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Mol Cell Biol. 1985 April; 5(4): 698-704

Transfection by genomic DNA of cytochrome P1-450 enzymatic activity and inducibility.

ABSTRACT

An aryl hydrocarbon hydroxylase (AHH)-deficient gene A- mutant of the mouse line Hepa-1 was treated with calcium phosphate precipitates of DNA from Hepa-1, the rat line H4IIEC3, or an A- -human hybrid in which the A- mutation is complemented by the corresponding human gene. AHH+ transfectants were isolated by selection with benzo[ghi]perylene plus near UV. In addition, a gene A- mutant which also carries a mutation for hypoxanthine phosphoribosyltransferase deficiency was treated with the above genomic DNAs together with pSV2-gpt DNA, and cotransfectants were isolated after treatment with both benzo[ghi]pereylene and HAT. All transfectants and cotransfectants were inducible for AHH by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Both transfectants and cotransfectants were unstable during culture, rapidly losing AHH activity. Rat DNA-derived transfectants were probed in Southern blots with a cDNA probe to mouse cytochrome P1-450 that cross-hybridizes to the corresponding rat gene. All rat DNA-derived transfectants contained the rat P1-450 gene. In half of the transfectants, the rat gene was amplified four- to sevenfold. In one transfectant, the rat gene was truncated at the 3' end. The proportion of rat DNA in different transfectants, as determined by hybridization to a rat repetitive sequence, ranged from less than 1% to 5%. AHH activity and the rat P1-450 gene segregated together in subclones of one of the transfectants. These results demonstrate that the A gene is either the structural gene for cytochrome P1-450, or another very closely linked gene. Previous results (O. Hankinson et al., J. Biol. Chem. 260:1790-1795, 1985) favor the former alternative.

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