MOLECULAR AND GENETIC ANALYSIS OF FLAGELLAR DYNEINS

这个项目同时使用了分子生物学和遗传学来研究 鞭毛动力蛋白ATPase的结构和功能，是一种大的、多细胞的、 支持沿细胞质和细胞质运动的亚基复合体 鞭毛微管骨架。这项工作的长期目标是 目的：探讨动力蛋白力的产生机制及其调控。 确定一级结构并预测高阶结构 衣藻的催化重链亚基和基因组克隆 Reinhardtii外臂动力蛋白α和β重链将被测序。 序列分析也将用于寻找核苷酸和微管 与其他机械力化学酶的结合域和同源性。 衣藻动力蛋白装配突变体的转化研究 作为一种表达克隆的动力蛋白基因的方法。杂交种在水稻中的表达 克隆的野生型和sup-1突变的β重链基因将用于 精确定位sup-1突变，它定义了这个重链的一个区域 径向辐条-中心对微管参与运动调节 互动。序列分析将揭示分子缺陷 负责抑制基因的表型。 将使用基因组α重链和70k中间链克隆 利用限制性片段定位编码每种蛋白质的遗传位点 长度多态(RFLP)分析。先前确定的动力蛋白组装 然后对突变体进行等位基因测试，以确定这些结构基因的等位基因。 将RFLP定位和转化与克隆基因相结合。

This project uses both molecular biology and genetics to study the structure and function of flagellar dynein ATPases, which are large, multi- subunit complexes that support motility along both cytoplasmic and flagellar microtubule frameworks. The long-term objective of this work are to determine the mechanism of dynein force generation and its regulation. To determine primary structure and predict higher-order structure in the catalytic heavy chain subunits, genomic clones for the Chlamydomonas reinhardtii outer arm dynein alpha and beta heavy chains will be sequenced. Sequence analysis will also be used to look for nucleotide and microtubule binding domains and homology with other mechanochemical enzymes. Transformation of Chlamydomonas dynein assembly mutants will be developed as a method to express cloned dynein genes. Expression of hybrids between cloned wild-type and sup-1 mutant beta heavy chain genes will be used to pinpoint the sup-1 mutation, which defines a region of this heavy chain involved in motility regulation by radial spoke-central pair microtubule interactions. Sequences analysis will reveal the molecular defect responsible for the suppressor phenotype. Genomic alpha heavy chain and 70 k intermediate chain clones will be used to map the genetic loci encoding each protein by restriction fragment length polymorphism (RFLP) analysis. Previously-identified dynein assembly mutants will then be tested for allelism to these structural gene loci by a combination of RFLP mapping and transformation with cloned genes.