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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, S. and Brice, A. and Lottspeich, F. and Braun, A. and Barde, Y. A. and Mallet, J.. (1987) cDNA cloning and complete sequence of porcine choline acetyltransferase : in vitro translation of the corresponding RNA yields an active protein. Proceedings of the National Academy of Sciences of the United States of America, Vol. 84, H. 24. S. 9280-9284.

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Official URL: http://edoc.unibas.ch/dok/A5259570

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Abstract

A cDNA clone encoding the complete sequence of porcine choline acetyltransferase (ChoAcTase; acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6.) has been identified. A cDNA library, constructed from poly(A)+ RNA of ventral spinal cord, was screened with a mixture of eight oligonucleotides corresponding to the N-terminal sequence of pig brain ChoAcTase. Among five positive clones, one, pChAT-1, was identified as a ChoAcTase cDNA clone based on the following criteria. (i) This clone has an open reading frame coding for a protein of the size expected for ChoAcTase (640 amino acids). (ii) The amino acid composition deduced from the nucleotide sequence of this open reading frame matches that of purified porcine ChoAcTase. (iii) When subcloned in the T7 expression system, the corresponding RNA directs the synthesis in the rabbit reticulocyte lysate of a protein that is specifically immunoprecipitated by antibodies raised against ChoAcTase. (iv) Finally and most important, this corresponding RNA, when translated in the reticulocyte lysate, as well as in the Xenopus oocyte system, directs the synthesis of a protein displaying ChoAcTase activity. This activity is inhibited by the specific ChoAcTase inhibitor 4-(1-naphthylvinyl)pyridine. Comparison of porcine ChoAcTase sequence with that of Drosophila reveals 32% identity between these proteins, when the sequences are suitably aligned. pChAT-1 probe hybridizes with a porcine mRNA species that is at least 7000 nucleotides long, whereas the equivalent rat mRNA species is 3700 nucleotides long.
Faculties and Departments:05 Faculty of Science > Departement Biozentrum > Former Organization Units Biozentrum > Pharmacology/Neurobiology (Barde)
UniBasel Contributors:Barde, Yves-Alain
Item Type:Article, refereed
Bibsysno:Link to catalogue
Publisher:National Academy of Sciences
ISSN:0027-8424
Note:Publication type according to Uni Basel Research Database: Journal article
Last Modified:22 Mar 2012 14:21
Deposited On:22 Mar 2012 13:22

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