences form a monophyletic group (Reza et al. 2006) (Figure 7). Within the reptilian
clade, group C and D prothrombin activators appear as two separate clades on the tree. This
indicates that, despite their similarities, group C and D prothrombin activators have
originated independently. Interestingly, the PFX2 sequence is found nested within the
group C prothrombin activators. This supports the hypothesis that PFX2 is an evolutionary
intermediate of PCCS from PFX1. Based on the topology of the phylogenetic tree, it is
suggested that these snake venom prothrombin activators have been recruited through
independent evolutionary events (Reza et al. 2006) (Figure 7).


Fig. 7. Phylogenetic relationships of snake venom and plasma prothrombin activators with
other known FX sequences (Reza et al. 2006). vPA is an abbreviation for venom
prothrombin activators. Arrows indicate the three independent recruitment events of
snake venom prothrombin activators.
5. Comparison of trocarin D and TrFX genes
In the previous sections, we have described how the venom prothrombin activators have
been modified to gain certain characteristics, such as resistance to inactivation, which
enables them to function better as toxins. However, differential and tissue-specific
expression of venom prothrombin activators and their plasma coagulation factors is also
important for their respective physiological roles. The expression of toxins should be venom
gland-specific and inducible to higher levels. This is so the snake can protect itself against its
own venom toxins and replenish its venom supply quickly. Conversely, plasma coagulant
factors are mainly expressed in the liver at constituently low levels so that they can be
activated to induce blood coagulation during vascular injuries.
To understand how the venom prothrombin activators are regulated for tissue specificity
and level of expression, we determined the gene structure of trocarin D and TrFX. Based on
the cDNA sequences of trocarin D and TrFX, and that of mammalian FX gene, primers were
Human_FX
Rat_FX
Murine_FX
99
80
Rabbit_FX
81
Bovine_FX
100
Trocarin_D
Hoplocepha
69
Notechis_s
100
Pseudechis
85
PFX1
T._carinat
100
Oxyuranus_microlepid
Oxyuranus_scutellatu
PCCS
PFX2
95
79
99
87
100
Zebrafish_
0.05
Pseudonaja textilis PFX2
Tropidechis carinatus FX
Pseutarin C (PCCS)
Trocarin D
Oxyuranus microlepidotus vPA
Oxyuranus scutellatus vPA
Zebrafish FX
Pseudonaja textilis PFX1
Pseudechis porphyriacus vPA
Bovine FX
Hoplocephalus stephensii vPA
Notechis scutatus vPA
Murine FX
Rabbit FX
Human FX
Rat FX
95
79
99
100
85
87
100
100
100
69
81
99
80
Mammalian Factor X
Group D Prothrombin
Activators
Group C Prothrombin
Activators
Reptilian Factor X

designed, and the gene sequences were determined using genomic DNA PCR and genome
walking strategies (Reza et al. 2006). The gene organizations of trocarin D and TrFX are
identical (eight exons and seven introns). The intron sequences were highly similar to each
other with only differences in the promoter and intron 1 regions (Figure 8). Such similarities
strongly support our findings that there are two closely related, parallel prothrombin
activator systems, and that the venom prothrombin activators are recruited from plasma
coagulation factors through gene duplication. The duplicated FX gene was subsequently
modified and recruited for expression in the venom gland as a venom prothrombin
activator.

Fig. 8. Gene organization comparison of trocarin D and TrFX. Exons are shown as numbered
boxes. The differences in the promoter and intron 1 regions are indicated in yellow
(insertions in trocarin D gene) and green (deletions in TrFX gene) (Reza et al., 2007).
5.1 Cis-elements in trocarin D promoter region
The overlapping promoter regions of trocarin D and TrFX were characterized by
comparing them against previously characterized human (Hung et al. 2001; Hung and
High 1996) and murine (Wilberding and Castellino 2000) FX promoter regions (Reza et al.
2007) (Figure 9). Based on these comparisons, four conserved cis-regulatory elements in
the trocarin D and TrFX promoter regions were identified (Figure 9): (i) a CCAAT box
(Hung et al. 2001; Hung and High 1996; Wilberding and Castellino 2000), (ii) a gut-specific
transcription factor GATA-4 binding site (Hung et al. 2001), (iii) a liver-specific
transcription factor HNF-4 (Hung and High 1996), and (iv) multiple Sp1/Sp3 binding
sites (Hung et al. 2001).
Comparison of the trocarin D and TrFX promoter regions reveals that trocarin D has a 264
bp insertion (Figure 8 and 9). This 264 bp is located from -33 to -297 bp upstream of the
trocarin D start codon (ATG) (Figure 9). This insertion is postulated to play a major role in
the recruitment of the duplicated TrFX gene by causing it to be exclusively expressed in the
venom gland as the procoagulant toxin, trocarin D. Hence, it was termed Venom
Recruitment/Switch Element (VERSE). This segment was characterized for its cis-elements
and gene-regulatory role using luciferase assays in primary venom gland cells and
mammalian cell lines (Kwong et al. 2009). The VERSE promoter was found to be responsible
for the elevation of expression levels, but not tissue-specific expression, of trocarin D. In
terms of cis-element characterization, besides confirming the presence of two TATA-boxes,
one GATA box and one Y-box, three novel cis-elements were also identified (Figure 9).
Functionally, it is found that both TATA boxes (TLB2 and TLB3) are functional. However,
TLB2 is the primary TATA box which initiates and directs transcription start site (Kwong et
al. 2009) (Figure 9).
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Intron 1 Promoter
Trocarin D
TrFX


Fig. 9. Comparison of promoter regions in mammalian and T. carinatus prothrombin
activator genes (Reza et al., 2007).
5.2 Comparison of trocarin D and TrFX first introns
The intron 1 size of trocarin D is 7911 bp, while that of TrFX is 5293 bp. The difference in
size is explained by three insertions and two deletions in the trocarin D intron 1 region
(Reza et al. 2007) (Figure 10). Bioinformatics analysis of these insertion/deletion segments
reveals that they are novel. The three insertion segments within intron 1 of trocarin D
region are 214 bp, 1975 bp and 2174 bp in size with respective positions at 128 bp, 914 bp
and 3300 bp on the trocarin D gene (Figure 10). Upon closer analysis of the insertion
segment sequences, it is observed that the first insert within intron 1 of trocarin D is
almost an exact repeat (96.33% identity) of the intron 1 segment spanning from 3082 bp to
3299 bp. The other two inserts seem to be inverted repeats of each other (~71% identity).
The third insert shows potential of being a Scaffold/Matrix Attachment Region (S/MAR)
due to: (i) a high AT content (Cockerill and Garrard 1986; Liebich et al. 2002; Zhou and
Liu 2001), (ii) a topoisomerase II (Boulikas 1993), (iii) a S/MAR consensus motif (van
Drunen et al. 1997), (iv) a significant over-representation of characteristic hexanucleotides
(Liebich et al. 2002), and (v) an ATTA motif and an AT-rich region with H-box (Will et al.
1998). The two deletion segments within intron 1 of trocarin D are 255 bp and 1406 bp in
size with respective positions at 2610 bp and 3770 bp on the TrFX gene (Reza et al. 2007)
(Figure 10). As the VERSE promoter of trocarin D does not regulate tissue-specific
expression (Kwong et al. 2009), it is postulated that these insertions and deletions in the
Human FX
SP1/SP3 SP1/SP3
CCAAT
box
GATA-4
SP1/SP3
HNF-4
-177 -149 -121 -96 -65 -58
-50
CCAAT
box GATA-4 HNF-4
-106 -91
Murine FX
SP1/SP3
CCAAT
box GATA-4
HNF-4
SP1/SP3
-336 -154 -101 -59 -47
TrFX
SP1/SP3
CCAAT
box GATA-4
HNF-4
SP1/SP3
-522 -416 -366 -325 -312
Trocarin D
GATA-4
-221
TLB3
Y box
TLB2 TLB1
-176 -167 -141 -91
VERSE
264 bp
-297 -33

intron 1 of trocarin D could contain cis-elements that are responsible for the venom gland-
specific expression of trocarin D. In summary, the characterization of the VERSE
promoter and intron 1 regions of trocarin D has increased our understanding regarding
gene regulation of venom prothrombin activators.

Fig. 10. Comparison of intron 1 regions in TrFX and trocarin D genes (Reza et al., 2007).
6. Gene duplication in snake venom toxin diversification
Besides recruitment, gene duplication also plays an important role in the diversification of
venom toxins. This diversification is essential for the development of novel toxins. This
diversification through gene duplication is evident from the many toxin isoforms present in
the snake venom. Interestingly, each isoform varies in its function and gene regulation.
Gene duplication has also led to neofunctionalization of venom toxins, which has led to the
new families of snake venom toxins (St Pierre et al. 2008) and addition of new members
within these families (Fry et al. 2003; Landan et al. 1991b; Lynch 2007; Moura-da-Silva et al.
1996). The three-finger toxin (3FTx) multigene family is a good example of
neofunctionalization by gene duplication (Fry et al. 2003). Structurally, all the members of
this family have very well-conserved cysteine residues and share a common structure of
three beta-stranded loops extending from a central core. However, they exhibit a wide
variety of pharmacological effects. For example, acetylcholinesterase inhibition (fasciculin
from Dendroaspis angusticeps venom), neurotoxicity (-bungarotoxin from Bungarus
multicinctus venom), cardiotoxicity ( cardiotoxin from Ophiophagus hannah venom), and
many others (for details, see (Kini and Doley 2010)). Neofunctionalization occurs when a
toxin gene undergoes gene duplication and the duplicated gene is mutated within the
functional sites, which often results in new ligand-binding specificities (Kini 2002).
Besides neofunctionalization, changes in gene regulation are also the outcomes of gene
duplication. This can be seen in two isoforms present in the venom of Naja sputatrix:
cardiotoxin and -neurotoxin (Ma et al. 2001). Besides varying in function, these two
isoforms have different expression levels in the venom gland. Cardiotoxin constitutes 60%
of the venom while the -neurotoxin makes up only 3% of the venom. Gene duplication is
evident from the gene comparison whereby the structures and amino acid sequence of these

two toxins are very well-conserved (Ma et al. 2002). The main difference lies in the promoter
segment, where it was found that the -neurotoxin promoter contains a stronger silencer
element, which is responsible for significantly reducing its expression level in the venom
(Ma et al. 2001; Ma et al. 2002).
In the case of venom prothrombin activators, we have shown that they have been
recruited from the gene of an ancestral plasma prothrombin activator protein through
gene duplication. The duplicated gene underwent modifications in its regulatory and
coding regions to gain toxin characteristics. VERSE segments were inserted in the promoter
regions of trocarin D and PCCS and are responsible for their elevated level of expression.
Insertion/deletion segments in their intron 1 regions are postulated to be responsible for
venom-gland specific expression. Modifications in the gene-coding regions enable
prothrombin activators to function better as toxin by gaining certain characteristics such as
resistance to inactivation.
7. Conclusion
Gene duplication has played a major role in the development of snake venom toxins. Our
findings on venom prothrombin activators and blood coagulation factors have captured
the first molecular evidence of gene duplication. The characterization of the differences in
their genes, i.e. VERSE segment and intron 1 of trocarin D, has increased our
understanding of gene regulation of snake venom toxins. It is shown that the VERSE
segment is responsible for the elevation of gene expression and that the intron 1 is
probably responsible venom gland-specific expression (unpublished observations). We
identified three novel cis-elements in the VERSE segment, and these play important roles
in gene regulation. It would be interesting to further characterize them and their trans-
factor partners to determine how various trans-factors interact with each other to regulate
gene expression. The answers to some of these questions will increase our overall
understanding of gene regulation.
8. References
Bos, M.H., M.Boltz, L.St Pierre, P.P.Masci, J.de Jersey, M.F.Lavin, and R.M.Camire. 2009.
"Venom factor V from the common brown snake escapes hemostatic regulation
through procoagulant adaptations." Blood. 114:686-692.
Boulikas, T. 1993. "Nature of DNA sequences at the attachment regions of genes to the
nuclear matrix." J.Cell Biochem. 52:14-22.
Braud, S., C.Bon, and A.Wisner. 2000. "Snake venom proteins acting on hemostasis."
Biochimie. 82:851-859.
Chester, A. and G.P.Crawford. 1982. "In vitro coagulant properties of venoms from
Australian snakes." Toxicon. 20:501-504.
Chow, G. and R.M.Kini. 2001. "Exogenous factors from animal sources that induce platelet
aggregation." Thromb.Haemost. 85:177-178.
Cockerill, P.N. and W.T.Garrard. 1986. "Chromosomal loop anchorage of the kappa
immunoglobulin gene occurs next to the enhancer in a region containing
topoisomerase II sites." Cell. 44:273-282.

Davie, E.W. 2003. "A brief historical review of the waterfall/cascade of blood coagulation."
J.Biol.Chem. 278:50819-50832.
Esmon, C.T. 2001. "Role of coagulation inhibitors in inflammation." Thromb.Haemost. 86:51-
56.
Filippovich, I., N.Sorokina, L.St Pierre, S.Flight, J.de Jersey, N.Perry, P.P.Masci, and
M.F.Lavin. 2005. "Cloning and functional expression of venom prothrombin
activator protease from Pseudonaja textilis with whole blood procoagulant
activity." Br.J.Haematol. 131:237-246.
Foster, W.B., M.E.Nesheim, and K.G.Mann. 1983. "The factor Xa-catalyzed activation of
factor V." J.Biol.Chem. 258:13970-13977.
Fry, B.G. 2005. "From genome to "venome": molecular origin and evolution of the snake
venom proteome inferred from phylogenetic analysis of toxin sequences and
related body proteins." Genome Res. 15:403-420.
Fry, B.G., K.Roelants, D.E.Champagne, H.Scheib, J.D.Tyndall, G.F.King, T.J.Nevalainen,
J.A.Norman, R.J.Lewis, R.S.Norton, C.Renjifo, and R.C.de la Vega. 2009. "The
toxicogenomic multiverse: convergent recruitment of proteins into animal
venoms." Annu.Rev.Genomics Hum.Genet. 10:483-511.
Fry, B.G., W.Wuster, R.M.Kini, V.Brusic, A.Khan, D.Venkataraman, and A.P.Rooney. 2003.
"Molecular evolution and phylogeny of elapid snake venom three-finger toxins."
J.Mol.Evol. 57:110-129.
Gao, R., R.M.Kini, and P.Gopalakrishnakone. 2002. "A novel prothrombin activator from the
venom of Micropechis ikaheka: isolation and characterization." Arch. Biochem.
Biophys. 408:87-92.
Hanover, J.A., C.K.Cohen, M.C.Willingham, and M.K.Park. 1987. "O-linked N-
acetylglucosamine is attached to proteins of the nuclear pore. Evidence for
cytoplasmic and nucleoplasmic glycoproteins." J.Biol.Chem. 262:9887-9894.
Hasson, S.S., R.D.Theakston, and R.A.Harrison. 2003. "Cloning of a prothrombin activator-
like metalloproteinase from the West African saw-scaled viper, Echis ocellatus."
Toxicon. 42:629-634.
Holt, G.D., R.S.Haltiwanger, C.R.Torres, and G.W.Hart. 1987. "Erythrocytes contain
cytoplasmic glycoproteins. O-linked GlcNAc on Band 4.1." J.Biol.Chem. 262:14847-
14850.
Holt, G.D. and G.W.Hart. 1986. "The subcellular distribution of terminal N-
acetylglucosamine moieties. Localization of a novel protein-saccharide linkage, O-
linked GlcNAc." J.Biol.Chem. 261:8049-8057.
Hung, H.L. and K.A.High. 1996. "Liver-enriched transcription factor HNF-4 and ubiquitous
factor NF-Y are critical for expression of blood coagulation factor X." J.Biol.Chem.
271:2323-2331.
Hung, H.L., E.S.Pollak, R.D.Kudaravalli, V.Arruda, K.Chu, and K.A.High. 2001. "Regulation
of human coagulation factor X gene expression by GATA-4 and the Sp family of
transcription factors." Blood. 97:946-951.
Hutton, R.A. and D.A.Warrell. 1993. "Action of snake venom components on the
haemostatic system." Blood Rev. 7:176-189.

Inoue, K. and T.Morita. 1993. "Identification of O-linked oligosaccharide chains in the
activation peptides of blood coagulation factor X. The role of the carbohydrate
moieties in the activation of factor X." Eur.J.Biochem. 218:153-163.
Joseph, J.S., M.C.Chung, K.Jeyaseelan, and R.M.Kini. 1999. "Amino acid sequence of
trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural
similarity to coagulation factor Xa." Blood. 94:621-631.
Joseph, J.S. and R.M.Kini. 2001. "Snake venom prothrombin activators homologous to blood
coagulation factor Xa." Haemostasis. 31:234-240.
Kalafatis, M., M.D.Rand, and K.G.Mann. 1994. "The mechanism of inactivation of human
factor V and human factor Va by activated protein C." J.Biol.Chem. 269:31869-
31880.
Kini, R.M. 2002. "Molecular moulds with multiple missions: functional sites in three-finger
toxins." Clin.Exp.Pharmacol.Physiol. 29:815-822.
Kini, R.M. 2004. "Platelet aggregation and exogenous factors from animal sources."
Curr.Drug Targets.Cardiovasc.Haematol.Disord. 4:301-325.
Kini, R.M. and G.Chow. 2001. "Exogenous inhibitors of platelet aggregation from animal
sources." Thromb.Haemost. 85:179-181.
Kini, R.M. and R.Doley. 2010. "Structure, function and evolution of three-finger toxins: mini
proteins with multiple targets." Toxicon. 56:855-867.
Kini, R.M. and H.J.Evans. 1990. "Effects of snake venom proteins on blood platelets."
Toxicon. 28:1387-1422.
Kini, R.M., T.Morita, and J.Rosing. 2001. "Classification and nomenclature of prothrombin
activators isolated from snake venoms." Thromb.Haemost. 86:710-711.
Kornalik, F. and B.Blomback. 1975. "Prothrombin activation induced by Ecarin - a
prothrombin converting enzyme from Echis carinatus venom." Thromb.Res. 6:57-
63.
Kwong, S., A.E.Woods, P.J.Mirtschin, R.Ge, and R.M.Kini. 2009. "The recruitment of blood
coagulation factor X into snake venom gland as a toxin: the role of promoter Cis-
elements in its expression." Thromb.Haemost. 102:469-478.
Landan, G., A.Bdolah, Z.Wollberg, E.Kochva, and D.Graur. 1991a. "Evolution of the
sarafotoxin/endothelin superfamily of proteins." Toxicon. 29:237-244.
Landan, G., A.Bdolah, Z.Wollberg, E.Kochva, and D.Graur. 1991b. "Evolution of the
sarafotoxin/endothelin superfamily of proteins." Toxicon. 29:237-244.
Liebich, I., J.Bode, I.Reuter, and E.Wingender. 2002. "Evaluation of sequence motifs found in
scaffold/matrix-attached regions (S/MARs)." Nucleic Acids Res. 30:3433-3442.
Lynch, V.J. 2007. "Inventing an arsenal: adaptive evolution and neofunctionalization of
snake venom phospholipase A2 genes." BMC.Evol.Biol. 7:2.
Ma, D., A.Armugam, and K.Jeyaseelan. 2001. "Expression of cardiotoxin-2 gene. Cloning,
characterization and deletion analysis of the promoter." Eur.J.Biochem. 268:1844-
1850.
Ma, D., A.Armugam, and K.Jeyaseelan. 2002. "Alpha-neurotoxin gene expression in Naja
sputatrix: identification of a silencer element in the promoter region."
Arch.Biochem.Biophys. 404:98-105.

Mann, K.G., M.F.Hockin, K.J.Begin, and M.Kalafatis. 1997. "Activated protein C cleavage of
factor Va leads to dissociation of the A2 domain." J.Biol.Chem. 272:20678-20683.
Markland, F.S. 1997. "Snake venoms." Drugs. 54 Suppl 3:1-10.
Markland, F.S. 1998. "Snake venoms and the hemostatic system." Toxicon. 36:1749-1800.
Marsh, N.A., T.L.Fyffe, and E.A.Bennett. 1997. "Isolation and partial characterization of a
prothrombin-activating enzyme from the venom of the Australian rough-scaled
snake (Tropidechis carinatus)." Toxicon. 35:563-571.
Masci, P.P., A.N.Whitaker, and J.de Jersey. 1988. "Purification and characterization of a
prothrombin activator from the venom of the Australian brown snake, Pseudonaja
textilis textilis." Biochem.Int. 17:825-835.
McMullen, B.A., K.Fujikawa, W.Kisiel, T.Sasagawa, W.N.Howald, E.Y.Kwa, and
B.Weinstein. 1983. "Complete amino acid sequence of the light chain of human
blood coagulation factor X: evidence for identification of residue 63 as beta-
hydroxyaspartic acid." Biochemistry. 22:2875-2884.
Minh, L.T., M.A.Reza, S.Swarup, and R.M.Kini. 2005. "Gene duplication of coagulation
factor V and origin of venom prothrombin activator in Pseudonaja textilis snake."
Thromb.Haemost. 93:420-429.
Morita, T. 2004. "C-type lectin-related proteins from snake venoms." Curr.Drug
Targets.Cardiovasc.Haematol.Disord. 4:357-373.
Morita, T. and S.Iwanaga. 1978. "Purification and properties of prothrombin activator from
the venom of Echis carinatus." J.Biochem.(Tokyo). 83:559-570.
Moura-da-Silva, A.M., R.D.Theakston, and J.M.Crampton. 1996. "Evolution of disintegrin
cysteine-rich and mammalian matrix-degrading metalloproteinases: gene
duplication and divergence of a common ancestor rather than convergent
evolution." J.Mol.Evol. 43:263-269.
Nesheim, M.E., W.M.Canfield, W.Kisiel, and K.G.Mann. 1982. "Studies of the capacity of
factor Xa to protect factor Va from inactivation by activated protein C." J.Biol.Chem.
257:1443-1447.
Nesheim, M.E., J.B.Taswell, and K.G.Mann. 1979. "The contribution of bovine Factor V and
Factor Va to the activity of prothrombinase." J.Biol.Chem. 254:10952-10962.
Nishida, S., T.Fujita, N.Kohno, H.Atoda, T.Morita, H.Takeya, I.Kido, M.J.Paine, S.Kawabata,
and S.Iwanaga. 1995. "cDNA cloning and deduced amino acid sequence of
prothrombin activator (ecarin) from Kenyan Echis carinatus venom." Biochemistry.
34:1771-1778.
Owen, W.G. and C.M.Jackson. 1973. "Activation of prothrombin with Oxyranus scutellatus
scutellatus (Taipain snake) venom." Thromb.Res. 3:705-714.
Pittman, D.D., K.N.Tomkinson, D.Michnick, U.Selighsohn, and R.J.Kaufman. 1994.
"Posttranslational sulfation of factor V is required for efficient thrombin
cleavage and activation and for full procoagulant activity." Biochemistry.
33:6952-6959.
Rao, V.S., J.S.Joseph, and R.M.Kini. 2003a. "Group D prothrombin activators from snake
venom are structural homologues of mammalian blood coagulation factor Xa."
Biochem.J. 369:635-642.

Rao, V.S. and R.M.Kini. 2002. "Pseutarin C, a prothrombin activator from Pseudonaja textilis
venom: its structural and functional similarity to mammalian coagulation factor
Xa-Va complex." Thromb.Haemost. 88:611-619.
Rao, V.S., S.Swarup, and R.M.Kini. 2003b. "The nonenzymatic subunit of pseutarin C, a
prothrombin activator from eastern brown snake (Pseudonaja textilis) venom,
shows structural similarity to mammalian coagulation factor V." Blood. 102:1347-
1354.
Rao, V.S., S.Swarup, and R.M.Kini. 2004. "The catalytic subunit of pseutarin C, a group C
prothrombin activator from the venom of Pseudonaja textilis, is structurally similar
to mammalian blood coagulation factor Xa." Thromb.Haemost. 92:509-521.
Reza, A., S.Swarup, and R.M.Kini. 2005a. "Two parallel prothrombin activator systems in
Australian rough-scaled snake, Tropidechis carinatus. Structural comparison of
venom prothrombin activator with blood coagulation factor X." Thromb.Haemost.
93:40-47.
Reza, M.A., L.T.Minh, S.Swarup, and R.M.Kini. 2006. "Molecular evolution caught in action:
gene duplication and evolution of molecular isoforms of prothrombin activators in
Pseudonaja textilis (brown snake)." J.Thromb.Haemost. 4:1346-1353.
Reza, M.A., S.Swarup, and R.M.Kini. 2005b. "Gene structures of trocarin D and coagulation
factor X, two functionally diverse prothrombin activators from Australian rough
scaled snake." Pathophysiol.Haemost.Thromb. 34:205-208.
Reza, M.A., S.Swarup, and R.M.Kini. 2007. "Structure of two genes encoding parallel
prothrombin activators in Tropidechis carinatus snake: gene duplication and
recruitment of factor X gene to the venom gland." J.Thromb.Haemost. 5:117-126.
Robin Doley, Xingding Zhou, and R.M.Kini. 2009. "Snake Venom Phospholipase A2
Enzymes." In Stephen P.Mackessy, editor, Handbook of Venoms and Toxins of Reptiles.
CRC Press. 173-205.
Rosing, J. and G.Tans. 1991. "Inventory of exogenous prothrombin activators. For the
Subcommittee on Nomenclature of Exogenous Hemostatic Factors of the Scientific
and Standardization Committee of the International Society on Thrombosis and
Haemostasis." Thromb.Haemost. 65:627-630.
Rosing, J. and G.Tans. 1992. "Structural and functional properties of snake venom
prothrombin activators." Toxicon. 30:1515-1527.
Schieck, A., E.Habermann, and F.Kornalik. 1972. "The prothrombin-activating principle
from Echis carinatus venom. II. Coagulation studies in vitro and in vivo." Naunyn
Schmiedebergs Arch.Pharmacol. 274:7-17.
Silva, M.B., M.Schattner, C.R.Ramos, I.L.Junqueira-de-Azevedo, M.C.Guarnieri,
M.A.Lazzari, C.A.Sampaio, R.G.Pozner, J.S.Ventura, P.L.Ho, and
A.M.Chudzinski-Tavassi. 2003. "A prothrombin activator from Bothrops
erythromelas (jararaca-da-seca) snake venom: characterization and molecular
cloning." Biochem.J. 369:129-139.
Snow, C.M., A.Senior, and L.Gerace. 1987. "Monoclonal antibodies identify a group of
nuclear pore complex glycoproteins." J.Cell Biol. 104:1143-1156.

Speijer, H., J.W.Govers-Riemslag, R.F.Zwaal, and J.Rosing. 1986. "Prothrombin activation by
an activator from the venom of Oxyuranus scutellatus (Taipan snake)." J.Biol.Chem.
261:13258-13267.
St Pierre, L., S.T.Earl, I.Filippovich, N.Sorokina, P.P.Masci, J.De Jersey, and M.F.Lavin. 2008.
"Common evolution of waprin and kunitz-like toxin families in Australian
venomous snakes." Cell Mol.Life Sci. 65:4039-4054.
St Pierre, L., P.P.Masci, I.Filippovich, N.Sorokina, N.Marsh, D.J.Miller, and M.F.Lavin. 2005.
"Comparative analysis of prothrombin activators from the venom of Australian
elapids." Mol.Biol.Evol. 22:1853-1864.
Stenflo, J., A.Lundwall, and B.Dahlback. 1987. "beta-Hydroxyasparagine in domains
homologous to the epidermal growth factor precursor in vitamin K-dependent
protein S." Proc.Natl.Acad.Sci.U.S.A. 84:368-372.
Stocker, K., H.Hauer, C.Muller, and D.A.Triplett. 1994. "Isolation and characterization of
Textarin, a prothrombin activator from eastern brown snake (Pseudonaja textilis)
venom." Toxicon. 32:1227-1236.
Suzuki, K., B.Dahlback, and J.Stenflo. 1982. "Thrombin-catalyzed activation of human
coagulation factor V." J Biol Chem. 257:6556-6564.
Tans, G., J.W.Govers-Riemslag, J.L.van Rijn, and J.Rosing. 1985. "Purification and properties
of a prothrombin activator from the venom of Notechis scutatus scutatus."
J.Biol.Chem. 260:9366-9372.
Thorelli, E., R.J.Kaufman, and B.Dahlback. 1998. "Cleavage requirements of factor V in
tissue-factor induced thrombin generation." Thromb.Haemost. 80:92-98.
van der Neut, K.M., R.J.Dirven, H.L.Vos, G.Tans, J.Rosing, and R.M.Bertina. 2004b. "Factor
Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306,
Arg-506, and Arg-679." J.Biol.Chem. 279:6567-6575.
van der Neut, K.M., R.J.Dirven, H.L.Vos, G.Tans, J.Rosing, and R.M.Bertina. 2004a. "Factor
Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306,
Arg-506, and Arg-679." J.Biol.Chem. 279:6567-6575.
van Drunen, C.M., R.W.Oosterling, G.M.Keultjes, P.J.Weisbeek, R.van Driel, and
S.C.Smeekens. 1997. "Analysis of the chromatin domain organisation around the
plastocyanin gene reveals an MAR-specific sequence element in Arabidopsis
thaliana." Nucleic Acids Res. 25:3904-3911.
Walker, F.J., W.G.Owen, and C.T.Esmon. 1980. "Characterization of the prothrombin
activator from the venom of Oxyuranus scutellatus scutellatus (taipan venom)."
Biochemistry. 19:1020-1023.
Wang, C., M.Eufemi, C.Turano, and A.Giartosio. 1996. "Influence of the carbohydrate
moiety on the stability of glycoproteins." Biochemistry. 35:7299-7307.
Welton, R.E. and J.N.Burnell. 2005. "Full length nucleotide sequence of a factor V-like
subunit of oscutarin from Oxyuranus scutellatus scutellatus (coastal Taipan)."
Toxicon. 46:328-336.
Wilberding, J.A. and F.J.Castellino. 2000. "Characterization of the murine coagulation factor
X promoter." Thromb.Haemost. 84:1031-1038.

Will, K., G.Warnecke, N.Albrechtsen, T.Boulikas, and W.Deppert. 1998. "High affinity MAR-
DNA binding is a common property of murine and human mutant p53." J.Cell
Biochem. 69:260-270.
Yamada, D. and T.Morita. 1997. "Purification and characterization of a Ca2+ -dependent
prothrombin activator, multactivase, from the venom of Echis multisquamatus."
J.Biochem.(Tokyo). 122:991-997.
Zhou, C.Z. and B.Liu. 2001. "Identification and characterization of a silkgland-related matrix
association region in Bombyx mori." Gene. 277:139-144.
15
A Puroindoline Mutigene Family
Exhibits Sequence Diversity in Wheat and is
Associated with Yield-Related Traits
Feng Chen
1,2
, Fuyan Zhang
1
,
Craig F. Morris
3
and Dangqun Cui
1,2

1
Department of Agronomy, Henan Agricultural University, Zhengzhou
2
Key Laboratory of Physiological Ecology and Genetic Improvement
of Food Crops in Henan Province, Zhengzhou
3
USDA-ARS, Western Wheat Quality Laboratory, E-202 Food Science and Human
Nutrition Facility East, Washington State University, Pullman,

1,2
China
3
USA
1. Introduction
Kernel texture (grain hardness) is a leading quality characteristic of bread wheat (Triticum
aestivum L.) as it dramatically influences the milling and processing properties, and
consequently determines the classification and marketing of grain (Bhave and Morris 2008a,
b). The word puroindoline is derived from the Greek word puros meaning wheat and
indoline describing the indole ring of tryptophan (Gautier et al. 1994). Puroindolines,
composed of puroindoline a and b, are amphipathic proteins of ca. 13,000 Da, and share
homology with grain softness protein (GSP), purothionins, lipid transfer proteins, and other
members of the prolamin super-family of proteins (Shewry and Halford 2002). Puroindoline
proteins possess a characteristic tryptophan-rich domain and cysteine backbone; isoforms
occur in the starchy endosperm of the Triticeae. Their secondary structure, determined by
infrared and Raman spectroscopies, is comprised of approximately 30% -helices, 30% -
sheets, and 40% unordered structure at pH 7.4 in solution (Bihan et al. 1996). Puroindoline
genes are present throughout the Triticeae tribe of the Poaceae (Gramineae), including
wheat (Triticum sp.), rye (Secale sp.), barley (Hordeum sp.), and the wild relatives of wheat
(Aegilops sp. and Triticum sp.). In Triticum aestivum, puroindolines exist as two expressed
genes, Puroindoline a and Puroindoline b, and are located on the distal end of the short arm
of chromosome 5 (5DS). An exception to this general situation lies with the tetraploid
(AABB) wheats (T. turgidum), which include cultivated durum (ssp. durum). Apparently
during the allotetraploidization formation of T. diccocoides, the wild ancestor of cultivated
durum, both the A- and B-genome Puroindoline loci were eliminated due to transposable
element insertion and two large deletions in the Hardness loci caused by illegitimate DNA
recombination (Chantret et al. 2005). Consequently, hexaploid wheat, T. aestivum, possesses

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280
puroindoline a and b on only the D-genome (contributed by Ae. tauschii during the
allohexaploidization of this taxon), but lacks homoeologous loci on the A- and B-genomes.
The expression of puroindoline genes is mainly associated with soft and hard grain
texture of bread wheat, whereas durum wheat is very hard due to the lack of puroindoline
genes (Capparelli et al. 2003). Extensive genetic surveys, cytological analysis, and
transformation experiments in wheat and rice (Oryza sativa) have demonstrated that
puroindoline a and b act to create soft kernel texture (Bhave and Morris 2008a, b, Morris
2002, Morris and Bhave 2008). However, puroindolines have also been extensively studied
regarding their multiple roles in the development and resistance of plants against biotic
stress, and their profound influence on the breeding and processing quality of food crops
(Luo et al. 2008; Giroux and Morris 1997, 1998; Ragupathy and Cloutier 2008; Chen et al.
2007a, b). Considerable allelic variation in puroindoline a and b have been identified in a
great number of genotypes stemming from different geographies. Gene sequence
polymorphism and allele designations have been recently reconciled and reviewed by
Morris and Bhave (2008) and Bhave and Morris (2008a, b).
Although clearly exerting a major role in wheat kernel texture variation, puroindolines do
not account for all of the variation observed in bread wheat. Several minor quantitative trait
loci (QTLs) for kernel texture have been mapped on different chromosomes (Sourdille et al.
1996; Turner et al. 2004). Four additional regions located on chromosomes 2A, 2D, 5B, and
6D were shown to have single-factor effects on hardness, while three others located on
chromosomes 5A, 6D and 7A had interaction effects (Sourdille et al. 1996).
Three new puroindoline gene-like sequences in hexaploid wheat were reported by
Wilkinson et al. (2008). Chen et al. (2010) renamed them Pinb-2v1, Pinb-2v2 and Pinb-2v3,
and reported the discovery of a fourth novel puroindoline variant in bread wheat,
designated Pinb-2v4. Physical mapping results of Chen et al. (2010) were not fully consistent
with the results reported by Wilkinson et al. (2008). Interestingly, Pinb-2v1 and Pinb-2v4
were present in all of the surveyed cultivars whereas Pinb-2v2 and Pinb-2v3 were
reciprocally present in different wheat cultivars (Chen et al. 2010). This result indicates that
Pinb-2v2 and Pinb-2v3 are likely allelic. In this study, we remapped the four known
Puroindoline b-2 variants using aneuploids of bread wheat and durum wheat in order to
confirm their physical location on the chromosomes of wheat, and report the discovery of a
novel Pinb-2v3 allele and a new Puroindoline b-2 variant, designated Pinb-2v5. Also, we
investigated the association of Puroindoline b-2 variants with yield-related traits. This study
provides useful information for illustrating the molecular and genetic basis of kernel
hardness and gene duplication events in wheat.
2.1 Wheat germplasm, DNA extraction and PCR amplification
A total of 109 bread wheat lines developed for the Yellow and Huai Valleys of China were
planted at the Zhengzhou Scientific Research and Education Center of Henan Agricultural
University during the 2009-10 cropping seasons according to local management practices.
Analysis of agronomic traits and measurement of SKCS hardness as well as identification of
puroindoline b-2 variants were performed. Each plot was comprised of four 200 cm-long
rows with 23 cm between neighboring rows and 10 cm between neighboring plants. All
surveyed cultivars grew well and no lodging was present in the trial. After harvest, all
wheat samples were cleaned.
A Puroindoline Mutigene Family Exhibits Sequence
Diversity in Wheat and is Associated with Yield-Related Traits

281
Durum wheat cultivar Langdon (LND), bread wheat cultivar Chinese Spring (CS), two
disomic substitution lines of Langdon 7D(7A) and Langdon 7D(7B) with substituted 7D
chromosomes of Chinese Spring and six nullisomic-tetrasomic lines of Chinese Spring
involving group 7 chromosomes (CS N7A-T7B, CS N7A-T7D, CS N7B-T7A, CS N7B-T7D, CS
N7D-T7A and CS N7D-T7B) were used in this study. DNA was extracted from two seeds
each of the 109 bread wheat lines, genetic stocks, and 15 Chinese, CIMMYT and Italian
durum wheat cultivars following the rapid extraction method of genomic DNA derived
from Chen et al. (2006).
PCR primer sequences were designed using Primer Premier 5.0 software. Reactions were
performed in 25 L containing 100 ng of genomic DNA, 10 pmol of each primer, 250 M of
each dNTP, 1x Taq DNA polymerase reaction buffer containing 1.5 M of MgCl
2
and 0.5
unit of Taq DNA polymerase (Promega, Madison, WI). The cycling conditions were 94C for
5 min followed by 35 cycles of 94C for 50 s, 45C to 65C for 50 s (primer-specific annealing
temperatures, see Table 1), 72C for 1 min, followed by a final 10-min extension at 72C. An
aliquot (8 L) of the PCR products was analyzed on 1.5% (w/v) agarose gels, stained with
ethidium bromide, and visualized with UV light. Forty-eight clones from three independent
PCR reactions using the universal primers on durum cv. Langdon were sequenced from
both strands by SinoGenoMax Co., Ltd.
Multiple alignments of sequences and translations of nucleotide into amino acid sequence
were performed by software DNAMAN Version 6.0. Sequence chromatograms were
analyzed by Chromas Version 1.4.5 and FinchTV Version 1.4.0.
2.2 Measurement of agronomic traits and hardness index
Before harvesting, ten plants of each genotype were randomly selected in each plot to
determine spikelet number per plant, flag leaf length and flag leaf width, as well as flag leaf
area calculated from the product of leaf length and maximum leaf width 0.75. Grains per
spike, grain weight per plant and grain weight per spike were determined after harvesting.
Kernel hardness, grain diameter and thousand-kernel weight were measured on 300-kernel
samples of each genotype using the Perten Single Kernel Characterization System (SKCS)
4100, following the manufacturers operation procedure (Perten Instruments North America
Inc., Springfield, IL). Mean, standard deviation, and distribution of SKCS hardness data
were used to classify the genotypes tested into soft, mixed, and hard types.
3. Results
3.1 Physical mapping of Pinb-2 variants
Physical mapping results with variant-specific primers (Table 1) showed that Pinb-2v1 was
present in LND 7D(7A), LND 7D(7B), Chinese Spring, CS N7A-T7B, CS N7A-T7D, CS N7B-
T7A and CS N7B-T7D, but absent in wild-type Langdon, CS N7D-T7A and CS N7D-T7B,
indicating that Pinb-2v1 is located on 7D of Chinese Spring. Pinb-2v2 was present in wild-
type Chinese Spring, CS N7A-T7B, CS N7A-T7D, CS N7D-T7A and CS N7D-T7B, but absent
in wild-type Langdon, LND 7D(7A), LND 7D(7B), CS N7B-T7A and CS N7B-T7D, indicating
that Pinb-2v2 is located on 7B of Chinese Spring. Pinb-2v3 was present in wild-type
Langdon, LND 7D(7A), but absent in LND 7D(7B), wild-type Chinese Spring and all of its
six nullisomic-tetrosomic lines, indicating that Pinb-2v3 is located on 7B of Langdon. Pinb-
2v4 was present in Langdon, LND 7D(7B), CS N7B-T7A, CS N7B-T7D, CS N7D-T7A and CS

Gene Duplication

282
N7D-T7B, but absent in LND 7D(7A), CS N7A-T7B and CS N7A-T7D, indicating that Pinb-
2v4 is located on 7A of Langdon and Chinese Spring.
Therefore, it is concluded that Pinb-2v1 is on 7D of Chinese Spring, Pinb-2v2 is on 7B of
Chinese Spring, Pinb-2v3 is on 7B of Langdon and Pinb-2v4 is on 7A of Langdon and
Chinese Spring. The results of mapping Pinb-2v in Chinese Spring and its derived
nullisomic-tetrosomic lines are consistent with the locations of the four Puroindoline b-2
variants reported by Chen et al. (2010a).
3.2 Variation in puroindoline b-2 variants and the discovery of a new puroindoline b-2
variant
Based on PCR amplification with four specific primers (Table 1), 35 of 36 durum wheat
cultivars surveyed possessed variant combination Pinb-2v3/Pinb-2v4, whereas the durum
wheat cultivar Norba, introduced from Italy, possessed combination Pinb-2v2/Pinb-2v4.
None of the surveyed durum cultivars possessed the Pinb-2v1 gene. Further sequencing of
PCR products with specific primers showed that all of the sequences of Pinb-2v4 were
exactly the same as the Pinb-2v4 sequence of bread wheat reported by Chen et al. (2010),
whereas the coding region of Pinb-2v3 in durum wheat cultivar Langdon had a single
nucleotide change from G to T at the 6th position compared with the Pinb-2v3 sequence
reported by Wilkinson et al. (2008) and Chen et al. (2010). Furthermore, 18 durum wheat
cultivars with the Pinb-2v3 variant were sequenced and all of them uniformly contained a
single nucleotide change from G to T at the 6th position. Sequencing of bread wheat cultivar
Wichita showed the same change at the 6th position, compared with a previous Pinb-2v3
sequence of Wichita reported by Wilkinson et al. (2008) and Chen et al. (2010). The
discrepancy may have resulted from the previously used primers due to a single nucleotide
change present in the forward primer sequence. However, after we modified the Pinb-2v3
sequence from G to T at the 6th position of Wichita, a one base-pair change from T to C at
the 311th position was found in the coding region of Pinb-2v3 in 12 durum wheat cultivars,
resulting in the deduced amino acid change from valine to alanine at position 104 in PINB-
2v3. According to the gene nomenclature for puroindoline a and b, this Pinb-2v3 allele in
wheat cultivars containing the sequence of AM99733 and GQ496618 with the modification
of G to T at the 6th position will be designated as Pinb-2v3a, and the Pinb-2v3 allele with a
nucleotide change from T to C at the 311th position in durum wheat cultivars will be
designated as Pinb-2v3b.
Three repeated PCR amplifications of the durum cultivar Langdon were performed with the
universal primer in order to reduce the influence of mismatching amplification of Taq
polymerase. Cloning and sequencing results showed that 40 sub-clones belonged to either
Pinb-2v3 or Pinb-2v4 whereas 7 other sub-clones did not belong to any known Puroindoline b-
2 variant. Further analysis indicated that a novel variant, designated as Pinb-2v5
(HM245236), shared high homology and was most closely related to Pinb-2v4 with only 5
SNPs (Fig. 1).
The overall alignment with Pinb-D1a with the five Pinb-2 variant genes showed that Pinb-2v5
has 74.3%, 95.8%, 94.0%, 92.3% and 98.8% identity with Pinb-D1a, Pinb-2v1, Pinb-2v2, Pinb-
2v3 and Pinb-2v4, respectively, at the DNA level. Moreover, all four puroindoline b variant
sequences were at least 91.8% homologous amongst themselves. Full alignment of deduced
amino acid sequences of Pinb-D1a and the five Puroindoline b-2 variant genes indicated that

283
Pinb-2v5 has 57.8%, 96.0%, 91.3%, 87.9% and 96.5% identity with Pinb-D1a, Pinb-2v1, Pinb-
2v2, Pinb-2v3 and Pinb-2v4, respectively, at the amino acid level (data not shown).

Gene Forward primer Reverse primer PCR
annealing
Temp
Fragment
size (bp)
Pinb-2vU ATGAAGACCTTATTCCTCCTA TCASTAGTAATAGCCATTAKT
A
54 453
Pinb-2v1 GGTTCTCAAAACTGCCCAT ACTTGCAGTTGGAATCCAG
57 319
Pinb-2v2 CTTGTAGTGAGCACAACCTTT
GCA
GTATGGACGAACTTGCAGCTG
GAG
65 401
Pinb-2v3 GCAGAAAAAGCCATTGCACCT
A
CATTAGTAGGGACGAACTTGC
AGCTA
65 528
Pinb-2v4 CCTTTCTCTTGTAGTGAGCAC
AACCA
GACGAACTTGCAGTTGGAATC
CAA
65 403
Pinb-D1b ATGAAGGCCCTCTTCCTCA CTCATGCTCACAGCCGCT
58 250
N-Pinb-
D1b
ATGAAGGCCCTCTTCCTCA CTCATGCTCACAGCCGCC
58 250
Table 1. PCR primers used in generating Puroindoline b-2 variant gene sequences in durum
wheat
3.3 Association of puroindoline b-B2 variants with SKCS grain hardness, other grain
traits, grain yield components, and flag leaf size
In order to investigate the influence of puroindoline b-2 variants on kernel hardness
independent of puroindoline a and b alleles, the average SKCS hardness index of two
different Puroindoline b-2 variant combinations, Pinb-2v1/Pinb-2v2/Pinb-2v4 and Pinb-
2v1/Pinb-2v3/Pinb-2v4, were compared by sub-setting the lines according to Pin-D1
haplotype. Puroindoline alleles are associated with dramatic effects on kernel hardness
(reviewed in Morris, 2002; Morris and Behave, 2008; Behave and Morris, 2008a, b). Results
indicated that in soft wheat cultivars with Pina-D1a/Pinb-D1a, the average of SKCS hardness
index of cultivars with Pinb-2v3 was 24.6, which was significantly higher than that of
cultivars with Pinb-2v2 (14.2). No significant difference was observed among cultivars with
Pinb-D1b, possibly suggesting that the Puroindoline b-2 variants had a larger impact in soft
wheat than in hard.
No significant association between the puroindoline D1 alleles and the 9 agronomic traits
relating to wheat yield were observed in the surveyed Chinese wheat cultivars of the Yellow
and Huai Valleys, suggesting that the puroindoline genes have no apparent relationship with
wheat grain yield. However, when the agronomic traits of cultivars with different Puroindoline
b-2 variant combinations were compared, the averages of grain number per spike, grain
weight per spike, width of flag leaf and flag leaf area of cultivars with Pinb-2v3 were
significantly higher than those cultivars with Pinb-2v2. Differences in thousand kernel weight,
grain length and spikelet number per spike were not different between these two Pinb-2v2 and

Gene Duplication

284
Pinb-2v3 genotypes (Table 2). The results indicated that Pinb-2v3 cultivars possessed superior
grain yield traits compared to Pinb-2v2, possibly suggesting that the Puroindoline b-2 gene
could modulate wheat grain yield to some extent, and that the grain yield of cultivars with
Pinb-2v3 is slightly higher than that of cultivars with Pinb-2v2. Another likely possibility is that
these Pinb-2 variants are identifying germplasm pools or founder effects.

