Substitution of a non-retinal phospholipase C in Drosophila phototransduction.

The Drosophila norpA gene encodes at least two subtypes of phospholipase C (PLC), one of which is essential for phototransduction and the other is utilized in signalling pathways other than phototransduction. The two subtypes of norpA-PLC differ by 14 amino acids that have been proposed as important for the function of PLC in different signalling pathways. The present study aimed to determine whether norpA subtype II enzyme can functionally substitute for the subtype I enzyme in the phototransduction pathway. We found that the non-retinal norpA-PLC enzyme can substitute for its retinal counterpart, but that there is a reduced rate of repolarization of photoreceptors following intense light stimuli. This reduced repolarization might be due to the inability of a regulatory component being able to interact with the non-retinal norpA-PLC enzyme.

Evidence for indirect control of phospholipase C (PLC-beta) by retinoids in Drosophila phototransduction.

PURPOSE: To determine how retinoids regulate the phospholipase C (PLC) gene in the Drosophila visual system. METHODS: Western blotting, activity analyses and immunocytochemistry were applied to Drosophila reared on various diets. RESULTS: Western blots and activity analyses showed that retinoid deprivation decreases PLC, the product of the norpA gene, by approximately 1/3 to 1/2 in Drosophila. Immunocytochemistry using standard and confocal fluorescence microscopy confirmed the expectation that PLC is localized to the photoreceptive rhabdomeres. Rhabdomeres of flies that were retinoid deprived, or reared on other diets devoid of chromophore precursors, fluoresced brightly. These observations are consistent with earlier morphometric analyses showing that retinoid deprivation decreases the size of rhabdomeres. In a separate control, rhabdomeric PLC was shown to be virtually eliminated by retinoid deprivation in transgenic Drosophila where the norpA coding sequence was driven by the opsin promoter. CONCLUSIONS: PLC is decreased by retinoid deprivation. Retinoid control of PLC is indirect, as expected, since the norpA promoter is so different from the promoter for rhodopsin's gene. PLC is not eliminated by deprivation but decreases in proportion to the associated decrease in rhabdomere size which, in turn, is caused by the opsin decrease. By contrast, opsin is controlled by retinoids both translationally by chromophore availability and transcriptionally. The fact that PLC is eliminated by retinoid deprivation when opsin's promoter drives the PLC gene is important evidence substantiating retinoid control via opsin's promoter.

Two distantly positioned PDZ domains mediate multivalent INAD-phospholipase C interactions essential for G protein-coupled signaling.

Drosophila INAD, which contains five tandem protein interaction PDZ domains, plays an important role in the G protein-coupled visual signal transduction. Mutations in InaD alleles display mislocalization of signaling molecules of phototransduction which include the essential effector, phospholipase C-beta (PLC-beta), which is also known as NORPA. The molecular and biochemical details of this functional link are unknown. We report that INAD directly binds to NORPA via two terminally positioned PDZ1 and PDZ5 domains. PDZ1 binds to the C-terminus of NORPA, while PDZ5 binds to an internal region overlapping with the G box-homology region (a putative G protein-interacting site). The NORPA proteins lacking binding sites, which display normal basal PLC activity, can no longer associate with INAD in vivo. These truncations cause significant reduction of NORPA protein expression in rhabdomeres and severe defects in phototransduction. Thus, the two terminal PDZ domains of INAD, through intermolecular and/or intramolecular interactions, are brought into proximity in vivo. Such domain organization allows for the multivalent INAD-NORPA interactions which are essential for G protein-coupled phototransduction.

Molecular characterization of a novel Drosophila gene which is expressed in the central nervous system.

We have isolated and characterized a novel Drosophila melanogaster gene (noe) that is specifically and abundantly expressed in the central nervous system (CNS). The gene, which maps to 74B on the left arm of third chromosome, encodes a protein of 74 amino acids with no significant similarity to known protein sequences. The deduced amino acid sequence of the gene product is rich in basic amino acids, especially the lysine, and contains five potential phosphorylation sites. The noe gene lacks introns and seems to produce two transcripts by alternative polyadenylations. The promoter region deduced from 5'-RACE analysis contains a sequence similar to the TATA-box consensus sequence. RNA blot analysis detected 1.0 kb noe transcripts that are expressed from the third-instar larval stage to the adult stage and which are predominantly found in the adult heads. In situ hybridizations to tissue sections showed that the gene is abundantly expressed in neuronal cell bodies as well as in the neurophiles of adult and larval CNS (brain, optic lobe, and thoracic ganglia of adults and larval brain).

