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  • 1
    Electronic Resource
    Electronic Resource
    Genomic Organization of Drosophila Choline Acetyltransferase (1991)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 57 (1991), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: : Choline acetyltransferase (ChAT; acetyl-CoA:cho-line-O-acetyltransferase; EC 2.3.1.6) is the enzyme responsible for the synthesis of the neurotransmitter acetylcholine and is thus the genetic determinant of neurons with a cholinergic phenotype. We have screened a Drosophila genomic library using a cRNA probe, transcribed from Drosophila ChAT cDNA, and isolated three independent clones representing all the exons of this gene. The gene spans more than 26 kb of DNA and is organized into eight exons containing 594, 80,192, 759, 408, 147, 201, and 1,612 nucleotides. All inserts that hybridized with a cRNA probe have been subcloned and the sequence of intron/exon boundaries determined. The only part of the ChAT gene not represented in our clones is a part of the first intron. A minimum size for this uncloned DNA has been deduced from Southern analysis of Drosophila genomic DNA. We also have probed the transcripts of the ChAT gene by northern analysis of total Drosophila RNA using two different exon-specific antisense RNA probes. An exon I probe detected two bands of RNA whereas an exon VHI probe hybridized with only the smaller band, previously identified as ChAT mRNA. These results indicate a complex transcription pattern for the ChAT locus in Drosophila.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1991.tb06362.x
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  • 2
    Electronic Resource
    Electronic Resource
    Production of Polyclonal Antisera to Choline Acetyltransferase Using a Fusion Protein Produced by a cDNA Clone Victor (1988)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 50 (1988), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a λ gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodal-ton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37°C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons. The previously reported molecular mass of 67 kilodaltons for native Drosophila ChAT as well as ChAT polypeptides in partially purified enzyme preparations may have resulted from proteolysis of the 75-kilodalton form of the enzyme. Recombinant DNA technology provides a good approach to raise high-titered, sequence-directed polyclonal antibodies to ChAT without the difficulties encountered in purifying the enzyme directly from Drosophila.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1988.tb13245.x
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  • 3
    Electronic Resource
    Electronic Resource
    Characterization of Nervana, a Drosophila melanogaster Neuron-Specific Glycoprotein Antigen Recognized by Anti-Horseradish Peroxidase Antibodies (1995)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 65 (1995), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: Antibodies to the plant glycoprotein horseradish peroxidase (HRP) are used extensively to identify neurons in Drosophila and other insects. We are interested in characterizing the gene product(s) recognized by anti-HRP antibodies because it may be important for nervous system function and/or development. Here we identify and purify from adult Drosophila heads an anti-HRP-reactive Mr 42K glycoprotein that is likely to be the major contributor to neuronal specific anti-HRP staining. Several different monoclonal antibodies to the purified 42K glycoprotein recognize up to three proteins with distinct mobilities between Mr 38K and 42K that vary as a function of developmental age. We have collectively named these components Nervana (nerve antigen), because the monoclonal antibodies also specifically stain cultured neurons and embryonic nervous system with a pattern indistinguishable from anti-HRP staining. Western blots indicate the presence of immunologically similar proteins in a wide variety of insect species and in nac (neurally altered carbohydrate) mutant Drosophila flies that lack anti-HRP staining in adult nervous system. It should now be possible to undertake a full biochemical and functional characterization of Nervana in Drosophila.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1995.65010434.x
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  • 4
    Electronic Resource
    Electronic Resource
    Functional Analysis and Tissue-Specific Expression of Drosophila Na+,K+-ATPase Subunits (1998)
    Oxford, UK : Blackwell Science Ltd
    Journal of neurochemistry 71 (1998), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract: We have previously purified and characterized a nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana [nerve antigen (NRV)] and identified two separate genes coding for three different proteins. All three proteins share homology with the β subunits of Na+,K+-ATPase from various other species. In this study we have isolated a new Drosophila Na+,K+-ATPase α subunit cDNA clone (PSα; GenBank accession no. AF044974) and demonstrate expression of functional Na+,K+-ATPase activity when PSα mRNA is coinjected into Xenopus oocytes along with any of the three different Nrv mRNAs. Western blotting, RNase protection assays, and immunocytochemical staining of adult fly sections indicate that NRV2 is expressed primarily in the nervous system. Staining is most intense in the brain and thoracic ganglia and is most likely associated with neuronal elements. NRV1 is more broadly expressed in muscle and excretory tissue and also shows diffuse distribution in the nervous system. Similar to other species, Drosophila expresses multiple isoforms of Na+,K+-ATPase subunits in a tissue- and cell type-specific pattern. It will now be possible to use the advantages of Drosophila molecular and classical genetics to investigate the phenotypic consequences of altering Na+,K+-ATPase expression in various cell and tissue types.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1046/j.1471-4159.1998.71010142.x
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  • 5
    Electronic Resource
    Electronic Resource
    [1251]2α-BUNGAROTOXIN AND [3H]QUINUCLIDINYLBENZILATE BINDING IN CENTRAL NERVOUS SYSTEMS OF DIFFERENT SPECIES (1979)
    Oxford, UK : Blackwell Publishing Ltd
    Journal of neurochemistry 32 (1979), S. 0 
    Details
    ISSN: 1471-4159
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Medicine
    Notes: Abstract— [125I]Diiodo α-bungarotoxin ([125I]2BuTx) and [3H]quinuclidinylbenzilate ([3H]QNB) binding sites were measured in post-nuclear membrane fractions prepared from whole brains or brain regions of several species. Species studied included Drosophila melanogaster (fruit fly), Torpedo californiea (electric ray), Carassius auratus (goldfish), Ram pipiens (grass frog), Kana cutesheiana (bullfrog), Rattus norvegicus (rat, Sprague-Dawley), Mus muscalus (mouse, Swiss random, C58/J, LG/J), Oryctolagus cuniculus (rabbit, New Zealand Whitc), and Bos (cow). Acetyl-CoA: choline O-acetyltransferase (EC 2.3.1.6) levels were also determined in the post nuclear supernatants and correlated with the number of binding sites.All species and regions except Drosophila had 16–150 fold more [3H]QNB binding sites than [125I]2BuTx binding sites. Brain regions with the highest levels of [125I]2BuTx binding were Drosophila heads (300 fmol/mg), goldfish optic tectum (80fmol/mg), and rat and mouse hippocampus (3040 fmol/mg). The highest levels of [3H]QNB binding were seen in rat and mouse caudate (1.3–1.6 pmol/mg). Lowest levels of [3H]QNB and [125I]2BuTx binding were seen in cerebellum. The utility of [125I]2BuTx and [3H]QNB binding as quantitative measures of nicotinic and muscarinic acetylcholine receptors in CNS is discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1111/j.1471-4159.1979.tb11092.x
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  • 6
    Electronic Resource
    Electronic Resource
    Developmental regulatory elements in the 5′ flanking DNA of the Drosophila choline acetyltransferase gene (1993)
    Springer
    Development genes and evolution 202 (1993), S. 159-169 
    Details
    ISSN: 1432-041X
    Keywords: Drosophila ; Choline acetyltransferase ; cis-Regulatory element ; lacZ reporter gene ; Colinergic neuron
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Choline acetyltransferase (ChAT, EC 2.3.1.6) catalyzes the production of the neurotransmitter acetylcholine, and is an essential factor for neurons to be cholinergic. We have analyzed regulation of the Drosophila ChAT gene during development by examining the β-galactosidase expression pattern in transformed lines carrying different lengths of 5′ flanking DNA fused to a lacZ reporter gene. The largest fragment tested, 7.4 kb, resulted in the most extensive expression pattern in embryonic and larval nervous system and likely reflects all the cis-regulatory elements necessary for ChAT expression. We also found that 5′ flanking DNA located between 3.3 kb and 1.2 kb is essential for the reporter gene expression in most of the segmentally arranged embryonic sensory neurons as well as other distinct cells in the CNS. The existence of negative regulatory elements was suggested by the observation that differentiating photoreceptor cells in eye imaginal discs showed the reporter gene expression in several 1.2 kb and 3.3 kb transformants but not in 7.4 kb transformants. Furthermore, we have fused the 5′ flanking DNA fragments to a wild type ChAT cDNA and used these constructs to transform Drosophila with a Cha mutant background. Surprisingly, even though different amounts of 5′ flanking DNA resulted in different spatial expression patterns, all of the positively expressing cDNA transformed lines were rescued from lethality. Our results suggest that developmental expression of the ChAT gene is regulated both positively and negatively by the combined action of several elements located in the 7.4 kb upstream region, and that the more distal 5′ flanking DNA is not necessary for embryonic survival and development to adult flies.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365306
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  • 7
    Electronic Resource
    Electronic Resource
    Choline acetyltransferase-like immunoreactivity in the brain of Drosophila melanogaster (1986)
    Springer
    Cell & tissue research 246 (1986), S. 57-62 
    Details
    ISSN: 1432-0878
    Keywords: Insect brain ; Neurotransmitters ; Immunocytochemistry ; Drosophila melanogaster
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: 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.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00218999
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  • 8
    Electronic Resource
    Electronic Resource
    Localization of choline acetyltransferase-expressing neurons in the larval visual system of Drosophila melanogaster (1995)
    Springer
    Cell & tissue research 282 (1995), S. 193-202 
    Details
    ISSN: 1432-0878
    Keywords: Key words: Choline acetyltransferase ; Cholinergic neuron ; Visual system ; Bolwig’s organ ; Immunocytochemistry ; In situ hybridization ; Drosophila melanogaster (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Choline acetyltransferase (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4  kb of 5′ flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig’s organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig’s nerve and a neuron close to the insertion site of the optic stalk. This neuron’s axon ran in parallel with Bolwig’s nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig’s organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004410050472
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  • 9
    Electronic Resource
    Electronic Resource
    Localization of choline acetyltransferase-expresing neurons in the larval vissual system of Drosophila melanogaster (1995)
    Springer
    Cell & tissue research 282 (1995), S. 193-202 
    Details
    ISSN: 1432-0878
    Keywords: Choline acetyltransferase ; Cholinergic neuron ; Visual system ; Bolwig's organ ; Immunocytochemistry ; In situ hybridization ; Drosophila melanogaster (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Choline acetyltransferease (ChAT) is the enzyme catalyzing the biosynthesis of acetylcholine and is considered to be a phenotypically specific marker for cholinergic neurons. We have examined the distribution of ChAT-expressing neurons in the larval nervous system of Drosophila melanogaster by three different but complementary techniques: in situ hybridization with a cRNA probe to ChAT messenger RNA, immunocytochemistry using a monoclonal anti-ChAT antibody, and X-gal staining of transformed animals carrying a reporter gene composed of 7.4 kb of 5′ flanking DNA from the ChAT gene fused to a lacZ reporter gene. All three techniques demonstrated ChAT-expressing neurons in the larval visual system. In embryos, the photoreceptor organ (Bolwig's organ) exhibited strong cRNA hybridization signals. The optic lobe of late third-instar larvae displayed ChAT immunoreactivity in Bolwig's nerve and a neuron close to the insertion site of the optic stalk. This neuron's axon ran in parallel with Bolwig's nerve to the larval optic neuropil. This neuron is likely to be a first-order interneuron of the larval visual system. Expression of the lacZ reporter gene was also detected in Bolwig's organ and the neuron stained by anti-ChAT antibody. Our observations indicate that acetylcholine may be a neurotransmitter in the larval photoreceptor cells as well as in a first-order interneuron in the larval visual system of Drosophila melanogaster.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00319111
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  • 10
    Electronic Resource
    Electronic Resource
    Developmental expression of choline acetyltransferase mRNA inDrosophila (1990)
    Springer
    Neurochemical research 15 (1990), S. 1089-1096 
    Details
    ISSN: 1573-6903
    Keywords: Choline acetyltransferase ; development ; mRNA ; Drosophila
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Notes: Abstract We have measured the steady state levels of choline acetyltransferase (ChAT, EC 2.3.1.6) mRNA during different developmental stages ofDrosophila melanogaster using a ChAT specific cRNA probe. ChAT mRNA was first detected approximately 6–7 h after oviposition, increased until the 1st–2nd larval instar, decreased into early pupal stages and increased again during late pupation, reaching a maximum in adults. Northern analysis showed a major RNA band with a Mr of 4.7 kilobases and Western analysis also showed a single major 75 kD protein band at all developmental stages. Our results support the hypothesis that a major point of regulation of ChAT expression may be at the transcriptional level.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01101709
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