80 Gsp-1
80 Pina-D1a
80 Pinb-D1a
80 Pinb-2v1
80 Pinb-2v2
80 Pinb-2v3
80 Pinb-2v4
80 Pinb-2v5
A
A
A
A
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T
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G
G
G
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157 Gsp-1
124 Pina-D1a
127 Pinb-D1a
127 Pinb-2v1
127 Pinb-2v2
127 Pinb-2v3
127 Pinb-2v4
127 Pinb-2v5
A
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A
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G
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T
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G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
T
T
T
G
T
T
T
T
T
T
T
C
C
C
C
C
C
C
T
T
T
T
T
T
T
T
G
C
C
C
C
C
C
C
G
A
A
A
A
A
A
A
G
A
A
A
A
A
A
A
G
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
C
C
C
C
C
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
G
C
T
C
C
C
C
C
G
C
C
C
C
C
C
C
T
C
C
C
C
C
C
C
T
T
G
A
A
A
A
A
G
G
C
T
C
C
C
C
C
T
A
C
C
C
C
C
G
A
G
G
G
G
G
G
A
G
G
G
G
G
G
G
T
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
T
.
.
.
.
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.
.
G
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
A
.
.
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.
G
.
.
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.
T
.
.
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.
G
.
.
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G
.
.
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.
.
T
.
.
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.
.
.
T
.
.
.
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.
.
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C
.
.
.
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.
.
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T
.
.
.
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G
.
.
.
.
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.
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A
.
.
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G
.
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.
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.
.
.
A
.
.
.
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.
.
.
A
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
231 Gsp-1
189 Pina-D1a
195 Pinb-D1a
195 Pinb-2v1
195 Pinb-2v2
195 Pinb-2v3
195 Pinb-2v4
195 Pinb-2v5
G
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
A
.
.
.
.
.
.
.
G
.
.
.
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.
.
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G
.
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.
.
.
.
.
A
.
.
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.
A
.
.
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.
.
.
G
.
.
.
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.
.
A
.
.
.
.
.
.
.
G
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
C
A
C
A
A
A
A
A
C
C
G
A
A
A
A
A
A
A
G
G
G
G
G
G
.
.
C
A
A
A
A
A
.
.
C
C
C
C
C
C
.
.
G
G
G
G
G
G
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
C
C
C
C
C
C
C
T
T
T
T
T
T
T
T
A
A
A
A
G
G
G
G
G
A
A
G
G
G
G
G
A
A
G
A
A
A
A
A
C
T
C
C
C
C
C
C
T
T
T
C
T
T
T
T
C
C
C
C
C
C
C
C
T
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
T
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
G
G
A
A
A
A
A
A
C
G
G
G
G
G
G
G
G
A
G
G
G
G
G
G
A
A
A
A
A
A
A
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
A
A
A
A
A
A
A
A
T
C
C
T
T
C
T
T
G
C
G
G
G
G
G
G
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
A
C
A
A
A
A
A
A
T
T
T
T
T
T
T
T
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
A
T
T
G
G
G
G
G
G
C
C
C
C
C
C
T
C
G
G
G
G
G
G
G
G
G
A
A
G
G
A
G
G
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
T
C
T
C
C
C
C
C
G
T
T
T
T
T
T
T
T
C
T
T
T
T
T
T
.
A
C
G
G
G
G
G
.
A
A
G
G
G
G
G
.
C
C
C
C
C
C
C
G
G
A
T
G
G
G
G
A
A
A
G
G
T
G
G
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
A
A
G
G
G
G
G
T
T
T
T
T
T
T
T
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
G
C
T
T
T
T
T
T
C
C
C
T
T
C
T
T
C
C
C
C
C
C
C
C
G
G
A
G
G
G
A
A
311 Gsp-1
266 Pina-D1a
272 Pinb-D1a
272 Pinb-2v1
272 Pinb-2v2
272 Pinb-2v3
272 Pinb-2v4
272 Pinb-2v5
C
G
G
A
A
A
A
A
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
T
A
A
G
G
T
G
G
C
C
C
C
C
C
C
C
T
C
C
T
C
C
C
C
T
T
T
A
A
A
A
A
G
G
G
G
G
C
G
G
G
G
G
G
A
G
G
G
T
C
C
C
C
C
C
C
T
G
C
T
T
T
T
T
C
T
C
T
T
T
T
T
T
T
A
T
T
T
T
T
T
G
C
T
T
T
T
T
T
G
A
G
G
G
G
G
C
T
A
A
A
A
A
A
C
.
.
.
.
.
.
.
T
.
.
.
.
.
.
.
C
.
.
.
.
.
.
.
G
G
A
A
A
A
A
A
G
G
A
A
A
A
A
A
A
A
T
T
T
T
T
T
C
A
G
G
G
G
G
G
T
A
G
G
G
G
G
G
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
T
A
A
A
A
A
A
G
G
A
A
A
A
A
A
G
G
G
G
G
G
G
G
A
A
G
G
G
G
G
G
A
A
G
G
G
G
G
G
G
G
C
C
C
T
C
C
A
G
G
G
G
G
G
G
G
G
G
C
C
C
C
C
A
A
C
C
C
C
C
C
A
G
T
T
T
T
T
T
G
G
G
G
G
G
G
G
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
A
A
A
A
A
A
T
T
G
A
A
G
A
A
G
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
G
A
T
A
A
A
A
A
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
A
G
G
G
A
G
G
G
G
G
C
G
G
G
G
G
G
T
T
T
C
C
T
C
C
C
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
G
T
G
T
T
T
T
T
A
T
G
A
A
A
A
A
A
G
G
G
G
G
G
G
A
G
A
A
A
A
A
A
C
G
G
C
C
C
C
C
C
G
A
C
C
C
C
C
A
A
A
A
A
A
A
A
G
G
G
G
A
G
G
G
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
T
T
C
T
T
T
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
T
C
C
C
C
C
C
C
A
A
A
C
C
C
C
C
A
G
A
A
A
T
A
A
G
T
G
G
G
G
G
G
C
C
C
C
C
C
C
C
A
G
A
A
A
A
A
A
A
G
G
A
A
A
A
A
T
C
C
C
C
C
C
C
T
T
T
T
T
T
T
T
G
C
G
A
A
A
A
A
A
G
A
C
C
C
C
T
G
G
G
G
G
G
G
G
G
C
C
A
C
C
C
C
C
C
C
C
C
C
C
C
A
A
A
C
C
C
C
C
391 Gsp-1
346 Pina-D1a
352 Pinb-D1a
352 Pinb-2v1
352 Pinb-2v2
352 Pinb-2v3
352 Pinb-2v4
352 Pinb-2v5
A
A
G
G
G
G
G
G
A
A
A
A
G
G
A
A
C
T
T
T
T
T
T
T
G
G
A
A
A
A
A
A
A
C
G
G
G
G
G
G
C
C
C
C
T
C
C
C
G
A
A
A
A
A
A
A
T
C
C
A
A
A
A
A
C
C
C
A
A
A
A
A
G
G
A
A
G
G
G
G
C
C
C
A
A
A
A
A
G
A
A
A
A
A
A
A
T
A
A
G
G
G
G
G
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
C
C
T
C
C
C
C
C
C
C
C
C
C
C
C
C
G
G
G
G
G
G
G
G
T
C
C
A
A
A
T
T
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
C
C
T
T
T
T
T
T
A
A
G
G
G
G
G
G
A
A
A
A
A
A
A
A
G
C
T
G
G
G
G
G
G
A
T
G
G
G
G
G
C
T
C
C
C
C
C
C
T
C
T
C
C
C
C
C
A
A
A
A
A
A
A
A
T
T
T
T
T
T
T
T
A
C
C
T
T
T
T
T
T
C
C
T
T
T
T
T
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
A
G
C
A
A
A
A
A
C
G
G
G
G
G
G
G
A
G
A
A
G
G
A
A
T
T
G
G
G
G
G
G
C
C
T
C
C
T
C
C
A
A
G
C
T
T
C
C
A
A
A
G
G
G
G
G
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
C
C
C
G
C
C
C
C
G
G
A
A
A
A
A
A
A
A
G
A
A
A
A
A
T
T
G
C
C
C
C
C
C
C
C
C
C
C
C
C
T
T
T
T
T
T
T
T
A
C
C
T
T
G
T
T
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
T
T
T
T
T
T
T
T
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
C
C
C
C
C
C
C
C
T
A
T
A
G
A
A
A
T
T
T
T
T
T
T
T
C
C
C
C
C
C
C
C
A
T
T
T
T
T
T
T
A
T
T
T
T
T
T
T
G
C
G
T
T
T
T
T
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
G
C
A
C
T
C
C
T
T
C
T
A
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
T
C
C
T
C
C
C
C
C
A
A
G
A
A
A
A
A
A
G
G
G
G
G
G
G
C
C
C
C
C
C
C
C
A
G
G
A
A
A
A
A
A
T
A
A
A
A
A
A
G
G
G
G
G
G
G
G
470 Gsp-1
422 Pina-D1a
427 Pinb-D1a
429 Pinb-2v1
429 Pinb-2v2
429 Pinb-2v3
429 Pinb-2v4
429 Pinb-2v5
G
A
G
G
G
G
G
G
T
T
T
C
C
T
C
C
C
C
G
C
A
A
C
C
T
G
A
A
A
A
A
A
T
G
G
G
G
G
G
G
A
G
G
A
A
A
A
A
A
C
T
T
T
T
T
T
A
A
A
A
A
A
A
A
G
A
T
A
A
A
A
A
C
G
T
C
C
C
C
C
C
C
C
C
G
C
C
C
A
A
A
A
A
A
A
A
G
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
G
C
C
C
C
C
C
C
T
A
A
A
A
A
A
G
G
A
A
A
A
A
A
G
A
C
A
A
A
A
A
T
T
T
T
T
T
T
T
G
A
T
T
T
T
T
T
C
C
C
C
C
C
C
C
A
A
A
A
A
A
A
A
G
A
G
G
G
G
G
G
A
G
A
A
A
A
A
A
C
A
G
G
G
G
G
G
G
A
G
G
G
G
G
G
G
G
G
G
G
G
G
G
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
C
A
A
C
A
A
A
A
A
A
A
A
T
T
T
T
T
G
G
G
G
G
G
G
G
A
A
A
A
A
A
A
A
G
A
G
T
T
T
T
T
C
C
C
G
G
G
G
G
C
C
C
T
T
T
T
T
T
T
T
T
T
T
T
T
T
G
C
G
G
G
G
G
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495 Gsp-1
447 Pina-D1a
447 Pinb-D1a
453 Pinb-2v1
453 Pinb-2v2
453 Pinb-2v3
453 Pinb-2v4
453 Pinb-2v5
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Fig. 1. DNA sequence alignment of Puroindoline b-2 gene variants Pinb-2v1, Pinb-2v2, Pinb-
2v3, Pinb-2v4, Pinb-2v5, Pinb-D1a, Pina-D1a as well as Gsp-1 from bread wheat.

285
Wild type Pinb-D1b Total
Pinb-2v2 Pinb-2v3 Pinb-2v2 Pinb-2v3 Pinb-2v2 Pinb-2v3
No. of sample 2 23 19 48 21 71
SKCS Hardness 14.2a 24.6b 67.5a 60.7b - -
1000-kernel weight 59.2a 54.7b 50.9a 52.2b 51.7a 52.9a
Grain length (mm) 6.85a 6.8a 6.61a 6.73a 6.63a 6.66a
Grain width (mm) 3.65a 3.57a 3.47a 3.55a 3.49a 3.50a
Spikelet number per
spike
20.1a 20.83a 20.09a 20.59a 20.10a 20.67a
Grain number per spike 42.1a 45.18b 40.7a 43.7b 40.86a 44.17b
Grain weight per spike
(g)
2.11a 2.27a 1.95a 2.17b 1.96a 2.21b
Length of flag leaf (cm) 16.9a 17.4a 15.7a 17.9b 15.77a 17.70b
Width of flag leaf (cm) 1.85a 1.96a 1.85a 1.92a 1.85a 1.93a
Area of flag leaf (cm
2
) 23.6a 25.6b 21.7a 25.7b 21.89a 25.69b
Different letters indicate significant difference at 5% probability level
Table 2. Comparison of two Puroindoline b-B2 variants on grain hardness and yield-related
traits
4. Discussion
Even though physical mapping of Puroindoline b-2 variants was conducted using aneuploids
of Chinese Spring by Chen et al. (2010), we re-mapped the Puroindoline b-2 variants in the
durum wheat cultivar Langdon and bread wheat cultivar Chinese Spring. The re-mapping
was performed using different stocks than those reported by Chen et al. (2010) due to the
controversial difference between the results of Chen et al. (2010) and Wilkinson et al. (2008).
According to this study and the previous report (Chen et al. 2010), Pinb-2v1 is located on
chromosome 7D and is present in all the bread wheat cultivars surveyed. Therefore, in this
study, the absence of Pinb-2v1 in durum wheat is expected. Pinb-2v2 and Pinb-2v3 are
reciprocally present on chromosome 7B in all of the bread wheat cultivars surveyed. Only
one durum cultivar possessed the Pinb-2v2/Pinb-2v4 haplotype combination, whereas 35
durum cultivars possessed the Pinb-2v3/Pinb-2v4 combination, suggesting that Pinb-2v3 and
Pinb-2v2 were likely allelic. In this set of durum germplasm, the Pinb-2v3/Pinb-2v4
combination was the predominant haplotype.
Wilkinson et al. (2008) mapped Puroindoline b-2 variant 2 to chromosome 7AL in three
doubled haploid populations and amplified sequences of Pinb-2v3 and Pinb-2v1 from the
genomic DNA of the durum wheat cultivar Ofanto. We did not find Pinb-2v1 in any of the
durum cultivars surveyed, including 48 sequencing results of cloned PCR amplicon in the

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durum wheat Langdon. The reason for this discrepancy is possibly due to primer sequence
and specificity (or lack thereof) for the various variant genes used in the two studies.
According to Chen et al. (2010), Pinb-2v1, Pinb-2v2, Pinb-2v3 and Pinb-2v4 are located on
chromosome 7DL, 7BL, 7B and 7AL in bread wheat, respectively. Coincidently, the strongest
QTL effects controlling grain yield, especially for grain weight, were found on chromosomes
7AL and 7BL in the report of Quarrie et al. (2005), and a QTL associated with grain yield has
also been identified on chromosome 7D in the study of Kuchel et al. (2007). More recently,
several QTLs including QTLs on 7A and 7B associated with grain yield and yield
components have been discovered in a recombinant inbred line population (McIntyre et al.
2009). However, further studies are required to determine if this is cause and effect or
simply linkage occurring.
Even though many QTLs controlling wheat grain yield and its components have been
studied for many years, no specific gene with detailed sequence has been reported so far.
Therefore, the possibility of the Puroindoline b-2 gene possessing some function in
modulating grain yield traits may provide useful information for MAS (Marker Assisted
Selection). Future studies should define the function of the Puroindoline b-2 genes by using
populations with defined genetic background.
5. Acknowledgements
This project was funded by the National Natural Science Foundation (31000708), Henan
Provincial Key Technologies R & D Program (114300510013) and Specialized Research Fund
for the Doctoral Program of Higher Education (20104105120003) of China.
6. References
Bhave M, Morris CF. 2008a. Molecular genetics of puroindolines and related genes: allelic
diversity in wheat and other grasses. Plant Molecular Biology 66, 205-219.
Bhave M Morris CF. 2008b. Molecular genetics of puroindolines and related genes:
regulation of expression, membrane binding properties and applications. Plant
Molecular Biology 66, 221-231.
Bihan TL, Blochet JE, Desormeaux A, Marion D, Pezolet M. 1996. Determination of the
secondary structure and conformation of puroindolines by infrared and Raman
spectroscopy. Biochemistry 35: 12712-12722.
Capparelli R, Borriello G, Giroux MJ, Amoroso MG. 2003. Puroindoline a-gene expression is
involved in association of puroindolines to starch. Theoretical and Applied
Genetics 107: 1463-1468.
Chantret N, Salse J, Sabot F, Rahman S, Bellec A, Laubin B, Dubois I, Dossat C, Sourdille P,
Joudrier P, Gautier MF, Cattolico L, Beckert M, Aubourg S, Weissenbach J,
Caboche M, Bernard M, Leroy P, Chalhoub B. 2005. Molecular basis of evolutionary
events that shaped the hardness locus in diploid and polyploid wheat species
(Triticum and Aegilops). Plant Cell 17: 1033-1045
Chen F, Beecher B, Morris CF. 2010a. Physical mapping and a new variant of Puroindoline b-2
genes in wheat. Theoretical and Applied Genetics 120:745-751.

287
Chen F, He ZH, Chen DS, Zhang CL, Zhang Y, Xia XC. 2007a. Influence of puroindoline
alleles on milling performance and qualities of Chinese noodles, steamed bread and
pan bread in spring wheats. Journal of Cereal Science 45: 59-66.
Chen F, He ZH, Xia XC, Xia LQ, Zhang XY, Lillemo M, Morris CF. 2006. Molecular and
biochemical characterization of puroindoline a and b alleles in Chinese
landraces and historical cultivars. Theoretical and Applied Genetics 112: 400-
409.
Chen F, Yu Y, Xia X, He Z. 2007b. Prevalence of a novel puroindoline b allele in Yunnan
endemic wheats (Triticum aestivum ssp. Yunnanense King). Euphytica 156: 39-46.
Giroux MJ, Morris CF. 1997. A glycine to serine change in puroindoline b is associated with
wheat grain hardness and low levels of starch-surface friabilin. Theoretical and
Applied Genetics 95, 857-864.
Giroux MJ, Morris CF. 1998. Wheat grain hardness results from highly conserved mutations
in the friabilin components puroindoline a and b. Proceedings of the National
Academy of the United States of America 95, 6262-6266.
Kuchel H, Williams JK., Langridge P, Eagles HA, Jefferies SP. 2007. Genetic dissection of
grain yield in bread wheat. I. QTL analysis, Theoretical and Applied Genetics 115:
1029-1041.
Luo LT, Zhang JR, Yang GX, Li Y, Li KX, He GY. 2008. Expression of puroindoline a
enhances leaf rust resistance in transgenic tetraploid wheat. Molecular Biology
Reports 35: 195-200.
McIntyre CL, Mathews KL, Rattey A, Chapman SC, Drenth J, Ghaderi, M, Reynolds M,
Shorter R. 2010. Molecular detection of genomic regions associated with grain
yield and yield-related components in an elite bread wheat cross evaluated
under irrigated and rainfed conditions. Theoretical and Applied Genetics, 120:
527-541.
Morris CF. 2002. Puroindolines, the molecular genetic basis of wheat grain hardness. Plant
Molecular Biology 48, 633-647.
Morris CF, Bhave M. 2008. Reconciliation of D-genome puroindoline allele designations
with current DNA sequence data. Journal of Cereal Science 48, 277-287.
Quarrie SA, Steed A, Calestani C, Semikbodskii A, Lebreton C, Chinoy C, Steele N,
Pljevlajakusic D, Habash DZ, Farmer P, Saker L, Clarkson DT, Abugalieva A,
Yessimbekova M, Turuspekov Y, Abugalieva S, Tuberosa R, Sanguineti M-C,
Hollington PA, Aragues R, Royo A, Dodig D. 2005. A high density genetic map of
hexaploid wheat (Triticum aestivum L.) from the cross Chinese Spring x SQ1 and its
use to compare QTLs for grain yield across a range of environments. Theoretical
and Applied Genetics 110: 865-880.
Ragupathy R, Cloutier S. 2008. Genome organization and retrotransposon driven molecular
evolution of the endosperm Hardness (Ha) locus in Triticum aestivum cv Glenlea.
Molecular Genetics Genomics 280: 467-481.
Shewry PR, Halford NG. 2002. Cereal seed storage proteins: structures, properties and role
in grain utilization. Journal of Experimental Botany 53: 947-958.

Gene Duplication

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Sourdille P, Perretant MR, Charmet G, Leroy P, Gautier MF, Joudrier P, Nelson JC, Sorrells
ME, Bernard M. 1996. Linkage between RFLP markers and genes affecting kernel
hardness in wheat. Theoretical and Applies Genetics 93: 580-586.
Turner AS, Bradburne RP, Fish L, Snape JW. 2004. New quantitative trait loci influencing
grain texture and protein content in bread wheat. Journal of Cereal Science 40: 51-
60.
Wilkinson M, Wan Y, Tosi P, Leverington M, Snape J, Mitchel RAC, Shewry PR. 2008.
Identification and genetic mapping of variant forms of puroindoline b expressed in
developing wheat grain. Journal of Cereal Science 48: 722-728.
16
Evolution of GPI-Aspartyl Proteinases
(Yapsines) of Candida spp
Berenice Parra-Ortega
1,2
, Lourdes Villa-Tanaca
1*

and Csar Hernndez-Rodrguez
*1

1
Departamento de Microbiologa,
Escuela Nacional de Ciencias Biolgicas del Instituto Politcnico Nacional
2
Centro de Diagnstico y Vigilancia Epidemiolgica de Distrito Federal
Instituto de Ciencia y Tecnologa
Mxico
1. Introduction
The Candida genus is a polyphyletic genus with at least 150 species. Nine are recognized
opportunistic pathogens of humans and animals. C. albicans is the species most frequently
isolated from human infections, followed by Candida non-Candida species (CNCA), as C.
glabrata, C. tropicalis, C. dubliniensis, C. parapsilosis, C. guilliermondii, C. lusitaniae, C. kefyr and
C. krusei (Man et al. 2008; Pfaller & Diekema, 2007; Almirante et al. 2005; Manzano-Gayosso
et al. 2000).
Some works describe the phylogenetic relationships of Candida genus and illustrate the
limited relationship between the pathogenic Candida spp. The genus has been divided into:
the CTG clade, which includes yeast that encodes CTG as serine instead of leucine (C.
albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. lusitaniae); and the WGD clade,
which includes yeast that has undergone a genome duplication event (Saccharomyces spp.,
Kluyveromyces spp. and C. glabrata). Evidently, C. glabrata is more related to non-pathogenic
yeasts, as Saccharomyces cerevisiae, than to the other pathogenic species (Scannell et al. 2007).
C. albicans is a normal microorganism in humans, and colonise up to 70% of skin, mucoses,
and faeces of individuals with no apparent detriment to health. However, in some
circumstances, either through environmental factors or a weakening of the host immune
system, a proliferation and infection by C. albicans arise inducing candidosis (Wei et al.
2011).
Biofilm formation, adhesion, cavitation, phenotypic switching, dimorphism, interaction with
the host immune system, invasion and tissue damage are virulence virulence factors for C.
albicans. All these factors are related to the secreted aspartyl proteases (Sap) family, which is
considered an important virulence factor and is studied as a possible target for therapeutic
drug design (Naglik et al. 2004; Chaffin et al. 1998; Hube, 1998; Naglik et al. 2003, 2004,
2008).
The topic of this chapter is to understand the molecular characteristics, evolution and
putative functions of glycosylphosphatidylinositol (GPI)-linked aspartyl proteases (Yps), a
protein superfamily distributed among all pathogenic Candida species. Cell location motifs,

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290
gene duplications, similitude, synteny, putative transcription factor binding sites and
genome traits of the Yps family members are analysed by bioinformatics tools in an
evolutionary context.
2. Aspartyl proteases
Aspartyl proteases or acid proteases (optimum activity at acidic pH) are proteins with a
signal peptide in the amino-terminal site, at least one aspartic residue in the active site, and
4 cysteins (Hube & Naglik, 2001). The signal peptide is processed in the endoplasmic
reticule and the protein is transported to their corresponding cell localization by the
secretory pathway. The active site is formed by different amino acids. The consensus pattern
described by PROSITE-EXPASY (http://expasy.org/prosite/) is [LIVMFGAC]-
[LIVMTADN]-[LIVFSA]-D-[ST]-G-[STAV]-[STAPDENQ]-{GQ}-[LIVMFSTNC]-{EGK}-
[LIVMFGTA], and the cysteins help the protein to the three dimensional structure by
intramolecular disulfide bond (Fig. 1). According to the cell localization, aspartyl proteases
could be secreted, or destined to vacuole or cell membrane by a GPI-linked site in the
carboxyl-terminal residues (Alberch et al. 2006; Jones, 1991; Naglik et al. 2003).

Fig. 1. Typical molecular structure of aspartyl proteases. SP: signal peptide; ASP: aspartic
residue in the active site, C: cystein.
2.1 Secreted aspartyl proteases (Sap)
The C. albicans secreted aspartyl protease family comprises ten members, eight of which are
proper secreted Sap1-Sap8, and two, Sap9 and Sap10, that have been reclassified as GPI-
anchored aspartyl proteases (Alberch et al. 2006). Nevertheless, Sap9 and Sap10 are clearly
more phylogenetically related to Sap than any GPI-anchored aspartyl proteases (Parra et al.
2009). The function of Sap in C. albicans has been widely studied, and these proteases are
important in proteolysis to get a source of nitrogen, and are differentially regulated
depending on the environmental conditions (Schaller et al. 1998; 2003; Taylor et al. 2005;
Naglik et al. 2008). SAP1-SAP3 are relevant in phenotypic switching during the opaque
phase and are not expressed in the WO-1 phase (Morrow et al. 1992; White et al. 1993). Also,
they are expressed when yeast colonize and damage reconstructed human epithelium, oral
and vaginal, which means that these Sap are important in superficial infections (Schaller et
al. 1998; 2003; Copping et al. 2005). SAP1-SAP8 are related to tissular damage (Taylor et al.
2005). SAP1, SAP3 and SAP8 are expressed in oral and vaginal infections. On the other
hand, SAP4-SAP6 are related to systemic infections and only they are expressed in yeast and
germ tube at pH 5-7 (Hube et al. 1997; Sanglard et al. 1997; White & Agabian, 1995).
Meanwhile SAP5 is important in epithelial colonization, invasion and infection (Naglik et al.
2008; Lermann & Morschhuser, 2008).
This kind of proteases are no exclusive of C. albicans. Orthologous genes have been
described in other closely related species, as C. dubliniensis (Sap1-4 and Sap7-10), C. tropicalis
(Sapt1-12), C. guilliermondii (Sapg1-8), C. parapsilosis (Sapp1-14) and C. lusitaniae (Sapl1-3)

Evolution of GPI-Aspartyl Proteinases (Yapsines) of Candida spp

291
(Parra et al. 2009). Particularly in C. dubliniensis, the expression of SAPD3 and SAPD4 genes
is related to the infection of keratinocyte (HaCAT cells) by yeast. The number and shape of
the keratinocyte cells was altered by the infection, but these effects decreased in the presence
of pepstatin A, an aspartyl protease inhibitor, suggesting that the Sapd3 and 4 of C.
dubliniensis could be considered as virulence factors, like their orthologous genes from C.
albicans (Loaiza-Loeza et al. 2009). The function of these proteases in metabolism and
pathogenesis in the rest of pathogenic species is unknown.
According to Dayhoff, protein superfamilies and families are defined as groups of related
proteins that exhibit less than 50% and greater than 50% similarity, respectively. Subfamilies
were defined as groups of proteins with at least 90% similarity and were often equivalent to
clusters of orthologous groups (COGs) (Dayhoff, 1979). Behind this idea, the phylogeny of
pathogenic Candida spp. Saps allows for the recognition of a superfamily with at least 12
paralogous families and nine orthologous subfamilies. In several Sap families, at least two
subfamilies or orthologous groups are proposed (Parra et al. 2009).
2.2 Vacuolar aspartyl proteases (PrA)
The vacuole is a hydrolytic organelle similar to lysosomes in animals and is the site of non-
specific degradation of cytoplasmic proteins (Robinson et al. 1988), proteins delivered via
autophagy (Klionsky & Emr, 2000), or plasma membrane proteins turned over via
endocytosis (Hicke, 1996). In S. cerevisiae the vacuole has been studied and possesses
different vacuolar proteases (Table 1).
One of the most important vacuolar proteins is the proteinase A (PrA), encoded by the PEP4
gene. Mutants in PEP4 (pep4) accumulate multiple zymogens, indicating that PrA initiates
processing, maturation and activation of multiple different precursors of PrB, DAP, CPY
and PrA, because of their autocatalytic activity and their lack of production of dead cells in
nutritional stress. Also, PrA is important in cellular response to starvation, microautophagy,
proteolysis involved in cellular and vacuolar protein catabolic process, and sporulation
(Palmer, 2007; Jones, 1991; Teichert et al. 1989).
The function of PrA, encoded by the CaPEP4 gene in the metabolism of C. albicans, has also
been studied. Null mutants of CaPEP4 maintain their hydrolytic activity intact, clearly
suggesting that C. albicans possesses an alternative system that compensates for the lack of
this gene (Palmer, 2007). In C. albicans, the vacuole is important in cell differentiation,
surviving into macrophages, and elimination of drugs as hygromicin B, orthovanadate and
rapamicine (Palmer, 2005).
In C. dubliniensis, this protein could be important in carbon and nitrogen metabolism and
might participate in protein degradation and precursor processing as occurs in S. cerevisiae
(Loaiza et al. 2007). The genome-wide environmental stress response expression profile of
C. glabrata revealed that CgPEP4 is induced in osmotic stress and glucose starved conditions.
Meanwhile, in S. cerevisiae no changes in the expression were observed in the same
conditions (Gash et al. 2000; Roetzer et al. 2008).
Bioinformatic genomic analysis of Candida pathogenic species exhibited that only one
version of PrA is harboured by yeast (Table 3), but apparently the CgPEP4 gene is
universally distributed among C. glabrata strains, as revealed by PCR multiplex in a
collection of 52 C. glabrata clinical strains (Table 5; Fig. 3; for PCR conditions see 2.3 section).
Phylogenetic analysis was performed by an aligment of PrA homologues identified in silico
and those previously characterized. The aligment was conducted using MUSCLE in
SeaView 2.4 program (Galtier et al. 1996) with default alignment parameter adjustments.

Gene Duplication

292
The phylogenetic analyses were performed in the MEGA4 program (Tamura et al. 2007)
using Maximun Parsimony evolution. A similitude and identity matrix were computed with
the MatGAT4.50.2 software (Campanella et al. 2003). The phylogenetic reconstruction and
similarity of PrA reproduce the phylogenetic tree topologies of Candida spp. obtained with
other genes, suggesting a common ancestral gene (Fig. 2; Table 2). In brief, C. albicans was
more related to C. dubliniensis, followed by C. tropicalis, C. parapsilosis, C. guilliermondi and C.
lusitaniae. Meanwhile, C. glabrata PrA was more related to S. cerevisiae PrA than other
Candida species.

Name/systematic
name
Gene/
Protein
Access number Function Reference
Proteinase A
YPL154C
PEP4/
PrA
NM_001183968
/ NP_015171
Activities of other yeast
vacuolar hydrolases
Parr et al., 2007
Carboxypeptidase
Y
YMR297W
CPY
NM_001182806
/NP_014026
Contributes to the
proteolytic function of the
vacuole
Wnschmann et
al., 2007
Proteinase B
YEL060C
PRB1
NM_001178875
/NP_010854
Involved in protein
degradation in the vacuole
and required for full
protein degradation during
sporulation
Teichert et al.,
1989
Carboxypeptidase S
YJL172W
CPS
X63068/
CAA44790
Nitrogen compound
metabolic process,
proteolysis involved in
cellular protein catabolic
processes
Bordallo &
Suarez-
Rendueles, 1993
Dipeptidyl
aminopeptidase B*
YHR028C
DAP-B
X15484/
CAA33512
Protein processing
Aminopeptidase
YKL103C
APEI
NM_001179669
/NP_012819
Catabolic processes
Table 1. Soluble and membrane-bound * vacuolar proteolytic system of S. cerevisiae.

C. albicans (Calorf19 1891)
C. dubliniensis (Cd36 21670)
C. tropicalis (CTRG 01724)
C. parapsilosis (CPAG 03663)
C. guilliermondii (PGUG 04145)
C. lusitaniae (CLUG 04124)
S. cerevisiae (YPL154C)
C. glabrata (CAGL0M02211g)

Fig. 2. Maximun Parsimony phylogenetic analysis of vacuolar aspartyl proteases (PrA)
superfamily from pathogenic Candida spp.


293
1 2 3 4 5 6 7 8
1. S. cerevisiae YPL154C 62 66 66 66 66 65 68
2. C. glabrata CAGL0M02211g 77 55 55 54 54 55 57
3. C. albicans orf19_1891 77 69 98 90 85 75 78
4. C. dubliniensis Cd36_21670 77 69 99 91 85 76 79
5. C. tropicalis CTRG_01724 76 69 97 97 86 75 77
6. C. parapsilosis CPAG_03663 76 67 91 92 93 74 78
7. C. guilliermondii PGUG_04145 79 69 84 85 85 85 78
8. C. lusitaniae CLUG_04124 78 69 86 86 87 86 87
Table 2. Similarity and identity (UP/down) between PrA proteins from pathogenic Candida
spp.

PrA (AN)
Amino
acid
residues
MM
(kDa)
IP MOTIF
Signal
peptide
(aa)
C
C. albicans
Calorf19_1891

419 45.4 4.5
119-130:
VILDTGSSNLWV
304-315:
AAIDTGTSLITL
20 7
C. dubliniensis
Cd36_21670
419 45.4 4.5 14 2
C. tropicalis
CTRG_01724
422 45.6 4.5
121-132:
VILDTGSSNLWV
306-317:
AAIDTGTSLITL
24

2: 1400605-
1401870-
C. parapsilosis
CPAG_03663
428 46 4.5
127-138:
VILDTGSSNLWV
312323:
AAIDTGTSLITL
25

130: 135636-
136919 -
C. guilliermondii
PGUG_04145
409 44 4.3
109-120:
VILDTGSSNLWV
294-305:
AAIDTGTSLITL
21 5: 355498-356724 -
C. glabrata
CLUG_04124

407 43 4.3
107-118:
VILDTGSSNLWV
292303:
AAIDTGTSLITL
19 5: 46984-48204 -
Table 3. Vacuolar aspartyl proteases in pathogenic Candida species. (AN): Access number in
the respective genome; MM: molecular mass; IP: Isoelectric Point; C: Chromosome or Contig
or supercontig.
C. glabrata is an opportunistic haploid yeast that suffered evident and extensive reductive
evolutionary events. A lot of genes involved in nitrogen metabolism, carbohydrate
assimilation (saccharose, galactose, etc.), as well as sulfur, phosphor, thiamine, pyridoxine
and nicotinic acid biosynthesis have been lost from the genome (Byrne & Wolfe, 2005;

Gene Duplication

294
Wolfe, 2006). This species produces between 15-20% of reported systemic yeast infections
(Almirante et al. 2005; Manzano-Gayosso et al. 2000; Trick et al. 2002; Man et al. 2008). C.
glabrata is the most common yeast species isolated from patients with cancer, organ
transplantation and fluconazole therapy (Safdar et al. 2001; Bodey et al. 2002). The mortality
associated with C. glabrata in systemic infections of cancer patients is 50% and almost 100%
in transplant patients (Anaissie et al. 1992; Goodman et al. 1992; Krcmery et al. 1998). This
scenario is related to indiscriminate antifungal use, and to the innate resistance of C. glabrata
(Sobel, 2006).
According to Table 4, virulence factors of C. albicans and C. glabrata are quite different.
However, an evident feature is the difference in number and kind of aspartyl proteases. A
total of 12 YPS genes, but no SAP genes have been detected in C. glabrata. Contrarily, a total
of 10 SAP genes, but no YPS genes have been recognized in C. albicans. Clearly, the
phylogenetic trees constructed with ribosomal or other gene groups include the majority of
the clinical relevant Candida species, with exception of C. glabrata, which is grouped in
another cluster with non-pathogenic yeasts, as S. cerevisiae and Kluyveromyces spp. This
evidence suggests that the aspartyl proteases in Candida spp. have evolved independently as
virulence factors at least two times, and possibly the amplification by duplication of SAP
and YPS gene superfamilies in clinically relevant species is an example of convergent
evolution.
A physiological approach could possibly contribute to the understanding of which C.
glabrata YPS (CgYPS) genes are covering the functions of each secreted aspartyl protease of
C. albicans under different conditions. Evidently, the comparison of virulence strategies,
expression profiles, complementation of mutants, among other experiments, could suggest
common and particular features and roles for all SAP and YPS genes. For now, the questions
remain open. Have the function of C. glabrata CgYPS and SAP C. albicans genes functionally
converged?
The transcription profile of 11 CgYPS was studied when yeast were ingested by
macrophages. Apparently, CgYPS are important in survival and virulence of the the yeast in
macrophages, damage to mousses, Epa1 protein processing, and cell wall integrity, as occur
in S. cerevisiae, which possesses 5 ScYPS (ScYPS1-ScYPS3, ScYPS6 and ScYPS7) (Kaur et al.
2007; Krysan et al. 2005). They are important to cell wall synthesis and glucan homeostasis,
mainly ScYPS1 and ScYPS7. It seems that ScYPS3 does not have functions associated with
the cell wall (Krysan et al. 2005).
C. albicans SAP9 and C. glabrata CgYPS1 genes complement the defects in the cell wall
provoked by yps1 of S. cerevisiae. One important difference is that SAP9 complement yps1
only when SAP9 is under a heterologous and constitutive promoter from S. cerevisiae, while
CgYPS1 complements the mutation, using its promoter (Krysan et al. 2005), evidence that
supports the othologous status proposed above for these gene pairs. As happened with
ScYPS1, SAP9 gene expression increases during the stationary phase and damage of the cell
wall (Monod et al. 1998; Copping et al. 2005), and protects the yeast from caspofungin (an
inhibitor of 1,3-glucan synthesis) (Lesage et al. 2004). Also, inhibitors of ScYps1p disable
the specificity of both proteins, ScYps1p and Sap9 (Cawley et al. 2003).
Distribution of the SAP gene superfamily among C. albicans strains is universal (Gilfillan et
al. 1998; Bautista et al. 2003; Parra et al. 2009), although one study concludes that the
distribution of SAP genes in clinical strains depends on infection associated with isolation


295
(Kalkanci et al. 2005). Given the number of CgYPS in C. glabrata and their potential role in
pathogenesis, it is important to establish the universality of CgYPS in C. glabrata
populations.

Factor C. glabrata C. albicans Reference
Infection sites Oral, vaginal, bloodstream Fidel et al. 1999
Mortality in systemic infection urinary tract
Abi-Said et al. 1997;
Krcmery, 1999
Virulence in animal models High Arendrup et al. 2002
Filamentation Present
Lachke et al. 2002; Csank
& Haynes, 2000
Biofilm formation High Castao et al. 2006
Adherence to oral
keratinocytes
Lower Higher
Nikawa et al. 1995;
Biasoli et al. 2002
Adherence to denture material Lower Higher
Luo & Samaranayake,
2002
Extracellular proteinase
activity
Absent Present Chakrabarti et al. 1991
Phospholipase activity
Isolation site
dependent
High
Samaranayake et al. 1994;
Ghannoum, 2000
Phenotypic switching Low High Brockert et al. 2003
IL-8 induction in oral
keratinocytes
Pseudohyphae
True hyphae
and
pseudohyphae
Schaller et al. 2002
GM-CSF induction in oral
keratinocytes
Weak Strong
Schaller et al. 2002; Li et
al. 2007a
Human-defensin resistance Strong Weak
Joly et al. 2004; Feng et al.
2005
Histatin resistance Partially resistant Susceptible Helmerhorst et al. 2005
Azole resistance High Low Sanglard et al. 1999
Molecules involved in
adherence
20 EPA genes ALS proteins
Castao et al. 2005; Hoyer
et al. 2001
SAP genes 0 10 Parra et al. 2009
YPS genes 12 0
Albrecht et al. 2006; Kaur
et al. 2007
This work
Table 4. Comparison of virulence factors of C. glabrata and C. albicans (modified from Li,
2007b).
Our group explored the CgYPS gene distribution among clinical isolates (n=52) and type
strains CBS138 and BG6 (N=2) by an original multiplex PCR procedure (Table 5). The yeasts
were routinely grown on YPD broth and DNA was extracted using a previously reported
protocol (Hoffman & Winston 1987). PCR was performed in a DNA thermal cycler 9600

Gene Duplication

296
(Applied Biosystems, Foster City, CA). Amplification reactions (25 L) were performed
using a buffer containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2 mM MgCl
2
, 0.2 mM of
each deoxynucleoside triphosphate, 0.6 M each primers, 4 ng/L of genomic DNA, and 1.5
U/L of Taq polymerase (Invitrogen). The PCR conditions included a denaturation step for
3 min at 94C, followed by 38 amplification cycles consisting of 1 min at 94C, 1 min
annealing temperature and 1 min at 72C. A final extension step was performed for 7 min at
72C. Fig. 3 shows the amplification products of CgYPS gene fragments of some
representative C. glabrata clinical strains electrophoresed in 1% agarose gels. Similar PCR
conditions were used to study the universal distribution of PrA.

Gene Primer Location
Expected
amplified
fragment
(bp)
Tm (C)
CgYPS1
CAGL0M04191g
F:5-TTCTGGTGACAGTTGTATCTTGG-3
R:5-GATAAATGAAACCAAAAGACCAGCG-3
+1326 a +1348
+1779 a +1803
477 55
CgYPS2
CAGL0E01419g
F:5-ACTCAACTTGTTTTTAACTTCGGTGGTGC-3
R:5-TAGCATGGAGAGTAGGATGTTAAACACC-3
+1234 a +1262
+1743 a +1770
536 61
CgYPS3
CAGL0E01727g
F:5-AAAGCAAGTCGTCGATGTCATCG-3
R:5-TTGCAACTAACACTAAAGTGGTGC-3
+951 a +973
+1580 a +1603
652 58
CgYPS4
CAGL0E01749g
F:5-TTCTGTGTTACCAGCAAAGGTTGC-3
R:5-TTAATGTAGTTCTCTTACGGAGAGC-3
+933 a +956
+1411 a +1435
502 55
CgYPS5
CAGL0E01771g
F:5-TATACATATATGCCAAGCAGCGTTGC-3
R:5-AACAAGGCAGTAACTGCTGATAAAGC-3
+934 a +959
+1528 a +1553
619
58

CgYPS6
CAGL0E01793g
F:5-ACCAGAAGGTAGCTGCATTAATCG-3
R:5-AATGGTAGCTAATATGGCAGCAACG-3
+887 a +910
+1542 a +1518
631
58

CgYPS7
CAGL0A02431g
F: 5-TATGGGACCAATCTATATAACGTCC-3
R: 5-TAAGTAGCATACGGTATGTAGCCC-3
+831 a +855
+1404 a +1427
596
55

CgYPS8
CAGL0E01815g
F: 5-TTGGGATTACAGGGTAATGATGC-3
R: 5-AACTCTTTTTTGAAGGTCAAAACGCG-3
+856 a +878
+1457 a +1482
626
58

CgYPS9
CAGL0E01837g
F: 5-TTCCGTAAATGTGACTGATTTCATGG-3
R: 5-ATCATAATGAGTATGGCAGAGTTGGC-3
+1071 a +1096
+1510 a +1535
464
58

CgYPS10
CAGL0E01859g
F: 5-TAATAAGACGGAAGCCATCAGACTGC-3
R: 5-TTGTAATTGCTGCTAGTACTAGGACG-3
+978 a +1003
+1479 a +1504
526
58

CgYPS11
CAGL0E01881g
F: 5-TTGGTGTCCCATACAAGGAAATGGTC-3
R: 5-AATCCACAAG ACCAGCAACA GGATAGC-3
+1100 a +1125
+1495 a +1521
421
61

CgYPS12
CAGL0J02288g
F: 5-AATTGCACATGAAGATTCCGTTGCG-3
R: 5-TATCAGTTATTGTAGCAGTTACTGGC-3
+1001 a +1025
+1542 a +1567
566
58

CgPEP4
CAGL0M02211g
F 5 -TATCTGAAGAGTGTCAATGACCCAGC-3
R 5-TACAGCCTCAGCTAAACTGACAACATTGG-3
+691 - +716
+1208 - +1236
545
58

Table 5. Primer pairs used for conventional multiplex PCR of C. glabrata YPS genes. Bp, Base
pair; Tm, Melting temperature.


297
The universality of the 12 CgYPS genes among all C. glabrata clinical isolates and type strains
was confirmed (Fig. 3), which suggests that all CgYPS are important to yeast life cycle as
pathogen or commensal, and probably are differentially regulated according to each
environmental condition, as occurs with C. albicans SAP.
C
G
L
1
C
G
L
3
C
G
L
4
C
G
L
5
C
G
L
6
C
G
L
7
1
0
0

p
b
C
G
L
2
1
0
0

p
b
653 CgYPS3
502 CgYPS4
C)
200
300
400
500
600
200
300
400
500
600
200
300
400
500
600
421 CgYPS11
536 CgYPS2
C
G
L
1
C
G
L
2
C
G
L
3
C
G
L
4
C
G
L
5
C
G
L
6
C
G
L
7
1
0
0

p
b
A)
C
G
L
2
C
G
L
4
C
G
L
2
200
300
400
500
600
1
0
0

p
b
B)
200
300
400
500
600
1
0
0

p
b
C
G
L
1
200
300
400
500
600
477 CgYPS1
596 CgYPS7
1
0
0

p
b
C
G
L
2
C
G
L
3
C
G
L
4
C
G
L
5
C
G
L
6
C
G
L
7
F)
526 CgYPS10
631 CgYPS6
1
0
0
p
b
C
G
L
1
C
G
L
2
C
G
L
3
C
G
L
4
C
G
L
5
C
G
L
6
C
G
L
7
200
300
400
500
600
200
300
400
500
600
200
300
400
500
600
545 CgPEP4
619 CgYPS5
1
0
0

p
b
C
G
L
1
1
0
0

p
b
C
G
L
2
C
G
L
3
C
G
L
4
C
G
L
5
C
G
L
6
C
G
L
7
D)
200
300
400
500
600
800
200
300
400
500
600
800
200
300
400
500
600
800
1
0
0

p
b
C
G
L
1
1
0
0

p
b
C
G
L
2
C
G
L
3
C
G
L
4
C
G
L
5
C
G
L
6
C
G
L
7
464 CgYPS9
526 CgYPS8
566 CgYPS12
E)

Fig. 3. Amplification of CgYPS C. glabrata gene fragments by multiplex PCR. A, CgYPS2 and
CgYPS11; B, CgYPS7 and CgYPS1; C, CgYPS3 and CgYPS4; D, CgYPS3 and CgPEP4; E,
CgYPS8, CgYPS12 and CgYPS9; F, CgYPS6 and CgPEP10.
2.3 YPS genes in clinically relevant Candida species
The genome sequence projects of Candida species allows for the exploration of whether YPS
genes are harboured in these opportunistic pathogen yeasts. C. dubliniensis sequences were
obtained from the Sanger Institute Microorganisms Sequencing Group
(http://www.sanger.ac.uk/sequencing/Candida/dubliniensis/). Sequences from C.
guilliermondii, C. lusitaniae, C. tropicalis and C. parapsilosis were obtained from
(http://www.broad.mit.edu/annotation/genome/candida_group/MultiHome.html). The
GenBank database (http://www.ncbi.nlm.nih.gov) was also used. The detection was made
by using the previous YPS and SAP genes detected in S. cerevisiae
(http://www.yeastgenome.org), C. glabrata (http://cbi.labri.fr/Genolevures/elt/CAGL)
and C. albicans (http://www.candidagenome.org) genomes, and the proteins detected by
BLAST analysis in NCBI. Also, the different patterns of motif that could be obtained were
used as a new query. In C. lusitaniae and C. guilliermondii only one YPS was detected.
Meanwhile in C. dubliniensis and C. albicans four YPS genes were detected, in C. tropicalis
two, and in C. parapsilosis six. Theoretical isoelectric point, molecular weight and amino acid
content were calculated using Antheprot 2000 version 5.2 (Table 6).
Prediction of motif sequences was performed with PROSITE (http://www.expasy.org)
(Falquet et al. 2002). Some of the proteins possess a typical molecular structure of aspartyl
proteases, but others have some differences in composition (Fig. 4; Table 6). Some of them
possess high Ser/Thr content in the amino terminal, suggesting that this zone is exposed at
the surface of the protein. The presence of Ser/Thr in the carboxyl terminal in almost all YPS

Gene Duplication

298
is postulated to be heavily O-glycosylated. The exact function of this Ser/Thr-rich domain in
yapsins has not been investigated. However, O-mannosylation is important for proper cell-
wall biogenesis and integrity. It has also been proposed that clustered O-glycans create rigid
stalks that keep protein domains away from membranes or wall surfaces (Lipke & Ovalle,
1998).

