RAN, A SMALL GTPASE--ROLE IN CELL CYCLE REGULATION

RAN，一种小 GTP 酶——在细胞周期调节中的作用

• 批准号: 2459517
• 负责人: IAN G MACARA
• 金额: $ 18.77万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2459517
• 关键词: CHO cells; DNA damage; DNA replication; animal genetic material tag; antiserum; cell cycle proteins; cell growth regulation; flow cytometry; fluorescence microscopy; genetic library; guanine nucleotide binding protein; guanosine diphosphate; guanosine triphosphate; guanosinetriphosphatases; human genetic material tag; immunoprecipitation; microinjections; tissue /cell culture; yeast two hybrid system

Environmental pollutants and carcinogens may cause genetic damage either by acting as direct mutagens or by disrupting the checkpoints that regulate the cell cycle and maintain genetic stability. Ran1 is the prototype of a novel class of small, Ras-like GTPases members of which play essential roles in the control of DNA replication and chromosome condensation/decondensation. Recent studies in the fission yeast S. pombe, suggest that partial loss of Ran function can cause genetic instability and aneuploidy. A similar loss may account for the aneuploidy frequently found in human tumors. Despite the importance of this class of small GTPase, however, it remains unknown how many members of the Ran family exist, what the functions of Ran and its homologs are, at the molecular level, how Ran GTPase activity is regulated, what factors Ran proteins interact with in the nucleus, and what role Ran may play in the mitotic checkpoints of the cell cycle. The purpose of this grant application is to answer these questions. cDNA libraries are being screened to identify Ran homologs. Antisera will be produced to GST-fusions of Ran homologs, and epitope-tagged versions of the proteins will be created. Subcellular localization and tissue distribution of the homologs will be determined. The hypothesis will be tested that Ran GTP/GDP ratios are regulated in a cell cycle-dependent fashion, and that this ratio changes either at The G1/S or the G2/M boundaries. A second hypothesis to be tested is that Ran proteins are components of cell cycle check points, and that disruption of Ran function can cause genetic instability. This hypothesis will be tested by micro- injection of Ran mutants, and by measuring the frequency of CAD gene amplification following treatment of Ran-transfected cells with the cytotoxic drug, PALA. Finally, to begin to elucidate the molecular mechanisms by which Ran exerts its effects on the cell cycle, proteins that interact specifically with Ran will be identified and cloned, by use of GST-fusion affinity matrices, and by use of the yeast two hybrid system. Overall, these studies will yield information on the functions and regulation of important GTP-binding proteins which play essential roles in the control of the cell cycle. The studies will also provide information on potential targets for xenobiotic toxins and carcinogens that induce genetic damage through mechanisms other than direct mutagenesis.

环境污染物和致癌物质也可能导致遗传损伤 作为直接诱变剂或破坏检查点， 调节细胞周期，维持遗传稳定性。Ran 1是 一个新的小类，Ras样GTP酶的成员， 在控制DNA复制和染色体中起重要作用 缩合/解缩合。对裂殖酵母S.粟酒， 这表明Ran功能部分丧失可导致遗传不稳定性， 和非整倍性。类似的丢失可能解释了非整倍体， 在人类肿瘤中发现。尽管这类小的重要性 然而，目前尚不清楚冉家有多少成员 存在，Ran及其同系物的功能是什么，在分子水平上 水平，Ran GT3活性如何调节，Ran蛋白质的影响因素 在细胞核中相互作用，以及Ran在有丝分裂中可能发挥什么作用， 细胞周期的检查点 本申请的目的就是回答这些问题。cDNA 筛选文库以鉴定Ran同源物。抗血清将 产生Ran同源物的GST融合物，以及Ran同源物的表位标记形式。 蛋白质就会产生。亚细胞定位和组织 将确定同系物的分布。假设是 测试Ran GTP/GDP比率在细胞周期依赖性 这一比例在G1/S或G2/M时都有变化。 边界第二个待检验的假设是Ran蛋白是 细胞周期检查点的组成部分，以及Ran功能的破坏 会导致基因不稳定这一假设将通过微- 注射Ran突变体，并通过测量CAD基因的频率 在RAN转染的细胞处理后的扩增 细胞毒性药物PALA。最后，为了开始阐明 Ran对细胞周期、蛋白质和细胞周期发挥作用的机制 与Ran特异性相互作用的基因将被识别和克隆， GST融合亲和基质，并使用酵母双杂交 系统 总的来说，这些研究将产生关于功能的信息， 调节重要的GTP结合蛋白，这些蛋白在 细胞周期的控制。 这些研究还将提供信息， 外源毒素和致癌物质的潜在目标， 通过直接诱变以外的机制造成的遗传损害。