Chromatin fractionation procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins.
Initial results of an approach to the isolation of functionally active chromatin are described. Slight digestion of mouse myeloma nuclei at 0 degrees C with micrococcal nuclease, followed by dialysis against near-physiological saline solution containing 1 mM Mg2+, caused release of up to 17% of the nuclear DNA as soluble nucleoproteins. This soluble (S) fraction was relatively depleted in H1 histones and methylated DNA (5-methylcytosine) but highly enriched in RNA, single-stranded DNA, and nonhistone chromosomal proteins, particularly two species of the high mobility group identified as HMG 1 and HMG 2. The S fraction released most rapidly (6--8% of the total DNA) consisted mainly of mono- and small oligonucleosomes. The mononucleosomes appeared normal in terms of sedimentation behavior, DNA length, and content of histones H2A, H2B, H3, and H4, but lacked H1, and instead were associated with approximately stoichiometric amounts of HMG 1 and HMG 2. Studies using isolated, fluorescence-labeled, total mouse HMG proteins indicated that added HMG 1 and HMG 2 do not bind strongly to S-fraction nucleoproteins but that two smaller HMG species (probably HMG 14 and HMG 17) do bind preferentially to S-fraction mono- and dinucleosomes. These results argue against artifactual redistribution of HMG 1 and HMG 2 during this fractionation but suggest caution in interpreting the distribution of smaller HMG proteins after digestion of chromatin. The potential relationship of this soluble fraction to transcriptionally active chromatin is discussed.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002843:Chromatin / Q000032:analysis*; D002868:Chromosomal Proteins, Non-Histone / Q000302:isolation & purification*; D004273:DNA, Neoplasm / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000302:isolation & purification; D006657:Histones / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D009101:Multiple Myeloma / Q000032:analysis; D009374:Neoplasms, Experimental / Q000032:analysis; D009691:Nucleic Acid Denaturation; D012334:RNA, Neoplasm / Q000302:isolation & purification; D012260:Ribonucleases; D014364:Tryptophan / Q000032:analysis; D014443:Tyrosine / Q000032:analysis
D000596:Amino Acids; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D004273:DNA, Neoplasm; D004277:DNA, Single-Stranded; D006657:Histones; D012334:RNA, Neoplasm; D014443:Tyrosine; D014364:Tryptophan; D012260:Ribonucleases