The long-term goal of this proposal is to understand the molecular and developmental biology of the mammalian photoreceptor cell. Based on information generated in the bovine retina during the initial funding period it is now known that (1) many, if not all, genes for rod phototransduction are transcriptionally induced simultaneously at about 6 months (2/3) gestation, corresponding to the initial emergence of rod outer segments, (2) the rod opsin gene experiences specific transcriptional enhancement not seen for other phototransduction genes, (3) a final switch in mechanism of transcriptional regulation occurs for rod opsin late in fetal development, and is most evident in the adult, (4) at least ten cis regulatory elements exist within the first 2 kb upstream domain of rod opsin, (5) among these are multiple tissue specific and developmentally regulated elements, (6) a retinoid- responsive transcriptional element(s) exists for rod opsin, (7) cone phototransduction genes are developmentally induced coordinately with corresponding rod genes, (8) rod cells are induced during the final trimester in a topographically reproducible central-to-peripheral pattern and (9) cone cells are analogously induced, both spatially and temporally. Using both in vitro and in vivo assays we propose to test numerous hypotheses regarding molecular features regulating the above elements and processes. Focusing predominately on rod opsin genes, further analysis of cis-elements regulating the efficiency of opsin transcription will involve a recently developed in vitro footprinting and transcription systems. To confirm and extend conclusions based on in vitro analysis, the opsin gene upstream sequence will be systematically surveyed for in vivo footprinting and for the ability of identified domains to support transcription of reporter genes delivered to adult and neonatal murine retina by injection of recombinant parvoviruses. Southwestern blotting, UV crosslinking, expression cloning and the in vitro transcription system will be employed to characterize trans-acting elements interacting with each important cis-element. Characterizing the retinoid-mediated transcriptional response will require both nuclear run- on and isolated whole retina assays. At all levels, resolution in terms of developmental stage will be attempted in order to integrate individual mechanisms of the regulation with overall development of the fetal retinal gene expression.
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