Figure 6

In vitro integration catalyzed by WT and delta NED PFV INs on naked and chromatinized p5S vectors. Concerted integration assay was performed with 10 ng of donor DNA and 100 ng of p5S naked plasmids (lanes 1), or p5S polynucleosomalp5S vectors assembled with a 1/1.1 DNA/histones ratio (μg/μg) either 90nM wt PFV IN or 90nM delta NED PFV IN. The reaction products were loaded on 1% agarose gel and the linear FSI and circular FSI + HSI products were quantified on gel autoradiography using the ImageJsoftware and the values are plotted respectively in (A) and (B). The circular FSI products were specifically quantified by cloning in bacteria and plotted as the number of ampicillin-, kanamycin- and tetracycline-resistant selected clones in percentage of integration reaction control performed with naked vectors (C). The positions of the different integration events were then identified by sequencing and plotted in region 1 or region 2 (D). Values correspond to the mean ± standard deviation (error bars) of 3 independent sets of experiments. The number of selected clones is also shown at the top of the histograms. Fifty integrants carrying the correct 4 bp target DNA duplication obtained after integration assay carried on the p5S vector chromatinized with a 1/1.3 DNA/histones ratio were localized on the p5S sequence (E) and compared to the nucleosome occupancy determined using the method previously described by [53] and used in [36]. The mean nucleosome occupancy found at the integration site on chromatinized or naked plasmid was calculated and plotted respectively in (F). A Student test was performed on serial values: *p < 0.05.