Construction and characterization of the chloramphenicol-resistance gene cartridge: A new approach to the transcriptional mapping of extrachromosomal elements

Timothy J. Close, R. L. Rodriguez

Research output: Contribution to journalArticle

204 Scopus citations

Abstract

A system has been developed to transcriptionally map plasmid DNA molecules in vivo. This system is based upon the activity of the Tn 9 Cmr gene whose product, chloramphenicol acetyltransferase (CAT), can be easily assayed enzymatically and identified phenotypically in Escherichia coli. The Cmr gene was excised as a single DNA fragment from the plasmid cloning vector, pBR328, and its ends were converted to hexamer-specific restriction enzyme recognition sites. These modified Cmr gene fragments were designated "CAT cartridges". The CAT cartridges were expressed at high levels only when inserted in the proper orientation, downstream of a plasmid promoter. Using DNA restriction endonuclease cleavage sites as insertion points, the CAT cartridges were inserted into plasmid pBR327, and the levels of CAT activity produced in cells containing the various plasmids were determined. The CAT activities at each position and orientation were used to construct a transcriptional map of plasmid pBR327. The positions and strengths of promoters demonstrated in vivo using the CAT cartridge coincided with those predicted by in vitro studies. Using the CAT cartridges it should be possible to study gene expression on any extrachromosomal element in the appropriate host.

Original languageEnglish (US)
Pages (from-to)305-316
Number of pages12
JournalGene
Volume20
Issue number2
DOIs
StatePublished - 1982

Keywords

  • pBR327 promoters
  • readthrough transcription
  • Tn9 CAT gene

ASJC Scopus subject areas

  • Genetics

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