CLONING AND SEQUENCING OF THE MAJOR RAINBOW-TROUT CONSTITUTIVE CYTOCHROME-P450 (CYP2K1) - IDENTIFICATION OF A NEW CYTOCHROME-P450 GENE SUBFAMILY AND ITS EXPRESSION IN MATURE RAINBOW-TROUT LIVER AND TRUNK KIDNEY

We have cloned and sequenced a full-length cDNA encoding the major con stitutive rainbow trout liver cytochrome P450 that we had earlier desi gnated as cytochrome P450 LMC2 (Arch. Biochem. Biophys. 268, 227, 1989 ). The 1859-bp cDNA was isolated from a male rainbow trout liver cDNA library and contained an open reading frame encoding for a protein of 504 amino acids and having a calculated molecular weight of 56,795. Fr om nucleotide and amino acid sequence comparisons, the trout P450 LMC2 has been assigned to a new cytochrome P450 gene subfamily designated P4502K1 or CYP2K1. Marked differences in the sex- and tissue-specific expression of this gene were found at both the transcriptional and tra nslational level in sexually mature rainbow trout liver and trunk kidn ey. Transcriptional expression investigated by Northern analysis of to tal RNA using a 440-bp 3'-terminal cDNA probe (2K1,7c) showed sexual a nd organ differences. Mature male trunk kidney expressed 2K1,7c-hybrid izable mRNA to a substantially greater extent than did female trunk ki dney, with multiple mRNA bands appearing in approximately the 2.8- and 1.9-kb region. While the livers of some males displayed separate 2.8- kb hybridizable bands, such bands were generally undetectable in femal e liver samples. By contrast, 1.9-kb mRNA bands were found at generall y similar concentrations in the livers of both sexes. Taking into cons ideration the individual biological variabilities, it was apparent tha t mature male trout expressed the 2.8-kb mRNA much more strongly in tr unk kidney than in liver. Translational expression, analyzed by Wester n blotting of microsomes separated on sodium dodecyl sulfate-polyacryl amide gel electrophoresis and probed with rabbit polyclonal antibody a nd two different monoclonal antibodies prepared against P450 LMC2, dem onstrated corresponding sex- and organ-related differences in P450 pro tein expression. Southern analysis of sexually mature male trout genom ic DNA suggested that CYP2K1 is not a single copy gene, thus providing additional evidence for the complexity of the CYP2K1 system in rainbo w trout. (C) 1994 Academic Press,Inc.