Molecular and genetic studies characterizing the role of ninaG in chromophore biogenesis, role of rhodopsin in maintenance of photoreceptor cell structure, and genome-wide transcript changes during norpA retinal degeneration in Drosophila

The work presented in this dissertation describes the following studies:; 1. The role of ninaG in visual pigment chromophore biogenesis. The Drosophila NinaG oxidoreductase mutant flies are characterized by low levels of Rh1 rhodopsin but have normal levels of minor opsins, Rh4 and Rh6. ninaG mutants ectopically expressing Rh4 as the major rhodopsin accumulate an anomalous retinoid, identified as 3-hydroxyretinol by HPLC-UV/VIS-MS analysis. Further, ninaG mutants fed on retinal as the sole chromophore precursor are able to synthesize 3-hydroxyretinoids. Thus, NinaG oxidoreductase acts subsequent to the hydroxylation step in the biogenesis of rhodopsin chromophore, (3S)-3-hydroxyretinol.; 2. Heterologous expression of bovine rhodopsin in Drosophila photoreceptor cells. Vertebrate and invertebrate rhodopsins are similar in amino acid sequence, structural organization, spectral properties, and mode of action, but bind different chromophores and trigger phototransduction through distinct pathways. Transgenic Drosophila expressing bovine rhodopsin were created to compare and contrast properties of bovine and Drosophila rhodopsin with respect to maturation (glycosylation, chromophore binding, and folding), trafficking, physiology (light response), and photoreceptor cell morphology. This analysis establishes Drosophila photoreceptor cells as a valuable model to study vertebrate and invertebrate visual pigments in a common cell type.; 3. Rh1 cytoplasmic tail and non-cytoplasmic interface domains play a structural role in development and integrity of rhabdomeres. Rh1 expression is essential for the structural development of rhabdomeres in photoreceptor cells. Flies expressing bovine rhodopsin (Rho) show severe disintegration of rhabdomeres 7-10 days post-eclosion. A series of Rh1-Rho chimera were expressed to show that both Rh1 cytoplasmic-tail and non-cytoplasmic-interface domains are essential for the structural development and maintenance of rhabdomeres.; 4. Genome-wide transcript analysis during norpA retinal degeneration. Drosophila norpA mutants show rapid light-dependent retinal degeneration. Among 13,000 genes present on the genome chip, 300 genes showed significant changes in transcript levels during the degeneration process. One of the downregulated genes was I, that when mutated cause light-dependent retinal degeneration due to accumulation of activated rhodopsin. norpA; sun double-mutants showed faster retinal degeneration rate than single-mutants, suggesting that downregulation of sun transcript in norpA mutants contributes towards the attainment of retinal degeneration. Large-scale changes in transcript levels of known apoptotic genes were not observed thereby suggesting that apoptotic genes are not transcriptionally regulated during norpA retinal degeneration.