MOLECULAR MECHANISM OF VISUAL EXCITATION

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The long term goal of this project is to understand the molecular processes underlying photoreceptor function using Drosophila melanogaster. The use of Drosophila as a model system to study visual function has the following merits: (1) many kinds of vision-defective mutants are available, (2) advanced genetic techniques are available, (3) in vivo as well as in vitro experiments can be performed relatively easily, and (4) Drosophila shows homology to higher mammals in the nucleotide sequence of genes and in the amino acid sequence of proteins. Therefore, careful studies on the Drosophila mutant system could give us a rare opportunity to dissect and understand each step of the visual pathway in biochemical and molecular biological terms. Moreover, the results thus obtained from Drosophila research might be applicable to mammalian systems. Seven classes of retina-specific polypeptides have been found in Drosophila. Three of these seven classes undergo light-induced phosphorylation in vivo. The accumulated results indicate that these retina-specific proteins and their light-dependent phosphorylation are likely to be involved in visual processes. One of these phosphoproteins is the major class of Drosophila rhodopsin. The other two, however, are not yet characterized. In order to characterize the molecular nature and functional identity of these retina-specific phosphoproteins, we propose to clone the genes encoding these phosphoproteins. The proposed project employs multi-step approaches represented by three major protocols. First, these two classes of retina-specific phosphoproteins will be purified to near homogeneity on two-dimensional gels and the proteins will be injected into rabbits in order to raise polyclonal antibodies. Second, with use of the antibodies, an expression library of Drosophila genomic DNA will be screened in order to identify the positive phage containing the DNA insert (or a part of it) encoding the phosphoprotein. Finally, cDNA libraries will be screened to obtain sequences homologous to the initial positive clones. The positive cDNA clones will be sequenced in order to deduce the amino acid sequence of the gene product. At the same time a partial amino acid sequence of these phosphoproteins will be determined to confirm the identity of the sequenced gene. These efforts would give us a clue to understand the identity of these retina-specific molecules and their function in visual excitation.