STRUCTURAL AND FUNCTIONAL ANALYSES OF YEAST RAS PROTEINS

ras癌基因产物的机制研究是至关重要的 重要性，因为任何关于生物化学活动差异的线索， 野生型ras蛋白及其致癌变异体只能在 通过这样的研究。 我们建议进行广泛的生化检查 分析酵母ras同源物（RAS 1和RAS 2）的产物， to approach处理this problem问题. 最近的遗传和生物化学研究 RAS基因支持使用酵母作为模型系统的有效性。 此外 在酵母中有强大的表达系统，这将使我们能够 为生化研究准备了大量真实的蛋白质。 在 此外，酵母遗传学可用于探测任何生物化学发现。 我们 我们将专注于以下实验。 (1)利用各种 酵母和E.大肠杆菌、野生型和突变型RAS 蛋白质将被过量生产，并且蛋白质将被纯化。 针对这些蛋白质的抗体将被提高。 (2)酶学性质 RAS蛋白的研究。 我们将首先进行动能分析 野生型和突变蛋白的GTP结合活性的研究。 其他可能的内在活性，如GT3和磷酸化， 被搜查 (3)RAS蛋白的结构研究将在 出去 我们将鉴定GTP结合结构域并剖析其功能结构域。 还将研究RAS蛋白的修饰。 研究 蛋白质构象将使用荧光探针进行。 (4)任何 将鉴定与RAS蛋白相互作用的蛋白， 这些蛋白质是否影响RAS蛋白的生化活性 将被调查。 （十）

Mechanistic studies of the products of ras oncogenes are of utmost importance since any clues on the difference in biochemical activities of wild-type ras protein and its oncogenic variants could only be obtained through such studies. We propose to carry out extensive biochemical analyses of the product of ras homologues of yeast (RAS1 and RAS2) in order to approach this problem. Recent genetic and biochemical studies of the RAS genes support the validity of using yeast as a model system. Moreover powerful expression systems are available in yeast which will enable us to prepare a large amount of authentic protein for the biochemical study. In addition, yeast genetics can be used to probe any biochemical findings. We will concentrate on the following experiments. (1) Utilizing various expression systems in yeast and in E. coli, wild-type and mutant RAS proteins will be overproduced and the proteins will be purified. Antibodies against these proteins will be raised. (2) Enzymatic properties of the RAS proteins will be investigated. We will first carry out kinetic studies of GTP binding activity of the wild-type and mutant proteins. Other possible intrinsic activities such as GTPase and phosphorylation will be searched. (3) Structural studies of the RAS proteins will be carried out. We will identify GTP binding domain and dissect functional domains. Modification of the RAS proteins will also be investigated. Studies on protein conformation will be carried out using fluorescent probes. (4) Any proteins which interact with the RAS proteins will be identified and whether these proteins affect biochemical activities of the RAS proteins will be investigated. (X)