Unit II

CHROMOSOMES STRUCTURE AND

ORGANISATION
Chromosomes in eukaryotes and prokaryotes are different

PROKARYOTES EUKARYOTES

Single chromosome plus plasmids Many chromosomes

Circular chromosome Linear chromosomes
Made only of DNA Made of chromatin, a nucleoprotein
(DNA coiled around histone
proteins)
Found in cytoplasm Found in a nucleus
Copies its chromosome and divides Copies chromosomes, then the cell
immediately afterwards grows, then goes through mitosis to
organise chromosomes in two equal
groups

© 2007 Paul Billiet ODWS

 Representative genome sizes

• Phage virus: 168 kb  65 nm phage head (~1,000 x

length)

• E. coli bacteria: 1,100 mm DNA  ~0.2 micron

space nucleoid region (5,500 x)

• Human cell: 7.5 feet of DNA  ~3 micron nucleus

(2.3 million times longer than the nucleus)
 Chromosome Characterization
• Chromosomes  linear with 2 telomeres and a constricted region called the centromere
• All human chromosomes have 2 arms -- a short arm and a long arm -- that are separated
from each other only by the centromere, the point at which the chromosome is attached to
the spindle during cell division. By international convention, the short arm is termed the
"p arm(" petit", "small" in French). while the long arm of the chromosome is termed the
"q arm.“ (next to p)

Centromere position
• Metacentric :

• Submetacentric :

• Acrocentric :

• Telocentric :
Chromosome Characterization cont..

• Chromosomes usually occur in pairs  homologous chromosomes 

gene sites called loci

Heterozygous locus

Homozygous locus

• Homologous chromosomes  not identical  on a molecular level

 Prokaryotic chromosome
• The bacterial chromosome must be tightly packed to fit into the small volume of the
bacterial cell. The contour length of the circular DNA molecule present in the
chromosome of the bacterium Escherichia coli is about 1500 µm. Because an E. coli cell
has a diameter of only 1 to 2 µm, the large DNA molecule must exist in a highly
condensed (folded or coiled) configuration.
• Compacting the DNA involves supercoiling, or further twisting the twisted chromosome.
• The chromosome's fifty or so DNA domains are held together by a scaffold of RNA and
protein, and the entire nucleoid is attached to the cell membrane.
• This membrane attachment aids in the segregation of the chromosomes after they
replicate in preparation for cell division.
• Bacteria lack the histone proteins that are found bound to DNA and that form a
nucleosomes of eukaryotic chromosomes.
Bacterial chromosome is normally supercoiled

(~ 40 kb)

Bacterial DNA released from supercoiling

Diagram of the structure of the functional state of the E. coli chromosome.
 Model for coiling activity of Topoisomerase II (Gyrase)
Upper jaws
DNA wraps around
DNA binds to the A subunits in a Upper jaws
the lower jaws. right-handed direction. clamp onto DNA.

DNA held in lower jaws is cut. DNA

Lower jaws DNA held in upper jaws is released and
passes downward through the
A subunits B subunits opening in the cut DNA (process
uses 2 ATP molecules).
(a) Molecular mechanism of DNA gyrase function

Circular Cut DNA is ligated back

2 negative
DNA DNA gyrase together, and the DNA is
supercoils
molecule 2 ATP released from DNA gyrase.

(b) Overview of DNA gyrase function

Model for genome organization

Genomes 3 (© Garland Science 2007)

 Chromosomes in eukaryotes
• Found in the nucleus
• Condensed and visible during cell division
• At the beginning of mitosis they can be seen to consist of two threads (sister
chromatids) joined by a centromere
• The sister chromatids are identical copies
• During mitosis the sister chromatids separate and are placed into two nuclei
Numbers of chromosomes

