We have generated two serum-and anchorage-dependent revertants from NIH 3T3 cells transformed with multiple copies of the human c-H-ras oncogene. In both revertants, the c-H-ras oncogene was fully expressed. Fusion of either revertant with untransformed cells or of the two revertants with one another resulted in transformed progeny. These results indicated that the two revertants were recessive and in different complementation groups. We believe that in our two revertants some of the genes mediating the transforming activity of the c-H-ras oncogene are defective; we are attempting to identify these mediator genes.We wish to identify genes that mediate the transforming activity of the c-H-ras oncogene (1). The approach chosen requires the isolation of recessive revertants from cells transformed with the oncogene (3,9,19). In the revertants sought, the oncogene is fully expressed, yet the transformed state is not manifested in consequence of a defect in a gene (mediator gene) required for mediating the transforming activity. We plan to retransform the revertants by transfection with DNA from normal cells and to identify the retransforming gene.For this purpose, we have transfected (7) mouse NIH 3T3 cells with a plasmid carrying an activated human c-H-ras oncogene from the T24 bladder carcinoma cell line (13) and, as a selectable marker, the neomycin (G418) resistance gene (12). From the resulting G418-resistant clones, we picked one (FT9) carrying five to seven copies of the integrated ras oncogene. Revertants (serum dependent) were obtained from FT9 (serum independent) by incubation in serum-free medium with bromodeoxyuridine and irradiation (17). The procedure was repeated with the survivors except that the incubation with bromodeoxyuridine was in the presence of 1% fetal calf serum (FCS) to eliminate cells with a low serum requirement. After screening of 162 revertant clones, 2 with flat morphology (R116 and R260) were chosen for further studies. R116 cells were similar in size to NIH 3T3 cells; R260 cells were smaller (Fig. 1A). Both revertants were serum dependent: in the absence of serum, they formed only a few small colonies in conditions in which FT9 cells reached confluency ( Fig. 2A). The efficiency of growth of the two revertants in 1% FCS was similar to that of NIH 3T3 cells and clearly lower than that of FT9 cells (Table 1)
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