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Promoter-glutathione S-transferase Ya cDNA hybrid genes were constructed and analyzed to determine the efficiency with which the Ya coding sequence was transcribed and also to determine the associated levels of Ya-specific enzyme activity in mammalian cells which had received the hybrid gene constructs via electroporation. Promoter-containing fragments from either the SV40 early region or the herpes simplex thymidine kinase gene were positioned 5' to the Ya cDNA present in the pGTB38 plasmid. Both promoters supported transcription in in vitro run-off incubations containing a rat cell extract. Efficient transcription was also observed in both monkey Cos cells and mouse C3H/10T1/2 cells. Constructs containing the SV40 promoter and a residual portion of the homopolymeric G tail used in the original Ya cDNA cloning consistently gave 4-50-fold higher levels of transcript than other promoter-cDNA configurations. Associated with transcription of the hybrid gene was the appearance of a glutathione S-transferase YaYa-specific enzyme activity (delta 5-androstene-3,17-dione isomerization) in cytosols of cells electroporated with the hybrid genes. 50-260-fold increases in Ya-specific enzyme activity were found in Cos or C3H/10T1/2 cells containing multiple, episomal copies of the plasmid constructs; enzyme levels dropped in cells containing fewer, integrated plasmid copies. When a mixed population of Cos cells containing YaYa overexpressing cells was treated with benzo(a)pyrene (+/-)-anti-diol epoxide, a cytotoxic alkylating molecule and known YaYa substrate, a 20-30-fold enrichment in clones of YaYa overexpressing cells was seen among those cells which survived the treatment. The results clearly indicate that glutathione S-transferase isozymes can be overexpressed in mammalian cells and that this is accompanied by significant biological resistance to a known alkylating molecule.