• Constant for each cell in the body (except sex cells

which only have half sets).
• Constant throughout the life of an individual (you
don’t lose or gain chromosomes)
• Constant for all members of a species
 Organism Chromosome
numbers
Human chromosomes
Human 46
Chimpanzee 48
House Mouse 40
Maize 20
Bread mold 14
Maize 20
Tomato 24

© 2007 Paul Billiet ODWS

 Identifying chromosomes

Chromosomes can be identified by:

• Their size
• Their shape (the position of the centromere)
NB Chromosomes are flexible
• Banding patterns produced by specific stains (Giemsa)

Chromosomes are analysed by organising them into a

KARYOTYPE

© 2007 Paul Billiet ODWS

 Development and chromosomes

• Differences in chromosomes are associated with difference in the way we grow.

• The karyotypes of males and females are not the same

• Females have two large X chromosomes

• Males have a large X and a small Y chromosome
• The X and the Y chromosomes are called sex chromosomes

• The sex chromosomes are placed at the end of the karyotype Unusual growth can be
associated with chromosome abnormalities

e.g. People who develop Down’s syndrome have trisomy 21

Female Male Down’s syndrome
 Chromosomal abnormalities

Trysomy-21  Down’s syndrome Trysomy-18  Edward’s syndrome

 The current model for progressive levels of DNA packing.
• Nucleosome  basic unit of DNA packing [formed from DNA wound around a protein
core that consists of 2 copies each of the 4 types of histone (H2A, H2B, H3, H4)].
• A 5th histone (H1) attaches near the bead when the chromatin undergoes the next level of
packing.
• 30 nm chromatin fiber  next level of packing; coil with 6 nucleosomes per turn.
• The 30 nm chromatin forms looped domains, which are attached to a nonhistone protein
scaffold (contains 20,000 – 100,000 base pairs).
• Looped domains attach to the inside of the nuclear envelope.

How histones influence folding in eukaryotic DNA.

• Histones  small proteins rich in basic amino acids that bind to DNA, forming
chromatin.
• Contain a high proportion of positively charged amino acids which bind tightly
to the negatively charged DNA.
Condensation of Chromosomes Nucleosome Structure
 Organization of prokaryotic and eukaryotic genomes.

Prokaryotic
• Usually circular
• Smaller
• Found in the nucleoid region
• Less elaborately structured and folded
Eukaryotic
• Complexed with a large amount of protein to form chromatin
• Highly extended and tangled during interphase Human metaphase chromosome showing the presence of 30-nm
chromatin fibers. Each chromatid contains one large, highly coiled
or folded 30-nm fiber.
• Found in the nucleus
Heterochromatin
• Chromatin that remains highly condensed during interphase and is NOT actively
transcribed
Euchromatin
• Chromatin that is less condensed during interphase and IS actively transcribed
• Becomes highly condensed during mitosis
Where satellite DNA is found and what role it may play in the cell.

