CHARACTERIZATION OF TRANSCRIPTIONALLY REGULATED GENES

我们的目标是了解热休克的作用和相关的 在正常生长和应激期间细胞中的蛋白质。 我们 研究将涉及遗传和生物化学相结合的方法， 作为模型系统，两种生物体，特别是 根据这种分析，S。cerevisiae和E.杆菌 酵母 基因组包含一个家族的基因相关的热休克蛋白70的其他 真核细胞和E.杆菌 这种基因编码的蛋白质 家庭将被分析。 对一种（或两种） 密切相关）将分离70 kDa蛋白质并用于纯化 单个70kDa蛋白质。 以确定功能 相关蛋白质之间的相似性和差异，我们将 确定这些酵母蛋白质的细胞位置， 它们的生化特性。 在大肠大肠杆菌热激基因dnaK和htpG，它们与大肠杆菌热激基因的表达相关， 真核生物的Hsp70和Hsp83基因。 分别将 研究了 dnaK缺失和点的第二位点抑制因子 突变将被分离和表征。 识别和 抑制基因的特征将提供信息 关于与DnaK相互作用或可以绕过DnaK的蛋白质 功能 将使用几种策略来分离额外的 dnaK突变。 由于先前分离的含有dnaK的菌株 突变具有一组复杂的表型，我们的目标是分离 新的dnaK突变体具有这些表型的子集。 如果 表型是可分离的。通过对突变体的分析， 能够定义功能域。 在htpG中含有突变的菌株在温度上稍低， 对增长敏感。 我们将尝试找出 只有在没有htpG的情况下，功能才是必需的。 识别 这些基因的研究将有助于更好地理解htpG 功能 此外，我们将确定是否由htPG编码的蛋白质。 C62.5，将补充酵母的hsp83突变体，以解决 问题是，是否功能，至少有一些，热休克 蛋白质在进化中是保守的。

Our goal is to understand the role of heat shock and related proteins in cells during normal growth and periods of stress. Our studies will involve a combined genetic and biochemical approach, using as model systems, the two organisms that are particularly amenable to such analysis, S. cerevisiae and E. coli. The yeast genome contains a family of genes related to Hsp70 of other eucaryotes and dnaK of E. coli. The proteins encoded by this gene family will be analyzed. Antibodies specific for a single (or two closely related) 70kDa proteins will be isolated and used to purify the individual 70kDa proteins. To determine functional similarities and differences amongst the related proteins, we will determine the cellular location of these yeast proteins and analyze their biochemical properties. In E. coli, the heat shock genes, dnaK and htpG, which are related to the Hsp70 and Hsp83 genes of eucaryotes. respectively. will be studied. Second-site suppressors of dnaK deletion and point mutations will be isolated and characterized. Identification and characterization of suppressor genes will provide information concerning the proteins that interact with DnaK or can bypass DnaK function. Several strategies will be used to isolate additional dnaK mutations. Since previously isolated strains containing dnaK mutations have a complex set of phenotypes, our goal is to isolate new dnaK mutants that have subsets of these phenotypes. If the phenotypes are separable. we may, by analysis of the mutants, be able to define functional domains. Strains containing mutations in htpG are slightly temperature sensitive for growth. We will attempt to identify genes whose function is essential only in the absence of htpG. Identification of such genes should lead to a better understanding of htpG function. Also, we will determine if the protein encoded by htPG. C62.5, will complement the hsp83 mutants of yeast, to address the question of whether the functions, of at least some, heat shock proteins have been conserved in evolution.