Yea-Huey Yang

Expression of a constitutive cytochrome P450 (CYP2K1) in livers of rainbow trout (Oncorhynchus mykiss) embryo and sac-fry

Abstract The hepatic expression of a recently reported cytochrome P450, P450 2K1 (CYP2K1), was studied in livers of rainbow trout ( Oncorhynchus mykiss ) 30 day embryos (1 day before hatching) and 2 day and 10 day sac-fry at both the transcriptional and translational levels using a CYP2K1 cDNA probe (2K1,7c, 440 bp) and antibodies (polyclonal and monoclonal) against rainbow trout cytochrome P450 LMC2, the presumed translational product of the CYP2K1 gene, as probes for Northern and Western blots, respectively. The P450 LMC2 isoform was previously identified as a constitutive carcinogen-activating P450 present in the livers of adult rainbow trout. Simultaneous recovery of RNA, DNA and protein was accomplished by extraction of the same pooled liver tissues removed from the trout embryos and sac-fry by microdissection. The concentrations of both hepatic CYP2K1 mRNA and protein (LMC2) increased with embryo and sac-fry development. Comparison of the levels of the LMC2-like protein (translational expression of CYP2K1) using mouse anti-trout LMC2 monoclonal antibody (3M1) as a probe in 30 day embryo, 2 day sac-fry, 10 day sac-fry and 1-year-old trout liver yielded values of 0.035 pmol μg −1 DNA, 0.08 pmol μg −1 DNA, 0.12 pmol μg −1 DNA and 0.90 pmol μg −1 DNA, respectively.

Cloning, sequencing and expression of the major rainbow trout constitutive cytochrome P450 (CYP2K1). Identification of a new cytochrome P450 gene subfamily

Abstract A full-length cDNA has been cloned and sequenced for the major constitutive rainbow trout liver cytochrome P450 that had earlier been designated as cytochrome P450 LMC2. This cDNA clone encodes for a protein of 504 amino acids with a calculated M r of 56,795. While the cDNA nucleotides for trout P450 LMC2 shared a 49 and 47% homology, respectively, with the two rainbow trout P450s previously sequenced (hepatic P4501A1 and an ovarian 17-hydroxylase), it showed a 54–56% nucleotide identity with several human or other mammalian P450 forms from family 2. From amino acid sequence comparisons, the trout P450 LMC2 has been assigned to a new cytochrome P450 gene family designated P4502K1 (CYP2K1). Northern blots of total RNA isolated from sexually mature male and female rainbow trout examined with a cDNA probe (2K1,7c), indicated that mRNA levels were substantially higher in the trunk kidney of male trout compared to that seen in females. Multiple 2K1,7c hybridizable bands at about 2.8 kb and 1.6–1.9 kb were found in the trunk kidney preparations, with the 2.8 kb band predominating. A major 1.9 kb mRNA band, detectable with the 2K1,7c probe, was present at similar concentrations in livers of both sexes.