Structured Review

TaKaRa dnase i
<t>DNase</t> I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.
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Images

1) Product Images from "Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis"

Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01398

DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.
Figure Legend Snippet: DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.

Techniques Used: Footprinting, Sequencing

2) Product Images from "Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *"

Article Title: Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M111.245605

Identification of novel HS sites by DNase I hypersensitivity assay. A , schematic diagram of the DNase I hypersensitivity assay of the Pcdh- γ gene. Upper part , the large colored boxes , Pcdh- γ or Diap1 exons. The small red boxes indicate
Figure Legend Snippet: Identification of novel HS sites by DNase I hypersensitivity assay. A , schematic diagram of the DNase I hypersensitivity assay of the Pcdh- γ gene. Upper part , the large colored boxes , Pcdh- γ or Diap1 exons. The small red boxes indicate

Techniques Used:

3) Product Images from "Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation"

Article Title: Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

Journal: BMC Microbiology

doi: 10.1186/1471-2180-11-146

S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I . (A) Attached SE1457 ΔatlE , SE1457 ΔsaeRS , SE1457 and SE1457 saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P
Figure Legend Snippet: S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I . (A) Attached SE1457 ΔatlE , SE1457 ΔsaeRS , SE1457 and SE1457 saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P

Techniques Used: Microscopy, Incubation, Cell Culture

Effect of DNaseI on SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilm formation . SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P
Figure Legend Snippet: Effect of DNaseI on SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilm formation . SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P

Techniques Used: Staining

4) Product Images from "AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region"

Article Title: AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region

Journal: The Plant Cell

doi:

Immunoblot Analysis of AHM1. (A) Immunodetection of AHM1. Wheat nuclear proteins and nuclear matrix proteins were extracted 40 hr after imbibition. Proteins were separated on an SDS–12% polyacrylamide gel for silver staining (lanes 1 to 3) or on an SDS–10% polyacrylamide gel for immunoblotting (lanes 4 to 9). Lane 1, molecular mass markers given at left in kilodaltons; lanes 2, 5, and 8, total nuclear proteins; lanes 3, 6, and 9, lithium diiodosalicylate (LIS)–extracted nuclear matrix proteins; and lanes 4 and 7, affinity-purified His-AH8. The amounts of proteins loaded were derived from 1 and 3 A 260 units of nuclei for silver staining and immunoblot, respectively. Anti-AHM1 antibodies were affinity-purified with His-MutJL (lanes 4 to 6) or His-MutR1Z (lanes 7 to 9) and used to detect AHM1. An arrowhead indicates the position of AHM1. (B) AHM1 in the nuclear matrix fraction. Nuclei were prepared 5 days (lanes 1 to 3) or 40 hr (lanes 4 to 6) after imbibition. Proteins were separated on an SDS–12% polyacrylamide gel for Coomassie blue staining (top, CBB) or on an SDS–10% polyacrylamide gel for immunoblotting with the IgG fraction of the anti-AHM1 antibody (bottom, Immunoblot). Lane 1, total nuclear proteins; lanes 2 and 3, LIS-extracted nuclear matrix proteins prepared with (lane 2) or without (lane 3) heat stabilization; lane 4, nuclear proteins after DNase I treatment; lane 5, nuclear matrix proteins prepared from DNase I–treated nuclei by the high-salt extraction method; and lane 6, nuclear proteins solubilized during the high-salt treatment. The amounts of proteins loaded for immunoblot were equivalent to 6 A 260 units (lanes 1 to 3) or 4 A 260 units (lanes 4 to 6) of nuclei; for CBB staining, they were equivalent to 2 A 260 units (lanes 1 to 3) or 1.3 A 260 units (lanes 4 to 6) of nuclei. An arrowhead indicates the position of AHM1. Positions of molecular mass markers are indicated at left in kilodaltons.
Figure Legend Snippet: Immunoblot Analysis of AHM1. (A) Immunodetection of AHM1. Wheat nuclear proteins and nuclear matrix proteins were extracted 40 hr after imbibition. Proteins were separated on an SDS–12% polyacrylamide gel for silver staining (lanes 1 to 3) or on an SDS–10% polyacrylamide gel for immunoblotting (lanes 4 to 9). Lane 1, molecular mass markers given at left in kilodaltons; lanes 2, 5, and 8, total nuclear proteins; lanes 3, 6, and 9, lithium diiodosalicylate (LIS)–extracted nuclear matrix proteins; and lanes 4 and 7, affinity-purified His-AH8. The amounts of proteins loaded were derived from 1 and 3 A 260 units of nuclei for silver staining and immunoblot, respectively. Anti-AHM1 antibodies were affinity-purified with His-MutJL (lanes 4 to 6) or His-MutR1Z (lanes 7 to 9) and used to detect AHM1. An arrowhead indicates the position of AHM1. (B) AHM1 in the nuclear matrix fraction. Nuclei were prepared 5 days (lanes 1 to 3) or 40 hr (lanes 4 to 6) after imbibition. Proteins were separated on an SDS–12% polyacrylamide gel for Coomassie blue staining (top, CBB) or on an SDS–10% polyacrylamide gel for immunoblotting with the IgG fraction of the anti-AHM1 antibody (bottom, Immunoblot). Lane 1, total nuclear proteins; lanes 2 and 3, LIS-extracted nuclear matrix proteins prepared with (lane 2) or without (lane 3) heat stabilization; lane 4, nuclear proteins after DNase I treatment; lane 5, nuclear matrix proteins prepared from DNase I–treated nuclei by the high-salt extraction method; and lane 6, nuclear proteins solubilized during the high-salt treatment. The amounts of proteins loaded for immunoblot were equivalent to 6 A 260 units (lanes 1 to 3) or 4 A 260 units (lanes 4 to 6) of nuclei; for CBB staining, they were equivalent to 2 A 260 units (lanes 1 to 3) or 1.3 A 260 units (lanes 4 to 6) of nuclei. An arrowhead indicates the position of AHM1. Positions of molecular mass markers are indicated at left in kilodaltons.

Techniques Used: Immunodetection, Silver Staining, Affinity Purification, Derivative Assay, Staining

5) Product Images from "Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis"

Article Title: Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00919-12

DNase I footprinting analysis to identify the YcnK binding site in the intergenic region between ycnK and ycnL . A DNA probe including the ycnK - ycnL intergenic region (P ycnK probe), 5′ end labeled at either the coding or noncoding strand, was prepared. The 5′-end-labeled probe (0.8 nM) was incubated in the reaction mixture with the YcnK protein (50 nM [lanes 2] and 25 nM [lanes 3]) as a dimer and without the YcnK protein (lanes 1 and 4). After partial digestion with DNase I, the resulting mixtures were subjected to urea-PAGE. Lanes G, A, T, and C contain the products of the dideoxy sequencing reactions with the corresponding 5′-end-labeled primers. Nucleotide sequences protected by YcnK are indicated on the right of each panel; the 16-bp direct repeat is indicated by tandem arrows.
Figure Legend Snippet: DNase I footprinting analysis to identify the YcnK binding site in the intergenic region between ycnK and ycnL . A DNA probe including the ycnK - ycnL intergenic region (P ycnK probe), 5′ end labeled at either the coding or noncoding strand, was prepared. The 5′-end-labeled probe (0.8 nM) was incubated in the reaction mixture with the YcnK protein (50 nM [lanes 2] and 25 nM [lanes 3]) as a dimer and without the YcnK protein (lanes 1 and 4). After partial digestion with DNase I, the resulting mixtures were subjected to urea-PAGE. Lanes G, A, T, and C contain the products of the dideoxy sequencing reactions with the corresponding 5′-end-labeled primers. Nucleotide sequences protected by YcnK are indicated on the right of each panel; the 16-bp direct repeat is indicated by tandem arrows.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Sequencing

6) Product Images from "PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa"

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00548

The PruR binding site is upstream of −35 box of the putA promoter. (A) Identification of the sequence of the PruR-protected region in the putA promoter by DNase I protection footprinting. Electropherograms are superimposed to show the region protected by different concentrations of rPruR (green, 1 μg; red, 2 μg;) or BSA (blue, 2 μg) within the putA promoter after digestion with DNase I. The DNA sequence protected by PruR is shown below the electropherograms. (B) The PruR binding site in the putA promoter. The sequence of F6 fragment is boxed with solid lines. The sequence protected by PruR is boxed with dash line. −35 box and −10 box are underlined. The transcriptional start site is bold and labeled with an arrow. Fragments F7, F7MU (the mutated nucleotides are in red letters and indicated by dotted lines), and F8 were inserted upstream of the promoter-less lacZ gene in pACYC184 for reporter gene assay. Designation and length of each fragment is shown on the left. (C) PruR binds to the protected region and activates the putA expression. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. * P
Figure Legend Snippet: The PruR binding site is upstream of −35 box of the putA promoter. (A) Identification of the sequence of the PruR-protected region in the putA promoter by DNase I protection footprinting. Electropherograms are superimposed to show the region protected by different concentrations of rPruR (green, 1 μg; red, 2 μg;) or BSA (blue, 2 μg) within the putA promoter after digestion with DNase I. The DNA sequence protected by PruR is shown below the electropherograms. (B) The PruR binding site in the putA promoter. The sequence of F6 fragment is boxed with solid lines. The sequence protected by PruR is boxed with dash line. −35 box and −10 box are underlined. The transcriptional start site is bold and labeled with an arrow. Fragments F7, F7MU (the mutated nucleotides are in red letters and indicated by dotted lines), and F8 were inserted upstream of the promoter-less lacZ gene in pACYC184 for reporter gene assay. Designation and length of each fragment is shown on the left. (C) PruR binds to the protected region and activates the putA expression. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. * P

Techniques Used: Binding Assay, Sequencing, Footprinting, Labeling, Reporter Gene Assay, Expressing

7) Product Images from "A multistep damage recognition mechanism for global genomic nucleotide excision repair"

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair

Journal: Genes & Development

doi: 10.1101/gad.866301

DNase I footprinting analysis of XPC–HR23B binding to a 3-base bubble. ( A,B ) The indicated substrates were 5′-end-labeled for top strands ( A ) or bottom strands ( B ), and subjected to the DNase I footprinting assay using various amounts of XPC–HR23B as indicated. The digested DNA samples were subjected to denaturing PAGE followed by autoradiography. The Maxam-Gilbert G ladder prepared from each probe was loaded in parallel, where indicated, below the gels. The position of the bubble in each substrate is shown by an asterisk. ( C ) A schematic representation of the protection patterns for the B3 substrates. In each panel, strongly and weakly protected regions are shown by solid and shaded bars, respectively. The sites that became hypersensitive to DNase I upon binding are indicated by arrowheads ( A,B ) or arrows ( C ). Size of the arrows in C corresponds to the observed degree of hypersensitivity.
Figure Legend Snippet: DNase I footprinting analysis of XPC–HR23B binding to a 3-base bubble. ( A,B ) The indicated substrates were 5′-end-labeled for top strands ( A ) or bottom strands ( B ), and subjected to the DNase I footprinting assay using various amounts of XPC–HR23B as indicated. The digested DNA samples were subjected to denaturing PAGE followed by autoradiography. The Maxam-Gilbert G ladder prepared from each probe was loaded in parallel, where indicated, below the gels. The position of the bubble in each substrate is shown by an asterisk. ( C ) A schematic representation of the protection patterns for the B3 substrates. In each panel, strongly and weakly protected regions are shown by solid and shaded bars, respectively. The sites that became hypersensitive to DNase I upon binding are indicated by arrowheads ( A,B ) or arrows ( C ). Size of the arrows in C corresponds to the observed degree of hypersensitivity.

Techniques Used: Footprinting, Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Autoradiography

8) Product Images from "Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow ▿"

Article Title: Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow ▿

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00202-11

Nucleic acids of ClorDNAV without enzyme or heat treatment (lane 1), treatment with DNase I (lane 2), RNase A (lane 3), S1 nuclease (lane 4), and 100°C for 2 min (lane 5). Samples were electrophoresed in an agarose gel with DNA molecular size
Figure Legend Snippet: Nucleic acids of ClorDNAV without enzyme or heat treatment (lane 1), treatment with DNase I (lane 2), RNase A (lane 3), S1 nuclease (lane 4), and 100°C for 2 min (lane 5). Samples were electrophoresed in an agarose gel with DNA molecular size

Techniques Used: Agarose Gel Electrophoresis

9) Product Images from "Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses"

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006234

SsMYRV4 confers hypovirulence on S . sclerotiorum . (A) Hypovirulent strain SX10 was dual-cultured with a virulent strain (such as Ep-1PNA367R). When the hyphae of the two strains contacted each other for 2 days, a mycelial plug potentially containing a newly transmitted mycovirus was picked up from the colony margin (marked with a blue star) of virulent strain (a site farthest from the hypovirulent strain). (B) Biological characteristics of strains Ep-1PNA367, SX10 and Ep-1PNA367T1 (derived from a dual culture of strains Ep-1PNA367 and SX10; as shown in A). All strains were cultured on PDA plates for 10 days prior to photography. Pathogenicity assay of S . sclerotiorum strains were carried out on detached leaves (left panels) and whole rapeseed plants (right panels). Photos and data were taken at 72 hour post inoculation. (C) Ultrastructure of fungal cells of strains Ep-1PNA367 and Ep-1PNA367T1 as observed under TEM. The two strains were cultured for 4 days and then examined by TEM. The right panel is an enlargement of nuclear area that is indicated by boxes in left panels. VLPs in the cell (right panel) were marked with blue arrows. N = Nucleus, W = cell wall. (D) TEM images of negatively stained SsMYRV4 particles. (E) PAGE analysis on 5% polyacrylamide gel of dsRNA directly extracted from mycelia of strains SX10, Ep-1PNA367T1 (A367T1), Ep-1PNA367 (A367), or extracted from purified virus particles from SX10, Ep-1PNA367T1. Lane M, λ Hind III-digested DNA used as size markers. All dsRNA samples were treated with DNase I and S1 nucleases prior to electrophoresis. (F) SDS-PAGE analysis of purified SsMYRV4 particles. Samples collected from sucrose gradient fractions were subjected to SDS-PAGE analysis on a 12% polyacrylamide gel. The sizes of the Coomassie blue-stained proteins were estimated by comparison with protein size markers (lane M). (G) Phylogenetic analysis of SsMYRV4. A neighbor-joining phylogenetic tree was constructed based on the complete amino acid sequences of viral RdRp. The virus name (Sclerotinia sclerotiorum mycoreovirus 4) was printed in blue color and pertinent information on other reoviruses selected for phylogenetic analysis is shown in S3 Table . Bootstrap values (%) obtained with 2000 replicates are indicated on the branches, and branch lengths correspond to genetic distance; scale bar at lower left corresponds to genetic distance.
Figure Legend Snippet: SsMYRV4 confers hypovirulence on S . sclerotiorum . (A) Hypovirulent strain SX10 was dual-cultured with a virulent strain (such as Ep-1PNA367R). When the hyphae of the two strains contacted each other for 2 days, a mycelial plug potentially containing a newly transmitted mycovirus was picked up from the colony margin (marked with a blue star) of virulent strain (a site farthest from the hypovirulent strain). (B) Biological characteristics of strains Ep-1PNA367, SX10 and Ep-1PNA367T1 (derived from a dual culture of strains Ep-1PNA367 and SX10; as shown in A). All strains were cultured on PDA plates for 10 days prior to photography. Pathogenicity assay of S . sclerotiorum strains were carried out on detached leaves (left panels) and whole rapeseed plants (right panels). Photos and data were taken at 72 hour post inoculation. (C) Ultrastructure of fungal cells of strains Ep-1PNA367 and Ep-1PNA367T1 as observed under TEM. The two strains were cultured for 4 days and then examined by TEM. The right panel is an enlargement of nuclear area that is indicated by boxes in left panels. VLPs in the cell (right panel) were marked with blue arrows. N = Nucleus, W = cell wall. (D) TEM images of negatively stained SsMYRV4 particles. (E) PAGE analysis on 5% polyacrylamide gel of dsRNA directly extracted from mycelia of strains SX10, Ep-1PNA367T1 (A367T1), Ep-1PNA367 (A367), or extracted from purified virus particles from SX10, Ep-1PNA367T1. Lane M, λ Hind III-digested DNA used as size markers. All dsRNA samples were treated with DNase I and S1 nucleases prior to electrophoresis. (F) SDS-PAGE analysis of purified SsMYRV4 particles. Samples collected from sucrose gradient fractions were subjected to SDS-PAGE analysis on a 12% polyacrylamide gel. The sizes of the Coomassie blue-stained proteins were estimated by comparison with protein size markers (lane M). (G) Phylogenetic analysis of SsMYRV4. A neighbor-joining phylogenetic tree was constructed based on the complete amino acid sequences of viral RdRp. The virus name (Sclerotinia sclerotiorum mycoreovirus 4) was printed in blue color and pertinent information on other reoviruses selected for phylogenetic analysis is shown in S3 Table . Bootstrap values (%) obtained with 2000 replicates are indicated on the branches, and branch lengths correspond to genetic distance; scale bar at lower left corresponds to genetic distance.

Techniques Used: Cell Culture, Derivative Assay, Transmission Electron Microscopy, Staining, Polyacrylamide Gel Electrophoresis, Purification, Electrophoresis, SDS Page, Construct

10) Product Images from "The Disruption of an OxyR-Like Protein Impairs Intracellular Magnetite Biomineralization in Magnetospirillum gryphiswaldense MSR-1"

Article Title: The Disruption of an OxyR-Like Protein Impairs Intracellular Magnetite Biomineralization in Magnetospirillum gryphiswaldense MSR-1

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.00208

OxyR-Like specifically binds to the oxyR-Like and pdh operators. (A,B) OxyR-Like specifically binds to the oxyR-Like (A) and pdh (B) promoters, and the interactions between the protein and DNA were dissociated by unlabeled probe. (C,D) The oxyR-Like (C) and pdh (D) promotor regions were protected by OxyR-Like in the DNase I footprinting assay. All experiments were independently repeated three times to ensure their reproducibility. The black arrows in the figure indicate the positions of free DNA probes.
Figure Legend Snippet: OxyR-Like specifically binds to the oxyR-Like and pdh operators. (A,B) OxyR-Like specifically binds to the oxyR-Like (A) and pdh (B) promoters, and the interactions between the protein and DNA were dissociated by unlabeled probe. (C,D) The oxyR-Like (C) and pdh (D) promotor regions were protected by OxyR-Like in the DNase I footprinting assay. All experiments were independently repeated three times to ensure their reproducibility. The black arrows in the figure indicate the positions of free DNA probes.

Techniques Used: Footprinting

11) Product Images from "Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis"

Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.01398

DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.
Figure Legend Snippet: DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.

Techniques Used: Footprinting, Sequencing

Binding of OhrR to aveR promoter region. (A) EMSA of His 6 -OhrR with aveR promoter region. (B) DNase I footprinting assay of aveR promoter region using His 6 -OhrR and nucleotide sequences of ohrR–ohrB2 intergenic region. EMSA and DNase I footprinting conditions as in Figures 1 , 2 .
Figure Legend Snippet: Binding of OhrR to aveR promoter region. (A) EMSA of His 6 -OhrR with aveR promoter region. (B) DNase I footprinting assay of aveR promoter region using His 6 -OhrR and nucleotide sequences of ohrR–ohrB2 intergenic region. EMSA and DNase I footprinting conditions as in Figures 1 , 2 .

Techniques Used: Binding Assay, Footprinting

12) Product Images from "Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors"

Article Title: Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors

Journal: Scientific Reports

doi: 10.1038/s41598-019-56627-0

DNase I and Proteinase K inhibit nicotine-induced biofilm formation. The biofilm formation of S. aureus USA300 strain was detected using a microtiter plate assay by measuring crystal violet stained biofilm at OD570. DNase I (25 U/well) and Proteinase K (2 ug/ml) was added to the well in both 2 mg/ml nicotine treatment group and control group. ( A ) Treated by DNaseI, the average thickness of biofilms varied from 1.506 ± 0.04 to 0.450 ± 0.07 in the nicotine-treated group (n = 3) and from 0.720 ± 0.14 to 0.328 ± 0.05 in untreated group. (n = 3) ( B ) Proteinase K(2 ug/ml) disrupted biofilm formation, in the nicotine treated group decreasing from 1.300 ± 0.06 to 0.393 ± 0.02 (n = 3), and in the control group decreasing from 0.721 ± 0.14 to 0.316 ± 0.08 (n = 3). *P
Figure Legend Snippet: DNase I and Proteinase K inhibit nicotine-induced biofilm formation. The biofilm formation of S. aureus USA300 strain was detected using a microtiter plate assay by measuring crystal violet stained biofilm at OD570. DNase I (25 U/well) and Proteinase K (2 ug/ml) was added to the well in both 2 mg/ml nicotine treatment group and control group. ( A ) Treated by DNaseI, the average thickness of biofilms varied from 1.506 ± 0.04 to 0.450 ± 0.07 in the nicotine-treated group (n = 3) and from 0.720 ± 0.14 to 0.328 ± 0.05 in untreated group. (n = 3) ( B ) Proteinase K(2 ug/ml) disrupted biofilm formation, in the nicotine treated group decreasing from 1.300 ± 0.06 to 0.393 ± 0.02 (n = 3), and in the control group decreasing from 0.721 ± 0.14 to 0.316 ± 0.08 (n = 3). *P

Techniques Used: Staining

13) Product Images from "DNA binding and helicase actions of mouse MCM4/6/7 helicase"

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki607

Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.
Figure Legend Snippet: Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.

