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Genetics Formal Lab Report.docx

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Colin Montpetit

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 2 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 Mutations Predicted ratio Observed Chi- DF P- H0a (♀ x ♂) (%) ratio (%) square valueb 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 50♀+:50♂+ 50M+50F+ 0.46 1 0.50 Accept recessive) P(EY) x P(+) 2 (sex link 50♀+:50♂EY 50M+:50F+ 12340572 1 0 Reject recessive) 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 25+:8EY:50 10858272 3 0 Reject Y CY:17EYCY P (EYCY) x P 1,2 (recessive 66EY:34 EYCY 66EY:34EY 0.24 3 0.66 Accept (EYCY) epistasis) CY F1 (EY) x P 1,2 (test 50EY:50EYCY 50EY:50EY 0.22 1 0.64 Accept (EYCY) cross) CY aThe 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 Mutations Genotype Phenotype Genotype Phenotype (♀ x ♂) P(CY) x P(+) 1 (lethality) CcEE x CCEE Curly wings CcEE, Curly wings and and wild CCEE wild F1 (CY) x F1 (CY) 1 (lethality) CcEE x CcEE Both have CCEE Wild type and curly wings CcEE curly wings P(+) x P(EY) 2 (sex link CCEE x CCee Wild type and CCEe Wild type recessive) eyeless P(EY) x P(+) 2 (reciprocal CCee x CCEE Eyeless and CCEe Wild type cross) wild type F1 (+) x F1 (+) 2 (autosomal) CCEe x CCEe Wild type CCEE, Wild type and CCEe, eyeless CCee P (EY) x P (CY) 1,2 (recessive CCee x CcEE Eyeless; curly CCEe, Wild type; curly epistasis) wings CcEe wings P (CYEY) x P 1,2 (further Ccee x Ccee Both are CCee, Eyeless; Curly (CYEY) proof) eyeless with Ccee wings and curly wings eyeless F1 (EY) x P 1,2 (test CCee x Ccee Eyeless; CCee, Eyeless; Curly (CYEY) cross) eyeless w/ Ccee wings and a_ curly wings eyeless 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 mutan
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