Isolation by Differential and Zonal Centrifugation of Minicells Segregated by Escherichia coli

Summary: Minicells segregated from Escherichia coli x925 carrying a drug-resistance plasmid were separated from nucleated cells by differential centrifugation and purified by rate-zonal centrifugation in sucrose gradients. Minicells purified in this way were capable of donating the plasmid to nucleated cells. They also incorporated thymidine, uridine and methionine into macromolecules. Methods are described for purification of plasmid-containing mini-cells on a scale large enough to allow isolation of DNA, DNA polymerase and RNA polymerase in sufficient quantities for studies of enzymes involved in replication and transcription of plasmid DNA.