Vertebrate genomes contain proto-oncogenes whose enhanced expression or alteration by mutation seems to be involved in the development of naturally occurring tumours. These activated genes, usually assayed by their ability to induce the malignant transformation of NIH 3T3 cells, are frequently related to the ras oncogene of Harvey (Ha-ras) or Kirsten (Ki-ras) murine sarcoma viruses, or a third member of this family (N-ras). Activation involves point mutation which often affect codon 12 (refs 16-26) of the encoded 21,000-molecular weight polypeptide (p21). To provide insight into structural requirements involved in p21 activation, we have now constructed 20 mutant c-Ha-ras1 genes by in vitro mutagenesis, each encoding a different amino acid at codon 12. Analysis of rat fibroblasts transfected with these altered genes demonstrates that all amino acids except glycine (which is encoded by normal cellular ras genes) and proline at position 12 activate p21, suggesting a requirement for an alpha-helical structure in this region of the polypeptide. The morphological phenotype of cells transformed by the activated genes can, however, depend on the particular amino acid at this position.

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The myelocytomatosis viruses are a family of replication-defective avian retroviruses that cause a variety of tumours in chickens and transform both fibroblasts and macrophages in culture through the activity of their oncogene v-myc. A closely related gene (c-myc) is found in vertebrate animals and is thought to be the progenitor of v-myc. Changes in the expression and perhaps the structure of c-myc have been implicated in the genesis of avian, murine and human tumours (for a review, see ref. 15). Elucidation of the mechanisms by which v-myc and c-myc might elicit tumorigenesis requires identification of the proteins encoded by these genes. To this end, we have expressed a portion of v-myc in a bacterial host and used the resulting protein to raise antisera that react with myc proteins. We report here that v-myc and c-myc encode closely related proteins with molecular weights (MWs) of approximately 58,000. Integration of retroviral DNA near or within c-myc in avian lymphomas apparently enhances expression of the gene. Here we have used cells from one such tumour to identify the protein encoded by c-myc and find that the coding domain for the gene is probably intact.

Avian myelocytomatosis virus MC29 is a replication-defective acute leukaemia virus which induces a variety of tumours in chickens including sarcomas, renal and hepatic carcinomas, and myelocytomatosis. The oncogenic potential of the virus is mediated by the gene v-myc, acquired from sequences (c-myc) present in normal uninfected chicken DNA. Sequences closely related to chicken c-myc have been highly conserved throughout evolution, from Drosophila to vertebrates. The hypothesis that c-myc may be involved in neoplastic transformation has been strengthened by the finding that B-cell lymphomas induced in chickens by avian leukosis virus (ALV) are often associated with increased expression of c-myc resulting from integration of the ALV provirus adjacent to the c-myc gene. More recently, it has been demonstrated that the malignant human cell line HL-60, derived from the peripheral blood leukocytes of a patient with acute promyelocytic leukaemia, expresses elevated levels of myc-related mRNA associated with an amplification of the c-myc gene. To explore the relationship of the human cellular myc gene with the corresponding viral oncogene from MC29, and to provide a framework for the analysis of the mechanism and significance of c-myc amplification in human tumours, we have isolated and determined the nucleotide sequence of a genomic clone prepared from a normal human library which contains all domains sharing homology with v-myc.

Segments of the simian virus 40 (SV40) genome that encode only fragments of large tumor antigen can facilitate immortalization of secondary rat embryo cells. The phenotypes of the immortalized cells range from nearly "normal" to fully transformed. All of the cell lines contain SV40 sequences and express unstable NH2-terminal fragments of large tumor antigen. SV40 small tumor antigen does not appear to be essential for either immortalization or transformation.Transformed cells and tumors induced by polyoma virus do not always contain the entire coding region for the viral large tumor antigen (T antigen) (1,2). Both transformants and tumors can be obtained by introducing segments of polyoma DNA that span only the proximal portion of the early region (3-5). In contrast, all simian virus 40 (SV40)-transformed rodent cells examined so far contain and express at least one complete copy of the early viral gene which encodes the T antigen (6). Transformed rodent cells that contain and express fragments of T antigen, but always in the presence of an apparently intact early gene, have been identified (7,8).SV40 and polyoma virus are not strictly comparable because their early regions are organized differently (6). Nevertheless, the polyoma-derived results raise the possibility that a portion of the SV40 early region encoding only the NH2-terminal half of T antigen might facilitate transformation. We have found this to be the case. We utilized an assay that required only that secondary rat embryo cells continue to grow after transfection with recombinant plasmids containing segments of the SV40 genome. The immortalized cells exhibited phenotypes ranging from nearly "normal" to fully transformed. The generation of a range of growth phenotypes implies a significant but nonuniform contribution to the growth potential of the immortalized lines by cellular gene products.MATERIALS AND METHODS Recombinant Plasmids. Recombinant plasmids [pBR322 (9)] used in this study are diagrammed in Fig. 1. pSV-7 was derived from SV40 wt830 (10) DNA and contains an intact early region. pSV-8 was derived from pSV-7 by removing a 1,067-base-pair (bp) Hpa I cleavage fragment (0.17 to 0.37 map units). This plasmid encodes the NH2-terminal 311 amino acids of T antigen. Its deletion extends beyond the normal termination codon for T antigen, and no termination signal occurs before the site for cleavage and polyadenylylation at the 3' end of the early mRNA. pSV-11 was derived from d11440 DNA (11). d11440 lacks 268 bp and cannot produce small tumor antigen (t antigen) because the splice acceptor site for the t antigen mRNA has been deleted; T antigen is not affected. pSV-12 was generated by deleting the Hpa I cleavage fragment at 0.17 to 0.37 map units from pSV-ll. Cells, Transfections, and Growth Assays. Primary Fischer rat embryo cells (Microbiological Associates, Bethesda, MD) were subcultured at a 1:10 dilution in Dulbecco's modified Eagle's medium supplemented with 10% calf serum. Secondary cultures at 50% confluence were transfected with ...