cDNA Cloning of a Serotonin 5-HT1C Receptor by Electrophysiological Assays of mRNA-Injected Xenopus Oocytes
Abstract
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
© 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.Attached Files
Published - LUBpnas87.pdf
Files
| Name | Size | Download all |
|---|---|---|
|
md5:b209f50b063c54127467effbfdae9be8
|
1.1 MB | Preview Download |
Additional details
- PMCID
- PMC305079
- Eprint ID
- 6751
- Resolver ID
- CaltechAUTHORS:LUBpnas87
- NIH
- GM-10991
- NIH
- NS-11756
- NIH
- NS-23048
- NIH
- CA-38757
- Deutsche Forschungsgemeinschaft (DFG)
- American Cancer Society, California Division
- American Heart Association, Greater Los Angeles Affiliate
- Natural Sciences and Engineering Research Council of Canada (NSERC)
- Created
-
2006-12-20Created from EPrint's datestamp field
- Updated
-
2019-10-02Created from EPrint's last_modified field