FIG. 2.

Pho4-induced generation of a hypersensitive site at the PHO5 promoter during de novo assembly in vitro. DNase I indirect end-labeling analysis of the PHO5 promoter region in chromatin assembled de novo with yeast extract (from pho4 strain YS22) in vitro with or without the addition of exogenous Pho4 and in free DNA. The schematic on the left denotes positioned nucleosomes as in the schematic of the PHO5 promoter in Fig. 1A. Black dots between lanes 2 and 3 mark the bands corresponding to the linker regions between the positioned nucleosomes. The vertical bar between lanes 4 and 5 highlights the hypersensitive region generated by the addition of Pho4. The stippled region interrupting the vertical bar points to a region of maintained protection from DNase I. Ramps above the lanes stand for increasing DNase I concentrations. Marker fragments were generated by double digests of DraI, ClaI, and BamHI, each with ApaI. All samples were electrophoresed in lanes of the same gel. Stippled lines between lanes show where lanes were moved next to each other using Adobe Photoshop CS2.