The broad,long-term objective of this research is to elucidate the molecular and cell biology of photoreceptors. The specific research described in this application is intended to establish the role in photoreceptor function of the transient receptor potential (trp) gene in Drosophila. The knowledge expected to be gained from this study is fundamentally important and has actual value for furthering our understanding of human hereditary eye diseases. The trp mutation affects specifically the photoreceptor; the phenotype is characterized by blindness in bright light and by progressive retinal degeneration. The trp mutation was mapped and the trp locus has been cloned. Results obtained to date reveal that the trp locus entails approximately 50 kb of DNA and is complex. At least five mRNAs are mapped to the trp locus. One mRNA (4.5 kb) is eye-specific, one is body-specific, one is expressed predominantly in the body, and the other two (3.0 kb and 1.5 kb) lack detectable tissue specificity. Compared with the wildtype fly, the trp mutant (Cosens-Manning allele) lacks the 3.0- kb and 1.5-kb mRNAs. These two mRNAs and the eye-specific mRNA are the main foci of current study, aimed at characterizing the molecular basis of the trp phenotype. The sequences of these mRNAs are being established; based on the deduced amino acid sequence, antibodies will be raised against these proteins and used for the cellular localization and biochemical characterization of the proteins. Organization of the genes in the trp locus is complex and novel, involving transcription from both strands of DNA. For example, a 442-bp DNA is transcribed in one direction to form the 3'-end of the 4.5-kb eye-specific mRNA and in the opposite direction to form the 5'-end of the mRNA that is expressed predominantly in the body. Therefore, these two mRNAs are complementary at their respective 3'- and 5'-ends and form anti-sense mRNAs. Organization of the other mRNAs in the trp locus also involves complex arrangements. It is reasonable to expect that the novel arrangement of genes in the trp locus is related to the mechanisms of gene regulation. For example, cross-regulation may take place by forming hybrid molecules of anti-sense mRNAs that are not transported out of the nucleus, therefore preventing the expression of the protein products. One of the main goals of this research is to unravel the organization and regulation of the genes in the trp locus, specifically the genes that are directly involved in the trp phenotype. We will study the regulation of these genes by manipulation of the cloned DNA and by introduction of the modified genes into flies by germline transformation.
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