nucleosomes  (Worthington Biochemical)


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    Name:
    Deoxyribonucleic Acid E coli
    Description:
    Supplied as a dried powder purified from E coli Type B cells ATCC 11303 as described by Marmur J Mol Biol 3 208 1961
    Catalog Number:
    ls004449
    Price:
    104
    Size:
    10 mg
    Source:
    E. coli
    Cas Number:
    9007.49.2
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    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.
    Supplied as a dried powder purified from E coli Type B cells ATCC 11303 as described by Marmur J Mol Biol 3 208 1961
    https://www.bioz.com/result/nucleosomes/product/Worthington Biochemical
    Average 92 stars, based on 456 article reviews
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    nucleosomes - by Bioz Stars, 2021-01
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    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 "DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides"

    Article Title: DNA Builds and Strengthens the Extracellular Matrix in Myxococcus xanthus Biofilms by Interacting with Exopolysaccharides

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0051905

    Mechanical strength, anti-disruptive properties and nanomechanical adhesive characteristics recorded using force-separation curves of the M. xanthus starvation biofilm matrix with or without eDNA. In panel A, DK1622 biofilms without DNase I (black bar) and with DNase I (grey bar) were established over a 24 hr time period in MOPS buffer and biomass was measured as crystal violet optical density (No-treatment control). The changes of biomass in these two kinds of biofilms after sonication or SDS treatment, respectively, are also shown. In panel B, representative force curves measured by AFM on DK1622 24 hr starvation biofilm matrix (curve I), matrix treated with DNase I (curve II) and bare portion of the substrate after the tip was used (curve III). Force-separation curves were recorded as “approach” (blue) and “retraction” (red) curves.
    Figure Legend Snippet: Mechanical strength, anti-disruptive properties and nanomechanical adhesive characteristics recorded using force-separation curves of the M. xanthus starvation biofilm matrix with or without eDNA. In panel A, DK1622 biofilms without DNase I (black bar) and with DNase I (grey bar) were established over a 24 hr time period in MOPS buffer and biomass was measured as crystal violet optical density (No-treatment control). The changes of biomass in these two kinds of biofilms after sonication or SDS treatment, respectively, are also shown. In panel B, representative force curves measured by AFM on DK1622 24 hr starvation biofilm matrix (curve I), matrix treated with DNase I (curve II) and bare portion of the substrate after the tip was used (curve III). Force-separation curves were recorded as “approach” (blue) and “retraction” (red) curves.

    Techniques Used: Sonication

    eDNA colocalized with EPS within M. xanthus starvation biofilm and fruiting body structures. Panel A, DK1622 starvation biofilm formed in MOPS buffer for 24 hr labeled with STYOX orange (red), Alexa 633-conjugated WGA (blue) and SYTO 9 (green). Panel B, DK1622 24 hr fruiting body structures with STYOX orange, Alexa 633-conjugated WGA and SYTO 9. Images in panel A were taken with a 40× objective using CLSM, and images in panel B were taken with a 63× objective. The bars in panels A and B represent 50 µM. Panel C showed the quantitative colocalization analysis results of STYOX orange (eDNA) and Alexa 633-WGA (EPS) signals from submerged 24 hr starvation biofilms (left) and 24 hr fruiting bodies (right). The PCC represents Pearson’s correlation coefficient, MOC represents overlap coefficients according to Manders, M1 represents colocalization coefficient M1 (fraction of eDNA overlapping EPS), M2 represents colocalization coefficient M2 (fraction of EPS overlapping eDNA), and ICQ represents intensity correlation quotient. Mean ± SD is plotted.
    Figure Legend Snippet: eDNA colocalized with EPS within M. xanthus starvation biofilm and fruiting body structures. Panel A, DK1622 starvation biofilm formed in MOPS buffer for 24 hr labeled with STYOX orange (red), Alexa 633-conjugated WGA (blue) and SYTO 9 (green). Panel B, DK1622 24 hr fruiting body structures with STYOX orange, Alexa 633-conjugated WGA and SYTO 9. Images in panel A were taken with a 40× objective using CLSM, and images in panel B were taken with a 63× objective. The bars in panels A and B represent 50 µM. Panel C showed the quantitative colocalization analysis results of STYOX orange (eDNA) and Alexa 633-WGA (EPS) signals from submerged 24 hr starvation biofilms (left) and 24 hr fruiting bodies (right). The PCC represents Pearson’s correlation coefficient, MOC represents overlap coefficients according to Manders, M1 represents colocalization coefficient M1 (fraction of eDNA overlapping EPS), M2 represents colocalization coefficient M2 (fraction of EPS overlapping eDNA), and ICQ represents intensity correlation quotient. Mean ± SD is plotted.

    Techniques Used: Labeling, Whole Genome Amplification, Confocal Laser Scanning Microscopy, Periodic Counter-current Chromatography

    eDNA in M. xanthus non-developmental starvation biofilms. M. xanthus DK1622 biofilm structures formed in MOPS buffer at 24 hr (panels A and B) and DNase I treated biofilm (panels C and D) were counterstained with SYTOX orange (red) and SYTO9 (green). Panels A and C are the single channel images (SYTOX orange), and panels B and D are the overlaid images. M. xanthus DK10547 with a GFP label (green) formed starvation biofilms (panels E and F) in MOPs buffer at 24 hr was counterstained with SYTOX orange (red) and FM 4-64 (blue). Panel E is the single channel image (FM 4-64), and panel F is the overlaid image. The bars represent 50 µm in panels A–D and 10 µm in panels E and F.
    Figure Legend Snippet: eDNA in M. xanthus non-developmental starvation biofilms. M. xanthus DK1622 biofilm structures formed in MOPS buffer at 24 hr (panels A and B) and DNase I treated biofilm (panels C and D) were counterstained with SYTOX orange (red) and SYTO9 (green). Panels A and C are the single channel images (SYTOX orange), and panels B and D are the overlaid images. M. xanthus DK10547 with a GFP label (green) formed starvation biofilms (panels E and F) in MOPs buffer at 24 hr was counterstained with SYTOX orange (red) and FM 4-64 (blue). Panel E is the single channel image (FM 4-64), and panel F is the overlaid image. The bars represent 50 µm in panels A–D and 10 µm in panels E and F.

    Techniques Used:

    3) Product Images from "?-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations"

    Article Title: ?-III spectrin underpins ankyrin R function in Purkinje cell dendritic trees: protein complex critical for sodium channel activity is impaired by SCA5-associated mutations

    Journal: Human Molecular Genetics

    doi: 10.1093/hmg/ddu103

    Ankyrin R recruited to membrane by β-III spectrin. ( A ) Sagittal cerebellar sections independently immunostained for β-III spectrin or ankyrin R at P3, 7 and 14. ( B ) Immunoblot analysis of cerebellar homogenates from P3, 7 and 14 animals. ( C ) Representative confocal images of HEK 293T cells transfected with SBD-GFP or AnkR-GFP only. ( D ) Representative confocal image of cell transfected with myc-tagged β-III spectrin only and immunostained using anti-c-myc antibody. ( E ) Immunoblot analysis of untransfected HEK 293T cells and ankyrin R transfected cell homogenates probed with anti-AnkR antibody. ( F ) Cells cotransfected with myc-tagged β-III spectrin and either SBD-GFP or AnkR-GFP. Cells immunostained using anti-c-myc antibody (red). Degree of colocalization shown both by residual map (right column), with cyan representing highest, and histogram of red and green fluorescence intensity through the cell. All images are representative of at least three independent experiments [PCL, Purkinje cell layer; scale bar, 50 μm (A), 10 μm (C, D, F)].
    Figure Legend Snippet: Ankyrin R recruited to membrane by β-III spectrin. ( A ) Sagittal cerebellar sections independently immunostained for β-III spectrin or ankyrin R at P3, 7 and 14. ( B ) Immunoblot analysis of cerebellar homogenates from P3, 7 and 14 animals. ( C ) Representative confocal images of HEK 293T cells transfected with SBD-GFP or AnkR-GFP only. ( D ) Representative confocal image of cell transfected with myc-tagged β-III spectrin only and immunostained using anti-c-myc antibody. ( E ) Immunoblot analysis of untransfected HEK 293T cells and ankyrin R transfected cell homogenates probed with anti-AnkR antibody. ( F ) Cells cotransfected with myc-tagged β-III spectrin and either SBD-GFP or AnkR-GFP. Cells immunostained using anti-c-myc antibody (red). Degree of colocalization shown both by residual map (right column), with cyan representing highest, and histogram of red and green fluorescence intensity through the cell. All images are representative of at least three independent experiments [PCL, Purkinje cell layer; scale bar, 50 μm (A), 10 μm (C, D, F)].

    Techniques Used: Transfection, Fluorescence

    Loss of ankyrin R in β-III spectrin-deficient mice. ( A ) Sagittal cerebellar sections from 6-week-old WT (+/+) and β-III −/− (−/−) mice immunostained for ankyrin R. ( B ) Dissociated Purkinje cell cultures from WT (+/+) and β-III −/− (−/−) mice 14 DIV immunostained for ITPR1 and ankyrin R. ( C ) Immunoblot analysis of cerebellar homogenates from 6-week-old WT (+/+) and β-III −/− (−/−) mice probed for ankyrin R, ankyrin G, calbindin and actin. All images are representative of at least three independent experiments. Scale bar, 20 μm.
    Figure Legend Snippet: Loss of ankyrin R in β-III spectrin-deficient mice. ( A ) Sagittal cerebellar sections from 6-week-old WT (+/+) and β-III −/− (−/−) mice immunostained for ankyrin R. ( B ) Dissociated Purkinje cell cultures from WT (+/+) and β-III −/− (−/−) mice 14 DIV immunostained for ITPR1 and ankyrin R. ( C ) Immunoblot analysis of cerebellar homogenates from 6-week-old WT (+/+) and β-III −/− (−/−) mice probed for ankyrin R, ankyrin G, calbindin and actin. All images are representative of at least three independent experiments. Scale bar, 20 μm.

    Techniques Used: Mouse Assay

    4) Product Images from "Natural Single-Nucleosome Epi-Polymorphisms in Yeast"

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

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000913

    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.
    Figure Legend 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.

    Techniques Used: 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
    Figure Legend 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

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Purification, Amplification, Transformation Assay, Expressing

    5) Product Images from "MBD4-Mediated Glycosylase Activity on a Chromatin Template Is Enhanced by Acetylation ▿"

    Article Title: MBD4-Mediated Glycosylase Activity on a Chromatin Template Is Enhanced by Acetylation ▿

    Journal:

    doi: 10.1128/MCB.00588-08

    (A) DNA mismatches do not alter the position of the histone octamer. (1) Native 4% PAGE analysis of nucleosomes reconstituted onto a native 164-bp fragment of the 5S rRNA gene (lane 1) and the same DNA fragment containing a T/G mismatch at position
    Figure Legend Snippet: (A) DNA mismatches do not alter the position of the histone octamer. (1) Native 4% PAGE analysis of nucleosomes reconstituted onto a native 164-bp fragment of the 5S rRNA gene (lane 1) and the same DNA fragment containing a T/G mismatch at position

    Techniques Used: Polyacrylamide Gel Electrophoresis

    MBD4 glycosylase activity in the presence of a nucleosome.
    Figure Legend Snippet: MBD4 glycosylase activity in the presence of a nucleosome.

    Techniques Used: Activity Assay

    Histone acetylation facilitates the glycosylase activity of MBD4 on nucleosome substrates. (A) Acetic acid-urea-Triton X-100-PAGE analysis of core histones used for the reconstitution of acetylated nucleosomes. CM, chicken erythrocyte histone marker;
    Figure Legend Snippet: Histone acetylation facilitates the glycosylase activity of MBD4 on nucleosome substrates. (A) Acetic acid-urea-Triton X-100-PAGE analysis of core histones used for the reconstitution of acetylated nucleosomes. CM, chicken erythrocyte histone marker;

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Marker

    MBD4 glycosylase activity on a nucleosome is unaffected by DNA methylation. (A) Native 4% PAGE analysis of the 164-bp DNA construct depicted in Fig. and the corresponding nucleosomes reconstituted using this DNA template. Lanes:
    Figure Legend Snippet: MBD4 glycosylase activity on a nucleosome is unaffected by DNA methylation. (A) Native 4% PAGE analysis of the 164-bp DNA construct depicted in Fig. and the corresponding nucleosomes reconstituted using this DNA template. Lanes:

    Techniques Used: Activity Assay, DNA Methylation Assay, Polyacrylamide Gel Electrophoresis, Construct

    MeCP2 and MBD4 preferentially bind to methylated nucleosomes. Nucleosomes were reconstituted using [γ 32 P]ATP-end-labeled unmethylated (control) and methylated versions of a 5S rRNA gene fragment (see Materials and Methods) and were titrated with
    Figure Legend Snippet: MeCP2 and MBD4 preferentially bind to methylated nucleosomes. Nucleosomes were reconstituted using [γ 32 P]ATP-end-labeled unmethylated (control) and methylated versions of a 5S rRNA gene fragment (see Materials and Methods) and were titrated with

    Techniques Used: Methylation, Labeling

    MBD4 glycosylase activity in the presence of a nucleosome. (A) Urea-denaturing PAGE analysis of the glycosylase activities on a 164-bp DNA construct with a mismatch at position 126 in its DNA and reconstituted nucleosome versions. Approximately 10 nM
    Figure Legend Snippet: MBD4 glycosylase activity in the presence of a nucleosome. (A) Urea-denaturing PAGE analysis of the glycosylase activities on a 164-bp DNA construct with a mismatch at position 126 in its DNA and reconstituted nucleosome versions. Approximately 10 nM

    Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Construct

    6) Product Images from "Biocomposite nanofiber matrices to support ECM remodeling by human dermal progenitors and enhanced wound closure"

    Article Title: Biocomposite nanofiber matrices to support ECM remodeling by human dermal progenitors and enhanced wound closure

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10735-x

    Second harmonic generation images of collagen deposited on nanofiber scaffolds seeded with TdTomato + hSKP. ( a ) Representative SHG images of scaffolds without cells and with cells at 2 and 4 weeks in culture. SHG is represented by white and TdT + cells in red. ( b ) Mean intensity of the SHG signal shows a decrease between 2 and 4 weeks for the PCL-cGE scaffold and an increase between 2 and 4 weeks for the PCL-bGE scaffold (asterisks). A significant increase at 2 and 4 weeks for the PCL-cGE compared to gauze control was also noted (letters). ( c ) Mean intensity of the SHG signal normalized to control shows a significant difference between PCL and PCL-cGE at 2 weeks and between PCL and PCL-bGE at both 2 and 4 weeks (asterisks). Bar = 100 μm. Data is presented as mean ± SEM for n = 3 for each group. **** represents p
    Figure Legend Snippet: Second harmonic generation images of collagen deposited on nanofiber scaffolds seeded with TdTomato + hSKP. ( a ) Representative SHG images of scaffolds without cells and with cells at 2 and 4 weeks in culture. SHG is represented by white and TdT + cells in red. ( b ) Mean intensity of the SHG signal shows a decrease between 2 and 4 weeks for the PCL-cGE scaffold and an increase between 2 and 4 weeks for the PCL-bGE scaffold (asterisks). A significant increase at 2 and 4 weeks for the PCL-cGE compared to gauze control was also noted (letters). ( c ) Mean intensity of the SHG signal normalized to control shows a significant difference between PCL and PCL-cGE at 2 weeks and between PCL and PCL-bGE at both 2 and 4 weeks (asterisks). Bar = 100 μm. Data is presented as mean ± SEM for n = 3 for each group. **** represents p

    Techniques Used:

