DNase 1 HS studies of the 5′- and 3-intergenic regions

. (A) Nuclei from megakaryocytic HEL and nonmegakaryocytic HeLa cells were digested with DNase I nuclease at increasing concentrations (wedges). The genomic DNA was then isolated, digested with EcoRI restriction enzyme, and analyzed by Southern blotting. The blots shown were hybridized to probe P1 covering hαIIb exon 1. The size marker is indicated as well as the detected HS sites, which are denoted with Roman numerals. (B) Same as in panel A but for SNU-1 and HEL cells, and the probe P3 covered hαIIb exon 30. (C) The hαIIb gene locus is shown schematically at the top with its transcriptional start site indicated as well as the orientation of the genes shown. Restriction sites of importance are indicated with E indicating EcoRI and B, BamHI. The full-length fragment anticipated after EcoRI digestion for the studies in panels A and B and a schema of the observed HS sites (vertical arrows) within the cell lines tested are shown. P1 indicates probe forEcoRI blot in panel A; P2, probe for BamHI blot (not shown); and P3, probe for EcoRI blot in panel B. Similar results were seen on at least 2 independent studies for each probe, cell line, and endonuclease restriction conditions.