GENETIC ANALYSIS OF EXIT FROM MITOSIS IN S CEREVISIAE

酿酒酵母有丝分裂退出的遗传分析

• 批准号: 2603528
• 负责人: WENDY E RAYMOND
• 金额: $ 11.36万
• 依托单位: WILLIAMS COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-05-01 至 2002-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2603528
• 关键词: Saccharomyces cerevisiae; cell cycle; fungal genetics; gene expression; genetic regulation; molecular cloning; suppressor mutations; western blottings

DESCRIPTION: (Adapted from the applicant's proposal) Cells exiting mitosis undergo a series of dramatic events: the mitotic spindle breaks down, p34CD28/cyclin B mitotic kinase activity diminishes, chromosomes decondense and reconfigure, cytokinesis occurs, and the cellular machinery is reset for entry into the ensuing G1 phase. The molecular mechanisms that regulate and coordinate these essential processes are poorly understood. This project seeks to identify and characterize genes that execute or regulate cellular activities at this critical stage of the cell cycle. A genetic method and accompanying molecular approaches will investigate the role that CDC14, a Saccharomyces cerevisiae cell-division cycle gene required for completion of telophase, play in exit from mitosis. The particular function of CDC14 in the telophase-to-G1 transition is unknown. To investigate the pathway in which CDC14 acts, dominant extragenic suppressor of temperature-sensitive alleles of cdc14 will be isolated. Dominant extragenic suppressors of cdc14 may identify genes whose products physically associate with CDC14 and are thus themselves important for late mitototic events. Genes which regulate expression of CDC14, or which act downstream of CDC14, may also be identified by this approach. Cloning the suppressor genes, along with cytological and additional genetic analyses of suppressor mutants, will further define the roles of these newly identified genes in exit from mitosis. CDC14 encodes a putative protein tyrosine phosphatase. Protein tyrosine phosphatases are important in cellular differentiation, development, and cancer, but their precise functions in signaling pathways that control these processes are not well understood. If protein dephosphorylation led by CDC14 regulates exit from mitosis, genes identified as suppressors of cdc14 may be components of such a signaling pathway. An additional specific aim of this proposal is to purify CDC14 protein produced in E. coli, for use in generating polyclonal antibodies against CDC14. These antibodies will be used in future experiments designed to identify CDC14 interactions with other polypeptides. In the current proposal, western blot analysis using anti-CDC14 antibodies will ascertain whether a subset of the cdc14-suppressor mutants may identify genes whose encoded proteins interact with CDC14.

描述：（改编自申请人的提案）退出有丝分裂的细胞 经历了一系列戏剧性的事件：有丝分裂纺锤体分解， p34 CD 28/细胞周期蛋白B有丝分裂激酶活性降低，染色体 去致密化和重组，胞质分裂发生，细胞机器 被重置以进入随后的G1阶段。的分子机制 调节和协调这些基本过程的能力很差， 明白该项目旨在识别和表征基因， 在细胞的这个关键阶段执行或调节细胞活动 周期遗传方法和伴随的分子方法将 研究CDC 14，酿酒酵母细胞分裂的作用 完成末期所需的周期基因，在退出时发挥作用， 分裂。 CDC 14在终末期至G1期转变中的特殊功能是 未知为了研究CDC 14的作用途径，显性CDC 14在细胞内的表达， CDC 14的温度敏感等位基因的基因外抑制基因将被 与世隔绝cdc 14的显性基因外抑制因子可以识别基因 其产物与CDC 14物理结合，因此其本身 对晚期有丝分裂事件很重要。调节以下基因表达的基因 CDC 14或作用于CDC 14下游的CDC 14也可以通过此来识别 approach.克隆抑癌基因，沿着细胞学和 对抑制突变体的额外遗传分析，将进一步确定 这些新发现的基因在退出有丝分裂中的作用。 CDC 14编码一种推定的蛋白酪氨酸磷酸酶。蛋白酪氨酸 磷酸酶在细胞分化、发育和分化中非常重要。 癌症，但它们在控制癌症的信号通路中的精确功能 这些过程还没有被很好地理解。如果蛋白质去磷酸化导致 由CDC 14调节退出有丝分裂，基因被鉴定为抑制因子， CDC 14可以是这种信号传导途径的组分。 本发明的另一个具体目的是纯化CDC 14蛋白 在E.大肠杆菌，用于产生抗 CDC 14.这些抗体将用于未来的实验， 鉴定CDC 14与其它多肽的相互作用。在当前 建议，使用抗CDC 14抗体的蛋白质印迹分析将确定 CDC 14抑制突变体的子集是否可以鉴定出 编码的蛋白质与CDC 14相互作用。