©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Phospholipase C Rescues Visual Defect in norpA Mutant of Drosophila melanogaster(*)

Richard R. McKay (1), De-Mao Chen (2), Karen Miller (1), Sunkyu Kim (1), William S. Stark (2), Randall D. Shortridge (1)(§)

From the (1) Department of Biological Sciences, State University of New York, Buffalo, New York 14260 and the (2) Department of Biology, Saint Louis University, St. Louis, Missouri 63103

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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 norpA 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 norpA 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.


INTRODUCTION

Phosphatidylinositol-specific phospholipase C (PLC) hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield two preeminent second messenger molecules, inositol trisphosphate (IP) and diacylglycerol, in one of the cornerstone signaling pathways of cellular communication (Rhee et al., 1989; Meldrum et al., 1991; Rhee and Choi, 1992a, 1992b). IP 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.

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., 1991 for references). The respective PLC enzymes have been classified into four major groups (designated PLC-, PLC-, PLC-, and PLC-) based on molecular weight, immunological, and structural differences (Rhee et al., 1989). The PLC-, -, and - enzymes have been further divided into subgroupings (PLC-1 through PLC-4, PLC-1 through PLC-2, and PLC-1 through PLC-3) based on differences in amino acid sequence and that each subtype is encoded by a separate gene (Meldrum et al., 1991; Rhee and Choi, 1992a, 1992b). 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., 1989; Fain, 1990; Meldrum et al., 1991). 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., 1986; Hakata et al., 1982; Hofmann and Majerus, 1982; Homma et al., 1988; Manne and Kung, 1987; Nakanishi et al., 1988; Meldrum et al., 1989; Rebecchi and Rosen, 1987; Takenawa and Nagai, 1981; Wang et al., 1986), 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, 1970; Pak et al., 1970). 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. This is in contrast to vertebrate phototransduction which is proposed to occur via activation of cGMP phosphodiesterase (reviewed by Kaupp and Koch, 1986; Stryer, 1986).

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., 1985; Zuker et al., 1985), its promoter will drive expression of norpA RNA in R1-6 photoreceptor cells. The norpA minigene chimera was transformed into norpA mutant by P-element-mediated germline transformation (Spradling and Rubin, 1982; Rubin and Spradling, 1982; Spradling, 1986). 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.


MATERIALS AND METHODS

Drosophila Strains

The D. melanogaster white (w ) mutant was used in all experiments as a control group because its genetic background is most similar to norpA mutant. norpA mutants were chosen for transformation experiments because they are strong mutants which completely lack detectable amounts of norpA protein (Zhu et al., 1993) and express severely reduced amounts of norpA mRNA as well (Bloomquist et al., 1988; Zhu et al., 1993).

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, 1986) supplemented with -carotene at 0.125 mg/ml. Separate experiments verified that Carolina Instant Medium has sufficient vitamin A to mediate normal vision (Lee, 1994). 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.

Construction and Transformation of the norpA Minigene Chimera

Standard molecular biological techniques (Sambrook et al., 1989) 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., 1985). 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., 1988) to create a chimeric ninaE/norpA minigene (Fig. 1). This norpA minigene chimera was subcloned into the pCaSpeR4 transformation vector (Thummel and Pirotta, 1991) for transformation into the germline of norpA /white mutant using the procedure decribed by Spradling(1986). Surviving adults were crossed back to norpA 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.


Figure 1: Schematic illustration of construction of a chimeric norpA minigene. A ninaE/norpA chimeric minigene was constructed by removing a fragment containing the ninaE open reading frame from ninaE genomic DNA and replacing it with a cDNA fragment that contains the norpA open reading frame (represented by the cross-hatched box at the top). The arrow at the top represents the transcription start site of the ninaE gene. Positions of translation start and stop codons in the norpA open reading frame are also labeled. The chimeric norpA minigene was cloned into the KpnI site in the polylinker region (black box) of the pCaSpeR4 P-element transformation vector (represented by the circle) (Thummel and Pirotta, 1991). Stipled boxes labeled 5`P and 3`P in the pCaSpeR4 vector represent the 5` and 3` P-element inverted repeats. DNA sequences in the pCaSpeR4 transformation vector that are bacterial plasmid derived (pUC) or comprise the Drosophila mini-white gene (large semicircular arrow) are indicated.



