50-kb chromatin fragmentation in the absence of apoptosis.
Treatment with ionic detergents of nuclei isolated from various continuously growing cell lines generally yields chromatin samples of high viscosity. Extensive treatment with nuclease-free proteinase K or pronase solubilized the viscous lysates with > 90% of the DNA migrating at approximately 50 kb. Freshly prepared human peripheral blood T cells also yield a substantial fraction of their DNA in an approximately 50- to 100-kb band. The cleavage sites may coincide with a class of DNase I-hypersensitive regions, since digestion of chromatin by DNase I at approximately 10 U/ml, without protease, also yields fragments of preferentially approximately 50-kb size. Occasionally, the oligonucleosomal ladder was also detected together with high molecular weight degradation products. Remarkably, all of these fragmentation patterns were seen in healthy, resting or proliferating cells, i.e., in the absence of apoptosis. Tritiated thymidine incorporation could be readily detected in the approximately 50-kb DNA fragments. The effect of an apoptotic intracellular milieu on the integrity of isolated chromatin is apparently imitated by the extensive protease treatment used in our DNA isolation protocol.
D000818:Animals; D017209:Apoptosis; D002455:Cell Division; D002467:Cell Nucleus; D002522:Chlorocebus aethiops; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism*; D004247:DNA / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003850:Deoxyribonuclease I; D003902:Detergents; D019286:Endopeptidase K; D018922:HL-60 Cells; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D011402:Pronase; D012521:Sarcosine / Q000031:analogs & derivatives; D012697:Serine Endopeptidases; D013601:T-Lymphocytes; D014407:Tumor Cells, Cultured; D014709:Vero Cells
D002843:Chromatin; D003902:Detergents; C025231:sarkosyl; D004247:DNA; D003850:Deoxyribonuclease I; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D011402:Pronase; D012521:Sarcosine