REGULATION AND FUNCTION OF THE YEAST HEAT SHOCK RESPONSE

本提案的长期目标是深入了解 基因表达的控制机制和应激蛋白在 细胞代谢 更具体地说，研究 组成HSP70多基因的基因的转录和功能 酿酒酵母（Saccharomyces cerevisiae） 诱导HSP70的必要和充分的序列将 通过体外构建缺失和点突变来确定， 体内分析改变的启动子的表达。 热休克蛋白70对自身启动子及转录的调控作用 其他热休克基因的表达将被确定。 HSP70的合成已被 通过将GAL1启动子连接到 HSP70结构基因。 初步结果表明， 热休克启动子在热休克蛋白70存在下不被诱导， 冲击. 观察将被扩展，并且本构的影响将被扩展。 HSP70的合成对其他热休克基因的表达有影响。 参与HSP70转录抑制的序列将是 划定的 启动子区域的突变将在体外构建 并在体内分析改变的启动子的表达。 对热休克反应的调节的洞察将通过以下方式获得： 分离和分析与糖尿病相关的非连锁基因中的突变， 调节热休克基因的转录。 我们将尝试 分离不能诱导转录突变体。 我们还将承担 一种更直接的尝试，分离蛋白质中的突变， 直接与HSP70启动子连接。 序列中的向下突变 我们将以归纳所需的内容为起点， 试图在其他基因中分离出可以克服这种影响的突变 第一个突变的基因，并允许增加合成。 HSP70基因的点突变将被分离出来，以深入了解HSP70基因的突变。 诱导型70K基因（YG100）的功能。 这些对温度敏感的 HSP70突变将被分离，以鉴定其蛋白质 产品直接或间接地与HSP70相互作用。 此外，表型 的点突变体将进行分析，试图分离的 蛋白质的其他活性的调节活性。

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.