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SG Dann, WT Allison, CW Hawryshyn; Upstream Sequence of Retinal Opsin Genes Capitulates Transcriptional Control of Those Genes in Oncorhynchus mykiss . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3639.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Salmonid fish such as rainbow trout (O. mykiss) experience loss of the ultraviolet sensitive photoreceptor (SWS1 cone) via apoptosis, and thus photosensitivity, during a metamorphosis-like event. This event, referred to as smoltification, is driven by release of thyroid hormone (T3) and results in physiological and anatomical changes in the fish. Our goal is to determine if opsin gene transcription is directly affected by T3 through its nuclear receptors (THR) or through other transcription factors (TF) whose expression is linked to T3. Methods: A simple method for attaining upstream sequence from known genes is genome walking. Briefly, genomic DNA from the organism of interest is digested with restriction endonucleases that produce blunt ends. Adapters of known sequence are ligated to blunt end DNA and nested PCR is preformed with two forward primers matching the adapter and two reverse primers matching the 5' end of the gene of interest. Data from cloned and sequenced PCR products are subjected to TF search (http://www.cbrc.jp/research/db/TFSEARCH.html), which identifies sites of known TF-DNA binding. Results: 500 to 2000bp of upstream sequence have been generated from each of the retinal opsin genes identified in O. mykiss i.e. SWS1, SWS2, LWS, RH1, RH2. TF binding sites have been identified in each of these gene's promoter regions, a summary of which will be presented in this poster. Of particular interest is a palindromic AP-1 binding site found in the upstream sequence of the SWS1 gene. Conclusion: Identifying the TFs involved in opsin gene expression is the first step in creating genomic and proteomic paradigms, which attempt to describe neurodegenerative events in the salmon retina. The AP-1 site upstream of the SWS1 opsin gene, for instance, binds activating protein - 1, a TF known to exert effects in cis with THR in the human malic enzyme and ChAT. In fact Schmidt et al. (1993) have shown that nuclear THR represses transcriptional activation by AP-1 in a T3-dependent fashion. AP-1 has also been shown to regulate expression of genes central to the apoptotic pathway, specifically Bcl-x, Bax, and Bcl-3. In order to understand mechanisms controlling apoptosis of SWS1 cones in salmon retina we are seeking the mechanisms controlling transcription of genes central to its cellular function. Similarly we are attempting to identify mechanisms that may confer proliferative events on adjacent photoreceptors in the salmon retina. This work supported by Fellowships from the Alzheimer Society of B.C./Canada (WTA) and NSERC (SGD), and by grants from NSERC (CWH).
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