An investigation of curly wings and eyeless mutations of

Drosophila melanogaster

By Anonymous

######

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Lab BIO2133, Section B7

Lab Demonstrators:

Yulia Konarsk and Rebecca Rochman

April 2nd, 2013

Department of Biology

University of Ottawa

Abstract

The purpose of the experiment was to determine the genotype and conditions associated with the

eyeless and curly wings mutation in the Drosophila melanogaster. The experiment was

conducted using the simulation software FlyLab. Multiple crosses were conducted in order to

compare the obtained ratios to predicted ratios using chi-squared analysis. Among the significant

results were the modified ratios of 2curly:1wild in the offspring of first generation curly wing

flies cross and 3eyeless:1wild in the F2 of eyeless flies generation cross of eyeless flies. These

results lead to conclusions about curly wing condition being lethal and the autosomal recessive

state of eyeless mutation.

Introduction

The purpose of this experiment is to investigate the nature of 2 mutations in Drosophila m. fruit

fly by simulation of parental and first generation crosses with FlyLab. FlyLab automatically

assigns the correct genotype to the mutated flies and takes into account any special conditions.

Hypotheses: One of the mutations is sex linked, one mutation is lethal in the homozygous state,

the other mutation is autosomal recessive and is epistatic to the other.

Assumptions:

H1 sex linked mutation:

- the mutation is located on the X chromosome, otherwise all boys will be afflicted

- sex-linked mutation is not lethal, otherwise all males will die

H2 lethal mutation:

- the dead flies do not appear in the ratio and instead the total of 10000 flies is redistributed

between the living

- the lethal mutation is not sex linked, elsewise only live females would possess the mutation

H3 Epistatic mutation:

- the genes are located on the same chromosome, therefore one will mask the expression of

the other

- epistatic mutation is not lethal

H4 Autosomal recessive mutation:

- the wild type genotype is dominant for the mutation

- the alleles separate according to Mendels laws of segregation and independent assortment.

-The mutations are controlled by a single gene

Predictions:

H1: Crossing an afflicted male with a non afflicted female, all of the males will be wild type, and

the females will be 100% afflicted or non-afflicted, depending whether the mutation is

dominant or recessive respectively.

H2: Parental cross of 2 mutants will result in a 2mutant:1wild ratio.

H3: F1 cross of the 2 mutations will result in a ratio of 9:4:3 or 12:3:1, depending whether

epistasis is dominant or recessive

H4: A cross of a wild fly and a mutated fly will give all wild offspring and the 2nd

generation

will

have a 3wild:1mutated ratio in the population.

Background

Epistasis- expression of one gene pair masks or modifies the expression of another gene pair.

Usually occurs if the genes involved influence the same general phenotypic characteristic. (Klug)

Sex-linked: the mutation is located on a chromosomal locus of an X or Y chromosome. Since

males contain an XY pair, only one mutated allele is needed for the phenotypic expression

Lethal mutation: the presence of mutated alleles in the homozygous state causes premature

death of the organism. (Klug)

Autosomal mutation: mutation located on a non sex-chromosomal locus

Recessive mutation: mutation only expressed in the homozygous state.

Experimental design overview:

The study is conducted using Flylab, a software for simulation of 3 generations of flies

possessing the mutations for a total of 10000 offspring.

Materials and Methods

Flylab software was used as outlined in BIO2133 Genetics Lab Manual. (Droun)

Modifications:

At steps 4-5, the mutations for parental generation were the following in the

corresponding order: curly wings (CY) and wild type (+), eyeless (EY) and wild type (+), eyeless

(EY) and curly wings (CY).

At step 12 the following flies have been selected for crossing of the first generation offspring in

the corresponding order: curly wings (CY) and curly wings (CY), wild type (+) and wild type

(+), curly wings (CY) with curly wings (CY).

Results

Table 1: Predicted and observed ratios (%) of various parental and first generations crosses for

the 2 mutations: curly wings (CY-1) and eyeless (EY-2), obtained using Flylab. A chi-square test

has been conducted to test the difference between predicted and observed ratios for appropriate

degrees of freedom (DF).

