dnase  (Worthington Biochemical)


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    • 99
    Name:
    Deoxyribonuclease I
    Description:
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    Catalog Number:
    LS002004
    Price:
    33
    Source:
    Bovine Pancreas
    Size:
    5 mg
    Cas Number:
    9003.98.9
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    Structured Review

    Worthington Biochemical dnase
    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the <t>DNA</t> at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with <t>DNase</t> and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Chromatographically purified A lyophilized powder with glycine as a stabilizer
    https://www.bioz.com/result/dnase/product/Worthington Biochemical
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase - by Bioz Stars, 2021-04
    99/100 stars

    Images

    1) Product Images from "Initiation-specific alleles of the Cdc45 helicase-activating protein"

    Article Title: Initiation-specific alleles of the Cdc45 helicase-activating protein

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0214426

    Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for CMG formation. (A) Proteins associated with the DNA at the end of the CMG formation assay. Bead-associated proteins were washed with Buffer H + 0.3M KCl, 0.02%NP-40, released with DNase and detected by immunoblot. (B) Relative association of Cdc45, Mcm2-7 and GINS with origin DNA after CMG-formation assay. Three experimental replicates were quantified and plotted. Error bars represent standard error from the mean. p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Tube Formation Assay

    Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).
    Figure Legend Snippet: Cdc45-ts mutants are defective for DNA replication. (A) DNA replication products produced with the indicated Cdc45 proteins were separated on a 0.8% alkaline agarose gel and imaged using a phosphoimager. Relative intensities of +DDK lanes were quantified and plotted using ImageJ (Cdc45 = red, Cdc45-124 = blue, Cdc45-238 = green, Cdc45-485 = purple). Horizontal lines indicate the most highly represented product length for each Cdc45 protein tested. (B) Relative levels of DNA replication for the indicated Cdc45 proteins from six experimental replicates of replication assays performed with the indicated Cdc45 mutant proteins were quantified and plotted. (C) Proteins associated with the DNA at the end of the replication reaction. Bead-associated proteins were washed with Buffer H + 0.3M K-Glut 0.02%NP-40, released with DNase, and detected by immunoblot. (D) Relative association of Cdc45, Mcm2-7, GINS and Pol ε with origin DNA after replication. Six (Cdc45 and GINS association) and five (Pol ε association) experimental replicates were quantified and plotted. For both (B) and (D), error bars represent standard error from the mean. Asterisks indicate the following p-values: p≤0.01(**), p≤0.001(***), p≤0.0001(****), not significant (n.s., p≥0.05).

    Techniques Used: Produced, Agarose Gel Electrophoresis, Mutagenesis

    Related Articles

    Concentration Assay:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: .. DNase I (Worthington Biochemical) was added to a final concentration of 130 μg/ml. .. To aid the removal of cut DNA, (NH4 )2 SO4 was added to a final concentration of 0.25 M and the samples were incubated for 5 min at room temperature.

    Article Title: STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells
    Article Snippet: Briefly, nuclei were isolated and treated with increasing concentrations of DNase I (Worthington Biochemical Co, Lakewood, NJ). .. Concentration ranges for DNase I were determined empirically for each lot and cell type by titrating DNase I for its ability to digest CR-C as the positive control but not regions previously found to be resistant to DNase I (e.g., +6.3 region). .. Following purification of the digested DNA, PCR was performed across the Pdcd1 locus using a set of 59 primer pairs ( ).

    End Labeling:

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The positions of the MNase cleavages within chromatin of the HPRT promoter region of permeabilized cells relative to a downstream Bcl I site in the first intron of the HPRT gene were mapped using a 400-bp hybridization probe located just upstream of the Bcl I site (Fig. ). .. To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe. .. Figure shows the Southern blot analysis of the DNase I and MNase cleavage patterns on the inactive and active HPRT promoters in permeabilized 8121 and 4.12 cells, respectively.

    Hybridization:

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The positions of the MNase cleavages within chromatin of the HPRT promoter region of permeabilized cells relative to a downstream Bcl I site in the first intron of the HPRT gene were mapped using a 400-bp hybridization probe located just upstream of the Bcl I site (Fig. ). .. To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe. .. Figure shows the Southern blot analysis of the DNase I and MNase cleavage patterns on the inactive and active HPRT promoters in permeabilized 8121 and 4.12 cells, respectively.

