Millet Wine Fission Yeast Coding Trna3 'end Processing Enzyme Gene Trz1 Back Mutation Gene Research

Endonuclease tRNase Z catalyzes the generation of the mature3’end of tRNA precursors through specific endonucleolytic cleavage,which belongs to the superfamily of metallo-(3-lactamases(MBL). Mutation of humans tRNase Z gene ELAC2is associated with an increased risk of prostate cancer,our lab trying to study the evolution and function of the candidate prostate cancer susceptibility gene ELAC2with Schizosaccharomyces pombe. The fission yeast Schizosaccharomyces pombe contains two tRNase Z genes, one is trzl+(SPAC1D4.10) encoding the nuclear tRNase Z and the other is trz2+(SPBC3D6.03c) encoding the mitochondria tRNase Z,both of they are essential genes.This study is mainly for screening and verifying the interaction genes with Sptrzl+and Sptrz2+. In eukaryotes,Schizosaccharomyces pombe is a model organism which provid us convenient genetic research methods after the budding yeast, which makes it as a model organismthis of cell cycle regulation, mitosis andamitosis, DNA repair and recombination.In this study,we create a system of temperature-sensitive strain of trzl(tsl (N408K,Y571L,D702G)) and trz2(yGWl(A623V)) to find the genes which have genetic interactions with this two gene.And we hope we can provide some help to the study of human cancer with our work.we found that human ELAC2and Saccharomyces cerevisiae ScTrzl can rescue our trzl temperature-sensitive strain tsl,indicate that they are highly homologous to the conservative in evolution. We also find some revertants which can fully suppress the temperature-sensitive phenotype like wild type strain At37℃, Reverse mutation rate is about9.4×10-5.We verified there is no additional mutation in trzl gene and promoter, then we use whole-genome sequencing in order to find the reverse gene. Whole-genome sequencing coverage is100%and depth of coverage is65.9. The results was compared with the NCBI S.pombe972h-genome,and we got20mutations,18mutations genes, six genes we interested were chosen to verify whether they can suppress the lethal phenotype at37℃. Ultimately they all did not save temperature-sensitive strains.We found that overexpression of trz2+results in severe growth defects and lethality in S.pombe.And then we used a cDNA library(SPLE1) trying to supress the lethal phenotype of overexpression of trz2+But we got8false positive results because of gene homologous recombination.