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.

Invertebrate phosphatidylinositol-specific phospholipases C and their role in cell signaling.

Phosphatidylinositol-specific phospholipase C (PLC) is a family of enzymes that occupy a pivotal role in one of the largest classes of cellular signaling pathways known. Mammalian PLC enzymes have been divided into four major classes and a variety of subclasses based on their structural characteristics and immunological differences. There have been five invertebrate PLC-encoding genes cloned thus far and these fall within three of the four major classes used in categorizing mammalian PLC. Four of these invertebrate genes have been cloned from Drosophila melanogaster and one is from Artemia, a brine shrimp. Structural characteristics of the invertebrate enzymes include the presence of highly conserved Box X and Box Y domains found in major types of mammalian PLC as well as novel features. Two of the invertebrate PLC genes encode multiple splice-variant subtypes which is a newly emerging level of diversity observed in mammalian enzymes. Studies of the invertebrate PLCs have contributed to the identification of the physiological functions of individual isozymes. These identified roles include cellular processes such as phototransduction, olfaction, cell growth and differentiation.

Phospholipase C rescues visual defect in norpA mutant of Drosophila melanogaster.

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.

Multiple subtypes of phospholipase C are encoded by the norpA gene of Drosophila melanogaster.

The norpA gene of Drosophila melanogaster encodes a phosphatidylinositol-specific phospholipase C that is essential for phototransduction. Besides being found abundantly in retina, norpA gene products are expressed in a variety of tissues that do not contain phototransduction machinery, implying that norpA is involved in signaling pathways in addition to phototransduction. We have identified a second subtype of norpA protein that is generated by alternative splicing of norpA RNA. The alternative splicing occurs at a single exon that is excluded from mature norpA transcripts when a substitute exon of equal size is retained. The net difference between the two subtypes of norpA protein is 14 amino acid substitutions occurring between amino acid positions 130 and 155 of the enzyme. Results from Northern analyses suggest that norpA subtype I transcripts are most abundantly expressed in adult retina, while subtype II transcripts are most abundant in adult body. Moreover, norpA subtype I RNA can be detected by the reverse transcription-polymerase chain reaction in extracts of adult head tissue but not adult body nor at earlier stages of Drosophila development. Conversely, norpA subtype II RNA can be detected by reverse transcription-polymerase chain reaction throughout development as well as in heads and bodies of adults. Furthermore, norpA subtype I RNA is easily detected in retina using tissue in situ hybridization analysis, while subtype II RNA is not detectable in retina but is found in brain. Since only norpA subtype I RNA is found in retina, we conclude that subtype I protein is utilized in phototransduction. Since norpA subtype II RNA is not found in retina but is expressed in a variety of tissues not known to contain phototransduction machinery, subtype II protein is likely to be utilized in signaling pathways other than phototransduction. The amino acid differences between the two subtypes of norpA protein may reflect the need for each subtype to interact with signaling components of different signal-generating pathways.

A Drosophila gene that encodes a member of the protein disulfide isomerase/phospholipase C-alpha family.

Screening of a Drosophila genomic DNA library at reduced stringency hybridization conditions using a rat PLC alpha cDNA probe yielded a gene which encodes a member of the protein disulfide isomerase/PLC alpha family. The gene has been localized to band 74C on the left arm of the third chromosome and has been designated dpdi. Northern analysis shows that the dpdi gene encodes a transcript that is 2.3 kb in length and is present throughout development as well as in both heads and bodies of adults. The deduced dpdi protein is 496 amino acids in length and contains two domains exhibiting high similarity to thioredoxin, two regions that are similar to the hormone binding domain of human estrogen receptor, and a sequence of four amino acids (KDEL) at the C-terminus which has been described by others as being responsible for retention of proteins in the endoplasmic reticulum. Overall, dpdi contains a higher similarity to rat protein disulfide isomerase (53% identical) than to rat PLC alpha (30% identical). However, it is unclear whether dpdi functions in vivo as a PDI or as a PLC, or both. Drosophila, with its well characterized genetics and the ability to generate mutants in a gene that has been cloned, provides an excellent system in which to resolve this issue.

Membrane association of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Severe mutations within the norpA gene of Drosophila abolish the photoreceptor potential and render the fly blind by deleting phospholipase C, an essential component of the phototransduction pathway. To study the membrane association of phospholipase C, we have utilized biochemical assays of phospholipase C activity, which predominant measurable phospholipase C activity in head homogenates has been shown to be encoded by norpA, as well as antisera generated against the major gene product of norpA to examine its subcellular distribution before and during phototransduction. We find that both phospholipase C activity and the norpA protein are predominantly associated with membrane fractions in heads of both light- and dark-adapted flies. Moreover, phospholipase C activity as well as norpA protein can be easily extracted from membrane preparations of light- or dark-adapted flies using high salt, indicating that the norpA protein is peripherally localized on the membrane. These data suggest that the norpA encoded phospholipase C of Drosophila is a permanent peripheral membrane protein. If this is indeed the case, then it would mean that the reversible redistribution of phospholipase C from the cytosol to the membrane, as observed in epidermal growth factor receptor stimulation of mammalian phospholipase C gamma, is not a universal mechanism utilized by all types of phosphatidylinositol-specific phospholipase C.

The rpa (receptor potential absent) visual mutant of the blowfly (Calliphora erythrocephala) is deficient in phospholipase C in the eye.

The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

Tissue-specific expression of phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene of Drosophila melanogaster severely affect the light-evoked photoreceptor potential with strong mutations rendering the fly blind. Molecular cloning of the norpA gene revealed that it encodes phosphatidylinositol-specific phospholipase C, which enzymes play a pivotal role in one of the largest classes of signaling pathways known. We have used Northern analysis, Western blots, phospholipase C activity assays, and immunohistochemical staining of tissues to examine the tissue-specific expression of the norpA gene and found that it is expressed in a variety of tissues besides the eye. Hybridization of norpA cRNA probe to blots of poly(A+) RNA reveals that the gene encodes at least four transcripts: a 7.5-kilobase (kb) transcript that is expressed in eye and 6.5-, 5.5-, and 5.0-kb transcripts that are expressed in adult body or early stages of development. Antiserum generated against the major gene product of norpA recognizes a 130-kDa protein that is abundant in eyes but severely reduced or absent in norpA mutants, a result which is consistent with previous conclusions that the norpA gene encodes an essential component of the visual system. However, the norpA antiserum also recognizes a 130-kDa protein in adult legs, thorax, and male abdomen, but not female abdomen. These localizations are supported by results of phospholipase C activity assays which show that the norpA mutation reduces phospholipase C activity in each of the tissues in which norpA protein can be detected. Furthermore, immunohistochemical staining of tissue sections with the norpA antiserum demonstrates that the norpA protein is abundant in the retina and ocelli and is present to a lesser extent in the brain and thoracic nervous system. Since some of the above mentioned tissues that express norpA (such as thorax, legs, and abdomen) have no known photoreceptor tissue, we conclude that the norpA gene product is also likely to have a role in signaling pathways other than phototransduction.

Distinctive subtypes of bovine phospholipase C that have preferential expression in the retina and high homology to the norpA gene product of Drosophila.

The Drosophila norpA gene encodes a phospholipase C involved in phototransduction. However, phospholipase C apparently is not directly involved in phototransduction in vertebrate photoreceptors, although light-activated phospholipase C activity has been reported in vertebrate rod outer segments. Conserved regions of norpA cDNA were used to isolate bovine cDNAs that would encode four alternative forms of phospholipase C of the beta class that are highly homologous to the norpA protein and expressed preferentially in the retina. Two of the variants are highly unusual in that they lack much of the N-terminal region present in all other known phospholipases C. The sequence conservation between these proteins and the norpA protein is higher than that between any other known phospholipases C. GTPase sequence motifs found in proteins of the GTPase superfamily are found conserved in all four variants of the bovine retinal protein as well as the norpA protein but not in other phospholipases C. Results suggest that these proteins together with the norpA protein constitute a distinctive subfamily of phospholipases C that are closely related in structure, function, and tissue distribution. Mutations in the norpA gene, in addition to blocking phototransduction, cause light-dependent degeneration of photoreceptors. In view of the strong similarity in structure and tissue distribution, a defect in these proteins may have similar consequences in the mammalian retina.

