Drosophila Lab Report

DROSOPHILA LAB REPORT INTRODUCTION: The Drosophila Melanogaster is a fruit fly that serves as a model organism that is commonly used in genetics to study mutations in genes passed from one generation to the next. Geneticists experiment with the fruit fly because the Drosophila are easy to breed, have simple genetic analysis that can be easily distinguished, produce a large amount of offspring, and have short life cycles. The four distinct stages in the life cycle of a Drosophila Melanogaster fruit fly include an egg, larvae, pupa and adult. It takes about 10 days for a fresh culture to become adults. The egg to larvae time period is only about a day. The larvae then grow while feeding on yeast cells and is at full size on the 7th day. It is then considered a pupa and remains in the pupa stage for 2 days. After the pupa stage it becomes an adult fruit fly. These flies lay many eggs at a time that grow extremely fast. They are small enough to keep the large amount needed for a good number of data but large enough to observe with the naked eye. Drosophila also has a low chromosome number of four. Three of these are autosomal and one is sex-linked. The purpose of this experiment is to help us have a better understanding of Mendel’s work. Mendel, the founder of genetics created two laws, which were Law of Segregation and the Law of independent assortment. Through this experiment we were able grasp the idea of Mendel’s work by setting up monohybrid crosses, sex-linked crosses, and 3- point mapping crosses. We performed these crosses to determine how our offspring will turn out so we can compare to the expected outcomes. In genetics different mutations and traits are studied so through these crosses we can determine how these mutations or traits will be passed on to offspring. A monohybrid cross is done to reveal how one trait or mutation is passed from one generation to the next. Another example is through dihybrid cross, we can determine how two traits or mutations are passed on to the offspring. Through a sex-linked cross we observe through the male and female fruit flies how a specific gene is carried on the X chromosome and how the inheritance patterns between the two sexes will be different. Distinguishing the sex of a fruit fly can be done very easily by observing key traits. The females are larger than the males and have an oval abdomen. The female fly’s tip is more sharp and pointy versus the male. The tip of the male fly is more round and contains a black color covered with hairy bristles. The male fruit fly also contains a round abdomen. In 3 point mapping three different genes are crossed with the wildtype. Through this cross we can figure out the gene distance and gene order of the fly’s genome. The normal wildtype Drosophila has red eyes, grey-brown bodies, and wings. The mutant Drosophila is classified by eye color, body shape, and wing shape. In our lab the mutants that our class experimented on was sephia, which have brown eyes, and apterous which are wingless flies. It takes several weeks for our experiment to come to an end because it is necessary to allow the fruit fly’s life cycles to occur before we could mate the flies. For example, for each cross we had to create virgin flies before we crossed the flies. It is important to start any cross with a virgin female fruit fly because female fruit flies hold on to sperm from all of their mating. We want to have an accurate cross with just our gene of interests so to create virgin flies we must isolate and remove all adult flies from the vial leaving just the larvae that are ready to hatch and become virgin fruit flies. These virgin flies will not have any previous stored genetic information so they will be well suited to cross and perform surely accurate results. PROCEDURE AND RESULTS: MONOHYBRID CROSS We began the monohybrid cross by choosing the phenotypes that each of the flies will be mating with. The phenotypes that our lab class was experimenting with were wildtype, apterous, and sephia. During this experiment it doesn’t matter whether the gene is on a female or male because it is not sex-linked. In our specific cross we crossed wildtype male and apterous female. Once the specific phenotype types were chosen we had to prepare virgin flies so that they could mate. We did this by anesitizing the flies through a chemical called Fly Nap and once the flies were asleep we removed all of the adults. We put the flies to sleep with the Fly Nap by using a small bristle and dipping it into the Fly Nap solution and into the vial. We left the bristle there for