MOLECULAR AND GENETIC ANALYSIS OF FLAGELLAR DYNEINS

鞭毛动力蛋白的分子和遗传分析

• 批准号: 6625078
• 负责人: DAVID R MITCHELL
• 金额: $ 26.07万
• 依托单位: UPSTATE MEDICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-06-01 至 2004-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6625078
• 关键词: Chlamydomonas; adenosinetriphosphatase; antibody; biochemistry; cilium /flagellum motility; complementary DNA; dynein ATPase; electron microscopy; enzyme activity; flagellum; gene expression; gene mutation; genetic mapping; immunoprecipitation; isozymes; laboratory rabbit; microtubules; molecular cloning; molecular genetics; nucleic acid sequence; protein biosynthesis; protein protein interaction; protein structure function

The long-term goals of this work are to understand the mechanisms that generate and regulate motility in cilia and flagella. Human mutations that inhibit motility of these organelles cause upper respiratory illnesses and infertility, and related forms of motility are important for mitosis and directed transport of many intracellular particles. Flagellar dynein ATPases are large, multi-subunit motor enzymes that power cilia and flagella. Most flagella have two non-identical rows of dyneins attached to each doublet microtubule, both of which contribute to microtubule sliding and flagellar motility. Dyneins of the outer row more closely resemble cytoplasmic dyneins in containing two or three ca. 500 kD catalytic heavy chains, two ca. 75 kD intermediate chains and multiple light chains. While the role of dyneins as microtubule-associated motors for both ciliary and cytoplasmic motility is well recognized, little is known about their regulation or their mode of attachment to the loads they carry (vesicles or chromosomes in the cytoplasm, doublet microtubules in cilia and flagella). Goals of the proposed experiments are to characterize interactions between dynein subunits and doublet microtubule-associated proteins that are important for dynein attachment during flagellar assembly and for regulation of outer row dynein motors. Using the outer row dynein of Chlamydomonas reinhardtii flagella as a model system, genes essential for normal dynein assembly and function are being cloned, sequenced, and genetically mapped. The function of each gene product in cytoplasmic assembly, targeting to flagella, and in situ regulation is examined using molecular cloning, electron microscopic and biochemical approaches. Experiments proposed here focus on the products of four genes identified through published and preliminary studies of assembly mutations as having unique roles in dynein assembly: oda7, oda8, oda15 and pfl3. All materials are now available for rapid progress in determining the products of these genes, which should provide key information about mechanisms that regulate the intracellular distribution and activity of flagellar dynein motors.

这项工作的长期目标是了解纤毛和鞭毛运动的产生和调节机制。抑制这些细胞器运动的人类突变会导致上呼吸道疾病和不孕不育，相关形式的运动对有丝分裂和许多细胞内颗粒的定向运输非常重要。鞭毛动力蛋白ATPase是为纤毛和鞭毛提供动力的大的、多亚单位的马达酶。大多数鞭毛有两排不同的动力蛋白附着在每一对微管上，这两者都有助于微管的滑动和鞭毛的运动。外排的动力蛋白更接近于细胞质动力蛋白，含有两条或三条约500kD的催化重链，两条约75kD的中间链和多条轻链。虽然动力蛋白作为纤毛和细胞质运动的微管相关马达的作用得到了很好的认识，但对它们的调节或它们与所携带的负荷(细胞质中的小泡或染色体、纤毛和鞭毛中的双微管)的附着方式知之甚少。拟议实验的目的是描述动力蛋白亚基和双线微管相关蛋白之间的相互作用，这些蛋白对鞭毛组装过程中动力蛋白的附着和对外排动力蛋白马达的调节非常重要。以莱茵衣藻鞭毛外排动力蛋白为模型系统，对动力蛋白正常组装和功能所必需的基因进行克隆、测序和基因定位。用分子克隆、电子显微镜和生物化学方法研究了每个基因产物在细胞质组装、针对鞭毛和原位调控中的功能。这里提出的实验重点放在四个基因的产物上，这些基因是通过已发表的和初步的组装突变研究确定的，它们在动力蛋白组装中具有独特的作用：oda7、oda8、oda15和pfl3。所有材料现在都可以用来快速确定这些基因的产物，这应该会提供关于调节鞭毛动力蛋白马达在细胞内分布和活性的机制的关键信息。