Regulation of cell cycle in the somatic cells

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This project is comprised of three parts, (1) the isolation of temperature-sensitive (ts) mutants for cell growth from hamster BHK21 cell line, (2) the identification of mutated genes in those mutants, and (3) the characterization of cloned mutated genes. I will review for each part. Part 1. Previously, I have established a method to isolate ts mutants of hamster BHK21 cell line. However, a majority of those mutants have a mutation in the X chromosome where a haploid dose of gene is expressed. In this project, I tried to isolate ts mutants of the autosome using a drug, benomyl. Finally, we isolated ts mutants belonging to a different complementation group. Part 2. We have identified mutated genes of ts mutants of hamster BHK21 cell line, which are genes encoding HCF (host cell factor for herpes virus proliferation), histidyl-tRNA synthetase and lysyl-tRNA synthetase, respectively. Part 3. I will review for each genes which have been cloned using ts mutants of hamster BHK21 cell line. H … More CF : Based on our finding, HCF was approved to be essential for host cell proliferation as well. The mutant defective in HCF gene, tsBN67, arrest in G0 phase after completion of the G1, S, G2 and M phase at the nonpermissive temperature. Thus, this mutant will be useful for characterizing the G0 phase at the molecular level. Aminoacyl-tRNA synthetase : mutants defective in histidyl- and lysyl-tRNA synthetases rapidly enter apoptosis under nonpermissive condition, indicating that there is a quality control for aminoacyl-tRNA synthesis. DAD1 : The mutant defective in this gene, tsBN7, has a defect in N-linked glycosylation, and mice defective in this gene die due to apoptosis. Our results indicate a tight relationship between apoptosis and cycle : 1) We have identified Ran-binding proteins, RanRP1/Yrb1p, RanBP3/Yrb2, RanBP2/Yrb2, Dis3p, RanBPM and Mog1. The finding of RanBPM introduced that Ran is directly involved in microtubule assembly. 2) We found that a dual phosphates, Cdc25B triggers mitosis. Due to the instability of Cdc25B, the entry of mitosis is blodked by cycloheximide, a protein synthesis inhibitor. 3) As a suppressor gene of prp20 and rna1-1, ts mutants of S. cerevisiae RCC1 and RanGAP homologue, we isolated GTR1 and GTR2. These are new G proteins which seems to function antagonistically to the Ran cycle. Less