4] Plasmids for the selection and analysis of prokaryotic promoters

Abstract

The chapter discusses plasmid vectors for selecting and analyzing prokaryotic promoters. Plasmid vectors that can select or screen for transcription promoters are vital tools for the isolation and characterization of DNA fragments, containing such regulatory sequences. The chapter describes two vectors (pKK175-6 and pKK232-8), which utilize antibiotic resistance structural genes whose natural promoters have been deleted. These promoter probe vectors are particularly useful, because they have very low background of antibiotic resistance when no promoter is present and they are constructed to protect against the creation of artifactual promoters. The chapter describes a number of applications of plasmid pKK232-8. Promoter-probe plasmid vectors allow constructing transcriptional gene fusions between cloned promoters and drug resistance structural genes. They are, therefore, useful tools for analyzing transcription initiation signals, because the protein products of these constructions are readily assayed. The vectors enable direct selection of promoter sequences and an easy method to evaluate the effects of mutations in cloned promoters. There are, however, limitations to the method of measuring a translational product for determining effects on a transcription initiation signal. Many of these limitations can be overcome by assaying a stable RNA product, which is transcribed by a cloned promoter. It should be noted that plasmid DNA copy number and DNA topology may introduce artifacts that do not truly represent events occurring in vivo on a single-copy chromosome. Nevertheless, this system allows a comparative analysis of promoters, especially from genes, which are vital to cell integrity and not so amenable to study by other means.