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., 1991), 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.

Western Blot Analysis

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.(1993).

Phospholipase C Activity Assays

PLC activity assays were carried out by incubating a 0.1 ml volume of 50 mM Tris-Cl, pH 7.5, 10M CaCl, 0.1 mg/ml BSA, 0.2 mM phosphatidylinositol (Sigma), 44,000 disintegrations/min phosphatidyl-[H]inositol 4,5-bisphosphate (New England Nuclear), and Drosophila tissue extract for 5 min at room temperature essentially as described Zhu et al.(1993). 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.

Immunocytochemistry

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, 1971). 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.

Electron Micrograph Immunohistochemistry

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., 1993) 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, 1988).

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.(1994). 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.

Electrophysiological Analyses

Electroretinograms (ERGs) and prolonged depolarizing afterpotential (PDA) analyses were carried out on adult flies essentially as described by Chen et al. (1992). 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/cm 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/cm s for 2 s each and followed by two of 570 nm stimulation at about 16.39 log quanta/cm s for 2 s each.


RESULTS

Expression of norpA Minigene in Photoreceptor Cells of norpA Mutant

A chimeric norpA minigene was constructed by replacing the open reading frame of the Drosophila ninaE gene (O'Tousa et al., 1985; Zuker et al., 1985) with that of the norpA gene (Bloomquist et al., 1988) 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 norpA 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).

Presence of norpA Protein in Head of Transformed Mutants

Antiserum generated against the major gene product of norpA has been previously described (Zhu et al., 1993). 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 norpA mutant, but is present in head homogenates of norpA 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 norpA 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 norpA mutant, is capable of directing the synthesis of norpA protein.


Figure 2: Immunodetection of norpA protein on Western blots. Lanes were loaded with protein from two heads. Blots were stained with antiserum generated against the major gene product of the norpA protein (Zhu et al., 1993) which detects the 130-kDa norpA protein (arrow) that is absent from head of norpA mutant. Primary antibody binding was detected using an alkaline phosphatase-conjugated secondary antibody with NBT/BCIP as chromogen (Zhu et al., 1993). The norpA protein is present in head of two progeny lines of norpA mutant (designated T-15 and T-35) which have been transformed with the chimeric norpA minigene. The intensity of staining of norpA protein in T-35 transformant head is consistently less in Western blots than that in T-15 transformant or white mutant. Numbers on the left indicate molecular mass (in kDa) and relative positions of migration of protein size standards.



Rescue of Phospholipase C Activity in Head of Transformed Mutants

Drosophila heads contain a high amount of PLC activity that is severely reduced by norpA mutations (Yoshioka et al., 1985; Inoue et al., 1988; Zhu et al., 1993; McKay et al., 1994). To determine whether PLC activity is rescued in heads of norpA transformants, head homogenates were tested for their ability to cleave PIP in an in vitro activity assay.

As shown in Fig. 3, norpA 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.()norpA 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.


Figure 3: Phospholipase C activity in head homogenates. Head homogenates of white (w) mutant, norpA mutant, and T-15 and T-35 transformants were assayed for the ability to cleave [H]PIP in an in vitro biochemical reaction. Results are expressed as specific activity of the homogenate (pmol of [H]PIP cleaved/5 min/mg of protein). Data shown are averaged from four or more determinations with error bars indicating standard deviation. PLC activity is severely reduced in the head of the norpA mutant, in comparison to that found in head of white controls, a strain selected because it matches the genetic background of norpA mutants used in this study. Transformation of the chimeric norpA minigene into the genome of the norpA mutant partially restores PLC activity in head. Transformants T-15 and T-35 exhibit 64 and 27%, respectively, of the amount PLC of activity found in white mutant.



