FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2054297318> ?p ?o ?g. }

• W2054297318 endingPage "13276" @default.
• W2054297318 startingPage "13271" @default.
• W2054297318 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. 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. Phosphatidylinositol-specific phospholipase C (PLC) 1The abbreviations used are: PLCphospholipase CBSAbovine serum albuminDTTdithiothreitolERGelectroretinogramPAGEpolyacrylamide gel electrophoresisPDAprolonged depolarizing afterpotentialPIP2phosphatidylinositol 4,5-bisphosphate. hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield two preeminent second messenger molecules, inositol trisphosphate (IP3) and diacylglycerol, in one of the cornerstone signaling pathways of cellular communication (Rhee et al., 1989Rhee S.G. Suh P.G. Ryu S.H. Lee S.Y. Science. 1989; 244: 546-550Crossref PubMed Scopus (698) Google Scholar; Meldrum et al., 1991Meldrum E. Parker P.J. Carozzi A. Biochim. Biophys. Acta. 1991; 1092: 49-71Crossref PubMed Scopus (157) Google Scholar; Rhee and Choi, 1992aRhee S.G. Choi K.D. J. Biol. Chem. 1992; 267: 12393-12396Abstract Full Text PDF PubMed Google Scholar, 1992b). IP3 elicits the release of calcium from intracellular stores, and diacylglycerol has been shown to activate protein kinase C. These, in turn, affect other cellular processes which, eventually, result in a cellular response. phospholipase C bovine serum albumin dithiothreitol electroretinogram polyacrylamide gel electrophoresis prolonged depolarizing afterpotential phosphatidylinositol 4,5-bisphosphate. PLC comprises a large family of enzymes. More than a dozen PLC-encoding genes have been identified and the cognate cDNAs cloned (see Meldrum et al., 1991Meldrum E. Parker P.J. Carozzi A. Biochim. Biophys. Acta. 1991; 1092: 49-71Crossref PubMed Scopus (157) Google Scholar for references). The respective PLC enzymes have been classified into four major groups (designated PLC-α, PLC-β, PLC-γ, and PLC-δ) based on molecular weight, immunological, and structural differences (Rhee et al., 1989Rhee S.G. Suh P.G. Ryu S.H. Lee S.Y. Science. 1989; 244: 546-550Crossref PubMed Scopus (698) Google Scholar). The PLC-β, -γ, and -δ enzymes have been further divided into subgroupings (PLC-β1 through PLC-β4, PLC-γ1 through PLC-γ2, and PLC-δ1 through PLC-δ3) based on differences in amino acid sequence and that each subtype is encoded by a separate gene (Meldrum et al., 1991Meldrum E. Parker P.J. Carozzi A. Biochim. Biophys. Acta. 1991; 1092: 49-71Crossref PubMed Scopus (157) Google Scholar; Rhee and Choi, 1992aRhee S.G. Choi K.D. J. Biol. Chem. 1992; 267: 12393-12396Abstract Full Text PDF PubMed Google Scholar, Rhee and Choi, 1992bRhee S.G. Choi K.D. Putney Jr., J.W. Advances in Second Messenger and Phosphoprotein Research. Vol. 26. Raven Press, NY1992: 35-61Google Scholar). Observations that the different subtypes of PLC differ in tissue distribution have led to the idea that they are coupled to different receptors and are utilized in different cellular processes (Rhee et al., 1989Rhee S.G. Suh P.G. Ryu S.H. Lee S.Y. Science. 1989; 244: 546-550Crossref PubMed Scopus (698) Google Scholar; Fain, 1990Fain J.N. Biochim. Biophys. Acta. 1990; 1053: 81-88Crossref PubMed Scopus (85) Google Scholar; Meldrum et al., 1991Meldrum E. Parker P.J. Carozzi A. Biochim. Biophys. Acta. 1991; 1092: 49-71Crossref PubMed Scopus (157) Google Scholar). However, very little is known about the identity of signaling pathways that utilize the enzymes or how they function in vivo. Even for the PLC enzymes that have been purified or extensively characterized biochemically (Banno et al., 1986Banno Y. Nakanishi S. Nozawa Y. Biochem. Biophys. Res. Commun. 1986; 136: 713-721Crossref PubMed Scopus (33) Google Scholar; Hakata et al., 1982Hakata H. Kambayashi J.-I. Kosaki G. J. Biochem. (Tokyo). 1982; 92: 929-935Crossref PubMed Scopus (31) Google Scholar; Hofmann and Majerus, 1982Hofmann S.L. Majerus P.W. J. Biol. Chem. 1982; 257: 6461-6469Abstract Full Text PDF PubMed Google Scholar; Homma et al., 1988Homma Y. Imaki J. Nakanishi O. Takenawa T. J. Biol. Chem. 1988; 263: 6592-6598Abstract Full Text PDF PubMed Google Scholar; Manne and Kung, 1987Manne V. Kung H.-F. Biochem. J. 1987; 243: 763-771Crossref PubMed Scopus (22) Google Scholar; Nakanishi et al., 1988Nakanishi O. Homma Y. Kawasaki H. Emori Y. Suzuki K. Takenawa T. Biochem. J. 1988; 256: 453-459Crossref PubMed Scopus (18) Google Scholar; Meldrum et al., 1989Meldrum E. Katan M. Parker P. Eur. J. Biochem. 1989; 182: 673-677Crossref PubMed Scopus (30) Google Scholar; Rebecchi and Rosen, 1987Rebecchi M.J. Rosen O.M. J. Biol. Chem. 1987; 262: 12526-12532Abstract Full Text PDF PubMed Google Scholar; Takenawa and Nagai, 1981Takenawa T. Nagai Y. J. Biol. Chem. 1981; 256: 6769-6775Abstract Full Text PDF PubMed Google Scholar; Wang et al., 1986Wang P. Toyoshima S. Osawa T. J. Biochem. (Tokyo). 1986; 100: 1015-1022Crossref PubMed Scopus (22) Google Scholar), the functions of the enzymes in vivo remain poorly described. A PLC enzyme for which a function in vivo has been proposed is encoded by the norpA gene of Drosophila melanogaster. 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, 1970Hotta Y. Benzer S. Proc. Natl. Acad. Sci. U. S. A. 1970; 67: 1156-1163Crossref PubMed Scopus (250) Google Scholar; Pak et al., 1970Pak W.L. Grossfield J. Arnold K. Nature. 1970; 227: 518-520Crossref PubMed Scopus (148) Google Scholar). 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., 1988Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (516) Google Scholar). These data, as well as a growing body of evidence suggesting that PLC is involved in invertebrate phototransduction (reviewed by Payne, 1986Payne R. Photobiochem. Photobiophys. 1986; 13: 373-397Google Scholar; Pak and Shortridge, 1991Pak W.L. Shortridge R.D. Photochem. and Photobiol. 1991; 53: 871-875Crossref Scopus (17) Google Scholar), have converged to suggest that the norpA gene encodes PLC that is utilized in phototransduction in Drosophila. This is in contrast to vertebrate phototransduction which is proposed to occur via activation of cGMP phosphodiesterase (reviewed by Kaupp and Koch, 1986Kaupp U.B. Koch K.-W. Trends Biochem. Sci. 1986; 11: 43-47Abstract Full Text PDF Scopus (20) Google Scholar; Stryer, 1986Stryer L. Annu. Rev. Neurosci. 1986; 9: 87-119Crossref PubMed Scopus (774) Google Scholar). To determine whether the norpA gene indeed encodes PLC activity that is required for phototransduction in Drosophila, we constructed a chimeric norpA minigene by fusing norpA cDNA to the ninaE gene promoter. Since ninaE gene encodes R1-6 photoreceptor cell-specific rhodpsin (O'Tousa et al., 1985O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (381) Google Scholar; Zuker et al., 1985Zuker C.S. Cowman A.F. Rubin G.M. Cell. 1985; 40: 851-858Abstract Full Text PDF PubMed Scopus (330) Google Scholar), its promoter will drive expression of norpA RNA in R1-6 photoreceptor cells. The norpA minigene chimera was transformed into norpAP24 mutant by P-element-mediated germline transformation (Spradling and Rubin, 1982Spradling A.C. Rubin G.M. Science. 1982; 218: 341-347Crossref PubMed Scopus (1169) Google Scholar; Rubin and Spradling, 1982Rubin G.M. Spradling A.C. Science. 1982; 218: 348-353Crossref PubMed Scopus (2335) Google Scholar; Spradling, 1986Spradling A.C. Roberts D.B. Drosophila: A Practical Approach. IRL Press, Oxford, United Kingdom1986: 175-197Google Scholar). Transformed flies were 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. The D. melanogaster white (wA35) mutant was used in all experiments as a control group because its genetic background is most similar to norpAP24 mutant. norpAP24 mutants were chosen for transformation experiments because they are strong mutants which completely lack detectable amounts of norpA protein (Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar) and express severely reduced amounts of norpA mRNA as well (Bloomquist et al., 1988Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (516) Google Scholar; Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar). Flies were grown at 21°C in a 12 h light/12 h dark cycle on Carolina Instant Medium (Carolina Biological) supplemented with dry yeast or on cornmeal/sucrose/agar medium (Roberts, 1986Roberts D.B. Roberts D.G. Drosophila: A Practical Approach. IRL Press, Oxford, United Kingdom1986: 1-38Google Scholar) supplemented with β-carotene at 0.125 mg/ml. Separate experiments verified that Carolina Instant Medium has sufficient vitamin A to mediate normal vision (Lee, 1994Lee, R. D., 1994, The effects of carotenoid and retinoids upon Drosophila photoreception: Regulation of the expression of opsin, phospholipase C, Drosophila retinol binding protein, and membrane buildup, M. S. Thesis, St. Louis University.Google Scholar). One- to 4-day-old (after eclosion) adults were used for Western blot analysis, PLC activity assays, and immunohistochemistry. Four-day-old