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)
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