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A.F. Wiechmann, E.W. Howard; Analysis of the Mel1c Melatonin Receptor Promoter Region in Xenopus Laevis . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3993.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The hormone melatonin is a circadian signal that acts as a paracrine and/or endocrine messenger in the retina and other ocular structures. Many of the functions of melatonin are thought to be mediated via a set of integral membrane receptors on target cells. Furthermore, cyclic changes in melatonin receptor expression may contribute to some of the circadian effects of melatonin in ocular tissues. The goal of this study was to identify regions of the Xenopus laevis Mel1c melatonin receptor gene that are involved in the regulation of expression of this receptor gene in the eye. Methods: The promoter region the Xenopus laevis Mel1c gene was cloned using a polymerase chain reaction (PCR)–based gene–walking method. Genomic DNA isolated from X. laevis tissues was digested with different restriction enzymes to prepare different PCR templates. A short synthetic adapter was ligated onto the cut genomic DNA, and two rounds of PCR were performed on the digest libraries, using adapter–specific primers and gene–specific primers. The second round PCR products, which resulted from the use of nested primers, were approximately 2.0 kb, 1.2 kb, and 230 bp in size. The PCR products were sequenced directly, and also cloned into cloning vectors and sequenced, and then cloned into expression vectors to drive the expression of EGFP in transgenic X. laevis. Results: The cloned PCR products contained approximately 2.0 kb, 1.2 kb, and 230 bp DNA sequence upstream of the 31 bp of previously published 5' untranslated region (UTR) of the X. laevis Mel1c gene. The 230 bp fragment displayed 100% identity with the known 31 bp 5' UTR, whereas the 2.0 and 1.2 kb fragments displayed only 90% identity with the known 31 bp 5' UTR and the 230 bp fragment. The source of genomic DNA that yielded the 230 bp fragment was multiple whole tadpoles, whereas the genomic DNA used to generate the 2.0 and 1.2 kb fragments was obtained from adult liver. Conclusions: The presence of two genes encoding 5' UTR putative promoter regions for the X. laevis Mel1c receptor supports previous reports of the existence of two isoforms of the X. laevis Mel1c receptor. The presence of cis–elements in the 5' UTR of the Mel1c receptor genes suggests that this region contains the promoter sites for these genes. The ability of specific fragments of this region to drive expression of EGFP in ocular cells will be analyzed in experiments utilizing transgenic frogs. Identification of the promoter region that drives cell–specific Mel1c receptor expression will facilitate studies on the functional role of melatonin receptors in ocular cells.
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