The specific aims of the research proposed are: (1) to identify, clone and characterize cellular proto-oncogenes that control differentiation and growth in response to exogenous morphogens and regulatory signals in the larvae activity of mutationally altered, cloned forms of such proto-oncogenes by transformation of these larvae; and (3) to define the dependence of the tumorigenic activity and phenotype(s) on specific sequences (alleles) of the cloned transforming oncogenes, using site-directed mutagenesis in conjunction with transformation assays. The applicants propose to accomplish these aims by taking advantage of the unique experimental tractability of molluscan and other marine invertebrate larvae, and the extensive information they have developed on the molecular signals, receptors and transducers controlling metamorphosis in these larvae. Potential proto-oncogenes controlling normal metamorphosis and early development in these larvae will be identified, amplified, cloned and sequenced using PCR and molecular hybridization procedures. Morphogen-induced transcription of the proto-oncogenes will be used to facilitate purification of the proto-oncogene mRNAs, and subsequent amplification and cloning of the cDNAs. C-ras homologs will be amplified and cloned using primers constructed from sequences the applicants recently obtained from a larval G protein involved in regulation of morphogenesis. Both site-directed and random (multi-site) mutagenesis will be used in efforts to convert these genes to tumorigenic transforming oncogenes. A unique transformation system will be developed to screen for transforming oncogenes, taking advantage of the high content of stem cells in the invertebrate larvae. Site-directed mutagenesis will be used in conjunction with the transformation assays to define the relationship between specific oncogene nucleotide sequences (alleles) and specific phenotypes of the neoplastic transformed cells. These experiments also may lead to the development of a system for transformed cell tissue culture, for further analyses of the processes controlling tumorigenesis in such marine animals. Results of the proposed studies should for the first time define the relationship between the molecular mechanisms of morphogen recognition/signal transduction controlling normal gene activation, differentiation and cellular proliferation and those controlling cancer in shellfish and other marine invertebrates.
