nucleosomes  (Worthington Biochemical)


Bioz Verified Symbol Worthington Biochemical is a verified supplier
Bioz Manufacturer Symbol Worthington Biochemical manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90

    Structured Review

    Worthington Biochemical nucleosomes
    Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S <t>nucleosome</t> positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.
    Nucleosomes, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleosomes/product/Worthington Biochemical
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nucleosomes - by Bioz Stars, 2020-07
    90/100 stars

    Images

    1) Product Images from "In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes"

    Article Title: In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.6.2364-2372.2004

    Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S nucleosome positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.
    Figure Legend Snippet: Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S nucleosome positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.

    Techniques Used: Binding Assay, Purification, Recombinant, Nucleic Acid Electrophoresis, Staining, Incubation, Agarose Gel Electrophoresis

    2) Product Images from "The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed"

    Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed

    Journal: Genome Research

    doi: 10.1101/gr.160150.113

    Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein
    Figure Legend Snippet: Nucleosome redistributions are determined by the underlying DNA sequence. ( A ) Western blots with the specified antibodies, at various times (in hours) after KSHV reactivation (hpr), of iSLK.219 cells treated with 0.2 µg/mL doxycycline. BRG1 protein

    Techniques Used: Sequencing, Western Blot

    Reactivation of KSHV resulted in widespread, transient nucleosome redistribution. ( A ) Boxplot of the correlation values for the 472 loci nucleosome distributions between the 0-h time point and the time points following KSHV reactivation. ( B ) Number of
    Figure Legend Snippet: Reactivation of KSHV resulted in widespread, transient nucleosome redistribution. ( A ) Boxplot of the correlation values for the 472 loci nucleosome distributions between the 0-h time point and the time points following KSHV reactivation. ( B ) Number of

    Techniques Used:

    DNA sequence determined the concerted, widespread, transient redistribution of nucleosomes. ( A ) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for 0 h (black) and 24 h (red). ( B
    Figure Legend Snippet: DNA sequence determined the concerted, widespread, transient redistribution of nucleosomes. ( A ) Average values for all genes identified as DNA-directed and DNA-independent, calculated by alignment of loci to the TSS for 0 h (black) and 24 h (red). ( B

    Techniques Used: Sequencing

    Model of chromatin regulation in which nucleosome distributions move from a basal state architecture, to a transient intermediate state, then return to the basal architecture, in response to a common stimulus. The transient intermediate state's architecture
    Figure Legend Snippet: Model of chromatin regulation in which nucleosome distributions move from a basal state architecture, to a transient intermediate state, then return to the basal architecture, in response to a common stimulus. The transient intermediate state's architecture

    Techniques Used:

    3) Product Images from "Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent manner"

    Article Title: Linker histone protects linker DNA on only one side of the core particle and in a sequence-dependent manner

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi:

    Chromatosome reconstitution on the 5S rDNA fragment from L. variegatus. ( A ) Reconstitution of DNA into core nucleosomes and chromatosomes as visualized by the band shift assay. The 243-bp fragment was reconstituted with core histone octamer and further with histone H1° (LH/core histone molar ratios of 1.0 and 1.3 in lanes 3 and 4, respectively). The resultant complexes were resolved in 0.9% agarose gels (see Materials and Methods ). Lane 1 contains pBSIISK + / Hin fI, Xba I marker. ( B ) Products of MNase digestion of the mixture of chromatosomes and naked DNA, presented in A , lane 4. Lanes labeled M contain pBR322/ Msp I size markers. The open triangle above the lanes indicates increasing levels of MNase digestion. The positions of core- and chromatosome-sized DNA fragments are indicated. The control pattern of digestion of free DNA is also presented. The free DNA present in the incubation mixture will not contribute to the patterns observed with the core or chromatosome reconstitutes, because under the conditions of digestion used to obtain DNA from these particles naked DNA is completely digested to small fragments unobservable on gels.
    Figure Legend Snippet: Chromatosome reconstitution on the 5S rDNA fragment from L. variegatus. ( A ) Reconstitution of DNA into core nucleosomes and chromatosomes as visualized by the band shift assay. The 243-bp fragment was reconstituted with core histone octamer and further with histone H1° (LH/core histone molar ratios of 1.0 and 1.3 in lanes 3 and 4, respectively). The resultant complexes were resolved in 0.9% agarose gels (see Materials and Methods ). Lane 1 contains pBSIISK + / Hin fI, Xba I marker. ( B ) Products of MNase digestion of the mixture of chromatosomes and naked DNA, presented in A , lane 4. Lanes labeled M contain pBR322/ Msp I size markers. The open triangle above the lanes indicates increasing levels of MNase digestion. The positions of core- and chromatosome-sized DNA fragments are indicated. The control pattern of digestion of free DNA is also presented. The free DNA present in the incubation mixture will not contribute to the patterns observed with the core or chromatosome reconstitutes, because under the conditions of digestion used to obtain DNA from these particles naked DNA is completely digested to small fragments unobservable on gels.

    Techniques Used: Electrophoretic Mobility Shift Assay, Marker, Labeling, Incubation

    4) Product Images from "VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells"

    Article Title: VivosX, a disulfide crosslinking method to capture site-specific, protein-protein interactions in yeast and human cells

    Journal: eLife

    doi: 10.7554/eLife.36654

    A cartoon depicting the proposed histone cycle. Z-B dimers are in green, A-B dimers in red and H3-H4 tetramers in gray. NFR: Nucleosome free region. Black arrows: transcription start site. +1: the nucleosome immediately downstream of a promoter. PIC: preinitiation complex. Balls in cyan: cysteine probes. Cysteine probes on H2A are not shown for simplicity.
    Figure Legend Snippet: A cartoon depicting the proposed histone cycle. Z-B dimers are in green, A-B dimers in red and H3-H4 tetramers in gray. NFR: Nucleosome free region. Black arrows: transcription start site. +1: the nucleosome immediately downstream of a promoter. PIC: preinitiation complex. Balls in cyan: cysteine probes. Cysteine probes on H2A are not shown for simplicity.

    Techniques Used:

    The thiol-disulfide interchange reaction between the cysteine thiols and 4-DPS. ( A ) A proposed mechanism of the thiol-disulfide interchange reaction between cysteine thiols and 4-DPS. Light and dark green loops represent the L1 regions of the opposite nucleosomal H2A.Z (or H2A) pair at the L1-L1’ interface. ( B ) The proposed clamshell opening of the nucleosome structure could facilitate the conformational dynamics of the L1-L1’ interface. Dotted arrows: direction of movement.
    Figure Legend Snippet: The thiol-disulfide interchange reaction between the cysteine thiols and 4-DPS. ( A ) A proposed mechanism of the thiol-disulfide interchange reaction between cysteine thiols and 4-DPS. Light and dark green loops represent the L1 regions of the opposite nucleosomal H2A.Z (or H2A) pair at the L1-L1’ interface. ( B ) The proposed clamshell opening of the nucleosome structure could facilitate the conformational dynamics of the L1-L1’ interface. Dotted arrows: direction of movement.

    Techniques Used:

    Relative Cα-Cα’ distances at the L1-L1’ interface of AA and ZZ nucleosomes. ( A,B ) Cartoons depicting the anti-parallel L1 and L1’ loops of yeast H2A.Z and H2A. ( C ) A table of Cα-Cα’ distances measured between the indicated amino acid positions in the AA and ZZ nucleosomes. The Cα-Cα’ distances for H2A.Z are based on the published mouse/frog ZZ nucleosome structure ( Suto et al., 2000 ) but the corresponding positions of yeast Htz1 were indicated. The Cα-Cα’ distances are based on the yeast AA nucleosome structure ( White et al., 2001 ).
    Figure Legend Snippet: Relative Cα-Cα’ distances at the L1-L1’ interface of AA and ZZ nucleosomes. ( A,B ) Cartoons depicting the anti-parallel L1 and L1’ loops of yeast H2A.Z and H2A. ( C ) A table of Cα-Cα’ distances measured between the indicated amino acid positions in the AA and ZZ nucleosomes. The Cα-Cα’ distances for H2A.Z are based on the published mouse/frog ZZ nucleosome structure ( Suto et al., 2000 ) but the corresponding positions of yeast Htz1 were indicated. The Cα-Cα’ distances are based on the yeast AA nucleosome structure ( White et al., 2001 ).

    Techniques Used:

    5) Product Images from "A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver"

    Article Title: A Mutation in Histone H2B Represents A New Class Of Oncogenic Driver

    Journal: Cancer discovery

    doi: 10.1158/2159-8290.CD-19-0393

    Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.
    Figure Legend Snippet: Expression of mutant H2B in yeast destabilizes nucleosomes, deregulates gene expression and reduces nucleosome occupancy at the PHO5 promoter. WT or E79K H2B (analogous to human H2B-E76K) was expressed in S. Cerevisiae. (A) Yeast cells expressing H2B-E79K are temperature sensitive. Limiting dilutions of yeast expressing WT, E79A, E79Q or E79K were plated and incubated at 30°C or 37°C. Cell growth was evaluated after 1 day. (B) Yeast doubling time is significantly increased in cells expressing E79K-H2B at 37°C. (C) Time course of MNase sensitivity from spheroplasted yeast grown in rich media. M, marker. (D) Chromatin pellets were extracted with increasing concentrations of salt as indicated. Immuno-blotting of the soluble fraction was performed with antibody to H4. (E) Cells expressing WT, E79Q or E79K H2B were maintained in either rich media (YPDA) or phosphate-free media and expression of the phosphate-inducible PHO5 gene was measured by RT-PCR. (F) Nucleosome scanning assay of the PHO5 promoter from cells expressing either WT or E79K grown in rich media. Chromatin was digested with MNase, mononucleosomal DNA was purified and MNase protection was determined by qPCR. H2B occupancy at −2 nucleosome position of PHO5 is reduced in E79K cells as indicated by the arrow.

    Techniques Used: Expressing, Mutagenesis, Incubation, Marker, Reverse Transcription Polymerase Chain Reaction, Purification, Real-time Polymerase Chain Reaction

    The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.
    Figure Legend Snippet: The E76K mutation in H2B destabilizes the histone octamer and fails to protect the nucleosome from nuclease treatment in vitro . (A) The H2B-E76K mutant was unable to form stable octamers in vitro . Recombinant human histones (H2A, H2B, H3, and H4) were mixed and histone octamers resolved from (H3/H4) 2 tetramers, H2A/H2B dimers and free histones by gel filtration chromatography. (B) Nucleosomes were reconstituted by mixing equimolar amounts of DNA (147bp) and octamers or in the case of E76K of tetramers and dimers (1:2 molar ratio) and resolved by Native PAGE. Nucleosomes containing H2B-E76K and E76Q have an altered migration pattern, intermediate between a tetrasome and a WT nucleosome. (C) Micrococcal nuclease (MNase) sensitivity assay performed on nucleosomes made with WT, E76Q and E76K H2B mutants shows more rapid digestion of E76K containing nucleosomes than those with WT H2B. A time course by gel (left) and densitometry quantification (right) of intact nucleosomes following MNase treatment. (D) The MNase susceptibility of E76K nucleosomes is distinct from nucleosomes formed only with tetrasomes.

