The long-term objective of the proposed research is to use a molecular genetic approach to study vision in the fruitfly, Drosophila melanogaster. The approach takes advantage of the combination of molecular, biochemical, genetic and physiological techniques available to Drosophila, to identify and characterize molecules important in visual physiology that have not been previously identified. The goal of the current proposal is to understand the functions of a gene, ninaC, which is required for formation of the photoreceptor cell cytoskeleton. This gene encodes two highly related proteins with linked domains homologous to protein kinases and the myosin heavy chain. Protein kinases play a role regulating many cell processes including signal transduction and cell growth. Myosins are proteins which convert the chemical energy in ATP into mechanical force used in a variety of cell movements. Two approaches to the study of ninaC are proposed. The first involves construction directed mutations to determine the functions of the putative kinase and myosin domains in vivo. The altered genes will be introduced into the genome of ninaC null mutant using P-element germline transformation. The second is to test the hypothesis that the ninaC proteins are genuine protein kinases joined to the myosin heavy chain. This will be investigated by assaying for the biochemical activities characteristic of protein kinases and the myosin heavy chain.
The specific aims are to: 1) determine whether the ninaC proteins have myosin activity, 2) determine whether the ninaC proteins have protein kinase activity, 3) construct and analyze the effect of a mutation which should eliminate the kinase activity, 4) construct and analyze the effects of mutations which should affect just the myosin activity, and 5) analyze the effects of mutations which should result in expression of just one or the other ninaC protein. The results from the experiments proposed here should contribute not only to understanding visual physiology but also to the structure/function relationships of myosins in general and are part of the long-term goal to identify and characterize new components important in vision in the fruitfly. Homologs of many of these molecules may be found in other organisms including humans and may lead to a better understanding of human and may lead to a better understanding of human retinal diseases caused by defects in these molecules.
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