Discovery of vestigial gene through classical and molecular genetics

Theo Tran
College of Natural Sciences and Mathematics
Department of Biology and Biochemistry
University of Houston

Semester Project Manuscript

Lab TA Instructor: Fahmi Mesmar

Abstract
Drosophila melanogaster are a species that can be heavily studied for genetics. Experiments in
the lab were executed to find and map an unknown gene and hypothesize what gene it could
be. Crosses performed in controlled environments allowed observations with little difficulty. Chi-

1
Square analysis are performed after scoring of the Drosophila melanogaster to show fitness of
the expected outcomes. These shows how genetic crosses are continued to prove theories of
classical and molecular genetics. To carry out these crosses, vials were made with virgin
females and males of the desired genotype. The vestigial gene was determined near the middle
of the semester. Further experiments were carried out to find its mode of inheritance through
classical genetics. The results show that it is recessive. Molecular genetics using
electrophoresis showed that the vestigial gene showed no band in the gel when compared sideby-side with wild-type DNA. Three-point crosses were carried out to demonstrate recombination
frequencies and helped determine the chromosomal map location of the vestigial gene.
Performing a Chi-Square test showed that results were skewed and was rejected. Since the
data was inconclusive, relying on the scoring of the progeny prevented from accurate
chromosomal mapping of the gene. Further inspection of vestigial shows an insertion of the 412transposon.
Introduction
Drosophila melanogaster has forever been utilized in history due to their advantageous
properties for scientific research. They have proved invaluable to the field of genetics to help
continue and further understanding of the inheritance of organisms. They are very suitable for
qualitative and quantitative environments, because of their abundance of progeny, short mating
and maturation period compared to other life forms. Drosophila melanogaster allow quick
studies that would normally take much longer, therefore increasing efficiency in how genes are
studied.
It takes approximately 10 days for D. melanogaster to become an adult, from the time
they hatch from their eggs into larva, to when they pupate. This makes Drosophila melanogaster
an ideal model organism for studies. Their short maturation period allows them to be studied
quickly, in a short amount of time. Temperature affects their maturation by elongating or
shortening their maturation period. This affects how long females stay virgins which are crucial

2
for any genetic crosses. In addition, they are very inexpensive to maintain and take up very little
space (2). Drosophila melanogaster are not large organisms compared to other organisms,
therefore, it allows them to be studied virtually anywhere, and in any condition. In a study
comparing human disease genes to Drosophila, 548 genes matched the 714 human disease
genes (4). This allows studies and experiments on an organism that closely matches humans,
therefore grants a better understanding of human disease without intrusiveness.
Mutations arise from Drosophila melanogaster, just as any other organism. As a result,
the mutations have been heavily studied in the field for better understanding of how genes
function. Not only are there mutations in each organism, but each mutation could have multiple
variations of them. Each group of lab partners are assigned a three-digit code of a mutant vial to
maintain and perform various crosses and experiments to determine the mutant gene. The gene
that will be focused on is the vestigial (vg) gene. Various testing allows identification of the gene
and how it operates. In this study, the vestigial gene is shown to have an insertion in the base,
which causes its mutation. Research shows that is a natural transposable element named
412(molecular organization of the vesgitial). The various experiments that were performed in the
lab, aided in identifying the vestigial gene and what causes this mutant expression.
Materials and Methods
Culturing: In the fly lab, Drosophila melanogaster are cultured specifically for the needs
of their survival, and to aid in helping students perform genetic crosses. Clear plastic vials and
cellulose acetate plugs are used in preparation for making food for the flies. Food from a jar, that
has a blue dye when wet, is measured with a plastic cup, and poured into the vial in which the
flies will be cultured. This food is flaky, and is used in combination with water to make a mushy
food, suitable for larvae and flies. From there, distilled water of about 8-10 mL is measured from
a separate vial that is specific for measuring the water to pour into the vial of food. After carefully
pouring the water into the vial, swirling the vial for 3-5 seconds will mix the food and ensure it is
evenly hydrated. If there are any droplets on the walls of the vial, firmly tapping the vial on the

3
table will eventually bring the droplets down. This will prevent the flies from either drowning or
becoming stuck after waking from the anaesthetizing; both will result in death of the fly. Adding
6-8 flakes of yeast to the vial will complete the food making process and help in prevention of
bacterial growth. Anaesthetizing flies require careful procedures. Using carbon dioxide (CO 2)
allows the flies to enter a sleep like state, rendering them unable to fly, or move about. Using a
gun-like device to anaesthetize the flies in the vial, they are then laid onto a pad that provides a
constant flow of CO2 to ensure the flies do not awaken from their immobile state. A brush is used
to manipulate the flies on the CO2 pad. Any flies that are not useful anymore are anaesthetized,
and deposited in a cup filled with mineral oil, preventing flies from ever recovering.
Crosses: Genetic crosses are performed in a timely manner. Beginning a cross require
female virgins of the certain phenotype. This makes a cross time sensitive, since virgins are
extracted before they are fecund and become inseminated from mating with any male flies that
are in the vial. Resetting, or clearing, the vial of any flies by transferring or killing, ensures that
any flies that pupate are relatively young. This depends on the time that they are pulled from the
vial. Flies that are cultured in a 25C environment have approximately 8 hours before females
become fecund. The time is nearly doubled when vials are placed in an 18C environment. After
virgins are collected, the cross can then proceed. About 5 virgins and 3 males should be placed
in a new vial and labelled to indicate the appropriate cross that is being performed. More flies
can be added, although, there should be more female virgins than males, as the females lay the
eggs required to progress the cross.
Scoring: When scoring phenotypes, it is important to be able to tell the difference in
minute details. Errors in scoring could alter the results, rendering the data useless, and making
the experiment unreliable. When the pupa start to get darker, the parental flies are either thrown
away or placed in a new vial to continue making progeny for scoring. Using a reference fly will
aid in scoring flies from crosses. In general, a wild-type fly is used as a reference fly. This allows
various phenotypes easier to differentiate. A main phenotype that would difficult to differentiate is

4
a wild-type eye, and a Plum eye. Another gene that is less difficult to score would be the Lobe
gene. Although some phenotypes are easily recognized in the vial, placing the flies onto the
CO2 pad will allow easier scoring since the flies are sedated.
Analysis: Scored flies are then used for analysis to make sure the data matches with
known ratios and outcomes of the crosses. If there is a mistake, then the data cannot be used
and must be rejected. Tallying the scored flies grants a superficial glance at the outcomes of the
progeny. If there are no females in the study of the gene of interest, it can be assumed that the
gene is sex-linked. Furthermore, if the gene does not show up at all in the cross, then it can be
predicted that the gene is recessive. Performing a Chi-Square analysis tests if the data fits the
hypothesis.
Polymerase

chain

reaction

and

electrophoresis:

In

PCR,

section

of

deoxyribonucleic acid (DNA) is amplified so that it can be further examined. The first step
involves extraction of the DNA. Since DNA is studied on a small-scale basis, micropipettes are
used to extract minute amounts of liquid. Homogeneous flies of wild-type and mutant are placed
in a small cone-shaped vial, and frozen over night to make sure they are ready for DNA
extraction. Grinding the flies and adding various solutions like Tris-HCl, ethylenediamine
tetraacetic acid (EDTA), sodium dodecyl sulfate (SDS), sodium hydroxide, and potassium
acetate are all used in extracting and isolating the DNA. The second step uses the PCR method
to amplify a small area of DNA. A PCR primer forward and reverse is used to isolate the part of
DNA of interest. Furthermore, Taq DNA polymerase and deoxynucleotide triphosphates (dNTPs)
are used to elongate the DNA, just as in DNA replication of the human body, but without the use
of any living organisms. With these components, the vial is placed in a machine that controls the
temperature. The PCR method requires various temperatures for amplification. High
temperatures are used to denature the DNA so that the PCR primers can place themselves onto
the DNA. This will then allow the Taq DNA polymerase to elongate and replicate the DNA

