Seven cellular transforming genes have been detected by transfection of human tumor DNAs into NIH 3T3 cells. Human ras-K genes were identified in primary 3T3 cell transformants derived from four of the tumor DNAs. The remaining three transforming genes, detected in a lung carcinoid tumor (CA1), breast carcinoma (BR2) and renal cell carcinoma (RC1), are not homologous to rasK, rasH, rasN, erb A, erb B, myb or MNNG-HOS probes. Each of these genes has been passaged in two additional rounds of NIH 3T3 cell transfection. DNAs of secondary and tertiary transformants from all three genes showed retained human repetitive sequences when probed with the Alu containing clone BLUR 8. Multiple independently derived secondary (and, from BR2 and CA1 only, tertiary) transformants from each of the three genes retained shared Alu bands in Southern blots, however, the pattern of shared bonds was different in transformants derived from the different genes. This indicates that the three genes are distinct from each other. It is now proposed that the three novel cellular transforming genes be cloned. Libraries of tertiary transformant cell DNA will be constructed in lambda phage vectors and screened by plaque hybridization using radiolabelled BLUR 8 as probe. Initially only part of the CA1 and RC1 genes will be cloned because of their sizes (over 30kb), as estimated by adding the sizes of the shared Alu bands in secondary or tertiary transformant DNAs. Overlapping clones spanning the entire BR2 gene, estimate to be 15-25kb, will be isolated. The clones will be hybridized to the retroviral oncogenes and cellular transforming genes which have not yet been screened for homology. If BR2, CA1 and RC1 are in fact unique genes their chromosomal map positions will be determined by in situ hybridization. Subsequently, the mRNAs transcribed from these genes will be analyzed by northern blotting to determine their size and expression in tertiary transformant cells and a variety of human tumor cell lines. A cDNA clone of the BR2 transforming gene will be produced to facilitate analysis of its mode of action. The cDNA clone will be sequenced using the Maxam-Gilbert method. Next, synthetic polypeptides corresponding to selected segments of the predicted amino acid sequence will be linked to a carrier protein and injected into rabbits to raise antisera against the protein encoded by the BR2 gene. The specificity of these atisera, which will be raised against at least two peptides from different regions of the predicted sequence, will be tested by comparison of the proteins they immunoprecipitate. The properties of the BR2 transforming gene product, including molecular weight, cellular localization and biochemistry, will be examined.
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