Immunochemical localization of the C-terminal hexapeptide of histone H3 at the surface of chromatin subunits.

Abstract

The C‐terminal hexapeptide of histone H3 of chicken erythrocytes (residues 130‐135) corresponding to the sequence Ile‐Arg‐Gly‐Glu‐Arg‐Ala (IRGERA) was prepared by solid‐phase peptide synthesis and, after coupling to bovine serum albumin, was used to elicit antibodies in rabbits. The antigenic activity of the synthetic peptide IRGERA was found to be very similar to that of the natural CN3 fragment (residues 121‐135), and it inhibited the H3‐anti H3 reaction in complement fixation, solid‐phase radioimmunoassay, and enzyme‐linked immunosorbent assay. Antibodies induced by IRGERA were found to bind equally well to IRGERA coupled to hemocyanin, to the intact H3 molecule, and to chromatin subunits (nucleosomes and core particles). The results demonstrate that the C‐terminal hexapeptide of histone H3 is located at the surface of chromatin subunits and agree with current models proposed for the spatial organization of the chromatin core particle.

Use of histone antibodies for studying chromatin topography and the phosphorylation of chromatin subunits.

“…Chicken erythrocyte core particles were prepared and characterized as previously described (Muller et al, 1982a). These particles contained the four core histones H3, H4, H2B and H2A in equal proportions but no Hl nor H5; the size of DNA was 145 : 3 bp.…”

“…All four histone antisera reacted strongly with the corresponding free histones in solution (Muller et al, 1982a(Muller et al, , 1983. The interaction between the chromatin and specific antibodies was measured in ELISA by coating the wells of microtiter plates with chromatin or core particles diluted in phosphate buffer pH 7.4 of 220 mM ionic strength, as described in previous studies (Muller et al, 1982b(Muller et al, , 1983.…”

“…Preparation of antisera and monoclonal antibodies Antibodies to histones H2A, H2B, H3, H4 and H5 were raised in rabbits by immunizing with histone-RNA complexes as described by Stollar and Ward (1970). Antisera specific for C-terminal hexapeptide 130-135 of H3, fragments 85-102 and 80-89 of H4 and 36-43 of H2B were obtained as previously described (Muller et al, 1982a(Muller et al, , 1983, and in preparation).…”

“…The usefulness of antibodies as analytical tools is enhanced when the antigenic determinants that they recognize have been identified. In recent years, about a dozen antigenic determinants (or epitopes) of the core histones have been located in their primary structures, within linear segments of 6-20 residues (Absolom and Van Regenmortel, 1977;Muller et al, 1982aMuller et al, , 1983and in preparation). The availability of several monoclonal antibodies and antisera specific for some of these histone epitopes makes it possible to determine whether these regions of the histone molecules are exposed at the surface of core particles and nucleosomes.…”

Immunocytochemical localization of histones H2B, H3 and H4 in pronuclei and four-cell stages of porcine embryos. Preliminary results

“…Rabbit specific anti-histone sera had been previously tested : H2B (Muller et al, 1985), H3 (Muller et al, 1982), and H4 (Muller et al, 1987 (Courtens et al, 1983(Courtens et al, , 1988 antisera.…”