CLONING MAMMALIAN GENES BY EXPRESSION SELECTION OF GENETIC SUPPRESSORELEMENTS - ASSOCIATION OF KINESIN WITH DRUG-RESISTANCE AND CELL IMMORTALIZATION

We describe a general strategy for cloning mammalian genes whose downr egulation results in a selectable phenotype. This strategy is based on expression selection of genetic suppressor elements (GSEs), cDNA frag ments encoding either specific peptides that act as dominant inhibitor s of protein function or antisense RNA segments that efficiently inhib it gene expression. Since GSEs counteract the gene from which they are derived, they can be used as dominant selectable markers for the phen otype associated with downregulation of the corresponding gene. A retr oviral library containing random fragments of normalized (uniform abun dance) cDNA expressed in mouse NIH 3T3 cells was used to select for GS Es inducing resistance to the anticancer drug etoposide. Three GSEs we re isolated, two of which are derived from unknown genes and the third encodes antisense RNA for the heavy chain of a motor protein kinesin. The kinesin-derived GSE induces resistance to several DNA-damaging dr ugs and immortalizes senescent mouse embryo fibroblasts, indicating th at kinesin is involved in the mechanisms of drug sensitivity and in vi tro senescence. Expression of the human kinesin heavy-chain gene was d ecreased in four of four etoposide-resistant HeLa cell lines, derived by conventional drug selection, indicating that downregulation of kine sin represents a natural mechanism of drug resistance in mammalian cel ls.