The histone 2A faily of proteins is shown to consist of eight protein species. In addition to the previously described mammalian 2A variants H2A.1 and H2A.2, we describe two variants which are separable from each other and from variants 1 and 2 on both sodium dodecyl sulfate and acetic acid-urea gels. These two proteins H2A.X and H2A.Z are termed heteromorphous variants to distinguish them from the predominating form and its homeomorphous variants which require nonionic detergents for their resolution. The two heteromorphous variants are present in nucleosomal core particles isolated from mouse L1210 cells. In addition, these variants are found in normal mouse tissues, human HeLa cells, and chicken erythrocytes. On sodium dodecyl sulfate gels, one variant, H2A.X, has an apparent molecular weight approximately 1000 larger than H2A.1 and comprises approximately 11% of the total 2A in mouse L1210 cells. The second variant, H2A.Z, has an apparent molecular weight approximately 600 smaller than H2A.1 and comprises approximately 4% of the total 2A in mouse L1210 cells. The two heteromorphous variants have the same arginine/lysine ratio as H2A.1. In addition, a fraction of each of the four variants (approximately 11% in L1210 cells) is combined with ubiquitin. The molar sum of these eight H2A species approximately equals the number of moles of H4, H2B, or H3 in chromatin.

Histone 2B in mouse and man can be modified by the post-translational addition of ubiquitin. In mouse L1210 cells, both H2B variants are modified, however only to the extent of 1-1.5% compared with about 11% of H2A. Analysis of cyanogen bromide peptides shows that ubiquitin is attached to the C-terminal part of the histone.

Two‐dimensional gel analysis of histones from extracts of nuclei, cells, and tissues is described. A discontinuous buffer system which concentrates the sample was used to increase resolution in both dimensions and also to allow the direct loading of HCl extracts of chromatin, nuclei, cells, and tissues. Stained one‐dimensional gels are used as sample gels for the second dimension, cetyltrimethylammonium bromide being used to solubilize the proteins in the dye‐protein complex. These methods enable one to purify proteins through acetic acid/urea/Triton, acetic acid/urea, and sodium dodecyl sulfate gels without eluting them from the gels. The method is also compatible with the use of protamine to displace histones from nuclei and nucleosomes separated in chromatin gels.