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 ca scade 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 activ ity analyses. ERG responses of the mutants show allele-dependent reduc tions in amplitudes and retardation in kinetics. The mutants also exhi bit allele-dependent reductions in in vitro PLC activity levels and gr eatly reduced or undetectable NorpA protein levels. Three carry a miss ense mutation and five carry a nonsense mutation within the norpA codi ng sequence. In missense mutants, the amino acid substitution occurs a t residues highly conserved among PLCs. These substitutions reduce the levels of both the NorpA protein and the PLC activity, with the reduc tion 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 ef fector (NorpA) protein level. Transgenic flies were generated by germl ine transformation of a null norpA mutant using a P-element construct containing the wild-type norpA cDNA driven by the ninaE promoter. Tran sformed 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.