Isolation of a lambda dv plasmid carrying the bacterial gal operon

Abstract

A lambdadvgal plasmid carrying genes for controlled plasmid replication from phage lambda and the bacterial gal operon was isolated as a deletion mutant of phage lambdagalq4, which carries the gal operon between lambda 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 EcoRI, sucrose gradient centrifugation, and electron microscopy. In one clone analyzed, the plasmid was a complete dimer (O(lambda)P(lambda)galO(lambda)P(lambda)gal); in a subclone derived from it, the plasmid was a partial dimer with only one copy of gal (O(lambda)P(lambda)O(lambda)P(lambda)gal). The partial dimer may be a recombination product of the complete dimer, since test crosses show that the gal and lambda sequences in the plasmid can be separated by recombination. Analyses of the EcoRI digests of plasmid DNAs indicated one cleavage site per lambda gene sequence and none in the gal operon. A lambdadvgal monomer was approximately 6.7 x 10(6) daltons and the lambda gene and gal components were 3.9 x 10(6) and 2.8 x 10(6) daltons, respectively. The lambdadvgal plasmid can be introduced into a new bacterial host by transfection at an efficiency of 10(-6) per DNA molecule.