Chromatin subunits elicit species-specific antibodies against nucleoprotein antigenic determinants.
Nucleosomes composed of 195 base pairs of DNA associated with histones H2A, H2B, H3, and H4 purified from chicken erythrocyte nuclei were used to elicit antibodies in rabbits. Specific serological reaction between the antisera and the nucleosomes is demonstrated by immunodiffusion, immunofluorescence, microcomplement fixation, solid-phase radioimmunoassay, immunosedimentation, and polyacrylamide gel electrophoresis of 5'-32P end-labeled nucleosomes. The antisera did not react with DNA extracted from these nucleosomes, core histones, or the cross-linked histone octamer from chicken erythocytes, calf thymus total histones, or chromosomal proteins HMG-1 or HMG-17. Nucleosome antigenicity was not affected by redigestion with micrococcal nuclease. Digestion with DNase I brought about 50% loss of reactivity while digestion with trypsin or proteinase K resulted in total loss of activity. The antisera reacted strongly with trimer, dimer, and monomer nucleosomes as well as with the core particle (145 base pairs of DNA) and subnucleosome (greater than 145 base pairs) obtained from chicken. It reacted less well with nucleosomes obtained from HeLa cells and was almost totally devoid of activity against chromatin particles obtained from rat liver or wheat germ. Experiments employing the technique of transferring proteins from a polyacrylamide gel to diazobenzyloxymethyl paper and visualization of antigens by autoradiography excluded the possibility that the serum contains antibodies against tissue-specific antigens which are found in small amounts but are very immunogenic. It is concluded that most of the anitbodies in the sera are directed against nucleoprotein antigenic determinants composed of the N-terminal portion of the histones and segments of DNA. Antibody binding is dependent on contact between the histone and DNA segments and is independent of the integrity of the entire nucleosome. Thus, certain histone DNA contacts remain intact even though the structure of the nucleosome has been disrupted.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D002467:Cell Nucleus / Q000032:analysis; D002645:Chickens; D002843:Chromatin / Q000276:immunology*; D003168:Complement Fixation Tests; D004247:DNA / Q000276:immunology*; D003852:Deoxyribonucleoproteins / Q000276:immunology*; D000939:Epitopes; D004912:Erythrocytes / Q000032:analysis; D006657:Histones / Q000276:immunology*; D007106:Immune Sera; D008099:Liver / Q000032:analysis; D009698:Nucleoproteins / Q000276:immunology*; D009707:Nucleosomes / Q000276:immunology*; D010944:Plants / Q000032:analysis; D011817:Rabbits / Q000276:immunology; D051381:Rats; D014908:Triticum / Q000032:analysis
D000906:Antibodies; D000936:Antigen-Antibody Complex; D002843:Chromatin; D003852:Deoxyribonucleoproteins; D000939:Epitopes; D006657:Histones; D007106:Immune Sera; D009698:Nucleoproteins; D009707:Nucleosomes; D004247:DNA