Lübbert, Hermann and Hoffman, Beth J. and Snutch, Terry P. and van Dyke, Terry and Levine, Arnold J. and Hartig, Paul R. and Lester, Henry A. and Davidson, Norman (1987) cDNA Cloning of a Serotonin 5-HT1C Receptor by Electrophysiological Assays of mRNA-Injected Xenopus Oocytes. Proceedings of the National Academy of Sciences of the United States of America, 84 (12). pp. 4332-4336. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:LUBpnas87
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We describe a strategy for the cloning of neurotransmitter-receptor and ion-channel cDNAs that is based on electrophysiological assays of mRNA-injected Xenopus oocytes. This procedure circumvents the purification of these membrane proteins, which is hindered by their low abundance and their hydrophobic nature. It involves methods for RNA fractionation by high-resolution gel electrophoresis, directional cDNA cloning in a single-stranded vector, and screening of the cDNA library by voltage-clamp measurements of currents induced by serotonin in mRNA-injected oocytes. The applicability of our approach is demonstrated by the isolation of a serotonin receptor cDNA clone from a mouse choroid plexus papilloma. The clone was identified by hybrid-depletion and hybrid-selection procedures. The receptor expressed in oocytes injected with hybrid-selected RNA is fully functional, indicating that it is composed of a single subunit encoded by a 5-kilobase RNA. The pharmacology of the hybrid-selected receptor confirms that we have successfully cloned a serotonin 5-HT1C receptor cDNA.
|Additional Information:||Copyright © 1987 by the National Academy of Sciences Contributed by Norman Davidson, March 16, 1987 We thank Hieu Nguyen for technical assistance, J. Vieira and J. Messing for supplying pUC119 and M13K07, and J. Kowalski and D.T. Denhardt for donating the bacterial strain E. coli R4. This research has been supported by National Institutes of Health grants GM-10991, NS-11756, NS-23048, and CA-38757 and by fellowship support from the Deutsche Forschungsgemeinschaft and the American Cancer Society, California Division to H.L.; by the National Science Foundation to B.J.H.; by the American Heart Association, Greater Los Angeles Affiliate, and the Natural Sciences and Engineering Research Council of Canada to T.P.S. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.|
|Subject Keywords:||RNA fractionation; hybrid depletion; hybrid selection; choroid plexus; voltage clamp|
|Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||Archive Administrator|
|Deposited On:||20 Dec 2006|
|Last Modified:||14 Nov 2014 19:19|
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