• Satellite DNA  highly repetitive DNA consisting of short unusual

nucleotide sequences that are tandemly repeated 1000’s of times.
• It is found at the tips of chromosomes and the centromere.
• Its function is not known, perhaps it plays a structural role during
chromosome replication and separation .
Giant chromosomes
Giant Chromosomes :
• Chromosomes are generally decondensed during interphase, so that their structure is
difficult to discern.
• Notable exceptions are the specialized lampbrush chromosomes of vertebrate oocytes
and the polytene chromosomes of insect giant secretory cells.
• Studies of these two types of interphase chromosomes suggest that each long DNA
molecule in a chromosome is divided into a large number of discrete domains that are
folded differently (euchromatin and heterochromatin).
• In both lampbrush and polytene chromosomes the regions that are actively synthesizing
RNA are least condensed.
• about 10% of the DNA in interphase vertebrate cells is in a relatively uncondensed
conformation that correlates with DNA transcription in these regions.
• These chromosomes are very long and thick (upto 200 times their size during mitotic
metaphase in the case of Drosophila).
• Hence they are known as Giant chromosomes .
• Such "active chromatin" is biochemically distinct from the more condensed inactive
regions of chromatin
 Lamp brush chromosomes
• The amphibian oocyte (germ cells in the ovary) has
certain periods of very active RNA synthesis.
• During the diplotene stage, certain chromosomes
stretch out large loops of DNA, causing the
chromosome to resemble a lamp brush
• These chromosomes show RNA synthesis and form
unusual chromatin loops.
• loops consist of transcriptionally active DNA which
can synthesize large amount of mRNA, necessary
for the synthesis of yolk
• They are covered with newly transcribed RNA and
are packed in dense RNA– Protein complexes.
• Lampbrush chromosomes in are unusually large
compared with their mitotic chromosomes, about
400–800 μm long as opposed to at the most 15–20
μm during later stages of meiosis.
• These are visible under the light microscope as the
so-called lampbrush chromosomes.
Lampbrush chromosomes
• Lampbrush chromosomes are greatly extended chromosome bivalents during the diplotene
phase of meiosis in oocytes of certain amphibians.
• This stage can last for several months.
• A meiotic bivalent of two pairs of sister chromatids can be seen under the light microscope.
• They are held together at point of chiasma formation.
• The loops of a paired chromosome form mirror-image structures .
Lamp brush chromosomes

• Transcription occurs either along the whole loop or at parts of a loop.

• The chromatin loops of a chromosome are paired, mirror-image structures , each corresponds to the loop
of a sister chromatid.
• The chromomere at the base of the loops consists of dense chromatin of the two sister chromatids
• At the beginning of meiosis, when DNA replication is complete, the homologous pairs lie immediately
next to each other and form characteristic structure composed of four chromatids.
• Lampbrush chromosomes are distinguished by an especially high rate of RNA
• transcription.
 Polytene Chromosomes

• Many of the cells of certain fly larvae grow to an enormous size through multiple cycles of DNA synthesis
without cell division.
• In several types of secretory cells of fly larvae, all the homologous chromosome copies are held side by
side, creating a single gaint polytene chromosome.
• Polytene chromosome have a distinct pattern of chromosome banding readily visible under the light
microscope.

• These chromosomes, first observed in cells of insect salivary glands of Drosophila .

• Found in salivary glands and other tissues of flies
• Seen in the nucleus during interphase
• Show linear series of alternating band and dark bands, distinctive banding for each chromosome in a given
species
• A polytene chromosome results from ten cycles of replication without division into daughter
chromosomes.
• Thus, there are about 1024 identical chromatid strands, which lie strictly side by side.
• It contains five long and one short arm radiating from a central point called chromocentre formed by the
fusion of centromeres of all the eight chromosomes found in the cell.

• Of the 6 arms, the short arm represents the fused IV chromosome and the longest represents the fused
sex chromosomes.
• About 80% of the DNA in polytene chromosomes is located in bands and about 15% in interbands.

• The chromatin in the darkly stained band is condensed to a much greater degree than the chromatin in the
interbands.

• Intensely stained chromosomal segments correspond to a high degree of packing and are genetically
inactive (heterochromatin);

• less tightly packed segments stain less distinctly and correspond to segments with genetic activity
(euchromatin).
 Functional stages in polytene chromosomes

• Polytene chromosomes form structures that

correlate with the functional state
• During the larval development of drosophila, a
series of expansions (puffs) appear in temporal
stages in the polytene chromosomes.
• Chromosome puffs are decondensed, expanded
segments that represent active chromosomal
regions, i.e., regions that are being transcribed.
• The location and duration of the puffs reflect
different stages of larval development
• The incorporation of radioactively labeled RNA
has been used to demonstrate that RNA
synthesis, a sign of gene activity (transcription),
occurs in these regions
Linkage and Crossing over
 LINKAGE
Definition:

Genetic marker located on the same chromosome thus tend to remain together during sexual reproduction . That is
they do not exhibit independent assortment. Such genetic marker are said to be linked, and the phenomenon, or
transmission pattern, of linked genes is called Linkage.

