ISOLATION OF REGULATORY KINASES FROM THE VISUAL SYSTEM OF DROSOPHILA

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We are interested in isolating and characterizing genes which modulate the visual response. Several members of the visual cascade are known to be phosphorylated in response to activation by light. By analogy to other systems, it is thought that this phosphorylation is responsible for a large part of the modulation of the response. We therefore plan to concentrate on the isolation of genes which encode regulatory kinases. Mutations which cause excessive activation of the visual cascade lead to retinal degeneration. Thus, regulation of the level of response of the visual system is critical not only for proper vision but also to maintain viability of the photoreceptor cells. Mutations which affect the level of phosphorylation of rhodopsin are also know to cause degeneration. Therefore, the serine/threonine kinases we are searching for may be key players in retinal degenerative diseases. In order to isolate serine/threonine protein kinase genes, we have designed two degenerate oligonucleotides which encode conserved amino acid domains VI and VIII found in all members of the kinase family. PCR reactions will be done on cDNA which is enriched for eye-specific sequences by multiple subtractive hybridizations using head mRNA from the mutant fly eya which does not develop eyes. Amplified sequences will be isolated and screened for kinases by sequencing. Any kinases found will be further characterized to assess their role in regulating the visual system.