MOLECULAR BIOLOGY OF MICROTUBULE INTERACTING PROTEINS

微管相互作用蛋白的分子生物学

• 批准号: 2838486
• 负责人: BERL Ray OAKLEY
• 金额: $ 31.47万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1983
• 资助国家: 美国
• 起止时间: 1983-04-01 至 2001-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2838486
• 关键词: Aspergillus; Caenorhabditis elegans; alleles; electron microscopy; gene expression; gene mutation; genetic manipulation; genetic mapping; ion exchange chromatography; microtubules; molecular cloning; posttranslational modifications; protein biosynthesis; protein purification; protein structure function; tubulin

DESCRIPTION: The long-term objective of the research proposed is to identify and characterize proteins that have essential functions in the assembly and activities of microtubules. Most of these proteins and their functions have not been described but they likely have roles in determining the many essential cellular activities of microtubules. The investigator's earlier work supported by this grant led to the discovery of one such protein known as gamma tubulin. Gamma tubulin is related to but distinct from the alpha and beta tubulins which interact to form the heterodimer known as tubulin. Since the discovery of gamma tubulin, data from several labs have shown that gamma tubulin is ubiquitous among eukaryotes and it is found primarily in the microtubule organizing centers (MTOCs) of fungal and animal cells. This has led to the hypothesis that gamma tubulin is essential for microtubule assembly from MTOCs but questions about the function of gamma tubulin, its role in regulating microtubule assembly, its interactions with microtubules, and its interactions with other components of MTOCs remain to be answered. The first aim of the proposed work is to isolate and characterize conditionally-lethal gamma tubulin mutations in the filamentous fungus Aspergillus nidulans. Such mutants have been difficult to isolate by conventional means so the "alanine scanning" approach is proposed. These conditional mutants should be useful tools for clarification of gamma tubulin function and for pseudoreversion analysis for identification of gamma tubulin interacting proteins. Mutants will be examined to determine the effects of mutations on nuclear division, mitosis, mitotic spindle formation, the cytoplasmic microtubule network, and localization and stability of gamma tubulin. The second aim is to identify and characterize proteins that interact with gamma tubulin. Two approaches will be taken. The first approach will involve a yeast two-hybrid screen while the second will use pseudoreversion analysis similar to that used in discovery of gamma tubulin. Genes identified by either of these complementary approaches will be characterized further to determine the potential roles for their products in microtubule assembly and function. The final aim of the proposal is to investigate a novel tubulin identified in the C. elegans genome project. This putative gene product was originally identified as a gamma tubulin but closer analysis shows that it shares only 40-44% identity with gamma tubulins and less with other tubulins. This is sufficiently different to propose that this represents a new tubulin family. This will be investigated by first determining whether C. elegans has a conventional gamma tubulin. If identified, the investigator then intents to search for homologs of this novel tubulin in other organisms and begin to dissect its function.

描述：拟议研究的长期目标是 鉴定和表征在细胞中具有重要功能的蛋白质， 微管的组装和活动。 大多数蛋白质和 它们的功能尚未描述，但它们可能在以下方面发挥作用： 决定微管的许多基本细胞活动。 的 一位研究人员在这项资助下的早期工作导致了这一发现 一种叫做γ微管蛋白的蛋白质。 γ微管蛋白与 但与α和β微管蛋白不同， 称为微管蛋白的异二聚体。 自从发现γ微管蛋白以来， 来自几个实验室的数据表明，γ微管蛋白在 它主要存在于微管组织中， 真菌和动物细胞的MTOC中心。 这导致 假设γ微管蛋白是微管组装所必需， 但是关于γ微管蛋白的功能，它在 调节微管组装及其与微管的相互作用，以及 它与多边技术选择委员会其他组成部分的相互作用仍有待回答。 拟议工作的第一个目标是分离和表征 丝状真菌中的条件致死γ微管蛋白突变 构巢曲霉 这样的突变体很难分离， 因此，提出了“丙氨酸扫描”方法。 这些 条件突变体应该是澄清伽马射线的有用工具。 微管蛋白功能和假逆转分析，以确定 γ微管蛋白相互作用蛋白质。 将对突变体进行检查， 确定突变对核分裂、有丝分裂、有丝分裂 纺锤体形成、细胞质微管网络和定位 和γ微管蛋白的稳定性。 第二个目标是识别和表征相互作用的蛋白质 与γ微管蛋白结合。 将采取两种办法。 第一种方法 将涉及酵母双杂交筛选，而第二个将使用 类似于发现伽马射线时所用的假回复分析 微管蛋白 通过这两种互补方法中的任何一种鉴定的基因 将进一步确定其潜在作用， 微管组装和功能中的产物。 该提案的最终目的是研究一种新的微管蛋白 在C.线虫基因组计划。 这种假定的基因产物 最初被鉴定为γ微管蛋白，但更近的分析显示， 它与γ微管蛋白只有40-44%的同一性，而与 其他tubulins 这一差异足以表明， 代表一个新的微管蛋白家族。 这将首先由 确定C. elegans具有常规的γ微管蛋白。 如果 鉴定，然后研究人员打算寻找这种同源物 新的微管蛋白在其他生物体和开始剖析其功能。