Yps (AN)
Amino
acid
residues
MM
(kDa)
IP MOTIF
Signal peptide
(aa)
C
ScYps1
YLR120C
569 60 4.5
98-109:
VLVDTGSSDLWI
368-379:
ALLDSGTTLTYL
21 XVII
ScYps2
YDR144C
596 64.2 4.3
96-107:
VLVDTGSSDLWV
356-368:
VLLDSGTTISYM
496-570:
SER
18 IV
ScYps3
YLR121C
508 54.5 8.4
78-89:
VLLDTGSADLWV
285-296:
ALLDSGTTLTYL
439-470:
THR
20 XVII
ScYps6
YLR039C
537 58.2 3.9
82-93: LQLDTGSSDMIV
321-32:
VMLDSGTTFSYL
24 IX
ScYps7
YDR349C
596 64.4 4.6
71-82:
LLVDVIIQPYINL
318-329:
ALLDSTSSVSYL
16 IV
ScBar1
YIL015W
587

60-71:
VLFDTGSADFWV
284-295:
VLLDSGTSLLNA

CgYps1
CAGL0M04191g
601 63.8 5.0
88-99: VLVDTGSSDLWI
375-386:
ALLDSGTTLTYL
18 M
CgYps2
CAGL0E01419g
591 63.2 4.4
82-92:
LLLDTGSSDMWV
366-377:
ALLDSGTTVSYL
18 E
CgYps3
CAGL0E01727g
539 58.9 6.4
66-79:
VQLDTGSSDLWF
305-316:
VLLDTGTTLAYA
14 E
CgYps4
CAGL0E01749g
482 53.2 8.4
65-76:
VQLDTGSSDLWF
303-14:
TLLDTGVTTSVL
15 E


299
Yps (AN)
Amino
acid
residues
MM
(kDa)
IP MOTIF
Signal peptide
(aa)
C
CgYps5
CAGL0E01771g
519 57.2 5.5
66-77:
VQLDTGSSDLWF
304-315:
ALLDTGTTYTYM
60-70:
LECT
15 E
CgYps6
CAGL0E01793g
516 55.9 4.6
65-76:
VQLDTGSADLWF
301-312: ALIDSGTTISEF
62-68:
LECT
15 E
CgYps7
CAGL0A02431g
587 63.4 4.7
64-75:
LGLGLAQPYVWV
302-313:
VLLDPSFALSYL
18 A
CgYps8
CAGL0E01815g
519 56.7 6.8
65-76:
VQLDTGSSDLWF
304-315:
ALLDSGTTLTVV
15 E
CgYps9
CAGL0E01837g
521 56.9 5.1
65-76: LQIDTGSSDLFV
300-311:
TLLDSGSTISLL
16 E
CgYps10
CAGL0E01859g
505 55.3 7.3
61-72:
AQLDTGSSDLWF
298-309:
ALFDSGTSYSYV
13 E
CgYps11
CAGL0E01881g
508 55.6 5.0
63-74:
LLVDTGSSDFWV
310-321:
ALLDTGSTDTHL
29 E
CgYps12
CAGL0J02288g
541 59.5 4.6
68-79:
LVLDTGSSDLWV
279-290:
ALLDTGSTLIEL
448-495:
SER
19 J
orf19_852 365 39.6 5.4
73-84: LAADTGSWLIQI
245-256:
YTIDTGGRYGFL
17 2
orf19.6481 702 75.9 4.4
159-170:
LRLDLIQPEIWV
406-417:
VLLDSRASNFYL
565-662:
SER
20 7

Gene Duplication

300
Yps (AN)
Amino
acid
residues
MM
(kDa)
IP MOTIF
Signal peptide
(aa)
C
orf19_853 364 39.1 5.7
72-83: LSIDTGSWLTHI
244-255:
YTLDTGGGTGFL
42-44:
RGD
17 2
orf19_2082* 436 47.7 3.8
53-64:
VIVDSGSSDLMI
229-240:
YQIDSGTNGFVP
14 2
Cd36_18360

72-82:
VVII-1DTGSWLTHI
848-859:
YTLDTGGGNGYL
17 2
Cd36_18370 365 40 5.6
73-84:
IAADTGSWLTQI
246-257:
YTMDTGGGYGYL
17 2
Cd36_72090

697 76.6 4.6
149-150:
LRLDLIQPEIWVM
402-412: VILDSRASNFY
13 7
Cd36_15430

442 48.7 4.2
60-71:
VII-1VDSGSSDLMI
236-47:
YQIDSGTNGFVP
27 2
CTRG_05014 690 74.8 4
151-162:
LRLDLIQPEIWV
401-412:
VLIDSRSSYFYL
20
7: 407395-
409464 -
CTRG_01112

432 47.9 3.8
55-66:
VII-1VDSGSSDLMI
232-243:
YQIDSGSNGFLP
392-423:
THR
20
2: 57814-
59109 -
CPAG_04785 369 40.5 4.5
72-83:
VMIDTGSWRLNV
245-256:
IGIDSGNPRLAF
20
139:296423
-297529 -
CPAG_04801 374 40.5 5.7
251-262:
LALDTGNPGIGL
76-77: VFIDTGSWALNF
19
139:334234
-335355 -
CPAG_04802 371 40.4 6.1
76-87:
VVII-1DTGSWALNF
248-259:
19
139:337822
-338934 +


301
Yps (AN)
Amino
acid
residues
MM
(kDa)
IP MOTIF
Signal peptide
(aa)
C
LAFDTGSAGLIL
CPAG_03253 366 40.9 6.4
74-85:
VLLDTASTVLNV
246-257:
VLHDSGTPTMEL
15
126: 70630-
71727 +
CPAG_02564 366 40.5 6.5
74-85:
VLLDTASIVLNV
246-255:
VLHDSGTPTMAL
15
116:138449
-139546 -
CPAG_04713 700 75.5 4.5
157-168:
LRLDLIQPEVWV
417-428:
VLLDSRILYSYL
19-55: SER
27
139:123872
-125971 +
PGUG_04882
583

63.5 4.1
81-92:
LRLDLTQPEIWV
224-235:
LVQQGVIIKSSAY
16
6: 496161-
497909
CLUG_00903 722 74 3
63-74:
VLLDTGSSDLWV
275-286:
ALLDSGTSLQYL
470-701: SER
540638: THR
14
1: 1836367-
1838532 +
Table 6. Aspartyl proteases GPI- linked to cell membrane in pathogenic Candida spp. AN:
Access number in the respective genome; MM: molecular mass in kilodaltons (kDa); IP:
Isoelectric Point; C: Chromosome/Contig or supercontig; the atypical amino acids in the
PROSITE motif are shown in black (Eukaryotic and viral aspartyl protease active site).
The presence of a GPI attachment site, a characteristic feature of the yapsin family, was
determined with big-PI predictor (http://mendel.imp.univie.ac.at/gpi/gpi_server.html),
and GPI-SOM. GPI-anchor signals were identified by a Kohonen Self Organizing Map
(http://gpi.unibe.ch/). A total of 36 protein sequences were analyzed, but GPI sites were
recognized only in 21 proteins. GPI sites were not detected in ScYps2 and CgYps2, although
both proteins have been previously confirmed as Yps proteins. The software programs must
be enhanced, but an experimental approach to confirm the cell location is necessary.
PSORTII (http://www.psort.org/) and Softberry (http://www.softberry.com) programs
were used to predict subcellular localization. All proteins detected seem to be extracellular,
which could be because of the presence of a signal peptide in the amino terminal extreme.
Nevertheless during their synthesis, yapsins are cotranslocated and modified by the
addition of GPI to the lumen of the endoplasmic reticulum (ER). Then proteins are
glycosylated in Golgi apparatus, associated to membrane vesicles and sent to plasma
membrane or the cell wall (Mayor & Rieaman, 2004; Caro et al. 1997). Softberry program
was also used to find exons, which were absent in all genes studied. A search was made for

Gene Duplication

302
internal protein sequence repeats to detect possible internal duplication events, but none
were detected by TRUST (Szklarczyk & Heringa, 2004) even though it is likely were not

Fig. 4. Motifs of Candida spp. GPI-anchored aspartyl proteases (Yps). Rectangle boxes (SP):
amine terminal signal peptide; pentagon (ASP): aspartyl protease domains in agreement
with PROSITE; circles (ASP): atypical aspartyl protease domains proposed as
[LIVMFGACTPSYF]-(LIVMTADNQSFH)-(LIVFSAE)-D-(STP)-(GS)-(STAV)-(STAPDENQY)-
X-(LIVMFSTNCGQ)-(LIVMFGTAW); hexagons: serine (SER), threonine (THR), lecithin
(LEC) rich regions; star: RGD motif; rhombus (C): cysteine residues, semicircles. Ca, C.
albicans; Cd, C. dubliniensis; Cg, C. glabrata; Cgu, C. guilliermondii; Cl, C. lusitaniae; Cp, C.
parapsilosis; Ct, C. tropicalis; Sc, S. cerevisiae. A) ScYps1 (YLR120C), ScYps6 (YLR139C),
CgYps1 (CAGL0M04191g), CgYps2 (CAGL0E01419g), CgYps11 (CAGL0E01881g); B) ScYps2
(YDR144C); C) ScYps3 (YLR121C); D) ScYps7 (YDR349C), CdYps (Cd36_18370), CpYps
(CPAG_04785), CpYps (CPAG_04801), CpYps (CPAG_04802), CpYps (CPAG_03253), CpYps
(CPAG_02564), CguYps (PGUG_04882), CgYps3 (CAGL0E01727g), CgYps4
(CAGL0E01749g), CgYps7 (CAGL0A02431g), CgYps9 (CAGL0E01837g), CaYps (orf19_852),
CdYps (Cd36_72090); E) CdYps (Cd36_15430), CaYps (orf19.2082); F) CtYps (CTRG_01112);
G) CpYps (CPAG_04713); H) CgYps8 (CAGL0E01815g), CgYps10 (CAGL0E01859g); I)
ClYps (CLUG_00903); J) CtYps (CTRG_05014); K) CgYps5 (CAGL0E01771g), CgYps6
(CAGL0E01793g); L) CgYps12 (CAGL0J02288g); M) CaYps (orf19.6481); N) CaYps
(orf19_853), CdYps (Cd36_18360).


303
detected by TRUST (Szklarczyk & Heringa, 2004) even when it is likely that the Yps and Sap
superfamilies have duplicated aspartyl protease motifs.
The analysis of possible evolutive and molecular events that has given place to the presence
of different numbers of YPS in each pathogenic Candida species was made to establish the
COGs between Yps. Phylogenetic analysis was performed by an alignment of YPS
homologues identified in silico and those of the previously characterized. The alignment was
carried out using MUSCLE in SeaView 2.4 program (Galtier et al. 1996) with default
alignment parameter adjustments. The phylogenetic analyses were performed in the
MEGA4 program (Tamura et al. 2007) using minimum evolution computed with the Poisson
correction. A similitude and identity matrix were computed with the MatGAT4.50.2
software (Campanella et al. 2003). To corroborate support for the branches on trees,
bootstrap analysis (1,000 replicates) was performed. Synteny analysis was made to
recognize the putative COGs (Fig. 5).
CgYps6 (CAGL0E01793g)
CgYps1 (CAGL0M04191g)
ScYap1 (YLR120C)
ScYap2 YDR144C
ScYap3 (YLR121C)
ScYap6 (YIR039C)
CgYps12 (CAGL0J02288g)
ScBar1 (YIL015W )
Cl (CLUG_00903)
Ca (orf19.6481)
Cd (Cd36_72090)
Ct (CTRG_05014)
Cp (CPAG_04713)
Cgu (PGUG_04882)
CgYps7 (CAGL0A02431g)
ScYap7 (YDR349C)
Ca (orf19.2082)
Cd (Cd36_15430)
Ct (CTRG_01112)
Ca (orf19.852)
Cd (Cd36_18370)
Ca (orf19.853)
Cd (Cd36_18360)
Cp (CPAG_03253)
Cp (CPAG_02564)
Cp (CPAG_04785)
Cp (CPAG_04801)
Cp (CPAG_04802)
100
100
99
100
100
97
99
100
100
100
100
76
100
100
100
98
98
93
89
74
60
55
26
25
20
30
63
79
89
35
39
73
57
0.1
A) CgYps2-6 y 8-11 (C. glabrata)
C) CgYps12 (C. glabrata) Bar1 (S. cerevisiae)
D) ClYps (C. lusitaniae)
E) Yps (C. albicans, C. dubliniensis,
C. tropicalis, C. parapsilosis,
C. guilliermondii); CgYps7 (C. glabrata);
ScYps7 (S. cerevisiae)
F) Yps (C. albicans, C.
dubliniensis, C. tropicalis)
G) Yps (C. albicans,
C. dubliniensis)
H) CpYps (C. parapsilosis)
Family
B) CgYps1 (C. glabrata);
ScYps1-3 y 6 (S. cerevisiae)

Fig. 5. Minimum evolution phylogenetic tree of GPI-anchored aspartyl proteinase (Yps)
superfamily of opportunistic pathogenic Candida species. Ca, C. albicans; Cd, C. dubliniensis;
Cg, C. glabrata; Cgu, C. guilliermondii; Cl, C. lusitaniae; Cp, C. parapsilosis; Ct, C. tropicalis; Sc,
S. cerevisiae. Bootstrap values > 50% are on branches. Curly brackets and arrows indicate the
Yps protein families defined by phylogenetic relationships, similitude percentage (> 50%),
synteny and motif array. Yps are grouped into 8 families. Family A, CgYps2-6 and 8-11;
family B, CgYps1, ScYps1-3 and ScYps6; family C, CgYps12 and ScBar1; family D, ClYps (C.
lusitaniae); family E, CgYps7, ScYps7, CaYps (orf19.6481), CdYps (Cd36_72090), CtYps
(CTRG_05014), CpYps (CPAG_04713) and CguYps (PGUG_04882); family F, CaYps
(orf19.2082), CdYps (Cd36_15430) and CtYps (CTRG_01112); family G, CaYps (orf19.852),
CdYps (Cd36_18370), CaYps (orf19.853) and CdYps (Cd36_18360); family H, CpYps
(CPAG_03253, CPAG_02564, CPAG_04785, CPAG_04801 and CPAG_04802).

Gene Duplication

304

Fig. 6. Synteny of YPS genes of S. cerevisiae (Sc), C. glabrata (Cg), C. albicans (Caand
C.dubliniensis (Cd). A) ScYPS1 and CgYPS1; B) ScYPS2 and CgYPS2; C) ScYPS7 and CgYPS7;
D) CaYPS7 (orf19.6481) and CdYPS7 (Cd36_72090); E) CaYPS (Sap99), Cd (orf19.853 and
Sap98, orf19.852); F) CaYPS and CdYPS (Bar1); G) CgYPS and ScBar1. CgYPS1, CgYPS7,
ScYPS1, ScYPS3, CaYPS7, CaSAP98, CaSAP99 and BAR1 are GPI anchored aspartyl
proteases; APC2 and CAGL0M04235g, subunit of the anaphase-promoting; APT1, acyl-
protein thioesterase; ATP22, mitochondrial inner membrane protein; CAGL0M04147g,
similar to low affinity vacuolar membrane, is a localized monovalent cation/H+ antiporter
protein; CAGL0M04169g, similar to cell wall glycoprotein involved in beta-glucan assembly;
CDH1, cell-cycle regulated activator of the anaphase-promoting complex/cyclosome
(APC/C); CLF1, crooked neck-like factor; DOP1, protein essential for viability; EKL1,
ethanolamine kinase; FAF1, protein required for pre-rRNA processing and 40S ribosomal
subunit assembly; HAT, histone acetyltransferase; HXT3, low affinity glucose transporter of
the major facilitator superfamily; LDG3 and LDG4, leucine, aspartic acid, glycine rich;
MNN42, putative positive regulator of mannosylphosphate transferase; MNT3, alpha-1,3-
mannosyltransferase; MTQ2, S-adenosylmethionine-dependent methyltransferase; MRP1,
mitochondrial ribosomal protein of the small subunit; NOP16, constituent of 66S pre-
ribosomal particles; NTA1, amidase; orf19.2088, shared subunit of DNA polymerase (II)
epsilon and of ISW2/yCHRAC chromatin accessibility complex; PDR11, ATP-binding
cassette transporter, PEX7, peroxisomal signal receptor; PFK27, 6-phosphofructo-2-kinase;
PHHB, transposon mutation affects filamentous growth; PMI40, mannose-6-phosphate


305
isomerase; POM152, nuclear pore membrane glycoprotein; RPL2B, protein component of the
large ribosomal subunit; SAN1, ubiquitin-protein-ligase; SBE2, protein involved in the
transport of cell wall components from the Golgi to the cell surface; SEC1, Sm-like protein
involved in docking and fusion of exocytic vesicles through binding to assembled SNARE
complexes at the membrane; SNL1, putative protein involved in nuclear pore complex
biogenesis and maintenance; SRN2, component of the ESCRT-I complex; SVF1, protein with
a potential role in cell survival pathways; SW15, transcription factor that activates
transcription of genes expressed at the M/G1 phase boundary and in G1 phase; TAF12,
subunit (61/68 kDa) of TFIID and SAGA complexes; TIR3, cell wall mannoprotein of the
Srp1p/Tip1p family of serine-alanine-rich proteins; TMA20, protein associated with
ribosomes with a putative RNA binding domain; UGA11: gamma-aminobutyrate
transaminase (4-aminobutyrate aminotransferase); VID28, protein involved in proteasome-
dependent catabolite degradation of fructose-1,6-bisphosphatase (FBPase); v-SNARE,
component of the vacuolar SNARE complex involved in vesicle fusion; YCF1, putative
glutathione S-conjugate transporter; YLR126C, protein with similarity to glutamine
amidotransferase proteins; YMD8, putative nucleotide sugar transporter; ORF, APM2, BSC6,
CAGL0M04125g, Cd36_72050, Cd36_72080, FM02, IFK2, orf19.6482, RTC1, tRNA-Glu,
YDR352W, YDR348C and YLR125W and ORF, unknown predicted open reading frame.
The lack of SAP genes and the expansion of 12 CgYPS genes in C. glabrata, and the extended
family of SAP genes in C. albicans support the hypothesis that both protein superfamilies are
an example of convergent evolution. Although more research is necessary to reach definite
conclusions, apparently YPS of C. glabrata and SAP of C. albicans have developed some
equivalent physiological functions and roles in virulence. The rest of pathogenic Candida
species are less virulent, and, curiously, harbour less genes in their genomes than C. albicans.
These facts lead to the supposition that SAP and YPS have evolved in an independent way
for at least 700 million years. However, more SAP duplication events have happened in C.
albicans (Parra et al. 2009).
Phylogenetic analyses of Yps deduced protein sequences of Candida spp. and S. cerevisiae
allow for the definition of 8 Yps families, A-H (Fig. 5). In particular, CgYps1-12 proteins of
C. glabrata were clustered in four families. Family A was constituted exclusively of nine Yps
of C. glabrata (CgYps2-6 and CgYps8-11) encoded in chromosome E. With exception of
CgYps2, all codifying genes of these proteins are organized in tandem, and possibly derived
from at least eight recent duplication events that occurred exclusively in the C. glabrata
genome. Apparently these recent duplications led to the emergence of a paralogous gene
family with novel or slightly different functions. No pseudogenes were detected in CgYPS1-
11 genes, but in their deduced proteins a moderate amino acid similitude (48-53%) and
identity (36-38%) were retained. Frequently, very high similitudes are maintained by
concerted evolution in paralogous members of some multigene families (Lszl, 1999).
However, in CgYPS genes, this evolutive phenomenon is not evident. Previously, CgYPS4
and CgYPS11 were recognized as GPI anchored aspartyl proteases (Kaur et al., 2007), but
comparative studies of the regulatory region and expression of each CgYPS genes are
necessary to clearly define the physiological role and orthology relationships of each gene.
Family B was formed by a set of Yps proteins, detected exclusively in S. cerevisiae (ScYps2-3
and ScYps6), and a highly similar putative orthologous pair (ScYPS1/CgYPS1) (Fig. 6A).
Also, the partial synteny observed between the ScYPS2/CgYPS2 gene pair supports the
hypothesis that those protein-coding genes are probable orthologous (Fig. 6B). Family C was

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306
integrated by CgYps12 and ScBar1 of S. cerevisiae, a putative orthologous pair with low
similitude synteny, but with a clear ancestor-descendant relationship (Fig. 6G). Finally,
family E was formed by a representative of each Candida spp. Yps, CgYps7 and ScYps7. This
family forms a sub tree with the same topology as those phylogenies constructed with
ribosomal and other protein sequences (Diezman et al., 2004). The CgYPS7 and ScYPS7
genes exhibited an extensive synteny (Fig. 6C), but no synteny with CaYPS (orf19.6481) and
CdYPS (Cd36_72090) was observed (Fig. 6D). In C. albicans and C. dubliniensis genome
databases these YPS are described as ScYPS7 orthologous genes (Schaefer et al., 2007).
Nevertheless, both YPS exhibited low similarity with ScYPS7 (37.2-38.7%) and no-synteny.
The final decision to consider family E as an orthologous family will depend on comparative
analyses of functional features not yet performed.
Families C, F, G and H have not any C. glabrata or S. cerevisiae Yps representative protein.
Families C and H were formed only by one ClYps gene of C. lusitaniae and seven CpYps
genes of C. parapsilosis, respectively (Fig. 5). Curiously, C. lusitaniae is the species that
harbours the fewest ClYPS (n=1) and SAP (n=3) genes, and its isolation frequency from
clinical samples ,as well as its virulence, are lower than the other Candida species (Abi-Said
et al. 1997). This evidence supports a hypothesis of relevance of aspartyl proteases in
virulence. That is, species with numerous aspartyl proteases in virulence; species with broad
aspartyl proteases are more virulent than those with a limited number of these proteins.
Family F harboured C. albicans, C. dubliniensis and C. tropicalis yapsins organized
congruently according to the ribosomal phylogenetic tree. The C. albicans CaBar1
(orf19.2082) and C. dubliniensis CdBar1 (Cd36_15430) gene, found in family F, has been
described as orthologous to S. cerevisiae BAR1 (Schaefer et al., 2007) found in family C. In
both species, C. albicans and S. cerevisiae, the protein is involved in alpha pheromone
degradation and secreted to the periplasmic space of mating alpha-type cells. These proteins
help cells find mating partners by cleaving and inactivating the alpha factor, which allows
cells to recover from alpha-factor-induced cell cycle arrest (Mackay et al., 1988). The in silico
analysis performed in this work established that these proteins and the Bar1 from C.
dubliniensis are extracellular, but anchored to the cell wall or cell membrane. Also,
phylogenetic analysis shows that Bar1 from C. albicans and C. dubliniensis belongs to the Yps
superfamily, with a similarity of 40%, and are not grouped with CgYps12 of C. glabrata
(CgYps12 or CgBar1) and Bar1 of S. cerevisiae. The reason for which an aspartyl protease,
that apparently is secreted, is groupedwith the yapsines superfamily could be a mistake in
the cell location method because almost all software use the signal peptide, transmembranal
regions, and the GPI site in the C-terminal, to predict the cell location. In C. albicans it has
been detected that aspartyl proteases are associated with the plasmatic membrane, or to
both the plasmatic membrane and cell wall. This makes the experimental corroboration of
the cell location necessary. The Bar1 protein of C. albicans has been described as a protein
with three domains: 2 aspartyl protease domains and another unidentified. Apparently, this
GPI-membrane anchored domain determines that Bar proteins are not secreted, but
anchored to cellular membranes, and their two actives sites are oriented to cellular
membranes, and their two actives sites are oriented to the exterior to inactivate alpha
pheromone, which is secreted by Mat-alpha cells. In C. albicans, the degradation of secreted
alpha pheromone is not exclusive to Bar1. CaYPS7 (orf19.6481) of family E also encodes for
this function with lesser efficiency (Schaefer et al., 2007). This physiological redundancy has
not been demonstrated in S. cerevisiae ScYps7. C. albicans can mate under some in vitro and in


307
vivo conditions when alpha pheromone is degraded (Hull et al., 2000; Magee & Magee, 2000)
and C. glabrata harbours homologous genes of S. cerevisiae that control the mating (Srikantha
et al., 2003). Nevertheless, in C. glabrata a cell cycle has not been demonstrated, and the
participation of CgYps7 of C. glabrata in alpha pheromone inactivation has not been
demonstrated. No possible gene orthologous to possible gene orthologous to ScBar1 was
detected in C. guilliermondii, C. lusitaniae, C. parapsilosis, C. tropicalis, C. guilliermondii or C.
lusitaniae. All these yeasts have a heterothallic sex cycle (cross-mating only), but C.
parapsilosis and C. tropicalis mating has never been observed (Butler et al., 2009).
Family G is formed by two C. albicans/C. dubliniensis Yps protein pairs with high similitude
(>88%), located in tandem in chromosome 2 and with very similar synteny. All this data is
evidence from the recent speciation of both species (Fig. 6E). According to the Candida
genome database (http://www.candidagenome.org/cgi-bin/locus.pl?locus=orf19.852) Cal
orf19.852 and Cdu Cd36_18370 sequences are described as CaSAP98 and CdSAP98 genes,
respectively, and have their best hits with PEP4 of S. cerevisiae (Pra protein). S. cerevisiae PrA
is a vacuolar protease, and clearly C. albicans/C. dubliniensis Yps are not phylogenetically
grouped with PrA. In our opinion no orthology relationship among these proteins exists.
Cal orf19.853 and Cdu Cd36_18360 formed a second pair, described as CaSAP99 and
CdSAP99 genes, which had their best hits with ScYPS3 of S. Cerevisiae. Similarly, it is clear
that CaSAP99 has no synteny, phylogenetic relationship, or possible common physiological
role with ScYPS3.
3. Conclusion
Why have C. albicans/C. dubliniensis and C. glabrata/S. cerevisiae been suffering some genetic
duplication events in their Sap and Yps superfamilies? This is something that has not been
resolved, but it is clear that the decrease in virulence in null mutants, in both CaSAP and
CgYPS, endorse the idea that the presence and expansion of SAP and YPS families is
necessary for adaptation to the host, and therefore for survival and virulence. Also, species
with broad aspartyl protease families are more virulent than those with a limited number of
these proteins. C. glabrata belongs to a phylogenetic group with no pathogenic yeast, and its
virulence attributes could be evolving independently from the CTG clade, where C. albicans
is the main opportunistic pathogenic species. The expansion of the CgYPS gene superfamily
of C. glabrata maintains a parallelism with the expansion of the SAP gene superfamily of C.
albicans, and constitutes a possible example of convergent evolution. The transition from a
commensally life style to a successful opportunistic pathogen could be related to gene
expansion that encodes for each kind of aspartyl protease. A lot of experimental
methodologies must be performed to recognize the orthologous gene families, as well as the
virulence, participation and transition commensal-pathogen roles of aspartyl proteases,
including Sap and Yps.
4. Acknowledgment
We are grateful for the financial support from CONACyT-CB-13695, CONACyT-69984,
SIP201005214 and SIP20113066. BPO is a fellow of CONACyT and PIFI-IPN. Thanks to
Dr. Bernard Dujon (Institut Pasteur and Universit Pierre et Marie Curie) for donating
strains. Thanks also to Bruce Allan Larsen for reviewing the use of English.

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5. References
Abi-Said, D., Anaissie, E., Uzun, O., Raad, I., Pinzcowski, H. & Vartivarian, S. (1997). The
epidemiology of hematogenous candidiasis caused by different Candida species.
Clin Infect Dis. Vol. (24): 1122-1128.
Albrecht, A., Felk, A., Pichova, I., Naglik, J., Schaller, M., de Groot, P., MacCallum, D., Odds,
F., Schafer, W., Klis, F., Monod M. & Hube, B. (2006).
Glycosylphosphatidylinositolanchored proteases of Candida albicans target proteins
necessary for both cellular processes and host pathogen interactions. J Biol Chem.
Vol. (281): 688-694.
Almirante, B., Rodrguez, D., Park, B., Cuenca-Estrella, M., Planes, A., Almela, M., Mensa, J.,
Snchez, F., Ayats, J., Gimnez, M., Saballs, P., Fridkin, S., Morgan, J., Rodrguez-
Tudela, J., Warnock, D. & Pahissa, A. (2005). Epidemiology and predictors of
mortality in cases of Candida bloodstream infection: results from population-based
surveillance, Barcelona, Spain, from 2002 to 2003. J Clin Microbiol. Vol. (43): 1829-
1835.
Anaissie, E., Vartivarian, S., Abi-Said, D., Uzun, O., Pinczowski, H. & Kontoyiannis, D.
(1992). Fluconazole versus amphotericin B in the treatment of hematogenous
candidiasis: a matched cohort study. Am J Med. Vol. (101): 170-176.
Arendrup, M., Horn, T. & Frimodt-Moller, N. (2002). In vivo pathogenicity of eight medically
relevant Candida species in an animal model. Infection. Vol. (30): 286-291.
Bautista, M., Boldo, X., Villa-Tanaca L. & Hernndez-Rodrguez, C. (2003). Identification of
Candida spp. by randomly amplified polymorphic DNA and differentiation
between Candida albicans and Candida dubliniensis by direct PCR methods. J Clin
Microbiol. Vol. (41): 414-420.
Biasoli, M., Tosello, M. & Magaro, H. (2002). Adherence of Candida strains isolated from the
human gastrointestinal tract. Mycoses. (45): 465-459.
Bordallo, J. & P. Suarez-Rendueles. (1993). Control of Saccharomyces cerevisiae
carboxypeptidase S (CPS1) gene expression under nutrient limitation. Yeast. Vol.
(9): 339349.
Bodey, G., Mardani, M., Hanna, H., Boktour, M., Abbas, J., Girgawy, E., Hachem, R.,
Kontoyiannis, D. & Raad II. (2002). The epidemiology of Candida glabrata and
Candida albicans fungemia in immunocompromised patients with cancer. Am J Med.
Vol. (112): 380-5.
Brockert, P., Lachke, S., Srikantha, T., Pujol, C., Galask, R. & Soll, D. (2003). Phenotypic
switching and mating type switching of Candida glabrata at sites of colonization.
Infect Immun. Vol. (71): 7109-7118.
Butler, G., Rasmussen, M., Lin, M., Santos, M., Sakthikumar, S., Munro, C., Rheinbay, E.,
Grabherr, M., Forche, A., Reedy, J., Agrafioti, I., Arnaud, M., Bates, S., Brown, A.,
Brunke, S., Costanzo, M., Fitzpatrick, D., de Groot, P., Harris, D., Hoyer, L., Hube,
B., Klis, F., Kodira, C., Lennard, N., Logue, M., Martin, R., Neiman, A., Nikolaou,
E., Quail, M., Quinn, J., Santos, M., Schmitzberger, F., Sherlock, G., Shah, P.,
Silverstein, K., Skrzypek, M., Soll, D., Staggs, R., Stansfield, I., Stumpf, M., Sudbery,
P., Srikantha, T., Zeng, Q., Berman, J., Berriman, M., Heitman, J., Gow, N., Lorenz,
M., Birren, B., Kellis, M. & C. A. Cuomo. (2009). Evolution of pathogenicity and
sexual reproduction in eight Candida genomes. Nature. Vol. (459): 657-662.


309
Byrne, K. & Wolfe, K. (2005). The Yeast Gene Order Browser: combining curated homology
and syntenic context reveals gene fate in polyploid species. Genome Res. Vol. (15):
1456-1461.
Campanella, J., Bitincka, L. & Smalley, J. (2003). MatGAT: an application that generates
similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics.
Vol. (4): 29.
Caro, L., Tettelin, H., Vossen, J., Ram, A., van den Ende, H. & Klis, F. (1997) In silicio
identification of glycosylphosphatidylinositol-anchored plasma-membrane and cell
wall proteins of Saccharomyces cerevisiae. Yeast. Vol. (13): 14771489.
Castao, I., Cormack, B. & De Las Peas, A. (2006). Virulence of the opportunistic pathogen
mushroom Candida glabrata. Rev Latinoam Microbiol. Vol. (48): 66-69.
Castao, I., Pan, S., Zupancic, M., Hennequin, C., Dujon, B., & Cormack, B. P. (2005).
Telomere length control and transcriptional regulation of subtelomeric adhesins in
Candida glabrata. Mol Microbiol. Vol. (55): 1246-1258.
Cawley, N., Chino M., Maldonado A., Rodriquez Y., Loh Y. & Ellman, J. (2003). Synthesis
and characterization of the first potent inhibitor of yapsin 1. J Biol Chem. Vol. (278):
5523-5530.
Chaffin, W., Lopez-Ribot J., Casanova, M., Gozalbo M. & Martinez J. (1998). Cell wall and
secreted proteins of Candida albicans: identification, function, and expression.
Microbiol Mol Biol Rev. Vol. (62): 130-180.
Chakrabarti, A., Nayak, N. & Talwar, P. (1991). In vitro proteinase production by Candida
species. Mycopathologia. Vol. (114): 163-168.
Copping, V., Barelle, C., Hube, B., Gow, N., Brown, A. & Odds, F. (2005). Exposure of
Candida albicans to antifungal agents affects expression of SAP2 and SAP9 secreted
proteinase genes. J Antimicrob Chemother. Vol. (55): 645-654.
Csank, C. & Haynes, K. (2000). Candida glabrata displays pseudohyphal growth. FEMS
Microbiol Lett. Vo. (189): 115-120.
Dayhoff, M. (1979). Atlas of protein sequence and structure, vol. 5, Suppl. 3, National
Biomedical Research Foundation, Silver Springs, 978-0912466071, Maryland, p.
353-358.
Diezmann, S., Cox, C., Schnian, G., Vilgalys, R. & Mitchell. T. (2004). Phylogeny and
evolution of medical species of Candida and elated taxa: a multigenic analysis. J Clin
Microbiol. Vol. (42): 5624-5635.
Falquet, L., Pagni, M., Bucher, P., Hulo, N., Sigrist, C., Hofmann, K. & Bairoch, A. (2002).
The PROSITE database. Nucleic Acids Res. Vol. (30): 235-238.
Feng, Z., Jiang, B., Chandra, J., Ghannoum, M., Nelson, S. & Weinberg, A. (2005). Human
beta-defensins: differential activity against Candida species and regulation by
Candida albicans. J Dent Res. Vol. (84): 445-450.
Fidel, P., Vazquez, J. & Sobel, J. (1999). Candida glabrata: review of epidemiology,
pathogenesis, and clinical disease with comparison to C. albicans. Clin Microbiol Rev.
Vol (12): 80-96.
Galtier, N., Gouy, M. & Gautier, C. (1996). SeaView and Phylowin, two graphic tools for
sequence alignment and molecular phylogeny. Comput Appl Biosci. Vol. (12): 543-
548.

Gene Duplication

310
Gasch, A., Spellman, P., Kao, C., Carmel-Harel, O., Eisen, M., Storz, G., Botstein, D. &
Brown, P. (2000). Genomic expression programs in the response of yeast cells to
environmental changes. Mol Biol Cell. Vol. (11): 4241-4257.
Ghannoum, M. (2000). Potential role of phospholipases in virulence and fungal
pathogenesis. Clin Microbiol Rev. Vol. (13): 122-143.
Gilfillan, G., Sullivan, D., Haynes, K., Parkinson, T., Coleman, D. & Grow, N. (1998).
Candida dubliniensis: phylogeny and putative virulence factors. Microbiology. Vol.
(144): 829-838.
Goodman, J., Winston, D., Greenfield, R., Chandrasekar, P., Fox, B. & Kaizer, H. (1992). A
controlled trial of fluconazole to prevent fungal infections in patients undergoing
bone marrow transplantation. N Engl J Med. Vol. (326): 845-851.
Helmerhorst, E., Venuleo, C., Beri, A. & Oppenheim, F. (2005). Candida glabrata is unusual
with respect to its resistance to cationic antifungal proteins. Yeast. Vol. (22): 705-714.
Hicke, L. & Riezman, H. (1996). Ubiquitination of a yeast plasma membrane receptor signals
its ligandstimulated endocytosis. Cell. Vol. (84): 27787.
Hoyer, L., Fundyga, R., Hecht, J., Kapteyn, J., Klis, F. & Arnold, J. (2001). Characterization of
Agglutinin-like Sequence Genes From Non-albicans Candida and Phylogenetic
Analysis of the ALS Family. Genetics. Vol. (157): 1555-1567.
Hube, B. (1998). Posible role of secreted proteinases in Candida albicans infections. Rev
Iberoam Micol. Vol. (15): 65-68.
Hube, B. & J. Naglik. (2001). Candida albicans proteinases: resolving the mystery of a gene
family. Microbiology. Vol. (147): 1997-2005.
Hube, B., Sanglard, D., Odds, F., Hess, D., Monod, M., Schfer, W., Brown, A. & Gow, N.
(1997). Disruption of each of the aspartyl proteinase genes SAP1, SAP2, and SAP3
of Candida albicans attenuates virulence. Infect Immun. Vol. (65): 3529-3538.
Hull, C., Raisner, R. & Johnson, A. (2000). Evidence for mating of the asexual yeast
Candida albicans in a mammalian host. Science. Vol. (289): 307-310.
Kalkanci, A., Bozdayi, G., Biri, A., & Kustimur, S. (2005). Distribution of secreted aspartyl
proteinase using a polymerase chain reaction assay with SAP specific primers in
Candida albicans isolates. Folia Microbiol. Vol. (50): 409-413.
Joly, S., Maze, C., McCray, P. & Guthmiller, J. (2004). Human betadefensins 2 and 3
demonstrate strain-selective activity against oral microorganisms. J Clin Microbiol.
Vol. (42): 1024-1029.
Jones, E. (1991). Three proteolytic system in the yeast Saccharomyces cerevisiae. J Biol Chem.
Vol. (266): 7963-7966.
Kaur, R., B. & Cormack, B. (2007). A family of glycosylphosphatidylinositollinked aspartyl
proteases is required for virulence of Candida glabrata. Proc Natl Acad Sci USA. Vol.
(104): 7628-7633.
Klionsky, D. & Emr, S. (2000). Autophagy as a regulated pathway of cellular Degradation.
Science. Vol. (290): 1717-1721
Krcmery, K. (1999). Torulopsis glabrataan emerging yeast pathogen in cancer patients. Int J
Antimicrob Agents. Vol. (11): 1-6.
Krcmery, V., Oravcova, E., Spanik, S., Mrazova-Studena, M., Trupl, J. & Kunova, A. (1998).
Nosocomial breakthrough fungaemia during antifungal prophylaxis or empirical
antifungal therapy in 41 cancer patients receiving antineoplastic chemotherapy:


311
analysis of etiology risk factors and outcome. J Antimicrob Chemother. Vol. (41): 373-
380.
Krysan, D., Ting, E., Abeijon, C., Kroos, L. & Fuller, R. (2005). Yapsins are a family of
Aspartyl Protease required for cell wall integrity in Saccharomyces cerevisiae.
Eukaryot Cell. Vol. (4): 1364-1374.
Lachke, S. Joly, S., Daniels, K. & Soll, D. (2002). Phenotypic switching and filamentation in
Candida glabrata. Microbiology. Vol. (148): 2661-2674.
Lszl Patthy (1999). Protein Evolution. Blackwell Science Ltd, ISBN 0-632-04774-7, London.
Lermann, U. & Morschhauser, J. (2008). Secreted aspartic proteases are not required for
invasion of reconstituted human epithelia by Candida albicans. Microbiology. Vol.
(154): 3281-3295.
Lesage, G., Sdicu, A., Menard, P., Shapiro, J., Hussein, S. & Bussey, H. (2004). Analysis of -
1,3-glucan assembly in Saccharomyces cerevisiae using a synthetic interaction
network and altered sensitivity to caspofungin. Genetics. Vol. (167): 35-49.
Li. L., Kashleva, H. & Dongari-Bagtzoglou, A. (2007a). Cytotoxic and cytokine inducing
properties of Candida glabrata in single and mixed oral infection models. Microb
Pathog. Vol. (42): 138-147.
Li, L., Redding, S. & Dongari-Bagtzoglou, A. (2007b). Candida glabrata, an emerging oral
opportunistic pathogen. J Dent Res. 86: 204-215.
Lipke, P. & Ovalle, R. (1998) Cell wall architecture in yeast: new structure and new
challenges. J Bacteriol. Vol. (180): 37353740.
Loaiza-Loeza, S., Parra-Ortega, B., Bautista-Muoz, C., Casiano-Rosas, C., Hernndez-
Rodrguez, C. H. & Villa-Tanaca, L. (2007). The proteolytic system of Candida
dubliniensis. Am J Infect Dis. Vol. (3): 76-83.
Loaiza-Loeza, S., Parra-Ortega, B., Cancino-Daz, J., Illades-Aguiar, B., Hernndez-
Rodrguez, C. & Villa-Tanaca, L. (2009). Differential expression of Candida
dubliniensis secreted aspartyl proteinase genes (CdSAP1-4) under different
physiological conditions and during the infection of a keratinocytes culture. FEMS
Immunol Med Microbiol. Vol. (56): 212-22.
Luo, G. & Samaranayake, L. (2002). Candida glabrata, an emerging fungal pathogen, exhibits
superior relative cell surface hydrophobicity and adhesion to denture acrylic
surfaces compared with Candida albicans. APMIS. Vol. (110): 601-610.
Manzano-Gayosso, P., Hernandez-Hernandez, F., Bazan-Mora, E., Mendez-Tovar, L.,
Gonzalez-Monroy, J. & Lpez-Martnez, R. (2000). Identification and typing of
yeast isolates from hospital patients in Mexico City. Rev Argent Microbiol. Vol. (32):
1-6.
MacKay, V., Welch, S., Insley, M., Manney, T., Holly, J., Saari, G. & Parker, M. (1988). The
Saccharomyces cerevisiae BAR1 gene encodes an exported protein with homology to
pepsin. Proc Natl Acad Sci USA. Vol. (85): 55-59.
Magee, B. & Magee, P. (2000). Induction of mating in Candida albicans by construction of
MTLa and MTLalpha strains. Science. Vol. (289): 310-313.
Mayor, S. & Riezman, H. (2004). Sorting GPI-anchored proteins. Nat Rev Mol Cell Biol. Vol.
(5): 110120.
Man, M., Marchetti, O. & Calandra, T. (2008). Bench-to-bedside review: Candida infections
in the intensive care unit. Crit Care. Vol. (12): 1-9.

Gene Duplication

312
Monod, M., Hube, B., Hess, D. & Sanglard, D. (1998). Differential regulation of SAP8 and
SAP9 which encode two new members of the secreted aspartyl protease family in
Candida albicans. Microbiology. Vol. (244): 2731-2737.
Morrow, B., Srikantha, T. & Soll, D. (1992). Transcription of the gene for a pepsinogen,
PEP1, is regulated by white-opaque switching in Candida albicans. Mol Cell Biol. Vol.
(12): 2997-3005.
Naglik, J., Albrecht, A., Bader, O. & Hube, B. (2004). Candida albicans proteinases and
host/pathogen interactions. Cell Microbiol. Vol. (6): 915-926.
Naglik, J., Challacombe, S. & Hube, B. (2003). Candida albicans secreted aspartyl proteinases
in virulence and pathogenesis. Microbiol Mol Biol Rev. Vol. (67): 400-428.
Naglik, J., Moyes, D., Makwana, J., Kanzaria, P., Tsichlaki, E., Weindl, G., Tappuni, A.,
Rodgers, C., Woodman, A., Challacombe, S., Schaller, M. & Hube, B. (2008).
Quantitative expression of the Candida albicans secreted aspartyl proteinase gene
family in human oral and vaginal candidiasis. Microbiology. Vol. (154): 3266-3280.
Nikawa, H., Nishimura, H., Yamamoto, T. & Samaranayake, L. (1995). A novel method to
study the hyphal phase of Candida albicans and to evaluate its hydrophobicity. Oral
Microbiol Immunol. Vol. (10): 110-114.
Palmer, G. (2007). Autophagy in the Invading Pathogen. Autophagy. Vol. (3): 251-253.
Addendum to: Palmer, G., Michelle, N. & Sturtevant, J. (2007). Autophagy in the
Pathogen Candida albicans. Microbiology. Vol. (153): 51-58.
Palmer, G., Kelly, M. & Sturtevant J. (2005). The Candida albicans vacuole is required for
differentiation and efficient macrophage killing. Eukaryot. Cell. Vol. (4): 1677-1686.
Parr, C., Keates, R., Bryksa, B., Ogawa, M. & Yada, R. (2007). The structure and function of
Saccharomyces cerevisiae proteinase A. Yeast. Vol. (24): 467-80.
Parra-Ortega, B., Cruz-Torres, H., Villa-Tanaca, L., Hernndez-Rodrguez, C. (2009).
Phylogeny and evolution of the aspartyl protease family from clinically relevant
Candida species. Mem Inst Oswaldo Cruz, Rio de Janeiro. Vol. (104): 505-512.
Pfaller, M. & Diekema, D. (2007). Epidemiology of Invasive Candidiasis: a Persistent Public
Health Problem. Clin Microbiol Rev. Vol. (20): 133-163.
Robinson, J., Klionsky, D., Banta, L. & cEmr, S. (1988). Protein sorting in Saccharomyces
cerevisiae: isolation of mutants defective in the delivery and processing of multiple
vacuolar hydrolases. Mol Cell Biol. Vol. (8): 4936-4948.
Roetzer, A., Gregori, C., Jennings, A., Quintin, J., Ferrandon, D., Butler, G. Kuchler, K.,
Ammerer, G. & Schller, C. (2008). Candida glabrata environmental stress response
involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors. Mol
Microbiol. Vol. (69): 603-620.
Safdar, A., Van Rhee, F., Henslee-Downey, J., Singhal, S., Mehta, J. & Bone, M. (2001).
Candida glabrata and Candida krusei fungemia after high-risk allogeneic marrow
transplantation: no adverse effect of low-dose fluconazole prophylaxis on incidence
and outcome. Bone marrow Transplant. Vol. (28): 873-8.
Samaranayake, Y., MacFarlane, T., Samaranayake, L. & Aitchison, T. (1994). The in vitro
proteolytic and saccharolytic activity of Candida species cultured in human saliva.
Oral Microbiol Immunol. Vol. (9): 229-235.


313
Sanglard, D., Hube, B., Monod, M., Odds, F. & Gow, N. (1997). A triple deletion of the
aspartyl proteinase genes SAP4, SAP5, and SAP6 of Candida albicans causes
attenuated virulence. Infect Immun. Vol. (65): 3539-3546.
Sanglard, D., Ischer, F., Calabrese, D., Majcherczyk, P. & Bille, J. (1999). The ATP binding
cassette transporter gene CgCDR1 from Candida glabrata is involved in the
resistance of clinical isolates to azole antifungal agents. Antimicrob Agents
Chemother. Vol. (43): 2753-2765.
Scannell, D., Butler, G. & Wolfe, K. (2007). Yeast genome evolution the origin of the species.
Yeast. Vol. (24): 929-942.
Schaefer, D., Cote, P., Whiteway, M., & Bennett, R. (2007). Barrier activity in Candida albicans
mediates pheromone degradation and promotes mating. Eukaryot cell. Vol. (6): 907-
918.
Schaller, M., Bein, M., Korting, H., Baur, S., Hamm, G., Monod, M., Beinhauer, S. & Hube, B.
(2003). The secreted aspartyl proteinases Sap1 and Sap2 cause tissue damage in an
in vitro model of vaginal candidiasis based on reconstituted human vaginal
epithelium. Infect Immun. Vol. (71): 3227-3234.
Schaller, M., Mailhammer, R., Grassl, G., Sander, C., Hube, B. & Korting, H. (2002). Infection
of human oral epithelia with Candida species induces cytokine expression
correlated to the degree of virulence. J Invest Dermatol. Vol. (118): 652-657.
Schaller, M., Schfer, W., Korting, H. & Hube, B. (1998). Differential expression of secreted
aspartyl proteinases in a model of human oral candidosis and in patient samples
from the oral cavity. Mol Microbiol. Vol. (29): 605-615.
Sobel, J. (2006). The emergence of non-albicans Candida species as causes of invasive
candidiasis and candidemia. Curr Infect Dis Rep. Vol. (8): 427-433.
Srikantha, T., Lachke, S. & Soll, D. (2003) Three mating type-like loci in Candida glabrata.
Eukaryot Cell. Vol. (2): 328-340.
Szklarczyk, R., & Heringa, J. (2004). Tracking repeats using significance and transitivity.
Bioinformatics. Vol. (20): 311-317.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular Evo-lutionary
Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol. Vol. (24): 1596-1599.
Taylor, B., Staib, P., Binder, A., Biesemeier, A. Sehnal, M., Rollinghoff, M., Morschhauser, J.
& Schroppel, K. (2005). Profile of Candida albicans-secreted aspartic proteinase
elicited during vaginal infection. Infect Immun. Vol. (73): 1828-1835.
Teichert, U., Mechler, B., Muller, H. & Wolf D. (1989). Lysosomal (vacuolar) proteinases of
yeast are essential catalysts for protein degradation, differentiation, and cell
survival. Rev. Microbiol. Vol. (6): 2500-2510.
Trick, W., Fridkin, S., Edwards, J., Hajjeh, R. & Gaynes, R. (2002). Secular trend of hospital-
acquired candidemia among intensive care unit patients in the United States during
1989-1999. Clin Infect Dis. Vol. (35): 627-630.
Wei, X., Rogers, H., Lewis, M., & Williams, D. (2011). The role of the IL-12 cytokine family in
directing T-cell responses in oral candidosis. Clin Dev Immunol. Vol. (2011): 1-10.
White, T. & Agabian, N. (1995). Candida albicans secreted aspartil proteinases: isoenzyme
pattern is determined by cell type, and levels are determined by enviromental
factors. J Bacteriol. Vol. (177): 5215-5221.

