ISOLATION AND ANALYSIS OF PROTEINS WHICH REGULATE RAS

哺乳动物细胞中细胞分裂的调节是一个复杂的过程; 协调跨细胞质转导的生长促进信号 膜由生长因子启动，信号由元件启动 监测细胞的营养状态。细胞的发现 癌基因提供了一种手段来研究，至少部分地， 控制细胞分裂的机制。其中一组癌基因ras 基因，被发现在大量和广泛的突变激活 各种人类肿瘤。 ras基因在进化中高度保守， 这表明它们在细胞生物学中起着重要作用。两 ras蛋白的功能以及ras蛋白是如何被调节的 对哺乳动物细胞来说仍然是个谜。酵母RAS基因的发现 使得系统研究RAS蛋白功能成为可能， 基因操控的可能性这项建议是 目的是研究酵母中的CDC 25蛋白，它不仅可以调节 酵母RAS蛋白的功能，但人H-ras蛋白也是如此。这 调节可能是由于蛋白质之间的直接蛋白质-蛋白质相互作用， CDC 25和RAS蛋白。蛋白质相互作用的研究， 它的保守性测试粗略的进化提供了一种独特的方法来识别 调节ras蛋白功能的哺乳动物分子。我们将开发 编码或识别所述结构域的DNA探针和免疫试剂 CDC 25蛋白是RAS激活所必需的。这些试剂将 用于克隆编码与CDC 25同源的结构域的cDNA。此外，我们将 使用遗传筛选来鉴定可以补充 CDC 25在酵母中的功能。分子机制的鉴定 人ras蛋白的作用可以允许设计分子上的 逆转突变ras蛋白致瘤作用的合理药物。

The regulation of cell division in mammalian cells is a complex process; coordinating growth promoting signals transduced across the cytoplasmic membrane initiated by growth factors, with signals initiated by elements monitoring the nutritional status of the cells. The discovery of cellular oncogenes provide a means to study, at least in part, the biochemical mechanisms controlling cell division. One group of these oncogenes, the ras genes, are found to be mutationally activated in a large number and wide variety of human tumors. The ras genes are highly conserved in evolution, suggesting they play a fundamental role in the biology of the cell. Both the function of the ras proteins and how the ras proteins are regulated remains a mystery for mammalian cells. The discovery of the yeast RAS genes made possible a systematic approach to study RAS protein function because of the genetic manipulation possible in that organism. This proposal is aimed at studying the CDC25 proteins in yeast which can regulate, not only yeast RAS protein function but the human H-ras protein as well. This regulation is likely due to a direct protein-protein interaction between CDC25 and RAS proteins. The study of this protein-protein interaction and its conservation t rough evolution provides a unique means to identify the mammalian molecule which regulates ras protein function. We will develop DNA probes and immunological reagents which encode or recognize the domain of the CDC25 protein essential for RAS activation. These reagents will be used to clone cDNAs encoding domains homologous to CDC25. Further, we will use a genetic screen to identify human cDNAs which can complement loss of CDC25 function in yeast. The identification of the molecular mechanism of action of the human ras proteins may allow the design of molecularly rational drugs to reverse the tumorigenic effect of mutant ras proteins.