VACUOLE DIVISION/SEGREGATION DURING THE YEAST CELL CYCLE

酵母细胞周期期间的液泡分裂/分离

• 批准号: 2179591
• 负责人: WILLIAM Tobey WICKNER
• 金额: $ 27.85万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-07-01 至 1996-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2179591
• 关键词: Golgi apparatus; Saccharomyces cerevisiae; cell cycle; cytogenetics; cytoskeleton; dynein ATPase; fluorescence microscopy; fluorescent dye /probe; freeze etching; gene mutation; immunoprecipitation; kinesin; laboratory rabbit; lethal genes; lysosomes; membrane proteins; microtubule associated protein; molecular cloning; monoclonal antibody; mutant; nucleic acid sequence; optics; organelles; protein kinase; protein transport; temperature sensitive mutant; vesicle /vacuole

The division and segregation of cytoplasmic organelles must be spatially and cell-cycle regulated to insure faithful inheritance through many cell divisions. Study of this process for the vacuole (lysosome) of S. cerevisiae during the last 5 years has 1. established the cytology of its division and inheritance pattern 2. led to vac mutants which are specifically defective in vacuole inheritance, and 3. provided an in vitro reaction, the formation of vacuole-derived tubulo-vesicular protuberances, which faithfully reflects the in vivo inheritance process. This in vitro reaction requires semi-intact cells or isolated vacuoles, cytosol, ATP, GTP, and incubation at 23 to 37 degrees C and depends on tubulin and the VAC1 and VAC2 proteins. Planned studies will combine this cytology, genetics and biochemistry. New, and old, vac mutants will be collected, combined genetically to explore interrelationships, VAC genes cloned and sequenced, and antibody prepared for immunolocalization and to aid in biochemical assay. The components of the in vitro reaction will be fractionated and purified, using complementation of vac extracts, nucleotide affinity matrices, and total fractionation of the yeast cytosol, assaying for the fractions required to support tubulovesicle formation. The relationships of this reaction to proteins such as cyclins and kinases which govern the cell cycle and to MAPS such as kinesin and dynein which drive microtubule-based organelle motion will be explored. These studies will provide basic information on an important, yet little studied, aspect of cell division.

细胞质细胞器的分裂和分离必须是空间的 和细胞周期受监管以确保通过许多细胞的忠实继承 部门。 研究S.液泡（溶酶体）的这一过程 在过去的5年中，酿酒酵母有1个。 划分和继承模式2。导致VAC突变体 在液泡继承方面特殊有缺陷，3。 体外反应，形成液泡衍生的微管叶子 突出，忠实地反映了体内遗传过程。 这种体外反应需要半直体细胞或分离的液泡， 细胞质，ATP，GTP和在23至37度的孵育，取决于 微管蛋白以及VAC1和VAC2蛋白。 计划研究将结合 这种细胞学，遗传学和生物化学。 新的和旧的Vac突变体将 收集，遗传结合以探索相互关系，vac 克隆和测序的基因以及制备用于免疫定位的抗体 并有助于生化测定。 体外反应的成分 使用真空提取物的互补，将被分馏和纯化， 核苷酸亲和力矩阵和酵母的总分数 细胞质，分析支撑管状的馏分所需的分数 形成。 这种反应与蛋白质的关系，例如 控制细胞周期和地图（例如）的细胞周期蛋白和激酶 驱动基于微管的细胞器运动的动力素和动力蛋白将是 探索。 这些研究将提供有关重要的基本信息 然而，细胞分裂方面的研究很少。