Search

Cytochrome P450 monooxygenases play a significant role in the detoxification of hostplant allelochemicals and synthetic insecticides in Lepidoptera. In the corn earworm Helicoverpa zea, a noctuid of considerable economic importance, metabolisms of xanthotoxin, a toxic furanocoumarin, and alpha-cypermethrin, an insecticide, are mediated by at least one P450 with a catalytic site capable of accepting both substrates. To further the characterization of P450s in this species, we have cloned three full-length cDNAs encoding two CYP4M subfamily members and a novel CYP321A subfamily member. RNA analyses have demonstrated that the CYP321A1 gene is highly induced (51-fold) in larval midguts in response to xanthotoxin but not cypermethrin. Both CYP4M genes are expressed at negligible levels that are not increased by xanthotoxin or cypermethrin. Baculovirus-mediated expression of the full-length CYP321A1 cDNA has demonstrated that the CYP321A1 protein metabolizes xanthotoxin and angelicin, like the CYP6B1 protein in the furanocoumarin specialist Papilio polyxenes, and alpha-cypermethrin, like the CYP6B8 protein previously characterized in H. zea. In contrast, the CYP4M7 protein does not metabolize xanthotoxin at any detectable level. We conclude that at least two xanthotoxin-inducible P450s from highly divergent subfamilies (CYP6B and CYP321A) contribute to the resistance of H. zea larvae to toxic furanocoumarins and insecticides. Genomic PCR analysis indicates that the CYP321A1 gene has evolved independently from the CYP6B genes known to be present in this insect.

The cDNAs of two novel P450s (CYP9E2 and CYP4C21) were isolated from German cockroaches, Blattella germanica . Both CYP9E2 and CYP4C21 are typical microsomal P450s and their deduced amino acid sequences share a number of common characteristics with other members of the P450 superfamily. Northern blot analyses using a CYP9E2 or CYP4C21 probe showed that ‘CYP9E2’ and ‘CYP4C21’ were expressed at all life stages. Two pseudogenes related to CYP9E2 and three pseudogenes related to CYP4C21 were also isolated. These represent the first P450 pseudogenes from an insect other than Drosophila melanogaster . The relative number of P450 pseudogenes in B. germanica is apparently higher than in D. melanogaster . The implications of these results for the molecular evolution, expression studies and nomenclature of P450s are discussed.

CYP6D1 is a cytochrome P450 found in the house fly, Musca domestica. Expression is greater in pyrethroid-resistant vs. -susceptible strains and can be induced by phenobarbital in adult susceptible flies. CYP6D1 is expressed only in adult flies. To gain information about possible regulatory elements involved in CYP6D1 expression, and to confirm the gene sequence that was previously determined by polymerase chain reaction (PCR), we screened a house-fly library prepared with genomic DNA from the pyrethroid-resistant LPR strain. A CYP6D1v1 clone was isolated and sequenced. This clone contained 887 nucleotides 5′ to the open reading frame and a previously unknown 2.4-kb intron. Using polymerase chain reaction with primers based on the CYP6D1v1 allele, the sequences 5′ to the ORF were obtained from five pyrethroid susceptible strains. The transcription initiation site (TIS) was identified at the same position in LPR and two susceptible strains (86 nucleotides upstream from the translation start site). A comparison of the 5′ flanking sequences revealed a high degree of similarity for most regions, although differences in the sequences were identified. The possible roles of these sequence differences in regulation of CYP6D1 expression are discussed.