    Properties of PCL and composite nanofiber mats. Top : SEM images of nanofibers. PCL 10% in CHCl 3 :MeOH (3:1), PCL immobilized with RGD (PCL-RGD), PCL-bGE (50:50) in TFE, PCL coated with gelatin (PCL-cGE). Bottom : Mechanical properties of PCL based nanofibrous scaffolds. ( a ) Tensile strength. ( b ) Strain at break ( c ) Young’s modulus. Data is presented as mean ± SEM for n = 3 for each group. **** represents p = 0.0001, *** represents p
    Figure Legend Snippet: Properties of PCL and composite nanofiber mats. Top : SEM images of nanofibers. PCL 10% in CHCl 3 :MeOH (3:1), PCL immobilized with RGD (PCL-RGD), PCL-bGE (50:50) in TFE, PCL coated with gelatin (PCL-cGE). Bottom : Mechanical properties of PCL based nanofibrous scaffolds. ( a ) Tensile strength. ( b ) Strain at break ( c ) Young’s modulus. Data is presented as mean ± SEM for n = 3 for each group. **** represents p = 0.0001, *** represents p

    Techniques Used:

    ECM components produced by hSKPs. ( a ) Cell proliferation determined by trypan blue at 1, 3 and 7 days. The initial cell density at day 0 was 30,000 cells/gel as indicated by the dotted line. ( b ) DNA content at 3, 14 and 28 days of hSKPs culture measured by CyQuant TM showing the influence of RGD and gelatin on cell proliferation. The dotted line represents the DNA content immediately after seeding. ( c ) Glycosaminoglycan quantification of various nanofiber meshes at 3, 14 and 28 days determined by DMMB ( d ) GAG content normalized to total DNA content. # represents p
    Figure Legend Snippet: ECM components produced by hSKPs. ( a ) Cell proliferation determined by trypan blue at 1, 3 and 7 days. The initial cell density at day 0 was 30,000 cells/gel as indicated by the dotted line. ( b ) DNA content at 3, 14 and 28 days of hSKPs culture measured by CyQuant TM showing the influence of RGD and gelatin on cell proliferation. The dotted line represents the DNA content immediately after seeding. ( c ) Glycosaminoglycan quantification of various nanofiber meshes at 3, 14 and 28 days determined by DMMB ( d ) GAG content normalized to total DNA content. # represents p

    Techniques Used: Produced, CyQUANT Assay

    7) Product Images from "Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources"

    Article Title: Control of hmu Heme Uptake Genes in Yersinia pseudotuberculosis in Response to Iron Sources

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2018.00047

    E. coli IscR-C92A DNase I footprinting reveals IscR Motif II binding sites in the intergenic region between hmuR and hmuS . (A) DNase I footprinting of IscR-C92A binding to the intergenic region between hmuR and hmuS . The labeled DNA is the top strand from −123 to−1 relative to the hmuS translational start site. The region protected by IscR-C92A is marked by a black line and −40 denotes the position from the hmuS translational start site. (B) Sequence of the intergenic region between hmuR and hmuS . The region of protection by IscR-C92A is underlined. Possible Motif II Sites are marked below the sequence and the number of nucleotides contained in each sequence shown to be important for IscR binding are denoted. The first residue of each possible motif are capitalized and bolded.
    Figure Legend Snippet: E. coli IscR-C92A DNase I footprinting reveals IscR Motif II binding sites in the intergenic region between hmuR and hmuS . (A) DNase I footprinting of IscR-C92A binding to the intergenic region between hmuR and hmuS . The labeled DNA is the top strand from −123 to−1 relative to the hmuS translational start site. The region protected by IscR-C92A is marked by a black line and −40 denotes the position from the hmuS translational start site. (B) Sequence of the intergenic region between hmuR and hmuS . The region of protection by IscR-C92A is underlined. Possible Motif II Sites are marked below the sequence and the number of nucleotides contained in each sequence shown to be important for IscR binding are denoted. The first residue of each possible motif are capitalized and bolded.

    Techniques Used: Footprinting, Binding Assay, Labeling, Sequencing

    E. coli Fur binds to the promoter upstream of hmuR and E. coli IscR-C92A binds to the intergenic region between hmuR and hmuSTUV . (A) Electrophoretic mobility shift assays (EMSAs) using DNA from the promoter regions shown in Figure 1 . Concentrations of Fur protein used in the gel shift assays are denoted above the gel lanes. (B) EMSAs using DNA from the intergenic region between hmuR and hmuSTUV or control DNA within the hmuR coding region. Concentrations of Apo-locked IscR-C92A protein used in the gel shift assays are denoted above the gel lanes.
    Figure Legend Snippet: E. coli Fur binds to the promoter upstream of hmuR and E. coli IscR-C92A binds to the intergenic region between hmuR and hmuSTUV . (A) Electrophoretic mobility shift assays (EMSAs) using DNA from the promoter regions shown in Figure 1 . Concentrations of Fur protein used in the gel shift assays are denoted above the gel lanes. (B) EMSAs using DNA from the intergenic region between hmuR and hmuSTUV or control DNA within the hmuR coding region. Concentrations of Apo-locked IscR-C92A protein used in the gel shift assays are denoted above the gel lanes.

    Techniques Used: Electrophoretic Mobility Shift Assay

    E. coli IscR-C92A cannot activate transcription from the intergenic promoter between hmuR and hmuS . In vitro transcription reactions contain plasmids harboring the intergenic promoter between hmuR and hmuS , Eσ 70 RNA polymerase, and increasing concentrations of IscR-C92A protein. Transcripts from the intergenic promoter are marked with an arrow and transcripts from the control RNA-1 promoter are indicated.
    Figure Legend Snippet: E. coli IscR-C92A cannot activate transcription from the intergenic promoter between hmuR and hmuS . In vitro transcription reactions contain plasmids harboring the intergenic promoter between hmuR and hmuS , Eσ 70 RNA polymerase, and increasing concentrations of IscR-C92A protein. Transcripts from the intergenic promoter are marked with an arrow and transcripts from the control RNA-1 promoter are indicated.

    Techniques Used: In Vitro

    8) Product Images from "DNA binding by the MATα2 transcription factor controls its access to alternative ubiquitin-modification pathways"

    Article Title: DNA binding by the MATα2 transcription factor controls its access to alternative ubiquitin-modification pathways

    Journal: Molecular Biology of the Cell

    doi: 10.1091/mbc.E17-10-0589

    Reduced DNA binding is not the sole cause of stabilization for the α2(N182A, R185A) mutant. (A) Degradation rates, reported as half-life, for the indicated α2 variant following pulse-chase analysis in matα2 Δ cells (MHY1147) bearing plasmid pRS314-α2 or variant. Half-life ranges shown for α2-WT and the α2(N182A, R185A) mutant are from at least three replicates. All other half-life values are from a single degradation assay in which α2-WT was also tested and had a half-life of 4–6 min. Each α2 variant was tested at least twice with similar results. (B) Assay for repression of a-specific gene transcription in MHY481 cells by α2 or the indicated variant, selected from those characterized in degradation assays (A). Error bars denote SD ( N = 3). * p = 0.025. No other α2 variant tested, besides α2(N182D, R185A), yielded statistically poorer repression than the α2(N182A, R185A) variant. (C) Representative EMSA data for the interaction of purified α2 103-210 -6His, or the indicated variants of this protein, with synthetic, Cy5-labeled DNA corresponding to regulatory sequence upstream of BAR1 (an a-specific gene). DNA used was a half operator (a single α2 binding site and single Mcm1 binding site), and no Mcm1 protein was included in assays shown. Two previous experiments, using independent protein preparations, showed comparable DNA binding efficiencies for the different α2 103-210 -6His variants.
    Figure Legend Snippet: Reduced DNA binding is not the sole cause of stabilization for the α2(N182A, R185A) mutant. (A) Degradation rates, reported as half-life, for the indicated α2 variant following pulse-chase analysis in matα2 Δ cells (MHY1147) bearing plasmid pRS314-α2 or variant. Half-life ranges shown for α2-WT and the α2(N182A, R185A) mutant are from at least three replicates. All other half-life values are from a single degradation assay in which α2-WT was also tested and had a half-life of 4–6 min. Each α2 variant was tested at least twice with similar results. (B) Assay for repression of a-specific gene transcription in MHY481 cells by α2 or the indicated variant, selected from those characterized in degradation assays (A). Error bars denote SD ( N = 3). * p = 0.025. No other α2 variant tested, besides α2(N182D, R185A), yielded statistically poorer repression than the α2(N182A, R185A) variant. (C) Representative EMSA data for the interaction of purified α2 103-210 -6His, or the indicated variants of this protein, with synthetic, Cy5-labeled DNA corresponding to regulatory sequence upstream of BAR1 (an a-specific gene). DNA used was a half operator (a single α2 binding site and single Mcm1 binding site), and no Mcm1 protein was included in assays shown. Two previous experiments, using independent protein preparations, showed comparable DNA binding efficiencies for the different α2 103-210 -6His variants.

    Techniques Used: Binding Assay, Mutagenesis, Variant Assay, Pulse Chase, Plasmid Preparation, Degradation Assay, Purification, Labeling, Sequencing

    9) Product Images from "Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1"

    Article Title: Nucleosomes around a mismatched base pair are excluded via an Msh2-dependent reaction with the aid of SNF2 family ATPase Smarcad1

    Journal: Genes & Development

    doi: 10.1101/gad.310995.117

    Nucleosomes are excluded from a > 1-kb region surrounding a mismatch. ( A ) The DNA substrate used in this study. The 3011-base-pair (bp) DNA carries an A:T base pair (pMM1 homo ) or an A:C mispair (pMM1 AC ) at position 1. Positions of restriction enzyme sites used in this study, the site of biotin modification, and amplicons for quantitative PCR (qPCR) (P1: 2950–61, P2: 253–383, P3: 476–602, P4: 728–860, P5: 1498–1628, P6: 2266–2397, and P7: 2413–2537) are indicated. ( B ) Supercoiling assay in NPE. Covalently closed pMM1 homo (lanes 2 – 8 ) or pMM1 AC (lanes 9 – 15 ) was incubated in NPE and sampled at the indicated times. (Lane 1 ) Supercoiled pMM1 homo purified from Escherichia coli was used as a size standard. (oc/r) Open circular or relaxed DNA; (sc) supercoiled DNA. ( C ) pMM1 homo (lanes 1 – 4 ) or pMM1 AC (lanes 5 – 8 ) was incubated in NPE for 60 min and digested by micrococcal nuclease (MNase). DNA samples stained with SYBR Gold ( top ) and Southern blotting with the PvuII–PvuII probe ( middle ) and the DraI–DraI probe ( bottom ) are shown. ( D – F ) The MNase assay described in C was repeated in the presence of a control plasmid (pControl), and undigested DNA was quantified by qPCR. The amount of DNA relative to the input ( D ) and normalized to pControl ( E ) and pMM1 homo ( F ) is presented. Mean ± one standard deviation (SD) is shown. n = 3.
    Figure Legend Snippet: Nucleosomes are excluded from a > 1-kb region surrounding a mismatch. ( A ) The DNA substrate used in this study. The 3011-base-pair (bp) DNA carries an A:T base pair (pMM1 homo ) or an A:C mispair (pMM1 AC ) at position 1. Positions of restriction enzyme sites used in this study, the site of biotin modification, and amplicons for quantitative PCR (qPCR) (P1: 2950–61, P2: 253–383, P3: 476–602, P4: 728–860, P5: 1498–1628, P6: 2266–2397, and P7: 2413–2537) are indicated. ( B ) Supercoiling assay in NPE. Covalently closed pMM1 homo (lanes 2 – 8 ) or pMM1 AC (lanes 9 – 15 ) was incubated in NPE and sampled at the indicated times. (Lane 1 ) Supercoiled pMM1 homo purified from Escherichia coli was used as a size standard. (oc/r) Open circular or relaxed DNA; (sc) supercoiled DNA. ( C ) pMM1 homo (lanes 1 – 4 ) or pMM1 AC (lanes 5 – 8 ) was incubated in NPE for 60 min and digested by micrococcal nuclease (MNase). DNA samples stained with SYBR Gold ( top ) and Southern blotting with the PvuII–PvuII probe ( middle ) and the DraI–DraI probe ( bottom ) are shown. ( D – F ) The MNase assay described in C was repeated in the presence of a control plasmid (pControl), and undigested DNA was quantified by qPCR. The amount of DNA relative to the input ( D ) and normalized to pControl ( E ) and pMM1 homo ( F ) is presented. Mean ± one standard deviation (SD) is shown. n = 3.

    Techniques Used: Modification, Real-time Polymerase Chain Reaction, Incubation, Purification, Staining, Southern Blot, Plasmid Preparation, Standard Deviation

    10) Product Images from "Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis"

    Article Title: Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2020.01247

    Digestion pattern of archaeal chromosomes treated with micrococcal nuclease (MNase). Purified chromosomes of (A) T. kodakarensis , (B) T. acidophilum , (C) P. calidifontis , and (D) S. solfataricus were digested with increasing concentrations of MNase and separated on 2.5% agarose gels in 1X TBE. The accumulation of DNA of particular sizes was observed with T. kodakarensis (arrows) and T. acidophilum (curly brackets) but not with P. calidifontis and S. solfataricus chromosomes. MNase concentration was 0.3, 1, 3, 10 U MNase in 100 μl reaction (A) or 0, 0.3, 1, 3, 10, and 30 U MNase in 100 μl reaction (B–D) .
    Figure Legend Snippet: Digestion pattern of archaeal chromosomes treated with micrococcal nuclease (MNase). Purified chromosomes of (A) T. kodakarensis , (B) T. acidophilum , (C) P. calidifontis , and (D) S. solfataricus were digested with increasing concentrations of MNase and separated on 2.5% agarose gels in 1X TBE. The accumulation of DNA of particular sizes was observed with T. kodakarensis (arrows) and T. acidophilum (curly brackets) but not with P. calidifontis and S. solfataricus chromosomes. MNase concentration was 0.3, 1, 3, 10 U MNase in 100 μl reaction (A) or 0, 0.3, 1, 3, 10, and 30 U MNase in 100 μl reaction (B–D) .

    Techniques Used: Purification, Concentration Assay

    In vitro reconstitution of chromosome structures with recombinant Alba and HTa. (A–C) Reconstitution on a linear 3-kb plasmid using recombinant Alba from T. acidophilum and P. calidifontis at different Alba to DNA weight ratios. (A) AFM images showing various structures formed with Alba and DNA. Fibers are indicated with arrow heads. Alba:DNA weight ratios are indicated. The asterisk indicates a structure in which two 3-kb DNA molecules are joined together by Alba binding. (B) Histograms show the diameters of the fibrous structures formed (indicated with arrow heads in A ). (C) Histograms show the respective contour lengths of DNA at a 15:1 Alba:DNA ratio. The theoretical length of a linear 3-kb DNA (∼1000 nm) is indicated with a dashed line. See Figure 6A for a histogram of unbound DNA length. (D–G) Reconstitution with histidine-tagged HTa from T. acidophilum at varying protein to DNA ratio. (D) AFM images showing beaded (arrows) and filamentous (arrow heads) structures. (E) Diameter of the beads formed at a relatively lower HTa concentration. (F) Width of the filaments formed at relatively higher HTa concentration. (G) Histogram shows the contour DNA lengths of the structures formed at a 15:1 HTa:DNA ratio. Dashed line indicates the theoretical length of a 3-kb unbound DNA. Scale bars: 100 nm.
    Figure Legend Snippet: In vitro reconstitution of chromosome structures with recombinant Alba and HTa. (A–C) Reconstitution on a linear 3-kb plasmid using recombinant Alba from T. acidophilum and P. calidifontis at different Alba to DNA weight ratios. (A) AFM images showing various structures formed with Alba and DNA. Fibers are indicated with arrow heads. Alba:DNA weight ratios are indicated. The asterisk indicates a structure in which two 3-kb DNA molecules are joined together by Alba binding. (B) Histograms show the diameters of the fibrous structures formed (indicated with arrow heads in A ). (C) Histograms show the respective contour lengths of DNA at a 15:1 Alba:DNA ratio. The theoretical length of a linear 3-kb DNA (∼1000 nm) is indicated with a dashed line. See Figure 6A for a histogram of unbound DNA length. (D–G) Reconstitution with histidine-tagged HTa from T. acidophilum at varying protein to DNA ratio. (D) AFM images showing beaded (arrows) and filamentous (arrow heads) structures. (E) Diameter of the beads formed at a relatively lower HTa concentration. (F) Width of the filaments formed at relatively higher HTa concentration. (G) Histogram shows the contour DNA lengths of the structures formed at a 15:1 HTa:DNA ratio. Dashed line indicates the theoretical length of a 3-kb unbound DNA. Scale bars: 100 nm.

