nucleosome repeat length  (Worthington Biochemical)


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    Name:
    Nuclease Micrococcal
    Description:
    Chromatographically purified to be essentially homogeneous chromatographically and electrophoretically SDS PAGE A lyophilized powder
    Catalog Number:
    ls004796
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    Staphylococcus aureus (Strain Foggi)
    Cas Number:
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    Structured Review

    Worthington Biochemical nucleosome repeat length
    Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) <t>Nucleosome</t> repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.
    Chromatographically purified to be essentially homogeneous chromatographically and electrophoretically SDS PAGE A lyophilized powder
    https://www.bioz.com/result/nucleosome repeat length/product/Worthington Biochemical
    Average 85 stars, based on 131 article reviews
    Price from $9.99 to $1999.99
    nucleosome repeat length - by Bioz Stars, 2020-10
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    Images

    1) Product Images from "Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability"

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01567-13

    Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.
    Figure Legend Snippet: Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.

    Techniques Used: Staining, Marker, Quantitation Assay, DNA Synthesis, Labeling, BrdU Incorporation Assay, Periodic Counter-current Chromatography, Nucleic Acid Electrophoresis, Software

    2) Product Images from "Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability"

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01567-13

    Conditional ablation of transcription factor HINFP inactivates histone H4 expression. (A) Schematic diagram showing targeted Hinfp locus to generate conditional Hinfp knockout mice. The arrow indicates the recombined locus that generates a conditional Hinfp -null mutation (−, null). Ovals indicate right- and left-arm probes for Southern blotting. Arrowheads represent genotyping primers for PCR. (B) Autoradiographs of Southern blot analysis of mouse ES cell clones (wild type [+/+] and +/FN) and mouse tail DNA (+/FN, FN/FN, and +/+) that were hybridized to either left- or right-arm probes: 18.0-kb WT allele and either 10.5-kb (LA probe) or 9.6-kb (RA probe) targeted allele. (C) PCR genotyping analysis of DNA from MEFs of wild-type (+/+) and Hinfp -null pups (F/F) with or without infection with Ad5CMVCre-EGFP virus using primers a and b shown in panel A. Lane 1, marker; lane 2, WT MEFs; lane 3, F/F MEFs without Cre infection; and lane 4, Hinfp -null MEFs after Cre treatment. (D and E) RT-qPCR analysis of WT and cKO MEFs without (No Inf.) or with Cre infection (d0 to d2) showing expression of Hinfp mRNA for multiple exons (exons 2-3, 5, 6-7, 7-8) (D) and two histone H4 genes ( Hist2H4 [H4] and Hist1H4m [H4m]) (E). Removal of Hinfp causes a marked decrease in histone H4 gene expression. (F) Western blot analysis of WT and cKO MEFs at d2 and d4 shows reduction of total H4 protein in Hinfp -null cells (see also Fig. S2 in the supplemental material).
    Figure Legend Snippet: Conditional ablation of transcription factor HINFP inactivates histone H4 expression. (A) Schematic diagram showing targeted Hinfp locus to generate conditional Hinfp knockout mice. The arrow indicates the recombined locus that generates a conditional Hinfp -null mutation (−, null). Ovals indicate right- and left-arm probes for Southern blotting. Arrowheads represent genotyping primers for PCR. (B) Autoradiographs of Southern blot analysis of mouse ES cell clones (wild type [+/+] and +/FN) and mouse tail DNA (+/FN, FN/FN, and +/+) that were hybridized to either left- or right-arm probes: 18.0-kb WT allele and either 10.5-kb (LA probe) or 9.6-kb (RA probe) targeted allele. (C) PCR genotyping analysis of DNA from MEFs of wild-type (+/+) and Hinfp -null pups (F/F) with or without infection with Ad5CMVCre-EGFP virus using primers a and b shown in panel A. Lane 1, marker; lane 2, WT MEFs; lane 3, F/F MEFs without Cre infection; and lane 4, Hinfp -null MEFs after Cre treatment. (D and E) RT-qPCR analysis of WT and cKO MEFs without (No Inf.) or with Cre infection (d0 to d2) showing expression of Hinfp mRNA for multiple exons (exons 2-3, 5, 6-7, 7-8) (D) and two histone H4 genes ( Hist2H4 [H4] and Hist1H4m [H4m]) (E). Removal of Hinfp causes a marked decrease in histone H4 gene expression. (F) Western blot analysis of WT and cKO MEFs at d2 and d4 shows reduction of total H4 protein in Hinfp -null cells (see also Fig. S2 in the supplemental material).

    Techniques Used: Expressing, Knock-Out, Mouse Assay, Mutagenesis, Southern Blot, Polymerase Chain Reaction, Clone Assay, Infection, Marker, Quantitative RT-PCR, Western Blot

    Loss of Hinfp causes deregulation of cell proliferation. WT and cKO MEFs (GFP sorted) were cultured for 4 days. (A) WT and cKO MEFs plated at 0.35 × 10 6 /60-mm dish were harvested at different time points (d0 to d4) to analyze proliferation in culture. cKO cells show severe delay in proliferation. (B) Cell cycle analysis by flow cytometry for DNA content shows increased sub-G 1 population, altered S phase, as well as polyploid cells in cKO MEFs. (C) Senescence-associated β-galactosidase (SA β-Gal) activity was measured at d2 and d4. There is an obvious increase in SA β-Gal-specific staining in cKO MEFs that is enhanced at d4. (D) The distribution of HLBs was determined by staining for NPAT (red). IF microscopy revealed an increase in the fraction of cells with multiple NPAT foci (white arrowheads) or diffused NPAT staining (red arrowheads) in cKO MEFs at d2 and d4. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (E) Quantitation of NPAT staining patterns in WT and cKO MEFs at d2 and d4. Bar graph shows the percentage of cells with specific numbers of NPAT foci. A total of 200 nuclei were counted from two biological replicates per sample for each time point.
    Figure Legend Snippet: Loss of Hinfp causes deregulation of cell proliferation. WT and cKO MEFs (GFP sorted) were cultured for 4 days. (A) WT and cKO MEFs plated at 0.35 × 10 6 /60-mm dish were harvested at different time points (d0 to d4) to analyze proliferation in culture. cKO cells show severe delay in proliferation. (B) Cell cycle analysis by flow cytometry for DNA content shows increased sub-G 1 population, altered S phase, as well as polyploid cells in cKO MEFs. (C) Senescence-associated β-galactosidase (SA β-Gal) activity was measured at d2 and d4. There is an obvious increase in SA β-Gal-specific staining in cKO MEFs that is enhanced at d4. (D) The distribution of HLBs was determined by staining for NPAT (red). IF microscopy revealed an increase in the fraction of cells with multiple NPAT foci (white arrowheads) or diffused NPAT staining (red arrowheads) in cKO MEFs at d2 and d4. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (E) Quantitation of NPAT staining patterns in WT and cKO MEFs at d2 and d4. Bar graph shows the percentage of cells with specific numbers of NPAT foci. A total of 200 nuclei were counted from two biological replicates per sample for each time point.

    Techniques Used: Cell Culture, Cell Cycle Assay, Flow Cytometry, Cytometry, Activity Assay, Staining, Microscopy, Quantitation Assay

    Hinfp is required for genomic stability and maintenance of DNA replication. WT and cKO MEFs were analyzed for factors associated with double-strand DNA damage by IF microscopy. (A) γ-H2AX S-139 (red); scale bar, 20 μm; (B) 53BP1 (red); scale bar, 20 μm. Nuclei were counterstained with DAPI (blue). cKO MEFs show a higher percentage of cells with both γ-H2AX and 53BP1 foci. (C) DNA fiber assay. WT and cKO MEFs were consecutively labeled with iododeoxyuridine (IdU; green) and chlorodeoxyuridine (CldU; red) at d2 and d4. Representative images of elongating and stalled forks are shown. Hinfp -null MEFs have a higher incidence of stalled replication forks than WT. (D) The bar graph represents percentage of stalled replication forks observed in WT and cKO MEFs at d2 and d4. There is increased frequency of stalled forks in Hinfp -null MEFs.
    Figure Legend Snippet: Hinfp is required for genomic stability and maintenance of DNA replication. WT and cKO MEFs were analyzed for factors associated with double-strand DNA damage by IF microscopy. (A) γ-H2AX S-139 (red); scale bar, 20 μm; (B) 53BP1 (red); scale bar, 20 μm. Nuclei were counterstained with DAPI (blue). cKO MEFs show a higher percentage of cells with both γ-H2AX and 53BP1 foci. (C) DNA fiber assay. WT and cKO MEFs were consecutively labeled with iododeoxyuridine (IdU; green) and chlorodeoxyuridine (CldU; red) at d2 and d4. Representative images of elongating and stalled forks are shown. Hinfp -null MEFs have a higher incidence of stalled replication forks than WT. (D) The bar graph represents percentage of stalled replication forks observed in WT and cKO MEFs at d2 and d4. There is increased frequency of stalled forks in Hinfp -null MEFs.

    Techniques Used: Microscopy, Labeling

    Hinfp ablation results in atypical nuclear and chromosomal morphology. WT and cKO MEFs (post-GFP sorting) were harvested at d1, d2, or d4 in culture. (A) IF microscopy shows obvious differences in nuclear size and shape between WT and cKO MEFs from d2 onwards. Nuclei stained with DAPI (gray) show clear increase in size after removal of Hinfp . Scale bar, 50 μm. (B) IF microscopy of MEFs stained with α-tubulin (red) show increased presence of binucleated cells (arrowheads) in cKO MEFs. The insets indicate percentage of binucleate cells. Scale bar, 50 μm. (C) Mitotic preparations of MEFs at d2 were subjected to DNA-FISH with probes against mouse minor satellite (top) or major satellite (bottom) regions. Hinfp -ablated cells show obvious increase in the chromosome complement. Scale bar, 20 μm. (D) Quantitation of mitotic cells from WT and cKO MEFs at d2 probed with satellite DNA was performed to calculate percent distribution of diploid (∼2n) and polyploid ( > 2n) chromosome complement. A total of 50 metaphases were counted per sample. The bar graph shows higher percentage of polyploidy in cKO MEFs than in WT MEFs. (E) The microtubules of asynchronous WT and cKO MEFs at d2 were stained with α-tubulin (red) and analyzed by IF microscopy. The micrograph shows the presence of mitotic cells with multipolar spindles in cKO MEFs.
    Figure Legend Snippet: Hinfp ablation results in atypical nuclear and chromosomal morphology. WT and cKO MEFs (post-GFP sorting) were harvested at d1, d2, or d4 in culture. (A) IF microscopy shows obvious differences in nuclear size and shape between WT and cKO MEFs from d2 onwards. Nuclei stained with DAPI (gray) show clear increase in size after removal of Hinfp . Scale bar, 50 μm. (B) IF microscopy of MEFs stained with α-tubulin (red) show increased presence of binucleated cells (arrowheads) in cKO MEFs. The insets indicate percentage of binucleate cells. Scale bar, 50 μm. (C) Mitotic preparations of MEFs at d2 were subjected to DNA-FISH with probes against mouse minor satellite (top) or major satellite (bottom) regions. Hinfp -ablated cells show obvious increase in the chromosome complement. Scale bar, 20 μm. (D) Quantitation of mitotic cells from WT and cKO MEFs at d2 probed with satellite DNA was performed to calculate percent distribution of diploid (∼2n) and polyploid ( > 2n) chromosome complement. A total of 50 metaphases were counted per sample. The bar graph shows higher percentage of polyploidy in cKO MEFs than in WT MEFs. (E) The microtubules of asynchronous WT and cKO MEFs at d2 were stained with α-tubulin (red) and analyzed by IF microscopy. The micrograph shows the presence of mitotic cells with multipolar spindles in cKO MEFs.

    Techniques Used: Microscopy, Staining, Fluorescence In Situ Hybridization, Quantitation Assay

    Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.
    Figure Legend Snippet: Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.

    Techniques Used: Staining, Marker, Quantitation Assay, DNA Synthesis, Labeling, BrdU Incorporation Assay, Periodic Counter-current Chromatography, Nucleic Acid Electrophoresis, Software

    Related Articles

    DNA Extraction:

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast
    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. .. ChIP was performed using 3 µl of anti-H3K14Ac polyclonal antibody (Upstate, 07–353).

    Amplification:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification ..

    Sample Prep:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification ..

    Isolation:

    Article Title: Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching
    Article Snippet: .. Nuclei were isolated, digested with micrococcal nuclease or DNaseI (Worthington), and DNA was purified as described ( ; ) with modifications detailed in . .. Naked DNA controls were obtained by digesting a PCR product.

    Purification:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification ..

    Article Title: Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching
    Article Snippet: .. Nuclei were isolated, digested with micrococcal nuclease or DNaseI (Worthington), and DNA was purified as described ( ; ) with modifications detailed in . .. Naked DNA controls were obtained by digesting a PCR product.

    Concentration Assay:

    Article Title: Histone hypoacetylation-activated genes are repressed by acetyl-CoA- and chromatin-mediated mechanism
    Article Snippet: .. Micrococcal nuclease (MNase; Worthington) was added immediately to a final concentration of 0, 1, 2.5, 10, 20, 50 U/ml. .. The samples were incubated for 5 min at 28°C with occasional gentle tapping of the microfuge tube.

    Incubation:

    Article Title: Ribosomal Pausing at a Frameshifter RNA Pseudoknot Is Sensitive to Reading Phase but Shows Little Correlation with Frameshift Efficiency
    Article Snippet: .. Unprotected mRNA was degraded by incubation with micrococcal nuclease (1 or 2 U/μl; Worthington) and RNase V1 (0.02 U/μl; Pharmacia) at 26°C for 30 min in the presence of 3.5 mM Mg(OAc)2 and 3 mM CaCl2 in a final reaction volume of 40 μl. .. Following addition of 60 μl of buffer T (20 mM HEPES, 150 mM KOAc, 10 mM Mg(OAc)2 , 5 mM EGTA, 2 mM dithiothreitol), the reaction mixture was overlayered onto a 60-μl cushion of 0.25 M sucrose in buffer T and subsequently centrifuged at 30 lb/in2 for 30 min in an A-110 rotor in a Beckman airfuge.

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast
    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. .. ChIP was performed using 3 µl of anti-H3K14Ac polyclonal antibody (Upstate, 07–353).

    Polymerase Chain Reaction:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification ..

    Gel Extraction:

    Article Title: Genome-Wide Analysis of Nucleosome Positions, Occupancy, and Accessibility in Yeast: Nucleosome Mapping, High-Resolution Histone ChIP, and NCAM
    Article Snippet: .. Overnight saturated culture of S. cerevisiae strain 37% Formaldehyde 2.5 M Glycine Spheroblast solution (see recipe) β-mercaptoethanol (14.3 M) 100T zymolyase (AMSBIO) Micrococcal Nuclease (Worthington): Stored −80°C, 20U/μL in 10 mM Tris pH 7.4 Exonuclease III (NEB) MNase Digestion Buffer (See Recipe) MNase Stop Buffer (See Recipe) Proteinase K (20 mg/ml) 37°C incubator/water bath 65°C incubator Phenol:Chloroform:Isoamyl Alcohol (25:24:1) Ethanol (100%) 3M Sodium Acetate pH 5.2 Glycogen (20 mg/ml) NEB Buffer 2 RNase A (DNase-Free, 10 mg/ml) PCR Clean-up Kit (Qiagen) Alkaline Phosphatase (NEB) NEB Buffer 3 Low-melt Agarose (GeneMate) Gel Extraction Kit (Qiagen) TruSeq Sample Prep Kit (Illumina) [ or other desired library preparation kit ] MinElute PCR Purification Kit (Qiagen) Thermal Cycler for PCR Amplification ..

    Chromatin Immunoprecipitation:

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast
    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. .. ChIP was performed using 3 µl of anti-H3K14Ac polyclonal antibody (Upstate, 07–353).

    Immu-Puri:

    Article Title: Natural Single-Nucleosome Epi-Polymorphisms in Yeast
    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. .. ChIP was performed using 3 µl of anti-H3K14Ac polyclonal antibody (Upstate, 07–353).

