THE GENETICS OF MICROTUBULE ASSEMBLY AND FUNCTION

微管组装和功能的遗传学

• 批准号: 3276792
• 负责人: N. R MORRIS
• 金额: $ 23.39万
• 依托单位: RUTGERS, THE STATE UNIV OF N.J.
• 依托单位国家: 美国
• 财政年份: 1981
• 资助国家: 美国
• 起止时间: 1981-06-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3276792
• 关键词: Aspergillus; DNA; developmental genetics; frameshift mutation; fungal genetics; gel electrophoresis; gene deletion mutation; gene expression; genetic library; genetic manipulation; genetic mapping; genetic recombination; immunocytochemistry; immunofluorescence technique; laboratory mouse; microtubules; molecular cloning; monoclonal antibody; nucleic acid hybridization; nucleic acid sequence; protein biosynthesis; protein sequence; radiotracer; structural genes; temperature sensitive mutant; tubulin

Microtubules (MTs) are important cellular organelles that are structurally and functionally diverse. Moreover, different MTs are differentially sensitive to different depolymerizing agents and bind different MT associated proteins. It has been hypothesized that MT diversity may be caused by differences in the tubulin composition of MTs. Tubulin, the major structural protein of MTs, is composed of two subunits, alpha and beta tubulin. There are multiple genes for each of these subunits in all of the organisms that have been examined. Not only are there genetic isotypes of alpha and beta tubulin, but there are also posttranscriptionally modified tubulin isotypes, some of which have been shown to be localized to specific MT populations. In this application we propose to analyze the relationship between tubulin genetic isotypes and MT structure and function in the filamentous fungus Aspergillus nidulans. Three of the four Aspergillus tubulin genes, two beta tubulins and one alpha tubulin have been cloned and sequenced. The beta tubulins have been assigned to their genetic loci by DNA mediated integrative transformation, and the function of one of these genes has been examined by DNA mediated "gene disruption." We propose to clone the remaining alpha tubulin gene, to use DNA mediated integrative transformation to assign the two as yet unassigned alpha tubulin sequences to their genetic loci and to use "gene disruption" and in vitro mutagenesis to examine the functions of all of these genes. In addition we propose to use isotype specific antitubulin antibodies to localize specific tubulin to specific MTs by fluorescence immunocytochemistry and to construct chaemeric tubulin genes to determine whether tubulin genetic isotypes are interchangeable.

微管（MT）是重要的细胞器，其在结构上 功能多样。 此外，不同的MT是不同的。 对不同解聚剂敏感，结合不同MT 相关蛋白质 据推测，MT多样性可能是 这是由MT的微管蛋白组成的差异引起的。 微管蛋白 MT的主要结构蛋白由两个亚基α和β组成， β微管蛋白 在所有的细胞中，每个亚基都有多个基因。 已经检查过的微生物的数量。 不仅有基因 α和β微管蛋白的同种型，但也有 转录后修饰的微管蛋白同种型，其中一些已经被 显示其定位于特定的MT群体。 在本申请中，我们 建议分析微管蛋白遗传同种型与MT的关系 结构和功能的丝状真菌构巢曲霉。 四个曲霉微管蛋白基因中的三个，两个β微管蛋白和一个 α微管蛋白已被克隆和测序。 β微管蛋白已经被 通过DNA介导的整合转化分配到它们的遗传基因座， 其中一个基因的功能已经通过DNA介导的 “基因破坏。“我们建议克隆剩下的α微管蛋白基因， 利用DNA介导的整合转化来分配这两个尚未 未将α微管蛋白序列分配到其遗传基因座并使用“基因 破坏”和体外诱变，以检查所有 这些基因。 此外，我们建议使用同种型特异性抗微管蛋白 通过荧光将特异性微管蛋白定位于特异性MT的抗体 免疫细胞化学和构建chaemerictubulin基因， 微管蛋白基因同种型是否可以互换。