The phototransduction cascade initiates with the photoexcitation of rhodopsin and terminated with the polarization of the cell by sodium ions in vertebrates and invertebrates. In Drosophila, an electroretinogram (ERG) measures the mass electrical graded potential across the retina, thus measuring the functional and structural integrity of the compound eye. Several ERG mutations have been isolated and have been shown to be involved in phototransduction. This proposal describes a neurogenic analysis of three of these mutations. Retinal degeneration B(rdgB) negatively interacts with phospholipase C or its enzymatic product, inositol 1,4,5-triphosphate, which is a second messenger in the cascade which activates the sodium channel to produce the graded potential. No on transient A (nonA) affects the transduction of the photoreceptor potential signal to the laminal interneurons, while slow receptor potential (slrp) exhibits a defect in returning to the ground state after the depolarization of the photoreceptor. 56 Drosophila visual system-specific cDNA clones were isolated, and two cDNAs mapped near rdgB and nonA or slrp. I will isolate the corresponding genomic clones and transform them into the Drosophila germline to examine if they are capable of rescuing the mutant ERG phenotype, demonstrating that the clone contains the wild-type gene. These genes will then be analyzed at a molecular level by intron/exon mapping, RNA 5' end analysis, and sequencing. The deduced amino acid sequence will be determined from the DNA sequence and will be used to search for homology to known functional protein domains and examined by hydrophobicity plots for transmembrane regions. These data should reveal potential roles of the molecules in the cascade. In vitro mutagenesis will be used to introduce point mutations within the deduced functional regions. The mutagenized gene will be introduced into flies and the phenotype will be observed. Failure to observe a wild-type ERG with the introduced gene will suggest that the mutagenized domain plays a vital role in the protein's function. Additionally, antibodies will be raised to examine the temporal and spatial localization of protein expression and immuno-electron microscopy will be used to localize the protein's function. It is the goal of this work to better understand the mechanism of signal transduction, particularly how the second messengers regulate the cellular response, such as photoreceptor depolarization, and how the second messengers are regulated, thereby also regulating the response.
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