DNA Topology and the Initiation of Virus DNA Packaging
Fig 5
Effects of I2 mutations on in vivo cos cleavage.
A. Rationale of the in vivo cos cleavage assay. Total phage nucleic acids were isolated from λ-infected cells. AccI digestion of intracellular DNA not cut at cos results in a 7681 bp-long joint DNA fragment (J). cosN cleavage generates 5591 bp right (R) and 2190 bp left (L) end pieces. In an infection by a mutant that is able to cut cos and form complex I, but is unable package DNA, the uncut J and cut L fragments can be detected. AccI-cut total DNA was electrophoresed on a 0.8% agarose gel and transferred to a membrane for southern blotting. B. AccI digested intracellular DNAs: lane 1 is the DNA probe used for Southern blot assay (λ bp 177–2099). Lanes 2 and 3 are AccI-cut λ DNA loaded on the gel before (lane 2) and after heat treatment at 70°C for 10 min to melt the cohesive ends (lane 3). Phage DNAs from positive and negative control phages λ-P1 I2+ and λ-P1 cos2 (ΔcosN), respectively are in lanes 4 and 5. In lanes 6, 7, and 8 are DNAs from λ-P1 I2+ (a second sample), λ-P1 I2re30-35 and λ-P1 I2re18-50, respectively.