5
whenever it is in a lower temperature. Replicating part of the DNA strand and amplifying it will
help the understanding of the DNA of interest.
After the DNA is extracted and amplified, it is then subjected to electrophoresis. This will
move the DNA band to certain. There will be a purified and unpurified versions of the DNA to
show any differences in the band after electrophoresis. Purifying the DNA removes all of the
polymerase and dNTPs, thus purifying and isolating the DNA. After the DNA is subjected to the
purification process, it is then put in wells of agarose gel that has been prepared with the
ethidium bromide (EtBr), a substance that attaches to DNA and fluoresces under ultra-violet
(UV) light. The speed at which the DNA travels depends on the amount of agarose that is
used; .5% was used in this case. With this, DNA will be able to be seen under UV and the
thickness of the band after electrophoresis will show how concentrated the DNA is. There are 7
wells that will be loaded. A control ladder to compare to the other DNA to see an approximation
of how many base pairs there in the DNA. Second, both purified and unpurified DNA of the wildtype and mutant will be loaded. Lastly the positive control and negative control will be loaded so
that there is a reference to what a band with DNA and no DNA looks like.
BLAST Analysis: A primer forward and reverse were provided for Basic Local Alignment
Search Tool (BLAST) sequencing. This will then be used on a reputable web-site that has a
database of the known sequences of the certain organisms. The site will then search its
database to align the sequencing to the query given. If going to a more detailed view of the
sequence that was put under BLAST, more information is given to help interpret what is shown,
including possible insertions, and deletions, which ultimately lead to comprehending the mutant.
Using BLAST for a protein analysis through the website is available as well, and show if there is
any change in the protein sequences compared to the wild-type Drosophila melanogaster.
Results
At the beginning of the project the group was assigned to mutant number 979 of the
Drosophila melanogaster, which later identified as the vestigial gene. Differences in the vestigial

6
Drosophila melanogaster against the wild-typed were easily discernable. Comparing the two
vials of the mutant and wild-type D. melanogaster, it can be seen that in the vestigial vial the D.
melanogaster were not as active as the wild-type D. melanogaster. Only under closer inspection
can one start to speculate why the D. melanogaster were not as active. Looking under a
microscope, it is immediately noticeable that the wings are not normal. More specifically, the
wings are in a crushed manner. It is difficult to see the veins that are on the wings of the mutant,
because of the nature of the scrunched wings; little could be deduced from initial observations.
The wings seem to not have furthered in development during or after the pupa stage. After a
group member plucked the wing and took pictures of the mutant types wings, we could see that
the veins were thicker, but were not able to discern all of the veins. At first, the bristles were
noticed to be thicker, but upon further inspection proved that wild-type were the same. This
could be due to the environment that the flies were cultured in, like temperature. The abdomen
was then inspected for differences between the two fly cultures. During mutant types
maturation, the female bodies look similar to the wild-type. It is only noticeable in their adult
stage that a larger abdomen and were prominent. During this project, subcultures of the vestigial
flies were maintained. As a result, longer wings started appearing in the progeny of the
subculture. A study shows that heat affect the vestigial gene and could be a possibility in the
result of long wings. Another possibility of the longer vestigial wings could be the expressivity of
the gene. Figures 1 and 2 show the differences in the wing types.
First genetic cross: DC1
Setting up a cross, referenced as discriminant cross 1 (DC1), required mutant virgins and
marker flies. Marker flies were Curly, Plum; Dichaete, Stubble. The marker flies genes are
located on chromosome 2 and 3 respectively. This cross is to determine the mode of
inheritance. With the unknown mutant type, it is predicted that it is recessive. 100 progeny were
scored, but discriminant cross 2 was started when a trend of no unknown mutant were showing.