    Techniques Used: Mutagenesis, In Vitro, Recombinant, Filtration, Chromatography, Clear Native PAGE, Migration, Sensitive Assay

    6) Product Images from "Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming"

    Article Title: Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming

    Journal: Cell

    doi: 10.1016/j.cell.2015.03.017

    The contribution of non-specific binding to nucleosome targeting in vitro (A) Representative EMSA showing the affinity of recombinant O, S, K, M proteins (bact. top panels and mamm. bottom panels) to LIN28B -DNA (left panels) and LIN28B . (B) DNase-I footprinting showing the protection of LIN28B -DNA (left panels) and LIN28B -nuc (right panels) in the absence (blue lines) or presence (red lines) of O, S, K, and M. Electropherograms of 5’-6FAM end-labeled LIN28B (top strand) oligonucleotides generated by DNase-I digestion of DNA (0.006 U) and nucleosomal DNA (0.06 U). Dashed boxes and stars represent specific and non-specific sites protected by O, S, K, and M, respectively. (C) A cartoon representation of the 162 bp LIN28B DNA (left) and nucleosome (right) highlighting the binding sites of O, S, K, and M in vitro in blue, red, orange, and green, respectively, as measured by DNase-I footprining. The protected DNA sequences are indicated.
    Figure Legend Snippet: The contribution of non-specific binding to nucleosome targeting in vitro (A) Representative EMSA showing the affinity of recombinant O, S, K, M proteins (bact. top panels and mamm. bottom panels) to LIN28B -DNA (left panels) and LIN28B . (B) DNase-I footprinting showing the protection of LIN28B -DNA (left panels) and LIN28B -nuc (right panels) in the absence (blue lines) or presence (red lines) of O, S, K, and M. Electropherograms of 5’-6FAM end-labeled LIN28B (top strand) oligonucleotides generated by DNase-I digestion of DNA (0.006 U) and nucleosomal DNA (0.06 U). Dashed boxes and stars represent specific and non-specific sites protected by O, S, K, and M, respectively. (C) A cartoon representation of the 162 bp LIN28B DNA (left) and nucleosome (right) highlighting the binding sites of O, S, K, and M in vitro in blue, red, orange, and green, respectively, as measured by DNase-I footprining. The protected DNA sequences are indicated.

    Techniques Used: Binding Assay, In Vitro, Recombinant, Footprinting, Labeling, Generated

    O, S, K, and M display a range of nucleosome targeting in vivo (A) Read density heatmaps (in color scales) showing the intensity of O, S, K, and M ChIP-seq signal (blue) and MNase-seq (red) spanning ± 1 kb from the center of the O, S, K, and M peaks where each factor binds alone within 500 bp threshold. The analyzed sequences were organized in rank order, from high to low number ChIP-seq reads within the central 200 bp (double arrows). The number of targeted sites is indicated. . (C) The binding affinity of S, K, and M (1 nM) in the presence of Oct4 (0.3 nM) to LIN28B nucleosomal DNA (lanes; 4, 6, and 8, respectively) or absence of Oct4 (lanes; 3, 5, and 7). The binding of Oct4 on its own (lane 2) and free LIN28B .
    Figure Legend Snippet: O, S, K, and M display a range of nucleosome targeting in vivo (A) Read density heatmaps (in color scales) showing the intensity of O, S, K, and M ChIP-seq signal (blue) and MNase-seq (red) spanning ± 1 kb from the center of the O, S, K, and M peaks where each factor binds alone within 500 bp threshold. The analyzed sequences were organized in rank order, from high to low number ChIP-seq reads within the central 200 bp (double arrows). The number of targeted sites is indicated. . (C) The binding affinity of S, K, and M (1 nM) in the presence of Oct4 (0.3 nM) to LIN28B nucleosomal DNA (lanes; 4, 6, and 8, respectively) or absence of Oct4 (lanes; 3, 5, and 7). The binding of Oct4 on its own (lane 2) and free LIN28B .

    Techniques Used: In Vivo, Chromatin Immunoprecipitation, Binding Assay

    Related Articles

    Purification:

    Article Title: A novel histone H4 variant H4G regulates rDNA transcription in breast cancer
    Article Snippet: .. A total of 20 μl of the purified reconstituted nucleosome was digested by the indicated amount of MNase (Worthington) at room temperature for 5 min. .. Samples were loaded onto 6% non-denaturing acrylamide gel without DNA extraction.

    Footprinting:

    Article Title: Pioneer Transcription Factors Target Partial DNA Motifs on Nucleosomes to Initiate Reprogramming
    Article Snippet: .. DNase footprinting was carried out by treating free DNA or nucleosomes, 6FAM 5’ end-labelled, with DNase-I (Worthington) in the absence or presence of TFs. .. The end-labelled digested fragments were separated by capillary electrophoresis in ABI 96-capillary 3730XL Sequencer (Applied Biosystems).

    Titration:

    Article Title: The spring-loaded genome: Nucleosome redistributions are widespread, transient, and DNA-directed
    Article Snippet: .. All MNase nucleosome-mapping experiments were performed with two biological replicates from completely independent growths and treatments of cells (Supplemental Fig. 19). iSLK.219 nuclei or in vitro–assembled nucleosomes were digested with a titration of MNase (Worthington Biochemical Corp.) for nucleosome-distribution experiments. .. The concentrations for the MNase titration were 4 units/mL, 2 units/mL, 1 unit/mL, and 0.5 unit/mL of MNase in MNase cleavage buffer: 5 mM KCl, 4 mM MgCl2 , 1 mM CaCl2 , 12.5% glycerol, and 50 mM Tris-Cl (pH 7.4).

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 90
    Worthington Biochemical nucleosomes
    Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S <t>nucleosome</t> positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.
    Nucleosomes, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 90/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleosomes/product/Worthington Biochemical
    Average 90 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    nucleosomes - by Bioz Stars, 2020-07
    90/100 stars
      Buy from Supplier