• Genetic markers are said to be linked whenever over 50% of the gametes produced contain Parental combinations
of the markers and the less than 50% of the gametes contain recombinant combinations of the markers

• The effect of linkage were first evident in the results of a dihybrid cross in sweet peas that were reported by W.
Bateson and R.C. Punnett in 1906. However they did not interpret their results in terms of the behavior of genes
located on the same chromosome. T.H. Morgan was the first to relate linkage to the segregation of homologous
chromosomes and the occurrence of crossing over between homologous chromosomes during meiosis.
 ARRANGEMENT OF LINKED GENES
Coupling phase
The two dominant or recessive genes are coming from same
parent enter in to the same gamete and inherit together for
many generations called coupling phase linkage. In this case
two dominant genes located on one chromosome of
homologous pair and two recessive genes located in other
pair of homologous chromosome. This type of arrangement
is called cis arrangement.

Repulsion phase:
The dominant or recessive genes coming from two parent
tend to separate each other and enters in to the different
gametes called repulsion phase linkage. In this case one
dominant and recessive genes located on same chromosome
of homologous pair.This type of arrangement is called trans
arrangement.
 Example:
Sweet pea → Blue (B)>Red(b) Sweet pea → Blue (B)>Red(b)
Pollen shape → long type (L)>round type (l) Pollen shape → long type (L)>round type (l)
Repulsion phase:
Coupling phase: Blue round type X Red long
Blue long type X Red round The F1 resulted is Blue long type and then the test cross
resulted in 1:7:7:1 with more of parental type
The F1 resulted is Blue long type and then the test
cross resulted in 7:1:1:7 with more of parental type
FACTORS AFFECTING THE LINKAGE

• Distance between the genes:

Closely linked genes shows strong linkage while genes widely located shows deep linkage.
The factors like age, high temp, X-ray treatment decrease the strength of the linkage.
• Linkage group
The genes or loci on one chromosome comprise a linkage group. One linkage group corresponds to a
pair of homologous chromosomes. The number of linkage group corresponds to a number of pairs of
chromosomes in a species.
• Syntenic
The genes on the same chromosome whether they show linkage or not they are called as syntenic groups.
• Recombination:
Recombination is production of gene combination which are found in the parents. It is produced by:
(1) Assortment of Non-homologous chromosome.
(2) By crossing over between homologous chromosomes during meiosis.
Determining Linkage Distances
• By definition, one map unit is equal to one percent recombinant gametes or phenotypes
• In honor of Morgan, one map unit is also called one centimorgan (cM)
• To determine the distance between two genes, divide the number of recombinant gametes by the
total number of gametes
Coupling Data Repulsion Data

F1 Gamete Testcross F1 Gam Testcross

Gamete Type Gamete Type
Distribution ete Distribution
pr+ vg+ 1339 Parental pr+ vg+ 157 Recombinant
pr+ vg 151 Recombinant pr+ vg 965 Parental
pr vg+ 1067 Parental
pr vg+ 154 Recombinant
Recombinant
pr vg 1195 Parental pr vg 146

pr vg distance = (151 +154)/2839 = 10.7 m.u = 10.7 cM pr vg distance = (157 + 146)/2335 = 13.0 cM

•Remember these are estimates; the differences between the two estimates reflect random deviation
•Neither estimate is incorrect; repeated experimentation would give a more accurate estimate
CROSSING
Definition:
OVER
• It is a process by which the party of homologous chromosomes are interchanged and crossing over takes space
during prophase-1 of meiosis(tetrad stage).
• The cross-shaped structure in which the two of the four chromatid of homologous chromosomes were appear to
exchange the parts.it is detached in cytological studies of meiosis in many organisms. These cross-shaped structures
were first detected in amphibians by F-janssens and these structures are called chiasmate.
• Features of crossing over
 The loci of genes on a chromosome are arranged in a linear sequence.
 The two alleles of a gene in a heterozygote occupy corresponding positions in homologous chromosomes.
 Crossing over involves the breakage of each of two homologous chromosome and exchange of parts takes place.
 Chromosome with recombinant combinations of linked genes are formed by the occurance of crossing over in the
region between the two loci.
 The probability that crossing over will occur between the two loci increases with increasing distance b/w the two loci
on the chromosomes.
Experiments on crossing over