Techniques Used: Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

14) Product Images from "DNA binding and helicase actions of mouse MCM4/6/7 helicase"

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki607

Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.
Figure Legend Snippet: Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.

Techniques Used: Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

15) Product Images from "Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿ †"

Article Title: Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01468-10

DNase I footprinting of CsgD-binding sites. Fluorescently labeled DNA fragments of the indicated CsgD target promoters (1.0 pmol each) were incubated in the absence (lane 1) or presence of increasing concentrations of purified CsgD (lanes 2 to 5 contain
Figure Legend Snippet: DNase I footprinting of CsgD-binding sites. Fluorescently labeled DNA fragments of the indicated CsgD target promoters (1.0 pmol each) were incubated in the absence (lane 1) or presence of increasing concentrations of purified CsgD (lanes 2 to 5 contain

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation, Purification

16) Product Images from "Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity"

Article Title: Mycoplasma bovis MBOV_RS02825 Encodes a Secretory Nuclease Associated with Cytotoxicity

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17050628

NETs degradation by rMbovNase and its variant rMbovNase Δ181–342 . ( A ) NETs were generated by PMA induction; ( B ) NETs were induced by M. haemolytica . The rMbovNase and the variant, together with DNase I (positive control) and PBS (negative control) were added into activated neutrophils in both A and B, and only rMbovNase and DNase I apparently degraded NETs, while the variant and PBS did not. No treatment was performed in blank control. The fields in the small squares are zoomed in the large squares (magnifications: ×100; the zoomed images in the lower left boxes are ×3 magnifications of the images in middle small boxes).
Figure Legend Snippet: NETs degradation by rMbovNase and its variant rMbovNase Δ181–342 . ( A ) NETs were generated by PMA induction; ( B ) NETs were induced by M. haemolytica . The rMbovNase and the variant, together with DNase I (positive control) and PBS (negative control) were added into activated neutrophils in both A and B, and only rMbovNase and DNase I apparently degraded NETs, while the variant and PBS did not. No treatment was performed in blank control. The fields in the small squares are zoomed in the large squares (magnifications: ×100; the zoomed images in the lower left boxes are ×3 magnifications of the images in middle small boxes).

Techniques Used: Variant Assay, Generated, Positive Control, Negative Control

17) Product Images from "Mechanism for Regulation of the Putrescine Utilization Pathway by the Transcription Factor PuuR in Escherichia coli K-12"

Article Title: Mechanism for Regulation of the Putrescine Utilization Pathway by the Transcription Factor PuuR in Escherichia coli K-12

Journal: Journal of Bacteriology

doi: 10.1128/JB.00097-12

DNase I footprint analysis of the intergenic region between puuA and puuD . (A) Location of two [ 32 P]ATP-labeled DNA segments between puuA and puuD . Arrows indicate directions from 5′ to 3′ on 32 P-labeled DNA strands. Striped areas labeled
Figure Legend Snippet: DNase I footprint analysis of the intergenic region between puuA and puuD . (A) Location of two [ 32 P]ATP-labeled DNA segments between puuA and puuD . Arrows indicate directions from 5′ to 3′ on 32 P-labeled DNA strands. Striped areas labeled

Techniques Used: Labeling

18) Product Images from "PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa"

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00548

The PruR binding site is upstream of −35 box of the putA promoter. (A) Identification of the sequence of the PruR-protected region in the putA promoter by DNase I protection footprinting. Electropherograms are superimposed to show the region protected by different concentrations of rPruR (green, 1 μg; red, 2 μg;) or BSA (blue, 2 μg) within the putA promoter after digestion with DNase I. The DNA sequence protected by PruR is shown below the electropherograms. (B) The PruR binding site in the putA promoter. The sequence of F6 fragment is boxed with solid lines. The sequence protected by PruR is boxed with dash line. −35 box and −10 box are underlined. The transcriptional start site is bold and labeled with an arrow. Fragments F7, F7MU (the mutated nucleotides are in red letters and indicated by dotted lines), and F8 were inserted upstream of the promoter-less lacZ gene in pACYC184 for reporter gene assay. Designation and length of each fragment is shown on the left. (C) PruR binds to the protected region and activates the putA expression. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. * P
Figure Legend Snippet: The PruR binding site is upstream of −35 box of the putA promoter. (A) Identification of the sequence of the PruR-protected region in the putA promoter by DNase I protection footprinting. Electropherograms are superimposed to show the region protected by different concentrations of rPruR (green, 1 μg; red, 2 μg;) or BSA (blue, 2 μg) within the putA promoter after digestion with DNase I. The DNA sequence protected by PruR is shown below the electropherograms. (B) The PruR binding site in the putA promoter. The sequence of F6 fragment is boxed with solid lines. The sequence protected by PruR is boxed with dash line. −35 box and −10 box are underlined. The transcriptional start site is bold and labeled with an arrow. Fragments F7, F7MU (the mutated nucleotides are in red letters and indicated by dotted lines), and F8 were inserted upstream of the promoter-less lacZ gene in pACYC184 for reporter gene assay. Designation and length of each fragment is shown on the left. (C) PruR binds to the protected region and activates the putA expression. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. * P

Techniques Used: Binding Assay, Sequencing, Footprinting, Labeling, Reporter Gene Assay, Expressing

19) Product Images from "Human mitochondrial DNA is packaged with TFAM"

Article Title: Human mitochondrial DNA is packaged with TFAM

Journal: Nucleic Acids Research

doi:

DNase I digestion of P2. P2 was digested with DNase I and centrifuged. TFAM was detected by western blotting. W, before centrifugation; S, supernatant; P, pellet.
Figure Legend Snippet: DNase I digestion of P2. P2 was digested with DNase I and centrifuged. TFAM was detected by western blotting. W, before centrifugation; S, supernatant; P, pellet.

Techniques Used: Western Blot, Centrifugation

20) Product Images from "Human mitochondrial DNA is packaged with TFAM"

Article Title: Human mitochondrial DNA is packaged with TFAM

Journal: Nucleic Acids Research

doi:

DNase I digestion of P2. P2 was digested with DNase I and centrifuged. TFAM was detected by western blotting. W, before centrifugation; S, supernatant; P, pellet.
Figure Legend Snippet: DNase I digestion of P2. P2 was digested with DNase I and centrifuged. TFAM was detected by western blotting. W, before centrifugation; S, supernatant; P, pellet.

Techniques Used: Western Blot, Centrifugation

21) Product Images from "Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences"

Article Title: Molecular architecture of G-quadruplex structures generated on duplex Rif1-binding sequences

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.RA118.005240

Summary of nuclease sensitivity of the heat-treated Rif1BS-derived duplex DNAs. Portions of the sequences of the Rif1BS I:2663 and Rif1BS II:4255 are shown. Locations of S1 nuclease sensitivities and those of protection from DNase I digestion are shown for both strands, as indicated in the key .
Figure Legend Snippet: Summary of nuclease sensitivity of the heat-treated Rif1BS-derived duplex DNAs. Portions of the sequences of the Rif1BS I:2663 and Rif1BS II:4255 are shown. Locations of S1 nuclease sensitivities and those of protection from DNase I digestion are shown for both strands, as indicated in the key .

Techniques Used: Derivative Assay

22) Product Images from "PdhR (Pyruvate Dehydrogenase Complex Regulator) Controls the Respiratory Electron Transport System in Escherichia coli ▿"

Article Title: PdhR (Pyruvate Dehydrogenase Complex Regulator) Controls the Respiratory Electron Transport System in Escherichia coli ▿

Journal: Journal of Bacteriology

doi: 10.1128/JB.00229-07

PdhR-bound DNA fragments. Using genomic SELEX, PdhR-bound DNA fragments were isolated, the indicated four groups of which contained the promoter regions from the pdhR , cyoA , ndh , and yfiD genes. Open bars indicate the genomic DNA sequence, while thick bars indicate the regions of fragments isolated by SELEX. The dotted lines show the DNA fragments used for gel shift and DNase I footprinting assays. The vertical line shows the translation initiation site. Numbers on each line represent the distance (bp) from the respective initiation codon. Transcription initiation sites, shown by arrows, are from pdhR ), cyoA ), ndh ), and yfiD ).
Figure Legend Snippet: PdhR-bound DNA fragments. Using genomic SELEX, PdhR-bound DNA fragments were isolated, the indicated four groups of which contained the promoter regions from the pdhR , cyoA , ndh , and yfiD genes. Open bars indicate the genomic DNA sequence, while thick bars indicate the regions of fragments isolated by SELEX. The dotted lines show the DNA fragments used for gel shift and DNase I footprinting assays. The vertical line shows the translation initiation site. Numbers on each line represent the distance (bp) from the respective initiation codon. Transcription initiation sites, shown by arrows, are from pdhR ), cyoA ), ndh ), and yfiD ).

Techniques Used: Isolation, Sequencing, Electrophoretic Mobility Shift Assay, Footprinting

DNase I footprinting of the cyoA promoter. (A) The fluorescently labeled cyoA promoter was incubated with increasing amounts of purified PdhR (lane 1, 0 pmol; lane 2, 2.5 pmol; lane 3, 5 pmol; lane 4, 10 pmol) and subjected to DNase I footprinting assays. Lanes A, T, G, and C represent sequence ladders. (B) The black bar (positions −185 to −158) on the right indicates the PdhR-binding region located upstream of the cyoA ) (solid line). (C) Primer extension assay of the transcription initiation site of the cyoA operon in both wild-type KP7600 and its pdhR ).
Figure Legend Snippet: DNase I footprinting of the cyoA promoter. (A) The fluorescently labeled cyoA promoter was incubated with increasing amounts of purified PdhR (lane 1, 0 pmol; lane 2, 2.5 pmol; lane 3, 5 pmol; lane 4, 10 pmol) and subjected to DNase I footprinting assays. Lanes A, T, G, and C represent sequence ladders. (B) The black bar (positions −185 to −158) on the right indicates the PdhR-binding region located upstream of the cyoA ) (solid line). (C) Primer extension assay of the transcription initiation site of the cyoA operon in both wild-type KP7600 and its pdhR ).

Techniques Used: Footprinting, Labeling, Incubation, Purification, Sequencing, Binding Assay, Primer Extension Assay

23) Product Images from "Age-associated chromatin relaxation is enhanced in Huntington's disease mice"

Article Title: Age-associated chromatin relaxation is enhanced in Huntington's disease mice

Journal: Aging (Albany NY)

doi: 10.18632/aging.101193

DNase-I hypersensitive site profiling in aged Huntington's disease (HD) and wild-type splenic cells ( A ) Scatter plot of DNase-I hypersensitive (DHS) read counts (log 2 ) between HD and wt around transcription start site (TSS). DHS reads ± 2 kb around TSSs were counted. Colored dots (orange for HD-high and green for wt-high) indicate DHSs reads with 2-fold or more count differences. ( B ) Heatmaps of DHS signals around TSSs (± 2 kb) in HD and wt mice. DHS signals were clustered by the k -means algorithm ( k = 2). Sites are ordered by DHS signal intensity around TSS. ( C-D ) Read count distribution around TSSs (± 2 kb, left) and the mean DHS signal density around TSSs (± 300 bp, right) in cluster-1 (C) and cluster-2 (D). ( E ) Comparison of DHS signal density at PRG TSSs between HD and wt samples. Shown are the proportions of PRGs with HD-high (orange) and wt-high (gray) DHS signals. Of the 16 PRGs, 15 PRGs belong to cluster-2 and 10 (63%) have denser signals in HD sample than in wt.
Figure Legend Snippet: DNase-I hypersensitive site profiling in aged Huntington's disease (HD) and wild-type splenic cells ( A ) Scatter plot of DNase-I hypersensitive (DHS) read counts (log 2 ) between HD and wt around transcription start site (TSS). DHS reads ± 2 kb around TSSs were counted. Colored dots (orange for HD-high and green for wt-high) indicate DHSs reads with 2-fold or more count differences. ( B ) Heatmaps of DHS signals around TSSs (± 2 kb) in HD and wt mice. DHS signals were clustered by the k -means algorithm ( k = 2). Sites are ordered by DHS signal intensity around TSS. ( C-D ) Read count distribution around TSSs (± 2 kb, left) and the mean DHS signal density around TSSs (± 300 bp, right) in cluster-1 (C) and cluster-2 (D). ( E ) Comparison of DHS signal density at PRG TSSs between HD and wt samples. Shown are the proportions of PRGs with HD-high (orange) and wt-high (gray) DHS signals. Of the 16 PRGs, 15 PRGs belong to cluster-2 and 10 (63%) have denser signals in HD sample than in wt.

Techniques Used: Mouse Assay

24) Product Images from "Identification and Characterization of a Novel Hepta-Segmented dsRNA Virus From the Phytopathogenic Fungus Colletotrichum fructicola"

Article Title: Identification and Characterization of a Novel Hepta-Segmented dsRNA Virus From the Phytopathogenic Fungus Colletotrichum fructicola

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2018.00754

Double-stranded RNA (dsRNAs) extracted from strain FJ-4 and the genomic organization of Colletotrichum fructicola dsRNA virus 1 (CfCV1). (A) 1.2% agarose gel electrophoretic profiles of dsRNA preparations extracted from strains FJ-4 and FJ-85 after digestion with DNase I and S1 nuclease. (B) Genomic organization of dsRNA1–7 of CfCV1.
Figure Legend Snippet: Double-stranded RNA (dsRNAs) extracted from strain FJ-4 and the genomic organization of Colletotrichum fructicola dsRNA virus 1 (CfCV1). (A) 1.2% agarose gel electrophoretic profiles of dsRNA preparations extracted from strains FJ-4 and FJ-85 after digestion with DNase I and S1 nuclease. (B) Genomic organization of dsRNA1–7 of CfCV1.

Techniques Used: Agarose Gel Electrophoresis

25) Product Images from "Coordinated Regulation of gnd, Which Encodes 6-Phosphogluconate Dehydrogenase, by the Two Transcriptional Regulators GntR1 and RamA in Corynebacterium glutamicum"

Article Title: Coordinated Regulation of gnd, Which Encodes 6-Phosphogluconate Dehydrogenase, by the Two Transcriptional Regulators GntR1 and RamA in Corynebacterium glutamicum

Journal: Journal of Bacteriology

doi: 10.1128/JB.01635-12

DNase I footprinting analysis of GntR1 and RamA binding to the gnd promoter. The DNA fragment of the intergenic region between gnd and cgR_1514 fluorescently labeled at the gnd gene terminal site was incubated with the indicated amount of RamA and GntR1.
Figure Legend Snippet: DNase I footprinting analysis of GntR1 and RamA binding to the gnd promoter. The DNA fragment of the intergenic region between gnd and cgR_1514 fluorescently labeled at the gnd gene terminal site was incubated with the indicated amount of RamA and GntR1.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation

26) Product Images from "Bacillus subtilis IolQ (DegA) is a transcriptional repressor of iolX encoding NAD+-dependent scyllo-inositol dehydrogenase"

Article Title: Bacillus subtilis IolQ (DegA) is a transcriptional repressor of iolX encoding NAD+-dependent scyllo-inositol dehydrogenase

Journal: BMC Microbiology

doi: 10.1186/s12866-017-1065-8

DNase I foot printing of IolQ-His 6 on the iolX promoter region. DNase I foot printing of the upper a and lower b strands. Sequence data are shown on the top and below are fragment analysis data acquired using various concentrations of IolQ-His 6 as indicated on the right. c Summary of DNase I foot printing data. The nucleotide sequences (upper and lower strands) of the DNA fragment that correspond to the 200 bp iolX promoter region used for the electrophoretic gel mobility shift assay are shown. Transcription initiation sites +1 (P1) and +1 (P2) and their corresponding −35 and −10 regions are indicated. The protected regions with higher and lower affinities are indicated by black and gray bars, respectively. The conserved sequences within the protected regions are boxed. The cre sites are indicated by the dashed bars between the upper and lower strand sequences within the two regions for IolQ binding
Figure Legend Snippet: DNase I foot printing of IolQ-His 6 on the iolX promoter region. DNase I foot printing of the upper a and lower b strands. Sequence data are shown on the top and below are fragment analysis data acquired using various concentrations of IolQ-His 6 as indicated on the right. c Summary of DNase I foot printing data. The nucleotide sequences (upper and lower strands) of the DNA fragment that correspond to the 200 bp iolX promoter region used for the electrophoretic gel mobility shift assay are shown. Transcription initiation sites +1 (P1) and +1 (P2) and their corresponding −35 and −10 regions are indicated. The protected regions with higher and lower affinities are indicated by black and gray bars, respectively. The conserved sequences within the protected regions are boxed. The cre sites are indicated by the dashed bars between the upper and lower strand sequences within the two regions for IolQ binding

Techniques Used: Sequencing, Mobility Shift, Binding Assay

27) Product Images from "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 ▿ §"

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 ▿ §

Journal: Journal of Bacteriology

doi: 10.1128/JB.00215-10

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.
Figure Legend 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.

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

28) Product Images from "Discovery of Two Novel Viruses Expands the Diversity of Single-Stranded DNA and Single-Stranded RNA Viruses Infecting a Cosmopolitan Marine Diatom"

Article Title: Discovery of Two Novel Viruses Expands the Diversity of Single-Stranded DNA and Single-Stranded RNA Viruses Infecting a Cosmopolitan Marine Diatom

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02380-14

Nucleic acid analysis of CtenDNAV type II and CtenRNAV type II. (A) CtenDNAV type II genome. Nucleic acids of CtenDNAV type II without treatment (lane 1), treated at 100°C for 5 min (lane 2), treated with DNase I (lane 3), treated with RNase A (lane 4), and treated with S1 nuclease (lane 5) are shown. The samples were electrophoresed on a formaldehyde-agarose gel. (B) CtenRNAV type II genome without treatment (lane 1) and treated with DNase I (lane 2) and RNase A (lane 3).
Figure Legend Snippet: Nucleic acid analysis of CtenDNAV type II and CtenRNAV type II. (A) CtenDNAV type II genome. Nucleic acids of CtenDNAV type II without treatment (lane 1), treated at 100°C for 5 min (lane 2), treated with DNase I (lane 3), treated with RNase A (lane 4), and treated with S1 nuclease (lane 5) are shown. The samples were electrophoresed on a formaldehyde-agarose gel. (B) CtenRNAV type II genome without treatment (lane 1) and treated with DNase I (lane 2) and RNase A (lane 3).

Techniques Used: Agarose Gel Electrophoresis

29) Product Images from "Fluorescence Resonance Energy Transfer-Based DNA Tetrahedron Nanotweezer for Highly Reliable Detection of Tumor-Related mRNA in Living Cells"

Article Title: Fluorescence Resonance Energy Transfer-Based DNA Tetrahedron Nanotweezer for Highly Reliable Detection of Tumor-Related mRNA in Living Cells

Journal: ACS nano

doi: 10.1021/acsnano.7b00725

(A) Electrophoresis characterization for the degradation of DTNT nanoprobe treated with (a) low concentration of Dnase I (0.5 U/mL) and (b) high concentration of Dnase I (5 U/mL). (B) Emission intensity ratio ( F A / F D ) as a function of time treated with different concentrations of Dnase I.
Figure Legend Snippet: (A) Electrophoresis characterization for the degradation of DTNT nanoprobe treated with (a) low concentration of Dnase I (0.5 U/mL) and (b) high concentration of Dnase I (5 U/mL). (B) Emission intensity ratio ( F A / F D ) as a function of time treated with different concentrations of Dnase I.

Techniques Used: Electrophoresis, Concentration Assay

30) Product Images from "The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3"

Article Title: The Histone-Like Nucleoid Structuring Protein (H-NS) Is a Negative Regulator of the Lateral Flagellar System in the Deep-Sea Bacterium Shewanella piezotolerans WP3

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00297-16

Identification of the sequence of the H-NS-protected regions of the motY (A) and lafB (B) promoter by DNase I protection footprinting. A concentration of 0.075 μM probe P motY and P lafB covering the entire promoter region of motY and lafB was incubated with H-NS (at 0.9 μM and 3.6 μM) in the EMSA buffer. The promoter fragments were labeled with 6-carboxyfluorescein (FAM) dye. The regions protected by H-NS from DNase I cleavage are indicated with red dotted boxes. The sequences of the high-affinity H-NS binding region are shown at the bottom (with the protected region in gray shading), and the start codons are underlined. The black box denotes the conserved H-NS binding motif.
Figure Legend Snippet: Identification of the sequence of the H-NS-protected regions of the motY (A) and lafB (B) promoter by DNase I protection footprinting. A concentration of 0.075 μM probe P motY and P lafB covering the entire promoter region of motY and lafB was incubated with H-NS (at 0.9 μM and 3.6 μM) in the EMSA buffer. The promoter fragments were labeled with 6-carboxyfluorescein (FAM) dye. The regions protected by H-NS from DNase I cleavage are indicated with red dotted boxes. The sequences of the high-affinity H-NS binding region are shown at the bottom (with the protected region in gray shading), and the start codons are underlined. The black box denotes the conserved H-NS binding motif.

