Construction and characterization of E. coli promoter-probe plasmid vectors III. pBR322 derivatives with deletions in the tetracycline resistance promoter region

Robert W. West, R. L. Rodriguez

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

Deletions of the promoter region for the tetracycline-resistance (Tcr) gene(s) of pBR322 were constructed in order to generate new promoter-probe plasmid cloning vectors. The deletions were constructed in vitro by exonuclease digestion at the HindIII site and blunt-end ligation of the digestion products. Plasmids which lost the HindIII site but retained the EcoRI site carried deletions ranging from 5 to 60 bp. Some of the plasmids lacked the nucleotide sequences required for initiation of transcription from the Tcr promoter and "anti-Tcr" promoter. Three of the promoter-deletion plasmids (containing deletions of 5-29 bp) formed tight-binding complexes with RNA polymerase in vitro, despite their tetracycline sensitive phenotype. One deletion plasmid, pPV33, retained three out-of-phase stop codons located between the promoter-cloning site (EcoRI) and the translational start codon for the tetracycline resistance gene. These features give pPV33 several advantages over previously described promoter-cloning vehicles.

Original languageEnglish (US)
Pages (from-to)291-304
Number of pages14
JournalGene
Volume20
Issue number2
DOIs
StatePublished - 1982

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Keywords

  • Antibiotic resistance
  • gene cartridge
  • insertional activation
  • overlapping promoters
  • pPV33
  • RNA polymerase binding

ASJC Scopus subject areas

  • Genetics

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