Genetic analysis of novel mutations on the ckcr and kdbr genes in Drosophila melanogaster

Zachary Bibb and Kirsten Pierce

Professor Jim Smart

BIOL 350A – Genetics

December 7, 2014

George Fox University

Abstract

The objective of this experiment was to identify mutations in Drosophila melanogaster labeled

D and U, and determine the inheritance patterns for both mutations. Drosophila has been

considered an ideal model organism due to a large amount of offspring being generated in a short

amount of time. In addition, the Drosophila genome is remarkably similar to the human

genome, making it a valuable model organism when studying diseases. In this experiment, the D

mutation was determined to cause small dents along the wings was named cookie­cutter and

determined to be on the ckcr gene. The U mutation was determined to result in shorter, bent

bristles in mutants, was termed kinked­bristles, and was determined to be located on the kdbr

gene. The results of the crosses made in this experiment led to inconclusive results. While

reciprocal crosses suggested that the cookie­cutter and kinked­bristle mutations were sex­linked

recessive, the F2 progeny for both strains (and the unexpected ratios of kinked­bristle F1s), did

not correspond to the expected ratios for this mode of inheritance. However, it is still likely that

these mutations are sex­linked recessive due to incomplete penetrance.

Introduction

The fruit fly Drosophila melanogaster is one of the most widely used genetic model organisms

and has been used for a variety of purposes, from complementation testing to comparison of

disease­associated gene sequences to studying the p53 tumor­suppressor gene (2). Numerous

studies have made it clear that the fruit fly is one of the most useful genetic model organisms due

to similarities between genes in its genome and crucial genes in the human genome. In fact, it

has been determined that Drosophila have the most similar genome to humans, compared to

other invertebrates Because of this, fruit flies have served as a valuable model organism for drug

testing (6). The use of D. melanogaster as a model organism has been crucial to understanding

the human genome and how genes are passed down from one generation to the next.

The first notable use of Drosophila as a model organism was by Thomas Hunt Morgan in the

early 1900s, when he demonstrated that the white gene for eye color was linked to the X

chromosome (7). Drosophila was the ideal choice of a model organism because hundreds of

progeny could be produced from one female, a new generation could be produced in

approximately 10 days, and the flies were relatively easy to take care of (4). One of Morgan’s

students, A. H. Sturtevant, experimented with D. melanogaster to show that genes are located

linearly along a chromosome (7). D. melanogaster has also been used in complementation

testing, specifically with regards to eye color in order to determine which eye color mutations are

located on the same genes (7).

More recently, fruit flies have also been used in a study on disease­associated gene sequences in

humans. It was found that 77% of the 929 genes analyzed had almost identical genes in

Drosophila, the majority of these counterparts being genes that affected the visual,

cardiovascular, auditory, skeletal, and endocrine systems. In addition, there were genes in which

a mutation caused neurological disorders as well as cancer (5). It has been hypothesized that

these genes can be extremely useful if studied in Drosophila since the processes involving these

genes are controlled in very similar ways between fruit flies and humans (5). Additionally, a

functional and structural homolog of the human p53 gene has been found in Drosophila, with

similarities in the secondary structures as well as the structures of the tetramerization domains of

their respective proteins. This gene was also seen to induce apoptosis (2).

The above experiments have demonstrated that experimenting with Drosophila as a model

organism can result in findings that have high levels of significance with regard to human health

and well­being. These experiments, as well as the short generation time and the propensity for

Drosophila to produce large numbers of offspring, led it to be the model organism of choice for

the conduction of mutation analysis. In this experiment, two Drosophila mutations were studied

to determine their effect on offspring phenotype as well as their inheritance patterns. These

Drosophila were in Group 4, and the mutations were labeled D and U. The first of these

mutations resulted in small chunks taken out of the wings of mutant flies, and was termed

cookie­cutter and determined to be on the ckcr gene. The second, termed kinked bristles, caused

slightly smaller, bent bristles in mutant flies, and was determined to be located on the kdbr gene.