Techniques Used: Sequencing, Footprinting, Concentration Assay, Incubation, Labeling, Binding Assay

31) Product Images from "Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation"

Article Title: Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

Journal: BMC Microbiology

doi: 10.1186/1471-2180-11-146

S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I . (A) Attached SE1457 ΔatlE , SE1457 ΔsaeRS , SE1457 and SE1457 saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P
Figure Legend Snippet: S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I . (A) Attached SE1457 ΔatlE , SE1457 ΔsaeRS , SE1457 and SE1457 saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P

Techniques Used: Microscopy, Incubation, Cell Culture

Effect of DNaseI on SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilm formation . SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P
Figure Legend Snippet: Effect of DNaseI on SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilm formation . SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P

Techniques Used: Staining

32) Product Images from "The expression and construction of engineering Escherichia coli producing humanized AluY RNAs"

Article Title: The expression and construction of engineering Escherichia coli producing humanized AluY RNAs

Journal: Microbial Cell Factories

doi: 10.1186/s12934-017-0800-z

The estimation of yield of pure AluY RNAs. a The results of northern detection. RNAs were extracted from pET-AluY × 14 DE3 bacteria induced by IPTG and digested with DNase I. 5 μg RNAs digested with DNase I were loaded in lane 1, 0.2 μg Alu reference was loaded in lane 2; b IOD values of each lane of the northern blotting results (means of three independent experiments)
Figure Legend Snippet: The estimation of yield of pure AluY RNAs. a The results of northern detection. RNAs were extracted from pET-AluY × 14 DE3 bacteria induced by IPTG and digested with DNase I. 5 μg RNAs digested with DNase I were loaded in lane 1, 0.2 μg Alu reference was loaded in lane 2; b IOD values of each lane of the northern blotting results (means of three independent experiments)

Techniques Used: Northern Blot, Positron Emission Tomography

Effects of digestion of DNA enzyme and RNA enzyme. a The result of northern detection. pET-AluY × 8 DE3 bacteria were induced with IPTG (final concentration 0.2 mg/ml) at 37 °C for 4 h. RNAs were extracted using SDS-hot phenol method and digested with DNase I (lane 1), without DNase I (lane 2), or with DNase I plus RNase A (lane 3). AluY RNAs were detected using northern blotting. b Dyeing results of methylene blue; c IOD values of each lane of the northern blotting results (means of three independent experiments). “*”, the AluY RNA yield of pET-AluY × 8 DNase I digestion and pET-AluY × 8 without DNase I digestion was significantly higher than that of pET-AluY × 8 DNase I plus RNase A digestion (p
Figure Legend Snippet: Effects of digestion of DNA enzyme and RNA enzyme. a The result of northern detection. pET-AluY × 8 DE3 bacteria were induced with IPTG (final concentration 0.2 mg/ml) at 37 °C for 4 h. RNAs were extracted using SDS-hot phenol method and digested with DNase I (lane 1), without DNase I (lane 2), or with DNase I plus RNase A (lane 3). AluY RNAs were detected using northern blotting. b Dyeing results of methylene blue; c IOD values of each lane of the northern blotting results (means of three independent experiments). “*”, the AluY RNA yield of pET-AluY × 8 DNase I digestion and pET-AluY × 8 without DNase I digestion was significantly higher than that of pET-AluY × 8 DNase I plus RNase A digestion (p

Techniques Used: Northern Blot, Positron Emission Tomography, Concentration Assay

33) Product Images from "Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1"

Article Title: Sulfolobus Replication Factor C Stimulates the Activity of DNA Polymerase B1

Journal: Journal of Bacteriology

doi: 10.1128/JB.01552-14

Interaction between RFC and PolB1. (A) Pulldown analysis of the interaction between RFC and PolB1 from  S. islandicus .  S. islandicus  (PolB1-Tag) was grown to the exponential phase and harvested. The cell extract was incubated with Ni 2+  beads. The beads were subsequently washed and treated with DNase I, if indicated. Proteins retained by the beads were subjected to SDS-PAGE and identified by immunoblotting with antibodies against RFC S . Lane C, a cell extract from  S. islandicus , instead of  S. islandicus  PolB1-Tag, was used in the assay as a control for nonspecific binding of RFC to Ni 2+  beads. (B) Blue native PAGE. RFC (30 pmol) and PolB1 (30 pmol) were mixed individually or in combination with loading buffer for blue native PAGE. Samples were subjected to electrophoresis in a 5 to 15% gradient blue native polyacrylamide gel at 10°C. Molecular mass markers are indicated on the left. RFC-PolB1 complexes are indicated by arrows. (C) Bio-layer interferometry. Amine-reactive (AR) biosensors, covered with immobilized PolB1, were treated in RFC solutions of increasing concentration (160, 320, 650, 1,300, and 2,600 nM), as represented by curves from bottom to top, respectively. The rate constants  k a  and  k d  were determined, and the equilibrium dissociation constant  K D  was calculated as described by the manufacturer.
Figure Legend Snippet: Interaction between RFC and PolB1. (A) Pulldown analysis of the interaction between RFC and PolB1 from S. islandicus . S. islandicus (PolB1-Tag) was grown to the exponential phase and harvested. The cell extract was incubated with Ni 2+ beads. The beads were subsequently washed and treated with DNase I, if indicated. Proteins retained by the beads were subjected to SDS-PAGE and identified by immunoblotting with antibodies against RFC S . Lane C, a cell extract from S. islandicus , instead of S. islandicus PolB1-Tag, was used in the assay as a control for nonspecific binding of RFC to Ni 2+ beads. (B) Blue native PAGE. RFC (30 pmol) and PolB1 (30 pmol) were mixed individually or in combination with loading buffer for blue native PAGE. Samples were subjected to electrophoresis in a 5 to 15% gradient blue native polyacrylamide gel at 10°C. Molecular mass markers are indicated on the left. RFC-PolB1 complexes are indicated by arrows. (C) Bio-layer interferometry. Amine-reactive (AR) biosensors, covered with immobilized PolB1, were treated in RFC solutions of increasing concentration (160, 320, 650, 1,300, and 2,600 nM), as represented by curves from bottom to top, respectively. The rate constants k a and k d were determined, and the equilibrium dissociation constant K D was calculated as described by the manufacturer.

Techniques Used: Incubation, SDS Page, Binding Assay, Blue Native PAGE, Electrophoresis, Concentration Assay

34) Product Images from "Identification and characterization of DNA endonucleases in Plasmodium falciparum 3D7 clone"

Article Title: Identification and characterization of DNA endonucleases in Plasmodium falciparum 3D7 clone

Journal: Malaria Journal

doi: 10.1186/s12936-018-2388-0

Transcription of the seven DNA endonucleases genes of the  P. falciparum  3D7 clone. The transcriptions of the seven DNA endonuclease genes at 8, 16, 24, 32, 40 and 48 h post invasion are shown. Transcript levels relative to that of gene PF3D7_0305600 at 16 h post invasion were calculated as 2 −∆∆Ct . A log-scale was calculated and used on the y-axis
Figure Legend Snippet: Transcription of the seven DNA endonucleases genes of the P. falciparum 3D7 clone. The transcriptions of the seven DNA endonuclease genes at 8, 16, 24, 32, 40 and 48 h post invasion are shown. Transcript levels relative to that of gene PF3D7_0305600 at 16 h post invasion were calculated as 2 −∆∆Ct . A log-scale was calculated and used on the y-axis

Techniques Used:

35) Product Images from "Negative Regulation of DNA Repair Gene (ung) Expression by the CpxR/CpxA Two-Component System in Escherichia coli K-12 and Induction of Mutations by Increased Expression of CpxR"

Article Title: Negative Regulation of DNA Repair Gene (ung) Expression by the CpxR/CpxA Two-Component System in Escherichia coli K-12 and Induction of Mutations by Increased Expression of CpxR

Journal: Journal of Bacteriology

doi: 10.1128/JB.186.24.8317-8325.2004

DNase I footprinting assay. Probe A was incubated with various amounts of the purified CpxR (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 30 pmol; lane 5, 40 pmol; lane 6, 50 pmol; lane 7, 60 pmol; lane 8, 70 pmol; lane 9, 80 pmol) and subjected to DNase I footprinting assays. Lanes AG represent the Maxam-Gilbert sequence ladder. The black boxes and bold arrows indicate the CpxR binding region and the direct repeat, respectively.
Figure Legend Snippet: DNase I footprinting assay. Probe A was incubated with various amounts of the purified CpxR (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 30 pmol; lane 5, 40 pmol; lane 6, 50 pmol; lane 7, 60 pmol; lane 8, 70 pmol; lane 9, 80 pmol) and subjected to DNase I footprinting assays. Lanes AG represent the Maxam-Gilbert sequence ladder. The black boxes and bold arrows indicate the CpxR binding region and the direct repeat, respectively.

Techniques Used: Footprinting, Incubation, Purification, Sequencing, Binding Assay

36) Product Images from "Transcriptional Regulation of the CmeABC Multidrug Efflux Pump and the KatA Catalase by CosR in Campylobacter jejuni"

Article Title: Transcriptional Regulation of the CmeABC Multidrug Efflux Pump and the KatA Catalase by CosR in Campylobacter jejuni

Journal: Journal of Bacteriology

doi: 10.1128/JB.01636-12

Determination of the CosR binding sites in katA and cmeABC promoters by DNase I footprinting. The CosR binding regions in the promoter regions of katA (A) and cmeABC ). The PerR binding region (C) in the katA promoter and the CmeR binding region (D) in cmeABC promoter are marked with boldface letters and arrows. The start codon, the transcriptional start site (+1), and the −10, −16, and −35 elements are underlined. The regions protected by rCosR from DNase I cleavage are indicated by a black background.
Figure Legend Snippet: Determination of the CosR binding sites in katA and cmeABC promoters by DNase I footprinting. The CosR binding regions in the promoter regions of katA (A) and cmeABC ). The PerR binding region (C) in the katA promoter and the CmeR binding region (D) in cmeABC promoter are marked with boldface letters and arrows. The start codon, the transcriptional start site (+1), and the −10, −16, and −35 elements are underlined. The regions protected by rCosR from DNase I cleavage are indicated by a black background.

Techniques Used: Binding Assay, Footprinting

37) Product Images from "Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation"

Article Title: Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation

Journal: BMC Microbiology

doi: 10.1186/s12866-018-1317-2

DNase I footprints of PhaR on the phaP1 ( a ) and phaP4 ( b ) promoter regions. DNaseI footprints of PhaR-His 6 binding found in DNA fragment patterns are shown for the phaP1 ( a ) and phaP4 ( b ) promoter regions; the fragment patterns on upper and lower strands are in the left and right side of each panel, respectively. Each of the panels contain four fragment charts for respective upper and lower strands; from the top to down, the first is the sequencing ladders in four colors, the second is the negative control without PhaR-His 6 , and the third and fourth are the two different concentrations of PhaR-His 6 as indicated. At the bottom of each panel, nucleotide sequences of the promoter regions of phaP1 ( a ) and phaP4 ( b ) are shown. The sequence stretches protected from DNase I digestion by PhaR-His 6 binding are shown in hatched squares. Hocked arrowheads and the labels “+ 1” indicate the transcriptional start point (shown in blue letters in the upper strands). The −35 and − 10 regions are underlined and the ATG initiating codons are shown in red letters in the upper strands
Figure Legend Snippet: DNase I footprints of PhaR on the phaP1 ( a ) and phaP4 ( b ) promoter regions. DNaseI footprints of PhaR-His 6 binding found in DNA fragment patterns are shown for the phaP1 ( a ) and phaP4 ( b ) promoter regions; the fragment patterns on upper and lower strands are in the left and right side of each panel, respectively. Each of the panels contain four fragment charts for respective upper and lower strands; from the top to down, the first is the sequencing ladders in four colors, the second is the negative control without PhaR-His 6 , and the third and fourth are the two different concentrations of PhaR-His 6 as indicated. At the bottom of each panel, nucleotide sequences of the promoter regions of phaP1 ( a ) and phaP4 ( b ) are shown. The sequence stretches protected from DNase I digestion by PhaR-His 6 binding are shown in hatched squares. Hocked arrowheads and the labels “+ 1” indicate the transcriptional start point (shown in blue letters in the upper strands). The −35 and − 10 regions are underlined and the ATG initiating codons are shown in red letters in the upper strands

Techniques Used: Binding Assay, Sequencing, Negative Control

38) Product Images from "Regulation of Oxidative Stress Response by CosR, an Essential Response Regulator in Campylobacter jejuni"

Article Title: Regulation of Oxidative Stress Response by CosR, an Essential Response Regulator in Campylobacter jejuni

Journal: PLoS ONE

doi: 10.1371/journal.pone.0022300

DNase I footprinting of the sodB and ahpC promoter regions. The CosR binding sites in the promoter regions of sodB (A) and ahpC (B) were determined by DNase I footprinting assays. Based on previous studies [6] , [9] , the start codon (ATG), transcriptional start site (+1), and −10 and −35 regions of sodB and ahpC are indicated on the left. The CosR binding sites are indicated with dotted lines and labeled “BsodB” for the binding site in the sodB promoter and “BahpC-1 and BahpC-2” for the two binding sites in the ahpC promoter. (C) Alignment of CosR binding sequences for sodB and ahpC . Nucleotide sequences of the CosR binding sites determined from panel (A) and (B) were compared. (D) Prediction of putative CosR binding sites in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence described in panel (C). Highly conserved nucleotides are shaded on black background, and identical nucleotides are indicated in capital letters.
Figure Legend Snippet: DNase I footprinting of the sodB and ahpC promoter regions. The CosR binding sites in the promoter regions of sodB (A) and ahpC (B) were determined by DNase I footprinting assays. Based on previous studies [6] , [9] , the start codon (ATG), transcriptional start site (+1), and −10 and −35 regions of sodB and ahpC are indicated on the left. The CosR binding sites are indicated with dotted lines and labeled “BsodB” for the binding site in the sodB promoter and “BahpC-1 and BahpC-2” for the two binding sites in the ahpC promoter. (C) Alignment of CosR binding sequences for sodB and ahpC . Nucleotide sequences of the CosR binding sites determined from panel (A) and (B) were compared. (D) Prediction of putative CosR binding sites in the regulatory region of dps and luxS genes based on homology to the CosR-binding sequence described in panel (C). Highly conserved nucleotides are shaded on black background, and identical nucleotides are indicated in capital letters.

Techniques Used: Footprinting, Binding Assay, Labeling, Sequencing

39) Product Images from "Isolation and Characterization of a Single-Stranded DNA Virus Infecting the Marine Diatom Chaetoceros sp. Strain SS628-11 Isolated from Western JAPAN"

Article Title: Isolation and Characterization of a Single-Stranded DNA Virus Infecting the Marine Diatom Chaetoceros sp. Strain SS628-11 Isolated from Western JAPAN

Journal: PLoS ONE

doi: 10.1371/journal.pone.0082013

Nucleic acids analysis of Csp07DNAVgenome. (A) Csp07DNAV genome. Extracts of DNA (lane 1) and RNA (lane 2). (B) Nucleic acids of Csp07DNAV without treatment (lane 1), 100°C for 5 min (lane 2), treated with DNase I (lane 3), RNase A (lane 4), and S1 nuclease (lane 5). The samples were electrophoresed on a formaldehyde-agarose gel.
Figure Legend Snippet: Nucleic acids analysis of Csp07DNAVgenome. (A) Csp07DNAV genome. Extracts of DNA (lane 1) and RNA (lane 2). (B) Nucleic acids of Csp07DNAV without treatment (lane 1), 100°C for 5 min (lane 2), treated with DNase I (lane 3), RNase A (lane 4), and S1 nuclease (lane 5). The samples were electrophoresed on a formaldehyde-agarose gel.

Techniques Used: Agarose Gel Electrophoresis

40) Product Images from "Designing a Biostable L-DNAzyme for Lead(II) Ion Detection in Practical Samples"

Article Title: Designing a Biostable L-DNAzyme for Lead(II) Ion Detection in Practical Samples

Journal: Analytical methods : advancing methods and applications

doi: 10.1039/C6AY01791F

Fluorescence spectra of D-S (A) and L-S (B) incubated in regular reaction buffer (50 mM HEPES, 50 mM NaCl and 5 mM MgCl 2  at pH 7.2) from 0 to 31 days. (C) Denatured PAGE of the Pb 2+ -dependent L-/D-DNAzyme in the absence or presence of DNase I. L-E with (Lane 1) or without (Lane 2) DNase I; D-E with (Lane 3) or without (Lane 4) DNase I. RFI: relative fluorescence intensity.
Figure Legend Snippet: Fluorescence spectra of D-S (A) and L-S (B) incubated in regular reaction buffer (50 mM HEPES, 50 mM NaCl and 5 mM MgCl 2 at pH 7.2) from 0 to 31 days. (C) Denatured PAGE of the Pb 2+ -dependent L-/D-DNAzyme in the absence or presence of DNase I. L-E with (Lane 1) or without (Lane 2) DNase I; D-E with (Lane 3) or without (Lane 4) DNase I. RFI: relative fluorescence intensity.

Techniques Used: Fluorescence, Incubation, Polyacrylamide Gel Electrophoresis

41) Product Images from "DNA binding and helicase actions of mouse MCM4/6/7 helicase"

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki607

Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.
Figure Legend Snippet: Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.

Techniques Used: Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

42) Product Images from "SIRT1 increases YAP- and MKK3-dependent p38 phosphorylation in mouse liver and human hepatocellular carcinoma"

Article Title: SIRT1 increases YAP- and MKK3-dependent p38 phosphorylation in mouse liver and human hepatocellular carcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.7022

Mechanism underlying SIRT1-induced increases in MKK3 transcription via YAP A-D. Pol II enrichment at −2k (upstream of TSS), +1 (near the TSS), and +2k (downstream of the gene) regions of the mYAP and mMKK3 promoters respectively, as measured by ChIP-qPCR in the livers of paired WT-I and mSIRT1-KI mice (A and C) or paired WT-O and mSIRT1-LKO mice (B and D). E-F. YAP enrichment at the −2k, +1, and +2k regions of the mMKK3 gene in the livers of paired WT-I and mSIRT1-KI mice (E) and paired WT-O and mSIRT1-LKO mice (F), as measured by ChIP-qPCR. G-H. YAP occupancy at the −2k, +1, and +2k regions of the hMKK3 gene in Bel-7402 (G) and SMMC-7721 (H) cells, as measured by ChIP-qPCR using anti-YAP antibodies. Non-specific IgG antibodies were also used as a control. I-J. YAP enrichment at the +1 region within the hMKK3 promoter as measured by ChIP-qPCR in control and Bel-7402 or SMMC-7721 cells with hSIRT1 overexpression (I) or knockdown (J). K-L. Pol II enrichment at the +1 region of the hMKK3 promoter in Bel-7402 (K) and SMMC-7721 cells (L) after the indicated treatments. M. DNase I accessibility of the +1 region of the mMKK3 promoter in the livers of WT, mSIRT1-KI, and mSIRT1-LKO mice, as measured by CHART-PCR. N. DNase I accessibility of the +1 region of the hMKK3 promoter in control and Bel-7402 or SMMC-7721 cells with either SIRT1 overexpression or knockdown, as measured by CHART-PCR. The data are shown as mean±SD from three independent experiments. **, p
Figure Legend Snippet: Mechanism underlying SIRT1-induced increases in MKK3 transcription via YAP A-D. Pol II enrichment at −2k (upstream of TSS), +1 (near the TSS), and +2k (downstream of the gene) regions of the mYAP and mMKK3 promoters respectively, as measured by ChIP-qPCR in the livers of paired WT-I and mSIRT1-KI mice (A and C) or paired WT-O and mSIRT1-LKO mice (B and D). E-F. YAP enrichment at the −2k, +1, and +2k regions of the mMKK3 gene in the livers of paired WT-I and mSIRT1-KI mice (E) and paired WT-O and mSIRT1-LKO mice (F), as measured by ChIP-qPCR. G-H. YAP occupancy at the −2k, +1, and +2k regions of the hMKK3 gene in Bel-7402 (G) and SMMC-7721 (H) cells, as measured by ChIP-qPCR using anti-YAP antibodies. Non-specific IgG antibodies were also used as a control. I-J. YAP enrichment at the +1 region within the hMKK3 promoter as measured by ChIP-qPCR in control and Bel-7402 or SMMC-7721 cells with hSIRT1 overexpression (I) or knockdown (J). K-L. Pol II enrichment at the +1 region of the hMKK3 promoter in Bel-7402 (K) and SMMC-7721 cells (L) after the indicated treatments. M. DNase I accessibility of the +1 region of the mMKK3 promoter in the livers of WT, mSIRT1-KI, and mSIRT1-LKO mice, as measured by CHART-PCR. N. DNase I accessibility of the +1 region of the hMKK3 promoter in control and Bel-7402 or SMMC-7721 cells with either SIRT1 overexpression or knockdown, as measured by CHART-PCR. The data are shown as mean±SD from three independent experiments. **, p

Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Mouse Assay, Over Expression, Polymerase Chain Reaction

43) Product Images from "Activation of the chicken Ig-? locus by the collaboration of scattered regulatory regions through changes in chromatin structure"

Article Title: Activation of the chicken Ig-? locus by the collaboration of scattered regulatory regions through changes in chromatin structure

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkl469

DNase I general sensitivity of the chromatin in cells with combinations of DHS deletions. Isolated nuclei were treated with DNase I for 3 min at 20°C. The concentrations of DNase I for treatment of nuclei from left to right: 100, 200, 400, 600 and 800 U/ml. The DNA was purified from nuclei and digested with BamHI for the olfactory receptor domain, PstI for the Ig-β gene or KpnI/EcoRI for the G3PDH gene. The digests (3.0 μg) were subjected to Southern hybridization with probe P10 ( 30 ) for the olfactory receptor domain. The same Ig-β and G3PDH cDNA probes were used as in Figure 3 for the analysis of Ig-β and G3PDH genes. The analyzed region, DHS, and the position of the probe are shown at the bottom. The intensity of bands was determined by using a Typhoon 9210 microarray imager and Image Quant software. Relative values are shown in the right figures, in which the value at 100 U/ml DNase I treatment is plotted as 100%. Closed triangle, olfactory receptor domain; open square, Ig-β gene; closed circle, G3PDH gene.
Figure Legend Snippet: DNase I general sensitivity of the chromatin in cells with combinations of DHS deletions. Isolated nuclei were treated with DNase I for 3 min at 20°C. The concentrations of DNase I for treatment of nuclei from left to right: 100, 200, 400, 600 and 800 U/ml. The DNA was purified from nuclei and digested with BamHI for the olfactory receptor domain, PstI for the Ig-β gene or KpnI/EcoRI for the G3PDH gene. The digests (3.0 μg) were subjected to Southern hybridization with probe P10 ( 30 ) for the olfactory receptor domain. The same Ig-β and G3PDH cDNA probes were used as in Figure 3 for the analysis of Ig-β and G3PDH genes. The analyzed region, DHS, and the position of the probe are shown at the bottom. The intensity of bands was determined by using a Typhoon 9210 microarray imager and Image Quant software. Relative values are shown in the right figures, in which the value at 100 U/ml DNase I treatment is plotted as 100%. Closed triangle, olfactory receptor domain; open square, Ig-β gene; closed circle, G3PDH gene.

