Construction and characterization of E. coli promoter-probe plasmid vectors. III. pBR322 derivatives with deletions in the tetracycline resistance promoter region.
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.
D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004352:Drug Resistance, Microbial; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics*; D009154:Mutation; D009876:Operon; D010957:Plasmids / Q000187:drug effects*; D014176:Protein Biosynthesis; D013752:Tetracycline / Q000494:pharmacology*
D004262:DNA Restriction Enzymes; D013752:Tetracycline