Purchase this article with an account.
T. Masuda, J.S. Friedman, J. Qian, A. Swaroop, D.J. Zack; Identification of Transcription Factors Regulating Cone Photoreceptor Gene Expressions . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4907.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The goal of this study is to identify transcription factors that regulate cone photoreceptor gene expression.
We are taking a yeast one–hybrid (YOH) approach to screen for proteins that bind to DNA elements upstream of cone–specific genes. In advance of the screening, in order to define candidate "baits" for the YOH screening, we examined the 5’–upstream regions of vertebrate genes for short– (SWS) and middle–wave–sensitive (MWS) opsin and cone arrestin genes for conserved DNA sequences. We then tested these candidate bait sequences by electrophoretic mobility shift assay (EMSA) using nuclear extracts from Nrl–/– mouse retina (which is enriched for cone photoreceptor cells), and wild–type mouse retina, brain, and liver.
Our bioinformatics analysis identified 9 candidate regulatory (bait) sequences within the cone opsin and arrestin gene upstream regions (4 from SWS, 4 from MWS, and 1 from cone arrestin). EMSA revealed retina specific binding with 3/4, 4/4, and 0/1 of the sequences that were designed from the SWS, MWS, and cone arrestin genes, respectively. Among these probes, one which was designed from the proximal region of SWS opsin gave a stronger signal with nuclear extract from Nrl–/– mouse retina compared to that from wild–type mouse. YOH screening with these probes is currently underway. For the screening we are using a retinal cDNA library from cone–dominant Nrl–/– mice because such a library should be enriched for cDNAs encoding proteins that regulate cone gene expression.
Our results suggest that at least several of the bait sequences that we have identified should be suitable for YOH screening. The screening, which is now in progress, will hopefully identify transcription factors that are of importance for the regulation of cone photoreceptor gene expression.
This PDF is available to Subscribers Only