branch migration assays helicase assay substrates  (New England Biolabs)


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    Structured Review

    New England Biolabs branch migration assays helicase assay substrates
    ComM exhibits <t>helicase</t> activity in vitro . ( A ) A representative forked substrate helicase assay with increasing concentrations of purified ComM. This forked substrate has 20 bp of annealed sequence and 25 bp tails. Concentrations of ComM used (in hexamer) were 0, 10, 25, 37.5, 50, 75, 100, 150, 200 and 250 nM. Images were quantified and plotted as indicated. ( B ) Helicase assay using forked DNA substrate with the indicated purified protein (100 nM Pif1 and 250 nM ComM/ComM K224A hexamer) in the presence of 5 mM ATP (Columns 1, 2 and 3) or ATPγS (Column 4). ( C ) Helicase assays using forked DNA substrates that accommodate enzymes of either directionality or that can only be unwound by 5′ to 3′ or 3′ to 5′ activity. Directional substrates contained one ssDNA tail that is inverted relative to the remainder of the strand (inverted portion indicated in gray on the schematic above bars), thus, preventing helicase activity in one direction. Substrates were incubated with 100 nM purified ComM (hexamer), 10 nM Pif1, or 50 nM SftH. All data are shown as the mean ± SD and are the result of at least three independent replicates. *** P
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    Images

    1) Product Images from "ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species"

    Article Title: ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky343

    ComM exhibits helicase activity in vitro . ( A ) A representative forked substrate helicase assay with increasing concentrations of purified ComM. This forked substrate has 20 bp of annealed sequence and 25 bp tails. Concentrations of ComM used (in hexamer) were 0, 10, 25, 37.5, 50, 75, 100, 150, 200 and 250 nM. Images were quantified and plotted as indicated. ( B ) Helicase assay using forked DNA substrate with the indicated purified protein (100 nM Pif1 and 250 nM ComM/ComM K224A hexamer) in the presence of 5 mM ATP (Columns 1, 2 and 3) or ATPγS (Column 4). ( C ) Helicase assays using forked DNA substrates that accommodate enzymes of either directionality or that can only be unwound by 5′ to 3′ or 3′ to 5′ activity. Directional substrates contained one ssDNA tail that is inverted relative to the remainder of the strand (inverted portion indicated in gray on the schematic above bars), thus, preventing helicase activity in one direction. Substrates were incubated with 100 nM purified ComM (hexamer), 10 nM Pif1, or 50 nM SftH. All data are shown as the mean ± SD and are the result of at least three independent replicates. *** P
    Figure Legend Snippet: ComM exhibits helicase activity in vitro . ( A ) A representative forked substrate helicase assay with increasing concentrations of purified ComM. This forked substrate has 20 bp of annealed sequence and 25 bp tails. Concentrations of ComM used (in hexamer) were 0, 10, 25, 37.5, 50, 75, 100, 150, 200 and 250 nM. Images were quantified and plotted as indicated. ( B ) Helicase assay using forked DNA substrate with the indicated purified protein (100 nM Pif1 and 250 nM ComM/ComM K224A hexamer) in the presence of 5 mM ATP (Columns 1, 2 and 3) or ATPγS (Column 4). ( C ) Helicase assays using forked DNA substrates that accommodate enzymes of either directionality or that can only be unwound by 5′ to 3′ or 3′ to 5′ activity. Directional substrates contained one ssDNA tail that is inverted relative to the remainder of the strand (inverted portion indicated in gray on the schematic above bars), thus, preventing helicase activity in one direction. Substrates were incubated with 100 nM purified ComM (hexamer), 10 nM Pif1, or 50 nM SftH. All data are shown as the mean ± SD and are the result of at least three independent replicates. *** P

    Techniques Used: Activity Assay, In Vitro, Helicase Assay, Purification, Sequencing, Incubation

    Proposed model for the role of ComM during natural transformation. ComM is shown as a hexameric ring that promotes integration of tDNA via its bidirectional helicase and/or branch migration activity. This can support integration of tDNA with a heterologous region, which is indicated by a gray box.
    Figure Legend Snippet: Proposed model for the role of ComM during natural transformation. ComM is shown as a hexameric ring that promotes integration of tDNA via its bidirectional helicase and/or branch migration activity. This can support integration of tDNA with a heterologous region, which is indicated by a gray box.

    Techniques Used: Transformation Assay, Migration, Activity Assay

    2) Product Images from "Multiple RNA–RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome"

    Article Title: Multiple RNA–RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky966

    The Hoogsteen interactions of U6 nucleotides A59 and G60 are not essential for splicing. ( A – E ) Kinetics of splicing of actin pre-mRNA in U6-depleted yeast extract supplemented with different synthetic U6 snRNAs. Splicing performed with (A) wild-type U6 (wt), (B) N7-deaza-A59 U6 (7cA59), (C) N7-deaza-G60 U6 (7cG60) or (D) N7-deaza-A59/N7-deaza-G60 U6 (7cA59/7cG60). The positions of the pre-mRNA, and of the splicing intermediates and products are indicated on the right. (E) Quantification of spliced mRNA production (left panel) and intron lariat-3′ exon (denoted as intermediate) production (right panel). The average amount of mRNA or intermediate present (plus the standard error) at each time point was calculated from three independent experiments. The production of mRNA at all time points was normalized to the amount of mRNA produced at the 60 min time point with wt U6. Similarly, lariat-3′ exon production (intermediate) was normalized to the amount of splicing intermediate produced at the 60 min time point with wt U6.
    Figure Legend Snippet: The Hoogsteen interactions of U6 nucleotides A59 and G60 are not essential for splicing. ( A – E ) Kinetics of splicing of actin pre-mRNA in U6-depleted yeast extract supplemented with different synthetic U6 snRNAs. Splicing performed with (A) wild-type U6 (wt), (B) N7-deaza-A59 U6 (7cA59), (C) N7-deaza-G60 U6 (7cG60) or (D) N7-deaza-A59/N7-deaza-G60 U6 (7cA59/7cG60). The positions of the pre-mRNA, and of the splicing intermediates and products are indicated on the right. (E) Quantification of spliced mRNA production (left panel) and intron lariat-3′ exon (denoted as intermediate) production (right panel). The average amount of mRNA or intermediate present (plus the standard error) at each time point was calculated from three independent experiments. The production of mRNA at all time points was normalized to the amount of mRNA produced at the 60 min time point with wt U6. Similarly, lariat-3′ exon production (intermediate) was normalized to the amount of splicing intermediate produced at the 60 min time point with wt U6.

    Techniques Used: Produced

    3) Product Images from "Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403"

    Article Title: Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.187.11.3762-3778.2005

    Substitution analysis of the metB2 promoter region. Upper panel: sequence of metB2 wild-type and mutated promoter region. Position +1 corresponds to the experimental transcriptional start. The deduced extended −10 box is underlined, and putative FhuR-DNA-binding box 1 and box 2 are shaded. Shifts + and − indicate the observation of a band shift or not, respectively, of the corresponding DNA fragment in the presence of the purified FhuR protein. Letters A to F on the right refer to the corresponding lower panel. Lower panels: gel mobility shift assay for FhuR binding to metB2 wild-type and mutated promoter regions. The corresponding fragments were PCR amplified and labeled. DNA probes (10 × 10 −15 mole) were incubated without (lane 1) or with 3.6 μM FhuR (lane 2) in the presence of 60 mM OAS and analyzed by nondenaturing polyacrylamide gel electrophoresis. (A) metB2 wild-type promoter region. (B to F) Mutated sequences indicated on the right column of the upper panel. In each binding assay, the bands for the probe (free) and the FhuR-probe complex (bound) are indicated by an arrow and an arrow with an asterisk, respectively.
    Figure Legend Snippet: Substitution analysis of the metB2 promoter region. Upper panel: sequence of metB2 wild-type and mutated promoter region. Position +1 corresponds to the experimental transcriptional start. The deduced extended −10 box is underlined, and putative FhuR-DNA-binding box 1 and box 2 are shaded. Shifts + and − indicate the observation of a band shift or not, respectively, of the corresponding DNA fragment in the presence of the purified FhuR protein. Letters A to F on the right refer to the corresponding lower panel. Lower panels: gel mobility shift assay for FhuR binding to metB2 wild-type and mutated promoter regions. The corresponding fragments were PCR amplified and labeled. DNA probes (10 × 10 −15 mole) were incubated without (lane 1) or with 3.6 μM FhuR (lane 2) in the presence of 60 mM OAS and analyzed by nondenaturing polyacrylamide gel electrophoresis. (A) metB2 wild-type promoter region. (B to F) Mutated sequences indicated on the right column of the upper panel. In each binding assay, the bands for the probe (free) and the FhuR-probe complex (bound) are indicated by an arrow and an arrow with an asterisk, respectively.

    Techniques Used: Sequencing, Binding Assay, Electrophoretic Mobility Shift Assay, Purification, Mobility Shift, Polymerase Chain Reaction, Amplification, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

    Gel mobility shift assay for FhuR binding to different regulated gene promoter regions. The promoter regions of FhuR target genes were PCR amplified and labeled. Radiolabeled DNA probes (10 × 10 −15 mole) for metB2 (A), plpA (B), yjgC (C), cysM (D), cysD (E), and metA (F) were incubated with purified FhuR protein at various concentrations and analyzed on nondenaturing polyacrylamide gel electrophoresis (see Materials and Methods for details). (A and B) Lane 1, no protein; lanes 2 to 5, 0.6, 1.2, 2.4, and 3.6 μM final concentrations of the FhuR protein, respectively; lane 6, 3.6 μM final concentration of the FhuR protein with an excess of unlabeled DNA probe (10 × 10 −13 mole). As a negative control, lanes 7 and 8 contain no protein and 3.6 μM (final) of the FhuR protein, respectively, with a purH gene 32 P-labeled internal fragment as DNA probe. (C to F) Lanes 1 and 2, no protein and 3.6 μM (final) of the FhuR protein, respectively; lane 3, 3.6 μM (final) of the FhuR protein with an excess of unlabeled DNA probe (10 × 10 −13 mole). In all panels, the bands for the probe (free) and the FhuR-probe complex (bound) are indicated by an arrow and an arrow with an asterisk, respectively.
    Figure Legend Snippet: Gel mobility shift assay for FhuR binding to different regulated gene promoter regions. The promoter regions of FhuR target genes were PCR amplified and labeled. Radiolabeled DNA probes (10 × 10 −15 mole) for metB2 (A), plpA (B), yjgC (C), cysM (D), cysD (E), and metA (F) were incubated with purified FhuR protein at various concentrations and analyzed on nondenaturing polyacrylamide gel electrophoresis (see Materials and Methods for details). (A and B) Lane 1, no protein; lanes 2 to 5, 0.6, 1.2, 2.4, and 3.6 μM final concentrations of the FhuR protein, respectively; lane 6, 3.6 μM final concentration of the FhuR protein with an excess of unlabeled DNA probe (10 × 10 −13 mole). As a negative control, lanes 7 and 8 contain no protein and 3.6 μM (final) of the FhuR protein, respectively, with a purH gene 32 P-labeled internal fragment as DNA probe. (C to F) Lanes 1 and 2, no protein and 3.6 μM (final) of the FhuR protein, respectively; lane 3, 3.6 μM (final) of the FhuR protein with an excess of unlabeled DNA probe (10 × 10 −13 mole). In all panels, the bands for the probe (free) and the FhuR-probe complex (bound) are indicated by an arrow and an arrow with an asterisk, respectively.

    Techniques Used: Mobility Shift, Binding Assay, Polymerase Chain Reaction, Amplification, Labeling, Incubation, Purification, Polyacrylamide Gel Electrophoresis, Concentration Assay, Negative Control

    In vivo and in vitro effects of OAS on FhuR-dependent regulation in L. lactis . Upper panel: effect of OAS on expression of FhuR-regulated genes of L. lactis . Luciferase activities (10 4 lux/OD 600 ) measured from JIM8499, JIM8543, and JIM8545 strains carrying the P metB2 - lux , P yjgC - lux , and P yhcE - lux fusions, respectively, grown in M17 medium with or without OAS. The x axis shows time of incubation relative to the addition of OAS (time zero). Symbols: ⋄, without OAS; ▪, with OAS at a final concentration of 1 mM; •, at 2 mM; ▴, at 3 mM; ♦, at 4 mM; ×, at 5 mM. Lower panel: OAS effect on FhuR- metB2 complex migration. The labeled DNA fragment containing the metB2 promoter region (10 × 10 −15 mole) was incubated with different concentrations of purified FhuR protein (0 μM, 1.2 μM, and 3.6 μM) with an increasing concentration of OAS (0 mM, 20 mM, 40 mM, 60 mM, and 80 mM) and analyzed on nondenaturing polyacrylamide gel electrophoresis.
    Figure Legend Snippet: In vivo and in vitro effects of OAS on FhuR-dependent regulation in L. lactis . Upper panel: effect of OAS on expression of FhuR-regulated genes of L. lactis . Luciferase activities (10 4 lux/OD 600 ) measured from JIM8499, JIM8543, and JIM8545 strains carrying the P metB2 - lux , P yjgC - lux , and P yhcE - lux fusions, respectively, grown in M17 medium with or without OAS. The x axis shows time of incubation relative to the addition of OAS (time zero). Symbols: ⋄, without OAS; ▪, with OAS at a final concentration of 1 mM; •, at 2 mM; ▴, at 3 mM; ♦, at 4 mM; ×, at 5 mM. Lower panel: OAS effect on FhuR- metB2 complex migration. The labeled DNA fragment containing the metB2 promoter region (10 × 10 −15 mole) was incubated with different concentrations of purified FhuR protein (0 μM, 1.2 μM, and 3.6 μM) with an increasing concentration of OAS (0 mM, 20 mM, 40 mM, 60 mM, and 80 mM) and analyzed on nondenaturing polyacrylamide gel electrophoresis.

    Techniques Used: In Vivo, In Vitro, Expressing, Luciferase, Incubation, Concentration Assay, Migration, Labeling, Purification, Polyacrylamide Gel Electrophoresis

    4) Product Images from "Poly(ADP-ribose) polymerase 1 regulates both the exonuclease and helicase activities of the Werner syndrome protein"

    Article Title: Poly(ADP-ribose) polymerase 1 regulates both the exonuclease and helicase activities of the Werner syndrome protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkh721

    Activated and poly(ADP-ribosyl)ated PARP-1 does not inhibit WRN catalytic activities. ( A ) PARP-1 (500 ng) and NAD + (100 μM) as indicated were incubated with an unlabeled forked duplex for 10 min at room temperature. Western blot analysis was performed using anti-PARP-1 (left panel) or anti-PAR (right panel) antibodies. ( B ) WRN (7.5 nM, lanes 2 to 4) and a radiolabeled 34 bp forked duplex (10 fmol, 34ForkA/34ForkB) were incubated with aliquots from the reactions performed in (A) containing PARP-1 (lane 2), PARP-1 and NAD + (lane 3), or NAD + (lane 4) for 15 min at 37°C. Lane 1, no enzyme. Products were heat-denatured for 5 min at 95°C, run on a 14% denaturing polyacrylamide gel, and visualized using a PhosphorImager. ( C ) Aliquots from the reactions performed in (A) containing PARP-1 (lanes 3 and 6), PARP-1 and NAD + (lanes 4 and 7), or NAD + (lanes 5 and 8) were incubated with a radiolabeled 22 bp forked duplex (20 fmol, 22Fork3/22Fork4) in the absence (lanes 3 to 5) or presence (lanes 6 to 8) of WRN (1 nM) for 15 min at 37°C. Lane 1, heat-denatured substrate. Lane 2, no enzyme. Products were run on a 12% native polyacrylamide gel and visualized using a PhosphorImager.
    Figure Legend Snippet: Activated and poly(ADP-ribosyl)ated PARP-1 does not inhibit WRN catalytic activities. ( A ) PARP-1 (500 ng) and NAD + (100 μM) as indicated were incubated with an unlabeled forked duplex for 10 min at room temperature. Western blot analysis was performed using anti-PARP-1 (left panel) or anti-PAR (right panel) antibodies. ( B ) WRN (7.5 nM, lanes 2 to 4) and a radiolabeled 34 bp forked duplex (10 fmol, 34ForkA/34ForkB) were incubated with aliquots from the reactions performed in (A) containing PARP-1 (lane 2), PARP-1 and NAD + (lane 3), or NAD + (lane 4) for 15 min at 37°C. Lane 1, no enzyme. Products were heat-denatured for 5 min at 95°C, run on a 14% denaturing polyacrylamide gel, and visualized using a PhosphorImager. ( C ) Aliquots from the reactions performed in (A) containing PARP-1 (lanes 3 and 6), PARP-1 and NAD + (lanes 4 and 7), or NAD + (lanes 5 and 8) were incubated with a radiolabeled 22 bp forked duplex (20 fmol, 22Fork3/22Fork4) in the absence (lanes 3 to 5) or presence (lanes 6 to 8) of WRN (1 nM) for 15 min at 37°C. Lane 1, heat-denatured substrate. Lane 2, no enzyme. Products were run on a 12% native polyacrylamide gel and visualized using a PhosphorImager.

    Techniques Used: Incubation, Western Blot

    The effect of PARP-1 and PARP-2 on WRN helicase activity. ( A ) Reactions containing WRN (0.25 nM, lanes 3 to 7; or 0.5 nM, lanes 8 to 12) in the absence or presence of PARP-1 were incubated with a 22 bp forked substrate (0.5 nM, 22Fork3/22Fork4) for 15 min at 37°C. The concentrations of PARP-1 were 4 nM (lane 2), 0.25, 0.5, 1 and 2 nM (lanes 4 to 7, respectively), and 0.5, 1, 2 and 4 nM (lanes 9 to 12, respectively). Lane 1, substrate only. Lane 13, heat-denatured substrate. Reaction products were run on a 12% native gel and visualized using a PhosphorImager. %D, percentage of single-stranded product displaced. ( B ) WRN (0.25 nM, lanes 3 to 7; or 0.5 nM, lanes 8 to 12) was incubated in the absence or presence of PARP-2 for 15 min at 37°C with a 22 bp forked duplex substrate (0.5 nM, 22Fork3/22Fork4). Concentrations of PARP-2 were 4 nM (lane 2), 0.25, 0.5, 1 and 2 nM (lanes 4 to 7, respectively), and 0.5, 1, 2 and 4 nM (lanes 9 to 12, respectively). Lane 1, substrate only. Lane 13, heat-denatured substrate. Reactions were analyzed as above.
    Figure Legend Snippet: The effect of PARP-1 and PARP-2 on WRN helicase activity. ( A ) Reactions containing WRN (0.25 nM, lanes 3 to 7; or 0.5 nM, lanes 8 to 12) in the absence or presence of PARP-1 were incubated with a 22 bp forked substrate (0.5 nM, 22Fork3/22Fork4) for 15 min at 37°C. The concentrations of PARP-1 were 4 nM (lane 2), 0.25, 0.5, 1 and 2 nM (lanes 4 to 7, respectively), and 0.5, 1, 2 and 4 nM (lanes 9 to 12, respectively). Lane 1, substrate only. Lane 13, heat-denatured substrate. Reaction products were run on a 12% native gel and visualized using a PhosphorImager. %D, percentage of single-stranded product displaced. ( B ) WRN (0.25 nM, lanes 3 to 7; or 0.5 nM, lanes 8 to 12) was incubated in the absence or presence of PARP-2 for 15 min at 37°C with a 22 bp forked duplex substrate (0.5 nM, 22Fork3/22Fork4). Concentrations of PARP-2 were 4 nM (lane 2), 0.25, 0.5, 1 and 2 nM (lanes 4 to 7, respectively), and 0.5, 1, 2 and 4 nM (lanes 9 to 12, respectively). Lane 1, substrate only. Lane 13, heat-denatured substrate. Reactions were analyzed as above.

    Techniques Used: Activity Assay, Incubation

    5) Product Images from "The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis ▿"

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis ▿

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00849-08

    Regulation of transcripts initiating at the P sdh and P sodB promoters in response to iron, Fur, and NrrF by S1 nuclease protection assays. Total RNA from wild-type MC58, the NrrF mutant (MC-sRN2), the Fur mutant (MC-Fko), and the Fur-NrrF double mutant (Fko-sRN2) cells grown to mid-log phase under iron-replete conditions (−) or then exposed to iron-limiting conditions: 15 min with 100 μM (+), 15 min with 250 μM (++), or 45 min with 250 μM (+++). The results of the S1 nuclease assay with a sdhC -specific probe (probe C) (A) or a sodB -specific probe (B) are shown. Bands corresponding to S1-resistant products are indicated. The band corresponding to the +1 nucleotide of transcriptional initiation is labeled accordingly (P sdh or P sodB ), and the lower band is thought to be degradation products. The total levels of the two transcripts relating to the +1 of transcription (P sdh or P sodB ) and the putative shorter degradation product (*) were measured by phosphorimaging, and the ImageQuant software results indicating relative quantities are shown in graphic form.
    Figure Legend Snippet: Regulation of transcripts initiating at the P sdh and P sodB promoters in response to iron, Fur, and NrrF by S1 nuclease protection assays. Total RNA from wild-type MC58, the NrrF mutant (MC-sRN2), the Fur mutant (MC-Fko), and the Fur-NrrF double mutant (Fko-sRN2) cells grown to mid-log phase under iron-replete conditions (−) or then exposed to iron-limiting conditions: 15 min with 100 μM (+), 15 min with 250 μM (++), or 45 min with 250 μM (+++). The results of the S1 nuclease assay with a sdhC -specific probe (probe C) (A) or a sodB -specific probe (B) are shown. Bands corresponding to S1-resistant products are indicated. The band corresponding to the +1 nucleotide of transcriptional initiation is labeled accordingly (P sdh or P sodB ), and the lower band is thought to be degradation products. The total levels of the two transcripts relating to the +1 of transcription (P sdh or P sodB ) and the putative shorter degradation product (*) were measured by phosphorimaging, and the ImageQuant software results indicating relative quantities are shown in graphic form.

