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  • 99
    New England Biolabs dnase i
    AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap <t>DNase</t> I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value
    Dnase I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8772 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dnase i enzyme
    Absolute quantification of phlD and hcnC gene transcripts from liquid bacterial culture of Pseudomonas sp. LBUM300. Bars are standard error of the mean. X-axis indicates the no. of rounds of <t>DNase</t> I treatment performed on the RNA sample. Values followed by a different letter are significantly different using Tukey-Kramer test (P ≤ 0.05).
    Dnase I Enzyme, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 487 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnaase i
    CLEC5A is critical for restriction of bacterial spreading in vivo. a , b Wild type ( WT ), <t>WT/DNase-I</t> (4 KU per mouse) and mutant mice were injected with the live luminescent L. monocytogenes ( Xen32 ) (3 × 10 8 CFUs per mouse) intravenously, and luminescence intensity in liver was determined at 1 h, 2 h, and 4 h post- infection (10 mice per group) by IVIS system (Xenogen). Bacterial loads in liver c and blood d were determined 4 h post-infection. The data were collected and expressed as mean ± s.e.m. from four independent experiments ( n = 20). One-way ANOVA test performed. * P
    Dnaase I, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 235 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher dnase i
    ssRNAs from CVB3-Infected Cells Do Not Activate MDA5 (A) ssRNA fraction from mock- or CVB3-infected HeLa cells were transfected into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU <t>DNase</t> I at 37°C for 15 min. Resulting RNA samples equivalent to 500 ng starting material were transfected into approximately 200,000 MAVS +/+ MEFs and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. Data presented as mean ± SD. (C) The same samples as in (B) were analyzed on agarose gel. (D) CVB3 vRNA was isolated from pelleted viral particles. Indicated amounts of vRNA were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml), and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.. Data presented as mean ± SD. (E) Indicated amounts of mock-treated or unlinkase-treated poliovirus vRNAs were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). Total RNA was extracted at 8 hr.p.t. and IFN-b mRNA induction was determined by real-time qPCR. Data presented as mean ± SD. See also Figure S3 .
    Dnase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 74048 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dnaase i type ii
    ssRNAs from CVB3-Infected Cells Do Not Activate MDA5 (A) ssRNA fraction from mock- or CVB3-infected HeLa cells were transfected into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU <t>DNase</t> I at 37°C for 15 min. Resulting RNA samples equivalent to 500 ng starting material were transfected into approximately 200,000 MAVS +/+ MEFs and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. Data presented as mean ± SD. (C) The same samples as in (B) were analyzed on agarose gel. (D) CVB3 vRNA was isolated from pelleted viral particles. Indicated amounts of vRNA were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml), and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.. Data presented as mean ± SD. (E) Indicated amounts of mock-treated or unlinkase-treated poliovirus vRNAs were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). Total RNA was extracted at 8 hr.p.t. and IFN-b mRNA induction was determined by real-time qPCR. Data presented as mean ± SD. See also Figure S3 .
    Dnaase I Type Ii, supplied by Millipore, 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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    Thermo Fisher dnaase i grade ii
    ssRNAs from CVB3-Infected Cells Do Not Activate MDA5 (A) ssRNA fraction from mock- or CVB3-infected HeLa cells were transfected into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU <t>DNase</t> I at 37°C for 15 min. Resulting RNA samples equivalent to 500 ng starting material were transfected into approximately 200,000 MAVS +/+ MEFs and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. Data presented as mean ± SD. (C) The same samples as in (B) were analyzed on agarose gel. (D) CVB3 vRNA was isolated from pelleted viral particles. Indicated amounts of vRNA were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml), and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.. Data presented as mean ± SD. (E) Indicated amounts of mock-treated or unlinkase-treated poliovirus vRNAs were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). Total RNA was extracted at 8 hr.p.t. and IFN-b mRNA induction was determined by real-time qPCR. Data presented as mean ± SD. See also Figure S3 .
    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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    92
    FUJIFILM dnase i
    ssRNAs from CVB3-Infected Cells Do Not Activate MDA5 (A) ssRNA fraction from mock- or CVB3-infected HeLa cells were transfected into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU <t>DNase</t> I at 37°C for 15 min. Resulting RNA samples equivalent to 500 ng starting material were transfected into approximately 200,000 MAVS +/+ MEFs and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. Data presented as mean ± SD. (C) The same samples as in (B) were analyzed on agarose gel. (D) CVB3 vRNA was isolated from pelleted viral particles. Indicated amounts of vRNA were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml), and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.. Data presented as mean ± SD. (E) Indicated amounts of mock-treated or unlinkase-treated poliovirus vRNAs were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). Total RNA was extracted at 8 hr.p.t. and IFN-b mRNA induction was determined by real-time qPCR. Data presented as mean ± SD. See also Figure S3 .
    Dnase I, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 130 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Roche dnase i
    Impact of <t>DNase</t> I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p
    Dnase I, supplied by Roche, used in various techniques. Bioz Stars score: 94/100, based on 33815 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Boehringer Mannheim dnase i
    Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark <t>DNase</t> I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.
    Dnase I, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 93/100, based on 2033 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Applichem dnase i
    Extracellular DNA is a component of the V. cholerae biofilm matrix. A. Analysis of nuclease sensitivity of wild type and Δ dns Δ xds mutant biofilms. Biofilms grown for 21 h under static conditions were treated 3 h with <t>DNase</t> I (diagonally shaded bars), λExonuclease (dotted bars) or with a combination of DNase I and λExonuclease (horizontally shaded bars). Biofilm mass remaining after nuclease treatment was quantified by crystal violet staining. Significant differences compared with untreated biofilms of the wild type or Δ dns Δ xds mutant are indicated (* P
    Dnase I, supplied by Applichem, used in various techniques. Bioz Stars score: 94/100, based on 727 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega dnase i
    Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to <t>DNase</t> I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.
    Dnase I, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 14080 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    TaKaRa dnase i
    <t>DNase</t> I footprinting of LigR binding to the ligK and ligJ promoter regions. The 238-bp fragment or the 251-bp fragment containing the ligK (A) or ligJ (B) promoter region, respectively, was labeled with DIG on the coding (left) or noncoding (right) strands. The presence (+) or absence (−) of purified LigR (100 nM), PCA, and GA in the reaction mixture is indicated. Inducers were added at a concentration of 1 mM in each reaction. Thicker bars indicate strong protection by LigR, and thinner bars show weakly protected regions. The DNase I-hypersensitive sites are shown with arrowheads. The colors of bars and arrowheads indicate the reactions performed in the absence (black) or presence (gray) of PCA or GA, respectively.
    Dnase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 92/100, based on 11356 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    US Biological Life Sciences dnaase i
    <t>DNase</t> I footprinting of LigR binding to the ligK and ligJ promoter regions. The 238-bp fragment or the 251-bp fragment containing the ligK (A) or ligJ (B) promoter region, respectively, was labeled with DIG on the coding (left) or noncoding (right) strands. The presence (+) or absence (−) of purified LigR (100 nM), PCA, and GA in the reaction mixture is indicated. Inducers were added at a concentration of 1 mM in each reaction. Thicker bars indicate strong protection by LigR, and thinner bars show weakly protected regions. The DNase I-hypersensitive sites are shown with arrowheads. The colors of bars and arrowheads indicate the reactions performed in the absence (black) or presence (gray) of PCA or GA, respectively.
    Dnaase I, supplied by US Biological Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap DNase I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value

