micrococcal nuclease mnase Search Results


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  • 99
    Worthington Biochemical micrococcal nuclease
    Micrococcal Nuclease, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 1072 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Zymo Research micrococcal nuclease
    Micrococcal Nuclease, supplied by Zymo Research, used in various techniques. Bioz Stars score: 95/100, based on 92 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs micrococcal nuclease
    Micrococcal Nuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2731 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore micrococcal nuclease
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
    Micrococcal Nuclease, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1988 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher micrococcal nuclease
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
    Micrococcal Nuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Cell Signaling Technology Inc micrococcal nuclease
    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to <t>MNase.</t> (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.
    Micrococcal Nuclease, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 291 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    TaKaRa micrococcal nuclease
    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) <t>MNase</t> sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested <t>DNA</t> was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).
    Micrococcal Nuclease, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 417 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare micrococcal nuclease
    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) <t>MNase</t> sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested <t>DNA</t> was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).
    Micrococcal Nuclease, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim micrococcal nuclease
    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) <t>MNase</t> sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested <t>DNA</t> was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).
    Micrococcal Nuclease, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega micrococcal nuclease treated rabbit reticulocyte lysate
    IRES-mediated translation and IRES inhibition in a cell-free system and in cells. (A) Diagrammatic comparison of general protein synthesis to IRES-mediated translation. General protein synthesis is mediated by cap-dependent ribosomal scanning from the 5′-end of the mRNA and may be modulated by mTOR inhibitors. Internal ribosome entry sites (IRESs) allow the 40S ribosome to engage the mRNA at a position much closer (in many cases immediately adjacent to) the AUG initiation codon. IRES-mediated translation is independently regulated and serves as a fail-safe mechanism ensuring the synthesis of proteins most critical for cell survival. (B) Structure of IRES inhibitor lead compound W (cpd_W): Ethyl 2-{[2-(1,3-benzoxazol-2-ylthio)butanoyl]amino}-4-methyl-1,3-thiazole-5-carboxylate, MW 405. (C) In vitro translation assays: <t>Rabbit</t> <t>reticulocyte</t> <t>lysate</t> was programmed with a bicistronic reporter RNA in which translation of the second cistron (firefly luciferase coding sequence) is mediated by the IGF1R IRES, while translation of the first cistron ( Renilla luciferase coding sequence) is mediated by ribosomal scanning. IRES inhibitor cpd_W (or vehicle control) was included in the reaction in increasing concentrations as indicated. The result is indicative of selective inhibition of IRES-mediated translation. A structural analog of cpd_W (W-7) in which a single atom has been modified (converting the benzoxazole to a benzimidazole) was completely inactive in this assay, indicative of the chemical specificity of IRES inhibition. Cycloheximide (5 µg/ml, chx) and puromycin (250 µg/ml, puro) were included as reference standards for non-specific translational inhibition (far right). (D) IRES inhibitor cpd_W completely blocked de novo synthesis of IGF1R in breast tumor cells under adverse conditions (serum-deprivation, loss of adhesion) relevant to the microenvironment of the tumor. T47D breast tumor cells were seeded in 6-well plates and allowed 48 h to recover and resume proliferation, then incubated in the presence of IRES inhibitor cpd_W (10 µg/ml) or vehicle control (0.1% DMSO) as indicated. The cells were simultaneously subjected to acute serum deprivation (0.5% fetal calf serum, no added insulin) to increase dependence on IRES-mediated translation. After 24 h, the cells were harvested and whole cell lysates prepared, equivalent aliquots separated by SDS-PAGE and immunoblotted for IGF1R-β and α-tubulin. In lanes 7–12, the cells were trypsinized and seeded into 6-well plates and immediately incubated in the presence of IRES-inhibitor cpd_W or vehicle control as indicated. Robust regeneration of trypsin-catabolized IGF1R was observed within 24 h in <t>vehicle-treated</t> cells, however, this was completely blocked in the presence of cpd_W (10 µg/ml as shown; IC 50 , 2 µg/ml). The asterisk (*) marks the position of trypsin-catabolized IGF1R. In lanes 13–17, the cells were treated as described for lanes 7–12, except that following trypsinization, cells were transferred to low-adherence plates, forcing cells to adapt to a state of anchorage-independence. The results confirmed the activity of cpd_W against the endogenous IRES in genetically-unmodified tumor cells. Similar results were obtained with IRES inhibitor lead cpd_P ( 11 ).
    Micrococcal Nuclease Treated Rabbit Reticulocyte Lysate, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher micrococcal nuclease s7
    VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of <t>MNase</t> or the indicated concentrations of <t>NaCl.</t> Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N
    Micrococcal Nuclease S7, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher micrococcal nuclease solution 100 u µl
    VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of <t>MNase</t> or the indicated concentrations of <t>NaCl.</t> Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N
    Micrococcal Nuclease Solution 100 U µl, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Illumina Inc micrococcal nuclease mnase
    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of <t>MNase-protected</t> fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic <t>DNA,</t> and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.
    Micrococcal Nuclease Mnase, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to MNase. (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.

    Journal: Molecular and Cellular Biology

    Article Title: Genetic Inactivation of ATRX Leads to a Decrease in the Amount of Telomeric Cohesin and Level of Telomere Transcription in Human Glioma Cells

    doi: 10.1128/MCB.01317-14

    Figure Lengend Snippet: shRNA-mediated inactivation of ATRX does not alter subtelomeric chromatin accessibility to MNase. (A) Chromatin isolated from 8-MG-BA glioma cells in which ATRX had been inactivated (shATRX) or not (shscrambled [shSCR]) was digested with MNase for the indicated times. (Left gel) Ethidium bromide (EtBr) staining of bulk chromatin. (Right gel) Southern blot with subtelomeric probe. (Far right) Quantification of the data. The signals obtained for mononucleosomes were normalized to the total signals measured for each time point (EtBr or Southern blot). (B) Chromatin samples from shATRX or shSCR 8-MG-BA cells were digested for 5 min with the indicated amounts of MNase (milliunits per microgram of DNA). (Far right) Quantification of the data.

    Article Snippet: Nuclei isolated from 107 cells were digested with 8 mU of micrococcal nuclease (MNase) (Sigma)/μg DNA at 37°C for the indicated times, as described previously ( ).

    Techniques: shRNA, Isolation, Staining, Southern Blot

    Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) MNase sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested DNA was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).

    Journal: Genetics

    Article Title: Correlation of Meiotic DSB Formation and Transcription Initiation Around Fission Yeast Recombination Hotspots

    doi: 10.1534/genetics.116.197954

    Figure Lengend Snippet: Chromatin structure around ade6-3049 is more open than around ade6-3057 , and HRA is involved in the chromatin regulation. (A) MNase sensitivity of chromatin around ade6-3049/3057 . Indicated cells of the pat1-114 background were induced to enter meiosis by temperature-shift method, and harvested 3 hr after the induction. MNase-digested DNA was prepared by MNase (0, 20, or 30 units) treatment of chromatin fraction and subsequent purification. DNA was further cut with Sac I, fractionated on 1.2% agaraose gel, and analyzed by Southern blotting using the probe indicated by the open box. The vertical arrow and the open arrowhead show the ade6 ORF and the 3049/3057 site, respectively. The numbers on the left and the right side of the panel indicate the positions of λ/ Eco T14 I fragments used for electrophoresis marker and the positions from the first A of the ade6 ORF, respectively. The dotted lines indicate a region in which MNase-sensitive sites are clustered around ade6-3049 . Presented is an example from two independent experiments, whose results were similar to each other. (B) HRA deletion reduced acetylation level of H3K9 around ade6-3049 . Indicated cells of the pat1-114 ). DNA isolated from immunoprecipitates and whole-cell extracts was analyzed by real-time qPCR, where fragments corresponding to ade6-3049/3057 and the prp3 + promoter were amplified. Relative enrichment at ade6-3049/3057 over prp3 is shown. Bar graphs are created based on mean values of two independent experiments (shown by ○).

    Article Snippet: Genomic DNA was treated with 0, 20, or 30 units of micrococcal nuclease (MNase) (Takara), digested with Sac I, and separated on a 1.2% agarose gel.

    Techniques: Purification, Southern Blot, Electrophoresis, Marker, Isolation, Real-time Polymerase Chain Reaction, Amplification

    IRES-mediated translation and IRES inhibition in a cell-free system and in cells. (A) Diagrammatic comparison of general protein synthesis to IRES-mediated translation. General protein synthesis is mediated by cap-dependent ribosomal scanning from the 5′-end of the mRNA and may be modulated by mTOR inhibitors. Internal ribosome entry sites (IRESs) allow the 40S ribosome to engage the mRNA at a position much closer (in many cases immediately adjacent to) the AUG initiation codon. IRES-mediated translation is independently regulated and serves as a fail-safe mechanism ensuring the synthesis of proteins most critical for cell survival. (B) Structure of IRES inhibitor lead compound W (cpd_W): Ethyl 2-{[2-(1,3-benzoxazol-2-ylthio)butanoyl]amino}-4-methyl-1,3-thiazole-5-carboxylate, MW 405. (C) In vitro translation assays: Rabbit reticulocyte lysate was programmed with a bicistronic reporter RNA in which translation of the second cistron (firefly luciferase coding sequence) is mediated by the IGF1R IRES, while translation of the first cistron ( Renilla luciferase coding sequence) is mediated by ribosomal scanning. IRES inhibitor cpd_W (or vehicle control) was included in the reaction in increasing concentrations as indicated. The result is indicative of selective inhibition of IRES-mediated translation. A structural analog of cpd_W (W-7) in which a single atom has been modified (converting the benzoxazole to a benzimidazole) was completely inactive in this assay, indicative of the chemical specificity of IRES inhibition. Cycloheximide (5 µg/ml, chx) and puromycin (250 µg/ml, puro) were included as reference standards for non-specific translational inhibition (far right). (D) IRES inhibitor cpd_W completely blocked de novo synthesis of IGF1R in breast tumor cells under adverse conditions (serum-deprivation, loss of adhesion) relevant to the microenvironment of the tumor. T47D breast tumor cells were seeded in 6-well plates and allowed 48 h to recover and resume proliferation, then incubated in the presence of IRES inhibitor cpd_W (10 µg/ml) or vehicle control (0.1% DMSO) as indicated. The cells were simultaneously subjected to acute serum deprivation (0.5% fetal calf serum, no added insulin) to increase dependence on IRES-mediated translation. After 24 h, the cells were harvested and whole cell lysates prepared, equivalent aliquots separated by SDS-PAGE and immunoblotted for IGF1R-β and α-tubulin. In lanes 7–12, the cells were trypsinized and seeded into 6-well plates and immediately incubated in the presence of IRES-inhibitor cpd_W or vehicle control as indicated. Robust regeneration of trypsin-catabolized IGF1R was observed within 24 h in vehicle-treated cells, however, this was completely blocked in the presence of cpd_W (10 µg/ml as shown; IC 50 , 2 µg/ml). The asterisk (*) marks the position of trypsin-catabolized IGF1R. In lanes 13–17, the cells were treated as described for lanes 7–12, except that following trypsinization, cells were transferred to low-adherence plates, forcing cells to adapt to a state of anchorage-independence. The results confirmed the activity of cpd_W against the endogenous IRES in genetically-unmodified tumor cells. Similar results were obtained with IRES inhibitor lead cpd_P ( 11 ).

    Journal: Oncology Reports

    Article Title: Translational control of the undifferentiated phenotype in ER-positive breast tumor cells: Cytoplasmic localization of ERα and impact of IRES inhibition

    doi: 10.3892/or.2018.6332

    Figure Lengend Snippet: IRES-mediated translation and IRES inhibition in a cell-free system and in cells. (A) Diagrammatic comparison of general protein synthesis to IRES-mediated translation. General protein synthesis is mediated by cap-dependent ribosomal scanning from the 5′-end of the mRNA and may be modulated by mTOR inhibitors. Internal ribosome entry sites (IRESs) allow the 40S ribosome to engage the mRNA at a position much closer (in many cases immediately adjacent to) the AUG initiation codon. IRES-mediated translation is independently regulated and serves as a fail-safe mechanism ensuring the synthesis of proteins most critical for cell survival. (B) Structure of IRES inhibitor lead compound W (cpd_W): Ethyl 2-{[2-(1,3-benzoxazol-2-ylthio)butanoyl]amino}-4-methyl-1,3-thiazole-5-carboxylate, MW 405. (C) In vitro translation assays: Rabbit reticulocyte lysate was programmed with a bicistronic reporter RNA in which translation of the second cistron (firefly luciferase coding sequence) is mediated by the IGF1R IRES, while translation of the first cistron ( Renilla luciferase coding sequence) is mediated by ribosomal scanning. IRES inhibitor cpd_W (or vehicle control) was included in the reaction in increasing concentrations as indicated. The result is indicative of selective inhibition of IRES-mediated translation. A structural analog of cpd_W (W-7) in which a single atom has been modified (converting the benzoxazole to a benzimidazole) was completely inactive in this assay, indicative of the chemical specificity of IRES inhibition. Cycloheximide (5 µg/ml, chx) and puromycin (250 µg/ml, puro) were included as reference standards for non-specific translational inhibition (far right). (D) IRES inhibitor cpd_W completely blocked de novo synthesis of IGF1R in breast tumor cells under adverse conditions (serum-deprivation, loss of adhesion) relevant to the microenvironment of the tumor. T47D breast tumor cells were seeded in 6-well plates and allowed 48 h to recover and resume proliferation, then incubated in the presence of IRES inhibitor cpd_W (10 µg/ml) or vehicle control (0.1% DMSO) as indicated. The cells were simultaneously subjected to acute serum deprivation (0.5% fetal calf serum, no added insulin) to increase dependence on IRES-mediated translation. After 24 h, the cells were harvested and whole cell lysates prepared, equivalent aliquots separated by SDS-PAGE and immunoblotted for IGF1R-β and α-tubulin. In lanes 7–12, the cells were trypsinized and seeded into 6-well plates and immediately incubated in the presence of IRES-inhibitor cpd_W or vehicle control as indicated. Robust regeneration of trypsin-catabolized IGF1R was observed within 24 h in vehicle-treated cells, however, this was completely blocked in the presence of cpd_W (10 µg/ml as shown; IC 50 , 2 µg/ml). The asterisk (*) marks the position of trypsin-catabolized IGF1R. In lanes 13–17, the cells were treated as described for lanes 7–12, except that following trypsinization, cells were transferred to low-adherence plates, forcing cells to adapt to a state of anchorage-independence. The results confirmed the activity of cpd_W against the endogenous IRES in genetically-unmodified tumor cells. Similar results were obtained with IRES inhibitor lead cpd_P ( 11 ).

    Article Snippet: In vitro translation Standard in vitro translation reactions were set up using micrococcal nuclease treated rabbit reticulocyte lysate (Promega) at a final concentration of 50% (vol/vol).

    Techniques: Inhibition, In Vitro, Luciferase, Sequencing, Modification, Incubation, SDS Page, Activity Assay

    VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of MNase or the indicated concentrations of NaCl. Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N

    Journal: BMC Veterinary Research

    Article Title: Identification of the NLS and NES motifs of VP2 from chicken anemia virus and the interaction of VP2 with mini-chromosome maintenance protein 3

    doi: 10.1186/1746-6148-8-15

    Figure Lengend Snippet: VP2 binds to chromatin and interacts with MCM3 but this does not require dual phosphatase activity . ( A ) The soluble (S) and chromatin (C) fractions were prepared by using CSK buffer containing 0.5% Triton X-100 in CHO cells of transfected with VP2-GFP plasmid. The fractions were treated with 0 U (-) and 150 U (+) of MNase or the indicated concentrations of NaCl. Nuclear extracts (N) containing VP2-GFP were as positive control and all fractions were subjected to immunoblotting against with lamin B receptor, MCM3, and VP2 antibodies. ( B ) At 48 h post-transfection, the GFP (as Control) and Flag-VP2-GFP (as wild-type; WT) were found in the CHO cells. Next the lysates were immunoprecipitated using Flag M2 beads and immunoblotted against VP2 and MCM3 antibodies. ( C ) The WT and mutants (C95S, C97S, and C95S/C97S) of the Flag-VP2-GFP in CHO cells were also examined at 48 h post-transfection. The cell lysates were immunoprecipitated using Flag M2 beads and immunoblotted against with VP2 and MCM3 antibodies. The arrow head was indicated a none-specific band. The nuclear extracts containing VP2-GFP were designated as N

    Article Snippet: For micrococcal nuclease (MNase) (Fermentas, Canada) or NaCl treatment, the chromatin was resuspended in 100 μl of CSK buffer supplemented with 0.1 M, 0.3 M of NaCl or 0 U (-) and 150 U (+) MNase with 2 mM CaCl2 and incubated for 30 min at 37°C.

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Positive Control, Immunoprecipitation

    Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of MNase-protected fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic DNA, and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.

    Journal: Genome Research

    Article Title: Regulation of nucleosome positioning by a CHD Type III chromatin remodeler and its relationship to developmental gene expression in Dictyostelium

    doi: 10.1101/gr.216309.116

    Figure Lengend Snippet: Genome-wide nucleosome positioning in Dictyostelium . ( A ) Normalized read midpoint frequency distributions of MNase-protected fragments (nucleosome dyads) of all 12,750 genes in growth-stage WT cells were aligned relative to their ATG codons. Peaks (arrows) correspond to dyad midpoints for globally phased nucleosomes in the 5′ region of intragenic DNA, and distances between mapped read peaks correspond to ∼170 bp NRL. The protein coding DNA sequence (cds) region is shaded. ( B ) Normalized read midpoint frequency distributions of all genes in growth-stage WT cells were aligned relative to their translational termination sites (stop codons). Peaks (arrows) in the mean normalized frequency distribution correspond to globally phased nucleosomes in the 3′ region of intragenic DNA. The protein cds region is shaded. ( C ) Normalized dyad read midpoint frequency distributions for WT chromatin (CHR; dotted line) (see A ) were adjusted for sequence mappability by dividing with equivalent control data from MNase-digested naked (protein free) WT DNA (DNA; red line) and replotted as the ratio (CHR/DNA; thick black line) within 1.2 kb of flanking chromatin relative to ATG sites of all 12,750 genes. An ∼170-bp nucleosome-depleted (“free”) region (NDR) is centered near the AT-rich regions of Dictyostelium TSS. Positioned nucleosomes upstream (+) and downstream (−) to the NDR are indicated by arrows. The protein cds region is shaded.

    Article Snippet: Chromatin was digested with micrococcal nuclease (MNase) to create nuclease-resistant DNA ladders with a fragment spectrum of < 1 kb and was analyzed by Illumina paired-end DNA sequencing.

    Techniques: Genome Wide, Sequencing