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D.F. Woodward, Y. Liang, C. Li, W. Chang, V. Guzman; IDENTIFICATION OF NOVEL, INDUCIBLE CYCLO–OXYGENASE 1 (COX–1) ISOFORMS IN HUMAN OCULAR TISSUE . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3656.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Previous studies have identified novel canine and zebra fish COX–1 isoforms. The canine isoform has been claimed acetominophen–sensitive. The purpose of these studies was to investigate the existence of human COX–1 mRNA splicing variants. Methods: Database search and RT–PCR resulted in the discovery of four novel, alternatively spliced human COX–1 variants, using COX–1 GGTTCTTGCTGTTCCTGCTC (forward) and TCACACTGGTAGCGGTCAAG (reverse) primers. The final PCR products were subcloned and sequenced. Tissue distribution was determined by PCR on total RNA from a commercial source or isolated from human ocular tissues. Results: Four novel COX–1 isoforms, containing two novel exons that reside between exons 2 and 3 of wildtype COX–1, were discovered. Each alternative COX–1 variant (designated alt 1–4) contained a novel N–terminal sequence. In alt 2 the novel exon was spliced to exon 6, which resulted in a considerably truncated protein that lacked Arg 120. A further variant (alt–3) lacked the catalytic site. The other two variants (alt1 and 4) retained the key residues Arg 120, His 207 and 388, Tyr 385 and Ser 530 and accepted arachidonic acid as a substrate. Under normal conditions these alternative splicing variants were barely detectible in non–ocular tissue but were highly inducible in response to TGFIß and serum. Alt 1 and alt 4 were detected in the cornea, retina, and conjunctiva but not in the ciliary body. Alt 4 COX–1 was highly inducible and, although no message could be detected in untreated neuronal cells, a clear band was present after serum treatment. Conclusions: The discovery of new alternative splicing variants may have implications in ocular diseases and ACAID.
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