Materials and Methods

Preparing the Drosophila culture medium

One full scoop of banana flakes was added to a plastic vial. An equal amount of water was then

added until all of the flakes were saturated, which was signified when they turned blue in color.

Once this had occurred, five grains of baker’s yeast were added on top to prevent contamination.

The vial was then plugged with a foam cork to prevent the flies from escaping, and the flies were

added five minutes later.

Checking for new hatches

When checking for parental and F1 progeny, the vials containing the flies were checked four

times a day at approximately six hour intervals. This was due to the fact that female Drosophila

are not sexually mature for the first eight hours of adulthood (1). Checking every six hours

reduced the risk of collecting non­virgin females, giving enough time to collect all the flies if a

large number of hatches occurred. The shifts were scheduled around 6:00AM, 12:00PM,

6:00PM, and 10:30 PM (due lab access only being allowed until 11:00PM). Once a substantial

amount of F2 progeny had begun to hatch for D and U strains of Drosophila, the checks were

reduced to twice a day, with shifts at 10:30 and 6:00. This routine continued until the

experiment’s completion.

Anesthetization of Drosophila specimens

Two different methods were used to anesthetize D. melanogaster, involving the use of carbon

dioxide (CO2) as well as ice when CO2 was not available. The first method involved lightly

tapping the tube to be anesthetized against a hard edge to prevent the flies from getting trapped

in the culture medium, and then inserting the nozzle of the CO2 gun into the vial without

removing the foam stopper. The trigger was squeezed until all of the flies were anesthetized, and

the vial was emptied onto either a pad connected to a CO2 tank or onto a clean Petri dish on top

of a deeper glass dish filled with ice to sort the flies. When there was no CO2 available, the vials

to be analyzed were put in a freezer for 3­4 minutes to sufficiently anesthetize the flies, and were

then emptied onto the Petri dish.

Sorting the Drosophila specimens

After the vial was emptied, the Drosophila specimens were analyzed under a dissecting

microscope (3x magnification). A small camel hair paintbrush was used to sort the fruit flies by

phenotype and sex. The traits used to distinguish sex were the presence of sex combs on the front

pair of legs in males and the shape of the posterior segment of the abdomen. This body part was

rounder in the males and more pointed in the females. Mutant phenotypes (kinked bristle or

cookie­cutter) were looked for as well. After sorting, the flies were brushed back into their

respective vials with a camel hairbrush.

Cleaning out the culture medium vials

When it was determined that a vial needed to be cleaned out, all live specimens were

anesthetized with CO2 (or frozen as previously described). The flies were then either dumped

into a bottle of ethanol to kill them or were transferred to a new vial. Cleaning out the vials was

done for three different reasons: there were no live specimens remaining, there was

contamination in the vial, or the specimens in the vial were no longer needed for experimental

purposes. Following this step, the culture medium was scraped out of the vial using a plastic

spatula. Any culture medium that could not be cleaned out using this method was rinsed out

using cold water. The clean vials were then put on a tube rack to dry.

Crossing the parental generations to produce F1 flies

A vial with culture medium was prepared, and 3­4 virgin female flies as well as 1­2 male flies

were anesthetized and transferred to the CO2 pad to keep them anesthetized. The specimens were

then brushed into the previously prepared vial and the vial was labeled with the date and

phenotypes of the male and female specimens. Two different types of crosses were made for this

experiment. One consisted of exclusively wild­type males crossed with mutant virgin female

flies. A reciprocal cross was also made; 3­4 wild­type females were crossed with 1­2 mutant

male flies. Both types of crosses maintained an approximate ratio of 3 female flies to 1 male fly.

