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PMID: 9630253 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

A dual-luciferase reporter system for studying recoding signals.

RNA (New York, N.Y.) ·Vol. 4 ·No. 4 ·1998-04-00 ·Pages 479-86

Grentzmann G, Ingram JA, Kelly PJ, Gesteland RF, Atkins JF

Abstract

A new reporter system has been developed for measuring translation coupling efficiency of recoding mechanisms such as frameshifting or readthrough. A recoding test sequence is cloned in between the renilla and firefly luciferase reporter genes and the two luciferase activities are subsequently measured in the same tube. The normalized ratio of the two activities is proportional to the efficiency with which the ribosome "reads" the recoding signal making the transition from one open reading frame to the next. The internal control from measuring both activities provides a convenient and reliable assay of efficiency. This is the first enzymatic dual reporter assay suitable for in vitro translation. Translation signals can be tested in vivo and in vitro from a single construct, which allows an intimate comparison between the two systems. The assay is applicable for high throughput screening procedures. The dual-luciferase reporter system has been applied to in vivo and in vitro recoding of HIV-1 gag-pol, MMTV gag-pro, MuLV gag-pol, and human antizyme.

MeSH Terms
Amino Acid Sequence Animals Base Sequence Cnidaria/enzymology Coleoptera/enzymology Fusion Proteins, gag-pol/genetics Genes, Reporter Genetic Code Genetic Vectors HIV-1/genetics Humans Luciferases/biosynthesis Mammary Tumor Virus, Mouse/genetics Molecular Sequence Data Ornithine Decarboxylase Inhibitors Protein Biosynthesis Reading Frames Simian virus 40/genetics
Chemicals
Fusion Proteins, gag-pol Ornithine Decarboxylase Inhibitors Luciferases
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Grentzmann G
Howard Hughes Medical Institute, University of Utah, Salt Lake City 84112, USA.
Ingram J A
Kelly P J
Gesteland R F
Atkins J F
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Article Info
Journal
RNA (New York, N.Y.)
Abbr.
RNA
ISSN
1355-8382
Published
1998-04-00
Pages
479-86
Language
English
Region
United States
NLM ID
9509184
PMCID
PMC1369633
Subset
IM
Grants
NIGMS NIH HHS · R01-GM48152-05 · United States
Databases
GENBANK
AF043450
External Links
PubMed source PMC Full Text View journal details
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