    Journal: bioRxiv

    Article Title: Histone chaperone Nucleophosmin regulates transcription of key genes involved in oral tumorigenesis

    doi: 10.1101/852095

    Figure Lengend Snippet: AcNPM1 co-occupies with RNA Pol II, chromatin remodeling factors and transcription factors at transcriptional regulatory elements. (A) Plot showing the percent number of AcNPM1 peaks overlapped with ChromHMM + Segway combined segmentation for HeLa S3 genome from the UCSC genome browser. (Key: TSS, predicted promoter region including TSS; PF, predicted promoter flanking region; E, enhancer; WE, predicted weak enhancer or open chromatin cis-regulatory element; CTCF, CTCF enriched element; T, predicted transcribed region; R, predicted repressed or low activity region; None, unclassified). (B) Percent number of TSS and enhancer regions identified by ChromHMM + Segway combined segmentation for HeLa S3, overlapped with AcNPM1 peaks. (C) UCSC genome browser snapshot showing AcNPM1 enrichment at TSS and enhancer regions defined by ChromHMM + Segway combined segmentation for HeLa S3 genome. (Key: TSS, predicted promoter region including TSS; E, enhancer). (D) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap DNase I hypersensitive sites (DHSs). (E) Boxplots showing AcNPM1 read density on AcNPM1 peaks with high or low enrichment of H3K27ac. (F-G) Boxplots showing AcNPM1 read density on AcNPM1 peaks that overlap or do not overlap (F) p300 and (G) RNA Pol II (Pol2). (H) Transcription factor binding motifs enriched in AcNPM1 peaks and broadly grouped by transcription factor family. P -value

    Article Snippet: RNA was treated with DNase I (NEB, Ipswich, MA, USA) according to the manufacturer’s instructions followed by re-precipitation.

    Techniques: Activity Assay, Binding Assay

    Absolute quantification of phlD and hcnC gene transcripts from liquid bacterial culture of Pseudomonas sp. LBUM300. Bars are standard error of the mean. X-axis indicates the no. of rounds of DNase I treatment performed on the RNA sample. Values followed by a different letter are significantly different using Tukey-Kramer test (P ≤ 0.05).

    Journal: BMC Biotechnology

    Article Title: Development of a versatile TaqMan(TM) real-time quantitative PCR (RT-qPCR) compliant anchor sequence to quantify bacterial gene transcripts from RNA samples containing carryover genomic DNA

    doi: 10.1186/1472-6750-13-7

    Figure Lengend Snippet: Absolute quantification of phlD and hcnC gene transcripts from liquid bacterial culture of Pseudomonas sp. LBUM300. Bars are standard error of the mean. X-axis indicates the no. of rounds of DNase I treatment performed on the RNA sample. Values followed by a different letter are significantly different using Tukey-Kramer test (P ≤ 0.05).

    Article Snippet: To rule out RNA hydrolysis due to heat/cation [ ] or organic based procedure (phenol/proteinase K), two of the commonly used procedures to inactivate the DNase I enzyme, we in our study made use of a commonly used proprietary, non-thermal/cationic inactivation system ( http://tools.invitrogen.com/content/sfs/manuals/cms_055740.pdf ).

    Techniques:

    PCR amplification of  hcnC  and  phlD  genes.  Agarose gel electrophoresis of  hcnC  and  phlD  genes, PCR amplified from RNA (non-DNase I treated) extracted from liquid culture of  Pseudomonas  sp. LBUM300. Primer system: phlD-F/MYT4 (Lane 1-2); phlD-F/phlD-R: (Lane 3-4); hcnC-F/MYT4 (Lane 5-6) and hcnC-F/R: Lane (7-8). Template: Non-reverse Transcribed RNA (Lane 1, 3, 5, 7); cDNA (Lane 2, 4, 6, 8). M: Quick-Load DNA marker, Broad Range (New England Biolabs, Mississauga, ON).

    Journal: BMC Biotechnology

    Article Title: Development of a versatile TaqMan(TM) real-time quantitative PCR (RT-qPCR) compliant anchor sequence to quantify bacterial gene transcripts from RNA samples containing carryover genomic DNA

    doi: 10.1186/1472-6750-13-7

    Figure Lengend Snippet: PCR amplification of hcnC and phlD genes. Agarose gel electrophoresis of hcnC and phlD genes, PCR amplified from RNA (non-DNase I treated) extracted from liquid culture of Pseudomonas sp. LBUM300. Primer system: phlD-F/MYT4 (Lane 1-2); phlD-F/phlD-R: (Lane 3-4); hcnC-F/MYT4 (Lane 5-6) and hcnC-F/R: Lane (7-8). Template: Non-reverse Transcribed RNA (Lane 1, 3, 5, 7); cDNA (Lane 2, 4, 6, 8). M: Quick-Load DNA marker, Broad Range (New England Biolabs, Mississauga, ON).

    Article Snippet: To rule out RNA hydrolysis due to heat/cation [ ] or organic based procedure (phenol/proteinase K), two of the commonly used procedures to inactivate the DNase I enzyme, we in our study made use of a commonly used proprietary, non-thermal/cationic inactivation system ( http://tools.invitrogen.com/content/sfs/manuals/cms_055740.pdf ).

    Techniques: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Marker

    CLEC5A is critical for restriction of bacterial spreading in vivo. a , b Wild type ( WT ), WT/DNase-I (4 KU per mouse) and mutant mice were injected with the live luminescent L. monocytogenes ( Xen32 ) (3 × 10 8 CFUs per mouse) intravenously, and luminescence intensity in liver was determined at 1 h, 2 h, and 4 h post- infection (10 mice per group) by IVIS system (Xenogen). Bacterial loads in liver c and blood d were determined 4 h post-infection. The data were collected and expressed as mean ± s.e.m. from four independent experiments ( n = 20). One-way ANOVA test performed. * P

    Journal: Nature Communications

    Article Title: CLEC5A is a critical receptor in innate immunity against Listeria infection

    doi: 10.1038/s41467-017-00356-3

    Figure Lengend Snippet: CLEC5A is critical for restriction of bacterial spreading in vivo. a , b Wild type ( WT ), WT/DNase-I (4 KU per mouse) and mutant mice were injected with the live luminescent L. monocytogenes ( Xen32 ) (3 × 10 8 CFUs per mouse) intravenously, and luminescence intensity in liver was determined at 1 h, 2 h, and 4 h post- infection (10 mice per group) by IVIS system (Xenogen). Bacterial loads in liver c and blood d were determined 4 h post-infection. The data were collected and expressed as mean ± s.e.m. from four independent experiments ( n = 20). One-way ANOVA test performed. * P

    Article Snippet: The livers were collected and minced into pieces followed by being digested with the buffer containing 1 mg/ml collagenase type IV (Sigma C5138) and 300 U/ml DNAase I (Sigma D5025) at 37 °C for 30 min. Liver homogenates were passed through cell strainer (40 μm), and lysed with RBC lysis buffer to remove the remaining red blood cells.

