DYNEIN ISOFORMS IN PARAMECIUM

草履虫中的动力蛋白异构体

• 批准号: 2187451
• 负责人: DAVID J ASAI
• 金额: $ 19.45万
• 依托单位: PURDUE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2187451
• 关键词: Paramecium; binding proteins; cell motility; chimeric proteins; dynein ATPase; enzyme linked immunosorbent assay; fluorescence microscopy; gene expression; intracellular; laboratory mouse; messenger RNA; microtubules; molecular cloning; nucleic acid sequence; polymerase chain reaction; protein isoforms; protein structure function; site directed mutagenesis; southern blotting; western blottings

Dynein is of fundamental importance in the processes of fertilization and early embryonic development as well as in intracellular molecular movements including cell division and nerve cell axonal transport. The various tasks which dynein mediates are produced by different dynein heavy chain isoforms. A key element in the understanding of dynein function and regulation is the elucidation of the mechanisms that specify the intracellular location of each dynein isoform. The goal of this project is to determine the molecular basis for the accumulation of specific isoforms in specific places in the cell. This analysis will be made in Paramecium, an experimental model system particularly well suited for the proposed studies. There are five specific aims: (1) clone and sequence the ATP-binding domains of the dynein heavy chains expressed in Paramecium in order to be able to describe as completely as possible the structures and functions of the dynein isoforms in a single organism; (2) measure the steady state mRNA levels of each of the heavy chain isoforms after deciliation in order to classify the isoforms into those that are ciliary and those that are cytoplasmic; (3) determine the intracellular location of individual dynein isoforms by using site-directed antibodies in order to establish directly the linkage between heavy chain sequence and isoform function; (4) sequence the complete tail domains and complete motor domains of the functionally divergent ciliary and cytoplasmic isoforms in order to identify conserved regions which may represent common functional domains, such as the microtubule binding site, and nonconserved regions which may represent sequences that specify the intracellular location of dynein; and (5) construct and express in Paramecium chimeric heavy chain genes in which the tail domains will be interchanged between ciliary and cytoplasmic isoforms in order to determine the roles of particular sequences in directing the intracellular location of the dyneins.

动力素在受精过程中至关重要 早期胚胎发育以及细胞内分子 运动包括细胞分裂和神经细胞轴突运输。这 动力蛋白介导的各种任务是由不同的动力蛋白重制产生的 链同工型。理解动力蛋白功能和 调节是阐明指定的机制 每个动力蛋白同工型的细胞内位置。这个项目的目标是 确定特定同工型积累的分子基础 在细胞中的特定位置。该分析将在Pragemium中进行， 实验模型系统特别适合提议 研究。有五个具体目标： （1）克隆并对动力蛋白重链的ATP结合域进行序列 以甲状腺的形式表达，以便能够完全描述 单个动力蛋白同工型的结构和功能可能 生物; （2）测量每个重链的稳态mRNA水平 分解后的同工型为了将同工型分类为 纤毛和细胞质的纤毛； （3）确定单个动力蛋白同工型的细胞内位置 使用定点抗体以直接建立链接 在重链序列和同工型功能之间； （4）序列完整的尾部域和完整的电机域 在功能上发散的睫状和细胞质同工型，以便 确定可能代表常见功能域的保守区域， 例如微管结合位点和未经保护的区域 表示指定动力蛋白细胞内位置的序列；和 （5）在芯片嵌合重链基因中的构建和表达 哪个尾部域将在睫状和 细胞质同工型为了确定特定的作用 指导动力蛋白的细胞内位置的序列。