    85
    Worthington Biochemical nucleosomal dna isolation
    Abundance and genomic distribution of Single Nucleosome Epi-Polymorphisms. (A) The fraction of nucleosomes that were called SNEP at FDR = 0.0001 was computed in every 1Kb-segment along each chromosome. Density ranged from 0 (white) to 100% (red). Grey denotes regions where nucleosomes could not be aligned. (B) Enrichment of H3K14ac SNEPs upstream Ty insertions and rDNA repeats. The fraction of BYac SNEPs among all nucleosomes was counted in 10 kb intervals upstream the rDNA region (brown triangles). The 7 fold enrichment of BYac SNEPs in the first 10 kb was significant (grey area, Chi-square test P = 0.01). Upstream regions of all Ty insertions present in BY and absent from RM were analyzed similarly (black points), and their fractions of BYac SNEPs were averaged. The 1.3 fold enrichment in the 10 kb interval directly upstream the insertions was significant (grey area, Chi-square test P = 0.014). (C) Local correlation between H3K14ac SNEPs. Ten nucleosomes were interrogated upstream and downstream each SNEP (x-axis). For each one, cases where the nucleosome was a SNEP similar to the centered one (either BYac or RMac) were counted and divided by the total number of nucleosomes interrogated at that position (brown histogram). Control values were obtained from the same procedure applied after re-assigning SNEPs to random nucleosomes (grey histogram). (D) Density of H3K14 acetylation and SNEPs relative to gene position. Every gene was divided by segmenting the coding sequence in 10 bins (average bin size of 160 bp) and its upstream and downstream regions in 100 bp bins. For every gene and every bin, log(acBY/nucBY) was averaged across replicated experiments and across all probes matching <t>intra-nucleosomal</t> <t>DNA</t> to produce the top green profile. Similarly, averaged log(acRM/nucRM) values generated the top black profile. Here acBY and acRM refer to H3K14ac ChIP-CHIP experiments on BY and RM samples, respectively, while nucBY and nucRM refer to nucleosomal mapping experiments on BY and RM samples, respectively. Note that probes matching inter-nucleosome linkers do not contribute to the profiles, which are therefore corrected for nucleosome abundance. Bottom profiles were obtained by counting the fraction of BYac SNEPs (green) and RMac SNEPs (black) among all nucleosomes that overlapped at least partially the bin, and averaging these fractions across all genes.
    Nucleosomal Dna Isolation, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleosomal dna isolation/product/Worthington Biochemical
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    nucleosomal dna isolation - by Bioz Stars, 2020-07
    85/100 stars
      Buy from Supplier

    88
    Worthington Biochemical nucleosomal arrays
    TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of <t>nucleosomal</t> fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .
    Nucleosomal Arrays, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/nucleosomal arrays/product/Worthington Biochemical
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    nucleosomal arrays - by Bioz Stars, 2020-07
    88/100 stars
      Buy from Supplier

    Image Search Results


    Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S nucleosome positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.

    Journal: Molecular and Cellular Biology

    Article Title: In Vitro Targeting Reveals Intrinsic Histone Tail Specificity of the Sin3/Histone Deacetylase and N-CoR/SMRT Corepressor Complexes

    doi: 10.1128/MCB.24.6.2364-2372.2004

    Figure Lengend Snippet: Reconstitution and analysis of the nucleosomal template. (A) Schematic representation of the DNA template containing eight LexA binding sites and a 5S nucleosome positioning element. (B) Analysis of purified recombinant (Rec.) Xenopus octamers and hyperacetylated (Hyperac.) core histones purified from HeLa cells on SDS-polyacrylamide (15%) gel electrophoresis gel stained with Coomassie brilliant blue. (C) Partial micrococcal nuclease digestion. Nucleosomal templates were incubated with 10 mU micrococcal nuclease at 37°C for 0, 20, 40, 60, and 180 s. Reactions were stopped by adding 10 mM EGTA. DNA was phenol chloroform extracted, precipitated, and loaded onto a 1.5% agarose gel. DNA size markers are indicated on the left. An arrow indicates mononucleosomal DNA.

    Article Snippet: Approximately 750 ng of DNA reconstituted into nucleosomes was digested with 10 mU of micrococcal nuclease (Worthington Biochemicals) for 0, 20, 40, 60, and 180 s at 37°C in buffer F containing 50 mM KCl and 3 mM CaCl2 .

    Techniques: Binding Assay, Purification, Recombinant, Nucleic Acid Electrophoresis, Staining, Incubation, Agarose Gel Electrophoresis

    Remodeling of dinucleosomes by ISWI complexes stimulates histone methylation by SET domain proteins. (A) The SET domain of SET7 binds histones, but not nucleosomes. GST pulldown experiments were conducted with immobilized GST-SET7 polypeptides (residues

    Journal: Molecular and Cellular Biology

    Article Title: SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

    doi: 10.1128/MCB.00775-09

    Figure Lengend Snippet: Remodeling of dinucleosomes by ISWI complexes stimulates histone methylation by SET domain proteins. (A) The SET domain of SET7 binds histones, but not nucleosomes. GST pulldown experiments were conducted with immobilized GST-SET7 polypeptides (residues

    Article Snippet: The remodeled products were analyzed by native gel electrophoresis or by probing nucleosome structures with micrococcal nuclease, DNase I, or restriction endonuclease (Worthington Biochemical and New England Biolabs) ( , , ).

    Techniques: Methylation

    Analysis of ISW2 and SWI/SNF remodeled dinucleosomes. 32 P-end-labeled dinucleosomes were remodeled by ISW2 (A and B) or SWI/SNF (C and D) as described in the legend for Fig. . Remodeling was terminated by apyrase treatment, and nucleosomes

    Journal: Molecular and Cellular Biology

    Article Title: SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

    doi: 10.1128/MCB.00775-09

    Figure Lengend Snippet: Analysis of ISW2 and SWI/SNF remodeled dinucleosomes. 32 P-end-labeled dinucleosomes were remodeled by ISW2 (A and B) or SWI/SNF (C and D) as described in the legend for Fig. . Remodeling was terminated by apyrase treatment, and nucleosomes

    Article Snippet: The remodeled products were analyzed by native gel electrophoresis or by probing nucleosome structures with micrococcal nuclease, DNase I, or restriction endonuclease (Worthington Biochemical and New England Biolabs) ( , , ).

    Techniques: Labeling

    The SET domain of trithorax binds core histones and altered nucleosomal structures but not intact nucleosomes. (A) Schematic of the domain structure of trithorax. Positions of highly conserved blocks of homology with methyltransferases, a C-terminal cysteine-rich

    Journal: Molecular and Cellular Biology

    Article Title: SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

    doi: 10.1128/MCB.00775-09

    Figure Lengend Snippet: The SET domain of trithorax binds core histones and altered nucleosomal structures but not intact nucleosomes. (A) Schematic of the domain structure of trithorax. Positions of highly conserved blocks of homology with methyltransferases, a C-terminal cysteine-rich

    Article Snippet: The remodeled products were analyzed by native gel electrophoresis or by probing nucleosome structures with micrococcal nuclease, DNase I, or restriction endonuclease (Worthington Biochemical and New England Biolabs) ( , , ).

    Techniques:

    The SET domain of ALL1 does not bind remodeled mononucleosomes. (A, top) Analysis of remodeled mononucleosomes by native PAGE. Remodeling assay mixtures (50 μl) contained 1.2 μg of nucleosomes and 2.5 ng of ISWI or 5 ng of Swi-Snf complexes

    Journal: Molecular and Cellular Biology

    Article Title: SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

    doi: 10.1128/MCB.00775-09

    Figure Lengend Snippet: The SET domain of ALL1 does not bind remodeled mononucleosomes. (A, top) Analysis of remodeled mononucleosomes by native PAGE. Remodeling assay mixtures (50 μl) contained 1.2 μg of nucleosomes and 2.5 ng of ISWI or 5 ng of Swi-Snf complexes

    Article Snippet: The remodeled products were analyzed by native gel electrophoresis or by probing nucleosome structures with micrococcal nuclease, DNase I, or restriction endonuclease (Worthington Biochemical and New England Biolabs) ( , , ).

    Techniques: Clear Native PAGE

    The SET domain of ALL1 binds dinucleosomes remodeled by the ISWI class of chromatin remodeling enzymes. (A) Example of dinucleosome assembly. Dinucleosomes were reconstituted onto DNA containing two 601 minimal nucleosome positioning sequences (the orientation

    Journal: Molecular and Cellular Biology

    Article Title: SET Domains of Histone Methyltransferases Recognize ISWI-Remodeled Nucleosomal Species ▿

    doi: 10.1128/MCB.00775-09

    Figure Lengend Snippet: The SET domain of ALL1 binds dinucleosomes remodeled by the ISWI class of chromatin remodeling enzymes. (A) Example of dinucleosome assembly. Dinucleosomes were reconstituted onto DNA containing two 601 minimal nucleosome positioning sequences (the orientation

    Article Snippet: The remodeled products were analyzed by native gel electrophoresis or by probing nucleosome structures with micrococcal nuclease, DNase I, or restriction endonuclease (Worthington Biochemical and New England Biolabs) ( , , ).

    Techniques:

    Abundance and genomic distribution of Single Nucleosome Epi-Polymorphisms. (A) The fraction of nucleosomes that were called SNEP at FDR = 0.0001 was computed in every 1Kb-segment along each chromosome. Density ranged from 0 (white) to 100% (red). Grey denotes regions where nucleosomes could not be aligned. (B) Enrichment of H3K14ac SNEPs upstream Ty insertions and rDNA repeats. The fraction of BYac SNEPs among all nucleosomes was counted in 10 kb intervals upstream the rDNA region (brown triangles). The 7 fold enrichment of BYac SNEPs in the first 10 kb was significant (grey area, Chi-square test P = 0.01). Upstream regions of all Ty insertions present in BY and absent from RM were analyzed similarly (black points), and their fractions of BYac SNEPs were averaged. The 1.3 fold enrichment in the 10 kb interval directly upstream the insertions was significant (grey area, Chi-square test P = 0.014). (C) Local correlation between H3K14ac SNEPs. Ten nucleosomes were interrogated upstream and downstream each SNEP (x-axis). For each one, cases where the nucleosome was a SNEP similar to the centered one (either BYac or RMac) were counted and divided by the total number of nucleosomes interrogated at that position (brown histogram). Control values were obtained from the same procedure applied after re-assigning SNEPs to random nucleosomes (grey histogram). (D) Density of H3K14 acetylation and SNEPs relative to gene position. Every gene was divided by segmenting the coding sequence in 10 bins (average bin size of 160 bp) and its upstream and downstream regions in 100 bp bins. For every gene and every bin, log(acBY/nucBY) was averaged across replicated experiments and across all probes matching intra-nucleosomal DNA to produce the top green profile. Similarly, averaged log(acRM/nucRM) values generated the top black profile. Here acBY and acRM refer to H3K14ac ChIP-CHIP experiments on BY and RM samples, respectively, while nucBY and nucRM refer to nucleosomal mapping experiments on BY and RM samples, respectively. Note that probes matching inter-nucleosome linkers do not contribute to the profiles, which are therefore corrected for nucleosome abundance. Bottom profiles were obtained by counting the fraction of BYac SNEPs (green) and RMac SNEPs (black) among all nucleosomes that overlapped at least partially the bin, and averaging these fractions across all genes.

    Journal: PLoS Genetics

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast

    doi: 10.1371/journal.pgen.1000913

    Figure Lengend Snippet: Abundance and genomic distribution of Single Nucleosome Epi-Polymorphisms. (A) The fraction of nucleosomes that were called SNEP at FDR = 0.0001 was computed in every 1Kb-segment along each chromosome. Density ranged from 0 (white) to 100% (red). Grey denotes regions where nucleosomes could not be aligned. (B) Enrichment of H3K14ac SNEPs upstream Ty insertions and rDNA repeats. The fraction of BYac SNEPs among all nucleosomes was counted in 10 kb intervals upstream the rDNA region (brown triangles). The 7 fold enrichment of BYac SNEPs in the first 10 kb was significant (grey area, Chi-square test P = 0.01). Upstream regions of all Ty insertions present in BY and absent from RM were analyzed similarly (black points), and their fractions of BYac SNEPs were averaged. The 1.3 fold enrichment in the 10 kb interval directly upstream the insertions was significant (grey area, Chi-square test P = 0.014). (C) Local correlation between H3K14ac SNEPs. Ten nucleosomes were interrogated upstream and downstream each SNEP (x-axis). For each one, cases where the nucleosome was a SNEP similar to the centered one (either BYac or RMac) were counted and divided by the total number of nucleosomes interrogated at that position (brown histogram). Control values were obtained from the same procedure applied after re-assigning SNEPs to random nucleosomes (grey histogram). (D) Density of H3K14 acetylation and SNEPs relative to gene position. Every gene was divided by segmenting the coding sequence in 10 bins (average bin size of 160 bp) and its upstream and downstream regions in 100 bp bins. For every gene and every bin, log(acBY/nucBY) was averaged across replicated experiments and across all probes matching intra-nucleosomal DNA to produce the top green profile. Similarly, averaged log(acRM/nucRM) values generated the top black profile. Here acBY and acRM refer to H3K14ac ChIP-CHIP experiments on BY and RM samples, respectively, while nucBY and nucRM refer to nucleosomal mapping experiments on BY and RM samples, respectively. Note that probes matching inter-nucleosome linkers do not contribute to the profiles, which are therefore corrected for nucleosome abundance. Bottom profiles were obtained by counting the fraction of BYac SNEPs (green) and RMac SNEPs (black) among all nucleosomes that overlapped at least partially the bin, and averaging these fractions across all genes.