    Techniques Used: Mutagenesis, Nuclease Assay, Labeling, Software

    6) Product Images from "Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid"

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    Journal: Nucleic Acids Research

    doi:

    N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.
    Figure Legend Snippet: N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Techniques Used: Activity Assay, High Performance Liquid Chromatography, Incubation, Labeling

    7) Product Images from "Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression *"

    Article Title: Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.420521

    LASV NP exoribonuclease activity preferentially degrades dsRNA substrates. Various concentrations of purified full-length NP, C-terminal domain of NP ( NP-C ), and a catalytic mutant ( NP-D389A ) were incubated at 37 °C for 60 min with a 5′ 32 P-labeled 15-nt RNA oligo of either ss or ds forms (“Experimental Procedures”). The products were separated in a 17% urea-polyacrylamide gel and exposed to films. A representative gel of at least three independent experiments was shown.
    Figure Legend Snippet: LASV NP exoribonuclease activity preferentially degrades dsRNA substrates. Various concentrations of purified full-length NP, C-terminal domain of NP ( NP-C ), and a catalytic mutant ( NP-D389A ) were incubated at 37 °C for 60 min with a 5′ 32 P-labeled 15-nt RNA oligo of either ss or ds forms (“Experimental Procedures”). The products were separated in a 17% urea-polyacrylamide gel and exposed to films. A representative gel of at least three independent experiments was shown.

    Techniques Used: Activity Assay, Purification, Mutagenesis, Incubation, Labeling

    8) Product Images from "Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter"

    Article Title: Kinetic resolution of bimolecular hybridization versus intramolecular folding in nucleic acids by surface plasmon resonance: application to G-quadruplex/duplex competition in human c-myc promoter

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki750

    Sensorgrams analyzed with simple versus QC hybridization models. Sensorgrams were obtained by hybridization with immobilized G1B ( a–c ) or C1B ( d ) using 16, 32, 64, 128, 256, 512 or 1024 nM of the respective complementary strand in the mobile phase. Fitted curves (red) were obtained by fitting the sensorgrams (black) with either the QC model using Equation 8 (a and b) or simple hybridization model using Equation 2 (c and d) as in Material and Methods. Sensorgrams were obtained at 25°C and pH 7.4 in either 150 mM KCl (a) or NaCl (b–d). Hybridization of C1 and G1 shows no triplex formation ( e ). Reactions had 10 nM of 5′ end labeled and 0.5 μM unlabeled C1 (lane 1) with either, 0.5 μM G1 (lane 2) and 5 U DNase I (lane 3) or 1.5 μM G1 (lane 4) and 5 U DNase I (lane 5). Samples were incubated at 4°C for 18 h before 15 min DNase I treatment (lanes 3 and 5). G1 shows multiple folded conformations ( f ). An aliquot of 10 nM of 5′ end labeled and 0.5 μM unlabeled G1 (lane 1), C1 (lane 2) and 31mer control dT31 (lane 3) were incubated for 4 h at 25°C before separation. Both (e) and (f) were incubated in 10 mM HEPES, 150 mM NaCl, 3mM MgCl 2 and pH 7.4. Bands were separated in a 20% non-denaturing PAGE in 0.5× TBE buffer (pH 8.0) at 4°C for 6 h at 90 V and visualized using autoradiography on phosphorimager (Fujifilm FLA 2000).
    Figure Legend Snippet: Sensorgrams analyzed with simple versus QC hybridization models. Sensorgrams were obtained by hybridization with immobilized G1B ( a–c ) or C1B ( d ) using 16, 32, 64, 128, 256, 512 or 1024 nM of the respective complementary strand in the mobile phase. Fitted curves (red) were obtained by fitting the sensorgrams (black) with either the QC model using Equation 8 (a and b) or simple hybridization model using Equation 2 (c and d) as in Material and Methods. Sensorgrams were obtained at 25°C and pH 7.4 in either 150 mM KCl (a) or NaCl (b–d). Hybridization of C1 and G1 shows no triplex formation ( e ). Reactions had 10 nM of 5′ end labeled and 0.5 μM unlabeled C1 (lane 1) with either, 0.5 μM G1 (lane 2) and 5 U DNase I (lane 3) or 1.5 μM G1 (lane 4) and 5 U DNase I (lane 5). Samples were incubated at 4°C for 18 h before 15 min DNase I treatment (lanes 3 and 5). G1 shows multiple folded conformations ( f ). An aliquot of 10 nM of 5′ end labeled and 0.5 μM unlabeled G1 (lane 1), C1 (lane 2) and 31mer control dT31 (lane 3) were incubated for 4 h at 25°C before separation. Both (e) and (f) were incubated in 10 mM HEPES, 150 mM NaCl, 3mM MgCl 2 and pH 7.4. Bands were separated in a 20% non-denaturing PAGE in 0.5× TBE buffer (pH 8.0) at 4°C for 6 h at 90 V and visualized using autoradiography on phosphorimager (Fujifilm FLA 2000).

    Techniques Used: Hybridization, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    9) Product Images from "Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination"

    Article Title: Preferential D-loop Extension by a Translesion DNA Polymerase Underlies Error-Prone Recombination

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2573

    Pol IV is highly proficient and error-prone in recombination-directed replication (a) Model of DSB repair. DNA ends are resected by nucleases resulting in 3’ ssDNA tails. RecA promotes strand invasion resulting in a D-loop. Pol extends the D-loop (red arrow). The second DNA end is captured then Holliday junctions are formed which are subsequently resolved by an endonuclease. (b) Scheme for reconstitution of RDR (D-loop extension). A 5’- 32 P labeled ssDNA is incubated with RecA, ATP and dNTPs which promotes RecA filament formation. A supercoiled plasmid containing the same sequence as the ssDNA is then added which facilitates D-loop formation. The β-clamp, which confers processivity onto pols, is then assembled at the D-loop by adding β along with its clamp-loader (γ-complex) and SSB. Last, DNA polymerase is added which initiates RDR by extending the D-loop. (c) RDR was performed with 500 nM pol IV (lanes 1–4) or pol V (lanes 5–8) for the indicated times. (d) Controls for pol IV RDR activity. RDR was performed as in (c) in the presence or absence of the indicated reagents. (e) RDR was performed with 500 nM pol V in the presence of increasing amounts of ssDNA and 3.3 µM RecA. (f) Primer extension was performed with 500 nM pol V and 2 µM RecA in the presence (lane 3) and absence (lane 2) of 160 nM trans ssDNA. * indicates 32 P. (g) RDR was performed with pol IV at relative concentrations corresponding to SOS-induced cells in the presence (lane 3, left) and absence (lane 2, left; right) of β with (right) or without (left) increasing amounts of sodium glutamate (NaGlu). Relative D-loop extension (RE) was determined by dividing the fraction of D-loop extension observed in lane 2 by that observed in lane 3 (left). Fraction of D-loop extension was determined by dividing the intensity of the extended D-loop product by the sum of the intensities of the unextended and extended D-loop products. (h) Primer (left) and D-loop (right) extension were performed with pol IV and the indicated dNTP. D-loops were purified and DNA products were resolved in denaturing urea polyacrylamide gels. RE was determined by dividing the fraction of extension products for each lane by the fraction of extension products in lane 2 for each panel. (i) D-loop extension was performed as in (h). (j) RDR was performed with pol IV in the presence of 50 µM dGTP and 10 µM 2',3'-dideoxyadenosine triphosphate (ddATP)(lane 1). Incorporation of the ddAMP chain terminator opposite the thymidine base (T) prevents further extension of the D-loop. DNA products were analyzed as in (h). The DNA sequence of the product in lane 1 was determined by comparison to the DNA markers in lanes 2 and 3. The mobility of the product in lane 1 (upper band) corresponds to the marker in lane 2 which indicates that the D-loop was extended by the incorporation of 3 dGMPs and 1 ddAMP demonstrating a −1 frameshift mutation (see schematic at bottom). Partial DNA sequences of the invading ssDNA and markers are indicated. β-clamp, clamp-loader, and SSB were present in all reactions except where indicated.
    Figure Legend Snippet: Pol IV is highly proficient and error-prone in recombination-directed replication (a) Model of DSB repair. DNA ends are resected by nucleases resulting in 3’ ssDNA tails. RecA promotes strand invasion resulting in a D-loop. Pol extends the D-loop (red arrow). The second DNA end is captured then Holliday junctions are formed which are subsequently resolved by an endonuclease. (b) Scheme for reconstitution of RDR (D-loop extension). A 5’- 32 P labeled ssDNA is incubated with RecA, ATP and dNTPs which promotes RecA filament formation. A supercoiled plasmid containing the same sequence as the ssDNA is then added which facilitates D-loop formation. The β-clamp, which confers processivity onto pols, is then assembled at the D-loop by adding β along with its clamp-loader (γ-complex) and SSB. Last, DNA polymerase is added which initiates RDR by extending the D-loop. (c) RDR was performed with 500 nM pol IV (lanes 1–4) or pol V (lanes 5–8) for the indicated times. (d) Controls for pol IV RDR activity. RDR was performed as in (c) in the presence or absence of the indicated reagents. (e) RDR was performed with 500 nM pol V in the presence of increasing amounts of ssDNA and 3.3 µM RecA. (f) Primer extension was performed with 500 nM pol V and 2 µM RecA in the presence (lane 3) and absence (lane 2) of 160 nM trans ssDNA. * indicates 32 P. (g) RDR was performed with pol IV at relative concentrations corresponding to SOS-induced cells in the presence (lane 3, left) and absence (lane 2, left; right) of β with (right) or without (left) increasing amounts of sodium glutamate (NaGlu). Relative D-loop extension (RE) was determined by dividing the fraction of D-loop extension observed in lane 2 by that observed in lane 3 (left). Fraction of D-loop extension was determined by dividing the intensity of the extended D-loop product by the sum of the intensities of the unextended and extended D-loop products. (h) Primer (left) and D-loop (right) extension were performed with pol IV and the indicated dNTP. D-loops were purified and DNA products were resolved in denaturing urea polyacrylamide gels. RE was determined by dividing the fraction of extension products for each lane by the fraction of extension products in lane 2 for each panel. (i) D-loop extension was performed as in (h). (j) RDR was performed with pol IV in the presence of 50 µM dGTP and 10 µM 2',3'-dideoxyadenosine triphosphate (ddATP)(lane 1). Incorporation of the ddAMP chain terminator opposite the thymidine base (T) prevents further extension of the D-loop. DNA products were analyzed as in (h). The DNA sequence of the product in lane 1 was determined by comparison to the DNA markers in lanes 2 and 3. The mobility of the product in lane 1 (upper band) corresponds to the marker in lane 2 which indicates that the D-loop was extended by the incorporation of 3 dGMPs and 1 ddAMP demonstrating a −1 frameshift mutation (see schematic at bottom). Partial DNA sequences of the invading ssDNA and markers are indicated. β-clamp, clamp-loader, and SSB were present in all reactions except where indicated.

