Identification of two novel Drosophila melanogaster histamine-gated chloride channel subunits expressed in the eye.

Histamine has been shown to play a role in arthropod vision; it is the major neurotransmitter of arthropod photoreceptors. Histamine-gated chloride channels have been identified in insect optic lobes. We report the first isolation of cDNA clones encoding histamine-gated chloride channel subunits from the fruit fly Drosophila melanogaster. The encoded proteins, HisCl1 and HisCl2, share 60% amino acid identity with each other. The closest structural homologue is the human glycine alpha3 receptor, which shares 45 and 43% amino acid identity respectively. Northern hybridization analysis suggested that hisCl1 and hisCl2 mRNAs are predominantly expressed in the insect eye. Oocytes injected with in vitro transcribed RNA, encoding either HisCl1 or HisCl2, produced substantial chloride currents in response to histamine but not in response to GABA, glycine, and glutamate. The histamine sensitivity was similar to that observed in insect laminar neurons. Histamine-activated currents were not blocked by picrotoxinin, fipronil, strychnine, or the H2 antagonist cimetidine. Co-injection of both hisCl1 and hisCl2 RNAs resulted in expression of a histamine-gated chloride channel with increased sensitivity to histamine, demonstrating coassembly of the subunits. The insecticide ivermectin reversibly activated homomeric HisCl1 channels and, more potently, HisCl1 and HisCl2 heteromeric channels.     

Properties of photoreceptor-specific phospholipase C encoded by the norpA gene of Drosophila melanogaster.

Mutations in the norpA gene drastically affect the phototransduction process in Drosophila. To study the biochemical characteristics of the norpA protein and its cellular and subcellular distributions, we have generated antisera against the major gene product of norpA. The antisera recognize an eye-specific protein of 130-kDa relative molecular mass that is present in wild-type head extracts but not in those of strong norpA mutants. The protein is associated with membranes and can be extracted with high salt. Immunohistochemical analysis at the light and electron microscopic levels indicates that the protein is expressed in all adult photoreceptor cells and specifically localized within the rhabdomeres, preferentially adjacent to, but not within, the rhabdomeric membranes. The results of the present study strongly support the previous suggestion that the norpA gene encodes the major phosphoinositol-specific phospholipase C in the photoreceptors. Moreover, insofar as the rhabdomeres are specialized structures for photoreception and phototransduction, specific localization of the norpA protein within these structures, in close association with the membranes, is consistent with the proposal that it has an important role in phototransduction.     

Two nitridergic peptides are encoded by the gene capability in Drosophila melanogaster

A Drosophila gene (capability, capa) at 99D on chromosome 3R potentially encodes three neuropeptides: GANMGLYAFPRV-amide (capa-1), ASGLVAFPRV-amide (capa-2), and TGPSASSGLWGPRL-amide (capa-3). Capa-1 and capa-2 are related to the lepidopteran hormone cardioacceleratory peptide 2b, while capa-3 is a novel member of the pheromone biosynthesis-activating neuropeptide/diapause hormone/pyrokinin family. By immunocytochemistry, we identified four pairs of neuroendocrine cells likely to release the capa peptides into the hemolymph: one pair in the subesophageal ganglion and the other three in the abdominal neuromeres. In the Malpighian (renal) tubule, capa-1 and capa-2 increase fluid secretion rates, stimulate nitric oxide production, and elevate intracellular Ca(2+) and cGMP in principal cells. Capa-stimulated fluid secretion, but not intracellular Ca(2+) concentration rise, is inhibited by the guanylate cyclase inhibitor methylene blue. The actions of capa-1 and capa-2 are not synergistic, implying that both act on the same pathways in tubules. The capa gene is thus the first to be shown to encode neuropeptides that act on renal fluid production through nitric oxide.     

Identification of two proteins encoded by the Saccharomyces cerevisiae GAL4 gene.