    Article Snippet: We followed the protocol of Liu et al. for both nucleosomal DNA isolation and ChIP, except that incubation time with micrococcal nuclease (Worthington Biochemical) prior to immunopurification was increased to 30 min at 37°C to obtain mononucleosomes.

    Techniques: Sequencing, Generated, Chromatin Immunoprecipitation

    SNEPs are not associated with transcriptional differences but are enriched at conserved regulatory sites. (A) Display from microarray data directly. Density plots representing the distribution of genes with respect to H3K14 acetylation differences (y-axis) and gene expression differences (x-axis). For every gene, three regions were considered as indicated above the panels. For each region, H3K14ac inter-strain difference was estimated as log(acBY/nucBY)−log(acRM/nucRM) (as defined in legend of Figure 2D ), averaged across replicated experiments and across all probes interrogating nucleosomal DNA of the region. Gene expression inter-strain differences are represented by their t -statistic computed from data of Brem et al. [20] . ρ, Pearson correlation coefficient. A similar picture was obtained when using fold change of expression instead of t -statistics ( Figure S10 ). (B) Display from SNEP locations. For every gene, the fraction of H3K14ac SNEPs correlated to expression was defined as the number of SNEPs acetylated in the strain with highest expression, divided by the total number of nucleosomes in the region. Curves represent the density distribution of genes according to this measure, from actual data (colored) and data where indexes of expression ratios were permuted (black). Colored curves are not significantly shifted to the right (as compared to black curves), ruling out association between SNEP and gene expression differences. (C) BYac but not RMac SNEPs are more abundant at conserved regulatory sites. Nucleosomes were divided in three categories: nucleosomes that covered entirely a conserved regulatory site from the list of MacIsaac et al. [35] , nucleosomes that did not contain such sites but were located in highly conserved non-coding sequences (see Methods ), and nucleosomes excluded from the first two categories. The fraction of SNEPs within each category is presented. Error bars, 95% C.I. The 3.2 and 2.6 fold enrichment at regulatory sites and other conserved regions, respectively, were highly significant ( P

    Journal: PLoS Genetics

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast

    doi: 10.1371/journal.pgen.1000913

    Figure Lengend Snippet: SNEPs are not associated with transcriptional differences but are enriched at conserved regulatory sites. (A) Display from microarray data directly. Density plots representing the distribution of genes with respect to H3K14 acetylation differences (y-axis) and gene expression differences (x-axis). For every gene, three regions were considered as indicated above the panels. For each region, H3K14ac inter-strain difference was estimated as log(acBY/nucBY)−log(acRM/nucRM) (as defined in legend of Figure 2D ), averaged across replicated experiments and across all probes interrogating nucleosomal DNA of the region. Gene expression inter-strain differences are represented by their t -statistic computed from data of Brem et al. [20] . ρ, Pearson correlation coefficient. A similar picture was obtained when using fold change of expression instead of t -statistics ( Figure S10 ). (B) Display from SNEP locations. For every gene, the fraction of H3K14ac SNEPs correlated to expression was defined as the number of SNEPs acetylated in the strain with highest expression, divided by the total number of nucleosomes in the region. Curves represent the density distribution of genes according to this measure, from actual data (colored) and data where indexes of expression ratios were permuted (black). Colored curves are not significantly shifted to the right (as compared to black curves), ruling out association between SNEP and gene expression differences. (C) BYac but not RMac SNEPs are more abundant at conserved regulatory sites. Nucleosomes were divided in three categories: nucleosomes that covered entirely a conserved regulatory site from the list of MacIsaac et al. [35] , nucleosomes that did not contain such sites but were located in highly conserved non-coding sequences (see Methods ), and nucleosomes excluded from the first two categories. The fraction of SNEPs within each category is presented. Error bars, 95% C.I. The 3.2 and 2.6 fold enrichment at regulatory sites and other conserved regions, respectively, were highly significant ( P

    Article Snippet: We followed the protocol of Liu et al. for both nucleosomal DNA isolation and ChIP, except that incubation time with micrococcal nuclease (Worthington Biochemical) prior to immunopurification was increased to 30 min at 37°C to obtain mononucleosomes.

    Techniques: Microarray, Expressing

    Nucleosome positioning in two unrelated natural S. cerevisiae strains. (A) Example of raw signals and nucleosome positioning inference in the region of the PER1 gene. Nucleosomal DNA was purified from each strains in triplicate, amplified linearly and hybridized to whole genome oligonucleotide Tiling arrays. Data were log-transformed and normalized using the quantile-quantile method and averaged across replicates to produce the probe-level signal intensities shown on the top panels. A Hidden Markov Model (HMM) similar to the one previously described [25] was applied to each strain independently to infer nucleosomal positioning (blue rectangles). Faded and plain colors represent ‘delocalized’ and ‘well-positioned’ nucleosomes, respectively, as defined previously [24] . Signal intensities are colored according to the HMM posterior probability to be within a nucleosome (cumulating delocalized and well-positioned). Nucleosome positions from the published atlas of Lee et al. [24] , who used a strain isogenic to BY, are indicated by green rectangles and are also faded when reported as ‘delocalized’. (B) Genes (rows) were clustered based on profiles of nucleosome occupancy at their promoter in the BY strain (see Methods ). Their order was then used to plot heatmaps of nucleosome occupancy around transcriptional start site in BY and RM, respectively, as well as expression divergence between the two strains (according to statistical significance at FDR 5% from the dataset of Brem et al. [20] ). Left curves represent mean occupancy profiles of the six main classes of promoters. (C) Absence of correlation between promoter occupancy and expression divergence. Each dot represents one gene. X-axis: inter-strain difference in expression measured as log2(RM/BY) from Brem et al. [20] . Y-axis: inter-strain dissimilarity of promoter occupancy profiles. For each promoter region, the RM/BY dissimilarity was estimated as 1 - R, where R is the Spearman correlation coefficient between the BY and RM occupancy profiles shown in (B). ρ: Spearman correlation between the resulting X and Y data.