    Techniques Used: Labeling, Incubation, Plasmid Preparation, Sequencing, Activity Assay, Purification, Marker, Mutagenesis

    10) Product Images from "Dodecamer d-AGATCTAGATCT and a Homologous Hairpin form Triplex in the Presence of Peptide REWER"

    Article Title: Dodecamer d-AGATCTAGATCT and a Homologous Hairpin form Triplex in the Presence of Peptide REWER

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0065010

    KMnO 4 and DEPC sequencing results show protection of DNA bases due to triplex formation in the presence of REWER. Prior to electrophoresis all chemical modifications were done at 4°C. A ) KMnO 4 panel shows the reactivity of the free RY28 and RY28+RY12 (1∶1) mixture with radiolabeled RY28 in the presence and absence of the pentapeptide, REWER. [P]/[N] ratio in all experiments was 3.8. Lane1, RY28 alone; lane 2, RY28+RY12 mixture; Lane 3, RY28+REWER; lane 4, RY28+RY12+REWER. Bands corresponding to thymines and adenines are indicated. B ) DEPC reactivity of the triplex. Lane 1 represents probe only (without treatment) and was used as control for both KMnO 4 and DEPC experiments. Lane 2, RY28; lane 3, mixture of radiolabeled RY12+RY28; lane 4, RY28+RY12+REWER. The bands corresponding to adenines and guanines are marked. * represents 5′ radiolabeled oligonucleotide.
    Figure Legend Snippet: KMnO 4 and DEPC sequencing results show protection of DNA bases due to triplex formation in the presence of REWER. Prior to electrophoresis all chemical modifications were done at 4°C. A ) KMnO 4 panel shows the reactivity of the free RY28 and RY28+RY12 (1∶1) mixture with radiolabeled RY28 in the presence and absence of the pentapeptide, REWER. [P]/[N] ratio in all experiments was 3.8. Lane1, RY28 alone; lane 2, RY28+RY12 mixture; Lane 3, RY28+REWER; lane 4, RY28+RY12+REWER. Bands corresponding to thymines and adenines are indicated. B ) DEPC reactivity of the triplex. Lane 1 represents probe only (without treatment) and was used as control for both KMnO 4 and DEPC experiments. Lane 2, RY28; lane 3, mixture of radiolabeled RY12+RY28; lane 4, RY28+RY12+REWER. The bands corresponding to adenines and guanines are marked. * represents 5′ radiolabeled oligonucleotide.

    Techniques Used: Sequencing, Electrophoresis

    Schematic representation of the proposed triplex structure. A ) Base sequence of RY12 dodecamer. B ) RY28 hairpin. C D ) Schematics of base pairing in the triplex formed between RY12 and RY28. It show the representation diagram of the two putative triplex formations via alternate strand recognition (–) represent Watson-Crick base pair and (•) represent non Watson- Crick hydrogen bonds. The third strand is shown in lowercase. Open and solid arrows depict the polarity of third strand and hairpin duplex, respectively. Numbers 1–4 represent base triads in triplex structure. A parallel triplex is formed when three purines of the third strand form hydrogen bonds with underlying purine of the hairpin (Pu•PuPy) it then changes strand and binds to the other strand of the hairpin (Py•PuPy) in an antiparallel orientation. Please note that unlike more commonly found orientation in Pu•PuPy triplex where chemically homologous strands show antiparallel polarity our model suggests homologous strands in parallel orientation. E ) A three dimensional rendition for a triplex of type C ) in which the third strand recognizes alternate strands of a hairpin duplex. Shaded bars in the hairpin structure represent Watson-Crick hydrogen bonding. The third strand is shown in the middle as black ribbon. The purine triplet of the third strand (e.g., aga) forms base pairs (dotted bars) with the purine tract (e.g., AGA) of one strand of the Watson-Crick hairpin, whereas the pyrimidine triplet of the third strand (e.g., tct) base pairs (vertical bars) with the purine tract (e.g., AGA) of the other Watson-Crick strand.
    Figure Legend Snippet: Schematic representation of the proposed triplex structure. A ) Base sequence of RY12 dodecamer. B ) RY28 hairpin. C D ) Schematics of base pairing in the triplex formed between RY12 and RY28. It show the representation diagram of the two putative triplex formations via alternate strand recognition (–) represent Watson-Crick base pair and (•) represent non Watson- Crick hydrogen bonds. The third strand is shown in lowercase. Open and solid arrows depict the polarity of third strand and hairpin duplex, respectively. Numbers 1–4 represent base triads in triplex structure. A parallel triplex is formed when three purines of the third strand form hydrogen bonds with underlying purine of the hairpin (Pu•PuPy) it then changes strand and binds to the other strand of the hairpin (Py•PuPy) in an antiparallel orientation. Please note that unlike more commonly found orientation in Pu•PuPy triplex where chemically homologous strands show antiparallel polarity our model suggests homologous strands in parallel orientation. E ) A three dimensional rendition for a triplex of type C ) in which the third strand recognizes alternate strands of a hairpin duplex. Shaded bars in the hairpin structure represent Watson-Crick hydrogen bonding. The third strand is shown in the middle as black ribbon. The purine triplet of the third strand (e.g., aga) forms base pairs (dotted bars) with the purine tract (e.g., AGA) of one strand of the Watson-Crick hairpin, whereas the pyrimidine triplet of the third strand (e.g., tct) base pairs (vertical bars) with the purine tract (e.g., AGA) of the other Watson-Crick strand.

    Techniques Used: Sequencing

    UV mixing curves show the stoichiometry of interaction between RY28 and RY12 in the presence of REWER. Both the oligonucleotide solutions contained REWER at [P]/[N] ratio of 30 prior to mixing. The abscissa is in mole fraction of RY28. There is a clear inflection point at 0.5 mole fraction indicating that single strand of RY12 interact with RY28 hairpin.
    Figure Legend Snippet: UV mixing curves show the stoichiometry of interaction between RY28 and RY12 in the presence of REWER. Both the oligonucleotide solutions contained REWER at [P]/[N] ratio of 30 prior to mixing. The abscissa is in mole fraction of RY28. There is a clear inflection point at 0.5 mole fraction indicating that single strand of RY12 interact with RY28 hairpin.

    Techniques Used:

    EMSA shows the formation of triplex between RY12 and RY28. Triple helix formation between dodecamer RY12 and RY28 hairpin in the presence of the peptide REWER detected on 15% native polyacrylamide gel electrophoresis. First four lanes show the results of experiments using radiolabeled RY12, last four lanes used radiolabeled RY28. * represents 5′ radiolabeled oligonucleotide.
    Figure Legend Snippet: EMSA shows the formation of triplex between RY12 and RY28. Triple helix formation between dodecamer RY12 and RY28 hairpin in the presence of the peptide REWER detected on 15% native polyacrylamide gel electrophoresis. First four lanes show the results of experiments using radiolabeled RY12, last four lanes used radiolabeled RY28. * represents 5′ radiolabeled oligonucleotide.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    Melting profiles of oligonucleotides RY12 and RY28 and their mixtures in the presence and absence of pentapeptide REWER. A ) Heat-induced denaturation of the RY12 duplex (curve 1), RY28 hairpin (curve 2) and equimolar mixture of RY12 and RY28. B ) Melting curves of equimolar mixtures of RY12 and RY28 in the presence of the peptide REWER at the [P]/[N] ratio of 30 (curve 1) and 60 (curve 2). To maintain clarity melting curves at other [P]/[N] ratios are not shown.
    Figure Legend Snippet: Melting profiles of oligonucleotides RY12 and RY28 and their mixtures in the presence and absence of pentapeptide REWER. A ) Heat-induced denaturation of the RY12 duplex (curve 1), RY28 hairpin (curve 2) and equimolar mixture of RY12 and RY28. B ) Melting curves of equimolar mixtures of RY12 and RY28 in the presence of the peptide REWER at the [P]/[N] ratio of 30 (curve 1) and 60 (curve 2). To maintain clarity melting curves at other [P]/[N] ratios are not shown.

    Techniques Used:

    11) Product Images from "Involvement of CD11b integrin in the alteration of metabolic factors after phorbol ester stimulation of human myeloid leukemia cells"

    Article Title: Involvement of CD11b integrin in the alteration of metabolic factors after phorbol ester stimulation of human myeloid leukemia cells

    Journal: Cell Communication and Signaling : CCS

    doi: 10.1186/1478-811X-10-13

    Flow cytometry analysis of cell surface markers. a ) Wildtype U937 cells, pMTH1-U937 and asCD11b-U937 were compared with respect to the expression of the CD11b antigen in both, steady-state culture and following a 72 h exposure to 5nM TPA. An IgG1 subclass staining serves as a control which was not altered in the different populations. b ) Untreated pMTH1-U937 (pMTH1) and untreated asCD11b-U937 (asCD11b) cells were cultured in either uncoated or 2% (w/v) agarose-precoated cell culture dishes. Following 72 h of incubation with 5nM TPA, respectively, the cells were harvested and flow cytometry analysis was performed in the different populations with the β2-integrin subunit antibodies CD11a, CD11c and CD18 as well as with the monocytic differentiation markers CD14 and F4-80. The relative fluorescence intensity represents the fold induction which has been normalized separately for the appropriate control cell population. Data represent the mean ± s.d. of three measurements.
    Figure Legend Snippet: Flow cytometry analysis of cell surface markers. a ) Wildtype U937 cells, pMTH1-U937 and asCD11b-U937 were compared with respect to the expression of the CD11b antigen in both, steady-state culture and following a 72 h exposure to 5nM TPA. An IgG1 subclass staining serves as a control which was not altered in the different populations. b ) Untreated pMTH1-U937 (pMTH1) and untreated asCD11b-U937 (asCD11b) cells were cultured in either uncoated or 2% (w/v) agarose-precoated cell culture dishes. Following 72 h of incubation with 5nM TPA, respectively, the cells were harvested and flow cytometry analysis was performed in the different populations with the β2-integrin subunit antibodies CD11a, CD11c and CD18 as well as with the monocytic differentiation markers CD14 and F4-80. The relative fluorescence intensity represents the fold induction which has been normalized separately for the appropriate control cell population. Data represent the mean ± s.d. of three measurements.

