GENETIC ANALYSIS OF THE UPSTREAM CONTROL OF YEAST RAS

本研究方案中的一组实验探索了 涉及的生理信号和特定的产物 酿酒酵母中RAS/cAMP途径的上游调控。这个 长期目标是了解生物化学的细节 这一感觉/信号转导途径涉及(A) 感受到的分子，以及(B)基因产物和机制 参与将这些感官信息传递给RAS。 对控制酵母RAS活性的见解是有潜力的 因其结构和功能而具有医学意义 酵母和哺乳动物ras蛋白的同源性以及ras的作用 人类肿瘤中的基因。 有相当多的间接证据表明 酵母菌RAS基因产物活性反映营养状态 在牢房里。我将开发RAS活性的生化分析方法 反映活体条件，并与这些分析直接 确定哪些营养素控制RAS的活性。突变者 这些营养物质的新陈代谢缺陷将被用来 帮助确定代谢物或其他分子实际上是 受RAS途径的监控。 人们对蛋白质的特性或功能知之甚少 来控制RAS的活动。CDC25基因产物是唯一的 已在酵母中发现的控制RAS的蛋白质 活动。将生产和使用针对CDC25蛋白的抗体 检查任何可能的共价修饰，这些修饰可能 参与CDC25功能的调节。要分析 CDC25的功能结构域，我将过度表达各个片段 并分析了它们的表型和生化效应。 将在遗传筛选中利用表型效应来识别 调节CDC25功能的基因产物。 为了确定参与调节RAS的新基因产物， 我将使用新的基因筛选器，它采用了特定的突变体 RAS2的等位基因引发了强烈的表型，但不同于 RAS等位基因目前用于基因筛查。活动 相应的改变的蛋白质或过度生产的蛋白质 已知由这些特定等位基因编码的 受CDC25活性调节，原则上受CDC25活性调节 其他上游监管机构。感兴趣的基因将通过以下方式克隆 互补性，其特点是结构和 突变等位基因的表型检测。以确定生化指标 对于基因产物的功能，我会追求不同的方法 包括测序，测试突变等位基因对 营养控制的RAS活性的生化分析，以及 检测突变等位基因对RAS和RAS状态的影响 CDC25蛋白。

The set of experiments in this research proposal explore the physiological signals and specific products involved in the upstream control of the RAS/cAMP pathway in S. cerevisiae. The long-term objective is to understand the biochemical details of this sensory/signal transduction pathway in terms of (a) the molecules that are sensed, and (b) the gene products and mechanisms involved in communicating this sensory information to RAS. Insights into the control of yeast RAS activity are of potential medical significance because of the structural and functional homology of yeast and mammalian ras proteins, and the role of ras genes in human tumors. There is considerable indirect evidence suggesting that the activity of yeast RAS gene products reflects the nutritional state of the cell. I will develop biochemical assays for RAS activity that reflect in vivo conditions, and with these assays directly determine which nutrients control the activity of RAS. Mutants defective in the metabolism of these nutrients will be employed to help identify the metabolites or other molecules that actually are being monitored by the RAS pathway. Very little is known about the identity or function of proteins that control RAS activity. The CDC25 gene product is the only protein that has been identified in yeast which controls RAS activity. Antibodies to CDC25 protein will be produced and used to examine any possible covalent modifications that might be involved in the regulation of CDC25 function. To analyze the functional domains of CDC25, I will overexpress various segments of CDC25 and analyze their phenotypic and biochemical effects. Phenotypic effects will be exploited in genetic screens to identify gene products that regulate CDC25 function. To identify new gene products involved in the regulation of RAS, I will use novel genetic screens that employ particular mutant alleles of RAS2 that elicit a strong phenotype, but are unlike the RAS alleles currently employed in genetic screens. The activity of the corresponding altered protein or overproduced protein encoded by these particular alleles is known to be significantly modulated by CDC25 activity, and in principle by the activity of other upstream regulators. Genes of interest will be cloned by complementation and characterized by the construction and phenotypic testing of mutant alleles. To ascertain the biochemical function of the gene products, I will pursue different approaches including sequencing, testing the effect of mutant alleles on biochemical assays for nutrient-controlled RAS activity, and examining the effect of mutant alleles on the state of RAS and CDC25 proteins.