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Cloning and sequence analysis of benzo-a-pyreneinducible cytochrome P450 1A in Nile tilapia (Oreochromis niloticus)
Abstract
Polycyclic aromatic hydrocarbons (PAHs), dioxins, dibenzofurans and polychlorinated biphenyls (PCBs) present in polluted environment induce cytochrome P4501A (CYP1A) isozyme in fish which in
turn results in a marked increased production of carcinogenic metabolites. The induction of hepatic CYP1A in fish by certain classes of chemicals has been suggested as an early warning system, a “most
sensitive biological response” for assessing environmental contamination conditions. This has implications for human fish consumption as well as for the health status of aquatic organisms.
Considering the importance of Oreochromis niloticus fish as a laboratory animal, the common CYP1A sequence was determined from cDNA and genomic DNA after intraperitoneal injection with benzo-apyrene (BaP). The full-length cDNA was 2530 bp long and contained an open reading frame of 1566 bp encoding a protein of 521 amino acids and a stop codon. The sequence exhibited 5' and 3' noncoding
regions of 134 and 830 bp, respectively. The deduced amino acid sequence of O. niloticus CYP1A shows similarities of 80.5, 79.3, 79.1, 77.8, 77.6, 74.3, 72.4, 77.2, 71.8, 70.7 and 50.8% with European
flounder CYP1A, scup CYP1A, killifish CYP1A, butterfly fish CYP1A, European sea bass CYP1A, rainbow trout CYP1A, Japanese eel CYP1A, toad fish CYP1A, European eel CYP1A, red sea bream CYP1A and common carp CYP1A, respectively. The phylogenetic tree based on the amino acid sequences clearly shows tilapia CYP1A and killifish CYP1A to be more closely related to each other than to the other CYP1A subfamilies. Sequence analysis of 3727 bp of genomic DNA showed that the clone obtained was the structural gene of CYP1A which consists of seven exons and six introns, the initiation codon was not found in the first exon but in the second one as was reported for the CYP1A genes of fish and mammals.
turn results in a marked increased production of carcinogenic metabolites. The induction of hepatic CYP1A in fish by certain classes of chemicals has been suggested as an early warning system, a “most
sensitive biological response” for assessing environmental contamination conditions. This has implications for human fish consumption as well as for the health status of aquatic organisms.
Considering the importance of Oreochromis niloticus fish as a laboratory animal, the common CYP1A sequence was determined from cDNA and genomic DNA after intraperitoneal injection with benzo-apyrene (BaP). The full-length cDNA was 2530 bp long and contained an open reading frame of 1566 bp encoding a protein of 521 amino acids and a stop codon. The sequence exhibited 5' and 3' noncoding
regions of 134 and 830 bp, respectively. The deduced amino acid sequence of O. niloticus CYP1A shows similarities of 80.5, 79.3, 79.1, 77.8, 77.6, 74.3, 72.4, 77.2, 71.8, 70.7 and 50.8% with European
flounder CYP1A, scup CYP1A, killifish CYP1A, butterfly fish CYP1A, European sea bass CYP1A, rainbow trout CYP1A, Japanese eel CYP1A, toad fish CYP1A, European eel CYP1A, red sea bream CYP1A and common carp CYP1A, respectively. The phylogenetic tree based on the amino acid sequences clearly shows tilapia CYP1A and killifish CYP1A to be more closely related to each other than to the other CYP1A subfamilies. Sequence analysis of 3727 bp of genomic DNA showed that the clone obtained was the structural gene of CYP1A which consists of seven exons and six introns, the initiation codon was not found in the first exon but in the second one as was reported for the CYP1A genes of fish and mammals.
