Journal: Journal of Virology
Article Title: Regulatory Mechanisms by Which Barrier-to-Autointegration Factor Blocks Autointegration and Stimulates Intermolecular Integration of Moloney Murine Leukemia Virus Preintegration Complexes
Figure Lengend Snippet: MoMLV PIC is compacted by BAF. (A) Sedimentation properties of initial (panel 1), salt-stripped (panel 2), BAF-reconstituted (panel 3), HMGA1-reconstituted (panels 4 and 5), and HMGB1-reconstituted (panels 6 and 7) PICs. The MoMLV PIC fraction was salt stripped with 750 mM KCl and then incubated with BAF, HMGA1, or HMGB1 protein in the presence of 400 mM KCl (panels 1, 2, 3, 4, and 6) or 150 mM KCl (panels 5 and 7). After gel filtration, each reconstituted PIC fraction was layered on a 15 to 30% sucrose gradient and centrifuged at 30,000 rpm for 1 h with a Beckman rotor. The gradient was fractionated from the top to bottom into 22 fractions, and viral DNA in each fraction was detected by Southern blotting. (B) Intermolecular integration activities of initial, salt-stripped, and reconstituted PICs. Salt-stripped PICs were incubated with BAF (lane 3), HMGA1 (lanes 4 and 6), or HMGB1 (lanes 5 and 7) in the presence of 400 mM KCl (lanes 1 to 5) or 150 mM KCl (lanes 6 and 7) and then added to the integration reaction mixture containing φX174 RFI DNA as the target DNA to check integration activities. After incubation, DNA products from the reaction were digested with Bam HI and detected by Southern blotting with a 32 ).
Article Snippet: Integration activities of reconstituted PICs were evaluated by the previously described integration activity assay with φX174 replicative form I (RFI) DNA (Gibco BRL) as the target DNA ( ).
Techniques: Sedimentation, Incubation, Filtration, Southern Blot