We placed the Saccharomyces cerevisiae GAL4 gene under control of the galactose regulatory system by fusing it to the S. cerevisiae GAL1 promoter. After induction with galactose, GAL4 is now transcribed at about 1,000-fold higher levels than in wild-type S. cerevisiae. This regulated high-level expression has enabled us to tentatively identify two GAL4-encoded proteins.     

DNA polymerase-primase from embryos of Drosophila melanogaster. DNA primase subunits

The primase associated with the DNA polymerase-primase of Drosophila melanogaster fails to show enzymatic turnover. However, it does show turnover when dissociated from the intact polymerase-primase. Both forms of the enzyme can catalyze the synthesis of primers that are not complementary to the DNA template. Like the intact enzyme, the isolated primase synthesizes primers of a unique chain length; however, they are twice as long as those synthesized by the polymerase-primase. The activity of the primase separated from the polymerase-primase is similar in all other respects to the intact polymerase-primase.     

Molecular cloning and characterization of the genes encoding the two subunits of Drosophila melanogaster calcineurin.

Genomic clones containing the full coding sequences of the two subunits of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin, were isolated from a Drosophila melanogaster genomic library using highly conserved human cDNA probes. Three clones encoded a 19.3-kDa protein whose sequence is 88% identical to that of human calcineurin B, the Ca(2+)-binding regulatory subunit of calcineurin. The coding sequences of the Drosophila and human calcineurin B genes are 69% identical. Drosophila calcineurin B is the product of a single intron-less gene located at position 4F on the X chromosome. Drosophila genomic clones encoding a highly conserved region of calcineurin A, the catalytic subunit of calcineurin, were used to locate the calcineurin A gene at position 21 EF on the second chromosome of Drosophila and to isolate calcineurin A cDNA clones from a Drosophila embryonic cDNA library. The structure of the calcineurin A gene was determined by comparison of the genomic and cDNA sequences. Twelve exons, spread over a total of 6.6 kilobases, were found to encode a 64.6-kDa protein 73% identical to either human calcineurin A alpha or beta. At the nucleotide level Drosophila calcineurin A cDNA is 67 and 65% identical to human calcineurin A alpha and beta cDNAs, respectively. Major differences between human and Drosophila calcineurins A are restricted to the amino and carboxyl termini, including two stretches of repetitive sequences in the carboxyl-terminal third of the Drosophila molecule. Motifs characteristic of the putative catalytic centers of protein phosphatase-1 and -2A and calcineurin are almost perfectly conserved. The calmodulin-binding and auto-inhibitory domains, characteristic of all mammalian calcineurins A, are also conserved. A remarkable feature of the calcineurin A gene is the location of the intron/exon junctions at the boundaries of the functional domains and the apparent conservation of the intron/exon junctions from Drosophila to man.     

The multifunctional Drosophila melanogaster V-ATPase is encoded by a multigene family.

In animals, V-ATPases are believed to play roles in the plasma membrane, as well as endomembrane. To understand these different functions, it is necessary to adopt a genetic approach in a physiologically tractable model organism. For this purpose, Drosophila melanogaster is ideal, because of the powerful genetics associated with the organism and because of the unusually informative epithelial phenotype provided by the Malpighian tubule. Recently, the first animal "knockouts" of a V-ATPase were described in Drosophila. The resulting phenotypes have general utility for our understanding of V-ATPase function and suggest a screen for novel subunits and associated proteins. Genome project resources have accelerated our knowledge of the V-ATPase gene family size and the new Drosophila genes vhaSFD, vha100-1, vha100-2, vha100-3, vha16-2, vha16-3, vha16-4, vhaPPA1, vhaPPA2, vhaM9.7.1, and vhaM9.7.2 are described. The Drosophila V-ATPase model is thus well-suited to both forward and reverse genetic analysis of this complex multifunctional enzyme.     

