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Intracellular development of bacteriophage phi-R. II. Fractionation of replicative form deoxyribonucleic acid
Abstract:
When Escherichia coli is infected with bacteriophage phiR, 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 phiX174. 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, (32)P-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.
Insights
Bacteriophage phiR infection of Escherichia coli attaches parental DNA to cellular structures. This DNA separates into dense and light bands upon centrifugation, indicating distinct replication and binding sites.
Area of Science:
- Microbiology
- Molecular Biology
- Virology
Background:
- Bacteriophages, such as phiR and phiX174, infect bacteria like Escherichia coli.
- Parental deoxyribonucleic acid (DNA) from infecting phages can associate with host cellular structures.
- Understanding phage-host DNA interactions is crucial for molecular biology research.
Purpose of the Study:
- To investigate the association of bacteriophage phiR parental DNA with cellular structures in Escherichia coli.
- To characterize the location and behavior of phage and host DNA during infection using density gradient centrifugation.
- To differentiate between active DNA replication sites and nonfunctional binding sites.
Main Methods:
- Infection of Escherichia coli with bacteriophage phiR.
- Isolation of phage-associated cellular structures containing parental DNA.
- Equilibrium density gradient centrifugation to separate DNA components.
- Radioactive labeling (32P) of parental DNA to track its location.
Main Results:
- Parental DNA forms a rapidly sedimenting component upon phage infection.
- Centrifugation reveals two infective DNA peaks at densities of 1.30 g/ml (dense band) and 1.15 g/ml (light band).
- At low multiplicities, parental DNA is in the dense band; at higher multiplicities, it appears in the light band as well. Actively replicating host DNA is exclusively in the dense band, while progeny DNA associates with the light band.
Conclusions:
- The dense band represents primary sites of DNA replication, binding both replicating host DNA and, initially, parental DNA.
- The light band contains nonfunctional binding sites where progeny DNA and, at higher multiplicities, parental DNA can associate.
- Buoyant density fractionation effectively separates active replication sites from inactive binding sites during phage infection.