Identification of multiple isoforms of cytochrome P450 3A in medaka Oryzias latipes: characterization and ontogeny

Abstract

In this study, two isoforms of cytochrome P450 3A have been cloned from medaka Oryzias latipes. Screening of a cDNA library to medaka liver resulted in the identification of two full length cDNA clones containing 1750 and 2366 base pair inserts with open reading frames encoding two peptides of 500 and 501 amino acids, respectively. Amino acid sequence comparisons indicate that these gene products belong to the cytochrome P450 superfamily and display a high degree of sequence homology to the CYP3A subfamily, designated CYP3A38 and CYP3A39. Northern blot analysis identified two abundant gender specific CYP3A related transcripts in liver of both male and female adults. Western blot analysis confirmed the mRNA data suggesting that Oryzias latipes contains multiple CYP3A isoforms. Expression analysis was used to examine the ontogeny of each CYP3A isoform. Western blots demonstrate the presence of two abundant cytochrome P450 3A like proteins in later stages of development including larva and adults and only a single 3A protein for embryonic stages 11–35. RT-PCR was consistent with the protein data and suggests that CYP3A39 is expressed early in embryonic development and continues throughout adult stages while CYP3A38 is expressed only in larva and adults.