    Techniques Used: Flow Cytometry, Cytometry, Expressing, Staining, Cell Culture, Incubation, Fluorescence

    Zymographic assay of gelatinase activity. Medium supernatants of 20 ml culture medium (medium control) as well as 20 ml conditioned medium from 10 7 U937 cells, pMTH1-U937 cells and asCD11b-U937 cells in the absence or presence of 5nM TPA for 72 h, respectively, were 18-fold concentrated and subjected to SDS-PAGE containing 2 mg/ml of gelatine. Following incubation with MMP enzyme buffer, the gels were stained with 0.4% Coomassie blue and afterwards destained again to visualize the appearance of gelatinase activity by light bands against the dark background. The molecular weight markers on both sides of the gels indicate the size of the MMPs exhibiting gelatinase activities.
    Figure Legend Snippet: Zymographic assay of gelatinase activity. Medium supernatants of 20 ml culture medium (medium control) as well as 20 ml conditioned medium from 10 7 U937 cells, pMTH1-U937 cells and asCD11b-U937 cells in the absence or presence of 5nM TPA for 72 h, respectively, were 18-fold concentrated and subjected to SDS-PAGE containing 2 mg/ml of gelatine. Following incubation with MMP enzyme buffer, the gels were stained with 0.4% Coomassie blue and afterwards destained again to visualize the appearance of gelatinase activity by light bands against the dark background. The molecular weight markers on both sides of the gels indicate the size of the MMPs exhibiting gelatinase activities.

    Techniques Used: Activity Assay, SDS Page, Incubation, Staining, Molecular Weight

    a: Telomerase activity. Telomerase activity was detected by TRAPeze telomerase detection kit (Millipore, Beverly, MA, USA). Thus, radiolabeled telomere adducts were measured in the wild-type U937 control cells as compared to pMTH1-U937 and asCD11b-U937 cells following culture in the absence and in the presence of 5nM TPA for 24 h up to 72 h, respectively. One representative telomerase assay out of three similar ones is presented. b: Proliferation assay. About 2 × 10 4 cells (either U937, pMTH1-U937 or asCD11b-U937) were incubated in 96-well microtiter plates in the presence or absence of 5nM TPA for up to 72 h. The cells were radiolabelled with about 0.5 μCi/well [ 3 H] thymidine for 15 h and at the time points indicated, the cells were harvested and the radioactivity was quantified in a β-scintillation counter. The incorporated [ 3 H] thymidine was calculated as percentage of the appropriately untreated control cells at each time point which were set to 100%. Data represent the mean ± s.d. (n = 10).
    Figure Legend Snippet: a: Telomerase activity. Telomerase activity was detected by TRAPeze telomerase detection kit (Millipore, Beverly, MA, USA). Thus, radiolabeled telomere adducts were measured in the wild-type U937 control cells as compared to pMTH1-U937 and asCD11b-U937 cells following culture in the absence and in the presence of 5nM TPA for 24 h up to 72 h, respectively. One representative telomerase assay out of three similar ones is presented. b: Proliferation assay. About 2 × 10 4 cells (either U937, pMTH1-U937 or asCD11b-U937) were incubated in 96-well microtiter plates in the presence or absence of 5nM TPA for up to 72 h. The cells were radiolabelled with about 0.5 μCi/well [ 3 H] thymidine for 15 h and at the time points indicated, the cells were harvested and the radioactivity was quantified in a β-scintillation counter. The incorporated [ 3 H] thymidine was calculated as percentage of the appropriately untreated control cells at each time point which were set to 100%. Data represent the mean ± s.d. (n = 10).

    Techniques Used: Activity Assay, Telomerase Assay, Proliferation Assay, Incubation, Radioactivity

    Western blot analysis. The pMTH1-U937 (pMTH1) and asCD11b-U937 (asCD11b) cells were cultured in the absence and in the presence of 5nM TPA for 24 h up to 72 h, respectively. Thereafter, the different populations were harvested, lyzed and 40 μg of total cellular protein was separated by 10% SDS-PAGE followed by Western blotting. a ) For the expression of the metabolic enzymes MnSOD, p97/VCP and the 20 S proteasome (α-subunits), gels were subsequently stripped corresponding to a β-actin loading control. b ) Analysis of the expression patterns of different extracellular matrix proteolytic enzymes were tested using antibodies against MMP-1, MMP-7 and MMP-9 for matrix restructuring. The unaltered expression level of β-actin serves as a loading control.
    Figure Legend Snippet: Western blot analysis. The pMTH1-U937 (pMTH1) and asCD11b-U937 (asCD11b) cells were cultured in the absence and in the presence of 5nM TPA for 24 h up to 72 h, respectively. Thereafter, the different populations were harvested, lyzed and 40 μg of total cellular protein was separated by 10% SDS-PAGE followed by Western blotting. a ) For the expression of the metabolic enzymes MnSOD, p97/VCP and the 20 S proteasome (α-subunits), gels were subsequently stripped corresponding to a β-actin loading control. b ) Analysis of the expression patterns of different extracellular matrix proteolytic enzymes were tested using antibodies against MMP-1, MMP-7 and MMP-9 for matrix restructuring. The unaltered expression level of β-actin serves as a loading control.

    Techniques Used: Western Blot, Cell Culture, SDS Page, Expressing

    20S proteasomal proteolytic activity. The pMTH1-U937 and asCD11b-U937 cells were treated with 5nM TPA for 4 h up to 72 h. At the time points indicated the cells were harvested and nuclear extracts were measured by a fluorometric proteolytic assay to obtain the 20 S proteasomal activity. The relative proteasomal activity of pMTH1-U937 control cells in steady-state was set to 100%. Data represent the mean ± s.d. of three independent experiments.
    Figure Legend Snippet: 20S proteasomal proteolytic activity. The pMTH1-U937 and asCD11b-U937 cells were treated with 5nM TPA for 4 h up to 72 h. At the time points indicated the cells were harvested and nuclear extracts were measured by a fluorometric proteolytic assay to obtain the 20 S proteasomal activity. The relative proteasomal activity of pMTH1-U937 control cells in steady-state was set to 100%. Data represent the mean ± s.d. of three independent experiments.

    Techniques Used: Activity Assay

    12) Product Images from "Regulation of Enteric vapBC Transcription: Induction by VapC Toxin Dimer-Breaking"

    Article Title: Regulation of Enteric vapBC Transcription: Induction by VapC Toxin Dimer-Breaking

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks029

    The vapBC promoter is controlled by conditional cooperativity. ( A ) Binding of VapB and VapBC complex to a vapO -encoding DNA fragment analysed by gel shifting. Purified VapB and VapC were added to a 302-bp 32 P-labelled vapO probe (lanes 1–11; numbers below the gel are in nM). Protein–DNA complexes were separated by 5% native PAGE. U denotes unbound vapO DNA and C1 and C2 VapBC O complexes. ( B ) DNase I protection assay of vapO . VapB and VapC were incubated with vapO DNA as in (A) and subsequently incubated with DNase I (lanes 1–9; numbers are pmol). A DNA sequencing ladder was generated using 5′-end labelled vapBC_EMSA_down primer. Inverted repeats sites 1 and 2 and promoter sequences are indicated by arrows. DNAse I protected bases are enclosed by boxes. ( C ) Ectopic expression of VapC D7A in vivo induces vapBC transcription. TB28 (MG1655 ΔlacIZYA ) pKW512TFZD7A ( vapBC D7A :: lacZYA ) containing either pKW3353HC (pBAD::SD opt :: vapC D7A ) or pBAD33 were streaked to single colonies on LB plates containing X-gal and 0.2% arabinose. ( D ) VapC D7A induced transcription quantified by qPCR. TB28 (MG1655 ΔlacIZYA ) pKW512TFZD7A ( vapBC D7A :: lacZYA ) with pKW3353HC (pBAD::SD opt :: vapC D7A -H6) or pBAD33 (empty vector plasmid) were grown exponentially in LB medium. At 0′, arabinose was added to induce transcription from the pBAD promoter. Samples were taken at time points indicated (min) and total RNA extracted. Fold-of-changes relative to house keeping gene rpsA mRNA were measured by quantitative RT-PCR.
    Figure Legend Snippet: The vapBC promoter is controlled by conditional cooperativity. ( A ) Binding of VapB and VapBC complex to a vapO -encoding DNA fragment analysed by gel shifting. Purified VapB and VapC were added to a 302-bp 32 P-labelled vapO probe (lanes 1–11; numbers below the gel are in nM). Protein–DNA complexes were separated by 5% native PAGE. U denotes unbound vapO DNA and C1 and C2 VapBC O complexes. ( B ) DNase I protection assay of vapO . VapB and VapC were incubated with vapO DNA as in (A) and subsequently incubated with DNase I (lanes 1–9; numbers are pmol). A DNA sequencing ladder was generated using 5′-end labelled vapBC_EMSA_down primer. Inverted repeats sites 1 and 2 and promoter sequences are indicated by arrows. DNAse I protected bases are enclosed by boxes. ( C ) Ectopic expression of VapC D7A in vivo induces vapBC transcription. TB28 (MG1655 ΔlacIZYA ) pKW512TFZD7A ( vapBC D7A :: lacZYA ) containing either pKW3353HC (pBAD::SD opt :: vapC D7A ) or pBAD33 were streaked to single colonies on LB plates containing X-gal and 0.2% arabinose. ( D ) VapC D7A induced transcription quantified by qPCR. TB28 (MG1655 ΔlacIZYA ) pKW512TFZD7A ( vapBC D7A :: lacZYA ) with pKW3353HC (pBAD::SD opt :: vapC D7A -H6) or pBAD33 (empty vector plasmid) were grown exponentially in LB medium. At 0′, arabinose was added to induce transcription from the pBAD promoter. Samples were taken at time points indicated (min) and total RNA extracted. Fold-of-changes relative to house keeping gene rpsA mRNA were measured by quantitative RT-PCR.

