MOLECULAR ANALYSIS OF MICROTUBULE FUNCTION IN YEAST

微管是微管蛋白的聚合物，它参与了一系列 包括染色体分离在内的运动过程， 轴浆运输和细胞形态的建立。不适当 染色体分离导致多种遗传性疾病 比如唐氏综合症和自然流产。这个 拟议中的研究旨在从分子水平上确定 微管在酿酒酵母中起作用。 酵母菌是一种很容易通过基因和 应该能够对这些复杂的过程进行快速分析。这些 具体问题将会得到解答： 1.酵母中微管的特殊功能是什么？ TUB1和TUB2基因的条件性致死突变 分别编码α和β微管蛋白，将产生 体外诱变。全谱的表型分析 突变体将允许对微管进行详细的表征 在体内发挥作用。 2.哪些非微管蛋白对微管是必需的 功能呢？ 微管功能的多样性必须由 调控微管组装的非微管蛋白，建立 微管和其他细胞结构之间的相互作用，以及 产生机动性所需的力量。我们将使用抑制器 分析以确定其产物与之相互作用的基因 微管。不同类型微管蛋白的抑制因子 突变应该代表所涉及的不同类别的蛋白质 在微管功能方面。 3.非微管蛋白对微管功能有何影响？ 将获得抑制基因的条件致死等位基因 通过直接选择或通过体外诱变 克隆的基因。对突变体的表型分析将决定 这些蛋白质在体内的作用。将使用特定的抗体 以确定它们是否与微管或其他 细胞结构。纯化的蛋白质将被检测其 在体外影响微管组装的能力。

Microtubules are polymers of tubulin that are involved in a range of motile processes including chromosome segregation, axoplasmic transport, and establishment of cell shape. Improper chromosome segregation causes a variety of genetic disorders such as Down's syndrome and spontaneous fetal abortion. The proposed research is designed to determine at molecular level how microtubules function in the yeast Saccharomyces cerevisiae. Yeast is an organism which is easily manipulated genetically and should allow rapid analysis of these complex processes. These specific questions will be addressed: 1. What are the specific functions of microtubules in yeast? Conditional-lethal mutations in TUB1 and TUB2, the genes encoding alpha- and beta-tubulin respectively, will be produced by in vitro mutagenesis. Phenotypic analysis of a full spectrum of mutants will allow a detailed characterization of microtubule function in vivo. 2. What nontubulin proteins are essential for microtubule function? The diversity of microtubule function must be mediated by nontubulin proteins that regulate microtubule assembly, establish interactions between microtubules and other cell structures, and generate the force required for mobility. We will use suppressor analysis to identify genes whose products interact with microtubules. Suppressors of different classes of tubulin mutations should represent different classes of proteins involved in microtubule function. 3. How do nontubulin proteins influence microtubule function? Conditional-lethal alleles of suppressor genes will be obtained either by selecting for them directly or by in vitro mutagenesis of the cloned genes. Phenotypic analysis of mutants will determine the role of these proteins in vivo. Specific antibodies will be used to establish whether they associate with microtubules or other cellular structures. Purified proteins will be assayed for their ability to influence microtubule assembly in vitro.