This proposal aims to study retinal development using expression of rod and cone visual pigment genes as a model system. The goal is to understand the mechanisms responsible for the selection by an individual photoreceptor of the expression of a single visual pigment gene. The DNA sequences of a number of opsin and other genes will be compared looking for conserved sequences which could represent regulatory elements. Electrophoretic mobility-shift assays will be used to determine the presence of specific DNA binding proteins and to explore their tissue specificity. DNA """"""""footprinting"""""""" will be used to characterize the binding sites. The effects of mutations in these sequences will be assessed. If such putative factors can be identified, their purification, characterization, and cloning will be begun. Efforts will also be made to develop a homologous in vitro transcription system for use in analyzing these putative regulatory sequences and factors. These studies will hopefully contribute to the understanding of normal retinal development and retinal specific gene expression. As such, they may help form the basis for future studies of the molecular mechanisms responsible for abnormal development such as is seen in some of the congenital retinal degenerations. They may also be helpful for the development of ocular genetic engineering, which has the potential of some day providing treatment for many sight threatening ocular disorders.
