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B.E. Knox, S. Babu, C.M. Kuemmel, S.L. Whitaker, V.A. McIlvan; Conserved cis–Elements in the Xenopus Red Cone Opsin Promoter Necessary for Cone–Specific Expression . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4887.
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© ARVO (1962-2015); The Authors (2016-present)
To understand the molecular mechanisms driving vertebrate red cone opsin (LWS) gene expression, we characterized cis–acting elements in the 5' flanking sequence responsible for the precise spatial and temporal regulation of transcription.
We prepared nested deletions or mutations in the Xenopus LWS 5' flanking region (–1792/+127) and cloned them upstream of a GFP reporter. Functional consequences on both tissue– and cone–specific expression were investigated in transgenic Xenopus. The identity of GFP–expressing cells within the retina was characterized by immunolabeling of fixed cryosections and light microscopy.
Within the 1.8 kb flanking region, we identified a small region close to the transcription start site (–117 to –45) that was highly conserved among tetrapods but not with teleosts. We term this the tetrapod homology domain. In addition, we found an extended region in the promoter (–725 to +127) that was specifically conserved between Xenopus laevis and Xenopus tropicalis. A construct containing these regions (–725 to +127) drove expression only in cones and pinealocytes. Removal of a part of the Xenopus conserved sequences (–725 to –174) resulted in widespread extraocular expression as well as misexpression within the retina, not only in cones but also in rods and bipolar cells. Two mutations within the tetrapod homology domain were prepared to alter putative short red opsin promoter elements (ROPs). Mutation of ROP2 (–92/–82) resulted in expression of the reporter transgene throughout many retinal and extraocular cell types, while mutation of ROP3 (–120/–112) caused extraocular expression in some animals. Mutation of both ROP2 and ROP3 dramatically reduced expression in all cells.
These results identify novel conserved elements that regulate spatial expression of tetrapod red cone opsin genes. Moreover, our results suggest an important role for repressive mechanism(s) in controlling cell–specific transcription.
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