    Techniques Used: In Vitro, Recombinant, Plasmid Preparation, Binding Assay, Concentration Assay

    In vitro reconstitution using a combination of archaeal proteins. Chromatin structures were reconstituted on a linear 3-kb plasmid using recombinant proteins at varying Alba concentrations. (A) AFM images showing reconstitution using histone and Alba from T. kodakarensis . Histograms below the AFM images show the contour DNA length in each condition. Dashed line indicates the theoretical length of a 3-kb unbound DNA. (B) AFM images showing reconstitution using HTa and Alba from T. acidophilum . Scale bars: 100 nm. Note that histograms are not shown in (B) because of the inability to accurately measure the DNA contour length due to a high degree of folding or joining of the fiber structures.
    Figure Legend Snippet: In vitro reconstitution using a combination of archaeal proteins. Chromatin structures were reconstituted on a linear 3-kb plasmid using recombinant proteins at varying Alba concentrations. (A) AFM images showing reconstitution using histone and Alba from T. kodakarensis . Histograms below the AFM images show the contour DNA length in each condition. Dashed line indicates the theoretical length of a 3-kb unbound DNA. (B) AFM images showing reconstitution using HTa and Alba from T. acidophilum . Scale bars: 100 nm. Note that histograms are not shown in (B) because of the inability to accurately measure the DNA contour length due to a high degree of folding or joining of the fiber structures.

    Techniques Used: In Vitro, Plasmid Preparation, Recombinant

    11) Product Images from "3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine"

    Article Title: 3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

    Journal: Nucleic Acids Research

    doi:

    MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.
    Figure Legend Snippet: MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Techniques Used: Modification, Sequencing

    12) Product Images from "Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿ §"

    Article Title: Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿Dynamic BRG1 Recruitment during T Helper Differentiation and Activation Reveals Distal Regulatory Elements ▿ §

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00920-10

    Novel BRG1 binding sites in the Gata3 locus are in open chromatin and possess enhancer-like activity. (A, B, and C) Distal BRG1 binding sites have an open chromatin conformation. Nuclei from stimulated Th1 and Th2 cells were digested with DNase I; DNA was quantified using Q-PCR primers detecting the indicated regions. For each region, a representative dose response is shown; similar results were obtained by averaging three biological replicates digested with 8 μg/ml DNase I (data not shown). (A) Distal BRG1 binding regions are DHS in Th2 cells but not in Th1 cells. (B) Control regions lacking BRG1 binding are not DHS. (C) A control region with BRG1 binding in Th1 and Th2 cells is DHS in both. (D and E) Distal BRG1 binding sites possess enhancer-like activity. The indicated elements were placed immediately upstream (D) or 2.2 kb downstream (E) of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (F) Enhancer-like activity is BRG1 dependent. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Cells were treated with BRG1 shRNA or a control, as indicated. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (G) Distal BRG1 binding sites lack enhancer-like activity in a nonepisomal vector. The indicated elements were placed upstream of the SV40 promoter in the pGL3 promoter vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). These same elements exhibited increased expression using episomal reporters (D and E). None of the five tested regions were positive for enhancer-like activity in this assay. (H) Distal regions without BRG1 binding in Th2 cells lack enhancer-like activity. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values. All four tested regions lacking BRG1 binding were negative for enhancer-like activity in this assay.
    Figure Legend Snippet: Novel BRG1 binding sites in the Gata3 locus are in open chromatin and possess enhancer-like activity. (A, B, and C) Distal BRG1 binding sites have an open chromatin conformation. Nuclei from stimulated Th1 and Th2 cells were digested with DNase I; DNA was quantified using Q-PCR primers detecting the indicated regions. For each region, a representative dose response is shown; similar results were obtained by averaging three biological replicates digested with 8 μg/ml DNase I (data not shown). (A) Distal BRG1 binding regions are DHS in Th2 cells but not in Th1 cells. (B) Control regions lacking BRG1 binding are not DHS. (C) A control region with BRG1 binding in Th1 and Th2 cells is DHS in both. (D and E) Distal BRG1 binding sites possess enhancer-like activity. The indicated elements were placed immediately upstream (D) or 2.2 kb downstream (E) of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (F) Enhancer-like activity is BRG1 dependent. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. Cells were treated with BRG1 shRNA or a control, as indicated. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). (G) Distal BRG1 binding sites lack enhancer-like activity in a nonepisomal vector. The indicated elements were placed upstream of the SV40 promoter in the pGL3 promoter vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with (black bars) and without (white bars) stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values (also indicated as a horizontal line). These same elements exhibited increased expression using episomal reporters (D and E). None of the five tested regions were positive for enhancer-like activity in this assay. (H) Distal regions without BRG1 binding in Th2 cells lack enhancer-like activity. The indicated elements were placed upstream of the SV40 promoter in the pRep4-luc episomal vector and transfected into CEM cells (human lymphoblastoid T cell line), and the reporter was assayed with stimulation. The horizontal line at 1 on the y axis indicates the value obtained with the promoter alone. Averages and standard deviations of the results from two biological replicate experiments are shown; all values are shown relative to the promoter values. All four tested regions lacking BRG1 binding were negative for enhancer-like activity in this assay.

    Techniques Used: Binding Assay, Activity Assay, Polymerase Chain Reaction, Plasmid Preparation, Transfection, shRNA, Expressing

    13) Product Images from "Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo"

    Article Title: Role of the Promoter in Maintaining Transcriptionally Active Chromatin Structure and DNA Methylation Patterns In Vivo

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.12.4150-4161.2003

    Analysis of DNase I hypersensitivity. HT-1080 (male human fibrosarcoma) cells and Δ3B (HT-1080 cells carrying the promoter mutation) cells were treated with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg of DNase I/ml. Southern blot analysis was performed on DNA digested with Eco RI and was hybridized to the HPRT promoter probe HPRT A. “HS site” indicates the positions of HPRT -hypersensitive sites; “globin” indicates the hybridization band detected by the control human β-globin probe.
    Figure Legend Snippet: Analysis of DNase I hypersensitivity. HT-1080 (male human fibrosarcoma) cells and Δ3B (HT-1080 cells carrying the promoter mutation) cells were treated with 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μg of DNase I/ml. Southern blot analysis was performed on DNA digested with Eco RI and was hybridized to the HPRT promoter probe HPRT A. “HS site” indicates the positions of HPRT -hypersensitive sites; “globin” indicates the hybridization band detected by the control human β-globin probe.

    Techniques Used: Mutagenesis, Southern Blot, Hybridization

    Physical map of the human  HPRT  gene. The thick horizontal line represents the  HPRT  gene. Open horizontal boxes represent exons, and “ATG” indicates the translation initiation site. The bent arrow represents the major transcription initiation sites, and a putative initiator element is shaded. The gray box indicates the position of probe “HPRT A.” The bracket above the line indicates the position of the 5′ flanking DNase I-hypersensitive site. The positions of CpG-dinucleotides within the gene are shown as vertical lines below the gene map. Numbers indicate the position of CpGs relative to the translation initiation site. The horizontal bracket below the line indicates the region deleted in Δ2B and Δ3B cells.
    Figure Legend Snippet: Physical map of the human HPRT gene. The thick horizontal line represents the HPRT gene. Open horizontal boxes represent exons, and “ATG” indicates the translation initiation site. The bent arrow represents the major transcription initiation sites, and a putative initiator element is shaded. The gray box indicates the position of probe “HPRT A.” The bracket above the line indicates the position of the 5′ flanking DNase I-hypersensitive site. The positions of CpG-dinucleotides within the gene are shown as vertical lines below the gene map. Numbers indicate the position of CpGs relative to the translation initiation site. The horizontal bracket below the line indicates the region deleted in Δ2B and Δ3B cells.

    Techniques Used:

    14) Product Images from "Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter"

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Summary of the nucleosomal organization of the active and inactive HPRT ) (vertical rectangles, their binding sites); bent arrow, position of the two major transcription initiation sites on the HPRT promoter; white box, first exon of the HPRT gene; ATG, position of the translation initiation site; thick vertical arrows, approximate positions and relative intensities of the major MNase cleavage sites in the HPRT promoter; clusters of thin triangular dashed arrows and barbed arrows, positions of the high-resolution DNase I cleavage ladders suggestive of rotationally positioned nucleosomes on the active and inactive HPRT promoters, respectively, in permeabilized cells (the slightly longer arrows on the lower strand in the inactive allele indicate that this ladder was unusually prominent); hatched bars, approximate locations of the DNase I-hypersensitive sites on the active HPRT promoter in permeabilized cells; All position numbers are relative to the translation initiation site.
    Figure Legend Snippet: Summary of the nucleosomal organization of the active and inactive HPRT ) (vertical rectangles, their binding sites); bent arrow, position of the two major transcription initiation sites on the HPRT promoter; white box, first exon of the HPRT gene; ATG, position of the translation initiation site; thick vertical arrows, approximate positions and relative intensities of the major MNase cleavage sites in the HPRT promoter; clusters of thin triangular dashed arrows and barbed arrows, positions of the high-resolution DNase I cleavage ladders suggestive of rotationally positioned nucleosomes on the active and inactive HPRT promoters, respectively, in permeabilized cells (the slightly longer arrows on the lower strand in the inactive allele indicate that this ladder was unusually prominent); hatched bars, approximate locations of the DNase I-hypersensitive sites on the active HPRT promoter in permeabilized cells; All position numbers are relative to the translation initiation site.

    Techniques Used: Binding Assay

    Locations of probes and primers for analysis of the HPRT promoter region. Horizontal line bounded by Bcl I sites, 4.3-kb Bcl I fragment containing the HPRT promoter; gray box, potential AP-2 site; five black boxes, cluster of GC boxes in the HPRT promoter; white box, first exon of the HPRT gene including the region of multiple transcription initiation sites in the promoter; ATG, translation initiation site; Bam HI, position of a reference Bam HI site in the first intron 100 bp downstream of the translation initiation site; hatched box, position of the 400-bp hybridization probe used to map DNase I and MNase cleavage sites in the HPRT promoter by indirect end labeling; black rectangles above and below the line, positions of the LMPCR primer sets used to map the high-resolution DNase I cleavage pattern of the HPRT minimal promoter; arrows extending from the black boxes, strand and region analyzed with each primer set.
    Figure Legend Snippet: Locations of probes and primers for analysis of the HPRT promoter region. Horizontal line bounded by Bcl I sites, 4.3-kb Bcl I fragment containing the HPRT promoter; gray box, potential AP-2 site; five black boxes, cluster of GC boxes in the HPRT promoter; white box, first exon of the HPRT gene including the region of multiple transcription initiation sites in the promoter; ATG, translation initiation site; Bam HI, position of a reference Bam HI site in the first intron 100 bp downstream of the translation initiation site; hatched box, position of the 400-bp hybridization probe used to map DNase I and MNase cleavage sites in the HPRT promoter by indirect end labeling; black rectangles above and below the line, positions of the LMPCR primer sets used to map the high-resolution DNase I cleavage pattern of the HPRT minimal promoter; arrows extending from the black boxes, strand and region analyzed with each primer set.

    Techniques Used: Hybridization, End Labeling

    DNase I in vivo footprint analysis of the human HPRT promoter. Active, samples from cells containing an active HPRT gene on the active human X chromosome; inactive, samples from cells containing an inactive HPRT gene on the inactive human X chromosome; DNA , naked DNA treated with DNase I; cells, DNA from permeabilized cells treated with DNase I; GC boxes, position of a DNase I in vivo footprint over the five GC boxes in the human HPRT promoter; AP-2, position of a DNase I in vivo footprint over a putative consensus AP-2 site in the human HPRT promoter. All position numbers (left and right) are relative to the translation initiation site of the HPRT gene. (A) DNase I in vivo footprint analysis of the upper strand of the HPRT promoter using LMPCR primer set E. Ladder of arrows, apparent 10-bp ladder of DNase I cleavages in permeabilized cells consistent with rotationally positioned nucleosomes on the inactive HPRT promoter. (B) DNase I in vivo footprinting analysis of the lower strand of the HPRT promoter using LMPCR primer set A. All designations and symbols are as described above. This analysis identifies footprints over both a cluster of five GC boxes and a putative AP-2 site in the active HPRT promoter. (C) DNase I in vivo footprinting analysis of the upper strand using LMPCR primer set C. All designations and symbols are as described above. This analysis identifies a DNase in vivo footprint over a putative AP-2 site on the active HPRT promoter.
    Figure Legend Snippet: DNase I in vivo footprint analysis of the human HPRT promoter. Active, samples from cells containing an active HPRT gene on the active human X chromosome; inactive, samples from cells containing an inactive HPRT gene on the inactive human X chromosome; DNA , naked DNA treated with DNase I; cells, DNA from permeabilized cells treated with DNase I; GC boxes, position of a DNase I in vivo footprint over the five GC boxes in the human HPRT promoter; AP-2, position of a DNase I in vivo footprint over a putative consensus AP-2 site in the human HPRT promoter. All position numbers (left and right) are relative to the translation initiation site of the HPRT gene. (A) DNase I in vivo footprint analysis of the upper strand of the HPRT promoter using LMPCR primer set E. Ladder of arrows, apparent 10-bp ladder of DNase I cleavages in permeabilized cells consistent with rotationally positioned nucleosomes on the inactive HPRT promoter. (B) DNase I in vivo footprinting analysis of the lower strand of the HPRT promoter using LMPCR primer set A. All designations and symbols are as described above. This analysis identifies footprints over both a cluster of five GC boxes and a putative AP-2 site in the active HPRT promoter. (C) DNase I in vivo footprinting analysis of the upper strand using LMPCR primer set C. All designations and symbols are as described above. This analysis identifies a DNase in vivo footprint over a putative AP-2 site on the active HPRT promoter.

    Techniques Used: In Vivo, Footprinting

    Summary of the 10-base DNase I cleavage ladders of chromatin from the active and inactive HPRT promoters. Boldface letters, protein-coding region of the first exon; lowercase letters, nucleotides within the first intron; partial ovals, approximate positions of the translationally positioned nucleosomes on the active HPRT promoter as determined by MNase cleavage; open boxes, positions of transcription factor (TF) binding sites. From top to bottom, left to right, the TF binding sites are a putative AP-1 site (−271 to −264), five GC boxes (centered at −213, −201, −187, −177, and −166), and a putative initiator element (−94 to −86). Bent arrows, positions of the two major transcription initiation sites identified by Kim et al. (16); line between the nucleotide sequence of the upper and lower strands, region of multiple transcription initiation sites described by Patel et al. (32); black triangles above the sequence, positions of DNase I cleavage sites on the upper strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; gray triangles below the sequence, positions of DNase I cleavages on the lower strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; white triangles, positions of DNase I cleavages on the lower strand making up the 10-bp ladder, suggestive of rotational positioning of a nucleosome on the active promoter region in permeabilized cells; vertical ovals, positions of three CpG dinucleotides whose methylation is strongly correlated with transcriptional repression of the HPRT ).
    Figure Legend Snippet: Summary of the 10-base DNase I cleavage ladders of chromatin from the active and inactive HPRT promoters. Boldface letters, protein-coding region of the first exon; lowercase letters, nucleotides within the first intron; partial ovals, approximate positions of the translationally positioned nucleosomes on the active HPRT promoter as determined by MNase cleavage; open boxes, positions of transcription factor (TF) binding sites. From top to bottom, left to right, the TF binding sites are a putative AP-1 site (−271 to −264), five GC boxes (centered at −213, −201, −187, −177, and −166), and a putative initiator element (−94 to −86). Bent arrows, positions of the two major transcription initiation sites identified by Kim et al. (16); line between the nucleotide sequence of the upper and lower strands, region of multiple transcription initiation sites described by Patel et al. (32); black triangles above the sequence, positions of DNase I cleavage sites on the upper strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; gray triangles below the sequence, positions of DNase I cleavages on the lower strand comprising the 10-bp ladder suggestive of rotationally positioned nucleosomes in the inactive promoter; white triangles, positions of DNase I cleavages on the lower strand making up the 10-bp ladder, suggestive of rotational positioning of a nucleosome on the active promoter region in permeabilized cells; vertical ovals, positions of three CpG dinucleotides whose methylation is strongly correlated with transcriptional repression of the HPRT ).

