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PMID: 3906652 Published · ppublish English Journal Article Research Support, U.S. Gov't, Non-P.H.S. Research Support, U.S. Gov't, P.H.S.

Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 82 ·No. 23 ·1985-12-00 ·Pages 7870-3

de Wet JR, Wood KV, Helinski DR, DeLuca M

Abstract

A cDNA library was constructed from firefly (Photinus pyralis) lantern poly(A)+ RNA, using the Escherichia coli expression vector lambda gt11. The library was screened with anti-P. pyralis luciferase (Photinus luciferin:oxygen 4-oxidoreductase, EC 1.13.12.7) antibody, and several cDNA clones expressing luciferase antigens were isolated. One clone, lambda Luc1, contained 1.5 kilobase pairs of cDNA that hybridized to a 1.9- to 2.0-kilobase band on a nitrocellulose blot of electrophoretically fractionated lantern RNA. Hybridization of the cloned cDNA to lantern poly(A)+ RNA selected an RNA that directed the in vitro synthesis of a single polypeptide. This polypeptide comigrated with luciferase on NaDodSO4/PAGE and produced bioluminescence upon the addition of luciferin and ATP. A 1.8-kilobase-pair cDNA was isolated by probing the firefly cDNA library with the cDNA from lambda Luc1. This cDNA contained sufficient coding information to direct the synthesis of active firefly luciferase in E. coli.

MeSH Terms
Animals Cloning, Molecular Coleoptera/genetics DNA/genetics Escherichia coli/genetics Gene Expression Regulation Luciferases/genetics Plasmids Promoter Regions, Genetic Protein Biosynthesis RNA, Messenger/genetics
Chemicals
RNA, Messenger DNA Luciferases
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
de Wet J R
Wood K V
Helinski D R
DeLuca M
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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.82.23.7870
Published
1985-12-00
Pages
7870-3
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC390871
Subset
IM
Grants
NIAID NIH HHS · AI07194 · United States
External Links
PubMed source DOI: 10.1073/pnas.82.23.7870 PMC Full Text View journal details
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