Activation of oncogenes is responsible for cellular transformation. C-src, found in normal cells, represents a cellular proto-oncogene progenitor of the Rous sarcoma virus (RSV) transforming gene v-src. The product of v-src gene is a kinase designated pp60v-src. At low levels of pp60v-src cells grow in a normal manner, at higher concentrations the cells become transformed, and at much higher concentrations the cells die. For effective expression, pp60v-src must be expressed from mRNAsrc in a regulated fashion. This investigation concerns the mechanism of initiation of protein synthesis on mRNAsrc. We propose to investigate whether the short open reading frames in the 5' leader sequence modulate the translational efficiency of mRNAsrc. Translation of mRNAsrc is very unusual when compared to the translation of most other eukaryotic mRNAs. Eukaryotic mRNAs are generally monocistronic whereas mRNAsrc appears to be polycistronic, probably directing the synthesis of one or more poly-peptides as well as pp60v-src. Moreover, translation of most eukaryotic mRNA begins at the first AUG codon downstream of the 5' end of the mRNA whereas initiation of pp60v-src occurs at the fifth or sixth AUG codon from the 5' end. This proposal describes experiments designed to elucidate the translational properties of mRNAsrc in order 1) to determine whether the upstream AUG codons are critical for the regulation of pp60v-src expression and 2) to characterize further the mechanisms by which ribosomes recognize translational initiation sites on mRNAsrc. Two approaches will be used to resolve the mechanism of mRNAsrc translation, in vitro translation of mRNAsrc's mutated at specific sites and in vivo analyses of pp60v-src produced in cells transfected with modified v-src genes. Both the in vitro and the in vivo assays will be comprised of i) ribosome binding site analysis, ii) leader peptide analysis (assays for peptides encoded in the short open reading frames that precede the src open reading frame), and iii) translational efficiency of the src gene on mRNAs containing mutations in the 5' leader. The results of the experiments should indicate how a viral oncogene, v-src, is regulated in infected cells and how finely tuned expression of the gene must be for neoplastic transformation.
