Bacterial acid phosphatase gene fusions useful as targets for cloning-dependent insertional inactivation.
The Morganella morganii phoC gene, encoding a class A acid phosphatase, was used to generate gene fusions with modified amino-terminal moieties of the Escherichia coli lacZ gene carrying a multiple-cloning site flanked by phage-specific promoters and recognition sites for universal sequencing primers. The corresponding hybrid proteins retained a PhoC-like enzymatic activity which is easily detectable by a plate histochemical assay, rendering similar gene fusions potentially useful as targets for cloning-dependent insertional inactivation. Cloning experiments performed in plasmids carrying similar lacZ-phoC fusions confirmed their usefulness as cloning vectors for direct screening of recombinants. As compared to conventional lacZ alpha-complementation-based vectors, which can only be used in E. coli hosts carrying specific lacZ mutations, the lacZ-phoC fusion-based vectors can be used in combination with any E. coli host and require a less expensive histochemical assay for screening of recombinants, while retaining all the advantageous features that made the former so popular as general purpose cloning vehicles.
Thaller
M C
MC
Dipartimento di Biologia, II Università di Roma "Tor Vergata", 00133 Rome, Italy. PEZZI@axrma.uniroma1.it
Berlutti
F
F
Schippa
S
S
Selan
L
L
Rossolini
G M
GM
D000135:Acid Phosphatase / Q000235:genetics*; D019677:Artificial Gene Fusion; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D005815:Genetic Code; D005822:Genetic Vectors; D007763:Lac Operon; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics
D004274:DNA, Recombinant; D000135:Acid Phosphatase