    Journal: PLoS Genetics

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast

    doi: 10.1371/journal.pgen.1000913

    Figure Lengend Snippet: Nucleosome positioning in two unrelated natural S. cerevisiae strains. (A) Example of raw signals and nucleosome positioning inference in the region of the PER1 gene. Nucleosomal DNA was purified from each strains in triplicate, amplified linearly and hybridized to whole genome oligonucleotide Tiling arrays. Data were log-transformed and normalized using the quantile-quantile method and averaged across replicates to produce the probe-level signal intensities shown on the top panels. A Hidden Markov Model (HMM) similar to the one previously described [25] was applied to each strain independently to infer nucleosomal positioning (blue rectangles). Faded and plain colors represent ‘delocalized’ and ‘well-positioned’ nucleosomes, respectively, as defined previously [24] . Signal intensities are colored according to the HMM posterior probability to be within a nucleosome (cumulating delocalized and well-positioned). Nucleosome positions from the published atlas of Lee et al. [24] , who used a strain isogenic to BY, are indicated by green rectangles and are also faded when reported as ‘delocalized’. (B) Genes (rows) were clustered based on profiles of nucleosome occupancy at their promoter in the BY strain (see Methods ). Their order was then used to plot heatmaps of nucleosome occupancy around transcriptional start site in BY and RM, respectively, as well as expression divergence between the two strains (according to statistical significance at FDR 5% from the dataset of Brem et al. [20] ). Left curves represent mean occupancy profiles of the six main classes of promoters. (C) Absence of correlation between promoter occupancy and expression divergence. Each dot represents one gene. X-axis: inter-strain difference in expression measured as log2(RM/BY) from Brem et al. [20] . Y-axis: inter-strain dissimilarity of promoter occupancy profiles. For each promoter region, the RM/BY dissimilarity was estimated as 1 - R, where R is the Spearman correlation coefficient between the BY and RM occupancy profiles shown in (B). ρ: Spearman correlation between the resulting X and Y data.

    Article Snippet: We followed the protocol of Liu et al. for both nucleosomal DNA isolation and ChIP, except that incubation time with micrococcal nuclease (Worthington Biochemical) prior to immunopurification was increased to 30 min at 37°C to obtain mononucleosomes.

    Techniques: Purification, Amplification, Transformation Assay, Expressing

    TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of nucleosomal fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: TRF2-dependent changes in surface charge density (µ' o ) and effective radius (R e from dilute gels) of nucleosomal fibers determined by analytical agarose gel electrophoresis (AAGE). Multi-gels of telomeric nucleosomal array fibers (NA) in the absence ( A ) or presence ( B ) of 200 nM TRF2 prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. TRF2-induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or non-telomeric (non-Tel) fragments ( C ). The µ' o (black bars) or R e (grey bars) of NA in the presence of 200 nM TRF2 was normalized to 0 nM TRF2. Bars represent the mean ±1 SD from 3 separate experiments. The data were derived from multi-gels of 0.25–1% agarose concentrations while the R e bars represent the average from 0.25–0.6% agarose concentrations according to Materials and Methods .

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Derivative Assay

    TRF2 stimulates self-association of DNA and nucleosomal arrays. Differential centrifugation assay as described in Materials and Methods . 1% agarose gel of samples with indicated amounts of TRF2 in nM where “T” refers to telomeric and “NT” refers to non-telomeric fragments ( A ). Quantification of experiments with TRF2 ( B ) TRF2 BH ( C ) and TRF2 B ( D ). Each data point represents the mean ± 1 SD from 3 separate experiments.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: TRF2 stimulates self-association of DNA and nucleosomal arrays. Differential centrifugation assay as described in Materials and Methods . 1% agarose gel of samples with indicated amounts of TRF2 in nM where “T” refers to telomeric and “NT” refers to non-telomeric fragments ( A ). Quantification of experiments with TRF2 ( B ) TRF2 BH ( C ) and TRF2 B ( D ). Each data point represents the mean ± 1 SD from 3 separate experiments.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Centrifugation, Agarose Gel Electrophoresis

    The role of the TRF2 basic N-terminus alone (TRF2 B ) or with the TRFH domain (TRF2 BH ) in TRF2-dependent negative charge reduction and compaction of nucleosomal arrays (NA). TRF2 BH -induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or largest non-telomeric (non-Tel) fragment ( A ). Bars represent the mean ±1 SD of 3 multi-gel experiments. The µ' o (black bars) or R e (grey bars) of NA in the presence of 1 µM TRF2 BH was normalized to 0 µM TRF2 BH . Multi-gels of telomeric nucleosomal array fibers (NA) in the absence or presence of 1 µM TRF2 BH ( B ) prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. Multi-gels of telomeric nucleosomal array fibers (NA) in the presence of indicated amounts of TRF2 B ( C ). TRF2 B -induced changes in surface charge density (µ' o ). ( D ) and effective radius (R e from dilute gels) ( E ) of DNA and nucleosomal arrays (NA). The µ' o or R e for each TRF2 B concentration was normalized to 0 µM TRF2 B . Each data point represents the mean ±1 SD of 3–4 multi-gel experiments.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: The role of the TRF2 basic N-terminus alone (TRF2 B ) or with the TRFH domain (TRF2 BH ) in TRF2-dependent negative charge reduction and compaction of nucleosomal arrays (NA). TRF2 BH -induced change in surface charge density (µ' o ) and effective radius (R e ) of nucleosomal arrays derived from the telomeric (Tel) or largest non-telomeric (non-Tel) fragment ( A ). Bars represent the mean ±1 SD of 3 multi-gel experiments. The µ' o (black bars) or R e (grey bars) of NA in the presence of 1 µM TRF2 BH was normalized to 0 µM TRF2 BH . Multi-gels of telomeric nucleosomal array fibers (NA) in the absence or presence of 1 µM TRF2 BH ( B ) prepared and subjected to electrophoresis according to Materials and Methods . “S” refers to carboxylate-coated microsphere standards (35 nm radius). “T” refers to the telomeric fragments liberated by SfaNI/PvuII/BspHI digestion of pRST5 and “NT” refers to the non-telomeric DNA fragments. Multi-gels of telomeric nucleosomal array fibers (NA) in the presence of indicated amounts of TRF2 B ( C ). TRF2 B -induced changes in surface charge density (µ' o ). ( D ) and effective radius (R e from dilute gels) ( E ) of DNA and nucleosomal arrays (NA). The µ' o or R e for each TRF2 B concentration was normalized to 0 µM TRF2 B . Each data point represents the mean ±1 SD of 3–4 multi-gel experiments.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Derivative Assay, Electrophoresis, Concentration Assay

