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1.
R T Simpson D W Stafford 《Proceedings of the National Academy of Sciences of the United States of America》1983,80(1):51-55
Chicken erythrocyte inner histones associate with a cloned 260-base-pair (bp) segment of Lytechinus variegatus DNA in a unique location. The fragment contains a 120-bp segment encoding 5S rRNA, a 90-bp flanking sequence to the 5' side of the transcribed segment, and a 50-bp downstream flanking sequence. Association of DNA, uniquely labeled at one end or the other and at either the 3' or the 5' terminus of a given strand, with histones at 0.1 M ionic strength leads to formation of a compact complex which sediments at about 13 S. Analysis of cutting of the complex by DNase I shows that protection from the nuclease is confined to a region beginning 20 bp from the left end of the segment and extending to about 165 bp from the left end. Within the protected region, the two DNA strands differ in their susceptibilities to the nuclease, the precise location of nuclease cutting sites and the spacing between these sites, and the relative susceptibilities of specific cutting locations. It seems that information present in DNA and the histone octomer is sufficient to create a precisely phased nucleosome in which interactions of the two DNA strands with histones are not the same. The structure of this unique nucleosome is not predicted by the intellectual model based on studies of mixed populations of nucleosome core particles. 相似文献
2.
Preferential and asymmetric interaction of linker histones with 5S DNA in the nucleosome. 总被引:12,自引:0,他引:12 下载免费PDF全文
J J Hayes A P Wolffe 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(14):6415-6419
We establish that linker histones H1 and H5 bind preferentially to a Xenopus borealis somatic 5S RNA gene associated with an octamer of core histones rather than to naked 5S DNA. This preferential binding requires free linker DNA to either side of the nucleosome core. Incorporation of a single linker histone molecule into the nucleosome protects an additional 20 bp of linker DNA from micrococcal nuclease digestion. This additional DNA is asymmetrically distributed with respect to the nucleosome core. Incorporation of linker histones causes no change to the cleavage of DNA in the nucleosome by hydroxyl radical or DNase I. 相似文献
3.
Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro 总被引:4,自引:0,他引:4
Yoda K Ando S Morishita S Houmura K Hashimoto K Takeyasu K Okazaki T 《Proceedings of the National Academy of Sciences of the United States of America》2000,97(13):7266-7271
Centromere protein A (CENP-A) is a variant of histone H3 with more than 60% sequence identity at the C-terminal histone fold domain. CENP-A specifically locates to active centromeres of animal chromosomes and therefore is believed to be a component of the specialized centromeric nucleosomes on which the kinetochores are assembled. Here we report that CENP-A, highly purified from HeLa cells, can indeed replace histone H3 in a nucleosome reconstitution system mediated by nucleosome assembly protein-1 (NAP-1). The structure of the nucleosomes reconstituted with recombinant CENP-A, histones H2A, H2B, and H4, and closed circular DNAs had the following properties. By atomic force microscopy, "beads on a string" images were obtained that were similar to those obtained with nucleosomes reconstituted with four standard histones. DNA ladders with repeats of approximately 10 bp were produced by DNase I digestion, indicating that the DNA was wrapped round the protein complex. Mononucleosomes isolated by glycerol gradient sedimentation had a relative molecular mass of approximately 200 kDa and were composed of 120-150 bp of DNA and equimolar amounts of CENP-A, and histones H4, H2A, and H2B. Thus, we conclude that CENP-A forms an octameric complex with histones H4, H2A, and H2B in the presence of DNA. 相似文献
4.
Modulation of nucleosome structure by histone subtypes in sea urchin embryos. 总被引:7,自引:2,他引:7 下载免费PDF全文
R T Simpson 《Proceedings of the National Academy of Sciences of the United States of America》1981,78(11):6803-6807
Switches of the types of histones synthesized and incorporated into chromatin occur during sea urchin embryogenesis. In an attempt to define the possible effects of these variant histones on chromatin structure, I have isolated and characterized nucleosome core particles from Strongylocentrotus purpuratus blastula (nearly 100% early histones) and pluteus (75% late histones). Both particles contain 146-base-pair lengths of DNA wrapped around an octamer of H2A, H2B, H3, and H4. Although sharing these similarities with the canonical core particle, the nucleosome structures have certain features that differ from those of typical adult tissues. Both the reversible and the irreversible conformational transitions occurring on heating core particles are destabilized in the embryonic particles vs. "typical" core particles. The blastula core particle unfolds more easily than pluteus (or other) nucleosomes under the stress of low ionic strength. The rate of DNase I digestion of pluteus core particles is about half that of particles from blastula; certain cutting sites differ in their susceptibility between the two embryonic particles and between these two and the canonical core particle. The data demonstrate that the variant histones synthesized during early embryogenesis have demonstrable effects on chromatin structure, even at this basic level. 相似文献
5.
Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNase I sensitivity of the associated DNA sequences. 总被引:42,自引:1,他引:42 下载免费PDF全文
G Vidali L C Boffa E M Bradbury V G Allfrey 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(5):2239-2243
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D Lohr K E Van Holde 《Proceedings of the National Academy of Sciences of the United States of America》1979,76(12):6326-6330
Detailed analysis of the DNA fragment patterns produced by DNase I digestion of yeast, HeLa, and chicken erythrocyte nuclei reveals surprising features of nucleosome phasing. First, the spacer regions in phased yeast chromatin must be of lengths (10m + 5) base pairs, where m = 0, 1, 2,....This feature is not seen in parallel studies of chicken erythrocyte chromatin. The 5-base pair increment in the yeast spacer imposes interesting restraints on the higher order structure of yeast chromatin. Second, we have been able to simulate the DNase I cutting patterns and get good agreement with the observed yeast patterns. Third, three different chromatins show a long range periodicity in the DNase I digest pattern, with a period half that of the staphylococcal nuclease repeat. These results suggest that the amount of chromatin observed in discrete extended-ladder bands is a minimum estimate of phasing and in fact phasing may be a more general feature. 相似文献
8.
T E Shrader D M Crothers 《Proceedings of the National Academy of Sciences of the United States of America》1989,86(19):7418-7422
We have used the emerging rules for the sequence dependence of DNA bendability to design and test a series of DNA molecules that incorporate strongly into nucleosomes. Competitive reconstitution experiments showed the superiority in histone octamer binding of DNA molecules in which segments consisting exclusively of A and T or G and C, separated by 2 base pairs (bp), are repeated with a 10-bp period. These repeated (A/T)3NN(G/C)3NN motifs are superior in nucleosome formation to natural positioning sequences and to other repeated motifs such as AANNNTTNNN and GGNNNCCNNN. Studies of different lengths of repetitive anisotropically flexible DNA showed that a segment of approximately 40 bp embedded in a 160-bp fragment is sufficient to generate nucleosome binding equivalent to that of natural nucleosome positioning sequences from 5S RNA genes. Bending requirements along the surface of the nucleosome seem to be quite constant, with no large jumps in binding free energy attributable to protein-induced kinks. The most favorable sequences incorporate into nucleosomes more strongly by 100-fold than bulk nucleosomal DNA, but differential bending free energies are small when normalized to the number of bends: a free energy difference of only about 100 cal/mol per bend (1 cal = 4.184 J) distinguishes the best bending sequences and bulk DNA. We infer that the distortion energy of DNA bending in the nucleosome is only weakly dependent on DNA sequence. 相似文献
9.
Deoxyribonuclease I sensitivity of plasmid genomes in teratocarcinoma-derived stem and differentiated cells. 总被引:2,自引:1,他引:2 下载免费PDF全文
K Huebner A Linnenbach S Weidner G Glenn C M Croce 《Proceedings of the National Academy of Sciences of the United States of America》1981,78(8):5071-5075
The DNase I (EC 3.1.21.1) sensitivities of the simian virus 40 (SV40) genome, the pBR322 genome, and the herpes simplex virus type 1 thymidine kinase (HSV-1 tk) gene have been compared in teratocarcinoma-derived stem (12-1) and differentiated (12-1a) cell lines established by transfection of thymidine kinase (ATP:thymidine 5'-phosphotransferase, EC 2.7.1.21)-deficient F9 cells with DNA from a tripartite plasmid genome consisting of the pBR322 genome, the SV40 genome, and the HSV-1 tk gene. HSV-1 tk is present in both stem and differentiated cells; SV40 early proteins are present in differentiated cells but not in stem cells; the pBR322 genome is not expressed in either cell type. The SV40 and pBR322 genomes are more sensitive to DNase I digestion in stem cells than in differentiated cells, reflecting the DNase I-hypersensitivity of total stem-cell chromatin. The HSV-1 tk gene is the least sensitive to DNase I digestion in both cell types. 相似文献
10.
R T Simpson 《Proceedings of the National Academy of Sciences of the United States of America》1976,73(12):4400-4404
Isolated HeLa cell nucleosomes (core particles) were labeled at the 5'-termini of their DNA with 32P using [gamma-32P]ATP and polynucleotide kne by sequential methylation, depurination, Schiff base formation, and reduction with sodium borhydride. After digestion of the noncrosslinked DNA by DNase I and venom phosphodiesterase, histones were separated by gel electrophoresis and those crosslinked to the 5'-termini were identified by 32P-autoradiography. Histones H3 and H4 occur with equeal frequency as the nearest protein neighbors to the end of the DNA in nucleosomes. Histone arrangements within the core particle compatible with these results are discussed. 相似文献
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Specific DNA sequences associated with the nuclear matrix in synchronized mouse 3T3 cells. 总被引:4,自引:0,他引:4 下载免费PDF全文
G I Goldberg I Collier A Cassel 《Proceedings of the National Academy of Sciences of the United States of America》1983,80(22):6887-6891
Eukaryotic chromatin appears to be organized into arrays of supercoiled loops anchored to the scaffolding structure of the mitotic chromosome core or to the nuclear matrix of interphase nuclei. To reveal whether specific DNA sequences are involved in this level of chromatin organization, we isolated and cloned a population of DNA molecules [average length of 150 base pairs (bp)] closely associated with the nuclear matrix after exhaustive DNase digestion and subsequent extensive protease digestion. The nuclear matrix was obtained from murine BALB/c 3T3 cells synchronized at the G1/S border of the cell cycle. We report the structure of two sequences, designated G4 and G5, which are highly enriched in the matrix DNA. Sequence G4, of 152 bp, contains three 31-bp direct head-to-tail repeats. An 11-bp sequence at the end of each repeat is homologous to the first large tumor antigen recognition site of human papova virus. Sequence G5, of 135 bp, consists of two well-defined domains, in which the first domain is a fragment of the B1 repetitive sequence. The results suggest the possibility that the loops of histone-depleted chromatin are connected to the scaffold of the nuclear matrix, with specific DNA sequences at the anchorage sites. 相似文献
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Suzette L. Pereira Rowan A. Grayling Rudi Lurz John N. Reeve 《Proceedings of the National Academy of Sciences of the United States of America》1997,94(23):12633-12637
Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect ≈60 bp of DNA and multiples of ≈60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones. 相似文献
16.
Multiple nucleosome positioning with unique rotational setting for the Saccharomyces cerevisiae 5S rRNA gene in vitro and in vivo. 总被引:6,自引:1,他引:5 下载免费PDF全文
M Buttinelli E Di Mauro R Negri 《Proceedings of the National Academy of Sciences of the United States of America》1993,90(20):9315-9319
A simple no-background assay was developed for high-resolution in vivo analysis of yeast chromatin. When applied to Saccharomyces cerevisiae 5S rRNA genes (5S rDNA), this analysis shows that nucleosomes completely cover this chromosomal region, occupying alternative positions characterized by a unique helical phase. This supports the notion that sequence-intrinsic rotational signals are the major determinant of nucleosome localization. Nucleosomal core particles reconstituted in vitro occupy the same positions and have the same helically phased distribution observed in vivo, as determined by mapping of exonuclease III-resistant borders, mapping by restriction cleavages, and by DNase I and hydroxyl-radical digestion patterns. 相似文献
17.
Selective association of the trout-specific H6 protein with chromatin regions susceptible to DNase I and DNase II: possible location of HMG-T in the spacer region between core nucleosomes. 总被引:25,自引:2,他引:23 下载免费PDF全文
B Levy W N C Wong G H Dixon 《Proceedings of the National Academy of Sciences of the United States of America》1977,74(7):2810-2814
Nuclei and chromatin from trout testis cells were digested with three different nucleases (DNase I, DNase II, and micrococcal nuclease), and the acid-soluble proteins that were solubilized and those remaining bound to the nuclease-resistant DNA were compared electrophoretically. With the conditions described by H. Weintraub and M Groudine [(1976) science, 193, 848-856], which we previously found to be selective in digesting actively transcribed regions in trout testis chromatin, a single chromosomal protein, H6, was solubilized. The nucleosomal histones and H1 remained insoluble, bound to the resistant DNA. In contrast, digestion with micrococcal nuclease led to a preferential solubilization of a second protein, HMG-T, together with the release of some nucleosomal histones and H1 into the soluble fraction. DNase II also discriminated between "active" and "inactive" chromatins; when a DNase II-solubilized "active" chromatin fraction was prepared, it too was enriched in H6 and HMG-T. Thus, both H6 and HMG-T, the two major low-salt extractable chromosomal nonhistone the two major low-salt extractable chromosomal nonhistone proteins from trout testis, are associated with chromatin regions selectively sensitive to nucleases. The preferential solubilization of HMG-T by micrococcal nuclease action suggests that it might be located at the internucleosomal "spacer" region. 相似文献
18.
Altered nucleosome structure containing DNA sequences complementary to 19S and 26S ribosomal RNA in Physarum polycephalum. 总被引:10,自引:0,他引:10
E M Johnson V G Allfrey E M Bradbury H R Matthews 《Proceedings of the National Academy of Sciences of the United States of America》1978,75(3):1116-1120
The localization of DNA sequences coding for ribosomal RNA was studied by hybridization of purified ribosomal RNA to DNA from chromatin fragments prepared by limited digestion of Physarum nuclei with staphylococcal nuclease. The 32P-labeled 19S and 26S RNA hybridized to DNA from nucleosome monomers, dimers, trimers, and higher oligomers, separated by sucrose gradient centrifugation, although the level of hybridization to DNA from nucleosome fractions was less than the level of hybridization to undigested nuclear DNA. The distribution of 19S and 26S rDNA sequences in the nucleosome fractions differed from the distribution of bulk DNA in that the rDNA sequences were recovered primarily in two fractions containing monomer-sized DNA lengths (140-160 base pairs). The percentage of DNA hybridizing to 19S plus 26S RNA was greater in peak A, the more slowly sedimenting monomer peak, than in any other chromatin fraction at all stages of digestion. Peak A and monomer particles differed in protein content and distribution. The presence of ribosomal cistrons in an altered nucleosome configuration may be related to changes in functional states of rDNA chromatin. 相似文献
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UV-induced formation of pyrimidine dimers in nucleosome core DNA is strongly modulated with a period of 10.3 bases. 总被引:14,自引:1,他引:14
J M Gale K A Nissen M J Smerdon 《Proceedings of the National Academy of Sciences of the United States of America》1987,84(19):6644-6648
We have determined the distribution of the major UV-induced photoproducts in nucleosome core DNA using the 3'----5' exonuclease activity of T4 DNA polymerase, which has been shown to stop digestion immediately 3' to UV-induced pyrimidine dimers. This assay is extremely sensitive since all DNA fragments without photoproducts (background) are reduced to small oligonucleotides, which can be separated from those fragments containing photoproducts. The results show that the distribution of UV-induced photoproducts (primarily cyclobutane dipyrimidines) is not uniform throughout core DNA but displays a striking 10.3 (+/- 0.1) base periodicity. Furthermore, this characteristic distribution of photoproducts was obtained regardless of whether nucleosome core DNA was isolated from UV-irradiated intact chromatin fibers, histone H1-depleted chromatin fibers, isolated mononucleosomes, or cells in culture. The yield of pyrimidine dimers along the DNA seems to be modulated in a manner that reflects structural features of the nucleosome unit, possibly core histone-DNA interactions, since this pattern was not obtained for UV-irradiated core DNA either free in solution or bound tightly to calcium phosphate crystals. Based on their location relative to DNase I cutting sites, the sites of maximum pyrimidine dimer formation in core DNA mapped to positions where the phosphate backbone is farthest from the core histone surface. These results indicate that within the core region of nucleosomes, histone-DNA interactions significantly alter the quantum yield of cyclobutane dipyrimidines, possibly by restraining conformational changes in the DNA helix required for formation of these photoproducts. 相似文献
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Sequence-specific interactions of nuclear factors with conserved sequences of human class II major histocompatibility complex genes 总被引:15,自引:9,他引:15 下载免费PDF全文
K Miwa C Doyle J L Strominger 《Proceedings of the National Academy of Sciences of the United States of America》1987,84(14):4939-4943
All class II major histocompatibility complex genes contain two highly conserved sequences, termed X and Y, within the promoter regions(s), which may have a role in regulation of expression. To study trans-acting factors that interact with these sequences, sequence-specific DNA binding activity has been examined by the gel electrophoresis retardation assay using the HLA-DQ2 beta gene 5' flanking DNA and nuclear extracts derived from various cell types. Several specific protein-binding activities were found using a 45-base-pair (bp) HinfI/Sau96I (-142 to -98 bp) and a 38-bp Sau96I/Sau96I (-97 to -60 bp) fragment, which include conserved sequence X (-113 to -100 bp) and conserved sequence Y (-80 to -71 bp), respectively. Competition experiments, methylation interference analysis, and DNase I foot-printing demonstrated that distinct proteins in a nuclear extract of Raji cells (a human B lymphoma line) bind to sequence X, to sequence Y, and to DNA 5' of the X sequence (termed sequence W). The factor binding site in the W sequence is also found to be conserved among beta-chain genes and is suggested to be a gamma-interferon control region. 相似文献