Release of discrete subunits after nuclease and trypsin digestion of chromatin.
Digestion of chromatin with DNase (nucleate 3'-oligonucleotidohydrolase, EC 3.1.4.7) releases 11-12S nucleoprotein particles. After extensive nuclease digestion, the DNA in these particles consists of a collection of eight discrete DNA fragments. When these nuclease-particles are treated with trypsin (EC 3.4.21.4), only 20 to 30 amino-acid residues are cleaved from histone N-terminals, the histone C-terminal segments being resistant. The resulting 5S nucleoprotein particles have now been shown on acrylamide gels to consist of a series of eight discrete DNA-containing bands. Four of these bands contain C-terminal cleavage fragments from four histones (III, IV, IIb2, and IIb1) tightly bound to them; a fifth contains fragments from only histones III and IV. The remaining three bands contain only DNA. Since these protein-free DNA bands were resistant to nuclease prior to trypsin treatment, they were presumably associated with histone N-terminal segments in the native structure. Trypsin, therefore, appears to split nuclease-particles, releasing two subfractions of DNA--one associated with protein, the other not. The data is compatible with a model in which the majority of DNA in the eukaryotic nucleus is folded into hairpin loops of double-stranded helix, each created by the concerted cross-linking action of 6 to 10 histones which interact to form a trypsin-resistant complex composed, for the most part, of all four major histones. These loops may further fold upon themselves to form the "nu" bodies that have been visualized by electron microscopy.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002645:Chickens; D002843:Chromatin / Q000032:analysis*; D003851:Deoxyribonucleases; D004912:Erythrocytes / Q000032:analysis; D006657:Histones / Q000032:analysis; D006868:Hydrolysis; D008715:Methionine / Q000032:analysis; D009698:Nucleoproteins / Q000032:analysis; D014357:Trypsin
D000596:Amino Acids; D002843:Chromatin; D006657:Histones; D009698:Nucleoproteins; D008715:Methionine; D003851:Deoxyribonucleases; D014357:Trypsin