Conversion of the lac repressor into an allosterically regulated transcriptional activator for mammalian cells.
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.
Labow
M A
MA
Department of Biology, Lewis Thomas Laboratory, Princeton University, New Jersey 08544.
Baim
S B
SB
Shenk
T
T
Levine
A J
AJ
D000494:Allosteric Regulation; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D009876:Operon; D010750:Phosphoproteins / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000276:immunology; D018139:Simplexvirus / Q000235:genetics; D015534:Trans-Activators / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D000952:Antigens, Polyomavirus Transforming; D015345:Oligonucleotide Probes; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors