Isolation of a λd v Plasmid Carrying the Bacterial gal Operon

Abstract

A λ dvgal plasmid carrying genes for controlled plasmid replication from phage λ and the bacterial gal operon was isolated as a deletion mutant of phage λ gal q4, which carries the gal operon between λ genes P and Q . The plasmid DNA was found in cell extracts as covalently closed circular molecules. The plasmid was characterized by using genetic crosses, digestion with the specific endonuclease Eco RI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer ( O ^λ P ^λ gal O ^λ P ^λ gal ); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal ( O ^λ P ^λ O ^λ P ^λ gal ). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and λ sequences in the plasmid can be separated by recombination. Analyses of the Eco RI digests of plasmid DNAs indicated one cleavage site per λ gene sequence and none in the gal operon. A λ dvgal monomer was approximately 6.7 × 10 ⁶ daltons and the λ gene and gal components were 3.9 × 10 ⁶ and 2.8 × 10 ⁶ daltons, respectively. The λ dvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10 ⁻⁶ per DNA molecule.