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PMID: 2162473 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.

Molecular and cellular biology ·Vol. 10 ·No. 7 ·1990-07-00 ·Pages 3343-56

Labow MA, Baim SB, Shenk T, Levine AJ

Abstract

A novel mammalian regulatory system was created by using the Escherichia coli lac repressor. The lac repressor was converted into a mammalian transcriptional activator by modifying the lac repressor coding region to include a nuclear localization signal from the simian virus 40 (SV40) large tumor antigen and the transcription activation domain from the herpes simplex virus type 1 virion protein 16. The lac activator protein (LAP) fusions were potent activators of several promoters containing lac operator sequences positioned either upstream or downstream of the transcription unit. A single lac operator allowed for transactivation, whereas multiple operators acted synergistically when separated by a small distance. Promoters containing 14 or 21 operator sequences were induced at least 1,000-fold in response to LAP, reaching levels of activity 20 to 30 times greater than that of the SV40 early promoter in HeLa cells. Activation was strongly inhibited by isopropyl-beta-D-thiogalactoside (IPTG), indicating that LAP retained the functions needed for allosteric regulation. LAP was bifunctional, also acting as a repressor of expression of an SV40 promoter containing an operator immediately downstream of the TATA box. Finally, genetic selection schemes were developed such that LAP-expressing cell lines can be generated at high frequency from either established or primary cells in culture.

MeSH Terms
Allosteric Regulation Antigens, Polyomavirus Transforming/genetics Base Sequence Escherichia coli/genetics HeLa Cells/metabolism Humans Molecular Sequence Data Oligonucleotide Probes Operon Phosphoproteins/genetics Promoter Regions, Genetic Recombinant Fusion Proteins/metabolism Repressor Proteins/genetics,metabolism Simian virus 40/genetics,immunology Simplexvirus/genetics Trans-Activators/genetics Transcription Factors/genetics,metabolism Transcription, Genetic Transcriptional Activation
Chemicals
Antigens, Polyomavirus Transforming Oligonucleotide Probes Phosphoproteins Recombinant Fusion Proteins Repressor Proteins Trans-Activators Transcription Factors
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Labow M A
Department of Biology, Lewis Thomas Laboratory, Princeton University, New Jersey 08544.
Baim S B
Shenk T
Levine A J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/mcb.10.7.3343-3356.1990
Published
1990-07-00
Pages
3343-56
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC360756
Subset
IM
Grants
NCI NIH HHS · CA41086 · United States
External Links
PubMed source DOI: 10.1128/mcb.10.7.3343-3356.1990 PMC Full Text View journal details
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