Regulation of cell cycle in the somatic cells

本项目包括三个部分，(1)从仓鼠BHK21细胞系中分离细胞生长的温度敏感(Ts)突变体，(2)在这些突变体中鉴定突变基因，(3)克隆突变基因的特征。我会针对每个部分进行复习。第一部分：以前，我已经建立了一种分离仓鼠BHK21细胞系ts突变体的方法。然而，这些突变体中的大多数在X染色体上有突变，在X染色体上表达单倍体的基因。在这个项目中，我试图使用一种名为苯菌灵的药物来分离常染色体的ts突变体。最后，我们分离出属于不同互补群的ts突变体。第二部分，我们鉴定了仓鼠BHK21细胞的ts突变基因，分别是编码疱疹病毒增殖的宿主细胞因子hcf、组氨酰-tRNA合成酶和赖氨酰-tRNA合成酶的基因。第三部分，我将对利用仓鼠BHK21细胞的ts突变体克隆的每个基因进行综述。H…更多的CF：根据我们的发现，HCF对宿主细胞的增殖也是必不可少的。突变的人绒毛膜促性腺激素基因缺陷株tsBN67在不允许的温度下，在G1、S、G2和M期完成后，停滞在G0期。因此，该突变体将有助于在分子水平上表征G0相。氨酰-tRNA合成酶：组氨酰-tRNA合成酶和赖氨酰-tRNA合成酶缺陷的突变体在不允许的条件下迅速进入细胞凋亡，这表明氨酰-tRNA合成是有质量控制的。DAD1：该基因缺陷的突变体tsBN7在N-连接糖基化方面存在缺陷，该基因缺陷的小鼠死于细胞凋亡。我们的结果表明细胞凋亡与细胞周期密切相关：1)我们发现了Ran结合蛋白RanRP1/Yrb1p、RanBP3/Yrb2、RanBP2/Yrb2、Dis3p、RanBPM和Mog1。RanBPM的发现指出Ran直接参与了微管的组装。2)我们发现双磷酸盐，Cdc25B可以触发有丝分裂。由于CDC25B的不稳定性，有丝分裂的进入被蛋白质合成抑制剂放线菌亚胺阻断。3)从酿酒酵母RCC1和RanGAP同源物的ts突变体中分离到了GTR1和GTR2，作为prp20和rna 1-1的抑制基因。这些是新的G蛋白，它们的功能似乎与RAN周期相反。较少

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