共查询到20条相似文献,搜索用时 31 毫秒
1.
To examine the distribution of 5-methylcytosine in chromatin DNA, DNA of HeLa cells was labeled with [3H-methyl]methionine and [14C] thymidine and analyzed after extensive digestion of the nuclei with micrococcal nuclease. When the chromatin solubilized with the nuclease was fractionated on a sucrose density gradient, DNA in mononucleosomes was considerably depleted in 5-methylcytosine, as compared with polynucleosomes. Electrophoretic separation of DNA from the chromatin also revealed the depletion of 5-methylcytosine in the mononucleosomal size of DNA. This was confirmed by the chromatographic analysis of 5-methyldeoxycytidine after enzymatic digestion of the DNA to nucleosides. Thus the DNA in mononucleosomes solubilized by extensive micrococcal nuclease digestion is depleted in 5-methylcytosine, suggesting that 5-methylcytosine is preferentially missing from the DNA in the nucleosome core particles. 相似文献
2.
DNase I sensitivity of ribosomal genes in isolated nucleosome core particles 总被引:13,自引:7,他引:6
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The level of chromatin structure at which DNase I recognizes conformational differences between inert and activated genes has been investigated. Bulk and ribosomal DNA's of Tetrahymena pyriformis were differentially labeled in vivo with [14C]- and [3H]-thymidine, respectively, utilizing a defined starvation-refeeding protocol. The 3H-labeled ribosomal genes were shown to be preferentially digested by DNase I in isolated nuclei. Staphylococcal nuclease digested the ribosomal genes more slowly than bulk DNA, probably owing to the higher GC content of rDNA. DNase I and staphylococcal nuclease digestions of purified nucleosomes and of nucleosome core particles isolated from dual-labeled, starved-refed nuclei were indistinguishable from those of intact nuclei. We conclude from these studies that DNase I recognizes an alteration in the internal nucleosome core structure of activated ribosomal genes. 相似文献
3.
A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation. 总被引:6,自引:3,他引:3
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Hepatoma tissue culture (HTC) cell nuclei were digested with either DNase I or micrococcal nuclease and the nucleohistone digestion products fractionated by gel electrophoresis or exclusion chromatography. Under appropriate conditions, gel electrophoresis demonstrates that for both nucleases, only cleavages within the nucleosome spacer regions and not within the nucleosome core lead to freely migrating nucleohistone particles. These particles consist of nucleosome cores, nucleosomes and nucleosome oligomers. Following DNase I digestion and fractionation by exclusion chromatography, analysis of the histones indicates a direct relationship between increased spacer region susceptibility to nuclease and increased nucleosomal histone acetylation. Evidently digestion sites outside the regions of DNA protected by core histones can reflect the degree of acetylation of core histones. Such a relationship is not found when micrococcal nuclease is used to digest the samples. 相似文献
4.
The effects of salt concentration and H-1 depletion on the digestion of calf thymus chromatin by micrococcal nuclease.
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We have removed histone H1 specifically from calf thymus nuclei by low pH treatment, and studied the digestion of such nuclei in comparison with undepleted nuclei. By a number of criteria the nuclei do not appear damaged. The DNA repeat-length in nuclear chromatin is found to be the same (192 +/- 4 bp) in the presence or absence of H1. These experiments demonstrate that the core histone complex of H2A, H2B, H3, and H4 can itself protect DNA sequences as long as 168 bp from nuclease. Our interpretation is that this represents an important structural element in chromatin, carrying two full turns of superhelical DNA. Depending on conditions of digestion this 168 bp fragment may be metastable and is normally rapidly converted by exonucleolytic trimming to the well-known "core-particle" containing 145 bp. Larger stable DNA fragments observed indigestion of H-1 depleted nuclei appear to arise from oligomers assembled from 168 bp cores in close contact exhibiting trimming of 0-20 bp at the ends. Electrophorograms of undepleted nuclear digests reveal oligomer bands in several size classes, each corresponding to one or more combinations of 168 bp particles, H1-protected spacers of about 20 bp length, and particles with ends trimmed to varying degrees. 相似文献
5.
Structure of chromatin containing extensively acetylated H3 and H4 总被引:39,自引:0,他引:39
R T Simpson 《Cell》1978,13(4):691-699
I have grown HeLa cells in 5 mM sodium n-butyrate leading to extensive in vivo histone acetylation, and have characterized the structure of chromatin containing the modified histones. Nuclear DNA in butyrate-treated cells is digested 5-10 fold more rapidly by DNAase I than the DNA of control cells. Staphylococcal nuclease degrades the two nuclear samples to acid-soluble material with identical rates; this nuclease, however, does excise nucleosomes with extensively acetylated histones from the nucleoprotein chain preferentially. The physical properties of unsheared chromatin and isolated core particles from control and butyrate-treated cells are closely similar, as are the rates of digestion of core particles from the two cell preparations by DNAase I. Determination of the relative susceptibilities of cleavage sites for DNAase I demonstrates that the site 60 bases from the ends of the DNA resistant in control cells, becomes susceptible to the nuclease in core particles containing acetylated histones. Similarly, the 5' terminal phosphate at the end of DNA in core prticles is removed by staphylococcal nuclease 2-3 fold faster in particles containing acetylated histones than in particles from control cells. 相似文献
6.
The interaction of high mobility proteins HMG14 and 17 with nucleosomes. 总被引:41,自引:22,他引:19
下载免费PDF全文
![点击此处可从《Nucleic acids research》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The interaction of the high mobility group proteins, HMG14 and HMG17, with nucleosome core particles has been studied. The results show that two molecules of HMG14/17 can be bound tightly but reversibly to each core particle and that their affinity for core particles is greater than their affinity for histone-free DNA of core size. Thermal denaturation and nuclease digestion studies suggest that major sites of interaction are located near the ends of the nucleosome core DNA. When nucleosome preparations from chicken erythrocyte nuclei stripped of HMG proteins are partially titrated with HMG14/17, the nucleosome-HMG complex fraction is enriched in beta-globin gene sequences. 相似文献
7.
Nucleosome core particles and oligonucleosomes were isolated by digesting rat testis nuclei with micrococcal nuclease to 20% acid-solubility, followed by fractionation of the digest on a Bio-Gel A-5m column. The core particles thus isolated were characterized on the basis of their DNA length of 151 +/- 5 base-pairs and sedimentation coefficient of 11.4S. Analysis of the acid-soluble proteins of the core particles indicated that histones TH2B and X2 are constituents of the core particles, in addition to the somatic histones H2A, H2B, H3 and H4. The acid-soluble proteins of the oligonucleosomes comprised all the histones, including both the somatic (H1, H2A, H2B, H3, H4 and X2) and the testis-specific ones (TH1 and TH2B). It was also observed that histones TH1 and H1 are absent from the core particles and were readily extracted from the chromatin by 0.6 M-NaCl, which indicated that both of them are bound to the linker DNA. 相似文献
8.
Pachytene nuclei were isolated from rat testes by the unit gravity sedimentation technique and contained histone variants H1a, H1t, TH2A, TH2B, and X2 in addition to the somatic histones H1bde, H1c, H2A, H2B, H3, and H4. The basic organization of the pachytene chromatin namely the nucleosome repeat length and the accessibility to micrococcal nuclease, was similar to that of rat liver interphase chromatin. However, when digested by DNase I, the susceptibility of pachytene chromatin was 25% more than liver chromatin under identical conditions. Nucleosome core particles were isolated from both liver and pachytene nuclei and were characterized for their DNA length and integrity of the nucleoprotein on low ionic strength nucleoprotein gels. While liver core particles contained all the somatic histones H2A, H2B, H3, and H4, in the pachytene core particles, histone variants TH2A, X2, and TH2B had replaced nearly 60% of the respective somatic histones. A comparison of the circular dichroism spectra obtained for pachytene and liver core particles indicated that the pachytene core particles were less compact than the liver core particles. Studies on the thermal denaturation properties of the two types of core particles revealed that the fraction of the pachytene core DNA melting at the premelting temperature region of 55-60 degrees C was significantly higher than that of the liver core DNA. 相似文献
9.
The assembly of an H2A2,H2B2,H3,H4 hexamer onto DNA under conditions of physiological ionic strength 总被引:2,自引:0,他引:2
A novel nucleohistone particle is generated in high yield when a complex of DNA with the four core histones formed under conditions that are close to physiological (0.15 M NaCl, pH 8) is treated with micrococcal nuclease. The particle was found to contain 102 base pairs of DNA in association with six molecules of histones in the ratio 2H2A:2H2B:1H3:1H4 after relatively brief nuclease treatment. Prolonged nuclease digestion resulted in a reduction in the DNA length to a sharply defined 92-base pair fragment that was resistant to further degradation. Apparently normal nucleosome core particles containing two molecules each of the four core histones in association with 145 base pairs of DNA and a particle containing one molecule each of histones H2A and H2B in association with approximately 40 base pairs of DNA were also generated during nuclease treatment of the histone-DNA complexes formed under physiological ionic strength conditions. Kinetic studies have shown that the hexamer particle is not a subnucleosomal fragment produced by the degradation of nucleosome core particles. Furthermore, the hexamer particle was not found among the products of nuclease digestion when histones and DNA were previously assembled in 0.6 M NaCl. The high sedimentation coefficient of the hexameric complex (8 S) suggests that the DNA component of the particle has a folded conformation. 相似文献
10.
The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes. 相似文献
11.
Cellular DNA replication was stimulated in confluent monolayers of CV-1 monkey kidney cells following infection with SV40. Nuclei were isolated from CV-1 cells labeled with [3H]thymidine and then incubated in the presence of [alpha-32P]deoxyribonucleoside triphosphates under conditions that support DNA replication. To determine whether or not the cellular DNA synthesized in vitro was assembled into nucleosomes the DNA was digested in situ with either micrococcal nuclease or pancreatic DNase I, and the products were examined by electrophoretic and sedimentation analysis. The distribution of DNA fragment lengths on agarose gels following micrococcal nuclease digestion was more heterogeneous for newly replicated than for the bulk of the DNA. Nonetheless, the state of cellular DNA synthesized in vitro (32P-labeled) was found to be identical with that of the DNA in the bulk of the chromatin (3H-labeled) by the following criteria: (i) The extent of protection against digestion by micrococcal nuclease of DNase I. (ii) The size of the nucleosomes (180 base pairs) and core particles (145 base pairs). (iii) The number and sizes of DNA fragments produced by micrococcal nuclease in a limit digest. (iv) The sedimentation behavior on neutral sucrose gradients of nucleoprotein particles released by micrococcal nuclease. (v) The number and sizes of DNA fragments produced by DNase I digestion. These results demonstrate that cellular DNA replicated in isolated nuclei is organized into typical nucleosomes. Consequently, subcellular systems can be used to study the relationship between DNA replication and the assembly of chromatin under physiological conditions. 相似文献
12.
The granular particles of chromatin peripheral layer, were isolated together, with the nuclear envelope by treatment of nuclei with nuclease. These particles differ from total chromatin by a decreased content of histone H1, a specific set of minor acid-soluble proteins and a low DNA methylation level. Taking account of the fact that these particles facilitate chromatin interaction with the nuclear envelope, the latter were termed as "anchorosomes". Using UV-induced cross-linking of DNA to the proteins, it was found that all anchorosome-specific acid-soluble proteins can directly interact with anchorosomal DNA. Treatment of anchorosomes with staphylococcal nuclease and electron microscopic data showed that anchorosomes have a nucleosomal organization. Five to ten per cent of anchorosomal DNA appear to be firmly bound to nuclear lamina. This DNA cannot be separated from the lamina by treatment with 2 M NaCl, 1% SDS or heparin (1 mg/ml). The bulk of DNA in the laminal fraction after treatment with the above reagents is protected from hydrolysis with DNAase I by anchorosomal proteins and thus has a high molecular weight (10,000-30,000 base pairs). After treatment of anchorosomes with 0.6 M or 2 M NaCl, DNAase I splits this DNA, predominantly to minor fragments. 相似文献
13.
Multiple structural features are responsible for the nuclease sensitivity of the active ovalbumin gene 总被引:5,自引:0,他引:5
The ovalbumin gene in chick oviduct nuclei or nucleosomes is digested preferentially by either DNase I or staphylococcal nuclease. Staphylococcal nuclease preferentially cuts between and within core particles of the oviduct ovalbumin gene; thus, the ovalbumin gene is more quickly degraded to mononucleosomes and the DNA within these monomers is digested to a nonhybridizable size significantly faster than the chicken globin gene. Mono- and oligonucleosomes generated by partial staphylococcal nuclease digestion at 0 degrees C, but not at 37 degrees C, retain equal sensitivity to DNase I. Most of this sensitivity persists when histone H1 and most of the non-histone chromosomal proteins are removed with 0.6 M NaCl. On the basis of these observations, we propose that nuclease sensitivity of the oviduct ovalbumin gene is due to covalent modifications of the core histones and that this sensitivity is amplified by interaction of other chromosomal proteins with these modified histones. 相似文献
14.
This paper describes the distribution of DNA-lesions generated by the potent carcinogen benzo(a)pyrene (BP) or its ultimate metabolic derivative 7 alpha, 8 8 beta, di-hydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) within mammalian chromatin using the enzymic probe micrococcal nuclease. We have shown that the progress of the nuclease on naked DNA is unaffected by the presence of the hydrocarbon lesion at moderate extents of digestion. Digestion of nuclei isolated from murine erythroleukaemic cells immediately following BPDE treatment, and analysis of micrococcal nuclease resistant DNA by TCA precipitation, hydroxyapatite chromatography and gel electrophoresis demonstrates a non-random distribution of lesions. Approximately three times more binding occurs on the linker DNA regions between nucleosome cores than on the nucleosome core DNA itself. A similar result was obtained with BPDE treated primary mouse embryo cells; however nuclei isolated from these cells after prolonged treatment with BP (to allow metabolic activation) showed no such preferential binding. Post-treatment incubation of BPDE-treated cells shows that this difference can be accounted for by the loss of preferential localisation with time. 相似文献
15.
The influence of cis-diamminedichloroplatinum (II) (cis-DDP) binding to chromatin in chicken erythrocyte nuclei and the nucleosomal core particle is investigated. The cis-DDP modifications alter DNA-protein interactions associated with the higher order structure of chromatin to significantly inhibit the rate of micrococcal nuclease digestion and alter the digestion profile. However, cis-DDP modification of core particle has little effect on the digestion rate and the relative distribution of DNA fragments produced by microccocal nuclease digestion. Analysis of the monomer DNA fragments derived from the digestion of modified nuclei suggests that cis-DDP binding does not significantly disrupt the DNA structure within the core particle, with its major influence being on the internucleosomal DNA. Together these findings suggest that cis-DDP may preferentially bind to the internucleosomal region and/or that the formation of the intrastrand cross-link involving adjacent guanines exhibits a preference for the linker region. Sucrose gradient profiles of the modified nucleoprotein complexes further confirm that the digestion profile for micrococcal nuclease is altered by cis-DDP binding and that the greatest changes occur at the initial stages of digestion. The covalent cross-links within bulk chromatin fix a sub-population of subnucleosomal and nucleosomal products, which are released only after reversal by NaCN treatment. Coupled with our previous findings, it appears that this cis-DDP mediated cross-linking network is primarily associated with protein-protein crosslinks of the low mobility group (LMG) proteins. 相似文献
16.
Chromatin in isolated rat liver nuclei was compared with chromatin in (i) nuclei depleted of H1 by acid extraction; (ii) nuclei treated at pH 3.2 (without removal of H1), and (iii) depleted nuclei following reassociation of H1. Electron microscopy and digestion by DNase I, micrococcal nuclease and endogenous Ca/Mg endonuclease were used for this comparative examination. Electron micrographs of H1-depleted nuclei showed a dispersed and finely granular appearance. The rate of DNA cleavage by micrococcal nuclease or DNase I was increased several-fold after H1 removal. Discretely sized intermediate particles produced by Ca/Mg endonuclease in native nuclei were not observed in digests of depleted nuclei. Digestion by micrococcal nuclease to chromatin particles soluble in 60 mM NaCl buffer appeared not to be affected in depleted nuclei. When nuclei were treated at pH 3.2, neither the appearance of chromatin in electron micrographs nor the mode or rate of nuclease digestion changed appreciably. Following reassociation of H1 to depleted nuclei, electron micrographs demonstrated the reformation of compacted chromatin, but the lower rate of DNA cleavage in native nuclei was not restored. Further, H1 reassociation produced a significant decrease in the solubility of nuclear chromatin cleaved by micrococcal nuclease or Ca/Mg endonuclease. In order to evaluate critically the reconstitution of native chromatin from H1-depleted chromatin we propose the use of digestion by a variety of nucleases in addition to an electron microscopic examination. 相似文献
17.
Mononucleosomes were released from both isolated mammalian (hog thyroid) and protozoan (Tetrahymena) nuclei by the bleomycin-induced DNA-strand breaking reaction. Trout sperm nuclei, on the other hand, were protected from the bleomycin-mediated DNA degradation. The mononucleosomes released from the bleomycin-treated nuclei contained the core histones H2A, H2B, H3, and H4; while HMG1 and HMG2 proteins, in addition to the core histones, were detected in the mononucleosomes obtained by micrococcal nuclease digestion of nuclei. HMGs, but not H1 histone, were dissociated into the supernatant by cleavage of chromatin DNA with bleomycin, whereas both HMGs and H1 were found in that fraction by digestion of nuclei with micrococcal nuclease. HMG1 and HMG2 were exclusively dissociated from chromatin with 1 mM bleomycin under the solvent condition where the DNA strand-breaking activity of the drug is repressed. These observations suggest the possibility that bleomycin preferentially binds to linker DNA regions not occupied by H1 histone in chromatin and exclusively dissociates HMG proteins and breaks the DNA strand. The results of the effects on bleomycin-induced DNA cleavage of nuclei of various drugs including polyamines, chelating agents, intercalating antibiotics such as mitomycin C or adriamycin, and radical scavengers are also presented. 相似文献
18.
19.
S G Bavykin S I Usachenko A O Zalensky A D Mirzabekov 《Journal of molecular biology》1990,212(3):495-511
We have compared the mononucleosomal pattern produced by micrococcal nuclease digestion of condensed and unfolded chromatin and chromatin in nuclei from various sources with the repeat length varying from 165 to 240 base-pairs (bp). Upon digestion of isolated H1-containing chromatin of every tested type in a low ionic strength solution (unfolded chromatin), a standard series of mononucleosomes (MN) was formed: the core particle, MN145, and H1-containing, MN165, MN175, MN185, MN195, MN205 and MN215 (the indexes give an approximate length of the nucleosomal DNA that differs in these particles by an integral number of 10 bp). In addition to the pattern of unfolded chromatin, digestion of whole nuclei or condensed chromatin (high ionic strength of Ca2+) gave rise to nuclei-specific, H1-lacking MN155. Digestion of H1-lacking chromatin produced only MN145, MN155 and MN165 particles, indicating that the histone octamer can organize up to 165 bp of nucleosomal DNA. Although digestion of isolated sea urchin sperm chromatin (repeat length of about 240 bp) at a low ionic strength gave a typical "unfolded chromatin pattern", digests of spermal nuclei contained primarily MN145, MN155, MN235 and MN245 particles. A linear arrangement of histones along DNA (primary organization) of the core particle was found to be preserved in the mononucleosomes, with the spacer DNA length from 10 to 90 bp on one (in MN155) or both sides of core DNA being a multiple of about 10 bp. In MN235, the core particle occupies preferentially a central position with the length of the spacer DNA on both sides of the core DNA being usually about 30 + 60 or 40 + 50 bp. Histone H1 is localized at the ends of these particles, i.e. close to the centre of the spacer DNA. The finding that globular part of histones H3 and sea urchin sperm H2B can covalently bind to spacer DNA suggests their involvement in the organization of chromatin superstructure. Our data indicate that decondensation of chromatin is accompanied by rearrangement of histone H1 on the spacer DNA sites adjacent to the core particle and thus support a solenoid model for the chromatin superstructure in nuclei in which the core DNA together with the spacer DNA form a continuous superhelix. 相似文献