2 Unique CYP1 Genes Are Expressed in Response to 3-Methylcholanthrene Treatment in Rainbow Trout

Abstract

In animals, exposure to polyaromatic hydrocarbons (PAHs) such as 3-methylcholanthrene (3-MC) is known to induce the expression of two unique cytochrome P450 genes, CYP1A1 and CYP1A2. These genes are thought to have originated by a gene duplication event and diverged no more than 250 million years ago (D. W. Nerbert and F. J. Gonzalez, 1987, Annu. Rev. Biochem. 56,945-993). Lower vertebrates, such as fish, diverged from land animals before this time and are thought to express only a single CYP1 gene. In this paper, we present evidence to refute this hypothesis and report the isolation and complete genomic nucleotide sequence of two distinct CYP1 genes in rainbow trout. Genomic clones encoding the entire CYP1A1 and CYP1A2 genes were characterized. DNA sequence analysis revealed that both genes contained seven exons and six introns. Exons 1-7 of CYP1A1 and CYP1A2 were highly similar in length and nucleotide sequence. In contrast, the 5′-flanking region and introns 1, 2, 5, and 6 of both genes were significantly less conserved. Two xenobiotic regulatory elements (XREs) were identified in the 5′ flanking region of CYP1A1 but not in that of CYP1A2. The 5′-most start site of transcription was determined to begin at a cytosine residue 27 bases downstream of the putative TATA box of both genes. Northern blot analysis demonstrated that exposure to 3-MC resulted in an increase in CYP1 mRNA levels in the liver. RNase protection assays conducted with riboprobes specific for either CYP1A1 or CYP1A2 confirmed that the transcripts of both genes were expressed in rainbow trout liver in response to 3-MC treatment.