adults were used for most electrophysiological analyses although adults of various ages were also tested. Standard molecular biological techniques (Sambrook et al., 1989Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) were used to excise a 1.7-kilobase SpeI/BglI restriction fragment that contains the ninaE open reading frame from ninaE genomic DNA (O'Tousa et al., 1985O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (381) Google Scholar). The excised ninaE DNA fragment was replaced with a 3.8-kilobase BglI/SalI restriction fragment that contains the norpA open reading frame derived from the norpA cDNA (nucleotides 519-4331 in Bloomquist et al., 1988Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (516) Google Scholar) to create a chimeric ninaE/norpA minigene (Fig. 1). This norpA minigene chimera was subcloned into the pCaSpeR4 transformation vector (Thummel and Pirotta, 1991Thummel C.S. Pirotta V. Dros. Information Newsletter. 1991; Vol. 2Google Scholar) for transformation into the germline of norpAP24/white mutant using the procedure decribed by Spradling, 1986Spradling A.C. Roberts D.B. Drosophila: A Practical Approach. IRL Press, Oxford, United Kingdom1986: 175-197Google Scholar. Surviving adults were crossed back to norpAP24 mutants. Transformed flies were identified in the progeny of this cross by their red eye color caused by expression of the mini-white gene which is contained within the pCaSpeR4 transformation vector. For identification of the insertion site of the norpA minigene chimera in the genome, chromosome squashes were prepared from salivary glands of transformed flies as described previously (Shortridge et al., 1991Shortridge R.D. Yoon J. Lending C. Bloomquist B.T. Perdew M. Pak W.L. J. Biol. Chem. 1991; 266: 12474-12480Abstract Full Text PDF PubMed Google Scholar), except that the cDNA probe was prepared by labeling with digoxygenin-dUTP (Boehringer Mannheim) according to manufacturer's instructions. Visualization of the digoxygenin-labeled DNA after hybridization was by immunostaining in sheep anti-digoxygenin antibody (Boehringer Mannheim) followed by incubation with 0.4 mM 4-nitro blue tetrazolium chloride, and 0.4 mM 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) according to the manufacturer's instructions. Tissues from 0- to 4-day-old flies were homogenized in 50 mM Tris-HCl, pH 7.4, 250 mM KCl, 0.05% sodium deoxycholate, and 0.1 mM phenylmethylsulfonyl fluoride in 1.5-ml microfuge tubes using Teflon pestles. Homogenates were centrifuged briefly at 12,000 × g to remove particulate matter. These crude homogenates were fractionated by 7% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE), electroblotted onto a nitrocellulose membrane, stained using antiserum generated against the major gene product of norpA using conditions described by Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar. PLC activity assays were carried out by incubating a 0.1 ml volume of 50 mM Tris-Cl, pH 7.5, 10−7 M CaCl2, 0.1 mg/ml BSA, 0.2 mM phosphatidylinositol (Sigma), 44,000 disintegrations/min phosphatidyl-[3H]inositol 4,5-bisphosphate (New England Nuclear), and Drosophila tissue extract for 5 min at room temperature essentially as described Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar. Crude tissue extracts were prepared by grinding tissue in a buffer of 50 mM Tris-Cl, pH 7.5, 250 mM KCl, 0.05% sodium deoxycholate, 0.1 mM DTT, and 0.1 mM phenylmethylsulfonyl fluoride, using a Teflon pestle in a 1.5-ml microfuge tube on ice. These homogenates were then centrifuged briefly at 12,000 × g to remove particulate matter. Protein concentration in homogenates were determined using the BCA protein assay (Pierce) with BSA as a standard and an appropriate amount of extract (amount empirically determined to yield linear results with respect to time) added to the reaction mixture. Reactions were stopped by precipitating in 5% trichloroacetic acid and quantifying emissions in the supernatant by liquid scintillation. Drosophila heads were embedded in O.C.T. compound (Tissue-Tek) and frozen on dry ice. Ten μm thick tissue sections were cut on a Reichert cryostat and transferred onto gelatin-subbed slides (Gall and Pardue, 1971Gall J.G. Pardue M.L. Methods Enzymol. 