We were interested in cloning activated oncogenes that play a causative role in a specific tumor type that may have escaped detection by the conventional genomic DNA transfection-transformation assay. To achieve this, we developed a cDNA vector for expression cloning in mammalian cells. This system provides a high proportion of full-length cDNAs that could be inserted directionally downstream of a strong LTR promoter. Following transfection into NIH/3T3 indicator cells, morphologically transformed cells were isolated and plasmid DNA were rescued by standard procedures. Three cDNA libraries were constructed separately from a human mammary epithelial cell line (B5/589), a human soft-tissue sarcoma cell line (HA1095/RD-ES-1) and a human neuroblastoma cell line (SK-N-MC). We were able to identify a novel transforming gene from each library in addition to several previously characterized oncogenes. Two oncogenes, tim and nep, encode proteins that show significant sequence similarity to a region that is homologous among several growth regulatory genes such as dbl, bcr, and CDC24. The second oncogene codes for the alpha-subunit of a recently identified GTP-binding protein, G alpha 12, for which transforming activity has not been described. We are currently characterizing the potential role of these genes in human malignancies.