    Techniques Used: Binding Assay, Sequencing, Methylation

    15) Product Images from "Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity"

    Article Title: Heparan Sulfate Modulates Neutrophil and Endothelial Function in Antibacterial Innate Immunity

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00545-15

    Reduced extracellular-trap (NET) formation in Hs2st-deficient neutrophils. (A) Human neutrophils were treated with 25 nM PMA for 3 h to allow NET formation, treated with heparan lyase III (5 mU/ml), fixed, and stained with mouse anti-stub heparan sulfate MAb, rabbit anti-myeloperoxidase PAb, and DAPI, followed by appropriate fluorochrome-conjugated secondary antibodies. (B) Human neutrophils were treated with 25 nM PMA for 3 h to induce NET formation and then treated with DNase I (10 U/ml) or heparan lyase I and III (5 mU/ml) for 30 min at 37°C, followed by incubation with GBS for 30 min. Surviving GBS were enumerated by serial plating. Differences between groups were calculated by unpaired t test. **, P
    Figure Legend Snippet: Reduced extracellular-trap (NET) formation in Hs2st-deficient neutrophils. (A) Human neutrophils were treated with 25 nM PMA for 3 h to allow NET formation, treated with heparan lyase III (5 mU/ml), fixed, and stained with mouse anti-stub heparan sulfate MAb, rabbit anti-myeloperoxidase PAb, and DAPI, followed by appropriate fluorochrome-conjugated secondary antibodies. (B) Human neutrophils were treated with 25 nM PMA for 3 h to induce NET formation and then treated with DNase I (10 U/ml) or heparan lyase I and III (5 mU/ml) for 30 min at 37°C, followed by incubation with GBS for 30 min. Surviving GBS were enumerated by serial plating. Differences between groups were calculated by unpaired t test. **, P

    Techniques Used: Staining, Incubation

    16) Product Images from "Structural and functional conservation at the boundaries of the chicken ?-globin domain"

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.10.2315

    Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).
    Figure Legend Snippet: Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).

    Techniques Used: Derivative Assay

    Fig. 4.  Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with  Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from  Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.
    Figure Legend Snippet: Fig. 4. Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.

    Techniques Used: End Labeling, Migration, Derivative Assay, Mutagenesis

    Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.
    Figure Legend Snippet: Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.

    Techniques Used: Southern Blot, Hybridization, Isolation, Derivative Assay

    Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.
    Figure Legend Snippet: Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.

    Techniques Used: Blocking Assay, Activity Assay, Stable Transfection, Transfection, Construct, Expressing

    17) Product Images from "Structural and functional conservation at the boundaries of the chicken ?-globin domain"

    Article Title: Structural and functional conservation at the boundaries of the chicken ?-globin domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.10.2315

    Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).
    Figure Legend Snippet: Fig. 2. A constitutive hypersensitive site (3′HS) within the 3′ chromatin boundary. Nuclei from various types of chicken cells were treated with increasing amounts of DNase I (from left to right lanes: 0, 0.06, 0.1, 0.2, 0.4 and 0.6 U/ml for RBCs and brain nuclei; 0, 8.0, 10, 20, 40 and 60 U/ml for DT40 and 6C2 cell nuclei). Genomic DNA was extracted and digested with Kpn I. In addition to a parental fragment, a hypersensitive site, 3′HS, was detected as marked by arrowheads, in all cell types tested. Cells tested are a chicken erythroid precursor derived cell line (6C2), erythroid cells from either 11-day-old embryo (11D RBC) or adult blood (Adult RBC), brain from 11-day-old embryos (Brain) and a lymphoma-cell derived cell line (DT40).

    Techniques Used: Derivative Assay

    Fig. 4.  Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with  Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from  Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.
    Figure Legend Snippet: Fig. 4. Sequences homologous to the 5′ insulator element of the chicken β-globin locus are found at the site of the 3′HS. The position of the 3′HS was measured by the indirect end-labeling method and the strategy is shown in ( A ). Nuclei from 11-day-old chick embryos were treated with 0.4 U/ml DNase I, from which genomic DNA was extracted and digested with Kpn I. In ( B ), the position of the 3′HS (arrow) was compared with the migration of genomic fragments of known length. The 3′HS hypersensitive fragment co-migrates with a fragment derived from Bgl II digestion. ( C ) are found at or close to the sites of 3′HS, 3′HS-A and 3′HS-B, respectively. Alignment of the sequences 3′HS-A and 3′HS-B with the sequences of the 5′FII is shown. Conserved bases are shaded. Bases altered to generate a mutant site are underlined.

    Techniques Used: End Labeling, Migration, Derivative Assay, Mutagenesis

    Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.
    Figure Legend Snippet: Fig. 1. A transition in DNase I sensitivity defines the 3′ boundary of the chicken β-globin domain. The 3′ boundary of generalized DNase I sensitivity is located between regions C and D. ( A ). Restriction fragments A–F detected in DNase I sensitivity assays in (B) are shown below the map. Probes used are indicated as thin lines. A detailed description of DNA fragments A–F and probes is given in Materials and methods. ( B ) Generalized DNase I sensitivity of DNA fragments A–F visualized by Southern blot hybridization. Erythrocyte nuclei isolated from 11-day-old chick embryos were treated with increasing amounts of DNase I (from right to left lanes, 0, 0.2, 0.4, 0.6, 1.0, 2.0 and 5.0 U/ml). Restriction fragments A–F were detected by Southern blot hybridizations. Relative sensitivities to DNase I correspond to the extent of loss of signal intensities of each band. DNA fragments B and C are relatively sensitive to DNase I, while fragments D–F are resistant to DNase I. A DNA fragment derived from the ovalbumin gene, which is transcriptionally inactive in this cell type, and fragment A, which is located farther upstream of the 5′ chromatin boundary, were used as DNase I resistant controls. ( C ) and plotted on a graph: S = log ( G D / G U )/log ( O D / O U ) × T , where G and O are β-globin and ovalbumin band intensities for the undigested (U) or digested (D) samples and T is the size ratio of the ovalbumin to globin fragments. DNase I sensitivity drops significantly between C and D.

    Techniques Used: Southern Blot, Hybridization, Isolation, Derivative Assay

    Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.
    Figure Legend Snippet: Fig. 3. Directional enhancer-blocking activity of the 3′HS. ( A ) The human erythroleukemic cell line K562 was stably transfected with the constructs shown on the left. Each construct has the neomycin resistance gene (NEO) driven by a human β A -globin promoter with mouse β-globin HS2 as an enhancer. The DNA fragments 3′HS and 3′HS-2 include the DNase I HS 3′HS. 3′HS-6 does not contain the HS. For each construct, the 1.2 kb chromatin insulator fragment (5′Ins) including the 5′HS4 was placed upstream of the promoter in order to block influence from regulatory elements at the site of integration. The level of expression of each construct was measured as the number of neomycin-resistant colonies. Colony numbers obtained from construct 1, which does not have a DNA fragment between the promoter and the enhancer, were set at 100. Relative numbers of neomycin-resistant colonies are shown in the bar graph. We present the mean of five independent experiments. Enhancer-blocking activity resides in a DNA fragment containing the 3′HS. ( B ) Enhancer-blocking assays were performed using constructs shown to the left. In construct 1, a 2.3 kb fragment of λ DNA was inserted, as a spacer control, between the enhancer and the reporter. Thick bars show means of at least four independent experiments.

    Techniques Used: Blocking Assay, Activity Assay, Stable Transfection, Transfection, Construct, Expressing

    18) Product Images from "A Cellular Protein Binds Vaccinia Virus Late Promoters and Activates Transcription In Vitro"

    Article Title: A Cellular Protein Binds Vaccinia Virus Late Promoters and Activates Transcription In Vitro

    Journal: Journal of Virology

    doi:

    DNase I footprinting of protein bound to the I1L promoter. I1L promoter 120-nucleotide DNA uniquely labeled on the template (A) or nontemplate (B) strand was treated with DNase I in the absence (lanes 1) or presence (lanes 2) of glycerol gradient-purified LPBP. Cleavage products were resolved on a 6% polyacrylamide DNA sequencing gel. Lanes 3 contain probe DNA chemically cleaved at purine residues. The nucleotide sequence of each strand of the I1L promoter is shown on the right.
    Figure Legend Snippet: DNase I footprinting of protein bound to the I1L promoter. I1L promoter 120-nucleotide DNA uniquely labeled on the template (A) or nontemplate (B) strand was treated with DNase I in the absence (lanes 1) or presence (lanes 2) of glycerol gradient-purified LPBP. Cleavage products were resolved on a 6% polyacrylamide DNA sequencing gel. Lanes 3 contain probe DNA chemically cleaved at purine residues. The nucleotide sequence of each strand of the I1L promoter is shown on the right.

    Techniques Used: Footprinting, Labeling, Purification, DNA Sequencing, Sequencing

    19) Product Images from "Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting"

    Article Title: Constitutive Nucleosome Depletion and Ordered Factor Assembly at the GRP78 Promoter Revealed by Single Molecule Footprinting

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0020160

    Transcription Factor Binding and Nucleosome Depletion at the Human GRP78 Promoter (A) ChIP analysis of the GRP78 promoter was performed on noninduced or TG-induced LD419 cells using antibodies against TBP, RNA polII, NF-Y, total H3, and acetylated H3-K9/14. Precipitated DNA was quantified by real-time PCR using primers specific for the indicated four regions of the promoter. The enrichment at each region is plotted as percentage of input. The data are representatives of experiments performed from two or three independent chromatin preparations. (B, C) MNase assay. (B) A mixture of oligonucleosomes (lane 2) from LD419 nuclei digested with MNase was fractionated through a sucrose gradient to obtain mononucleosomal, dinucleosomal, and trinucleosomal DNA (lanes 4, 5, and 6). Purified genomic DNA partially digested with MNase was used as naked DNA control (D1 and D2, lanes 7 and 8; M in lanes 1 and 9 indicates size marker). (C) Relative enrichment of nucleosomal and naked DNA at four regions of the GRP78 promoter. The values plotted are normalized with respect to the value at the R1 region, arbitrarily defined as 1. The four regions are depicted on the promoter diagram at the top of the figure. The TATA box (T), TIS (bent arrow), and ERSEs (E1–E3) are marked. (D) DNase I hypersensitivity assay. Top: EtBr staining. Bottom: Southern blot. The assay was performed on nuclei extracted from noninduced or TG-induced cells. Genomic DNA was included as a control to show a lack of sequence specificity of the enzyme. DNase I–digested samples were resolved by gel electrophoresis, showing varying extents of digestion (EtBr staining). Southern blot performed after RsaI digestion revealed a hypersensitive region in the GRP78 promoter similar in size in both noninduced and induced samples. On the left, a map of the GRP78 promoter region is shown indicating the 1,802-bp DNA fragment generated by RsaI digestion, transcription start site (bent arrow), and probe fragment (black box). Numbers next to the marker indicate size in bp.
    Figure Legend Snippet: Transcription Factor Binding and Nucleosome Depletion at the Human GRP78 Promoter (A) ChIP analysis of the GRP78 promoter was performed on noninduced or TG-induced LD419 cells using antibodies against TBP, RNA polII, NF-Y, total H3, and acetylated H3-K9/14. Precipitated DNA was quantified by real-time PCR using primers specific for the indicated four regions of the promoter. The enrichment at each region is plotted as percentage of input. The data are representatives of experiments performed from two or three independent chromatin preparations. (B, C) MNase assay. (B) A mixture of oligonucleosomes (lane 2) from LD419 nuclei digested with MNase was fractionated through a sucrose gradient to obtain mononucleosomal, dinucleosomal, and trinucleosomal DNA (lanes 4, 5, and 6). Purified genomic DNA partially digested with MNase was used as naked DNA control (D1 and D2, lanes 7 and 8; M in lanes 1 and 9 indicates size marker). (C) Relative enrichment of nucleosomal and naked DNA at four regions of the GRP78 promoter. The values plotted are normalized with respect to the value at the R1 region, arbitrarily defined as 1. The four regions are depicted on the promoter diagram at the top of the figure. The TATA box (T), TIS (bent arrow), and ERSEs (E1–E3) are marked. (D) DNase I hypersensitivity assay. Top: EtBr staining. Bottom: Southern blot. The assay was performed on nuclei extracted from noninduced or TG-induced cells. Genomic DNA was included as a control to show a lack of sequence specificity of the enzyme. DNase I–digested samples were resolved by gel electrophoresis, showing varying extents of digestion (EtBr staining). Southern blot performed after RsaI digestion revealed a hypersensitive region in the GRP78 promoter similar in size in both noninduced and induced samples. On the left, a map of the GRP78 promoter region is shown indicating the 1,802-bp DNA fragment generated by RsaI digestion, transcription start site (bent arrow), and probe fragment (black box). Numbers next to the marker indicate size in bp.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Purification, Marker, Staining, Southern Blot, Sequencing, Nucleic Acid Electrophoresis, Generated

    20) Product Images from "Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins "

    Article Title: Prefoldin-Nascent Chain Complexes in the Folding of Cytoskeletal Proteins

    Journal: The Journal of Cell Biology

    doi:

    The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.
    Figure Legend Snippet: The actin–PFD complex forms cotranslationally. Reticulocyte lysate was programmed with mRNA encoding either a 336– or a 257–amino acid NH 2 -terminal fragment of actin. After 8 min, edeine and 7- Me GMP were added, and the reaction continued for an additional 9 min. Nascent chain–ribosome complexes were stabilized by addition of 0.5 mM cycloheximide, and polysomes isolated by gradient centrifugation. Aliquots from each fraction were incubated with puromycin to release nascent chains from the ribosome. (A and B) SDS-PAGE and native-PAGE analyses of the gradient fractions obtained from the 336–amino acid actin translation product. S denotes the starting material for the gradient. Shown are fluorograms of the gels with molecular mass markers on the left. The position of sedimentation of the standards catalase (11.3 S) and α2-macroglobulin (18.6 S) are indicated at the bottom of A. (C) The proteins in the starting material for the gradient and each of the gradient fractions were analyzed for their content of CCT by an immunoblot using anti-CCT antibodies. (D) The polysome-bound actin complex contains PFD. The purified polysomes shown in A and B (lane 7) were treated with puromycin and apyrase, and then incubated at 4°C for 30 min with immobilized preimmune antibody, immobilized PFD antibody, or DNase I coupled to Sepharose 4B. After clarification, the supernatant fractions from each of the reactions were analyzed by native-PAGE (top). The antibody beads were washed four times with PBS and the material retained by the beads was eluted by heating in Laemmli sample buffer, then analyzed by SDS-PAGE (bottom). The starting material is shown in lane 1; immobilized preimmune antibodies, lane 2; immobilized anti-PFD antibody, lane 3; immobilized DNase I, lane 4. The position of actin PFD is indicated on the left. (E and F) SDS-PAGE and native-PAGE analysis of the gradient fractions obtained from the 257–amino acid actin translation product, performed as in A and B. S denotes the starting material for the gradient which was immediately frozen while S′ denotes the starting material which was stored at 4°C during the centrifugation run.