1971; 21: 470-480Crossref Scopus (301) Google Scholar). The sections were fixed for 30 min in a freshly made solution of 150 mM sodium phosphate, pH 7.0, 75 μM lysine, 10 μM sodium metaperiodate, and 2% paraformaldehyde (McLean and Nakane, 1974). Following tissue fixation, sections were washed two times in PBS for 5 min each. The sections were blocked, incubated with antibodies, washed, and developed in the same way as Western blots mentioned above except primary antibody incubations were carried out overnight at 4°C. The primary antibody used in the immunohistochemical preparations was affinity purified from norpA antiserum by cross-linking a bacterially expressed norpA fusion protein (Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar) to cyanogen bromide-activated Sepharose beads (Pharmacia) according to manufacturer's instructions followed by passing the crude antiserum across the cross-linked beads packed loosely in a column. The column with bound antibodies was washed in 1 × PBS and the antibodies eluted in glycine-Cl buffer, pH 2.3 (Harlow and Lane, 1988Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1988Google Scholar). Immunogold decoration of sites in photoreceptor cells that are stained by the norpA antiserum was carried out using minor modifications of the methods of Long et al., 1994Long J.J. Wang J.-L. Berry J.O. J. Biol. Chem. 1994; 269: 2827-2833Abstract Full Text PDF PubMed Google Scholar. Briefly, flies were decapitated manually and the heads fixed overnight at 4°C in 2% paraformaldehyde, 0.2% picric acid, and 0.1 M sodium cacodylate, pH 7.2. After rinsing in 0.1 M sodium cacodylate, heads were incubated in a 0.5 M ammonium chloride, 0.1 M sodium cacodylate, at room temperature for 1 h to block any remaining free aldehyde groups. The heads were washed twice in 50% ethanol and twice in 70% ethanol at room temperature for 5 min each to dehydrate the sample. Heads were then infiltrated and embedded with LR white resin (London Resin Company, Ltd., Hampshire, United Kingdom) according to manufacturer's directions. One-hundred-nm sections were prepared and placed on Formvar-coated nickel grids for immunolocalization. Grids were incubated in a blocking solution of 0.5% BSA, 0.05% Tween 20, 1 × PBS, supplemented with 2.5% normal goat sera for 15 min prior to incubation with affinity purified norpA antibody. Primary antibody staining was done by incubating the grid preparations for 30 min at room temperature in blocking solution containing affinity purified primary antibody at a final concentration of 20 μg/ml. Samples were washed four times for 30 min each in a washing solution of 0.5% BSA, 0.1% Tween 20, 1 × PBS. Primary antibody was detected with a 1:20 dilution of 15 nm gold Auroprobe (Amersham) according to manufacturer's instructions. After washing the preparations in washing solution, as described above, the sample was stained with uranyl acetate and lead citrate. Specimens were observed with an electron microscope (H500:Hitachi Ltd, Tokoyo) operating at 75 kV. Electroretinograms (ERGs) and prolonged depolarizing afterpotential (PDA) analyses were carried out on adult flies essentially as described by Chen et al., 1992Chen D.-M. Christianson J.S. Sapp R.I. Stark W.S. Vis. Neurosci. 1992; 9: 125-135Crossref PubMed Scopus (49) Google Scholar. The fly's compound eye was carefully located at the focal plane of an optical stimulator using 625-nm light at an average intensity of 16.43 log quanta/cm2 × s. A glass micropipette was inserted into the retinal cell layer under 625-nm light. After the fly was dark adapted for 40 min, the eye was stimulated by 470-nm light and the ERG was recorded, amplified, and fed into a MacLab/2e-Macintosh LC II computer system for storage, viewing, and analysis. PDA was induced by two stimuli of 470 nm at about 16.04 log quanta/cm2 × s for 2 s each and followed by two of 570 nm stimulation at about 16.39 log quanta/cm2 × s for 2 s each. A chimeric norpA minigene was constructed by replacing the open reading frame of the Drosophila ninaE gene (O'Tousa et al., 1985O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (381) Google Scholar; Zuker et al., 1985Zuker C.S. Cowman A.F. Rubin G.M. Cell. 1985; 40: 851-858Abstract Full Text PDF PubMed Scopus (330) Google Scholar) with that of the norpA gene (Bloomquist et al., 1988Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (516) Google Scholar) such that expression of the norpA protein would come under control of the ninaE gene promoter (Fig. 1). Since the ninaE gene encodes the major form of rhodopsin in R1-6 photoreceptor cells, the expression of the norpA protein, derived from the chimeric minigene, should occur in the retina, more specifically in R1-6 photoreceptor cells. The