Techniques Used: Isolation, Purification, Hybridization, Microarray, Software

DNase I hypersensitive sites in cells with combinations of DHS deletions. Isolated nuclei were treated with DNase I for 3 min at 20°C. The concentrations of DNase I for treatment of nuclei from left to right: 0, 25, 50, 75 and 100 U/ml. The DNA was purified from nuclei and digested with HindIII ( A ), ScaI ( B ) or EcoT22I ( C ). The digests (3.0 μg) were subjected to Southern hybridization with 0.7 kb SacI/SacI DNA (−12.1 to −11.4 kb) (A), 0.9 kb ScaI/KpnI DNA (−4.6 to −3.7 kb; #849–1751 in AB066568) (B) or 0.2 kb GH exon 5 (#3515–3744 in CHKGHG/D10484) (C). The analyzed region, DHS, and the position of the probe are shown at the right side.
Figure Legend Snippet: DNase I hypersensitive sites in cells with combinations of DHS deletions. Isolated nuclei were treated with DNase I for 3 min at 20°C. The concentrations of DNase I for treatment of nuclei from left to right: 0, 25, 50, 75 and 100 U/ml. The DNA was purified from nuclei and digested with HindIII ( A ), ScaI ( B ) or EcoT22I ( C ). The digests (3.0 μg) were subjected to Southern hybridization with 0.7 kb SacI/SacI DNA (−12.1 to −11.4 kb) (A), 0.9 kb ScaI/KpnI DNA (−4.6 to −3.7 kb; #849–1751 in AB066568) (B) or 0.2 kb GH exon 5 (#3515–3744 in CHKGHG/D10484) (C). The analyzed region, DHS, and the position of the probe are shown at the right side.

Techniques Used: Isolation, Purification, Hybridization

Positions of the DT40-specific DNase I hypersensitive sites (DHSs) and deleted regions in the chicken Ig-β locus. Exons are shown by gray or closed rectangles, introns by open rectangles and intergenic regions by solid lines. Horizontal arrows represent transcriptional orientations. Exon numbers are indicated. Sizes of the intergenic regions are shown in kb. Upward vertical arrows indicate DHSs; distances from the transcriptional starting site of the Ig-β gene in kb are indicated underneath. Closed arrow, DT40-specific; open arrow, common to both DT40 cells and liver-derived LMH cells. DHS arrows with enhancing activity are enclosed by rectangles. Positions of the arm sequences used for the targeting constructs are shown by closed bars and regions deleted are shown by dotted lines. Numbers above the 5′ and 3′ ends of the region deleted are the distances from the transcriptional starting site of the Ig-β gene.
Figure Legend Snippet: Positions of the DT40-specific DNase I hypersensitive sites (DHSs) and deleted regions in the chicken Ig-β locus. Exons are shown by gray or closed rectangles, introns by open rectangles and intergenic regions by solid lines. Horizontal arrows represent transcriptional orientations. Exon numbers are indicated. Sizes of the intergenic regions are shown in kb. Upward vertical arrows indicate DHSs; distances from the transcriptional starting site of the Ig-β gene in kb are indicated underneath. Closed arrow, DT40-specific; open arrow, common to both DT40 cells and liver-derived LMH cells. DHS arrows with enhancing activity are enclosed by rectangles. Positions of the arm sequences used for the targeting constructs are shown by closed bars and regions deleted are shown by dotted lines. Numbers above the 5′ and 3′ ends of the region deleted are the distances from the transcriptional starting site of the Ig-β gene.

Techniques Used: Derivative Assay, Activity Assay, Construct

44) Product Images from "Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis"

Article Title: Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00856-15

DNase I footprinting analysis to identify the binding sites of the RhaR protein and the CcpA/P-Ser-HPr complex in the regulatory region of the rhaEWRBMA operon. A 5′-end- 32 P-labeled DNA probe (0.04 pmol), corresponding to the region around the
Figure Legend Snippet: DNase I footprinting analysis to identify the binding sites of the RhaR protein and the CcpA/P-Ser-HPr complex in the regulatory region of the rhaEWRBMA operon. A 5′-end- 32 P-labeled DNA probe (0.04 pmol), corresponding to the region around the

Techniques Used: Footprinting, Binding Assay, Labeling

45) Product Images from "PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator"

Article Title: PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

Journal: Genes & Development

doi: 10.1101/gad.242644.114

Mitotic activation of PKR depends on dsRNA. ( A ) Treating cells with 5 µg/mL Act D for 3 h resulted in a significant decrease in pPKR signal. ( B ) As a positive control, disruption of nucleolus localization of NPM1 was observed under the same treatment conditions. ( C ) The effect of different enzyme treatments on the pattern of pPKR and its downstream targets. We used enzymes that can digest dsDNA (DNase I); ssRNA (RNase T1); ssRNA and dsRNA (RNase A); and dsDNA, ssRNA, and dsRNA (MNase). H3 and NPM1 were used as controls. Bars, 20 µm.
Figure Legend Snippet: Mitotic activation of PKR depends on dsRNA. ( A ) Treating cells with 5 µg/mL Act D for 3 h resulted in a significant decrease in pPKR signal. ( B ) As a positive control, disruption of nucleolus localization of NPM1 was observed under the same treatment conditions. ( C ) The effect of different enzyme treatments on the pattern of pPKR and its downstream targets. We used enzymes that can digest dsDNA (DNase I); ssRNA (RNase T1); ssRNA and dsRNA (RNase A); and dsDNA, ssRNA, and dsRNA (MNase). H3 and NPM1 were used as controls. Bars, 20 µm.

Techniques Used: Activation Assay, Activated Clotting Time Assay, Positive Control

46) Product Images from "Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis"

Article Title: Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00919-12

DNase I footprinting analysis to identify the YcnK binding site in the intergenic region between ycnK and ycnL . A DNA probe including the ycnK - ycnL intergenic region (P ycnK probe), 5′ end labeled at either the coding or noncoding strand, was prepared. The 5′-end-labeled probe (0.8 nM) was incubated in the reaction mixture with the YcnK protein (50 nM [lanes 2] and 25 nM [lanes 3]) as a dimer and without the YcnK protein (lanes 1 and 4). After partial digestion with DNase I, the resulting mixtures were subjected to urea-PAGE. Lanes G, A, T, and C contain the products of the dideoxy sequencing reactions with the corresponding 5′-end-labeled primers. Nucleotide sequences protected by YcnK are indicated on the right of each panel; the 16-bp direct repeat is indicated by tandem arrows.
Figure Legend Snippet: DNase I footprinting analysis to identify the YcnK binding site in the intergenic region between ycnK and ycnL . A DNA probe including the ycnK - ycnL intergenic region (P ycnK probe), 5′ end labeled at either the coding or noncoding strand, was prepared. The 5′-end-labeled probe (0.8 nM) was incubated in the reaction mixture with the YcnK protein (50 nM [lanes 2] and 25 nM [lanes 3]) as a dimer and without the YcnK protein (lanes 1 and 4). After partial digestion with DNase I, the resulting mixtures were subjected to urea-PAGE. Lanes G, A, T, and C contain the products of the dideoxy sequencing reactions with the corresponding 5′-end-labeled primers. Nucleotide sequences protected by YcnK are indicated on the right of each panel; the 16-bp direct repeat is indicated by tandem arrows.

Techniques Used: Footprinting, Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Sequencing

47) Product Images from "WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch"

Article Title: WblAch, a Pivotal Activator of Natamycin Biosynthesis and Morphological Differentiation in Streptomyces chattanoogensis L10, Is Positively Regulated by AdpAch

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.01849-14

DNase I footprinting assay for determination of the AdpA ch -binding site. (A) A 5′-FAM-labeled wblA ch probe was used in the DNase I footprinting assay without or with purified AdpA ch (10 μg). The protected regions are underlined. (B) Sequences
Figure Legend Snippet: DNase I footprinting assay for determination of the AdpA ch -binding site. (A) A 5′-FAM-labeled wblA ch probe was used in the DNase I footprinting assay without or with purified AdpA ch (10 μg). The protected regions are underlined. (B) Sequences

Techniques Used: Footprinting, Binding Assay, Labeling, Purification

48) Product Images from "Two Novel Relative Double-Stranded RNA Mycoviruses Infecting Fusarium poae Strain SX63"

Article Title: Two Novel Relative Double-Stranded RNA Mycoviruses Infecting Fusarium poae Strain SX63

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms17050641

Identification of dsRNA isolated from Fusarium poae . ( A ) Colony morphology of strain SX63 after three days of culture on PDA at 25 °C in the dark; ( B ) Agarose gel electrophoresis of dsRNA isolated from Fusarium poae strain SX63. The nucleic acid was fractionated on 1.0% agarose gel and stained with ethidium bromide. Lane M, DNA marker (15-kb ladder, TaKaRa); lane 1, dsRNA sample with treatment of both RNase-free DNase I and S1 nuclease.
Figure Legend Snippet: Identification of dsRNA isolated from Fusarium poae . ( A ) Colony morphology of strain SX63 after three days of culture on PDA at 25 °C in the dark; ( B ) Agarose gel electrophoresis of dsRNA isolated from Fusarium poae strain SX63. The nucleic acid was fractionated on 1.0% agarose gel and stained with ethidium bromide. Lane M, DNA marker (15-kb ladder, TaKaRa); lane 1, dsRNA sample with treatment of both RNase-free DNase I and S1 nuclease.

Techniques Used: Isolation, Agarose Gel Electrophoresis, Staining, Marker

49) Product Images from "Characterization of SAV7471, a TetR-Family Transcriptional Regulator Involved in the Regulation of Coenzyme A Metabolism in Streptomyces avermitilis"

Article Title: Characterization of SAV7471, a TetR-Family Transcriptional Regulator Involved in the Regulation of Coenzyme A Metabolism in Streptomyces avermitilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00716-13

DNase I footprinting of the coding strand of the SAV7472 promoter region identified using His 6 -SAV7471. (A) The fluorograms correspond to the control DNA (with 10 μM bovine serum albumin [BSA]) and to the protection reactions (with 2 μM
Figure Legend Snippet: DNase I footprinting of the coding strand of the SAV7472 promoter region identified using His 6 -SAV7471. (A) The fluorograms correspond to the control DNA (with 10 μM bovine serum albumin [BSA]) and to the protection reactions (with 2 μM

Techniques Used: Footprinting

50) Product Images from "New single-stranded DNA virus with a unique genomic structure that infects marine diatom Chaetoceros setoensis"

Article Title: New single-stranded DNA virus with a unique genomic structure that infects marine diatom Chaetoceros setoensis

Journal: Scientific Reports

doi: 10.1038/srep03337

Nucleic acids of CsetDNAV untreated (lane 1) or treated with S-1 nuclease (lane 2), DNase I (lane 3), RNase A (lane 4), and heated at 100°C for 2 min (lane 5).
Figure Legend Snippet: Nucleic acids of CsetDNAV untreated (lane 1) or treated with S-1 nuclease (lane 2), DNase I (lane 3), RNase A (lane 4), and heated at 100°C for 2 min (lane 5).

Techniques Used:

51) Product Images from "Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis"

Article Title: Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00856-15

DNase I footprinting analysis to identify the binding sites of the RhaR protein and the CcpA/P-Ser-HPr complex in the regulatory region of the rhaEWRBMA operon. A 5′-end- 32 P-labeled DNA probe (0.04 pmol), corresponding to the region around the
Figure Legend Snippet: DNase I footprinting analysis to identify the binding sites of the RhaR protein and the CcpA/P-Ser-HPr complex in the regulatory region of the rhaEWRBMA operon. A 5′-end- 32 P-labeled DNA probe (0.04 pmol), corresponding to the region around the

Techniques Used: Footprinting, Binding Assay, Labeling

52) Product Images from "PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator"

Article Title: PKR is activated by cellular dsRNAs during mitosis and acts as a mitotic regulator

Journal: Genes & Development

doi: 10.1101/gad.242644.114

Mitotic activation of PKR depends on dsRNA. ( A ) Treating cells with 5 µg/mL Act D for 3 h resulted in a significant decrease in pPKR signal. ( B ) As a positive control, disruption of nucleolus localization of NPM1 was observed under the same treatment conditions. ( C ) The effect of different enzyme treatments on the pattern of pPKR and its downstream targets. We used enzymes that can digest dsDNA (DNase I); ssRNA (RNase T1); ssRNA and dsRNA (RNase A); and dsDNA, ssRNA, and dsRNA (MNase). H3 and NPM1 were used as controls. Bars, 20 µm.
Figure Legend Snippet: Mitotic activation of PKR depends on dsRNA. ( A ) Treating cells with 5 µg/mL Act D for 3 h resulted in a significant decrease in pPKR signal. ( B ) As a positive control, disruption of nucleolus localization of NPM1 was observed under the same treatment conditions. ( C ) The effect of different enzyme treatments on the pattern of pPKR and its downstream targets. We used enzymes that can digest dsDNA (DNase I); ssRNA (RNase T1); ssRNA and dsRNA (RNase A); and dsDNA, ssRNA, and dsRNA (MNase). H3 and NPM1 were used as controls. Bars, 20 µm.

Techniques Used: Activation Assay, Activated Clotting Time Assay, Positive Control

53) Product Images from "Evaluation of the Functional Involvement of Human Immunodeficiency Virus Type 1 Integrase in Nuclear Import of Viral cDNA during Acute Infection"

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

Journal: Journal of Virology

doi: 10.1128/JVI.78.21.11563-11573.2004

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

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

54) Product Images from "Molecular Characterization of a Novel Positive-Sense, Single-Stranded RNA Mycovirus Infecting the Plant Pathogenic Fungus Sclerotinia sclerotiorum"

Article Title: Molecular Characterization of a Novel Positive-Sense, Single-Stranded RNA Mycovirus Infecting the Plant Pathogenic Fungus Sclerotinia sclerotiorum

Journal: Viruses

doi: 10.3390/v7052470

Agarose gel electrophoresis of dsRNA isolated from strain JMTJ14 of S. sclerotiorum . The dsRNA segment was treated with DNase I and S1 nuclease prior to fractionation on a 1.0% agarose gel (lane JMJT14). The size of the dsRNA was estimated using DNA size markers generated by digestion of λ DNA with Hin d III (lane M).
Figure Legend Snippet: Agarose gel electrophoresis of dsRNA isolated from strain JMTJ14 of S. sclerotiorum . The dsRNA segment was treated with DNase I and S1 nuclease prior to fractionation on a 1.0% agarose gel (lane JMJT14). The size of the dsRNA was estimated using DNA size markers generated by digestion of λ DNA with Hin d III (lane M).

Techniques Used: Agarose Gel Electrophoresis, Isolation, Fractionation, Generated

55) Product Images from "CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer"

Article Title: CREB up-regulates long non-coding RNA, HULC expression through interaction with microRNA-372 in liver cancer

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq285

Analysis of chromatin accessibility around the Hulc promoter. ( A ) 5′RACE PCR products suggest the putative TSS is ∼146 bp upstream of the HULC reverse 5′RACE primer (left panel). Mapping the TSS of Hulc in HCC (right panel). ( B ) Schematic representation of the DNA regions around Hulc promoter, which can be amplified by corresponding primer sets. ( C ) DNase I accessibility of Hulc gene in Hep3B and HL-7702 cells. Nuclei from two cell lines were harvested and treated with three units (left panel) or increasing amount (1–7 units, right panel) of DNase I for 5 min at room temperature. Then the genomic DNA was purified and quantitated relative to DNA from undigested nuclei using the primers described in (B) by quantitative PCR and listed as percent protected. ( D ) Restriction enzymes accessibility of Hulc gene in Hep3B and HL-7702 cells. Nuclei was treated with 25–150 units of restriction enzymes for 30 min at 37°C. Then the genomic DNA was purified and quantitated relative to DNA from undigested nuclei using the primers described in (B) by quantitative PCR and listed as percent protected. Chromatin from Hep3B and HL-7702 cells were harvested and precipitated with anti-diacetyl-H3 ( E ), anti-tetra-acetyl-H4 ( F ), anti-tri-methyl-H3-K4 ( G ) and anti-dimethyl-H3-K9 ( H ) antibodies. After DNA recovery, the precipitates were evaluated by real-time PCR for the level of enrichment over negative control antibody using primer sets described in (B). All results are the means ± SD of three independent experiments. The asterisk indicates statistical significance using t -test, * P
Figure Legend Snippet: Analysis of chromatin accessibility around the Hulc promoter. ( A ) 5′RACE PCR products suggest the putative TSS is ∼146 bp upstream of the HULC reverse 5′RACE primer (left panel). Mapping the TSS of Hulc in HCC (right panel). ( B ) Schematic representation of the DNA regions around Hulc promoter, which can be amplified by corresponding primer sets. ( C ) DNase I accessibility of Hulc gene in Hep3B and HL-7702 cells. Nuclei from two cell lines were harvested and treated with three units (left panel) or increasing amount (1–7 units, right panel) of DNase I for 5 min at room temperature. Then the genomic DNA was purified and quantitated relative to DNA from undigested nuclei using the primers described in (B) by quantitative PCR and listed as percent protected. ( D ) Restriction enzymes accessibility of Hulc gene in Hep3B and HL-7702 cells. Nuclei was treated with 25–150 units of restriction enzymes for 30 min at 37°C. Then the genomic DNA was purified and quantitated relative to DNA from undigested nuclei using the primers described in (B) by quantitative PCR and listed as percent protected. Chromatin from Hep3B and HL-7702 cells were harvested and precipitated with anti-diacetyl-H3 ( E ), anti-tetra-acetyl-H4 ( F ), anti-tri-methyl-H3-K4 ( G ) and anti-dimethyl-H3-K9 ( H ) antibodies. After DNA recovery, the precipitates were evaluated by real-time PCR for the level of enrichment over negative control antibody using primer sets described in (B). All results are the means ± SD of three independent experiments. The asterisk indicates statistical significance using t -test, * P

Techniques Used: Polymerase Chain Reaction, Amplification, Purification, Real-time Polymerase Chain Reaction, Negative Control

56) Product Images from "Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors"

