Molecular, biochemical, and electrophysiological characterization of Drosophila norpA mutants.
Inositol phosphate signaling has been implicated in a wide variety of eukaryotic cellular processes. In Drosophila, the phototransduction cascade is mediated by a phosphoinositide-specific phospholipase C (PLC) encoded by the norpA gene. We have characterized eight norpA mutants by electroretinogram (ERG), Western, molecular, and in vitro PLC activity analyses. ERG responses of the mutants show allele-dependent reductions in amplitudes and retardation in kinetics. The mutants also exhibit allele-dependent reductions in in vitro PLC activity levels and greatly reduced or undetectable NorpA protein levels. Three carry a missense mutation and five carry a nonsense mutation within the norpA coding sequence. In missense mutants, the amino acid substitution occurs at residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduction in PLC activity being greater than can be accounted for simply by the reduction in protein. The effects of the mutations on the amount and activity of the protein are much greater than their effects on the ERG, suggesting an amplification of the transduction signal at the effector (NorpA) protein level. Transgenic flies were generated by germline transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Transformed flies show rescue of the electrophysiological phenotype in R1-R6 photoreceptors, but not in R7 or R8. The degeneration phenotype of R1-R6 photoreceptors is also rescued.
Pearn
M T
MT
Department of Biological Science, Purdue University, West Lafayette, Indiana 47907, USA.
Randall
L L
LL
Shortridge
R D
RD
Burg
M G
MG
Pak
W L
WL
D000483:Alleles; D000818:Animals; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000235:genetics / Q000502:physiology*; D004596:Electroretinography; D005020:Ethyl Methanesulfonate; D017344:Genes, Insect; D007700:Kinetics; D008027:Light; D016296:Mutagenesis; D043265:Phosphatidylinositol Diacylglycerol-Lyase; D054799:Phospholipase C beta; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D017956:Photoreceptor Cells, Invertebrate / Q000502:physiology*; D016133:Polymerase Chain Reaction; D012160:Retina / Q000201:enzymology / Q000502:physiology*; D017384:Sequence Deletion; D015398:Signal Transduction; D010738:Type C Phospholipases
D029721:Drosophila Proteins; D005020:Ethyl Methanesulfonate; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; C468371:NorpA protein, Drosophila; D054799:Phospholipase C beta; D043265:Phosphatidylinositol Diacylglycerol-Lyase