Synthesis of catalytically active choline acetyltransferase in Xenopus oocytes injected with messenger RNA from rat central nervous system

Berrard, Sylvie; Biguet, Nicole Faucon; Gregoire, Danielle; Blanot, Francois; Smith, Julian C.; Mallet, Jacques

doi:10.1016/0304-3940(86)90625-7

Cited by 14 publications

(6 citation statements)

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“…To establish that pChAT-1 cDNA encodes an active ChoAcTase, sense RNA was first injected into frog oocytes, which have been shown to be a convenient system in which to express active rat ChoAcTase (24). Injection of pChAT-1 RNA yielded a high level of ChoAcTase activity that was inhibited by NVP, a specific ChoAcTase inhibitor (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…This was an important point since the codons corresponding to the N terminus of porcine ChoAcTase turned out to be mostly of low frequency (33). (iii) The XgtlO cDNA library was derived from the ventral spinal cord, a region shown (24) to be a suitable source of ChoAcTase mRNA in the central nervous system. Oocytes injected with mRNA from ventral spinal cord generated about 10 times more ChoAcTase activity than those injected with striatal mRNA, although the latter structure was found to contain the highest ChoAcTase activity in the central nervous system (24).…”

Section: Discussionmentioning

confidence: 99%

“…(iii) The XgtlO cDNA library was derived from the ventral spinal cord, a region shown (24) to be a suitable source of ChoAcTase mRNA in the central nervous system. Oocytes injected with mRNA from ventral spinal cord generated about 10 times more ChoAcTase activity than those injected with striatal mRNA, although the latter structure was found to contain the highest ChoAcTase activity in the central nervous system (24). (iv) The synthesis of the first-strand cDNA was performed with random primers to ensure that the 5' portions of the mRNAs would be included in the library.…”

Section: Discussionmentioning

confidence: 99%

“…Proteins were separated by isochratic (8%) NaDodSO4/polyacrylamide gel electrophoresis (23). Oocytes were treated and injected as described (24).…”

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confidence: 99%

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cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

Berrard

Brice

Lottspeich

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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(3, 4). The analysis, in molecular terms, of the mechanisms underlying this plasticity requires the study of the genes encoding these two enzymes.We have identified (5-7) cDNA clones corresponding to rat and human tyrosine hydroxylases. Here, we describe the isolation of a cDNA clone-pChAT-1-that encodes an active porcine ChoAcTase enzyme. The nucleotide and complete amino acid sequence is reported. ¶ Some structural characteristics of porcine ChoAcTase are discussed, and the sequence is compared with that of Drosophila melanogaster reported by Itoh et al. (8). MATERIALS AND METHODSConstruction of a Randomly Primed cDNA Library in the XgtlO Vector. Total RNA from porcine ventral spinal cord was extracted as described by Lomedico and Saunders (9). Poly(A)+ RNA was purified by oligo(dT)-cellulose chromatography. Random DNA sequences 20-50 nucleotides in length were prepared by sonication and DNase I digestion of calf thymus DNA (10) and used as primers for cDNA synthesis. First-strand cDNA was synthesized from 2.5 ,g of ventral spinal cord poly(A)+ RNA with 30-fold excess of random primer. The second-strand synthesis and following steps were carried out using standard procedures (11, 12). The longest cDNAs [_500 base pairs (bp)] were selected on a 5-20o (wt/vol) sucrose gradient and ligated to the XgtlO vector. The amplified library contained =1.2 x 106 independent recombinant phages.Oligonucleotide Screening. The N-terminal sequence of porcine brain ChoAcTase was determined as described (13). A mixture of oligodeoxynucleotides, each containing eight different chains of 29 nucleotides, was prepared with a Biosearch DNA synthesizer model 8600 by the phosphoramidite method and purified by PAGE. The probes were end-labeled to a minimal specific activity of 8 x 108 cpm/,ug. About 106 recombinant phages were plated at 50,000-70,000 plaques per 13-cm plate, and duplicate filters were prepared. Filters were hybridized at 35°C with oligonucleotides in 6x SSC (lx SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH 7.0), 5x Denhardt's solution (lx Denhardt's solution = 0.02% Ficoll/0.02% polyvinylpyrrolidone/0.02% bovine serum albumin), 10% (wt/vol) dextran sulfate, 0.05% sodium pyrophosphate, herring sperm DNA at 0.1 mg/ml, and Escherichia coli tRNA at 0.1 mg/ml. Filters were then washed at 35°C, 40°C, and 45°C in 6x SSC containing 0.05% NaDodSO4. Positive clones were isolated after three successive rounds of screening. Phage DNA was prepared as Abbreviations: ChoAcTase, choline acetyltransferase; NVP, 4-(1-naphthylvinyl)pyridine. §To whom reprint requests should be addressed.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