Article Title: Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors

Journal: Scientific Reports

doi: 10.1038/s41598-019-56627-0

DNase I and Proteinase K inhibit nicotine-induced biofilm formation. The biofilm formation of S. aureus USA300 strain was detected using a microtiter plate assay by measuring crystal violet stained biofilm at OD570. DNase I (25 U/well) and Proteinase K (2 ug/ml) was added to the well in both 2 mg/ml nicotine treatment group and control group. ( A ) Treated by DNaseI, the average thickness of biofilms varied from 1.506 ± 0.04 to 0.450 ± 0.07 in the nicotine-treated group (n = 3) and from 0.720 ± 0.14 to 0.328 ± 0.05 in untreated group. (n = 3) ( B ) Proteinase K(2 ug/ml) disrupted biofilm formation, in the nicotine treated group decreasing from 1.300 ± 0.06 to 0.393 ± 0.02 (n = 3), and in the control group decreasing from 0.721 ± 0.14 to 0.316 ± 0.08 (n = 3). *P
Figure Legend Snippet: DNase I and Proteinase K inhibit nicotine-induced biofilm formation. The biofilm formation of S. aureus USA300 strain was detected using a microtiter plate assay by measuring crystal violet stained biofilm at OD570. DNase I (25 U/well) and Proteinase K (2 ug/ml) was added to the well in both 2 mg/ml nicotine treatment group and control group. ( A ) Treated by DNaseI, the average thickness of biofilms varied from 1.506 ± 0.04 to 0.450 ± 0.07 in the nicotine-treated group (n = 3) and from 0.720 ± 0.14 to 0.328 ± 0.05 in untreated group. (n = 3) ( B ) Proteinase K(2 ug/ml) disrupted biofilm formation, in the nicotine treated group decreasing from 1.300 ± 0.06 to 0.393 ± 0.02 (n = 3), and in the control group decreasing from 0.721 ± 0.14 to 0.316 ± 0.08 (n = 3). *P

Techniques Used: Staining

57) Product Images from "The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair *"

Article Title: The C-terminal Region and SUMOylation of Cockayne Syndrome Group B Protein Play Critical Roles in Transcription-coupled Nucleotide Excision Repair *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M115.683235

Effects of the deletion in CSB on TC-NER. A , interaction of CSB with Pol II.  Top panel , solubilized chromatin fractions were prepared from UV-irradiated and non-irradiated cells, and CSB was immunoprecipitated with anti-FLAG-agarose. Immunoprecipitated samples were analyzed by Western blotting ( WB ) using anti-Pol II and anti-CSB antibodies.  Bottom panel , the intensities of Pol II and CSB bands were quantified using ImageQuant TL software (GE Healthcare). The intensities in UV-irradiated cells wer compared with those in non-irradiated cells.  B , UV-induced translocation of CSA to the nuclear matrix. CSK-ppt fractions were prepared from UV-irradiated and non-irradiated cells expressing CSB mutants, incubated with CSK-sup fraction containing FLAG-HA epitope-tagged CSA, and treated with DNase I. CSA retained in DNase I-insoluble fractions was detected by Western blotting with an anti-HA antibody. Lamin B is a loading control.  C , association of CSB with chromatin. Insoluble fractions were prepared from cells at the indicated times after UV irradiation and analyzed by Western blotting with an anti-CSB antibody. Samples from the same number of cells were applied to each lane of the gel. Lamin B is a loading control. XPA is associated with chromatin after UV irradiation. The  asterisk  represents shifted bands of CSB.  D , quantification of CSB bands in  C . The intensities of CSB bands were quantified using ImageQuant TL software. The intensity in UV-irradiated cells was compared with that in non-irradiated cells.  Points  are the averages of two independent experiments.
Figure Legend Snippet: Effects of the deletion in CSB on TC-NER. A , interaction of CSB with Pol II. Top panel , solubilized chromatin fractions were prepared from UV-irradiated and non-irradiated cells, and CSB was immunoprecipitated with anti-FLAG-agarose. Immunoprecipitated samples were analyzed by Western blotting ( WB ) using anti-Pol II and anti-CSB antibodies. Bottom panel , the intensities of Pol II and CSB bands were quantified using ImageQuant TL software (GE Healthcare). The intensities in UV-irradiated cells wer compared with those in non-irradiated cells. B , UV-induced translocation of CSA to the nuclear matrix. CSK-ppt fractions were prepared from UV-irradiated and non-irradiated cells expressing CSB mutants, incubated with CSK-sup fraction containing FLAG-HA epitope-tagged CSA, and treated with DNase I. CSA retained in DNase I-insoluble fractions was detected by Western blotting with an anti-HA antibody. Lamin B is a loading control. C , association of CSB with chromatin. Insoluble fractions were prepared from cells at the indicated times after UV irradiation and analyzed by Western blotting with an anti-CSB antibody. Samples from the same number of cells were applied to each lane of the gel. Lamin B is a loading control. XPA is associated with chromatin after UV irradiation. The asterisk represents shifted bands of CSB. D , quantification of CSB bands in C . The intensities of CSB bands were quantified using ImageQuant TL software. The intensity in UV-irradiated cells was compared with that in non-irradiated cells. Points are the averages of two independent experiments.

Techniques Used: Irradiation, Immunoprecipitation, Western Blot, Software, Translocation Assay, Expressing, Incubation

58) Product Images from "Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of the gapA Gene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase of Corynebacterium glutamicum ▿ ▿ †"

Article Title: Involvement of the LuxR-Type Transcriptional Regulator RamA in Regulation of Expression of the gapA Gene, Encoding Glyceraldehyde-3-Phosphate Dehydrogenase of Corynebacterium glutamicum ▿ ▿ †

Journal: Journal of Bacteriology

doi: 10.1128/JB.01425-08

DNase I footprint analysis of the interaction between RamA and the gapA promoter regions examined on the coding and the noncoding strands. A DNA fragment (4 nM) was incubated with different concentrations of RamA: lanes 1 and 7, no protein; lane 2, 200 nM; lane 3, 400 nM; lane 4, 800 nM; lane 5, 1,600 nM; lane 6, 3,200 nM. The three RamA binding sites are indicated with brackets on the left side of each panel. Protected regions are indicated by bars, and hypersensitive sites are indicated by arrows. The DNA sequencing reactions were set up using the same labeled primer and plasmid as for generating labeled footprinting probes.
Figure Legend Snippet: DNase I footprint analysis of the interaction between RamA and the gapA promoter regions examined on the coding and the noncoding strands. A DNA fragment (4 nM) was incubated with different concentrations of RamA: lanes 1 and 7, no protein; lane 2, 200 nM; lane 3, 400 nM; lane 4, 800 nM; lane 5, 1,600 nM; lane 6, 3,200 nM. The three RamA binding sites are indicated with brackets on the left side of each panel. Protected regions are indicated by bars, and hypersensitive sites are indicated by arrows. The DNA sequencing reactions were set up using the same labeled primer and plasmid as for generating labeled footprinting probes.

Techniques Used: Incubation, Binding Assay, DNA Sequencing, Labeling, Plasmid Preparation, Footprinting

59) Product Images from "Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation"

Article Title: Bradyrhizobium diazoefficiens USDA110 PhaR functions for pleiotropic regulation of cellular processes besides PHB accumulation

Journal: BMC Microbiology

doi: 10.1186/s12866-018-1317-2

DNase I footprints of PhaR on the phaP1 ( a ) and phaP4 ( b ) promoter regions. DNaseI footprints of PhaR-His 6 binding found in DNA fragment patterns are shown for the phaP1 ( a ) and phaP4 ( b ) promoter regions; the fragment patterns on upper and lower strands are in the left and right side of each panel, respectively. Each of the panels contain four fragment charts for respective upper and lower strands; from the top to down, the first is the sequencing ladders in four colors, the second is the negative control without PhaR-His 6 , and the third and fourth are the two different concentrations of PhaR-His 6 as indicated. At the bottom of each panel, nucleotide sequences of the promoter regions of phaP1 ( a ) and phaP4 ( b ) are shown. The sequence stretches protected from DNase I digestion by PhaR-His 6 binding are shown in hatched squares. Hocked arrowheads and the labels “+ 1” indicate the transcriptional start point (shown in blue letters in the upper strands). The −35 and − 10 regions are underlined and the ATG initiating codons are shown in red letters in the upper strands
Figure Legend Snippet: DNase I footprints of PhaR on the phaP1 ( a ) and phaP4 ( b ) promoter regions. DNaseI footprints of PhaR-His 6 binding found in DNA fragment patterns are shown for the phaP1 ( a ) and phaP4 ( b ) promoter regions; the fragment patterns on upper and lower strands are in the left and right side of each panel, respectively. Each of the panels contain four fragment charts for respective upper and lower strands; from the top to down, the first is the sequencing ladders in four colors, the second is the negative control without PhaR-His 6 , and the third and fourth are the two different concentrations of PhaR-His 6 as indicated. At the bottom of each panel, nucleotide sequences of the promoter regions of phaP1 ( a ) and phaP4 ( b ) are shown. The sequence stretches protected from DNase I digestion by PhaR-His 6 binding are shown in hatched squares. Hocked arrowheads and the labels “+ 1” indicate the transcriptional start point (shown in blue letters in the upper strands). The −35 and − 10 regions are underlined and the ATG initiating codons are shown in red letters in the upper strands

Techniques Used: Binding Assay, Sequencing, Negative Control

60) Product Images from "Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR"

Article Title: Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

Journal: Journal of Bacteriology

doi: 10.1128/JB.00453-15

DNase I footprinting analysis of the ald control region bound by AldR. The DNA fragments containing the noncoding (A) and coding (B) strands labeled with TAMRA at their 5′ ends were incubated with increasing concentrations of purified AldR (0.5
Figure Legend Snippet: DNase I footprinting analysis of the ald control region bound by AldR. The DNA fragments containing the noncoding (A) and coding (B) strands labeled with TAMRA at their 5′ ends were incubated with increasing concentrations of purified AldR (0.5

Techniques Used: Footprinting, Labeling, Incubation, Purification

Model for the regulation of ald expression by AldR. The numbers between the two adjacent AldR-binding sites indicate the distances between their central T nucleotides in base pairs. The hypersensitive sites detected by DNase I footprinting are marked
Figure Legend Snippet: Model for the regulation of ald expression by AldR. The numbers between the two adjacent AldR-binding sites indicate the distances between their central T nucleotides in base pairs. The hypersensitive sites detected by DNase I footprinting are marked

Techniques Used: Expressing, Binding Assay, Footprinting

61) Product Images from "Production of HIV-1 vif mRNA Is Modulated by Natural Nucleotide Variations and SLSA1 RNA Structure in SA1D2prox Genomic Region"

Article Title: Production of HIV-1 vif mRNA Is Modulated by Natural Nucleotide Variations and SLSA1 RNA Structure in SA1D2prox Genomic Region

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.02542

Effects of nSNVs in SA1D2prox on  vif  expression. Proviral clones indicated were transfected into 293T cells, and at 20 h post-transfection, total RNAs were prepared. After DNase I-treatment, samples were subjected to qRT-PCR analysis by a specific primer set for  vif  transcript ( Figure   1 ). For clones designated K240aaa, L242ctt, A248gcg, V249gtg, N254aac, and V259gtg, cDNA samples synthesized using DNase I-treated RNA and oligo (dT) primer were subjected to qRT-PCR analysis. Expression levels of all HIV-1 transcripts/mRNAs and GAPDH transcript/mRNA were analyzed by qRT-PCR in parallel as transfection and internal controls, respectively. A  vif  expression level in each sample was normalized by those of all HIV-1 transcript/mRNA species and GAPDH transcript/mRNA.  Vif  expression levels relative to that by NL4-3 are presented. Blue letters/bar, red letters/bars, and green letters/bar indicate low, high, and excessive  vif  types (  Nomaguchi et al., 2016 ), respectively. Mean values ± SD from four independent experiments are shown.
Figure Legend Snippet: Effects of nSNVs in SA1D2prox on vif expression. Proviral clones indicated were transfected into 293T cells, and at 20 h post-transfection, total RNAs were prepared. After DNase I-treatment, samples were subjected to qRT-PCR analysis by a specific primer set for vif transcript ( Figure 1 ). For clones designated K240aaa, L242ctt, A248gcg, V249gtg, N254aac, and V259gtg, cDNA samples synthesized using DNase I-treated RNA and oligo (dT) primer were subjected to qRT-PCR analysis. Expression levels of all HIV-1 transcripts/mRNAs and GAPDH transcript/mRNA were analyzed by qRT-PCR in parallel as transfection and internal controls, respectively. A vif expression level in each sample was normalized by those of all HIV-1 transcript/mRNA species and GAPDH transcript/mRNA. Vif expression levels relative to that by NL4-3 are presented. Blue letters/bar, red letters/bars, and green letters/bar indicate low, high, and excessive vif types ( Nomaguchi et al., 2016 ), respectively. Mean values ± SD from four independent experiments are shown.

Techniques Used: Expressing, Clone Assay, Transfection, Quantitative RT-PCR, Synthesized

Characterization of proviral clones with a naturally occurring full-length SA1D2prox sequence. Various proviral clones were examined for  vif  mRNA production, Vif expression, and  vpr  mRNA production.  (A)  Expression levels of  vif  mRNAs by various variants. Proviral clones indicated were transfected into 293T cells, and at 20 h post-transfection, cells were collected for extraction of total RNA. DNase I-treated total RNAs were used to synthesize cDNAs with oligo (dT) primer, and the cDNA samples were subjected to qRT-PCR analysis using a specific primer set of  vif  mRNA. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by qRT-PCR in parallel for transfection and internal controls, respectively. A  vif  expression level in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA.  Vif  expression levels relative to that by NL-pC3 are presented. Blue and red bars indicate clones that exhibit lower and higher  vif  expression levels, respectively, relative to that by NL-pC3. Mean values ± SD from three independent experiments are shown. All clones were simultaneously assayed for  vif  production, but results obtained are separately shown for clarity.  (B)  Expression of Vif proteins by various variants. 293T cells were transfected with proviral clones indicated. On day 1 post-transfection, cell lysates were prepared, and analyzed by Western blotting using anti-Vif and anti-β-actin antibodies. Migration positions of mass standards are shown on the left. Representative data from at least two independent experiments are shown. Blue letters indicate clones that exhibit lower  vif  expression level relative to that by NL-pC3. NL-p11807 ∗ , undetectable.  (C)  Expression levels of  vpr3  mRNA by various variants. Semiquantitative PCR was carried out using cDNA samples prepared as described in  (A)  and a specific primer set for  vpr  mRNAs. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by semiquantitative PCR in parallel for transfection and internal controls, respectively. Signal intensities of semiquantitative RT-PCR products were quantitated by Amersham Imager 600 instrument. Intensity of  vpr3  mRNA in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA. Normalized mRNA intensity in each sample relative to that by NL-pC3 is presented. Blue bars show clones with decreased  vif  expression level relative to NL-pC3. Mean values ± SD from three independent experiments are shown.
Figure Legend Snippet: Characterization of proviral clones with a naturally occurring full-length SA1D2prox sequence. Various proviral clones were examined for vif mRNA production, Vif expression, and vpr mRNA production. (A) Expression levels of vif mRNAs by various variants. Proviral clones indicated were transfected into 293T cells, and at 20 h post-transfection, cells were collected for extraction of total RNA. DNase I-treated total RNAs were used to synthesize cDNAs with oligo (dT) primer, and the cDNA samples were subjected to qRT-PCR analysis using a specific primer set of vif mRNA. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by qRT-PCR in parallel for transfection and internal controls, respectively. A vif expression level in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA. Vif expression levels relative to that by NL-pC3 are presented. Blue and red bars indicate clones that exhibit lower and higher vif expression levels, respectively, relative to that by NL-pC3. Mean values ± SD from three independent experiments are shown. All clones were simultaneously assayed for vif production, but results obtained are separately shown for clarity. (B) Expression of Vif proteins by various variants. 293T cells were transfected with proviral clones indicated. On day 1 post-transfection, cell lysates were prepared, and analyzed by Western blotting using anti-Vif and anti-β-actin antibodies. Migration positions of mass standards are shown on the left. Representative data from at least two independent experiments are shown. Blue letters indicate clones that exhibit lower vif expression level relative to that by NL-pC3. NL-p11807 ∗ , undetectable. (C) Expression levels of vpr3 mRNA by various variants. Semiquantitative PCR was carried out using cDNA samples prepared as described in (A) and a specific primer set for vpr mRNAs. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by semiquantitative PCR in parallel for transfection and internal controls, respectively. Signal intensities of semiquantitative RT-PCR products were quantitated by Amersham Imager 600 instrument. Intensity of vpr3 mRNA in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA. Normalized mRNA intensity in each sample relative to that by NL-pC3 is presented. Blue bars show clones with decreased vif expression level relative to NL-pC3. Mean values ± SD from three independent experiments are shown.

Techniques Used: Clone Assay, Sequencing, Expressing, Transfection, Quantitative RT-PCR, Western Blot, Migration, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction

Relationship between the  vif  expression level and SLSA1 RNA stability. SLSA1 variants in  Figure   9  were analyzed for  vif  expressions and SLSA1 stabilities. Free energy ( d G) for each SLSA1 RNA is predicted by mfold program (  Zuker, 2003 ). Stability ( dd G) of SLSA1 RNA structure is presented as difference of free energy between mutants and NL4-3. For determination of  vif  production level, proviral clones indicated were transfected into 293T cells, and total RNA was prepared from cells collected at 20 h post-transfection. DNase I-treated RNAs were used for cDNA synthesis with oligo (dT) primer, and resultant cDNA samples were subjected to qRT-PCR analysis using a specific primer set for  vif  mRNA. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by qRT-PCR in parallel for transfection and internal controls, respectively. A  vif  expression level in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA.  Vif  expression levels relative to those by NL4-3 are presented. Mean values ± SD from three independent experiments are shown. Scatter diagram on the right was created by plotting  vif  expression levels and SLSA1 stabilities [–( dd G)] of the test clones relative to those of NL4-3. Exponential trendline and coefficient of determination ( R 2  = 0.084,  p  = 0.161 by  F -test) are shown. Blue, red, and green letters indicate low, high, and excessive  vif  types (  Nomaguchi et al., 2016 ), respectively.
Figure Legend Snippet: Relationship between the vif expression level and SLSA1 RNA stability. SLSA1 variants in Figure 9 were analyzed for vif expressions and SLSA1 stabilities. Free energy ( d G) for each SLSA1 RNA is predicted by mfold program ( Zuker, 2003 ). Stability ( dd G) of SLSA1 RNA structure is presented as difference of free energy between mutants and NL4-3. For determination of vif production level, proviral clones indicated were transfected into 293T cells, and total RNA was prepared from cells collected at 20 h post-transfection. DNase I-treated RNAs were used for cDNA synthesis with oligo (dT) primer, and resultant cDNA samples were subjected to qRT-PCR analysis using a specific primer set for vif mRNA. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by qRT-PCR in parallel for transfection and internal controls, respectively. A vif expression level in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA. Vif expression levels relative to those by NL4-3 are presented. Mean values ± SD from three independent experiments are shown. Scatter diagram on the right was created by plotting vif expression levels and SLSA1 stabilities [–( dd G)] of the test clones relative to those of NL4-3. Exponential trendline and coefficient of determination ( R 2 = 0.084, p = 0.161 by F -test) are shown. Blue, red, and green letters indicate low, high, and excessive vif types ( Nomaguchi et al., 2016 ), respectively.

