The immediate goal of this research is to provide a deeper understanding of the similarities and differences between the biological properties of v-src, the transforming gene of Rous sarcoma virus (RSV), and its normal homologue, c-src. Ultimately, the hope is to obtain insight into the molecular mechanisms of src-induced transformation. A rat cell line, 2A-2, that expresses an exogenously added chicken c-src gene will be used to analyze the properties of pp60?c-src?. Additionally, cell lines containing higher levels of rp60?c-src? will be made. This will be done by cloning c-src into efficient expression vectors and adding the chimeric molecules to cells as part of a calcium-phosphate precipitate. Among the properties of pp60?c-src? that are of interest are its: half-life, sites of phosphorlation, protein kinase activity, and interactions with other cellular proteins. Not only will pp60?c-src? be studied, but also chimeric genes that are part c-src and part v-src. These genes will provide insight into the differences that enable one gene (v-src) to transform cells (and cause tumors) while the other cannot. Cell lines will also be created that contain mutant v-src genes. These cell lines will provide further insight into the life cycle of pp60?src? if they contain proteins with identifiable behavioral changes (e.g., inability to bind to a membrane or lack of phosphorylation). The cell lines will also be the targets for mutagenesis experiments. These experiments are designed to alter cellular constitutents that interact with pp60?src?. An appropriate mutation will complement the deficiency in pp60?src? and restore the wild-type transforming behavior. Such genes that interact with pp60?src? will be isolated for further study. Finally, a retrovirus vector will be made that will be used to identify promoters turned on or off in a scr-transformed cell. This vector will allow for the selection of cells containing promoters that signal a beginning or an and to transcription after src-mediated transformation. Those promoters (and accordingly the genes with which they are associated) will be isolated for further study. (X)