Stern’s experiment -Drosophila • Stern studies two X chromosomes the differed from the
normal X chromosome of Drosophila.
• One X chromosome had part of a Y Chromosome
attached to one end.
• The second X chromosome was shorter the Normal.
• Crosses were done to produce female flies heterozygous .
• Stern monitored crossing over between two genes, the
recessive carnation eye color (car) and the dominant bar-
shaped eye (B), on chromosomes with physical
peculiarities visible under a microscope.
• Whenever these genes recombined through crossing over,
the chromosomes recombined as well. Therefore, the
recombination of genes reflects a physical exchange of
chromosome arms.
• The “+” notation on the alleles refers to the wild-type
allele, the most common allele at a particular gene.
• Stern’s Experiment demonstrate that crossing over
involoves the interachnge of parts of homologous
chromosomes.
• The last bottom left cross indicates the occurrence of
crossing over between the car and B loci.
 Postreplication Tetrad Stage

• Each pair of synapsed homologues is called a tetrad, because it consists of four

chromatids.

• Crossing over refers to the ex- change of segments of chromosomes whereas

recombination implies the formation of new combinations of genes.

• Thus crossing over occurs in completely homozygous organisms, but recombination

can occur only in organisms that are heterozygous at two or more loci

• Neurospora crassa has been of particular importance in the study of crossing over.
Sexual and Asexual reproduction in Neurospora crassa Pattern Prediction in Neurospora crassa
Gene Mapping and Recombinant Frequency
• Gene mapping determines the order of genes and the relative distances
between them in map units

1 map unit = 1 cM (centimorgan)

• Gene mapping methods use recombination frequencies between alleles in

order to determine the relative distances between them.

• Recombination frequencies between genes are inversely proportional to

their distance apart

• Distance measurement: 1 map unit = 1 percent recombination (true for

short distances)
Recombinant Frequencies

• Genes with recombination frequencies less than 50 percent are on the same chromosome = linked

• Linkage group - a group of genes in a chromosome that tends to be inherited as a unit.

• Two genes that undergo independent assortment have recombination frequency of 50 percent and
are located on nonhomologous chromosomes or far apart on the same chromosome = unlinked
Two Factor cross and Three Factor
cross
• Linked genes can be mapped on a chromosome by
studying how often their alleles recombine.
• Crossing over during the prophase of the first
meiotic division has two observable outcomes:
1. Formation of chiasmata in late prophase.
2. Recombination between genes on opposite sides of
the crossover point.
• The second outcome can only be seen in the next
generation, when the genes on the recombinant
chromosomes are expressed.
CROSSING OVER AS A MEASURE OF GENETIC
DISTANCE
• The distance between two points on the genetic map
of a chromosome is the average number of
crossovers between them.
• Linkage maps are made quantitative by defining one map unit as the distance that yields 1 percent
recombination.
• Linkage map distances are determined by the frequencies of crossover or recombinant chromosomes.
• Each meiotic crossing over event yields two crossover chromosomes.
• If one assumes that the probability of a crossover occurring between two loci is directly proportional to the
distance between the two loci, that is.
Probability of crossover = K (distance)
Where, K is a proportionality constant, then one would predict that map distances would be additive.
Example, If loci P and Q are linked and are 8 map units apart (yield 8 percent recombinant gametes), and loci
P and R are linked and are 3 map units apart, then loci Q and R are also linked and are either (1) 5 map units
apart or (2) 11 map units apart. That is, additivity can be achieved only by the following two linkage
arrangements:
• Wild-type Drosophila females were mated to males
homozygous for two autosomal mutations—
vestigial (vg), which produces short wings, and
black (b), which produces a black body.
• All the F1 flies had long wings and gray bodies;
thus, the wild-type alleles (vg and b) are dominant.
• The F1 females were then testcrossed to vestigial,
black males, and the F2 progeny were sorted by
phenotype and counted
• simple analysis indicates that, on average, 18 out of
100 chromosomes recovered from meiosis had a
crossover between vg and b. Thus, vg and b are
separated by 18 units on the genetic map.
 Double crossover can occur in three different ways:

