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

Abstract

We have cloned and sequenced a full-length cDNA encoding the major constitutive rainbow trout liver cytochrome P450 that we had earlier designated 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. From 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 translational level in sexually mature rainbow trout liver and trunk kidney. Transcriptional expression investigated by Northern analysis of total RNA using a 440-bp 3′-terminal cDNA probe (2K1,7c) showed sexual and organ differences. Mature male trunk kidney expressed 2K1,7c-hybridizable mRNA to a substantially greater extent than did female trunk kidney, 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 female liver samples. By contrast, 1.9-kb mRNA bands were found at generally similar concentrations in the livers of both sexes. Taking into consideration the individual biological variabilities, it was apparent that mature male trout expressed the 2.8-kb mRNA much more strongly in trunk kidney than in liver. Translational expression, analyzed by Western blotting of microsomes separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and probed with rabbit polyclonal antibody and two different monoclonal antibodies prepared against P450 LMC2, demonstrated corresponding sex- and organ-related differences in P450 protein expression. Southern analysis of sexually mature male trout genomic DNA suggested that CYP2K1 is not a single copy gene, thus providing additional evidence for the complexity of the CYP2K1 system in rainbow trout.