Type of cross

(♀ x ♂) Mutations Predicted ratio

(%)

Observed

ratio (%)

Chi-

square

DF P-

valueb

H0a

P(CY) x P(+) 1 (lethality) 50CY:50+ 50CY:50+ 0.18 1 0.66 Accept

F1 (CY) x F1 (CY) 1 (lethality) 67CY:33+ 67CY:33+ 0.76 1 0.38 Accept

P(+) x P(EY) 2 (sex link

recessive)

50♀+:50♂+ 50M+50F+ 0.46 1 0.50 Accept

P(EY) x P(+) 2 (sex link

recessive)

50♀+:50♂EY 50M+:50F+

12340572 1 0 Reject

F1 (+) x F1 (+) 2 (autosomal) 75+:25EY 75+:25EY 0.77 1 0.38 Accept

P (EY) x P (CY) 1,2 (epistasis) 50+:50CY 50+:50CY 0.12 1 0.73 Accept

F1 (CY) x F1 (CY) 1,2 (epistasis) 56+:25EY:19C

Y

25+:8EY:50

CY:17EYCY

10858272 3 0 Reject

P (EYCY) x P

(EYCY)

1,2 (recessive

epistasis)

66EY:34 EYCY 66EY:34EY

CY

0.24 3 0.66 Accept

F1 (EY) x P

(EYCY)

1,2 (test

cross)

50EY:50EYCY 50EY:50EY

CY

0.22 1 0.64 Accept

a_The null hypothesis that states the differences between observed and expected ratios are due to

chances and not an external factor.

b_The P value is 0 when some of the phenotype weren’t present in the prediction.

Table 2: Genotypes and phenotypes of parents and their offspring for various crosses of

Drosohpila m. carrying eyeless (E-2) and curly wings (C-1) alleles, stimulated using Flylab.

Parents Offspring

Type of cross

(♀ x ♂)

Mutations Genotype Phenotype Genotype Phenotype

P(CY) x P(+) 1 (lethality) CcEE x CCEE Curly wings

and wild

CcEE,

CCEE

Curly wings and

wild

F1 (CY) x F1 (CY) 1 (lethality) CcEE x CcEE Both have

curly wings

CCEE

CcEE

Wild type and

curly wings

P(+) x P(EY) 2 (sex link

recessive)

CCEE x CCee Wild type and

eyeless

CCEe Wild type

P(EY) x P(+) 2 (reciprocal

cross)

CCee x CCEE Eyeless and

wild type

CCEe Wild type

F1 (+) x F1 (+) 2 (autosomal) CCEe x CCEe Wild type CCEE,

CCEe,

CCee

Wild type and

eyeless

P (EY) x P (CY) 1,2 (recessive

epistasis)

CCee x CcEE Eyeless; curly

wings

CCEe,

CcEe

Wild type; curly

wings

P (CYEY) x P

(CYEY)

1,2 (further

proof)

Ccee x Ccee Both are

eyeless with

curly wings

CCee,

Ccee

Eyeless; Curly

wings and

eyeless

F1 (EY) x P

(CYEY)

1,2 (test

cross)

CCee x Ccee Eyeless;

eyeless w/

curly wings

CCee,

Ccee

Eyeless; Curly

wings and

eyeless a_

Wild type flies (+) are all CCEE as they do not carry the mutations.

Results description

The second generation offspring from a cross between 2 curly winged flies resulted in a 2 curly :

1 wild phenotypic ratio. The cross of first generation 2 wild flies resulted in a ratio of 3 wild : 1

eyeless flies. The cross between 2 flies carrying individual mutations yields a ratio 1 wild to 1

curly. The cross between these curly first generation offspring gave an unusual ratio of 6:3:2:1.

Discussion:

A total of 9 crosses have been performed of flies with various phenotypes. The purpose of the

first cross was to test for lethality of the curly wings mutation. The crossing was of a curly wings

fly with a normal fly. If the mutation in question is indeed lethal, it will only be expressed in the

heterozygote state. If the organism has been assorted both alleles with the mutation, it will die

prematurely or soon after birth. This is due to the homozygous state not being able to produce

enough of a protein or enzyme vital for the survival of the organism. In that case, half of the

offspring will have curly wings and half will be normal, because the wild type will only provide

wild type alleles (C) while the mutant will have a 50:50 chance of giving the mutated allele (c) or

the wild type allele. That was indeed the observed ratio giving strong support for the

hypothesis. In the first generation cross, 2 offspring carrying the mutation have been selected.

The standard ratio for a heterozygote monohybrid cross is 1:2:1, however the observed was

only 1wild:2curly wings. This satisfies the hypothesis about lethality of the gene, because the

homozygote mutant offspring didn’t hatch or all died. One of the assumptions was that a lethal

gene cannot be located on a sex chromosome because none of the males would express the

mutation and die, instead of being present in equal proportions with female flies like they were.

From the first conclusion, the eyeless mutation is then predicted to be located on the X-

chromosome and is tested for sex linkage by a cross between a fly carrying the mutation and a

wild type fly. If the mutation is X-linked recessive mutant male fly only contains one mutated

allele given by the only X chromosome, thus there will be an equal proportion of wild male to

female flies. This ratio is observed and therefore a reciprocal cross is done for further evidence.

If the mutation is indeed recessive, the female eyeless fly will be a homozygote for the

condition so it will only give Xe alleles, making all boys eyeless and all girls normal. Instead the

same ratio of male to female wild offspring as the first cross is achieved, successfully disproving

the hypothesis that the mutation is located on an X-chromosome. Since this hypothesis failed

and the gene is determined to be autosomal, we move onto the next hypothesis that the gene

is recessive and not lethal like the first mutation. The result of the first cross and its reciprocal is

of strong for this hypothesis because the wild type allele masks the expression of mutant allele

and 100% of the offspring are normal, but all carry the mutation (Ee). A first generation cross of

these flies gave the predicted 3:1 ratio because the only a quarter of the flies carry both

recessive alleles (ee) by mendels laws of segregation and independent assortment. These

findings prove the hypothesis right and the eyeless mutation is autosomal recessive.

Finally we must test our last hypothesis that there is an epistatic relationship between

the 2 mutations. Since the eyeless condition was determined to be recessive, the only type of

epistasis that can occur is also recessive, where the eyeless mutation (ee), masks the expression

of the curly wings condition. Realistically, since we already know that the curly wings condition

is lethal, the ability to express a parental fly containing both mutations suggests theres no

epistasis, because the phenotypic expression of no eyes would mask the expression of curly

wings phenotype, or the other way around in case of dominant epistasis. However a cross of

the 2 mutations is conducted anyway, for further proof. Since we know we are crossing CCee

with CcEE, we get the predicted ratio of equal amounts of wild and curly winged flies. From the

dihybrid cross of the 2 curly wing fly carrying both mutations (CcEe), we predict the 9:4:3 ratio

in the case of recessive epistasis, where instead of a fly expressing both mutations, the fly

would only be eyeless, if curly wing mutation is hypostatic. However instead we obtain a ratio

of 3:1:6:2. This ratio is supportive of our findings since its out of 12, meaning the quarter of flies

possessing homozygous curly wings alleles have all died. From the actual cross conducted on

the next page, we can see the 1/16th

offspring with just the eyeless mutation, from the eeCC

genotype due to the recessives of the gene.

In order to fully prove the observed findings, a cross of 2 flies carrying both mutations

was conducted resulted in another 2:1 ratio characteristic to a homozygote lethal gene. Since the

flies are both carrying autosomal recessive eyeless allele pair, all of the offspring are also

missing eyes. The chance of segregating C or c allele of curly wings is 50:50, however since the

condition is heterozygous, there are more eyeless curly winged flies than just eyeless. A

testcross between one of the parents with both conditions (Ccee) and an eyeless offspring (CCee)

again results in only eyeless and eyeless with curling wings flies, however this time in an equal

ratio because there is no way a lethal state can be achieved due to the wild C alleles of the F1

offspring.

According to a study on curly wings conducted in 1923, the mutation maintains itself in

a dominant heterozygote condition of a lethal stock and not sex linked (Ward). A more recent

study of 2010 suggests a reason for the lethality of homozygous gene. The mutation itself is a

result of 102 base pair deletion at the syt-daw genetic linkage. Among these base pairs are the

TAAT and ATTA regions – transcription binding sites coding for synthesis of homeoproetins,

which have a major role in developmental processes of most multicellular organisms (Hongwei).

Another study of 1921 determined that the eyeless mutation is indeed an autosomal recessive

mutation, and segregates freely by Mendels laws. One source of error is that in very rare cases

the homozygous individuals can survive as very curly but fertile flies (Ward). However one very

curly fly would not make a significant impact on the ratio of 10000 flies.

Another study of 1921 determined that the eyeless mutation is indeed an

autosomal recessive mutation, and segregates freely by Mendels laws (Hyde). A different article

states that the mutation is due to the presence of the ey gene containing transcription factors for

paired domain and homeodomain (Halder). Ey gene functions in eye morphogenesis and absence

of it leads to absence of compound eyes. In the heterozygote state, the threshold for the domains

ismet, therefore no phenotypic expression is observed.

One important source of error is that since the crosses were obtained using a simulation,

some of the real life external factors aren’t taken into account. Natural selection is one factor that

would affect real life ratios of large fly populations. Eyeless flies are much less likely to survive

due to the absence of their compound eyes, their field of view is limited by their simple eyes

(Halder).This applies to the curly flies as well, because it affects flight of the organism and could

be a detrimental risk for escaping from predators. Another external factor which has an effect on

mating is temperature. Flies kept at a reduced temperature had eyes just like the wild type flies

(Hyde). This suggest that temperature and moisture has an impact on the expression of speck

eyes and reduced temperature promotes larger speck eyes instead of their complete absence.

A temperature of 25-30 degrees is optimal for mating flies and absence of compound

eyes (Hyde), therefore a real life experience suggestion of improvement would be optimal

temperature regulation. Since access to flies and necessary laboratory equipment is unavailable,

the experiment can be repeated in another stimulation software, in order to verify the results.

More chi square tests can be conducted for verification as well as more crosses. We should also

consider the possibility of the phenotype being controlled by multiple gene interactions.

In conclusion, only 2 out of 4 hypotheses turned out correct. The curly wing mutation

was determined to be lethal in the homozygous state, while the eyeless mutation was disproved

to be sex-linked, but instead autosomal recessive. Finally, neither condition was found to be

epistatic to the other.

References

1Droun, G. et al, BIO2133 Genetics Lab Manual, Ottawa, 2013, p. 10-12

2Halder, G. et al, W. Induction of Ectopic Eyes by

Targeted Expression of the eyeless Gene in Drosophila, Basel, 1995, p1788

3Hongwei L. et al, A Deletion is Associated with Cy Mutant Chromosome in Drosophila

anogaster. Asian Journal of Animal and Veterinary Advances, 6, 2011, p391-396.

4Hyde, R. An eyeless mutant in Drosophila hydei, Baltimore, 1921, p323

5Klug, W. et al, Concepts of Genetics 9

th Ed., San Fransico, 2009, p79

6Ward, L. The genetics of curly wing in Drosophila. Another case of balanced lethal factors,

Sioux City, 1923, p276,283.

Appendix

♀CCee /♂ CcEE CE cE

Ce CCEe (wild phenotype) CcEe (curly wings)

Figure 1: A simplified punnet square of the parental generation used to test for epistasis, between

an eyeless fly and a fly with curly wings. The reduced ratio is 1:1 of normal and curly flies.

♀CcEe /♂ CcEe CE Ce cE ce

CE CCEE CCEe CcEE CcEe

Ce CCEe CCee CcEe Ccee

cE CcEE CcEe ccEE ccEe

ce CcEe Ccee ccEe ccee

Figure 2: A punnet square of curly wings offspring cross from Figure 1. Results in a 6:3:2:1

ratio, where green is curly wings, yellow is wild type, red is eyeless with curly wings and cyan is

eyeless. Crossed out are the dead flies.

Table 3: Calculation of chi-squared value for parental cross of Cc x CC in testing for lethality.

Phenotype + CY Total

Observed value 5048 5006 10054

Predicted value 5027 5027 10054

Obs – Exp 21 -21 -

(Obs – Exp)2

441 441 -

(Obs – Exp)2

/ Exp 0.088 0.088 0.18

Table 4: Calculation of chi-squared value for first generation cross of Cc x Cc in testing for

lethality.

Phenotype + Cy Total

Observed value 3285 6691 9998

Predicted value 3332 6665 9997

Obs – Exp -47 26 -

(Obs – Exp)2

2209 676 -

(Obs – Exp)2

/ Exp 0.66 0.10 0.76