Techniques Used: Expressing, Clone Assay, Transfection, Quantitative RT-PCR

Changes in  vif  expression level and SLSA1 RNA stability by nSNVs within SLSA1. Free energy ( d G) for the SLSA1 sequence carrying an nSNV was predicted by mfold program (  Zuker, 2003 ). RNA stability ( dd G) is presented as difference of free energy between each nSNV and NL4-3. For determination of  vif  production level, proviral clones indicated were transfected into 293T cells, and at 20 h post-transfection, DNase I-treated total RNAs were prepared. After synthesis of cDNA using DNase I-treated RNA and oligo (dT) primer, samples were subjected to qRT-PCR analysis using a specific primer set for  vif  mRNA. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by qRT-PCR in parallel for transfection and internal controls, respectively. A  vif  expression level in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA.  Vif  expression levels relative to that by NL4-3 are presented. Mean values ± SD from three independent experiments are shown. Scatter diagram on the right was created by plotting  vif  expression levels and SLSA1 stabilities [–( dd G)] of variant clones with nSNVs relative to those of NL4-3. Exponential trendline and coefficient of determination ( R 2  = 0.024,  p  = 0.549 by  F -test) are shown. Blue, red, and green letters indicate low, high, and excessive  vif  types (  Nomaguchi et al., 2016 ), respectively. In this figure, the data of relative  vif  expression levels in our previous report (  Nomaguchi et al., 2016 ) were used for clones R224cgc, Y226tac, R228aga, D229gat, R231Kaaa, D232gac, and K236aag for easy comparison.
Figure Legend Snippet: Changes in vif expression level and SLSA1 RNA stability by nSNVs within SLSA1. Free energy ( d G) for the SLSA1 sequence carrying an nSNV was predicted by mfold program ( Zuker, 2003 ). RNA stability ( dd G) is presented as difference of free energy between each nSNV and NL4-3. For determination of vif production level, proviral clones indicated were transfected into 293T cells, and at 20 h post-transfection, DNase I-treated total RNAs were prepared. After synthesis of cDNA using DNase I-treated RNA and oligo (dT) primer, samples were subjected to qRT-PCR analysis using a specific primer set for vif mRNA. Expression levels of all HIV-1 mRNA species and GAPDH mRNA were analyzed by qRT-PCR in parallel for transfection and internal controls, respectively. A vif expression level in each sample was normalized by those of all HIV-1 mRNA species and GAPDH mRNA. Vif expression levels relative to that by NL4-3 are presented. Mean values ± SD from three independent experiments are shown. Scatter diagram on the right was created by plotting vif expression levels and SLSA1 stabilities [–( dd G)] of variant clones with nSNVs relative to those of NL4-3. Exponential trendline and coefficient of determination ( R 2 = 0.024, p = 0.549 by F -test) are shown. Blue, red, and green letters indicate low, high, and excessive vif types ( Nomaguchi et al., 2016 ), respectively. In this figure, the data of relative vif expression levels in our previous report ( Nomaguchi et al., 2016 ) were used for clones R224cgc, Y226tac, R228aga, D229gat, R231Kaaa, D232gac, and K236aag for easy comparison.

Techniques Used: Expressing, Sequencing, Clone Assay, Transfection, Quantitative RT-PCR, Variant Assay

62) Product Images from "Monoclonal Antibodies against Accumulation-Associated Protein Affect EPS Biosynthesis and Enhance Bacterial Accumulation of Staphylococcus epidermidis"

Article Title: Monoclonal Antibodies against Accumulation-Associated Protein Affect EPS Biosynthesis and Enhance Bacterial Accumulation of Staphylococcus epidermidis

Journal: PLoS ONE

doi: 10.1371/journal.pone.0020918

EPS biosynthesis in biofilm of S. epidermidis . (A) Extracellular DNA quantification. Extracellular DNA was isolated from the biofilm matrices of S. epidermidis RP62A, and Q-PCRs of four chromosomal loci ( gyrA (gyrase A), serp0306 (ferrichrome transport ATP-binding protein A), lysA (diaminopimelate decarboxylase A), and leuA (2-isopropylmalate synthase)) were performed for eDNA quantification in each biofilm. The biomass that represented the biofilm density was quantified at A 600 , and the eDNA measurement was normalized to biofilm biomass as described previously [27] . Data are depicted as averages of three Q-PCR detections with the standard deviation, and the results represent one of three independent experiments. (B, C) Biofilm stability against DNase treatment. When exposed to DNase I (0.14 U/µL), the biofilm was more severely disintegrated in the presence of each MAb compared with that formed in the absence of the MAbs. The data are means ± SD of three independent experiments. The biofilm density ratios of the untreated biofilms and biofilms treated with DNase I were plotted (C). (D) PIA synthesis in biofilm of S. epidermidis . PIA synthesis in biofilm of S. epidermidis RP62A was detected using the WGA-HRP dot blot assay. Serial dilutions of the PIA extractions from biofilm bacteria were spotted onto nitrocellulose transfer membranes, and the HRP activity was visualized using chromogenic detection. The data represent one of three independent experiments. “RP62A”: untreated; “Mock”: normal mouse IgG-treated.
Figure Legend Snippet: EPS biosynthesis in biofilm of S. epidermidis . (A) Extracellular DNA quantification. Extracellular DNA was isolated from the biofilm matrices of S. epidermidis RP62A, and Q-PCRs of four chromosomal loci ( gyrA (gyrase A), serp0306 (ferrichrome transport ATP-binding protein A), lysA (diaminopimelate decarboxylase A), and leuA (2-isopropylmalate synthase)) were performed for eDNA quantification in each biofilm. The biomass that represented the biofilm density was quantified at A 600 , and the eDNA measurement was normalized to biofilm biomass as described previously [27] . Data are depicted as averages of three Q-PCR detections with the standard deviation, and the results represent one of three independent experiments. (B, C) Biofilm stability against DNase treatment. When exposed to DNase I (0.14 U/µL), the biofilm was more severely disintegrated in the presence of each MAb compared with that formed in the absence of the MAbs. The data are means ± SD of three independent experiments. The biofilm density ratios of the untreated biofilms and biofilms treated with DNase I were plotted (C). (D) PIA synthesis in biofilm of S. epidermidis . PIA synthesis in biofilm of S. epidermidis RP62A was detected using the WGA-HRP dot blot assay. Serial dilutions of the PIA extractions from biofilm bacteria were spotted onto nitrocellulose transfer membranes, and the HRP activity was visualized using chromogenic detection. The data represent one of three independent experiments. “RP62A”: untreated; “Mock”: normal mouse IgG-treated.

Techniques Used: Isolation, Binding Assay, Polymerase Chain Reaction, Standard Deviation, Whole Genome Amplification, Dot Blot, Activity Assay

63) Product Images from "Rab2A is a pivotal switch protein that promotes either secretion or ER-associated degradation of (pro)insulin in insulin-secreting cells"

Article Title: Rab2A is a pivotal switch protein that promotes either secretion or ER-associated degradation of (pro)insulin in insulin-secreting cells

Journal: Scientific Reports

doi: 10.1038/srep06952

Polyubiquitinated proinsulin aggregates at the LUb-ERGIC. (a) ERGIC53 and ubiquitinated proteins (Ub-proteins) were subjected to double staining in MIN6 cells. Dotted lines indicate the outline of a cell. (b) ERGIC53 and proinsulin were subjected to double staining in MIN6 cells (Intact) or MIN6 cells treated with 1% Triton X-100 alone (1% Tx) or sequentially with 1% Tx, DNase I and 2 M NaCl (LUb-ERGIC enriched fraction) (See Methods and Supplementary Fig. S2 in detail). High mag indicates high magnification images of the white rectangles in the upper hand panels. Arrowhead indicates proinsulin localized to the LUb-ERGIC. Arrows indicate high intensity dots of proinsulin in the LUb-ERGIC. (c) ERGIC53 and proinsulin were subjected to double staining in MIN6 cells expressing ubiquitin tagged with RFP (RFP-Ubiquitin). (d) MIN6 cells were treated with 10 μg/ml CHX or 1 μM MG132 for 2 hr. Left: Ub-proteins and proinsulin in the 1% Tx soluble fraction (1% Tx) were immunoblotted to evaluate the effect of the inhibitors, using the GAPDH signal as a loading control. Right: proinsulin in the 1% Tx/2 M NaCl resistant fraction (LUb-ERGIC enriched fraction) was immunoblotted with anti-proinsulin antibody, using the actin signal as a loading control. Ub 1 and Ub n represent mono and polyubiquitinated proinsulin, respectively. (e) MIN6 cells were transfected with scramble or insulin siRNA. The cells were lysed, and the lysates were immunoblotted with anti-proinsulin antibody, using the actin signal as a loading control. (f) Left: 36 hr after MIN6 cells were transfected with scramble or insulin siRNA, the cells were exposed to low glucose for 24 hr and then low glucose (Low glu) or high glucose (High glu) for 12 hr, and immunostained with antibodies against ERGIC53 and Ub-proteins. Images were derived by three-dimensional reconstruction of the cells (3D reconstruction). Arrows indicate the LUb-ERGIC. Right: the mean fluorescent intensities of Ub-proteins at the LUb-ERGIC were quantified (mean ± s.d., n = 18 in each condition). Indicated P values were calculated with Student's t -test, followed by the Bonferroni correction. Scale bars: 5 μm. (a and c) Insets show high magnification images of the corresponding white rectangles. Scale bars: 5 μm (low magnification) and 2 μm (high magnification). Full-length blots are presented in Supplementary Fig. S10 .
Figure Legend Snippet: Polyubiquitinated proinsulin aggregates at the LUb-ERGIC. (a) ERGIC53 and ubiquitinated proteins (Ub-proteins) were subjected to double staining in MIN6 cells. Dotted lines indicate the outline of a cell. (b) ERGIC53 and proinsulin were subjected to double staining in MIN6 cells (Intact) or MIN6 cells treated with 1% Triton X-100 alone (1% Tx) or sequentially with 1% Tx, DNase I and 2 M NaCl (LUb-ERGIC enriched fraction) (See Methods and Supplementary Fig. S2 in detail). High mag indicates high magnification images of the white rectangles in the upper hand panels. Arrowhead indicates proinsulin localized to the LUb-ERGIC. Arrows indicate high intensity dots of proinsulin in the LUb-ERGIC. (c) ERGIC53 and proinsulin were subjected to double staining in MIN6 cells expressing ubiquitin tagged with RFP (RFP-Ubiquitin). (d) MIN6 cells were treated with 10 μg/ml CHX or 1 μM MG132 for 2 hr. Left: Ub-proteins and proinsulin in the 1% Tx soluble fraction (1% Tx) were immunoblotted to evaluate the effect of the inhibitors, using the GAPDH signal as a loading control. Right: proinsulin in the 1% Tx/2 M NaCl resistant fraction (LUb-ERGIC enriched fraction) was immunoblotted with anti-proinsulin antibody, using the actin signal as a loading control. Ub 1 and Ub n represent mono and polyubiquitinated proinsulin, respectively. (e) MIN6 cells were transfected with scramble or insulin siRNA. The cells were lysed, and the lysates were immunoblotted with anti-proinsulin antibody, using the actin signal as a loading control. (f) Left: 36 hr after MIN6 cells were transfected with scramble or insulin siRNA, the cells were exposed to low glucose for 24 hr and then low glucose (Low glu) or high glucose (High glu) for 12 hr, and immunostained with antibodies against ERGIC53 and Ub-proteins. Images were derived by three-dimensional reconstruction of the cells (3D reconstruction). Arrows indicate the LUb-ERGIC. Right: the mean fluorescent intensities of Ub-proteins at the LUb-ERGIC were quantified (mean ± s.d., n = 18 in each condition). Indicated P values were calculated with Student's t -test, followed by the Bonferroni correction. Scale bars: 5 μm. (a and c) Insets show high magnification images of the corresponding white rectangles. Scale bars: 5 μm (low magnification) and 2 μm (high magnification). Full-length blots are presented in Supplementary Fig. S10 .

Techniques Used: Double Staining, Expressing, Transfection, Derivative Assay

64) Product Images from "Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins"

Article Title: Expression of Genes Involved in Bacteriocin Production and Self-Resistance in Lactobacillus brevis 174A Is Mediated by Two Regulatory Proteins

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.02707-17

Binding sites of BreD and BreG to the promoter region of breED and breBC , respectively. (A) DNase I footprinting for the determination of BreD and BreG binding sites upstream of breED and breBC , respectively. The amounts of BreD (10 to 40 pmol) and N-BreG (300 to 1,200 pmol) used for the assay are indicated at the top of each lane. A probe amplified with a 5′-end-biotinylated primer was used. The nucleotide corresponding to the first nucleotide of the transcribed RNA is numbered +1. (B) Nucleotide sequences of the predicted binding site of BreD or BreG. The numbers indicate positions from the transcription starting point, beginning with +1. The putative RBS, −10, and −35 regions are underlined. The regions protected by both regulators are in bold. The arrows indicate direct repeats (DR) and inverted repeats (IR). The dotted line indicates spacer sequences. The transcriptional start point is marked with a bent arrow.
Figure Legend Snippet: Binding sites of BreD and BreG to the promoter region of breED and breBC , respectively. (A) DNase I footprinting for the determination of BreD and BreG binding sites upstream of breED and breBC , respectively. The amounts of BreD (10 to 40 pmol) and N-BreG (300 to 1,200 pmol) used for the assay are indicated at the top of each lane. A probe amplified with a 5′-end-biotinylated primer was used. The nucleotide corresponding to the first nucleotide of the transcribed RNA is numbered +1. (B) Nucleotide sequences of the predicted binding site of BreD or BreG. The numbers indicate positions from the transcription starting point, beginning with +1. The putative RBS, −10, and −35 regions are underlined. The regions protected by both regulators are in bold. The arrows indicate direct repeats (DR) and inverted repeats (IR). The dotted line indicates spacer sequences. The transcriptional start point is marked with a bent arrow.

Techniques Used: Binding Assay, Footprinting, Amplification

65) Product Images from "A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth conditions"

Article Title: A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth conditions

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkq077

Identification of the Dan box sequence. (A) Fluorescent-labeled SELEX segment (1.0 pmol) from the dan-ttdA spacer was incubated in the absence or presence of increasing concentrations of purified Dan (10, 20 and 40 pmol from left to right) and then subjected to DNase-I foot-printing assays. Lanes A, T, G and C represent the respective sequence ladders. ( B ) Four Dan-binding sites were identified on the dan-ttdA spacer sequence. ( C ) The Dan-box sequence GTTAAT was predicted after sequence comparison of four Dan-binding sites. Similar sequences were identified among a total of 688 Dan-binding fragments selected by Genomic SELEX. Using this Dan-box sequence, a total of about 1860 sites were predicted to be present in the entire E. coli genome. ( D ) Logo representation of Dan-binding site derived from sequences in in silico analysis. Logos were generated using weblogo ( http://weblogo.berkeley.edu/ ).
Figure Legend Snippet: Identification of the Dan box sequence. (A) Fluorescent-labeled SELEX segment (1.0 pmol) from the dan-ttdA spacer was incubated in the absence or presence of increasing concentrations of purified Dan (10, 20 and 40 pmol from left to right) and then subjected to DNase-I foot-printing assays. Lanes A, T, G and C represent the respective sequence ladders. ( B ) Four Dan-binding sites were identified on the dan-ttdA spacer sequence. ( C ) The Dan-box sequence GTTAAT was predicted after sequence comparison of four Dan-binding sites. Similar sequences were identified among a total of 688 Dan-binding fragments selected by Genomic SELEX. Using this Dan-box sequence, a total of about 1860 sites were predicted to be present in the entire E. coli genome. ( D ) Logo representation of Dan-binding site derived from sequences in in silico analysis. Logos were generated using weblogo ( http://weblogo.berkeley.edu/ ).

Techniques Used: Sequencing, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, In Silico, Generated

66) Product Images from "DNA binding and helicase actions of mouse MCM4/6/7 helicase"

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase

Journal: Nucleic Acids Research

doi: 10.1093/nar/gki607

Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.
Figure Legend Snippet: Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. ( A ) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32 P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. ( B ) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder.

Techniques Used: Binding Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

67) Product Images from "Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis"

Article Title: Regulation of the rhaEWRBMA Operon Involved in l-Rhamnose Catabolism through Two Transcriptional Factors, RhaR and CcpA, in Bacillus subtilis

Journal: Journal of Bacteriology

doi: 10.1128/JB.00856-15

DNase I footprinting analysis to identify the binding sites of the RhaR protein and the CcpA/P-Ser-HPr complex in the regulatory region of the rhaEWRBMA operon. A 5′-end- 32 P-labeled DNA probe (0.04 pmol), corresponding to the region around the
Figure Legend Snippet: DNase I footprinting analysis to identify the binding sites of the RhaR protein and the CcpA/P-Ser-HPr complex in the regulatory region of the rhaEWRBMA operon. A 5′-end- 32 P-labeled DNA probe (0.04 pmol), corresponding to the region around the

Techniques Used: Footprinting, Binding Assay, Labeling

68) Product Images from "Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR"

Article Title: Regulation Mechanism of the ald Gene Encoding Alanine Dehydrogenase in Mycobacterium smegmatis and Mycobacterium tuberculosis by the Lrp/AsnC Family Regulator AldR

Journal: Journal of Bacteriology

doi: 10.1128/JB.00453-15

DNase I footprinting analysis of the ald control region bound by AldR. The DNA fragments containing the noncoding (A) and coding (B) strands labeled with TAMRA at their 5′ ends were incubated with increasing concentrations of purified AldR (0.5
Figure Legend Snippet: DNase I footprinting analysis of the ald control region bound by AldR. The DNA fragments containing the noncoding (A) and coding (B) strands labeled with TAMRA at their 5′ ends were incubated with increasing concentrations of purified AldR (0.5

Techniques Used: Footprinting, Labeling, Incubation, Purification

Model for the regulation of ald expression by AldR. The numbers between the two adjacent AldR-binding sites indicate the distances between their central T nucleotides in base pairs. The hypersensitive sites detected by DNase I footprinting are marked
Figure Legend Snippet: Model for the regulation of ald expression by AldR. The numbers between the two adjacent AldR-binding sites indicate the distances between their central T nucleotides in base pairs. The hypersensitive sites detected by DNase I footprinting are marked

Techniques Used: Expressing, Binding Assay, Footprinting

69) Product Images from "Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation"

Article Title: Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation

Journal: BMC Microbiology

doi: 10.1186/1471-2180-11-146

S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I . (A) Attached SE1457 ΔatlE , SE1457 ΔsaeRS , SE1457 and SE1457 saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P
Figure Legend Snippet: S. epidermidis attachment to polystyrene surfaces in the presence or absence of DNase I . (A) Attached SE1457 ΔatlE , SE1457 ΔsaeRS , SE1457 and SE1457 saec cells were observed by microscopy. Briefly, cell suspensions from the mid-exponential phase were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Roskilde, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. S. epidermidis cells attached to the polystyrene surface were counted under microscope (400× magnification). (B) The number of attached bacteria per field was then counted. Results represent the mean ± SD of three independent experiments. *, P

Techniques Used: Microscopy, Incubation, Cell Culture

Effect of DNaseI on SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilm formation . SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P
Figure Legend Snippet: Effect of DNaseI on SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilm formation . SE1457 ΔsaeRS , SE1457, and SE1457 saec biofilms were washed and then stained with crystal violet. Their retained biomass was quantified by measuring the absorbance of each well at 570 nm. Biofilms were formed in the absence (black bars) or presence of DNase I (28 U/200 μL/well) (white bars). Mean values and standard deviations from three independent experiments are shown. (*), P

Techniques Used: Staining

70) Product Images from "Involvement of Cyclic AMP Receptor Protein in Regulation of the rmf Gene Encoding the Ribosome Modulation Factor in Escherichia coli"

Article Title: Involvement of Cyclic AMP Receptor Protein in Regulation of the rmf Gene Encoding the Ribosome Modulation Factor in Escherichia coli

Journal: Journal of Bacteriology

doi: 10.1128/JB.02279-12

DNase I footprinting assay of CRP-binding sequences on the ribosome-related gene promoters. FITC-labeled DNA probes (1.0 pmol) of the ribosome-related gene promoters were subjected to a DNase I footprinting assay in the absence (−) or presence (+) of CRP (5 μM)-cAMP (1 mM). The region protected from DNase I digestion is indicated on the right.
Figure Legend Snippet: DNase I footprinting assay of CRP-binding sequences on the ribosome-related gene promoters. FITC-labeled DNA probes (1.0 pmol) of the ribosome-related gene promoters were subjected to a DNase I footprinting assay in the absence (−) or presence (+) of CRP (5 μM)-cAMP (1 mM). The region protected from DNase I digestion is indicated on the right.

Techniques Used: Footprinting, Binding Assay, Labeling

71) Product Images from "The LacI-Type Transcriptional Regulator AraR Acts as an l-Arabinose-Responsive Repressor of l-Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831"

Article Title: The LacI-Type Transcriptional Regulator AraR Acts as an l-Arabinose-Responsive Repressor of l-Arabinose Utilization Genes in Corynebacterium glutamicum ATCC 31831

Journal: Journal of Bacteriology

doi: 10.1128/JB.01655-14

DNase I footprinting analysis with the AraR protein and the araB-galM intergenic region (A) or the araE upstream region (B). Each 20-μl binding reaction mixture, containing 80 ng (A) or 120 ng (B) of the DNA probe and the AraR protein at various
Figure Legend Snippet: DNase I footprinting analysis with the AraR protein and the araB-galM intergenic region (A) or the araE upstream region (B). Each 20-μl binding reaction mixture, containing 80 ng (A) or 120 ng (B) of the DNA probe and the AraR protein at various

Techniques Used: Footprinting, Binding Assay

The AraR-binding motif. (A) The three AraR-binding sites determined by DNase I footprinting analyses, located in the araB (BS B ) and araE (BS E1 and BS E2 ) upstream regions, are aligned with the consensus sequence, in which the nucleotides in capital and
Figure Legend Snippet: The AraR-binding motif. (A) The three AraR-binding sites determined by DNase I footprinting analyses, located in the araB (BS B ) and araE (BS E1 and BS E2 ) upstream regions, are aligned with the consensus sequence, in which the nucleotides in capital and

Techniques Used: Binding Assay, Footprinting, Sequencing

72) Product Images from "The Small Protein HemP Is a Transcriptional Activator for the Hemin Uptake Operon in Burkholderia multivorans ATCC 17616"

Article Title: The Small Protein HemP Is a Transcriptional Activator for the Hemin Uptake Operon in Burkholderia multivorans ATCC 17616

Journal: Applied and Environmental Microbiology

doi: 10.1128/AEM.00479-17

Binding of HemP 6×His to the fragment upstream of hmuR . (A) EMSA for HemP 6×His binding to the FAM-labeled 221-bp fragment. The mobility shift was not affected by the addition of a 100-fold excess of salmon sperm DNA and was almost completely abolished by the addition of a 100-fold excess of the nonlabeled fragment (data not shown). (B) Sequence of the 221-bp fragment used for this analysis. The protected region (HemP binding) is shown by red shading and an inverted repeat by arrows. (C) DNase I footprinting analysis. The FAM-labeled 221-bp fragment was digested by DNase I in a reaction mixture supplemented with (red) or without (blue) the purified HemP 6×His protein. The data obtained were analyzed with TraceViewer software. See Materials and Methods for details.
Figure Legend Snippet: Binding of HemP 6×His to the fragment upstream of hmuR . (A) EMSA for HemP 6×His binding to the FAM-labeled 221-bp fragment. The mobility shift was not affected by the addition of a 100-fold excess of salmon sperm DNA and was almost completely abolished by the addition of a 100-fold excess of the nonlabeled fragment (data not shown). (B) Sequence of the 221-bp fragment used for this analysis. The protected region (HemP binding) is shown by red shading and an inverted repeat by arrows. (C) DNase I footprinting analysis. The FAM-labeled 221-bp fragment was digested by DNase I in a reaction mixture supplemented with (red) or without (blue) the purified HemP 6×His protein. The data obtained were analyzed with TraceViewer software. See Materials and Methods for details.