a minute until all of the flies were asleep. We then removed the foam stopper and took the flies out of the vial. Once we isolated the adults out of the vial we killed them. We then took the larvae out and put it into a new vial. This step is essential because it will create new wildtype virgin flies. We did the same procedure for the apterous flies. Once we had both true-breeding homozygous wildtype virgin flies and homozygous recessive apterous virgin flies we mated them. The following week we would examine the results under a microscope and determined if they were male or female. After examining the cross we separated and killed the parents and left the larvae in the vial in order to have an F2 generation. The following week we would observe our F2 results the same way we did for the F1 generation. MONOHYBRID CROSS-EXPECTED RESULTS P1 Ap x wt (+) The phenotypes that were used in the monohybrid cross were wildtype and apterous flies. Drosophila is classified by eye color, body shape, and wing shape. The wildtype fly has red eyes, grayish-brown bodies, and wings. The mutant apterous fly is wingless. F1 Ap Ap(+) Ap(+) + + ap Ap(+) Ap(+) The ratios we expected to see are the following Punnet square’s analysis Wild type is dominant so all should be wild type in this generation. Genotype: Heterozygous dominant Phenotype: Red eyes with wing F1 cross Ap(+) x Ap(+) Heterozygous dominant x Heterozygous dominant F2 Ap Ap ApAp + Ap+ The phenotypic ratio here is 3:1. Wildtype to Apterous + Ap+ ++ The genotypic ratio is 1:2:1. Homozygous recessive : Heterozygous dominant : Homozygous dominant In the F1 cross the parental wildtype flies are homozygous dominant and the parental apterous flies are homozygous recessive and the results of this cross are all heterozygous Ap(+). In this genotype, wildtype is dominant because all of our F1 generation were wildtype. When the heterozygous flies for Ap(+) cross the F2 generation will have a 3:1 ratio, which will have ¾ wildtype and ¼ apterous. The ¼ that is apterous will have the genotype of homozygous recessive for apterous. MONOHYBRID CROSS OBSERVED RESULTS P1 Ap x wt (+) 4 wild type male x 5 apterous female F1 All Wildtype Ap(+)  42 male and 20 female F2 69 wildtype and 20 apterous NULL HYPOTHESIS In order to confirm that our genetic cross represents a monohybrid cross and is similar to the expected we calculated our observed results with the expected results. CALCULATIONS Observed Expected (Observed - (Observed - (Observed – Expected) Expected)2 Wild Type 69 ¾ x 89 = 66.75 (69-66.75) = 2.25 (2.25)2 = 5.0625 Apterous 20 ¼ x 89 = 22.25 (20-22.25) = -2.25 (-2.25)2= 5.0625 Total 89 89 Expected)2 / (Expected) (5.0625) / 66.75 = .08 (5.0625) / 22.25 = .23 .31 Degrees of Freedom = n-1 = 2-1= 1 Critical value = 3.841 Test Statistic = .31 The test statistic is lower than the critical value so it fails to reject the null hypothesis and we can assertively come to a conclusion that there isn’t a difference between the observed and expected results. PROCEDURE AND RESULTS: SEX-LINKED CROSS Drosophila female flies are XX and male flies are XY. In sex linkage alleles are located on sex chromosomes and if a gene is carried on a sex chromosome the phenotypic ratios will depend particularly on the sex of the fly that is being crossed with that sex-linked gene. In the sex linkage cross we mated homozygous dominant wildtype male flies with homozygous recessive white flies. White is sex-linked and is located on the X-chromosome. Before we mated the flies we used Fly Nap just as we did for the monohybrid and put the flies asleep and killed them. We transferred each larva for wildtype and white genes into a new vial in order to make virgin flies just as we did in the monohybrid cross. We used Fly Nap again to put our virgin flies asleep and under the microscope we determined the sex of each fly. Once we observed and counted our P1 generation our flies were then ready to be mated. The following week we observed our F1 generation under the microscope. We separated and counted the flies according to their sex and phenotype. After counting we killed the F1 generation and the following week we did the same and observed the F2 generation. SEX-LINKED CROSS EXPECTED RESULTS P1  X+X+ Xw Y x Homozygous dominant female x Hemizygous recessive male F1 X+ Xw X+ Xw Y X+Y Wild type is Dominant so all should be wild type in this generation. F1 cross X+Xw x X+ X+ Xw X+Y X+Y Heterozygous dominant female x Hemizygous dominant male F2 Xw X+ Xw Xw Y X+ X+ X+X+ Y X+Y The phenotypic ratio here is 3:1. Wild type : Male White Eyed The genotypic ratio is 