Distribution of two GABA receptor-like subunits in theDrosophila CNS

Previously we have described the distribution of theRdl GABA receptor subunit in theDrosophila CNS. Knowing thatRdl can coassemble with LCCH3 (aDrosophila GABA receptor-like subunit showing sequence similarity to vertebrate subunit GABA_A receptors) in baculovirus infected insect cells, we compared the localization of these two receptor subunits in order to identify any potential overlap in their spatial or temporal distribution. The two subunits show very different patterns of localization. Early in development LCCH3 is found in the majority of developing neuroblasts and later is localized to the cell bodies of the embryonic nerve cord and brain, and the neuronal cell bodies surrounding the adult brain. In contrast,Rdl receptor subunits appear confined to the neuropil in all developmental stages. These results have two important implications. Firstly, they suggest that although these two subunits can coassemble in heterologous expression systems, they may not be found in the same tissues in the nervous system. Secondly, production of LCCH3 before neuronal differentiation leads us to speculate on the role of that LCCH3 containing receptors in the developing nervous system.     

Characterization and comparative pharmacological studies of a functional γ-aminobutyric acid (GABA) receptor cloned from the tobacco budworm,Heliothis virescens (Noctuidae:Lepidoptera)

This paper reports the functional expression and pharmacological characterization of a full length complementary deoxyribonucleic acid (cDNA) (pIVY12) cloned from aHeliothis virescens fertilized egg cDNA library that encodes for a γ-aminobutyric acid (GABA) receptor subunit (HVRDL-Ser 285). Two electrode voltage clamp recordings ofXenopus oocytes expressing the HVRDL GABA-gated chloride channel revealed robust chloride ion conductance in response to GABA and the GABA_A receptor agonist, muscimol. Baclofen, a GABA_B agonist had no effect. Phenobarbital showed a positive dose-dependent allosteric modulatory effect, whereas the benzodiazepine, flunitrazepam, had no effect. Chloride conductance was depressed by the novel insecticide, fipronil ((±)-5-amino-1-(2,6 dichloro-α, α, α-trifluoro-p-tolyl)-4-trifluoromethyl-sulfinylpyrazole-3-carbonitrile) and the GABA_A antagonist, picrotoxinin. The HVRDL GABA receptor was insensitive to blockage by dieldrin and the GABA_A antagonist, bicuculline. The comparative actions of fipronil, picrotoxinin and dieldrin were examined on oocytes expressing theH. virescens wild-type (HVRDL-Ser 285), the site-directed mutant (HVRDL-Ala 285), theDrosophila melanogaster Rdl wild-type (DMRDL-Ala 302) and theRdl dieldrin resistant (DMRDL-Ser 302) homo-oligomeric GABA receptors. HVRDL-Ala 285 was 15-fold more sensitive to blockage by fipronil than HVRDL-Ser 285. DMRDL-Ala 302 and DMRDL-Ser-302 showed a similar level of sensitivity to blockage by fipronil. HVRDL-Ser 285 and DMRDL-Ser 302 exhibited a similar level of insensitivity to picrotoxinin. HVRDL-Ala 285 and DMRDL-Ala 302 showed a similar range of picrotoxinin sensitivity. DMRDL-Ala 302 and HVRDL-Ala 285 showed some sensitivity to blockage by dieldrin. Fipronil sensitivity was significantly altered by the serine to alanine mutation at position 285 in the M2 region of the HVRDL subunit, whereas no difference was observed between the DMRDL-Ser 302 and DMRDL-Ala 302 receptors.     

Duplication of the Rdl GABA receptor subunit gene in an insecticide-resistant aphid, Myzus persicae

