Journal:

Article Title: Topoisomerase IV, alone, unknots DNA in E. coli

doi: 10.1101/gad.872301

Figure Lengend Snippet: Metabolism of Int recombination knots in vivo. (A) The extent of Int recombination for each strain is shown. Cells were grown as described for Figure ​Figure2.2. Plasmid DNA was isolated and submitted to restriction digestion with EcoRI and BamHI. Samples were analyzed by agarose gel electrophoresis. Shown is the percent of total plasmid DNA recombined with time. No recombination occurred before Int was induced (Time = 0). Error bars are from two experiments that were averaged. (B) Unknotting of Int recombination products. Representative agarose gels of Int recombination in the isogenic strains. In this and subsequent figures, the grid above the gels shows which strain was used and whether gyrase and topoisomerase IV were active (+) or not (−). The positions of nicked circular (○) and linear (wavy line) plasmid, as well as the trefoil (3) and pentafoil (5) knots are shown. (C) Graphic representation of the data. Shown is the percent of the total plasmid DNA that was knotted with time for each strain. Data were quantified by Scanning densitometry with Nucleovision or TechSoftware PhosphorImage analyses using Storm. Each time point was repeated 2–3 times with similar results. (D) A fraction of the purified plasmid DNA used in A and B was subjected to electrophoresis on 1%–1.2% agarose gels that contained (2–20 μg/mL) chloroquine to visualize DNA supercoiling (Zechiedrich et al. 2000). The mean ς was calculated and is shown for each strain over time. Where not shown, the error was smaller than the symbol (except for the gyrA+ parCr strain at 8, 20, and 60 min, each of which was performed once).

Article Snippet: Quantification of bands on agarose gels was carried out using either the Storm PhosphorImaging system or Nucleovision densitometry software from Nucleotech.

Techniques: In Vivo, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Electrophoresis