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Type of Document Dissertation Author Hibbard, Karen Louise URN etd-03242007-162901 Title A GENETIC SCREEN TO IDENTIFY GENES AFFECTING THE BIOGENESIS OF RHODOPSIN Degree Doctor of Philosophy Department Biological Sciences Advisory Committee
Advisor Name Title Joseph Edward OTousa Committee Chair Crislyn DSouza-Schorey Committee Member David R Hyde Committee Member Lei Li Committee Member Mary Ann McDowell Committee Member Keywords
- Drosophila
- RH1 maturation
- protein trafficking
- photoreceptors
Date of Defense 2007-02-20 Availability restricted Abstract In Drosophila melanogaster, RH1 rhodopsin is the primary photopigment in the photoreceptor cells. Photoisomerization of rhodopsin to metarhodopsin triggers the activation of the phototransduction cascade which results in the depolarization of the photoreceptor cell. Additionally, RH1 is required to maintain the structural integrity of the rhabdomeres. Mutations in components of the phototransduction cascade or the maintenance of the rhabdomere usually result in retinal degeneration. My research focuses on identifying the genetic control of RH1 biogenesis.
Through a FLP/FRT mutagenesis screen, six mutants were isolated that exhibit a dim fluorescent pseudopupil phenotype. One identified mutant, kh1, has low RH1 protein levels and shows signs of rapid retinal degeneration. Visualization of RH1::GFP fusion proteins within the photoreceptors show that the RH1::GFP fails to progress from the cell body to the rhabdomere and that the GFP signal colocalizes with an endoplasmic reticulum cellular marker. Cytological and molecular mapping experiments identified the kh1 gene as Dbp21E2.
Protein sequence analysis of Dbp21E2 revealed that it belongs to the DEAD/DExH box family of RNA helicases. The DEAD-box family of RNA helicases has roles in the numerous steps of RNA metabolism, which include transcription, splicing, translation, ribosome formation, and RNA degradation. In particular, Dbp21E2 shares distinct homology with the DEAD-box family which includes the genes: vasa, eif4a, and PL10. As a result of this screen, I identified a previously uncharacterized gene, Dbp21E2, and showed that it has an essential role in the synthesis of proteins within the photoreceptor cells.
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