Reconstitution of glucose transport using human erythrocyte band 3

A chromosomal histone, H2S, specific to the mouse testis has been purified. Amino acid analysis indicated lack of cysteine and a high basic amino acid content typical of histones. Specific antibodies against histones H2S have been generated in rabbits and partially purified using (NH4)2SO4 precipitation and ion-exchange chromatography. Protein transfer experiments indicate presence of antigenically similar histones in the rat and rabbit testes but not in the guinea pig and dog testes. In addition, histone complement of somatic tissues such as lung, kidney, liver and spleen lacked antigenically similar proteins. Immunocytochemical studies using peroxidase-antiperoxidase complex indicated presence of immunoreactive cells in the seminiferous epithelium which were lacking in the interstitium. These data demonstrate histone H2S to be a unique histone associated with spermatogenesis in the mouse.     

Biochemical and immunological characterization of an H2A variant from the mouse testis

A histone H2A variant, protein 'A', has been isolated and purified from the mouse testis. Amino acid composition analysis and electrophoretic properties indicate it to be apparently similar to H2A X X variant present in the mouse L1210 cells. Specific antibodies against protein 'A' have been generated in rabbits and used to study tissue and species distribution in mammals. Protein transfer experiments indicate the presence of antigenically similar proteins in somatic tissues of the mouse. Immunologically similar proteins were also detected in other mammalian testes. The data further indicate that protein A is antigenically distinct from the other members of the H2A family, H2A X 1, H2A X 2 and H2A X Z.     

Histones from Saccharomyces cerevisiae

Total histones and histone fractions isolated from Saccharomyces cerevisiae chromatin were analysed by polyacrylamide gel electrophoresis. The presence of the four histone fractions H2a, H2b, H3 and H4 was demonstrated. In addition, yeast chromatin contained a protein similar to histone H1 from mammals in molecular weight, charge and association properties with Triton X-100. However, it had a much lower lysine to arginine ratio, equal to about 3, as compared with H1 histones from higher eukaryotes. The order of electrophoretic mobilities of yeast histone fractions in acidic urea-polyacrylamide gels was similar to that observed for histones from plant sources, i.e. H4>H3>H2a>H2b>H1. Previously undetected protein (protein X) was extracted from yeast chromatin with 5 % HClO₄. The properties of this protein are under investigation.     

Differential mode of antimicrobial actions of arginine-rich and lysine-rich histones against Gram-positive Staphylococcus aureus

We previously reported the activities and modes of action of arginine (Arg)-rich histones H3 and H4 against Gram-negative bacteria. In the present study, we investigated the properties of the Arg-rich histones against Gram-positive bacteria in comparison with those of lysine (Lys)-rich histone H2B. In a standard microdilution assay, calf thymus histones H2B, H3, and H4 showed growth inhibitory activity against Staphylococcus aureus with minimum effective concentration values of 4.0, 4.0, and 5.6 μM, respectively. Laser confocal microscopic analyses revealed that both the Arg-rich and Lys-rich histones associated with the surface of S. aureus. However, while the morphology of S. aureus treated with histone H2B appeared intact, those treated with the histones H3 and H4 closely resembled each other, and the cells were blurred. Electrophoretic mobility shift assay results revealed these histones have binding affinity to lipoteichoic acid (LTA), one of major cell surface components of Gram-positive bacteria. Scanning electron microscopic analyses demonstrated that while histone H2B elicited no obvious changes in cell morphology, histones H3 and H4 disrupted the cell membrane structure with bleb formation in a manner similar to general antimicrobial peptides. Consequently, our results suggest that bacterial cell surface LTA initially attracts both the Arg- and Lys-rich histones, but the modes of antimicrobial action of these histones are different; the former involves cell membrane disruption and the latter involves the cell integrity disruption.     

Analysis of histones from the yeast Saccharomyces carlsbergensis.

Basic chromosomal proteins were isolated from the chromatin of the yeast Saccharomyces carlsbergensis by extraction with H2SO4 and were purified by ion-exchange chromatography. Electrophoresis of the purified fraction on acetic acid/urea gels revealed the presence of four main components. These four proteins were identified as histones H2A, H2B, H3 and H4 on the basis of their amino acid composition, molecular weight and solubility properties, all of which are very similar to the corresponding properties of the various histone proteins from other eukaryotic organisms. A fifth basic protein could be isolated from yeast chromatin by extraction with HClO4. The available evidence indicates this protein to be an H1-type histone. Yeast thus appears to contain a complete set of histone proteins which are strongly homologous to the histones occurring in higher eukaryotes.     

Affinity labeling of rabbit skeletal muscle phosphorylase kinase by 5′-(p-fluorosulfonylbenzoyl)adenosine

Simple mixing of acid purified histones H₃ and H₄ in equimolar quantities at low ionic strength near pH 7 does not yield the tetramer but rather a high M_r aggregate. Dialysis of acid extracted total or core histones into 2 M NaCl 150 mM phosphate (pH 7.4) followed by fractionation of the histone complexes at lower ionic strength (150 mM NaCl) results in an H₃H₄ tetramer of a structure identical to that derived from salt-extracted histones. Dialysis of acid extracted total or core histones directly into the lower ionic strength buffer with subsequent fractionation, results in H₃H₄ tetramer of closely similar structure.     

Uncoordinate synthesis of histone H1 in cells arrested in the G1 phase

It has been known for several years that DNA replication and histone synthesis occur concomitantly in cultured mammalian cells. Normally all five classes of histones are synthesized coordinately. However, mouse myeloma cells, synchronized by starvation for isoleucine, synthesize increased amounts of histone H1 relative to the four nucleosomal core histones. This unscheduled synthesis of histone H1 is reduced within 1 h after refeeding isoleucine, and is not a normal component of G1. The synthesis of H1 increases coordinately again with other histones during the S phase. The DNA synthesis inhibitors, cytosine arabinoside and hydroxyurea, block all histone synthesis in S-phase cells. The levels of histone H1 mRNA, relative to the other histone mRNAs, is increased in isoeleucine-starved cells and decreases rapidly after refeeding isoleucine. The increased incorporation of histone H1 is at least partially due to the low isoleucine content of histone H1. Starvation of cells for lysine resulted in a decrease in H1 synthesis relative to core histones. Again the ratio was altered on refeeding the amino acid. 3T3 cells starved for serum also incorporated only H1 histones into chromatin. The ratio of different H1 proteins also changed. The synthesis of the H1⁰ protein was predominant in G₀ cells, and reduced in S-phase cells. These data indicate the metabolism of H1 is independent of the other histones when cell growth is arrested.     

Unambiguous identification of histone H1 in Trypanosoma cruzi

The existence of histone H₁ has been questioned in Trypanosomatids. We report here the presence of a histone H₁ in the chromatin of Trypanosoma cruzi. This protein was purified by narrow-bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H₁ from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H₁.     

Identification of minor tightly bound H1 histone subfractions which fail to cleave their initiator methionine

Groups of CBA mice were administered [³⁵S] methionine (1 mCi/mouse). Non-histone proteins, H1 and H1⁰ histones and nucleosomal core histones were isolated from different issues by selective extractions. The measurements of radioactivity of individual bands and autoradiography of dry gels were used to identify methionine-containing and methionine-free histone variants. H1A and H1B histone variants extracted with 5% perchloric acid were methionine-free. However, minor sub-fractions of these histones which are more tightly bound to DNA (and which can be extracted only with 0.25 N HC1) contained [³⁵S] methionine and did show a higher specific activity than methionine-containing nucleosomal hitones. Cyanogen Bromide reaction which destroys non-histone proteins and methionine-containing nucleosomal histones removes radioactivity but does not alter the position of methionine-containing H1 minor bands. This indicates that the radioactive methionine occupies only the N-terminus of the H1 molecules. It is suggested that this methionine is an uncleaved initiator methionine. The presence of these methionine-containing minor H1 subfractions varies in different tissues.     

H1 histone variants in Xenopus laevis

The H1 histones from erythrocytes, livers, intestines, testes, and embryos of Xenopus laevis have been examined electrophoretically. This species has been found to contain at least five electrophoretically resolvable lysine-rich histones in addition to the presumptive H5 histone of erythrocytes. Quantitative and qualitative distinctions between the H1 histones from each source were readily observed. Three H1 histones (H1A, H1B, and H1C) were found in both embryos and adult tissues, although in varying amounts. Two other H1 histones (H1D and H1E) were found only in adult tissues. Comparative SDS gel V8 protease cleavage maps of the lysine-rich histones from testes and erythrocytes have demonstrated that the “adult-specific” H1D and H1E are not artifacts of proteolysis and may be closely related to the presumptive H5 histone. Spermatogenic cells were found to be similar to embryonic cells in being deficient in H1D and H1E. These observations suggest that H1D and H1E are enriched in cell types with low rates of cell division similar to the mammalian H1° histone. The results presented here demonstrate a previously unrecognized degree of developmental and cell-specific variance in the H1 histones of Xenopus laevis.     

