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1.
Conformation of reconstituted mononucleosomes and effect of linker histone H1 binding studied by scanning force microscopy
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The conformation of mononucleosome complexes reconstituted with recombinant core histones on a 614-basepair-long DNA fragment containing the Xenopus borealis 5S rRNA nucleosome positioning sequence was studied by scanning/atomic force microscopy in the absence or presence of linker histone H1. Imaging without prior fixation was conducted with air-dried samples and with mononucleosomes that were injected directly into the scanning force microscopy fluid cell and visualized in buffer. From a quantitative analysis of approximately 1,700 complexes, the following results were obtained: i), In the absence of H1, a preferred location of the nucleosome at the X. borealis 5S rRNA sequence in the center of the DNA was detected. From the distribution of nucleosome positions, an energy difference of binding to the 5S rRNA sequence of DeltaDeltaG approximately 3 kcal mol(-1) as compared to a random sequence was estimated. Upon addition of H1, a significantly reduced preference of nucleosome binding to this sequence was observed. ii), The measured entry-exit angles of the DNA at the nucleosome in the absence of H1 showed two maxima at 81 +/- 29 degrees and 136 +/- 18 degrees (air-dried samples), and 78 +/- 25 degrees and 137 +/- 25 degrees (samples imaged in buffer solution). In the presence of H1, the species with the smaller entry-exit angle was stabilized, yielding average values of 88 +/- 34 degrees for complexes in air and 85 +/- 10 degrees in buffer solution. iii), The apparent contour length of the nucleosome complexes was shortened by 34 +/- 13 nm as compared to the free DNA due to wrapping of the DNA around the histone octamer complex. Considering an 11 nm diameter of the nucleosome core complex, this corresponds to a total of 145 +/- 34 basepairs that are wound around the nucleosome. 相似文献
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It is shown in our FRET studies that both chromatosomes and histone H1-depleted chromatosomes exist in their arm-closed forms in the absence of spermidine. In the presence of spermidine, however, these two types of structural assemblies are converted into their arm-open forms. In addition, ATP as polyanion is capable of suppressing the polycationic effect of spermidine, thus facilitating re-formation of arm-closed forms of these two types of structural assemblies. Our studies therefore illustrate that conversion between arm-closed and arm-open forms of chromatosomes and histone H1-depleted chromatosomes can be manipulated by varying concentrations of polycationic spermidine and polyanionic ATP. 相似文献
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Contributions of linker histones and histone H3 to chromatin structure: scanning force microscopy studies on trypsinized fibers. 总被引:6,自引:1,他引:5
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Little is known about the mechanisms that organize linear arrays of nucleosomes into the three-dimensional structures of extended and condensed chromatin fibers. We have earlier defined, from scanning force microscopy (SFM) and mathematical modeling, a set of simple structural determinants of extended fiber morphology, the critical parameters being the entry-exit angle between consecutive linkers and linker length. Here we study the contributions of the structural domains of the linker histones (LHs) and of the N-terminus of histone H3 to extended fiber morphology by SFM imaging of progressively trypsinized chromatin fibers. We find that cleavage of LH tails is associated with a lengthening of the internucleosomal center-to-center distance, and that the somewhat later cleavage of the N-terminus of histone H3 is associated with a flattening of the fiber. The persistence of the "zigzag" fiber morphology, even at the latest stages of trypsin digestion, can be attributed to the retention of the globular domain of LH in the fiber. 相似文献
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We investigated the relationship between linker histone stoichiometry and the acetylation of core histones in vivo. Exponentially growing cell lines induced to overproduce either of two H1 variants, H1(0) or H1c, displayed significantly reduced rates of incorporation of [(3)H]acetate into all four core histones. Pulse-chase experiments indicated that the rates of histone deacetylation were similar in all cell lines. These effects were also observed in nuclei isolated from these cells upon labeling with [(3)H]acetyl-CoA. Nuclear extracts prepared from control and H1-overexpressing cell lines displayed similar levels of histone acetylation activity on chromatin templates prepared from control cells. In contrast, extracts prepared from control cells were significantly less active on chromatin templates prepared from H1-overexpressing cells than on templates prepared from control cells. Reduced levels of acetylation in H1-overproducing cell lines do not appear to depend on higher order chromatin structure, because it persists even after digestion of the chromatin with micrococcal nuclease. The results suggest that alterations in chromatin structure, resulting from changes in linker histone stoichiometry may modulate the levels or rates of core histone acetylation in vivo. 相似文献
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Lipopolysaccharides (LPS; endotoxin) activate immunocompetent cells of the host via a transmembrane signaling process. In this study, we investigated the function of the LPS-binding protein (LBP) in this process. The cytoplasmic membrane of the cells was mimicked by lipid liposomes adsorbed on mica, and the lateral organization of LBP in these membranes and its interaction with LPS aggregates were characterized by atomic force microscopy. Using cantilever tips functionalized with anti-LBP antibodies, single LBP molecules were localized in the membrane at low concentrations. At higher concentrations, LBP formed clusters of several molecules and caused cross-linking of lipid bilayers. The addition of LPS to LBP-containing liposomes led to the formation of LPS domains in the membranes, which could be inhibited by anti-LBP antibodies. Thus, LBP mediates the fusion of lipid membranes and LPS aggregates. 相似文献
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Considerable progress has been made recently in defining the interactions of linker histones (H1s) within nucleosomes. Major advancements include atomic resolution structures of the globular domain of full-length H1s in the context of nucleosomes containing full-length linker DNA. Although these studies have led to a detailed understanding of the interactions and dynamics of H1 globular domains in the canonical on-dyad nucleosome binding pocket, more information regarding the intrinsically disordered N-terminal and C-terminal domains is needed. In this review, we highlight studies supporting our current understanding of the structures and interactions of the N-terminal, globular, and C-terminal domains of linker histones within the nucleosome. 相似文献
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Suppression of homologous recombination by the Saccharomyces cerevisiae linker histone 总被引:1,自引:0,他引:1
The basic unit of chromatin in eukaryotes is the nucleosome, comprising 146 bp of DNA wound around two copies of each of four core histones. Chromatin is further condensed by association with linker histones. Saccharomyces cerevisiae Hho1p has sequence homology to other known linker histones and interacts with nucleosomes in vitro. However, disruption of HHO1 results in no significant changes in the phenotypes examined thus far. Here, we show that Hho1p is inhibitory to DNA repair by homologous recombination (HR). We find Hho1p is abundant and associated with the genome, consistent with a global role in DNA repair. Furthermore, we establish that Hho1p is required for a full life span and propose that this is mechanistically linked to its role in HR. Finally, we show that Hho1p is inhibitory to the recombination-dependent mechanism of telomere maintenance. The role of linker histones in genome stability, aging, and tumorigenesis is discussed. 相似文献
9.
Liu CH Horng JT Chang JS Hsieh CF Tseng YC Lin S 《Biochemical and biophysical research communications》2012,417(1):109-115
Atomic force microscopy (AFM) is a vital instrument in nanobiotechnology. In this study, we developed a method that enables AFM to simultaneously measure specific unbinding force and map the viral glycoprotein at the single virus particle level. The average diameter of virus particles from AFM images and the specificity between the viral surface antigen and antibody probe were integrated to design a three-stage method that sets the measuring area to a single virus particle before obtaining the force measurements, where the influenza virus was used as the object of measurements. Based on the purposed method and performed analysis, several findings can be derived from the results. The mean unbinding force of a single virus particle can be quantified, and no significant difference exists in this value among virus particles. Furthermore, the repeatability of the proposed method is demonstrated. The force mapping images reveal that the distributions of surface viral antigens recognized by antibody probe were dispersed on the whole surface of individual virus particles under the proposed method and experimental criteria; meanwhile, the binding probabilities are similar among particles. This approach can be easily applied to most AFM systems without specific components or configurations. These results help understand the force-based analysis at the single virus particle level, and therefore, can reinforce the capability of AFM to investigate a specific type of viral surface protein and its distributions. 相似文献
10.