Crossing the F1 generation to produce F2 flies

The vials containing F1 eggs were checked 4 times per day for F1 hatches. When it was observed

that the eggs containing F1 flies were at either the second or third instar stage of the fly life

cycle, the parental flies were transferred to a separate vial. This was done to prevent mating

between generations. When F1 flies were observed, a vial was prepared to hold the first hatches.

The newly hatched flies were then anesthetized, sorted by phenotype and sex, and then brushed

into the vial to breed with each other. When it was determined that a specific vial was getting too

full, a new one was prepared.

Results

cookie­cutter F1 progeny

To obtain F1 progeny, 2­3 females expressing the cookie­cutter mutation and 1­2 wild type

males were sorted into the same vial. After the cross was made, the vials were checked for F1

progeny four times a day at approximately six hour intervals. A total of 694 F1 progeny were

bred, with the results displayed in Table 1 below.

Table 1: cookie­cutter Drosophila F1 progeny

Sex and Phenotype Number of Progeny

Wild­type males 12

Wild­type females 287

ckcr males 302

ckcr females 53

Unknown males 14

Unknown females 26

T1 displays the different phenotypes present among male and female F1 progeny. The number

of F1’s expressing the various phenotypes are also recorded.

According to T1, the most common phenotype expressed by male Drosophila was the mutant

cookie­cutter phenotype (302). The most common phenotype expressed by female Drosophila

was wild type. (287). Other sex­phenotype combinations observed were: wild type males (12)

and ckcr females (53). Some specimens were categorized as “unknown” due to under developed

wings or their wings being too damaged to distinguish a phenotype upon inspection.

cookie­cutter F2 progeny

When the F1 progeny first hatched they were placed in a previously prepared vial. More F1s

were added to the vial as they hatched, and additional crosses were made in a new vial when

overcrowding seemed to occur in the first vial. F2’s were obtained by checking the F1 crosses

four times a day at approximately six hour intervals. The F2s were then separated in a similar

matter to the F1s. A total of 302 ckcr males were crossed with 287 wild­type females to

produce a total of 1210 F2 progeny.

Table 2: cookie­cutter F2 progeny

Sex and Phenotype Number of Progeny

Wild­type males 207

Wild­type females 228

ckcr males 307

ckcr females 343

Unknown males 71

Unknown females 54

T2 shows phenotypes present among the cookie­cutter F2 progeny and the numbers of male or

female Drosophila with each phenotype. Male and female flies with an unknown phenotype are

also shown.

According to T2, the most common phenotype within the F2 generation was the ckcr mutant

phenotype, with 307 males and 343 females exhibiting this phenotype. The remaining

phenotypes were wild type females (228) and wild type males (207). Male and female

Drosophila of unknown phenotype occurred at lower frequencies than known phenotypes (71

unknown males and 54 unknown females).

cookie­cutter reciprocal F1 progeny

The cookie­cutter reciprocal F1 progeny were bred in a similar manner as the cookie­cutter F1s.

About 2­3 females expressing wild type phenotype were crossed with 1­2 males expressing the

cookie­cutter mutation. More P generation male and female Drosophila expressing the

previously described phenotypes were also placed into the cross as they hatched, to produce

more reciprocal F1 progeny. A total of 301 cookie­cutter reciprocal F1 progeny were bred for

this experiment.

Table 3: cookie­cutter Drosophila reciprocal F1 progeny

Sex and Phenotype Number of Progeny

Wild­type males 133

Wild­type females 150

ckcr males 4

ckcr females 1

Low expressivity ckcr female 1

Unknown / under developed winged females 12

T3 indicates the different phenotypes expressed among the F1 progeny of the reciprocal cross,

and indicates whether males or female Drosophila expressed those phenotypes. Drosophila of

unknown phenotypes were also observed, due to under developed wings, wings being smashed

prior to inspection, or other reasons.

