Identification and characterization of a cDNA encoding cytochrome P450 3A from the fresh water teleost medaka (Oryzias latipes).
A new member of the CYP3A gene family has been cloned from the teleost fish medaka (Oryzias latipes) by reverse-transcriptase polymerase chain reaction (RT-PCR). Degenerate primers homologous to highly conserved regions of known CYP3A sequences were used for initial RT-PCRs. Individual PCR products were cloned, sequenced, and identified as those belonging to the cytochrome P450 superfamily based on amino acid sequence similarity and the presence of the highly conserved heme-binding region. PCR products were subsequently used as probes to screen a complementary DNA library. A full-length cDNA clone was identified containing a 1758-base-pair (bp) insert with an open reading frame encoding a single peptide of 500 amino acids. Comparisons of the deduced amino acid sequence to other known cytochrome P450 sequences indicate that this gene product is most similar to the CYP3A gene family and has been designated as CYP3A38 by the cytochrome P450 nomenclature committee. Northern blot analysis identified two abundant CYP3A related transcripts in liver of both male and female adults and demonstrated quantitative differences in abundance according to gender. Similarly, Western blot analysis demonstrated the presence of two abundant cytochrome P450 related proteins in liver of both male and female adults. These results suggests that O. latipes contains multiple forms of CYP3A. Heterologous expression of CYP3A38 cDNA in HEK 293 cells produced a single protein that was reactive with anti-scup P450A (CYP3A) polyclonal antibody. Microsomes of HEK 293 cells expressing recombinant CYP3A38 protein actively catalyzed the hydroxylation of testosterone.
Kullman
S W
SW
School of Veterinary Medicine, University of California Toxic Substance Research and Training Program, Lead Campus in Ecotoxicology, Davis, California, 95616-8732, USA. swkullman@ucdavis.com
Hamm
J T
JT
Hinton
D E
DE
D000595:Amino Acid Sequence; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism*; D005260:Female; D005399:Fishes / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008099:Liver / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000235:genetics*; D012313:RNA / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012737:Sex Factors; D013739:Testosterone / Q000378:metabolism
D018076:DNA, Complementary; D007527:Isoenzymes; D011994:Recombinant Proteins; D013739:Testosterone; D012313:RNA; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating