THE DROSOPHILA CYTOCHROME-P450 GENE CYP6A2 - STRUCTURE, LOCALIZATION,HETEROLOGOUS EXPRESSION, AND INDUCTION BY PHENOBARBITAL

The cytochrome P450 gene Cyp6a2 from Drosophila melanogaster is locate d on the right arm of chromosome 2 at position 43A1-2 and comprises tw o exons separated by a 69-bp intron. Phenobarbital treatment of flies leads to a rapid increase in the level of CYP6A2 mRNA and to an increa sed production of the CYP6A2 protein, DNA from the Cyp6a2 promoter reg ion was functional when linked to a luciferase reporter gene and trans fected into D. melanogaster Schneider cells, Moreover, a dose-dependen t induction of luciferase activity by phenobarbital indicated that ele ments necessary for phenobarbital induction are located within 428 bp of the translation start site. Heterologous expression of the CYP6A2 p rotein in lepidopteran cells infected with a Cyp6a2-recombinant baculo virus was observed by Western blotting of cell lysates and by spectral characterization of the reduced-CO complex of the P450. The CYP6A2 pr otein produced in this system metabolized aldrin and heptachlor to the ir epoxides and metabolized the insecticide diazinon by desulfuration to diazoxon and by oxidative ester cleavage to 2-isopropyl-4-methyl-6- hydroxypyrimidine. Metabolism in lysates of cells infected with recomb inant baculovirus was greatly enhanced by the addition of purified hou sefly NADPH cytochrome P450 reductase and cytochrome b(5). These resul ts show that CYP6A2 catalyzes the metabolism of organophosphorus insec ticides and they implicate Cyp6a2 overexpression in metabolic resistan ce. The Cyp6a2 gene appears to be a suitable model for a genetic analy sis of the phenobarbital induction process.