GAMMA TUBULIN AND CENTROSOME STRUCTURE AND FUNCTION

伽玛微管蛋白和中心体的结构和功能

• 批准号: 6019043
• 负责人: Tim Stearns
• 金额: $ 18.9万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-08-01 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019043
• 关键词: Schizosaccharomyces pombe; Xenopus oocyte; antibody; binding proteins; cell cycle; centrosome; enzyme linked immunosorbent assay; gene mutation; immunoprecipitation; laboratory rabbit; microtubules; molecular cloning; phenotype; polymerase chain reaction; protein biosynthesis; protein purification; protein structure function; tissue /cell culture; tubulin

The long term objectives are to understand the molecular mechanisms of microtubule organizing center function, including microtubule nucleation, microtubule organization, and cell cycle regulated duplication. These objectives are significant in that the microtubule organizing center is essential to cytoplasmic organization, morphological differentiation and mitotic spindle assembly and function. The general strategy is to focus on one important molecular, gamma-tubulin, characterize its role, and use it to identify other important molecules. The microtubule organizing center is a complex organelle, and gamma-tubulin is one of the only highly conserved, functionally important components known. Two experimental systems with distinct nd non-overlapping advantages for study of the organizing center will be used; the egg of the frog Xenopus laevis, and the fission yeast Schizosaccharomyces pombe. Four specific aims are proposed to address important questions of organizing center function and the role of gamma-tubulin in those functions: 1) Purify gamma-tubulin and characterize its activities on microtubules. gamma-Tubulin will be purified from frog eggs, a rich source. The effects of gamma-tubulin on microtubule nucleation, and the capping of microtubule ends will be examined,a nd athe molecular details of gamma-tubulin microtubule binding determined. gamma-Tubulin exists as a large complex in the cytoplasm: the composition of this complex will be determined. 2) Determine the requirements for assembly of the microtubule organizing center. An in vitro assay that reconstitutes the organizing center formation reaction that takes place during fertilization in Xenopus will be used to determine what components of the egg and sperm are required for the reaction. gamma-Tubulin is one of the components required for the reactions. The relationship of this assembly process to the cell cycle- regulation change in microtubule nucleation capacity will be examined. 3) Study in vivo role of gamma-tubulin in microtubule organization. Conditional-lethal mutations will be isolated int he S. pombe gamma-tubulin gene, and the phenotypes of these mutations will be analyzed in detail. These phenotypes will reveal the in vivo role of gamma-tubulin in a simple microtubule system. 4) Identify proteins that interact with gamma-tubulin and assess their function. The conditional-lethal gamma-tubulin mutations will be used in genetic screens to identify new components of the organizing center, and these new components will be studied genetically to determine their function and their relationship to gamma-tubulin.

长期的目标是了解的分子机制， 微管组织中心功能，包括微管成核， 微管组织和细胞周期调控的复制。 这些 目的是重要的，因为微管组织中心是 对细胞质组织、形态分化和 有丝分裂纺锤体组装和功能。 总的战略是把重点放在 一个重要的分子，γ-微管蛋白，表征其作用，并使用它 来识别其他重要的分子。 微管组织中心 是一个复杂的细胞器，而γ-微管蛋白是唯一一个高度 保守的，功能上重要的已知成分。 两个实验 具有独特和非重叠优势的系统，用于研究 组织中心将被使用;青蛙非洲爪蟾的卵， 裂殖酵母 四个具体目标是 建议解决组织中心职能的重要问题， γ-微管蛋白在这些功能中的作用： 1)纯化γ-微管蛋白并表征其在微管上的活性。 γ-微管蛋白将从青蛙卵中纯化，这是一个丰富的来源。 的影响 γ-微管蛋白对微管成核和微管帽的影响 末端进行检查，并观察γ-微管蛋白的分子细节 微管结合测定。 γ-微管蛋白作为一种大的复合物存在于 细胞质：将确定该复合物的组成。 2)确定微管组装的要求 中心 一种重建组织中心的体外试验 在非洲爪蟾受精过程中发生的形成反应将是 用于确定需要卵子和精子的哪些成分， 反应 γ-微管蛋白是一种必需的成分， 反应. 组装过程与细胞周期的关系- 将检查微管成核能力的调节变化。 3)研究γ-微管蛋白在微管组织中的体内作用。 条件致死突变将在S.粟酒γ-微管蛋白 基因，并将详细分析这些突变的表型。 这些表型将揭示γ-微管蛋白在体内的作用，在一个简单的 微管系统 4)鉴定与γ-微管蛋白相互作用的蛋白质，并评估其 功能 条件致死性γ-微管蛋白突变将用于遗传学研究。 屏幕来识别组织中心的新组件，这些新组件 将对这些成分进行遗传学研究，以确定它们的功能， 与γ-微管蛋白的关系