Frequency of replication from alternative origins in the composite R plasmid NR1

The relative frequency of initiation of DNA replication within the RTF-Tc and r-determinant components of the composite drug resistance plasmid NR1 in Proteus mirabilis was evaluated. Using fractionated radioactively labeled plasmid DNA, analytical procedures that distinguished between the two components of the composite plasmid were carried out. A mixture of uniformly 14C-labeled and 3H-pulse-labeled plasmid DNA (pulse-labeled origin[s] of replication) was used in each of three experiments. First, shear products of the DNA were analyzed using CsCl density gradient centrifugation. Second, fragmented DNA was hybridized to nonradioactive RTF-Tc and r-determinant DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNAs immobilized on nitrocellulose filters. Third, the radioactive plasmid DNA was digested with restriction enzyme (EcoRI), producing a set of RTF-Tc and r-determinant fragments with differing 3H/14C isotpe ratios. The three experiments suggested that under the conditions used to accumulate replicating plasmid DNA molecules (DNA substrate limitation), the r-determinant origin of replication was preferentially utilized in the composite plasmid.