Molecular analysis of the constitutive and barbiturate-induced expression of a Drosophila melanogaster cytochrome P450 gene, Cyp6a2

Detoxification of endogenous compounds and environmental pollutants is mediated by cytochrome P450 monooxygenases (CYPs). In insects, CYPs may also play a role in the resistance to insecticides because the levels and enzymatic activities are generally higher in resistant strains than in susceptible ones. However, little is known about the regulation of resistance-associated insect CYP genes. Therefore, in the present investigation the Cyp6a2 gene of D. melanogaster was used as a prototype to understand this phenomenon. The 3'-untranslated region of the Cyp6a2 gene of the underproducer 91C strain, but not the overproducer 91R strain, contains the long terminal repeat (LTR) of transposable element 17.6. In the present study, the hypothesis that the LTR insertion influences the expression of Cyp6a2 was tested. Quantitative RNA blot analysis of Cyp6a2 expression in adults of different underproducer and overproducer strains showed that (i) the presence or absence of the LTR insertion does not correlate with the steady- state level of CYP6A2 mRNA; (ii) a constitutively low level of CYP6A2 mRNA is the wild-type phenotype and (iii) the level of CYP6A2 mRNA is higher in males than in females. Chromosome substitution experiments showed that the 2R-linked Cyp6a2 gene is regulated by a locus (loci) on the third chromosome. Using P-element mediated germ line transformation, the cis-regulatory elements required for constitutive expression of Cyp6a2 were localized between —985 and —129 relative to +1 of ATG. DNA sequence analysis of the -1331 to +1 region in an underproducer and overproducer strain showed that the upstream DNA sequences are identical except for six bases. This same region also contains several interesting sequence motifs: an ecdysone response element, three binding sites for the ecdysone-inducible transcription factor. Broad Complex, and six "barbie boxes," cis elements that are found in the upstream DNA of barbiturate-inducible CYPs. Both barbital and phenobarbital induced the expression of Cyp6a2, but the pattern of induction with both compounds was different in underproducer and overproducer strains. Using germ line transformation, the sequences required for barbital-mediated induction were localized between -985 and -129. Collectively, these data shed new light on the molecular and genetic mechanisms that regulate insect CYP genes.