    Techniques Used: Isolation, Gradient Centrifugation, Incubation, SDS Page, Clear Native PAGE, Sedimentation, Purification, Clarification Assay, Centrifugation

    Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.
    Figure Legend Snippet: Actin species I contains PFD. (A) HeLa cell lysate or rabbit reticulocyte lysate was examined for content of PFD 6 by SDS-PAGE and Western blot analysis. Lane 1, HeLa cells grown at 37°C; lane 2, HeLa cells 12 h after a 43°C/60 min heat shock treatment; lane 3, rabbit reticulocyte lysate. The position of PFD 6 is shown on the right of the panel. (B) Purified bovine PFD (lane 1) and in vitro translated [ 35 S]methionine-labeled actin (lane 2) were analyzed by native-PAGE, the proteins transferred to nitrocellulose, and the position of purified PFD was determined by immunoblot using the PFD 6 antibody. After extensive washing, the nitrocellulose was placed on film and the position of actin species I and II revealed by autoradiography. The positions of actin species I and II are indicated on the right. (C) Identification of actin-containing complex components by electrophoretic mobility shift assays. Full-length actin mRNA (left) was translated for 15 and 50 min. The reactions were mixed together to create a pool of all actin-containing species. The mRNA encoding 336–amino acid actin was translated for 30 min (right). Before native-PAGE analysis, equal aliquots of the reaction mixtures were incubated with: PBS (control, lane 1); preimmune antiserum (lane 2); anti–PFD 6 serum (lane 3); anti–PFD 6 serum supplemented with purified actin (lane 4); purified anti-CCT mAb (lane 5); and DNase I (lane 6). DNase shift (lane 6) was omitted for the 336–amino acid actin translation products. The different protein complexes were then analyzed by native-PAGE. A fluorogram of the gel is shown. Molecular mass markers are shown at the left, and the positions of the different actin complexes are indicated in the center. The arrowhead indicates the shift in full-length actin migration due to the presence of actin-binding proteins present in the crude rabbit antisera. (D and F) Identification of actin-containing complex components by immunodepletion with immobilized antichaperone specific antibodies and DNase I. The full-length [ 35 S]methionine-labeled actin translation reaction products (C) were incubated with antichaperone antibodies first bound to protein A–Sepharose, or with DNase I coupled to Affigel-10. After incubation, the samples were clarified and the corresponding supernatants analyzed for the presence of the different actin species by native-PAGE as shown in D. In parallel, the corresponding pellets containing the immobilized antibodies or DNase I were resuspended in Laemmli sample buffer and analyzed for their relative content of radiolabeled actin by SDS-PAGE as shown in F. An aliquot of the in vitro translation products (i.e., starting material) is shown in lane 1; protein A–Sepharose, lane 2; immobilized preimmune antibodies, lane 3; PFD 6 antibody, lane 4; anti-CCT antibody, lane 5; immobilized DNase, lane 6. The positions of the different actin complexes are indicated in the center. (E and G) The [ 35 S]methionine-labeled 336–amino acid actin translation reaction products (C) were incubated with the immobilized antichaperone antibodies or immobilized DNase. Subsequently, the samples were clarified and the supernatants and pellets analyzed by native-PAGE and SDS-PAGE, respectively. Lane designations are the same as in D and F.

    Techniques Used: SDS Page, Western Blot, Purification, In Vitro, Labeling, Clear Native PAGE, Autoradiography, Electrophoretic Mobility Shift Assay, Incubation, Migration, Binding Assay

    21) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).
    Figure Legend Snippet: Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).

    Techniques Used: Staining, SDS Page, Western Blot, Protein Purification, Incubation, Protease Inhibitor

    Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.
    Figure Legend Snippet: Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.

    Techniques Used:

    22) Product Images from "Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans"

    Article Title: Neutrophil Extracellular Traps Contain Calprotectin, a Cytosolic Protein Complex Involved in Host Defense against Candida albicans

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000639

    Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).
    Figure Legend Snippet: Identification of NET-associated proteins. (A) Silver stained SDS-PAGE and (B) immunoblots with samples from NET protein purification procedure. Human neutrophils were stimulated to form NETs. Supernatants from unstimulated (lane 1) and stimulated (lane 2) neutrophils; first wash (lane 3); second wash (lane 4); medium containing DNase-1 incubated with unstimulated neutrophils (lane 5); DNase-1-free medium incubated with washed NETs (lane 6); medium containing DNase-1 incubated with washed NETs (lane 7); medium containing DNase-1 incubated with washed NETs including protease inhibitor cocktail (lane 8).

    Techniques Used: Staining, SDS Page, Western Blot, Protein Purification, Incubation, Protease Inhibitor

    Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.
    Figure Legend Snippet: Histones are altered during NET formation. NETs from human neutrophils were washed and digested with DNase-1. (A) The NET-fraction (N) and the remaining pellet after DNase-1 digest (P) were analyzed by immunoblotting at the indicated time points. Unstimulated neutrophils served as controls. All core histones have a reduced molecular mass (2–5 kDa less) in NETs compared to the pellet fraction and the unstimulated control. A representative experiment out of three in total is shown. (B) High-resolution SEM analysis of NETs which consist of smooth fibers (white box) and globular domains (diameter 25–50 nm, arrows), scale bar = 100 nm. (C) High-resolution FESEM analysis of smooth stretch of a singular NET-fiber. Signal intensities were profiled vertically and horizontally showing similar diameters to nucleosomes (depicted as cartoon structure models taken from [41] , with approximate horizontal and vertical diameters of 5 nm and 10 nm, respectively). One experiment out of two is shown.

    Techniques Used:

    23) Product Images from "Remodeling of chromatin structure within the promoter is important for bmp-2-induced fgfr3 expression"

    Article Title: Remodeling of chromatin structure within the promoter is important for bmp-2-induced fgfr3 expression

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp261

    Mapping of the BMP-2-induced DNase I hypersensitivity within the proximal FGFR3 promoter. C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml) and DNA was purified and cleaved with Nco I. After Southern blotting, the filter was hybridized with the 32 P-labeled probe used in the MNase digestion assay. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I. Size markers were generated as described in ‘Material and Methods’ section. The diagrams at the right of each panel indicate the positions of the transcription start site and the probes used for hybridization (solid bars). The large signal near +1 in the figure on the left is an autoradiograph artifact.
    Figure Legend Snippet: Mapping of the BMP-2-induced DNase I hypersensitivity within the proximal FGFR3 promoter. C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml) and DNA was purified and cleaved with Nco I. After Southern blotting, the filter was hybridized with the 32 P-labeled probe used in the MNase digestion assay. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I. Size markers were generated as described in ‘Material and Methods’ section. The diagrams at the right of each panel indicate the positions of the transcription start site and the probes used for hybridization (solid bars). The large signal near +1 in the figure on the left is an autoradiograph artifact.

    Techniques Used: Purification, Southern Blot, Labeling, In Vitro, Generated, Hybridization, Autoradiography

    Alteration in the restriction enzyme accessibility at the FGFR3 proximal promoter induced by BMP-2 treatment. ( A ) C3H10T1/2 cells were incubated in the presence or absence of 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with 25–200 U of restriction enzyme/ml, and purified DNA was digested to completion with Nco I. Products were detected by Southern blotting (top panel). A tentative assignment of the nucleosome positions in this region based on nuclease digestion is shown and is aligned with the nuclease-hypersensitive sites. Sites of cutting by DNase I and MNase and restriction enzyme are depicted by solid bars for basal conditions. Because MNase preferentially digests DNA in linker regions between nucleosomes, it is possible to locate nucleosomes within the FGFR3 proximal promoter. Hollow arrow heads indicate increases in the nuclease hypersensitivity upon BMP-2 exposure. ( B ) The relative nuclease sensitivity of restriction enzyme sites was quantitated, and the intensities of the radioactive bands were used to calculate the percentage of DNA digested. All the results are the means of three independent experiments ± standard deviation. * P
    Figure Legend Snippet: Alteration in the restriction enzyme accessibility at the FGFR3 proximal promoter induced by BMP-2 treatment. ( A ) C3H10T1/2 cells were incubated in the presence or absence of 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with 25–200 U of restriction enzyme/ml, and purified DNA was digested to completion with Nco I. Products were detected by Southern blotting (top panel). A tentative assignment of the nucleosome positions in this region based on nuclease digestion is shown and is aligned with the nuclease-hypersensitive sites. Sites of cutting by DNase I and MNase and restriction enzyme are depicted by solid bars for basal conditions. Because MNase preferentially digests DNA in linker regions between nucleosomes, it is possible to locate nucleosomes within the FGFR3 proximal promoter. Hollow arrow heads indicate increases in the nuclease hypersensitivity upon BMP-2 exposure. ( B ) The relative nuclease sensitivity of restriction enzyme sites was quantitated, and the intensities of the radioactive bands were used to calculate the percentage of DNA digested. All the results are the means of three independent experiments ± standard deviation. * P

    Techniques Used: Incubation, Purification, Southern Blot, Standard Deviation

    Inducible DNase I hypersensitivity within the FGFR3 promoter. ( A ) Schematic representation of the probe used to map the DNase-hypersensitive sites within the FGFR3 promoter by the indirect end-labeling technique. ( B ) C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml). DNA was purified and cleaved with Hind III. After Southern blotting, the filter was hybridized with the 32 P-labeled probe. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I (0, 0.05, 0.1, 0.2 U/ml).
    Figure Legend Snippet: Inducible DNase I hypersensitivity within the FGFR3 promoter. ( A ) Schematic representation of the probe used to map the DNase-hypersensitive sites within the FGFR3 promoter by the indirect end-labeling technique. ( B ) C3H10T1/2 cells were treated with or without 200 ng/ml BMP-2 with 5% FBS for 1 day. Nuclei were then purified and digested with increasing concentrations of DNase I (0, 1, 3, 5 U/ml). DNA was purified and cleaved with Hind III. After Southern blotting, the filter was hybridized with the 32 P-labeled probe. The DNase I-hypersensitive sites are indicated by arrowheads. As a control, purified genomic DNA from C3H10T1/2 cells was digested in vitro with increasing concentrations of DNase I (0, 0.05, 0.1, 0.2 U/ml).

    Techniques Used: End Labeling, Purification, Southern Blot, Labeling, In Vitro

    24) Product Images from "Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells"

    Article Title: Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells

    Journal:

    doi: 10.1042/BJ20041024

    Summary of in vivo DMS, UVC and DNase I footprints of the mouse Smn proximal promoter
    Figure Legend Snippet: Summary of in vivo DMS, UVC and DNase I footprints of the mouse Smn proximal promoter

    Techniques Used: In Vivo

    25) Product Images from "Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock"

    Article Title: Controller protein of restriction–modification system Kpn2I affects transcription of its gene by acting as a transcription elongation roadblock

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky880

    Genetic organization of restriction–modification system Kpn2I. In the middle of the figure, the Kpn2I genes are schematically shown by colored arrows, with arrow direction matching the direction of transcription. The DNA sequence upstream of the kpn 2I R gene (both strands) is expanded at the top. The initiating codon of the kpn 2I. R reading frame is indicated; the transcription start points of kpn 2I .R promoters are shown by arrows, likely -10 and -35 promoter elements are underlined, nucleotides matching promoter element consensus are shown in bold. The sequence of the intergenic region between and the beginnings of oppositely transcribed kpn 2I. M and kpn 2I .C genes is expanded below. Initiating codons of both ORFs, transcription start points, and promoter consensus elements are indicated. Sequences downstream of initiating codons are colored to match the coloring scheme of the genes as shown in the middle of the figure (dark blue for kpn 2I .R , red for kpn 2I .M , dark green for kpn 2I .C ). Arrows indicating transcription start points are also colored to match the same coloring scheme. Arrows of darker shades indicate stronger promoters. The binding site of C.Kpn2I (as determined by DNase I and Exo III footprinting) is shown by an orange-colored horizontal line and marked ‘C box’). The likely UP element of kpn 2I .M is marked.
    Figure Legend Snippet: Genetic organization of restriction–modification system Kpn2I. In the middle of the figure, the Kpn2I genes are schematically shown by colored arrows, with arrow direction matching the direction of transcription. The DNA sequence upstream of the kpn 2I R gene (both strands) is expanded at the top. The initiating codon of the kpn 2I. R reading frame is indicated; the transcription start points of kpn 2I .R promoters are shown by arrows, likely -10 and -35 promoter elements are underlined, nucleotides matching promoter element consensus are shown in bold. The sequence of the intergenic region between and the beginnings of oppositely transcribed kpn 2I. M and kpn 2I .C genes is expanded below. Initiating codons of both ORFs, transcription start points, and promoter consensus elements are indicated. Sequences downstream of initiating codons are colored to match the coloring scheme of the genes as shown in the middle of the figure (dark blue for kpn 2I .R , red for kpn 2I .M , dark green for kpn 2I .C ). Arrows indicating transcription start points are also colored to match the same coloring scheme. Arrows of darker shades indicate stronger promoters. The binding site of C.Kpn2I (as determined by DNase I and Exo III footprinting) is shown by an orange-colored horizontal line and marked ‘C box’). The likely UP element of kpn 2I .M is marked.

    Techniques Used: Modification, Sequencing, Binding Assay, Footprinting

    In vitro transcription and RNAP promoter complex formation in the presence of C.Kpn2I. ( A ). Results of multiple-round transcription by σ 70 RNAP holoenzyme with wild-type (‘wt’) α subunit or with α lacking the CTD domain (‘-αCTD’) from a DNA fragment containing the intergenic region between kpn 2I .M and kpn 2I .C in the presence or in the absence of added C.Kpn2I. ( B ) The same DNA fragment was combined with σ 70 RNAP holoenzyme, C.Kpn2I, or both and subjected to DNase I footprinting. ( C ) As in B but showing the results of KMnO 4 probing of complexes formed by σ 70 RNAP holoenzymes with wild-type α or α lacking the CTD. ( D ) Results of multiple-round transcription in the presence or in the absence of C.Kpn2I by RNAP holoenzymes containing wild-type σ 70 or σ 1–565 lacking the region 4 domain. In addition to transcription from the kpn 2I .M promoter, results of transcription from strong -10/-35 class promoter T7 A1 and extended –10 class gal P1 promoter are shown.
    Figure Legend Snippet: In vitro transcription and RNAP promoter complex formation in the presence of C.Kpn2I. ( A ). Results of multiple-round transcription by σ 70 RNAP holoenzyme with wild-type (‘wt’) α subunit or with α lacking the CTD domain (‘-αCTD’) from a DNA fragment containing the intergenic region between kpn 2I .M and kpn 2I .C in the presence or in the absence of added C.Kpn2I. ( B ) The same DNA fragment was combined with σ 70 RNAP holoenzyme, C.Kpn2I, or both and subjected to DNase I footprinting. ( C ) As in B but showing the results of KMnO 4 probing of complexes formed by σ 70 RNAP holoenzymes with wild-type α or α lacking the CTD. ( D ) Results of multiple-round transcription in the presence or in the absence of C.Kpn2I by RNAP holoenzymes containing wild-type σ 70 or σ 1–565 lacking the region 4 domain. In addition to transcription from the kpn 2I .M promoter, results of transcription from strong -10/-35 class promoter T7 A1 and extended –10 class gal P1 promoter are shown.

