Construction of a lacZ-kanamycin-resistance cassette, useful for site-directed mutagenesis and as a promoter probe.

A lacZ gene without a promoter, but containing its ribosome-binding site, was cloned next to the kanamycin-resistance (KmR) gene of plasmid pUC4K, yielding a lacZ-KmR cassette. From the resulting plasmid, pKOK5, the lacZ-KmR cassette was recloned by means of BamHI into plasmid pKOK4, a mobilizable derivative of pBR322 which mediates ampicillin, chloramphenicol and tetracycline resistance. The lacZ-KmR cassette can be excised from pKOK5 or pKOK6 by digestion with BamHI, SalI or PstI. It can be used for insertion mutagenesis by ligation of the cassette to target DNA that has been linearized by one of these enzymes. Insertions can be selected by the KmR phenotype and mapped by digestion, e.g., with PstI and SalI. The orientation of the inserted cassette can be determined by digestion, e.g., with EcoRI or HindIII. Within the lacZ-KmR cassette, the transcription of the lacZ and the KmR genes are directed towards each other, and the two genes are separated by the bidirectionally active terminator from phage fd. In Escherichia coli, no transcription emanating from the cassette was detected. Transcription within DNA mutagenized by the cassette can be monitored by the promoterless lacZ gene. The lacZ-KmR cassette is currently used by us for the site-directed mutagenesis of hydrogen uptake gene-specific DNA from Rhizobium leguminosarum B10.