A NEW PLASMID CLONING VECTOR FOR DIRECT-DETECTION OF RECOMBINANT CLONES, BASED ON INACTIVATION OF A BACTERIAL ACID PHOSPHATASE-ENCODING GENE

A new plasmid cloning vector for Escherichia coli (pPhoR) suitable for direct detection of recombinant clones has been constructed. The plas mid is a multicopy of a vector which carries a pUC-derived origin of r eplication and beta-lactamase gene, and the phoN acid phosphatase-enco ding gene from Providencia stuartii. Foreign DNA fragments can be clon ed 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 easil y detected by a plate histochemical assay, recombinant clones carrying foreign DNA fragments inserted in the phoN gene can be easily detecte d as PhoN-negative clolonies on the above medium. The efficiency of th e pPhoR-based cloning system for direct cloning of PCR amplimers of a variable region of the HIV-1 genome was comparable to that of conventi onal cloning systems for direct detection of recombinant based on beta -galactosidase inactivation. Advantage of the pPho-R-based system incl ude reduced costs for histochemical assays and the possibility of bein g used with any E. coli host.