T3 illustrates that the most common phenotype expressed among both male and female

reciprocal F1 progeny was wild type, with 133 wild type males and 150 wild type females, with

the ckcr mutant phenotype also being expressed. Additional phenotypes that were expressed by

female reciprocal F1 progeny were the cookie­cutter phenotype, low expressivity cookie­cutter

phenotype, and unknown phenotypes due to reasons previously described.

cookie­cutter reciprocal F2 progeny

The cookie­cutter reciprocal F2 progeny were bred in a similar manner to the cookie­cutter F2

progeny. As the reciprocal F1s hatched, they were placed into a prepared vial with no other

Drosophila present, and their sex was recorded for future reference. The vials were then

checked four times a day at approximately 6 hour intervals. When reciprocal F2 progeny

hatched, they were separated into newly prepared empty vials and their sex was also recorded. A

total of 218 cookie­cutter reciprocal F2 progeny were bred, expressing wild type, ckcr (with

various levels of expressivity), as well as unknown phenotypes.

Table 4: cookie­cutter Drosophila reciprocal F2 progeny results

Sex and Phenotype Number of Progeny

Wild­type males 49

Wild­type females 93

ckcr males 36

ckcr females 18

Low expressivity males 1

Extremely low expressivity males 1

Unknown / under developed wings: males 10

unknown / under developed wings: females 10

T4 displays the phenotypes expressed among the reciprocal F2 progeny, as well as the number of

male and female Drosophila expressing that phenotype.

As indicated by T4, the most common phenotype expressed by male reciprocal F2 progeny was

wild type with a frequency of 49. The most common phenotype expressed by female reciprocal

F2 offspring was wild type at 93 progeny. Additional phenotypes expressed among male

reciprocal F2 progeny were the ckcr phenotype (36 progeny), low expressivity ckcr wings (1),

extremely low expressivity ckcr wings (1), and unknown / underdeveloped wings (10). Other

phenotypes that were expressed among females were ckcr females (18 progeny), unknown /

underdeveloped wings (10 progeny), and crumpled wings.

kinked bristle F1 progeny

When enough P generation flies had hatched, 2­3 females expressing the kinked bristle mutation

were crossed with 1­2 wild type males. As more P generation Drosophila hatched, they were

added to the cross and additional crosses were made when overcrowding started to occur in the

original cross. These crosses were checked four times a day at approximately 6 hour intervals.

When F1 progeny hatched, their sex was recorded and all F1 progeny were placed into a newly

prepared vial, making a kinked bristle F1 cross. 97 F1 males were crossed with 121 females to

produce 496 F1 progeny. These progeny expressed wild type, mutant kdbr, and unknown

phenotypes.

Table 5: kinked bristle Drosophila F1 progeny

Sex and Phenotype Number of Progeny

kdbr males 189

Wild­type females 305

kdbr females 1

Unknowns 1

T5 establishes which phenotypes were expressed among the kinked bristle F1 progeny, and

whether that phenotype was expressed by male or female Drosophila. The number of male or

female Drosophila expressing a particular phenotype is also recorded.

According to T5, the most common phenotype among male F1 progeny was the kinked bristle

mutation (189 male F1 progeny expressed), and the most common phenotype expressed among

female F1 progeny was wild type ( 305 female F1 progeny expressed). Other phenotypes present

were kdbr females (1), and a Drosophila specimen of unknown gender and phenotype.

kinked bristle F2 progeny

The kinked bristle F1 crosses that were made were used to produce the kinked bristle F2

progeny. The F1 crosses were checked 4 times a day at six hour intervals, and F2 progeny were

separated into a newly prepared vials upon hatching. Their sex was recorded but both sexes

were placed into the same vial. A total of 189 male F1s were crossed with 306 female F1s to

produce a total of 218 F2 progeny. These F2 progeny expressed wild type, kdbr, and unknown

phenotypes.

