MOLECULAR AND GENETIC ANALYSIS OF FLAGELLAR DYNEINS

鞭毛动力蛋白ATPase是一种大的、多亚单位的运动酶。多数 纤毛和鞭毛上各有两排不同的动力蛋白 二重微管，两者都有助于微管滑动和 鞭毛运动。外排的动力蛋白更接近于 胞质动力蛋白中含有两个或三个约500kd的催化重 链条，两条。75kd中间链和多个轻链。 而动力蛋白作为微管相关马达对两者的作用 纤毛和细胞质的运动性是公认的，但对此知之甚少。 他们的规则或他们对所承载的负荷的依附方式 (细胞质中的小泡，纤毛和鞭毛中的双微管)。 拟议实验的长期目标是确定 鞭毛动力蛋白重链、中间链和轻链 鞭毛运动，并表征动力蛋白之间的相互作用 重要的亚基和二联体微管相关蛋白 用于鞭毛组装过程中的动力蛋白附着和调节 外排动力马达。衣藻外排动力蛋白的利用 雷氏鞭毛作为模式系统，正常动力所必需的基因 组装和功能正在被克隆、测序和基因定位。 各亚基在动力蛋白生成和调节中的作用 然后通过表型特征对运动活动进行分析 每个基因座的突变、体外改变和体内表达 克隆的基因。这里提出的实验旨在探索 重于α和β的结构-功能关系。链和 70kd中间链，采用插入失活诱变 克隆额外的动力蛋白组装基因。

Flagellar dynein ATPases are large, multi-subunit motor enzymes. Most cilia and 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 in the cytoplasm, doublet microtubules in cilia and flagella). Long term goals of the proposed experiments are to determine the roles of flagellar dynein heavy chains, intermediate chains and light chains in flagellar motility, and 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 subunit in the generation and regulation of dynein motor activity is then being analyzed by phenotypic characterization of mutations at each locus and by in vitro alteration and in vivo expression of cloned genes. Experiments proposed here are designed to explore structure-function relationships in the alpha and beta heavy. chains and 70 kd intermediate chain, and to use insertional inactivation mutagenesis to clone additional dynein assembly genes.