    Techniques Used: In Vitro, Footprinting

    Mapping of the C.Kpn2I binding site. DNase I ( A ) and Exo III ( B ) footprinting of C.Kpn2I complexes formed on a DNA fragment separating kpn 2I .M and kpn 2I .C . Results obtained with DNA fragment labeled at either top or bottom strands (see Figure 1 ) in the presence or in the absence of C.Kpn2I are shown. Areas protected by C.Kpn2I from DNase I digestion are indicated by blue-colored brackets at both sides of the gel shown in panel A (also shown by orange line at the bottom of Figure 1 ). The positions of Exo III stalling points during DNA digestion in B are shown by horizontal arrows. ( C ) Summary of footprinting results. The positions of Exo III stalls and areas of DNA protection on both strands are shown by vertical arrows and horizontal blue lines, respectively. The –35 element of the kpn 2I .M promoter is underlined. The initiating TTG (Leu 1 ) codon of kpn 2I .C and the Met 9 ATG are indicated (see text for det ails). The left- and right half sites fragments used in EMSA experiments are indicated. ( D ) A double-stranded radioactively-labeled Kpn2I DNA fragment shown in C (‘full C-box’) or shorter fragments corresponding to its left- and right-hand side halves were combined with increasing amounts of C.Kpn2I and reaction products were resolved by native PAGE. ‘F’ indicates free DNA, ‘D’—a complex likely bound to C.Kpn2I dimer, ‘T’—a complex bound to C.Kpn2I tetramer.
    Figure Legend Snippet: Mapping of the C.Kpn2I binding site. DNase I ( A ) and Exo III ( B ) footprinting of C.Kpn2I complexes formed on a DNA fragment separating kpn 2I .M and kpn 2I .C . Results obtained with DNA fragment labeled at either top or bottom strands (see Figure 1 ) in the presence or in the absence of C.Kpn2I are shown. Areas protected by C.Kpn2I from DNase I digestion are indicated by blue-colored brackets at both sides of the gel shown in panel A (also shown by orange line at the bottom of Figure 1 ). The positions of Exo III stalling points during DNA digestion in B are shown by horizontal arrows. ( C ) Summary of footprinting results. The positions of Exo III stalls and areas of DNA protection on both strands are shown by vertical arrows and horizontal blue lines, respectively. The –35 element of the kpn 2I .M promoter is underlined. The initiating TTG (Leu 1 ) codon of kpn 2I .C and the Met 9 ATG are indicated (see text for det ails). The left- and right half sites fragments used in EMSA experiments are indicated. ( D ) A double-stranded radioactively-labeled Kpn2I DNA fragment shown in C (‘full C-box’) or shorter fragments corresponding to its left- and right-hand side halves were combined with increasing amounts of C.Kpn2I and reaction products were resolved by native PAGE. ‘F’ indicates free DNA, ‘D’—a complex likely bound to C.Kpn2I dimer, ‘T’—a complex bound to C.Kpn2I tetramer.

    Techniques Used: Binding Assay, Footprinting, Labeling, Clear Native PAGE

    26) Product Images from "Differential Nucleosome Spacing in Neurons and Glia"

    Article Title: Differential Nucleosome Spacing in Neurons and Glia

    Journal: Neuroscience letters

    doi: 10.1016/j.neulet.2019.134559

    DRG neurons have shorter nucleosome spacing than OPCs and astrocytes. (A) Gel electrophoresis of DNA purified from a typical MNase titration (example: astrocytes). The oligo-nucleosomes become progressively shorter as more linkers are cut by MNase, eventually resulting in mono-nucleosomes. The repeat length is measured using samples with multiple nucleosome bands (as indicated). Nucleosome positioning (MNase-seq) data are obtained using samples containing predominantly mono-nucleosomes, avoiding over-digestion (indicated by the smearing below the mono-nucleosome band in the last 3 samples). 100-bp marker. (B) Typical gel used for repeat length measurement. Specific samples from titrations like that in A are analyzed side-by-side. M1: 100-bp marker; M2: mixture of pBR322 MspI and λ-DNA Bst ).
    Figure Legend Snippet: DRG neurons have shorter nucleosome spacing than OPCs and astrocytes. (A) Gel electrophoresis of DNA purified from a typical MNase titration (example: astrocytes). The oligo-nucleosomes become progressively shorter as more linkers are cut by MNase, eventually resulting in mono-nucleosomes. The repeat length is measured using samples with multiple nucleosome bands (as indicated). Nucleosome positioning (MNase-seq) data are obtained using samples containing predominantly mono-nucleosomes, avoiding over-digestion (indicated by the smearing below the mono-nucleosome band in the last 3 samples). 100-bp marker. (B) Typical gel used for repeat length measurement. Specific samples from titrations like that in A are analyzed side-by-side. M1: 100-bp marker; M2: mixture of pBR322 MspI and λ-DNA Bst ).

    Techniques Used: Nucleic Acid Electrophoresis, Purification, Titration, Marker

    27) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    28) Product Images from "DNase I aggravates islet β-cell apoptosis in type 2 diabetes"

    Article Title: DNase I aggravates islet β-cell apoptosis in type 2 diabetes

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2016.5102

    DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I knockdown can reduce the apoptosis of cells cultured with high glucose. (A) Knockdown efficiency examined by western blotting. Expression of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Apoptotic rate examined by flow cytometry [(a), normal; (b), high glucose; and (c), siRNA group]. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Culture, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Cytometry, Standard Deviation

    Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.
    Figure Legend Snippet: Immunohistochemistry of the human pancreas. Pancreatic tissues from patients with pancreatic cancer, with or without type 2 diabetes were stained with insulin, glucagon, and DNase I. (A) Insulin, (B) glucagon and (C) DNase I staining of tissues from patients without type 2 diabetes. (D) Insulin, (E) glucagon and (F) DNase I staining of tissues from patients with type 2 diabetes. DNase I, deoxyribonuclease I.

    Techniques Used: Immunohistochemistry, Staining

    DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P
    Figure Legend Snippet: DNase I combined with high glucose induced cell apoptosis. (A) Cell viability assessed by the Cell Counting Kit-8 assay. The expression levels of DNase I, Bcl-2 and caspase-3 in the three groups were examined by (B) western blotting and (C) reverse transcription-quantitative polymerase chain reaction. (D) Cell apoptosis results from (a) the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and (b) flow cytometry. Data are expressed as the mean ± standard deviation from three independent experiments. * P

    Techniques Used: Cell Counting, Expressing, Western Blot, Real-time Polymerase Chain Reaction, TUNEL Assay, Flow Cytometry, Cytometry, Standard Deviation

    DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P
    Figure Legend Snippet: DNase I activity in human serum. (A) DNase I activity in human serum. (B) The correlation of DNase I activity with calcium. (C) DNase I activity Spearman correlation coefficient. All data are presented as the mean ± standard deviation. *** P

    Techniques Used: Activity Assay, Standard Deviation

    29) Product Images from "Transcriptional regulatory logic of the diurnal cycle in the mouse liver"

    Article Title: Transcriptional regulatory logic of the diurnal cycle in the mouse liver

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001069

    DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.
    Figure Legend Snippet: DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.

    Techniques Used: Chromatin Immunoprecipitation, DNA Sequencing, Activation Assay

    Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].
    Figure Legend Snippet: Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].

    Techniques Used: Binding Assay

    Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p
    Figure Legend Snippet: Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, DNA Sequencing, Binding Assay, Expressing

    BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.
    Figure Legend Snippet: BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, DNA Sequencing, Mouse Assay

    Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p
    Figure Legend Snippet: Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p

    Techniques Used: Mouse Assay, Western Blot

    Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p
    Figure Legend Snippet: Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p

    Techniques Used: Genome Wide

    30) Product Images from "Transcriptional regulatory logic of the diurnal cycle in the mouse liver"

    Article Title: Transcriptional regulatory logic of the diurnal cycle in the mouse liver

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001069

    DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.
    Figure Legend Snippet: DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.

    Techniques Used: Chromatin Immunoprecipitation, DNA Sequencing, Activation Assay

    Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].
    Figure Legend Snippet: Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].

    Techniques Used: Binding Assay

    Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p
    Figure Legend Snippet: Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, DNA Sequencing, Binding Assay, Expressing

    BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.
    Figure Legend Snippet: BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, DNA Sequencing, Mouse Assay

    Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p
    Figure Legend Snippet: Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p

    Techniques Used: Mouse Assay, Western Blot

    Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p
    Figure Legend Snippet: Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p

    Techniques Used: Genome Wide

    31) Product Images from "Transcriptional regulatory logic of the diurnal cycle in the mouse liver"

    Article Title: Transcriptional regulatory logic of the diurnal cycle in the mouse liver

    Journal: PLoS Biology

    doi: 10.1371/journal.pbio.2001069

    DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.
    Figure Legend Snippet: DNase I hypersensitivity is rhythmic during diurnal cycles in mouse liver. A. DNase I hypersensitivity, RNA polymerase II (Pol II) density, and H3K27ac enrichment at the Dbp locus. The DNase I track shows the frequency at which nucleotide-resolved DNase I cuts, while H3K27ac and Pol II chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signals are smoothed over 100 bp. All time points are overlaid. The center of each DNase I hypersensitive site (DHS)-enriched region is indicated by vertical ticks (three sites near the TSS are numbered). B. Zoom-in around the transcription start site (TSS) of Dbp (the three TSSs in A are marked) reveals DNase I cuts in between H3K27ac-marked nucleosomes. Both DNase I and H3K27ac signals are maximal at ZT6–ZT10 and minimal at ZT22, consistent with BMAL1-mediated activation of Dbp transcription (absolute signal is highest for site 2, while amplitude is highest for site 3; see panel D). The red and green lines (identical in all subgraphs) show the max signal over the time points at each position and serve as a guide to the eye. C. Quantification of read counts (in log 2 units) for DNase I cuts (in windows of ±300 bp) and Pol II and H3K27Ac ChIP-seq data (in windows of ±1,000 bp) centered on the Dbp TSS using cosine fits. Cosine fits show a common estimated peak time around ZT10 (marked by the inverted triangles). Peak-to-trough amplitudes are about 16-fold for Pol II and approximately 4-fold for both DNase I and H3K27ac. D. Phases and amplitudes of all DHS sites located in the neighborhood of the Dbp gene (nearest TSS association according to annotation). Distances from the center of the plot indicate fitted log 2 amplitudes, and angles (clockwise from ZT0) indicate peak times. We observed that all regions oscillate around a common phase of ZT10. Of the three sites near the TSS (numbered 1–3), site 3 has the highest amplitude. E–H. Idem as A–D but for Npas2 , which has an opposite phase to Dbp (i.e., Npas2 peaks near ZT22). Oscillatory amplitudes are generally larger for Npas2 compared to Dbp . G shows quantification of the signal at the TSS as in panel C.

    Techniques Used: Chromatin Immunoprecipitation, DNA Sequencing, Activation Assay

    Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].
    Figure Legend Snippet: Location-dependent footprint characteristics of DNase I Hypersensitive Sites (DHSs). A. Visualization of DNase I signal (red) around the Rev-erbα promoter with the footprints (detected by Wellington) annotated in black, on top. This region contains BMAL1-binding sites (blue) with E-box motifs, annotated on the bottom line, which is marked by a characteristic footprint. The DNase I cleavage pattern is lower at the binding site, reflecting protection of the DNA from digestion, whereas high signals are observed on the edges of the binding site. B. Number of footprints within DHSs (±300 bp around the peak center). TSS regions contain more footprints on average. More than half of distal regions contain a footprint. C. Number of footprints detected in DHSs in function of (relative) H3K36me3 signal [ 12 ].

    Techniques Used: Binding Assay

    Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p
    Figure Legend Snippet: Chromatin accessibility in Bmal1 -/- mice at ZT6 is generally similar as in the Wild-Type (WT) mice but is lower at BMAL1 sites. A. The Rev-erbα (left) and Gsk3a (right) promoters. DNase I signal (in red) is strongly reduced in Bmal1 -/- mice at sites bound by CLOCK:BMAL1 in WT mice (BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) signal in blue) in the Rev-erbα promoter but is similar in WT and Bmal1 -/- mice at the Gsk3a promoter that are not bound by BMAL1. The vertical scale is the same for all three DNase I tracks, as well as for both BMAL1 ChiP-seq tracks. Wild-type ZT18 signals are lower (about half) than at ZT6 in both genes but not as low as in the Bmal1 -/- mice. B. Comparison of DNase I signals at ZT6 in Bmal1 - /- versus WT mice. All DNase I hypersensitive sites (DHSs) overlapping BMAL1 ChIP-seq peaks in [ 17 ] are shown ( n = 1,555). The dashed lines indicate 4-fold difference. C. Boxplots showing DNase I intensity at the same sites as in B, at peak (ZT6) and trough (ZT18) activities of BMAL1 in the WT, and at ZT6 in Bmal1 -/- mice for all BMAL1-binding sites (green), BMAL1 sites with an associated expression phase between ZT2 and ZT10 (orange), and with a tandem E-box (grey). All pairwise comparisons (within the same color) between either ZT6 versus ZT18 or ZT6 versus ZT6 Bmal1 -/- are significant ( p

    Techniques Used: Mouse Assay, Chromatin Immunoprecipitation, DNA Sequencing, Binding Assay, Expressing

    BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.
    Figure Legend Snippet: BMAL1 footprints indicate temporally changing protein–DNA complexes, consistent with binding of a heterotetramer to DNA. A. Genomic profiles of DNase I cuts around double E-boxes with a spacer of 6 bp (E1-E2 sp6). We selected n = 249 E1-E2 sp6 motifs overlapping a BMAL1 chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) peak and show the average of profiles for loci classified as bound by the mixture model (posterior probability > 0.5). At ZT6, we observed that nucleotides around both E-boxes are protected. In contrast, at ZT18, the width of the protected region is reduced by approximately half, with the second E-box no longer protected from digestion. The signals are anchored to the motif position. Orientation of sites and signals is according to the best match to the E1-E2 sp6 motif. In Bmal1 -/- , only one E-box appears occupied. B. Width (left-side y -axis, green) of the protected region in WT and in Bmal1 -/- mice for E1-E2 sp6 motifs occupied by BMAL1. Fraction of predicted occupied sites is shown in blue (right-side y -axis). C. Two views of the 3-D computational model of the CLOCK:BMAL1 heterotetramer showing two heterodimers of CLOCK:BMAL1 occupying an E1-E2 sp6 site. The two heterodimers are shown in green and blue, while darker green and darker blue correspond to BMAL1 and lighter colors to CLOCK proteins. Information content along the DNA strands is shown in grey with highly constrained nucleotides of the motif in red. D. Zoom on the interacting residuals on the PAS-B domain of CLOCK implicated in the heterotetramer formation.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, DNA Sequencing, Mouse Assay

    Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p
    Figure Legend Snippet: Distal DNase I Hypersensitive Sites (DHSs) help identify diurnally active transcription regulators. A. Scheme of the linear model to infer active transcription regulators: transcription factor (TF) motifs in DHSs within a symmetric window around active transcription start sites (TSSs) are used to explain diurnal rhythms in transcription. B. Fraction of explained temporal variance (deviance ratio) in RNA polymerase II (Pol II) loading (at the TSS of all actives genes) for WT and Bmal1 -/- mice, in function of the window size (radius) for DHS inclusion, shows a maximum at around 50 kb. Here, α = 0 was used in the glmnet ( Materials and methods ). C–D. Inferred TF motif activities for WT and in Bmal1 -/- mice shown with amplitudes (distance from center) and peak times (clockwise, ZT0 at the top) using a window size of 50 kb. All 819 (WT) and 629 ( Bmal1 -/- ) motifs (overlap is 427) with nonzero activities are shown. Note though that most activities are very small and cluster in the center. Certain families of TFs are indicated in colors (full results are provided in S4 Table ). Radial scale for activities is arbitrary but comparable in C and D. E. Quantification of western blots for pCREB (Ser 133 phosphorylation) and CREB in WT and Bmal1 - /- genotypes (log 2 (pCREB/CREB)). Nuclear extracts from four independent livers were harvested every 2 h. Both genotypes showed a significant oscillation ( p

    Techniques Used: Mouse Assay, Western Blot

    Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p
    Figure Legend Snippet: Genome-wide rhythms in DNase I signals are synchronous with RNA Polymerase II (Pol II) transcription and histone acetylation. A. Number of DNase I hypersensitive sites (DHSs) with statistically significant cycling DNase I signals (left), H3K27ac signals (middle), or Pol II signals (right) at three different thresholds ( p

    Techniques Used: Genome Wide

    32) Product Images from "Further Unraveling the Regulatory Twist by Elucidating Metabolic Coinducer-Mediated CbbR-cbbI Promoter Interactions in Rhodopseudomonas palustris CGA010"

