MOLECULAR ANALYSIS OF FLAGELLAR AUTOTOMY

鞭毛自切的分子分析

• 批准号: 2184397
• 负责人: JONATHAN W JARVIK
• 金额: $ 14.1万
• 依托单位: CARNEGIE-MELLON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2184397
• 关键词: Chlamydomonas; G protein; alternatives to animals in research; calcium binding protein; cilium /flagellum motility; electron microscopy; flagellum; gene expression; gene mutation; light microscopy; microtubules; molecular cloning; nucleic acid sequence; phenotype; protein structure function

The investigation proposed in this application is aimed towards a molecular understanding of the process of flagellar shedding, also called flagellar autotomy, in the unicellular eucaryote Chlamydomonas reinhardtii. Autotomy occurs at a specific site - the cell-distal end of the flagellar transition region - and it results in the release of the flagellum without compromising the integrity of the cell. At first acquaintance, autotomy seems to be a peculiar process with little general cell biological relevance. In fact, just the opposite is true: autotomy is a property of ciliated cells in general, and it involves a number of familiar and fundamental processes, including signalling and membrane fusion, as well as at least one unfamiliar and therefore quite remarkable process -microtubule severing. The experiments proposed in this application make use of recent advances in Chlamydomonas molecular genetic technology to test specific hypotheses about the structural and biochemical nature of autotomy. And, because we can readily assay for autotomy in vitro using detergent extracted cell models or isolated nucleo-basal body apparatuses, the prospect exists for a direct biochemical test of any model(s) favored by the genetic and molecular data. The proposed project has three major foci. The first focus is on the calcium binding protein centrin and its role in autotomy. The gene encoding centrin has been cloned and sequenced, and we have already obtained and analyzed a number of mutations in the gene. We will construct a centrin gene disruption and isolate new centrin mutants, both by selection and by in vitro mutagenesis, to identify those with specific autotomy defects. By examining these mutants at the biochemical functional and structural levels, we hope to acquire a molecular understanding of centrin's role'(s) in autotomy. The second focus is on proteins that interact with centrin; we will attempt to identify centrin binding proteins, and the genes that encode them, using a screen for lambda gt11 clones that encode centrin-binding fusion proteins. The third focus is on genes identified by mutations with autotomydefective phenotypes. We will clone, sequence and analyze a known gene,fa-1, that is required for microtubule severing, and we will identify - and when appropriate isolate, clone and sequence -additional autotomy genes. In all of these cases, once genes of interest have been isolated, we will prepare specific antibodies to the proteins that they encode and use them as probes for protein localization and function.

本申请中提出的调查旨在 对鞭毛脱落过程的分子理解，也称为 单细胞真核生物衣藻的鞭毛自切 莱茵哈蒂。自切发生在特定部位——细胞远端 鞭毛过渡区 - 它导致释放 鞭毛而不损害细胞的完整性。起初 相识后，自切似乎是一个特殊的过程，没有什么通用性 细胞生物学相关性。事实上，事实恰恰相反：自切是 一般而言，纤毛细胞的一种特性，它涉及许多 熟悉的基本过程，包括信号传导和膜 融合，以及至少一种不熟悉的、因此相当引人注目的融合 过程-微管切断。本文提出的实验 应用利用衣藻分子遗传学的最新进展 测试有关结构和结构的具体假设的技术 自切的生化性质。而且，因为我们可以很容易地分析 使用去污剂提取的细胞模型或分离的体外自切术 核基底体装置，存在直接的前景 遗传和分子支持的任何模型的生化测试 数据。 拟议项目有三个主要重点。第一个重点是 钙结合蛋白中心蛋白及其在自切术中的作用。基因 编码中心蛋白已经被克隆和测序，我们已经 获得并分析了该基因的一些突变。我们将建设 中心蛋白基因破坏并分离新的中心蛋白突变体，均通过 通过选择和体外诱变，鉴定具有特定特征的 自切缺陷。通过检查这些突变体的生化功能 和结构水平，我们希望获得分子上的理解 centrin 在自切术中的作用。第二个重点是蛋白质 与中心蛋白相互作用；我们将尝试识别中心蛋白结合 使用 lambda gt11 筛选蛋白质以及编码它们的基因 编码中心蛋白结合融合蛋白的克隆。第三个重点是 通过具有自切缺陷表型的突变鉴定出的基因。我们将 克隆、测序和分析已知基因 fa-1，这是 微管切断，我们将识别 - 并在适当时分离， 克隆和测序-额外的自切基因。在所有这些情况下，一旦 感兴趣的基因已分离，我们将制备特异性抗体 到它们编码的蛋白质并将其用作蛋白质探针 定位和功能。