    Techniques Used: Binding Assay, Purification, Clear Native PAGE, Incubation, DNA Sequencing, Generated, Expressing, In Vivo, Real-time Polymerase Chain Reaction, Plasmid Preparation, Quantitative RT-PCR

    13) Product Images from "LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro"

    Article Title: LEDGF/p75 interacts with divergent lentiviral integrases and modulates their enzymatic activity in vitro

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl885

    Time course of LEDGF-dependent HIV-1, BIV and EIAV integration reactions with radiolabeled donor DNA substrates. HIV-1 (lanes 1–9), BIV (lanes 10–18) and EIAV (lanes 19–27) RU5 substrates were incubated with their respective INs (0.6 μM) for 5–90 min (as indicated), in the presence (lanes 2–6, 11–15 and 20–24) or absence (lanes 7–9, 16–18 and 25–27) of 0.4 μM LEDGF. Deproteinized reaction products separated in 1.5% agarose gels were detected and quantified by phosphor autoradiography. Lanes 1, 10 and 19 contained mock samples where IN and LEDGF were omitted. Migration positions of DNA standards (kb) are indicated. The plot below each gel shows accumulation of the concerted (filled circles) or half-site (triangles) integration products in the presence (discontinuous line) or absence (dashed line) of LEDGF in terms of relative band intensity. Error bars represent SDs calculated from duplicate measurements.
    Figure Legend Snippet: Time course of LEDGF-dependent HIV-1, BIV and EIAV integration reactions with radiolabeled donor DNA substrates. HIV-1 (lanes 1–9), BIV (lanes 10–18) and EIAV (lanes 19–27) RU5 substrates were incubated with their respective INs (0.6 μM) for 5–90 min (as indicated), in the presence (lanes 2–6, 11–15 and 20–24) or absence (lanes 7–9, 16–18 and 25–27) of 0.4 μM LEDGF. Deproteinized reaction products separated in 1.5% agarose gels were detected and quantified by phosphor autoradiography. Lanes 1, 10 and 19 contained mock samples where IN and LEDGF were omitted. Migration positions of DNA standards (kb) are indicated. The plot below each gel shows accumulation of the concerted (filled circles) or half-site (triangles) integration products in the presence (discontinuous line) or absence (dashed line) of LEDGF in terms of relative band intensity. Error bars represent SDs calculated from duplicate measurements.

    Techniques Used: Incubation, Autoradiography, Migration

    Strand transfer activities of HIV-1, BIV and EIAV INs in the presence of LEDGF. ( A ) Schematic of in vitro integration with circular DNA target and the expected linear concerted and circular half site strand transfer products. Coordinated insertion of a pair of substrate DNA molecules into opposing strands of target DNA results in a linear concerted integration product (left). Uncoupled insertion of a single substrate molecule into one strand of the target plasmid gives a circular half site product (right). Thick lines are substrate and thin are target-derived DNA. ( B ) HIV-1 (lanes 1–6), BIV (lanes 7–11), or EIAV (lanes 12–16) RU5 substrates and supercoiled pGEM target DNA were incubated with 0.6 μM (∼20 μg/ml) recombinant HIV-1, BIV, or EIAV INs and 0–0.4 μM LEDGF, as indicated. Deproteinized reaction products were separated in 1.5% agarose gels and detected by staining with ethidium bromide. Lanes 17 and 18 contained nicked circular and linearized pGEM DNA respectively. Migration positions of DNA markers (kb), pGEM DNA forms [supercoiled multimer (s.c. mult.), nicked circular, linear, and supercoiled (s.c.)], substrate RU5 DNAs, and the reaction products (concerted, half-site, and multiple half-site) are indicated. ( C ) BIV (lanes 1–6) and EIAV (lanes 7–12) integration reactions were carried out with the respective RU5 or/and RU5 +300 substrates. Lanes 13 and 14 contained nicked circular and linearized pGEM DNA, respectively. Migration positions of the two circular half site and three linear concerted integration products derived from RU5 and RU5 +300 substrates are indicated.
    Figure Legend Snippet: Strand transfer activities of HIV-1, BIV and EIAV INs in the presence of LEDGF. ( A ) Schematic of in vitro integration with circular DNA target and the expected linear concerted and circular half site strand transfer products. Coordinated insertion of a pair of substrate DNA molecules into opposing strands of target DNA results in a linear concerted integration product (left). Uncoupled insertion of a single substrate molecule into one strand of the target plasmid gives a circular half site product (right). Thick lines are substrate and thin are target-derived DNA. ( B ) HIV-1 (lanes 1–6), BIV (lanes 7–11), or EIAV (lanes 12–16) RU5 substrates and supercoiled pGEM target DNA were incubated with 0.6 μM (∼20 μg/ml) recombinant HIV-1, BIV, or EIAV INs and 0–0.4 μM LEDGF, as indicated. Deproteinized reaction products were separated in 1.5% agarose gels and detected by staining with ethidium bromide. Lanes 17 and 18 contained nicked circular and linearized pGEM DNA respectively. Migration positions of DNA markers (kb), pGEM DNA forms [supercoiled multimer (s.c. mult.), nicked circular, linear, and supercoiled (s.c.)], substrate RU5 DNAs, and the reaction products (concerted, half-site, and multiple half-site) are indicated. ( C ) BIV (lanes 1–6) and EIAV (lanes 7–12) integration reactions were carried out with the respective RU5 or/and RU5 +300 substrates. Lanes 13 and 14 contained nicked circular and linearized pGEM DNA, respectively. Migration positions of the two circular half site and three linear concerted integration products derived from RU5 and RU5 +300 substrates are indicated.

    Techniques Used: In Vitro, Plasmid Preparation, Derivative Assay, Incubation, Recombinant, Staining, Migration

    14) Product Images from "Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6"

    Article Title: Multiple transcription factors directly regulate Hox gene lin-39 expression in ventral hypodermal cells of the C. elegans embryo and larva, including the hypodermal fate regulators LIN-26 and ELT-6

    Journal: BMC Developmental Biology

    doi: 10.1186/1471-213X-14-17

    Direct transcriptional regulators of lin-39 in the embryo and larva. A) Horizontal lines represent 20 kb of genomic DNA surrounding the lin-39 locus. The lin-39 transcript is shown below the top line, with boxes representing exons. The next horizontal line shows evolutionarily-conserved regions (ECRs; thin vertical lines), the PCR fragments used in the yeast one hybrid assays containing the ECRs (boxes labeled 1–12), and two fragments ( pJW3.9 shown, JW5 unlabeled) identified previously using an enhancerless GFP assay [ 47 ]. Transcription factors that bind the lin-39 gene are shown above the line (previously reported) or below the line (reported in this work). B) Model for positive feedback loop between egl-18 / elt-6 and lin-39 . EGL-18 and ELT-6 act via the GATA site in enhancer pJW3.9 to facilitate initiation of lin-39 expression in the embryo, and then LIN-39 acts to positively regulate egl-18 / elt-6 expression via the Hox/Pbx binding site in the intron of egl-18 [ 55 ].
    Figure Legend Snippet: Direct transcriptional regulators of lin-39 in the embryo and larva. A) Horizontal lines represent 20 kb of genomic DNA surrounding the lin-39 locus. The lin-39 transcript is shown below the top line, with boxes representing exons. The next horizontal line shows evolutionarily-conserved regions (ECRs; thin vertical lines), the PCR fragments used in the yeast one hybrid assays containing the ECRs (boxes labeled 1–12), and two fragments ( pJW3.9 shown, JW5 unlabeled) identified previously using an enhancerless GFP assay [ 47 ]. Transcription factors that bind the lin-39 gene are shown above the line (previously reported) or below the line (reported in this work). B) Model for positive feedback loop between egl-18 / elt-6 and lin-39 . EGL-18 and ELT-6 act via the GATA site in enhancer pJW3.9 to facilitate initiation of lin-39 expression in the embryo, and then LIN-39 acts to positively regulate egl-18 / elt-6 expression via the Hox/Pbx binding site in the intron of egl-18 [ 55 ].

    Techniques Used: Polymerase Chain Reaction, Labeling, Activated Clotting Time Assay, Expressing, Binding Assay

    15) Product Images from "Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells"

    Article Title: Selective Release of MicroRNA Species from Normal and Malignant Mammary Epithelial Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0013515

    Extracellular miR-16 Levels Correlate with Nanovesicle Abundance. A Plot of extracellular to intracellular levels of miRNAs quantitated by qRT-PCR using stem-loop primers of cells lines BT-20, MCF 10A, MCF7, MDA-MB-231 and SK-BR-3. MiRNA levels were calculated using standard curves, and corrected for recovery by normalization to a spiked synthetic RNA (INT-RNA) introduced at the time of RNA-extraction ( Materials and Methods and Table S4 ). Error bars indicate the measure of one standard deviation of 3 independent experiments. B Relative levels of miR-16 and absolute levels of CD81 were measured in P70 fractions of seven independent experiments and plotted. The line indicates a best-fit power curve (r 2 = 0.95). C The P70 was collected daily from media conditioned by MCF7 cells (solid symbols), or from MCF 10A cells (open symbol) cultures and miRNAs were quantified in triplicate. The release rates were linear; r 2 = 0.79 for miR-1246 (inset), r 2 = 0.98 for miR-16, r 2 = 0.85 for miR-451; and r 2 = 0.87 for miR-720 of MCF7 cells.
    Figure Legend Snippet: Extracellular miR-16 Levels Correlate with Nanovesicle Abundance. A Plot of extracellular to intracellular levels of miRNAs quantitated by qRT-PCR using stem-loop primers of cells lines BT-20, MCF 10A, MCF7, MDA-MB-231 and SK-BR-3. MiRNA levels were calculated using standard curves, and corrected for recovery by normalization to a spiked synthetic RNA (INT-RNA) introduced at the time of RNA-extraction ( Materials and Methods and Table S4 ). Error bars indicate the measure of one standard deviation of 3 independent experiments. B Relative levels of miR-16 and absolute levels of CD81 were measured in P70 fractions of seven independent experiments and plotted. The line indicates a best-fit power curve (r 2 = 0.95). C The P70 was collected daily from media conditioned by MCF7 cells (solid symbols), or from MCF 10A cells (open symbol) cultures and miRNAs were quantified in triplicate. The release rates were linear; r 2 = 0.79 for miR-1246 (inset), r 2 = 0.98 for miR-16, r 2 = 0.85 for miR-451; and r 2 = 0.87 for miR-720 of MCF7 cells.

    Techniques Used: Quantitative RT-PCR, Multiple Displacement Amplification, RNA Extraction, Standard Deviation

    Differential Cellular Release and Retention of Small RNAs. Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star : Extracellular enriched RNA; Circle : Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2 ). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.
    Figure Legend Snippet: Differential Cellular Release and Retention of Small RNAs. Medium conditioned by MCF7 cells for 5 days was enriched for exosomes by a filtration and ultracentrifugation protocol producing a P70 preparation. A The P70 was subjected to negative-staining EM. B The abundance of tetraspanin CD81, an exosome-marker was assessed in the filtered conditioned medium, the P70 pellet obtained by ultracentrifugation, and the supernatant (S70) using slot-blot (inset, n = 2). C The surface antigens CD81, CD63 and Mucin-1 were detected in the P70 fraction of the mammary epithelial cells using slot-blot. The absolute amount of bound antibody was quantified using standard-curves of antibody dilutions, and expressed as a percent of total antigenicity for the P70 of each cell line. The data of two replicate experiments for the indicated cell lines are shown. D Radiolabeled small RNAs isolated from MCF7 cells (c) and the extracellular preparation P70 (x) were separated by PAGE on a 12% denaturing gel. Star : Extracellular enriched RNA; Circle : Some extracellular RNAs identified by sequencing (see text and Tables S1 and S2 ). E Quantitation of labeled RNA species of D. The thin line indicates abundance of cellular small RNAs, whereas the thick line indicates the abundance of the extracellular miRNAs.