    Techniques: In Vivo, Mutagenesis, Mouse Assay, Injection, Infection

    ssRNAs from CVB3-Infected Cells Do Not Activate MDA5 (A) ssRNA fraction from mock- or CVB3-infected HeLa cells were transfected into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU DNase I at 37°C for 15 min. Resulting RNA samples equivalent to 500 ng starting material were transfected into approximately 200,000 MAVS +/+ MEFs and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. Data presented as mean ± SD. (C) The same samples as in (B) were analyzed on agarose gel. (D) CVB3 vRNA was isolated from pelleted viral particles. Indicated amounts of vRNA were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml), and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.. Data presented as mean ± SD. (E) Indicated amounts of mock-treated or unlinkase-treated poliovirus vRNAs were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). Total RNA was extracted at 8 hr.p.t. and IFN-b mRNA induction was determined by real-time qPCR. Data presented as mean ± SD. See also Figure S3 .

    Journal: Cell Reports

    Article Title: MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

    doi: 10.1016/j.celrep.2012.10.005

    Figure Lengend Snippet: ssRNAs from CVB3-Infected Cells Do Not Activate MDA5 (A) ssRNA fraction from mock- or CVB3-infected HeLa cells were transfected into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) ssRNA fractions (ssPools) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU DNase I at 37°C for 15 min. Resulting RNA samples equivalent to 500 ng starting material were transfected into approximately 200,000 MAVS +/+ MEFs and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. Data presented as mean ± SD. (C) The same samples as in (B) were analyzed on agarose gel. (D) CVB3 vRNA was isolated from pelleted viral particles. Indicated amounts of vRNA were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml), and IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t.. Data presented as mean ± SD. (E) Indicated amounts of mock-treated or unlinkase-treated poliovirus vRNAs were transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). Total RNA was extracted at 8 hr.p.t. and IFN-b mRNA induction was determined by real-time qPCR. Data presented as mean ± SD. See also Figure S3 .

    Article Snippet: CHX and DIP were purchased from Sigma-Aldrich, and RNase A, RNase III, and DNase I from Ambion.

    Techniques: Infection, Transfection, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Isolation

    Picornavirus RF Is a Potent MDA5 Agonist (A) dsRNA fractions from mock- or CVB3-infected HeLa cells were transfected-into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) dsRNA fractions (dsPool) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU DNase I at 37°C for 15 min, and analyzed on agarose gel. (C) DNA and RF bands observed in CVB3 dsRNA fraction were gel purified and analyzed on agarose gel. CVB3 dsRNA fraction (10 ng per well in 24-well format), and the gel-purified RNAs (10, 2, or 0.4 ng/well) were transfected into WT, RIG-I −/− , MDA5 −/− , or MAVS −/− MEFs in the presence of CHX (10 μg/ml). IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. (D) dsRNA fractions of mock-, CVB3-, or mengovirus-infected cells were analyzed on agarose gel. (E) DNA and RF bands from mengovirus dsRNA fraction were gel purified and transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). IFN-β response at 8 hr.p.t. was measured by real-time qPCR. (F) Gel-purified RF and DNA bands from CVB3 dsRNA fraction, as well as a in vitro transcribed dsRNA of CVB3 sequence (ivt dsRNA) (0.3 μg/ml), were incubated with recombinant MDA5 (0.3 μM) at 37°C in the presence of 2mM ATP, and free Pi was measured using Green Reagent at 0, 30, and 60 min after reaction was started. M, dsDNA marker with indicated size in kbp. Bands number 1 and 2 on gel corresponds to the samples used in RNA transfection. Data presented as mean ± SD.

    Journal: Cell Reports

    Article Title: MDA5 Detects the Double-Stranded RNA Replicative Form in Picornavirus-Infected Cells

    doi: 10.1016/j.celrep.2012.10.005

    Figure Lengend Snippet: Picornavirus RF Is a Potent MDA5 Agonist (A) dsRNA fractions from mock- or CVB3-infected HeLa cells were transfected-into MEFs of indicated genotypes, and IFN-β mRNA level at 2 hr.p.t. was measured by real-time qPCR. Data presented as mean ± SD. (B) dsRNA fractions (dsPool) of mock- and CVB3-infected cells were treated with 10 ng/μl RNase A, 10 mU RNase III, or 10 mU DNase I at 37°C for 15 min, and analyzed on agarose gel. (C) DNA and RF bands observed in CVB3 dsRNA fraction were gel purified and analyzed on agarose gel. CVB3 dsRNA fraction (10 ng per well in 24-well format), and the gel-purified RNAs (10, 2, or 0.4 ng/well) were transfected into WT, RIG-I −/− , MDA5 −/− , or MAVS −/− MEFs in the presence of CHX (10 μg/ml). IFN-β mRNA induction was determined by real-time qPCR at 8 hr.p.t. (D) dsRNA fractions of mock-, CVB3-, or mengovirus-infected cells were analyzed on agarose gel. (E) DNA and RF bands from mengovirus dsRNA fraction were gel purified and transfected into RIG-I −/− MEFs in the presence of CHX (10 μg/ml). IFN-β response at 8 hr.p.t. was measured by real-time qPCR. (F) Gel-purified RF and DNA bands from CVB3 dsRNA fraction, as well as a in vitro transcribed dsRNA of CVB3 sequence (ivt dsRNA) (0.3 μg/ml), were incubated with recombinant MDA5 (0.3 μM) at 37°C in the presence of 2mM ATP, and free Pi was measured using Green Reagent at 0, 30, and 60 min after reaction was started. M, dsDNA marker with indicated size in kbp. Bands number 1 and 2 on gel corresponds to the samples used in RNA transfection. Data presented as mean ± SD.

    Article Snippet: CHX and DIP were purchased from Sigma-Aldrich, and RNase A, RNase III, and DNase I from Ambion.

    Techniques: Infection, Transfection, Real-time Polymerase Chain Reaction, Agarose Gel Electrophoresis, Purification, In Vitro, Sequencing, Incubation, Recombinant, Marker

    α-subunits of RNAP bind to A-boxes upstream of the T-tract. A) DNA sequence of the P sabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes). Red, blue and green lines mark the interaction sites of σ 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B–C . B–C) Mapping of the binding site for σ 70 -RNAP to P sabA DNA using DNase I footprint assay. 10 nM of [γ 32 P]ATP-labeled P sabA DNA (−166 to +74) were mixed with increasing concentrations of σ 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM). The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines. The positions of the T-tract, predicted −35 and −10, and +1 transcriptional start site, are indicated to the left. The stars mark the region of the promoter that was deleted in Δ 46 variants (−97 to −49, see also Fig. S2 and S6A ). Nucleotide positions, relative to the transcriptional start site, are shown to the right. D) Binding of σ 70 -RNAP (55 nM) to P sabA DNA (−166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR. The sensorgrams show values normalized to that of the full-length T 13 -variant. Binding to a sabA CDS-fragment, also used in Fig. 4 , is shown as a background curve in the top diagram. The bottom diagram is an enlargement of the dotted-lined square in the top diagram. E) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing P sabA with proximal UP-like element deleted. The constructs contain different tract lengths and compositions (see Fig. 5B–C and S6A ). Black bars represent wt promoters and white bars Δ 46 variants, respectively. β-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods . Data is presented as relative values with activity of P sabA T 13 wt set to 1. F) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements. β-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of P sabA wt set to 1.