Extracellular histones inhibit efferocytosis

The uptake and clearance of apoptotic cells by macrophages and other phagocytic cells, a process called efferocytosis, is a major component in the resolution of inflammation. Increased concentrations of extracellular histones are found during acute inflammatory states and appear to contribute to organ system dysfunction and mortality. In these studies, we examined the potential role of histones in modulating efferocytosis. We found that phagocytosis of apoptotic neutrophils or thymocytes by macrophages was significantly diminished in the presence of histones H3 or H4, but not histone H1. Histone H3 demonstrated direct binding to macrophages, an effect that was diminished by preincubation of macrophages with the opsonins growth arrest–specific gene 6 (Gas6) and milk fat globule–epidermal growth factor (EGF) 8 (MFG-E8). Incubation of histone H3 with soluble α_vβ₅ integrin and Mer, but not with α_vβ₃, diminished its binding to macrophages. Phagocytosis of apoptotic cells by alveolar macrophages in vivo was diminished in the presence of histone H3. Incubation of histone H3 with activated protein C, a treatment that degrades histones, abrogated its inhibitory effects on efferocytosis under both in vitro and in vivo conditions. The present studies demonstrate that histones have inhibitory effects on efferocytosis, suggesting a new mechanism by which extracellular histones contribute to acute inflammatory processes and tissue injury.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

Expression and purification of the full murine NPM2 and study of its interaction with protamines and histones

Mouse nucleoplasmin M.NPM2 was recombinantly expressed and the protein consisting of the complete sequence was purified and characterized. Similar to its Xenopus laevis X.NPM2 counterpart, the protein forms stable pentameric complexes and exhibits an almost undistinguishable hydrodynamic ionic strength-dependent unfolding behavior. The interaction of N.PM2 with histones and mouse P1/P2 protamines revealed that these chromosomal proteins bind preferentially to the distal part of the nucleoplasmin pentamer. Moreover, the present work highlights the critical role played by histones H2B and H4 in the association of the histone H2A-H2B dimers and histone octamer with nucleoplasmin.     

Age-related decline in histone H1 fraction in human diploid fibroblast cultures

The age-related increase in cell volume and nuclear size of cultured human diploid fibroblasts reflected the accumulation of proteins in cytoplasm and nuclei of growth-retarded fibroblasts.Determination of the amount of nuclear proteins, which were fractionated into 0.15 M NaCl-soluble proteins, 0.4 N H₂SO₄-extractable proteins and residual acidic proteins, indicated that age-related increase in nuclear proteins was due mainly to the accumulation of residual acidic proteins.However, electrophoretic fractionation of histones from various passages of fibroblast cultures on acid urea polyacrylamide gel revealed that the relative amount of H1 fraction decreased with in vitro aging. This was further confirmed by mixing experiments examining the distribution of radioactivity of the histones from cell mixtures of young and senescent cultures labeled with [³H]lysine or [¹⁴C]lysine.A pulse label and chase experiment indicated that the observed decrease in the amount of histone H1 was mainly due to decrease in synthesis of histone H1 in senescent human fibroblast cultures.     