7
Progeny included four different classes, none that comprised of both genes from the same
chromosome appearing. The reason is that these genes are lethal, and that having a Curly,
Plum or Dichaete, Stubble fly would not be possible (Table 1). Performing a Chi-Square test will
show the fit of the data to the theoretical data (Table 2).
Second genetic cross: DC2
After performing DC1, the progeny is used for the second discriminant cross (DC2).
Since the progeny did not show any of the mutant type from DC1, it is hypothesized that the
mutant gene is recessive. Using one class of phenotypes from the progeny of DC1, DC2 is set
up with the males from DC1 and mutant virgins. In this case, Plum and Dichaete were used to
prevent error in future scoring due to the mutant being a wing mutation. 213 flies were scored
(Table 3). Unfortunately, 18 of the scored flies do not fit the expected classes. This is due to
error in scoring, and will not be used in the Chi-Square analysis.
Third genetic cross: MCC1 and MCC2
This cross is the mapping control cross 1 (MCC1). This cross helps with the next set of
crosses, mapping cross (MC1 and MC2). It will help assure that the crosses will be set up
correctly to determine the recombination frequencies. In the group assignments, if the mutant
types mode of inheritance was either recessive or dominant, then the group would use a sexlinked for their mapping control crosses. In this case, sex-linked recessive flies were used for
the cross. A fly stock of white (w), singed (sn), miniature (m) was provided. Females of the w,
sn, m were collected and crossed with male wild-type flies. This cross was not scored because it
is not informative on recombination frequencies.
The second mapping control cross (MCC2) consists of sibling mating since they are sexlinked recessive. A ratio of five females and three males were used in the cross. About ten vials
were made so that 1000 progeny could be scored. Table 5 shows the amount of flies that were
scored and will used to determine the recombination frequencies. Calculations of the
recombination frequencies between the white and singed, white and miniature, and singed
miniature, were 14.83%, 29.46%, and 17.95% respectively. These recombination frequency
show the distances in map units (m.u.) between each gene. When comparing this data to Figure

8
3 below and the recombination frequencies in Flybase, the results are relatively close ( ) . dfs
blah blah shows a chi-square table of the results and its chi-square value. A figure below ( figure)
shows the mapping of the genes shows how approximately how far each gene are from each
other. The chromosomal drawings (figure) shows the possible combinations of the MCC2 cross.
Fourth genetic cross: MC1 and MC2
Near the completion of scoring the MCC2 flies, the set of mapping crosses (MC1) was
started. Bristle (Bl), Lobe (L), Punch (Pu) were provided after a proposal of them were submitted
to the professor. Male Bl, L, Pu flies were used to cross with the female mutant virgins. Since
this cross is autosomal, virgin females from the progeny after mating are required to be
collected for the next cross.
The second mapping cross (MC2) requires the heterozygous female virgins from the
MC1 cross and male mutant virgins. From there, the progeny will be scored and used for ChiSquare analysis and mapping the genes on the chromosome. Figures blah blah will show them.
Agar analysis
After subjecting the amplified DNA through electrophoresis by loading each of the wells
with various samples, the agar gel was observed through a UV light. Comparing each sample of
the loaded wells to the control ladder will show an approximation of the size of the DNA. The
results show that each sample is around the 750bp band of the control ladder. Both the wildtype of purified and unpurified DNA were clearly seen, the purified DNA showing a thinner band.
The positive control showed the band approximately as thick as the wild-type unpurified band.
The mutant type DNA however, did not show a band in either the purified or unpurified portions
of the agar gel. A picture of the finished electrophoresis with the agar gel provides a visual of the
missing bands in the mutant type. ()
BLAST analysis
The site, FlyBase, will be used for the BLAST sequencing and amino acid sequencing as
well. Using the nucleotide sequence between the forward and reverse primer, it is then entered
into the FlyBase BLAST search, where it will search and match the sequence. The sequence
will show its best alignment against known nucleotide sequences in the database. Many results