chimeric norpA minigene was subcloned into the pCaSpeR4 transformation vector (Thummel and Pirrotta, 1991) and the resulting DNA injected into a total of 533 norpAP24 mutant embryos. Forty-five of the injected embryos developed into fertile adults, and of these, two different transformant lines were isolated (designated T-15 and T-35). To determine the position where the norpA minigene had inserted into the genome of the T-15 and T-35 transformant lines, labeled norpA cDNA was hybridized in situ to squashes of salivary chromosomes prepared from the transformants. The insertion site was at 46A in the T-15 transformant line and at 44A in the T-35 transformant, both on the right arm of the second chromosome (data not shown). Antiserum generated against the major gene product of norpA has been previously described (Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar). As shown in Fig. 2, norpA antiserum detects the 130-kDa norpA protein in head homogenates of the white strain (which is wild-type for norpA). This protein is missing in head homogenates of norpAP24 mutant, but is present in head homogenates of norpAP24 mutants after they have been transformed with the norpA minigene chimera. This 130-kDa protein is not detectable in head homogenates of white-eyed siblings of norpAP24 transformants (data not shown), which eye color indicates that the particular flies do not express the mini-white gene, and thus do not harbor norpA mini-gene inserts on their chromosomes. These data demonstrate that the norpA mini-gene chimera, when transformed into heads of norpAP24 mutant, is capable of directing the synthesis of norpA protein. Drosophila heads contain a high amount of PLC activity that is severely reduced by norpA mutations (Yoshioka et al., 1985Yoshioka T. Inoue H. Hotta Y. J. Biochem. (Tokyo). 1985; 97: 1251-1254Crossref PubMed Scopus (63) Google Scholar; Inoue et al., 1988Inoue H. Yoshioka T. Hotta Y. J. Biochem. (Tokyo). 1988; 103: 91-94Crossref PubMed Scopus (42) Google Scholar; Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar; McKay et al., 1994McKay R.R. Zhu L. Shortridge R.D. Neuroscience. 1994; 61: 141-148Crossref PubMed Scopus (7) Google Scholar). To determine whether PLC activity is rescued in heads of norpAP24 transformants, head homogenates were tested for their ability to cleave PIP2 in an in vitro activity assay. As shown in Fig. 3, norpAP24 mutants exhibit less than 1% of PLC activity that is measured in white strain (control) heads. The amount of PLC activity in white heads has been determined to be comparable to that found in wild-type heads.2( 2R. R. McKay, D-M. Chen, K. Miller, S. Kim, W. S. Stark, and R. D. Shortridge, unpublished results.) norpAP24 transformants exhibit 64% (T-15) and 27% (T-35) of the amount of PLC activity found in white heads (Fig. 3), demonstrating that the severe reduction of PLC activity exhibited by norpA mutants is capable of being at least partially reversed by transforming the norpA minigene chimera into the germline. To localize the expression of norpA protein in tissues, norpA antiserum was used to immunostain the norpA protein in tissue sections of heads. Since the expression of norpA RNA in transformants is driven by the ninaE gene promoter, which gene normally encodes R1-6 photoreceptor cell-specific rhodopsin (O'Tousa et al., 1985O'Tousa J.E. Baehr W. Martin R.L. Hirsh J. Pak W.L. Applebury M. Cell. 1985; 40: 839-850Abstract Full Text PDF PubMed Scopus (381) Google Scholar; Zuker et al., 1985Zuker C.S. Cowman A.F. Rubin G.M. Cell. 1985; 40: 851-858Abstract Full Text PDF PubMed Scopus (330) Google Scholar), norpA protein would be expected to localize in retina, more specifically in R1-6 photoreceptor cells. In agreement with prior work (Zhu et al., 1993Zhu L. McKay R.R. Shortridge R.D. J. Biol. Chem. 1993; 268: 15994-16001Abstract Full Text PDF PubMed Google Scholar), norpA antiserum stains retina of white strain controls, but there is a definite lack of staining of retina of norpAP24 mutant (Fig. 4). The retina of norpAP24 mutants that have been transformed with the norpA minigene stain darkly by norpA antiserum, demonstrating that expression of the norpA minigene chimera occurs in the retina, as expected. To localize norpA expression at the subcellular level in retina, immunogold labeling of norpA protein in tissue sections of retina were visualized using electron microscopy. As shown in Fig. 5, staining of retina in transformants by norpA antiserum occurs over the rhabdomeres