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    Thermo Fisher dnase i
    Fig. 7. <t>DNase</t> I and hydroxyl radical footprinting of the R2 protein bound to the 49 END nucleosome. ( A ) Hydroxyl radical footprint. Top: gel mobility shift assay. Both wild-type (wt) R2 and an endonuclease mutant (Endo – ). After pre-incubating the R2 protein with its substrate, the complexes were treated with hydroxyl radical and resolved on 5% native PAGE gels. Bottom: eluted bands from the native gel were analyzed in 8% denaturing gels. The lane number corresponds to the eluted band from the native gel. D, naked DNA; G, DNA sequence G tract. The R2 cleavage site is labeled by an arrow. ( B ) The same sets of experiments as in (A), except the complex was digested with DNase I. Altered DNase I sensitivity of the DNA compared with the nucleosome well downstream of the cleavage site is indicated by circles to the left of the denaturing gel.
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 74631 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnaase i grade ii
    Fig. 7. <t>DNase</t> I and hydroxyl radical footprinting of the R2 protein bound to the 49 END nucleosome. ( A ) Hydroxyl radical footprint. Top: gel mobility shift assay. Both wild-type (wt) R2 and an endonuclease mutant (Endo – ). After pre-incubating the R2 protein with its substrate, the complexes were treated with hydroxyl radical and resolved on 5% native PAGE gels. Bottom: eluted bands from the native gel were analyzed in 8% denaturing gels. The lane number corresponds to the eluted band from the native gel. D, naked DNA; G, DNA sequence G tract. The R2 cleavage site is labeled by an arrow. ( B ) The same sets of experiments as in (A), except the complex was digested with DNase I. Altered DNase I sensitivity of the DNA compared with the nucleosome well downstream of the cleavage site is indicated by circles to the left of the denaturing gel.
    Dnaase I Grade Ii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Fig. 7. DNase I and hydroxyl radical footprinting of the R2 protein bound to the 49 END nucleosome. ( A ) Hydroxyl radical footprint. Top: gel mobility shift assay. Both wild-type (wt) R2 and an endonuclease mutant (Endo – ). After pre-incubating the R2 protein with its substrate, the complexes were treated with hydroxyl radical and resolved on 5% native PAGE gels. Bottom: eluted bands from the native gel were analyzed in 8% denaturing gels. The lane number corresponds to the eluted band from the native gel. D, naked DNA; G, DNA sequence G tract. The R2 cleavage site is labeled by an arrow. ( B ) The same sets of experiments as in (A), except the complex was digested with DNase I. Altered DNase I sensitivity of the DNA compared with the nucleosome well downstream of the cleavage site is indicated by circles to the left of the denaturing gel.

    Journal: The EMBO Journal

    Article Title: R2 retrotransposition on assembled nucleosomes depends on the translational position of the target site

    doi: 10.1093/emboj/cdf665

    Figure Lengend Snippet: Fig. 7. DNase I and hydroxyl radical footprinting of the R2 protein bound to the 49 END nucleosome. ( A ) Hydroxyl radical footprint. Top: gel mobility shift assay. Both wild-type (wt) R2 and an endonuclease mutant (Endo – ). After pre-incubating the R2 protein with its substrate, the complexes were treated with hydroxyl radical and resolved on 5% native PAGE gels. Bottom: eluted bands from the native gel were analyzed in 8% denaturing gels. The lane number corresponds to the eluted band from the native gel. D, naked DNA; G, DNA sequence G tract. The R2 cleavage site is labeled by an arrow. ( B ) The same sets of experiments as in (A), except the complex was digested with DNase I. Altered DNase I sensitivity of the DNA compared with the nucleosome well downstream of the cleavage site is indicated by circles to the left of the denaturing gel.

    Article Snippet: In the case of the DNase I footprinting, after incubation with the R2 protein, the nucleosomes were treated with 0.2 U of DNase I (Ambion) for 3 min.

