Diversity and characterization of novel Cytochrome P450 2 genes in the marine teleost Fundulus Heteroclitus
Oleksiak, Marjorie F.
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The multiplicity and roles of cytochrome P450 CYP genes in the 2 family are not well known in fish. Characterizations of CYP2 genes were done in the killifish, Fundulus heteroclitus. Multiple cDNAs from three CYP2 subfamilies were isolated from killifish cDNA libraries. Sequence analyses showed that one cloned from liver is related to trout CYP2K1. The other cDNAs were classified into two new subfamilies, CYP2N and CYP2P. CYP2N1, CYP2P2, and CYP2P3 were cloned from liver, and CYP2N2 was cloned from heart CYP2P1 was cloned from a killifish genomic DNA library. Northern analyses showed that CYP2P transcripts are expressed strongly in gut and liver. Likewise, CYP2N1 transcripts are expressed strongly in liver and gut and also in heart and brain while CYP2N2 transcripts are expressed strongly in heart and brain and also in liver, gut, eye, kidney, and gill. The CYP2Ns and CYP2Ps are phylogenetically most closely related to the mammalian CYP2Js, and their transcripts also have similar tissue specific sites of expression as the CYP2Js. These phylogenetic and expression site similarities suggest functional similarities as well. To evaluate the functions of the killifish CYP, full length CYP2N 1, CYP2N2, and CYP2P3 proteins were expressed in Sf9 insect cells using a baculovirus system, and their metabolism of arachidonic acid was examined. CYP2N 1, CYP2N2, and CYP2P3 metabolized arachidonic acid at respective rates of 435, 95, and 135 pmol/nmol CYP/min forming a variety of eicosanoids including epoxyeicosatrienoic acids 14,15-, 11,12-, and 8,9- and hydroxyeicosatrienoic acids 5-, 9-, 11-, 12-, 16-, 19-, and 20-. Eicosanoids, especially arachidonic acid metabolites, have been shown to modulate epithelial salt and water transport in a wide variety of organisms including mammals, frogs, toads, fishes, molluscs and insects. They also have been shown to act in various aspects of reproductive biology in sea stars, sea urchins, molluscs, insects, fishes, reptiles, and mammals. Arachidonic acid metabolites thus are involved in the regulation of cellular processes that are fundamental to organisms in general, and their synthesis is of primary interest. These results suggest that the CYP2Ns and CYP2Ps may be early vertebrate arachidonic acid catalysts.
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution September 1997
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