of R1-6 photoreceptor cells, but not R7 or R8 photoreceptor cells. This is what is expected, since the expression of norpA is being driven by the promoter of the ninaE gene, which gene encodes R1-6 photoreceptor cell-specific rhodopsin. ERG and PDA analyses were performed to determine whether transformants exhibit light-induced depolarizing responses in the retina. white mutant exhibits a normal ERG and PDA while norpAP24 mutant exhibits no ERG or PDA (Fig. 6). norpAP24 transformants indeed exhibit electrophysiological responses to light as measured by ERGs (Fig. 6), demonstrating that expression of the norpA minigene is sufficient to rescue the lack of light-induced photoreceptor response exhibited by norpAP24 mutant. ERG tracings of T-15 transformant resemble that of a wild-type response to light stimuli, while those of T-35 transformant take longer to return to base line after high intensity light stimulation than do wild-type or T-15 transformant (Fig. 6). There are no observable differences in ERGs between male, and females (data not shown). Moreover, ERG sensitivity does not appear to change with age (not shown). PDA tracings of both T-15 and T-35 transformants resemble that of control flies except that there is no or very little response induced by a second (blue light) or third (orange light) stimuli (Fig. 6). The observed responses to the second and third stimuli in control PDA tracings are known to be derived from depolarization of R7 and R8 photoreceptor cells (eg. Stark and Zitzmann, 1976Stark W.S. Zitzmann W.G. J. Comp. Physiol. 1976; 105: 15-27Crossref Scopus (29) Google Scholar), indicating that the R7 and R8 photoreceptor cells are not functioning in transformants. These data correlate exactly with the immunohistochemical data, shown above, which demonstrate the presence of norpA protein in rhabdomeres of R1-6 photoreceptor cells, but its absence from R7 and R8 photoreceptor cells (Fig. 5). This finding was also expected, since the ninaE gene should drive expression of the norpA coding sequence only in R1-6 photoreceptor cells. norpA mutants have long been known to exhibit defects in vision and, more recently, it has been proposed that the norpA gene encodes a phospholipase C enzyme that is essential for phototransduction (Bloomquist et al., 1988Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (516) Google Scholar; Schneuwly et al., 1991Schneuwly S. Burg M. Lending C. Perdew M. Pak W.L. J. Biol. Chem. 1991; 266: 24314-24319Abstract Full Text PDF PubMed Google Scholar; Pak and Shortridge, 1991Pak W.L. Shortridge R.D. Photochem. and Photobiol. 1991; 53: 871-875Crossref Scopus (17) Google Scholar). The question addressed here is whether expression of norpA protein in mutants is sufficient to rescue the low amount of PLC activity in head of norpA mutant concomitant with rescuing the phototransduction defect. Indeed, our results show that expression of a norpA minigene in norpA mutant results in restoration of norpA protein in retina as well as a concomitant partial restoration of PLC activity and light-evoked responses of photoreceptor cells. This is the best evidence thus far supporting the hypothesis that the norpA gene encodes a PLC that is utilized in phototransduction. There are several possible explanations why expression of the norpA minigene in mutant fails to fully rescue 100% of the amount of PLC activity observed in wild-type retina. First, the ninaE promoter, which was used to drive the expression of norpA, targets expression only in a subset of photoreceptor cells while wild-type expression of norpA occurs in all of the photoreceptor cells of the compound eye and ocelli (eg. Schneuwly et al., 1991Schneuwly S. Burg M. Lending C. Perdew M. Pak W.L. J. Biol. Chem. 1991; 266: 24314-24319Abstract Full Text PDF PubMed Google Scholar). Second, the minigene was constructed by combining parts of two heterologous genes (ninaE and norpA), and this may have a drastic affect on amounts of viable RNA produced or amount of translated product that appears in photoreceptor cells. Third, position effects of the inserted DNA on the chromosome or presumptive changes occurring in the transposon during insertion into the genome may adversely affect efficiency of expression of the gene (Spradling, 1986Spradling A.C. Roberts D.B. Drosophila: A Practical Approach. IRL Press, Oxford, United Kingdom1986: 175-197Google Scholar). Considering that any or all of these could lead to a reduction in the amount of norpA protein that is expressed in transformed mutants when compared to that in wild-type flies, the observed rescue is quite convincing. More importantly, the quantity of PLC activity exhibited by transformants appears to correlate well with the extent to which photoreceptor cells respond to light as well as the amount of norpA protein that appears to be present. The T-35 transformant exhibits less PLC activity than found in normal heads (27%) as well as less than T-15 transformant head (Fig. 3). This correlates well with results of the ERG analyses which show the response amplitude for T-35 transformant is less than that of white mutant or T-15 transformant under conditions of identical intensity stimulation (Fig. 6). Moreover, the amount of norpA protein in head of T-35 transformant, as judged by the darkness of staining of norpA protein in Western analyses, appears to be less than the amount in white heads or the T-15 transformant head (Fig. 2). Thus, there appears to be a positive correlation between reduction in PLC activity, reduction in the amount of norpA protein, and reduction in ERG amplitude of T-35 transformants, which results argue for a direct relationship between norpA protein, PLC activity, and photoreceptor cell responses. The significance of the present work is underscored by the fact that the norpA gene product is a PLC, a pivotal enzyme in one of the largest classes of signaling pathways known. Very little is known about the identity of signaling pathways that utilize any of the subtypes of PLC. The norpA-encoded PLC is one of the few for which a specific receptor and signaling pathway has been identified. Moreover, rescue of the phototransduction defect by expression of a norpA minigene proves that the gene identified by Bloomquist et al., 1988Bloomquist B.T. Shortridge R.D. Schneuwly S. Perdew M. Montell C. Steller H. Rubin G. Pak W.L. Cell. 1988; 54: 723-733Abstract Full Text PDF PubMed Scopus (516) Google Scholar is indeed the norpA gene as well as provides a means to carry out structure-to-function analyses on the norpA-encoded PLC. Alterations can now be made in norpA DNA prior to introducing it into the germline and the results can be examined in vivo. Furthermore, the significance of the conclusion that the norpA-encoded PLC is essential for phototransduction in Drosophila is strengthened by the recent identification of a bovine homologue of norpA that is found in the retina (Ferreira et al., 1993Ferreira P.A. Shortridge R.D. Pak W.L. Proc. Natl. Acad. Sci., U. S. A. 1993; 90: 6042-6046Crossref PubMed Scopus (50) Google Scholar; Lee et al., 1993Lee C.-W. Park D.J. Lee K.-H. Kim C.G. Rhee S.G. J. Biol. Chem. 1993; 268: 21318-21327Abstract Full Text PDF PubMed Google Scholar). This bovine retinal PLC has been shown to localize in the outer segments of cone cells, but not rods (Ferreira and Pak, 1994Ferreira P.A. Pak W.L J. Biol. Chem. 1994; 269: 3129-3131Abstract Full Text PDF PubMed Google Scholar). Inasmuch as the outer segments of cones are specialized structures for phototransduction, there might be an inositol phosphate-based phototransduction pathway in cones that is mediated by a norpA-like PLC (Ferreira and Pak, 1994Ferreira P.A. Pak W.L J. Biol. Chem. 1994; 269: 3129-3131Abstract Full Text PDF PubMed Google Scholar). Thus, further studies designed to elucidate the function of the norpA-encoded PLC in the Drosophila visual system may yield important clues needed to develop models of an analogous phototransduction process in mammals. We thank Dr. Linda Hall for providing helpful advice, Dr. Guoping Feng for technical help in microinjection of Drosophila embryos, and Alan Siegal for technical help in immunohistochemistry." @default.
• W2054297318 created "2016-06-24" @default.
• W2054297318 creator A5000850685 @default.
• W2054297318 creator A5003701670 @default.
• W2054297318 creator A5022557246 @default.
• W2054297318 creator A5046466053 @default.
• W2054297318 creator A5056872905 @default.
• W2054297318 creator A5068428299 @default.
• W2054297318 date "1995-06-01" @default.
• W2054297318 modified "2023-10-16" @default.
• W2054297318 title "Phospholipase C Rescues Visual Defect in norpA Mutant of Drosophila melanogaster" @default.
• W2054297318 cites W1482441851 @default.
• W2054297318 cites W1490293082 @default.
• W2054297318 cites W1493830810 @default.
• W2054297318 cites W1510233002 @default.
• W2054297318 cites W1515430029 @default.
• W2054297318 cites W1524012432 @default.
• W2054297318 cites W1527359679 @default.