Techniques Used: Binding Assay, Labeling, Mobility Shift, Sequencing, Footprinting, Purification, Software

73) Product Images from "RTL-P: a sensitive approach for detecting sites of 2?-O-methylation in RNA molecules"

Article Title: RTL-P: a sensitive approach for detecting sites of 2?-O-methylation in RNA molecules

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks698

The RTL-P strategy for the detection of the exact location of a 2'-O-methylated site in RNA. ( A ) A schematic diagram of the primer design for the RT reaction and the PCR. m = 2'-O-methylation. ( B ) A schematic representation of the RTL-P procedure used to detect the exact location of the 2'-O-methylation. (1) RNA preparation by DNase I treatment to remove the DNA contaminants. (2) RT with the MeUA-RT or MeA-RT primers in different reaction tubes (①–④) at low or high dNTP concentrations, respectively. At a low dNTP concentration, the RT reaction with the UA-RT primer (①) is impeded by the 2'-O-methyl group and produces less cDNA products than that the MeA-RT primer (②). At a high dNTP concentration, the cDNA products generated with MeUA-RT primer (③) is equal to the MeA-RT primer (④). (3) PCR with the same PCR primer pair P F /P R to amplify the RT products. The quantity of PCR product generated from the MeUA-RT cDNA (①) is less than the quantity from the MeA-RT cDNA (②) when the residue of interest is 2'-O-methylated. In contrast, the quantity of PCR product generated from the MeUA-RT (③) and MeA-RT cDNAs (④) are equal when the residue analyzed is unmethylated. (4) Gel electrophoresis of the RT-PCR products. If the analyzed nucleotide is 2'-O-methylated, the band of the RT-PCR products generated from the MeUA-RT cDNA (①) is weaker than the band of the products generated from the MeA-RT cDNA (②) when a low concentration of dNTPs is used in the RT; however, the bands of the RT-PCR products generated from the MeUA-RT cDNA (③) and the MeA-RT cDNA (④) are equal when a high concentration of dNTPs is used. If the analyzed nucleotide is unmodified, no differences in band intensities of the RT-PCR products are observed regardless of the concentration of dNTPs used. N = any nucleotide base.
Figure Legend Snippet: The RTL-P strategy for the detection of the exact location of a 2'-O-methylated site in RNA. ( A ) A schematic diagram of the primer design for the RT reaction and the PCR. m = 2'-O-methylation. ( B ) A schematic representation of the RTL-P procedure used to detect the exact location of the 2'-O-methylation. (1) RNA preparation by DNase I treatment to remove the DNA contaminants. (2) RT with the MeUA-RT or MeA-RT primers in different reaction tubes (①–④) at low or high dNTP concentrations, respectively. At a low dNTP concentration, the RT reaction with the UA-RT primer (①) is impeded by the 2'-O-methyl group and produces less cDNA products than that the MeA-RT primer (②). At a high dNTP concentration, the cDNA products generated with MeUA-RT primer (③) is equal to the MeA-RT primer (④). (3) PCR with the same PCR primer pair P F /P R to amplify the RT products. The quantity of PCR product generated from the MeUA-RT cDNA (①) is less than the quantity from the MeA-RT cDNA (②) when the residue of interest is 2'-O-methylated. In contrast, the quantity of PCR product generated from the MeUA-RT (③) and MeA-RT cDNAs (④) are equal when the residue analyzed is unmethylated. (4) Gel electrophoresis of the RT-PCR products. If the analyzed nucleotide is 2'-O-methylated, the band of the RT-PCR products generated from the MeUA-RT cDNA (①) is weaker than the band of the products generated from the MeA-RT cDNA (②) when a low concentration of dNTPs is used in the RT; however, the bands of the RT-PCR products generated from the MeUA-RT cDNA (③) and the MeA-RT cDNA (④) are equal when a high concentration of dNTPs is used. If the analyzed nucleotide is unmodified, no differences in band intensities of the RT-PCR products are observed regardless of the concentration of dNTPs used. N = any nucleotide base.

Techniques Used: Methylation, Polymerase Chain Reaction, Microelectrode Array, Concentration Assay, Generated, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction

The RTL-P approach for detecting the presence of 2'-O-methylation in RNA. ( A ) A schematic of the primer-design strategy for RTL-P. The orientations of the primers are indicated by the arrows. ( B and C ) A schematic illustration of the RTL-P procedure used to detect the presence (B) or absence (C) of 2'-O-methylated sites in RNA. (1) RNA preparation by DNase I treatment to remove the DNA contaminants. (2) RT with a RT primer at a low or high dNTP concentration. At a low dNTP concentration, the RT reaction is impeded by the 2'-O-methyl groups, resulting in shorter RT products. At a high dNTP concentration, the RT reaction is not impeded and therefore does not produce shorter cDNA products. (3) PCR with different PCR primer pairs to amplify the RT products. The PCR reaction with the R/F U primer pair only amplifies the longer cDNA products, but amplification with the R/F D primer pair generates both the longer and shorter cDNA products. For RNAs with methylation sites, the quantity of the R/F U PCR product is less than the R/F D PCR product when the RT products generated at a low dNTP concentration are used as the amplification template. However, the quantity of the PCR product generated by the R/F U primers is equal to the R/F D PCR product when the RT products generated at a high dNTP concentration are used as the template. (4) Gel electrophoresis of the RT-PCR products. If the 2'-O-methylated site is present in the RNA analyzed, the R/F U band is weaker than the R/F D band in the low dNTP lane. In contrast, the R/F U band is nearly equal to or stronger than the R/F D band in the high dNTP lane. The intensity difference depends on the length of the PCR products generated from the R/F U primers versus the R/F D primers. m = 2'-O-methylation.
Figure Legend Snippet: The RTL-P approach for detecting the presence of 2'-O-methylation in RNA. ( A ) A schematic of the primer-design strategy for RTL-P. The orientations of the primers are indicated by the arrows. ( B and C ) A schematic illustration of the RTL-P procedure used to detect the presence (B) or absence (C) of 2'-O-methylated sites in RNA. (1) RNA preparation by DNase I treatment to remove the DNA contaminants. (2) RT with a RT primer at a low or high dNTP concentration. At a low dNTP concentration, the RT reaction is impeded by the 2'-O-methyl groups, resulting in shorter RT products. At a high dNTP concentration, the RT reaction is not impeded and therefore does not produce shorter cDNA products. (3) PCR with different PCR primer pairs to amplify the RT products. The PCR reaction with the R/F U primer pair only amplifies the longer cDNA products, but amplification with the R/F D primer pair generates both the longer and shorter cDNA products. For RNAs with methylation sites, the quantity of the R/F U PCR product is less than the R/F D PCR product when the RT products generated at a low dNTP concentration are used as the amplification template. However, the quantity of the PCR product generated by the R/F U primers is equal to the R/F D PCR product when the RT products generated at a high dNTP concentration are used as the template. (4) Gel electrophoresis of the RT-PCR products. If the 2'-O-methylated site is present in the RNA analyzed, the R/F U band is weaker than the R/F D band in the low dNTP lane. In contrast, the R/F U band is nearly equal to or stronger than the R/F D band in the high dNTP lane. The intensity difference depends on the length of the PCR products generated from the R/F U primers versus the R/F D primers. m = 2'-O-methylation.

Techniques Used: Methylation, Concentration Assay, Polymerase Chain Reaction, Amplification, Generated, Nucleic Acid Electrophoresis, Reverse Transcription Polymerase Chain Reaction

74) Product Images from "Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade ▿"

Article Title: Genomic SELEX Search for Target Promoters under the Control of the PhoQP-RstBA Signal Relay Cascade ▿

Journal:

doi: 10.1128/JB.00319-07

DNase I footprinting assay of the asr promoter. Fluorescence-labeled DNA probe of the asr promoter fragment was incubated with increasing concentrations of the purified RstA (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 40 pmol; lane 5, 80
Figure Legend Snippet: DNase I footprinting assay of the asr promoter. Fluorescence-labeled DNA probe of the asr promoter fragment was incubated with increasing concentrations of the purified RstA (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 40 pmol; lane 5, 80

Techniques Used: Footprinting, Fluorescence, Labeling, Incubation, Purification

DNase I footprinting assay of the csgD promoter. Fluorescence-labeled DNA probe of the csgD promoter fragment was incubated with increasing concentrations of the purified RstA (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 40 pmol; lane 5,
Figure Legend Snippet: DNase I footprinting assay of the csgD promoter. Fluorescence-labeled DNA probe of the csgD promoter fragment was incubated with increasing concentrations of the purified RstA (lane 1, 0 pmol; lane 2, 10 pmol; lane 3, 20 pmol; lane 4, 40 pmol; lane 5,

Techniques Used: Footprinting, Fluorescence, Labeling, Incubation, Purification

75) Product Images from "Regulation of c-maf gene expression by Pax6 in cultured cells"

Article Title: Regulation of c-maf gene expression by Pax6 in cultured cells

Journal: Nucleic Acids Research

doi:

Identification of c-Maf-binding sequences on the c- maf gene. ( A ) Schematic representation of probes used for EMSA and footprinting analyses. ( B ) EMSA of c- maf promoter probes with c-Maf–MBP protein. EMSA was carried out as in the legend to Figure 6 and Materials and Methods. ( C ) Footprinting analysis of the c- maf gene promoter with c-Maf–MBP protein. Probe B or C was mixed with increasing amounts of c-Maf–MBP and digested with DNase I as in the legend to Figure 6. c-Maf–MBP protein was used at 0.3 and 1 µg for lanes 2 and 3 with probe B, at 0.1, 0.3 and 1 µg for lanes 2–4 with probe C. A and G residues were modified and cleaved and used as markers (lanes 1 for both probes). ( C ) c-Maf-binding sequences are aligned with the MARE consensus sequence. TRE-type consensus and CRE-type consensus (G in the middle position) sequences are presented.
Figure Legend Snippet: Identification of c-Maf-binding sequences on the c- maf gene. ( A ) Schematic representation of probes used for EMSA and footprinting analyses. ( B ) EMSA of c- maf promoter probes with c-Maf–MBP protein. EMSA was carried out as in the legend to Figure 6 and Materials and Methods. ( C ) Footprinting analysis of the c- maf gene promoter with c-Maf–MBP protein. Probe B or C was mixed with increasing amounts of c-Maf–MBP and digested with DNase I as in the legend to Figure 6. c-Maf–MBP protein was used at 0.3 and 1 µg for lanes 2 and 3 with probe B, at 0.1, 0.3 and 1 µg for lanes 2–4 with probe C. A and G residues were modified and cleaved and used as markers (lanes 1 for both probes). ( C ) c-Maf-binding sequences are aligned with the MARE consensus sequence. TRE-type consensus and CRE-type consensus (G in the middle position) sequences are presented.

Techniques Used: Binding Assay, Footprinting, Modification, Sequencing

Identification of Pax6-binding sites of the c- maf gene. ( A ) Schematic representation of the probes used for EMSA and footprinting analyses. ( B ) EMSA of c- maf promoter fragments with Pax6–MBP fusion protein. The terminally labeled fragments indicated were bound with Pax6–MBP protein (100 ng) and analyzed as described in Materials and Methods. ( C ) Footprinting analysis of c- maf promoter fragments. Probe A, E or F was mixed with Pax6–MBP protein (1 µg), treated with DNase I and analyzed by denatured polyacrylamide gel electrophoresis. The same probes, modified and digested by the Maxam–Gilbert method, were used as makers (M). (+) and (–) indicate with or without Pax6–MBP protein, respectively. Footprinting regions are indicated by vertical bars and lined under the sequences. ( D ) Pax6-binding sequences. Pax6 paired domain binding consensus sequence obtained by PCR-based oligonucleotide selection (33) and that of the promoter region of the ζ-crystallin gene (28) are aligned together with binding sequences of the c- maf promoter.
Figure Legend Snippet: Identification of Pax6-binding sites of the c- maf gene. ( A ) Schematic representation of the probes used for EMSA and footprinting analyses. ( B ) EMSA of c- maf promoter fragments with Pax6–MBP fusion protein. The terminally labeled fragments indicated were bound with Pax6–MBP protein (100 ng) and analyzed as described in Materials and Methods. ( C ) Footprinting analysis of c- maf promoter fragments. Probe A, E or F was mixed with Pax6–MBP protein (1 µg), treated with DNase I and analyzed by denatured polyacrylamide gel electrophoresis. The same probes, modified and digested by the Maxam–Gilbert method, were used as makers (M). (+) and (–) indicate with or without Pax6–MBP protein, respectively. Footprinting regions are indicated by vertical bars and lined under the sequences. ( D ) Pax6-binding sequences. Pax6 paired domain binding consensus sequence obtained by PCR-based oligonucleotide selection (33) and that of the promoter region of the ζ-crystallin gene (28) are aligned together with binding sequences of the c- maf promoter.

Techniques Used: Binding Assay, Footprinting, Labeling, Polyacrylamide Gel Electrophoresis, Modification, Sequencing, Polymerase Chain Reaction, Selection

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Concentration Assay:

Article Title: Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow ▿
Article Snippet: Polyethylene glycol 6000 (Wako Pure Chemical Industries, Ltd., Japan) was added to the filtrate to a final concentration of 10% (wt/vol), and the suspension was stored at 4°C in the dark overnight. .. Aliquots (7 μl) of the nucleic acids solution were digested with 0.025 μg of RNase A (Nippon Gene Co., Ltd., Japan) at 37°C for 1 h, 0.5 U of DNase I (Takara Bio, Inc., Japan) at 37°C for 1 h, or 0.7 U of S1 nuclease (Takara Bio, Inc.) μl−1 at 23°C for 15 min according to the manufacturer's protocol.

Article Title: The Disruption of an OxyR-Like Protein Impairs Intracellular Magnetite Biomineralization in Magnetospirillum gryphiswaldense MSR-1
Article Snippet: The remaining DNA in the RNA was digested using DNase I (Takara, Shiga, Japan). cDNA was synthesized by reverse transcription using M-MLV reverse transcriptase (Promega Corp., San Luis Obispo, CA, USA), dNTPs and random primers (Takara, Shiga, Japan) according to the manufacturers’ instructions. qRT-PCR analysis was then performed to determine the transcription levels of the selected pdh operon, the tricarboxylic acid cycle-related genes and the mam genes using the obtained cDNA as a template and the corresponding primers. .. The total volume of each reaction was 20 μL, the template cDNA content in each reaction mixture was approximately 50 ng, and the concentration of each oligonucleotide was 0.5 μM.

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase
Article Snippet: .. At an optimum concentration of DNase I, strong cleavages were detected on the 21 bp duplex regions at both ends of the substrates, whereas weaker cleavages were detected on the central single-stranded region, consistent with preference of DNase I to double-stranded DNA. .. With the Bub66/T-rich substrate, strong protection was detected on the entire single-stranded regions of both top and bottom strands with increasing concentration of Mcm4/6/7, consistent with high affinity of Mcm4/6/7 to T-rich bubble sequences ( ).

Article Title: AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region
Article Snippet: .. Briefly, nuclei were suspended in buffer A (50 mM Mes, 5 mM MgCl2 , 0.25 M sucrose, and 10% glycerol, pH 6.0) plus 1 mM PMSF and incubated at 12°C for 1 hr with DNase I (Takara Shuzo) at a final concentration of 50 μg/mL. ..

Footprinting:

Article Title: Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿ †
Article Snippet: Paragraph title: DNase I footprinting assay. ... After incubation for 30 min, DNA digestion was initiated by adding 5 ng DNase I (Takara).

Article Title: Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis
Article Snippet: Paragraph title: DNase I footprinting analysis. ... The P ycnK probe (0.04 pmol), labeled at the 5′ end, was mixed with the YcnK protein to obtain a DNA-protein complex, which was then partially digested with DNase I (TaKaRa-bio) in 50 μl of a reaction mixture and subjected to urea-PAGE with sequencing ladders prepared using genomic DNA of strain 168 and the primer pair PycnKF/PycnKR.

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase
Article Snippet: After incubation at 30°C for 30 min, the indicated amount of DNase I (TAKARA Biomedical, Japan) was added, and the mixtures were incubated at room temperature for 1 min. DNase I was inactivated by the addition of 250 μl of stop solution (20 mM EDTA, 0.2M sodium chloride, 1% SDS and 12.5 μg/ml yeast RNA). .. For nuclease P1 footprinting assay, binding reactions were carried out under the same conditions except that CaCl2 was omitted.

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair
Article Snippet: Paragraph title: DNase I footprinting assay ... After incubation at 30°C for 30 min, the reactions were diluted with 25 μL of a 5 mM CaCl2 solution and then digested at 30°C for 2 min with DNase I (0.004 unit; Takara Shuzo).

Cell Culture:

Article Title: Role of the SaeRS two-component regulatory system in Staphylococcus epidermidis autolysis and biofilm formation
Article Snippet: .. Briefly, mid-exponential phase cells were diluted to OD600 = 0.1 in PBS and then incubated in wells (1 mL per well) of cell-culture polystyrene chambers (Nunc, Denmark) with DNase I (140 U/mL) for 2 h at 37°C. .. Detection of Aap expression Concentrations of lysostaphin-treated whole bacterial proteins from SE1457ΔsaeRS , SE1457, and SE1457saec were determined by the Bradford method.

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses
Article Snippet: Briefly, all fungal strains were cultured on PDA plates covered with cellophane membrane (CM-PDA) at 20°C for 6 days. .. All dsRNA samples were further treated with DNase I and S1 nuclease (TaKaRa, Dalian, China), and then fractioned by agarose gel electrophoresis (1% agarose) or polyacrylamide gel electrophoresis (5% polyacrylamide).

Transmission Assay:

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses
Article Snippet: Virus particles were purified and visualized using transmission electron microscopy (TEM) as previously described [ ]. .. All dsRNA samples were further treated with DNase I and S1 nuclease (TaKaRa, Dalian, China), and then fractioned by agarose gel electrophoresis (1% agarose) or polyacrylamide gel electrophoresis (5% polyacrylamide).

Sequencing:

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa
Article Snippet: Based on the results of DNase I (Takara, Dalian, China) optimization experiments, 0.09 U of DNase I was added to each reaction mixture and incubated at 25°C for 5 min. .. The sequences were then analyzed with Peak Scanner software v1.0 (Applied Biosystems, CA, USA) to convert the DNase I digestion maps into sequencing data to identify the exact sequences that were protected.

Article Title: Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis
Article Snippet: .. The P ycnK probe (0.04 pmol), labeled at the 5′ end, was mixed with the YcnK protein to obtain a DNA-protein complex, which was then partially digested with DNase I (TaKaRa-bio) in 50 μl of a reaction mixture and subjected to urea-PAGE with sequencing ladders prepared using genomic DNA of strain 168 and the primer pair PycnKF/PycnKR. .. Gel retardation analysis was performed essentially as described previously ( ).

Binding Assay:

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase
Article Snippet: After incubation at 30°C for 30 min, the indicated amount of DNase I (TAKARA Biomedical, Japan) was added, and the mixtures were incubated at room temperature for 1 min. DNase I was inactivated by the addition of 250 μl of stop solution (20 mM EDTA, 0.2M sodium chloride, 1% SDS and 12.5 μg/ml yeast RNA). .. For nuclease P1 footprinting assay, binding reactions were carried out under the same conditions except that CaCl2 was omitted.

