FIG. 3.

DNase I hypersensitivity test. (A) DNase I hypersensitivity of DHS-S1 on the nuclear episomal plasmids replicated in HEK293 cells and in the AAVS1 locus of HEK293 cells. Nuclei were extracted from HEK293 cells transfected with pEnh-bl1 containing the AAVS1-B/B fragment or from normal HEK293 cells. The nuclei were digested with 2.5, 5, 10, 20, and 40 U of DNase I per ml. Total DNA was isolated, digested with BamHI plus XhoI (upper panel) or with BamHI (lower panel), and electrophoresed in an agarose gel. After DNA samples were transferred to a nylon membrane, DNA fragments containing firefly luciferase gene (upper panel) or the AAVS1 region from the BamHI site (nt −1k) to the DNase I hypersensitive site (lower panel) were detected by ³²P-labeled probes hybridizing with the DNA region indicated. (B) DNase I hypersensitivity of DHS-S1 and chicken β-globin insulator (cHS4) on the nuclear episomal plasmids replicated in HEK293 cells. Nuclei were extracted from HEK293 cells transfected with pEnh-bl1 containing AAVS1-B/B, cHS4, and λ HindIII/594 fragments. The nuclei were digested with 10, 20, and 40 U of DNase I per ml. Total DNA was isolated, digested with BamHI plus XhoI, and electrophoresed in an agarose gel. After DNA samples were transferred to a nylon membrane, DNA fragments containing firefly luciferase gene were detected by ³²P-labeled probes hybridizing to DNA carrying the luciferase gene.