Abstract
Replicating T5 or lambda phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA. Two procedures are described for separating T5 replicating DNA from DNA of intracellular phage particles. Both T5 and lambda replicating DNA had the same bouyant density in cesium chloride as DNA from phage particles but sedimented faster when centrifuged in sucrose density gradients. The fast sedimentation did not appear to be caused by DNA protein or DNA-RNA complexes or by aggregation of DNA, but is probably due to DNA molecules of unusual structure. Experiments involving hydrodynamic shear and sucrose density gradient centrifugation at alkaline pH have suggested that with lambda the replicating form of DNA is a linear molecule considerably longer than the DNA molecules of lambda-phage particles. The constituent polynucleotide chains of lambda but not T5 replicating DNA also appear to be longer than those of phage DNA.
Highlights
Replicating T5 or X phage DNA has been labeled by adding tritiated thymidine for short periods to cultures of phage-infected Escherichia coli before isolation of intracellular DNA
By subtracting the amount of DNA in infective intracellular phage from the total phage DNA, Hershey and coworkers measured the size of the precursor pool in T2-infected bacteria [2, 3]
We have studied intracellular DNA from Escherichia coli infected with T5 and Xb+c1
Summary
DNA STRUCTURE min after infection, of dense particles similar in shape to phage heads [4]. DNA replicating during the interval of the "pulse" becomes labeled in this type of experiment For analytical purposes it was compared with the DNA of purified phage particles. In other experiments the bacteria were infected with 32P-labeled phage and pulsed with tritiated thymidine, so that the parental DNA could be recognized and compared with the newly synthesized DNA in the same culture. Column Chromatography of Pulse-Labeled DNA When DNA from pulselabeled T5-infected bacteria was passed through a column of kieselguhr coated with methylated serum albumin, two major components were resolved (Fig. 2) [11]. Concentration of Pulse-Labeled DNA at the Interface during Phenol Extraction When a culture of T5-infected bacteria, pulse-labeled at 35 min after infec-.
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