Absence of histone H2B in nucleosomes containing histone TH2B and interaction of immunoglobulin with nucleosomes.
Nucleosomes containing histone TH2B were isolated from chromatin subunits of rat testis nuclei (MNT) by incubating with anti-TH2B immunoglobulin (IgTH2B) which was covalently attached to agarose gels. Electrophoretic separation of histones of these isolated nucleosomes revealed that histone H2B was completely absent, suggesting that histone TH2B, the variant of H2B, existed in nucleosomes only as TH2B X TH2B and that TH2B X H2B was not likely to exist in chromatin. Sucrose gradient ultracentrifugation of mixtures of MNT and IgTH2B revealed that when excess amounts of immunologically active IgTH2B were present, complexes of higher sedimentation coefficients than MNT X IgTH2B were formed, but with limited amounts of active IgTH2B, only MNT X IgTH2B was formed. When purified IgTH2B was coated on polystyrene tubes and incubated with MNT, those MNT immobilized by the tube-coated IgTH2B adsorbed IgTH2B from diluted antiserum during subsequent incubation. Those results suggested the absence of steric hindrance in the binding of IgTH2B to MNT X IgTH2B. When MNT was coated on polystyrene tubes and incubated with DNase and then with dilute anti-TH2B antiserum, it was found that DNase digestion increased the binding of immunoglobulin to the tubes approximately 76%. Interaction of chromatin subunits of rat liver nuclei (MNL) with anti-TH2B antiserum was negligible, but DNase digestion of MNL coated on tubes was followed by considerable interaction with anti-TH2B antiserum. Those results indicated DNase unmasked at least part of the determinants encased by DNA. Anti-H2B immunoglobulin (IgH2B) interacted with histone H2B and TH2B to the same extent, and interacted significantly to a lesser extent with either MNT or MNL. DNase digestion of MNT and MNL increased binding of IgH2B approximately 170 and 117%, respectively.
D000818:Animals; D000915:Antibody Affinity; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000378:metabolism; D003850:Deoxyribonuclease I; D004586:Electrophoresis / Q000379:methods; D006657:Histones / Q000145:classification / Q000302:isolation & purification*; D007120:Immunochemistry; D007136:Immunoglobulins; D008297:Male; D009707:Nucleosomes / Q000032:analysis* / Q000276:immunology; D051381:Rats; D013737:Testis
D002843:Chromatin; D006657:Histones; D007136:Immunoglobulins; D009707:Nucleosomes; D003850:Deoxyribonuclease I