The histones of rainbow trout erythrocytes include an erythrocyte-specific histone.

The erythrocyte histones of rainbow trout were compared with those of goose by polyacrylamide gel electrophoresis. A band analogous to goose erythrocyte-specific histone V, but not identical in relative mobility or quantity, was found to be a component of trout erythrocyte histone. A similar component was also found in carp erythrocyte histone, but it was absent from trout liver histone. To reveal this band clearly, it was advantageous to displace the histone III monomer by oxidation. To verify the character of this protein, each of the main erythrocyte histones of trout were purified by chromatography on Amberlite CG-50, eluted with guanidinium chloride, and then further purified by exclusion chromatography on Bio-Gel P-60. Amino acid compositions of corresponding trout and goose histones, including that of the erythrocyte-specific histone, were sufficiently similar to establish their analogous identities. In general, the chromatographic and electrophoretic properties of histones I, IIb1, IIb2, and V from trout differed more from those of goose, than did their gross amino acid compositions. Comprehensive fractionation and characterization is necessary to extablish identities of corresponding histone fractions, An extensive quantitative variability was found among erythrocyte-specific histones of fish. This must be reconciled with hypothetical roles for this histone in erythropoiesis.     

Tissue-specific histones in the erythrocytes of chicken and turtle

Mature erythrocytes from Leghorn chickens contain lysine-rich histone F1 and a tissue-specific histone F2c. The composition of the F1 fraction was found to be similar to the F1 histones in higher vertebrates. In the erythrocytes of a sea turtle (Chelonia mydas), only lysine-rich histones F1 could be detected. One of these fractions (F1b) differed in amino acid composition from the typical F1 histones described in the literature. The F1b histone fraction was not found in turtle liver. Chromatographic analysis of tryptic peptides of the chicken erythrocyte F1 and F2c histones and of the turtle erythrocyte F1a and F1b histones revealed considerable similarities between these four fractions, thus indicating their possible phylogenetic relationships.     

Histone-like protein fractions of Kluyveromyces fragilis and their relation to the cell cycle.

Protein fractions were obtained from Kluyveromyces fragilis by pH titration, a technique used for histone extraction, following the inclusion of a distilled water extraction stage, and the fractions partially characterized. The inclusion of a distilled water step resulted in a tenfold purification of the fraction obtained at pH 2.20 as compared with pH titration alone. In synchronous cultures induced with 2'-deoxyadenosine or prepared by selection this fraction displayed a stepwise accumulation, and doubled in quantity at a point about one third of a cycle after cell division. The fraction obtained at pH 1.35 resembled calf thymus f2a2 histone in its extraction properties and amino acid composition, and also showed a possible stepwise accumulation. Other fractions appear to accumulate exponentially.     

Characterization of post-translational modifications of the linker histones H1 and H5 from chicken erythrocytes using mass spectrometry

Histone linker proteins H1 and H5 were purified from chicken erythrocyte cell nuclei under nondenaturing conditions. The purified linker histones were analyzed using in-solution enzymatic digestions followed by nanoflow reverse-phase high-performance liquid chromatography tandem mass spectrometry. We have identified all six major isoforms of the chicken histone H1 (H101, H102, H103, H110, H11R and H11L) and, in addition, the specialist avian isoform H5. In all the histone variants, both the acetylated and nonacetylated N (alpha)-terminal peptides were identified. Mass spectrometry analysis also enabled the identification of a wide range of post-translational modifications including acetylation, methylation, phosphorylation and deamidation. Furthermore, a number of amino acids were identified that were modified with both acetylation and methylation. These results highlight the extensive modifications that are present on the linker histone proteins, indicating that, similar to the core histones, post-translational modifications of the linker histones may play a role in chromatin remodelling and gene regulation.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

Histone phosphokinase activity in nuclear and cytoplasmic cell fractions from normal and regenerating rat livers

1. Liver cell fractions were prepared by non-aqueous procedures and nuclei were also obtained in a hyperosmotic sucrose medium. Histone phosphokinase activity, assayed with histone F1 as substrate, was present in the soluble fraction of the cytoplasm and also bound on to the chromatin fraction of the nucleus. 2. The activity of the enzyme increased sixfold in nuclei from regenerating livers 22h after partial hepatectomy. 3. The enzyme bound in the nucleus was only marginally activated by 1mum-3':5'-cyclic AMP which stimulated the cytoplasmic soluble enzyme fourfold. 4. Nuclei prepared by the non-aqueous technique were also able to phosphorylate histones F2a and F3 and showed histone phosphatase activity with histone F1 phosphate as substrate.     

Studies on the primary structure of chicken erythrocyte histone fraction V

Histone fraction V was prepared by selective acid extraction from chicken erythrocyte nuclei. Amino acid sequence studies on the tryptic and thermolytic peptides are reported. Only a limited amount of overlapping sequence data was obtained.     

GLUTARALDEHYDE FIXATION OF ISOLATED EUCARYOTIC NUCLEI : Evidence for Histone-Histone Proximity

Isolated chicken erythrocyte nuclei have been incubated with dilute concentrations of the bifunctional cross-linking agent glutaraldehyde (0–20 mM) in order to stabilize histone-histone interactions within the native nucleus. The kinetics of the disappearance of acid-soluble histones, free amino groups, and of individual histones have been observed to be pseudo first-order. Apparent first-order rate constants for the disappearance of individual histones correlate with the lysine mole percent of that fraction and follow the ranking, k_app: F1 > F2C > F2B ≥ F2A2, F2A1, F3. Histone polymers were observed to form very rapidly during the fixation reaction. Partial fractionation and amino acid analyses of these polymers support the view that they are composed principally of cross-linked (F2C)_n molecules (where n = 2 to ~8). The rate of glutaraldehyde reaction with free amino groups in histones is drastically reduced in solvents that promote chromatin decondensation (i.e., low ionic strengths in the absence of divalent cations) whereas the formation of cross-linked F2C polymers is less severely reduced. It is proposed that some F2C histones exist in close proximity within the isolated erythrocyte nucleus.     

Fractionation of whole histone by partition chromatography on Sephadex G-25.

Whole histone from calf thymus was fractionated by partition chromatography on the basis of distribution between an aqueous phase immobilized on Sephadex G-25 beads and mobile organic phases containing various concentrations of trichloroacetic acid. The chromatography was carried out by stepwise elution with five upper phases from water and butanol-2 solvent systems containing 4 M urea and 0.1-1.5% trichloroacetic acid, and finally water. Of the six peaks obtained, two (peaks 1 and 2) contained arginine-rich histones. Although these peaks were still heterogeneous electrophoretically, the band corresponding to F2al was observed only in the electrophoretic pattern of peak 1 and the main fraction in peak 2 was F3. A histone fraction having nearly equimolar amounts of arginine and lysine was obtained from peak 3. Its amino acid composition was similar to that of F2a2. Slightly lysine-rich histone obtained from peak 4 showed an amino acid composition typical of F2b. Peak 5 contained a histone fraction with a ratio of lysine/arginine of 6.14, showing a single band on gel-electrophoresis. Very lysine-rich histone (F1) was obtained from peak 6, and the electrophoretic pattern of this fraction showed a single band.     

The amino acid composition and some properties of histones

Some of the properties and the amino acid compositions of the histones of calf thymus, calf liver, fowl erythrocytes, and of a protamine-like material isolated from rooster sperm were described. The amino acid compositions of the histones were rather similar except that no methionine was found in the fowl erythrocyte histone. In the fowl, histones are found in the somatic chromosomes and protamines are found in the sperm chromosomes. This shows that great variations in chromosome composition can exist in an organism. Histone is digested by pepsin both when isolated and when in the chromosome.     

Characterization of the six chicken histone H1 proteins and alignment with their respective genes

Six histone H1 subtypes and histone H5, isolated from chicken erythrocyte nuclei, were visualized on acid/urea polyacrylamide gels. Four of the H1 subtypes have been purified to homogeneity by fast protein liquid chromatography on a strong cation exchange column. The other two subtypes were obtained as enriched fractions from the same fast protein liquid chromatography experiments. Because six chicken H1 genes have been completely sequenced (Coles, L.S., Robins, A. J., Madley, L.K., and Wells, J. R. E. (1987) J. Biol. Chem. 262, 9656-9663), it was possible to assign each of the six H1 proteins to a specific gene after amino acid sequence analysis of peptides derived from the subtypes.     

Histones from Trypanosoma lewisi nuclei]

A preparation of total histones has been isolated for the first time from the purified fractions of T. lewisi cell nuclei and characterized in terms of its chemical composition and RNA-polymerase activity. A special attention during the isolation procedure was given to the repression of proteolytic degradation of the histones. The amount of protein in the chromatin is equivalent to that of DNA. The amino acid composition and heterogeneity of the protein during polyacrylamide gel electrophoresis in an acid system and in the presence of sodium dodecyl sulfate are typical for histones. Using two-dimensional electrophoresis, differential staining of electrophoregrams and ion-exchange chromatography on CM-cellulose the total preparation has been found to be made up of five fractions: two -- arginine-rich (one of them identical to histone H4, the other being similar to histone H3 from calf thymus); two -- moderately lysine-rich fractions, slightly differing in their properties from histones H2A and H2B from calf thymus, and one specific fraction with mol. weight of 16 000 and an extremely high positive charge. The above methods in combination with specific extraction have been used to demonstrate the absence of a typical lysine histone in the preparation, which is correlated with the absence of typical methaphase chromosomes during mitosis in T. lewisi.     

Association of proteinases with histones from rat thymus nuclei]

Histone H2A, H2B, and H1--specific proteinases tightly associated with histones were shown to be present in rat thymus nuclei. The activity of proteinases tightly associated with histones increases after exposure of animals to gamma-rays. The denatured DNA activated the histone H1-specific proteinase. These proteinase dissociated from histones in the presence of dithiothreitol. The histones and proteinases were divided into fractions by chromatography on DEAE-cellulose in the presence of 5 mM dithiothreitol.     

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.     

Isolation and characterization of histones and other acid-soluble chromosomal proteins from Physarum polycephalum

Chromosomal basic proteins were isolated from amoebal and plasmodial stages of the acellular slime mold Physarum polycephalum. Polyacrylamide electrophoresis on high resolution acid-urea gels separated the five histone fractions in the sequence H1, H2A, H2B, H3, and H4. Under these electrophoretic conditions Physarum histones migrated more like plant (rye) than animal (calf) histones. Furthermore, Physarum histones H1, H2A, and H2B have higher molecular weights on sodium dodecyl sulfate (SDS) gels than the corresponding calf fractions. No differences were detected between amoebal and plasmodial histones on either acid-urea or SDS-polyacrylamide gel electrophoresis. Amoebal basic proteins were fractionated by exclusion chromatography. The five histone fractions plus another major acid-soluble chromosomal protein (AS) were isolated. The Physarum core histones had amino acid compositions more closely resembling those of the calf core histones than of rye, yeast, or Dictyostelium. Although generally similar in composition to the plant and animal H1 histones, the Physarum H1 had a lower lysine content. The AS protein was extracted with 5% perchloric acid or 0.5 M NaCl, migrated between histones H3 and H4 on acid-urea polyacrylamide gels, and had an apparent molecular weight of 15 900 on SDS gels. It may be related to a protein migrating near H1. Both somewhat resembled the high mobility group proteins in amino acid composition.     

Esters of serine and threonine in hydrolysates of histones and protamines, and attendant errors in amino acid analyses of proteins

1. Partial acid hydrolysates of histones from various origins and of protamine were analysed by a two-dimensional ionophoretic procedure to reveal strongly acidic ninhydrin-positive components. 2. Histone fractions prepared by extraction with sulphuric acid gave rise to spots identified as serine O-sulphate and threonine O-sulphate. These two compounds, which were not found in hydrolysates of corresponding fractions prepared by extraction with hydrochloric acid, were artifacts. 3. Hydrolysis of proteins in the presence of traces of sulphate can lead to the formation of the O-sulphates of serine and threonine. This can cause errors, which may sometimes be serious, in amino acid analyses of proteins. 4. O-Phosphoserine was obtained in small amounts from some histone fractions and from protamine, but was undetectable in other histone fractions, notably those of lower lysine content.     

