REGULATION AND FUNCTION OF THE YEAST HEAT SHOCK RESPONSE

这项建议的长期目标是深入了解 基因表达调控机制及应激蛋白在植物生长中的作用 细胞新陈代谢。更具体地说，要研究监管 HSP70多基因组成基因的转录和功能 酿酒酵母一科。 对于HSP70的诱导性而言，相应且充分的序列将 在体外通过构建缺失和点突变来确定 分析改变后的启动子在体内的表达。 热休克蛋白70在调节自身启动子和转录中的作用 其他热休克基因的数量将会被确定。热休克蛋白70的合成 通过将GAL1启动子连接到 HSP70结构基因。初步结果表明，转录 热休克促进剂在受热时不能在HSP70存在时被诱导 令人震惊。观察范围将扩大和本构效应 HSP70合成对其他热休克基因表达的测定。 参与抑制HSP70转录的序列将是 勾勒出来的。启动子区域的突变将在体外构建 并对改变的启动子在体内的表达进行了分析。 对热休克反应的调节的洞察将通过 分离和分析非连锁基因的突变 热休克基因转录的调控。我们将尝试 分离不能诱导转录的突变体。我们还将承诺 分离相互作用的蛋白质的突变的一种更直接的尝试 直接与HSP70启动子结合。序列中的向下突变 我们将以归纳所需的信息为起点， 尝试分离其他基因的突变，以克服这种影响 第一个突变，并允许增加合成。 将分离HSP70基因点突变，以深入了解 可诱导70K基因(YG100)的功能。这些对温度敏感的 将分离HSP70突变，以识别其蛋白质 产品直接或间接地与HSP70相互作用。另外，表型 将对点突变进行分析，以尝试分离出 调节活动有别于蛋白质的其他活动。

The long-term objective of this proposal is to gain insight into the mechanisms of control of gene expression and the role of stress proteins in cellular metabolism. More specifically, to study the regulation of transcription and the function of the genes composing the HSP70 multigene family of Saccharomyces cerevisiae. The sequences assential and sufficient for the inducibility of HSP70 will be determined by constructing deletion and point mutations in vitro and analyzing the expression of the altered promoters in vivo. The role of HSP70 in regulating its own promoter as well as transcription of other heat shock genes will be determined. Synthesis of HSP70 has been made independent of heat shock control by linking the GAL1 promoter to the HSP70 structural gene. Preliminary results indicate that transcription of the heat shock promoter is not induced in the presence of HSP70 upon a heat shock. The observation will be extended and the effect of constitutive HSP70 synthesis on the expression of other heat shock genes determined. The sequences involved in this repression of HSP70 transcription will be delineated. Mutations in the promoter region will be constructed in vitro and the expression of the altered promoters analyzed in vivo. Insight into the regulation of the heat shock response will be gained by isolating and analyzing mutations in unlinked genes involved in the regulation of transcription of the heat shock genes. We will attempt to isolate mutants which fail to induce transcription. We will also undertake a more directed attempt to isolate mutations in proteins which interact directly with the HSP70 promoter. A down mutation in the sequences required for induction will be used as a starting point, and we will attempt to isolate mutations in other genes which can overcome the effect of the first mutation and allow increased synthesis. Point mutations in the HSP70 gene will be isolated to gain insight into the function of the inducible 70K gene (YG100). These temperature-sensitive HSP70 mutations will be isolated in order to identify genes whose protein products directly or indirectly interact with HSP70. Also, the phenotypes of the point mutants will be analyzed in an attempt to separate the regulatory activities from other activities of the protein.