Abstract
Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. The norpA gene has been proposed to encode phosphatidylinositol-specific phospholipase C (PLC), which enzymes play a pivotal role in one of the largest classes of signaling pathways known. A chimeric norpA minigene was constructed by placing the norpA cDNA behind an R1-6 photoreceptor cell-specific rhodopsin promoter. This minigene was transferred into norpAP24 mutant by P-element-mediated germline transformation to determine whether it could rescue the phototransduction defect concomitant with restoring PLC activity. Western blots of head homogenates stained with norpA antiserum show that norpA protein is restored in heads of transformed mutants. Moreover, transformants exhibit a large amount of measurable PLC activity in heads, whereas heads of norpAP24 mutant exhibit very little to none. Immunohistochemical staining of tissue sections using norpA antiserum confirm that expression of norpA protein in transformants localizes in the retina, more specifically in rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. Furthermore, electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in R1-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells. This is the strongest evidence thus far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction.
Highlights
From the Wepartment of Biological Sciences, State University of New York, Buffalo, New York 14260 and the §Department of Biology, Saint Louis University, St
This is the strongest evidence far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction
Strong mutations in the norpA gene of Drosophila have long been known to abolish the light-evoked photoreceptor potential, rendering the fly blind (Hotta and Benzer, 1970; Pak et al, 1970). norpA mutants have been shown to be deficient in phospholipase C (PLC) activity in head (Yohsioka et al, 1985), and molecular cloning of the norpA gene has shown that it encodes a protein that is similar in structure and amino acid sequence to vertebrate PLC (Bloomquist et al, 1988)
Summary
Vol 270, No 22, Issue of June 2, pp. 13271-13276, 1995 Printed in U.S.A. Phospholipase C Rescues Visual Defect in norpA Mutant of Drosophila melanogaster*. Electrophysiological analyses reveal that transformants exhibit a restoration of light-evoked photoreceptor responses in Rl-6 photoreceptor cells, but not in R7 or R8 photoreceptor cells This is the strongest evidence far supporting the hypothesis that the norpA gene encodes phospholipase C that is utilized in phototransduction. NorpA mutants have been shown to be deficient in PLC activity in head (Yohsioka et al, 1985), and molecular cloning of the norpA gene has shown that it encodes a protein that is similar in structure and amino acid sequence to vertebrate PLC (Bloomquist et al, 1988) These data, as well as a growing body of evidence suggesting that PLC is involved in invertebrate phototransduction (reviewed by Payne, 1986; Pak and Shortridge, 1991), have converged to suggest that the norpA gene encodes PLC that is utilized in phototransduction in Drosophila. Rescue of Visual Defect in norpA Mutant examined to see whether the expression of the norpA protein in R1-6 photoreceptor cells is sufficient to rescue the phototransduction defect and the accompanying lack of PLC activity exhibited by norpA mutants
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