    Techniques: Footprinting, Mobility Shift, Mutagenesis, Clear Native PAGE, Sequencing, Labeling

    Fig. 3. DNase I and hydroxyl radical mapping of reconstituted nucleosomes. ( A ) Mapping of 49 END DNA and nucleosome. Lane 1, naked DNA; lane 2, DNA digested by DNase I; lane 3, reconstituted nucleosome digested by DNase I; lane 4, naked DNA cleaved by hydroxyl radical; lane 5, nucleosome cleaved by hydroxyl radical; lane 6, DNA cleaved by R2 to reveal the insertion site. Procedures for DNase I and hydroxyl radical mapping are described in the Materials and methods. ( B ) Hydroxyl radical mapping of bottom strand-labeled 39 END, 49 END, 74 END, 79 END and 130 END nucleosomes. For each fragment, free DNA (D) and/or nucleosome (N) substrates were exposed to hydroxyl radical and the nicked products resolved by 8% PAGE alongside a DNA sequence G track (G) of the same fragment. Arrows indicate the site of R2 cleavage. ( C ) Schematic representation of the rotational phase of the DNA around the R2 target site while the DNA is assembled into nucleosomes. The height of each bar above or below each nucleotide indicates the accessibility of hydroxyl radical to that site. The cleavage sites of R2 and Bst 98I on both strands are indicated by arrows. Two positions of difference between the D.melanogaster and B.mori 28S rRNA genes are circled. In B.mori , the top strand contains a C at position +43 and an A at position +32.

    Journal: The EMBO Journal

    Article Title: R2 retrotransposition on assembled nucleosomes depends on the translational position of the target site

    doi: 10.1093/emboj/cdf665

    Figure Lengend Snippet: Fig. 3. DNase I and hydroxyl radical mapping of reconstituted nucleosomes. ( A ) Mapping of 49 END DNA and nucleosome. Lane 1, naked DNA; lane 2, DNA digested by DNase I; lane 3, reconstituted nucleosome digested by DNase I; lane 4, naked DNA cleaved by hydroxyl radical; lane 5, nucleosome cleaved by hydroxyl radical; lane 6, DNA cleaved by R2 to reveal the insertion site. Procedures for DNase I and hydroxyl radical mapping are described in the Materials and methods. ( B ) Hydroxyl radical mapping of bottom strand-labeled 39 END, 49 END, 74 END, 79 END and 130 END nucleosomes. For each fragment, free DNA (D) and/or nucleosome (N) substrates were exposed to hydroxyl radical and the nicked products resolved by 8% PAGE alongside a DNA sequence G track (G) of the same fragment. Arrows indicate the site of R2 cleavage. ( C ) Schematic representation of the rotational phase of the DNA around the R2 target site while the DNA is assembled into nucleosomes. The height of each bar above or below each nucleotide indicates the accessibility of hydroxyl radical to that site. The cleavage sites of R2 and Bst 98I on both strands are indicated by arrows. Two positions of difference between the D.melanogaster and B.mori 28S rRNA genes are circled. In B.mori , the top strand contains a C at position +43 and an A at position +32.

    Article Snippet: In the case of the DNase I footprinting, after incubation with the R2 protein, the nucleosomes were treated with 0.2 U of DNase I (Ambion) for 3 min.

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis, Sequencing

    Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*,  p

    Journal: PLoS Pathogens

    Article Title: Mono-ubiquitylated ORF45 Mediates Association of KSHV Particles with Internal Lipid Rafts for Viral Assembly and Egress

    doi: 10.1371/journal.ppat.1005332

    Figure Lengend Snippet: Effect of the K297R mutation on progeny virus release. iSLK-BAC16 and iSLK-BAC-K297R cells were induced with Dox and butyrate for indicated time. The extracellular virions were collected from culture media and treated with Turbo DNase I. Viral DNAs were extracted and KSHV genomic DNA copy numbers were estimated by qPCR along with external standards of known concentrations of the viral DNA with primers against the ORF73 gene (A). Intracellular KSHV genomic DNAs were extracted from harvested cells and quantitated by qPCR as above (B). (*, p

    Article Snippet: For the preparation of DNA from intact virions, 200 μl of virus stocks was pretreated with 2 μl of Turbo DNase I (Ambion) for 1 h at 37°C.

    Techniques: Mutagenesis, BAC Assay, Real-time Polymerase Chain Reaction

    DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: DNase I cleavage patterns and footprint distribution for overrepresented footprints surrounding TSSs. ( A ) Mean per-nucleotide DNase I cleavage profile from aligning the annotated TSSs of 5050 genes (+/− 1 kb regions). ( B ) Top heat map plotted for DNase I cleavage patterns of 5050 genes at +/− 1 kb TSS flanking regions by K-means clustering, which were subsequently divided into four distinct clusters, marked with red, blue, green and purple bars. The bottom mean DNase I cleavage patterns derived from four distinct clusters, where the line colors correspond to the marked colors of the heatmap. ( C ) Distribution of digital footprints (FDR

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: Diversity of DNase I cleavage patterns and function annotation of target genes for the overrepresented motifs in genomic footprints. ( A ) DNase I cleavage density per nucleotide calculated for footprint instances from two culture conditions. Shaded regions delineate the overrepresented motifs derived from the footprint region. The MEME logo of overrepresented motifs derived from footprints is shown below the graph. ( B ) GO function enrichment for the target genes under the DPY_motif 3 and DPY_motif 7. The genes containing at least one motif instance inside the 1-kb region of the annotated TSSs were selected. The genes under the same motif were analyzed using ClueGo. Functional group networks are represented by nodes linked with each other based on their kappa score level ( > 0.3). The node size represents the percentage of associated genes with the enrichment significance of the term (Term P -value

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay, Functional Assay

    The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Journal: Nucleic Acids Research

    Article Title: Survey of protein–DNA interactions in Aspergillus oryzae on a genomic scale

    doi: 10.1093/nar/gkv334

    Figure Lengend Snippet: The DNase I cleavage patterns of five family types of TFs parallel the co-crystal structures of protein and DNA interaction. ( A ) Strand-specific DNase-seq signal for DNase I cleavage imbalance between the plus and minus motif sequences of five family types of the TFs independent of strand orientation. The upper panels show the heat maps of per-nucleotide DNase I cleavage derived from all instances of plus (red) and minus (blue) TFBS motifs within DHSs under DPY conditions ranked according to the probability of MILLIPEDE (FIMO P

    Article Snippet: RT-qPCR reactions, including 10 μl of reaction mixture with 50 ng DNase I digestion product, 2 × 0.4 μl primers (forward and reverse, 10 μM), 0.2 μl ROX reference dye II (50×), 5 μl SYBR Premix Ex Taq II (2×) and dH2 O, were amplified using an Applied Biosystems 7500 Real-time PCR System for 1 min at 95°C, followed by 40 cycles of 95°C for 5 s, 55°C for 20 s and 72°C for 34 s. The degree of DNase I digestion was determined based on changes in Ct values.

    Techniques: Derivative Assay

    DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I treatment is effective against C. jejuni biofilms on stainless steel surfaces and in the presence of organic materials in aerobic conditions . The ability of DNase I to inhibit biofilm formation of C. jejuni NCTC 11168 on sterile, stainless steel coupons (A) or in the presence of chicken juice, mimicking a conditioned surface (B) . TTC staining was used to measure biofilm formation in the presence of chicken juice (B) . DNase I is able to significantly decrease biofilm formation in both conditions. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Staining, Standard Deviation, MANN-WHITNEY

    DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: DNase I is able to rapidly degrade C. jejuni NCTC 11168 biofilms . (A) DNase I (4 units/ml) was added at defined intervals to aerobically incubated NCTC 11168 cultures over a 48 h static incubation and biofilm degradation assessed by crystal violet staining. (B) Following a 48 h static incubation to allow biofilm formation, DNase I was added to biofilms for between 5 and 120 min before biofilm degradation was assessed. (C) The concentration of DNase I required for biofilm control was also assessed using DNase I concentrations of between 0.01 and 5 U/ml. In each graph, “11168” represents an untreated biofilm culture of C. jejuni NCTC 11168 and “control” represents a tube containing sterile Brucella medium only. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Incubation, Staining, Concentration Assay, Standard Deviation, MANN-WHITNEY

    Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Treatment of pre-existing biofilms with DNase I leads to inhibition of biofilm regrowth . C. jejuni NCTC 11168 biofilms were allowed to form for 48 h in sterile borosilicate glass test tubes. To assess biofilm re-growth following DNase I treatment, two sets of tubes were treated with 4 U/ml DNase I for 15 min then washed with sterile PBS. Tubes were then supplemented with either fresh Brucella media (fifth bar) or fresh C. jejuni NCTC 11168 culture (sixth bar) and incubated for a further 48 h. The following controls were also prepared: C. jejuni NCTC 11168 biofilm formation following primary culture (first bar, white), tubes supplemented with sterile Brucella media (second bar, black), C. jejuni NCTC 11168 biofilm formation following only secondary culture (third bar, light gray), and 48 h-old C. jejuni NCTC 11168 biofilm, washed with PBS, then supplemented with fresh C. jejuni NCTC 11168 culture (fourth bar, dark gray). Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Inhibition, Incubation, Standard Deviation, MANN-WHITNEY

    Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Journal: Frontiers in Microbiology

    Article Title: Campylobacter jejuni biofilms contain extracellular DNA and are sensitive to DNase I treatment

    doi: 10.3389/fmicb.2015.00699

    Figure Lengend Snippet: Restriction endonuclease treatment of C. jejuni biofilms reduces biofilm formation . Static cultures of C. jejuni NCTC 11168 (A,B) and 81116 (C,D) were prepared then supplemented with either DNase I, RNase, or a single restriction endonuclease. Cultures were incubated for 48 h at 37°C in aerobic conditions. A range of restriction enzymes was selected, based on varying levels of DNA fragmentation following digestion of C. jejuni NCTC 11168 (B) and 81116 (D) genomic DNA. Restriction enzyme and DNase I treatment of NCTC 11168 biofilms lead to a reduction in biofilm formation. The same trend was observed for C. jejuni 81116, although only DNase I and Hae III digestion were significantly different from the control. Error bars show standard deviation. Statistically significant results, as determined using the Mann–Whitney U test, are indicated using an asterisk ( * P

    Article Snippet: Enzyme treatment of C. jejuni biofilms For DNase I treatments, unless otherwise stated, a volume of 4 μl DNase I enzyme (Fermentas), giving a final concentration within the biofilm of 4 U/ml v/v and 4 μl of DNase I buffer (Fermentas) were added to each test tube, along with 1 ml of diluted cell suspension at either the start of the static incubation or after 12, 24, 36, or 48 h of static incubation.

    Techniques: Incubation, Standard Deviation, MANN-WHITNEY

    Genome viewer snapshot shows domains of open and closed chromatin detected by GCSDI. An approximately 250 kbp window demonstrates the relationship of differential GCSDI signal, 2CM, and 3CM models to the genes, predicted 9 chromatin states, and lamina-associated domains (LADs). DNase I hypersensitive site assay signal (DHS) is shown for comparison. Open chromatin domains are shown in green and closed – in red in 2CM and 3CM traces. Discrepancies between chromatin structure predictions (repressed) and chromatin compactness (open) are outlined by arrows.

    Journal: BMC Genomics

    Article Title: Map of open and closed chromatin domains in Drosophila genome

    doi: 10.1186/1471-2164-15-988

    Figure Lengend Snippet: Genome viewer snapshot shows domains of open and closed chromatin detected by GCSDI. An approximately 250 kbp window demonstrates the relationship of differential GCSDI signal, 2CM, and 3CM models to the genes, predicted 9 chromatin states, and lamina-associated domains (LADs). DNase I hypersensitive site assay signal (DHS) is shown for comparison. Open chromatin domains are shown in green and closed – in red in 2CM and 3CM traces. Discrepancies between chromatin structure predictions (repressed) and chromatin compactness (open) are outlined by arrows.

    Article Snippet: To generate a GCSDI profile across genome, amplified DNA samples from DNase I-treated and control untreated cells (n = 2) were hybridized with tiling Affymetrix microarrays, signal intensities for each probe were averaged within the experimental groups and fold differences between the groups were calculated.

    Techniques:

    RNase Y allows differential expression between genes co-expressed in the saePQRS operon. ( A ) Schematic representation of the saePQRS operon, with its primary and mature RNA molecules (T1–T4), promoters (P1 and P3), terminator (Term), cleavage site (CS) and putative stem loops. ( B ) Schematic representation of sae-gfp constructs carrying different deletions (the deleted sequence is indicated in the panel with a cross). The RNAs observed in the northern blot analyses are indicated with their names and lengths below each constructs. ( C ) Northern blot analyses to examine sae processing in strains carrying the sae-gfp constructs. Newman saeP mutant and saeP rny double mutant strains carrying different constructs were grown to exponential phase. RNA was then harvested and hybridized with DIG-labeled DNA probes specific for gfp, saeP and rny . As a loading control, 16S rRNA detected in the ethidium bromide-stained gel was used, which is shown at the bottom of the panel. For clarity, lane numbers are indicated in the panel. ( D ) RT-qPCR to assess the ratio between saeR and saeP copy numbers. Newman wild-type, rny mutant and complemented strains were grown (in triplicate) to late exponential phase and RNA was extracted. After DNase I treatment, one-step RT-qPCR was performed. saeR and saeP copy numbers were calculated by reference to a standard curve. Statistically significant differences between the samples are indicated: ** P = 0.001 to 0.01; *** P

    Journal: Nucleic Acids Research

    Article Title: Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus

    doi: 10.1093/nar/gkx296

    Figure Lengend Snippet: RNase Y allows differential expression between genes co-expressed in the saePQRS operon. ( A ) Schematic representation of the saePQRS operon, with its primary and mature RNA molecules (T1–T4), promoters (P1 and P3), terminator (Term), cleavage site (CS) and putative stem loops. ( B ) Schematic representation of sae-gfp constructs carrying different deletions (the deleted sequence is indicated in the panel with a cross). The RNAs observed in the northern blot analyses are indicated with their names and lengths below each constructs. ( C ) Northern blot analyses to examine sae processing in strains carrying the sae-gfp constructs. Newman saeP mutant and saeP rny double mutant strains carrying different constructs were grown to exponential phase. RNA was then harvested and hybridized with DIG-labeled DNA probes specific for gfp, saeP and rny . As a loading control, 16S rRNA detected in the ethidium bromide-stained gel was used, which is shown at the bottom of the panel. For clarity, lane numbers are indicated in the panel. ( D ) RT-qPCR to assess the ratio between saeR and saeP copy numbers. Newman wild-type, rny mutant and complemented strains were grown (in triplicate) to late exponential phase and RNA was extracted. After DNase I treatment, one-step RT-qPCR was performed. saeR and saeP copy numbers were calculated by reference to a standard curve. Statistically significant differences between the samples are indicated: ** P = 0.001 to 0.01; *** P

    Article Snippet: After DNase I treatment, RNA was recovered using the MEGAclear Kit (Invitrogen), and RNA quantification was performed spectrophotometrically.

    Techniques: Expressing, Construct, Sequencing, Northern Blot, Mutagenesis, Labeling, Staining, Quantitative RT-PCR

    Transfection of total RNA harvested from poly(dA–dT) transfected cells results in IFN- β expression. 293T cells were transfected with 12 μg total RNA extracted from 293T cells that had been transfected with 12 μg poly(dA–dT) for 24 h. Induction of IFN- β mRNA was confirmed to be specific for an immunostimulatory RNA species by treating total RNA with either (a) 0.1 mg RNase A ml −1 or (b) 0.1 U amplification grade DNase I μl −1 . Total RNA was then extracted 24 h post-transfection and real-time qPCR performed for IFN- β . These data were normalized against GAPDH mRNA levels. Error bars indicate the mean± sem (* P

    Journal: The Journal of General Virology

    Article Title: Inhibition of the RNA polymerase III-mediated dsDNA-sensing pathway of innate immunity by vaccinia virus protein E3

    doi: 10.1099/vir.0.021998-0

    Figure Lengend Snippet: Transfection of total RNA harvested from poly(dA–dT) transfected cells results in IFN- β expression. 293T cells were transfected with 12 μg total RNA extracted from 293T cells that had been transfected with 12 μg poly(dA–dT) for 24 h. Induction of IFN- β mRNA was confirmed to be specific for an immunostimulatory RNA species by treating total RNA with either (a) 0.1 mg RNase A ml −1 or (b) 0.1 U amplification grade DNase I μl −1 . Total RNA was then extracted 24 h post-transfection and real-time qPCR performed for IFN- β . These data were normalized against GAPDH mRNA levels. Error bars indicate the mean± sem (* P

    Article Snippet: Nucleic acids were treated either with RNase A using the conditions described previously ( ) or with amplification grade DNase I following the manufacturer's instructions (Invitrogen).

    Techniques: Transfection, Expressing, Amplification, Real-time Polymerase Chain Reaction

    Northern analysis and RNase H and DNase I sensitivity of R-loops within mtDNA coding regions. Mitochondrial RNA remains bound to CsCl-purified mtDNA. Samples were prepared as described in the legend for Fig. 2 and treated with nucleases as indicated by (+) and (-). pA is poly(A)+-purified RNA. Gene-specific riboprobes were generated from PCR products as described under “Experimental Procedures.”

    Journal: The Journal of Biological Chemistry

    Article Title: Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome *Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome * S⃞

    doi: 10.1074/jbc.M806174200

    Figure Lengend Snippet: Northern analysis and RNase H and DNase I sensitivity of R-loops within mtDNA coding regions. Mitochondrial RNA remains bound to CsCl-purified mtDNA. Samples were prepared as described in the legend for Fig. 2 and treated with nucleases as indicated by (+) and (-). pA is poly(A)+-purified RNA. Gene-specific riboprobes were generated from PCR products as described under “Experimental Procedures.”

    Article Snippet: 500 ng of LA9 mtDNA-mtRNA were treated with 1.4 units of RNase-free DNase I (Ambion) or RNase H (Stratagene), denatured with glyoxal/DMSO, and separated on a 1% agarose gel using the NorthernMax-Gly kit (Ambion).

    Techniques: Northern Blot, Purification, Generated, Polymerase Chain Reaction

    Northern analysis and DNase and RNase sensitivity of mtDNA-bound nascent H-strand RNA and DNA at O H . EtBr-CsCl-purified closed circular mtDNA was analyzed by Northern analysis to detect stable R-loops. RNA size markers are in lane 1 . Poly(A)+-purified RNA is in the lane denoted by pA +. DNase I and RNase H sample treatments are indicated above the panels. A , probing for CSB-proximal RNA with T3#4 riboprobe (shown in D ). B , less exposed film of view shown in A , revealing the increased intensity of ∼150-nt species in lane 3 after DNase treatment. C , probing for CSB-distal RNA with T3#1 riboprobe as shown in D. D , reference diagram showing the major noncoding region of mtDNA. This region is identified in Fig. 1 as the area encompassing O H . The transcription start site is shown with a bent arrow followed by several relevant DNA sequence features, including CSBs III, II, and I. The termination-associated sequences ( TAS ) region is shown at the promoter distal end of the DNA. T3#4 and T#31 riboprobe positions are shown above. Below the DNA map are nucleic acids identified in the Northern blots shown in A-C . RNA is shown by thick black lines , and RNA primers in transition with DNA are shown in gray . DNA alone is in shown by thin black lines . The lines are to scale, with size interruptions shown by breaks .

    Journal: The Journal of Biological Chemistry

    Article Title: Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome *Native R-loops Persist throughout the Mouse Mitochondrial DNA Genome * S⃞

    doi: 10.1074/jbc.M806174200

    Figure Lengend Snippet: Northern analysis and DNase and RNase sensitivity of mtDNA-bound nascent H-strand RNA and DNA at O H . EtBr-CsCl-purified closed circular mtDNA was analyzed by Northern analysis to detect stable R-loops. RNA size markers are in lane 1 . Poly(A)+-purified RNA is in the lane denoted by pA +. DNase I and RNase H sample treatments are indicated above the panels. A , probing for CSB-proximal RNA with T3#4 riboprobe (shown in D ). B , less exposed film of view shown in A , revealing the increased intensity of ∼150-nt species in lane 3 after DNase treatment. C , probing for CSB-distal RNA with T3#1 riboprobe as shown in D. D , reference diagram showing the major noncoding region of mtDNA. This region is identified in Fig. 1 as the area encompassing O H . The transcription start site is shown with a bent arrow followed by several relevant DNA sequence features, including CSBs III, II, and I. The termination-associated sequences ( TAS ) region is shown at the promoter distal end of the DNA. T3#4 and T#31 riboprobe positions are shown above. Below the DNA map are nucleic acids identified in the Northern blots shown in A-C . RNA is shown by thick black lines , and RNA primers in transition with DNA are shown in gray . DNA alone is in shown by thin black lines . The lines are to scale, with size interruptions shown by breaks .

    Article Snippet: 500 ng of LA9 mtDNA-mtRNA were treated with 1.4 units of RNase-free DNase I (Ambion) or RNase H (Stratagene), denatured with glyoxal/DMSO, and separated on a 1% agarose gel using the NorthernMax-Gly kit (Ambion).

    Techniques: Northern Blot, Purification, Sequencing

    Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Journal: BMC Evolutionary Biology

    Article Title: Opossum carboxylesterases: sequences, phylogeny and evidence for CES gene duplication events predating the marsupial-eutherian common ancestor

    doi: 10.1186/1471-2148-8-54

    Figure Lengend Snippet: Expression of CES genes in opossum. Liver and intestinal cDNAs were reverse transcribed from DNase I-treated RNA, and they were used as templates in RT-PCR to analyze CES gene expression. Lanes 2 and 3 are RT-PCR products amplified from liver (L) and intestine (I) cDNAs for the CES1 gene; lanes 4 and 5, RT-PCR products from liver (L) and intestine (I) cDNAs for the CES2.1 gene; lanes 6 and 7, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.2 gene; and lanes 8 and 9, RT-PCR products from liver (L) and intestine (I) for cDNAs for the CES2.3 gene. M shows the DNA size ladder.

    Article Snippet: DNase I-treated RNA was reverse transcribed into cDNA using a High Capacity cDNA Reverse Transcription kit (Applied Biosystems).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Amplification