title: A new plasmid cloning vector for direct detection of recombinant clones, based on inactivation of a bacterial acid phosphatase-encoding gene.
abstract: A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plasmid is a multicopy of a vector which carries a pUC-derived origin of replication and beta-lactamase gene, and the phoN acid phosphatase-encoding gene from Providencia stuartii. Foreign DNA fragments can be cloned into unique restriction sites located within the phoN gene causing a loss of the acid phosphatase activity which is normally overproduced by E. coli strains carrying pPhoR. Since PhoN production can be easily detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detected as PhoN-negative clolonies on the above medium. The efficiency of the pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventional cloning systems for direct detection of recombinant based on beta-galactosidase inactivation. Advantage of the pPho-R-based system include reduced costs for histochemical assays and the possibility of being used with any E. coli host.
authors.affiliation: Istituto di Microbiologia, Università Cattolica del S. Cuore, Rome, Italy.
authors.forename: R
authors.initials: R
authors.lastname: Burioni
authors.forename: P
authors.initials: P
authors.lastname: Plaisant
authors.forename: M L
authors.initials: ML
authors.lastname: Riccio
authors.forename: G M
authors.initials: GM
authors.lastname: Rossolini
authors.forename: G
authors.initials: G
authors.lastname: Satta
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D015497:HIV-1 / Q000235:genetics; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011532:Providencia / Q000201:enzymology; D018741:Replication Origin / Q000235:genetics; D001618:beta-Lactamases / Q000235:genetics
chemical_list: D000135:Acid Phosphatase; D001618:beta-Lactamases