Resistance to cyclodiene insecticides is associated with replacements of a single amino acid (alanine 302) in a γ-aminobutyric acid (GABA) receptor subunit encoded by the single-copy gene Resistance to dieldrin (Rdl). Alanine 302 is predicted to reside within the second membrane-spanning region of the Rdl receptor, a region that is thought to line the integral chloride ion channel pore. In all cyclodiene-resistant insects studied to date, this same alanine residue is replaced either by a serine, or, in some resistant strains of Drosophila simulans, a glycine residue. Therefore, individuals can carry only two different Rdl alleles. In contrast, here we report the presence of up to four different Rdl-like alleles in individual clones of the green peach aphid, Myzus persicae. In addition to the wild-type copy of Rdl gene (encoding A302 or allele A), M. persicae carries three other alleles with the following amino acid replacements: A302 → Glycine (allele G), A302 → Serine^TCG (allele S) and A302 → Serine^AGT (allele S′). Evidence from direct nucleotide sequencing and Single Stranded Conformational Polymorphism (SSCP) analysis shows that at least three of these different Rdl alleles (i.e. A, G and S) are commonly present in individual aphids or aphid clones. Southern analysis using allele-specific probes and analysis of sequences downstream of the exon containing the resistance-associated mutation confirm the presence of two independent Rdl-like loci in M. persicae. One locus carries the susceptible alanine (A) and/or resistant glycine (G) allele while the other carries the two serine alleles (S or S′). Whereas resistance levels are correlated with the glycine replacement, the S allele was present in all aphid clones, regardless of their resistance status. These results suggest that target site insensitivity is associated with replacements at the first (A/G) but not the second (S/S′) locus. Phylogenetic analysis of nucleotide sequences indicates that both putative aphid Rdl loci are monophyletic with respect to other insect Rdl genes and may have arisen through a recent gene duplication event. The implications of this duplication with respect to insecticide resistance and insect GABA receptor subunit diversity are discussed. Received: 10 March 1998 / Accepted: 21 July 1998     

Bicuculline-insensitive GABA-gated Cl<Superscript>−</Superscript> channels in the larval nervous system of the moth<Emphasis Type="Italic"> Manduca sexta</Emphasis>

GABA-gated Cl^– channels were studied in the nervous system of the larval tobacco hawk moth, Manduca sexta, using electrophysiology, ³⁶Cl^– uptake into membrane microsacs and immunocytochemistry. A GABA-induced increase in Cl^– conductance was recorded from a visually identifiable neurone (fg1) in the desheathed frontal ganglion. The response was insensitive to the vertebrate GABA_A receptor antagonist, bicuculline, but was blocked by picrotoxinin. Bicuculline-insensitive, picrotoxinin-sensitive, GABA-stimulated ³⁶Cl^– uptake was also detected in membrane microsacs prepared from the isolated larval M. sexta nervous system. Such receptors appear to be the major type of GABA receptor in larval nervous system membrane microsac preparations. An antibody raised against a 17 amino acid peptide, based on the predicted C-terminus of the Drosophila GABA receptor subunit (RDL), stained not only cell bodies, including that of fg1, but also the neuropile in the frontal ganglion, indicating the existence of RDL-like GABA receptor subunits in neurones of this ganglion. Thus, bicuculline-insensitive GABA-gated Cl^– channels are present in the larval nervous system of M. sexta.     

Drosophila “enhancer-trap” transposants: Gene expression in chemosensory and motor pathways and identification of mutants affected in smell and taste ability

We have isolated about a thousandDrosophila P-element transposants that allow thein situ detection of genomic enhancer elements by a histochemical assay for β-galactosidase activity. We summarize the β-galactosidase staining patterns of over 200 such transposants in the adult. Our aim was to identify genes that are likely to be involved in the chemosensory and motor pathways ofDrosophila. Based on β-galactosidase expression patterns in the tissues of our interest, we have chosen some strains for further analysis. Behavioral tests on a subset of the transposants have, in addition, identified several strains defective in their chemosensory responses.     

Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster

Summary Using a monoclonal antibody selective for the acetylcholine (ACh)-synthesizing enzyme choline acetyltransferase (ChAT) of Drosophila melanogaster we find ChAT-like immunoreactivity in specific synaptic regions throughout the brain of Drosophila melanogaster apart from the lobes and the peduncle of the mushroom body and most of the first visual neuropile (lamina). Several anatomically well-defined central brain structures exhibit particularly strong binding. Characteristic differential staining patterns are observed for each of the four neuromeres of the optic lobes. Cell bodies appear not to bind this antibody. The prominent features of the distribution of ChAT-like immunoreactivity are paralleled by the distribution of acetylcholine hydrolyzing enzymatic activity as revealed by histochemical staining for acetylcholine esterase (AChE). These results are discussed in comparison with published data on enzyme distribution, choline uptake and ACh receptor binding in the nervous system of Drosophila melanogaster.     

