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

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.

• Authors: M A Labow, S B Baim, T Shenk, A J Levine
• Journal: Molecular and cellular biology (Mol Cell Biol) Vol. 10 Issue 7 Pg. 3343-56 (Jul 1990) ISSN: 0270-7306 [Print] United States
• PMID: 2162473 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Antigens, Polyomavirus Transforming
• Oligonucleotide Probes
• Phosphoproteins
• Recombinant Fusion Proteins
• Repressor Proteins
• Trans-Activators
• Transcription Factors

Topics

• 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