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Intracellular Development of Bacteriophage R

II. Fractionation of Replicative Form Deoxyribonucleic Acid Associated with Rapidly Sedimenting Host Cell Components

Biology Department, Brookhaven National Laboratory, Upton, New York 11973

ABSTRACT

When Escherichia coli is infected with bacteriophage R, parental deoxyribonucleic acid (the single- or double-stranded DNA containing the isotopic label of the infecting phage) becomes firmly attached to a cellular structure and can be isolated as a rapidly sedimenting component as described earlier for X174. If this component is centrifuged to equilibrium, two peaks of infective DNA are observed at densities of 1.30 and 1.15 g/ml. At low multiplicities of infection, ³²P-labeled parental DNA is found associated with only the cellular components in the dense band; as the multiplicities of infection are increased, the dense band becomes saturated and parental DNA molecules are then found at the light density as well. Actively replicating host DNA is found only in the dense band, whereas progeny DNA, which does not replicate semiconservatively, can become associated with cellular components in the light band. This fractionation of cellular components on the basis of their buoyant density separates primary sites of DNA replication associated with the dense band from nonfunctional binding sites in the light band.