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    • micrococcal nuclease mnase  (Worthington Biochemical)
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    Worthington Biochemical micrococcal nuclease mnase
    Micrococcal Nuclease Mnase, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    nucleosome repeat length  (Worthington Biochemical)
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    Worthington Biochemical nucleosome repeat length
    Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) <t>Nucleosome</t> repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.
    Nucleosome Repeat Length, 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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    hinfp cko mef nuclei  (Worthington Biochemical)
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    Worthington Biochemical hinfp cko mef nuclei
    Conditional ablation of transcription factor <t>HINFP</t> inactivates histone H4 expression. (A) Schematic diagram showing targeted Hinfp locus to generate conditional Hinfp knockout mice. The arrow indicates the recombined locus that generates a conditional Hinfp -null mutation (−, null). Ovals indicate right- and left-arm probes for Southern blotting. Arrowheads represent genotyping primers for PCR. (B) Autoradiographs of Southern blot analysis of mouse ES cell clones (wild type [+/+] and +/FN) and mouse tail DNA (+/FN, FN/FN, and +/+) that were hybridized to either left- or right-arm probes: 18.0-kb WT allele and either 10.5-kb (LA probe) or 9.6-kb (RA probe) targeted allele. (C) PCR genotyping analysis of DNA from MEFs of wild-type (+/+) and Hinfp -null pups (F/F) with or without infection with Ad5CMVCre-EGFP virus using primers a and b shown in panel A. Lane 1, marker; lane 2, WT MEFs; lane 3, F/F MEFs without Cre infection; and lane 4, Hinfp -null MEFs after Cre treatment. (D and E) RT-qPCR analysis of WT and <t>cKO</t> MEFs without (No Inf.) or with Cre infection (d0 to d2) showing expression of Hinfp mRNA for multiple exons (exons 2-3, 5, 6-7, 7-8) (D) and two histone H4 genes ( Hist2H4 [H4] and Hist1H4m [H4m]) (E). Removal of Hinfp causes a marked decrease in histone H4 gene expression. (F) Western blot analysis of WT and cKO MEFs at d2 and d4 shows reduction of total H4 protein in Hinfp -null cells (see also Fig. S2 in the supplemental material).
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    Conditional ablation of transcription factor <t>HINFP</t> inactivates histone H4 expression. (A) Schematic diagram showing targeted Hinfp locus to generate conditional Hinfp knockout mice. The arrow indicates the recombined locus that generates a conditional Hinfp -null mutation (−, null). Ovals indicate right- and left-arm probes for Southern blotting. Arrowheads represent genotyping primers for PCR. (B) Autoradiographs of Southern blot analysis of mouse ES cell clones (wild type [+/+] and +/FN) and mouse tail DNA (+/FN, FN/FN, and +/+) that were hybridized to either left- or right-arm probes: 18.0-kb WT allele and either 10.5-kb (LA probe) or 9.6-kb (RA probe) targeted allele. (C) PCR genotyping analysis of DNA from MEFs of wild-type (+/+) and Hinfp -null pups (F/F) with or without infection with Ad5CMVCre-EGFP virus using primers a and b shown in panel A. Lane 1, marker; lane 2, WT MEFs; lane 3, F/F MEFs without Cre infection; and lane 4, Hinfp -null MEFs after Cre treatment. (D and E) RT-qPCR analysis of WT and <t>cKO</t> MEFs without (No Inf.) or with Cre infection (d0 to d2) showing expression of Hinfp mRNA for multiple exons (exons 2-3, 5, 6-7, 7-8) (D) and two histone H4 genes ( Hist2H4 [H4] and Hist1H4m [H4m]) (E). Removal of Hinfp causes a marked decrease in histone H4 gene expression. (F) Western blot analysis of WT and cKO MEFs at d2 and d4 shows reduction of total H4 protein in Hinfp -null cells (see also Fig. S2 in the supplemental material).
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    Image Search Results


    Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.

    Journal: Molecular and Cellular Biology

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    doi: 10.1128/MCB.01567-13

    Figure Lengend Snippet: Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.

    Article Snippet: Nucleosome repeat length was assessed by digestion of WT and Hinfp cKO MEF nuclei (d4 after sorting) with micrococcal nuclease (MNase) (NFCP; Worthington Biochemical Corp).

    Techniques: Staining, Marker, Quantitation Assay, DNA Synthesis, Labeling, BrdU Incorporation Assay, Periodic Counter-current Chromatography, Nucleic Acid Electrophoresis, Software

    Conditional ablation of transcription factor HINFP inactivates histone H4 expression. (A) Schematic diagram showing targeted Hinfp locus to generate conditional Hinfp knockout mice. The arrow indicates the recombined locus that generates a conditional Hinfp -null mutation (−, null). Ovals indicate right- and left-arm probes for Southern blotting. Arrowheads represent genotyping primers for PCR. (B) Autoradiographs of Southern blot analysis of mouse ES cell clones (wild type [+/+] and +/FN) and mouse tail DNA (+/FN, FN/FN, and +/+) that were hybridized to either left- or right-arm probes: 18.0-kb WT allele and either 10.5-kb (LA probe) or 9.6-kb (RA probe) targeted allele. (C) PCR genotyping analysis of DNA from MEFs of wild-type (+/+) and Hinfp -null pups (F/F) with or without infection with Ad5CMVCre-EGFP virus using primers a and b shown in panel A. Lane 1, marker; lane 2, WT MEFs; lane 3, F/F MEFs without Cre infection; and lane 4, Hinfp -null MEFs after Cre treatment. (D and E) RT-qPCR analysis of WT and cKO MEFs without (No Inf.) or with Cre infection (d0 to d2) showing expression of Hinfp mRNA for multiple exons (exons 2-3, 5, 6-7, 7-8) (D) and two histone H4 genes ( Hist2H4 [H4] and Hist1H4m [H4m]) (E). Removal of Hinfp causes a marked decrease in histone H4 gene expression. (F) Western blot analysis of WT and cKO MEFs at d2 and d4 shows reduction of total H4 protein in Hinfp -null cells (see also Fig. S2 in the supplemental material).

    Journal: Molecular and Cellular Biology

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    doi: 10.1128/MCB.01567-13

    Figure Lengend Snippet: Conditional ablation of transcription factor HINFP inactivates histone H4 expression. (A) Schematic diagram showing targeted Hinfp locus to generate conditional Hinfp knockout mice. The arrow indicates the recombined locus that generates a conditional Hinfp -null mutation (−, null). Ovals indicate right- and left-arm probes for Southern blotting. Arrowheads represent genotyping primers for PCR. (B) Autoradiographs of Southern blot analysis of mouse ES cell clones (wild type [+/+] and +/FN) and mouse tail DNA (+/FN, FN/FN, and +/+) that were hybridized to either left- or right-arm probes: 18.0-kb WT allele and either 10.5-kb (LA probe) or 9.6-kb (RA probe) targeted allele. (C) PCR genotyping analysis of DNA from MEFs of wild-type (+/+) and Hinfp -null pups (F/F) with or without infection with Ad5CMVCre-EGFP virus using primers a and b shown in panel A. Lane 1, marker; lane 2, WT MEFs; lane 3, F/F MEFs without Cre infection; and lane 4, Hinfp -null MEFs after Cre treatment. (D and E) RT-qPCR analysis of WT and cKO MEFs without (No Inf.) or with Cre infection (d0 to d2) showing expression of Hinfp mRNA for multiple exons (exons 2-3, 5, 6-7, 7-8) (D) and two histone H4 genes ( Hist2H4 [H4] and Hist1H4m [H4m]) (E). Removal of Hinfp causes a marked decrease in histone H4 gene expression. (F) Western blot analysis of WT and cKO MEFs at d2 and d4 shows reduction of total H4 protein in Hinfp -null cells (see also Fig. S2 in the supplemental material).

    Article Snippet: Nucleosome repeat length was assessed by digestion of WT and Hinfp cKO MEF nuclei (d4 after sorting) with micrococcal nuclease (MNase) (NFCP; Worthington Biochemical Corp).

    Techniques: Expressing, Knock-Out, Mouse Assay, Mutagenesis, Southern Blot, Polymerase Chain Reaction, Clone Assay, Infection, Marker, Quantitative RT-PCR, Western Blot

    Loss of Hinfp causes deregulation of cell proliferation. WT and cKO MEFs (GFP sorted) were cultured for 4 days. (A) WT and cKO MEFs plated at 0.35 × 10 6 /60-mm dish were harvested at different time points (d0 to d4) to analyze proliferation in culture. cKO cells show severe delay in proliferation. (B) Cell cycle analysis by flow cytometry for DNA content shows increased sub-G 1 population, altered S phase, as well as polyploid cells in cKO MEFs. (C) Senescence-associated β-galactosidase (SA β-Gal) activity was measured at d2 and d4. There is an obvious increase in SA β-Gal-specific staining in cKO MEFs that is enhanced at d4. (D) The distribution of HLBs was determined by staining for NPAT (red). IF microscopy revealed an increase in the fraction of cells with multiple NPAT foci (white arrowheads) or diffused NPAT staining (red arrowheads) in cKO MEFs at d2 and d4. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (E) Quantitation of NPAT staining patterns in WT and cKO MEFs at d2 and d4. Bar graph shows the percentage of cells with specific numbers of NPAT foci. A total of 200 nuclei were counted from two biological replicates per sample for each time point.

    Journal: Molecular and Cellular Biology

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    doi: 10.1128/MCB.01567-13

    Figure Lengend Snippet: Loss of Hinfp causes deregulation of cell proliferation. WT and cKO MEFs (GFP sorted) were cultured for 4 days. (A) WT and cKO MEFs plated at 0.35 × 10 6 /60-mm dish were harvested at different time points (d0 to d4) to analyze proliferation in culture. cKO cells show severe delay in proliferation. (B) Cell cycle analysis by flow cytometry for DNA content shows increased sub-G 1 population, altered S phase, as well as polyploid cells in cKO MEFs. (C) Senescence-associated β-galactosidase (SA β-Gal) activity was measured at d2 and d4. There is an obvious increase in SA β-Gal-specific staining in cKO MEFs that is enhanced at d4. (D) The distribution of HLBs was determined by staining for NPAT (red). IF microscopy revealed an increase in the fraction of cells with multiple NPAT foci (white arrowheads) or diffused NPAT staining (red arrowheads) in cKO MEFs at d2 and d4. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (E) Quantitation of NPAT staining patterns in WT and cKO MEFs at d2 and d4. Bar graph shows the percentage of cells with specific numbers of NPAT foci. A total of 200 nuclei were counted from two biological replicates per sample for each time point.

    Article Snippet: Nucleosome repeat length was assessed by digestion of WT and Hinfp cKO MEF nuclei (d4 after sorting) with micrococcal nuclease (MNase) (NFCP; Worthington Biochemical Corp).

    Techniques: Cell Culture, Cell Cycle Assay, Flow Cytometry, Cytometry, Activity Assay, Staining, Microscopy, Quantitation Assay

    Hinfp is required for genomic stability and maintenance of DNA replication. WT and cKO MEFs were analyzed for factors associated with double-strand DNA damage by IF microscopy. (A) γ-H2AX S-139 (red); scale bar, 20 μm; (B) 53BP1 (red); scale bar, 20 μm. Nuclei were counterstained with DAPI (blue). cKO MEFs show a higher percentage of cells with both γ-H2AX and 53BP1 foci. (C) DNA fiber assay. WT and cKO MEFs were consecutively labeled with iododeoxyuridine (IdU; green) and chlorodeoxyuridine (CldU; red) at d2 and d4. Representative images of elongating and stalled forks are shown. Hinfp -null MEFs have a higher incidence of stalled replication forks than WT. (D) The bar graph represents percentage of stalled replication forks observed in WT and cKO MEFs at d2 and d4. There is increased frequency of stalled forks in Hinfp -null MEFs.

    Journal: Molecular and Cellular Biology

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    doi: 10.1128/MCB.01567-13

    Figure Lengend Snippet: Hinfp is required for genomic stability and maintenance of DNA replication. WT and cKO MEFs were analyzed for factors associated with double-strand DNA damage by IF microscopy. (A) γ-H2AX S-139 (red); scale bar, 20 μm; (B) 53BP1 (red); scale bar, 20 μm. Nuclei were counterstained with DAPI (blue). cKO MEFs show a higher percentage of cells with both γ-H2AX and 53BP1 foci. (C) DNA fiber assay. WT and cKO MEFs were consecutively labeled with iododeoxyuridine (IdU; green) and chlorodeoxyuridine (CldU; red) at d2 and d4. Representative images of elongating and stalled forks are shown. Hinfp -null MEFs have a higher incidence of stalled replication forks than WT. (D) The bar graph represents percentage of stalled replication forks observed in WT and cKO MEFs at d2 and d4. There is increased frequency of stalled forks in Hinfp -null MEFs.

    Article Snippet: Nucleosome repeat length was assessed by digestion of WT and Hinfp cKO MEF nuclei (d4 after sorting) with micrococcal nuclease (MNase) (NFCP; Worthington Biochemical Corp).

    Techniques: Microscopy, Labeling

    Hinfp ablation results in atypical nuclear and chromosomal morphology. WT and cKO MEFs (post-GFP sorting) were harvested at d1, d2, or d4 in culture. (A) IF microscopy shows obvious differences in nuclear size and shape between WT and cKO MEFs from d2 onwards. Nuclei stained with DAPI (gray) show clear increase in size after removal of Hinfp . Scale bar, 50 μm. (B) IF microscopy of MEFs stained with α-tubulin (red) show increased presence of binucleated cells (arrowheads) in cKO MEFs. The insets indicate percentage of binucleate cells. Scale bar, 50 μm. (C) Mitotic preparations of MEFs at d2 were subjected to DNA-FISH with probes against mouse minor satellite (top) or major satellite (bottom) regions. Hinfp -ablated cells show obvious increase in the chromosome complement. Scale bar, 20 μm. (D) Quantitation of mitotic cells from WT and cKO MEFs at d2 probed with satellite DNA was performed to calculate percent distribution of diploid (∼2n) and polyploid ( > 2n) chromosome complement. A total of 50 metaphases were counted per sample. The bar graph shows higher percentage of polyploidy in cKO MEFs than in WT MEFs. (E) The microtubules of asynchronous WT and cKO MEFs at d2 were stained with α-tubulin (red) and analyzed by IF microscopy. The micrograph shows the presence of mitotic cells with multipolar spindles in cKO MEFs.

    Journal: Molecular and Cellular Biology

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    doi: 10.1128/MCB.01567-13

    Figure Lengend Snippet: Hinfp ablation results in atypical nuclear and chromosomal morphology. WT and cKO MEFs (post-GFP sorting) were harvested at d1, d2, or d4 in culture. (A) IF microscopy shows obvious differences in nuclear size and shape between WT and cKO MEFs from d2 onwards. Nuclei stained with DAPI (gray) show clear increase in size after removal of Hinfp . Scale bar, 50 μm. (B) IF microscopy of MEFs stained with α-tubulin (red) show increased presence of binucleated cells (arrowheads) in cKO MEFs. The insets indicate percentage of binucleate cells. Scale bar, 50 μm. (C) Mitotic preparations of MEFs at d2 were subjected to DNA-FISH with probes against mouse minor satellite (top) or major satellite (bottom) regions. Hinfp -ablated cells show obvious increase in the chromosome complement. Scale bar, 20 μm. (D) Quantitation of mitotic cells from WT and cKO MEFs at d2 probed with satellite DNA was performed to calculate percent distribution of diploid (∼2n) and polyploid ( > 2n) chromosome complement. A total of 50 metaphases were counted per sample. The bar graph shows higher percentage of polyploidy in cKO MEFs than in WT MEFs. (E) The microtubules of asynchronous WT and cKO MEFs at d2 were stained with α-tubulin (red) and analyzed by IF microscopy. The micrograph shows the presence of mitotic cells with multipolar spindles in cKO MEFs.

    Article Snippet: Nucleosome repeat length was assessed by digestion of WT and Hinfp cKO MEF nuclei (d4 after sorting) with micrococcal nuclease (MNase) (NFCP; Worthington Biochemical Corp).

    Techniques: Microscopy, Staining, Fluorescence In Situ Hybridization, Quantitation Assay

    Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.

    Journal: Molecular and Cellular Biology

    Article Title: Fidelity of Histone Gene Regulation Is Obligatory for Genome Replication and Stability

    doi: 10.1128/MCB.01567-13

    Figure Lengend Snippet: Loss of Hinfp causes S-phase delay. (A) IF staining of Ki-67 (red) as the cell cycle marker was carried out on WT and cKO MEFs. The nuclei were counterstained with DAPI (blue). Scale bar, 50 μm. (B) Quantitation of different Ki-67 patterns revealed that Hinfp -null MEFs show a higher percentage of G 0 cells and persistence of cells with the S-phase pattern. (C) Active DNA synthesis was measured by pulse-labeling of WT and cKO MEFs with BrdU (red). cKO cells at d4 show a substantial decrease in BrdU incorporation. Scale bar, 50 μm. (D) Quantitation of percentage of S phase (BrdU-positive cells) shows a marked decrease in active DNA synthesis (5% in cKO versus 21% in WT) at d4. A total of 400 nuclei were counted from two biological replicates for panels B and D. (E) WT and cKO MEFs were subjected to drug-induced PCC assay. DNA was stained with DAPI (blue). A higher incidence of S-phase-specific (arrowheads) PCC pattern was observed in cKO MEFs. Insets indicate percentage of S-phase-specific PCC patterns. (F) Nucleosome repeat length (NRL) assay of MEF DNA at d4 shows differences in the nucleosome ladder by gel electrophoresis. The images were analyzed using ImageJ software. Line scan analysis revealed broadening of bands and increased nucleosomal spacing in cKO MEFs.

    Article Snippet: Nucleosome repeat length was assessed by digestion of WT and Hinfp cKO MEF nuclei (d4 after sorting) with micrococcal nuclease (MNase) (NFCP; Worthington Biochemical Corp).

    Techniques: Staining, Marker, Quantitation Assay, DNA Synthesis, Labeling, BrdU Incorporation Assay, Periodic Counter-current Chromatography, Nucleic Acid Electrophoresis, Software