Localization of Histone H5 in the Subunit Organization of Chromatin Using Immunoelectron Microscopy

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Abstract
In avian erythroid cells the erythrocyte-specific histone H5 is involved, like H1, in the packing of nucleosomes in the 25-nm chromatin fibers. The distribution of histone H5 along the polynucleosomal chains was visualized here by immunoelectron microscopy. Trinucleosomes from chicken erythrocytes and liver were used to test the specificity of the reaction with purified rabbit anti-H5 antibodies at various ionic strengths (5-80 mM). Long-chain chromatin was then reacted with anti-H5 antibodies and with sorted monomeric ferritin conjugate under chosen conditions. The antigenic determinants of histone H5 in the 25-nm fiber of long-chain chromatin (at 80 mM NaCl) are as accessible to the specific antibodies as in trinucleosomes. When the immunocomplexes were examined by EM in a low-ionic-strength buffer, permitting maximum extension of the chromatin structure on the grid, clusters of compacted nucleosomes were seen, separated by short regions of relaxed nucleosomes. Single nucleosomes enlarged by the antibodies are sometimes visible in the extended domains. Histone H5 is located primarily on series of adjacent nucleosomes but it can also be found on single nucleosomes located in the H1-enriched extended domains.