Metacarpophalangeal pattern (MCPP) profile analysis in a family with triphalangeal thumb

A new two-step high-performance liquid chromatography (HPLC) procedure has been developed to separate modified histone H1 subtypes. Reversed-phase (RP) HPLC followed by hydrophilic-interaction liquid chromatography (HILIC) was used for analytical and semi-preparative scale fractionation of multi-phosphorylated H1 histone subtypes into their non-phosphorylated and distinct phosphorylated forms. The HILIC system utilizes the weak cation-exchange column PolyCAT A and an increasing sodium perchlorate gradient in a methanephosphonic acid-triethylamine buffer (pH 3.0) in the presence of 70% (v/v) acetonitrile. The identity and purity of the individual histone subfractions obtained was assayed by capillary electrophoretic analysis. The results demonstrate that application of the combined RP-HPLC-HILIC procedure to the analysis and isolation of modified H1 histone subtypes provides an innovative and important alternative to traditional separation techniques that will be extremely useful in studying the biological function of histone phosphorylation.     

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.     

The effect of adrenalectomy on stress-induced c-fos mRNA expression in the rat brain

Hydrophilic-interaction liquid chromatography (HILIC) has recently been introduced as a highly efficient chromatographic technique for the separation of a wide range of solutes. The present work was performed with the aim of evaluating the potential utility of HILIC for the separation of postranslationally acetylated histones. The protein fractionations were generally achieved by using a weak cation-exchange column and an increasing sodium perchlorate gradient system in the presence of acetonitrile (70%, v/v) at pH 3.0. In combination with reversed-phase high-performance liquid chromatography (RP-HPLC) we have successfully separated various H2A variants and posttranslationally acetylated forms of H2A variants and H4 proteins in very pure form. An unambiguous assignment of the histone fractions obtained was performed using high-performance capillary and acid-urea-Triton gel electrophoresis. Our results demonstrate that for the analysis and isolation of modified core histone variants HILIC provides a new and important alternative to traditional separation techniques and will be useful in studying the biological function of histone acetylation.     

Isolation and study of the properties of the catalytic subunit of cAMP-dependent protein kinase of the small intestinal mucosa of the rabbit

cAMP-dependent and casein proteinkinase were found in cytosol of the rabbit small intestine mucosa. cAMP-dependent proteinkinase of cytosol is represented by two forms of types I and II. The activity of enzymes of types I and II constitutes 10 and 90%, respectively. Casein proteinkinase is represented by a single form. The catalytic subunit of cAMP-dependent proteinkinase of type II was isolated in a homogenous state. The catalytic subunit phosphorylates histones H1, H2a, H2b and protamine and to a far less degree histones H3, H4 and casein (H2b greater than H1 greater than H2a greater than protamine much greater than H3 greater than casein). The Km value for histone H1 is equal to 65 mkM, and that for Mg-ATP 12 mkM. Chloromethylpyrophosphonate and adenosine p-fluorosulfobenzoate were studied as affine modifiers of the active center of the catalytic subunit from the small intestine mucosa. It was shown that only adenosine p-fluorosulfonate is an irreversible inhibitor of the catalytic subunit.     

Histone neighbors in nuclei and extended chromatin

Histone neighbors in compact and extended chromatin have been investigated by cross-linking histones in nuclei and in nucleohistone extended with 6 M urea, using the bifunctional reversible reagent methyl-4-mercaptobutyrimidate (MMB). Similar histone dimers are found in both conformational states of chromatin. The dimers most frequently found are H2b-H2a, H2b-H3 and H3-H2a; dimers found less frequently are H3-H4, H3-H3 and H2b-H4. More H3-H3 is found in nuclei than in extended chromatin. H1 is found predominantly as poly-H1, although it can be cross-linked to H2b or H3. After reaction with MMB, native compact chromatin is no longer extendable in 6 M urea, which shows that the reagent is capable of linking together histones holding the chromatin in a compact conformation. Thus the histone propinquity in extended chromatin mimics and intimate histone associations in compact chromatin.     

Treatment of mice with a novel antineoplastic agent taxol before irradiation increases the frequency of micronuclei in the bone marrow

Prothymosin alpha (Pro Talpha) is a polypeptide which appears to be involved in cell proliferation, though its precise function has yet to be identified. Here, we report experiments which show that calf Pro Talpha selectively binds to core histones and histone H1 in vitro. Characterization of these interactions by various procedures (including affinity chromatography on Pro T alpha-Sepharose columns, immunoblotting assay and investigation of the behaviour of mixtures of Pro T alpha and histones in solution) indicated that Pro T alpha has higher affinity for core histones (particularly H3 and H4) than for H1. Similarities between the histone-binding patterns of Pro T alpha and of poly(glutamic acid) suggest that the observed histone-binding capacity resides largely in the acidic central region of Pro T alpha. However, all five histones were also bound by T alpha 1 (a peptide corresponding to the first 28 amino acids of Pro T alpha); histone binding by the N-terminal region of Pro T alpha thus cannot be ruled out. Phosphorylation of Pro T alpha does not appear to affect these interactions. In accordance with the observed capacity for histone binding, Pro T alpha (in conjunction with ATP and some Pro T alpha-binding factor/s in a thymocyte extract) was able to induce in vitro nucleosome assembly. We discuss the possibility that Pro T alpha plays a role in chromatin remodelling.     

Structure and binding properties of monoclonal antibodies to core histones from autoimmune mice

Histones are frequent targets of self-reactive antibodies during autoimmune syndromes. We report the specificities and V region genes of three IgG anti-histone MAbs obtained from autoimmune mice. Each of the MAbs, named LG2-1, LG2-2 and BWA3, is directed against a different determinant located in the basic amino-terminal domain of core histones. LG2-1 reacts with a peptide from histone H3 (residues 30-45), LG2-2 recognizes the amino-terminus of H2B (residues 1-13) and BWA3 binds an epitope corresponding to a region of high sequence similarity between H2A and H4 (residues 1-20 and 1-29, respectively). The analysis of their V region sequences indicates that the H chain CDRs of these MAbs are remarkable for the presence of negatively charged amino acid residues that may play a role in the binding to cationic histones. The H chain importance in conferring reactivity to histones is corroborated by the observation that each of the VH gene segments of these MAbs is very similar to VH genes of previously described murine anti-histone antibodies.     

Physicochemical properties of calcium phosphate coatings produced by pulsed laser deposition at different water vapour pressures

Conformational peculiarities of DNA complexes with histones of the H1 family have been studied by the method of circular dichroism (CD). The H1 histones were isolated from spermatozoa of the sea urchin Strongylocentrotus intermedius, starfish Aphelasterias japonica, and bivalve mollusc Chlamis islandicus and also from the rat thymus. It is shown that these sperm-specific histones do not compact DNA in low ionic strength solution. At physiologic conditions H1 from the sea urchin and starfish sperms compact DNA more intensively than other histones. The H1 from rat thymus has a minimum ability to compact DNA. This histone does not change the structure of DNA double helix. It was supposed that it could be associated with interactions of this histone with DNA in the major groove of its helix. At the same time sperm-specific H1 can interact with DNA not only in the major groove but also in the minor groove, and this induces changes in DNA structure. This DNA-protein interaction is specific for the sperm chromatin and may support the supercompact organization of the sperm chromatin.     

Interactions between arginine-rich histones and deoxyribonucleic acids. II. Circular dichroism

Circular dichroism (CD) was used to investigate the conformations of arginine-rich histones, H3 (III or f3) and H4 (IV or f2a1), and DNA in the complexes prepared by four different methods: (A) NaCl gradient dialysis with urea; (B) NaCl gradient dialysis without urea; (C) direct mixing in 2.5 x 10(-4) M EDTA, pH 8.0; and (D) direct mixing in 0.01 M sodium phosphate, pH 7.0. Using the CD spectrum of native chromatin as a criterion to judge the closeness of a complex to its native state, it was observed that a complex made by direct mixing at low ionic strength (methods C and D) is better than the ones made by NaCl gradient dialysis with or without urea (methods A and B). It is explained as a result of lack of ordered secondary structures in histones due to the presence of urea in method A or due to nonspecific aggregation in NaCl without urea (method B). Compared with all the earlier reports in literature on the CD of histone-DNA complexes, the CD spectra of arginine-rich histone-DNA complexes prepared by methods C and D are closest to that of native chromatin both in shape and in amplitude. These results imply (a) that arginine-rich histones play an important role in maintaining the conformation of chromatin and (b) that the binding of these two histones to DNA prepared by methods C and D are close to that in native chromatin. Noticeable variation in conformation of free and bound histone and histone-bound DNA has also been observed in histone H3 with one or two cysteine residues, and in reduced or oxidized state even when the complexes were prepared and examined in the same condition. CD spectra of arginine-rich histones in 0.01 M phosphates, pH 7.0, indicate the presence of alpha-helix which could be responsible for a favorable binding of the less basic regions of these histones to DNA under this condition as demonstrated by thermal denaturation (Yu, S. .S, Li H. J., and Shih, T. Y. (1976), Bio-chemistry, the preceding paper in this issue). To preserve or generate alpha-helical structures in histones seems to be a critical step in reconstituting good histone-DNA complexes.     

Changes in size and shape of chromatin particles after successive removal of histones

The preparations of whole chromatin, chromatin selectively depleted of histone f1, depleted of all lysine-rich histones (f1, f2b, f2a2), and DNA was studied by viscosimetric and light scattering methods. The obtained results were used for calculation of the dimensions and packing ratios of DNA for the preparations studied. The packing ratio in whole chromatin is 7.2 and is almost unaffected by selective removal of histone f1 (6.9), but decreases on successive removal of the remaining four histones, the decrease being dependent more on the quantity than the kind of the dissociated histones.     

Second-site mutation of Ala-220 to Glu or Asp suppresses the mutation of Asp-285 to Asn in the transposon Tn10-encoded metal-tetracycline/H+ antiporter of Escherichia coli

A carboxyl group of Asp-285 is essential for tetracycline/H+ antiport mediated by the transposon Tn10-encoded metal-tetracycline/H+ antiporter (TetA) of Escherichia coli (Yamaguchi, A., Akasaka, T., Ono, N., Someya, Y., Nakatani, M., and Sawai, T. (1992) J. Biol. Chem. 267, 7490-7498). Spontaneous tetracycline resistance revertants were isolated from E. coli cells carrying the Asn-285 mutant tetA gene. All of the revertants were due to the second-site mutation at codon 220 of GCG (Ala) to GAG (Glu). The Km value of the tetracycline transport mediated by the revertant TetA protein was about 4-fold higher than that of the wild-type, indicating that the revertant is a low affinity mutant. A Glu-220 and Asn-285 double mutant constructed by site-directed mutagenesis showed the same properties as the revertants, confirming that the mutation of Ala-220 is solely responsible for the suppression. The Asp-220 mutation of the Asn-285 mutant resulted in a lower level of restoration of the tetracycline resistance and the transport activity than in the case of the Glu-220 mutation. A single mutation replacing Ala-220 with Glu or Asp caused about a 2-4-fold decrease in the tetracycline resistance, but no crucial change in the transport activity. It is not likely that Glu-220 is required for a charge-neutralizing salt bridge because an unpaired negative charge in a Glu-220 or Asp-220 single mutant did not cause a serious change in the activity. An alternative explanation is reasonable; Asp-285 directly contributes to the binding of a cationic substrate, metal-tetracycline chelation complex, or proton, and an acidic residue at position 220 can take over the role of Asp-285.     