Gene Duplication

314
White, T., Miyasaki S. & Agabian, N. (1993). Three distinct secreted aspartyl proteinases in
Candida albicans. J Bacteriol. Vol. (175): 6126-6133.
Wolfe, K. (2006). Comparative genomics and genome evolution in yeasts. Philos Trans R Soc
Lond B Biol Sci. Vol. (361): 403-412.
Wnschmann, J., Beck, A., Meyer, L., Letzel, T., Grill, E. & Lendzian, K. (2007)
Phytochelatins are synthesized by two vacuolar serine carboxypeptidases in
Saccharomyces cerevisiae. FEBS Lett. Vol. 17:1681-7.
17
Clues to Evolution of the SERA Multigene
Family in the Genus Plasmodium
Nobuko Arisue, Nirianne M. Q. Palacpac,
Kazuyuki Tanabe and Toshihiro Horii
Research Institute for Microbial Diseases, Osaka University
Japan
1. Introduction
Malaria, one of the most serious infectious diseases prevalent in the tropics, is caused by the
genus Plasmodium. Despite considerable global efforts to control this parasitic disease at least
90% of deaths still occur in sub-Saharan Africa (WHO, 2010). The rising threat of drug-
resistant parasites, together with key interventions dependent on the use of a limited class of
insecticides, underscore the fragility of malaria control. A better understanding of the
parasite biology is required to gain insights for cost-effective tools or strategies including
malaria vaccines and new antimalarial drugs that can be instrumental for sustained control,
if not elimination, of malaria.
Malaria parasites comprise a diverse group of over 200 Plasmodium species that infect
mammals, birds and reptiles (Levine, 1988). Each Plasmodium species exhibits a restricted
host range, such that primate parasite cannot infect rodent, bird or reptile hosts. To find
genomic factors that determine host range transcends the interest of malaria researchers. It
is essential for control and conservation of wildlife. Recently, genome projects on several
Plasmodium species from different hosts have been completed (Gardner et al., 2002; Carlton
et al., 2002, 2008; Pain et al., 2008), with gene information available in the public database
(http://plasmodb.org/plasmo/). By comparing the genomes from different species we
obtain basic information at the molecular level on how Plasmodium has evolved and allows
us to infer the function of genes and noncoding regions in the genome. One of the
prominent features of Plasmodium genomes is the presence of various unique multigene
families. Multigene families are a group of related genes that are presumed to share a
common ancestor and are derived from each other by duplication and subsequent
divergence. One such example, the largest family identified so far in human, primate and
rodent malaria, is the Plasmodium interspersed repeat, pir (Janssen et al., 2004). The pir gene
family members are highly species-specific, suggesting evolution of lineage-specific immune
evasion mechanisms. P. falciparum var gene family is by far the best documented multigene
family of the most virulent human malaria parasite. Products of var genes appear on the
surface of infected erythrocytes and are involved in antigenic variation to evade host
immunity. Other species-specific gene families encode proteins involved in host cell
invasion, e.g. rhoptry proteins and parasite surface antigens, merozoite surface protein-3
and -7. There are also examples of families with few gene members. In sharp contrast to
several hundreds of tandem arrayed rRNA gene family members in other eukaryotes,

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Plasmodium has 4-7 gene units physically separated in the genome (Nei & Rooney, 2005;
Carlton et al., 2008). Thus, Plasmodium possesses unique multigene families with distinctive
evolutionary conundrums.
For more than 10 years after the first description of a gene family member, the existence of
the Plasmodium serine repeat antigen (SERA) multigene family has been overlooked. Serine
repeat antigen family proteins share homology with the papain family of cysteine proteases
(Kiefer et al., 1996; Gor et al., 1998; Bourgon et al., 2004; Arisue et al., 2007, 2011). Almost all
SERA genes are clustered in a head-to-tail manner and the number of SERA genes in the
clustered region varies among parasite species (Bourgon et al., 2004; McCoubrie et al., 2007;
Arisue et al., 2007, 2011). This leads us to infer that gene duplication occurred repeatedly
during evolution. Some SERA genes were confirmed to play essential role(s) in the parasite
life cycle (Miller et al., 2002; Aly & Matuschewski, 2005; McCoubrie et al., 2007). In addition,
a gene family member in P. falciparum, SERA5, is a vaccine candidate now on phase Ib
clinical trial (Horii et al., 2010). Two observations promise SERA5 as a vaccine candidate: (1)
SERA genes are not differently expressed like other antigen encoding gene families such as
var and rifin that show antigenic variation to evade host immune response (Aoki et al., 2002;
Miller et al., 2002; Palacpac et al., 2006; Schmidt- Christensen et al., 2008; Putrianti et al.,
2010; Arisue et al., 2011); and (2) P. falciparum SERA5 is less polymorphic (Fox et al., 1997:
Morimatsu et al., 1997; Liu et al., 2000) than other vaccine candidate genes such as merozoite
surface protein 1 (McBride et al., 1985) and apical membrane protein 1 (Polley et al., 2003;
Corts et al., 2003). These characteristics are indeed appealing and show the unique
biological features of Plasmodium SERA. Here, we summarize current reports and our recent
findings to understand the evolution of the SERA gene family.
2. SERA gene repertories in Plasmodium species
We refer briefly to the research history of the SERA multigene family: (i) the identification of
SERA5 and the proteolytic processing of the protein; (ii) the discovery of the gene family
following chromosome 2 sequencing of the P. falciparum genome; (iii) currently known
SERA genes from different species; and (iv) the resulting analyses of the multigene family in
various malaria parasites.
2.1 Research history of the SERA multigene family
SERA was first found in P. falciparum as an abundant, exported, soluble late-trophozite to
schizont stage protein (Perrin et al., 1984). The protein, independently isolated by different
groups, was described under various names as Pf140 (Perrin et al., 1984), p113 (Chulay et al.,
1987), p126 (Deplace et al., 1987) or SERP (Knapp et at., 1989). All identified a gene with a
long stretch of repeated serine residues in the N-terminal domain to which the family owes
its name (Bzik et al., 1988). At the central domain, SERA possess a motif which align with
two active site-determining regions of cysteine proteinases. The secreted protein was
described to accumulate in the parasitophorous vacuole, and released into the culture
medium at the time of schizont rupture. Notably, before the sequence of P. falciparum
Chromosome 2 was opened, Knapp et al. (1991) discovered a SERA homolog (serp H)
lacking the characteristic serine homopolymer, and subsequently Fox and Bzik (1994)
reported SERA as one of three consecutive series of homologous genes. The complete
genome sequence of P. falciparum revealed that SERA belongs to a multigene family
(Gardner et al., 1998). The originally described SERA gene was renamed SERA5 according to

Clues to Evolution of the SERA Multigene Family in the Genus Plasmodium

317
the gene arrangement order in P. falciparum (Aoki et al., 2002; Miller et al., 2002). It is
interesting to note that SERA5 is the only member with repeated serine residues among nine
gene members. The characteristic of the family is not the richness in serine residues but
motifs that generate the framework of a cysteine protease. SERA homologs were identified
in other Plasmodium species. Kiefer et al. (1996) found five SERA genes from another human
parasite, P. vivax and Gor et al. (1998) identified three SERA genes from the rodent parasite
of P. vinckei. Completed or ongoing genome projects of eight Plasmodium species: two
human malaria parasites, P. falciparum and P. vivax; chimpanzee parasite P. reichenowi;
macaque parasite P. knowlesi; three rodent parasites P. berghei, P. yoelii and P. chabaudi; and
avian parasite P. gallinaceum, confirmed the gene organization and allowed phylogenetic
analysis of the SERA gene family (Burgon et al., 2005; Arisue et al., 2007). In addition, Arisue
et al. (2011) newly identified SERA genes in 11 Plasmodium species that further elaborate the
genome organization of the gene family.
2.2 Processing of P. falciparum SERA5
The in vitro observation that P. falciparum SERA5 was released into the culture supernatant
at the time of schizont rupture/melozoite release corresponds to its specific processing into
several polypeptides. The full-length 120 kDa precursor accumulates in the parasitophorous
vacuole during late trophozoite and schizont stages. As shown in Fig. 1., during the course
of schizont rupture/merozoite release, SERA is proteolytically processed into a 47 kDa N-
terminal (P47), a 50 kDa central (P50), an 18 kDa C-terminal (P18) and a 6 kDa domain
(Delplace et al., 1987, 1988; Debrabant et al., 1992; Li et al., 2002a). The N-terminal P47
fragment is further processed into two 25 kDa fragments (P25n and P25c) in some allelic
types (Li et al., 2002a). P47 is linked with the C-terminal P18 via disulfide bond that is
localized at the merozoite surface that is localized at the merozoite surface (Delplace et al.,
1987; Li et al., 2002a; Okitsu et al., 2007). The proteolytic processing is mediated by
subtilisin-like serine protease subtilase 1 or SUB1 (Yeoh et al., 2007). Inhibition of SERA
maturation blocks parasite egress from the host erythrocyte (Li et al., 2002b; Yeoh et al.,
2007).

Fig. 1. Processing of P. falciparum SERA5 during parasite egress from host erythrocyte.

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2.3 SERA genes in the database
The discovery of the SERA gene family in P. falciparum sparked the subsequent
identification of a number of SERA genes in different Plasmodium species. Currently known
SERA genes in the PlasmoDB database (http://plasmodb.org/plasmo/) are summarized in
Table 1 and accession numbers of SERA genes found in the public database (NCBI,
http://www.ncbi.nlm.nih.gov/) are summarized in Table 2.
We opted not to list SERA genes of P. reichenowi and P. gallinaceum in either Table, but for
our analysis, their SERA gene sequences were assembled from various reads in the partial
genome shotgun database of Plasmodium at The Sanger Institute. Blast programs in the
following web sites were used to search SERA coding reads for: P. reichenowi:
http://www.sanger.ac.uk/cgi-bin/blast/submitblast/p_reichenowi; and P. gallinaceum:
http://www.sanger.ac.uk/cgi-bin/blast/submitblast/p_gallinaceum). Identified SERA
gene sequences could be referred to in Arisue et al. (2007).
2.4 Characteristic features of the SERA multigene family
Almost all SERA genes were aligned in a tandem cluster between the conserved
hypothetical protein gene and the iron-sulfur assembly protein gene. According to genetic
background, SERA genes can be categorized into Groups I to IV (Arisue et al., 2007, 2011).
Characteristic features of each Group are summarized in Fig. 2. Group I to Group III SERA
genes possess the protease motif that includes an active site cysteine residue, in contrast to
Group IV SERA genes where the cysteine residue is replaced by a serine (Bourgon et al.,
2005; Arisue et al., 2007, 2011). The mRNA transcription and/or protein expression of Group
I SERA genes were observed in the mosquito vector, while those of Group II to Group IV
SERA genes were observed in the vertebrate host (Ali & Matuschewski, 2005; Arisue et al.,
2007, 2011; Putrianti et al., 2010). The difference in the gene repertoire among species is due
to the number of Group IV SERA genes. SERA gene repertories are summarized in Table 3.

*The gene region of PY02062 and PY00294 was re-annotated and used as P. yoelii SERA4.
Table 1. GeneID of SERA genes in the PlasmoDB (http://plasmodb.org/plasmo/).
Species P. vivax P. falciparum P. knowlesi P. berghei P. yoelii P. chabaudi
(strain) (SalI) (3D7) (H) (ANKA) (17XNL) (AS)
SERA1 PVX_003850 PFB0360C PKH_041200 PB000108.03.0 PY00291 PCAS_030730
PY00294
SERA6 PVX_003825 PFB0335C PKH_041270
SERA7 PVX_003820 PFB0330C
SERA8 PVX_003810 PFB0325C
SERA9 PVX_003805 PFI0135C
SERA10 PVX_003800
SERA11 PVX_003795
SERA12 PVX_003790


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Species (strain) Accession No. Gene Reference
P. vivax (SalI) U51723 V_SERA1-V_SERA5 Kiefer et al., 1996
P. vinckei vinckei U59860 SERA
vin
-1 Gor et al., 1998
vin
-2
vin
-3
P. malariae (Kisii67) AB576870 SERA1-SERA10 Arisue et al., 2011
P. ovale (Nigeria II) AB576871 SERA1-SERA7
P. cynomolgi (Mulligan) AB576872 SERA1-SERA11
P. fieldi (N-3) AB576873 SERA1-SERA9
P. simiovale AB576874 SERA1-SERA9
P. inui (Celebes) AB576875 SERA1-SERA7
P. hylobati (WAK) AB576876 SERA1-SERA7
P. coatneyi (CDC) AB576877 SERA1-SERA7
P. knowlesi (ATCC30158) AB576878 SERA1-SERA6
P. fragile (Hackeri) AB576879 SERA1-SERA5
P. gonderi AB576880 SERA1-SERA4
P. gonderi AB576881 SERA5-SERA9

Table 2. Accession numbers of SERA genes in the public NCBI database.

Fig. 2. Organization of Plasmodium SERA multigene family and the characteristic features of
each SERA group.
Because of limited information for P. reichenowi, P. gonderi and P. vinckei, gene numbers are
tentative for these species. The total number of SERA pseudogenes, truncated gene and gene
fragments in Group IV SERA gene region are still undetermined in primate parasites
(Arisue et al., 2011). Except for P. gallinaceum, all parasite species have one each of Group I
to III SERA genes. The number of Group IV SERA genes remarkably increased in the
primate parasite lineage. The bird parasite, P. gallinaceum, has the least number of SERA
genes: two from Group I, and one from a branched common ancestor of Group II to IV
SERA gene.
Conserved
hypothetical
protein gene

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Species Natural host I II III IV Degenerated*
P. falciparum human 1 1 1 6 0
P. vivax human 1 1 1 9 2
P. malariae human 1 1 1 7 3
P. ovale human 1 1 1 4 1
P. knowlwsi human/macaque 1 1 1 3 2
P. cynomolgi macaque 1 1 1 8 3
P. coatneyi macaque 1 1 1 4 3
P. fragile macaque 1 1 1 2 3
P. simiovale macaque 1 1 1 6 3
P. fieldi macaque 1 1 1 6 3
P. inui macaque 1 1 1 4 5
P. hylobati gibbon 1 1 1 4 1
P. berghei rat 1 1 1 2 0
P. yoelii rat 1 1 1 2 0
P. chabaudi rat 1 1 1 2 0
P. gallinaceum bird 2 0
P. gonderi mangabey, guenon 1 1 1 6 ?
P. reichenowi chimpanzee 1 1 1 5? ?
P. vinckei rat ? 1 1 1? ?
Number of SERA gene in each group
1

Table 3. The number of SERA genes that belong to each group from several Plasmodium
species. 'Degenerate'(*) denotes defective gene copies, i.e., the total number of pseudogenes,
truncated gene and gene fragments found.
2.5 Primary structure of SERA molecules and genes
Schematic representation of SERA gene structure is shown in Fig. 3A. Group I SERA genes
code for around 700 amino acids. They share a similar six exon and five intron structure,
except for P. falciparum SERA8 and P. vivax SERA12 that both lack one intron. Group II to
Group IV SERA genes code for about 1000 amino acid residues, and similar to Group I,
share a common four exon/three intron structure with few exceptions. All SERA genes
have the structural context of cysteine proteinases, however, it is important to note that the
canonical Cys His Asn triad of the active proteinase is not present in all. The relatively small
number of amino acid residues in Group I SERA resulted to a shorter N-terminal region
when compared to Group II to IV SERA. Multiple amino acid sequence alignments revealed
the consensus primary structure of SERA, which consists of six putative domains shown in
Fig. 3B.
Amino acid sequences of the putative pro-enzyme and enzyme domains are remarkably
conserved, but extensive sequence variations are found in variable domains 1 and 2. In the
C-terminal cysteine rich conserved domain, seven cysteine residues are perfectly conserved
in all SERA genes.
The pro-enzyme and enzyme domains of P. falciparum SERA5 was identified by functional
genetic and structural analyses (Hodder et al., 2003, 2009). These domains, corresponding to
*


321
P50 in Fig. 1, are flanked by the reported SUB1 cleavage sites (Yeoh et al., 2007). The
consensus sequence of the cleavage site is (Val/Leu/Ile)-Xaa-(Gly/Ala)-Paa, in which Xaa is
any amino acid residue and Paa is any non-polar residue except for Leu (Yeoh et al., 2007).
This consensus sequence is well conserved with slight modifications in all Group II to IV
Plasmodium SERA genes which we have analyzed. In vitro, P. falciparum SERA4 (Group IV)
and SERA6 (Group II) were cleaved by recombinant PfSUB1 (Yeoh et al., 2007). Peculiarly,
Group I SERA genes lack most of the N-terminal variable domain 1 and SUB1 cleavage sites.

Fig. 3. Schematic representation of the SERA gene structure (A) and their putative domain
organization (B).
3. Phylogenetic relationships of SERA genes
The categorization of SERA genes into four groups, Group I to IV, was based on phylogenetic
analysis (Arisue et al., 2007, 2011). SERA amino acid sequences from 18 Plasmodium species
were aligned using CLUSTAL W program (http://clustalw.ddbj.nig.ac.jp/top-j.html) under
default options with manual corrections. Unambiguously aligned amino acid positions
corresponding to the putative pro-enzyme domain, enzyme domain and cysteine rich
conserved domain were selected and used for the phylogenetic analysis. Maximum likelihood
tree was inferred using the PROML program in PHYLIP version 3.69 (Felsenstein, 1996).
Except for the number of genes and number of amino acid sequences included in the analysis,
the same method was used to infer the phylogenetic tree shown in Fig. 4 and 5.
A simplified maximum likelihood tree inferred from 134 SERA genes with 392 amino acid
positions is shown in Fig. 4. Bootstrap proportion values were placed only on the common
ancestor branch of each group. The monophyletic grouping of Group I SERA genes was
supported with a bootstrap value of 100%. The long internal branch separating Group I
from Groups II to IV suggests that the root of the tree is located on the branch leading to the
common ancestor of Group I SERA genes. It is thus likely that Group I genes have appeared
early in the evolution of the SERA gene family. P. gallinaceum SERA1 branches at the
common ancestor of Group II to IV, suggesting that gene duplication events which
produced Groups II, III and IV likely occurred after the divergence of P. gallinaceum from the
common ancestral lineage of Plasmodium.

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322

Fig. 4. Phylogenetic tree based on the alignment of 134 SERA genes from 18 Plasmodium
species.

Fig. 5. Phylogenetic tree based on the alignment of 115 SERA genes from 18 Plasmodium
species.
(i) P. falciparum + P. reichenowi
P. falciparum


323
Group IV SERA genes which diverged after the cysteine to serine substitution in the
catalytic site were further divided to five monophyletic sub-Groups: (i) P. falciparum and P.
reichenowi, (ii) P. ovale, (iii) rodent Plasmodium species, (iv) P. vivax and P. vivax-related
monkey parasite species, and (v) P. malariae. These indicate that genes have duplicated
independently in each of the sub-group lineages. To increase the resolution of the tree, the
long branched Group I genes were excluded from the dataset and the maximum likelihood
tree was re-constructed from 115 SERA genes categorized into Group II to Group IV. The
resultant tree is shown in Fig. 5.
Interestingly, Group IV SERA genes of P. vivax and P. vivax-related monkey parasites (10
species) were further categorized into six orthologous gene groups, namely, Clade 1 to
Clade 6; and each clade has 5 (Clade 6) to 10 (Clade 5) parasite species. The number of SERA
genes analyzed varied from 5 (P. fragile) to 12 (P. vivax). This does suggest that a common
ancestor of P. vivax and related monkey malaria parasites had at least 6 SERA genes of
Group IV; and that gene duplications and gene deletions occurred in each lineage.
Orthologous relationships between SERA gene members and their relative arrangement are
shown in Fig. 6.

Fig. 6. Organization and phylogenetic relationship of SERA gene in 18 Plasmodium species.

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324
In addition to several gene members characterizing Group IV, there are multiple SERA gene
fragments and pseudogenes containing multiple stop codons. Taken together, these
extensive gene duplications, gene deletions as well as pseudogenization/truncation are
evident only in the serine type SERA gene (Group IV) of P. vivax and related monkey
malaria parasites.
4. Transcription analyses of SERA genes
Transcription analyses revealed, likewise, some discordance among Plasmodium species.
Transcription profile of the SERA gene family was analyzed first by Aoki et al. (2002) in P.
falciparum. Genes were most actively transcribed at the late trophozoite to schizont stages of
the parasite with SERA5 predominantly transcribed among the family (Fig. 7.).

Fig. 7. The relative abundance of mRNA for each P. falciparum SERA gene during late
trophozoite to schizont stages of the parasite.
Similar to P. falciparum, multiple number of SERA genes were transcribed at late trophozoite
to schizont stages when transcription analysis was done for the SERA gene family in other
Plasmodium parasites: human parasite P. vivax (Palacpac et al., 2006); rodent parasite P.
berghei (Arisue et al., 2011); and three monkey parasites P. knowlesi, P. cynomolgi and P.
coatneyi (Arisue et a.l, 2011). A representative summary of SERA gene transcription analysis
is shown in Fig. 8. In malaria parasite species infecting humans, one of Group IV SERAs of
P. falciparum (SERA5) and P. vivax (SERA4) showed the highest transcription level among
other gene members. In the rodent malaria parasite P. berghei, Group III SERA gene (SERA3)
was predominantly expressed. In three monkey malaria parasites, the abundantly expressed
genes are members of Group IV Clade 3: SERA3 and SERA5, in P. cynomolgi; SERA3 in P.
coatneyi; and SERA2 in P. knowlesi. These results show that SERA genes were differently
expressed between rodent and primate parasites. Based on the malaria parasite
mitochondrial genome, P. falciparum belongs to the primate parasite group 1 lineage
whereas P. vivax and the three macaque parasites belong to primate parasite lineage 2.
Phylogenetic analysis showed that these two lineages are not closely related; and the rodent
parasite lineage is positioned between them (Hayakawa et al., 2008). Note, however, that


325
both primate lineages showed similar transcription pattern of SERA gene which might
suggest a possible relationship between SERA function and host specificity.

Fig. 8. Representation summary of SERA gene transcription analyses in primate and rodent
Plasmodium spp. Abundantly transcribed SERA genes are differentiated using enlarged solid
circles.
5. Duplication in the multigene family
In general, duplicated genes undergo either (i) concerted evolution or (ii) birth-and-death
evolution (Nei & Rooney, 2005). In the concerted evolution model, all members of a gene
family evolve as a unit rather than independently. When a mutation occurs in a gene,
mutation spreads to all other gene members by unequal crossover or gene conversion. As a
result, all members of the gene family show identical sequence to each other. The evolution
of rRNA multigene families in vertebrates is a classic example of concerted evolution.
Analysis of MHC genes in mammals (Hughes & Nei, 1989; Nei et al., 1997; Nei & Hughes,
1992), other immune system related genes (Hughes & Nei, 1990; Ota & Nei, 1994) and
disease-related genes (Zhang et al., 2000) show a quite different evolutionary pattern. The
birth-and-death evolution model was proposed to explain differential
duplication/independent diversification processes that result to subsequent loss or
maintenance of genes in a multigene family. Thus, some duplicated genes are maintained in
the genome for a long time while others are deleted or became pseudogenes through
deleterious mutations. This model applies to rRNAs of Plasmodium species in marked
contrast to the concerted evolution of rRNAs in most organisms. The model aptly explains
the observation that rRNA genes in Plasmodium were structurally and functionally distinct
(Rooney, 2004; Nishimoto et al., 2008).
The observed gene duplication and gene deletion found in the Plasmodium SERA genes are
clearly in concordance with the birth-and-death model, although traits of gene conversion
are detected in a few of Group IV SERA genes. The birth-and-death model has, likewise,
been recently proposed for gene duplication/gene deletion of merozoite surface protein 7,
an immune target parasite surface antigen gene (Garzn-Ospina et al., 2010). It, thus, seem

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326
most probable that diversification of Plasmodium SERA multigene family was also driven by
the birth-and-death evolution. Inferred gene duplication events in the evolution of the
Plasmodium SERA gene family are shown in Fig. 9.

Fig. 9. Inferred gene duplication events in the evolution of the Plasmodium SERA gene
family. Asterisk in P. reichenowi denotes that the SERA gene number in this species is
tentative.
Theileria is the only other genus to possess a SERA homolog gene. The apicomplexan
parasite is closely related to Plasmodium. Sequence similarity search against Theileria genome
database at The Sanger Institute identified a single gene from both T. parva and T. annulata
which has similarity with cysteine-type SERA (Arisue et al., 2007; McCoubrie et al., 2007).
Based on Fig. 9, as all Plasmodium species have multiple SERA genes, we likely infer that the
first duplication occurred at the common ancestor lineage of Plasmodium. Because every
Plasmodium species has Group I SERA gene, the first duplication event is from Group I
SERA gene. The duplication events which gave rise to Group II and IV SERA genes occurred
after the divergence of P. gallinaceum from the branch leading to a common ancestral species
of other Plasmodium species since P. gallinaceum has no Group II to IV SERA gene. The rest of
the 17 Plasmodium species might have diverged into five lineages of (i) P. falciparum and P.
reichenowi, (ii) P. ovale, (iii) rodent Plasmodium species, (iv) P. vivax and P. vivax-related
monkey parasite species, and (v) P. malariae, and duplications of Group IV SERA genes
occurred independently on each lineage. In addition, gene deletions as well as
pseudogenization/truncation occurred frequently in P. vivax and P. vivax-related primate
parasite lineage.
6. Conclusion and open issues in SERA study
Multigene families are believed to provide an organism with a set of related genes that
allow fine tuning of its biological function with possibly different temporal or topologic
expression patterns. SERA gene duplications during Plasmodium evolution generated four
types of SERA genes: Group I to Group IV. The speculated function of SERA during the
parasite life cycle is summarized in Fig. 10.


327

Fig. 10. The life cycle of the malaria parasite and inferred role(s) of SERA.
Group I SERA gene, bearing the canonical cysteine, was shown to be transcribed in the
oocyst stage and its gene product was a protease required for sporozoite egress from the
oocyst (Aly & Matuschewski, 2005). Group III SERA gene was suggested to play an essential
role during schizont rupture/merozoite release in the mammalian host (Yeoh et al., 2007;
Arastu-Kapur et al., 2008; Putrianti et al., 2010). Group IV SERA genes bear the characteristic
replacement of active-site cysteine to a serine residue. Perhaps importantly, only
mammalian parasites have Group IV SERA gene; and Group IV SERA gene of primate
parasite was suggested to play an essential role in schizont rupture/merozoite release
together with Group III SERA gene (Yeoh et al., 2007; Arisue et al., 2011). The duplication of
Group IV SERA gene occurred particularly frequent in two evolutionarily distinct primate
lineages and it is intriguing to assume that duplications of SERA genes were associated with
host range expansion.
The study of the SERA gene family points to its unique features reinforcing the importance
of investigating other uncharacterized gene families of Plasmodium to further understand the
evolutionary history and biology of this harmful parasite. Many questions still remain in the
analysis of SERA. SERA genes are thought to be subject to birth-and-death evolution, and
thus, a pattern of interspecific gene clustering is expected to characterize the SERA family
whereby functional genes are maintained in the genome for a long time and others are
deleted or become non-functional. Group I and Group III SERA genes are highly conserved
in Plasmodium species. For Group II SERA genes, although maintained among Plasmodium
species with significant sequence similarity, no function has yet been predicted. Gene
disruption studies with Group II SERA gene of P. berghei showed no apparent phenotypic
change (Arisue et al., unpublished data). Group II is similar to Group I and Group III in
being a cysteine-type SERA gene which has been suggested to have proteolytic activity to
cleave host membrane structure (Aly & Matuschewski, 2005; Yoeh et al., 2007). The papain-
like cysteine protease motif in its amino acid sequence suggests the possibility that Group II
SERA act as a protease sometime in the parasite life cycle. Parasite egress from the host cell
is an important process that remains poorly understood.
P. falciparum SERA5 is a vaccine candidate molecule now on clinical trial in Uganda (Horii et
al., 2010). Serum antibodies against the N-terminal domain of P. falciparum SERA5 in
individuals living in malaria endemic area protect infants from clinical malaria and inhibit
in vitro parasite growth (Okech et al., 2001, 2006; Aoki et al., 2002; Horii et al., 2010). During

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blood stage growth, all SERA gene family member of P. falciparum are transcribed most
actively at trophozoite and schizont stages. SERA5 is the most abundantly expressed gene
family member, with expression levels estimated to be approximately 0.5-1.5% of the whole
mRNA at schizont stage (Aoki et al, 2002). However, sero-positivity rate against the N-
terminal domain of P. falciparum SERA5 was observed to be relatively low (Fig. 11.; Aoki et
al., 2002; Horii et al., 2010).

Fig. 11. Relative sero-conversion rates of P. falciparum SERA3 to SERA6 and merozoite
surface protein 1 (MSP1) in a malaria endemic area of Uganda.
Since the SERA gene family does not show antigenic variation to evade host immune
response (Fig. 7.), there may possibly be another mechanism of host parasite
evasion/molecular mimicry/interference or competition.
The host range or host specificity of Plasmodium is believed to be restricted, although,
primate malaria parasites generally infect multiple hosts. For example, it has been reported
that, P. knowlesi and P. cynomolgi have the ability to infect a wide variety of macaques and
human (Coatney et al., 1971); additionally, two human parasites P. malariae and P. ovale have
been detected in chimpanzees (Hayakawa et al., 2009; Duval et al., 2009). It may be probable
that duplications of Group IV SERA genes that occurred frequently in both primate parasite
lineages may be associated with host range expansions. To date, no experimental support
lends credence to this speculation.
As described above, the molecular function of SERA genes in each group, the relationship of
immune evasion mechanism and the SERA gene family, and the association of host range
with Group IV SERA genes remain important issues that needs to be addressed. The
importance of SERA genes in parasite egress and their role in host-parasite interactions
serve to propel further studies in understanding this multigene family.
7. Acknowledgment
This work was supported by KAKENHI (18073013 and 20390120) from the Japanese
Ministry of Education, Science, Sports, Culture and Technology.


329
8. References
Aly, ASI. & Matuschewski K. (2005) A malarial cysteine protease is necessary for Plasmodium
sporozoite egress from oocysts, The Journal of Experimental Medicine, Vol. 202, No. 2,
pp. 225-230.
Aoki, S.; Li J, Itagaki S et al. (2002) Serine repeat antigen (SERA5) is predominantly
expressed among the SERA multigene family of Plasmodium falciparum, and the
acquired antibody titers correlate with serum inhibition of the parasite growth, The
Journal of Biological Chemistry, Vol. 277, No 49, pp. 47533-47540.
Arastu-Kapur, S.; Ponder EL, Fonovic UP et al. (2008) Identification of proteases that
regulate erythrocyte rupture by the malaria parasite Plasmodium falciparum, Nature
Chemical Biology, Vol. 4, No. 3, pp. 203-210.
Arisue, N.; Hirai M, Arai M et al. (2007) Phylogeny and evolution of the SERA multigene
family in the genus Plasmodium, Journal of Molecular Evolution, Vol. 65, No. 1, pp. 82-
91.
Arisue, N.; Kawai S, Hirai M et al. (2011) Clues to evolution of the SERA multigene family in
18 Plasmodium species, PLoS One, Vol. 6, No. 3, e17775.
Bourgon, R.; Delorenzi M, Sargeant T et al. (2004) The serine repeat antigen (SERA) gene
family phylogeny in Plasmodium: the impact of GC content and reconciliation of
gene and species trees, Molecular Biology and Evolution, Vol. 21, No. 11, pp. 2161-
2171.
Bzik, DJ.; Li WB, Horii T et al. (1988) Amino acid sequence of the serine-repeat antigen
(SERA) of Plasmodium falciparum determined from cloned cDNA, Molecular and
Biochemical Parasitology, Vol. 30, No. 3, pp. 279-288.
Carlton, JM.; Angiuoli SV, Suh BB et al. (2002) Genome sequence and comparative analysis
of the model rodent malaria parasite Plasmodium yoelii yoelii, Nature, Vol. 419, No.
6906, pp. 512-519.
Carlton, JM.; Adams JH, Silva JC et al. (2008) Comparative genomics of the neglected human
malaria parasite Plasmodium vivax, Nature, Vol. 455, No. 7214, pp. 757-763.
Chulay, JD.; Lyon JA, Haynes JD et al. (1987) Monoclonal antibody characterization of
Plasmodium falciparum antigens in immune complexes formed when schizonts
rupture in the presence of immune serum. The Journal of Immunology, Vol. 139, No.
8, pp. 27682774.
Corts, A.; Mellombo M, Mueller I et al. (2003) Geographical structure of diversity and
differences between symptomatic and asymptomatic infections for Plasmodium
falciparum vaccine candidate AMA1. Infection and Immunity, Vol. 71, No. 3, pp. 1416-
1426.
Coatney, GR.; Collins WE, Warren M et al. (1971) The Primate Malarias, US Government
Printing Office, Washington DC, USA.
Debrabant, A.; Maes P, Delplace P et al. (1992) Intramolecular mapping of Plasmodium
falciparum P126 proteolytic fragments by N-terminal amino acid sequencing,
Molecular and Biochemical Parasitology, Vol. 53, No. 1-2, pp. 89-95.
Delplace, P.; Bhatia A, Cagnard M et al. (1988) Protein p126: a parasitophorous vacuole
antigen associated with the release of Plasmodium falciparum merozoites, Biology of
the Cell, Vol. 64, No. 2, pp. 215-221.

Gene Duplication

330
Delplace, P.; Fortier B, Tronchin G et al. (1987) Localization, biosynthesis, processing and
isolation of a major 126 kDa antigen of the parasitophorous vacuole of Plasmodium
falciparum, Molecular and Biochemical Parasitology, Vol. 23, No. 3, pp. 193-201.
Duval, L.; Nerrienet E, Rousset D et al. (2009) Chimpanzee malaria parasites related to
Plasmodium ovale in Africa, PLoS One, Vol. 4, No. 5, e5520.
Felsenstein, J. (1996) Inferring phylogenies from protein sequences by parsimony, distance,
and likelihood methods, Methods in Enzymology, Vol. 266, pp. 418-427.
Fox, BA. & Bzik DJ. (1994) Analysis of stage-specific transcripts of the Plasmodium falciparum
serine repeat antigen (SERA) gene and transcription from the SERA locus,
Molecular and Biochemical Parasitology, Vol. 68, No. 1, pp. 133-144.
Fox, BA.; Xing-Li P, Suzue K et al. (1997) Plasmodium falciparum: an epitope within a highly
conserved region of the 47-kDa amino-terminal domain of the serine repeat antigen
is a target of parasite-inhibitory antibodies, Experimental Parasitology, Vol. 85, No. 2,
pp. 121-134.
Gardner, MJ.; Tettelin H, Carucci DJ et al. (1998) Chromosome 2 sequence of the human
malaria parasite Plasmodium falciparum, Science, Vol. 282, No. 5391, pp. 1126-1132.
Gardner, MJ.; Hall N, Fung E et al. (2002) Genome sequence of the human malaria parasite
Plasmodium falciparum, Nature, Vol. 419, No. 6906, pp. 498-511.
Garzn-Ospina, D.; Cadavid LF & Patarroyo MA. (2010) Differential expansion of the
merozoite surface protein (msp)-7 gene family in Plasmodium species under a birth-
and-death model of evolution, Molecular Phylogenetics and Evolution, Vol. 55, No. 2,
pp. 399-408.
Gor, DO.; Li AC & Rosenthal PJ. (1998) Protective immune responses against protease-like
antigens of the murine malaria parasite Plasmodium vinckei, Vaccine, Vol 16, No. 11-
12, pp. 1193-1202.
Hayakawa, T.; Culleton R, Otani H et al. (2008) Big bang in the evolution of extant malaria
parasites, Molecular Biology and Evolution, Vol. 25, No. 10, pp. 2233-2239.
Hayakawa, T.; Arisue N, Udono T et al. (2009) Identification of Plasmodium malariae, a
human malaria parasite, in imported chimpanzees, PLoS One, Vol. 4, No. 10, e7412.
Hodder, AN.; Drew DR, Epa VC et al. (2003) Enzymic, phylogenetic, and structural
characterization of the unusual papain-like protease domain of Plasmodium
falciparum SERA5, The Journal of Biological Chemistry, Vol. 278, No. 48, pp. 48169-
48177.
Hodder, AN.; Malby RL, Clarke OB et al. (2009) Structural insights into the protease-like
antigen Plasmodium falciparum SERA5 and its noncanonical active-site serine, Journal
of Molecular Biology, Vol. 392, No. 1, pp. 154-165.
Horii, T.; Shirai H, Li J et al. (2010) Evidences of protection against blood-stage infection of
Plasmodium falciparum by the novel protein vaccine SE36, Parasitology International,
Vol. 59, No. 3, pp. 380-386.
Hughes, AL. & Nei M. (1989) Evolution of the major histocompatibility complex:
independent origin of nonclassical class I genes in different groups of mammals,
Molecular Biology and Evolution, Vol. 6, No. 6, pp. 559-579.
Hughes, AL. & Nei M. (1990) Evolutionary relationships of class II major-histocompatibility-
complex genes in mammals, Molecular Biology and Evolution, Vol. 7, No. 6, pp. 491-514.
Janssen, CS.; Phillips RS, Turner CM et al. (2004) Plasmodium interspersed repeats: the major
multigene superfamily of malaria parasites, Nucleic Acids Research, Vol. 32, No. 19,
pp. 5712-5720.


331
Kiefer, MC.; Crawford KA, Boley LJ et al. (1996) Identification and cloning of a locus of
serine repeat antigen (sera)-related genes from Plasmodium vivax, Molecular and
Biochemical Parasitology, Vol. 78, No. 1-2, pp. 55-65.
Knapp, B.; Hundt E, Nau U et al. (1989) Molecular cloning, genomic structure and
localization of a blood stage antigen of Plasmodium falciparum characterized by a
serine stretch. Molecular and Biochemical Parasitology, Vol. 32, No. 1, pp. 73-83.
Knapp, B.; Nau U, Hundt E et al. (1991) A new blood stage antigen of Plasmodium falciparum
highly homologous to the serine-stretch protein SERP, Molecular and Biochemical
Parasitology, Vol. 44, No. 1, pp. 1-13.
Levin, ND. (1988) The protozoan phylum Apicomplexa-volume II, CRC Press, ISBN 0-8493-4654-
1, Florida, USA.
Li, J.; Mitamura T, Fox BA et al. (2002a) Differential localization of processed fragments of
Plasmodium falciparum serine repeat antigen and further processing of its N-terminal
47 kDa fragment, Parasitology International, Vol. 51, No. 4, pp. 343-352.
Li J.; Matsuoka H, Mitamura T et al. (2002b) Characterization of proteases involved in the
processing of Plasmodium falciparum serine repeat antigen (SERA), Molecular and
Liu, Q.; Ferreira MU, Ndawi BT et al. (2000) Sequence diversity of serine repeat antigen gene
exon II of Plasmodium falciparum in worldwide collected wild isolates. Southeast
Asian Journal of Tropical Medicine and Public Health, Vol. 31, No. 4, pp. 808-817.
McBride, JS.; Newbold CI & Anand, R. (1985) Polymorphism of a high molecular weight
schizont antigen of the human malaria parasite Plasmodium falciparum, The Journal of
Experimental Medicine, Vol. 161, No. 1, pp. 160-180.
McCoubrie, JE.; Miller SK, Sargeant T et al. (2007) Evidence for a common role for the serine-
type Plasmodium falciparum serine repeat antigen proteases: Implications for vaccine
and drug design, Infection and Immunity, Vol. 75, No. 12, pp. 5565-5574.
Miller, SK.; Good RT, Drew DR et al. (2002) A subset of Plasmodium falciparum SERA genes
are expressed and appear to play an important role in the erythrocytic cycle, The
Journal of Biological Chemistry, Vol. 277, No. 49, pp. 47524-47532.
Morimatsu, K.; Morikawa T, Tanabe K et al. (1997) Sequence diversity in the amino-terminal
47 kDa fragment of the Plasmodium falciparum serine repeat antigen. Molecular and
Nei, M. & Hughes AL. (1992) Balanced polymorphism and evolution by the birth-and-death
process in the MHC loci. In: 11th Histocompatibility Workshop and Conference, Tsuji,
K., Aizawa, M., Sasazuki, T., editors. Vol. 2, pp. 27-38, Oxford University Press,
ISBN 0-19-262217-X, Oxford, UK.
Nei, M.; Gu, X. & Sitnikova T. (1997) Evolution by the birth-and-death process in multigene
families of the vertebrate immune system, Proceedings of the National Academy of
Sciences of the United States of America, Vol. 94, No. 15, pp. 7799-7806.
Nei, M. & Rooney AP. (2005) Concerted and birth-and death evolution of multigene
families, Annual Review of Genetics, Vol. 39, pp. 121-152.
Nishimoto, Y.; Arisue N, Kawai S et al. (2008) Evolution and phylogeny of the
heterogeneous cytosolic SSU rRNA genes in the genus Plasmodium, Molecular
Phylogenetics and Evolution, Vol. 47, No. 1, pp. 45-53.
Okech, BA.; Nalunkuma A, Okello D et al. (2001) Natural human immunoglobulin G
subclass responses to Plasmodium falciparum serine repeat antigen in Uganda, The
American Journal of Tropical Medicine and Hygiene, Vol. 65, No. 6, pp. 912-917.

Gene Duplication

332
Okech, B.; Mujuzi G, Ogwal A et al. (2006) High titers of IgG antibodies against Plasmodium
falciparum serine repeat antigen 5 (SERA5) are associated with protection against
severe malaria in Ugandan children, The American Journal of Tropical Medicine and
Hygiene, Vol. 74, No. 2, pp. 191-197.
Okitsu, SL.; Boato F, Mueller MS et al. (2007) Antibodies elicited by a virosomally
formulated Plasmodium falciparum serine repeat antigen-5 derived peptide detect
the processed 47 kDa fragment both in sporozoites and merozoites, Peptides, Vol.
28, No. 10, pp. 2051-2060.
Ota, T. & Nei M. (1994) Divergent evolution and evolution by the birth-and-death process in
the immunoglobulin VH gene family. Molecular Biology and Evolution, Vol. 11, No. 3,
pp. 469-482.
Pain, A.; Bohme U, Berry AE et al. (2008) The genome of the simian and human malaria
parasite Plasmodium knowlesi, Nature, Vol. 455, No. 7214, pp. 799-803.
Palacpac, NM.; Leung BW, Arisue N et al. (2006) Plasmodium vivax serine repeat antigen
(SERA) multigene family exhibits similar expression patterns in independent
infections, Molecular and Biochemical Parasitology, Vol. 150, No. 2, pp. 353-358.
Perrin, LH.; Merkli B, Loche M et al., (1984) Antimalarial immunity in Saimiri monkeys.
Immunization with surface components of asexual blood stages, The Journal of
Experimental Medicine, Vol. 160, No. 2, pp. 441-451.
Polley, SD.; Chokejindachai W & Conway DJ. (2003) Allele frequency-based analyses
robustly map sequence sites under balancing selection in a malaria vaccine
candidate antigen, Genetics, Vol. 165, No. 2, pp. 555-561.
Putrianti, ED.; Schmidt-Christensen A, Arnold I et al. (2010) The Plasmodium serine-type
SERA proteases display distinct expression patterns and non-essential in vivo roles
during life cycle progression of the malaria parasite, Cellular Microbiology, Vol. 12,
No. 6, pp. 725-739.
Rooney, AP. (2004) Mechanism underlying the evolution and maintenance of functionally
heterogeneous 18S rRNA genes in apicomplexans, Molecular Biology and Evolution,
Vol. 21, No. 9, pp. 1704-1711.
Schmidt-Christensen, A.; Sturm A, Horstmann S et al. (2008) Expression and processing of
Plasmodium berghei SERA3 during liver stages, Cellular Microbiology, Vol. 10, No. 8,
pp. 1723-1734.
WHO (2010) World Malaria Report 2010, WHO Press, ISBN 978 92 4 156410 6, Geneva,
Switzerland.
Yeoh, S.; O'Donnell RA, Koussis K et al. (2007) Subcellular discharge of a serine protease
mediates release of invasive malaria parasites from host erythrocytes, Cell, Vol. 131,
No. 6, pp. 1072-1083.
Zhang J.; Dyer KD. & Rosenberg HF. (2000) Evolution of the rodent eosinophil-associated
RNase gene family by rapid gene sorting and positive selection, Proceedings of the
National Academy of Sciences of the United States of America, Vol. 97, No. 9, pp. 4701-
4706.
18
Molecular Evolution of
Juvenile Hormone Signaling
Aaron A. Baumann and Thomas G. Wilson
The Ohio State University
United States of America
1. Introduction
Insect development proceeds through a series of discrete developmental stages called
instars. During hexapod evolution, the development of complete metamorphosis introduced
a novel mechanism for separating feeding and reproductive stages (Truman & Riddiford,
2002), facilitating the tremendous evolutionary success of holometabolous insects. In
contrast to hemimetabolous insects, which progress through a series of instars that appear
as smaller iterations of the adult form, holometabolous insects proceed from egg to adult
through a progression of isomorphic larval instars and a pupal transitory stage. In each case,
the physical boundary for growth during an instar is established by a chitinous exoskeleton,
which must be periodically shed. This molting process is under the control of two
counteracting hormones.
Toward the end of an instar, a pulse of the insect molting hormone, 20-hydroxyecdysone
(20E) initiates a transcriptional cascade that carries the molt to a subsequent instar.
However, it is the interaction of 20E and the sesquiterpenoid juvenile hormone (JH) that
governs the developmental outcome of each molt. During larval development, an elevated
JH titer and 20E directs the sequential progression through larval development until the
final larval instar, when the JH titer substantially declines. The removal of circulating JH
facilitates a 20E-directed developmental switch that initiates the metamorphic molt. Thus, it
was proposed that JH can modulate 20E activity, maintaining the status quo during pre-
adult development.
1.1 JH and JHAs: Insecticidal use of hormone agonists
Since the first chemical analysis resolved the sesquiterpenoid structure of endogenous JH
(Rller et al., 1967), several homologs have been identified, each bearing opposing, terminal
epoxide and methyl ester functions. Variation in the degree and identity of alkyl group
substitution at C3, C7, and C11 along the carbon skeleton defines the homologs. The
evolutionary importance of multiple JH homologs is unclear. JH 0, I, II, and III have all been
isolated from lepidopteran insects, whereas JH III, the presumed evolutionary precursor to
the higher homologs, is found in all insects. JH bisepoxide (JHB3) has been identified as a
product of the corpus allatum (CA) in higher Diptera including Drosophila melanogaster and
Sarcophaga bullata (Richard et al., 1989; Bylemans et al., 1998). Nearly identical in structure to
JH III, JHB3 is distinguished by an additional epoxide group spanning C6-C7.

