development of a chromosomally integrated metabolite-inducible leu3p-α-ipm “off-on” gene switch开发一个染色体整合metabolite-inducible leu3p-α-ipmu201d在u201c基因开关.pdfVIP

Development of a Chromosomally Integrated Metabolite-Inducible Leu3p-a-IPM ‘‘Off-On’’ Gene Switch 1 2 3 3¤a 4¤b Maria Poulou , Donald Bell , Kostas Bozonelos , Maria Alexiou , Anthony Gavalas , Robin Lovell-Badge2, Eumorphia Remboutsika 1,2* 1 Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics, Biomedical Sciences Research Center ‘‘Alexander Fleming,’’ Attica, Greece, 2 Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, London, United Kingdom, 3 Transgenics Unit, Institute of Immunology, Biomedical Sciences Research Center ‘‘Alexander Fleming,’’ Attica, Greece, 4 Division of Developmental Neurobiology, MRC National Institute for Medical Research, London, United Kingdom Abstract Background: Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression. Methodology/Principal Findings: Here, we show that a chromosomally integrated yeast ‘Leu3p-a-IRM’ system constitutes a ligand-inducible regulatory ‘‘off-on’’ genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of a-isopropylmalate (a-IRM) in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable a-IPM presents an EC50 equal to 0.8837 mM and fast ‘‘OFF-ON’’ kinetics (t50ON = 43 min, t50OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cel