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.     

DNA repeat lengths of erythrocyte chromatins differing in content of histones H1 and H5

Among the erythrocytes of chicken, trout, carp, and sucker, the relative proportion of the lysine-rich histone H5 varied from 20 to 0% of the total histones. Following digestion of nuclear chromatin with micrococcal nuclease, each of them displayed a longer DNA repeat length and greater repeat length heterogeneity than found in liver chromatin. Fish erythrocytes possessed similar repeat lengths of 207-209 base pairs which was 10-12 base pairs shorter than in chicken erythrocyte chromatin and approximately 10 base pairs longer than in liver chromatin. No correlation existed between the DNA repeat length or repeat length heterogeneity and the relative proportion of H5.     

Octamer reconstitution from acid-extracted chicken erythrocyte histones

Histone octamers have been reconstituted from acid-extracted chicken erythrocyte histones. By the criteria of molecular size on exclusion chromatography as well as sedimentation velocity and conformational properties established by circular dichroism, fluorescence spectroscopy and imido-ester cross-linking, the reconstituted octamers have a structure identical to that of salt-extracted octamers.     

The stepwise removal of histones from chicken erythrocyte nucleoprotein

1. A fractionation of chicken erythrocyte histones was achieved simultaneously with their extraction from saline-washed nuclei by stepwise titrations to progressively lower pH values. 2. Different acids and dilute buffer solutions of comparable pH behaved similarly in stepwise extractions of histones. 3. The histone preparations so obtained were characterized by their amino acid composition and behaviour on zone electrophoresis in starch gels. 4. The fractionation by titration was quite sharp at appropriate pH ranges, and the histone fraction that is apparently unique to avian erythrocytes was obtained without contamination by other histone fractions. 5. Histones prepared by stepwise titration were fractionated further by cation-exchange and exclusion chromatography. The chromatographic behaviour and amino acid composition of the components permitted comparison with histones prepared by other methods. 6. Histone fraction IIb was resolved into its subfractions IIb(1) and IIb(2) by exclusion chromatography on Bio-Gel P-60. 7. Histone fractions III and IV, previously reported to be absent from chicken erythrocyte nuclei, were found in extracts made at pH1.     

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.     

Phosphate incorporation into "nuclear" residual proteins of goose erythrocytes

Minor fractions of “residual proteins” from erythroblast-enriched avian erythroid cells were found to incorporate significantly larger amounts of radiophosphate than similar fractions from mature erythrocytes. This higher level of incorporation could not be detected in the bulk, unfractionated residual proteins from cell populations, respectively, enriched in erythroblasts, reticulocytes, or mature erythrocytes, probably because of variable amounts of phosphate-free protein. It was confirmed that when avian erythroid cells incorporated radiophosphate, specific activities of chromosomal “residual proteins” were orders of magnitude greater than those of histones, although levels of alkali-labile phosphate were only a fewfold higher.     

Radioiodination of chicken erythrocyte histones H4 and H5 in chromatin.

The conformational state of histones in isolated chicken erythrocyte chromatin was studied using procedures developed for probing surface proteins on membranes. Under controlled conditions, only exposed tyrosyl residues react with iodide radicals, generated either by the oxidant, chloramine-T (paratoluenesulfonyl chloramide), or the enzyme lactoperoxidase, giving monoidotyrosine. Using 125-iodine, this study compared the reactive tyrosines in free and bound histones H4, and H5. The relative extent of iodination of these histones within (H4) and outside (H5) of the nucleosomes was measured after extraction and gel electrophoresis. Each of the histones was further analyzed for the extent of specific tyrosine iodination by separating the tryptic peptides by high voltage electrophoresis. The identity of the labeled peptide was determined by dansylation of the amino acids present in each hydrolyzed peptide. The results show that there is a difference in the conformational arrangement of these histones on chromatin and in the free forms, since in chromatin not all tyrosine residues are as accessible for iodination as in the denatured state. Residue 53 of histone H5 for instance is more reactive than residues 28 and 58, indicating that the segments containing the latter residues are involved in either protein-DNA or protein-protein interactions. In histone H4, preferential labeling of 2 of the 4 tyrosines present was also observed.     

Gene conversion drives GC content evolution in mammalian histones

To examine the evolutionary influence of gene conversion on DNA base composition, I analysed an exhaustive dataset of histone paralogous genes from human and mouse. I show that those gene copies that belong to subfamilies of very similar sequences (presumably undergoing gene conversion) have a higher GC content than unique gene copies (presumably not undergoing gene conversion). Thus, it seems that gene conversion is a biased process that tends to increase the DNA GC content, a conclusion that has implications for the evolution of isochores in vertebrates.