1:1:1:1. Hemizygous recessive male, Hemizygous dominant male, heterozygous dominant female, homozygous dominant female SEX-LINKED CROSS OBSERVED RESULTS P1 F1 F2 Wild Type MALE 0 17 38 White Eyed MALE 3 0 18 Wild type FEMALE 6 27 43 Total 9 43 99 Our results seem to have similarities to the expected results because in the F1 ratio we saw all wildtype flies and in the F2 ratio we saw ¾ wildtype and ¼ white eyed. We did not see any white eyed females showing that wild type is dominant respectively to females hence their extra X chromosome. PROCEDURE AND RESULTS: THREE POINT MAPPING The purpose of this cross is to locate and order of the three genes of the flies genome. A male fly that is homozygous dominant for wildtype will cross with a female fly that is homozygous recessive for 3 mutations which are yellow miniature white eyed female. Before we mated the flies we used Fly Nap just as we did for the monohybrid and sex-linked and put the flies asleep and killed them afterwards. We transferred each larva for wildtype and ymw genes into a new vial in order to make virgin flies just as we did in the monohybrid and sex-linked cross. We used Fly Nap again to put our virgin flies asleep and under the microscope we determined the sex and phenotype of each fly. Once we observed and counted our P1 generation our flies were then ready to be mated. The following week we observed the results of our cross under the microscope. We separated and counted the flies according to their sex and phenotype. 3-PT MAPPING CROSS EXPECTED RESULTS CROSS +++ ymw X +++ ymw The phenotypes that were used in the 3-Point Mapping cross were wildtype and yellow flies. The wildtype fly has red eyes, grayish-brown bodies, and wings. The mutant yellow fly is has white eyes, yellow miniature body, and wings. In the cross the parents that are mating are wildtype flies that are homozygous dominant and the yellow miniature white eyed flies that are homozygous recessive for each of the genes for eye color, body color, and body size. ymw 300 PARENTAL +++ +mw 250 100 STRAIN SINGLE y++ y+w 90 25 CROSSOVER SINGLE +m+ ym+ 30 2 CROSSOVER DOUBLE ++w Total 4 801 CROSSOVER In the results there should be 8 different phenotypes with 8 different phenotypes. The most frequent offspring are the parental genotypes. The double crossover genotypes are always in lowest frequency. In order to find the gene order we can see from the table that the w gene must be in the middle because the + allele is now on the same chromosome as the y and m alleles, and the w allele is on the same chromosome as the recessive + and + alleles. In order to figure the gene distance of the Drosophila genome we must calculate as done below. CALCULATIONS y-w w-m Double crossover 100+90/801 x 100% = 25+30/801 x 100% = 2+4/801 x 100% = 23.7% 6.8% 0.75% GENE ORDER AND DISTANCE y---------------w--------------m 23.7+0.75 6.8+0.75 =24mu =7mu 3 PT MAPPING CROSS OBSERVED RESULTS CROSS 6 Wildtype Male x 10 Yellow Miniatures White-eyed Female +++ 6 +++ ymw  78 +++ 14 +m+2 ymw X 10 ymw The experiment did not work for us because we did not have 8 different phenotypes and genotypes. Instead we had a majority of the parental strain, which was ymw, and +++ and 2 flies that were red eyed winged miniature flies. Reasons why our experiment did not work properly may have been due to the prolonged period of time in the vial during Hurricane Sandy. This could have allowed mating between different generations, which would affect our results. Another reason could be because the flies might have still been growing so we could not tell which is a miniature and which is still not a complete adult fly. Another reason is some of the larvae fell in the nutritional medium and this would affect our results because it will decrease the amount of flies we can have. SUMMARY Our intentions during this experiment were to observe the genetic crosses and compare them to the expected in order to have a better understanding of Mendelian genetics. Through the Drosophila experiment and the genetic crosses of the monohybrid, sex-linked, and 3 genes we were able to gain knowledge on how certain traits are inherited. In this experiment we studied the physical characteristics of the flies in order to observe which trait was passed along to the next generation. We became familiar with the chi-square, 3 point mapping and many of the ratios of the crosses. Through this experiment, genotype, which is the genetic makeup of the fly, and phenotype, which is physical appearance of a fly, became two significant key words that were continuously used throughout the experiment.