Table 6: kinked bristle Drosophila F2 progeny

Sex and Phenotype Number of Progeny

Wild­type males 64

Wild­type females 69

kdbr males 49

kdbr females 35

Unknown males 1

T6 displays the various phenotypes expressed by male and female F2 progeny from the kinked

bristle F1 cross. The number of offspring expressing each phenotype (by gender) is also

displayed.

According to T6 the most common phenotype expressed by the male F2 offspring and the female

F2 offspring from the kinked bristle F1 cross was wild type (64 male progeny, 69 female

progeny). The other phenotype expressed by male and female F2 offspring was the kinked

bristle mutation (49 male progeny and 35 female progeny). One male of unknown phenotype

was also observed.

kinked bristle reciprocal F1 progeny

Reciprocal F1 progeny for the kinked bristle mutation were produced by crossing 2­3 P

generation wild type females with 1­2 P generation kinked bristle males. As more P generation

Drosophila with these specific sex­phenotype combination hatched, they were added to the cross

and additional crosses were made when overcrowding occurred. These crosses were checked

four times a day in approximately six hour intervals. When F1s hatched they were placed into a

newly prepared vial and their sex recorded. A total 171 kinked bristle reciprocal F1 progeny

were bred during the experiment, and exhibiting wild type phenotypes, with the exception of one

kinked bristle male.

Table 7: kinked bristle Drosophila reciprocal F1 progeny

Sex and Phenotype Number of Progeny

Wild­type males 84

Wild­type females 86

kdbr males 1

T7 establishes the phenotypes that were expressed among the reciprocal F1 progeny, and also

displays which sex of Drosophila had that particular phenotype. The frequency of phenotypic

expression among male and female Drosophila is also displayed.

According to T7, the most common phenotype expressed by kinked bristle reciprocal F1s was

wild type, with only 1 male F1 expressing a mutant phenotype.

kinked bristle reciprocal F2 progeny

There were no reciprocal F2 progeny produced for the kinked bristle mutation during this

experiment, although kinked bristle F1 crosses were prepared by placing the reciprocal F1

progeny into a newly prepared vial as they hatched, and additional crosses were made as

overcrowding occurred.

Discussion

The original purpose of this experiment was to analyze two different mutations in Drosophila in

order to determine their inheritance patterns from the phenotypic ratios of the F1s and F2s. The

original hypothesis was that both mutations were sex­linked.

Analysis of the cookie­cutter F1 generation

Almost all observed hatches for the cookie­cutter F1 generation were either ckcr male or

wild­type female flies, strongly suggestive of the sex­linked recessive hypothesis. If

cookie­cutter was sex­linked dominant, then it can be expected that at least half of both the males

and females would display the mutant ckcr phenotype. However, this was not the case, as the

parental cross for this mutation yielded an almost perfect 1:1 ratio of wild­type females to ckcr

males, with well under half of the females (53 out of 340, or 15.6%) displaying the mutant ckcr

phenotype. It was then reasoned that cookie­cutter must be sex­linked recessive. A chi­square

analysis of the ckcr males and wild­type females using this hypothesis yielded a value of 0.38.

With one degree of freedom in this case, this value falls well within the range of acceptable p

values and so cannot be rejected.

This hypothesis is further supported by the results of the reciprocal cross. A sex­linked recessive

mutation would be expected to yield a 1:1 ratio of wild­type males to wild­type females. The F1

results of the reciprocal cross in this case almost perfectly conformed to this ratio. In this case, a

chi­square analysis yielded a value of 1.02. With one degree of freedom, this value yields a p

value within the acceptable range, further supporting the sex­linked recessive hypothesis.