    Journal: PLoS Pathogens

    Article Title: A Repetitive DNA Element Regulates Expression of the Helicobacter pylori Sialic Acid Binding Adhesin by a Rheostat-like Mechanism

    doi: 10.1371/journal.ppat.1004234

    Figure Lengend Snippet: α-subunits of RNAP bind to A-boxes upstream of the T-tract. A) DNA sequence of the P sabA upstream region showing the predicted UP-like elements and multiple A-boxes (red boxes). Red, blue and green lines mark the interaction sites of σ 70 -RNAP found by Footprint analysis, correspondingly, see Fig. 5B–C . B–C) Mapping of the binding site for σ 70 -RNAP to P sabA DNA using DNase I footprint assay. 10 nM of [γ 32 P]ATP-labeled P sabA DNA (−166 to +74) were mixed with increasing concentrations of σ 70 -RNAP (0, 6.25, 12.5, 25, or 50 nM). The regions protected from DNase I cleavage are marked by red (core promoter), blue (proximal UP-like element) and green (distal UP-like element) lines. The positions of the T-tract, predicted −35 and −10, and +1 transcriptional start site, are indicated to the left. The stars mark the region of the promoter that was deleted in Δ 46 variants (−97 to −49, see also Fig. S2 and S6A ). Nucleotide positions, relative to the transcriptional start site, are shown to the right. D) Binding of σ 70 -RNAP (55 nM) to P sabA DNA (−166 to +74), with different repeat tract compositions and promoter mutant variants, analyzed by SPR. The sensorgrams show values normalized to that of the full-length T 13 -variant. Binding to a sabA CDS-fragment, also used in Fig. 4 , is shown as a background curve in the top diagram. The bottom diagram is an enlargement of the dotted-lined square in the top diagram. E) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing P sabA with proximal UP-like element deleted. The constructs contain different tract lengths and compositions (see Fig. 5B–C and S6A ). Black bars represent wt promoters and white bars Δ 46 variants, respectively. β-galactosidase assays were performed in the E. coli strain AAG1, with cultures grown to OD 600 of 2 and analyzed as described in Materials and Methods . Data is presented as relative values with activity of P sabA T 13 wt set to 1. F) Promoter activity of P sabA :: lacZ transcriptional fusion plasmids, containing sabA promoter with scrambled UP-like elements. β-galactosidase assays were performed as described in Fig. 5E and data is presented as relative values with activity of P sabA wt set to 1.

    Article Snippet: Before cDNA synthesis the total RNA (250 µg/µl) was treated an extra time with Turbo DNase I (Ambion) to remove any residual DNA. cDNA synthesis was performed in 20 µl reactions using 500 ng Turbo DNase treated total RNA, Transcriptor First Strand cDNA Synthesis kit (Roche Applied Science) and random hexamers (60 µM) provided with the kit, according to the manufacturer's protocol. cDNA synthesis was performed at 25°C for 10 min and at 55°C for 30 min.

    Techniques: Sequencing, Binding Assay, Labeling, Mutagenesis, SPR Assay, Variant Assay, Activity Assay, Construct

    sav_4189 promoter structure and SAV4189-binding sites. (A) Determination of sav_4189 TSS by 5′-RACE PCR. Box: complementary sequence of oligo(dT) anchor primer. Arrow: complementary base of TSS. (B) DNase I footprinting assay of SAV4189 on its own promoter region. Top fluorogram: reaction with 10 μM BSA (control). Protection patterns were acquired with increasing His 6 -SAV4189 concentrations. (C) Nucleotide sequences of sav_4189 promoter region and SAV4189-binding sites. Numbers: distance (nt) from sav_4189 TSS. Shading: translational start codons. Bent arrow: sav_4189 TSS and transcription orientation. Boxes: potential –10 and –35 regions. Underlining: SAV4189-binding sites. Straight arrows: inverted repeats. (D) EMSAs of His 6 -SAV4189 with probes A, B1, and B2. Each lane contained 0.3 nM labeled probe. Each probe was 59 bp. Non-specific DNA sequences from hrdB ORF (shaded) were fused with intact 13-bp palindromic sequences a, b1, and b2 (boldfaced) to generate probes A, B1, and B2, respectively. Lanes –: EMSAs without His 6 -SAV4189. Lanes 2, 3, and 4: 62.5, 125, and 250 nM His 6 -4189. (E) Consensus sequence analysis of SAV4189-binding sites by WebLogo program. Asterisks: consensus bases. Arrows: inverted repeats. Height of each letter is proportional to appearance frequency of corresponding base.

    Journal: Frontiers in Microbiology

    Article Title: SAV4189, a MarR-Family Regulator in Streptomyces avermitilis, Activates Avermectin Biosynthesis

    doi: 10.3389/fmicb.2018.01358

    Figure Lengend Snippet: sav_4189 promoter structure and SAV4189-binding sites. (A) Determination of sav_4189 TSS by 5′-RACE PCR. Box: complementary sequence of oligo(dT) anchor primer. Arrow: complementary base of TSS. (B) DNase I footprinting assay of SAV4189 on its own promoter region. Top fluorogram: reaction with 10 μM BSA (control). Protection patterns were acquired with increasing His 6 -SAV4189 concentrations. (C) Nucleotide sequences of sav_4189 promoter region and SAV4189-binding sites. Numbers: distance (nt) from sav_4189 TSS. Shading: translational start codons. Bent arrow: sav_4189 TSS and transcription orientation. Boxes: potential –10 and –35 regions. Underlining: SAV4189-binding sites. Straight arrows: inverted repeats. (D) EMSAs of His 6 -SAV4189 with probes A, B1, and B2. Each lane contained 0.3 nM labeled probe. Each probe was 59 bp. Non-specific DNA sequences from hrdB ORF (shaded) were fused with intact 13-bp palindromic sequences a, b1, and b2 (boldfaced) to generate probes A, B1, and B2, respectively. Lanes –: EMSAs without His 6 -SAV4189. Lanes 2, 3, and 4: 62.5, 125, and 250 nM His 6 -4189. (E) Consensus sequence analysis of SAV4189-binding sites by WebLogo program. Asterisks: consensus bases. Arrows: inverted repeats. Height of each letter is proportional to appearance frequency of corresponding base.

    Article Snippet: Following DNase I digestion, DNA samples were purified and subjected to capillary electrophoresis with 3730XL DNA analyzer (Applied Biosystems).

    Techniques: Binding Assay, Polymerase Chain Reaction, Sequencing, Footprinting, Labeling

    DNase I footprint analyses of MtsR complex with the promoter region of mtsR/A (A) ska (B) and aroE (C) genes. MtsR footprint generated as described in with DNA fragments labeled with the reverse primers ZE251 ( mtsA ), ZE361 ( ska ), and the forward

    Journal: Molecular microbiology

    Article Title: MtsR is a dual regulator that controls virulence genes and metabolic functions in addition to metal homeostasis in GAS

    doi: 10.1111/j.1365-2958.2010.07157.x

    Figure Lengend Snippet: DNase I footprint analyses of MtsR complex with the promoter region of mtsR/A (A) ska (B) and aroE (C) genes. MtsR footprint generated as described in with DNA fragments labeled with the reverse primers ZE251 ( mtsA ), ZE361 ( ska ), and the forward

    Article Snippet: Briefly, total RNA was isolated from each strain using the TritonX-100 isolation protocol ( ) and 25 ng was DNase I-treated, added to a SYBR Green Master mix (Applied Biosystems) containing 5 μg of each specific real-time primer , and combined with 6.25 units of Multiscribe reverse transcriptase (Applied Biosystems) in a 25 μl volume for a one step real-time RT-PCR reaction.

    Techniques: Generated, Labeling

    MtsR binding to shr promoter region. A. Schematic representation of shr promoter region. The thin solid lines represent the 3 DNA fragments used in the EMSAs: F1 (89 bp), F2 (159 bp), and F3 (160 bp). The DNA fragment used in the DNase I footprinting

    Journal: Molecular microbiology

    Article Title: MtsR is a dual regulator that controls virulence genes and metabolic functions in addition to metal homeostasis in GAS

    doi: 10.1111/j.1365-2958.2010.07157.x

    Figure Lengend Snippet: MtsR binding to shr promoter region. A. Schematic representation of shr promoter region. The thin solid lines represent the 3 DNA fragments used in the EMSAs: F1 (89 bp), F2 (159 bp), and F3 (160 bp). The DNA fragment used in the DNase I footprinting

    Article Snippet: Briefly, total RNA was isolated from each strain using the TritonX-100 isolation protocol ( ) and 25 ng was DNase I-treated, added to a SYBR Green Master mix (Applied Biosystems) containing 5 μg of each specific real-time primer , and combined with 6.25 units of Multiscribe reverse transcriptase (Applied Biosystems) in a 25 μl volume for a one step real-time RT-PCR reaction.

    Techniques: Binding Assay, Footprinting

    DNase I footprinting of MtsR complex with shr promoter. DNA fragments (1 μM) were incubated with DNase I in the presence of increasing concentration of purified rMtsR protein. The 5′end of the nontemplate strand was radiolabeled on the

    Journal: Molecular microbiology

    Article Title: MtsR is a dual regulator that controls virulence genes and metabolic functions in addition to metal homeostasis in GAS

    doi: 10.1111/j.1365-2958.2010.07157.x

    Figure Lengend Snippet: DNase I footprinting of MtsR complex with shr promoter. DNA fragments (1 μM) were incubated with DNase I in the presence of increasing concentration of purified rMtsR protein. The 5′end of the nontemplate strand was radiolabeled on the

    Article Snippet: Briefly, total RNA was isolated from each strain using the TritonX-100 isolation protocol ( ) and 25 ng was DNase I-treated, added to a SYBR Green Master mix (Applied Biosystems) containing 5 μg of each specific real-time primer , and combined with 6.25 units of Multiscribe reverse transcriptase (Applied Biosystems) in a 25 μl volume for a one step real-time RT-PCR reaction.

    Techniques: Footprinting, Incubation, Concentration Assay, Purification

    IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Journal: Oncotarget

    Article Title: Cancer therapies activate RIG-I-like receptor pathway through endogenous non-coding RNAs

    doi: 10.18632/oncotarget.8420

    Figure Lengend Snippet: IR induces RIG-I binding to endogenous double-stranded RNAs A. HEK293 reporter cells were irradiated after transfection with either an empty vector, a full length human RIG-I, a RIG-I lacking CARD domains (RIG-I helicase/CTD), or a RIG-I harboring K858A and K861A mutations in the C-terminal domain (RIG-I K858A-K861A), in addition to an IFN-beta promoter-driven luciferase construct. A Renilla reporter construct served as a transfection control. Data are presented as mean fold-change relative to the non-irradiated empty vector control. B. Donor HEK293 cells were either unirradiated or treated with IR (3 or 6 Gy). Total RNA was purified and transferred to independent batches of HEK293 reporter cells transfected by RIG-I constructs as described in (A). A synthetic double-stranded RNA construct comprised of 5′-triphosphorylated dsRNA and an unphosphorylated counterpart served as positive and negative controls, respectively (inset). C. Experimental design for isolation and purification of RNA bound to RIG-I after exposure to IR. *To validate RNA sequencing data by qPCR experiments, UV crosslinking was performed prior to cell lysis and immunoprecipitation of RIG-I. See methods for further details. D. Purified RNA from total cellular extracts (Lanes 2 and 3) and complexes with RIG-I (Lanes 4 and 5). Lane 1 is the marker. Data are representative of at least 3 independent experiments. E. HEK293 cells over-expressing the HA-tagged full length RIG-I (Lanes 2 and 3), the RIG-I helicase-CTD mutant (Lanes 4 and 5) and the RIG-I K858A-K861A CTD mutant (Lanes 6 and 7) were either un-irradiated or exposed to IR (6 Gy), lysed and incubated with anti-HA monoclonal antibody to pulldown the respective WT and mutant RIG-I proteins. RIG-I diagrams illustrate the mechanism of RIG-I activation (adapted from [ 57 ]). In the inactive/unbound conformation, the CARD domain of RIG-I is folded to block the helicase domain from RNA binding RNA, but allows the CTD to search for its ligand. Upon binding of the blunt end of a dsRNA molecule to the CTD, the CARD domain opens to allow the helicase domain to bind the remaining dsRNA molecule. Absence of the CARD domain in the helicase/CTD mutant enables higher affinity binding to dsRNA ligands as compared to the full length RIG-I. The lysine residues at amino acid positions 858 and 861 have previously demonstrated importance in latching onto the 5′-triphosphorylated end of viral dsRNA ligands. F. RNA bound to RIG-I after exposure to IR (6 Gy) was treated with: RNase A (lane 3), dsRNA-specific RNase III (lane 4), single-strand specific nuclease S1 (lane 5) and DNase I (lane 7). Lane 2 shows the input and lanes 1 and 6 display markers.

    Article Snippet: qRT-PCR analysis 1 μg total RNA was subjected to DNase I treatment in a 30 μL reaction volume using DNase I, RNase-free (Thermo Scientific) following the manufacturer's protocol. cDNA was synthesized from 10 μL of the DNase treated RNA using the High-Capacity cDNA Reverse Transcription Kit (LifeTechnologies) following the manufacturer's protocol.

    Techniques: Binding Assay, Irradiation, Transfection, Plasmid Preparation, Luciferase, Construct, Purification, Isolation, RNA Sequencing Assay, Real-time Polymerase Chain Reaction, Lysis, Immunoprecipitation, Marker, Expressing, Mutagenesis, Incubation, Activation Assay, Blocking Assay, RNA Binding Assay

    B23.1 is released from nuclear structure by RNase treatment. (A) Cell fractionation experiment using RNase A or DNase I. The experimental scheme is shown in the upper panel. Exponentially growing HeLa cells were first treated with 0.5% Triton X-100 in CSK buffer and were then subjected to digestion with DNase I (3 unit/μl) or RNase A (200 μg/ml). Released proteins by nuclease treatment were recovered by centrifugation as supernatant fraction, and the cell pellet was further extracted with 0.25 M ammonium sulfate on ice for 5 min. Remaining insoluble proteins were dissolved in 8 M urea. Proteins in each fraction were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting with anti-B23 antibody (NB23). (B) RNA-dependent nuclear retention of B23.1. Cells treated with Triton X-100 were digested with increasing amounts of RNase A (0, 2, 20, and 200 μg/ml for lanes 2–5, respectively) or DNase I (3 units/μl for lane 6) at 37°C for 20 min. Proteins were separated from insoluble materials by centrifugation, and the remaining pellet was dissolved in 8 M urea. Insoluble (Ppt, upper panel) and released (Sup, bottom panel) proteins were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting. Insoluble proteins after treatment with Triton X-100 are shown in lane 1 (Input). Positions of B23.1 are indicated by arrowheads at the left sides of panels.