The dissociation of chicken erythrocyte deoxyribonucleoprotein and some properties of its partial nucleoproteins

Histones were completely dissociated from their native complex with DNA in 2.0m-sodium chloride. Histone fractions IIb, V and I were dissociated in 1.2m-sodium chloride, fractions V and I in 0.7m-sodium chloride and fraction I in 0.45m-sodium chloride. Repeated extraction of partial dRNP (deoxyribonucleoprotein) preparations with sodium chloride of the same concentration as that from which they were prepared resulted in release of histones that previously had remained associated with the DNA of the complex. Gradual removal of histones from dRNP was paralleled by an improvement in solubility, a decrease in wavelength of the u.v.-absorption minimum, and a fall in sedimentation coefficient of the remaining partial dRNP. X-ray diffraction patterns of partial dRNP preparations showed that removal of histone fractions I and V from dRNP did not destroy the super-coil structure of the dRNP, but further removal of histones did. Infrared spectra of partial dRNP preparations showed that in native dRNP histone fraction I was present in the form of extended, isolated polypeptide chains, and that the other histone fractions probably contain a helical component that lies roughly parallel to the polynucleotide chains in the double helix and an extended polypeptide component that is more nearly parallel to the DNA helix axis. An analysis of the sedimentation of partial dRNP preparations on sucrose gradients showed that native dRNP consists of DNA molecules each complexed with histone fractions of all types.     

Separation of histones by reverse-phase high-performance liquid chromatography: analysis of the binding of carcinogens to histones

Reverse-phase high-performance liquid chromatography (RP-HPLC) has been examined as an approach to the rapid analysis of carcinogen-modified histones. H1 and core histone fractions were prepared by differential acid extraction of 0.35 M NaCl-extracted rat liver nuclei previously exposed to [3H]-7r,8t-dihydroxy-9t, 10t-oxy-7,8,9, 10-tetrahydrobenzo(a)pyrene [( 3H]BPDE-I). Using a sodium perchlorate-phosphate (PCP)/acetonitrile solvent system, the H1 histone fraction was eluted from an Aquapore RP-300 column in five peaks (P1-P5). The core histone fraction was resolved into eight peaks (C1-C8) using a PCP/acetonitrile-methanol solvent system. The histones of each peak were identified by sodium dodecyl sulfate and Triton/acid/urea gel electrophoresis or amino acid analysis as follows: P1, H1 degrees; P2-P5, four different H1 variant fractions; C1, H4 + A24; C2, H2B; C3, H2A X 2 + to one H2A variant; C4, H2A.1; C5, H2A.1 + two H2A variants; C6, H3.2; C7, H3.3; C8, H3.1. The bulk of radioactivity was covalently bound to histone H2A, which had higher specific activities of BPDE-I than other histones. Significant amounts of radioactivity were observed in histones H3 and H1, but not in histones H2B and H4. These RP-HPLC systems have the advantages of an analysis time within 60 min, the identification of H1, H2A, and H3 variants, and the quantitative analysis of radioactive histones. These results indicate that these RP-HPLC systems are very useful to analyze the binding of carcinogens to histones.     

Histones of the fungi, Endomyces magnusii and Neurospora crassa]

Histones of Endomyces magnusii and Neurospora crassa were found to consist of four main fractions similar to calf thymus histones in their electrophoretic mobilities, molecular sizes and chromatographic behaviour on Akrilex P-60. Two of them are homologous to the most conservative histones H3 and H4. Other two fractions correspond to the histones H2A and H2B; however, they have some pecularities. A fraction of N. crassa histones corresponding to the H2B was isolated in a homogeneous state by means of gel filtration. It appeared to be very similar to calf thymus histone H2B in its amino acid composition.