A new h.p.l.c. isolation procedure for chicken and goose erythrocyte histones

Total chicken erythrocyte histones were separated by reversed-phase h.p.l.c. using a multi-step acetonitrile gradient in a very short time (35 min). The proteins were eluted in the following order: H1, H5, H2B, H2A.2, H4, H2A.1 and H3.2. Applying a special gradient system adapted for the separation of very-lysine-rich histones, chicken erythrocyte H5 was resolved into two subfractions. Their electrophoretic mobilities were identical in both SDS and acetic acid/urea/Triton polyacrylamide-gel electrophoresis, but different in free-flow electrophoresis. Amino-acid-sequence analyses revealed that the two components only differ with respect to position 15, one having glutamine in that position and the other arginine. A separation of histones prepared from goose erythrocytes disclosed no H5 subfractionation. Furthermore, histones obtained from anaemic-chicken blood were analysed by the above-mentioned h.p.l.c. conditions. An alteration in the relation of H1 to H5 was detected, but no further differences in the number and quantity of the histones and histone variants were observed as compared with the corresponding proteins processed from normal-chicken blood.

Visualization of thermally denatured nucleosomes

Electron images of biological specimens composed of periodic structures are generally spurious. The reason is that unit structures may superimpose along the direction of view, and a “see through” image is formed especially from negatively stained and unstained objects. Methods to detail features of such objects from two dimensional images included optical and computer processing or combination of optical Fourier transformation and filtering. However, rotational superimposition may be employed when objects have simple symmetry.We described here the use of stroboscope illumination photography (stroboscopy) to demonstrate the distribution of globular units along nucleoprotein fibers of thermally denatured mononucleosomes. The information was further used to predict the structure of intact nucleosomes as seen from bright field (BF) and dark field (DF) electron micrographs.We previously obtained coSnsitometric scans from high resolution DF electron micrographs of partially relaxed unstained nucleosomes isolated from nuclease (EC. 3.1.4.7) digested goose erythrocyte nuclei and showed the presence of high density, periodic, contiguous globular regions (3-4 nm) along the DMA moiety.

Evidence for Erythrocyte-Specific Histone Modification and Structural Changes in Chromatin During Goose Erythrocyte Maturation

Regenerating blood of geese suffering from phenylhydrazine anaemia was separated into ‘mature’ and ‘immature’ cell populations by centrifugation through a barrier of BSA. Socalled ‘mature cells’ consisted of mainly mature erythrocytes and ‘immature cells’ included two-thirds polychromatic and younger erythroblasts. Histone proteins, dissociated from isolated nuclei of both populations of cells by sequential extraction with citric acid and hydrochloric acid, were compared and the nuclei were examined by electron microscopy. Erythrocyte-specific histone V (f2c) was fully extracted from immature nuclei at pH 2.0, but only partially extracted at the same pH from mature nuclei. An inverse correlation was found between relative case of extraction and alkali-labile phosphate content of purified samples of histone V. The more readily dissociated fraction of histone V from immature nuclei had a higher phosphate content than the less readily dissociated component V from immature and mature nuclei. Chromatin in mature nuclei became tightly congealed after only partial extraction of histone V at pH 2.0, but loosened visibly after subsequent full extraction of histone V at pH 1.8. In contrast, chromatin in immature nuclei never became totally congealed. Histone V may be a tissue-specific agent involved in packing of DNA within chromatin fibrils. During erythropoiesis, progressive decrease of histone V phosphate may lead to its increased binding affinity for chromatin and thus to the gradual transformation of the erythrocyte genome into a permanently repressed state.

Plasma Membrane and Nuclear Proteins of the Goose Erythrocyte

Plasma membrane and nuclear fractions have been prepared from mature goose erythrocytes. Examination of the nonhistone protein of the nuclear fraction by sodium dodecyl sulphate – polyacrylamide gel electrophoresis reveals a limited number of molecular weight species some of which are peculiar to the nuclear fraction.Electrophoretic comparison of the goose plasma membrane proteins with those of the human erythrocyte reveals many similarities. In particular, three large molecular weight species occur in both cells. Their function appears to predate the evolutionary loss of the nucleus.

Phosphate content of goose erythrocyte histones

Although chromatographic profiles and amino acid compositions of erythrocyte histones from normal and regenerating goose blood did not differ, characteristic fractions from regenerating blood contained more alkali-labile phosphate than did their counterparts from normal blood. Furthermore, the phosphate content of the erythrocyte-specific component was appreciably greater than that of any other histone.The problem of distinguishing between contamination and valid histone phosphate is considered; complex formation between nuclear phosphoprotein and specific histone fractions could not be unequivocally eliminated. The pronounced phosphate levels in the erythrocyte-specific histone V are discussed in relation to the prospective roles of histone phosphorylation.