Genetic Analysis of Alpha-Tubulin Function in Drosophila

微管蛋白作为微管的主要组成部分，在形态发生中起着至关重要的作用。我们建议使用果蝇的四个a-微管蛋白来解决关于这些重要结构蛋白的两个基本问题：a-微管蛋白亚型中有任何一个是功能专门化的，以及什么调节机制负责在每种细胞类型中实现正确的a-微管蛋白亚型的比例和浓度？将通过确定一种同型修复与另一种同型突变相关的发育缺陷的能力来测试每一种a-微管蛋白同种类型的多功能性。携带每个α-微管蛋白基因的异位拷贝和两个α-微管蛋白突变的转基因菌株是可用的。将尝试使用P元素突变和聚合酶链式反应在剩余的两个基因中产生突变，这种方法允许恢复没有检测到表型的突变。维持组织适宜的a-微管蛋白库的调节机制将通过检测细胞的能力来确定：1)消除嵌合基因产生的错误表达的a-微管蛋白亚型；2)对额外微管蛋白基因中适当的亚型过度表达做出反应。通过逆转与母体微管蛋白过度表达相关的雌性不育，将寻找编码α-微管蛋白调节因子的基因。微管蛋白是大多数类型细胞的重要结构成分。它以多种不同的形式存在，有些存在于所有细胞中，另一些仅存在于某些特定类型的细胞中。不同形式的微管蛋白可能具有不同的功能，但对这些功能是什么知之甚少。这是一项研究不同形式微管蛋白不同功能的建议。

Tubulin plays a crucial role in morphogenesis as the primary component of microtubules. We propose to address two basic questions about these important structural proteins using the four a-tubulins of Drosophila: are any of the a-tubulin isotypes functionally specialized, and what regulatory mechanisms are responsible for achieving the correct ratios and concentrations of a-tubulins isotypes in each cell type? The multifunctionality of each a-tubulin isotype will be tested by determining the ability of one isotype to rescue developmental defects associated with mutations in another isotype. Transgenic strains carrying ectopic copies of each a-tubulin gene and mutations in two of the a- tubulins are available. An attempt will be made to generate mutations in the two remaining genes using P-element mutagenesis and PCR, an approach that allows the recovery of mutations with no detectable phenotype. Regulatory mechanisms operating to maintain tissue-appropriate a-tubulin pools will be identified by examining the ability of cells to 1) eliminate misexpressed a-tubulin isotypes produced from chimeric genes, and 2) respond to overexpression of appropriate isotypes from supernumerary tubulin genes. Genes encoding transacting a-tubulin regulatory factors will be sought by reverting the female sterility associated with overexpression of a maternal tubulin. The protein tubulin is an important structural component of most types of cells. It exists in a number of different forms, some present in all cells, others present only in certain specialized types of cells. The different forms of tubulin presumably serve different functions, but very little is known about what these functions are. This is a proposal to investigate the different functions of the different forms of tubulin.