A. V. Lyubitelev K. S. Kudryashova M. S. Mikhaylova N. V. Malyuchenko O. V. Chertkov V. M. Studitsky A. V. Feofanov M. P. Kirpichnikov 《Moscow University Biological Sciences Bulletin》2016,71(2):108-113
A method for fluorescently labeled DNA synthesis, which makes it possible to assemble mononucleosomes with 40 bp linkers, was developed. Cy3 and Cy5 labels were introduced into the linkers at distances of 10 bp before the first nucleotide and 15 bp after the last nucleotide of the nucleosome positioning DNA sequence, respectively. Without histone H1.5, f luorescence microscopy of single complexes revealed two equally probable states of nucleosomes. The states were different in the linker conformation: the open one with the energy transfer efficiency (E) between the labels of 0.06 and the closed one with E = 0.37, when the linkers are brought together. Binding of histone H1.5 with nucleosomes occurs in a range of nanomolar concentrations, and the complex formation rate is significantly higher as compared with its dissociation rate. The significant convergence of the DNA linkers (E = 0.73) is observed in these complexes together with the higher conformation uniformity in the region where the labels are localized. The developed nucleosomal constructs represent highly sensitive f luorescent sensors that can be used for the analysis of structural linker rearrangements. Also, in combination with microscopy of single complexes, they are suitable for studying the structure of complexes of nucleosomes with different chromatin architectural proteins. 相似文献
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Phytochrome was localized by immunoelectron microscopy in cells of the coleoptile tip of etiolated and irradiated oat (Avena sativa L., cv. Konata) seedlings. By using ultrathin frozen sections and immunopurified, monospecific antibodies, both the sensitivity and resolution of the immunocytochemical assay were increased. The results with etiolated plants agree with and extend previously published data. A brief red light illumination caused the redistribution of phytochrome from a diffuse to a more particulate appearance. Areas that accumulated phytochrome were identified as small vacuoles into which phytochrome was sequestered following illumination. In seedlings illuminated for several hours and in normal light-grown plants, the cellular distribution of phytochrome is qualitatively similar to that of nonirradiated, dark-grown material, except that in green plants the nucleus shows a positive immunocytochemical reaction. 相似文献
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A variable migration of linker histone H1.b and H1.c spots in two-dimensional polyacrylamide gel patterns of total erythrocyte histone H1 has been detected during population screening in two differently plumaged Guinea fowl strains. Alloforms, H1.b1 and H1.b2 as well as H1.c1 and H1.c2, differing in apparent molecular weights tended to form only phenotypes b1 and b2 or c1 and c2 in a white-feathered strain while all phenotypes (b1, b2 and b1b2 or c1, c2 and c1c2, respectively) were present in a black-feathered population. Accordingly, the white-feathered population significantly deviated from the Hardy-Weinberg principle (chi-square test, d.f=1, p<0.001) due to a lack of heterozygotes while the black-feathered population conformed to the Hardy-Weinberg equilibrium (p>0.05) at both H1.b and H1.c loci. Differential electrophoretic mobilities of the C-peptides from a partial chemical cleavage (N-bromosuccinimide) or limited enzymatic digestion (α-chymotrypsin and protease V8) of the histone H1.b and H1.c alloforms seem to indicate that altered amino acid sequence segments might be located either at the C-terminal end of globular domain or in the C-terminal domain itself. 相似文献
19.
《Epigenetics》2013,8(6):353-356
Maintenance of intact heterochromatin structure through epigenetic mechanisms is essential for cell survival. Defects in heterochromatin formation caused by loss of chromatin-modifying enzymes lead to genomic instability and cellular senescence. The NAD+-dependent histone deacetylase SIR-2 and the H1 linker histone are intriguing chromatin elements that are connected to chromatin regulation and cell viability in the single cellular eukaryotic organism yeast. However, it remains an open question how SIR-2 and H1 mediate heterochromatin formation in simple multi-cellular organisms such as C. elegans and in even more complex organisms such as mammals. Recently we have identified SIR-2.1 and the H1 histone subtype, HIS-24 as factors involved in heterochromatin regulation at subtelomeric regions in C. elegans. In addition we show that SIR-2.1, HIS-24, and MES-2, a ortholog to Enhancer of zeste E(Z) are functionally related in heterochromatin formation contributing to fertility and embryogenesis. Here we discuss the interplay between SIR-2, H1 histone and histone methyltransferases in modulation of chromatin structure in further detail. 相似文献
20.
Linker histone tails and N-tails of histone H3 are redundant: scanning force microscopy studies of reconstituted fibers. 总被引:4,自引:1,他引:4
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The mechanisms responsible for organizing linear arrays of nucleosomes into the three-dimensional structure of chromatin are still largely unknown. In a companion paper (Leuba, S. H., et al. 1998. Biophys. J. 74:2823-2829), we study the contributions of linker histone domains and the N-terminal tail of core histone H3 to extended chromatin fiber structure by scanning force microscopy imaging of mildly trypsinized fibers. Here we complement and extend these studies by scanning force microscopy imaging of selectively reconstituted chromatin fibers, which differ in subtle but distinctive ways in their histone composition. We demonstrate an absolute requirement for the globular domain of the linker histones and a structural redundancy of the tails of linker histones and of histone H3 in determining conformational stability. 相似文献