MOLECULAR ANALYSIS OF MICROTUBULE FUNCTION IN YEAST

微管是微管蛋白的聚合物， 包括染色体分离在内的运动过程， 轴浆运输和细胞形状的建立。 不当 染色体分离导致多种遗传疾病 例如唐氏综合症和自然流产。 的 拟议的研究旨在确定在分子水平上如何 微管在酿酒酵母中起作用。 酵母是一种易于遗传操纵的生物体， 可以快速分析这些复杂的过程。 这些 将讨论以下具体问题： 1. 酵母中微管的具体功能是什么？ TUB1和TUB2中的条件性致死突变， 分别编码α-微管蛋白和β-微管蛋白，将由 体外诱变。 全谱表型分析 突变体将允许微管的详细表征 在体内发挥作用。 2. 什么样的非微管蛋白质对微管是必需的 功能？ 微管功能的多样性必须通过 调节微管组装的非微管蛋白， 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 微管 不同类别微管蛋白的抑制因子 突变应该代表不同种类的蛋白质 在微管功能中。 3. 非微管蛋白如何影响微管功能？ 将获得抑制基因的条件致死等位基因 通过直接选择它们或通过体外诱变 克隆的基因 突变体的表型分析将确定 这些蛋白质在体内的作用。 将使用特异性抗体 以确定它们是否与微管或其他 细胞结构 将测定纯化蛋白的 影响体外微管组装的能力。

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.