Abstract The main purpose of this experiment was to find which chromosome our unknown gene mutation is presented on and the exact location on that chromosome. In order to do so many cycles of crosses were completed using linkage analysis on Drosophila melanogaster, a type of fruit fly used in this experiment to identify our unknown gene mutation, unknown. Using virgin bar females crossed with Curly/Plum; Dichaete/Stubble male fruit flies represented our Discriminant Cross one (DC1). The DC1 piloted that the unknown mutation is sex linked dominant.
Knowing the mutation is sex linked gives evidence that it is located on chromosome 1. Discriminant Cross two (DC2) is then performed basically to execute a check for DC1 by crossing a wild type female with a Plum/Stubble male fruit fly. In our case if the cross was done correctly all the females should carry the uknown mutation and none of the male progeny should show the unknown phenotype. Following DC2, another cross, called Mapping cross one (MC1), is then executed using a virgin unknown female with males carrying the dominant marker genes for the phenotypes yellow (y), crossveinless (cv), vermillion (v), and forked (f).
The final cross that was performed was the Mapping cross two (MC2). The MC2 was carried out by crossing an F1 female from MC1 with an F1 male from MC1. The final step of the procedure is to validate the data obtained in order to confirm our results. This is done using the Chi-square analysis test. In conclusion, by using the data obtained from MC1 and MC2, unknown is approximated to be located at 45. 57 m. u. According to the book of mutations in the lab the exact location for unknown is at 57 m. u. Unknown is then found to be bar (b). Introduction
Drosophila melanogaster is most frequently used due to its small size, a conveniently short life cycle of about two weeks, and a small enough genome that helps us understand new mutations compared to our own genome. The goal in performing these many crosses is to figure out the exact map location of the unknown mutation and which chromosome unknown lies on. Over 11,000 species of both wild-type and mutant Drosophila melanogaster exist today that are accessible for research. (Malik, 2010). An example in today’s society shows a study that has been done on a Drosophila melanogaster gene hat carries both box and homeobox pairs. The Pax-6 (small eye) gene is a gene located on chromosome IV. It is located close to the locus that carries the eyeless mutation. Ey2 and eyR are two mutations used that widely affect the Drosophila gene expression, especially in the eye primordia. In the finding of the gene ey, human Aniridia, and the Pax-6 mutation in the mouse genome by homologous genes wraps up the conclusion that gene that causes change in the eye is the same for insects and invertebrates (Quiring, 1994).
The Drosophila melanogaster has been so frequently used due to the fact that we are acquainted with so many parts of the genome which have already been learned and studied widely. The Drosophila melanogaster is also conveniently a very diminutive size, large amounts are able to be held in a limited amount of space which makes the fruit fly one of the easiest organisms that can be studied to handle. In conclusion, the fruit fly’s genome is most easily studied in a small amount of time. (Manning, 2008). Materials and methods Several different materials were used in order to carry out this experiment.
A subculture of mutant flies and a subculture of wild-type flies were given to our group. The D. melanogaster were contained in plastic vials carrying their food made from sterilized water added to a dehydrated medium. Each of these plastic vials were then stored at twenty-five degrees Celsius in incubators inside the lab. CO2 was used to ‘knock out’ the flies when the time came to observe and sort the flies. When the flies became immobilized they would be placed onto the viewing plate under the microscope that kept carbon dioxide circulating the area where the flies were being observed.
These fruit flies were observed at 10X or 15X magnification and gently moved around with a fine brush. Four crosses had to be carried out in order to complete this project. Discriminant cross one (DC1) was first finished. Discriminant cross one was prepared by crossing a mutant virgin unknown female crossed with a wild type male (++). The wild type male carries the mutations of Curly/Plum; Dichaete/Stubble. One hundred F1 progeny from this cross are scored. The second cross, Discriminant cross two, was done by crossing a wild type female with a Plum/Stubble male fruit fly.
One hundred F1 progeny were also collected and scored from this cross. Mapping cross one (MC1) is the third cross to be carried out. The main goal of this cross is to produce heterozygous female. Ten virgin female from the unknown stock subculture are obtained and mated with given male D. melanogaster carrying the marker genes yellow, crossveinless, vermillion, and forked. The progeny from Mapping cross one is collected and used to carry out Mapping cross two (MC2). A male from the MC1 progeny is crossed with a female from the MC2 progeny. About two dozen vials were kept holding MC2 subcultures.
One thousand progeny were scored mainly focusing on scoring the male progeny since they carry the marker phenotypes and the female do not. Chi-square values and chromosomal drawings were made after the final cross validating the interpret data collected. The recombination frequency is calculated using the data collected from MC2 using the formula; (# of progeny in recombination class / total # of progeny) X 100. This enables us to locate the exact map unit of the unknown phenotype. Fly base, http://flybase. bio. indiana. edu/genes/fbgquery. hform is then used in order to discover what mutation unknown actually is. Results
Discriminant cross one The stock vial given to our group was labeled STOCK SUBCULTURE. The STOCK SUBCULTURE obtained had flies with long, flat eyes as opposed to the round eyes on the wild type Drosophila melanogaster. This phenotype is called bar. Hypothesis If the BAR mutation is a dominant gene, the DC1 F1 phenotypic classes will be: Curly Dichaete BAR maleCurly Dichaete BAR female Curly Stubble BAR maleCurly Stubble BAR female Plum Dichaete BAR malePlum Diachate BAR female Plum Stubble BAR malePlum Stubble BAR female If the BAR mutation is autosomal recessive, the DC1 F1 phenotypic classes will be: Curly Dichaete maleCurly Dichaete female
Curly Stubble maleCurly Stubble female Plum Dichaete malePlum Dichaete female Plum Stubble malePlum Stubble female If the BAR mutation is sex-linked recessive, the DC1 F1 phenotypic classes will be: Curly Dichaete BAR maleCurly Dichaete BAR female Curly Stubble BAR maleCurly Stubble BAR female Plum Dichaete BAR malePlum Dichaete BAR female Plum Stubble BAR malePlum Stubble BAR female Data: Discriminant cross one: Table 1 DISCRIMINANT CROSS ONE ClassPhenotypeFemaleMale 1X Cy D1411 2X Cy Sb1212 3X Pm D1510 4X Pm Sb818 According to the hypothesis our results show that the mutation BAR is sex-linked dominant. Discriminant cross two:
Table 2 DISCRIMINANT CROSS 2 Female Progeny ClassPhenotypeNumber of progeny 1X Cy16 2X D11 3X Cy D17 4X 5 Male Progeny 5Cy14 6D13 7Cy D16 8++ 8 Only the female progeny show the BAR mutation proving that the Discriminant cross one has been done correctly and we are ready to initiate mapping cross one. Mapping cross one: Table 3 MAPPING CROSS ONE Phenotype# of Female offspring # of Male offspring Lobe eyes300 Mutant X 060 The mutant phenotype BAR only appears on the male progeny. The lobe eyes shown on the female progeny represent a phenotype somewhere between a completely wild type eye and the mutant BAR phenotype eye.
Mapping cross two: An F1 female from Mapping cross one is crossed with an F1 male also from Mapping cross one. A total of one thousand F1 progeny are used for the Mapping cross two data. Table 4: MAPPING CROSS TWO MALE PROGENY ClassPhenotype# of Male Progeny 1X211 2y cv v X28 3y cv v f122 4f30 5v f70 6y X29 7y f7 8y cv X51 9cv v f15 10y cv f63 11cv f8 12v X5 13y v f10 14y cv v3 15v3 16y v x1 17cv x1 18y cv4 19cv1 A total of 662 male progeny have been scored with the shown phenotypes. Table 5: Chi-square analysis for Mapping cross two ClassOE(O-E)^2[(O-E)^2]/E X246225. 5103419. 2781. 86167 Cv/f208225. 5103306. 61061. 35963 Cv/x8098. 3351336. 17593. 41867 F11798. 3351348. 37853. 54276 f/x03. 834614. 704163. 8346 Cv83. 834617. 350564. 52473 Cv/f/x03. 3211. 02243. 32 wildtype33. 320. 1024. 030804 Total 662662X^2=21. 8929 X^2 = 21. 8929 > 14. 07 therefore the chi-square test does not confirm our data. This can be due to human error, by chance not having virgin females for our crosses, or not scoring enough male progeny. By calculating the recombination frequency we are able to outline how far bar is from vermillion and how far bar is from forked.
The formula for Recombination frequency is as follows: Recombination frequency = (# of double recombinants) + (# of single recombinants) X 100 (total # of progeny) Recombination frequency between vermillion and bar = ((3+0)+ (80+117))/662 = 30. 21 Double recombinant = (0. 3021)(0. 0166)*662 = 3. 32 Single recombinant = (1. 66) = ((3. 32)+(x1+x2))/662 X 100 = 3. 8346 Single recombinant = (30. 21) = ((3. 32)+ (x1+x2))/662 X 100 = 98. 3351 Non-recombinant = 225. 5103 Table 6 MC2 CONDENSED CLASSES FOR RECOMBINATION FREQUENCY CALCULATION BETWEEN VERMILLION AND X Phenotype# of male progeny
X292 v223 Xv34 ++ 113 Table 7 MC2 CONDENSED CLASSES FOR RECOMBINATION FREQUENCY CALCULATION BETWEEN FORKED AND X Phenotype# of male progeny X326 f325 X f0 ++ 11 By using tables 4, 6, and 7 we can calculate the approximate map location of the mutant gene. The approximate map location of the gene can be determined by adding the distance between crossveinless and forked, forked and the mutant X, and vermillion and the mutant X. Unknown mutation location = 30. 21 + 1. 66 + 13. 7 = 45. 57 The actual location of the bar phenotype is 57 m. u. Therefore our data shows an 11. 43 difference in mapping units. Discussion
The Discriminant cross one was the first to be completed in the series of crosses. DC1 showed whether the unknown mutation was dominant or recessive, and autosomal or sex-linked. According to our data the unknown phenotype only appeared in both male progeny and female progeny proving that the mutation is dominant. In conclusion our mutation, bar, is sex-linked dominant. The principle idea to performing Discriminant cross two was to determine the location of the unknown phenotype on chromosome one. The main idea in performing Mapping cross one is to create the two mating parent phenotypes needed to carry out Mapping cross two.
The MC1 progeny did need to be scored in order to know in definite that the female progeny did show the mutation. MC2 is carried out in order to evaluate the exact location of the mutation on the first chromosome of D. melanogaster. The progeny from MC1 is crossed in order to obtain the data for MC2 (table 4). The marker genes yellow, crossveinless, vermillion, and forked are used to calculate what map unit the mutation is located using recombination frequency. By adding up the recombination frequencies found we find that the unknown mutation is approximated at 45. 7 m. u. After obtaining this location we look on flybase finding that the phenotype closest to the one we have been observing is bar, at 57 mapping units. According to the chi-square analysis the data does not support the hypothesis. This proves that many different errors may have been made throughout the experiment. Human error is the most probable to have occurred. In scoring many flies at one time it is possible that any group member could have missed a phenotype or even counted and extra phenotype that did not exist on the fly itself.
Another possibility could be that we did not score enough male flies to calculate the recombination frequencies correctly. Also, if at any time in the experiment a virgin female had not been used the results could have been altered by a great amount. FlyBase creates a huge step in the process of finding the map location of the gene. Without the Fly Base computer program experiments like this one would take a much greater amount of time to complete. Although not all computer programs are one hundred percent accurate it still brings society ahead by a great amount in regards mostly to time.
A question that frequently crossed my mind was if it would be possible to cross our unknown phenotypes with less marker mutations and come up with the same map location that was obtained. This semester long project uses the Drosophila melanogaster species to teach the concept in finding map locations of genes and determining which chromosome these genes are located on using linkage analysis, which is an eternally used method in today’s laboratory society. The experiment proved the possibilities of how many genes can be discovered just by crossing different genes on different flies from different generations.
I have gone through a series of crosses and achieved the goal of finding the exact map location of the mutation bar. Bibliography Diaz-Benjumea, F. J. , Garcia-Bellido, A. (1990). Genetic analysis of the wing vein pattern of Drosophila. Rouxs Arch. Dev. Biol. 198(6): 336–354. September 20, 2011. Diaz-Benjumea, J. , Gonzalez-Gaitan, M. A. F. , Garcia-Bellido, A. (1989). Developmental genetics of the wing vein pattern of Drosophila. Genome 31(2): 612—619. September 20, 2011 Drosophila Gene Disruption Project: Progress Using Transposons With Distinctive Site Specificities Genetics July 1, 2011 188:731-743.
September 26, 2011 Homology of the eyeless gene of Drosophila to the Small eye gene in mice and Aniridia in humans R Quiring, U Walldorf, U Kloter, and WJ Gehring Science 5 August 1994: 265 (5173), 785-789. [DOI:10. 1126/science. 7914031] Lindsley, D. L. , Zimm, G. G. (1992). The Genome of Drosophila melanogaster. : viii + 1133pp. September 24, 2011 Malik, Nusrat. “Drosophila Melanogastern Biology and Cultering Techniques. ” Biol 3311 Genetics Manual 2010. 2010. 14. Print. Manning, G. (2008, July/August). Introduction to Drosophila. Services on this machine. Retrieved October 07, 2010, from http://www.
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