Identification, characterization, and ontogeny of a second cytochrome P450 3A gene from the fresh water teleost medaka (Oryzias latipes).

Abstract

Multiple copies of cytochrome P450 gene family 3 have been identified from numerous mammalian species. Often these genes exhibit differential catalytic activities and gene regulation. To date however, little information is available regarding multiple forms of this gene family in teleost fishes. In this study, a second isozyme of cytochrome P450 3A has been cloned from the teleost fish Oryzias latipes and designated CYP3A40. Screening of a cDNA library to medaka liver resulted in the identification of a full length cDNA clone containing a 2316 base pair (bp) insert with an open reading frame encoding a single peptide of 502 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 shares a 90% identity to CYP3A38 previously identified from medaka liver. Consistent with Northern blot and Western blot analysis, Southern blots of medaka genomic DNA demonstrated the presence of two CYP3A genes. Gene expression studies demonstrated that CYP3A38 and CYP3A40 are differentially regulated according to embryonic development. Northern blot analysis, using a probe to a conserved region of both CYP3A genes, demonstrated the presence of a single CYP3A transcript for early and late embryonic stages and two CYP3A transcripts in larvae and adult liver. Similarly, Western blots show a single faint immunoreactive cytochrome P450 3A protein in microsomes from early and late embryos and two abundant protein bands in microsomes from larval and adult liver. To further examine the transcriptional differences in CYP3A expression, RT-PCR analysis was performed on embryonic stages 11-35, 1- and 14-day-old larvae, and adult liver using primer sets specific for CYP3A38 and CYP3A40. These results demonstrate that CYP3A40 is expressed early in embryonic development and continues throughout adult stages. CYP3A38, however, is tightly regulated during embryonic development and is only expressed post-hatch.