A cluster of cytochrome P450 genes of the CYP6 family in the house fly

Abstract

A cluster of genes of the CYP6 family was found in a series of overlapping lambda DASH clones from a genomic library of the house fly, Musca domestica. Four complete genes, CYP6A3, CYP6A4, CYP6A5, and CYP6C1, and fragments of two other genes, CYP6A6 and CYP6C2, were closely linked on a 24-kb segment of DNA. Restriction fragment length polymorphism (RFLP) analysis of PCR-amplified segments of two of the genes showed that the cluster is localized on chromosome V of the house fly. Each gene contained a short intron of 57 to 125 bp interrupting a conserved Glu codon, as in the previously described CYP6A1 gene. The gene fragment CYP6A6 consisted only of the coding region downstream from this intron, i.e., about one-third of the complete P450. The gene fragment CYP6C2 was missing a short amino-terminal part of the coding region, and may represent the two last exons of a larger gene. Gene duplication and chromosomal inversion events may explain the origin of this cluster. The P450 proteins deduced from the nucleotide sequences shared 39-71% amino acid identity with each other. This low identity and the lack of evidence of recent gene conversion events suggested that this cluster may be evolutionarily ancient and that homologous clusters may be found in other holometabolous insects. Evidence for transcription of the genes and for correct splicing of the introns was obtained by northern blotting and reverse transcription polymerase chain reaction (RT-PCR) experiments. No overexpression was observed in any of three insecticide-resistant house fly strains. RT-PCR and sequencing also revealed the existence of other genes or alleles closely related to the members of this cluster.