Development of Molecular Tools, cDNA Cloning, and Sequence Analysis of Aromatase Cytochrome P45019A in Atlantic Tomcod, Microgadus tomcod

Cytochrome P450 induction and expression is recognized as a useful biomarker to compare toxicological effects of environmental contaminants such as PAHs and PCBs in fish.  Molecular tools have been developed for the cloning and characterization of ovarian cytochrome 19A (CYP19A), of Atlantic tomcod (Microgadus tomcod).  This species is a common estuarine-inhabiting cod (Gadidae) important as a forage fish in estuaries throughout the NE USA and Atlantic Canada.  Degenerate primers were designed for RT-PCR based on CYP19A sequences from the closely related Gadus morhua along with other most closely related teleosts species (Accession #s: DQ402370, AY859425, AY510711, AY547354, AY684256, FJ800566, and DQ184486).  RT-PCR cloning led to isolation of a 1.3 kbp cDNA, that encodes a gene product with a predicted molecular weight of 39.7, representing a partial CYP19A sequence.  5’ and 3’ rapid amplification of cDNA ends (RACE)-PCR was used to construct the full length cDNA sequence.  The cloned CYP19A sequence shares 86 % identity with Atlantic cod ovarian aromatase and 65–78 % with other fish species including Mugil cephalus, Micropogonias undulatus, Larimichthys crocea, Epinephelus coioides, Epinephelus akaara, and Lates calcarifer.  This new molecular tool developed here has application as a biomarker for the monitoring of environmental pollution.