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.

微丝是所有桉树的主要细胞骨架元素 细胞。 与肌动蛋白相关的大量蛋白质 较高的细胞已被鉴定和表征，但是它们在 体内角色经常理解不足。 无处不在的本质 微丝及其在肿瘤中的改变状态 转化的细胞强调研究其的重要性 生物功能。 现在是时候分析所有 一种细胞类型的微丝相关蛋白，并精确 每种蛋白质的生物学作用。 对于这样一个项目，是 希望使用合并的生化，遗传和细胞 生物学方法。 因此，我们选择了萌芽的酵母， 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 必需的肌动蛋白基因。 我们已经开始分离微丝 - 来自该生物的相关蛋白质，包括肌动蛋白，A 滋养素样蛋白和推定的Ca2+调节的F-肌动蛋白 切断的蛋白质。 我们建议在 细节并将它们与较高的细胞对应物进行比较 确定它们的特性以及酵母有用的用作 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 然后建议准备针对这些蛋白质的每一种抗体 将它们定位在酵母细胞中并分离它们各自的基因 来自lambda GT11表达载体中的DNA库。 什么时候 这些基因已被分离为基因破坏实验 用于确定蛋白质是否执行必不可少的 功能。 如果单倍体中的这些干扰会导致细胞死亡，我们 将启动一个隔离温度敏感的项目 每个基因的突变。 然后，我们将探索单元格 温度敏感突变的生物学后果 这些细胞在限制性温度下。 这将导致 未来对基因抑制器的遗传分析以识别 相互作用的微丝相关蛋白。 通过这项工作，我们 期望在生化和遗传上确定 酵母是研究功能的良好模型系统 微丝组织。 如果是这样，它应该为 对这些重要的细胞骨架元素的透彻分析。