MICROFILAMENTS IN THE YEAST SACCHAROMYCES CEREVISIAE

微丝基本上存在于所有的真核细胞中，在这些细胞中 为基本蜂窝网络提供结构性、动态支持的功能 流程。我们已经启动了一个项目来识别分子 酵母菌微丝细胞骨架的组成 并利用可用于酿酒的优秀分子遗传学 生物体来探索每种成分的功能。我们发现， 酵母只有一个基因，编码一种蛋白质，它与许多 高等细胞原肌球蛋白的特性。扰乱了 酵母中的原肌球蛋白基因，命名为TPM1，不是致死的，但提供 生长减慢，肌动蛋白细胞骨架异常，以及 细胞内有丰富的小泡。我们建议使用基因和 生物化学方法鉴定与之共同发挥功能的蛋白质 酵母中的原肌球蛋白。基因筛查将识别哪些突变 只有在原肌球蛋白耗尽的细胞中才会变得致命。这些合成材料 致命突变将识别一组将被克隆的基因， 对它们进行了测序，并分析了它们被破坏的生理后果。 我们还将在TPM1基因上产生突变，使 蛋白质，原肌球蛋白参与的中毒过程。这将是 允许直接识别这些过程。相互作用的蛋白质 将对原肌球蛋白进行生化鉴定、提纯和 特色化的。指定这些蛋白质的基因将被克隆并 测序并进行遗传分析。积聚的小泡 在原肌球蛋白缺陷的细胞中将被纯化并鉴定为 确定它们所在的细胞内囊泡路径。它也是 提议继续开展另外两个项目。我们对学习很感兴趣 调节肌动蛋白单体与聚合体比例的蛋白质。我们会 扩展我们对AMB1基因的分析，AMB1基因的产物与单体肌动蛋白结合。 我们还将利用单个肌动蛋白的过度表达这一事实 基因，ACT1，是致命的。结合单体的蛋白质的过度表达 肌动蛋白可能能够抑制这种致命性。我们已经建造了和 描述了一个酵母调控的cdna表达文库；我们将使用 筛选过表达抑制酵母基因克隆的文库 与肌动蛋白过度表达相关的致命性。我们还将使用 该文库用于识别其过度表达干扰的蛋白质 肌动蛋白细胞骨架的正常功能。这些研究应该 大大推进了我们揭示肌动蛋白及其功能的目标 酵母中的相关蛋白质。

Microfilaments are found In essentially all eucaryotic cells where they function to provide structural, yet dynamic, Support for basic cellular processes. We have initiated a project to identify the molecular components of the microfilament-based cytoskeleton in Saccharomyces cerevisiae and to use the excellent molecular genetics available in this organism to probe the functions of each component. We have found that yeast contains a single gene encoding a protein with many of the characteristic properties of higher cell tropomyosins. Disruption of the tropomyosin gene, designated TPM1, in yeast is not lethal but confers slower growth, an aberrant actin cytoskeleton, and the accumulation of abundant Intracellular vesicles. We propose to use genetic and biochemical approaches to identify proteins that function together with tropomyosin in yeast. Genetic screens will identify mutations which become lethal only in cells depleted of tropomyosin. These synthetic lethal mutations will identify a set of genes that will be cloned, sequenced and the physiological consequences of their disruption analyzed. We shall also generate mutations in the TPM1 gene that make a lethal protein, poisoning processes in which tropomyosin is involved. This shall allow a direct identification of these processes. Proteins that interact physically with tropomyosin will be identified biochemically, purified and characterized. Genes specifying these proteins will be cloned and sequenced and subjected to genetic analysis. The vesicles that accumulate in tropomyosin-deficient cells will be purified and characterized to determine in which intracellular vesicular pathway they lie. It is also proposed to continue two other projects. We are interested in studying proteins that modulate the ration of actin monomer to polymer. We will extend our analysis of the AMB1 gene, whose product binds monomeric actin. We will also make use of the fact that over-expression of the single actin gene, ACT1, is lethal. Over-expression of proteins that bind monomeric actin may be able to suppress this lethality. We have constructed and characterized a yeast regulated cDNA expression library; we shall use this library to identify yeast cDNA clones whose over-expression can suppress the lethality associated with actin over-expression. We shall also use this library to identify proteins whose over-expression interferes with the normal function of the actin cytoskeleton. These studies should greatly advance our goal of uncovering the function of actin and its associated proteins in yeast.