A Tn3 lacZ Transposon for the Random Generation of Beta-Galactosidase Gene Fusions: Application to the Analysis of Gene Expression in Agrobacterium

EMBO J. 1985 Apr;4(4):891-8.

Abstract

The construction and use of a Tn3-lac transposon, Tn3-HoHo1, is described. Tn3-HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta-galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3-HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta-galactosidase activity. Tn3-HoHo1 carries a non-functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3-HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3-HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3-HoHo1 generated lac fusions.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Arginine / analogs & derivatives
  • Arginine / pharmacology
  • Base Sequence
  • DNA Restriction Enzymes
  • DNA Transposable Elements*
  • Enzyme Induction / drug effects
  • Galactosidases / genetics*
  • Genes*
  • Genes, Bacterial*
  • Kinetics
  • Lac Operon
  • Protein Biosynthesis
  • Rhizobium / enzymology
  • Rhizobium / genetics*
  • Transcription, Genetic*
  • beta-Galactosidase / biosynthesis
  • beta-Galactosidase / genetics*

Substances

  • DNA Transposable Elements
  • octopine
  • Arginine
  • DNA Restriction Enzymes
  • Galactosidases
  • beta-Galactosidase