title: Development of a chromosomally integrated metabolite-inducible Leu3p-alpha-IPM "off-on" gene switch.
abstract: BACKGROUND
  Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression.
  
  
  RESULTS
  Here, we show that a chromosomally integrated yeast 'Leu3p-alpha-IotaRhoMu' 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 alpha-isopropylmalate (alpha-IotaRhoMu) 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 alpha-IPM presents an EC(50) equal to 0.8837 mM and fast "OFF-ON" kinetics (t(50)ON = 43 min, t(50)OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo.
  
  
  CONCLUSIONS
  Our results demonstrate that the 'Leu3p-alpha-IotaRhoMu' constitutes a simpler and safer system for inducible gene expression in biomedical applications.
authors.affiliation: Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics, Biomedical Sciences Research Center "Alexander Fleming," Attica, Greece.
authors.forename: Maria
authors.initials: M
authors.lastname: Poulou
authors.forename: Donald
authors.initials: D
authors.lastname: Bell
authors.forename: Kostas
authors.initials: K
authors.lastname: Bozonelos
authors.forename: Maria
authors.initials: M
authors.lastname: Alexiou
authors.forename: Anthony
authors.initials: A
authors.lastname: Gavalas
authors.forename: Robin
authors.initials: R
authors.lastname: Lovell-Badge
authors.forename: Eumorphia
authors.initials: E
authors.lastname: Remboutsika
mesh_terms: D000818:Animals; D001483:Base Sequence; D033481:Chromosomes, Mammalian / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005818:Genetic Engineering / Q000379:methods*; D008293:Malates / Q000378:metabolism*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D011247:Pregnancy; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics* / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
chemical_list: C063667:LEU3 protein, S cerevisiae; D008293:Malates; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C005906:alpha-isopropylmalate
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