    Journal: Molecular Biology of the Cell

    Article Title: The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

    doi: 10.1091/mbc.02-03-0036

    Figure Lengend Snippet: B23.1 is released from nuclear structure by RNase treatment. (A) Cell fractionation experiment using RNase A or DNase I. The experimental scheme is shown in the upper panel. Exponentially growing HeLa cells were first treated with 0.5% Triton X-100 in CSK buffer and were then subjected to digestion with DNase I (3 unit/μl) or RNase A (200 μg/ml). Released proteins by nuclease treatment were recovered by centrifugation as supernatant fraction, and the cell pellet was further extracted with 0.25 M ammonium sulfate on ice for 5 min. Remaining insoluble proteins were dissolved in 8 M urea. Proteins in each fraction were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting with anti-B23 antibody (NB23). (B) RNA-dependent nuclear retention of B23.1. Cells treated with Triton X-100 were digested with increasing amounts of RNase A (0, 2, 20, and 200 μg/ml for lanes 2–5, respectively) or DNase I (3 units/μl for lane 6) at 37°C for 20 min. Proteins were separated from insoluble materials by centrifugation, and the remaining pellet was dissolved in 8 M urea. Insoluble (Ppt, upper panel) and released (Sup, bottom panel) proteins were separated on a 10% SDS-PAGE, and B23 proteins were detected by western blotting. Insoluble proteins after treatment with Triton X-100 are shown in lane 1 (Input). Positions of B23.1 are indicated by arrowheads at the left sides of panels.

    Article Snippet: The cell pellet that contained the chromatin DNA was digested by 2 units/μl RNase-free DNase I (Invitrogen, Carlsbad, CA) at 37°C for 15 min and was extracted with (NH 4 )2 SO4 at a final concentration of 0.25 M in CSK buffer.

    Techniques: Cell Fractionation, Centrifugation, SDS Page, Western Blot

    Cellular localization of B23 splicing variants. HeLa cells grown on coverslips were transfected with pEGFPC1-hB23.1 (upper panels) or -hB23.2 (bottom panels). Thirty hours after transfection, cells on coverslips were fixed with 3% paraformaldehyde (first column) or were treated with 0.5% Triton X-10 0 followed by fixation with 3% paraformaldehyde (second column). Triton X-100–treated cells were further digested with DNase I (third column) or RNase A (fourth column) followed by extraction with 0.25 M ammonium sulfate before fixation with paraformaldehyde. DNA stained with Hoechst 33258 (blue) and GFP-tagged proteins (green) were visualized under a fluorescent microscope. The bar at the right bottom indicates 10 μm.

    Journal: Molecular Biology of the Cell

    Article Title: The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

    doi: 10.1091/mbc.02-03-0036

    Figure Lengend Snippet: Cellular localization of B23 splicing variants. HeLa cells grown on coverslips were transfected with pEGFPC1-hB23.1 (upper panels) or -hB23.2 (bottom panels). Thirty hours after transfection, cells on coverslips were fixed with 3% paraformaldehyde (first column) or were treated with 0.5% Triton X-10 0 followed by fixation with 3% paraformaldehyde (second column). Triton X-100–treated cells were further digested with DNase I (third column) or RNase A (fourth column) followed by extraction with 0.25 M ammonium sulfate before fixation with paraformaldehyde. DNA stained with Hoechst 33258 (blue) and GFP-tagged proteins (green) were visualized under a fluorescent microscope. The bar at the right bottom indicates 10 μm.

    Article Snippet: The cell pellet that contained the chromatin DNA was digested by 2 units/μl RNase-free DNase I (Invitrogen, Carlsbad, CA) at 37°C for 15 min and was extracted with (NH 4 )2 SO4 at a final concentration of 0.25 M in CSK buffer.

    Techniques: Transfection, Staining, Microscopy

    Impact of DNase I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: Impact of DNase I treatment on B . pseudomallei biofilm formation. Static biofilms of B . pseudomallei strains L1, P1 and H777 in LB were treated with DNase I (0.01, 0.1 and 1 U/mL) at 0 h, 24 h or 45 h after inoculation and maintained for up to 48 h. Biofilm formation and eDNA concentration of the 2-day biofilms were assessed using crystal-violet absorbance (OD 620 ) and the QuantiFluor dsDNA System, respectively. DNase I buffer acted as control. Biofilm formation of each strain was examined in eight replicates and eDNA was quantified in duplicates, on three independent occasions. Data represents mean ± SD. * p

    Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

    Techniques: Concentration Assay

    Exogenous chromosomal DNA did not alter either untreated biofilm or DNase I-treated biofilm of B . pseudomallei H777. (A) Amount of 2-day B . pseudomallei H777 biofilm formed in LB, treated with DNase I, supplemented with either salmon sperm DNA (SS DNA) or B . pseudomallei genomic DNA (Bp DNA) compared to the controls. Data represents mean ± SD from three independent experiments. (B) Amount of 2-day B . pseudomallei H777 biofilm formed in LB after treatment with 0.01 U/mL DNase I for 3 h, followed by washing steps to remove DNase, and then supplemented with exogenous salmon sperm DNA or B . pseudomallei genomic DNA. Data represents mean ± SD from three independent experiments. ** p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: Exogenous chromosomal DNA did not alter either untreated biofilm or DNase I-treated biofilm of B . pseudomallei H777. (A) Amount of 2-day B . pseudomallei H777 biofilm formed in LB, treated with DNase I, supplemented with either salmon sperm DNA (SS DNA) or B . pseudomallei genomic DNA (Bp DNA) compared to the controls. Data represents mean ± SD from three independent experiments. (B) Amount of 2-day B . pseudomallei H777 biofilm formed in LB after treatment with 0.01 U/mL DNase I for 3 h, followed by washing steps to remove DNase, and then supplemented with exogenous salmon sperm DNA or B . pseudomallei genomic DNA. Data represents mean ± SD from three independent experiments. ** p

    Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

    Techniques:

    DNase I treatment affects initial attachment and biofilm formation of B . pseudomallei . B . pseudomallei L1, P1 and H777 biofilms were grown in LB at 37°C. The biofilms were treated with DNase I (0.01 U/mL) at 0 h and 24 h post-seeding and maintained until 48 h. (A) CLSM images of DNase I treated biofilm structure and eDNA on coverslips. The 2-day biofilm architecture and quantity of eDNA were examined after staining with FITC-ConA (green) and TOTO-3 (red), respectively. The scale bars indicate 10 μm. The images were taken using a Zeiss 800 CLSM microscope (63× magnification). (B) COMSTAT image analysis of DNase I-treated B . pseudomallei biofilms and eDNA. Data represents mean ± SD of 18 images from three independent experiments. * p

    Journal: PLoS ONE

    Article Title: Extracellular DNA facilitates bacterial adhesion during Burkholderia pseudomallei biofilm formation

    doi: 10.1371/journal.pone.0213288

    Figure Lengend Snippet: DNase I treatment affects initial attachment and biofilm formation of B . pseudomallei . B . pseudomallei L1, P1 and H777 biofilms were grown in LB at 37°C. The biofilms were treated with DNase I (0.01 U/mL) at 0 h and 24 h post-seeding and maintained until 48 h. (A) CLSM images of DNase I treated biofilm structure and eDNA on coverslips. The 2-day biofilm architecture and quantity of eDNA were examined after staining with FITC-ConA (green) and TOTO-3 (red), respectively. The scale bars indicate 10 μm. The images were taken using a Zeiss 800 CLSM microscope (63× magnification). (B) COMSTAT image analysis of DNase I-treated B . pseudomallei biofilms and eDNA. Data represents mean ± SD of 18 images from three independent experiments. * p

    Article Snippet: DNase I noticeably lowered eDNA concentrations in biofilm if the enzyme was added into the starting inoculum (0 h).

