lab bio2133, section b7 - amazon...
Post on 08-Apr-2020
2 Views
Preview:
TRANSCRIPT
An investigation of curly wings and eyeless mutations of
Drosophila melanogaster
By Anonymous
######
Say no to plagiarism ;)
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
top related