    Article Title: Further Unraveling the Regulatory Twist by Elucidating Metabolic Coinducer-Mediated CbbR-cbbI Promoter Interactions in Rhodopseudomonas palustris CGA010

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.06418-11

    Summary of DNase I protection results in the absence (A) or presence (B to E) of coinducers, including (B) RuBP, (C) NADPH, (D) ATP, and (E) FBP, each at 500 μM. Results are compared to a BSA control. Putative CbbR binding sites are indicated
    Figure Legend Snippet: Summary of DNase I protection results in the absence (A) or presence (B to E) of coinducers, including (B) RuBP, (C) NADPH, (D) ATP, and (E) FBP, each at 500 μM. Results are compared to a BSA control. Putative CbbR binding sites are indicated

    Techniques Used: Binding Assay

    DNase I footprinting of the CbbR binding sites on the cbbLS region in the presence of coinducer. The DNase I-digested reactions were prepared and analyzed by capillary electrophoresis with a 3730 DNA analyzer (Applied Biosystems). The differences in the
    Figure Legend Snippet: DNase I footprinting of the CbbR binding sites on the cbbLS region in the presence of coinducer. The DNase I-digested reactions were prepared and analyzed by capillary electrophoresis with a 3730 DNA analyzer (Applied Biosystems). The differences in the

    Techniques Used: Footprinting, Binding Assay, Electrophoresis

    33) Product Images from "STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells"

    Article Title: STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    doi: 10.4049/jimmunol.1302750

    The Pdcd1 locus contains multiple inducible DNase I hypersensitive sites
    Figure Legend Snippet: The Pdcd1 locus contains multiple inducible DNase I hypersensitive sites

    Techniques Used:

    34) Product Images from "Association of Herpes Simplex Virus Type 1 ICP8 and ICP27 Proteins with Cellular RNA Polymerase II Holoenzyme"

    Article Title: Association of Herpes Simplex Virus Type 1 ICP8 and ICP27 Proteins with Cellular RNA Polymerase II Holoenzyme

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.12.5893-5904.2002

    Role of nucleic acids in association of HSV proteins with the Pol II holoenzyme. HEp-2 cells were infected with wt HSV-1 or mock-infected and harvested at 8 h p.i. (A) Cell lysates were untreated or treated with an various amounts of RNases or with DNase I and then were subjected to immunoprecipitation (IP) with rabbit anti-Pol II antibody C21. Proteins in cell lysates and the Pol II immunoprecipitates were resolved by SDS-PAGE and detected by Western blotting with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:13 for lanes 6, 8, 10, 12, and 14 and 1:26 for lanes 7, 9, 11, 13, and 15. 1x, cell lysate was treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min; 3x, cell lysate was treated with 75 U of RNase A/ml and 3,000 U of RNase T 1 /ml at 30°C for 30 min; DNase, cell lysate was treated with 10 μg of DNase I/ml. No, no nuclease during incubation. (B) Cell lysates were untreated or treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min. Immunoprecipitations were performed with monoclonal anti-ICP27 antibody H1113 on RNase-treated and untreated cell lysates in the absence or presence of ethidium bromide. Proteins in cell lysates and immunoprecipitates were resolved by SDS-PAGE and detected with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:40. EtBr, immunoprecipitation was done on untreated HSV-infected cell lysate in the presence of 50 μg of ethidium bromide/ml.
    Figure Legend Snippet: Role of nucleic acids in association of HSV proteins with the Pol II holoenzyme. HEp-2 cells were infected with wt HSV-1 or mock-infected and harvested at 8 h p.i. (A) Cell lysates were untreated or treated with an various amounts of RNases or with DNase I and then were subjected to immunoprecipitation (IP) with rabbit anti-Pol II antibody C21. Proteins in cell lysates and the Pol II immunoprecipitates were resolved by SDS-PAGE and detected by Western blotting with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:13 for lanes 6, 8, 10, 12, and 14 and 1:26 for lanes 7, 9, 11, 13, and 15. 1x, cell lysate was treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min; 3x, cell lysate was treated with 75 U of RNase A/ml and 3,000 U of RNase T 1 /ml at 30°C for 30 min; DNase, cell lysate was treated with 10 μg of DNase I/ml. No, no nuclease during incubation. (B) Cell lysates were untreated or treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min. Immunoprecipitations were performed with monoclonal anti-ICP27 antibody H1113 on RNase-treated and untreated cell lysates in the absence or presence of ethidium bromide. Proteins in cell lysates and immunoprecipitates were resolved by SDS-PAGE and detected with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:40. EtBr, immunoprecipitation was done on untreated HSV-infected cell lysate in the presence of 50 μg of ethidium bromide/ml.

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot, Incubation

    35) Product Images from "Association of Herpes Simplex Virus Type 1 ICP8 and ICP27 Proteins with Cellular RNA Polymerase II Holoenzyme"

    Article Title: Association of Herpes Simplex Virus Type 1 ICP8 and ICP27 Proteins with Cellular RNA Polymerase II Holoenzyme

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.12.5893-5904.2002

    Role of nucleic acids in association of HSV proteins with the Pol II holoenzyme. HEp-2 cells were infected with wt HSV-1 or mock-infected and harvested at 8 h p.i. (A) Cell lysates were untreated or treated with an various amounts of RNases or with DNase I and then were subjected to immunoprecipitation (IP) with rabbit anti-Pol II antibody C21. Proteins in cell lysates and the Pol II immunoprecipitates were resolved by SDS-PAGE and detected by Western blotting with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:13 for lanes 6, 8, 10, 12, and 14 and 1:26 for lanes 7, 9, 11, 13, and 15. 1x, cell lysate was treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min; 3x, cell lysate was treated with 75 U of RNase A/ml and 3,000 U of RNase T 1 /ml at 30°C for 30 min; DNase, cell lysate was treated with 10 μg of DNase I/ml. No, no nuclease during incubation. (B) Cell lysates were untreated or treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min. Immunoprecipitations were performed with monoclonal anti-ICP27 antibody H1113 on RNase-treated and untreated cell lysates in the absence or presence of ethidium bromide. Proteins in cell lysates and immunoprecipitates were resolved by SDS-PAGE and detected with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:40. EtBr, immunoprecipitation was done on untreated HSV-infected cell lysate in the presence of 50 μg of ethidium bromide/ml.
    Figure Legend Snippet: Role of nucleic acids in association of HSV proteins with the Pol II holoenzyme. HEp-2 cells were infected with wt HSV-1 or mock-infected and harvested at 8 h p.i. (A) Cell lysates were untreated or treated with an various amounts of RNases or with DNase I and then were subjected to immunoprecipitation (IP) with rabbit anti-Pol II antibody C21. Proteins in cell lysates and the Pol II immunoprecipitates were resolved by SDS-PAGE and detected by Western blotting with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:13 for lanes 6, 8, 10, 12, and 14 and 1:26 for lanes 7, 9, 11, 13, and 15. 1x, cell lysate was treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min; 3x, cell lysate was treated with 75 U of RNase A/ml and 3,000 U of RNase T 1 /ml at 30°C for 30 min; DNase, cell lysate was treated with 10 μg of DNase I/ml. No, no nuclease during incubation. (B) Cell lysates were untreated or treated with 25 U of RNase A/ml and 1,000 U of RNase T 1 /ml at 30°C for 30 min. Immunoprecipitations were performed with monoclonal anti-ICP27 antibody H1113 on RNase-treated and untreated cell lysates in the absence or presence of ethidium bromide. Proteins in cell lysates and immunoprecipitates were resolved by SDS-PAGE and detected with anti-Pol II antibody 8WG16 (1:1,000 dilution), anti-ICP8 antiserum 3-83 (1:1,000 dilution), and anti-ICP27 antibody H1113 (1:200 dilution). The ratio of cell lysate loaded on the gel to the amount of lysate used in immunoprecipitation was 1:40. EtBr, immunoprecipitation was done on untreated HSV-infected cell lysate in the presence of 50 μg of ethidium bromide/ml.

    Techniques Used: Infection, Immunoprecipitation, SDS Page, Western Blot, Incubation

    36) Product Images from "NET formation can occur independently of RIPK3 and MLKL signaling"

    Article Title: NET formation can occur independently of RIPK3 and MLKL signaling

    Journal: European Journal of Immunology

    doi: 10.1002/eji.201545615

    The formation of human NETs occurs independently of MLKL signaling . Human blood neutrophils were isolated from healthy donors by Ficoll‐Hypaque centrifugation. (A) Confocal microscopy. NET formation following short‐term stimulation (total 45 min) of human neutrophils with the indicated triggers in the presence and absence of 5 μM NSA. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields ( n = 5). Representative original data (right). Co‐localization of elastase (green) with released DNA (PI, red) is indicated by the yellow color. Bars, 10 μM. (B) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye ( n = 3). (C) Total ROS production by activated human neutrophils in the presence and absence of 5 μM NSA was measured by flow cytometry. During cell activation, cells were incubated with 1 μM DHR123 ( n = 3). (D) Bacterial killing by colony forming unit (cfu) assay. Human neutrophils exert antibacterial activity against E. coli that can be partially blocked by 100 U/mL DNase I both in the presence and absence of 5 μM NSA ( n = 3). (E) Viability of Jurkat cells was analyzed by ethidium bromide exclusion assay. In parallel experiments, 5 μM NSA blocked cell death induced by 20 ng/mL TNF‐α plus 100 nM Smac mimetic AT‐406 (cIAP1/2 selective IAP‐antagonist) in caspase‐8 deficient and 20 μM zVAD‐FMK–treated Jurkat cells ( n = 3), demonstrating that the inhibitor was pharmacologically active. All data are shown as mean ± SEM of the indicated number of independent experiments. * p
    Figure Legend Snippet: The formation of human NETs occurs independently of MLKL signaling . Human blood neutrophils were isolated from healthy donors by Ficoll‐Hypaque centrifugation. (A) Confocal microscopy. NET formation following short‐term stimulation (total 45 min) of human neutrophils with the indicated triggers in the presence and absence of 5 μM NSA. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields ( n = 5). Representative original data (right). Co‐localization of elastase (green) with released DNA (PI, red) is indicated by the yellow color. Bars, 10 μM. (B) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye ( n = 3). (C) Total ROS production by activated human neutrophils in the presence and absence of 5 μM NSA was measured by flow cytometry. During cell activation, cells were incubated with 1 μM DHR123 ( n = 3). (D) Bacterial killing by colony forming unit (cfu) assay. Human neutrophils exert antibacterial activity against E. coli that can be partially blocked by 100 U/mL DNase I both in the presence and absence of 5 μM NSA ( n = 3). (E) Viability of Jurkat cells was analyzed by ethidium bromide exclusion assay. In parallel experiments, 5 μM NSA blocked cell death induced by 20 ng/mL TNF‐α plus 100 nM Smac mimetic AT‐406 (cIAP1/2 selective IAP‐antagonist) in caspase‐8 deficient and 20 μM zVAD‐FMK–treated Jurkat cells ( n = 3), demonstrating that the inhibitor was pharmacologically active. All data are shown as mean ± SEM of the indicated number of independent experiments. * p

    Techniques Used: Isolation, Centrifugation, Confocal Microscopy, Flow Cytometry, Cytometry, Activation Assay, Incubation, Colony-forming Unit Assay, Activity Assay, Exclusion Assay

    The formation of mouse NETs is independent of RIPK3 . Mature mouse neutrophils were isolated from bone marrow of wild‐type and  Ripk3 ‐deficient mice. (A) Immunoblotting. Bone marrow cells from wild‐type and  Ripk3 ‐deficient mice were analyzed for RIPK3 protein expression. Three mice per genotype are shown. (B) Quantification of NET‐forming neutrophils by confocal microscopy. NET formation following short‐term stimulation (total 45 min) of mouse neutrophils with the indicated triggers. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields (Supporting Information Fig. 1A and Supporting Information Movie 1). No statistical differences were observed between wild‐type and  Ripk3 ‐deficient cells ( n  = 3). (C) Representative microscopy. Co‐localization (arrows) of elastase (green) with released DNA (PI, red) assessed by confocal microscopy. Bars, 10 μM. (D) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye. A significant difference in dsDNA release was detected between control and activated cells, but not between wild‐type and  Ripk3 ‐deficient neutrophils ( n  = 3). (E) Total ROS activity assessed by DHR123 fluorescence and flow cytometry ( n  = 3). (F) Quantification of H 2 O 2  production upon activation of neutrophils was performed using luminescent ROS‐Glo that measures H 2 O 2  levels directly in cell culture. Again, ROS activity is increased in activated mouse neutrophils, but no statistical differences were observed between wild‐type and  Ripk3 ‐deficient cells ( n  = 3). (G) Bacterial killing by colony formation unit (cfu) assay ( n  = 3). Mouse neutrophils exert antibacterial activity against  E. coli  that can be partially blocked by 100 U/mL DNase I. All data are shown as mean ± SEM of the indicated number of independent experiments. * p
    Figure Legend Snippet: The formation of mouse NETs is independent of RIPK3 . Mature mouse neutrophils were isolated from bone marrow of wild‐type and Ripk3 ‐deficient mice. (A) Immunoblotting. Bone marrow cells from wild‐type and Ripk3 ‐deficient mice were analyzed for RIPK3 protein expression. Three mice per genotype are shown. (B) Quantification of NET‐forming neutrophils by confocal microscopy. NET formation following short‐term stimulation (total 45 min) of mouse neutrophils with the indicated triggers. The number of NET‐forming neutrophils was determined by counting the DNA‐releasing cells in ten high power fields (Supporting Information Fig. 1A and Supporting Information Movie 1). No statistical differences were observed between wild‐type and Ripk3 ‐deficient cells ( n = 3). (C) Representative microscopy. Co‐localization (arrows) of elastase (green) with released DNA (PI, red) assessed by confocal microscopy. Bars, 10 μM. (D) Quantification of dsDNA in supernatants of activated neutrophils using PicoGreen fluorescent dye. A significant difference in dsDNA release was detected between control and activated cells, but not between wild‐type and Ripk3 ‐deficient neutrophils ( n = 3). (E) Total ROS activity assessed by DHR123 fluorescence and flow cytometry ( n = 3). (F) Quantification of H 2 O 2 production upon activation of neutrophils was performed using luminescent ROS‐Glo that measures H 2 O 2 levels directly in cell culture. Again, ROS activity is increased in activated mouse neutrophils, but no statistical differences were observed between wild‐type and Ripk3 ‐deficient cells ( n = 3). (G) Bacterial killing by colony formation unit (cfu) assay ( n = 3). Mouse neutrophils exert antibacterial activity against E. coli that can be partially blocked by 100 U/mL DNase I. All data are shown as mean ± SEM of the indicated number of independent experiments. * p

    Techniques Used: Isolation, Mouse Assay, Expressing, Confocal Microscopy, Microscopy, Activity Assay, Fluorescence, Flow Cytometry, Cytometry, Activation Assay, Cell Culture, Colony-forming Unit Assay

    37) Product Images from "Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis"

    Article Title: Extracellular traps are associated with human and mouse neutrophil and macrophage mediated killing of larval Strongyloides stercoralis

    Journal: Microbes and infection / Institut Pasteur

    doi: 10.1016/j.micinf.2014.02.012

    NETs are required for larval killing by human neutrophils in vitro. A) Human macrophages (Mϕ) and neutrophils (PMN) cultured in vitro with 50 larvae (L3) for 48 h. The cultures were treated with 100 U/ml of DNase I to block NET formation and assessed
    Figure Legend Snippet: NETs are required for larval killing by human neutrophils in vitro. A) Human macrophages (Mϕ) and neutrophils (PMN) cultured in vitro with 50 larvae (L3) for 48 h. The cultures were treated with 100 U/ml of DNase I to block NET formation and assessed

    Techniques Used: In Vitro, Cell Culture, Blocking Assay

    38) Product Images from "A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group"

    Article Title: A cell-cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf450

    Fig. 8. ( A ) β-galactosidase activity of the B.thuringiensis 407 Cry – A ′ Z Δ papR mutant strain. The cells were grown at 37°C in LB medium and each peptide (OS5, OS5-I1, OS5-M1 and OS5-V1; all 0.5 µM) was added at t 1 (1 h after the onset of the stationary phase). The amino acid sequences of the peptides are as follows: OS5, LPFEF; OS5-I1, IPFEF; OS5-M1, MPFEF; OS5-V1, VPFEF. ( B ) DNase I footprinting analysis of PlcR binding to the plcA promoter region in the presence of various pentapeptides. Lane 1, G+A Maxam and Gilbert reaction; lane 2, no protein; lane 3, 1.2 µg of PlcR (35 pmol); lane 4, 1.2 µg of PlcR and of OS5 10 µg; lane 5, 1.2 µg of PlcR and 10 µg of OS5-I1; lane 6, 1.2 µg of PlcR and 10 µg of OS5-M1; lane 7, 1.2 µg of PlcR and 10 µg of OS5-V1. The region protected by PlcR is indicated by a bracket and corresponds to the PlcR box upstream from the plcA gene.
    Figure Legend Snippet: Fig. 8. ( A ) β-galactosidase activity of the B.thuringiensis 407 Cry – A ′ Z Δ papR mutant strain. The cells were grown at 37°C in LB medium and each peptide (OS5, OS5-I1, OS5-M1 and OS5-V1; all 0.5 µM) was added at t 1 (1 h after the onset of the stationary phase). The amino acid sequences of the peptides are as follows: OS5, LPFEF; OS5-I1, IPFEF; OS5-M1, MPFEF; OS5-V1, VPFEF. ( B ) DNase I footprinting analysis of PlcR binding to the plcA promoter region in the presence of various pentapeptides. Lane 1, G+A Maxam and Gilbert reaction; lane 2, no protein; lane 3, 1.2 µg of PlcR (35 pmol); lane 4, 1.2 µg of PlcR and of OS5 10 µg; lane 5, 1.2 µg of PlcR and 10 µg of OS5-I1; lane 6, 1.2 µg of PlcR and 10 µg of OS5-M1; lane 7, 1.2 µg of PlcR and 10 µg of OS5-V1. The region protected by PlcR is indicated by a bracket and corresponds to the PlcR box upstream from the plcA gene.