Gene Duplication

334
The major JHs and some juvenile hormone analogs (JHAs) are presented in Figure 1.

Methyl farnesoate JH III
JHB3 JH II

JH I JH 0

Methoprene Pyriproxyfen

Fig. 1. Structures of endogenous JH molecules and two synthetic JHAs, methoprene and
pyriproxyfen.
The physiology and chemistry of JH prompted intense research into the synthesis and
commercial-scale production of JH analogs, or juvenoids, for agricultural use. The allure
of these compounds was at least twofold. First, juvenoids exhibit extremely low non-
target (in particular, mammalian) toxicity. Second, it was originally thought that insect
resistance to JHAs would be unlikely, since an insect was not likely to become refractory
to an endogenous hormone (Williams, 1967). Methoprene, a juvenoid structurally similar
to endogenous JH, has enjoyed success in the management of larval mosquito
populations. However, JHAs need not mimic the chemical structure of endogenous JH, as
exemplified by the pyridine-based pyriproxyfen, whose activity exceeds JH by two orders
of magnitude in dipteran white puparial and larval assays (Riddiford and Ashburner,
1991).
Exogenous JH exposure can elicit classic antimetamorphic activity in both Lepidoptera and
Coleoptera (Srivastava & Srivastava, 1983; Konopova & Jindra, 2007), extending larval
development through one or more supernumerary instars. Also in these insects, exposure to
exogenous JH or to its chemical analogs (JHA) can result in the deposition of a second pupal
cuticle (Zhou & Riddiford, 2002). Thus, in Lepidoptera and Coleoptera, JH exposure at an
inappropriate time inhibits 20E-directed developmental progression.
In Diptera, treatment with exogenous JH produces dose-dependent lethality at the pharate
adult stage. All adult structures arise from imaginal discs in flies, and these discs are
insensitive to JH during development, unlike Lepidoptera and Coleoptera, in which the

Molecular Evolution of Juvenile Hormone Signaling

335
polymorphic larval epidermis gives rise to pupal and adult structures. When flies are
challenged with JHAs, the adult structures that differentiate from imaginal discs remain
unaffected (Postlethwait, 1974). In D. melanogaster, only the abdominal histoblasts are JH
sensitive; diagnostic (sublethal) doses of methoprene disrupt abdominal bristle formation in
female flies (Madhavan, 1973).
2. Molecular mechanism of JH signal transduction
The molecular events underlying 20E signaling are relatively well understood. Ecdysone
released from the prothoracic glands is converted to its active metabolite 20E in target
tissues, where it regulates transcription through a heterodimeric receptor complex
comprised of Ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. When bound with 20E,
ECR-USP recognizes and binds ecdysone response elements located in the promoter region
of target genes, inducing transcription of a hierarchical network of early and late genes. The
early genes either repress their own expression or induce expression of late genes
(Ashburner et al., 1974). In this manner, the expression of genes involved in the 20E
transcriptional cascade is tightly controlled. In contrast, the nature of JH signal transduction
has been difficult to elucidate, largely due to the enigmatic nature of the JH receptor. A body
of ever-increasing experimental evidence strongly supports the product of the Methoprene
tolerant (Met) gene as the prime candidate for a JH receptor component (Wilson & Fabian,
1986; Konopova & Jindra, 2007; Yang et al., 2011).
Met was originally discovered by screening progeny of ethyl methanesulfonate (EMS)-
mutagenized D. melanogaster for resistance to methoprene (Wilson & Fabian, 1986). Met
mutants show dramatically enhanced (~100 fold) resistance to both the toxicity and
morphogenetic defects caused by methoprene exposure, but not to other classes of
insecticides (Wilson & Fabian, 1986). Such resistance is not restricted to compounds with
high structural similarity to JH; Met mutants are also resistant to the more potent,
structurally distinct JHA pyriproxyfen (Riddiford & Ashburner, 1991).
Cloning and sequence analysis identified Met as a member of the basic Helix-Loop-Helix
Period Ahr Sim (bHLH PAS) family of transcriptional regulators (Ashok et al., 1998). PAS
proteins function as dimers in a diverse array of functions in development, xenobiotic
binding, and detection of environmental signals (Crews, 1993). Both the bHLH domain and
the PAS repeats (PAS A and B) facilitate dimerization between PAS proteins (Huang, et al.,
1993). Additionally, the PAS domains function in small molecule ligand binding and target
gene specificity. Each dimerization partner recognizes and binds one half of a palindromic
E-box consensus sequence CANNTG in the promoter region of target genes via the stretch
of basic residues immediately N-terminal to the HLH motif. Examples of PAS proteins with
ligand binding activity include the bacterial photoreactive yellow protein (PYP), and the
vertebrate aryl hydrocarbon receptor (Ahr).
Genetic and biochemical data show that MET binds JH with nanomolar affinity
(Shemshedini & Wilson, 1990) and that MET product is present in the nuclei of several
known JH target tissues, including ovary, MAG, and larval fat body (Pursley et al., 2000). In
addition, MET can drive the expression of a reporter gene in a JH-sensitive manner (Miura
et al., 2005). All of the above data satisfy criteria for a hormone receptor.
Analysis of the Met
27
null allele provided the first demonstration of insecticide resistance
due to the absence of a target macromolecule (Wilson & Ashok, 1998). Even though Met
27

flies are viable, Met deficiency carries reproductive consequences, namely substantially

Gene Duplication

336
reduced oogenesis (~20% compared to Met
+
), consistent with a role for JH in this
physiology. However, since absence of a JH receptor is expected to preclude normal
development, the viability of Met
27
flies challenged the notion of Met as a bona fide JH
receptor. Some evidence supports alternative mechanism(s) of JH signaling (see Flatt et al.,
2008; Riddiford et.al., 2010). In this chapter, we review data that support the notion of germ
cell expressed (gce), the paralog of Met in higher Diptera, as conferring at least partial
functional redundancy.
3. Met homologs across holometabola
Reports of methoprene resistance in mosquito populations (Dame et al., 1998; Cornel et al.,
2000; Cornel et al., 2002) led us to investigate the Met orthologs of three mosquito species:
Aedes aegypti, Culex pipiens, and Anopheles gambiae. Using a combination of degenerate RT-
PCR and genomic database mining, we isolated a single Met homolog from each of these
mosquitoes. Sequence analysis of these genes showed that they share high identity with
both Met and the closely related gce from Drosophila, as expected. However, a comparison of
the genomic structures among DmMet, Dmgce, and the three putative mosquito Met genes
revealed higher structural conservation between each mosquito Met and Dmgce.
Importantly, the intron number of these genes is more consistent with that of Dmgce than
DmMet (from six to nine, versus one in DmMet). Furthermore, several introns in each
mosquito gene are positionally conserved with those in Dmgce. This led to our proposal that
the Met gene of higher Diptera originated via retrotransposition of a basal, gce-like gene of
lower Diptera (Wang et al., 2007).
Retrotransposition, or retroposition is a mechanism of gene duplication that proceeds
through an mRNA intermediate. Following post-transcriptional splicing, the parental
message is reintegrated into the genome. Ultimately, for the duplicate copy to escape the
fate of becoming a pseudogene, it must reintegrate with associated regulatory elements
intact or incorporate into a suitable transcriptional environment elsewhere in the genome.
Following duplication, the increase in copy number of the parental gene affords a relaxation
of selective constraint, facilitating functional divergence. This may manifest as
subfunctionalization, in which a modification of the parental function evolves, or
neofunctionalization, which refers to attainment of a novel function (MacCarthy & Bergman,
2007). DmMet retains a strong diagnostic feature of retroposition: a paucity of introns
relative to gce, which is consistent with splicing and genomic reintegration of an ancestral
gce-like transcript.
A conserved gce-like gene appears to be conserved across holometabolan genomes,
including the red flour beetle, Tribolium castaneum, and the honeybee, Apis mellifera. An
independent gene duplication within the Lepidoptera has given rise to two Met-like
proteins, presently called Methoprene tolerant proteins I and II, whose functions are
currently under investigation (i.e. Li et al., 2010). Despite a demonstrated sequence
conservation favoring the Met-like genes of more primitive Holometabola as ancestral to gce,
we will continue to refer to these genes as Met-like in this text.
3.1 Met and gce within the genus Drosophila
When the genomes of 12 representative Drosophila species became available (Ashburner,
2007), we chose to examine the molecular evolution of Met and gce within this genus of flies.
Both paralogs are conserved in each species, indicating that the origin of Met predates that


337
of the genus Drosophila, some 63 million years ago (Tamura et al., 2004). The architecture of
these genes is generally conserved in each species, with a few notable exceptions. A single
conserved intron is present in Met in the PAS B domain of 11 species. In addition to this
conserved intron, independent intron gains have occurred in the lineages leading to D.
simulans and D. willistoni. A single Met ortholog exists in each Drosophila genome examined,
but D. persimilis harbors two separate, consecutive loci on the X chromosome, currently
called GL13106 and GL13107, that align to distinct regions of DmMet. The 5 putative gene
GL13106 contains a complete PAS A domain followed by a severely truncated PAS B
domain. We performed RT-PCR across these two genes and failed to obtain a single PCR
product, suggesting that GL13106 and GL13107 indeed code for two distinct open reading
frames. Eleven of the 12 representative gce orthologs contain at least six conserved introns,
with independent intron gains evident in the lineages leading to D. melanogaster, D.
pseudoobscura, and D. mojavensis, whereas a substantial deletion in D. persimilis gce has
eliminated the central portion of this gene, including the PAS repeats.
In addition to the bHLH, PAS, and PAC domains, putative transactivation domains (TAD)
are evident in Met and gce orthologs. TADs are glutamine and/or aspartic acid-rich motifs
whose amino acid sequences are broadly defined and generally reside in the C-terminal
region of PAS proteins (Ramadoss & Perdew, 2005). Met homologs show Q- and D-rich
motifs between the PAS B and PAC domains, while alignments of gce homologs indicate a
D-rich region C-terminal to the PAC domain. Miura et al. (2005) suggest the presence of a C-
terminal TAD in recombinant MET protein, but this region has yet to be functionally
defined.
Using DmMet and Dmgce as query sequences, we conducted homology searches under
tBLASTx criteria (translated nucleotide query to search a translated nucleotide database)
against the publicly available EST library of Glossina morsitans, the tsetse fly. Our search
recovered several clones, which were imported into the Sequencher program to produce
two independent contigs. These composite nucleotide sequences were used to infer a gene
tree with other holometabolan Met and gce orthologs, including those of two representative
Drosophila species (Figure 2). This preliminary analysis reveals the presence of distinct Met
and gce orthologs in the G. morsitans genome, indicating that the origin of Met predates the
divergence of the Aschiza and Schizophora. These two taxonomic groups, which are
estimated to have diverged more than 85 million years ago (Bertone & Wiegmann, 2009),
reside within the brachyceran infraorder Muscomorpha.
3.2 Evidence for differential selective constraint imposed on Met and gce
Based on an a priori hypothesis that Met and gce were subject to differential post-duplication
selective constraint, we performed analyses of nonsynonymous-to-synonymous (dN/dS)
substitution ratios on codon alignments of these Drosophila paralogs. Datasets were analyzed
using the DataMonkey tool (Kosakovsky-Pond & Frost, 2005), a web-based implementation
of the HyPhy package (Kosakovsky Pond et al., 2005). dN/dS analyses can be used to infer
the relative selective pressure along entire coding sequences or in a site-specific manner. A
substantially depressed dN/dS ratio (i.e. zero or close to zero) implies purifying (negative)
selection. That is, nonsynonymous changes are stringently selected against. In contrast,
when dN/dS is nearly one, neutral evolution is inferred. A dN/dS value far in excess of one
implies positive selection, or adaptive evolution. In this case, nonsynonymous substitutions
confer a selective advantage.

Gene Duplication

338
The results of our dN/dS analyses showed dramatic dissimilarity in the relative selective
pressures that have shaped the coding sequences of Met and gce. In the case of Met, dN/dS
was generally suppressed along the entirety of the coding sequence, indicating strong
selection against nonsynonymous codon substitution. This is perhaps surprising, since MET
deficiency has no effect on viability (Wilson & Ashok, 1998). Possibly, mutations that alter
amino acid identities are selected against in Met due its involvement in reproduction. In the
absence of methoprene selection, Met mutants are quickly out-competed by wild type flies
despite the seemingly slight fitness cost of Met loss (Minkhoff III & Wilson, 1992). In
contrast, dN/dS values close to one dominate the C-terminal half of gce, indicating a
substantial relaxation of selective constraint in this region. The N-terminal region of this
gene, containing the canonical bHLH and PAS functional domains, shows a strongly
depressed dN/dS. Based on functional data from other PAS proteins, this region is assumed
to harbor DNA and ligand binding activity, whereas the C-terminal region contains putative
TADs. C-terminal degeneracy was shown to confer differential target gene specificity
between the Ahr homologs of mice and humans (Ramadoss & Perdew, 2005; Flaveny et al.,
2010). Similarly, the disparate selective constraints evident in the C-terminal regions of Met
and gce may partially define these genes functions.

Fig. 2. A gene tree of some holometabolous Met-like genes, showing placement of two
distinct G. morsitans sequences as putative Met and gce orthologs. D. melanogaster Tango (tgo),
the homolog of the vertebrate Aryl hydrocarbon receptor (Ahr), is used as an outgroup
sequence.


339
4. Toward a functional definition of Dmgce
A functional characterization of gce, named for its expression in a subset of embryonic germ
cells (Moore et al., 2000), is in its infancy. Column pulldown assays showed MET, in addition
to forming homodimers, forms heterodimers with GCE, and addition of JH or either of two
JHAs significantly impaired these interactions (Godlewski et al., 2006). It is unknown
whether GCE forms homodimers, like MET, or whether GCE can bind JH or its analogs.
GAL4/UAS-driven (Brand & Perrimon, 1993) overexpression of Met
+
from actin or tubulin
promoters results in larval lethality in the absence of methoprene (Barry et al., 2008), perhaps
by upsetting the stoichiometry of MET and GCE dimers, favoring MET homodimerization at
inappropriate times or in inappropriate tissues. Recently, JH was shown to inhibit MET and
GCE in D. melanogaster by preventing caspase-driven programmed cell death (PCD) and
histolysis of the larval fat body. DRONC and DRICE, evolutionarily conserved caspase
genes involved in this physiology at the onset of metamorphosis, were shown to be
downregulated in Met and gce deficient flies (Liu et al., 2009). Similarly, methoprene
interferes with caspase-driven midgut remodeling in A. aegypti (Nishiura et al., 2003; Wu et
al., 2006) and T. castaneum (Parthasarathy et al., 2008; Parthsarathy et al., 2009), showing that
this mechanism of JH action is evolutionarily conserved. It is noteworthy that recombinant
MET can repress reporter gene expression in the absence of JH (presumably, MET forms
homodimers in this system; Miura et al., 2005); transcriptional repression has previously
been reported in other PAS proteins (Dolwick et al., 1993). Therefore, the JH-dependent,
stage-specific formation of alternative MET/GCE dimers may have unique regulatory
consequences on distinct suites of target genes.
4.1 Dmgce substitution for DmMet
To evaluate the notion that gce might confer viability to Met null flies, we manipulated gce
expression using a binary UAS/GAL4 system to drive either a gce cDNA or an RNAi
construct designed to target gce transcript. We carried these experiments out in a variety of
genotypic contexts in order to examine the effect of gce transcript abundance on several
methoprene conditional and non-conditional phenotypes (Baumann et al., 2010b). First, we
explored the effect of gce over- and under-expression on a Met-specific non-conditional
phenotype that manifests as a variable number of grossly malformed posterior facets of the
compound eye (Figure 3). This phenotype is visible in Met
27
and Met
w3
flies, and is enhanced
in the latter genotype. In our experiments, we found that gce overexpression in a Met
w3
genetic background can rescue the Met-specific eye phenotype, suggesting functional
overlap of gce and Met. Notably, when gce was overexpressed in a Met
27
background from
the GawB}dan[AC116] promoter, targeting transgene expression to the compound eye, the
eye phenotype was completely rescued (Baumann et al., 2010b).
The Met
27
phenotype mimics a set of defects resulting from genetic ablation of the JH-
producing corpus allatum (CAX), including a heterochronic shift in EcR-B1 expression in the
optic lobe (Riddiford et.al., 2010). Exogenous JH application rescues the entire suite of
defects in CAX prepupae, while JH provision to Met
27
flies rescues only a subset of these
defects, suggesting an alternate mechanism of JH signal transduction (Riddiford et al., 2010).
Based on our findings that gce can substitute for Met in the compound eye, further study of
GCE involvement in eye development may provide a link between these phenomena. For
instance, GCE may partially substitute for MET as a ligand binder to mediate JH signaling
when this hormone is supplied in excess.

Gene Duplication

340

Fig. 3. Left: malformed facets in the posterior compound eye of Met
w3
flies appear dark
under light microscopy. Right: EMS-induced production of an unidentified enhancer gene
dramatically intensifies the Met
w3
phenotype (T.G.W., unpublished).
We also explored the effect of gce overexpression on several methoprene-conditional
phenotypes. Overexpressed gce rescued both the diagnostic malrotation of male genitalia
and sensitivity to the toxic effects of methoprene exposure. Sublethal doses of methoprene
can induce malrotation of the male genital disc in D. melanogaster, resulting in terminalia
that are improperly oriented for copulation (Bouchard & Wilson, 1987). Met
27
males are
resistant to this phenotype. We found that global gce overexpression in a Met
27
background
rescues blockage of the malrotation phenotype in Met
27
; UAS-gce/ tubulin-GAL4 flies. When
these flies were exposed to methoprene, we observed malrotation close to levels seen in
Met
+
flies (Baumann et al., 2010b).
Met and gce are generally co-expressed in JH target tissues, but we detected insignificant
amounts of gce transcript in late third instar larval fat body. When gce was expressed from
a construct targeting expression to this tissue, partial rescue of JH-induced pupal lethality
was achived, perhaps as a result of supplying gce to a tissue in which its expression is
normally depressed at this time in development. gce expression in the larval fat body was
unable to rescue either the eye phenotype or to prevent methoprene-induced malrotation
of the male genitalia, indicating that gce substitution for Met is tissue specific (Baumann et
al., 2010b).
4.2 Functional partitioning of DmMet and Dmgce in D. melanogaster reproduction
Following metamorphosis, the interaction of 20E and JH is crucial in insect reproduction.
JH was first isolated in large quantities from the MAG of Hyalophora cecropia (Williams,


341
1956), suggesting a role in male reproductive biology. In D. melanogaster, JH controls MAG
protein accumulation (Yamamoto et al., 1988) and male apterous (ap) mutants court females
less vigorously than wild-type flies (Tompkins, 1990). In females, the activity of these
counteracting hormones is critical for ovarian development and oocyte maturation.
Development of the D. melanogaster oocyte is under the control of JH through
previtellogenic stages 8-9. Female D. melanogaster apterous
4
mutants are sterile owing to
reduced levels of JH synthesis (Bownes, 1989); provision of exogenous JH rescues
vitellogenic oocyte development in ap females (Postlethwait & Weiser, 1973). In A. aegypti,
JH also controls previtellogenic ovarian development (Clements, 1992). In this case, JH
signaling is necessary to promote 20E competence in the fat body, the site of post-blood
meal vitellogenin synthesis. In contrast, vitellogenesis is retarded by JH treatment in the
gypsy moth, Lymantria dyspar (Davis et al., 1990). Thus, there is variation in hormonal
control in insects.
GCE clearly compensates for MET deficiency in preadult development (Baumann et al.,
2010b). In our experiments, over-expressed gce failed to rescue both the documented
behavior of reduced courtship in Met
27
; UAS-gce/tubulin-GAL4 males and the reduction in
oocyte development and oviposition in these females. Therefore, it appears that excess gce
cannot compensate for Met-induced reduction of reproductive capacity. This result suggests
that the functional roles for MET and GCE are incompletely partitioned between preadult
development and reproduction in adults.
In A. aegypti, AaMet regulates the transcription of several JH target genes in newly eclosed,
previtellogenic adult females (Zhu et al., 2010). Presumably, the MET-like gene product in
lower Diptera serves an analogous function both MET and GCE in JH signaling, but through
the action of a single gene. This is perhaps accomplished by virtue of its modular
architecture of DmMet- and Dmgce-specific domains. Higher sequence identity exists
between the bHLH and PAS B of Dmgce and more primitive holometabolous Met-like genes,
while the PAS A and PAC domains share higher sequence identity with DmMet. These
domains may confer a discriminating Met-like function that may partially underlie the
functional divergence of Met and gce in higher Diptera.
4.3 Dmgce is a vital gene
Overepxression studies demonstrated that gce can substitute for Met in a tissue specific
manner to rescue several preadult Met mutant phenotypes. Hence, our results empirically
support the notion of functional redundancy between Met and its paralog gce. To further
explore the relationship between Met and gce in JH signaling, we carried out
underexpression studies in Met
+
and Met mutant backgrounds by driving the expression of
a gce RNAi construct.
First, we examined the consequence of gce deficiency in a Met mutant background under the
justification that, if gce is responsible for Met
27
viability, then concomitant reduction of Met
and gce could result in lethality. Interestingly, Met
27
; UAS-gce-dsRNA/tubulin-GAL4 flies
died as early pupae (0-2 days), whereas expression of the dsRNA construct from an actin-
GAL4 promoter caused lethality in the pharate adult stage. Next, we assessed the effects of
gce reduction in Met
+
flies. Surprisingly, Met
+
; UAS-gce-dsRNA/tubulin-GAL4 flies died as
pharate adults, indicating that even in the presence of functional MET, gce is a vital gene.
Driving the transgene from an actin-GAL4 promoter allowed some degree of adult survival,
but these adults were clearly affected by insufficient gce, dying within two to three days.

Gene Duplication

342
Differential intensity of transgene expression from actin and tubulin promoters was
previously reported in our lab (Barry et al., 2008).
gce underexpression had no observable effect on embryonic development, a stage during
which no role for JH has been demonstrated. We have shown that gce transcription begins
after about eight hours in early embryos, in contrast to Met, which is supplied as a maternal
message (Baumann et al., 2010a). The importance of such divergence in temporal expression
profiles is unclear.
5. Evolutionary conservation of JH signaling mechanisms
Numerous JH target genes have been identified throughout Holometabola. Importantly,
many of these genes are known components of the early 20E response. Table 1 lists some
representative JH-inducible genes.

Symbol Gene name Molecular function Reference
JhI-1 JH inducible protein 1 Endoribonuclease Dubrovsky et al.,
2000 JhI-26 JH inducible protein 26 Unknown
Br Broad-Complex (BR-C) BTB POZ zinc finger
transcription factor
Zhou et al., 1998;
Zhou &
Riddiford, 2002
mnd Minidisks Amino acid
transmembrane
transporter
Dubrovsky et al.,
2002
JhI-21 JH inducible protein 21 Amino acid
transmembrane
transporter
JHE JH esterase JH-specific esterase Kethidi et al., 2005
E75A Ecdysone-induced
protein 75B
Heme binding Dubrovsky et al.,
2004
E74B Ecdysone-induced
protein 74EF
RNA polymerase II
transcription factor
activity
Beckstead et al.,
2007

pepck Phosphoenolpyruvate
carboxykinase
Phosphoenolpyruvate
carboxykinase (GTP)
activity
CG14949 CG14949 Unknown
Table 1. Representative JH-inducible genes. Many of these genes have evolutionarily
conserved roles in JH signaling in holometabolous insects. In addition, several are known
components of the 20E transcriptional cascade.
The majority of the work done in our lab has been carried out on D. melanogaster, in which
DmMet clearly plays a role in JH signaling: its absence both interferes with methoprene
toxicity (Wilson & Fabian, 1986) and hinders JH-driven reproductive physiology (Wilson,
1992; Wilson et al., 2003). However, Met involvement in metamorphosis has been difficult to
demonstrate in Drosophila (Riddiford, 2008). As previously stated, JH exposure has no effect
on dipteran entry into metamorphosis, unlike other insects (Williams, 1961; Zhou &
Riddiford, 2002). In recent years, researchers have turned to the model coleopteran, T.


343
castaneum. These beetles are both amenable to genetic manipulation and gene knockdown
owing to the dramatic effects of systemic RNAi, and the larvae of this species are very
sensitive to JH, unlike D. melanogaster larvae. Exposure to JH or a number of its chemical
analogs precipitates supernumerary larval instars, similar to the effects of JH on the model
lepidopteran, Manduca sexta (Parthasarathy & Palli, 2009). T. castaneum, like mosquitoes, has
a single Met-like gene. In their seminal paper, Konopova and Jindra (2007) demonstrated
that RNAi-mediated knockdown of TcMet results not only in a methoprene resistance
phenotype, but also in the precocious metamorphosis of early instar larvae. A long sought-
after result, the genetic reduction of TcMet provided the phenotype frustratingly absent in
D. melanogaster: metamorphic disruption. Reproductive roles for TcMet have also been
shown; TcMet knockdown results in a substantial decrease in vitellogenin transcription,
(Parthasarathy, et al., 2010) consistent with Met deficiency in D. melanogaster females (Wilson
& Ashok, 1998). These results demonstrate that the single Met-like genes in primitive
Holometabola function in both development (metamorphosis) and reproduction. Further
functional characterization of TcMet (and the single Met-like gene of lower Diptera) could
lead to a better understanding of how DmMet has apparently co-opted reproductive
functional roles from a gce-like ancestor in higher Diptera
5.1 JH regulation of the E-20 transcriptional cascade
The molecular networks that link JH and 20E signaling pathways form the foundation of
multiple aspects of insect physiology, as evidenced by the criticality of both hormones in
development, reproduction, and diapause (Zhou & Riddiford, 2002; Soller et al., 1999;
Denlinger, 1985). Broad Complex (Broad or BR-C) is an early gene in the 20E cascade that
encodes a family of alternatively spliced zinc finger transcription factors (four in D.
melanogaster, Z1-Z4) fused to a common core protein. Certain Broad alleles phenocopy the
morphogenetic defects incurred by methoprene exposure in D. melanogaster. Wilson et al
(2006) showed phenotypic synergism in Met and broad double mutants, demonstrating JH-
sensitive MET and BROAD interaction (BROAD protein accumulation is comparable to that
of wild type flies, suggesting physical interaction with, rather than transcriptional regulation
by Met), and providing a link between JH and 20E signaling (Wilson et al., 2006).
In a hemimetabolous insect, Oncopeltus fasciatus, continuous Broad expression directs
progressive development through nymphal instars (Erezyilmaz et al., 2006). In
Holometabola, Broad expression is confined to the prepupal stage, acting as a pupal specifier
(Zhou & Riddiford, 2002). Loss of Broad expression, characteristic of the npr1 mutant (non-
pupariating; a deletion of the entire complementation group), results in the namesake
phenotype of failure to enter the pupal program. Consequently, a restriction of Broad
expression during this developmental stage may have contributed to the evolution of
complete metamorphosis. During larval development in D. melanogaster, JH represses broad.
At pupariation, exogenous JH induces a second wave of broad expression in the abdominal
epidermis, resulting in the deposition of a second pupal cuticle (Zhou & Riddiford, 2002),
demonstrating that the networks underlying these signaling mechanisms are complex.
In T. castaneum, methoprene exposure induces Broad expression and this upregulation is
ablated upon TcMet knockdown. Therefore, TcMet is upstream of Broad in JH signaling in
these beetles (Konopova & Jindra, 2008). Krppel homolog 1 (Kr-h1) is upstream of Broad in D.
melanogaster JH signaling, where its expression in abdominal epidermis produces sternal
bristle disruption similar to that seen following low dose JHA exposure (Minakuchi et al.,

Gene Duplication

344
2008). Genetic suppression of TcKr-h1 induces precocious metamorphosis, similar to TcMet
deficiency; TcMet knockdown in combination with JHA treatment demonstrated that TcKr-
h1 exists downstream of TcMet and upstream of TcBroad (Minakuchi et al., 2009). Similarly,
Kr-h1 upregulation in newly eclosed A. aegypti females depends on AaMet expression (Zhu
et al., 2010). Therefore, the relationships among these genes are generally conserved within
holometabolan evolution.
While Kr-h1 also has demonstrated roles in JH-influenced social behavior of honeybees, it
has not been reported whether AmKr-h1 is under the transcriptional control of an A. mellifera
Met-like protein. However, there is evidence for conservation in the sets of genes regulated
by JH between flies and bees. This is perhaps unsurprising given the deep evolutionary
conservation of these genetic mechanisms; Kr-h1 and Broad expression profiles in two
species of hemimetabolous thrips, whose life histories involve pupa-like, quiescent or non-
feeding stages, are compatible with the expression profiles of Broad and Kr-h1 in
holometabolous insects (Minakuchi et al., 2011).
Microarray data from D. melanogaster and A. mellifera identified a subset of conserved, JH-
inducible genes (Li et al., 2007). In the promoter region of 16 of the D. melanogaster orthologs, a
conserved JH response element (JHRE) was identified. RNAi-driven reduction of the
expression of two proteins identified as JHRE binders, FKBP39 and Chd64, inhibits JHIII-
induced expression of a reporter construct, suggesting their involvement in JH-dependent
transcriptional machinery. Bitra and Palli (2009) demonstrated physical interaction of MET
with both ECR and USP. Furthermore, column pulldown assays showed FKBP39 and CHD64
as binding partners of D. melanogaster ECR, USP, and MET, providing a more robust
framework for a protein complex involving constituents of both JH and 20E signaling
pathways (Li et al., 2007). FKBP39, which is present at the onset of metamorphosis (Riddiford,
2008), is an inhibitor of autophagy in D. melanogaster; FKBP39 overexpression precludes the
developmental autolysis of larval fat body cells in wandering third instar larvae (Juhsz et al.,
2007), a physiology shown to be partially dependent on MET/GCE regulation of caspase gene
expression (Liu et al., 2009). A role for GCE in any of these protein complexes has yet to be
reported. Chd64 is expressed during larval molts, but not in the third instar or during
metamorphosis (Riddiford, 2008). Accordingly, putative regulatory complexes consisting of
different combinations of these elements may assemble in a stage- or tissue-specific manner.
Assembly of differential protein complexes in response to JH, 20E, or both could be a strategy
for the tight regulation of the activities of these counteracting hormones.
The Met-like genes of Tribolium and Drosophila appear to act in similar genetic
environments to regulate the expression of members of the 20E induced transcriptional
cascade, including EcR (Riddiford et al., 2010) and USP (Xu et al., 2010), the heterodimeric
components of the ecdysone receptor, various orphan nuclear receptors involved in 20E
activity, and 20E-induced caspase genes involved in PCD (Liu et al., 2009). Knockdown of
seven nuclear receptors (E75, HR3, EcR, USP, SVP, FTZ-F1, and HR4) results in a
significant reduction of vitellogenin prouction in T. castaneum (Xu et al., 2010), a
phenotype similar to that obtained via TcMet knockdown (Parthasarathy & Palli, 2009).
The data presented in this section therefore strongly support for the action of Met-like
genes as crucial to 20E/JH crosstalk.
5.2 Discovery of an evolutionarily conserved Met binding partner
Recent biochemical data from A. aegypti indicate that AaMet binds another bHLH PAS gene,
AaFISC, and that this interaction requires a high JH titer. FISC is a coactivator of EcR/USP


345
(Chen, J.D., 2000; Zhu, et al., 2006), providing yet another link between 20E and JH signaling.
The authors also report that coexpression of DmMet or Dmgce with DmTaiman (the D.
melanogaster AaFISC ortholog) in the presence of JH III induced reporter gene expression in
L57 cells (Li et al., 2011). Furthermore, this interaction has also been demonstrated in T.
castaneum between TcMet and the FISC/TAI homolog, TcSRC (Steroid receptor coactivator;
Zhang et al., 2011). This gene has previously been implicated in metamorphic activity. T.
castaneum larvae treated with SRC RNAi fail to achieve critical weight and consequently die
before the larval-pupal transition (Bitra et al., 2009). Therefore, MET interaction with
FISC/SRC/TAIMAN underpins key transcriptional events of JH signaling throughout
holometabolous insects.
Structure-function analyses performed using site-directed mutagenesis identified regions of
MET that are necessary for homodimerization and GCE binding. Point mutations in the
bHLH and PAS A domains (Met
1
and Met
w3
alleles, respectively) had no effect on partner
binding, whereas N- and C-terminal truncations, deletions in the HLH or PAS A domains,
and a point mutation in the PAS B domain (Met
128
allele) all inhibited dimerization
(Godlewski, et al., 2006). Structure-function data for AaMET and AaFISC binding illustrates
that the criticality of PAS domains for protein-protein interaction. Interestingly, two-hybrid
assays showed that MET/FISC interaction increased when AaMET lacked a bHLH domain
(Li et al., 2011). Therefore, this domain is unnecessary for MET-FISC interaction, suggesting
that the sole function of the AaMet bHLH may be in DNA binding. In contrast, deletion of
the bHLH domain in FISC hindered the JH-induced interaction with AaMET.
Mixespression of DmTaiman in a variety of Met/gce genetic backgrounds will be valuable
from both physiological and evolutionary perspectives. How do these proteins interact in
the context of hormonal control of D. melanogaster development? Presumably, during larval
development JH secreted from the CA inhibits MET and GCE interaction while promoting
MET and GCE binding with TAIMAN. Are MET:TAI and GCE:TAI dimers functionally
congruent in D. melanogaster or do these complexes preferentially regulate disparate target
genes? Is the Met-like gene in A. aegypti and T. castaneum functionally analogous to Met/gce
or are other, unidentified proteins involved? How has the interaction of these proteins
changed during dipteran evolution following the origin of Met?
6. Conclusions
RT-PCR analysis with degenerate primers identified a single Met-like homolog in the
genome of each of the three mosquito species, Aedes aegypti, Anopheles gambiae, and Culex
pipiens. Likewise, a single Met-like ortholog exists in the beetle, T. castaneum, (Konopva &
Jindra, 2007). Phylogenetic analysis and comparison of intron number and position in each
of the identified mosquito genes indicates that the mosquito Met orthologs share higher
sequence identity with Dmgce than DmMet, suggesting that DmMet arose from the
duplication of an ancestral, gce-like gene in lower Diptera. To examine the evolutionary
history of Met and gce within the Diptera, we mined the public G. morsitans EST library,
recovering unique putative Met and gce orthologs in this fly, showing conservation of a Met
homolog within the Schizophora. We also recently isolated a putative gce homolog from a
Bombyliid, Bombylius major (A.A.B., unpublished). Taxonomically, this group of flies exists
in the Asilomorpha, a paraphyletic sister taxon to the Muscomorpha within the dipteran
infraorder, Brachycera. While this study is in its preliminary stages, only a single Met-like
gene has thus far been obtained from this fly using degenerate PCR with cDNA and

Gene Duplication

346
genomic DNA templates, suggesting the possibility that the Met/gce duplication occurred
within the Brachycera. Met function is evolutionarily conserved in Diptera; consistent with
independent reports (Zhu et al., 2010) we observed that RNAi-driven reduction of AaMet
results in concomitant reduction of JH-inducible genes (Figure 4).

0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
Met JHE (AAEL012886) JhI-1(AAEL006600) JhI-26 (AAEL000516)
dsB-gal dsMet

Fig. 4. Expression of three JH-inducible genes following RNAi-induced knockdown of
AaMet (dsMet: orange bars) vs. controls (dsB-gal: red bars). AaMet reduction produced
concomitant suppression of the A. aegypti homologs of DmJHE, DmJhI-1, and DmJhI-26.
Analysis of the nonsynonymous-to-synonymous substitution ratios (dN/dS) of Met and gce
orthologs within the genus Drosophila indicates a substantial relaxation of selective
constraint on the C-terminal half of gce, downstream of the functional domains. Conversely,
nonsynonymous substitutions in the N-terminal half are stringently selected against.
Depressed dN/dS values across the Met coding sequence indicate strong selective constraint
over the entire open reading frame (Baumann et al., 2010b).
RT-PCR analysis of selected D. melanogaster tissues shows that gce is generally co-expressed
with Met in known JH target tissues, including ovary and MAG. Overexpression of gce in a
Met mutant background results in a dramatic enhancement of methoprene-conditional toxic
and morphogenetic defects, similar to those seen in wild type (Met
+
) flies after methoprene
exposure. Met mutant flies overexpressing gce show rescue of a non-conditional adult
phenotype, that of defective development of posterior facets in the compound eye. Our
results therefore support the notion of functional redundancy that has been hypothesized to
account for Met
27
viability flies. On the other hand, we have also shown that these paralogs
have undergone evolutionary subfunctionalization since their origin; gce overexpression


347
fails to rescue the phenotypes of deficient oogenesis or reduced male courtship characteristic
of Met adults, showing that Met has co-opted therole as the mediator of JH-regulated
reproductive functions in Drosophila.
RNAi-driven reduction of gce expression from either an actin or tubulin promoter
demonstrates that unlike Met, gce is a vital gene. Gce underexpression in both Met
+
and Met
genetic backgrounds results in lethality. Met
+
; UAS-gce-dsRNA / tubulin-GAL4 homo-
/hemizygotes do not survive to adulthood, and die primarily at the pharate adult stage,
while the same gce RNAi construct expressed in a Met mutant background shifts lethality to
early pupae (Baumann et al., 2010a).
6.1 Directions
Previously, USP was proposed as a candidate JH receptor (Jones, et al., 2001). Yet, USP only
binds JH with micromolar affinity, requiring a hormone concentration that exceeds
endogenous titers by orders of magnitude (Bownes & Rembold, 1987). It is now known that
USP binds methyl farnesoate (MF), a precursor in the biological synthesis of JH III (Figure
1), with nanomolar affinity both in D. melanogaster and in A. aegypti (Jones et al., 2006; Jones
et al., 2010). Recent studies on natural farnesoid derivatives including MF, JH III, and JHB3
(the main farnesoid secretion product of dipteran ring glands cultured in vitro) have teased
out the relative activities of each of these compounds during development in a series of
biological assays. Two recent studies have demonstrated that the activity series of these
three compounds changes during development. Dietary MF and JH III (MF > JH III) were
both more active than JHB3 in delaying larval attainment of the wandering stage. In
contrast, JH III applied to prepupae (white puparial assay; Riddiford & Ashburner, 1991)
showed much higher activity than MF or JHB3 in blocking adult eclosion (Jones et al., 2010;
Harshman et al., 2010).
Topical application or dietary provision of these compounds adds to endogenous hormone
titers. Therefore, just as USP binds JH III at concentrations exceeding physiological levels, it
is possible that MET nonspecifically binds MF or JHB3 under these conditions. It is an
intriguing proposition that MET and USP, which interact both with each other and JHRE
binding proteins (Bitra & Palli, 2009), may partner in a stage-specific manner throughout
development in response to a fluctuating mlange of methyl farnesoids. Does GCE
participate in the assembly of the molecular machinery that facilitates the crosstalk between
JH and 20E signaling? This protein has been largely ignored in studies regarding the
molecular interaction of these hormones. Further, it is unknown whether GCE binds any of
the farnesoid products of the CA. There appears to be a correlation between the presence of
paralogous Met-like genes and multiple JH isoforms in higher Diptera. If each of the
farnesoids JH III, MF, and JHB3 indeed has a unique receptor protein, the possibility arises
that GCE fills the role of JHB3 binder. Or perhaps in MET/GCE dimers, MET is the sole
ligand binder, while GCE and MET are both necessary for target gene transcription. Clearly,
further functional characterization of GCE is necessary to unravel the mechanisms through
which JH signaling has evolved from the basal holometabola to the most evolutionarily
diverged insects, the higher Diptera.
7. Acknowledgments
We extend thanks to Drs. John Freudenstein and H. Lisle Gibbs for their guidance in our
various evolutionary analyses. Additionally, we would like to recognize Dr. Shaoli Wang

Gene Duplication

348
for her expertise and contributions to much of the work presented herein, and for her
mentorship of AAB. Work presented in this text was supported by NIH grant AI052290 to
T.G.W.
8. References
Ashburner, M., 2007. Drosophila Genomes by the Baker's Dozen. Preface. Genetics. 177:1263
1268.
Ashburner, M., Chihara, C., Meltzer, P., Richards, G. 1974. Temporal control of puffing
activity in polytene chromosomes. Cold Spring Harbor Symposia on Quantitative
Biology. 38:655-662.
Ashok M., Turner, C., Wilson, T.G., 1998. Insect juvenile hormone resistance gene homology
with the bHLH-PAS family of transcriptional regulators. Proceedings of the
National Academy of Sciences, USA. 95:2761-2766.
Barry, J., Wang, S., Wilson, T.G., 2008. Overexpression of Methoprene-tolerant, a Drosophila
melanogaster gene that is critical for juvenile hormone action and insecticide
resistance. Insect Biochemistry and Molecular Biology. 3:346-353.
Baumann, A., Fujiwara, Y., Wilson, T.G. 2010a. Evolutionary divergence of the paralogs
Methoprene tolerant (Met) and germ cell expressed (gce) within the genus Drosophila.
Journal of Insect Physiology. 56:1445-1455.
Baumann, A., Wilson, T.G., Barry, J., Wang, S. 2010b. Juvenile hormone action requires
paralogous genes in Drosophila melanogaster. Genetics. 185:1327-1336.
Bertone, M.A., Wiegmann, B.M. 2009. Diptera (true flies). In: Hedges, S.B., Kumar, S. (Eds.),
The Timetree of Life. Oxford, New York. 270-277.
Bitra, K., Palli, S.R. 2009. Interaction of proteins involved in ecdysone and juvenile hormone
signal transduction. Archives of Insect Biochemistry and Physiology. 70:90-105.
Bitra, K., Tan, A., Downling, A., Palli, S.R. 2009. Functional characterization of PAS and HES
family bHLH transcription factors during the metamorphosis of the red flour
beetle, Tribolium castaneum. Gene. 448:74-87.
Bouchard B.L., Wilson, T.G. 1987. Effects of sublethal doses of Methoprene on reproduction
and longevity of Drosophila melanogaster (Diptera: Drosophilidae). Journal of
Economic Entomology. 80:317-21.
Bownes, M., Rembold, H. 1987. The titure of juvenile hormone during the pupal and adult
stages of the life cycle of Drosophila melnoagaster. European Journal of Biochemistry.
164:709-712.
Brand, A.H., Perrimon, N. l993. Targeted gene expression as a means of altering cell fates
and generating dominant phenotypes. Development. 118:401-415.
Bylemans, D., Borovsky, D., Ujvary, I., DeLoof, A. 1998. Biosynthesis and regulation of
juvenile hormone III, juvenile hormone III bisepoxide, and methyl farnesoate
during the reproductive cycle of the grey flesh fly, Neobellieria (Sarcophaga) bullata.
Archives of Insect Biochemistry and Physiology 37:248-256.
Chen. J.D. 2000. Steroid/nuclear receptor coactivators. Vitamins and Hormones. 58:391-448.
Cornel, A., Stanich, M., McAbee, R., Mulligan, F., 2002. High level methoprene resistance in
the mosquito Ochlerotatus nigromaculis Ludlow in Central California. Pest
Management Science. 58:791-798.
Cornel, A.J., Stanich, M.A., Farley, D., Mulligan III, F.S., Byde, G. 2000. Methoprene
tolerance in Aedes nigromaculis in Fresno County, California, Journal of American
Mosquito Control Association. 16:223228.


349
Crews, S.T., 2003. PAS Proteins, Regulators and Sensors of Development and Physiology.
Boston, Kluwer.
Dame, D.A., Wichterman, G.J., Hornby, J.A., 1998. Mosquito Aedes taeniorhynchus resistance
to methoprene in an isolated habitat. Journal of the American Mosquito Control
Association. 14:200-203.
Davis, R.E., Kelly, T.J., Masler, E.P., Fescemyer, H.W., Thyagaraja, B.S., Borkovec, A.B. 1990.
Hormonal control of vitellogenesis in the gypsy moth, Lymantria dispar (L.):
suppression of haemolymph vitellogenein by the juvenile hormone analogue,
methoprene. Journal of Insect Physiology, 36:231-238.
Denlinger, D.L., 1985. Hormonal control of diapause. In: Kerkut, G.A., Gilbert, L.I. (Eds.),
Comprehensive Insect Physiology, Biochemistry and Pharmacology, vol. 8.
Pergamon, New York. 37-84.
Dubrovsky E.B., Dubrovskaya ,V.A., Bilderback, A. L., Berger, E.M. 2000. The isolation of
two juvenile hormone-inducible genes in Drosophila melanogaster. Developmental
Biology. 224:486-495.
Dubrovsky, E.B., Dubrovskaya, V.A., Berger, E.M., 2002. Juvenile hormone signaling during
oogenesis in Drosophila. Insect Biochemistry and Molecular Biology. 32:1555-1565.
Dubrovsky, E.B., Dubrovskaya V.A., Berger E.M. 2004. Hormonal regulation and functional
role of Drosophila E75A orphan nuclear receptor in the juvenile hormone signaling
pathway. Developmental Biology. 268:258-270.
Erezyilmaz, D.F., Riddiford, L.M., Truman, J.W. 2006. The pupal specifier broad directs
progressive morphogenesis in a direct-developing insect. Proceedings of the
National Academy of Sciences, USA. 103:6925-6930.
Flatt, T., Heyland, A., Rus, F., Porpiglia, E., Sherlock, C., Yamamoto, R., Garbuzov, A., Palli,
S.R., Tatar, M., Silverman, N. 2008. Hormonal regulation of the humoral innate
immune response in Drosophila melanogaster. Journal of Experimental Biology.
211:2712-24.
Flaveny, C.A., Murray, I.A., Perdew, G.H., 2010. Differential gene regulation by the human
and mouse aryl hydrocarbon receptor. Toxicological Sciences. 114:217-225.
Godlewski, J., Wang, S., and Wilson, T.G., 2006. Interaction of bHLH-PAS proteins involved
in juvenile hormone reception in Drosophila. Biochemical and Biophysical Research
Communications. 3424:1305-1311.
Harshman, L.G., Song, K.D., Casas, J., Schuurmans, A., Kuwano, E., Kachman, S.D.,
Riddiford, L.M., Hammock, B.D. 2010. Bioassays of compounds with potential
juvenoid activity on Drosophila melanogaster: juvenile hormone III, bisepoxide
juvenile hormone III and methyl farnesoates. Journal of Insect Physiology. 56:1465-
1470.
Huang, Z. J., Edery, I., Rosbash, M. l993. PAS is a dimerization domain common to
Drosophila Period and several transcription factors. Nature. 364: 259-262.
Juhsz, G., Pusks, L.G., Komonyi, O., Erdi, B., Mary, P., Neufeld, T.P., Sass, M. 2007. Gene
expression profiling identifies FKBP39 as an inhibitor of autophagy in larval
Drosophila fat body. Cell Death and Differentiation. 14:1181-1190.
Kethidi, D.R., Xi, Z., Palli, S.R. 2005. Developmental and hormonal regulation of juvenile
hormone esterase gene in Drosophila melanogaster. Journal of Insect Physiology.
51:393-400.
Konopova, B., Jindra, M., 2007. Juvenile hormone resistance gene Methoprene-tolerant
controls entry into metamorphosis in the beetle Tribolium castaneum. Proceedings of
the National Academy of Sciences, USA. 10419:10488-10493.

Gene Duplication

350
Kosakovsky Pond, S., Frost, S., 2005. DataMonkey: rapid detection of selective pressure on
individual sites of codon alignments. Bioinformatics. 21:2531-2533.
Kosakovsky Pond, S., Muse, S., 2005. HyPhy, Hypothesis Testing Using Phylogenies. In:
Nielsen, R (Ed.), Statistical Methods in Molecular Evolution. Springer, New York,
pp. 125-182.
Li, Y., Zhang, Z., Robinson, G.E., Palli, S.R., 2007. Identification and characterization of a
juvenile hormone response element and its binding proteins. Journal of Biological
Chemistry. 52:37605-37617.
Li, Z., Cheng, D., Wei, L., Zhao, P., Shu, X., Tang, L., Xiang, Z., Xia, Q. 2010. The silkworm
homolog of Methoprene-tolerant (Met) gene reveals sequence conservation but
function divergence. Insect Science, 17:313-324.
Liu, Y., Sheng, Z., Liu, H., Wen, D., He, Q., Wang, S., Shao, W., Jiang, R.J., An, S., Sun, Y.,
Bendena, W.G., Wang, J., Gilbert, L.I., Wilson, T.G., Song, Q., Li, S., 2009. Juvenile
hormone counteracts the bHLH-PAS transcription factors MET and GCE to prevent
caspase-dependent programmed cell death in Drosophila. Development. 13612:2015-
2025.
Jones, G., Jones, D., Li, X., Tang, L., Ye, L., Teal, P., Riddiford, L., Sandifer, C., Borovsky, D.,
Martin, J. 2010. Activities of natureal methyl farnesoids on pupariation and
metamorphosis of Drosophila melanogaster. Journal of Insect Physiology. 56:1456-1464.
Jones, G., Jones, D., Teal, P., Sapa, A., Wozniak, M. 2006. The retinoid-X receptor ortholog,
ultraspiracle, binds with nanomolar affinity to an endogenous morphogenetic
ligand. FEBS Journal. 273:4983-4996
Jones, G., Wozniak, M., Chu, Y.X., Dhar, S., Jones, D. 2001. Juvenile hormone III-dependent
conformational changes of the nuclear receptor ultraspiracle. Insect Biochemistry
and Molecular Biology. 32:33-49.
Konopova, B., Jindra, M. 2008. Broad-Complex acts downstream of Met in hjuvenile hormone
signalling to coordinate primitive holometabolan metamorphosis. Development.
135:559-568.
MacCarthy, T., Bergman, A., 2007. The limits of subfunctionalization. BMC Evolutionary
Biology. 7:213-226.
Madhavan, K., 1973. Morphogenetic effects of juvenile hormone and juvenile hormone mimics
on adult development of Drosophila. Journal of Insect Physiology. 19:441-453.
Minakuchi, C., Namiki, T., Shinoda, T. 2009. Krppel homolog 1, an early juvenile hormone-
response gene downstream of Methoprene-tolerant, mediates its anti-metamorphic
action in the red flour beetle, Tribolium castaneum. Developmental Biology. 325:341-350.
Minakuchi, C., Tanaka, M., Miura, K., Tanaka, T. 2011. Developmental progile and
hormonal regulation of the transcription factors broad and Krppel homolog 1 in
hemimetabolous thrips. Insect Biochemistry and Molecular Biology. 41:125-134.
Minakuchi, C., Zhou, X., Riddiford, L.M. 2008. Krppel homolog 1 (Kr-h1) mediates juvenile
hormone action during metamorphosis of Drosophila melanogaster. Mechanisms of
Development. 125:91-105.
Minkhoff III, C., Wilson, TG. 1992. The competitive ability and fitness components of the
Methoprene-tolerant (Met) Drosophila mutant resistant to juvenile hormone analog
insecticides. Genetics, 131:91-97.
Miura, K., Oda, M., Makita, S., Chinzei, Y., 2005. Characterization of the Drosophila
Methoprene-tolerant gene product: juvenile hormone binding and ligand-dependent
gene regulation. FEBS Journal. 2725:1169-1178.