“…Proteins were separated by isochratic (8%) NaDodSO4/polyacrylamide gel electrophoresis (23). Oocytes were treated and injected as described (24).…”

mentioning

confidence: 99%

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cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

Berrard

Brice

Lottspeich

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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“…Heterologous expression of ChAT was first demonstrated by Gundersen et al (1 985), who were able to quantify a catalytically active form of the enzyme following injection of poly(A)+ mRNA, obtained from the electric lobes of Torpedo rnarrnorata and Torpedo ocellata, into Xenopus oocytes. Using this Xenopus oocyte system Berrard et al (1986) compared ChAT activity in different regions of the rat CNS and found that the ventral part of the spinal cord contains the highest level of ChAT mRNA, whereas the striatum, which exhibits maximal enzymatic activity, contained 10 times less ChAT mRNA. The Xenopus oocyte system has also been used to confirm cDNA clones for pig, rat, and Drosophila ChAT (Berrard et al, 1987;McCaman et al, 1988;Brice et al, 1989), whereas expression of the enzyme in African green monkey kidney (COS) cells and in Chinese hamster ovary cells has been used for the confirmation of ChAT cDNA clones from mouse, rat, and human spinal cords (Ishii et al, 1990;Oda et al, 1992).…”

Section: Heterologous Expression and Structure-function Studies Of Chatmentioning

confidence: 99%

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Hersh

1994

Journal of Neurochemistry

154

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It is well known that the regulation of choline acetyltransferase (ChAT) activity under physiological and pathological conditions is important for the development and neuronal activities of cholinergic systems involved in many fundamental brain functions. This review focuses on recent progress in understanding the regulation of ChAT at the levels of both the protein and the mRNA. A deficiency in ChAT activity has been reported for neurodegenerative conditions such as Alzheimer's disease, amyotrophic lateral sclerosis, and schizophrenia. Although a major feature of ChAT regulation is likely to involve the spatial and temporal control of transcription, regulation of expression can also be at the level of RNA processing, transport/ translocation, turnover, or translation. In addition, there is increasing evidence that ChAT might be regulated at the posttranslational level by compartmentation and/or covalent modification, i.e., phosphorylation, as well as noncovalent modification (protein‐protein interaction, etc.). Synaptic activity and the state of neuronal transmission may also involve the regulation of ChAT at different levels via both positive and negative feedback loops, as was demonstrated in the characterization of two ChAT mutant Drosophila strains. Clearly, identification of cholinergic‐specific elements and the characterization of the trans‐acting factors that bind to them represent an important area of future research. Equally important is research on the mechanisms governing ChAT as an enzymatic entity. The future should be an exciting time during which we look forward to the elucidation of the cholinergic signal and its regulation as well as the determination of the three‐dimensional structure of the enzyme.

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Complete sequence of a cDNA encoding an active rat choline acetyltransferase: A tool to investigate the plasticity of cholinergic phenotype expression

Brice¹,

Berrard²,

Raynaud³

et al. 1989

J of Neuroscience Research

186

102

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A cDNA clone encoding the complete sequence of an active rat choline acetyltransferase (ChoAcTase; acetyl-CoA:choline O-acetyltransferase, EC 2.3.1.6) has been isolated. Analysis of the deduced amino acid sequence reveals 85% and 31% identity with the porcine and Drosophila melanogaster enzymes, respectively. To further elucidate the molecular basis of neurotransmitter-related phenotypic plasticity, the expression of ChoAcTase mRNA was compared with that of tyrosine hydroxylase [TH; tyrosine 3-monooxygenase, L-tyrosine, tetrahydropteridine:oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2], in neurons from superior cervical ganglia grown in the following conditions: 1) normal medium, 2) high K+ medium, and 3) normal medium supplemented with 50% muscle-conditioned medium (CM). TH mRNA was expressed in all three media; its level rose in high K+ and decreased strikingly in the presence of CM. ChoAcTase mRNA could be visualized in CM, but fell to undetectable levels in normal and high K+ media. These results suggest that translational or post-translational mechanisms do not play a major role for the modulation of neurotransmitter-associated phenotype.

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Synthesis of catalytically active choline acetyltransferase in Xenopus oocytes injected with messenger RNA from rat central nervous system

Cited by 14 publications

References 14 publications

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

cDNA cloning and complete sequence of porcine choline acetyltransferase: in vitro translation of the corresponding RNA yields an active protein.

Choline Acetyltransferase: Celebrating Its Fiftieth Year

Complete sequence of a cDNA encoding an active rat choline acetyltransferase: A tool to investigate the plasticity of cholinergic phenotype expression

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