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1.
The vertebrate kinetochore complex assembles at the centromere on α-satellite DNA. In humans, α-satellite DNA has a repeat length of 171bp slightly longer than the DNA in the chromatosome containing the linker histone H1. The centromere-binding protein CENP-B binds specifically to α-satellite DNA with properties of a centromeric-linker histone. Here, we analysed if linker histone H1 is present at or excluded from centromeric chromatin by CENP-B. By immunostaining we detected the presence, but no enrichment or depletion of five different H1 subtypes at centromeric chromatin. The binding dynamics of H1 at centromeric sites were similar to that at other locations in the genome. These dynamics did not change in CENP-B depleted cells, suggesting that CENP-B and H1 co-exist in centromeric chromatin with no or little functional overlap. By bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET), we revealed that the linker histone H1 subtypes H1° and H1.2 bind to centromeric chromatin in interphase nuclei in direct neighbourhood to inner kinetochore proteins.  相似文献   

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Rat liver chromatin reconstituted from fractionated histones, chromosomal non-histone proteins, and DNA is extensively degraded by chromatin-bound protease.  相似文献   

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Summary The non-histone chromatin proteins (NHCp) from pig liver and kidney have been partially fractionated in non-denaturing conditions by the use of histone H3 immobilized on agarose and the fractions obtained have been analysed by SDS-polyacrylamide gel electrophoresis and amino acid analysis. At least six different fractions have been obtained by successive increases of the ionic strength of the medium. Few NHCp have been evidentiated with subunit molecular weights 55,000 and less than 30,000, which display a remarkably high affinity for histone H3, and require 5 M urea to be displaced from the immobilized histone. The elution patterns of the NHCp from liver and kidney, although very similar, reveal some significant differences between the two tissues, which are undetectable by SDS-electrophoresis and which are most likely due to tissue specific proteins. This histone-affinity chromatography appears to be a promising approach for the analysis of specific histone-NHCp interactions, and as a first step for NHCp purification.  相似文献   

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We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a 'beads on a string' structure with each nucleosome trapping 158 +/- 27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.  相似文献   

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Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.  相似文献   

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Histone H1 and the high-mobility group (HMG) proteins are chromatin binding proteins that regulate gene expression by modulating the compactness of the chromatin fiber and affecting the ability of regulatory factors to access their nucleosomal targets. Histone H1 stabilizes the higher-order chromatin structure and decreases nucleosomal access, while the HMG proteins decrease the compactness of the chromatin fiber and enhance the accessibility of chromatin targets to regulatory factors. Here we show that in living cells, each of the three families of HMG proteins weakens the binding of H1 to nucleosomes by dynamically competing for chromatin binding sites. The HMG families weaken H1 binding synergistically and do not compete among each other, suggesting that they affect distinct H1 binding sites. We suggest that a network of dynamic and competitive interactions involving HMG proteins and H1, and perhaps other structural proteins, constantly modulates nucleosome accessibility and the local structure of the chromatin fiber.  相似文献   

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The condensation of chromatin and histone H1-depleted chromatin by spermine   总被引:2,自引:0,他引:2  
At low ionic strength, spermine induces aggregation of native and H1-depleted chromatin at spermine/phosphate (Sp/P) ratios of 0.15 and 0.3, respectively. Physico-chemical methods (electric dichroism, circular dichroism and thermal denaturation) show that spermine, at Sp/P less than 0.15, does not appreciably alter the conformation of native chromatin and interacts unspecifically with all parts of chromatin DNA (linker as well as regions slightly or tightly bound to histones). In chromatin, the role of spermine could be more important in the stabilization of higher-order structure than in the condensation of the 30 nm solenoid. The addition of spermine to H1-depleted chromatin revealed two important features: (i) spermine can partially mimic the role of histone H1 in the condensation of chromatin; (ii) the core histone octamer does not appear to play any role in the aggregation process by spermine as DNA and H1-depleted chromatin aggregate at the same Sp/P ratio.  相似文献   

11.
Heterogeneity of chromatin subunits in vitro and location of histone H1.   总被引:74,自引:40,他引:34       下载免费PDF全文
Chromatin subunits ("nucleosomes") which were purified by sucrose gradient centrifugation of a staphylococcal nuclease digest of chromatin have been studied. We found that such a preparation contains nucleosomes of two discrete types which can be separated from each other by polyacrylamide gel electrophoresis. Nucleosome of the first type contains all five histones and a DNA segment of approximately 200 base pairs long, whereas nucleosome of the second type lacks histone H1 and its DNA segment is approximately 170 base pairs long, i.e., about 30 base pairs shorter than the DNA segment of the nucleosome of the first type. Purified dimer of the nucleosome also can be fractionated by gel electrophoresis into three discrete bands which correspond to dinucleosomes containing two molecules of histone H1, one and no H1. These and related findings strongly suggest that the H1 molecule is bound to a short (approximately 30 base pairs) terminal stretch of the nucleosomal DNA segment which can be removed by nuclease (possibly in the form of H1-DNA complex) without any significant disturbance of main structural features of the nucleosome.  相似文献   

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Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.  相似文献   

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The dynamic interaction of chromatin-binding proteins with their nucleosome binding sites is an important element in regulating the structure and function of chromatin in living cells. Here we review the major factors regulating the intranuclear mobility and chromatin binding of the linker histone H1, the most abundant family of nucleosome-binding proteins. The information available reveals that multiple and diverse factors modulate the interaction of H1 with chromatin at both a local and global level. This multifaceted mode of modulating the interaction of H1 with nucleosomes is part of the mechanism that regulates the dynamics of the chromatin fiber in living cells.  相似文献   

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The massive nonselective and reversible phosphorylation of histone H1 during mitosis is a universal phenomenon among eukaryotes. The growth-associated kinase responsible for this phosphorylation is identical to the maturation promoting factor, a key regulator of the cell cycle. Here we showed that growth-associated kinase, isolated from mitotic HeLa cells which were capable of phosphorylating HeLa H1 in vitro with high activity and mostly at the same sites phosphorylated during mitosis in vivo (assayed by two-dimensional analysis of tryptic phosphopeptides), did not significantly phosphorylate chromatin-bound or nuclear H1 in vitro. Its inability to phosphorylate chromatin-bound H1 did not change when the amount of kinase was increased or the incubation was prolonged. The resistance of chromatin-bound H1 to phosphorylation did not result from chromatin aggregation. Rapid phosphorylation of H1 in vitro, as well as in a nuclear system, was restored when NaCl concentrations were raised above 200 mM where H1:DNA interactions are weakened. At 300 mM NaCl, chromatin-bound H1 was phosphorylated in a subset of the sites observed for free H1 phosphorylated in vitro. These results suggest that active displacement of H1 from chromatin DNA may take place before H1 can be fully phosphorylated during mitosis.  相似文献   

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Five non-allelic histone H3 variants, H3.1, H3.2, H3.3, H3t and CENP-A, have been identified in mammals. H3t is robustly expressed in the testis, and thus was assigned as the testis-specific H3 variant. However, recent proteomics and tissue-specific RT-PCR experiments revealed a small amount of H3t expression in somatic cells. In the present study, we purified human H3t as a recombinant protein, and showed that H3t/H4 forms nucleosomes with H2A/H2B by the salt-dialysis method, like the conventional H3.1/H4. We found that H3t/H4 is not efficiently incorporated into the nucleosome by human Nap1 (hNap1), due to its defective H3t/H4 deposition on DNA. In contrast, human Nap2 (hNap2), a paralog of hNap1, promotes nucleosome assembly with H3t/H4. Mutational analyses revealed that the Ala111 residue, which is conserved among H3.1, H3.2 and H3.3, but not in H3t, is the essential residue for the hNap1-mediated nucleosome assembly. These results suggest that H3t may be incorporated into chromatin by a specific chaperone-mediated pathway.  相似文献   

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A comparative study of the condensation of reconstituted complexes of circular SV40 DNA with core histones from calf thymus and sea urchin sperm was performed using sedimentation and electron microscopic techniques. It is shown that in low ionic strength solutions both types of complexes are similar to native minichromosomes. In the region from 0.08 to 0.16 M NaCl the complexes of SV40 DNA with thymus histones form small compact particles. By contrast, the compaction of the SV40 DNA complexes with sperm histones results in the formation of giant intermolecular associates. The results obtained may mean that histone H2B of sea urchin sperm participates in the formation of a higher order structure in sperm chromatin.  相似文献   

20.
Phasing of nucleosomes in SV40 chromatin reconstituted in vitro   总被引:4,自引:0,他引:4  
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