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair
Article Snippet: Binding reactions were carried out under the same conditions as used for the gel mobility shift assay, except that the reactions were scaled up to 25 μL in which 10 fmole of the end-labeled DNA fragments were included. .. After incubation at 30°C for 30 min, the reactions were diluted with 25 μL of a 5 mM CaCl2 solution and then digested at 30°C for 2 min with DNase I (0.004 unit; Takara Shuzo).

Transmission Electron Microscopy:

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses
Article Snippet: Virus particles were purified and visualized using transmission electron microscopy (TEM) as previously described [ ]. .. All dsRNA samples were further treated with DNase I and S1 nuclease (TaKaRa, Dalian, China), and then fractioned by agarose gel electrophoresis (1% agarose) or polyacrylamide gel electrophoresis (5% polyacrylamide).

Isolation:

Article Title: Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *
Article Snippet: Total RNA was isolated using TRIzol (Invitrogen), according to the supplier's recommendations. .. For qPCR analysis, 1 μg of total RNA was treated with DNase I (Takara) and reverse transcribed with random primers (Invitrogen) and SuperscriptIII reverse transcriptase (Invitrogen). qPCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems) using ABI 7900HT (Applied Biosystems).

Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis
Article Snippet: RNA was isolated from S. avermitilis mycelia grown in FM-I or YEME, using TRIzol reagent (Tiangen, China) as described previously ( ). .. The chromosomal DNA contamination of RNA samples was removed by adding DNase I (TaKaRa, Japan).

Article Title: AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region
Article Snippet: Paragraph title: Isolation of Nuclei and Nuclear Matrix ... Briefly, nuclei were suspended in buffer A (50 mM Mes, 5 mM MgCl2 , 0.25 M sucrose, and 10% glycerol, pH 6.0) plus 1 mM PMSF and incubated at 12°C for 1 hr with DNase I (Takara Shuzo) at a final concentration of 50 μg/mL.

Labeling:

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa
Article Snippet: A 184-bp DNA fragment upstream of the putA gene was amplified by PCR with primers shown in Table , except that the forward primer was labeled with 6-carboxyfluorescein (FAM). .. Based on the results of DNase I (Takara, Dalian, China) optimization experiments, 0.09 U of DNase I was added to each reaction mixture and incubated at 25°C for 5 min.

Article Title: Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis
Article Snippet: .. The P ycnK probe (0.04 pmol), labeled at the 5′ end, was mixed with the YcnK protein to obtain a DNA-protein complex, which was then partially digested with DNase I (TaKaRa-bio) in 50 μl of a reaction mixture and subjected to urea-PAGE with sequencing ladders prepared using genomic DNA of strain 168 and the primer pair PycnKF/PycnKR. .. Gel retardation analysis was performed essentially as described previously ( ).

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair
Article Snippet: The 5′-end labeled, ∼270-bp DNA fragments containing the damaged site were purified by nondenaturing PAGE. .. After incubation at 30°C for 30 min, the reactions were diluted with 25 μL of a 5 mM CaCl2 solution and then digested at 30°C for 2 min with DNase I (0.004 unit; Takara Shuzo).

Purification:

Article Title: Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿ †
Article Snippet: In brief, 1.0 pmol each of FITC-labeled probes was incubated at 37°C for 30 min with purified CsgD in 25 μl of 10 mM Tris-HCl (pH 7.8), 150 mM NaCl, 3 mM magnesium acetate, 5 mM CaCl2 , and 25 mg/ml bovine serum albumin (BSA). .. After incubation for 30 min, DNA digestion was initiated by adding 5 ng DNase I (Takara).

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses
Article Snippet: Paragraph title: Intracellular localization, electron microscopy, virus particles purification and transfection ... All dsRNA samples were further treated with DNase I and S1 nuclease (TaKaRa, Dalian, China), and then fractioned by agarose gel electrophoresis (1% agarose) or polyacrylamide gel electrophoresis (5% polyacrylamide).

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair
Article Snippet: The 5′-end labeled, ∼270-bp DNA fragments containing the damaged site were purified by nondenaturing PAGE. .. After incubation at 30°C for 30 min, the reactions were diluted with 25 μL of a 5 mM CaCl2 solution and then digested at 30°C for 2 min with DNase I (0.004 unit; Takara Shuzo).

Article Title: AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region
Article Snippet: Further purification of nuclei by Percoll gradient centrifugation and preparation of the nuclear matrix with lithium diiodosalicylate (either with or without heat stabilization) were performed as described previously ( ). .. Briefly, nuclei were suspended in buffer A (50 mM Mes, 5 mM MgCl2 , 0.25 M sucrose, and 10% glycerol, pH 6.0) plus 1 mM PMSF and incubated at 12°C for 1 hr with DNase I (Takara Shuzo) at a final concentration of 50 μg/mL.

Polymerase Chain Reaction:

Article Title: Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *
Article Snippet: .. For qPCR analysis, 1 μg of total RNA was treated with DNase I (Takara) and reverse transcribed with random primers (Invitrogen) and SuperscriptIII reverse transcriptase (Invitrogen). qPCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems) using ABI 7900HT (Applied Biosystems). .. The primer sequences used for qPCR are shown in .

Article Title: The Disruption of an OxyR-Like Protein Impairs Intracellular Magnetite Biomineralization in Magnetospirillum gryphiswaldense MSR-1
Article Snippet: Paragraph title: Quantitative Real-Time Reverse Transcriptase PCR (qRT-PCR) ... The remaining DNA in the RNA was digested using DNase I (Takara, Shiga, Japan). cDNA was synthesized by reverse transcription using M-MLV reverse transcriptase (Promega Corp., San Luis Obispo, CA, USA), dNTPs and random primers (Takara, Shiga, Japan) according to the manufacturers’ instructions. qRT-PCR analysis was then performed to determine the transcription levels of the selected pdh operon, the tricarboxylic acid cycle-related genes and the mam genes using the obtained cDNA as a template and the corresponding primers.

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa
Article Snippet: A 184-bp DNA fragment upstream of the putA gene was amplified by PCR with primers shown in Table , except that the forward primer was labeled with 6-carboxyfluorescein (FAM). .. Based on the results of DNase I (Takara, Dalian, China) optimization experiments, 0.09 U of DNase I was added to each reaction mixture and incubated at 25°C for 5 min.

Article Title: Direct and Indirect Regulation of the ycnKJI Operon Involved in Copper Uptake through Two Transcriptional Repressors, YcnK and CsoR, in Bacillus subtilis
Article Snippet: Prior to PCR amplification, the 5′ terminus of only one of the primers was labeled with [γ-32 P]ATP using a Megalabel kit. .. The P ycnK probe (0.04 pmol), labeled at the 5′ end, was mixed with the YcnK protein to obtain a DNA-protein complex, which was then partially digested with DNase I (TaKaRa-bio) in 50 μl of a reaction mixture and subjected to urea-PAGE with sequencing ladders prepared using genomic DNA of strain 168 and the primer pair PycnKF/PycnKR.

Polyacrylamide Gel Electrophoresis:

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses
Article Snippet: .. All dsRNA samples were further treated with DNase I and S1 nuclease (TaKaRa, Dalian, China), and then fractioned by agarose gel electrophoresis (1% agarose) or polyacrylamide gel electrophoresis (5% polyacrylamide). .. Purified virus particles were used for infectivity assays.

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair
Article Snippet: The 5′-end labeled, ∼270-bp DNA fragments containing the damaged site were purified by nondenaturing PAGE. .. After incubation at 30°C for 30 min, the reactions were diluted with 25 μL of a 5 mM CaCl2 solution and then digested at 30°C for 2 min with DNase I (0.004 unit; Takara Shuzo).

Staining:

Article Title: Effect of nicotine on Staphylococcus aureus biofilm formation and virulence factors
Article Snippet: The cultures were then diluted 1:200 with TSBG supplemented with or without nicotine, and 200 μL of bacterial suspension was added to each well of a 96-well microplate and incubated at 37 °C for 24 h. To determine the effect of DNase I and proteinase K on biofilm formation, 5 μL DNase I (5 U/μL, Takara, Shanghai, China) and 2 μg/mL Proteinase K (Sango, Shanghai, China) were added to the wells. .. Thereafter, the wells were washed three times with phosphate-buffered saline (PBS) to remove unattached bacteria and then 200 μL of 100% methanol was added to each well to fix the attached cells at room temperature for 20 min. After removal of the methanol, the biofilms were air-dried and stained with 2% crystal violet at room temperature for 8 min.

Article Title: Isolation and Characterization of a Single-Stranded DNA Virus Infecting Chaetoceros lorenzianus Grunow ▿
Article Snippet: Aliquots (7 μl) of the nucleic acids solution were digested with 0.025 μg of RNase A (Nippon Gene Co., Ltd., Japan) at 37°C for 1 h, 0.5 U of DNase I (Takara Bio, Inc., Japan) at 37°C for 1 h, or 0.7 U of S1 nuclease (Takara Bio, Inc.) μl−1 at 23°C for 15 min according to the manufacturer's protocol. .. Nucleic acids were visualized by using SYBR-Gold staining (Molecular Probes, Inc., Oregon).

Software:

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa
Article Snippet: Based on the results of DNase I (Takara, Dalian, China) optimization experiments, 0.09 U of DNase I was added to each reaction mixture and incubated at 25°C for 5 min. .. The sequences were then analyzed with Peak Scanner software v1.0 (Applied Biosystems, CA, USA) to convert the DNase I digestion maps into sequencing data to identify the exact sequences that were protected.

SYBR Green Assay:

Article Title: Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *
Article Snippet: .. For qPCR analysis, 1 μg of total RNA was treated with DNase I (Takara) and reverse transcribed with random primers (Invitrogen) and SuperscriptIII reverse transcriptase (Invitrogen). qPCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems) using ABI 7900HT (Applied Biosystems). .. The primer sequences used for qPCR are shown in .

Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis
Article Snippet: The chromosomal DNA contamination of RNA samples was removed by adding DNase I (TaKaRa, Japan). .. Each reaction system (20 μl) contains template cDNA, forward and reverse primers (each 300 nM) and 10 μl FastStart Universal SYBR Green Master (ROX).

Article Title: The Disruption of an OxyR-Like Protein Impairs Intracellular Magnetite Biomineralization in Magnetospirillum gryphiswaldense MSR-1
Article Snippet: The remaining DNA in the RNA was digested using DNase I (Takara, Shiga, Japan). cDNA was synthesized by reverse transcription using M-MLV reverse transcriptase (Promega Corp., San Luis Obispo, CA, USA), dNTPs and random primers (Takara, Shiga, Japan) according to the manufacturers’ instructions. qRT-PCR analysis was then performed to determine the transcription levels of the selected pdh operon, the tricarboxylic acid cycle-related genes and the mam genes using the obtained cDNA as a template and the corresponding primers. .. The qRT-PCR assay was performed using a LightCycler 480 Instrument II (Roche, South San Francisco, CA, USA) and the SYBR Green I Master kit (Roche), according to the manufacturer’s recommendations.

Negative Control:

Article Title: PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa
Article Snippet: The FAM-labeled probe (300 ng) was incubated with 1 or 2 μg rPruR or 2 μg BSA (negative control) under the conditions described previously. .. Based on the results of DNase I (Takara, Dalian, China) optimization experiments, 0.09 U of DNase I was added to each reaction mixture and incubated at 25°C for 5 min.

Agarose Gel Electrophoresis:

Article Title: Virus-mediated suppression of host non-self recognition facilitates horizontal transmission of heterologous viruses
Article Snippet: .. All dsRNA samples were further treated with DNase I and S1 nuclease (TaKaRa, Dalian, China), and then fractioned by agarose gel electrophoresis (1% agarose) or polyacrylamide gel electrophoresis (5% polyacrylamide). .. Purified virus particles were used for infectivity assays.

Electrophoresis:

Article Title: Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿Role of the Biofilm Master Regulator CsgD in Cross-Regulation between Biofilm Formation and Flagellar Synthesis ▿ †
Article Snippet: After incubation for 30 min, DNA digestion was initiated by adding 5 ng DNase I (Takara). .. DNA was precipitated from the aqueous layer by ethanol, dissolved in formamide dye solution, and analyzed by electrophoresis on a 6% polyacrylamide gel containing 8 M urea.

Ethanol Precipitation:

Article Title: DNA binding and helicase actions of mouse MCM4/6/7 helicase
Article Snippet: After incubation at 30°C for 30 min, the indicated amount of DNase I (TAKARA Biomedical, Japan) was added, and the mixtures were incubated at room temperature for 1 min. DNase I was inactivated by the addition of 250 μl of stop solution (20 mM EDTA, 0.2M sodium chloride, 1% SDS and 12.5 μg/ml yeast RNA). .. Proteins were removed by phenol–chloroform extraction, and DNAs were collected by ethanol precipitation followed by wash with 70% ethanol.

Spectrophotometry:

Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis
Article Snippet: The chromosomal DNA contamination of RNA samples was removed by adding DNase I (TaKaRa, Japan). .. The concentrations of RNA were measured by NanoVue Plus spectrophotometer (GE Healthcare).

Mobility Shift:

Article Title: A multistep damage recognition mechanism for global genomic nucleotide excision repair
Article Snippet: Binding reactions were carried out under the same conditions as used for the gel mobility shift assay, except that the reactions were scaled up to 25 μL in which 10 fmole of the end-labeled DNA fragments were included. .. After incubation at 30°C for 30 min, the reactions were diluted with 25 μL of a 5 mM CaCl2 solution and then digested at 30°C for 2 min with DNase I (0.004 unit; Takara Shuzo).

Lysis:

Article Title: AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region
Article Snippet: The crude nuclei were collected by centrifugation and washed twice in the lysis buffer without Triton X-100. .. Briefly, nuclei were suspended in buffer A (50 mM Mes, 5 mM MgCl2 , 0.25 M sucrose, and 10% glycerol, pH 6.0) plus 1 mM PMSF and incubated at 12°C for 1 hr with DNase I (Takara Shuzo) at a final concentration of 50 μg/mL.

Gradient Centrifugation:

Article Title: AHM1, a Novel Type of Nuclear Matrix-Localized, MAR Binding Protein with a Single AT Hook and a J Domain-Homologous Region
Article Snippet: Further purification of nuclei by Percoll gradient centrifugation and preparation of the nuclear matrix with lithium diiodosalicylate (either with or without heat stabilization) were performed as described previously ( ). .. Briefly, nuclei were suspended in buffer A (50 mM Mes, 5 mM MgCl2 , 0.25 M sucrose, and 10% glycerol, pH 6.0) plus 1 mM PMSF and incubated at 12°C for 1 hr with DNase I (Takara Shuzo) at a final concentration of 50 μg/mL.

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    TaKaRa dnase i
    Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to <t>DNase</t> I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.
    Dnase I, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 580 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to DNase I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.

    Journal: Journal of Insect Science

    Article Title: Differentially Expressed Genes in the Cuticle and Hemolymph of the Silkworm, Bombyx mori, Injected with the Fungus Beauveria bassiana

    doi: 10.1673/031.013.13801

    Figure Lengend Snippet: Variations in expression levels of fungus-responsive genes in the hemolymph of silkworms, Bombyx mori , infected with Beauveria bassiana . The third-day larvae of the fifth instar of Dazao strains were infected with B. bassiana . Total RNA was extracted from the hemolymph at the indicated time points after infection and subjected to DNase I treatment and reverse transcription. Two microliters of each 10-fold diluted first strand cDNA (20 ng) was analyzed in each real-time qPCR reaction. The reaction was performed with specific primers for amplifying each of the six genes. The relative expression level of each gene at each time point was normalized using the Ct values obtained for the Bm GAPDH amplifications run in the same plate. In each assay, the expression level is shown relative to the lowest expression level, which was set to one. All samples were tested in triplicate. The mean value ± SD was used for the analysis of the relative transcript levels for each time point using the △△Ct method. The B. bassiana injected and water-treated individuals are shown on the left (blue) and right (purple), respectively. A. Chemosensory protein 11; B. Muscle LIM protein isoform 1; C. Transferrin; D. Sex-specific storage-protein SP1 precursor; E. Arylphorin; F. Low molecular lipoprotein 30K pBmHPC-6 (Lp-c6); G. lysozyme; H. Moricin. High quality figures are available online.

    Article Snippet: The RNA was treated with DNase I following the manufacturer' s instructions.

    Techniques: Expressing, Infection, Real-time Polymerase Chain Reaction, Injection

    Identification of the Dan box sequence. (A) Fluorescent-labeled SELEX segment (1.0 pmol) from the dan-ttdA spacer was incubated in the absence or presence of increasing concentrations of purified Dan (10, 20 and 40 pmol from left to right) and then subjected to DNase-I foot-printing assays. Lanes A, T, G and C represent the respective sequence ladders. ( B ) Four Dan-binding sites were identified on the dan-ttdA spacer sequence. ( C ) The Dan-box sequence GTTAAT was predicted after sequence comparison of four Dan-binding sites. Similar sequences were identified among a total of 688 Dan-binding fragments selected by Genomic SELEX. Using this Dan-box sequence, a total of about 1860 sites were predicted to be present in the entire E. coli genome. ( D ) Logo representation of Dan-binding site derived from sequences in in silico analysis. Logos were generated using weblogo ( http://weblogo.berkeley.edu/ ).

    Journal: Nucleic Acids Research

    Article Title: A novel nucleoid protein of Escherichia coli induced under anaerobiotic growth conditions

    doi: 10.1093/nar/gkq077

    Figure Lengend Snippet: Identification of the Dan box sequence. (A) Fluorescent-labeled SELEX segment (1.0 pmol) from the dan-ttdA spacer was incubated in the absence or presence of increasing concentrations of purified Dan (10, 20 and 40 pmol from left to right) and then subjected to DNase-I foot-printing assays. Lanes A, T, G and C represent the respective sequence ladders. ( B ) Four Dan-binding sites were identified on the dan-ttdA spacer sequence. ( C ) The Dan-box sequence GTTAAT was predicted after sequence comparison of four Dan-binding sites. Similar sequences were identified among a total of 688 Dan-binding fragments selected by Genomic SELEX. Using this Dan-box sequence, a total of about 1860 sites were predicted to be present in the entire E. coli genome. ( D ) Logo representation of Dan-binding site derived from sequences in in silico analysis. Logos were generated using weblogo ( http://weblogo.berkeley.edu/ ).

    Article Snippet: After brief treatment with DNase I, at least four protected regions were identified ( A), of which three (Dan-I, Dan-II and Dan-III) covered 14-bp-long sequences and one (Dan-IV) covered 29-bp sequence, suggesting that two molecules of Dan are associated with Dan-IV site, designated as Dan-IV-1 and Dan-IV-2 ( B).

    Techniques: Sequencing, Labeling, Incubation, Purification, Binding Assay, Derivative Assay, In Silico, Generated

    DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.

    Journal: Frontiers in Microbiology

    Article Title: Organic Peroxide-Sensing Repressor OhrR Regulates Organic Hydroperoxide Stress Resistance and Avermectin Production in Streptomyces avermitilis

    doi: 10.3389/fmicb.2018.01398

    Figure Lengend Snippet: DNase I footprinting assay of ohrR–ohrB2 intergenic region using His 6 -OhrR. (A) Fluorograms corresponding to control DNA and to protected reactions with 0.4 and 0.8 μM His 6 -OhrR. (B) Nucleotide sequences of ohrR–ohrB2 intergenic region. Non-shaded boxes: presumed –35 and –10 regions of ohrR and ohrB2 . Shaded boxes: regions protected by His 6 -OhrR. Underlining: OhrR motif (site a and site b). Gray bent arrows: translational start codons. Black bent arrows: TSSs. (C) Consensus sequence of OhrR motif.

    Article Snippet: The chromosomal DNA contamination of RNA samples was removed by adding DNase I (TaKaRa, Japan).

    Techniques: Footprinting, Sequencing

    Identification of novel HS sites by DNase I hypersensitivity assay. A , schematic diagram of the DNase I hypersensitivity assay of the Pcdh- γ gene. Upper part , the large colored boxes , Pcdh- γ or Diap1 exons. The small red boxes indicate

    Journal: The Journal of Biological Chemistry

    Article Title: Identification of the Cluster Control Region for the Protocadherin-? Genes Located beyond the Protocadherin-? Cluster *

    doi: 10.1074/jbc.M111.245605

    Figure Lengend Snippet: Identification of novel HS sites by DNase I hypersensitivity assay. A , schematic diagram of the DNase I hypersensitivity assay of the Pcdh- γ gene. Upper part , the large colored boxes , Pcdh- γ or Diap1 exons. The small red boxes indicate

    Article Snippet: For qPCR analysis, 1 μg of total RNA was treated with DNase I (Takara) and reverse transcribed with random primers (Invitrogen) and SuperscriptIII reverse transcriptase (Invitrogen). qPCR was performed with the SYBR Green PCR Master Mix (Applied Biosystems) using ABI 7900HT (Applied Biosystems).

    Techniques: