DNA transfection of a gene repressing aryl hydrocarbon hydroxylase induction.
High mol. wt genomic DNA from a genetically dominant aryl hydrocarbon hydroxylase (AHH)-deficient mutant cell line derived from the mouse hepatoma cell line Hepa-1 was used to transfect the parent cell line. AHH-deficient transfectants were recovered following single-step selection in medium containing the carcinogen benzo[a]pyrene. The transfectants arose at a frequency of 2 x 10(-7). This frequency was at least 4-fold greater than the frequency of spontaneous forward mutation in this cell line. In another set of experiments, dominant mutant DNA was co-transfected along with the selectable plasmid pSV2ecogpt into parental Hepa-1 cells. The frequency of co-transfection was determined to be 3 x 10(-8). This frequency was approximately 150 times greater than that expected on the basis of coincident but unrelated spontaneous mutation and plasmid uptake. Both types of transfectants were judged, following somatic cell hybridizations, to possess the dominant phenotype of the mutant cell line, demonstrating that a trans-acting dominant negative regulator of AHH was transferred in these experiments. DNA transfection should therefore provide a means for the molecular cloning of the gene that encodes the dominant regulator.
Watson
A J
AJ
Laboratory of Biomedical and Environmental Sciences, School of Public Health, University of California, Los Angeles 90024.
Hankinson
O
O
D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000172:deficiency / Q000378:metabolism*; D001564:Benzo(a)pyrene / Q000378:metabolism / Q000494:pharmacology; D004247:DNA / Q000032:analysis; D004351:Drug Resistance; D004794:Enzyme Repression; D005799:Genes, Dominant; D005809:Genes, Regulator; D006822:Hybrid Cells; D008114:Liver Neoplasms, Experimental; D051379:Mice; D014162:Transfection; D014407:Tumor Cells, Cultured
D001564:Benzo(a)pyrene; D004247:DNA; D001189:Aryl Hydrocarbon Hydroxylases