    Cell Culture:

    Article Title: Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection
    Article Snippet: Pseudotype viruses were prepared by cotransfection of 293T cells with the pNL43lucΔenv vector together with the MuLV envelope expression vector (pJD-1). .. After treatment with DNase I (40 μg/ml; Worthington), each virus (70 ng of p24) was inoculated into HeLa cells and cultured at 37°C for 6 h. The cells were washed with PBS and resuspended in fresh medium (DMEM plus 10% FBS). ..

    Lysis:

    Article Title: Preparation of Soluble Proteins from Escherichia coli
    Article Snippet: The purification process time described can be shortened through the use of chromatography systems and fast-flow column matrices (see and Time Considerations). .. DEAE Sepharose CL-4B resin GE Heathcare Life Sciences) Anion-exchange buffer (see recipe) 0.26% (w/v) sodium hypochlorite/70% ethanol or 5% (v/v) bleach (e.g., Clorox)/70% ethanol E. coli cells (~50 g wet weight) from fermentation ( UNIT 5.3 ) containing IL-1β Lysis buffer (see recipe) Bovine pancreas DNase I and RNase A (Worthington Biochemical; optional, for reducing solution viscosity) 2 N sodium hydroxide Ammonium sulfate, ground with mortar and pestle Cation-exchange buffer (see recipe) CM Sepharose CL-4B (GE Heathcare Cation-exchange buffer/250 mM NaCl (see recipe) Tris base Gel-filtration buffer (see recipe) Ultrogel AcA54 gel-permeation resin (Pall Corporation; Sigma-Aldrich)) Lyophilization buffer (see recipe; optional) 2- or 3-liter sintered glass funnel with fritted disc (coarse porosity) and 5-liter filter flask Chromatography columns (preferably glass) with adjustable flow adapters: one (or optionally two) 5 × 50 cm and one 2.5 × 100 cm (GE Healthcare), Amicon, or equivalent) RK50 packing reservoir GE Heathcare) Peristaltic pump, UV monitor, and fraction collector (GE Heathcare; check on-line for other suppliers of equivalent items) 16 × 150–mm culture tubes 40-ml French pressure cell and rapid-fill kit (Thermo Fisher Scientific – see Commentary section on breaking cells).) .. French laboratory press (Thermo Fisher Scientific)) 1-liter Waring commercial blender.

    Chromatography:

    Article Title: Preparation of Soluble Proteins from Escherichia coli
    Article Snippet: The purification process time described can be shortened through the use of chromatography systems and fast-flow column matrices (see and Time Considerations). .. DEAE Sepharose CL-4B resin GE Heathcare Life Sciences) Anion-exchange buffer (see recipe) 0.26% (w/v) sodium hypochlorite/70% ethanol or 5% (v/v) bleach (e.g., Clorox)/70% ethanol E. coli cells (~50 g wet weight) from fermentation ( UNIT 5.3 ) containing IL-1β Lysis buffer (see recipe) Bovine pancreas DNase I and RNase A (Worthington Biochemical; optional, for reducing solution viscosity) 2 N sodium hydroxide Ammonium sulfate, ground with mortar and pestle Cation-exchange buffer (see recipe) CM Sepharose CL-4B (GE Heathcare Cation-exchange buffer/250 mM NaCl (see recipe) Tris base Gel-filtration buffer (see recipe) Ultrogel AcA54 gel-permeation resin (Pall Corporation; Sigma-Aldrich)) Lyophilization buffer (see recipe; optional) 2- or 3-liter sintered glass funnel with fritted disc (coarse porosity) and 5-liter filter flask Chromatography columns (preferably glass) with adjustable flow adapters: one (or optionally two) 5 × 50 cm and one 2.5 × 100 cm (GE Healthcare), Amicon, or equivalent) RK50 packing reservoir GE Heathcare) Peristaltic pump, UV monitor, and fraction collector (GE Heathcare; check on-line for other suppliers of equivalent items) 16 × 150–mm culture tubes 40-ml French pressure cell and rapid-fill kit (Thermo Fisher Scientific – see Commentary section on breaking cells).) .. French laboratory press (Thermo Fisher Scientific)) 1-liter Waring commercial blender.

    Flow Cytometry:

    Article Title: Preparation of Soluble Proteins from Escherichia coli
    Article Snippet: The purification process time described can be shortened through the use of chromatography systems and fast-flow column matrices (see and Time Considerations). .. DEAE Sepharose CL-4B resin GE Heathcare Life Sciences) Anion-exchange buffer (see recipe) 0.26% (w/v) sodium hypochlorite/70% ethanol or 5% (v/v) bleach (e.g., Clorox)/70% ethanol E. coli cells (~50 g wet weight) from fermentation ( UNIT 5.3 ) containing IL-1β Lysis buffer (see recipe) Bovine pancreas DNase I and RNase A (Worthington Biochemical; optional, for reducing solution viscosity) 2 N sodium hydroxide Ammonium sulfate, ground with mortar and pestle Cation-exchange buffer (see recipe) CM Sepharose CL-4B (GE Heathcare Cation-exchange buffer/250 mM NaCl (see recipe) Tris base Gel-filtration buffer (see recipe) Ultrogel AcA54 gel-permeation resin (Pall Corporation; Sigma-Aldrich)) Lyophilization buffer (see recipe; optional) 2- or 3-liter sintered glass funnel with fritted disc (coarse porosity) and 5-liter filter flask Chromatography columns (preferably glass) with adjustable flow adapters: one (or optionally two) 5 × 50 cm and one 2.5 × 100 cm (GE Healthcare), Amicon, or equivalent) RK50 packing reservoir GE Heathcare) Peristaltic pump, UV monitor, and fraction collector (GE Heathcare; check on-line for other suppliers of equivalent items) 16 × 150–mm culture tubes 40-ml French pressure cell and rapid-fill kit (Thermo Fisher Scientific – see Commentary section on breaking cells).) .. French laboratory press (Thermo Fisher Scientific)) 1-liter Waring commercial blender.

    Positive Control:

    Article Title: STAT3, STAT4, NFATc1, and CTCF regulate PD-1 through multiple novel regulatory regions in murine T cells
    Article Snippet: Briefly, nuclei were isolated and treated with increasing concentrations of DNase I (Worthington Biochemical Co, Lakewood, NJ). .. Concentration ranges for DNase I were determined empirically for each lot and cell type by titrating DNase I for its ability to digest CR-C as the positive control but not regions previously found to be resistant to DNase I (e.g., +6.3 region). .. Following purification of the digested DNA, PCR was performed across the Pdcd1 locus using a set of 59 primer pairs ( ).

    Staining:

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium
    Article Snippet: To verify that the DNase I treatment mentioned in the previous paragraph indeed affected chromatin components, we analyzed the staining pattern of chromatin markers acetyl-H4 and H4K20m ( ; ) after DNase I treatment. .. Staining for these proteins was almost totally eliminated in cells treated with DNase I, whereas the staining pattern for SC35, indicative of nonchromatin splicing speckles, seemed unaltered ( , G–J). .. The quantitative analysis of the distribution of NuMA in differentiated S1 cells that have not undergone DNase I treatment reveals that NuMA staining foci are abundant in the midnuclear region ( D; ).

    other:

    Article Title: Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells
    Article Snippet: To increase the reproducibility and minimize experimental variations, we chose to digest the nuclei of CD4 T cells with various amounts of DNase I on ice for a relatively long period of time (1 hr).

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    • dnase i  (Worthington Biochemical)
    99
    Worthington Biochemical dnase i
    Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5 RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.
    Dnase I, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnase i/product/Worthington Biochemical
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dnase i - by Bioz Stars, 2021-04
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    Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5 RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.

    Journal: Journal of Virology

    Article Title: Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection

    doi: 10.1128/JVI.78.21.11563-11573.2004

    Figure Lengend Snippet: Effects of HIV-1 IN mutations on viral infectivity. Viruses were prepared by cotransfection of COS-7 cells with the pNL43lucΔenv vector containing either WT IN or mutant IN together with an amphotropic Moloney MuLV envelope expression vector (pJD-1) or a macrophage-tropic HIV-1 envelope vector (pJR-FL) by using Lipofectamine. At 48 h posttransfection, culture supernatants of the transfected COS-7 cells were harvested. DNase I-treated supernatants were inoculated into 10 5 RD cells, PBLs, and MDMs. At 4 days postinfection, the cells were washed with PBS and lysed with 200 μl of cell lysis buffer. Ten microliters of each cell lysate was subjected to the luciferase assay. Mean values from five independent experiments are shown with the error bars.

    Article Snippet: After treatment with DNase I (40 μg/ml; Worthington), each virus (70 ng of p24) was inoculated into HeLa cells and cultured at 37°C for 6 h. The cells were washed with PBS and resuspended in fresh medium (DMEM plus 10% FBS).

    Techniques: Infection, Cotransfection, Plasmid Preparation, Mutagenesis, Expressing, Transfection, Lysis, Luciferase

    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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend 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.

    Article Snippet: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend 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.

