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K. Rengarajan, E. Stodulkova, J.H. Boatright, J.M. Nickerson; Partial Purification and Characterization of a Mammalian IRBP Tissue Restricting Factor . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3545.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Transcription of the IRBP gene is limited to photoreceptor cells of the retina and pinealocytes. The 5' flanking region from -140 to -156 of the IRBP gene confers tissue-restricted promoter activity (Boatright et al. FEBS Lett 2001; 504: 27-30 and present work). We previously found nuclear proteins that bind to this cis element using EMSAs and UV crosslinking. Here, we sought to partially purify and characterize nuclear proteins that bind to the cis element. Methods:Bovine retina nuclear extract was run on heparin-, phenyl-, and DNA- sepharose columns. Protein fractions and radiolabeled probe (from -140 to –156) were crosslinked, run on SDS-PAGE or 2D gels, and visualized by autoradiography and silver staining. A supershift assay was performed by addition of Nrl antibodies (a kind gift from A. Swaroop, Swain et al. J Biol Chem 2001;276:36824) to a protein-nucleic acid complex. Mass spectrometry (MS) was carried out on tryptic peptides from a RecA-DNA crosslinked complex to validate the crosslinking-PAGE separation-MS approach to identify the trans factor. Results:The -140 to -156 DNA specifically bound nuclear proteins in EMSA. 2D analysis of the crosslinked DNA protein complex revealed a spot of ~45 kDa and pI of ~5.2. Without UV treatment or with specific DNA competitors, no band appeared. The protein-probe did not show any supershift with Nrl antibodies. Fractions from all columns revealed the presence of a 45 kDa protein. UV crosslinked RecA-oligo complex resolved by SDS-PAGE and analyzed by MS revealed 12 peptides matching RecA with a Z score of 2.18 or 2.38 in the all taxa and bacterial databases, respectively. Conclusions:High Z-scores of RecA MS data validate the principle that UV crosslinking of a protein-DNA complex is specific and can be used to identify a protein bound to a specific DNA sequence. There was no shift in mobility of the restricting factor on addition of Nrl antibody, suggesting it is not Nrl. A substantial purification of the factor was achieved by chromatography. UV crosslinking of fractions from the 3 columns revealed a single 45 kDa protein with a pI of 5.2 that binds to the restricting cis element.
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