Mycoplasma detection in cell culture by concomitant use of bisbenzamide and fluoresceinated antibody

Summary Conditions are presented for application of both bisbenzamide (Hoechst 33258) stain and a specific fluoresceinated anti-Mycoplasma hyorhinis IgG to a single cell culture preparation. This allows the same field on a slide to be viewed for presumptive diagnosis of any cell culture contaminant mycoplasma by bisbenzamide staining and for definitive diagnosis ofM. hyorhinis strains using fluoresceinated antibody. The use of this method plus a cultural procedure will permit identification of the “noncultivable”M. hyorhinis strain DBS 1050.     

Receptor modifiers indicate that 4-aminobutyric acid (GABA) is a potential modulator of ion transport in plants

GABA (4-aminobutyric acid) is a ubiquitousnon-protein amino acid that accumulates rapidly inplants in response to stress. GABA was firstidentified in plants (potato tubers) and animals(brain tissue) 50 years ago. Although GABA is nowrecognized as the most important inhibitoryneurotransmitter in the mammalian central nervoussystem (CNS), the role of GABA in plants remainsunclear. Studies were performed using Lemna toinvestigate the possibility that GABA elicits aresponse in plants that may be related to that of asignaling molecule as described for GABA effects onthe CNS. Lemna growth was increased 2 to 3-foldby 5 mM GABA, but growth was strongly inhibited by 0.5mM of the isomers 3-aminobutyric acid and2-aminobutyric acid. Growth promotion by GABA wasrapidly terminated by addition of 2-aminobutyric acidto the culture medium, but inhibitory effects of2-aminobutyric acid were not reversed by GABAregardless of amounts added. Promotion of Lemnagrowth by GABA was associated with an increase inmineral content of treated plants in a dose dependentmanner. Results support the hypothesis that GABAactivity in plants involves an effect on ion transportand an interaction with a receptor. Evidence for GABAreceptors in Lemna was obtained from experimentswith pharmacological agents that have been used toidentify GABA receptors in animals. GABA mediatedpromotion of Lemna growth was inhibited bybicuculline and picrotoxin, which are respectivelycompetitive and non-competitive antagonists of GABAreceptors in the CNS. Growth inhibition bybicuculline was not relieved by increasing the amountsof GABA in the medium, indicating that the alkaloid isnot acting, as in the CNS, by competitive antagonismof GABA at GABA receptor sites. Baclofen, a GABAagonist that promotes GABA activity in animalssignificantly increased GABA mediated promotion ofLemna growth. These findings and the knownaction of GABA in regulating ion channels in animalssuggests a way that GABA could amplify the stressresponse in plants.     

The Drosophila cinnamon gene is functionally homologous to Arabidopsis cnx1 and has a similar expression pattern to the mammalian gephyrin gene.

Molybdoenzymes are involved in a variety of essential pathways including nitrate assimilation, sulfur and/or purine metabolism and abscisic acid biosynthesis. Most organisms produce several such enzymes requiring a molybdopterin cofactor for catalytic function. Mutations that result in a lack of the molybdopterin cofactor display a pleiotropic loss of molybdoenzyme activities, and this phenotype has been used to identify genes involved in cofactor biosynthesis or utilization. Although several cofactor genes have been analyzed in prokaryotes, much less is known concerning eukaryotic molybdenum cofactor (MoCF) genes. This work is focused on the Drosophila MoCF gene cinnamon (cin) which encodes a multidomain protein, CIN, that shows significant similarity to three proteins encoded by separate prokaryotic MoCF genes. These domains are also present in the product of cnx1, an Arabidopsis MoCF gene, and in GEPHYRIN, a rat protein thought to organize the glycine receptor, GlyR, within the postsynaptic membrane. Since this apparent consolidation of separate prokaryotic genes into a single eukaryotic gene is a feature of other conserved metabolic pathways, we wished to determine whether the protein's function is also conserved. This report shows that the plant gene cnx1 can rescue both enzymatic and physiological defects of Drosophila carrying cin mutations, indicating that the two genes serve similar or identical functions. In addition, we have investigated the relationship between CINNAMON and GEPHYRIN, using immunohistochemical methods to localize the CIN protein in Drosophila embryos. Most of the CIN protein, like GEPHYRIN in the rat CNS, is localized to the cell borders and shows a tissue-specific pattern of expression. In a parallel study, antibody to GEPHYRIN revealed the same tissue-specific expression pattern in fly embryos. Both antibodies show altered staining patterns in cin mutants. Taken together, these results suggest that GEPHYRIN may also carry out a MoCF-related function. Received: 14 September 1998 / Accepted: 8 March 1999     

Evidence for a Diverse Cys-Loop Ligand-Gated Ion Channel Superfamily in Early Bilateria

The genome sequences of Caenorhabditis elegans and Drosophila melanogaster reveal a diversity of cysteine-loop ligand-gated ion channels (Cys-loop LGICs) not found in vertebrates. To better understand the evolution of this gene superfamily, I compared all Cys-loop LGICs from rat, the primitive chordate Ciona intestinalis, Drosophila, and C. elegans. There are two clades of GABA receptor subunits that include both verterbate and invertebrate orthologues. In addition, I identified nine clades of anion channel subunits found only in invertebrates, including three that are specific to C. elegans and two found only in Drosophila. One well-defined clade of vertebrate cation channel subunits, the α7 nicotinic acetylcholine receptor subunits (nAChR), includes invertebrate orthologues. There are two clades of invertebrate nAChRs, one of α-type subunits and one of non-α subunits, that are most similar to the two clades of vertebrate neuronal and muscle α and non-α subunits. There is a large group of divergent C. elegans nAChR-like subunits partially resolved into clades but no orthologues of 5HT3-type serotonin receptors in the invertebrates. The topology of the trees suggests that most of the invertebrate-specific Cys-loop LGIC clades were present in the common ancestor of chordates and ecdysozoa. Many of these disappeared from the chordates. Subsequently, selected subunit genes expanded to form large subfamilies. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Rafael Zardoya]     

Development of the Deformed protein pattern in the embryo of the honeybee Apis mellifera L. (Hymenoptera)

Summary We have raised antiserum against part of the Deformed (Dfd) protein of the honeybee and describe here the expression pattern of the Dfd protein during honeybee embryogenesis. Dfd protein is first stained in the prospective gnathal region of the cellular blastoderm. This circumferential band corresponds to the distribution of Dfd mRNA described earlier, and to the blastodermal Dfd expression pattern in Drosophila. Using an antibody against the engrailed (en) protein of Drosophila, we found that at the beginning of gastrulation Dfd expression in the honeybee, as in Drosophila, is restricted to the future intercalary, mandibular and maxillary segments. During gastrulation, the mesodermal nuclei loose the Dfd label gradually from anterior to posterior, and in the ectoderm the most posterior ventral cells loose Dfd while retaining en staining; thus, in contrast to what has been described for Drosophila, the posterior Dfd expression border seems to move forward ventrally to the parasegmental boundary within the maxillary segment. In the late germ band, the lateral tips of the Dfd-expressing band are connected across the dorsal side by a row of amnion cells with strongly staining large nuclei. After dorsal closure, a narrow stripe of Dfd-staining dorsal cells behind the neck region may indicate that the maxillary segment contributes to the dorsal body wall posterior to the head capsule. Thus, apart from some minor deviations, the Dfd expression pattern in the honeybee strongly resembles that in Drosophila prior to head involution. This is compatible with the assumption that head involution (which is a special adaption in higher dipterans) ensues after a rather conserved course of early head development in which Dfd appears to play a basic role. Offprint requests to: R. Fleig     

Screening of a specific monoclonal antibody against and detection ofListeria monocytogenes whole cells using a surface plasmon resonance biosensor

In this study, a specific monoclonal antibody againstListeria monocytogenes was screened using an SPR biosensor Monoclonal antibodies were bound to protein L, after which theL. monocytogenes cells were subjected to an affinity assay. Protein L was immobilized on a carboxymethyl dextran (CM-Dex) surface via an amine coupling method and utilized repeatedly by regeneration. The monoclonal antibody, ‘A18’, was selected and employed for the high-sensitivity detection ofL. monocytogenes. Under optimized conditions, 10³ cells/ml or 50 cells were detected by the SPR biosensor.     

