Journal: Molecular and Cellular Biology
Article Title: NF-Y Associates with H3-H4 Tetramers and Octamers by Multiple Mechanisms
Figure Lengend Snippet: ]; labeled on the top strand [lanes 10 to 15] and bottom strand [lanes 16 to 23]). After reconstitutions with stoichiometric amounts of the indicated HFM protein combinations, aliquots were digested with DNase I and analyzed in sequencing gels. Asterisks denote bands that were diminished (lanes 8 and 9) or increased (lanes 20 and 21) when NF-YB–NF-YC was added to reconstitutions. To locate the position of the NF-Y footprinted area, samples in lanes 2, 11, 17, and 23 contained only the NF-Y trimer, without histones. Asterisks indicate protections (lane 9) and hypersensitivities (lanes 19 and 21) upon addition of NF-YB–NF-YC to histones. F, Free DNA; Nuc, nucleosomes. (B) The indicated complexes were DNase I digested, purified from gels (see Materials and Methods), eluted, and run on sequencing gels. Lane 1, free DNA; lane 2, DNA and NF-Y; lanes 3 to 6, H3-H4, H3–H4–NF-YB–NF-YC, nucleosome, and nucleosome–NF-YB–NF-YC, respectively. Asterisks indicate protections (lane 4) and hypersensitivity (lane 6).
Article Snippet: In this procedure, 20,000 cpm of reconstituted tetramer or octamer particles was digested with 0.1 U of DNase I (grade I; Boehringer Mannheim) supplemented with 5 mM MgCl2 and 10 mM CaCl2 at 37°C for 3 min, while free DNA was digested with 0.001 U of DNase I.
Techniques: Labeling, Sequencing, Purification