title: Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene.
abstract: We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes. The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D. discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter. The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA. When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA. Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis. The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region. Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding. This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.
authors.affiliation: Institut für Biochemie der Medizinischen Fakultät, Universität Erlangen-Nürnberg, Germany.
authors.forename: T
authors.initials: T
authors.lastname: Dingermann
authors.forename: H
authors.initials: H
authors.lastname: Werner
authors.forename: A
authors.initials: A
authors.lastname: Schütz
authors.forename: I
authors.initials: I
authors.lastname: Zündorf
authors.forename: K
authors.initials: K
authors.lastname: Nerke
authors.forename: D
authors.initials: D
authors.lastname: Knecht
authors.forename: R
authors.initials: R
authors.lastname: Marschalek
mesh_terms: D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004257:DNA Polymerase II / Q000378:metabolism; D004274:DNA, Recombinant; D004023:Dictyostelium / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D016153:Genes, Suppressor; D007700:Kinetics; D008969:Molecular Sequence Data; D009875:Operator Regions, Genetic; D012343:RNA, Transfer / Q000235:genetics*; D013753:Tetracycline Resistance / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D004274:DNA, Recombinant; D012343:RNA, Transfer; D004257:DNA Polymerase II