L10H Midterm, Spring Quarter By Toni Lee (undergraduate student)

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1 L10H Midterm, Spring Quarter 2004 By Toni Lee (undergraduate student) I. Specific Aim Through the use of reverse genetics, methods of disrupting gene function when ony the sequence and position in the genome are known, it is possibe to decipher the function of newy discovered genes (Adams, 189). This mode, when appied to severa stocks of fies carrying P-eement mutations in minimay scrutinized genes wi accompish four goas: 1) creating sma and arge P-eement mutated Drosophia meanogaster cones capabe of mitotic recombination ocaized in the eye 2) observing and recording phenotypes produced by such mitotic recombination and 3) inking these observabe phenotypes to the function of the mutated gene within the eye. Depending on the function of the disrupted gene, homozygous etha fy ommatidia wi dispay different phenotypes. A crucia piece of information about these mutations has previousy been determined; a mutations covered in this study are homozygous recessive organismic etha. Fies carrying two mutated aees for the particuar gene of interest utimatey die and cannot be studied, necessitating the use of the fip mitotic recombination system ocaized in a non-essentia area of the fy (the eye). However, the nature of this ethaity has heretofore escaped exporation. This study wi determine whether each mutation can be characterized as ce etha, having a roe in eye deveopment, or having no roe in eye deveopment. Mutations having a roe in eye deveopment wi be examined in great detai to determine precisey the mutant gene s norma function in this process. The resuts of this assessment wi then end a hand in decoding the roe of the gene in which the mutation occurred. The phenotypic information geaned from observing mutant stocks wi be compared to rudimentary gene function information avaiabe from Fy Base and BLAST searches to carify the precise roe of the gene. Some of these genes may have functions inked to diseases and disorders found in humans. BLAST searches may be performed to search for homoogous sequences in the human genome for ater experimentation. Additionay, creating stocks of P-eement mutated fies is crucia to the deveopment of further experiments invoving the mutated gene. Once the genes have been inked to a specific function, further studies may proceed using these baanced stocks. II. Background Simpicity is the gateway to understanding. As far as functiona genomic research goes, Drosophia meanogaster seems to fit this statement ike a key in a ock. Labeed a premiere mode invertebrate system by the Nationa Institutes of Heath (NIH), fruit fies fit the bi with their simpe genome, easy of care, and spectacuar reproductive capabiities. With ony four pairs of chromosomes, the drosophia genome constitutes a tangibe, manipuatabe entity. The fies are sma; approximatey 100 individuas can fit in a via whie 600 fies can survive in a stock botte. Typica Drosophia ife cyces ast approximatey 10 days when progeny are incubated at 25ºC. Each femae can produce thousands of eggs with a singe mating, aowing propagation of a desired trait to severa fies in a brief time period. Distinct traits and a ack of meiotic crossing over in mae gametes further faciitate genetic research. Gene reguation is responsibe for great compexity of ife without onerous maintenance of mutitudinous genes. By manipuating this reguation, deviations from

2 natura expressivity patterns may arise which are often usefu in the aboratory. Determining the function of a gene can be carried out through empoying reverse genetics, which invoves disrupting gene function and observing the effect of its absence to derive function. Introducing mutations can be accompished through exposure to chemica mutagens, such as ethy methanesuphonate (EMS), or exposure to x-ray radiation. However, these methods are not ony dangerous, but aso ow in efficiency and difficut to track in the genome. An inordinatey safer and efficient way of introducing mutations invoves the P- eement, a transposabe sequence, or jumping gene, existing naturay in the genome. The P-eement consists of two P ends fanking a transposase gene. This gene codes for an enzyme that both ceaves the gene from the P ends and aids in inserting the sequence into another set of P ends. Mutations arise when a P-eement exists within a sequence necessary for the expression of a gene. When the transposase gene is suppied outside of the P ends, the enzyme may insert aternative sequences, such as P{PZ} (rosy) and P{LacW}(mini-white), between the P ends (Adams, 191). Remova of the transposase gene by performing a cross permanenty abandons the new gene within the P ends and norma transcription of the fanking gene is disrupted. Even after creating a mutation in a drosophia gene, if the mutant aee is recessive it cannot be studied unti a homozygous mutant is created. Unfortunatey, if heterozygous fies are crossed and the mutant aee is recessive for ethaity, a homozygous mutants woud perish. However, if homozygous mutant ces arose in a nonessentia structure, such as the eye, a phenotype coud be observed in viabe aduts. Mitotic recombination, a process invoving the yeast fip gene (fp) and fippase responsive target sequences (s), can be empoyed to create patches of homozygous mutant ces during mitosis rather than just during meiosis. When fp presides in the genome, its enzymatic product, fippase, binds to sites catayzing a recombination event between the two ocations. Each homoogous chromosome must contain an site; pieces of the chromosome can ony move to the extent that they have a pace to go. An additiona probem concerns generating mitotic recombination ony within the eye. When couped with the eyeess (ey) enhancer, fp wi ony be transcribed during eary eye deveopment, ocaizing mitotic recombination within the organ (Tapon, 345). Mitotic recombination resuts in three types of daughter ces: parenta heterozygous, recombinant wid type, and recombinant mutants (see fig. 1). Depending on the nature of the mutation, the eye wi dispay mutifarious phenotypes. Coor markers such as ry or w+, aow particuar phenotypes to visiby refect genotype. For exampe, if the mini-white marker is inserted in the P-eement to create a etha mutation () in a white-eyed fy background, recombinant mutant ces wi dispay a deep orangered hue. Parenta ces, those with one copy of the mutant aee and thus one copy of w+ wi appear orange. Recombinants receiving two norma aees wi show the white background coor. Coor combinations of this sort give rise to mosaic eyes. The arrangement of these bocks of coor depends on fy eye deveopment. Ideay, most of the eye ces wi be mutant recombinants. Athough a ces in the eye are capabe of undergoing mitotic recombination, they do not. Fippase production and function is attached to a certain frequency of success. Additionay, for every mitotic recombination that produces a homozygous mutant, there wi be a homozygous widtype ce (see figure 1). This resuts in the formation of twin spots, adjacent areas of differing coor usefu for detecting areas of recombination. To minimize the number of homozygous wid-type and heterozygous ces, the minute gene (M) aong with a uniwhite copy for wid-type eye coor is crossed into a stock. The M gene is homozygous etha and sows growth in the heterozygote. Ony ces homozygous for the etha aee

3 of interest (with no copies of the M gene) wi exhibit norma growth. The eye wi dispay primariy homozygous etha ces, which faciitates phenotype anaysis. With the minute gene screening out unwanted genotypes, one can readiy ascertain the nature of the mutation. Eyes with ce etha mutations wi show no ces of the correct recombination coor. Eyes wi mutations of genes invoved in eye deveopment wi possess ces of the correct coor with an atypica phenotype. Eyes with mutations that do not affect eye deveopment wi be the correct coor but norma in a other respects. Occasionay, an interesting phenotype wi arise in a fy that is prime for further study. Unike bacteria, fies cannot be frozen for future use. Instead, stocks requiring continuous (and inevitaby tedious) maintenance act as substitutes. However, when a stock possesses a homozygous recessive etha, simpy tossing fies together and transferring them over time wi resut in a diminished aee frequency of the etha aee. Baancer chromosomes eiminate this hurde. By couping the etha aee with the widtype of another etha aee on one chromosome and pairing the wid-type of the etha of interest with a etha aee of the marker, any anima homozygous for either the etha of interest or the etha marker wi perish. A dominant marker, usuay the same extra etha aee, aows one to visiby ensure that this process occurs. This process can easiy be disrupted during meiotic recombination, so as a preventative measure, an inversion is created in the region of interest. Crossing over with inversions produces chromosomes with either two teomeres or two centromeres, both etha situations. Essentiay, homozygous recessive etha mutant fy stocks can be maintained when another etha marker with some dominant phenotype is combined with an inversion. III. Significance As with most research, this project seeks to add to the coective knowedge of the drosophia genome as a means of unraveing the genetic mystery behind human diseases and disorders. Athough the approximatey 14,000 genes of the Drosophia genome have been sequenced, many of these genes have not been inked to specific functions. A too, no matter how usefu, cannot be expoited without knowing its purpose. By screening the Drosophia genome using the successfu /fp mitotic recombination technique, the scientific community wi gain insight to the roe these genes pay in fy eye formation. Determination of gene function is difficut to accompish in humans due to ethica and practica constraints. Drosophia serves as an adequate substitute organism. Like humans, fruit fies are eukaryotes with transcription and transationa methods homoogous to mammas. Essentia genes may show great conservation among a species, making their study in fies even more vaid. Studying Drosophia through reverse genetics wi aid in feshing out databases devoted to fy genetics. These databases can in turn be used to identify homoogies in human or other organisma DNA sequences to revea the moecuar mechanisms behind diseases and disorders. Severa genes inked to neuroogica disease, cardiovascuar maformation, rena, hematoogica, and metaboic syndromes were discovered in Drosophia and C. eegans. Aso, mutipe genes invoved in cancers have been isoated from invertebrate studies. Two genes, BRCA 1 and BRCA 2, were first identified in Drosophia. Additiona cancer genes may be unearthed as such research continues. This research wi not ony generate quaity information for incorporation into databases, such as Fybase, but wi aso generate stocks of mutant fies for additiona experiments reating to our own research or the research of others.

4 IV. Research Methods The proposed procedures are as foows: 1) generate homozygous mutant fies for each ine of mutations, 2) examine eye phenotype of sma and arge cones and take pictures of mosaic phenotypes, 3) cacuate the recombination distance between the miniwhite insertion and, and 4) create a baanced stock for each ine. In order to create homozygous mutants, a series of crosses must be performed. The particuar chromosoma segment of interest in this case is the eft arm of the second chromosome. Stocks from Boomington, Indiana with pre-made P-eement insertions of mini-white containing a cury oster (CyO) dominant marker wi be obtained (+/Y; P{w+}/CyO). In order to combine the x-inked yeow-bodied, eyeess-flp (yw ey-flp) traits with the P-eement, maes from the origina Boomington stock wi be crossed with femaes homozygous for yw ey-flp and containing scutoid (Sco) and CyO, y+ markers on the 2L chromosome. Cross 1: yw ey-flp/yw ey-flp; Sco/CyO, y+ x +/Y; P[w+]/CyO Mae progeny possessing yw ey-flp on the x-chromosome and the P-eement with CyO, y+ on the 2L chromosome wi have red eyes, gray bodies, and cury wings. These maes wi be coected and mated to femaes homozygous for both yw ey-flp and 40A. Cross 2: yw ey-flp/ yw ey-flp; 40A/40A x yw ey-flp/y; P[w+]/ CyO, y+ The goa of this cross is to create fies containing both the P-eement and an site. A progeny from this cross wi possess the yw-ey-flp and haf wi contain the P-eement. However, we wi ony coect femaes possessing the P-eement and 40A, which are coor-eyed (coor depends on mini-white expression), yeow-bodied, and straight-winged. Ony the femae genome recombines during meiosis, a step which is crucia to generating fies with sites on both 2L chromosomes. This is ony possibe through meiotic recombination in the femae to produce gametes with mini-white and 40A on the same arm. When these femaes are crossed with yw ey-flp, homozygous 40A maes, some of the resuting progeny wi have two sites and possiby a recombination event. Cross 3: yw ey-flp/ yw ey-flp; P[w+], 40A x yw ey-flp/y; 40A/40A Once fies with mosaic eyes have been isoated, scanning eectron and ight microscope pictures of mutant phenotypes wi be obtained. The number of progeny obtained with mutant phenotypes wi be counted aong with the tota number of progeny produced in order to cacuate the recombination frequency between the P-eement and 40A. Mosaic maes from the ast cross wi be mated to red-eyed, gray-bodied, curywinged femaes carrying the uniwhite, minute (w+ M) marker and a cury baancer: Cross 4: yw ey-flp/ yw ey-flp; w+ M, 40A/ CyO, y+ x yw ey-flp/y; P[w+], 40A/40A

5 The resuting progeny wi be used for two purposes: setting up a baanced stock for future use and examining eye phenotype once again. The baanced stock wi consist of mae and femae yw ey-flp; P-eement, 40A/CyO, y+ fies. CyO, y+ serves as a baancer chromosome, kiing off fies homozygous wid type for the etha mutation of interest. As for the second phenotype examination, both mae and femae fies with yw ey-flp, the P-eement, w+ M, and sites on each chromosome wi be examined. The addition of the minute aee ensures that ces with ony one P-eement wi experience sow growth and ces without a P-eement wi die. A arge cone resuts and more ight and SEM pictures wi be taken. Additiona recombination data wi be used to correct or corroborate data from the sma cones. V. Timeine The experiment wi occupy 10 weeks time. An instructor cross of gray-bodied, white-eyed, scutoid, cury-winged femaes carrying ey-flp with gray-bodied, red-eyed, cury-winged maes wi be performed during the week prior to the commencement of the experiment. During week 1, the gray-bodied, red-eyed, cury-winged mae progeny from week -1 carrying the P-eement wi be mated to yeow-bodied, white-eyed femaes carrying. In week 3, yeow-bodied, red-eyed femae progeny carrying both a P-eement and an wi be mated to yeow-bodied, white-eyed maes carrying two sites. During weeks 5 and 6, mosaic eyed maes from the week 3 cross wi be examined under a ight and scanning eectron microscope (SEM). Pictures wi be taken of particuary interesting mosaics and recombination frequencies wi be determined between the P-eement and 40A using progeny genotype numbers. These mosaic maes wi be crossed with gray-bodied, red-eyed, cury winged femaes carrying the minute gene. In weeks 7 and 8, baanced stocks wi be constructed using gray-bodied, cooreyed, cury-winged maes and femaes containing the P-eement and one site. Aso, mosaic eye progeny from week 5 and 6 wi be examined under the ight and scanning eectron microscope. Again, recombination frequencies wi be cacuated from progeny genotype numbers. Weeks 9 and 10 wi consist of anayzing data obtained throughout the experiment to make a concusion as to the function of each gene examined.

6 Fig 1 Heterozygous ce for etha aee + Fippase Recombination event + Fippase Post-Recombination Event + Fippase Recombinant Mutant Daughter Ce Recombinant Wid Type Daughter Ce Parenta Daughter Ce VI. References- Adams, M. D., Sekesky, J. J. From Sequence to Phenotype: Reverse Genetics in Drosophia Meanogaster. Genetics 3, (2002). Tapon, N., Ito, N., Dickson, B. J., Treisman, J.E., Hariharan, I. K. The Drosophia Tuberous Scerosis Compex Gene Homoogs Restrict Ce Growth and Ce Proiferation. Ce 105, (2001).