Mutagenesis of the tRNAse Z Gene in Drosophila Bill Maughan, Dr. Edward Dubrovsky Department of Biological Sciences, Fordham University INTRODUCTION MATERIALS AND METHODS Certain defects in the ELAC2 tRNAse Z enzyme in humans have been shown to increase prostate cancer susceptibility. The gene for this enzyme is well conserved through all three kingdoms of life, indicating its importance. The goal of this project was to create mutations in the Drosophila melanogaster form of the tRNAse Z enzyme and study its effects on cell growth and proliferation. Ethyl methane sulfonate, or EMS, was used to produce essentially random point mutations in the flies’ genomes. The following cross was then used to identify which flies had mutations in the tRNAse Z gene. tRNAse Z can be found in two general forms. The short form, also known as ELAC1, is sufficient for tRNA 3’ processing. The long form of the enzyme, ELAC2, which is not found in bacteria, contains additional domains. The function of these domains is not yet fully understood. Offspring of the final cross were scored for presence of Cy+ flies. Absence of this class of flies indicates a recessive lethal mutation within the Df12 chromosomal deficiency. ELAC2 is found in both humans and D. melanogaster. The domains unique to ELAC2 can be mutated in D. melanogaster and studied in order to better understand their function in both species. Those flies testing positive for a mutation within the Df12 deficiency were then crossed to each other and to the strain ED24, a strain of flies containing a deficiency of the tRNAse Z gene, in order to determine complementation. It has been shown that knocking out ELAC2 in D. melanogaster causes a build up of immature tRNA molecules with the 3’ trailers still attached, yet the amount of mature tRNA does not change. Though they had normal levels of mature tRNA, larvae died once the supply of ELAC2, suggesting that it has an essential function other than tRNA processing. RESULTS Of the nearly 3,000 mutagenized males, roughly 1,000 of them produced enough viable offspring to be scored. Of the nearly 1,000 mutants screened, 9 had recessive lethal mutations within the Df12 deficiency. One of those 9 mutations, BM128, is noncomplementary to the ED24 null allele. A different tRNAse Z mutant, dRNAseZ83 was also shown to be lethal, but it was late larvae lethal. Instead of taking the normal 4 days to mature to third instar larvae, it took them 9. Almost all larvae also developed tumors around day 15. None reached adulthood. Males Key: Cy:Cy+ ED24 BM23 BM47 BM105 BM106 BM119 BM127 BM128 x 112:61 117:74 99:63 94:62 87:50 101:44 137:0 129:68 141:61 138:59 88:44 184:102 124:59 134:71 126:83 123:64 148:89 184:0 104:59 117:54 115:63 Females 94:43 121:68 120:68 128:61 128:0 112:0 122:54 140:75 112:51 149:0 145:65 121:70 122:60 124:70 98:63 114:47 92:57 74:0 129:69 120:0 141:67 110:56 114:65 101:46 111:0 118:74 105:58 113:0 110:65 149:67 101:66 136:65 83:57 125:52 A B C D 3’ 5’ CONCLUSIONS AND NEXT STEP Those crosses which only produce offspring bearing the curly phenotype exhibit noncomplementation in their second chromosomes. Therefore the two parents strains have recessive lethal mutations in the same gene. Four complementation groups have been identified thus far. The BM128 strain is noncomplementary to the ED24 strain. This indicates that it contains a mutation that effectively knocks out the tRNAse Z gene. 14 more strains that do not complement to the Df12 deficiency have been identified. They will also undergo a complementation test with each other and with the mutant strains already identified. The Df12 deficiency omits a large part of chromosome 2, including the tRNAse Z gene. B) D. melanogaster larvae homozygous for tRNAse Z deletion do not develop properly. C) tRNAse Z performs the final cleavage at the 3’ end of an immature tRNA molecule. D) tRNAse Z mutants are unable to cleave the 3’ trailer sequence of an immature tRNA molecule. All strains that bear mutations in the tRNAse Z gene will be sequenced in order to determine the location of the mutation. The effects of the mutation on cell growth and proliferation will then be studied.