FUNCTIONAL IDENTIFICATION AND MOLECULAR-CLONING OF A RAT GENE MEDIATING GROWTH-INHIBITION AND PROGRAMMED CELL-DEATH IN NORMAL AND TRANSFORMED RAT-CELL LINES

We wished to identify DNA sequences conferring suppression of prolifer ation and transformed phenotypes. Thus, we have transfected DNA from n ormal rat cells, covalently linked to neo DNA coding for neomycin resi stance into a tumorigenic, HRAS transformed rat cell line. Phenotypic revertants were selected after the first cycle of transfection by enri chment procedures that served to eliminate transformed cells. The reve rtant clones continued to express the HRAS oncogene, but exhibited a l ower tumorigenicity, loss of anchorage-independent proliferation, flat morphology, and retardation of growth in monolayer culture. The rever ted phenotype could be transferred in a second cycle of transfection i nto the HRAS transformed rat cells. Neo DNA ligated to genomic donor D NA was used as a tagging sequence to molecularly clone the transferred DNA sequence in a recombinant phage. Fragments of the cloned DNA dete ct a 2.5 kb transcript in parental cells and revertants. Thus, the rec ombinant phage harbors a putative growth inhibitory gene, designated t rg, that is expressed at a higher level in rat embryo fibroblasts and in the REF52 cell line. Introduction of recombinant phage DNA into est ablished 208F and Rat-2 cells and into HRAS-, v-fgr-, v-fms- and v-raf -transformed rat cell lines resulted in inhibition of growth and induc tion of programmed cell death.