MOLECULAR MECHANISM OF FLAGELLAR MOTILITY

鞭毛运动的分子机制

• 批准号: 3278429
• 负责人: George B Witman
• 金额: $ 27.75万
• 依托单位: WORCESTER FOUNDATION FOR BIOMEDICAL RES
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-08-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3278429
• 关键词: Chlorophyta; SDS polyacrylamide gel electrophoresis; antisense nucleic acid; binding proteins; cilium /flagellum motility; complementary DNA; computer assisted sequence analysis; crosslink; dynein ATPase; gene mutation; genetic mapping; immunoelectron microscopy; immunoprecipitation; microtubule associated protein; molecular cloning; monoclonal antibody; nucleic acid hybridization; protein purification; protein structure function; restriction fragment length polymorphism; site directed mutagenesis; tubulin; western blottings

The long term goals are to determine the molecular structure of the outer arm dynein of cilia and flagella, and to elucidate the functions of the individual subunits, polypeptides, and domains of polypeptides in this important mechanochemical transducer. The work will concentrate on the outer arm dynein of Chlamydomonas, which structurally and compositionally is the best characterized of all dyneins. One of the dynein intermediate chains, IC 78, is located at the base of the arm and is the major polypeptide cross-linked to tubulin in the axoneme. A full-length cDNA clone encoding IC 78 has been isolated and sequenced; it will now be determined if there is an existing null mutant for IC 78. If not, one will be produced by homologous recombination or antisense-RNA technology. The cross-linked IC 78/tubulin peptides will be isolated and sequenced, and site-directed mutagenesis and transformation of the null mutant used to determine the significance of the IC 78/tubulin interaction. The IC 78 gene also will be randomly mutagenized and introduced into the null mutant to determine what roles other parts of IC 78 have in arm assembly and function. Existing monoclonal antibodies and immunoelectron microscopy will be used to locate regions of the dynein heavy chains in the isolated particles. An existing genomic clone encoding part of the gamma heavy chain will be used to isolate cDNA clones covering the entire gamma chain transcript; these will be sequenced and analyzed to sites of the gamma chain will be isolated and sequenced. Site-directed mutagenesis and transformation will be used to eliminate the gamma chain's ATP-hydrolytic activity and thus determine the role of that chain in outer arm function. Cells also will be transformed with gamma chain genes in which judiciously chosen regions have been deleted or randomly mutagenized, and selected transformants analyzed to identify other functionally important domains. The force-producing and microtubule-binding properties of individual dynein subunits will be examined under different physiological conditions to better understand the roles of each subunit in the generation and control of flagellar movement. Dynein arms generate the forces that are the basis for motility in all eukaryotic cilia and flagella. Because the basic structure and composition of dynein appears to have been conserved throughout evolution, the fundamental information obtained from these studies will be applicable to the dyneins of other organisms, including man. Consequently, knowledge obtained from these studies will increase our understanding of human diseases such as immotile cilia syndrome, in which the arms are frequently lacking. The studies will also provide a basis for understanding such important processes as sperm maturation and capacitation, which are necessary for fertilization and which ultimately must involve changes in the functioning of the arms.

长期目标是确定外部的分子结构 纤毛和鞭毛的臂动力蛋白，并阐明其功能 此中的各个亚基、多肽和多肽结构域 重要的机械化学传感器。 这项工作将集中于衣藻的外臂动力蛋白， 在结构和组成上，它是所有动力蛋白中最具特征的。 动力蛋白中间链之一 IC 78 位于 臂，是与轴丝中微管蛋白交联的主要多肽。 一个 编码 IC 78 的全长 cDNA 克隆已被分离并测序；它 现在将确定是否存在 IC 78 的现有无效突变体。如果 不是，同源重组或反义RNA会产生一个 技术。 交联的 IC 78/微管蛋白肽将被分离并 无效基因的测序、定点突变和转化 用于确定 IC 78/微管蛋白相互作用的重要性的突变体。 IC 78基因也将被随机诱变并引入 无效突变体以确定 IC 78 其他部分在手臂中的作用 组装和功能。 现有单克隆抗体和免疫电子 显微镜将用于定位动力蛋白重链的区域 孤立的颗粒。 编码部分伽马的现有基因组克隆 重链将用于分离覆盖整个γ的cDNA克隆 链转录本；这些将被测序并分析到 伽玛链将被分离并测序。 定点诱变和 转化将用于消除γ链的ATP水解 活动，从而决定该链在外臂功能中的作用。 细胞也将被伽马链基因转化，其中明智地 选定的区域已被删除或随机诱变，并被选择 分析转化体以确定其他重要的功能域。 单个动力蛋白的力产生和微管结合特性 亚单位将在不同的生理条件下进行检查 更好地理解每个子单元在生成和控制中的作用 鞭毛运动。 动力蛋白臂产生的力是所有运动的基础 真核细胞有纤毛和鞭毛。 因为基本结构和组成 动力蛋白似乎在整个进化过程中都是保守的， 从这些研究中获得的基本信息将适用于 其他生物体（包括人类）的动力蛋白。 因此，知识 从这些研究中获得的结果将增加我们对人类的了解 疾病，例如纤毛不动综合症，手臂经常受到影响 缺乏。 这些研究还将为理解此类问题提供基础 精子成熟和获能等重要过程 受精所必需的，最终必须涉及以下方面的变化 手臂的功能。