GENETIC ANALYSIS OF REGULATED TUBULIN SYNTHESIS

本提案的总体目标是调查 负调控系统的遗传控制， 微管蛋白合成对微管蛋白过度产生的反应 酿酒酵母 一般选择的 来自酵母基因组库的野生型基因的过表达 高拷贝数质粒上的DNA将用于鉴定 调节基因 此外，一个经典的遗传解剖， 微管蛋白调节将提供EMS诱导的条件性致死 调节突变体 条件致死突变将被用于 通过互补克隆野生型基因。 要么 这种方法将产生克隆的序列， 从生理和遗传两方面来描述基因。 的 条件致死突变体的特征在于 它们对微管蛋白表达的影响，以进一步了解它们的 功能 微管蛋白基因的顺式作用位点的边界将是 通过转座子诱变和缺失的组合定位 在体外构建的突变。 将绘制3'边界 通过转座子诱变，并将提供Lac Z融合， 监测微管蛋白表达。 描述了实验以绘制 通过构建通用三杂交表达的5'边界 含有Trp 1启动子、微管蛋白编码序列的载体 和一种乳酸菌Z融合物 该载体将用于研究所有三个 微管蛋白基因，并可以识别调控序列包含 在蛋白质编码序列中。 最后，一种研究微管蛋白调节的体外方法将在 使用酵母提取物开发的。 温度敏感调节 将对突变体进行分析，以获得额外的证据， 突变定义调控基因。 突变体提取物将 通过体外互补分析来确定哪个基因 产品是可扩散的，并提供其 洁净. 长期目标是了解分子 酵母中微管蛋白调节的细节。

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.