Expression and purification of recombinant human histones

Nucleosomes reconstituted from bacterially expressed histones are useful for functional and structural analyses of histone variants, histone mutants, and histone post-translational modifications. In the present study, we developed a new method for the expression and purification of recombinant human histones. The human histone H2A, H2B, and H3 genes were expressed well in Escherichia coli cells, but the human histone H4 gene was poorly expressed. Therefore, we designed a new histone H4 gene with codons optimized for the E. coli expression system and constructed the H4 gene by chemically synthesized oligodeoxyribonucleotides. The recombinant human histones were expressed as hexahistidine-tagged proteins and were purified by one-step chromatography with nickel-nitrilotriacetic acid agarose in the presence of 6 M urea. The H2A/H2B dimer and the H3/H4 tetramer were refolded by dialysis against buffer without urea, and the hexahistidine-tags of the histones in the H2A/H2B dimer and the H3/H4 tetramer were removed by thrombin protease digestion. The H2A/H2B dimer and the H3/H4 tetramer obtained by this method were confirmed to be proficient in nucleosome formation by the salt dialysis method. The human CENP-A gene, the centromere-specific histone H3 variant, contains 28 minor codons for E. coli. A new CENP-A gene optimized for the E. coli expression system was also constructed, and we found that the purified recombinant CENP-A protein formed a nucleosome-like structure with histones H2A, H2B, and H4.     

Histones and nucleosomes in Archaea and Eukarya: a comparative analysis

Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3+H4)₂ tetramer. Received: January 22, 1998 / Accepted: February 16, 1998     

H1° histones of normal and cancer human cells

Summary H1° histones were purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis from human lung carcinoma (line DMS79), human hepatoblastoma (HepG2), human adult lung and human adult and fetal liver. The purified human H1° histones were analyzed for their amino acid composition and terminal residues. The comparative analysis of the amino acid compositions of the different human H1° histones showed that: (a) all the H1° preparations have the characteristically high lysine content associated with a low arginine content, which distinguishes outer histones from core histones; (b) H1° is distinguishable from other H1 histones by the presence of methionine and histidine; (c) H1° histones from human adult, fetal and cancer cells are very similar in amino acid composition, and in cancer cells the level of the H1° histone is not inversely related with cell growth rate nor with the expression of the -fetoprotein gene.     

Histone H2A subfractions and their phosphorylation in cultured Peromyscus cells

Patterns of histone phosphorylation and histone H2A subfractionation have been compared in cultured cell lines from two species of deer mice, Peromyscus eremicus and Peromyscus boylii, which differ considerably in their content of heterochromatin but which contain essentially the same euchromatin content. DNA measurements by flow microfluorometry indicated that P. eremicus cells contained 34.2% more DNA than P. boylii cells, and C-band chromosome analysis indicated that the extra DNA in P. eremicus was present as constitutive heterochromatin. Subfraction of histone H2A by acid-urea polyacrylamide preparative gel electrophoresis in the presence of non-ionic detergent showed that each cell line contained two H2A subfractions. Incorporation of ³²PO₄ into these histones indicated that the steady state phosphorylation of the two H2A subfractions was not the same, the more hydrophobic H2A being greater than two times more phosphorylated than the less hydrophobic H2A in both cell lines. A comparison of the two cell lines indicated that the cell line with 34.2% greater constitutive heterochromatin contained a similar excess (29%) in its ratio of the more highly phosphorylated, more hydrophobic H2A subfraction to the less hydrophobic H2A subfraction. It is suggested that this enrichment of the more highly phosphorylated, more hydrophobic H2A subfraction may be related to the amount of constitutive heterochromatin present in the genome.     

Comparative electrophoretic properties of histones from cells of the mosquito Aedes aegypti and of the fruitfly Drosophila melanogaster

Electrophoretic mobility of histones from cell cultures of Drosophila melanogaster and of the mosquito Aedes aegypti was determined in polyacrylamide gels in the presence of different concentrations of urea. Great similarity in the electrophoretic behavior of H3, H2A, H2B and H4 histones from the two insect species was found. Histone H1 of Aedes under all conditions tested had a markedly higher electrophoretic mobility than H1 of Drosophila, but differed only slightly from H1 histones of mouse and of hamster.As can be deduced from the mobility of Aedes H1 in the presence of sodium dodecyl sulphate its molecular weight is smaller than that of Drosophila H1 and is very close to the molecular weight of the main component of mouse H1 histone. Heterogeneity of the H1 histone from Drosophila is demonstrated. This heterogeneity is due to phosphorylation of a part of H1 molecules, since it disappears after the treatment of H1 preparations by alkaline phosphatase. Phosphorylated components were not found in the H1 of Aedes.Thus two representatives of Diptera, Aedes and Drosophila possessing polytene chromosomes at the larval stage of development have H1 histones with markedly different primary structures. This pact demonstrates that the polytenization of chromosomes may occur in species with markedly different H1 histones.at Moscowat Nijmegen