FUNCTIONS OF CYTOPLASMIC DYNEIN

细胞质动力蛋白的功能

• 批准号: 3307450
• 负责人: IAN R GIBBONS
• 金额: $ 14.55万
• 依托单位: UNIVERSITY OF HAWAII AT MANOA
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1996-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3307450
• 关键词: Saccharomyces cerevisiae; cell cycle; cell migration; chemical binding; chromosome movement; dynein ATPase; electron microscopy; fluorescence microscopy; fungal genetics; gene expression; gene mutation; genetic transcription; meiosis; molecular cloning; nucleic acid probes; nucleic acid sequence; phenotype; polymerase chain reaction; protein structure function; site directed mutagenesis

The broad long-term objective of this project is to determine the cellular functions of cytoplasmic dynein by studying its role in yeast as a model for its role in cells of vertebrates and other higher organisms. More specifically, it is intended (i) to utilize the yeast DNA probe obtained from the gene encoding dynein to characterize the gene by determining whether it is a single or a multiple copy gene, obtaining its restriction map and base sequence at a fragment that encodes the hydrolytic site, and mapping it to its physical position on the chromosome, (ii) to localize the yeast cytoplasmic dynein during various stages of the cell cycle, (iii) to study the phenotypes of mutated forms of the gene in order to understand the general role of yeast cytoplasmic dynein, and the specific importance of different regions on the protein, (iv) to study the possible interaction between dynein and the other motor proteins of the cytoskeleton, (v) to study the relationship between dynein expression and the various mechanisms regulating the cell cycle. For the first goal, a genomic library of Saccharomyces cerevisiae will be screened with our probe in order to construct a plasmid that will carry the whole yeast dynein gene including its promoter, which is expected to contain more than 14 kb, based on the size of dynein from higher cells. This cloned gene will serve for later experiments of genetic manipulations. The localization of the yeast cytoplasmic dynein will be probed with methods of immunofluorescence microscopy using epitope-tagged antibodies. The variations in the cellular distribution of dynein at different stages of the cell cycle will clarify the processes in which dynein is involved. The extensive molecular genetic tools available in yeast will be exploited to study the phenotypic behavior of cells in which the dynein gene was entirely or conditionally disrupted. These targeted mutations will be induced, utilizing our sequenced yeast probe and methods that are widely used in yeast for these purposes. In observing the phenotypic behavior of conditional site-directed point mutations, special attention will be given to the effect on cellular processes that involve motility such as chromosome movement during cell division or nuclear migration during conjugation. The results of this study are expected to elucidate the involvement of cytoplasmic dynein in mitosis and meiosis and so to contribute to understanding of the health problems associated with improper chromosome segregation in higher organisms.

该项目的广泛长期目标是确定 细胞质动力蛋白的细胞功能通过研究其在酵母中的作用 它在脊椎动物和其他较高生物体中的作用的模型。 更具体地说，它旨在（i）使用酵母DNA探针 从编码动力蛋白的基因获得以表征该基因 确定它是单个或多个副本基因，获得其 限制图和基本序列，片段编码 水解部位，并将其映射到其在物理位置 染色体，（ii）在各种过程中定位酵母菌动力蛋白 细胞周期的阶段，（iii）研究突变形式的表型 该基因以了解酵母细胞质的一般作用 动力蛋白，以及不同区域对蛋白质的具体重要性， （iv）研究动力蛋白与其他电动机之间可能的相互作用 细胞骨架的蛋白质，（V）研究 动力蛋白表达和调节细胞周期的各种机制。 对于第一个目标，酿酒酵母的基因组库将是 用我们的探针筛选以构建一个携带的质粒 包括其启动子在内的整个酵母动力基因，预计将 基于来自较高细胞的动力蛋白的大小，包含超过14 kb。 该克隆的基因将用于以后的遗传实验 操纵。 酵母细胞质动力蛋白的定位将是 使用表位标签的免疫荧光显微镜探测 抗体。 动力蛋白的细胞分布的变化 细胞周期的不同阶段将阐明该过程 动力蛋白参与了。 可用的广泛的分子遗传工具 酵母将被利用以研究细胞的表型行为 动力蛋白基因完全或有条件地破坏了。 这些 将诱导靶向突变，利用我们的测序酵母探针 以及用于这些目的的酵母中广泛使用的方法。 在 观察条件定位点的表型行为 突变，将特别注意对细胞的影响 涉及运动性的过程，例如细胞期间的染色体运动 共轭过程中的分裂或核迁移。 结果的结果 预计研究将阐明细胞质动力蛋白参与 有丝分裂和减数分裂，因此有助于理解健康 与较高的染色体分离不当相关的问题 有机体。