    Techniques: Confocal Laser Scanning Microscopy, Staining, Microscopy

    Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark DNase I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Regulatory loci downstream of human Cα1 and Cα2. Lines A and C , based on this study, show an expanded map of the region downstream of Cα1 and Cα2, respectively, as well as available DNA clones, which are shown above (α1) or below (α2) the line: phage clones are marked with diagrammatic phage heads, while the subclones of PCR-amplified segments A1-HS3-12 and A1-HS4-3′ are drawn with hatched lines; and a BAC clone is drawn as a double line, containing a deletion ( dashed box ). Vertical ovals mark DNase I sites demonstrating enhancer activity and named according to the homologous murine HS sites. A series of small triangles identifies the 20-bp repeats located downstream from human Cα genes. X marks the position of a DNase I site which shows human/mouse sequence conservation. The position of a CpG island previously identified by Southern blotting is also shown ( oval ). The arrow under HS12 in line A indicates the orientation of this sequence, which is the same as that of the homologous mouse HS site, but opposite from the orientation of HS12 in the α2 locus (line C ). The thick black lines under the maps of lines A and C ( single lower case letters ) represent hybridization probes used in this study.

    Article Snippet: Since many regulatory regions accessible to DNase I are also accessible to restriction endonucleases, we digested HS Sultan nuclei with SspI and localized the cleavage sites by isolating the DNA, digesting with EcoRI, and hybridizing Southern blots with probe b′ as shown in Fig. B .

    Techniques: Clone Assay, Polymerase Chain Reaction, Amplification, BAC Assay, Activity Assay, Sequencing, Southern Blot, Hybridization

    Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference 13 (EMBL/GenBank/DDBJ accession numbers X96607 and X96608 ), while numbering for mouse HS4 is according to reference 12 (EMBL/DDBJ/GenBank accession number S74166 ). ( A ) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. ( B ) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. ( C ) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. ( D ) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE ( 41 , 42 ). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Sequence similarities between human and mouse 3′ α elements. Human-mouse alignments are shown between 3′α enhancers (H12, HS3, and HS4), as well as for the X DNase I site. Nucleotide matches between human α1 and α2 sequences and between one or both human sequences and mouse are indicated by shading. Core homology regions are indicated by a thick line above the sequences. Boxes denote motifs shown to function in mouse as transcription factor binding sites. For HS12, HS3, and HS4, 50–100 bp of sequence flanking the core homology regions are shown. Mouse sequence numbering is 5′ to 3′ with regard to the coding strand of the mouse heavy chain locus. Numbering for mouse HS12, HS3A, HS3B, and X segments is according to reference 13 (EMBL/GenBank/DDBJ accession numbers X96607 and X96608 ), while numbering for mouse HS4 is according to reference 12 (EMBL/DDBJ/GenBank accession number S74166 ). ( A ) HS12 sequences (α2 sequence inverted). Overlining highlights the striking 135-bp core segment which is 90% homologous between human and mouse. The sequence alignment has been extended downstream from the core to include additional transcription factor motifs which are functional in mouse. Vertical lines indicate the boundaries of the GC-rich 59-bp repeat units. ( B ) HS3. Comparison of the nearly identical human α1 and α2 HS3 sequences with mouse HS3A and HS3B sequences, which are also nearly identical, shows that there is a 200-bp core segment which is 74% homologous between the mouse and human sequences. ( C ) HS4. Excluding the 25-bp gap containing the mouse HS4 BSAP site, the 145 core HS4 region is 76% homologous between human and mouse. ( D ) X site. Near the center of a 61-bp segment which has 70% human-mouse homology, there is a 20-bp sequence which matches at 19 positions between humans and mice. In both mice and humans this segment contains a consensus HSE ( 41 , 42 ). The sequences of the human enhancers and X sites are available from EMBL/GenBank/DDBJ under accession numbers AF013718 (α1HS3), AF013719 (α2HS3), AF013720 (α1X), AF013721 (α2X), AF013722 (α1HS12T), AF013723 (α1HS12B), AF013724 (α2HS12), AF013725 (α1HS4), and AF013726 (α2HS4).

    Article Snippet: Since many regulatory regions accessible to DNase I are also accessible to restriction endonucleases, we digested HS Sultan nuclei with SspI and localized the cleavage sites by isolating the DNA, digesting with EcoRI, and hybridizing Southern blots with probe b′ as shown in Fig. B .

    Techniques: Sequencing, Binding Assay, Functional Assay, Mouse Assay

    Comparison of IgsH loci of mouse and human. Line A shows a map of the murine IgH locus, from which the region downstream from Cα is expanded in line B. The murine enhancers designated Cα3′E ( 11 ) and 3′αE ( 9 ) are shown as vertical ovals, along with the DNase I hypersensitivity site designations ( 12 ). We have distinguished the two copies of HS3 sequence as HS3A and HS3B; these are included in a large palindrome ( arrows ) that flanks HS12, according to the sequence analysis of Chauveau and Cogné ( 13 ). Line C shows the human IgH locus, illustrating the γ-γ-ε-α duplication units ( brackets ) and the possibility of two regions homologous to the murine LCR.

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Comparison of IgsH loci of mouse and human. Line A shows a map of the murine IgH locus, from which the region downstream from Cα is expanded in line B. The murine enhancers designated Cα3′E ( 11 ) and 3′αE ( 9 ) are shown as vertical ovals, along with the DNase I hypersensitivity site designations ( 12 ). We have distinguished the two copies of HS3 sequence as HS3A and HS3B; these are included in a large palindrome ( arrows ) that flanks HS12, according to the sequence analysis of Chauveau and Cogné ( 13 ). Line C shows the human IgH locus, illustrating the γ-γ-ε-α duplication units ( brackets ) and the possibility of two regions homologous to the murine LCR.

    Article Snippet: Since many regulatory regions accessible to DNase I are also accessible to restriction endonucleases, we digested HS Sultan nuclei with SspI and localized the cleavage sites by isolating the DNA, digesting with EcoRI, and hybridizing Southern blots with probe b′ as shown in Fig. B .

    Techniques: Sequencing

    Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

    Article Snippet: Since many regulatory regions accessible to DNase I are also accessible to restriction endonucleases, we digested HS Sultan nuclei with SspI and localized the cleavage sites by isolating the DNA, digesting with EcoRI, and hybridizing Southern blots with probe b′ as shown in Fig. B .

