Heterogeneity in the interaction of chromatin subunits with anti-histone sera visualized by immuno-electron microscopy

Drora Goldblatt*, Michael Bustin, Ruth Sperling

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

12 Scopus citations

Abstract

Chromatin repeating units were prepared by in situ digestion of rat liver nuclei with staphylococcal nuclease and fractionation on isokinetic sucrose gradients. The subunits were spread on electron microscope grids which had been precoated with bovine serum albumin. To these chromatin subunits antisera specific to either histone H2B, H3, H4 and H2A were added. Each antiserum contained antibodies which reacted with the chromatin repeating units, resulting in a significant increase in their diameter as detected by immuno-electron microscopy. The number of the chromatin subunits to which antibodies were bound was dependent on the concentration of sera used. At saturation 94% of the particles reacted with anti-H2B sera but only 73, 57 and 46% of the nucleosomes reacted with anti-H3, anti-H4 and anti-H2A sera, respectively. No increase in nucleosome diameter was obtained upon incubation with normal rabbit serum. These results indicate that the nucleosomes, spread on grids, are heterogeneous with respect to the availability of the antigenic determinants of their histones. The diameter of the nucleosome-antibodies complex is larger than that of the unreacted subunit. The maximum average diameters observed were 205 Å, 210 Å for anti-H2A and anti-H4, respectively, and 250 Å and 260 Å for anti-H3 and anti-H2B, respectively. Individual antibody molecules attached at two different sites on the nucleosomes have been observed. Anti-H2B molecules protrude from the periphery of the nucleosome more than anti-H4 and anti-H2A. The observed diameter of the reacted nucleosomes may reflect the spatial organization of the antigenic determinants of the various histones within the nucleosomes.

Original languageEnglish
Pages (from-to)1-14
Number of pages14
JournalExperimental Cell Research
Volume112
Issue number1
DOIs
StatePublished - 1 Mar 1978
Externally publishedYes

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