    Techniques Used: Activity Assay, Mutagenesis, Footprinting, Binding Assay

    Fig. 6. DNase I footprinting analysis of PlcR binding to the plcA promoter region. Radiolabeled fragments (50 000 c.p.m.) were incubated with various concentrations of PlcR and OS5. ( A ) Lane 1, G+A Maxam and Gilbert reaction; lane 2, no protein; lane 3, 0.1 µg of PlcR; lane 4, 10 µg of PlcR; lane 5, 20 µg of OS5; lane 6, 0.1 µg of PlcR and 10 µg of OS5; lane 7, 0.1 µg of PlcR and 20 µg of OS5; lane 8, 10 µg of PlcR and 10 µg of OS5; lane 9, 10 µg of PlcR and 20 µg of OS5. The region protected by PlcR is indicated by a bracket. ( B ) Lane 1, G+A Maxam and Gilbert reaction; lanes 2–8, 50 pmol of PlcR (1.7 µg) and OS5: lane 2, 1000 pmol (0.65 µg); lane 3, 500 pmol; lane 4, 150 pmol; lane 5, 70 pmol; lane 6, 35 pmol; lane 7, 10 pmol; lane 8, 5 pmol; lane 9, 50 pmol of PlcR; lane 10, neither protein nor peptide. ( C ) plcA promoter region. The DNase I-protected area is indicated by a bracket and the PlcR box is in bold. Positions are relative to the transcription start point. The –35 and –10 promoter regions, and the transcription start (+ 1) of the plcA ).
    Figure Legend Snippet: Fig. 6. DNase I footprinting analysis of PlcR binding to the plcA promoter region. Radiolabeled fragments (50 000 c.p.m.) were incubated with various concentrations of PlcR and OS5. ( A ) Lane 1, G+A Maxam and Gilbert reaction; lane 2, no protein; lane 3, 0.1 µg of PlcR; lane 4, 10 µg of PlcR; lane 5, 20 µg of OS5; lane 6, 0.1 µg of PlcR and 10 µg of OS5; lane 7, 0.1 µg of PlcR and 20 µg of OS5; lane 8, 10 µg of PlcR and 10 µg of OS5; lane 9, 10 µg of PlcR and 20 µg of OS5. The region protected by PlcR is indicated by a bracket. ( B ) Lane 1, G+A Maxam and Gilbert reaction; lanes 2–8, 50 pmol of PlcR (1.7 µg) and OS5: lane 2, 1000 pmol (0.65 µg); lane 3, 500 pmol; lane 4, 150 pmol; lane 5, 70 pmol; lane 6, 35 pmol; lane 7, 10 pmol; lane 8, 5 pmol; lane 9, 50 pmol of PlcR; lane 10, neither protein nor peptide. ( C ) plcA promoter region. The DNase I-protected area is indicated by a bracket and the PlcR box is in bold. Positions are relative to the transcription start point. The –35 and –10 promoter regions, and the transcription start (+ 1) of the plcA ).

    Techniques Used: Footprinting, Binding Assay, Incubation

    39) Product Images from "Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle"

    Article Title: Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00736-12

    Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene ( HIST2H4 ). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and
    Figure Legend Snippet: Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene ( HIST2H4 ). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and

    Techniques Used: Isolation

    40) Product Images from "Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle"

    Article Title: Epigenetic Control of Cell Cycle-Dependent Histone Gene Expression Is a Principal Component of the Abbreviated Pluripotent Cell Cycle

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00736-12

    Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene ( HIST2H4 ). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and
    Figure Legend Snippet: Chromatin architecture of the human histone H4 gene in hES cells. (A) DNase I hypersensitivity at the human histone H4 gene ( HIST2H4 ). Nuclei were isolated from hES cells (lanes 4 to 6) and normal diploid fibroblasts (TIG-1 and IMR-90; lanes 7 to 9 and

    Techniques Used: Isolation

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    Transfection:

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    Polymerase Chain Reaction:

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    Construct:

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    Incubation:

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    Sequencing:

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    Lysis:

    Article Title: Human Immunodeficiency Virus-Like Particles Activate Multiple Types of Immune Cells
    Article Snippet: .. After 9 days, spleens were collected from mice that received FL DNA and single cell suspensions were prepared after treatments with type IV collagenase (Worthington) and lysis of red blood cells. .. Single cell suspensions were incubated with CD11c (N418) microbeads and CD11c+ DC cells were obtained by passing through magnetic columns according to the manufacturer’s instructions (Miltenyi Biotec Inc. Auburn, CA).

    Plasmid Preparation:

    Article Title: Expression of metallothionein gene at different time in testicular interstitial cells and liver of rats treated with cadmium
    Article Snippet: .. PCR products were cloned using pUCm-T vector, The constructed plasmids were transfected into JM109, positive colonies were selected and the DNA sequence was analysed using a DNA sequencer (Worthington Biochemical Co.). .. Testicular interstitial cells were suspended in 500 μL of 10 mM Tris-HCL, pH7.4, and lysed by sonication (3 × 10 s) on ice.

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  • 93
    Worthington Biochemical e coli dna polymerase
    MALDI-TOF mass spectra of <t>SVPDE</t> digests of modified <t>DNA</t> 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.
    E Coli Dna Polymerase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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
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    92
    Worthington Biochemical genomic dna
    Digestion pattern of archaeal chromosomes treated with micrococcal nuclease (MNase). Purified chromosomes of (A) T. kodakarensis , (B) T. <t>acidophilum</t> , (C) P. calidifontis , and (D) S. solfataricus were digested with increasing concentrations of MNase and separated on 2.5% agarose gels in 1X TBE. The accumulation of <t>DNA</t> of particular sizes was observed with T. kodakarensis (arrows) and T. acidophilum (curly brackets) but not with P. calidifontis and S. solfataricus chromosomes. MNase concentration was 0.3, 1, 3, 10 U MNase in 100 μl reaction (A) or 0, 0.3, 1, 3, 10, and 30 U MNase in 100 μl reaction (B–D) .
    Genomic Dna, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Journal: Nucleic Acids Research

    Article Title: 3?-Exonuclease resistance of DNA oligodeoxynucleotides containing O6-[4-oxo-4-(3-pyridyl)butyl]guanine

    doi:

    Figure Lengend Snippet: MALDI-TOF mass spectra of SVPDE digests of modified DNA 16mers d(AACAGCCATATGXCCC): ( A ) X = O 6 -POB-dG, time-controlled digest; ( B ) X = O 6 -POB-dG, complete digest conditions; ( C ) O 6 -Me-dG-containing oligomers, controlled digest conditions. Arrows indicate the portion of the sequence represented in the spectra, and doubly charged ions are marked with #.

    Article Snippet: SVPDE and E.coli DNA polymerase I were purchased from Worthington Biochemicals (Lakewood, NJ).

    Techniques: Modification, Sequencing

    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

    Digestion pattern of archaeal chromosomes treated with micrococcal nuclease (MNase). Purified chromosomes of (A) T. kodakarensis , (B) T. acidophilum , (C) P. calidifontis , and (D) S. solfataricus were digested with increasing concentrations of MNase and separated on 2.5% agarose gels in 1X TBE. The accumulation of DNA of particular sizes was observed with T. kodakarensis (arrows) and T. acidophilum (curly brackets) but not with P. calidifontis and S. solfataricus chromosomes. MNase concentration was 0.3, 1, 3, 10 U MNase in 100 μl reaction (A) or 0, 0.3, 1, 3, 10, and 30 U MNase in 100 μl reaction (B–D) .

    Journal: Frontiers in Microbiology

    Article Title: Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

    doi: 10.3389/fmicb.2020.01247

    Figure Lengend Snippet: Digestion pattern of archaeal chromosomes treated with micrococcal nuclease (MNase). Purified chromosomes of (A) T. kodakarensis , (B) T. acidophilum , (C) P. calidifontis , and (D) S. solfataricus were digested with increasing concentrations of MNase and separated on 2.5% agarose gels in 1X TBE. The accumulation of DNA of particular sizes was observed with T. kodakarensis (arrows) and T. acidophilum (curly brackets) but not with P. calidifontis and S. solfataricus chromosomes. MNase concentration was 0.3, 1, 3, 10 U MNase in 100 μl reaction (A) or 0, 0.3, 1, 3, 10, and 30 U MNase in 100 μl reaction (B–D) .

    Article Snippet: MNase Digestion of Archaeal Chromosome T. acidophilum , P. calidifontis , T. kodakarensis , and S. solfataricus genomic DNA was incubated with MNase (Worthington Biochemical, Lakewood, NJ, United States) at various concentrations (0.3, 1, 3, 10 and 30 U MNase/100 μl) for 20 min at 37°C.

    Techniques: Purification, Concentration Assay

    In vitro reconstitution of chromosome structures with recombinant Alba and HTa. (A–C) Reconstitution on a linear 3-kb plasmid using recombinant Alba from T. acidophilum and P. calidifontis at different Alba to DNA weight ratios. (A) AFM images showing various structures formed with Alba and DNA. Fibers are indicated with arrow heads. Alba:DNA weight ratios are indicated. The asterisk indicates a structure in which two 3-kb DNA molecules are joined together by Alba binding. (B) Histograms show the diameters of the fibrous structures formed (indicated with arrow heads in A ). (C) Histograms show the respective contour lengths of DNA at a 15:1 Alba:DNA ratio. The theoretical length of a linear 3-kb DNA (∼1000 nm) is indicated with a dashed line. See Figure 6A for a histogram of unbound DNA length. (D–G) Reconstitution with histidine-tagged HTa from T. acidophilum at varying protein to DNA ratio. (D) AFM images showing beaded (arrows) and filamentous (arrow heads) structures. (E) Diameter of the beads formed at a relatively lower HTa concentration. (F) Width of the filaments formed at relatively higher HTa concentration. (G) Histogram shows the contour DNA lengths of the structures formed at a 15:1 HTa:DNA ratio. Dashed line indicates the theoretical length of a 3-kb unbound DNA. Scale bars: 100 nm.

    Journal: Frontiers in Microbiology

    Article Title: Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

    doi: 10.3389/fmicb.2020.01247

    Figure Lengend Snippet: In vitro reconstitution of chromosome structures with recombinant Alba and HTa. (A–C) Reconstitution on a linear 3-kb plasmid using recombinant Alba from T. acidophilum and P. calidifontis at different Alba to DNA weight ratios. (A) AFM images showing various structures formed with Alba and DNA. Fibers are indicated with arrow heads. Alba:DNA weight ratios are indicated. The asterisk indicates a structure in which two 3-kb DNA molecules are joined together by Alba binding. (B) Histograms show the diameters of the fibrous structures formed (indicated with arrow heads in A ). (C) Histograms show the respective contour lengths of DNA at a 15:1 Alba:DNA ratio. The theoretical length of a linear 3-kb DNA (∼1000 nm) is indicated with a dashed line. See Figure 6A for a histogram of unbound DNA length. (D–G) Reconstitution with histidine-tagged HTa from T. acidophilum at varying protein to DNA ratio. (D) AFM images showing beaded (arrows) and filamentous (arrow heads) structures. (E) Diameter of the beads formed at a relatively lower HTa concentration. (F) Width of the filaments formed at relatively higher HTa concentration. (G) Histogram shows the contour DNA lengths of the structures formed at a 15:1 HTa:DNA ratio. Dashed line indicates the theoretical length of a 3-kb unbound DNA. Scale bars: 100 nm.

    Article Snippet: MNase Digestion of Archaeal Chromosome T. acidophilum , P. calidifontis , T. kodakarensis , and S. solfataricus genomic DNA was incubated with MNase (Worthington Biochemical, Lakewood, NJ, United States) at various concentrations (0.3, 1, 3, 10 and 30 U MNase/100 μl) for 20 min at 37°C.

    Techniques: In Vitro, Recombinant, Plasmid Preparation, Binding Assay, Concentration Assay

    In vitro reconstitution using a combination of archaeal proteins. Chromatin structures were reconstituted on a linear 3-kb plasmid using recombinant proteins at varying Alba concentrations. (A) AFM images showing reconstitution using histone and Alba from T. kodakarensis . Histograms below the AFM images show the contour DNA length in each condition. Dashed line indicates the theoretical length of a 3-kb unbound DNA. (B) AFM images showing reconstitution using HTa and Alba from T. acidophilum . Scale bars: 100 nm. Note that histograms are not shown in (B) because of the inability to accurately measure the DNA contour length due to a high degree of folding or joining of the fiber structures.

    Journal: Frontiers in Microbiology

    Article Title: Different Proteins Mediate Step-Wise Chromosome Architectures in Thermoplasma acidophilum and Pyrobaculum calidifontis

    doi: 10.3389/fmicb.2020.01247

    Figure Lengend Snippet: In vitro reconstitution using a combination of archaeal proteins. Chromatin structures were reconstituted on a linear 3-kb plasmid using recombinant proteins at varying Alba concentrations. (A) AFM images showing reconstitution using histone and Alba from T. kodakarensis . Histograms below the AFM images show the contour DNA length in each condition. Dashed line indicates the theoretical length of a 3-kb unbound DNA. (B) AFM images showing reconstitution using HTa and Alba from T. acidophilum . Scale bars: 100 nm. Note that histograms are not shown in (B) because of the inability to accurately measure the DNA contour length due to a high degree of folding or joining of the fiber structures.

    Article Snippet: MNase Digestion of Archaeal Chromosome T. acidophilum , P. calidifontis , T. kodakarensis , and S. solfataricus genomic DNA was incubated with MNase (Worthington Biochemical, Lakewood, NJ, United States) at various concentrations (0.3, 1, 3, 10 and 30 U MNase/100 μl) for 20 min at 37°C.

    Techniques: In Vitro, Plasmid Preparation, Recombinant