    TRF2 binds to telomeric DNA (DNA) and nucleosomal array fibers (NA). TRF2 ( A ) or TRF2 BH ( B ) binding to substrates detected by electrophoresis on 0.3% agarose gels or 0.6% agarose gels to detect binding of TRF2 B ( C ). DNA and nucleosomal arrays pertain to pRST5 digested to obtain a 2 kb fragment containing the 580 bp telomeric DNA (Tel) with a 1 kb and smaller fragments being non-telomeric (NT). 0.6% agarose gel to detect binding of TRF2 to nucleosomal arrays derived from digestion of with SfaNI/PvuII/BspHI ( D ).The 0.3% agarose lanes in (A) and (B) were formed using a multi-gel apparatus as described in Materials and Methods . Red arrows point to mobility shifts produced by TRF2 or TRF2 BH complexes.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: TRF2 binds to telomeric DNA (DNA) and nucleosomal array fibers (NA). TRF2 ( A ) or TRF2 BH ( B ) binding to substrates detected by electrophoresis on 0.3% agarose gels or 0.6% agarose gels to detect binding of TRF2 B ( C ). DNA and nucleosomal arrays pertain to pRST5 digested to obtain a 2 kb fragment containing the 580 bp telomeric DNA (Tel) with a 1 kb and smaller fragments being non-telomeric (NT). 0.6% agarose gel to detect binding of TRF2 to nucleosomal arrays derived from digestion of with SfaNI/PvuII/BspHI ( D ).The 0.3% agarose lanes in (A) and (B) were formed using a multi-gel apparatus as described in Materials and Methods . Red arrows point to mobility shifts produced by TRF2 or TRF2 BH complexes.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Binding Assay, Electrophoresis, Agarose Gel Electrophoresis, Derivative Assay, Produced

    Atomic force microscopy of TRF2 B -nucleosomal array complexes. Nucleosomal array fibers (reconstituted with 1∶1 histone:DNA mass ratio) in the absence of TRF2 B ( A ). Nucleosomal arrays with 4 µM TRF2 B ( B ). An example of height measurements ( C ) of regions indicated by lines drawn on the fiber ( D ) expanded from in the boxed region in (B). Samples were prepared and analyzed according to Materials and Methods .

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: Atomic force microscopy of TRF2 B -nucleosomal array complexes. Nucleosomal array fibers (reconstituted with 1∶1 histone:DNA mass ratio) in the absence of TRF2 B ( A ). Nucleosomal arrays with 4 µM TRF2 B ( B ). An example of height measurements ( C ) of regions indicated by lines drawn on the fiber ( D ) expanded from in the boxed region in (B). Samples were prepared and analyzed according to Materials and Methods .

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Microscopy

    The effect of full-length TRF2, TRF2 BH , and TRF2 B on the insertion of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/µl). Samples were incubated with indicated amounts TRF2 or its truncated mutants and processed according to Materials and Methods . Agarose gel showing insertion of T7 (Free oligo) into increasing amounts of nucleosomal arrays (Oligo bound to NA), as indicated ( A ). The section of agarose gels showing T7 inserted into nucleosomal arrays (NA) or linear DNA (DNA) with increasing TRF2 or truncation mutants as indicated ( B, D and F ). Quantification of corresponding gels above where uptake was normalized to 0 nM TRF2 or truncation mutants ( C, E and G ). Each data point represents the mean ±1 SD from 3–4 separate experiments.

    Journal: PLoS ONE

    Article Title: The Telomere Binding Protein TRF2 Induces Chromatin Compaction

    doi: 10.1371/journal.pone.0019124

    Figure Lengend Snippet: The effect of full-length TRF2, TRF2 BH , and TRF2 B on the insertion of a 5′-[ 32 P]-labeled, single-stranded oligonucleotide, (dTTAGGG) 7 (T7), into nucleosomal arrays and DNA (20 ng/µl). Samples were incubated with indicated amounts TRF2 or its truncated mutants and processed according to Materials and Methods . Agarose gel showing insertion of T7 (Free oligo) into increasing amounts of nucleosomal arrays (Oligo bound to NA), as indicated ( A ). The section of agarose gels showing T7 inserted into nucleosomal arrays (NA) or linear DNA (DNA) with increasing TRF2 or truncation mutants as indicated ( B, D and F ). Quantification of corresponding gels above where uptake was normalized to 0 nM TRF2 or truncation mutants ( C, E and G ). Each data point represents the mean ±1 SD from 3–4 separate experiments.

    Article Snippet: Micrococcal Nuclease Digestion To verify proper reconstitution, an aliquot of reconstituted nucleosomal arrays (0.5 µg) was digested with 0.6 units/µl of micrococcal nuclease (Worthington) in 20 mM Tris-HCl and 2 mM CaCl2 (20 µl reaction volume).

    Techniques: Labeling, Incubation, Agarose Gel Electrophoresis