The project seeks to use Drosophila as a model for studying the molecular basis of inherited retinal diseases. The first specific aim is to conduct a phenotypic and molecular characterization of the gene responsible for the W108 mutant phenotype. This mutation was identified and partially characterized during the previous granting period. The applicant's high level of interest in this gene is because the mutant shows two phenotypes shared with genes involved in rhodopsin transport and retinal degeneration. These phenotypes are the accumulation of endoplasmic reticulum (ER) in the photoreceptors, and the reduction of the sustained response of the electroretinogram. The phenotypic analysis will be extended by examining if expression of proteins requiring transport through the ER are affected by the mutation, and if the phenotype becomes more severe when this mutation is combined with other mutations affecting trafficking components. The major effort to determine the role of the W108 mutant will be to clone this gene. Gene cloning will take advantage of the transposon tag within the gene. The gene transcript will be identified within the cloned sequence and sequenced. This information will be used to determine if the protein shares motifs with known proteins and, hence, may allow one to deduce its function in the cell. Antibodies will be generated against the protein and subsequently used to deduce the program for gene expression and determine whether related proteins are expressed in vertebrate photoreceptors. The second specific aim concerns the characterization of additional genes involved in intracellular transport. The applicant has used innovative techniques to biochemically purify a tagged form of rhodopsin from intact cells and plans to use this to find other proteins that copurify with rhodopsin. She has already shown that the ninaA cyclophilin will copurify with rhodopsin, and will use column chromatography to identify other associated proteins. These proteins will be identified and characterized, and then antibodies or peptide sequences generated from the protein will be used to clone the corresponding genes. Mutants will be sought in these genes and the fate of rhodopsin in mutant animals will be investigated.
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