Vector for IS element entrapment and functional characterization based on turning on expression of distal promoterless genes

Ildikó Szeverényi, Alois Hodel, Werner Arber, Ferenc Olasz

Research output: Contribution to journalArticle

20 Citations (Scopus)

Abstract

We constructed and characterized a novel trap vector for rapid isolation of insertion sequences. The strategy used for the isolation of IS elements is based on the ability of many IS elements to turn on the expression of otherwise silent genes distal to some sites of insertion. The simple transposition of an IS element can sometimes cause the constitutive expression of promoterless antibiotic resistance genes resulting in selectable phenotypes. The trap vector pAW1326 is based on a pBR322 replicon, it carries ampicillin and streptomycin resistance genes, and also silenced genes that confer chloramphenicol and kanamycin resistance once activated. The trap vector pAW1326 proved to be efficient and 85 percent of all isolated mutations were insertions. The majority of IS elements resident in the studied Escherichia coli strains tested became trapped, namely IS2, IS3, IS5, IS150, IS186 and Tn1000. We also encountered an insertion sequence, called IS1OL/R-2, which is a hybrid of the two IS variants IS1OL and IS1OR. IS1OL/R-2 is absent from most E. coli strains, but it is detectable in some strains such as JM109 which had been submitted to TnlO mutagenesis. The distribution of the insertion sequences within the trap region was not random. Rather, the integration of chromosomal mobile genetic elements into the offered target sequence occurred in element-specific clusters. This is explained both by the target specificity and by the specific requirements for the activation of gene transcription by the DNA rearrangement. The employed trap vector pAW1326 proved to be useful for the isolation of mobile genetic elements, for a demonstration of their transposition activity as well as for the further characterization of some of the functional parameters of transposition.

Original languageEnglish
Pages (from-to)103-110
Number of pages8
JournalGene
Volume174
Issue number1
DOIs
Publication statusPublished - Sep 26 1996

    Fingerprint

Keywords

  • Escherichia coli
  • IS10
  • IS150
  • IS186
  • IS2
  • IS3
  • IS5
  • Insertion sequences
  • Polar effect
  • Spontaneous mutation
  • Tn1000
  • Transposition

ASJC Scopus subject areas

  • Genetics

Cite this