Molecular and electrophysiological characterization of DrosophilanorpA mutants
Abstract
Inositol phosphate signalling has been implicated in a wide variety of vertebrate and invertebrate cellular processes. In Drosophila, the phototransduction cascade is mediated by an inositol-phosphate specific, $\beta$-class phospholipase C (PLC) encoded by the norpA gene. Here we have used electroretinogram (ERG), Western, molecular, ultrastructural and in vitro enzyme analyses to characterize several norpA mutants. ERG responses from these mutants are reduced in amplitude and show altered response kinetics. Responses to light stimuli are both slower and much more prolonged as compared to wild-type flies. All of the mutants characterized here show greatly reduced expression or total lack of the norpA-encoded protein on Western blots. Each was found to contain a mutation in the coding region of the gene. Of the eight alleles characterized, three have missense mutations, four have nonsense mutations, and one has a small deletion. This deletion creates a reading frame shift that results in 24 amino acid substitutions followed by a nonsense mutation. In vitro PLC activity of total protein extracted from wild-type or mutant eyes shows that the specific activity of the encoded enzyme is reduced in the missense mutants. The cloned wild-type norpA coding sequence was introduced into a null mutant by P-element-mediated germline transformation using the ninaE gene promoter to drive the expression. Transformed flies show full rescue of the R1-6 photoreceptor ERG and degeneration phenotypes. Since the ninaE gene encodes the R1-6 opsin of the compound eye, its promoter will permit transcription of the introduced sequence in R1-6 photoreceptors only. However, the electrophysiological phenotype of R7 and R8 cells is not rescued and the degeneration phenotype is only partially rescued. The degeneration of R7 rhabdomeres (R8 rhabdomeres were not examined in this study) appears to occur but more slowly than in the null norpA mutant, P24.
Degree
Ph.D.
Advisors
Pak, Purdue University.
Subject Area
Neurology|Molecular biology|Genetics
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