9
could appear, although, the highest percentage that matched the original sequence was used.
Looking through at a first glance it is seen that the gene of interest is on chromosome 2. Further
investigation by looking through a link labelled, GBrowse shows that the sequence is within the
vestigial gene and that is a sequence on a good portion of the intron. (figure) Within the
sequence that was searched shows a transposable element named 412, was inserted into the
gene.(figure)
Further analysis of the sequence can be performed through an amino acid alignment
analysis. The same process of sequencing is used through BLAST, but another option to
convert the nucleotide bases into amino acids will be used. The results give a weaker result
than with the nucleotide sequencing analysis, but the data can still be seen with a match that is
related to the vestigial gene. There are two amino acids missing from the polypeptide when
compared to the polypeptide provided by the database. This polypeptide is known named, vgPA. A picture is shown below to show the sequence.(). Looking up the polypeptide shows that
it is closely related to a gene in humans that affects human height and muscle growth (sources )
Discussion
Probably causes of the mutation could be of gene is the short wing (sw). It is located on
the X-chromosome, recombination map: 1-64.0(1). This could be a cause because since we
have not seen progeny of our DC1 crosses and cannot determine if it is sex-linked. Another
cause could be of the vein gene (vn). It is on chromosome 3L and its recombination map is 316.2(3). Obvious signs of larger veins of the wing can make one infer that this could be a
possible mutation. Last possible gene of mutation is the wrinkled gene (W). This gene is located
on 3L, and its recombination map is unknown (2). Because of the lack of development in the
wing, it could cause wrinkled wings.
None of the hypothesized genes could possibly be the gene, because they are either on
chromosome 1 or 3. After using BLAST with the sequence, it can be shown that the mutant
gene is on chromosome 2 of D. melanogaster.

10
In the study of genetics, time is a big factor in how it affects experiments. For one,
Drosophila melanogaster females will be fecund around 8 hours after eclosion ( ). This puts a
time restraint on when to collect virgin females, and must be collected at a non-leisurely pace.
Placing the flies in a colder environment, however, will close to double the time their fecundity.
This allows a lot more time in between coming into the lab to collect flies. If they are not
collected within those time frames, they could have been inseminated, therefore jeopardizing
future crosses and skewing results when scoring the progeny. A lot of flies need to be scored to
prevent and skewing of the data, therefore, it will take a longer time to score the flies. This is
only a few vials have been made. Second, approximately ten days are allotted to scoring the
flies from one vial. After 10 days, a second generation of flies will arise from the mating of the F1
progeny, and can no longer be used to score ( ).Other limitations that could affect the results of
the experiment include the amount of the progeny scored. If there are not a large amount of
progeny scored, then one phenotype could show up more than the other, not as a result of
classical genetics, but by chance. A large amount should be scored when carrying out statistical
analysis, like the Chi-Square test.
The DC2 cross that was performed is a clear example. Only 213 flies were scored. This could
affect the frequency of the phenotypes that show at the beginning, because one phenotype
could show up more dominantly at the beginning possibly because that phenotype allows faster
eclosion than the rest. Although DC1 fit the Chi-Square analysis well, DC2 did not. (figure)
Reasons behind this could be due to low amounts of scoring, error in scoring, or possibly and
error in the cross itself.
Mutations in the vestigial vary, there are many that could be explained in this gene, but
the ones in focus is the one that were searched from the BLAST sequence. The 412-transposon
is approximately 7kilo basepairs (kbp) in length. This could explain the reason why there was no
band in the agar gel during electrophoresis. The primers that were provided for the mutant most