    Techniques: Isolation, Generated, Labeling, Sequencing, Purification

    Enhancer activity of selected regions downstream of human Cα1 and Cα2 genes. ( A ) Analysis of the locus downstream of α2, which was studied in detail. The map shows the position of DNase I sites; below this are diagrammed the restriction sites defining the boundaries of each fragment tested for enhancer activity by insertion into pGL3-Vκ, transfection into the human myeloma HS Sultan, and assay of resulting luciferase activity, as described in the text. The enhancer activities are given for constructs in the A orientation (the same orientation with respect to transcribed strands of immunoglobulin and luciferase) or the opposite B orientation, where examined. The luciferase activities were normalized to β-galactosidase activity encoded by a cotransfected plasmid, and expressed as fold-increase over the activity of an enhancerless control plasmid. For fragments showing enhancer activity, assays were performed at least in triplicate, and standard deviations are given. ( B ) Comparable analysis of selected fragments amplified from the homologous locus downstream from Cα1.

    Journal: The Journal of Experimental Medicine

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    doi:

    Figure Lengend Snippet: Enhancer activity of selected regions downstream of human Cα1 and Cα2 genes. ( A ) Analysis of the locus downstream of α2, which was studied in detail. The map shows the position of DNase I sites; below this are diagrammed the restriction sites defining the boundaries of each fragment tested for enhancer activity by insertion into pGL3-Vκ, transfection into the human myeloma HS Sultan, and assay of resulting luciferase activity, as described in the text. The enhancer activities are given for constructs in the A orientation (the same orientation with respect to transcribed strands of immunoglobulin and luciferase) or the opposite B orientation, where examined. The luciferase activities were normalized to β-galactosidase activity encoded by a cotransfected plasmid, and expressed as fold-increase over the activity of an enhancerless control plasmid. For fragments showing enhancer activity, assays were performed at least in triplicate, and standard deviations are given. ( B ) Comparable analysis of selected fragments amplified from the homologous locus downstream from Cα1.

    Article Snippet: Since many regulatory regions accessible to DNase I are also accessible to restriction endonucleases, we digested HS Sultan nuclei with SspI and localized the cleavage sites by isolating the DNA, digesting with EcoRI, and hybridizing Southern blots with probe b′ as shown in Fig. B .

    Techniques: Activity Assay, Transfection, Luciferase, Construct, Plasmid Preparation, Amplification

    Extracellular DNA is a component of the V. cholerae biofilm matrix. A. Analysis of nuclease sensitivity of wild type and Δ dns Δ xds mutant biofilms. Biofilms grown for 21 h under static conditions were treated 3 h with DNase I (diagonally shaded bars), λExonuclease (dotted bars) or with a combination of DNase I and λExonuclease (horizontally shaded bars). Biofilm mass remaining after nuclease treatment was quantified by crystal violet staining. Significant differences compared with untreated biofilms of the wild type or Δ dns Δ xds mutant are indicated (* P

    Journal: Molecular Microbiology

    Article Title: Extracellular nucleases and extracellular DNA play important roles in Vibrio cholerae biofilm formation

    doi: 10.1111/j.1365-2958.2011.07867.x

    Figure Lengend Snippet: Extracellular DNA is a component of the V. cholerae biofilm matrix. A. Analysis of nuclease sensitivity of wild type and Δ dns Δ xds mutant biofilms. Biofilms grown for 21 h under static conditions were treated 3 h with DNase I (diagonally shaded bars), λExonuclease (dotted bars) or with a combination of DNase I and λExonuclease (horizontally shaded bars). Biofilm mass remaining after nuclease treatment was quantified by crystal violet staining. Significant differences compared with untreated biofilms of the wild type or Δ dns Δ xds mutant are indicated (* P

    Article Snippet: At the respective time points the wells were rinsed four times with spent LB (obtained from a 24 or 72 h old biofilm culture of the respective strain) before DNase I (AppliChem), λExonuclease (New England Biolabs) or in combination were added to a final concentration of 133 Kunitz units per ml.

    Techniques: Mutagenesis, Staining

    Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

    Journal: Nucleic Acids Research

    Article Title: Regulated chromatin domain comprising cluster of co-expressed genes in Drosophila melanogaster

    doi: 10.1093/nar/gki281

    Figure Lengend Snippet: Profiles of chromatin DNase I-resistance across the 60D cluster region and surrounding sequences. Chromatin resistance to DNase I [as normalized relative yield (NRY) for each amplicon] is plotted on the vertical axis; the length of 60D1-2 genomic region (in kb) is plotted on the horizontal. Positions of the genes in the region are shown at bottom. Circles (black for the regulated chromatin domain, and white for the rest of the region) indicate average NRY for each amplicon, the grey area corresponds to the calculated 95% confidence interval. Upper panel: in larval testes, the entire region shows nearly uniformal low resistance to DNase I typical for the ‘open’ chromatin. In contrast, in larval brains (middle panel) and in embryos (lower panel) regulated chromatin domain that contains the genes CG13589 through Pros28.1B shows significantly higher resistance to DNase I, indicative of ‘closed’ chromatin configuration.

    Article Snippet: Regulated chromatin domain in the 60D cluster region To study the organization of chromatin in the 60D cluster region, we examined the accessibility of chromatin to the DNase I using a modified PCR-based nuclease resistance assay ( ).

    Techniques: Amplification

    DNase I footprinting of LigR binding to the ligK and ligJ promoter regions. The 238-bp fragment or the 251-bp fragment containing the ligK (A) or ligJ (B) promoter region, respectively, was labeled with DIG on the coding (left) or noncoding (right) strands. The presence (+) or absence (−) of purified LigR (100 nM), PCA, and GA in the reaction mixture is indicated. Inducers were added at a concentration of 1 mM in each reaction. Thicker bars indicate strong protection by LigR, and thinner bars show weakly protected regions. The DNase I-hypersensitive sites are shown with arrowheads. The colors of bars and arrowheads indicate the reactions performed in the absence (black) or presence (gray) of PCA or GA, respectively.

    Journal: Journal of Bacteriology

    Article Title: Regulatory System of the Protocatechuate 4,5-Cleavage Pathway Genes Essential for Lignin Downstream Catabolism ▿Regulatory System of the Protocatechuate 4,5-Cleavage Pathway Genes Essential for Lignin Downstream Catabolism ▿ §

    doi: 10.1128/JB.00215-10

    Figure Lengend Snippet: DNase I footprinting of LigR binding to the ligK and ligJ promoter regions. The 238-bp fragment or the 251-bp fragment containing the ligK (A) or ligJ (B) promoter region, respectively, was labeled with DIG on the coding (left) or noncoding (right) strands. The presence (+) or absence (−) of purified LigR (100 nM), PCA, and GA in the reaction mixture is indicated. Inducers were added at a concentration of 1 mM in each reaction. Thicker bars indicate strong protection by LigR, and thinner bars show weakly protected regions. The DNase I-hypersensitive sites are shown with arrowheads. The colors of bars and arrowheads indicate the reactions performed in the absence (black) or presence (gray) of PCA or GA, respectively.

    Article Snippet: The binding reactions were carried out at 20°C in a final volume of 100 μl containing 0.5 pmol of DIG-labeled probe, 100 μM DTT, 2 μg of salmon sperm DNA, 5 μg of bovine serum albumin (BSA), and the LigR dimer (100 nM) in binding buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM CaCl2 , 3 mM MgCl2 , 100 μM EDTA, pH 7.9) for 20 min. After the reaction, samples were preincubated at 25°C for 5 min, and partial digestion of the DNA was initiated by the addition of 50 mU of DNase I (Takara Bio Inc.).

    Techniques: Footprinting, Binding Assay, Labeling, Purification, Concentration Assay