GENETIC ANALYSIS OF REGULATED TUBULIN SYNTHESIS

这项提案的总体目标是调查 负性调控系统的基因控制 微管蛋白合成对微管蛋白过度生产的响应 酿酒酵母。一份关于 酵母基因组库中野生型基因的高效表达 高拷贝数质粒上的DNA将用于鉴定 调控基因。此外，一个经典的基因解剖 微管蛋白调节将提供EMS诱导的条件性死亡 监管突变体。将使用条件致死突变 通过互补的方法克隆野生型基因。要么 该方法将产生克隆的序列，这些序列将用于进一步 从生理和基因两个方面描述这些基因。这个 条件致死突变体将根据以下方面进行描述 它们对微管蛋白表达的影响以进一步了解它们的 功能。 微管蛋白基因顺式作用位点的边界将是 利用转座子突变和缺失相结合的方法定位 在体外构建的突变。将绘制3‘边界 通过转座子突变，并将提供Lac Z融合 监测微管蛋白的表达。描述了实验以映射 通过构造一个通用的三杂交表达式来实现5‘边界 含有Trp1启动子、微管蛋白编码序列的载体 和Lac Z融合。向量将被用来研究所有这三个 微管蛋白基因，并能识别包含的调控序列 在蛋白质编码序列内。 最后，研究微管蛋白调节的体外方法将是 使用酵母提取物开发。温度敏感型调节 将对突变株进行化验，以获得进一步的证据 突变定义了调控基因。突变的提取物将是 通过体外互补分析来确定哪个基因 产品是可扩散的，并提供了一种检测其 净化。长期的目标是理解分子 酵母中微管蛋白调控的细节。

The overall objectives of this proposal are to investigate the genetic control of a negative regulatory system that regulates tubulin synthesis in response to tubulin overproduction in the yeast Saccharomyces cerevisiae. A general selection for the overexpression of wild type genes from genomic pools of yeast DNA on high copy number plasmids will be used to identify regulatory genes. In addition, a classical genetic dissection of tubulin regulation will provide EMS induced conditional lethal regulatory mutants. The conditional lethal mutations will be used to clone the wild type genes by complementation. Either approach will yield cloned sequences which will be used to further characterize the genes both physically and genetically. The conditional lethal mutants will be characterized with respect to their effect on tubulin expression to gain further insight into their function. The boundaries of the cis-acting sites of the tubulin genes will be mapped by a combination of transposon mutagenesis and deletion mutations constructed in vitro. The 3' boundaries will be mapped by transposon mutagenesis and will provide Lac Z fusions to monitor tubulin expression. Experiments are described to map the 5' boundaries by constructing a versatile trihybrid expression vector containing the Trp1 promoter, tubulin coding sequences and a Lac Z fusion. The vector will be used to study all three tubulin genes and can identify regulatory sequences contained within the protein coding sequence. Finally, an in vitro approach to studying tubulin regulation will be developed using yeast extracts. Temperature sensitive regulatory mutants will be assayed to obtain additional proof that the mutations define regulatory genes. Mutant extracts will be analysed by in vitro complementation to determine which gene products are diffusible and to provide an assay for their purification. The long term goal is to understand the molecular details of tubulin regulation in yeast.