(i) Two strand Double crossovers :

(i)
Both crossover involve the same two
chromatids.
(ii) Three strand Double crossovers :
(ii)
The second crossover involves one of
the same two chromatids as the first
crossover plus one different
chromatid.
(iii) Four-strand double crossovers :
occur when the second crossover (iii)

involves the two chromatids not

involved in the first crossover
RECOMBINATION MAPPING WITH A THREE-POINT
TESTCROSS
• We can also use the recombination mapping procedure with data from testcrosses involving more than
two genes

• Wild-type Drosophila males to females homozygous for three recessive X-linked mutations—scute
(sc) bristles, echinus (ec) eyes, and crossveinless (cv) wings.

• They are then intercrossed the F1 progeny to produce F2 flies, which they classified and counted.

Determining the Gene Order

There are three possible gene orders:

1. sc—ec—cv
2. ec—sc—cv
3. ec—cv—sc
• In our data, the rare, double
crossover classes are
7 (sc ec cv) and 8 (sc ec cv),
each containing a single fly.
• Comparing these to parental classes
1 (sc ec cv) and 2 (sc ec cv).
• we see that the allele has been
switched with respect to scute and
crossveinless.
• Hence the gene must be located
between the other two. The correct
gene order is therefore (1) sc ec cv.
Calculating the Distances between Genes

• Having established the gene order, we can now determine • Thus, in every 100 chromosomes coming from meiosis in
the distances between adjacent genes. Again, the procedure the F1 females, 9.1 had a crossover between sc and ec.
is to compute the average number of crossovers in each The distance between these genes is therefore 9.1 map
chromosomal region. units (or, if you prefer, 9.1 centiMorgans).
• Consequently, ec and cv are 10.5 map units apart.
Combining the data for the two regions, we obtain the map • We can also obtain this estimate by directly calculating
sc —9.1— ec —10.5— cv Map distances computed in this the average number of crossovers between these genes:
way are additive. Non–crossover Single crossover Double crossover classes
classes classes 1 and 2 3, 4, 5, and 6 7 and 8 (0) 0.805 (1)
• Thus, we can estimate the distance between sc and cv by 0.195 (2) 0.0006 0.196
summing the lengths of the two map intervals between
them: 9.1 cM + 10.5 cM =19.6 cM
Interference and the Coefficient of Coincidence
• A three-point cross has an important advantage over a two-point cross: it allows the detection of double crossovers,
permitting us to determine if exchanges in adjacent regions are independent of each other.
• For example, does a crossover in the region between sc and ec (region I on the map of the X chromosome) occur
independently of a crossover in the region between ec and cv (region II)? Or does one crossover inhibit the occurrence
of another nearby?
• I on Bridges and Olbrycht’s map, the crossover frequency was (163 +130+ 1+ 1)/ 3248= 0.091, and in region II, it was
(192 +148+ 1+ 1)/3248 =0.105.
• If we assume independence, the expected frequency of double crossovers in the interval between sc and cv would
therefore be 0.091x 0.105 =0.0095. We can now compare this frequency with the observed frequency, which was
2/3248 = 0.0006
• One crossover inhibited the occurrence of another nearby, a phenomenon called interference.
• The extent of the interference is customarily measured by the coefficient of coincidence, c, which is the ratio of the
observed frequency of double crossovers to the expected frequency.
• The level of interference, symbolized I, is calculated as I =1 – c = 0.937.
Practice Questions
• Drosophila females heterozygous for three recessive X-linked markers, y ( yellow body), ct
(cut wings), and m (miniature wings), and their wild-type alleles were crossed to y ct m males.
• The following progeny were obtained:

(a)Which classes are parental types?

