Histone H1(0) mapping using monoclonal antibodies.
Monoclonal antibodies (mAb) to ox liver histone H1 degree were produced and characterized. Two sets of mice were immunized either with pure H1(0) or with an H1(0)-yeast tRNA complex. Eleven hybridomas of various clonal origin were selected. Typing of the antibodies indicated that all but three IgM belonged to the IgG1 class and contained kappa light chains. Immunoblotting experiments using peptides derived from H1(0) or H5 treated by various proteolytic agents (trypsin, N-bromosuccinimide, cyanogen bromide, acetic acid), revealed that nine of the mAb reacted with the globular part of H1(0). More advanced characterization of the antigenic determinants allowed us to determine distinct regions within this globular part which are involved in the antigenic recognition. The peptopes could be subdivided into two groups. Three mAb bound to residues 24-27 and were specific for H1(0). Six mAb bound to residues 27-30 and were specific for H1(0) except one of them which strongly cross-reacted with H5 and GH5. Two mAb reacted with the entire histone H1(0) but failed to react with any of the peptides, suggesting that the corresponding epitope is a conformational antigenic determinant. In order to confirm the localization of the two distinct regions which are involved in the antigenic recognition, a synthetic decapeptide corresponding to the beginning of human H1(0) globular part (from residue 19 to residue 28) was synthesized. Inhibition experiments of the reaction between H1(0) and the various IgG1 mAb by increasing amounts of peptide-bovine serum albumin conjugates were then performed.
Dousson
S
S
Laboratoire de Biologie Moléculaire du Cycle Cellulaire, Unité INSERM 309, Département de Recherche Fondamentale, C.E.N., Grenoble, France.
Gorka
C
C
Gilly
C
C
Lawrence
J J
JJ
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D015153:Blotting, Western; D002417:Cattle; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D006657:Histones / Q000276:immunology*; D006801:Humans; D007132:Immunoglobulin Isotypes / Q000276:immunology; D008099:Liver; D008969:Molecular Sequence Data
D000911:Antibodies, Monoclonal; D000939:Epitopes; D006657:Histones; D007132:Immunoglobulin Isotypes