Spatial Localization of norpA Protein in Head

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., 1985; Zuker et al., 1985), norpA protein would be expected to localize in retina, more specifically in R1-6 photoreceptor cells.

In agreement with prior work (Zhu et al., 1993), norpA antiserum stains retina of white strain controls, but there is a definite lack of staining of retina of norpA mutant (Fig. 4). The retina of norpA 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.


Figure 4: Immunocytochemical staining of norpA protein in tissue sections of head. Frontal sections of heads were stained using antiserum generated against the norpA protein. Primary antibody binding was detected using an alkaline phosphatase-conjugated secondary antibody with NBT/BCIP as chromogen (Zhu et al., 1993). Staining of the norpA protein can be clearly seen in the retina of white controls (A), but no staining is seen in retina of the norpA mutant (B). Staining of retina of norpA transformants, T-15 (C) and T-35 (D), is clearly visible. Staining in transformants also appears over the lamina, which is potentially due to the additional presence of the norpA protein in axons of photoreceptor cells and likely caused by expressing norpA as a heterologous minigene chimera in the transformants.



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.


Figure 5: Subcellular localization of norpA protein in retina of transformed norpA mutant. Staining of the norpA protein in retinal tissue was visualized by immunogold decoration (15 nm diameter) and electron microscopy (60,000 magnification). Shown is a cross-section of the rhabdomeric region of a single retinal ommatidium of the T-35 transformant. Immunogold decoration of the norpA protein (shown by the presence of dense particles) appears over the rhabdomeric membranes of the peripheral (R1-6) photoreceptor cells, but is absent over the rhabdomeric region of the central photoreceptors (R7 and R8).



Electrophysiological Analysis Shows Rescue of Phototransduction

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 norpA mutant exhibits no ERG or PDA (Fig. 6). norpA 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 norpA 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).


Figure 6: ERG and PDA analysis. Electroretinographic waveforms (left column) and prolonged depolarizing after potentials (right column) were recorded from white mutant, norpA mutant, T-15 transformant, and T-35 transformant. Shown are typical results from 4-day-old (after eclosion) adult females, although no difference in ERG or PDA was observed between males and females (data not shown). All ERG stimuli were 470 nm (duration = 1 s) with relative intensities of -7.39, -6.43, -5.16, and -3.50 for white and -6.43, -5.16, -3.50, and -2.60 for T15 and T35. PDA stimuli (duration = 2 s) were either 470 or 570 nm as indicated along the bottom. white mutants show normal wild-type ERG and normal PDA, while norpA mutants completely lack electrophysiological responses to light. T-15 transformants exhibit an almost normal ERG while T-35 exhibit lower amplitudes and takes longer to return to base line. Both transformants (T-15 and T-35) exhibit no responses or very small responses induced by the second (blue, 470 nm) or the third (yellow, 570 nm) stimuli in the PDA waveforms.



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, 1976), 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.


DISCUSSION

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., 1988; Schneuwly et al., 1991; Pak and Shortridge, 1991). 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., 1991). 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, 1986). 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.(1988) 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., 1993; Lee et al., 1993). This bovine retinal PLC has been shown to localize in the outer segments of cone cells, but not rods (Ferreira and Pak, 1994). 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, 1994). 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant EY07192 (to W. S. S.) and National Science Foundation Grant IBN-9102866 (to R. D. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biological Sciences, Cooke Hall Rm. 109, State University of New York at Buffalo, Buffalo, NY 14260-1300. Tel.: 716-645-3122; Fax: 716-645-2975.

The abbreviations used are: PLC, phospholipase C; BSA, bovine serum albumin; DTT, dithiothreitol; ERG, electroretinogram; PAGE, polyacrylamide gel electrophoresis; PDA, prolonged depolarizing afterpotential; PIP, phosphatidylinositol 4,5-bisphosphate.

R. R. McKay, D-M. Chen, K. Miller, S. Kim, W. S. Stark, and R. D. Shortridge, unpublished results.


ACKNOWLEDGEMENTS

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.


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