MICROFILAMENTS IN THE YEAST SACCHAROMYCES CEREVISIAE

酿酒酵母中的微丝

• 批准号: 3295886
• 负责人: Anthony P. Bretscher
• 金额: $ 10.95万
• 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 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3295886
• 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; 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。与肌动蛋白相关的大量蛋白质 高等细胞已经被鉴定和表征，但它们在 人们对活体的作用往往知之甚少。无处不在的自然 微丝和它们在致癌过程中的变化状态 转化细胞强调研究它们的重要性 生物功能。现在是时候分析所有 一种细胞类型和Pinpoint中的微丝相关蛋白 每种蛋白质的生物学作用。对于这样的一个项目来说，是 希望使用生化、遗传和细胞的组合 生物学方法。因此，我们选择了萌芽酵母， 酿酒酵母，作为我们的实验有机体 服从详细的基因分析，并包含单一的 必需肌动蛋白基因。我们已经开始分离微丝- 来自这种有机体的相关蛋白质，包括肌动蛋白，一个 滋养肌素样蛋白和一个可能受钙离子调节的F-肌动蛋白 切断蛋白质。我们建议将这些蛋白质描述为 详细说明它们并将其与更高级别的单元格进行比较 确定它们的性质以及酵母作为一种 模型系统。我们还建议搜索和表征 酵母提取物中的其他微丝相关蛋白。我们 然后建议制备针对每种蛋白质的抗体以 在酵母细胞中定位它们并分离它们各自的基因 来自Lambda gt11表达载体中的DNA文库。什么时候 已经分离出的基因将进行基因破坏实验 用来确定蛋白质是否执行必要的 功能。如果单倍体的这些破坏导致细胞死亡，我们 将着手一项分离温度敏感物质的项目 每个基因都发生了突变。然后我们将探索细胞 温度敏感型突变的生物学后果 这些细胞在有限的温度下。这将导致一个 未来对基因外抑制基因的分析以确定 相互作用的微丝相关蛋白。通过这项工作，我们 预计将从生物化学和遗传角度确定 酵母菌是研究功能的良好模型系统 微丝的组织。如果是这样的话，它应该为 对这些重要的细胞骨架元素的彻底分析。