MOLECULAR ANALYSIS OF THE YEAST ACTIN CYTOSKELETON

酵母肌动蛋白细胞骨架的分子分析

• 批准号: 2183046
• 负责人: ALISON E ADAMS
• 金额: $ 12.94万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-05-01 至 1996-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2183046
• 关键词: Saccharomyces cerevisiae; actin binding protein; actins; autoradiography; cell motility; chickens; cytoskeleton; fluorescence microscopy; fungal genetics; gene complementation; gene expression; gene interaction; gene mutation; genetic manipulation; human tissue; laboratory rabbit; molecular cloning; molecular genetics; oligonucleotides; protein biosynthesis; protein sequence; protein structure function; radionuclides; site directed mutagenesis; temperature sensitive mutant; ultraviolet spectrometry

The actin cytoskeleton has been implicated in a large variety of motile functions, as well as in the structural and mechanical properties of the cytoplasmic matrix. Except in muscle cells, however, very little is known about its composition, roles, or mechanism of action. The proposed research is designed to determine at the molecular level how the actin cytoskeleton functions in the yeast Saccharomyces cerevisiae. Yeast can be readily manipulated genetically, and should allow rapid and detailed analysis of these complex processes. Because actin is highly conserved from yeast to man, what is learned from these studies is likely to apply to higher cells, where defective actin cytoskeleton function is implicated in pathological conditions such as muscular dystrophy and neoplastic transformation. The following specific questions will be addressed: 1. How does SAC6p, an actin-binding protein, function in the cytoskeleton? Conditional-lethal mutations in the SAC6 gene will be produced by in vitro mutagenesis. Biochemical and physiological analyses of a full spectrum of mutants will then allow activities observed in vitro to be correlated with functions seen in vivo. 2. What residues or domains are important for function of SAC6p and its higher-cell homologs? Fine-structure mapping of mutations will be used to identify regions of the gene associated with various C6p activities. Specific hypotheses concerning sequences such as the putative actin-binding domain (a highly conserved 25 amino acid sequence found in a number of actin-binding proteins) will be tested by site-specific mutagenesis. Information from these studies will be used in an analysis of human plastin and chicken fimbrin, which are 40% identical to SAC6p. 3. What other proteins are involved in the function of the actin cytoskeleton? The diversity of actin cytoskeletal function must be mediated by non-actin proteins that regulate actin assembly, establish interactions between the cytoskeleton and other cell structures, and generate the force for intracellular motility. A variety of genetic methods will be used to identify additional components of the actin cytoskeleton, and proteins that interact with it.

肌动蛋白细胞骨架参与了许多运动性的 功能，以及在结构和机械性能的 细胞质基质然而，除了肌肉细胞， 关于它的组成、作用或作用机制。拟议研究 旨在从分子水平上确定肌动蛋白细胞骨架 在酿酒酵母中发挥作用。酵母可以很容易地 基因操纵，并应允许快速和详细的分析， 这些复杂的过程。由于肌动蛋白从酵母到 伙计，从这些研究中学到的东西很可能适用于高等细胞， 其中有缺陷的肌动蛋白细胞骨架功能与病理性 例如肌肉萎缩症和肿瘤转化。的 将解决以下具体问题： 1. SAC6p是一种肌动蛋白结合蛋白，在细胞骨架中如何发挥作用？ SAC6基因中的条件性致死突变将通过体外培养产生。 诱变全谱的生化和生理分析， 然后，突变体将允许在体外观察到的活性与 在体内观察到的功能。 2.哪些残基或结构域对SAC6p及其 高级细胞同源物突变的精细结构定位将用于 鉴定与各种C6p活性相关的基因区域。 关于序列的特定假设，如假定的肌动蛋白结合 结构域（在许多人中发现的高度保守的25个氨基酸序列） 肌动蛋白结合蛋白）将通过位点特异性诱变进行测试。 来自这些研究的信息将用于分析人血浆蛋白 和鸡胚，与SAC6p有40%相同。 3.还有哪些蛋白质参与了肌动蛋白的功能 细胞骨架肌动蛋白细胞骨架功能的多样性必须通过 通过调节肌动蛋白装配的非肌动蛋白， 在细胞骨架和其他细胞结构之间， 用于细胞内运动。将采用多种遗传学方法， 确定肌动蛋白细胞骨架的其他成分，以及 与之互动。