Nicotinic acetylcholine receptors of Drosophila: three subunits encoded by genomically linked genes can co-assemble into the same receptor complex

The second beta-like subunit (SBD) is a putative structural subunit of Drosophila melanogaster nicotinic acetylcholine receptors (nAChRs). Here we have produced specific antibodies against SBD to study, which other nAChR subunits can co-assemble with SBD in receptor complexes of the Drosophila nervous system. Immunohistochemical studies in the adult optic lobe revealed that SBD has a distribution similar to that of the alpha-subunit ALS in the synaptic neuropil. The subunits ALS, D(alpha)2 and SBD can be co-purified by alpha-bungarotoxin affinity chromatography. Moreover, anti-SBD antibodies co-precipitate ALS and D(alpha)2 and, vice versa, ALS and D(alpha)2 antibodies co-immunoprecipitate SBD protein. Two-step immunoaffinity chromatography with immobilized antibodies against ALS and D(alpha)2 revealed the existence of nAChR complexes that include ALS, D(alpha)2 and SBD as integral components. Interestingly, the genes encoding these three subunits appear to be directly linked in the Drosophila genome at region 96 A of the third chromosome. In addition, SBD appears to be a component of a different receptor complex, which includes the ARD protein as an additional beta-subunit, but neither ALS nor D(alpha)2 nor the third alpha-subunit D(alpha)3. These findings suggest a considerable complexity of the Drosophila nicotinic receptor system.     

Differential gene silencing by trans-heterochromatin in Drosophila melanogaster.

The brown(Dominant) (bw(D)) allele contains a large insertion of heterochromatin leading to the trans-inactivation of the wild-type allele in bw(D)/bw(+) heterozygous flies. This silencing is correlated with the localization of bw(+) to a region of the interphase nucleus containing centric heterochromatin. We have used a series of transgene constructs inserted in the vicinity of the bw locus to demarcate both the extent of bw(D) influence along the chromosome and the relative sensitivities of various genes. Examples of regulatory regions that are highly sensitive, moderately sensitive, and insensitive were found. Additionally, by using the same transgene at increasing distances from the bw(D) insertion site in trans we were able to determine the range of influence of the heterochromatic neighborhood in terms of chromosomal distance. When the transgene was farther away from bw, there was, indeed, a tendency for it to be less trans-inactivated. However, insertion site also influenced silencing: a gene 86 kb away was trans-inactivated, while the same transgene 45 kb away was not. Thus location, distance, and gene-specific differences all influence susceptibility to trans-silencing near a heterochromatic neighborhood. These results have important implications for the ability of nuclear positioning to influence the expression of large blocks of a chromosome.     

Neuronal nicotinic acetylcholine receptors from Drosophila: two different types of alpha subunits coassemble within the same receptor complex

Although neuronal nicotinic acetylcholine receptors from insects have been reconstituted in vitro more than a decade ago, our knowledge about the subunit composition of native receptors as well as their functional properties still remains limited. Immunohistochemical evidence has suggested that two alpha subunits, alpha-like subunit (ALS) and Drosophila alpha2 subunit (Dalpha2), are colocalized in the synaptic neuropil of the Drosophila CNS and therefore may be subunits of the same receptor complex. To gain further understanding of the composition of these nicotinic receptors, we have examined the possibility that a receptor may imbed more than one alpha subunit using immunoprecipitations and electrophysiological investigations. Immunoprecipitation experiments of fly head extracts revealed that ALS-specific antibodies coprecipitate Dalpha2, and vice versa, and thereby suggest that these two alpha subunits must be contained within the same receptor complex, a result that is supported by investigations of reconstituted receptors in Xenopus oocytes. Discrimination between binary (ALS/beta2 or Dalpha2/beta2) and ternary (ALS/Dalpha2/beta2) receptor complexes was made on the basis of their dose-response curve to acetylcholine as well as their sensitivity to alpha-bungarotoxin or dihydro-beta-erythroidine. These data demonstrate that the presence of the two alpha subunits within a single receptor complex confers new receptor properties that cannot be predicted from knowledge of the binary receptor's properties.