A chimeric mammalian transactivator based on the lac repressor that is regulated by temperature and isopropyl beta-D-thiogalactopyranoside.
LAP267 is a lacI activator protein (LAP) containing an insertion of the transcriptional activation domain of the herpes simplex virus virion protein 16 within the inducer-binding and dimerization domain of the lac repressor protein. LAP267 strongly induces expression in a conditional manner from a minimal simian virus 40 early promoter linked to lac operator sequences. LAP267 is temperature-sensitive, activating expression at 32 degrees C but not at 39.5 degrees C. It is allosterically regulated in a manner opposite that of wild-type lac repressor, in that LAP267 activity is rescued at the nonpermissive temperature by isopropyl beta-D-thiogalactopyranoside (IPTG). Stable mouse cell lines containing both the LAP267 gene and a LAP-inducible chloramphenicol acetyltransferase (CAT) reporter gene were readily established and exhibited up to a 1200-fold increase in CAT activity within 24 hr upon addition of IPTG. Thus, LAP267 is a powerful inducible switch in mammalian cells, imparting a regulatory stringency similar to that observed with lac repressor in Escherichia coli.
Baim
S B
SB
Howard Hughes Medical Institute, Princeton, NJ 08544.
Labow
M A
MA
Levine
A J
AJ
Shenk
T
T
D000818:Animals; D002678:Chimera; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis / Q000235:genetics; D004247:DNA / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D006367:HeLa Cells; D006801:Humans; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology*; D007700:Kinetics; D008322:Mammals; D012097:Repressor Proteins / Q000235:genetics*; D013696:Temperature; D015534:Trans-Activators / Q000235:genetics*; D014162:Transfection
D012097:Repressor Proteins; D015534:Trans-Activators; D007544:Isopropyl Thiogalactoside; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase