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PMID: 3473504 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

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 ·Vol. 84 ·No. 12 ·1987-06-00 ·Pages 4332-6

Lübbert H, Hoffman BJ, Snutch TP, van Dyke T, Levine AJ, Hartig PR, Lester HA, Davidson N

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.

MeSH Terms
Animals Base Sequence Cerebral Ventricle Neoplasms/metabolism Choroid Plexus/metabolism Cloning, Molecular DNA/metabolism Female Genetic Vectors Mice Oocytes/physiology RNA, Messenger/analysis,genetics Receptors, Serotonin/genetics Xenopus
Chemicals
RNA, Messenger Receptors, Serotonin DNA
Authors & Affiliations
8 authors, click to expand affiliations / ORCID
Lübbert H
Hoffman B J
Snutch T P
van Dyke T
Levine A J
Hartig P R
Lester H A
Davidson N
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.84.12.4332
Published
1987-06-00
Pages
4332-6
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC305079
Subset
IM
Grants
NIGMS NIH HHS · GM-10991 · United States
NINDS NIH HHS · NS-11756 · United States
NINDS NIH HHS · NS-23048 · United States
External Links
PubMed source DOI: 10.1073/pnas.84.12.4332 PMC Full Text View journal details
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