We propose to study the expression of the interphotoreceptor retinoid-binding protein (IRBP) gene. IRBP is an extracellular protein located in high concentration in the interphotoreceptor space (IPS). IRBP facilitates the uptake of all-trans-retinol into the pigment epithelium, where this retinoid is isomerized and oxidized to 11-cis-retinal. IRBP mRNA appears about the time when outer segments begin to elongate, but the developmental expression of IRBP cannot be understood without defining the DNA sequence? near the gene and proteins and conditions in the cell that control IRBP transcription. Owing to our previous structural analyses of the IRBP gene and its flanking regions, we can now test hypotheses on how the promoter functions. For example, a highly conserved sequence of about 220 bases, which is located about 1200 bases from the start of transcription, has been identified in three species. We propose that this sequence plays a role in IRBP gene expression, possibly in coordinating smooth or gentle gradients of expression among adjacent photoreceptor cells. This is based in part on short opsin promoter transgenes causing a patchy or spotted expression in retinas of transgenic mice, whereas longer opsin promoters have shallow gradients of expression. Another sequence, closer to the start of transcription, is capable of binding a nuclear protein from the retina. We hypothesize that both this sequence and the protein are needed for the appropriate expression of IRBP in the retina. Other sequences in the IRBP promoter resemble consensus sequences found in the 5' flanking regions of other eukaryotic promoters. We propose that these are needed for expression. The 5' flanking regions of the IRBP gene appear to contain all the structural elements, DNA sequences near the gene (cis-elements), necessary for expression, based on our preliminary transient expression assays in Y-79 cells and transgenic mouse studies.
The aims of this proposal are to much more sharply define all the cis-elements and identify specific nuclear proteins (trans-factors) that are essential to express IRBP in its unique way. Continued and more detailed expression studies in Y-79 cells and transgenic mice, also gel-shift assays, DNase footprints, and southwestern blots will identify the important constituents and elucidate the mechanisms that control the expression of the IRBP gene.
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