• W2054297318 cites W1593225234 @default.
• W2054297318 cites W1594997102 @default.
• W2054297318 cites W1595716795 @default.
• W2054297318 cites W1850603239 @default.
• W2054297318 cites W1883220921 @default.
• W2054297318 cites W1896515542 @default.
• W2054297318 cites W1922623805 @default.
• W2054297318 cites W1969755572 @default.
• W2054297318 cites W1989141400 @default.
• W2054297318 cites W1994251246 @default.
• W2054297318 cites W1994528729 @default.
• W2054297318 cites W2006103165 @default.
• W2054297318 cites W2006464656 @default.
• W2054297318 cites W2006842412 @default.
• W2054297318 cites W2014318563 @default.
• W2054297318 cites W2023363801 @default.
• W2054297318 cites W2023930824 @default.
• W2054297318 cites W2029600228 @default.
• W2054297318 cites W2062748780 @default.
• W2054297318 cites W2067959181 @default.
• W2054297318 cites W2100973053 @default.
• W2054297318 cites W2103080800 @default.
• W2054297318 cites W2124923401 @default.
• W2054297318 cites W2150691484 @default.
• W2054297318 cites W2157674842 @default.
• W2054297318 cites W2160129162 @default.
• W2054297318 cites W2163438985 @default.
• W2054297318 cites W2164123016 @default.
• W2054297318 cites W2226570905 @default.
• W2054297318 cites W2418318517 @default.
• W2054297318 doi "https://doi.org/10.1074/jbc.270.22.13271" @default.
• W2054297318 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7768926" @default.
• W2054297318 hasPublicationYear "1995" @default.
• W2054297318 type Work @default.
• W2054297318 sameAs 2054297318 @default.
• W2054297318 citedByCount "49" @default.
• W2054297318 countsByYear W20542973182012 @default.
• W2054297318 countsByYear W20542973182017 @default.
• W2054297318 countsByYear W20542973182018 @default.
• W2054297318 countsByYear W20542973182019 @default.
• W2054297318 countsByYear W20542973182020 @default.
• W2054297318 countsByYear W20542973182023 @default.
• W2054297318 crossrefType "journal-article" @default.
• W2054297318 hasAuthorship W2054297318A5000850685 @default.
• W2054297318 hasAuthorship W2054297318A5003701670 @default.
• W2054297318 hasAuthorship W2054297318A5022557246 @default.
• W2054297318 hasAuthorship W2054297318A5046466053 @default.
• W2054297318 hasAuthorship W2054297318A5056872905 @default.
• W2054297318 hasAuthorship W2054297318A5068428299 @default.
• W2054297318 hasBestOaLocation W20542973181 @default.
• W2054297318 hasConcept C104317684 @default.
• W2054297318 hasConcept C143065580 @default.
• W2054297318 hasConcept C185592680 @default.
• W2054297318 hasConcept C2778597717 @default.
• W2054297318 hasConcept C2780104201 @default.
• W2054297318 hasConcept C55493867 @default.
• W2054297318 hasConcept C62478195 @default.
• W2054297318 hasConcept C78297661 @default.
• W2054297318 hasConcept C86803240 @default.
• W2054297318 hasConcept C95444343 @default.
• W2054297318 hasConceptScore W2054297318C104317684 @default.
• W2054297318 hasConceptScore W2054297318C143065580 @default.
• W2054297318 hasConceptScore W2054297318C185592680 @default.
• W2054297318 hasConceptScore W2054297318C2778597717 @default.
• W2054297318 hasConceptScore W2054297318C2780104201 @default.
• W2054297318 hasConceptScore W2054297318C55493867 @default.
• W2054297318 hasConceptScore W2054297318C62478195 @default.
• W2054297318 hasConceptScore W2054297318C78297661 @default.
• W2054297318 hasConceptScore W2054297318C86803240 @default.
• W2054297318 hasConceptScore W2054297318C95444343 @default.
• W2054297318 hasIssue "22" @default.
• W2054297318 hasLocation W20542973181 @default.
• W2054297318 hasOpenAccess W2054297318 @default.
• W2054297318 hasPrimaryLocation W20542973181 @default.
• W2054297318 hasRelatedWork W1819161614 @default.
• W2054297318 hasRelatedWork W1969421403 @default.
• W2054297318 hasRelatedWork W1993173008 @default.
• W2054297318 hasRelatedWork W2009733539 @default.
• W2054297318 hasRelatedWork W2054297318 @default.
• W2054297318 hasRelatedWork W2055617588 @default.

• next