May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
Use of a Yeast One-Hybrid Approach to Study Cone Photoreceptor Gene Regulation
Author Affiliations & Notes
  • W. Yan
    Ophthalmology, The Johns Hopkins University, Baltimore, MD, United States
  • C. Wang
    Ophthalmology, The Johns Hopkins University, Baltimore, MD, United States
  • D.J. Zack
    Ophthalmology, Neuroscience, Molecular Genetics and Biology, The Johns Hopkins University, Baltimore, MD, United States
  • Footnotes
    Commercial Relationships  W. Yan, None; C. Wang, None; D.J. Zack, None.
  • Footnotes
    Support  NEI, Macular Vision Foundation, Research to Prevent Blindness, Inc and Knights Templar Foundation
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3525. doi:
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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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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : 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.

Keywords: photoreceptors • transcription factors • age-related macular degeneration 

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