A Drosophila phospholipase C gene that is expressed in the central nervous system.

A Drosophila phospholipase C (PLC) gene, designated as plc-21, was isolated by screening a genomic DNA library using a cDNA for a previously isolated Drosophila PLC gene, norpA, as probe under reduced stringency hybridization conditions. The gene maps to 21C on the left arm of the second chromosome. Two proteins of 1305 and 1312 amino acids, respectively, were deduced from two classes of cDNA which were isolated. The two putative plc-21 proteins are similar in sequence and overall structure to the beta-class of PLCs found in mammals and differ from each other only by 7 amino acid residues that are present near the C terminus of one of the proteins but not the other. Hybridization of plc-21 cDNA probes to blots of poly(A)+ RNA revealed that the gene encodes a 7.0-kilobase transcript that could be detected in the head but not in the body of adult flies and a 5.6-kilobase transcript that could be detected throughout development and in both heads and bodies of adults. In situ hybridization of cDNA sequences to tissue sections showed that the gene is expressed in the neuronal cell bodies of the optic lobe, central brain, and thoracic ganglia of adults and the brain of larvae. This tissue distribution of plc-21 transcripts is identical to the distribution of transcripts from a Drosophila Go alpha-subunit gene that we reported previously.

Molecular characterization of Drosophila gene encoding G0 alpha subunit homolog.

A Drosophila melanogaster gene (dgo) encoding a G protein alpha subunit has been isolated by screening genomic and adult head cDNA libraries using bovine transducin alpha subunit cDNA as probe. The gene, which maps to 47A on the second chromosome, encodes two proteins which are both 354 amino acids long but differ in seven amino acids in the amino-terminal region. The deduced amino acid sequences of the two proteins are 81% identical to that of a rat Go alpha subunit. Analysis of genomic clones revealed that there are eight coding exons and that the putative transcripts for the two proteins differ in the 5'-noncoding regions and the first coding exons but share the remaining six coding exons. The arrangement of two different 5'-noncoding regions on the gene suggests that two different promoters regulate the expression of the transcripts encoding the two proteins. RNA blot analysis detected three transcripts: a 3.9-kilobase (kb) transcript found at all stages of development; a 5.4-kb transcript present predominantly in adult heads; and a 3.4-kb transcript present only in adult bodies. In situ hybridizations of a cDNA probe to adult tissue sections showed that the gene is expressed abundantly in neuronal cell bodies in the brain, optic lobe, and thoracic ganglia.

A human tRNA gene heterocluster encoding threonine, proline and valine tRNAs.

A cluster of three tRNA genes encoding a tRNA(UGUThr), a tRNA(UGGPro), and a tRNA(AACVal), and two Alu-elements occur in a 6.0-kb human DNA fragment. The tRNA(Thr) gene is 2.7-kb upstream from the tRNA(Pro) gene, which is separated by 367 bp from the tRNA(Val) gene. One Alu-element actually overlaps the tRNA(Val) gene and is of opposite polarity to all three tRNA genes. All three tRNA genes are accurately transcribed in a homologous HeLa cell extract, since the ribonuclease T1 fingerprints of the tRNA transcripts are consistent with the nucleotide sequences of the tRNAs. The upstream region flanking the tRNA(Thr) gene has two tracts of alternating purine/pyrimidine residues potentially capable of adopting the Z-DNA conformation, and presumptive binding sites for two RNA polymerase II transcription factors. The tRNA(Thr) gene apparently has a substantially higher in vitro transcriptional efficiency than the other two tRNA genes in this cluster, and a tRNA(GCCGly) gene from another human DNA segment. Deletion constructs of the tRNA(Thr) gene retaining 272, 168, and 33 bp of original 5'-flanking DNA had about the same in vitro transcriptional efficiency, whereas that of the construct with only 2 bp of 5'-flanking human DNA was drastically reduced. The tRNA(Thr) gene constructs with 272 and 168 bp of original 5'-flanking DNA apparently reduce the transcriptional efficiencies of the proline and glycine tRNA genes, implicating the upstream region from the tRNA(Thr) gene as being crucial for its high transcriptional efficiency.