11
likely did not code for the insertion, therefore no amplification occurred, leaving the well with no
noticeable band compared to the wild-type band. The 412-transoposon is placed with the third
intron of the vestigial transcript. This is also location that was sequenced. The 412-transposon
element in the vg gene reduces the amount of transcript in the gene, which could explain why
there are very little of the transcript that is expressed. When looking at the nucleotide sequence
of the forward and reverse primer, there are spaces in dashes. This means there is a
substitution, and thus makes a different polypeptide()figure.
When comparing this polypeptide, vg-pa, to the Homo sapiens, a similar function in body
growth occurs. While the transcription factors for the vestigial gene for Drosophila melanogaster
deals with wing shape, a vestigial-like protein 2 (VGLL2), affects height in humans. Overall the
general basis for the vestigial gen, is that it harms development of organism.()cite
Research in this gene could help with developmental issues and correct the issue in
Drosophila melanogaster, which could then lead to helping with issues in humans. The overall
experiment in determining the vestigial gene during the semester helped in the understanding of
how genetics is studied under classical and molecular. It has proved over again that with
multiple experiments can show how strong evidence can be to prove a hypothesis.

12

Tables and Figures

Figure 2. The wild-type wing is

Class
1

DC1 Progeny
Phenotypes
Cy,D
21

13
2

Cy,Sb

27

Pm,D

24

Pm,Sb

28

Total:

100

Table 1: This represents the scored data from discriminant cross 1. 100 progeny were scored

Chi-Square Analysis: DC1

Class

Phenotypes Observed Expected

O-E

(O - E)2

(O - E)2/E

1
2
3
4

Cy,D
21
25
-4
16
.64
Cy,Sb
27
25
2
4
.16
Pm,D
24
25
-1
1
.04
Pm,Sb
28
25
3
9
.36
2
Total
100
100
X =1.2
Table 2: This analysis shows that it cannot be rejected since the chi-square value is less than
the critical value given 3 degrees of freedom.

Class
1

DC2 Progeny
Phenotypes
Pm,Sb
37

Sb,un

61

Pm

20

Un

77

Pm,un

Pm,Sb,un

Sb

WT

Total:

195

Table 3: Data of scored flies from discriminant cross 2. Errors in scoring occurred and resulted
in unexpected phenotypic classes.

14

Chi-

Class

Phenotypes Observed Expected

1
2
3
4

Pm,Sb
Sb,un
Pm
Un
Total
Square Analysis: DC2

37
61
20
77
195

49
49
49
49
196

O-E

(O - E)

(O - E) /E

-12
12
-29
28

144
144
841
784
2
X =39.041

2.939
2.939
17.163
16.000

Table 4: Given the Chi-Square value is higher than the critical value in the table of critical
values, we reject the hypothesis. With the alpha value of .05, 39.041 is much higher than 7.815
making this data unreliable. To correct this, a larger amount of progeny could be scored.
Recombination frequencies: MCC2
eye

hair

wing

eye-wing

hair-wing

397

sn

309

56

56

56

sn

79

79

79

112

112

112

sn

55

55

55

sn

11

11

11

1025

152

302

184

14.83%

29.46%

17.95%

parenta
l
+
sco

dco

total

eye-hair

rf=

15
Table 5. Parental, single-recombinant, and double-recombinant classes are shown. The
recombination frequencies are determined by adding the single recombinants against the other
gene and dividing by the total amount

Chi-

Class
1
2
3
4
5
6
7
8

Phenotypes Observed Expected

w, sn, m
wild-type
m
w, sn
w
sn, m
w, m
sn
Total
Square Analysis: MCC2

309
397
112
55
56
79
6
11
1025

1025

O-E

(O - E)

(O - E) /E

X2=

Table 6. After performing the Chi-Square test, it can be shown that the data cannot be rejected
and subsequent should follow without any reason to reject it.

16

Figure3. This drawing of the w, sn, m genes shows its location in relation
to each other. By calculating the recombination frequencies after scoring
progeny, an approximation can be made depending on how reliable the
scoring was using a Chi-Square test.

Figure 4. The drawing above shows the locations of the genes relative to
each other on chromosome 2 of Drosophila melanogaster. The known
mutant at this point, vestigial, is shown.

17

18

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isolation

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