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W. Yan, C. Wang, D.J. Zack; Use of a Yeast One-Hybrid Approach to Study Cone Photoreceptor Gene Regulation . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3525.
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Purpose: Cone photoreceptors play an important role in human vision. Even in the rod-based photoreceptor degenerations, most clinically significant vision loss is cone-related. However, despite advances in our understanding of rod-specific gene regulation, relatively little is known about the mechanism that regulates cone gene expression. We have therefore initiated a yeast one-hybrid approach to clone and characterize transcription factors important in cone photoreceptors. Methods: We choose the 13-lined ground squirrel (13-lgs), a cone dominant vertebrate, as a model system. A retinal cDNA one-hybrid library was constructed by directionally inserting 13-lgs cDNA into the pGADT7 vector. The proximal upstream region of the 13-lgs green opsin gene was obtained by inverse PCR and several potential cis-elements in this region were chosen as baits for yeast one hybrid screening, which was carried out by stand methods. Results: The generated 13-lined ground squirrel retinal library contained approximately 8 million independent clones, with typical insert sizes ranging from 1.2 kb to 2.2 kb. The cloned 269 bp 13-lgs proximal promoter region showed 73.6% identity with the equivalent murine upstream region, and 84.7% identity with the human region. Several potential cis-elements within the 269 bp region were selected as baits for the one-hybrid screening. A total of 732 positive clones were identified in the initial yeast screenings. Amongst those clones that have been sequenced so far, several transcription factors have been identified (including a number of homeobox genes, nuclear receptors and sequences of unknown identity). Further characterization of these factors is underway, using a combination of DNA binding and trans-activation assays. Conclusions: We have identified several transcription factors that are candidate regulators of cone gene expression. Ongoing studies will hopefully help define their role in vivo and their possible involvement in retinal diseases.
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