351
Moore, A.W., Barbel, S., Jan, L.Y., Jan, Y.N., 2000. A genomewide survey of basic helix-loop-
helix factors in Drosophila. Proceedings of the National Academy of Sciences, USA.
9719:10436-10441.
Parthasarathy, R. and Palli, S. R. 2009. Molecular analysis of juvenile hormone action in
controlling the metamorphosis of the red flour beetle, Tribolium castaneum. Archives
of Insect Biochemistry and Physiology. 70:57-70.
Parthasarathy, R., Tan, A., Bai, H., Palli, S.R., 2008. bHLH-PAS family transcription factor
Methoprene-tolerant plays a key role in JH action in preventing the premature
development of adult structures during larval-pupal metamorphosis. Mechanisms
of Development. 1257:601-616.
Parthasarathy, R., Sun, Z., Bai, H., Palli, S.R. 2010. Juvenile hormone regulation of
vitellogenin synthesis in the red flour beetle, Tribolium castaneum. Insect
Biochemistry and Molecular Biology. 40:405-414.
Postlethwait, J.H. 1974. Juvenile hormone and the adult development of Drosophila.
Biological Bulletin. 147:119135.
Postlethwaite and Weiser, 1973. Vitellogenesis induced by juvenile hormone in the female
sterile mutant apterous-four in Drosophila melanogaster, Nature New Biology. :
284285.
Pursley, S., M. Ashok, Wilson, T.G. 2000. Intracellular localization and tissue specificity of
the Methoprene-tolerant (Met) gene product in Drosophila melanogaster. Insect
Biochemistry and Molecular Biology. 30: 839-845.
Ramadoss, P., Perdew, G.H., 2005. The transactivation domain of the Ah receptor is a key
determinant of cellular localization and ligand-independent nucelocytoplasmic
shuttling properties. Biochemistry. 4433:11148-11159.
Richard, D.S., Applebaum, S.W., Sliter, T.J., Baker, F.C., Schooley, D.A., Reuter, C.C., Henrich,
V.C., Gilbert, L.I. 1989. Juvenile hormone bisepoxide biosynthesis in vitro by the ring
gland of Drosophila melanogaster: a putative juvenile hormone in the higher Diptera.
Proceedings of the National Academy of Sciences, USA. 86:1421-1425.
Riddiford, L., 2008. Juvenile hormone action, a 2007 perspective. Journal of Insect
Physiology. 54:895-901.
Riddiford, L.M. and Ashburner, M. 1991. Effects of juvenile hormone mimics on larval
development and metamorphosis of Drosophila melanogaster. General and
Comparative Endocrinology. 82:172 -183.
Riddiford, L.M., 1994. Cellular and molecular actions of juvenile hormone. I. General
considerations and premetamorphic actions. Advances in Insect Physiology.
24:213274.
Riddiford, L.M., Truman, J.W., Mirth, C.K., Shen, Y.C. 2010. A role for juvenile hormone in
the prepupal development of Drosophila melanogaster. Development. 137:1117-1126.
Shemshedini, L., Wilson, T.G. 1990. Resistance to juvenile hormone and an insect growth
regulator in Drosophila is associated with an altered cytosolic juvenile hormone binding
protein. Proceedings of the National Academy of Sciences, USA. 87: 2072-2076.
Soller, M., Bownes, M., Kubli, E. 1999. Control of oocyte maturation in sexually mature
Drosophila females. Developmental Biology. 208:337-51.
Srivastava, U.S., Srivastava, R.C. 1983. Juvenoid-induced supernumerary larval instars in
certain stored grain insects. Proceedings: Animal Sciences. 92:263-276.
Tamura, K., Subramanian, S., and Kumar, S., 2004. Temporal patterns of fruit fly (Drosophila)
evolution revealed by mutation clocks. Molecular Biology and Evolution. 21:36-44.
Tomkins, L. 1990. Effects of the Apterous mutation on Drosophila melanogaster males
courtship. Journal of Neurogenetics. 6:221-227.

Gene Duplication

352
Truman, J.W., Riddiford, L.M. 2002. Endocrine insights into the evolution of metamorphosis
in insects. Annual Review of Entomology. 47:467-500.
Wang S., Baumann, A., Wilson, T.G., 2007. Drosophila melanogaster Methoprene- tolerant (Met)
gene homologs from three mosquito species, members of PAS transcriptional factor
family. Journal of Insect Physiology. 533:246-253.
Williams, C. M. 1956. The juvenile hormone of insects. Nature. 178: 212-213.
Williams, C.M., 1967. Third-generation pesticides. Scientific American. 21713-17.
Wilson TG, Wang S, Beno M, Farkas R. 2006. Wide mutational spectrum of a gene involved
in hormone action and insecticide resistance in Drosophila melanogaster. Molecular
Genetics and Genomics. 2763:294-303.
Wilson, T.G. 1982. A correlation between juvenile hormone deficiency and vitellogenic
oocyte degeneration in Drosophila melanogaster. Wilhelm Rouxs Archives
Entwicklungsmechanik der Organismen. 191:257-263.
Wilson, T.G., and Ashok, M., 1998. Insecticide resistance resulting from an absence of target-
site gene product. Proceedings of the National Academy of Sciences, USA.
9524:14040-14044.
Wilson, T.G., and Fabian, J. 1986. A Drosophila melanogaster mutant resistant to a chemical
analog of juvenile hormone. Developmental Biology. 118:190-201.
Wilson, T.G., DeMoor, S., Lei, J. 2003. Juvenile hormone involvement in Drosophila
melanogaster male reproduction as suggested by the Methoprene-tolerant
27
mutant
phenotype. Insect Biochemistry and Molecular Biology. 3312:1167-1175.
Wilson, T.G., Yerushalmi, Y., Donnell, D.M., Restifo, L.L. 2006. Interaction between
hormonal signaling pathways in Drosophila melanogaster as revealed by genetic
interaction between Methoprene-tolerant and Broad-Complex. Genetics 172:253264.
Xu, J., Tan, A., Palli, S.R. 2010. The function of nuclear receptors in regulation of female
reproduction and embryogenesis in the red flour beetle, Tribolium castaneum.
Journal of Insect Physiology. 56:1471-1480.
Yamamoto, K., Chadarevian, A., Pellegrini, M. 1988. Juvenile hormone action mediated in
male accessory glands of Drosophila by calcium and kinase C. Science. 239:916-919.
Yang, X.H., Liu, P.C., Zheng, W.W., Zhao, X.F. 2011. The juvenile hormone analogue
methoprene up-regulates the Ha-RNA-Binding protein. Molecular and Cellular
Endocrinology. 333:172-180.
Zhang, Z., Xu, J., Sheng, Z., Sui, Y., Palli, S.R. 2011. Steroid Receptor Co-activator is required
for juvenile hormone signal transduction through a bHLH-PAS transcription factor,
Methoprene tolerant. Journal of Biological Chemistry. 286: 8437-8447.
Zhou, B., Hiruma, K., Shinoda, T., Riddiford, L.M. 1998. Juvenile hormone prevents
ecdysteroid-induced expression of broad complex RNAs in the epidermis of the
tobacco hornworm, Manduca sexta. Developmental Biology. 203:233-44.
Zhou, X., Riddiford, L. M. 2002. Broad specifies pupal development and mediates the status
quo action of juvenile hormone on the pupal-adult transformation in Drosophila
and Manduca, Development. 129:2259-2269.
Zhu, J., Busche, J.M., Zhang, X. 2010. Identificatino of juvenile hormone target genes in the
adult female mosquitoes. Insect Biochemistry and Molecular Biology. 40:23-29.
Zhu, J., Chen, L., Sun, G., Raikhel, A.S. 2006. The competence factor beta Ftz-F1 potentiates
ecdysone receptor activity via recruiting a p160/SRC coactivator Molecular and
Cellular Biology. 26:9402-9412.
19
Gene Duplication and Subsequent
Differentiation of Esterases in
Cactophilic Drosophila Species
Rogrio P. Mateus
1
, Luciana P. B. Machado
1
and Carlos R. Ceron
2
1
Universidade Estadual do Centro-Oeste UNICENTRO
2
Instituto de Biocincias, Letras e Cincias Exatas IBILCE,
Universidade Estadual Paulista UNESP
Brazil
1. Introduction
Phytophagous insects are excellent model systems to study the genetic and ecological bases
of adaptation and population differentiation because the host plant constitutes an
immediate environmental factor that can affect the early stages of the life cycle (Matzkin,
2005; Matzkin et al., 2006). New host plant exploitation can result in genetic and biochemical
adjustments to the new resource and to chemically distinct niches, which can include
potentially toxic compounds, new mating environments, parasitoids, bacteria and fungi
(Kircher, 1982; Fogleman & Abril, 1990; Via, 1990; Fogleman & Danielson, 2001). These
adjustments are the result of a number of physiological changes, including those related to
biochemical systems associated with adaptation to the new environment.
The species of the Drosophila repleta group occupy different habitats, but their common
feature is that they are phytophagous; that is, they lay eggs in rotting cacti cladodes. The
developing larvae feed on the yeast that are part of the rotting process (Starmer & Gilbert,
1982; Pereira et al., 1983; Starmer et al., 1986), according to the cactus-Drosophila-yeast
system; therefore, they are considered specialists. However, adults are generalists because
they visit other food sources in their environment (Morais et al., 1994). This ecological
specificity of cactophilic Drosophila directly influences species distribution, as they are
always associated with the host cactus distribution (Tidon-Sklorz & Sene, 1995; Manfrin &
Sene, 2006; Mateus and Sene, 2007).
Drosophila has been used as a research model for more than a century, and the first report of
gene duplication was described by Bridges for the Bar locus in D. melanogaster over 70 years
ago (Bridges, 1936). Since that time, mainly after the advent of biochemical and molecular
biology techniques, several other examples of duplicated genes have been presented, and
pathways of evolution by gene duplication have been proposed (for example, Stephens,
1951; Nei, 1969). These pathways were thoroughly discussed in 1970 in Ohnos book
Evolution by gene duplication (Ohno, 1970). Subsequently, several other works have
reviewed the mechanisms and roles of gene duplication in the evolutionary process (A.
Wagner, 2002; Kondrashov et al., 2002; and Zhang, 2003).
Currently, the genomes of twelve Drosophila species have been completely sequenced
(Tweedie et al., 2009), but many aspects of the functional divergence of the products of a

Gene Duplication

354
gene duplication event cannot be answered through this method alone. A deeper
investigation of genetic differentiation after duplication is possible through molecular and
biochemical approaches. These approaches are extremely important because gene
duplication followed by functional divergence has been considered the primary mechanism
of molecular evolution (Lewis, 1951; Ohno, 1970). Analyses of isozymes have been crucial in
this process because they provide, along with cytological studies, evidence for the frequent
occurrence of gene duplication during the evolutionary process (Gottlieb, 1982; Hart, 1983).
Esterase is a polymorphic group of isozymes that play important biochemical roles in
insects. This group is composed of a heterogeneous set of hydrolytic enzymes that are
widely distributed among organisms and that catalyze the hydrolysis of esters, peptides,
amides and halides (Walker & Mackness, 1983). They are involved in digestive (Argentine &
James, 1995) and reproductive processes (Karotam et al., 1993), the degradation of
insecticides (Feyereisen, 1995) and female sex pheromones after male recognition (Vogt &
Riddiford, 1981) and in the regulation of juvenile hormone levels (Gu & Zera, 1994). In
Drosophila, esterases make up a diverse set of enzymes (G. B. Johnson, 1973, 1974), and gene
duplication has been used as one explanation for their evolution (Zouros et al., 1982; Pen et
al., 1990; Mateus et al., 2009).
Several studies on the changes in esterase activity during development in species of the D.
repleta group have detected two main -esterases that show different tissue-specific and
temporal expression patterns (Zouros et al., 1982; East, 1982; Pen et al., 1984; Pen et al.,
1986a, 1986b; Pen et al., 1990; Mateus et al., 2009). One esterase, named EST-4, is present
only in later third instar larvae and early pupae and has a high concentration in the carcass.
The other esterase, named EST-5, is present throughout the insect life cycle and occurs
predominantly in hemolymph and the fat body (Zouros et al., 1982).
According to Zouros et al. (1982), who studied these enzymes in D. mojavensis and D.
arizonae, the most likely hypothesis is that these enzymes are products of a gene duplication
as old as the D. repleta group that diverged later regarding their patterns of tissue-specific
and temporal expression. This hypothesis was suggested because these enzymes show
interlocus heterodimers, different patterns of expression (tissue and temporal) and 82%
identity in the N-terminal amino acid sequence (Pen et al., 1986a; Pen et al., 1990). More
recently, Robin et al. (2009) demonstrated that these enzymes are encoded by two genes that
are products of a gene duplication, Est-2a (EST-5) and Est-2c (EST-4), in D. mojavensis.
In this study, we investigated several genetic and biochemical features of EST-4 and EST-5
in six species of the D. repleta group, three belonging to the D. mulleri cluster (Drosophila
mulleri, D. aldrichi and D. wheeleri) and three belonging to the D. mojavensis cluster (D.
mojavensis, D. arizonae and D. navojoa) of the D. mulleri complex, as well as hybrids from
crosses involving some of these species. We aimed to establish the biochemical and genetic
differences among and possible physiological roles for these enzymes in the metabolic
processes of this group of Drosophila species.
2.1 Species
The materials used in this study included laboratory stocks of six Drosophila species (D.
arizonae - AR, D. mojavensis - MO, D. navojoa - NA, D. mulleri - MU, D. aldrichi - AL and, D.
wheeleri - WH) and two homozygote line stocks, one for the EST-5 "slow" allele of D. mulleri
(MU-S) and another for the EST-4 "fast" and EST-5 "slow" alleles of D. navojoa (NA-FS). All
Differentiation of Esterases in Cactophilic Drosophila Species

355
stocks were multifemales, except for the NA-FS stock, which was isofemale. The laboratory
stocks were obtained from Prof. Dr. Hermione E. M. C. Bicudo (Department of Biology,
IBILCE/UNESP, So Jos do Rio Preto, Brazil), who brought them to Brazil from stocks of
the Genetics Foundation (University of Texas, Austin, TX, US). The two line stocks were
prepared from laboratory stocks through endogamic crosses.
All laboratory and line stocks were maintained as mass cultures at a constant temperature of
20C 1C in culture vials with standard banana medium. The origin of each stock is listed in
Table 1.

Species Codes Locality
Drosophila mulleri MU Guayalejo, Tamazunchale, Mxico
Drosophila aldrichi AL Austin, Texas, US
Drosophila wheeleri WH Arroyo Solloro, Baja California, Mxico
Drosophila mojavensis MO Baja California, Mxico
Drosophila arizonae AR Guayalejo, Tamazunchale, Mxico
Drosophila navojoa NA Navojoa, Mxico
Table 1. List of analyzed species, with the respective codes and original localities of the
stocks (all stocks were obtained from the Genetics Foundation, University of Texas, Austin,
TX, US).
2.2 Obtaining late third instar larvae and adult flies for electrophoresis
Late third instar larvae and adult flies were collected directly from the vials and
immediately frozen at -20 C for further electrophoretic analyses. The larvae in that phase
show yellowish spiraculum and maximum EST-4 activity. Female adult flies were collected
at 5-10 days old and were used in electrophoresis for comparative analysis.
2.3 Obtaining hybrids
Mass crossings in both directions were performed in population boxes (16 cm
3
), using 200
couples, between NA-FS and MU-S, NA-FS and MO, NA-FS and AR and MU-S and MO.
After setting up a cross, the courtship behavior was observed for 10 minutes, as described
by Markow (1981). The culture media were placed in Petri plates at the bottom of the boxes
and were changed every three days. After every plate change, the plates were inspected to
detect eggs. The plates were maintained at a constant temperature of 20C 1C until late
third instar hybrid larvae were observed. These larvae were obtained directly from the
plates and frozen at -20C for further electrophoretic analyses.
2.4 Esterase detection
Esterase detection was performed using 10% polyacrylamide gel electrophoresis (PAGE),
adapted by Ceron (1988) from Davis (1964) and Laemmli (1970). All samples were prepared
in 25 L of 0.1 M Tris-HCl (pH 8.8) buffer containing 10% glycerol, where 10 L was used in
the gels. After electrophoresis, all gels were soaked in 0.1 M phosphate buffer (pH 6.2) for 1
hour at 25C. After this period, the gels were stained in solution containing 100 mL of
phosphate buffer, 10 ml of n-propanol and 120 mg of Fast Blue RR Salt, where 40 mg of -
naphthyl acetate and 30 mg of -naphthyl acetate, previously dissolved in 2 ml of acetone,
were added. After approximately 1 hour, the staining reactions were stopped in a solution
of acetic acid:ethanol:water (1:2.5:6.5 by v:v:v). Because the esterases hydrolyze substrates

Gene Duplication

356
differently, the bands in the gel stain differently: black when they hydrolyze -naphthyl
acetate, red when they hydrolyze -naphthyl acetate and magenta when they hydrolyze
both - and -naphthyl acetates. Polyacrylamide gels were air dried at room temperature
using gelatin and cellophane wound slab gels in an embroidering hoop (Ceron et al., 1992).
2.5 Characterization of esterases using inhibitors
Malathion, phenylmethylsulfonyl fluoride (PMSF), eserine sulfate, copper sulfate (CuSO
4
),
iodoacetamide (IAC), trans-epoxysuccinyl-L-leucyl-amido(4-guanidino) butane (E-64), p-
chloromercuribenzoate (pCMB) and mercuric chloride (HgCl
2
) were used as specific
inhibitors, all in 1 mM concentrations (with the exception of E-64, which was used at a
concentration of 5 mM) in the soaking and staining solution.
2.6 Determination of isoelectric point (I.P.)
The I.P. was determined in 10% PAGE containing 5% ampholyte solution (Sigma). The first
ampholyte formed a pH gradient between 3.0 and 10.0 after 1 hour of constant 100 V pre-
focusing. This experiment was performed to verify the best gradient to determine the I.P.
values of all enzymes in all species. After this verification, another ampholyte was used that
formed a pH gradient between 6.0 and 8.0 after 1 hour of constant 100 V pre-focusing. In
both cases, ampholyte solutions were added before gel polymerization. Samples of the six
Drosophila species and of the I.P. marker (hemoglobin) were prepared in a 10% glycerol in
water solution. Esterase isoelectric focusing was performed under constant 100 V conditions
in the power supply for 3 hours. After focusing, the gels were soaked in buffer for 1 hour,
followed by esterase staining for the same period, as described in section 2.5. Following
esterase identification, the gels were stained for total protein with Coomassie Blue G250
overnight. The staining reaction was stopped, and the gels were dried as described in
section 2.5. The I.P. was estimated by comparing the positions of EST-4 and EST-5 with the
position of human hemoglobin (I.P. = 7.1) after focusing.
2.7 Molecular weight (MW) estimation
The MW estimation was performed using the method adapted by Mateus et al. (2009) from
Hedrick and Smith (1968). The following standard MW proteins were used: myoglobin (17.8
kD), soybean trypsin inhibitor (24 kD), carbonic anhydrase (29 kD), ovalbumin (45 kD),
human serum albumin (66 kD) and phosphorylase-b (97.4 kD). All graphics were
constructed using Microcal Origin software, version 3.5 (Scientific and Technical Graphics in
Windows copyright 1991 1994, Microcal Software Inc.).
3. Results
3.1 Esterase pattern
Figure 1 shows the esterase patterns of larvae and adults (females) from six Drosophila
species. For all species, EST-4 always migrated slower than EST-5. The D. navojoa stock was
the only one that had more than one band for EST-4. EST-4 was more strongly stained than
EST-5 in D. mulleri, D. aldrichi and D. wheeleri (Figure 2A and B). The opposite was observed
for D. arizonae, with EST-5 more strongly stained than EST-4 (Figure 2A). Differences in the
staining intensity among EST-4 bands were also observed, with the D. mojavensis cluster
species (D. mojavensis, D. arizonae and D. navojoa) showing fainter bands than the species of
the D. mulleri cluster (D. mulleri, D. aldrichi and D. wheeleri).

357

Fig. 1. Esterase pattern in 10% PAGE for late third instar larvae and adult females of
Drosophila mulleri (1 and 2 = larvae; 3 = female), D. arizonae (4 and 5 = larvae; 6 = female), D.
mojavensis (7 and 8 = larvae; 9 = female), D. navojoa (10 and 11 = larvae; 12 = female), D.
wheeleri (13 and 14 = larvae; 15 = female), D. aldrichi (16 to 19 = larvae; 20 = female). All wells
contain individual samples, except for wells 18 and 19, which contain 2 larvae of D. aldrichi.
Arrow = EST-4; arrowhead = EST-5.

Fig. 2. A. 10% PAGE showing the electrophoretic staining differences for EST-4 and EST-5.
1- D. mojavensis; 2 - D. arizonae; 3 - D. navojoa; 4 - D. mulleri; 5 - D. aldrichi; 6 - D. wheeleri. B.
10% PAGE of late third instar larvae of D. navojoa, showing the different phenotypes
observed. Arrows in A and B indicate the interlocus heterodimer EST-4/EST-5. Larger
arrowhead = EST-4; smaller arrowhead = EST-5.

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358
Despite the observation of homozygotes for EST-4 in five out of six species analyzed, the
quaternary structure for this enzyme as a dimer could be deduced from the presence of a
heterodimer between EST-4 and EST-5 in D. mojavensis and D. arizonae (Figure 2A). This
dimeric structure for EST-4 and EST-5 was confirmed by hybrid analyses. In Drosophila
navojoa, in addition to the presence of the heterodimer, three phenotypes were observed in
gels for EST-4 and EST-5 (Figure 2B): homozygous for a slower band (EST-4
S
and EST-5
S
,
respectively); homozygous for a faster band (EST-4
F
and EST-5
F
, respectively); and
heterozygous, with a three-band pattern. These patterns reinforce the quaternary structure
of these enzymes for this species. The same results were observed for EST-5 of D. mulleri
(data not shown).
3.2 Pattern of esterase activity in the presence of inhibitors
Table 2 shows the results obtained for the esterase activity patterns of late third instar larvae
of the analyzed species in the presence of different inhibitors. All species showed the same
pattern for EST-4: they were inhibited only by PMSF. For EST-5, all species were inhibited
only by malathion. No other inhibitor affected the activity of either esterase.

Species Enzyme PMSF Malathion CuSO
4
IAC E-64 Eserine pCMB HgCl
2
D. mulleri EST-4 ++
EST-5 ++
D. aldrichi EST-4 ++
EST-5 ++
D. wheeleri EST-4 ++
EST-5 ++
D. mojavensis EST-4 ++
EST-5 ++
D. arizonae EST-4 ++
EST-5 ++
D. navojoa EST-4 ++
EST-5 ++
Table 2. Esterase activity patterns of EST-4 and EST-5 for the six Drosophila species analyzed
in the presence of different inhibitors. PMSF = phenylmethylsulfonyl fluoride; Eserine =
eserine sulfate; CuSO
4
= copper sulfate; IAC = iodoacetamide; E-64 = trans-epoxysuccinyl-L-
leucyl-amido(4-guanidino) butane; pCMB = p-chloromercuribenzoate; HgCl
2
= mercuric
chloride. ++ activity inhibited; activity not affected.
3.3 Isoelectric point (I.P.) determination
The I.P. determination was performed in two phases. In the first phase, we verified that the
best range for I.P. determination was 6.0 to 8.0. In the second phase, an ampholyte solution
was used for this pH range. Table 3 shows that all esterases presented I.P. between 6.0 and
7.0. As expected, the I.P. values for EST-5 in both larvae and adults of the same species were
equal, ranging from 6.47 (D. navojoa) to 6.64 (D. aldrichi). EST-4 showed a wider range of I.P.
variation than EST-5, with D. mulleri and D. navojoa showing the highest and lowest I.P.
values (6.88 and 6.37, respectively).

359
D. mulleri cluster I.P. D. mojavensis cluster I.P.
D. mulleri D. mojavensis
EST-4 6.88 EST-4 6.38
EST-5 (larvae and adult) 6.51 EST-5 (larvae and adult) 6.49
D. aldrichi D. arizonae
EST-4 6.55 EST-4 6.53
D. wheeleri D. navojoa
EST-4 6.59 EST-4 6.37
Table 3. Isoelectric points for EST-4 and EST-5 of larvae and adults of the six analyzed
Drosophila species, obtained through the comparison of esterase band mobility in gels with
an I.P. marker (hemoglobin; I.P. = 7.1) in a pH range between 6.0 and 8.0.
3.4 MW determination
To determine the MW of both enzymes in all six Drosophila species analyzed, the technique
described by Mateus et al. (2009) was applied using 6% to 12% PAGE and the same MW
markers. The results presented there are part of this study. Therefore, in this study, we
present the results that were not shown in Mateus et al. (2009), i.e., the MW determinations
of EST-4 and EST-5 for D. mulleri, D. aldrichi, D. wheeleri and D. navojoa. After
electrophoresis, the relative mobility (Rm) values for the esterases of these four species and
the molecular markers were obtained. The graphs of Rm versus gel concentration for each
MW marker resulted in a different slope. These slopes were plotted against the MW (Figure
1 Mateus et al., 2009). Fergusons plot (Log Rm versus gel concentrations) for EST-4 and
EST-5 of D. mulleri, D. aldrichi, D. wheeleri and D. navojoa are shown in Figure 3.
The plots for both esterases were parallel in all species, indicating that these enzymes have
different charges and/or tridimensional structures but very similar molecular weights.
From these graphs, the slope was obtained for each enzyme in each species. These values
were used to estimate the MW in each case, using the equation Y = A + BX, where A is the
intercept of the Y-axis (2.18766), and B is the slope (0.09452). The slopes and molecular
weights are presented in Table 4.
The slope values for both enzymes in all species were similar. EST-5 had more variation,
ranging from -10.05407 0.29546 for D. navojoa to -11.03429 0.30178 for D. mulleri. EST-4
was less variable, ranging from -10.08361 0.33581 for D. wheeleri to -10.52607 0.44878 for
D. mulleri. The MWs, estimated from these slope values (Table 4), were very close to each
other. For EST-4, the MW ranged from 83.537 kD in D. wheeleri to 88.218 kD in D. mulleri. For
EST-5, the MW ranged from 83.225 kD in D. navojoa to 93.595 kD in D. mulleri. The MWs
obtained, including standard deviations, were all approximately 80 kD to 96.8 kD.

Gene Duplication

360

Fig. 3. Log of Rm versus gel concentrations relationship (Fergusons plot) for EST-4 and EST-
5. A Drosophila mulleri; B Drosophila aldrichi; C Drosophila wheeleri; D Drosophila navojoa.

EST-4 EST-5
D. mulleri
cluster
Slope M.W. Slope M.W.
D. mulleri 10.52607 0.44878 88.218 4.748 11.03429 0.30178 93.595 3.193
D. aldrichi 10.29768 0.30168 85.802 3.192 10.34904 0.28362 86.346 3.000
D. wheeleri 10.08361 0.33581 83.537 3.553 10.32114 0.30252 86.050 3.201
D. mojavensis
cluster

1
D. mojavensis

10.45339 0.27581 87.450 2.918 10.33036 0.27437 86.148 2.903

1
D. arizonae

10.27775 0.32146 85.591 3.401 10.16554 0.30570 84.404 3.234
D. navojoa 10.24157 0.38515 85.209 4.074 10.05407 0.29546 83.225 3.126

Table 4. Slopes of Log Rm versus gel concentration relationships and MW estimates for EST-
4 and EST-5 of the six cactophilic Drosophila species analyzed.
1
Data obtained from Mateus et
al. (2009).

361
3.5 Interspecies crosses
3.5.1 Crosses between D. mulleri and D. mojavensis
The cross between D. mulleri and D. mojavensis showed asymmetric isolation, with many
descendents only in the direction of D. mulleri females and D. mojavensis males. The
reciprocal cross did not produce offspring despite the presence of courtship among couples
and eggs in the plate. The hybrid larvae were analyzed in 10% PAGE and showed three-
band patterns for EST-4 and EST-5 (Figure 4). For both enzymes, the slower band
corresponded to the enzyme encoded by D. mulleri and the faster to the enzyme encoded by
D. mojavensis. The intermediate band represented a hybrid enzyme, indicating that EST-4
and EST-5 are dimeric in both species.
For EST-4, the hybrid intermediate bands were located closer to the band encoded by D.
mulleri, which could be a result of differences in the I.P. values of theses enzymes (Table 3).
The same was not observed for EST-5, as this enzyme has nearly the same value for both of
these species.
3.5.2 Crosses between D. navojoa and D. mojavensis
Asymmetric isolation was also observed in the cross between D. navojoa and D. mojavensis.
No offspring were obtained in the direction of D. mojavensis females and D. navojoa males,
despite the fact that courtship between couples and eggs on the plate were observed. The
cross between D. navojoa females and D. mojavensis males was very fertile.

Fig. 4. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines and
the hybrids obtained from the cross of D. mulleri females and D. mojavensis males. 1 = D.
mulleri; 2 = D. mojavensis; 3-13 = hybrid larvae.
The hybrid larvae from this cross were analyzed in 10% PAGE and showed the same three-
band patterns observed for D. mulleri and D. mojavensis (Figure 5). For EST-4, the slower
band corresponded to the enzyme encoded by D. mojavensis, the faster band corresponded
to the enzyme encoded by D. navojoa, and the intermediate band corresponded to a hybrid
enzyme. The opposite was observed for EST-5: the slower band was from D. navojoa, the
faster band was from D. mojavensis, and an intermediate band was a hybrid enzyme. Again,
these results confirm the quaternary structure of both enzymes of these species. An
interesting observation was the absence of EST-5 expression in two samples (samples 12 and
13; Figure 5).

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3.5.3 Crosses between D. navojoa and D. arizonae
This cross was very fertile in both directions. However, larvae from the cross in the direction
of D. navojoa females and D. arizonae males had very slow development and took much
longer to achieve the late third instar stage; they also had a high mortality rate. The larvae
analyzed in 10% PAGE from both cross directions presented the same three-band patterns
for EST-5. For EST-4, as in both species of the cross, the enzymes had almost the same
migration speed under these electrophoretic conditions. One thicker band was observed in
the hybrid larvae, which must be the agglomeration of the three bands expected for this
enzyme (Figure 6).

Fig. 5. Esterase pattern in 10 % PAGE of late third instar larvae from the parental lines and
the hybrids obtained from the cross of D. navojoa females and D. mojavensis males. 1 = D.
navojoa; 2 = D. mojavensis; 3-14 = hybrid larvae.

Fig. 6. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines of D.
arizonae and D. navojoa and the hybrids obtained from crosses in both directions. 1 = D.
arizonae; 2 = D. navojoa; 3-15 = hybrid larvae.
3.5.4 Crosses between D. navojoa and D. mulleri
The cross between D. navojoa and D. mulleri was fertile in both directions. The larvae
analyzed by 10% PAGE showed three-band patterns for both EST-4 and EST-5, with the
slower enzyme from D. mulleri and the faster band from D. navojoa. The intermediate band
was a hybrid enzyme. These results confirm the dimeric quaternary structure of these

363
enzymes in these species (Figure 7). Again, the EST-4 hybrid band migrated closer to the
slower band from D. mulleri, which could be a consequence of the different I.P. values of
these enzymes. The same was not observed for EST-5, as they show similar I.P. values for
both species.

Fig. 7. Esterase pattern in 10% PAGE of late third instar larvae from the parental lines of D.
mulleri and D. navojoa and the hybrids obtained from crosses in both directions. 1 = D.
mulleri; 2 = D. navojoa; 3-9 = hybrid larvae.
4. Discussion
Isozymes are very important in insects and have been used to understand biological
problems in several fields of research, including population genetics and systematics, tissue
organization, development, metamorphosis, gene regulation and protein synthesis and gene
duplication (R. P. Wagner & Selander, 1974). The set of proteins known as esterases
constitute one of the most heavily studied groups of isozymes. In the Drosophila mulleri
complex, which is the subject of this study, esterases have been extensively studied in
several species, including D. serido (Lapenta et al., 1995, 1998), D. buzzatii (East, 1982; Barker,
1994; Lapenta et al., 1995, 1998), D. mojavensis (Zouros et al., 1982; Zouros & Van Delden,
1982; Pen et al., 1984, 1986a, 1986b; Mateus et al., 2009), D. arizonae (Zouros et al., 1982;
Ceron, 1988; Mateus et al., 2009), D. aldrichi (F. M. Johnson et al., 1968; Kambysellis et al.,
1968) and D. mulleri (F. M. Johnson et al., 1968; Kambysellis et al., 1968; Richardson &
Smouse, 1976; Ceron, 1988).
Zouros et al. (1982) detected two esterases with different patterns of temporal and tissue-
specific expression in Drosophila mojavensis and D. arizonae (formerly D. arizonensis). They
detected a specific -esterase of the late third instar phase of larval development and in the
carcass, named EST-4, in contrast to another -esterase, named EST-5, which is expressed
during all developmental phases and is found predominantly in hemolymph and the fat
body. They proposed that the most likely hypothesis is that both enzymes are products of a
gene duplication that occurred prior to the speciation of the D. repleta group, and their
patterns of tissue-specific and temporal expression diverged more recently. This hypothesis
was suggested because the enzymes show interlocus heterodimers, different patterns of
expression (Zouros et al., 1982) and 82% identity in their N-terminal amino acid sequences

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(Pen et al., 1986a; Pen et al., 1990). More recently, Robin et al. (2009) proposed that, in D.
mojavensis, these enzymes are most likely encoded by two genes, Est-2a (EST-5) and Est-2c
(EST-4), which are products of one gene duplication out of a total of eleven duplications that
explain the evolution of the catalytic -esterase cluster in the Drosophila genus (five in the
Sophophora and 6 in the Drosophila subgenus).
Our results reinforce the hypothesis proposed by Zouros et al. (1982), extending the
knowledge about these enzymes as products of a gene duplication to other D. mulleri
complex species. All six analyzed species show distinct temporal expression patterns for
EST-4 and EST-5, with EST-4 showing activity only at the end of the third instar larval stage
(Figure 1). The inhibition experiments (Table 2) showed that EST-4 has the same pattern for
all six species: it is inhibited by PMSF and not affected by malathion. The opposite was
observed for EST-5 in all six species: it was inhibited by malathion and not affected by
PMSF. The other inhibitors tested (eserine sulfate, copper sulfate, iodoacetamide and E-64)
had no effect on the activity of either enzyme. Moreover, the presence of homozygotes and
heterozygotes for EST-5 independent of the EST-4 genotype in D. navojoa (Figure 2) and D.
mulleri (data not shown) support the idea of an independent origin of these enzymes from
two distinct loci. Despite these differences, these enzymes display similar features, such as
structural similarities (Pen et al., 1986a; Pen et al. 1990) that allow the formation of dimers in
D. mojavensis, D. arizonae and D. navojoa (Figure 2).
The gene duplication process is considered one of the most important mechanisms of the
generation of new genes and functions during the evolutionary process. Jeffreys & Harris
(1982) suggested gene duplication mechanisms that could happen to genes during
evolution. Among the mechanisms presented, the most likely mechanism that could have
generated the EST-4 and EST-5 loci is the same mechanism that might have generated the
globin family, that is, in tandem gene duplication by pairing errors during meiosis that cause
unequal crossing-over because of the presence of short repeat sequences located in the 3
and 5 ends of the unduplicated ancestral gene.
The EST-5 gene in D. pseudoobscura is a good example of gene duplication with later
divergence (Brady & Richmond, 1992). The EST-5 enzyme is encoded by the Est-5B gene,
which is expressed during the life cycles of all insects and is linked to two other genes, Est-
5A and Est-5C, on the X chromosome (Brady et al., 1990). In D. melanogaster, the homologous
gene is Est-6, which codes for the enzyme EST-6 during the insects life cycle and has only
one grouped gene, Est-P (Collet et al., 1990). Both Est-5A of D. pseudoobscura and Est-P of D.
melanogaster are expressed only at the third instar larval stage, producing only one
transcript. On the other hand, Est-5C of D. pseudoobscura is not expressed in any
developmental phase (Brady et al., 1990). According to Brady & Richmond (1992), who
compared the DNA sequences of coding and flanking regions of all three D. pseudoobscura
and two D. melanogaster genes, only two genes, which are already products of a gene
duplication, were present before these two species diverged. These two ancestral genes were
probably Est-5A and Est-5B in the first species and Est-6 and Est-P in the second species. A
second duplication occurred later in D. pseudoobscura, giving rise to the Est-5C gene.
However, the findings of Robin et al. (2009) contrast with the evolutionary model proposed
by Brady & Richmond (1992); in their analyses, the Est-5A/Est-5B duplication (which they
call Est6/7) occurred after the melanogaster/obscura group divergence, whereas Brady &
Richmond (1992) place this duplication prior to the divergence.

365
In our case, Zouros et al. (1982) proposed that the genes coding for EST-4 and EST-5 (Est-2c
and Est-2a, respectively, according to Robin et al., 2009) were also products of a duplication
event prior to the divergence of the species that belong to the D. repleta group and that the
EST-4 gene was later inactivated in some species of this group, including D. tira, D. hydei
and D. eohydei. Moreover, the lower activity of EST-4 in D. mulleri, D. aldrichi, D. repleta and
D. peninsularis could indicate this EST-4 inactivation process. However, our results showed
that D. mulleri, D. aldrichi and D. wheeleri had high EST-4 activities compared to the other
species (Figure 2A). This difference in the level of activity of EST-4 for the same species in
these studies could be the result of differences in the origins of the lines used in each study.
Therefore, the populations of D. mulleri and D. aldrichi that were analyzed by Zouros et al.
(1982) could have a certain degree of EST-4 inactivation that was not observed in the present
study.
The enzymes analyzed in this study had biochemical differences compared to other
esterases of other Drosophila species. For example, the I.P. values for EST-4 and EST-5 for the
six Drosophila species analyzed were between 6.0 and 7.0 (Table 3). These values were
different from those of D. melanogaster obtained by Healy et al. (1991), as only 2 out of 15
esterases had I.P. values close to the values obtained in this study (between 6.0 and 7.0). All
others showed values below 6.0, with the majority of values between 4.0 and 5.0.
Regarding the MWs of these enzymes, our results are in agreement with previous studies
that estimated this parameter. EST-4 had MW values between 83 and 89 kD, and EST-5 had
MW values between 83 and 94 kD (Table 4), which are very close to the MWs obtained by
Pen et al. (1984), which were between 85 and 95 kD for a variant of the EST-4 (with altered
specificity to -naphthyl acetate) using gel filtration chromatography. Pen et al. (1984) also
used denaturing gel electrophoresis (SDS-PAGE) and obtained the MWs of the subunits of
EST-4 as 62-64 kD. In another study, Pen et al. (1986a) determined the MWs for the subunits
of EST-5 as 64-66 kD. Regardless of the method used and the different results obtained (for
the entire protein or for subunits), EST-4 had a smaller MW than EST-5, as observed in this
study.
The interspecies crosses performed in this study had results that were in accordance with
the known phylogenetic relationships among the species analyzed. This information is
based on the morphological work of Throckmorton (1982) and Vilela (1983), the cytological
work of Wasserman (1982, 1992 for reviews), several allozyme studies (Zouros, 1973;
Richardson et al., 1975; Richardson and Smouse, 1976; Richardson et al., 1977; Heed et al.,
1990), molecular studies (Sullivan et al., 1990; Russo et al., 1995; Spicer, 1995, 1996) and an
analysis using multiple sources of characters (Durando et al., 2000). The crosses between D.
mulleri and D. mojavensis showed the same results of those of Patterson & Crow (1940) and
Bicudo (1982), with offspring obtained only in the direction of D. mulleri females and D.
mojavensis males. For D. navojoa crossed with D. mojavensis, an F1 was produced only in the
direction of D. navojoa females and D. mojavensis males. In this case, Ruiz et al. (1990)
observed descendants in both directions but a very low percentage of offspring, depending
on the geographic lineage used, in the direction in which we detected isolation. In crosses
between D. navojoa and D. arizonae, both directions were fertile, which was also found by
Ruiz et al. (1990). Finally, in crosses between D. navojoa and D. mulleri, we detected
descendants in both directions, in contrast to the results of Bicudo (1982), who found fertility
only in the direction of D. mulleri females and D. navojoa males.

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In all of these crosses, the phenotypic observations of the esterase patterns from late third
instar hybrid larvae produced three bands for both EST-4 and EST-5 (Figures 4, 5, 6 and 7),
except for larvae from the cross between D. navojoa and D. arizonae, which produced a
thicker band because the parental bands have almost the same migration pattern under the
electrophoretic conditions used in this study. These results indicate that in all six Drosophila
species, EST-4 and EST-5 have dimeric quaternary structures. Another important
observation from some of these crosses was the presence of hybrid larvae with no EST-5
activity (D. navojoa x D. mojavensis Figure 5; D. navojoa x D. arizonae data not shown).
These results indicate that some hybrid larvae had problems with the regulation of Est-2a
gene expression, which most likely codes for the EST-5 enzyme, without affecting the
expression of its homologous gene, Est-2c, which most likely codes for the EST-4 enzyme
(Robin et al., 2009). These results reinforce the idea that these two loci are independent.
The possible role of EST-4 in these Drosophila species remains an open question. According
to Holmes & Masters (1967, as cited in Oakeshott et al., 1993), esterases can be classified into
four types through their specific inhibition patterns. Carboxylesterases are esterases that are
inhibited only by organophosphates, such as paraoxon, fenitrooxon and DFP
(diisopropylfluorophosphate). Cholinesterases are inhibited by organophosphates and
carbamates, such as eserine sulfate. Arylesterases are inhibited only by sulfhydrylic agents,
such as p-chloromercuribenzoate (pCMB). Acetylesterases are not inhibited by any of these
agents. Inhibition of EST-5 only by malathion, an organophosphate, suggests that this
enzyme belongs to the class of carboxylesterases. Inhibition of EST-4 by PMSF and the
absence of inhibition in the presence of all other inhibitors tested suggest that this enzyme
probably belongs to the class of acetylesterases.
According to Augustinsson (1968), esterases are closely related to the class of serine-
proteases, forming a multigenic family of serine-hydrolases. The main features that support
this hypothesis are the three consensus amino acid residues that are present in the active site
of esterases and serine-proteases, including an invariant serine, enzymatic inactivation by
DFP, which binds irreversibly to the serine residue of both enzymes, inhibition by
organophosphates and carbamates and the superposition of substrate preference
(Augusteyn et al., 1969; Krisch, 1971; Dayhoff et al., 1972; Heymann, 1980; Previero et al.,
1983; as cited in Myers et al., 1988). However, Myers et al. (1988) showed that some esterases
cannot be included in this multigenic family because they do not have the same amino acid
residues in the charge exchange system of the enzyme active site.
The absence of EST-4 and Est-5 inhibition by copper sulfate and iodoacetamide, combined
with data for E-64, which is a diagnostic inhibitor of cysteine-proteases, indicate that neither
enzyme has an essential cysteine residue in its active site. On the other hand, the inhibition
of EST-4 by PMSF, which is a diagnostic compound for serine-proteases and other enzymes
with a serine residue in the active site, and of EST-5 only by malathion indicated that both
enzymes have an important serine residue in the active site, suggesting that they belong to
the class of serine-hydrolases. As these enzymes display high esterase activity, we can
postulate that they are serine-esterases (Holmes & Masters, 1967). The multigenic family of
serine-esterases includes several enzymes with a wide range of functions, including
cholinesterases, lipases, lysophospholipases, cholesterol-esterases, non-specific
carboxylesterases and juvenile hormone esterases (Ryger et al., 1989; Doctor et al., 1990;
Shimada et al., 1990; as cited in Myers et al., 1993). Therefore, EST-4 and EST-5 probably
belong to this multigenic family, with EST-4 as an acetylesterase (E.C. 3.1.1.6) and EST-5 as a
non-specific carboxylesterase (E.C. 3.1.1.1).