Drosophila ninaA gene encodes an eye-specific cyclophilin (cyclosporine A binding protein).

Mutations in the ninaA gene of Drosophila severely reduce the amount of rhodopsin specifically in R1-6 photoreceptors. Isolation of the ninaA gene by chromosomal walking revealed that it is expressed only in the eye and encodes a 237-amino acid polypeptide that shows strong sequence similarity to cyclophilin, a putative molecular target for cyclosporine A, a potent immunosuppressant used in human organ transplantations. Unlike most cyclophilins characterized to date, the ninaA-encoded protein has a putative signal sequence and a transmembrane domain. Each of the three ehtyl methanesulfonate-induced ninaA mutant alleles analyzed shows a single nucleotide change in the mRNA coding region leading to either a nonsense or a missense mutation. We find no evidence that the ninaA-encoded protein is directly involved in phototransduction. The only detectable mutant phenotype that correlates with the severity of molecular defects in the three mutants is the amount of depletion of R1-6 rhodopsin. The above results and the recent findings that cyclophilin is a peptidylprolyl cis-trans-isomerase suggest that the ninaA-encoded protein may be required for proper folding and stability of R1-6 rhodopsin.

Isolation of a putative phospholipase C gene of Drosophila, norpA, and its role in phototransduction.

Severe norpA mutations in Drosophila eliminate the photoreceptor potential and render the fly completely blind. Recent biochemical analyses have shown that norpA mutants lack phospholipase C (PLC) activity in the eye. A combination of chromosomal walking and transposon-mediated mutagenesis was used to clone the norpA gene. This gene encodes a 7.5 kb RNA that is expressed in the adult head. In situ hybridizations of norpA cDNA to adult tissue sections show that this gene is expressed abundantly in the retina. The putative norpA protein is composed of 1095 amino acid residues and has extensive sequence similarity to a PLC amino acid sequence from bovine brain. We suggest that the norpA gene encodes a PLC expressed in the eye of Drosophila and that PLC is an essential component of the Drosophila phototransduction pathway.

IBM microcomputer programs that analyze DNA sequences for tRNA genes.

A set of four computer programs that search DNA sequence data files for transfer RNA genes have been written in IBM (Microsoft) BASIC for the IBM personal computer. These programs locate and plot predicted secondary structures of tRNA genes in the cloverleaf conformation. The set of programs are applicable to eukaryotic tRNA genes, including those containing intervening sequences, and to prokaryotic and mitochondrial tRNA genes. In addition, two of the programs search up to 150 residues downstream of tRNA gene sequences for possible eukaryotic RNA polymerase III termination sites comprised of at least four consecutive T residues. Molecular biologists studying a variety of gene sequence and flanking regions can use these programs to search for the additional presence of tRNA genes. Furthermore, investigators studying tRNA gene structure-to-function relationships would not need to do extensive restriction mapping to locate tRNA gene sequences within their cloned DNA fragments.

Nucleotide sequence and transcription of a human glycine tRNAGCC gene and nearby pseudogene.

A bacteriophage lambda clone containing a 15.4-kb human DNA fragment was isolated and found to contain a glycine tRNA gene and, 758 bp away, a pseudogene, both with an anticodon of GCC. The nucleotide (nt) sequence of a 1362-bp segment of this clone, encompassing the gene, pseudogene, and their flanking regions, was determined. The gene and pseudogene have an identical sequence of eight nt (5'-CAGCTGGA-3') in their 5'-flanking regions immediately preceding the coding regions, as well as characteristic transcription termination sites of five consecutive T nt in the 3'-flanking regions. Neither of these genes has intervening sequences. Only one of the two genes was efficiently transcribed in vitro by RNA polymerase III in a HeLa cell-free system. During the course of transcription, primary transcripts of one gene were processed to yield mature-sized products. In contrast, the level of transcription of the second gene was significantly less than that of the first, and no mature-sized products could be detected. The nt sequence of the inefficiently transcribed gene has two base substitutions compared to the sequence of the efficiently transcribed gene, and the DNA sequence predicted from the human placental tRNAGlyGCC sequence. One of these nt substitutions is a C to T transition in the TTCG sequence within the B block of the characteristic internal split promoter sequence. The precursor-product relationships of the tRNA transcripts were established by comparing the RNase T1 and RNase A fingerprints of the precursors and products.