ADH and phylogenetic relationships ofDrosophila lebanonensis (Scaptodrosophila)

Summary Increasing data onDrosophila alcohol dehydrogenase (ADH) sequences have made it possible to calculate the rate of amino acid replacement per year, which is 1.7×10^–9. This value makes this protein suitable for reconstructing phylogenetic relationships within the genus for those species for which no molecular data are available such asScaptodrosophila. The amino acid sequence ofDrosophila lebanonensis is compared to all of the already knownDrosophila ADHs, stressing the unique characteristic features of this protein such as the conservation of an initiating methionine at the N-terminus, the unique replacement of a glycine by an alanine at a very conserved position in the NAD domain of all dehydrogenases, the lack of a slowmigrating peptide, and the total conservation of the maximally hydrophilic peptide. The functional significance of these features is discussed.Although the percent amino acid identity of the ADH molecule inDrosophila decreases as the number of sequences compared increases, the conservation of residue type in terms of size and hydrophobocity for the ADH molecule is shown to be very high throughout the genusDrosophila. The distance matrix and parsimony methods used to establish the phylogenetic relationships ofD. lebanonensis show that the three subgenera,Scaptodrosophila, Drosophila, andSophophora separated at approximately the same time.     

BrdU incorporation reveals DNA replication in non dividing glial cells in the larval abdominal CNS ofDrosophila

Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) ofDrosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two otherDrosophila species,D. virilis andD. hydei.     

Molecular cloning,genomic structure,and genetic mapping of two Rdl‐orthologous genes of GABA receptors in the diamondback moth,Plutella xylostella

The Resistance to dieldrin (Rdl) gene encodes a subunit of the insect γ‐aminobutyric acid (GABA) receptor. Cyclodiene resistance in many insects is associated with replacement of a single amino acid (alanine at position 302) with either a serine or a glycine in the Rdl gene. Two Rdl‐orthologous genes of GABA receptors (PxGABARα1 and PxGABARα2) were cloned and sequenced from a susceptible strain (Roth) of Plutella xylostella. PxGABARα1 and PxGABARα2 showed 84% and 77% identity with the Rdl gene of Drosophila melanogaster at an amino acid level, respectively. The coding regions of PxGABARα1 and PxGABARα2 both comprise ten exons, with two alternative RNA‐splicing forms in exon 3 of both genes. At the orthologous position of alanine‐302 in D. melanogaster Rdl, PxGABARα1 has a conserved alanine at position 282. PxGABARα2 has a serine instead of an alanine at the equivalent position. With two informative DNA markers, both PxGABARα1 and PxGABARα2 were mapped onto the Z chromosome of P. xylostella. © 2010 Wiley Periodicals, Inc.     

A newScaevola (Goodeniaceae) from Kauai Hawaiian plant studies 92

Based on morphology, there is describedScaevola wahiawaensis, native to the mountains of Kauai Island, Hawaiian Islands. It has the blades tomentose below with erect, subequal hairs; corolla lateral lobes 12–14 mm long, 6–7 mm wide, and the thickened body 2.5–3 mm wide. Its closest relative,S. mollis, of Oahu, has the blades closely tomentose below and ascending pilose; corolla lateral lobes 7–11 mm long, 3–3.5 (−5) mm wide, and the thickened body 1.5 mm wide.     

Evolution of nested genes with special reference to cuticle proteins inDrosophila melanogaster

Summary One of the pupal cuticle protein (PCP) genes has been found within an intron of aDrosophila housekeeping gene (theGart locus) that encodes three enzymes involved in the purine pathway. This intronic gene has been described as a gene within a gene, and the gene is now called a “nested” gene. Because the intronic PCP gene has sequence similarity with the larval cuticle protein (LCP) gene, it may have been derived from one of the LCP genes or their ancestral gene. We have studied possible phylogenetic relationships among these five genes by comparing nucleotide sequences of four LCP genes with that of the PCP gene. The results obtained suggest that the PCP gene may have originated from an ancestral gene before duplication of the LCP genes occurred. Using the number of synonymous (silent) substitutions, we then estimated the divergence time between the PCP gene and the LCP genes to be about 70 million years (Myr). The divergence time estimated is much larger than that for the sibling species ofD. melanogaster (about 2.5 Myr), indicating that the “nested” gene structure can be seen not only inDrosophila melanogaster, but also in other distantly relatedDrosophila species.