(b) Which classes represent double crossovers?
(c) Which gene is in the middle of the other two?
(d) What was the genotype of the heterozygous
females used in the cross? (Show the correct
linkage phase as well as the correct order of the
markers along the chromosome.
Singed bristles (sn), crossveinless wings (cv), and vermilion eye color (v) are due to
recessive mutant alleles of three X-linked genes in Drosophila melanogaster. When a
female heterozygous for each of the three genes was testcrossed with a singed,
crossveinless, vermilion male.

The following progeny were obtained:

What is the correct order of these three
genes on the X chromosome?
What are the genetic map distances
between sn and cv, sn and v, and cv and v?
What is the coeffi cient of coincidence?
Somatic Hybridization
• The technique of hybrid production through the fusion of somatic cells under in vitro
conditions, is called Somatic Hybridization
• Somatic Cell Hybridization involves the fusion of two different cells to produce a
hybrid which is termed as Heterokaryon
• The product of fusion was called Homokaryon if the two parental cells come from
the same species
Somatic cell hybridization are of two types :
• Spontaneous Hybridization is very rare
• Induced Hybridization is mediated by either chemically with Polyethylene
Glycol, which effects the cell membrane, or with inactivated virus for
example the Sendai Virus
• Hybridization can either be performed between two same species which is
termed as interspecific or it can also be performed between two different
species Intraspecific.
• Somatic cells of different types can be fused to obtain hybrid cells. Hybrid
cells are useful in a variety of ways, e.g., –
(i) for gene or chromosome mapping
(ii) production of monoclonal antibodies by producing hybridoma
• The ability to distinguish each human chromosome is required to perform somatic-cell
hybridization, in which human and mouse (or hamster) cells are fused in culture to form a
hybrid.
• The fusion is usually mediated chemically with polyethylene glycol, which affects cell
membranes; or with an inactivated virus, for example the Sendai virus, that is able to fuse to
more than one cell at the same time.
• When two cells fuse, their nuclei are at first separate, forming a heterokaryon, a cell with
nuclei from different sources.
• The commonly used fusion agents for mapping of chromosome are Sendai virus,
lysolecithin, liposomes and polyethylene glycol (PEG).
• The most widely used fusion agent is Sendai virus, which is inactivated by UV light or β-
propiolactone.
• The process of fusion by Sendai virus involves 3 stages :
The virus particles cause cell agglutination
Cytoplasmic bridges are formed between cells
Cytoplasmic bridges expand to form spherical fused cells
• To isolate pure population of Human.

• Mouse Heterokaryon a selection procedure is used that kills both the parental cells and
the homokaryons but allows the human – mouse hybrid cells to survive and grow.

• In the medium contains a drug aminopterin, which blocks the de novo purine and
pyrimidine biosynthetic pathways of cells.

• However , the presence of hypoxanthine and thymidine the cells can overcome the
block by synthesizing their purines and pyrimidines using salvage pathways.
• For cells to grow in HAT medium, two enzymes, hypoxanthine-guanine phosphoribosyl transferase
(HGPRT) and thymidine kinase (TK) must be functional.
• Mouse cell is deficient in TK (TK-) and a human cell is deficient in HGPRT (HGPRT-)
• Heterokaryons , has normal HGPRT gene derived from the mouse genome and TK from gene from
the human genome. Therefore only hybrid cell grows.