title: Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say.
abstract: To analyze cytochrome P450s in the southern house mosquito, Culex quinquefasciatus, we quantified the content of P450s and b5 in larval microsomes of guts and carcasses. Results indicated that content was 30 times higher in guts than in carcasses. A conserved region in the alignment of insect P450 family 6 (CYP6) proteins served as a guide for the synthesis of degenerate oligonucleotide primers to clone P450 cDNAs. Primers were used in the reverse transcription-polymerase chain reaction (RT-PCR) of gut mRNA from 4th-instar larvae of the permethrin-susceptible or resistant C. quinquefasciatus. PCR products of ca. 250 base pairs (bp) were cloned, and nucleotide sequences of 35 clones from susceptible and 28 from resistant strains determined. Alignment of the deduced amino acid sequences from these clones showed them to be classifiable into six isoforms. We next screened a cDNA clone (CYP6F1) from a gut cDNA library and determined the nucleotide sequence. Northern blot analysis showed that the CYP6PF1 gene in the permethrin-resistant strain appeared to be expressed more strongly than in the susceptible strain. The deduced amino acid of CYP6F1 showed that it has conserved domains of a membrane-anchoring signal, reductase binding sites, a heme-binding site, ETLR motif and substrate recognition sites in P450s. Phylogenetic analysis showed that CYP6F1 is strongly related to CYP6D1 involved in pyrethroid detoxification.
authors.affiliation: Laboratory of Applied Zoology, University of Tsukuba, Ibaraki, Japan.
authors.forename: S
authors.initials: S
authors.lastname: Kasai
authors.forename: I S
authors.initials: IS
authors.lastname: Weerashinghe
authors.forename: T
authors.initials: T
authors.lastname: Shono
authors.forename: M
authors.initials: M
authors.lastname: Yamakawa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D003465:Culex / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D000072472:Cytochrome P450 Family 6; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D004351:Drug Resistance / Q000235:genetics; D015870:Gene Expression; D017344:Genes, Insect; D019476:Insect Proteins; D007306:Insecticides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics*; D010802:Phylogeny; D011722:Pyrethrins / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid
chemical_list: D017931:DNA Primers; D018076:DNA, Complementary; D019476:Insect Proteins; D007306:Insecticides; D011722:Pyrethrins; D003577:Cytochrome P-450 Enzyme System; D010105:Oxygenases; C406279:CYP6F1 protein, Culex pipiens pallens; D000072472:Cytochrome P450 Family 6