367
To establish the possible role of EST-4, the following information must be considered. Healy
et al. (1991) observed that all D. melanogaster acetylesterases are inhibited by OTFP (3-
octylthio-1,1,1-trifluoropropan-2-one), which is a powerful inhibitor of juvenile hormone
esterase activity in Lepidoptera, suggesting that all acetylesterases from this species have
similar properties as juvenile hormone esterase. Moreover, East (1982) proposed that
esterase-J from D. buzzatii, which is supposedly the enzyme from this species that
corresponds to EST-4 in this study, is a juvenile hormone esterase, acting together with EST-
1 in the larval phase to control the levels of this hormone. In the adult phase, only EST-1
would be responsible for this control. On the other hand, EST-2 could be the enzyme
responsible for digestive and detoxification processes and ester absorption in adults. EST-4
has a very tissue-specific and temporal pattern of expression, which indicates that there is a
specific regulatory system that controls its expression at a specific tissue (carcass) and
period of time (at the end of the larval phase, when all of the processes for pupation have
been initiated). Therefore, as an acetylesterase with a very specific temporal expression
pattern, EST-4 could be involved in these transformation processes, acting as an auxiliary
enzyme for the degradation of juvenile hormone esterase. The degradation of this hormone
in this phase allows the liberation of prothoracicotropic hormone by the brain, which
stimulates ecdysone production by the prothoracic gland, initiating metamorphosis
(Coundron et al., 1981). However, analyzing the EST-4 inhibition data alone could lead to
the hypothesis that this enzyme could be a serine-protease that also has esterase activity and
is involved in a proteolytic activity during the larva-pupae conversion process; it is likely to
be involved in this process. Regarding EST-5, considering the fact that it is expressed during
the entire life cycle of the insect and is found mainly in the hemolymph and fat body, it is a
non-specific carboxylesterase that is probably involved in digestive processes.
5. Conclusions
This study contributes to a better understanding of the differentiation of two enzymes that
are products of a gene duplication in six cactophilic Drosophila species. We present
additional evidence to support the gene duplication event that gave rise to the genes
responsible for the EST-4 and EST-5 enzymes, which are the main -esterases found in
several species of the D. mulleri complex of the D. repleta group. We also contribute to the
elucidation of the possible physiological roles of these esterases in this group. Further steps
in this investigation will be to determine specific biochemical parameters of both enzymes
after purification. We are also interested in identifying the changes that occur in the
regulatory system of gene expression that lead to differentiation in the patterns of tissue-
specific and temporal expression of these enzymes; that is, understanding what triggers
EST-4 expression only in the late third instar larvae and at the larval carcass. We are also
interested in determining the intra- and/or extracellular processes in which these enzymes
are involved and their interacting molecules. Thus, we will be able to complement this
initial step with an increased understanding of the differentiation of these two genes that
result from a gene duplication event.
6. Acknowledgments
We would like to thank CNPq for funding Rogrio P. Mateus (Masters degree fellowship).
We would also like to thank CAPES, FINEP and FUNDUNESP for supporting this work,

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Eliani Nobuco Ikeguchi Ohira for technical support and Dr. Hermione Elly Melara Campos
Bicudo for Drosophila stocks.
7. References
Argentine, J.A., & James, A.A. (1995). Characterization of a salivary gland-specific esterase
in the vector mosquito, Aedes aegypti. Insect Biochem. Molec. Biol., Vol. 25, No. 5, pp.
621-630
Augustinsson, K.B. (1968). The evolution of esterases in vertebrates, In: Homologous enzymes
and biochemical evolution, N.V. Their & J. Roche, pp. 299-311, Gordon & Breach,
New York
Barker, J.S.F. (1994). Sequential gel electrophoretic analysis of esterase-2 in two populations
of Drosophila buzzatii. Genetica, Vol. 92, No. 3, pp. 165-175
Bicudo, H.E.M.C. (1982). Estudo citogentico de espcies de Drosophila do complexo mulleri (grupo
repleta): A regulao da atividade organizadora nucleolar. Tese de Livre Docncia,
IBILCE/UNESP, So Jos do Rio Preto
Brady, J.P., & Richmond, R.C. (1992). An evolutionary model for the duplication and
divergence of esterase genes in Drosophila. J. Mol. Evol., Vol. 34, pp. 506-521
Brady, J.P., Richmond, R.C., & Oakeshott, J.G. (1990). Cloning of the esterase-5 locus from
Drosophila pseudoobscura and comparison with its homologue in D. melanogaster.
Mol. Biol. Evol., Vol. 7, pp. 525-546
Bridges, C.B. (1936). The Bar gene duplication. Science, Vol. 83, pp. 210-211
Ceron, C.R. (1988). Padro de esterases no desenvolvimento de Drosophila mulleri, D. arizonae e
seus hbridos. Tese de Doutorado, Departamento de Biologia, IB-USP, So Paulo
Ceron, C.R., Santos, J.R., & Bicudo, H.E.M.C. (1992). The use of gelatin to dry celophane
wound slab gels in an embroidering hoop. Braz. J. Genet., Vol. 15, No. 1, pp. 201203
Collet, C., Nielsen, K.M., Russell, R.J., Karl, M., Oakeshott, J.G., & Richmond, R.C. (1990).
Molecular analysis of duplicated esterase genes in Drosophila melanogaster. Mol. Biol.
Evol., Vol. 7, pp. 9-28
Coundron, T.A., Dunn, P.E., Seballos, H.L., Wharen, R.E., Sanburg, L.L., & Law, J.H. (1981).
Preparation of homogeneous juvenile hormone specific esterase from the
haemolymph of the tobacco hornworm, Manduca sexta. Insect Biochem., Vol. 11, No.
4, pp. 453-461
Davis, B.J. (1964). Disc electrophoresis. II. Methods and application to human serum
proteins. Annals N. Y. Acad. Sci., Vol. 121, pp. 404-427
Durando, C.M., Baker, R.H., Etges, W.J., Heed, W.B., Wasserman, M., & DeSalle, R. (2000).
Phylogenetic analysis of the repleta species group of the genus Drosophila using
multiple sources of characters. Molecular Phylogenetics and Evolution, Vol. 16, pp.
296307
East, P.D. (1982). Non-specific esterases of Drosophila buzzatii, In: Ecological genetics and
evolution. The cactus-yeast-Drosophila model system, J.S.F. Baker & W.T. Starmer, pp.
323-338, Academic Press, Australia
Feyereisen, R. (1995). Molecular biology of insecticide resistance. Toxicol. Lett., Vol. 82/83,
pp. 83-90
Fogleman, J.C., & Abril, J.R. (1990). Ecological and evolutionary importance of host plant
chemistry, In: Ecological and Evolutionary Genetics of Drosophila, J.S.F. Barker, W.T.
Starmer, & R. MacIntyre, pp. 121-143, Plenum Press, New York

369
Fogleman, J.C., & Danielson, P.B. (2001). Chemical interactions in the cactus-microorganism-
Drosophila model system of the Sonoran desert. Am. Zool., Vol. 41, pp. 877-889
Gottlieb, L.D. (1982). Conservation and duplication of isozymes in plants. Science, Vol. 216,
pp. 373-380
Gu, X., & Zera, A.J. (1994). Developmental profiles and characteristics of hemolymph
juvenile hormone esterase, general esterase and juvenile hormone binding in the
cricket, Gryllus assimilis. Comp. Biochem. Physiol. B Comp. Biochem. Mol Biol., Vol. 107,
No. 4, pp. 553-560
Hart, G.E. (1983). Genetics and evolution of multilocus isozymes in hexaploid wheat.
Isozymes Curr. Top. Biol. Med. Res., Vol. 10, pp. 365-380
Healy, M.J., Dumancic, M.M., & Oakeshott, J.G. (1991). Biochemical and physiological
studies of soluble esterases from Drosophila melanogaster. Biochem. Genet., Vol. 29,
pp. 365-388
Hedrick, J.L., & Smith, A.J. (1968). Size and charge isomer separation and estimation of
molecular weights of proteins by disc gel electrophoresis. Arch. Biochem. Bioph., Vol.
126, pp. 155-164
Heed, W.B., Sanchez, A., Armengol, R., & Fontdevila, A. (1990). Genetic differentiation
among island populations and species of cactophilic Drosophila in the West Indies,
In: Ecological and Evolutionary Genetics of Drosophila, J.S.F. Barker, W.T. Starmer, & R.
J. MacIntyre, pp. 447490, Plenum Press, New York
Holmes, R.S., Masters, C.J. (1967). The developmental multiplicity and isoenzyme status of
cavian esterases. Biochim. Biophys. Acta, Vol. 132, pp. 379-399
Jeffreys, A.J., Harris, S. (1982). Processes of gene duplication. Nature, Vol. 296, pp. 9-10
Johnson, G.B. (1973). Importance of substrate variability to enzyme polymorphism. Nature
New Biol., Vol. 243, pp. 151-153
Johnson, G.B. (1974). Enzyme polymorphism and metabolism. Science, Vol. 184, pp. 28-37
Johnson, F.M., Richardson, R.H., & Kambysellis, M.P. (1968). Isozyme variability in species
of the genus Drosophila. III. Qualitative comparision of esterases of D. aldrichi and
D. mulleri. Biochem. Genet., Vol. 1, pp. 239-247
Kambysellis, M.P., Johnson, F.M., & Richardson, R.H. (1968). Isozyme variability in species
of the genus Drosophila. IV. Distribution of the esterases in the body tissues of D.
aldrichi and D. mulleri adults. Biochem. Genet., Vol. 1, pp. 249-265
Karotam, J., Delves, A.C., & Oakeshott, J.G. (1993). Conservation and change in structural
and 5 flanking sequences of esterase 6 in sibling Drosophila species. Genetica, Vol
.88, pp. 11-28
Kircher, H.W. (1982). Chemical composition of cacti and its relationship to Sonoran desert
Drosophila, In: Ecological genetics and evolution: the cactus-yeast-Drosophila model
system, J.S.F. Barker & W.T. Starmer, pp. 143-158, Academic Press, Sydney,
Australia
Kondrashov, F.A., Rogozin, I.B., Wolf, Y.I., & Koonin, E.V. (2002). Selection in the evolution
of gene duplications. Genome Biology, Vol. 3, pp. 0008.1-0008.9
Laemmli, U.K. (1970). Cleavage of structural proteins during the assembly of the head of
bacteriophage T4. Nature, Vol. 227, pp. 680-685
Lapenta, A.S., Bicudo, H.E.M.C., Ceron, C.R., & Manzato, J.A. (1995). Esterase patterns of
species in the Drosophila buzzatii cluster. Cytobios, Vol. 84, pp. 13-29

Gene Duplication

370
Lapenta, A.S., Bicudo, H.E.M.C., Ceron, C.R., & Cordeiro, J.A. (1998). Esterase patterns and
phylogenetic relationships of species and strains included in the Drosophila buzzatii
cluster. Cytobios, Vol. 96, pp. 95-107
Lewis, E.B. (1951). Pseudoallelism and gene evolution. Cold Spring Harb. Symp. Quant. Biol.,
Vol. 16, pp. 159-174
Manfrin, M.H., & Sene, F.M. (2006). Cactophilic Drosophila in South America: a model for
evolutionary studies. Genetica, Vol. 126, pp. 5775
Markow, T. (1981). Courtship behavior and control of reproductive isolation between
Drosophila mojavensis and Drosophila arizonensis. Evolution, Vol. 35, No. 5, pp. 1022-
1026
Mateus, R.P., & Sene, F.M. (2007). Population genetic study of allozyme variation in natural
populations of Drosophila antonietae (Insecta, Diptera). Journal of Zoological
Systematics and Evolutionary Research, Vol. 45, pp. 136143
Mateus, R.P., Cabral, H., Bonilla-Rodriguez, G.O., & Ceron, C.R. (2009) Molecular weight
estimation of esterase isoenzymes in closely related Drosophila species (Diptera:
Drosophilidae) in non-denaturing polyacrylamide gel electrophoresis. Braz. Arch.
Biol. Technol., Vol. 52, pp. 1083-1089
Matzkin, L.M. (2005). Activity variation in alcohol dehydrogenase paralogs is associated
with adaptation to cactus host use in cactophilic Drosophila. Mol. Ecol., Vol. 14, pp.
22232231
Matzkin, L.M., Watts, H.D., Bitler, B.G., Machado, C.A., & Markow, T.A. (2006). Functional
genomics of cactus host shifts in Drosophila mojavensis. Mol. Ecol., Vol. 15, pp. 4635-
4643
Morais, P.B., Rosa, C.A., Hagler, A.N., Mendona-Hagler, L.C. (1994). Yeast communities of
the cactus Pilosocereus arrabidae as resources for larval and adult stages of Drosophila
serido. Antonie van Leeuwenhoek, Vol. 66, pp. 313-317
Myers, M.A., Richmond, R.C., & Oakeshott, J.G. (1988). On the origins of esterases. Mol. Biol.
Evol., Vol. 5, No. 2, pp. 113-119
Nei, M. (1969). Gene duplication and nucleotide substitution in evolution. Nature, Vol. 221,
pp. 40-42
Oakeshott, J.G., van Papenrecht, E.A., Boyce, T.M., Healy, M.J., & Russel, R.J. (1993).
Evolutionary genetics of Drosophila esterases. Genetica, Vol. 90, pp. 239-268
Ohno, S. (1970). Evolution by gene duplication, Springer-Verlag, New York
Patterson, J.T., & Crow, J.F. XII. Hybridization in the mulleri group of Drosophila. Univ. Texas
Publ., Vol. 4032, pp. 251-256
Pen, J., Rongen, H.A.H., & Beintema, J.J. (1984). Purification and properties of esterase-4
form Drosophila mojavensis. Biochem. Biophys. Acta, Vol. 789, pp. 203-209
Pen, J., van Beeumen, J., & Beintema, J.J. (1986a). Structural comparision of two esterases
from Drosophila mojavensis isolated by immunoaffinity chromatography. Biochem. J.,
Vol. 238, pp. 691-699
Pen, J., Schipper, A., Rongen, H.A.H., & Beintema, J.J. (1986b). Differences in specificity and
catalytic afficiency between allozymes of esterase-4 from Drosophila mojavensis. Mol.
Biol. Evol., Vol. 3, No. 4, pp. 366-373
Pen, J., Bolks, G.J., Hoeksema-Du Pui, M.L.L., & Beintema, J.J. (1990). Serine esterases:
structural conservation during animal evolution and variability in enzymic
properties in the genus Drosophila. Genetica, Vol. 81, pp. 125-131

371
Pereira, M.A.Q.R., Vilela, C.R., Sene, F.M. (1983). Notes on breeding and feeding sites on
some species of the repleta group of the genus Drosophila (Diptera, Drosophilidae).
Cincia e Cultura, Vol. 35, pp. 1313-1319
Richardson, R.H., & Smouse, P.E. (1976). Patterns of molecular variation. I. Interspecific
comparision of electromorphs in the Drosophila mulleri complex. Biochem. Genet.,
Vol. 14, pp. 447-466
Richardson, R.H., Richardson, M.E., & Smouse, P.E. (1975). Evolution of electrophoredtic
mobility in the Drosophila mulleri complex, In: Isozymes IV: Genetics and Evolution,
C.L. Markert, pp. 533545, Academic Press, New York
Richardson, R.H., Smouse, P.E., & Richardson, M.E. (1977). Patterns of molecular variation.
II. Associations of electrophoretic mobility and larval substrate within species of
the Drosophila mulleri complex. Genetics, Vol. 85, pp. 141154
Robin, C., Bardsley, L.M.J., Coppin, C., & Oakeshott, J.G. (2009). Birth and death of genes
and functions in the -esterase cluster of Drosophila. J. Mol. Evol., Vol. 69, pp. 10-21
Ruiz, A., Heed, W.B., & Wasserman, M. (1990). Evolution of the mojavensis cluster of
cactophilic Drosophila with descriptions of two new species. J. Hered., Vol. 81, pp.
30-42
Russo, C.A.M., Takezaki, N., & Nei, M. (1995). Molecular phylogeny and divergence times
of Drosophilid species. Mol. Biol. Evol., Vol. 12, pp. 391404
Spicer, G.S. (1995). Phylogenetic utility of the mitochondrial cytochrome oxidase gene:
Molecular evolution of the Drosophila buzzatii species complex. J. Mol. Evol., Vol. 41,
pp. 749759
Spicer, G.S., & Pitnick, S. (1996). Molecular systematics of the Drosophila hydei subgroup as
inferred from mitochondrial DNA sequences. J. Mol. Evol., Vol. 43, pp. 281286
Starmer, W.T., & Gilbert, D.G. (1982). A quick and reliable method for sterilizing eggs.
Drosophila Information Service, Vol. 58, pp. 170171
Starmer, W.T., Barker, J.S.F., Phaff, H.J., & Fogleman, J.C. (1986). Adaptations of Drosophila
and yeasts their interactions with the volatile 2-propanol in the cactus
microorganism Drosophila model system. Australian Journal of Biological Sciences,
Vol. 39, pp. 6977
Stephens, S.G. (1951). Possible significance of duplication in evolution. Adv. Genet., Vol. 4,
pp. 247-265
Sullivan, D.T., Atkinson, P.W., Bayer, C.A., & Menotti-Raymond, M. (1990). The evolution of
Adh expression in the repleta group of Drosophila, In: Ecological and Evolutionary
Genetics of Drosophila, J.S.F. Barker, W.T. Starmer, & R.J. MacIntyre, pp. 407418,
Plenum Press, New York
Tidon-Sklorz, R., & Sene, F. M. (1995). Evolution of the buzzatii cluster (Drosophila repleta
group) in the Northeastern South America. Evolucin Biolgica, Vol. 8/9, pp. 71-85
Throckmorton, L.H. (1982). Pathways of evolution in the genus Drosophila and the founding
of the repleta group, In: Ecological Genetics and Evolution: The CactusYeast
Drosophila Model System, J.S.F. Barker & W.T. Starmer, pp. 3348, Academic Press,
New York
Tweedie, S., Ashburner, M., Falls, K., Leyland, P., McQuilton, P., Marygold, S., Millburn, G.,
Osumi-Sutherland, D., Schroeder, A., Seal, R., Zhang, H., & The FlyBase
Consortium. (2009). FlyBase: enhancing Drosophila Gene Ontology annotations.
Nucleic Acids Research, Vol. 37, pp. D555-D559

Gene Duplication

372
Via, S. (1990). Ecological genetics and host adaptation in herbivorous insects: the
experimental study of evolution in natural and agricultural systems. Annu. Rev.
Entomol., Vol. 35, pp. 421-446
Vilela, C.R. (1983). A revision of the Drosophila repleta species group (Diptera,
Drosophilidae). Rev. Brasil. Entomol., Vol. 27, pp. 1114
Vogt, R.G., & Riddiford, L.M. (1981). Pheromone binding and inactivation by moth
antennae. Nature, Vol. 293, pp. 161-163
Wagner, A. (2002). Selection and gene duplication: a view from the genome. Genome Biology,
Vol. 3, pp. 1012.1-1012.3
Wagner, R.P., & Selander, R.K. (1974). Isozymes in insects and their significance. Annual
Review of Entomology, Vol. 19, pp. 117-138
Walker, C.H., & Mackness, M.I. (1983). Esterases: problems of identification and
classification. Biochem. Pharmacol., Vol. 32, pp. 3265-3269
Wasserman, M. (1982). Cytological evolution in the Drosophila repleta species group, In:
Ecological Genetics and Evolution: The CactusYeastDrosophila Model System, J.S.F.
Barker & W.T. Starmer, pp. 4964, Academic Press, New York
Wasserman, M. (1992). Cytological evolution of the Drosophila repleta species group. In:
Drosophila Inversion Polymorphism, C.B. Krimbas & J.R. Powell, pp. 455552, CRC
Press, Boca Raton, FL
Zhang, J. (2003). Evolution by gene duplication: an update. Trends in Ecology and Evolution,
Vol. 18, pp. 292-298
Zouros, E. (1973). Genetic differentiation associated with the early stages of speciation in the
mulleri subgroup of Drosophila. Evolution, Vol. 27, pp. 601621
Zouros, E., van Delden, W., Odense, R., van Dijk, H. (1982). An esterase duplication in
Drosophila: differences in expression of duplicate loci within and among related
species. Biochem. Genet., Vol. 20, pp. 929-942
20
SNCA Gene Multiplication:
A Model Mechanism of Parkinson Disease
Kenya Nishioka
1
, Owen A. Ross
2
and Nobutaka Hattori
1

1
Department of Neurology, Juntendo University School of Medicine, Tokyo
2
Division of Neurogenetics, Department of Neuroscience, Mayo Clinic Jacksonville FL
1
Japan
2
USA
1. Introduction
Parkinson disease (PD) is caused by several pathogenic mutations in genes such as alpha-
synuclein (SNCA; MIM#163890), Leucine-rich repeat kinase 2 (LRRK2; MIM#609007),
PARKIN (parkin; MIM#602544), PTEN-induced kinase 1 (PINK1; MIM#608309), and DJ-1
(MIM#602533) (Farrer, 2006). The alpha-synuclein protein is also a major component of
Lewy bodies (LB), the pathologic substrate that is observed in PD patients at autopsy
(Spillantini et al., 1997). LB are generally localized to the mid-brain in patients with PD,
however a widespread distribution of LB, including cortical regions, is seen in dementia
with Lewy bodies (DLB) (Braak et al., 2003, McKeith et al., 2005). The observation of SNCA
multiplications co-segregating with PD and dementia in families led to the hypothesis that
over-expression of the alpha-synuclein protein is an important mechanism of disease.
Herein, we place the gene dosage effect of SNCA in PD in perspective and describe the
recent molecular insights underlying them.
2. SNCA triplication and duplication in hereditary PD
PD is the second most frequent neurodegenerative disorder following Alzheimer disease in
the elderly. The main symptoms of PD are tremor, bradykinesia, and gait disturbance. PD
genetics is categorized into two groups; one is sporadic PD and the other is familial PD.
Familial PD has two forms; autosomal dominant heredity (ADPD) and autosomal recessive
heredity (ARPD). ADPD has been observed to be caused by mutations in SNCA and LRRK2.
ARPD is caused by homozygous or compound heterozygous mutations in PARKIN, PINK1,
and DJ-1(Farrer, 2006). This review will focus on SNCA which is located on chromosome
4q21-22 and encodes the 140 amino acid alpha-synuclein protein. SNCA has three point
mutations; c.88G>C (Ala30Pro), c.188G>A (Glu46Lys) and c.209G>A (Ala53Thr) (Kruger et
al., 1998, Zarranz et al., 2004), but they are very rare.
SNCA duplications and triplications have also been identified as a genetic cause of ADPD.
Duplication has two SNCA copies on one allele (50% dose increase) and triplication has
three, 100 percent dose increase (Figure 1). Rarely, compound heterozygote forms (two
duplication alleles) are seen as SNCA triplication events (Ikeuchi et al., 2008). These
multiplications generate higher SNCA expression of mRNA and protein, the so called gene

Gene Duplication

374
dosage effect. Increasing the levels of protein appears to influence the clinical manifestations
of PD patients. A subtle increase in alpha-synuclein expression may increase the risk of
developing typical sporadic PD, whereas higher expression may cause severe forms of
Parkinsonism similar to DLB. Pathologically, the burden of LB correlates with a PD or DLB
clinical diagnosis, and it is still unclear whether PD and DLB are a continuum within the
disease spectrum.

Fig. 1. A model for gene dosage of deletion, duplication and triplication
Singleton et al. first reported SNCA gene triplication within a large family with PD and
dementia family (the Iowa kindred) (Singleton et al., 2003). The family members had been
followed both clinically and pathologically by Mayo Clinic doctors for 100 years (Muenter et
al., 1998, Gwinn-Hardy et al., 2000). The pattern of inheritance is autosomal dominant. The
patients show a severe clinical course, early age at onset, complicating with dementia, or
Parkinsonism. The pathological features are similar to DLB; (1) widespread LB pathology in
the brain, (2) neuronal loss in the CA2/3, and (3) neuronal loss in the substantia nigra
(Muenter et al., 1998). SNCA triplication was confirmed with quantitative PCR and FISH
methodology. The size of the triplication region was over 2.0Mb. The expression values of
messenger RNA and protein in peripheral blood and brain were twice the amount that is
present in control subjects as predicted (Miller et al., 2004). Apart from the Iowan family, a

SNCA Gene Multiplication: A Model Mechanism of Parkinson Disease

375
Swedish-American family was also reported as a SNCA triplication family and the patients
had DLB-type prognosis and pathological findings. Their expression level of messenger
RNA and protein of alpha-synuclein was also double the dosage of normal subjects in
frontal cortex (Farrer et al., 2004).
These findings provided two novel insights regarding the underlying mechanisms of PD; (1)
over-expression of alpha-synuclein may cause a more severe form of Parkinsonism such as
DLB and (2) the gene dosage of alpha-synuclein may directly correlate with the clinical
features of PD. Furthermore, these findings give us hints into the development of potential
therapeutic avenues of treatment for PD by decreasing the expression of alpha-synuclein.
The suppression of alpha-synuclein by siRNA approaches has proven successful in
decreasing the levels of LB pathology in animal models (Lewis et al., 2008, McCormack et
al., 2010).
After the detection of SNCA locus triplication, SNCA duplications were also reported in two
families from France and Italy (Chartier-Harlin et al., 2004, Ibanez et al., 2004). Chartier-
Harlin et al detected one duplication family among nine ADPD. Ibanez et al detected two
families with SNCA duplication among 119 ADPD by 250K Affymetrix microarray and
semi-quantitative PCR method(Ibanez et al., 2004). The symptoms of the patients with
SNCA duplication are milder than that of SNCA triplication, younger age at onset and have
a good efficacy for levodopa therapy, similar to sporadic PD.
Following these findings, we started screening for SNCA multiplications within 216 ADPD
and 271 sporadic PD patients originating from Japan (Nishioka et al., 2006, Nishioka et al.,
2009). We found six ADPD families and one sporadic case with SNCA duplication.
Haplotype analysis showed these seven patients are derived from four common founders.
Interestingly, the clinical manifestations of these patients were quite diverse such as
sporadic PD, DLB-type and also many elderly asymptomatic carriers. The estimated
penetrance ratio is about 30-40%. One patient presented with a severe clinical course with
no efficacy for levodopa therapy. He progressed to Hoehn and Yahr stage V in a few years
and at autopsy demonstrated features similar to DLB (Obi et al., 2008).
In addition, we also detected five asymptomatic carriers over 65 years old. We therefore
focused our work on the reasons why the asymptomatic carriers at later ages do not present
any clinical features of parkinsonism. We started to assess Brain MRI, PET study with [
18
F]-
labeled 2-fluoro-2-deoxy-D-glucose (FDG) and [
11
C]-labeled 2-carbomethoxy-3-(4-
flurophenyl)-tropane (CFT), polysomnography, and Sniffin sticks to explore the differences
between the patients and the asymptomatic carriers with SNCA duplication. Against our
expectation, the assessments for asymptomatic carriers did not show any abnormal results
in our studies, with similar results as those obtained for normal individuals (Nishioka et al.,
2009). It is an area of intense research why the asymptomatic carriers do not develop
Parkinsonism at these late ages and its resolution will be a key in the puzzle regarding the
late-onset of PD.
Apart from our cases, other teams have reported two families from Japan with SNCA
duplication (Ikeuchi et al., 2008, Uchiyama et al., 2008). Interestingly, one family has both
heterozygote and homozygote duplication (producing a pseudo-triplication) (Ikeuchi et al.,
2008). The clinical features of the individual with the SNCA homozygote duplication
showed severe Parkinsonism similar to that of a triplication carrier. These findings also
confirm the gene dosage effect within the same family. Earlier studies had reported that the

Gene Duplication

376
Swedish family named the Lister family complex has both SNCA duplication and
triplication patients within different branches of the pedigree suggesting a primary
duplication event followed later by another resulting in the triplication (Fuchs et al., 2007).
In this family also the patient with SNCA triplication presented with more severe symptoms
than the patients with duplication. Recently one small pedigree with SNCA triplication was
detected in Japan (Sekine et al., 2010).
The breakpoint of SNCA multiplication is different in each family. The largest multiplication
about 4.9Mb is detected within a French family. The smallest one about 0.2 Mb is in a
Japanese family (Nishioka et al., 2009). The size and gene make-up of each multiplication
region does not seem to influence the clinical presentation of the carrier. The single common
determining factor that appears between all patients with SNCA multiplication is the
presence of the entire SNCA gene. To conclude, it is clear that SNCA multiplication alone is
sufficient to result in the parkinsonian phenotype.
2.1 Sporadic PD and SNCA duplication
In 2007, Ahn et al reported two sporadic patients with SNCA duplication from a screen of
906 PD patients (Ahn et al., 2008). The age at onset was 65 and 50 years old for the two
patients. Their clinical course was similar to typical sporadic PD without severe progression
or cognitive decline. The estimated penetrance ratio was 33.3% among the Korean patients.
Our studies have also detected one sporadic patient with SNCA duplication and this means
that the low penetrance SNCA duplications may give the appearance of sporadic disease.
2.2 The frequency of SNCA multiplications in PD
The prevalence of SNCA multiplications is relatively low (table 1). Bruggemann et al
reported one sporadic case among 403 PD cases from Germany (1/403=0.25%)
(Brueggemann et al., 2008). Troiano et al reported one sporadic cases among 101 young
onset PD cases from French (1/101=1%) (Troiano et al., 2008). Nuytemans et al reported one
duplication patient with dementia among 219 sporadic PD cases from Belgium (Nuytemans
et al., 2009). Sironi et al reported one duplication patient with dementia among 144 PD cases
from Italy (1/144=0.7%) (Sironi et al., 2009). Furthermore some reports did not detect SNCA
duplication; 0/50 and 0/290 (Hope et al., 2004, Xiromerisiou et al., 2007). To conclude,
SNCA multiplication is not a common cause of sporadic or hereditary PD.
2.3 SNCA multiplications in multiple system atrophy
Multiple system atrophy (MSA) is characterized by specific clinical features such as
Parkinsonism, autonomic dysfunction, poor response to levodopa therapy, and cerebellar
ataxia (Wenning et al., 2004). Glial cytoplasmic inclusions (GCIs) are the pathologic
hallmark of the disease. As alpha-synuclein is a major protein component of GCIs, MSA is
categorized within the group of neurodegenerative disorders classified as the alpha-
synucleinopathies. Interestingly common variation at the SNCA locus has been associated
with MSA risk (Scholz et al., 2009, Ross et al., 2010). Two studies did not identify any SNCA
multiplications in a combined total of 258 MSA patients (Lincoln et al., 2007, Ahn et al.,
2008). Although the number of assessed samples may be small, these findings suggest that
SNCA dosage is not a common cause of MSA. It is speculated that PD or DLB may be cause
by lysosomal dysfunction, however, MSA may be caused by the oligodendrocytic changes
in myelin basic protein (Wenning et al., 2008).


377
Country The number
of pedigrees
The number of
screening samples
Frequency
(%)
Average
ot age at
onset
SNCA duplication in
AD cases

Nishioka et al. 2006
and 2009
Japan
(Juntendo)
6 487 1.2 48.2
Ibanez et al. 2004 and
2009
France
and Italy
4 286 1.4 43.6
Sironi et al. 2009 Italy 1 144 0.7 41
Ikeuchi et al. 2008 Japan
(Niigata)
1 57
Uchiyama et al. 2008 Japan
(Niigata)
1 60
Fuchs et al. 2007 Sweden
(Lister
complex)
1 71
Ahn et al. 2007 Korea 1 906 0.1 40
Chartier-Harliln et al.
2004
France 1 9 11 50.8
total average 51.5
SNCA duplication in
sporadic cases

Nishioka et al. 2009 Japan
(Juntendo)
1 487 0.2 31
Nuytemans et al.2009 Belgium 1 219 0.5 71
Brueggemann et al.
2008
Germany 1 403 0.3 36
Troiano et al. 2008 France 1 101 1 35
Ahn et al. 2007 Korea 2 906 0.2 57.5
total average 46.1
SNCA triplication
Sekine et al. 2010 Japan
(Juntendo)
1 37
Ibanez et al. 2009 France 1 286 0.3 42
Fuchs et al. 2007 Sweden
(Lister
complex)
1
Farrer et al. 2004 Swedish-
American
1 31
Singlton et al. 2003 Iowa 1 33.2
SNCA homozygote
duplication

Ikeuchi et al. 2008 Japan
(Niigata)
1 28
total average 32.9
Table 1. The clinical manifestations and prevalence of SNCA duplication and triplication

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378
2.4 The Synuclein family in Parkinson disease
The SNCA gene is located on chromosome 4q21-22 and is associated with susceptibility to
PD and DLB. Alpha-synclein has two paralogous genes, beta- (SNCB; MIM#602569) and
gamma-synuclein (SNCG; MIM#602998) with which it shares a highly conserved N-terminal
domain. SNCB is located on chromosome 5q35, and SNCG is located on chromosome 10q23
associated with breast and ovarian cancer (Ji et al., 1997, Goedert, 2001). All three synuclein
genes are highly expressed in brain; thalamus, substantia nigra, caudate nucleus, and
amygdala (Lavedan, 1998, Lavedan et al., 1998). A phylogenic tree indicates that alpha- and
beta- synucleins are related more closely to each other than to gamma-synuclein (Lavedan,
1998). Interestingly, two putative pathogenic mutations in SNCB are reported to cause DLB,
however no significant co-segregation with disease could be shown and no other studies
have identified these variants (Ohtake et al., 2004). A murine model with over-expressed
gamma-synuclein is reported as a PD model with motor deficits (Ninkina et al., 2009). Our
recent studies on common variation in the synuclein family of genes also suggested
association for variants in both SNCA and SNCG with diffuse LB disease (Nishioka et al.,
2010). Given these findings, it is postulated that there is a connection between not only
SNCA, but also SNCB and SNCG and susceptibility to PD, however multiplications of the
SNCB and SNCG loci have not yet been observed.
3. Conclusion and future work
Research focused on copy number variation has made remarkable progress in recent years.
Genome-wide studies for copy number variants (CNV) indicate 1447 copy number variable
regions (CNVRs) (Redon et al., 2006). Presumably, many of these CNV polymorphisms
result in differential expression levels of proteins and dictate the phenotypic presentation at
the individual level. Interestingly in Alzheimer disease multiplications of the APP gene have
also been identified in families with autosomal dominantly inherited forms of the disease
(Cabrejo et al., 2006, Rovelet-Lecrux et al., 2006). Robust and comprehensive studies are now
warranted for CNV across the genome and may not only help develop new treatments for
PD but perhaps several other neurodegenerative diseases.
4. References
Ahn TB, Kim SY, Kim JY, Park SS, Lee DS, Min HJ, Kim YK, Kim SE, Kim JM, Kim HJ, Cho J,
Jeon BS (2008) alpha-Synuclein gene duplication is present in sporadic Parkinson
disease. Neurology 70:43-49.
Braak H, Rub U, Gai WP, Del Tredici K (2003) Idiopathic Parkinson's disease: possible routes
by which vulnerable neuronal types may be subject to neuroinvasion by an
unknown pathogen. J Neural Transm 110:517-536.
Brueggemann N, Odin P, Gruenewald A, Tadic V, Hagenah J, Seidel G, Lohmann K, Klein
C, Djarmati A (2008) Re: Alpha-synuclein gene duplication is present in sporadic
Parkinson disease. Neurology 71:1294; author reply 1294.
Cabrejo L, Guyant-Marechal L, Laquerriere A, Vercelletto M, De la Fourniere F, Thomas-
Anterion C, Verny C, Letournel F, Pasquier F, Vital A, Checler F, Frebourg T,
Campion D, Hannequin D (2006) Phenotype associated with APP duplication in
five families. Brain 129:2966-2976.


379
Chartier-Harlin MC, Kachergus J, Roumier C, Mouroux V, Douay X, Lincoln S, Levecque C,
Larvor L, Andrieux J, Hulihan M, Waucquier N, Defebvre L, Amouyel P, Farrer M,
Destee A (2004) Alpha-synuclein locus duplication as a cause of familial
Parkinson's disease. Lancet 364:1167-1169.
Farrer M, Kachergus J, Forno L, Lincoln S, Wang DS, Hulihan M, Maraganore D, Gwinn-
Hardy K, Wszolek Z, Dickson D, Langston JW (2004) Comparison of kindreds with
parkinsonism and alpha-synuclein genomic multiplications. Ann Neurol 55:174-
179.
Farrer MJ (2006) Genetics of Parkinson disease: paradigm shifts and future prospects. Nat
Rev Genet 7:306-318.
Fuchs J, Nilsson C, Kachergus J, Munz M, Larsson EM, Schule B, Langston JW, Middleton
FA, Ross OA, Hulihan M, Gasser T, Farrer MJ (2007) Phenotypic variation in a large
Swedish pedigree due to SNCA duplication and triplication. Neurology 68:916-922.
Goedert M (2001) Alpha-synuclein and neurodegenerative diseases. Nat Rev Neurosci 2:492-
501.
Gwinn-Hardy K, Mehta ND, Farrer M, Maraganore D, Muenter M, Yen SH, Hardy J,
Dickson DW (2000) Distinctive neuropathology revealed by alpha-synuclein
antibodies in hereditary parkinsonism and dementia linked to chromosome 4p.
Acta Neuropathol (Berl) 99:663-672.
Hope AD, Myhre R, Kachergus J, Lincoln S, Bisceglio G, Hulihan M, Farrer MJ (2004) Alpha-
synuclein missense and multiplication mutations in autosomal dominant
Parkinson's disease. Neuroscience letters 367:97-100.
Ibanez P, Bonnet AM, Debarges B, Lohmann E, Tison F, Pollak P, Agid Y, Durr A, Brice A
(2004) Causal relation between alpha-synuclein gene duplication and familial
Parkinson's disease. Lancet 364:1169-1171.
Ikeuchi T, Kakita A, Shiga A, Kasuga K, Kaneko H, Tan CF, Idezuka J, Wakabayashi K,
Onodera O, Iwatsubo T, Nishizawa M, Takahashi H, Ishikawa A (2008) Patients
homozygous and heterozygous for SNCA duplication in a family with
parkinsonism and dementia. Archives of neurology 65:514-519.
Ji H, Liu YE, Jia T, Wang M, Liu J, Xiao G, Joseph BK, Rosen C, Shi YE (1997) Identification
of a breast cancer-specific gene, BCSG1, by direct differential cDNA sequencing.
Cancer Res 57:759-764.
Kruger R, Kuhn W, Muller T, Woitalla D, Graeber M, Kosel S, Przuntek H, Epplen JT, Schols
L, Riess O (1998) Ala30Pro mutation in the gene encoding alpha-synuclein in
Parkinson's disease. Nat Genet 18:106-108.
Lavedan C (1998) The synuclein family. Genome Res 8:871-880.
Lavedan C, Leroy E, Torres R, Dehejia A, Dutra A, Buchholtz S, Nussbaum RL,
Polymeropoulos MH (1998) Genomic organization and expression of the human
beta-synuclein gene (SNCB). Genomics 54:173-175.
Lewis J, Melrose H, Bumcrot D, Hope A, Zehr C, Lincoln S, Braithwaite A, He Z,
Ogholikhan S, Hinkle K, Kent C, Toudjarska I, Charisse K, Braich R, Pandey RK,
Heckman M, Maraganore DM, Crook J, Farrer MJ (2008) In vivo silencing of alpha-
synuclein using naked siRNA. Mol Neurodegener 3:19.
Lincoln SJ, Ross OA, Milkovic NM, Dickson DW, Rajput A, Robinson CA, Papapetropoulos
S, Mash DC, Farrer MJ (2007) Quantitative PCR-based screening of alpha-synuclein

Gene Duplication

380
multiplication in multiple system atrophy. Parkinsonism & related disorders
13:340-342.
McCormack AL, Mak SK, Henderson JM, Bumcrot D, Farrer MJ, Di Monte DA (2010) Alpha-
synuclein suppression by targeted small interfering RNA in the primate substantia
nigra. PLoS One 5:e12122.
McKeith IG, Dickson DW, Lowe J, Emre M, O'Brien JT, Feldman H, Cummings J, Duda JE,
Lippa C, Perry EK, Aarsland D, Arai H, Ballard CG, Boeve B, Burn DJ, Costa D, Del
Ser T, Dubois B, Galasko D, Gauthier S, Goetz CG, Gomez-Tortosa E, Halliday G,
Hansen LA, Hardy J, Iwatsubo T, Kalaria RN, Kaufer D, Kenny RA, Korczyn A,
Kosaka K, Lee VM, Lees A, Litvan I, Londos E, Lopez OL, Minoshima S, Mizuno Y,
Molina JA, Mukaetova-Ladinska EB, Pasquier F, Perry RH, Schulz JB, Trojanowski
JQ, Yamada M (2005) Diagnosis and management of dementia with Lewy bodies:
third report of the DLB Consortium. Neurology 65:1863-1872.
Miller DW, Hague SM, Clarimon J, Baptista M, Gwinn-Hardy K, Cookson MR, Singleton AB
(2004) Alpha-synuclein in blood and brain from familial Parkinson disease with
SNCA locus triplication. Neurology 62:1835-1838.
Muenter MD, Forno LS, Hornykiewicz O, Kish SJ, Maraganore DM, Caselli RJ, Okazaki H,
Howard FM, Jr., Snow BJ, Calne DB (1998) Hereditary form of parkinsonism--
dementia. Ann Neurol 43:768-781.
Ninkina N, Peters O, Millership S, Salem H, van der Putten H, Buchman VL (2009) Gamma-
synucleinopathy: neurodegeneration associated with overexpression of the mouse
protein. Human molecular genetics 18:1779-1794.
Nishioka K, Hayashi S, Farrer MJ, Singleton AB, Yoshino H, Imai H, Kitami T, Sato K,
Kuroda R, Tomiyama H, Mizoguchi K, Murata M, Toda T, Imoto I, Inazawa J,
Mizuno Y, Hattori N (2006) Clinical heterogeneity of alpha-synuclein gene
duplication in Parkinson's disease. Ann Neurol 59:298-309.
Nishioka K, Ross OA, Ishii K, Kachergus JM, Ishiwata K, Kitagawa M, Kono S, Obi T,
Mizoguchi K, Inoue Y, Imai H, Takanashi M, Mizuno Y, Farrer MJ, Hattori N (2009)
Expanding the clinical phenotype of SNCA duplication carriers. Mov Disord
24:1811-1819.
Nishioka K, Wider C, Vilarino-Guell C, Soto-Ortolaza AI, Lincoln SJ, Kachergus JM,
Jasinska-Myga B, Ross OA, Rajput A, Robinson CA, Ferman TJ, Wszolek ZK,
Dickson DW, Farrer MJ (2010) Association of alpha-, beta-, and gamma-Synuclein
with diffuse lewy body disease. Archives of neurology 67:970-975.
Nuytemans K, Meeus B, Crosiers D, Brouwers N, Goossens D, Engelborghs S, Pals P, Pickut
B, Van den Broeck M, Corsmit E, Cras P, De Deyn PP, Del-Favero J, Van
Broeckhoven C, Theuns J (2009) Relative contribution of simple mutations vs. copy
number variations in five Parkinson disease genes in the Belgian population.
Human mutation 30:1054-1061.
Obi T, Nishioka K, Ross OA, Terada T, Yamazaki K, Sugiura A, Takanashi M, Mizoguchi K,
Mori H, Mizuno Y, Hattori N (2008) Clinicopathologic study of a SNCA gene
duplication patient with Parkinson disease and dementia. Neurology 70:238-241.
Ohtake H, Limprasert P, Fan Y, Onodera O, Kakita A, Takahashi H, Bonner LT, Tsuang DW,
Murray IV, Lee VM, Trojanowski JQ, Ishikawa A, Idezuka J, Murata M, Toda T,
Bird TD, Leverenz JB, Tsuji S, La Spada AR (2004) Beta-synuclein gene alterations
in dementia with Lewy bodies. Neurology 63:805-811.


381
Redon R, Ishikawa S, Fitch KR, Feuk L, Perry GH, Andrews TD, Fiegler H, Shapero MH,
Carson AR, Chen W, Cho EK, Dallaire S, Freeman JL, Gonzalez JR, Gratacos M,
Huang J, Kalaitzopoulos D, Komura D, MacDonald JR, Marshall CR, Mei R,
Montgomery L, Nishimura K, Okamura K, Shen F, Somerville MJ, Tchinda J,
Valsesia A, Woodwark C, Yang F, Zhang J, Zerjal T, Armengol L, Conrad DF,
Estivill X, Tyler-Smith C, Carter NP, Aburatani H, Lee C, Jones KW, Scherer SW,
Hurles ME (2006) Global variation in copy number in the human genome. Nature
444:444-454.
Ross OA, Vilarino-Guell C, Wszolek ZK, Farrer MJ, Dickson DW (2010) Reply to: SNCA
variants are associated with increased risk of multiple system atrophy. Ann Neurol
67:414-415.
Rovelet-Lecrux A, Hannequin D, Raux G, Le Meur N, Laquerriere A, Vital A, Dumanchin C,
Feuillette S, Brice A, Vercelletto M, Dubas F, Frebourg T, Campion D (2006) APP
locus duplication causes autosomal dominant early-onset Alzheimer disease with
cerebral amyloid angiopathy. Nat Genet 38:24-26.
Scholz SW, Houlden H, Schulte C, Sharma M, Li A, Berg D, Melchers A, Paudel R, Gibbs JR,
Simon-Sanchez J, Paisan-Ruiz C, Bras J, Ding J, Chen H, Traynor BJ, Arepalli S,
Zonozi RR, Revesz T, Holton J, Wood N, Lees A, Oertel W, Wullner U, Goldwurm
S, Pellecchia MT, Illig T, Riess O, Fernandez HH, Rodriguez RL, Okun MS, Poewe
W, Wenning GK, Hardy JA, Singleton AB, Del Sorbo F, Schneider S, Bhatia KP,
Gasser T (2009) SNCA variants are associated with increased risk for multiple
system atrophy. Ann Neurol 65:610-614.
Sekine T, Kagaya H, Funayama M, Li Y, Yoshino H, Tomiyama H, Hattori N (Clinical course
of the first Asian family with Parkinsonism related to SNCA triplication. Mov
Disord 25:2871-2875.2010).
Singleton AB, Farrer M, Johnson J, Singleton A, Hague S, Kachergus J, Hulihan M,
Peuralinna T, Dutra A, Nussbaum R, Lincoln S, Crawley A, Hanson M, Maraganore
D, Adler C, Cookson MR, Muenter M, Baptista M, Miller D, Blancato J, Hardy J,
Gwinn-Hardy K (2003) alpha-Synuclein locus triplication causes Parkinson's
disease. Science 302:841.
Sironi F, Trotta L, Antonini A, Zini M, Ciccone R, Della Mina E, Meucci N, Sacilotto G,
Primignani P, Brambilla T, Coviello DA, Pezzoli G, Goldwurm S (2009) alpha-
Synuclein multiplication analysis in Italian familial Parkinson disease.
Parkinsonism & related disorders.
Troiano AR, Cazeneuve C, Le Ber I, Bonnet AM, Lesage S, Brice A (2008) Re: Alpha-
synuclein gene duplication is present in sporadic Parkinson disease. Neurology
71:1295; author reply 1295.
Uchiyama T, Ikeuchi T, Ouchi Y, Sakamoto M, Kasuga K, Shiga A, Suzuki M, Ito M, Atsumi
T, Shimizu T, Ohashi T (2008) Prominent psychiatric symptoms and glucose
hypometabolism in a family with a SNCA duplication. Neurology 71:1289-1291.
Wenning GK, Colosimo C, Geser F, Poewe W (2004) Multiple system atrophy. Lancet
Neurol 3:93-103.
Wenning GK, Stefanova N, Jellinger KA, Poewe W, Schlossmacher MG (2008) Multiple
system atrophy: a primary oligodendrogliopathy. Ann Neurol 64:239-246.
Xiromerisiou G, Hadjigeorgiou GM, Gourbali V, Johnson J, Papakonstantinou I,
Papadimitriou A, Singleton AB (2007) Screening for SNCA and LRRK2 mutations

Gene Duplication

382
in Greek sporadic and autosomal dominant Parkinson's disease: identification of
two novel LRRK2 variants. Eur J Neurol 14:7-11.
Zarranz JJ, Alegre J, Gomez-Esteban JC, Lezcano E, Ros R, Ampuero I, Vidal L, Hoenicka J,
Rodriguez O, Atares B, Llorens V, Gomez Tortosa E, del Ser T, Munoz DG, de
Yebenes JG (2004) The new mutation, E46K, of alpha-synuclein causes Parkinson
and Lewy body dementia. Ann Neurol 55:164-173.
21
Bucentaur (Bcnt)
1
Gene Family:
Gene Duplication and
Retrotransposon Insertion
Shintaro Iwashita and Naoki Osada
Iwaki Meisei University / National Institute of Genetics
Japan
1. Introduction
Members of multiple gene families in higher organisms allow for more refined cellular
signaling networks and structural organization toward more stable physiological
homeostasis. Gene duplication is one the most powerful ways of providing an opportunity
to create a novel gene(s) because a novel function might be acquired without the loss of the
original gene function (Ohno, 1970). Gene duplication can result from unequal crossing over
by recombination, retroposition of cDNA, or whole-genome duplication. Furthermore, a
replication-based mechanism of change in gene copy number has been proposed recently
(Hastings et al., 2009). Gene duplication generated by retroposition is frequently
accompanied by deleterious effects because the insertion of cDNA into the genome is nearly
random or unlinks the original gene location resulting in an alteration of the original vital
functions of the target genes. Thus retroelements such as transposable elements and
endogenous retroviruses have been thought of as selfish. On the other hand, gene
duplication caused by unequal crossing over generally results in tandem alignment, which
less frequently disrupts the functions of other genes. Recent genome-wide studies have
demonstrated that retroelements can definitely contribute to the creation of individual novel
genes and the modulation of gene expression, which allows for the dynamic diversity of
biological systems, such as placental evolution (Rawn & Cross, 2008). It is now recognized
that tandem duplication and retroposition are among the key factors that initiate the
creation of novel gene family members (Brosius, 2005; Sorek, 2007; Kaessmann, 2010). By
these mechanisms, species-specific gene duplication can lead to species-specific gene
functions, which might contribute to species-specific phenotypes (Zhang, 2003). For
example, many genes derived from retroelements are expressed in mammalian placentas,
and species-specific gene duplication has occurred multiple times during placental
evolution (Rawn & Cross, 2008). If a combination of tandem gene duplication and
retroposition of cDNA occurs, there is a good possibility for the creation of a novel gene(s)

1
Although the vertebrate Bcnt (Bucentaur) gene is officially called Cfdp1 (craniofacial developmental
protein 1), its biological function remains unclear. So far, solid evidence that the gene is involved in
craniofacial development has not been provided except for its unique expression during mouse tooth
development (Diekwisch et al., 1999). The authors are concerned that a wrong naming may have
caused confusion concerning the function of the Bcnt/Cfdp1 gene. Thus we use the names Bcnt/Cfdp1,
p97Bcnt/Cfdp2 and p97Bcnt-2 in this article.

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384
because a novel function could be acquired with the guarantee that the original gene
functions will be retained. This type of evolutionary process has been described, e.g. in the
Jingwei gene of Drosophila, where segmental duplication of a certain gene followed by
retroposition of alcohol dehydrogenase (Adh) cDNA into one of the copied genes created a
new Adh with an altered substrate specificity (Zhang et al., 2004). Furthermore, since the
insertion of retrotransposons can speed up the natural mutation process tremendously
(Makalowski, 2003), the combined process of tandem duplication followed by
retrotransposon insertion has a greater potential to generate a novel gene(s) (Fig. 1). Indeed
we have identified an example of this type, the p97Bcnt protein (Nobukuni et al., 1997). The
p97Bcnt/Cfdp2 gene was created in a common ancestor of ruminants by a partial duplication
of the ancestral Bcnt/Cfdp1 gene followed by insertion of an order-specific retrotransposon,
Bov B-LINE (Iwashita et al., 2003, 2006). As a result, the paralog recruited an
apurinic/apyrimidinic (AP)-endonuclease domain in the middle of the protein. In this
article, we summarize the gene organization and protein structures of three Bcnt family
members, and describe their biochemical characteristics. We also argue that the process of
tandem duplication followed by retroelement insertion generates a high potential for
creating novel genes for expanding signaling networks.