Analysis of the cookie­cutter F2 generation

Since the results of the F1 generations for both the normal and reciprocal crosses supported the

hypothesis that cookie­cutter is sex­linked recessive, it is reasonable to expect equal ratios of all

four possible gender­phenotype combinations in the F2 generation for this mutation. However, it

was at this point in the experiment that results were obtained that did not support the sex­linked

recessive hypothesis. Crossing the F1 generation for this mutation yielded 307 ckcr males, 207

wild­type males, 343 ckcr females, and 228 wild­type females, or almost perfect 3:2 ratios

between mutants and wild­types for each sex. While these results appear to disprove the

sex­linked recessive hypothesis, with a chi­square value of 45.81, there may be an alternative

explanation of incomplete penetrance in the wild­type progeny. Since 1085 total offspring

having expected phenotypes were obtained, there should be approximately 271 offspring in each

phenotypic class. This would yield a penetrance of between 76.3% and 84.1%. This is not an

unreasonable expectation, since Drosophila mutations have previously been observed to display

incomplete penetrance, as seen in a study done by Lachance, et. al in 2013 (2).The original

hypothesis that the novel cookie­cutter mutation is sex­linked recessive can therefore be

maintained.

Analysis of the kinked bristles F1 generation

The F1 offspring for the kinked bristles mutation exhibited very different phenotypic ratios than

the cookie­cutter F1 offspring. 305 wild­type females along with 197 kdbr males were obtained,

in addition to a single kdbr female. This could be due to starting the kdbr female and wild type

male crosses later than originally planned due to difficulty in identifying the kinked bristle

mutation. Since females tend to hatch earlier than males, starting these crosses later could have

contributed to a larger proportion of wild­type females compared to kdbr males in the F1

generation. The original hypothesis was that this mutation was sex­linked recessive, since all the

female offspring (with one exception) were wild­type and all the males displayed the kdbr

phenotype. However, a chi­square analysis of the results obtained yields a value of 23.19, well

outside of the range for which the sex­linked recessive hypothesis can be accepted.

Analysis of the kinked bristles F2 generation

Crossing the F1 offspring for the kinked bristles mutation yielded a ratio of 4 wild­type males : 4

wild­type females : 3 kdbr males : 2 kdbr females, as shown in Table 6. While a chi­square

analysis of these data yields a value of 13.05, which would normally result in the rejection of a

hypothesis, an alternative explanation can be proposed. As was suggested with the cookie­cutter

mutation, it is likely that there is incomplete penetrance with both the F1 and F2 generations for

the kinked bristles mutation. If this is the case, then penetrance levels would be 78.3% for the F1

generation, and between 64.2% and 89.9% for the F2 generation.

The results of a reciprocal cross for this mutation also support the sex­linked recessive

hypothesis. If this hypothesis is correct, a reciprocal cross would be expected to yield a 1:1 ratio

of males to females, all wild­type. The reciprocal cross in this experiment yielded 86 wild­type

female and 84 wild­type male flies as well as 1 kdbr male fly. A chi­square analysis of these

offspring yields a value of 0.024; as such, the sex­linked recessive hypothesis is strongly

supported by the results of this reciprocal cross.

Conclusions

This experiment ultimately yielded inconclusive, yet intriguing results. Some of the offspring

results showed a near perfect conformation to the phenotypic ratios expected of a sex­linked

recessive mutation, while others yielded the expected phenotypes, but in unexpected ratios.

While a possible explanation for these results is incomplete penetrance, the approximate 9:7

phenotypic ratios of the F2 offspring could be suggestive of complementary gene action.

However, while the mechanism behind the unexpected ratios of the F2 offspring is unclear, it is

likely that the cookie­cutter and the kinked bristles mutations are both sex­linked recessive. A

sex­linked dominant mutation would cause at least half of the F1 females to display the mutant

phenotype, this number being dependent on whether or not the parent female is homozygous. In

this experiment, essentially all F1 females were wild­type. Additionally, a sex­linked dominant

mutation would result in 3 wild­type : 1 mutant fly among the males and 5 mutant : 3 wild­type

ratios among the F2 offspring. The results of both the F1 and F2 generation in this experiment

strongly suggest that neither the cookie­cutter nor the kinked bristles mutation is sex­linked

dominant.

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