    Article Snippet: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    Techniques: 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.

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend 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.

    Article Snippet: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    Techniques: 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 ).

    Journal: Molecular and Cellular Biology

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

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Figure Lengend 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 ).

    Article Snippet: To determine the positions of DNase I-hypersensitive sites relative to MNase cleavage sites, NP-40-permeabilized cells containing the active HPRT allele were treated with increasing concentrations of DNase I and the DNase I-hypersensitive sites in chromatin of the HPRT promoter relative to the same Bcl I site were also mapped by indirect end labeling using the same hybridization probe.

    Techniques: Binding Assay, Sequencing, Methylation

    NuMA distribution is altered by DNase I treatment. (A–J) S1 cells were cultured in 3D for 10 d to induce acinar differentiation. (A–F) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (A, C, and E) or treated with DNase I for 30 min (B, D, and F) before fixation and immunostaining for NuMA (red; A and B), PML (green; C and D), and lamin B (green; E and F). (G–J) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (G and I) or treated with DNase I for 30 min (H and J) before fixation and dual immunostaining for NuMA (red) and H4K20m (green) (G and H), or SC35 (red) and acetyl-H4 (green) (I and J). DAPI was used for DNA staining and is shown in images B–F. One nucleus is shown per image; in H and J, a dotted white circle indicates approximate nuclear boundary. Bar, 2.5 μm.

    Journal: Molecular Biology of the Cell

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

    doi: 10.1091/mbc.E06-06-0551

    Figure Lengend Snippet: NuMA distribution is altered by DNase I treatment. (A–J) S1 cells were cultured in 3D for 10 d to induce acinar differentiation. (A–F) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (A, C, and E) or treated with DNase I for 30 min (B, D, and F) before fixation and immunostaining for NuMA (red; A and B), PML (green; C and D), and lamin B (green; E and F). (G–J) Acinar cells were permeabilized with Triton X-100 without DNase I treatment (G and I) or treated with DNase I for 30 min (H and J) before fixation and dual immunostaining for NuMA (red) and H4K20m (green) (G and H), or SC35 (red) and acetyl-H4 (green) (I and J). DAPI was used for DNA staining and is shown in images B–F. One nucleus is shown per image; in H and J, a dotted white circle indicates approximate nuclear boundary. Bar, 2.5 μm.

    Article Snippet: DNase I (Worthington Biochemical) was added to a final concentration of 130 μg/ml.

    Techniques: Cell Culture, Immunostaining, Staining

    NuMA is associated with the chromatin compartment. (A–C) Western blot for NuMA, Lamin B, and MCM3. (A) S1 cells were cultured as a monolayer (2D) for 10 d. Cells were fractionated using a classical protocol to obtain nuclear matrices, including treatment with 130 μg/ml DNase I for 30 min. The entire content of each fraction [DNase I-sensitive (chromatin fraction) and nuclear matrix fractions] was loaded on the gel. (B and C) S1 cells were cultured as a monolayer (2D) (B) or in 3D (C) for 10 d, and fractionated using a classical protocol for chromatin isolation, including 5-min incubation with 1 U of micrococcal nuclease. Twenty micrograms of each fraction were loaded on the gel. CF, chromatin fraction; ND, nondigestible nuclear fraction; NMF, nuclear matrix fraction.

    Journal: Molecular Biology of the Cell

    Article Title: NuMA Influences Higher Order Chromatin Organization in Human Mammary Epithelium

    doi: 10.1091/mbc.E06-06-0551

    Figure Lengend Snippet: NuMA is associated with the chromatin compartment. (A–C) Western blot for NuMA, Lamin B, and MCM3. (A) S1 cells were cultured as a monolayer (2D) for 10 d. Cells were fractionated using a classical protocol to obtain nuclear matrices, including treatment with 130 μg/ml DNase I for 30 min. The entire content of each fraction [DNase I-sensitive (chromatin fraction) and nuclear matrix fractions] was loaded on the gel. (B and C) S1 cells were cultured as a monolayer (2D) (B) or in 3D (C) for 10 d, and fractionated using a classical protocol for chromatin isolation, including 5-min incubation with 1 U of micrococcal nuclease. Twenty micrograms of each fraction were loaded on the gel. CF, chromatin fraction; ND, nondigestible nuclear fraction; NMF, nuclear matrix fraction.

    Article Snippet: DNase I (Worthington Biochemical) was added to a final concentration of 130 μg/ml.

    Techniques: Western Blot, Cell Culture, Isolation, Incubation