The vertebrate linker histones H1 zero, H5, and H1M are descendants of invertebrate "orphon" histone H1 genes

We investigated the evolutionary history of the divergent vertebrate linker histones H1 zero, H5, and H1M. We observed that the sequence of the central conserved domain of these vertebrate proteins shares characteristic features with histone H1 proteins of plants and invertebrate animals which otherwise never appear in any vertebrate histone H1 protein. A quantitative analysis of 58 linker histone sequences also reveals that these proteins are more similar to invertebrate and plant histone H1 than to histone H1 of vertebrates. A phylogenetic tree deduced from an alignment of the central domain of all known linker histones places H1 zero, H5, and H1M in close vicinity to invertebrate sperm histone H1 proteins and to invertebrate histone H1 proteins encoded by polyadenylated mRNAs. We therefore conclude that the ancestors of the vertebrate linker histones H1 zero, H5, and H1M diverged from the main group of histone H1 proteins before the vertebrate type of histone H1 was established in evolution. We discuss this observation in the general context of linker histone evolution.     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

Distribution of allelic forms of erythrocyte H1 histones in Japanese quail populations divergently selected for amount of weight loss after transient starvation

Three polymorphic subtypes of erythrocyte histone H1 (H1.a, H1.b, and H1.z) were analyzed using a sodium dodecyl sulfate polyacrylamide gel in quail populations divergently selected for a high (line 1) or low (line 2) reduction in body mass following temporary food withdrawal. Both H1.b and H1.z histone alleles were found to be differently distributed in these populations during the selection period. The frequency of b1 in line 2 was approximately 1.9-2.8 times lower than in line 1 and approached the values in line 1 when the selection was suspended. Similarly, the frequency of allele z2 at locus H1.z increased significantly (about 1.6-2.3 times) in line 2 during selection and returned to the initial values when selection was stopped. On the other hand, allele a0 at locus H1.a was kept at relatively low levels (usually below 0.05) in both lines during selection. At that time its level was approximately three to four times lower than in a random mating control population. When selection was suspended, the frequency of a0 in line 1 increased significantly, approaching the values in the control line, and remained essentially unchanged in line 2. Thus, all three polymorphic histone H1 loci in quail responded through changes in allele frequencies to the breeding selection, which was directed at the amount of body weight loss upon transient starvation. It seems that either H1 histone locus could be linked to loci controlling the rate of body weight reduction following starvation or weight loss during fasting might be influenced by a panel of H1 histone alleles that can contribute to functional differences in avian chromatin.     

Antigen specificity of antihistone antibodies in systemic sclerosis

OBJECTIVES: The aim of the study was to determine the prevalence and clinical significance of antibodies to individual histone components in systemic sclerosis (SSc). METHODS: Serum samples from patients with limited cutaneous SSc (lSSc; n = 42) and diffuse cutaneous SSc (dSSc; n = 28) were examined for IgG and/or IgM antibodies to individual histone components and complexes by enzyme linked immunosorbent assay (ELISA). RESULTS: The level of IgG antibody to total histones was significantly higher in lSSc and dSSc than in normal controls. The level of IgM antibody to total histones was significantly higher in lSSc, but not in dSSc, than in normal controls. IgG antibody to total histones tended to be increased in dSSc when compared with that in lSSc. On the other hand, IgM antibody to total histones tended to be increased in lSSc when compared with that in dSSc. Although SSc showed various antihistone specificities, H2B, H2A-H2B, (H2A-H2B)-dsDNA were main antigens recognised by IgG antibodies in both lSSc and dSSc. Although IgM antibodies to H2B and H2A-H2B were also detected in both lSSc and dSSc, serum samples from lSSc patients exhibited highest IgM reactivity with H1. CONCLUSION: SSc may be included among conditions in which heterogeneous antihistone antibodies are produced. IgM antibodies to the most accessible histone H1 may be related to mild clinical features (lSSc) and IgG antibodies to the inner core molecules of native histone such as H2B or complexes including H2B may be associated with severe clinical features (dSSc) in Ssc.