MICROFILAMENTS IN THE YEAST SACCHAROMYCES CEREVISIAE

酿酒酵母中的微丝

• 批准号: 3295889
• 负责人: Anthony P. Bretscher
• 金额: $ 12.3万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295889
• 关键词: Saccharomyces cerevisiae; actins; alleles; antibody; cell biology; cytogenetics; fluorescence microscopy; fungal genetics; gel electrophoresis; gene expression; gene mutation; genetic library; genetic mapping; immunological substance; laboratory rabbit; microfilaments; molecular genetics; nucleic acid hybridization; nucleic acid sequence; temperature sensitive mutant; tropomyosin

Microfilaments are major cytoskeletal elements of all eucaryotic cells. A large number of proteins that associate with actin in higher cells have been identified and characterized, but their in vivo role is frequently poorly understood. The ubiquitious nature of microfilaments, and their altered state in oncogenically transformed cells stresses the importance of studying their biological functions. The time has come to analyze all the microfilament-associated proteins in one cell type and pinpoint the biological role of each protein. For such a project it, is desirable to use a combined biochemical, genetic and cell biological approach. We have therefore chosen the budding yeast, Saccharomyces cerevisiae, as our experimental organism as it is amenable to detailed genetic analysis and contains a single essential actin gene. We have begun to isolate microfilament- associated proteins from this organism, including actin, a trophomysin-like protein, and a putative Ca2+-regulated F-actin severing protein. We propose to characterize these proteins in detail and compare them with their higher cell counterparts to determine both their properties and how useful yeast will be as a model system. We also propose to search for and characterize other microfilament associated proteins in yeast extracts. We then propose to prepare antibodies to each of these proteins to localize them in yeast cells and to isolate their respective genes from a DNA library in the Lambda gt11 expression vector. When the genes have been isolated gene disruption experiments will be used to determine whether the proteins perform essential functions. If these disruptions in haploids result in cell death, we will embark on a project to isolate temperature-sensitive mutations in each of the genes. We will then explore the cell biological consequences of the temperature-sensitive mutations in these cells at the restrictive temperature. This will lead into a future genetic analysis of extragenic suppressors to identify interacting microfilament-associated proteins. By this work, we expect to determine, both biochemically and genetically, whether yeast is a good model system for studying the functional organization of microfilaments. If so, it should set the stage for the thorough analysis of these important cytoskeletal elements.

微丝是所有真核生物的主要细胞骨架元素。 细胞 大量与肌动蛋白结合的蛋白质在 高等细胞已被鉴定和表征，但它们的 体内作用常常知之甚少。 无处不在的自然 微丝，以及它们在致癌性中的改变状态， 转化细胞强调了研究其 生物功能。 是时候分析一下 一种细胞类型中的微生物相关蛋白， 每种蛋白质的生物学作用。 对于这样一个项目， 希望使用组合的生化、遗传和细胞 生物学方法。 因此我们选择了芽殖酵母， 酿酒酵母作为我们的实验生物 适合详细的遗传分析，并包含一个单一的 必需肌动蛋白基因 我们已经开始分离微丝- 相关蛋白质，包括肌动蛋白， 滋养细胞溶素样蛋白，和一个假定的钙离子调节的F-肌动蛋白 切断蛋白质。 我们建议将这些蛋白质的特征， 详细说明并将它们与更高级别的细胞对应物进行比较， 确定他们的属性和如何有用的酵母将作为一个 模型系统。 我们还建议寻找和表征 酵母提取物中的其他微丝相关蛋白。 我们 然后建议制备针对这些蛋白质的抗体， 将它们定位在酵母细胞中并分离它们各自的基因 来自λ gt 11表达载体中的DNA文库。 当 这些基因已经被分离出来， 用于确定蛋白质是否执行必需的 功能协调发展的 如果这些单倍体的破坏导致细胞死亡，我们 将着手进行一个项目， 每个基因的突变。 然后我们将探索细胞 温度敏感性突变的生物学后果 这些细胞处于限制性温度。 这将导致一个 未来的基因分析的基因外抑制，以确定 相互作用的微生物相关蛋白。 通过这项工作，我们 希望从生物化学和遗传学的角度来确定， 酵母是一个很好的研究功能的模型系统 微丝的组织。 如果是这样，它应该为 对这些重要的细胞骨架元素进行彻底的分析。