Fig. 1. Mechanism of novel gene creation by a combination of gene duplication and
retrotransposition
If segmental duplication followed by retrotransposon insertion occurs, it provides a good
opportunity to generate a paralogous gene, because a novel function could be acquired
under the guarantee that the original gene function will remain. The schematic is a
modification of the original (Makalowski, 2003)
2. Establishment of three Bcnt members
2.1 A bovine specific retrotransposon, Bov B-LINE
Autonomous non-long-terminal repeat (non-LTR) retrotransposons, also termed Long-
Interspersed Nuclear Elements (LINEs), have been identified in almost all eukaryotic
organisms. Based on their structures and type of endonuclease, non-LTR retrotransposons
are classified into two subtypes. The major subtype encodes an endonuclease with
homology to AP-endonuclease (APE), thus termed APE-type non-LTR retrotransposons.
These APE-type elements are now divided into four groups and eleven clades (Zingler et al.,
2005). The RTE clade is one of the most widespread and shortest APE-type non-LTR
retrotransposons, which are truncated forms of L1 (human LINE 1) lacking both the 5 and 3
regions (Fig. 2).

Bucentaur (Bcnt) Gene Family: Gene Duplication and Retrotransposon Insertion

385

Fig. 2. The structural relationships among the open reading frames of retrotransposable L1
and RTE elements and three Bcnt-related proteins
The L1 and RTE elements have apurinic/apyrimidic (AP)-endonuclease domains (yellow
boxes) and reverse transcriptase domains (green boxes). The L1 element has another restriction
enzyme-like endonuclease domain in the C-terminal region (dark blue bar). The square to the
left of RTE indicates the ambiguity of the 5 region. The assignment of the domains in L1 and
RTE is according to Malik & Eickbush, 1998. The numbers above the rectangles of the three
Bcnt-related proteins, Bcnt/Cfdp1, p97Bcnt/Cfdp2 and p97Bcnt2, indicate amino acid residue
numbers. The latter two contain a region derived from the AP-endonuclease domain of RTE
(termed the RTE domain) in the middle of their molecules. As described below, the three
proteins have common acidic N-terminal regions (grey boxes) and intramolecular repeat (IR)
units consisting of 40 amino acids each (orange boxes). The blue box at the C-terminus of
ancestral Bcnt/Cfdp1 indicates a conserved 82-amino acids region (Bcnt-C)
Retrotransposons spread through vertical transmission, but occasionally through horizontal
transmission (Gentles et al., 2007). Bov-B LINEs are order-specific RTEs that are found
specifically in ruminants, where they were initially reported as a bovine Alu-like dimer-
driven family; they potentially encode both an AP-enodnuclease domain and a reverse
transcriptase domain accompanied by a short interspersed repetitive element (SINE)
cassette (Szemraj et al., 1995). It has been suggested that BovB-LINEs were transferred
horizontally from squamata into an ancestral ruminant and expanded in all ruminants
(Zupunski et al., 2001). p97Bcnt/Cfdp 2 recruited the AP-endonuclease domain of Bov-B
LINE during the creation process in an ancient ruminant.
2.2 Discovery of a novel protein, p97Bcnt/Cfdp2
The p97Bcnt/Cfdp2 protein was discovered in bovine brain during screening for hybridoma
producing monoclonal antibodies (mAbs). In the course of a study on Ras GTPase-activating
proteins (GAPs, RAS p21 protein activators, Rasa), we had attempted to generate mAbs to
distinguish each GAP from among their family members (Kobayashi et al., 1993; Iwashita &
Song, 2008). We used a glutathione-S-transferase (GST) fusion protein of rat Rasa2 (GAP
1m
)
as an immunoantigen and screened for hybridomas by western blotting using bovine brain
extract. We isolated five independent clones, all of which showed a single broad band with
an apparent molecular mass of 97 kDa, exactly the expected size of rat Rasa2. At one time

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386
we thought we had obtained appropriate antibodies, but the target protein was entirely
different from Rasa as described below. Although we screened a bovine brain cDNA
expression library by western blotting with the obtained mAbs, we could not clone the
target molecule. Instead, a 97 kDa protein was isolated from bovine brain extract by affinity
chromatography with the antibodies, and the amino acid sequences of its protease-digested
fragments were determined. We used redundant primers designed based on the determined
peptide sequences as DNA probes, and cloned the target molecule by both rapid
amplification of cDNA ends (RACE) and screening of a bovine brain cDNA library
(Nobukuni et. al., 1997). The obtained clone, which had an open reading frame of 592 amino
acids, was named Bcnt after bucentaur, a Greek mythical creature that is half man and half
ox, implying a strange protein from bovine brain. The identified protein, named p97Bcnt,
Bcnt with a molecular mass of 97kDa, consists of an acidic N-terminal region, a
retrotransposon-derived 325-amino acid region (termed the RTE domain), and two 40-
amino acid intrarepeat (IR) units. The RTE domain is 72% identical to an order-specific
retrotransposon, Bov-B LINE (GenBank accession number AF332697). The relationship
between p97Bcnt and its estimated epitope in the mAbs, which enabled us to identify the
protein, is summarized in Fig. 3. It provides a reasonable explanation as to why the unique
protein was isolated by mAbs generated by a GST-fusion protein of rat Rasa2 as an
immunoantigen. The estimated epitope of five independent mAbs maps on a single site in
the N-terminal region of p97Bcnt/Cfdp2, and the antibodies recognize neither human
BCNT/CFDP1 (Nobukuni et al., 1997) nor bovine Bcnt/Cfdp1 (Iwashita et al., 2003). The
junction region of the fusion protein between GST and truncated Rasa2 codes a unique
amino acid sequence generated by the extra nucleotides of the multiple cloning sites and a
nucleotide linker for plasmid construction. Since Rasa is a highly conserved protein in
mammals, the junction region might present strong antigenicity. Generally, it is hard to
clone a target molecule by direct DNA screening when interspersed repetitive sequences are
involved. Therefore, we first isolated a 97kDa protein that was recognized by the
accidentally generated mAbs, determined its amino acid sequence, and then screened a
cDNA library with the designed oligonucleotide probes. This led to the identification of a
unique protein, p97Bcnt/Cfdp2.
2.3 Identification of three Bcnt-related proteins
Immediately after the identification of p97Bcnt/Cfdp2, we isolated its human and mouse
counterparts, and examined their differences from p97Bcnt/Cfdp2 at both the cDNA and
genome levels (Nobukuni et al., 1997; Takahashi et al., 1998). The counterparts, called
(ancestral) Bcnt/Cfdp1, have homologous acidic N-terminal regions and one IR unit of 40-
amino acids, but lack the RTE domain. Instead they contain a highly conserved 82-amino
acid region at the C-terminus that is not present in p97Bcnt/Cfdp2 (Fig. 2) as will be
described below. Subsequently, we found that ruminants have both ancestral Bcnt/Cfdp1 and
p97Bcnt/Cfdp2, while other animals have only Bcnt/Cfdp1. The pairwise sequence alignment
of bovine and human genome DNA revealed that the region encompassing the gene was
duplicated in two rounds in bovines (Iwashita et al., 2003). Although automated
computational annotation predicted another homolog of p97Bcnt (LOC514131) in the bovine
genome, its 5 UTR was different from the full-length cDNA that we isolated. Then we
identified another paralog, termed p97Bcnt-2, in the adjacent region (Iwashita et al., 2009).
The gene product, p97Bcnt2, is highly homologous to p97Bcnct/Cfdp2, comprising an acidic
N-terminal region, a 324-amino acid RTE domain, and three IR units instead of the two in
p97Bcnt/Cfdp2 in the C-terminal region (Fig. 2).


387

Fig. 3. Epitopes of the monoclonal antibodies that enabled the identification of the
p97Bcnt/Cfdp2 protein
A plasmid of a fusion protein of truncated rat Rasa2 (from Ile65 to Ser847) and glutathione S-
transferase was constructed using a linker (by Dr. S. Hattori), expressed in Escherichia coli, and its
protein was purified by glutathione-affinity column chromatography. mAbs against the fusion
protein were isolated according to a conventional method. Epitope mapping was carried out
using the full-length cDNA of the targeted molecule, hereafter p97Bcnt. Fragments of ~300 base
pairs in size were expressed in a protein expression vector and screened with the obtained mAbs.
Seven positive clones were isolated from among ~9 x 10
3
bacterial colonies, and the sequence
common to all clones was determined as the possible epitope for anti-p97Bcnt antibodies (13-
amino acids, RKQGRLSLDQEEE, represented by the red bar in the upper part) (Nobukuni et al.,
1997). Amino acid sequences corresponding to the epitope region of rat Rasa2, bovine
p97Bcnt/Cfdp2, human BCNT/CFDP1, bovine Bcnt/Cfdp1, and bovine p97Bcnt2 are aligned
and amino acid residues identical to those in the expected epitope are indicated in red
3. Tandem alignment of three Bcnt gene family members
The draft bovine genome sequence was published in 2009 (The Bovine Genome Sequencing
and Analysis Consortium, 2009). The initial analysis estimated that the bovine genome
contains about 22,000 genes, with a core set of 14,345 orthologs shared among seven
mammalian species. It has been shown that 3.1% of the bovine genome consists of recently
duplicated sequences (judged by sequences 1 kb in length and 90% identity), and more
than three-quarters (75-90%) of segmental duplications are organized into local tandem
duplication clusters (Liu et al., 2009). It is noteworthy that cattle-specific evolutionary
breakpoint regions in the chromosomes have a higher density of tandem duplications and
enrichment of repetitive elements. Furthermore, it has been pointed out that bovine tandem
gene duplication is significantly related to species-specific biological functions such as
immunity, digestion, lactation, and reproduction (Liu et al., 2009).

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388

Fig. 4. Bovine Bcnt/Cfdp1 locus and its corresponding region in the human genome
The organization of bovine Bcnt/Cfdp1-p97Bcnt/Cfdp2-p97Bcnt-2 is shown schematically.
Bcar1, Breast cancer anti-estrogen resistance 1 gene and Tmem170A, Transmembrane protein
170A gene, are located proximal and distal to the Bcnt-gene cluster or BCNT in both bovine
chromosome 18 (middle part) and human chromosome 16q23 (upper part), respectively. The
Bcnt/Cfdp1, p97Bcnt/Cfdp2, and p97Bcnt-2 genes comprise 7, 8, and 10 exons, respectively
(lower part); each exon is indicated by a vertical bar and is numbered
The three Bcnt-related genes are tandemly aligned on bovine chromosome 18 over a range of
more than 177 kb, a syntenic region of human chromosome 16q23 (Fig. 4) and mouse
chromosome 8. This gene cluster exists between the proximal breast cancer anti-estrogen
resistance 1 gene (Bcar1) and the distal transmembrane protein 170A gene (Tmem170A) in
bovines, as is the case of BCNT/CFDP1 in humans and Bcnt/Cfdp1 in mice. Therefore, the
cluster region was generated from an order-specific segmental duplication. It has been
suggested that Bov-B LINEs emerged by horizontal transfer from squamata to ancient
ruminants (Zupunski et al., 2001), and expanded just after the divergence of ruminantia and
Camelidae (Jobse et al., 1995). Bov-B LINEs have further expanded in different lineages
during the diversification of ruminant species after splitting from Tragulina, which was
confirmed by hybridization with DNA fragments of the RTE domain of Lesser Malay
chevrotain (Iwashita et al., 2006). This is consistent with the expansion of bovine SINEs (Jobse
et al., 1995). Tragulus javanicus, the living fossil of the basal ruminant stock, shares a similar
Bcnt/Cfdp1 and p97Bcnt/Cfdp2 gene organization to bovines (Iwashita et al., 2006). Thus the
partial gene duplication of the ancestral Bcnt/Cfdp1 followed by the Bov-B LINE insertion
occurred sometime after the Ruminantia-Suina-Tylopoda split and before the Pecora-
Tragulina divergence, ~50 million years ago. A phylogenic tree has been constructed based
on the N-terminal regions (~ 175 amino acids) encoded by the first four exons and shared
among three Bcnt-related members. The tree topology suggests p97Bcnt-2

was created from
duplication of ancestor p97Bcnt/Cfdp2 in an ancient ruminant prior to the Pecora-Tragulina
divergence. Furthermore, using the 120-bp sequence corresponding to 40 amino acid
residues in IR, duplication of the IR unit in p97Bcnt/Cfdp2 is estimated to have occured prior
to the creation of p97Bcnt-2, which has three IR units (Fig. 2). The two units in p97Bcnt-2 (IR-


389
II and IR-III) diverged from IR-II in p97Bcnt/Cfdp2. We propose a parsimonious scenario for
the creation of the three Bcnt-related genes in a process comprising 5 steps as shown in Fig. 5
(Iwashita et al., 2009). Self-BLAST search of the 120-kb region from Tmem 170A to exon 5 of
Bcnt/Cfdp1 confirms the two-round duplication of this gene cluster. Furthermore,
homologous fragments of Tmem170A 3 UTR, which is located 6.8-kb distal to p97Bcnt-2,
distribute at the 3-region of both Bcnt/Cfdp1 and p97Bcnt/Cfdp2. These data support the
above scenario that resulted in the creation of the two paralogs, p97Bcnt/Cfdp2 and p97Bcnt-
2. Furthermore, both the processed pseudogene of Bcnt/Cfdp1 and a 900-bp fragment
encompassing the IR-II exon of p9Bcnt-2 map on bovine chromosome 26 (Iwashita et al.,
2009). It is interesting to examine the relationship between the retrotransposon-mediated
creation of novel genes and the occurrence of processed pseudogenes.

Fig. 5. A scenario for the creation of the two paragolous genes, p97Bcnt/Cfdp2 and p97Bcnt-2
A parsimonious scenario for the creation of the three Bcnt-related family genes includes 5
steps as follows: (1) partial gene duplication of the ancestral Bcnt/Cfdp1, leaving the Bcnt-C
region by segmental duplication; (2) insertion of a Bov-B LINE in intron 5 of one of the
duplicated copies, recruitment of the AP-endonuclease domain of the retrotransposon, and
generation of the ancestor of p97Bcnt/Cfdp2 or p97Bcnt-2; (3) segmental duplication of the IR
unit of ancestor p97Bcnt; (4) further gene duplication of the ancestor p97Bcnt to generate the
nascent p97Bcnt-2; and, finally, (5) segmental duplication of the IR unit of the nascent
p97Bcnt-2 to create p97Bcnt-2. Nucleotide regions corresponding to the acidic N-terminal
regions, IR units, Bcnt-C, and Tmem170A, are symbolically indicated by boxes colored grey,
orange, dark blue, and purple, respectively. Bov-B LINE has an AP-endonuclease domain
(in yellow) and reverse transcriptase domain (in green)

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4. Characteristics of three Bcnt-related gene products
4.1 Ancestral Bcnt protein with highly conserved C-terminal region (Bcnt-C)
It has been proposed that duplicated genes yield genetic redundancy, which should result in
either the acquisition of a gene with a novel function or the degeneration of one of the
duplicated genes. Two paralogs, p97Bcnt/Cfdp2 and p97Bcnt-2 genes, were created via a
process of tandem duplication followed by retrotransposon insertion. We expect that the
three Bcnt-related proteins may play a role in more refined cellular signaling in ruminants.
The vertebrate Bcnt/Cfdp1 protein includes a highly conserved 82-amino acid region at the
C-terminus, termed Bcnt-C, which is not present in either p97Bcnt/Cfdp2 or p97Bcnt2
(Iwashita et al., 2003; 2009) (Fig. 2). Bcnt-C, known as the BCNT superfamily, is found in
most eukaryotes, including yeast, and is classified into Pfam 07572 in the Pfam database
(http://www.ncbi.nlm.nih.gov/Structure/cdd/cddsrv.cgi?uid=pfam07572). Although the
functions of the BCNT family members remain mostly unclear, a vertebrate Bcnt/Cfdp1 was
recently identified as a centromere protein, CENP-29, in DT40 cells, a chicken B cell line
transformed by avian leukosis virus (Ohta et al., 2010). Furthermore, a yeast ortholog, Swc5
/YBR231C/AOR1, is a component of the chromatin-remodeling complex SWR1 in
Saccharomyces cerevisiae (budding yeast)(Wu et al., 2009). The SWR1 complex mediates the
ATP-dependent exchange of histone H2A for the H2A variant HZT1, and the Swc5 null
mutant shows phenotypes of decreased resistance to macromolecule synthesis inhibitors
such as hydroxyurea and cycloheximide, and increased heat sensitivity in budding yeast.
These data indicate that the yeast Bcnt ortholog is not essential for survival, but contributes
to maintaining physiological homeostasis at the transcriptional level.
Whereas Bcnt-C is highly conserved among almost all eukaryotes, the N-terminal regions are
less conserved. For example, the amino acids in Drosophila Bcnt (YETI) are ~50% identitical to
those of bovine Bcnt/Cfdp1 in the C-terminal region, while the N-terminal region shows only
~22 % identity. Thus, although YETI is reported to bind to microtubule-based motor kinesin-I
(Wisniewski, et al., 2003), a reevaluation is needed to confirm whether vertebrate Bcnt
functions in intracellular trafficking because its interaction is mediated via its N-terminal
region. One characteristic of the three Bcnt-related proteins is their different numbers of IR
units: Bcnt/Cfdp1, p97Bcnt/Cfdp2, and p97Bcnt2 have one, two, and three IR units,
respectively. Sequences homologous to the 40 amino acid IR unit are found in zebra fish (Danio
rerio) and nematodes, but not in yeast. These IR units comprise intrinsically disordered regions
that might present scaffolds for protein-protein interaction as described later.
4.2 Intrinsic disorder of the three Bcnt-related proteins and cellular localization
The three Bcnt-related proteins move more slowly in sodium dodecyl sulfate acrylamide gel
electrophoresis (SDS-PAGE) than expected, resulting in apparently higher molecular masses
than those calculated. For example, bovine brain Bcnt/Cfdp1 has 297 amino acids and a
calculated molecular mass of 33.3 kDa, but appears around 45 kDa in SDS-PAGE (Fig. 6).
The situation is exactly the same for both the p97Bcnt/Cfdp2 and p97Bcnt2 proteins, which
have calculated molecular masses of 66. 3 and 70.8 kDa, respectively (Iwashita et al., 2003,
2009). This might be caused by their physical properties in that the three Bcnt-related
proteins are intrinsically disordered proteins (IDPs). It has been shown that many
biologically active proteins lack a stable three-dimensional (3-D) structure; such proteins are
referred to as IDPs (Dunker et al., 2008). IDPs are common to the three domains of life, and,
especially in multicellular eukaryotic proteins, account for more than 70% of total proteins.
They are involved in the regulation of various signalings through protein-protein


391

Fig. 6. Unique mobility of the Bcnt/Cfdp1 and p97Bcnt/Cfdp2 proteins in SDS-PAGE
Extracts of bovine brain (1), rat brain (2) and MDBK cells, a bovine kidney epithelial cell line
(3) were separated in SDS polyacrylamide gels and subjected to immunoblotting with anti-
Bcnt-C peptide antibody in the presence (a) or absence (b) of antigen peptide at a final
concentration of 100 M, or with anti-p97Bcnt monoclonal antibodies (c). The two small
black arrows indicate Bcnt/Cfdp1 with an apparent molecular mass of 45 kDa appearing as
a doublet, probably due to phosphorylation (Iwashita et. al., 2003); the red large arrow
indicates Bcnt/Cfdp1 with an apparent molecular mass of 53 kDa as described below
interactions that are frequently triggered by posttranslational modifications within the
regions of intrinsic disorder (Dunker et al., 2008). IDPs may function as hub proteins via the
formation of complexes with cellular proteins, which are then modulated by protein
modifications such as phosphorylation, acetylation, ubiquitination, or degradation.
By computational prediction, Bcnt/Cfdp1, p97Bcnt/Cfdp2, and p97Bcnt2 are all suggested to
comprise intrinsically disordered regions, except for the core of RTE domains that correspond to
AP-endonuclease in the two paralogs (Fig. 7). This computational prediction is partially
supported by an NMR study of the 3-D structure of the N-terminal 40 amino acid residues of the
Bcnt-C region prepared in Escherichia coli using
15
N-labeled amino acids. The spectrum revealed a
lack of fixed tertiary structure (courtesy of Dr. T. Kohno). Furthermore, the Bcnt/Cfdp1 protein
forms a tight protein complex with cellular proteins in bovine placenta even in the presence of a
detergent, CHAPSO, when evaluated by gel filtration chromatography on Sephacryl S-300 HR
followed by western blotting. Both Bcnt/Cfdp1 and p97Bcnt/Cfdp2 are phosphoproteins that
are potentially phosphorylated on serine residues by casein kinase II in vitro (Iwashita et. al.,
1999). Recently, the two phosphorylated serine residues in human BCNT,
116
S in the N-terminal
region and
250
S in the C-terminal region, were identified by mass spectrometric analysis
(Dephoure et al., 2008). This phosphorylation is cell cycle independent. It should be noted that
these two phosphorylated serine residues reside in amino acid sequences WASF and WESF,
respectively, which implies a unique motif for specific phosphorylation. Phosphorylation on
these motifs might be expected to play a role in switching, such as switching the cation-
mediated protein-ligand interaction (Zacharias & Dougherty, 2002). These characteristics
suggest that the three Bcnt-related family members are hub-like molecules.

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Fig. 7. Characteristics of intrinsic disorder of three Bcnt-related proteins
The Bcnt/Cfdp1, p97Bcnt/Cfdp2, and p97Bcnt2 proteins are predicted to comprise
intrinsically disordered regions. Amino acid sequences of the three Bcnt-related proteins
were subjected to analysis by a soft server of DisProt (Sickmeier et al., 2007), and individual
profiles were obtained. The data for p97Bcnt/Cfdp2 are not shown, but are quite similar to
those for p97Bcnt2. Vertical axes indicate the disorder probability of each amino acid
residue, and the horizontal axes indicate the number of amino acid residues. Schematic
domain structures of Bcnt/Cfdp1 and p97Bcnt2 are shown for each profile for comparision.
Similar results were obtained using another program, Anchor (Mszros et al., 2009)
We have found that the Bcnt/Cfdp1 protein from MDBK cells, a bovine kidney epithelial
cell line (Madin & Darby, 1965; Iwashita et al., 1999), migrates at around 53 kDa in SDS-
PAGE, significantly bigger than the rat or bovine brain proteins (Fig. 6). The same shift is
observed in many other ruminant organs such as bovine placenta, testis and goat kidney,
but not in all rat organs. Although we have yet not determined the cause, clarification of this
anomaly could shed light on the role of Bcnt/Cfdp1, because the modification may be
related to Bcnt/Cfdp1 function. Whereas the~175 amino acid N-terminal regions of the three
Bcnt-related proteins are acidic as a whole, they contain several arginine/lysine-rich
elements, including a putative nuclear targeting motif of Arg-Lys-Arg-Lys (~61-64
th
).
Therefore we examined the cellular distribution of the three Bcnt-related proteins in MDBK
cells. The three were localized in both the cytosolic and nuclear fractions, and, in addition,
both p97Bcnt/Cfdp2 and p97Bcnt2 were found significantly in the chromatin fractions (Fig.
8). These results suggest that Bcnt family members have the potential to function as shuttle
molecules between the cytosol and nuclei. The nuclear localizations of p97Bcnt/Cfdp2 and
p97Bcnt2 are consistent with their protein structure domains; the two paralogs include AP-
endonuclease domains in the middle of the molecule as described in more detail below. On
the other hand, either the 45 kDa (all rat organs and bovine brain) or 53 kDa (MDBK cells)
Bcnt/Cfdp1 is scarcely found in the chromatin fraction, although chicken Bcnt/Cfdp1 has
been reported as a centromere protein in a transformed cell line (Ohta et al., 2010).
4.3 RTE domains of p97Bcnt/Cfdp2 and p97Bcnt2
AP-endonuclease is well known to function as an abasic endonuclease in the base excision
repair pathway. It possesses multiple enzymic activities as a 3'-5' DNA exonuclease,


393

Fig. 8. Subcellular distribution of the three Bcnt-related proteins in MDBK cells
Subcellular fractionation of cultured MDBK cells was carried out successively using a
Subcellular Protein Fractionation Kit from Pierce. Constant volume amounts of each fraction
were assessed by immunoblotting. A: anti-Bcnt-C peptide antibody (Iwashita et al., 2003), B:
anti-p97Bcnt monoclonal antibodies (Nobukuni et al., 1997) and C: anti-p97Bcnt2 peptide
antibody (Iwashita et al., 2009). The right panel shows the Coomassie Brilliant Blue staining
pattern. The subcellular fractions are identified at the top of the panels. The effectiveness of
cellular fractionation was evaluated by immunoblotting using three antigens; anti-p120GAP
(a marker for the cytosolic fraction, Kobayashi et al., 1993), anti-Topoisomerase II (a marker
for the nuclear fraction, Iwashita et al., 1999), and anti-actin (a marker for the cytoskeleton).
The data are consistent with previously reported results (not shown)
3'-phosphodiesterase, 3'-phosphatase, and RNase H (Barzilay et al., 1995). Many organisms
possess two functional AP-endonucleases, which are thought to be important for cell
viability. In contrast to non-vertebrate AP-endonuclease, vertebrate AP-endonuclease,
which has an extra 6 kDa N-terminal region of intrinsic disorder, plays a role not only in
repairing DNA damage, but also in regulating the redox state of various proteins that
modulate transcription factors such as AP-1 (Fos/Jun), NF-B, HIF-1, and p53 (Tell et al.,
2009; Busso et al., 2010); thus it is termed APE/Ref-1 (AP-endonuclease/Redox effector
factor 1). This is natural considering that DNA damage is one of the most vital stresses faced
by living organisms. The extra N-terminal region of human AP-endonuclease (APE1)
contains multiple arginine/lysine rich elements, and provides a scaffold for protein-protein
interaction for DNA repair proteins such as Pol B and XRCC, and transcription factors
including STAT3, YB-1, and nucleophosmin (NPM1) (Vascotto et al., 2009; Busso et. al.,
2010). Although we have not yet found evidence that p97Bcnt/Cfdp2 and p97Bcnt2 possess
any of these activities, they have several characteristics common to mammalian AP-
endonuclease with intrinsic disorder regions at both the N- and C-termini. Amino acid
sequences in a part of the RTE domains are well conserved in all ruminants so far examined
including Lesser Malay chevrotain (Iwashita et al., 2009). The central 239-amino acid region of
the RTE domain (termed the core RTE domain) corresponds exactly to
Endonuclease/Exonuclease/Phosphatase family members (http://www.ncbi.nlm.nih.gov/

Gene Duplication

394
cdd?term=Pfam03372). The amino acid sequences of p97Bcnt/Cfdp2 and p97Bcnt2 were
compared with those of three canonical AP-endonucleases: human APEX1, Archaeoglobus
Af_Exo, and Neiserria Nape (Fig. 9). Although the comparison revealed low overall identity
(~20%) in the core RTE domains, eight amino acid residues involved in catalytic activity and
at least 6 amino acids participating in substrate binding are conserved among the molecules.
Furthermore, their 3-D structures could be remodeled with high accuracy, revealing the
characteristics of Exo III or AP-endonuclease.

Fig. 9. Highly conserved amino acid residues of the core RTE domains critical for
AP-endonuclease activity
The amino acid sequences of the core RTE domains of p97BcntCfdp (241-483
th
) and
p97Bcnt2 (243-485
th
), APEX1human APE, 61-318
th
, Nape (Neisseria, 1-259
th
, Carpenter et
al., 2007) and Af_Exo (Archaeoglobus fulgidus, 1-257
th
, Schmiedel et al., 2009) were aligned by
the ClustalW2 program of EMBL-EBI. Residues critical for the catalytic activity of canonical
AP-endonucleases are shown in red bold, and amino acid substitutions in the core RTE
domains between p97Bcnt/Cfdp2 and p97Bcnt2 are indicated in blue bold
The 3-D structure of AP-endonuclease is evolutionarily well conserved and comprises two
domains, each containing six-stranded sheets decorated by helixes on the concave site
(Barzilay et al., 1995). The predicted 3-D structures of both RTE domains of p97Bcnt/Cfdp2
and p97Bcnt2 are quite similar to each other, and present possible DNA-binding sites
between -helix domains and opposite both the N-terminal and C-terminal regions of the
RTE domain. Next we superimposed the structure of p97Bcnt2 onto that of p97Bcnt/Cfdp2
to examine the structural relationship between the two domains (Fig. 10). Whereas the N-
terminal ~60-amino acid region is variable between the two, there are only 12 amino acid
differences in the 239-amino acid core RTE domain. It is noteworthy that 7 of the 12 different
residues are located in the neighborhood of the predicted active sites. It is characteristic that
the enzymatic properties of AP-endonuclease change significantly with subtle changes in
the neighborhood of the active cavity. For example, a single amino acid substitution restores


395
Neisserial AP-endonuclease activity from the exonuclease (Carpenter et al., 2007), and a
spontaneous substitution of Val to Gly in the C-terminal Archaeglobus AP-endonuclease,
which participates in forming an abasic DNA binding pocket, is accompanied by an increase
in non-specific endonuclease activity (Schmiedel et al., 2009). This is probably because AP-
endonuclease possesses multiple enzymatic activities as described above. Thus it could be
expected that p97Bcnt/Cfdp2 and p97Bcnt2 would have different enzymatic properties,
with each compensating for the function of the other.

Fig. 10. 3-D comparison of the two core RTE domains of p97Bcnt/Cfdp2 and p97Bcnt2
3-D structures of the two core RTE domains of p97Bcnt/Cfdp2 and p97Bcnt2 were
remodeled by the I-TASSER server (Roy et al., 2010), and p97Bcnt2 was superimposed on
the p97Bcnt axis fixing the main alpha chain. RMSD (root mean square deviation) of 243
amino acid residues was 0.68 . From the data of the top templates of 2jc5A (Carpenter et al,
2007) and 2voaA (Schmiedel et al., 2009), the catalytic sites and DNA binding sites in red, or
a loop involved in the targeting site in orange from a template of 2v0rA (Repanas et al.,
2007) were identified. Twelve amino acid substitutions between the two domains are shown
in yellow. These drawings were obtained using PyMOL. Analysis was carried out courtesy
of Dr. M. Tanio, National Institutes of Natural Sciences, Okazaki
To explore further whether the nucleotide substitutions in p97Bcnt2 reflect natural selection
in the two paralogs, we examined the d
N
(non-synonymous substitution per site)/d
S

(synonymous substitution per site) values for both the core RTE domain (243-483
th
amino
acids) and the remaining regions (177-242
th
and 484-500
th
amino acids). The d
N
/d
S
values are
0.029/0.160 in the core region and 0.166/0.414 in the other regions. Although there are more
non-synonymous and synonymous substitutions outside the core RTE domain of p97bcnt2,
the d
N
/d
S
values are < 1, suggesting no definite attribution to positive selection. On the other
hand, the d
N
/d
S
value in the core RTE domain is much lower than that of the other regions,
suggesting that selective constraints have been substantially strong in the core RTE domain
(Iwashita etal., 2009). These data suggest that the recruited RTE domains in both
p97Bcnt/Cfdp2 and p97Bcnt2 have played a crucial role in the duplicated novel genes.

Gene Duplication

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5. Perspectives
Living organisms have evolved so as to acquire various anti-stress systems in response not
only to exogenous stresses, but also to the intrinsic stresses faced by multicelluar organisms
for physiological homeostasis. DNA repair systems in all organisms, immune systems in
vertebrates, and the placental systems in mammals are some of the most fruitfully acquired
systems. Several lines of evidence have shown that the expression of various integrated
retrotransposons is induced by environmental stimuli, such as ultraviolet light, heat shock,
or macromolecule synthesis inhibitors (Liu et al., 1995; Morales et al., 2003; Hsler et. al.,
2007). Although the induction mechanism of expression has not been fully elucidated, these
stresses may enhance promoter activity (Morales et al., 2003) or release the suppressive state
of expression, resulting in the creation of new genetic materials. If the retrotransposon
induction process is combined with tandem gene duplication, it is a much more efficient
way to create a novel gene. Under such stressful conditions, novel genetic materials may
play a role in adaptation to new environments.
Among the three Bcnt-related proteins in ruminants, the two paralogs p97Bcnt/Cfdp2 and
p97Bcnt2 were generated by partial segmental duplication of the ancestral Bcnt/Cfdp1 gene,
followed by the insertion of an order-specific retrotransposon, resulting in the recruitment of
the AP-endonuclease domain of the retrotransposon. Based on the 3-D remodeled structures of
these recruited RTE domains and comparison of their protein sequences, they probably retain
AP-endonuclease activity. Mammalian AP-endonuclease plays a role not only in direct DNA
repair, but also in stimulating pathways for anti-stress activities. Although the latter activity
depends mostly on the N-terminal 6-kDa region (Tell et al., 2009; Busso et al., 2010) which is
dissimilar to those of p97Bcnt/Cfdp2 and p97Bcnt2, they contain intrinsically disordered
regions other than the core RTE domains in both the N-terminal and C-terminal regions,
including outside of the core RTE domains. These regions may serve as scaffolds for cellular
protein-protein interactions and create novel functions as chimeric genes. The two paralogs
distribute in both the cytosolic and nuclear fractions. The properties of intrinsically disordered
proteins other than the AP-endonuclease domain and wide cellular distribution are similar to
those of the APE1/Ref-1 molecule. Based on these considerations, we conclude that
p97Bcnt/Cfdp2 and p97Bcnt2 have recruited the AP-endonuclease domain of a
retrotransposon, which originally played an essential role in the integration of the
retrotransposon into the genome, and that the two paralogs may have utilized AP-
endonuclease activity to suppress cellular stress for survival. The cellular stress that might
have induced the retrotransposition of Bov-B LINEs would increase the probability that the
newly created genes would become fixed in a population. In addition, because these novel
genes have chimeric origins, the original regulatory network of the ancestral Bcnt/Cfdp1 gene
may also have been modified to some extent. Therefore, we hypothesize that the two novel
genes have become additional components of pre-existing regulatory networks for anti-stress
activities. Although this hypothesis cannot explain why molecules containing the AP-
endocnuclease domain, such as p97Bcnt/Cfdp2 and p97Bcnt2, are so rare despite the
advantage of being able to regulate cellular activity, it will be intriguing to examine the
functions of the three Bcnt-related proteins based on this working hypothesis.
6. Conclusion
The Bcnt/Cfdp1 gene comprises a unique gene family with three members in ruminants. The
two paralogs, p97Bcnt/cfdp2 and p97Bcnt-2, were created in ancient ruminants by a partial


397
duplication of the ancestral Bcnt/Cfdp1 gene followed by the insertion of an order-specific
retrotransposon, Bov-B LINE. This type of combined process provides great potential to
generate a novel gene because a novel function can be acquired under the guarantee of the
original gene function. The ancestral Bcnt/Cfdp1 protein contains a highly conserved C-
terminus of 82-amino acids (Bcnt-C) that is not present in either p97Bcnt/Cfdp2 or
p97Bcnt2. Bcnt-C is found in all eukaryotes where it is known as the BCNT superfamily. A
chicken Bcnt/Cfdp1 is a centromere protein while the yeast ortholog is a component of the
chromatin-remodeling complex, suggesting that the ancestral Bcnt/Cfdp1 protein plays a
role in the regulation of gene expression. The two paralogs, p97Bcnt/Cfdp2 and p97Bcnt2,
recruited an AP-endonuclease domain of the retrotransposon during their generation
process as a ~325 amino acid region (RTE domain) in the middle of the molecule. The three
Bcnt-related proteins distribute in both the cytosolic and nuclear fractions, and include
intrinsically disordered regions other than the core of RTE domains of the two paralogs. The
3-D structures of the core RTE domains can be remodeled as canonical AP-endonucleases
with identical catalytic amino acid residues. Although as yet there is no direct evidence for
it, the two paralogs probably retain AP-endonuclease activity. Because AP-endonuclease/
Redox effector factor 1 is one of the major regulators of cellular responses to various
stresses, we propose that the recruited AP-endonuclease domains, which may have emerged
in response to cellular stresses, may be utilized by the paralogs in cellular regulation.
Therefore, the three Bcnt-related family members provide a good opportunity to examine
dynamic changes in signaling networks that accompany novel genes.
7. Acknowledgements
We thank all our colleagues for their contributions to the study over the last 15 years,
especially to Drs. K. Hashimoto and S. Hattori for their indispensable help in the early
stages. We are grateful to Dr. T. Kohno for providing unpublished data, to Drs. H. Ohmori,
M. Tanio, S-Y. Song, K. Nakashima, S. Imajo-Ohmi, E. B. Kuettner, M.B. Gerstein, G. Tell, Y.
Miyata, and Y. Ohno-Iwashita, and to The I-TASSER Server Team for useful discussion, and
to Dr. D. Izumi for providing unpublished data. We are also grateful to Dr. Y. Nagai, the
former president of Mitsubishi Kagaku Institute Life Sciences, for continuous
encouragement, and to Dr. M. Dooley-Ohto for patient editing. During the preparation of
this article, one of the authors in northern Japan suffered the disasters of a major earthquake
and resulting tsunami, which led to the Fukushima nuclear plant accident. In this, we
recognize the power of nature, and realize the importance of observing it carefully and
describing it correctly.
8. References
Barzilay, G., Walker, L.J., Robson, C.N., & Hickson, I.D. 1995. Site-directed mutagenesis of
the human DNA repair enzyme HAP1: identification of residues important for AP
endonuclease and RNase H activity. Nucleic Acids Res., 23, pp. 1544-1550
Brosius, J. 2005. Echoes from the pastare we still in an RNP world? Cytogenet. Genome Res.,
110, pp. 8-24
Busso, C.S., Lake, M.W., & Izumi, T. 2010. Posttranslational modification of mammalian AP
endonuclease (APE1). Cell. Mol. Life Sci., 67, pp. 3609-3620

Gene Duplication

398
Carpenter, E.P., Corbett, A., Thomson, H., Adacha, J., Jensen, K., Bergeron, J., Kasampalidis,
I., Exley, R., Winterbotham, M., Tang, C., Baldwin, G.S., & Freemont, P. 2007. AP
endonuclease paralogues with distinct activities in DNA repair and bacterial
pathogenesis. EMBO J., 26, pp. 1363-1372
Dephoure, N., Zhou, C., Villn, J., Beausoleil, S.A., Bakalarski, C.E., Elledge, S.J., & Gygi, S.P.
2008. A quantitative atlas of mitotic phosphorylation. Proc. Natl. Acad. Sci. USA,
105, pp. 10762-10767
Diekwisch, T.G., Marches, F., Williams, A., & Luan, X. 1999. Cloning, gene expression, and
characterization of CP27, a novel gene in mouse embryogenesis. Gene, 235, pp.
19-30
Dosztnyi, Z., Mszros, B., & Simon, I. 2009, ANCHOR: web server for predicting protein
binding regions in disordered proteins. Bioinformatics. 25, pp. 2745-2746
Dunker, A.K., Oldfield, C.J., Meng, J., Romero, P., Yang, J.Y., Chen, J.W., Vacic, V.,
Obradovic, Z., & Uversky, V.N. 2008. The unfoldomics decade: an update on
intrinsically disordered proteins. BMC Genomics 9, Suppl 2:S1.
Fritz, G., & Kaina, B., 1999. Phosphorylation of the DNA repair protein APE/REF-1 by CKII
affects redox regulation of AP-1. Oncogene, 18, pp. 1033-1040
Gentles, A.J., Wakefield, M.J., Kohany, O., Gu, W., Batzer, M.A., Pollock, D.D., & Jurka, J.
2007. Evolutionary dynamics of transposable elements in the short-tailed opossum
Monodelphis domestica. Genome Res., 17, pp. 992-1004
Hastings, P. J.. Lupski, J. R., Rosenberg,S.M., & Ira,G. 2009. Mechanisms of change in gene
copy number. Nature Reviews. Genetics, 10, pp. 551-564
Iwashita, S., Nobukuni, T., Tanaka, S., Kobayashi, M., Iwanaga, T., Tamate, H.B., Masui, T.,
Takahashi, I., & Hashimoto, K. 1999. Partial nuclear localization of a bovine
phosphoprotein, BCNT, that includes a region derived from a LINE repetitive
sequence in Ruminantia. Biochim. Biophys. Acta, 1427, pp. 408-416
Iwashita, S., Osada, N., Itoh, T., Sezaki, M., Oshima, K., Hashimoto, E., Kitagawa-Arita, Y.,
Takahashi, I., Masui, T., Hashimoto, K., & Makalowski, W. 2003. A transposable
element-mediated gene divergence that directly produces a novel type bovine Bcnt
protein including the endonuclease domain of RTE-1. Mol. Biol. Evol., 20, pp.
1556-1563
Iwashita, S., Ueno, S., Nakashima, K., Song, S.-Y., Ohshima, K., Tanaka, K., Endo, H.,
Kimura, J., Kurohmaru, M., Fukuta, K., David, L., & Osada, N. 2006. A tandem
gene duplication followed by recruitment of a retrotransposon created the
paralogous bucentaur gene (bcnt
p97
) in the ancestral ruminant. Mol. Biol. Evol., 23,
pp. 798-806
Iwashita, S., & Song, S.-Y. 2008. RasGAPs: a crucial regulator of extracellular stimuli for
homeostasis of cellular functions. Mol. BioSyst., 4, pp. 213-222
Iwashita, S., Nakashima, K., Sasaki, M., Osada, N., & Song, S.-Y. 2009. Multiple duplication
of the bucentaur gene family, which recruits the APE-like domain of
retrotransposon: Identification of a novel homolog and distinct cellular expression.
Gene, 435, pp. 88-95
Jobse, C., Buntjer, J.B., Haagsma, N., Breukelman, H.J., Beintema, J.J., & Lenstra, J.A. 1995.
Evolution and recombination of bovine DNA repeats. J. Mol. Evol., 41, pp. 277-283
Kaessmann, H. 2010. Origins, evolution, and phenotypic impact of new genes. Genome Res.
20, pp. 1313-1326


399
Kobayashi, M., Hashimoto, N., Hoshino, M., Hattori, S., & Iwashita, S. 1993. Differential
contribution of Mr 120 kDa rasGTPase-activating protein and neurofibromatosis
type 1 gene product during the transition from growth phase to arrested state in
human fibroblasts accompanied by a unique rasGTPase-activating activity. FEBS
Lett. 327, pp. 177-182
Liu, W.M., Chu, W.M., Choudary, P.V., & Schmid, C.W. 1995. Cell stress and translational
inhibitors transiently increase the abundance of mammalian SINE transcripts.
Nucleic Acids Res., 23, pp. 1758-1765
Liu, G.E., Ventura, M., Cellamare, A., Chen, L., Cheng, Z., Zhu, B., Li, C., Song, J., & Eichler,
E.E. 2009. Analysis of recent segmental duplications in the bovine genome. BMC
Genomics., 10, pp.571
Makalowski, W. 2003. Not junk after all. Science, 300, pp. 1246-1247
Malik, H.S., & Eickbush, T.H. 1998. The RTE class of non-LTR retrotransposons is widely
distributed in animals and is the origin of many SINEs. Mol. Biol. Evol. 15, pp.
1123-1134
Madin, S.H., & Darby, N.B.Jr. 1958. Established kidney cell lines of normal adult bovine and,
ovine origin. Proc. Soc. Exp. Biol. Med. 98, pp. 574-576
Morales, J.F., Snow, E.T., & Murnane, J.P. 2003. Environmental factors affecting transcription
of the human L1 retrotransposon. II. Stressors. Mutagenesis, 18, pp. 151- 158
Nobukuni, T., Kobayashi, M., Omori, A., Ichinose, S., Iwanaga, T., Takahashi, I., Hashimoto,
K., Hattori, S., Kaibuchi, K., Miyata, Y., Masui, T., & Iwashita, S. 1997. An Alu-
linked repetitive sequence corresponding to 280 amino acids is expressed in a novel
bovine protein, but not in its human homologue. J. Biol. Chem., 272, pp. 2801-2807
Ohno, S. 1970. Evolution by gene duplication. Springer-Verlag, New York
Ohta, S., Bukowski-Wills, J.-C., Sanchez-Pulido, L., Alves, de Lima Alves, F., Wood, L.,
Chen, Z.A. Platani, M., Fischer, L., Hudson, D. F., Ponting, C.P., Fukagawa, T.,
Earnshaw, W. C., & Rappsilber, J. 2010. The protein composition of mitotic
chromosomes determined using multiclassifier combinatorial proteomics. Cell, 142,
pp. 810821
Rawn, S.M., & Cross, J.C. 2008. The evolution, regulation, and function of placenta-specific
genes. Annu. Rev. Cell Dev. Biol., 24, pp. 159-181
Repanas, K., Zingler, N., Layer, L.E., Schumann, G.G., Perrakis, A., & Weichenrieder, O.
2007. Determinants for DNA target structure selectivity of the human LINE-1
retrotransposon endonuclease. Nucleic Acids Res., 35, pp. 4914-4926
Roy, A., Kucukural, A., & Zhang, Y. 2010, I-TASSER: a unified platform for automated
protein structure and function prediction. Nat. Protoc., 5, pp. 725-738
Schmiedel, R., Kuettner, E.B., Keim, A., Strter, N., & Greiner-Stffele, T. 2009. Structure and
function of the abasic site specificity pocket of an AP endonuclease from
Archaeoglobus fulgidus. DNA Repair (Amst), 8, pp. 219-231
Sickmeier, M., Hamilton, J.A., LeGall, T., Vacic, V., Cortese, M.S., Tantos, A., Szabo, B,
Tompa, P., Chen, J., Uversky, V.N., Obradovic, Z., & Dunker, A.K. 2007. DisProt:
the Database of Disordered Proteins. Nucleic Acids Res., 35, D786-793
Sorek, R. 2007. The birth of new exons: Mechanisms and evolutionary consequences. RNA,
13, pp. 16031608

Gene Duplication

400
Szemraj, J., Plucienniczak, G., Jaworski, J., & Plucienniczak, A. 1995. Bovine Alu-like
sequences mediate transposition of a new site-specific retroelement. Gene, 152, pp.
261-264
Takahashi, I., Nobukuni, T., Ohmori, H., Kobayashi, M., Tanaka, S., Ohshima, K., Okada, N.,
Masui, T., Hashimoto, K., & Iwashita, S. 1998. Existence of a bovine LINE repetitive
insert that appears in the cDNA of bovine protein BCNT in ruminant, but not in
human, genomes. Gene, 211, pp. 387-394
Tell, G., Quadrifoglio, F., Tiribelli, C., & Kelley, M.R. 2009. The many functions of
APE1/Ref-1: not only a DNA repair enzyme. Antioxid. Redox Signal., 11, pp.
601-620
The Bovine Genome Sequencing and Analysis Consortium. 2009. The Genome sequence of
taurine cattle: a window to ruminant biology and evolution. Science, 324, pp.
522-528
Vascotto, C., Cesaratto, L., Zeef, L.A., Deganuto, M., D'Ambrosio, C., Scaloni, A., Romanello,
M., Damante, G., Taglialatela, G., Delneri, D., Kelley, M.R., Mitra, S., Quadrifoglio,
F., & Tell, G. 2009. Genome-wide analysis and proteomic studies reveal APE1/Ref-
1 multifunctional role in mammalian cells. Proteomics, 9, pp. 1058-1074
Wisniewski, T.P., Tanzi, C.L., & Gindhart, J.G. 2003. The Drosophila kinesin-I associated
protein YETI binds both kinesin subunits. Biol. Cell, 95, pp. 595-602
Wu, W.-H., Wu, C.-H., Ladurner, A., Mizuguchi, G., Wei, D., Xiao, H., Luk, E., Ranjan, A., &
Wu, C. 2009. N-terminus of Swr1 binds to histone H2AZ and provides a platform
for subunit assembly in the chromatin remodeling complex. J. Biol. Chem., 284, pp.
62006207
Zacharias, N., & Dougherty, D.A. 2002. Cation- interactions in ligand recognition and
catalysis. Trends Pharmacol. Sci., 23, pp. 281-287
Zhang, J. 2003. Evolution by gene duplication: an update. Trends Ecol. Evol., 18, pp. 292-298
Zhang, J., Dean, A.M., Brunet, F., & Long, M. 2004. Evolving protein functional diversity in
new genes of Drosophila. Proc. Natl. Acad. Sci. USA, 101, pp. 16246-16250
Zingler, N., Weichenrieder, O., & Schumann, G.G. 2005. APE-type non-LTR
retrotransposons: determinants involved in target site recognition. Cytogenet.
Genome Res., 110, pp. 250-268
Zupunski, V., Gubensek, F., & Kordis, D. 2001. Evolutionary dynamics and evolutionary
history in the RTE clade of non-LTR retrotransposons. Mol. Biol. Evol.,18, pp.
1849-1863

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GENE DUPLICATION

Edited by Felix Friedberg

occurs as duplicons (see Fig. 2A).

0 if position i conserved within each group g

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