cDNA cloning, sequence analysis and expression of 3-methylcholanthrene-inducible cytochrome P450 1B1 in carp...

Cytochrome P450 (CYP) genes, which make up a large gene superfamily, are known to play an important role in drug metabolism. The levels of expression of CYP genes in the tissue of fish inhabiting polluted areas have been used extensively in biomonitoring studies as indicators of dioxin pollution. Complementary DNA of cytochrome CYP1B1 was isolated from carp (Cyprinus carpio) liver 24 h after the injection of 3-methylcholanthrene (3-MC). The full length cDNA obtained contained a 5' noncoding region of 178 bp, an open reading frame of 1593 bp coding for 530 amino acids and a stop codon, and a 3' noncoding region of 1599 bp. The predicted molecular weight of the encoded protein was approximately 60.2 kDa. The deduced amino acid sequence exhibited an identity of 60.6% with reported CYP1B sequences of plaice CYP1B, and of 52.4, 51.4, and 50.3% with human, rat, and mouse CYP1B1s, respectively. It exhibited similarities of 48.4 and 47.3% with scup CYP1C2 and -1C1 sequences. The percent identities with CYP1A sequences showed lower values in the range from 35.3 to 39.5% with mammals and teleosts. The phylogenetic tree of the CYP1 family members constructed by the protein maximum likelihood method indicates that the carp CYP1B1 and plaice CYP1B share a common ancestry with the mammalian CYP1B1s. Carp treated with 3-MC showed expression of CYP1B1 in liver, intestine and gills with distinct induction except for the gills that showed marked constitutive expression. The presence of two successive signals in treated gills at low stringency hybridization may suggest the existence of another CYP1B member that is expressed in the gills of carp.