CONTROL OF THE EXIT FROM MITOSIS IN FISSION YEAST

裂殖酵母有丝分裂退出的控制

• 批准号: 2415188
• 负责人: SHELLEY SAZER
• 金额: $ 19.45万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-05-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2415188
• 关键词: Schizosaccharomyces pombe; biological signal transduction; cell cycle; chromosomes; enzyme activity; fluorescent dye /probe; fungal genetics; gel electrophoresis; gene expression; genetic library; genetic mapping; guanosinetriphosphatases; immunofluorescence technique; immunoprecipitation; laboratory rabbit; molecular cloning; nucleic acid sequence; phenotype; protein structure function; southern blotting; suppressor mutations; western blottings

the long-term objective of the proposed research is to elucidate the mechanisms that regulate the exit from mitosis in the fission yeast Schizosaccharomyces pombe. The isolation and characterization of the fission yeast dcd1-1ts mutant has led to the identification of two evolutionarily conserved proteins that are required for the complex structural changes associated with the completion of mitosis and re- establishment of the interphase state (Sazer and Nurse, 1992. The dcd1 gene encodes a protein similar to the human chromatin binding protein RCC1. A suppressor of dcd1-1ts, called fyt1, is nearly identical to a human GTP-binding protein in the family typified by the human proto- oncogene p21ras. The similarity of these two S. pombe proteins to human proteins of known function, and in the case of p21ras of known structure as well, provide a framework within which to design experiments examining the role of these and related proteins in mitotic exit. The knowledge gained from the proposed research should further our understanding of the role these human proteins play in cell cycle control, signal transduction pathways, and ras-mediated cancer. The dcd1 and fyt1 proteins are the core of a molecular switch that plays a key role in chromosome decondensation (Sazer & Nurse, 1992). the proposed research is aimed at understanding what turns this switch on and off and what happens when the switch is flipped. The four Specific Aims of the proposed research are to: A) Characterize cell cycle stage specific changes in the behavior of dcd1 and fyt1 proteins. Antibodies to these two proteins will be used to monitor cell cycle changes in their intracellular distribution and their association with the chromatin and each other; B) Identify the proteins that convey the signal for the completion of mitosis to dcd1. Genetic screens will be used to isolate both extragenic suppressors of dcd1-1ts and additional mutants with the dcd1-1ts phenotype; C) Identify proteins that are the effectors of fyt1 and/or the activators of fyt1 GTPase activity. Mutations will be introduced into the fyt1 gene in regions comparable to those in p21ras that are known to be important for effector interaction and for nucleotide binding and hydrolysis. Aberrant phenotypes of cells expressing these genes will be characterized and extragenic suppressors isolated; D) Isolate additional mutants defective in the ability to re-enter the cell cycle following mitosis. The pilot screen in which the dcd1-1ts mutant was identified will be continued to isolate new mutants defective in the mitosis-to-interphase transition.

拟议研究的长期目标是阐明 在分裂酵母中调节退出有丝分裂的机制 裂殖酵母 的分离和表征。 裂殖酵母dcd 1 - 1 ts突变体导致了两个鉴定 进化上保守的蛋白质， 与有丝分裂完成相关的结构变化和重新 间期状态的建立（Sazer和Nurse，1992年）。 dcd1 基因编码一种类似于人类染色质结合蛋白的蛋白质 RCC 1。 dcd 1 - 1 ts的抑制因子fyt 1与dcd 1 - 1 ts的抑制因子fyt 1几乎相同。 人GTP结合蛋白家族，以人原 癌基因p21 ras。 这两个S.粟酒裂殖酵母蛋白质 已知功能的蛋白质，以及已知结构的p21 ras 同时，提供一个框架，在此框架内设计实验， 这些和相关蛋白在有丝分裂退出中的作用。 知识 从拟议的研究中获得的信息应该会进一步加深我们对 这些人类蛋白质在细胞周期控制、信号转导 途径和ras介导的癌症。 dcd 1和fyt 1蛋白是 在染色体中起关键作用的分子开关的核心 解凝聚（Sazer & Nurse，1992）。 拟议的研究旨在 了解是什么打开和关闭了这个开关， 开关已翻转。 拟议研究的四个具体目标是 目的：A）表征细胞周期阶段特异性的行为变化 dcd 1和fyt 1蛋白质。 将使用针对这两种蛋白质的抗体 监测细胞内分布的细胞周期变化， 它们与染色质的关联以及它们彼此的关联; B）鉴定 将完成有丝分裂的信号传递给dcd 1的蛋白质。 基因筛选将用于分离两种外源基因抑制因子， dcd 1 - 1 ts和具有dcd 1 - 1 ts表型的其他突变体; C）鉴定 作为Fyt 1的效应物和/或Fyt 1的激活物的蛋白质 GT活性。 突变将被引入fyt 1基因， 与p21 ras中的那些区域相当的区域，已知这些区域对于 效应物相互作用以及核苷酸结合和水解。 异常 将表征表达这些基因的细胞的表型， 分离的基因外抑制子; D）分离另外的缺陷突变体 在有丝分裂后重新进入细胞周期的能力。 试点 将继续进行鉴定dcd 1 - 1 ts突变体的筛选， 分离在有丝分裂到间期转变中有缺陷的新突变体。