GAMMA TUBULIN AND CENTROSOME STRUCTURE AND FUNCTION

长期的目标是了解其分子机制。 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 目标很重要，因为微管组织中心是 对细胞质组织、形态分化和 有丝分裂纺锤体的组装和功能。总体战略是将重点放在 一种重要的分子，伽马微管蛋白，描述了它的作用，并使用它 来识别其他重要的分子。微管组织中心 是一种复杂的细胞器，而伽马微管蛋白是仅有的高度 已知的保守的、功能重要的组件。两个实验性的 具有明显和非重叠优势的系统，用于研究 将使用组织中心；非洲爪蛙卵，以及 裂殖酵母：裂殖酵母。四个具体目标是 提出要解决组织中心职能和组织机构的重要问题 伽马微管蛋白在这些功能中的作用： 1)纯化γ-微管蛋白并鉴定其微管活性。 伽马微管蛋白将从青蛙卵中提纯，这是一种丰富的来源。其影响 γ-微管蛋白对微管成核和微管封端的影响 将检查末端，以及伽马微管蛋白的分子细节 确定了微管结合。γ-微管蛋白以一种大型复合体的形式存在于 细胞质：这个复合体的组成将被确定。 2)确定微管组织的组装要求 中间。一种重组组织中心的体外试验 在非洲爪哇受精过程中发生的形成反应将是 用来确定卵子和精子的哪些成分是 反应。伽马-微管蛋白是 反应。这种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)体内研究γ-微管蛋白在微管组织中的作用。 条件致死突变将在S.pombeγ-微管蛋白中分离出来 这些突变的表型将被详细分析。 这些表型将揭示γ-微管蛋白在体内的作用 微管系统。 4)确定与γ-微管蛋白相互作用的蛋白质，并评估其 功能。 条件性致命性伽马微管蛋白突变将用于遗传 屏幕以确定组织中心的新组成部分，以及这些新的 将对成分进行遗传研究，以确定其功能和 它们与伽马微管蛋白的关系。

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.