    Techniques Used: Filtration, Negative Staining, Marker, Dot Blot, Isolation, Polyacrylamide Gel Electrophoresis, Sequencing, Quantitation Assay, Labeling

    Some MicroRNAs are Released Disproportionately. Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods ). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing ( Table S5 ). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.
    Figure Legend Snippet: Some MicroRNAs are Released Disproportionately. Duplicate microRNA microarrays were hybridized with 1 µg of total cellular or 1 µg of extracellular miRNA from MCF7 cells. Results are plotted as A relative fluorescent intensities of extracellular (x, upper panel) and cellular (c, lower panel) miRNAs, or B ratio of extracellular to cellular miRNAs. The horizontal lines in B indicate the threshold of 2 fold-changes, whereas the red and the green marked populations indicate a greater than 4-fold enrichment in the released extracellular (A, upper panel, x), or in the cells (A, lower panel, c) respectively. C Scatter plot of average reads of the miRNAs quantitated by array. Only miRNAs with a fluorescent value of greater than 500 in the cellular or extracellular population are shown (see Materials and Methods ). The numbers next to dots indicate the miRNA the dot represents. D MCF7 cells were cultured for 5 days, and the total amount of specific cellular and extracellular miRNAs were measured by quantitative linker-ligation mediated RT-PCR, and the miRNA ratios were plotted. The average of 3 independent experiments is shown. E Native PAGE of products at end-point of quantitative RT-PCR. The major PCR-products between 32–48 ntds correspond to the mature miRNA (miRNA) as expected by size and determined by sequencing ( Table S5 ). The bands with a migration of less then 25 ntds are the PCR primers (primers) used in the reaction. Bands that retained in the well are amplification-independent reaction components (reaction components). Hsa-miR-923 has since been reclassified as a specific rRNA fragment.

    Techniques Used: Cell Culture, Ligation, Reverse Transcription Polymerase Chain Reaction, Clear Native PAGE, Quantitative RT-PCR, Polymerase Chain Reaction, Sequencing, Migration, Amplification

    Immature miRNAs are Released at miRNA-Species Specific Rates. A and B , pre-miRNAs of MCF7 cells (c), and miRNAs released from these cells (x) were amplified by rt-PCR and subjected to native PAGE. All the main bands correspond to the expected size of the amplification products of immature miRNAs (arrowheads). Some of the amplification products were confirmed by sequencing ( Table S7 ). C Those miRNAs that yielded single bands in 3 independent experiments were quantified by qRT-PCR. D Presence of upstream RNA sequences, corresponding to pri-miRNA sequences (Pri-s and Pri-l), and RNA corresponding to pre-miRNAs (Pre) were assed by PCR. The identity of the major products was confirmed by sequencing ( Table S7 ). Ntds: nucleotide size of sizing marker.
    Figure Legend Snippet: Immature miRNAs are Released at miRNA-Species Specific Rates. A and B , pre-miRNAs of MCF7 cells (c), and miRNAs released from these cells (x) were amplified by rt-PCR and subjected to native PAGE. All the main bands correspond to the expected size of the amplification products of immature miRNAs (arrowheads). Some of the amplification products were confirmed by sequencing ( Table S7 ). C Those miRNAs that yielded single bands in 3 independent experiments were quantified by qRT-PCR. D Presence of upstream RNA sequences, corresponding to pri-miRNA sequences (Pri-s and Pri-l), and RNA corresponding to pre-miRNAs (Pre) were assed by PCR. The identity of the major products was confirmed by sequencing ( Table S7 ). Ntds: nucleotide size of sizing marker.

    Techniques Used: Amplification, Reverse Transcription Polymerase Chain Reaction, Clear Native PAGE, Sequencing, Quantitative RT-PCR, Polymerase Chain Reaction, Marker

    Extracellular Mammary Epithelial Signature miRNAs are Present in Body Fluids. A . Abundance of indicated miRNAs quantitated using qRT-PCR from the plasma of mice injected subcutaneously with indicated breast-cancer cell lines as shown in Figure S3 , and normalized to miR-22, a microRNA of murine blood, but not found in the conditioned media of these cancer cell lines. B . Plot of quantities for indicated miRNAs in 3 samples each of human milk (milk), cell-free ductal lavages of breast cancer patients (lavages), extracellular (MCF7 X) and intracellular MCF7 (MCF7 C) preparations, as quantitated by the stem-loop-primer qRT-PCR approach. A: lavages of a patient with atypical ductal hyperplasia; B and C: lavages of patients with less severe epithelial hyperplasia. Inset: plot of ratios for miR-451 and miR-720. Dot shadings correspond to the same samples as labeled in Figure 6B . C . Additional miRNAs quantified in ductal lavages using the linker-ligation qRT-PCR method.
    Figure Legend Snippet: Extracellular Mammary Epithelial Signature miRNAs are Present in Body Fluids. A . Abundance of indicated miRNAs quantitated using qRT-PCR from the plasma of mice injected subcutaneously with indicated breast-cancer cell lines as shown in Figure S3 , and normalized to miR-22, a microRNA of murine blood, but not found in the conditioned media of these cancer cell lines. B . Plot of quantities for indicated miRNAs in 3 samples each of human milk (milk), cell-free ductal lavages of breast cancer patients (lavages), extracellular (MCF7 X) and intracellular MCF7 (MCF7 C) preparations, as quantitated by the stem-loop-primer qRT-PCR approach. A: lavages of a patient with atypical ductal hyperplasia; B and C: lavages of patients with less severe epithelial hyperplasia. Inset: plot of ratios for miR-451 and miR-720. Dot shadings correspond to the same samples as labeled in Figure 6B . C . Additional miRNAs quantified in ductal lavages using the linker-ligation qRT-PCR method.

    Techniques Used: Quantitative RT-PCR, Mouse Assay, Injection, Labeling, Ligation

    16) Product Images from "Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions"

    Article Title: Reactive oxygen species generated by thiopurine/UVA cause irreparable transcription-blocking DNA lesions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp070

    Inhibition of RNAPII transcription in vitro by oxidised template 6-TG. ( A ) In vitro transcription set-up. The transcription system comprises a radiolabelled 9-mer RNA primer and two 124-mer DNA oligonucleotides. The 124-mer transcribed strands contain either a single G or 6-TG at position 87. ( B ) Oxidation products of 6-TG block in vitro transcription. Transcribed strand oligonucleotides were treated with MMPP or UVA prior to formation of the ternary complexes. These were supplemented with GTP, ATP and UTP and transcription was initiated by purified S. cerevisiae RNAPII in the presence or absence of CTP. Transcription products were analysed by gel electrophoresis.
    Figure Legend Snippet: Inhibition of RNAPII transcription in vitro by oxidised template 6-TG. ( A ) In vitro transcription set-up. The transcription system comprises a radiolabelled 9-mer RNA primer and two 124-mer DNA oligonucleotides. The 124-mer transcribed strands contain either a single G or 6-TG at position 87. ( B ) Oxidation products of 6-TG block in vitro transcription. Transcribed strand oligonucleotides were treated with MMPP or UVA prior to formation of the ternary complexes. These were supplemented with GTP, ATP and UTP and transcription was initiated by purified S. cerevisiae RNAPII in the presence or absence of CTP. Transcription products were analysed by gel electrophoresis.

    Techniques Used: Inhibition, In Vitro, Blocking Assay, Purification, Nucleic Acid Electrophoresis

    17) Product Images from "HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression"

    Article Title: HOXB13 is co-localized with androgen receptor to suppress androgen-stimulated prostate-specific antigen expression

    Journal: Anatomy & Cell Biology

    doi: 10.5115/acb.2010.43.4.284

    HOXB13 physically interacts with androgen receptor (AR), not with DNA, to suppress androgen-stimulated AR activity. (A) To see if HOXB13's AR-suppressive function is due to DNA binding, electrophoretic mobility shift assay was performed. An androgen response element (ARE) was derived from MMTV and double-stranded oligonucleotides, tgtacaggatgttct, were labeled and used as a probe. First, LNCaP cells were grown in the presence of androgen followed by nuclear extraction. GST-HOXB13 protein was produced and purified. LNCaP nuclear extracts were used as a positive control (lane 1), whose signals were abolished by the addition of cold ARE (800X) (lane 2). Either GST (lane 3) or GST-HOXB13 (1-5 µg) (lanes 4-5) replaced nuclear extracts to see if HOXB13 binds to ARE. (B) For the in vivo interaction of HOXB13 and AR, co-immunoprecipitation was performed. LNCaP cells were grown under CDT-FBS for 3 days and treated with or without androgen and nuclear extracts were collected 48 hours after treatment. One hundred µg of proteins were mixed with 4 µg of anti-HOXB13 antibodies, followed by conjugation to agarose A/G beads. Normal IgG was used as negative control. Precipitated fractions (IP) were resolved by SDS-PAGE and analyzed by Western blot using anti-AR antibodies.
    Figure Legend Snippet: HOXB13 physically interacts with androgen receptor (AR), not with DNA, to suppress androgen-stimulated AR activity. (A) To see if HOXB13's AR-suppressive function is due to DNA binding, electrophoretic mobility shift assay was performed. An androgen response element (ARE) was derived from MMTV and double-stranded oligonucleotides, tgtacaggatgttct, were labeled and used as a probe. First, LNCaP cells were grown in the presence of androgen followed by nuclear extraction. GST-HOXB13 protein was produced and purified. LNCaP nuclear extracts were used as a positive control (lane 1), whose signals were abolished by the addition of cold ARE (800X) (lane 2). Either GST (lane 3) or GST-HOXB13 (1-5 µg) (lanes 4-5) replaced nuclear extracts to see if HOXB13 binds to ARE. (B) For the in vivo interaction of HOXB13 and AR, co-immunoprecipitation was performed. LNCaP cells were grown under CDT-FBS for 3 days and treated with or without androgen and nuclear extracts were collected 48 hours after treatment. One hundred µg of proteins were mixed with 4 µg of anti-HOXB13 antibodies, followed by conjugation to agarose A/G beads. Normal IgG was used as negative control. Precipitated fractions (IP) were resolved by SDS-PAGE and analyzed by Western blot using anti-AR antibodies.

    Techniques Used: Activity Assay, Binding Assay, Electrophoretic Mobility Shift Assay, Derivative Assay, Labeling, Produced, Purification, Positive Control, In Vivo, Immunoprecipitation, Conjugation Assay, Negative Control, SDS Page, Western Blot

    18) Product Images from "Methylation-Dependent Binding of the Epstein-Barr Virus BZLF1 Protein to Viral Promoters"

    Article Title: Methylation-Dependent Binding of the Epstein-Barr Virus BZLF1 Protein to Viral Promoters

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000356

    Z binds to the Na promoter, and binding is enhanced by Nap methylation. (A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of in vitro translated Z to bind to 32 P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the BRRF1 mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.
    Figure Legend Snippet: Z binds to the Na promoter, and binding is enhanced by Nap methylation. (A) Latently infected EBV+ 293 Z-KO cells were transfected with a BZLF1 expression vector (+) or an empty vector control (−). Chromatin immunoprecipitation assay was performed 20 hours after transfection using anti-Z and control goat IgG antibodies as indicated to examine Z binding to the Na promoter (Nap), ZREs within the EBV oriLyt (positive control) and EBV sequences in the 3′ end of the EBV BALF5 gene (negative control). (B) The ability of in vitro translated Z to bind to 32 P end-labeled probes (in either the methylated, or unmethylated forms) containing the BRRF1 promoter (Nap) sequences from −280 to −470, −108 to −301, and +77 to −128 (relative to the BRRF1 mRNA start site) was examined by EMSA. A probe containing the BRLF1 promoter (Rp) sequences from −1 to −270 (in the methylated form) served as a positive control.

    Techniques Used: Binding Assay, Methylation, Infection, Transfection, Expressing, Plasmid Preparation, Chromatin Immunoprecipitation, Positive Control, Negative Control, In Vitro, Labeling

    19) Product Images from "AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis"

    Article Title: AP endonucleases process 5-methylcytosine excision intermediates during active DNA demethylation in Arabidopsis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku834

    The 3′ phosphatase activity of Arabidopsis AP endonucleases. (A) Structure of 35-mer oligonucleotide duplex that mimics DME-catalyzed δ-elimination product for 3′ phosphatase assay. The radiolabeled upstream 17-mer oligonucleotide with a 3′-phosphate (F17F[3P]) and the downstream 17-mer with a 5′-phosphate ([5P]F17B) are annealed together to the complementary 35-mer strand (R35) to produce DNA substrate with a 1-nt gap in the middle. (B) The 3′ phosphatase activity of Arabidopsis AP endonuclease. DNA substrate depicted in (A) was reacted with purified MBP-APE1L, -APE2 or -ARP at 37°C for 1 h. Only MBP-ARP protein converted a δ-elimination product analog to 3′-OH, like E. coli Endonuclease IV (Endo IV). Either MBP-APE1L or -APE2 did not catalyze such conversion, like human APE1 (hAPE1). Methylated DNA substrate used for DNA glycosylase assay was reacted with MBP-DMEΔ and loaded alongside for size comparison. NE, no enzyme control. (C) Relative 3′ phosphatase activities of Arabidopsis AP endonucleases on a δ-elimination product. The amounts of 3′ phosphatase reaction products were measured by phosphorimager. (D) Kinetics analysis of ARP 3′ phosphatase activity. The above DNA substrate was reacted with MBP-ARP (5 nM) at 37°C in a time-course manner. The amounts of 3′ phosphatase reaction products at each time point were measured and plotted over time. Error bars represent standard deviations from three independent experiments (C and D).
    Figure Legend Snippet: The 3′ phosphatase activity of Arabidopsis AP endonucleases. (A) Structure of 35-mer oligonucleotide duplex that mimics DME-catalyzed δ-elimination product for 3′ phosphatase assay. The radiolabeled upstream 17-mer oligonucleotide with a 3′-phosphate (F17F[3P]) and the downstream 17-mer with a 5′-phosphate ([5P]F17B) are annealed together to the complementary 35-mer strand (R35) to produce DNA substrate with a 1-nt gap in the middle. (B) The 3′ phosphatase activity of Arabidopsis AP endonuclease. DNA substrate depicted in (A) was reacted with purified MBP-APE1L, -APE2 or -ARP at 37°C for 1 h. Only MBP-ARP protein converted a δ-elimination product analog to 3′-OH, like E. coli Endonuclease IV (Endo IV). Either MBP-APE1L or -APE2 did not catalyze such conversion, like human APE1 (hAPE1). Methylated DNA substrate used for DNA glycosylase assay was reacted with MBP-DMEΔ and loaded alongside for size comparison. NE, no enzyme control. (C) Relative 3′ phosphatase activities of Arabidopsis AP endonucleases on a δ-elimination product. The amounts of 3′ phosphatase reaction products were measured by phosphorimager. (D) Kinetics analysis of ARP 3′ phosphatase activity. The above DNA substrate was reacted with MBP-ARP (5 nM) at 37°C in a time-course manner. The amounts of 3′ phosphatase reaction products at each time point were measured and plotted over time. Error bars represent standard deviations from three independent experiments (C and D).

    Techniques Used: Activity Assay, Phosphatase Assay, Purification, Methylation

    AP site incision assay of APE1L, APE2 and ARP. (A) Schematic representation of APE1L, APE2 and ARP proteins. EEP, endonuclease-exonuclease-phosphatase; SAP, SAF-A/B, Acinus and PIAS; ZF, GRF-type zinc finger motif. (B) AP endonuclease activity on the AP site. Radiolabeled 35-nt double-stranded DNA containing a THF, an AP site analog, at position 18 (F35[AP]) was used as a substrate for AP endonuclease assay. Reactions were done with 5 nM each of MBP-APE1L, -APE2 and -ARP at 37°C for 30 min. As a reaction control, 0.5 unit of hAPE1 was used. AP endonuclease reaction product (17-nt with 3′-OH) is indicated at the right of the panel. NE, no enzyme control. (C) Kinetics analysis of Arabidopsis AP endonucleases. The incision activity of ARP on AP site was measured by reacting purified MBP-ARP (5 nM) with varying concentrations of substrate (0–100 nM) at 37°C for 4 min. Error bars represent standard deviations from three independent experiments.
    Figure Legend Snippet: AP site incision assay of APE1L, APE2 and ARP. (A) Schematic representation of APE1L, APE2 and ARP proteins. EEP, endonuclease-exonuclease-phosphatase; SAP, SAF-A/B, Acinus and PIAS; ZF, GRF-type zinc finger motif. (B) AP endonuclease activity on the AP site. Radiolabeled 35-nt double-stranded DNA containing a THF, an AP site analog, at position 18 (F35[AP]) was used as a substrate for AP endonuclease assay. Reactions were done with 5 nM each of MBP-APE1L, -APE2 and -ARP at 37°C for 30 min. As a reaction control, 0.5 unit of hAPE1 was used. AP endonuclease reaction product (17-nt with 3′-OH) is indicated at the right of the panel. NE, no enzyme control. (C) Kinetics analysis of Arabidopsis AP endonucleases. The incision activity of ARP on AP site was measured by reacting purified MBP-ARP (5 nM) with varying concentrations of substrate (0–100 nM) at 37°C for 4 min. Error bars represent standard deviations from three independent experiments.

    Techniques Used: Activity Assay, Purification

    20) Product Images from "The telomeric transcriptome of Schizosaccharomyces pombe"

    Article Title: The telomeric transcriptome of Schizosaccharomyces pombe

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1153

    ( A ) RNA isolated from the indicated strains was hybridized as in Figure 1 B. Two membranes obtained from blotting of gels run in parallel were first hybridized to detect TERRA and ARIA. After parallel exposures and signal detection, the same membranes were stripped and hybridized to detect ARRET and αARRET and successively to detect U6 snRNA (loading control). The asterisks indicate the hybridization bands used for ARRET and αARRET quantifications. Molecular weights are on the left in kb. ( B ) Quantifications of northern blots shown in (A). TERRA, ARIA, ARRET and αARRET hybridization signals were normalized through the relative U6 signals and expressed as fold increase over Δ cid12 samples for TERRA and ARIA and wt samples for ARRET and αARRET. nd: not determined.
    Figure Legend Snippet: ( A ) RNA isolated from the indicated strains was hybridized as in Figure 1 B. Two membranes obtained from blotting of gels run in parallel were first hybridized to detect TERRA and ARIA. After parallel exposures and signal detection, the same membranes were stripped and hybridized to detect ARRET and αARRET and successively to detect U6 snRNA (loading control). The asterisks indicate the hybridization bands used for ARRET and αARRET quantifications. Molecular weights are on the left in kb. ( B ) Quantifications of northern blots shown in (A). TERRA, ARIA, ARRET and αARRET hybridization signals were normalized through the relative U6 signals and expressed as fold increase over Δ cid12 samples for TERRA and ARIA and wt samples for ARRET and αARRET. nd: not determined.

    Techniques Used: Isolation, Hybridization, Northern Blot

    ( A ) Wt and Δ rap1 strains carrying intact Rpb7 or the G150D mutation were grown at permissive temperature (22°C) for several generations and then shifted to restrictive temperature (36°C) for 1 or 2 h. Total RNA was isolated and subjected to northern blot analyses as in Figure 1 B. After signal detection, membranes were stripped and hybridized using probes detecting 35S and 32S precursor rRNAs (unstable RNAPI transcripts), intron-containing U6 snRNA (U6 IN ; unstable RNAPIII transcript) and 18S rRNA (loading control). Molecular weights are on the left in kb. The asterisks indicate the ARRET and αARRET hybridization signals used for quantifications. ( B ) Quantifications of TERRA, ARIA, ARRET and αARRET levels at permissive and restrictive temperatures. Values were normalized through the relative 18S values and expressed as fold increase over Δ rap1 at 22°C. Values and error bars are averages and standard deviations from two independent experiments.
    Figure Legend Snippet: ( A ) Wt and Δ rap1 strains carrying intact Rpb7 or the G150D mutation were grown at permissive temperature (22°C) for several generations and then shifted to restrictive temperature (36°C) for 1 or 2 h. Total RNA was isolated and subjected to northern blot analyses as in Figure 1 B. After signal detection, membranes were stripped and hybridized using probes detecting 35S and 32S precursor rRNAs (unstable RNAPI transcripts), intron-containing U6 snRNA (U6 IN ; unstable RNAPIII transcript) and 18S rRNA (loading control). Molecular weights are on the left in kb. The asterisks indicate the ARRET and αARRET hybridization signals used for quantifications. ( B ) Quantifications of TERRA, ARIA, ARRET and αARRET levels at permissive and restrictive temperatures. Values were normalized through the relative 18S values and expressed as fold increase over Δ rap1 at 22°C. Values and error bars are averages and standard deviations from two independent experiments.

    Techniques Used: Mutagenesis, Isolation, Northern Blot, Hybridization

    ( A ) Schematic representation of S. pombe chromosome ends and telomeric transcriptome. Telomeric repeats are in black and subtelomeric sequences in white. Oligonucleotides used for RT–PCR and/or northern blotting are indicated by arrows. The sketch is not in scale. ( B ) RNA isolated from wt and Δ rap1 cells was hybridized with the indicated probes. Exposures and signal detection were performed in parallel for TERRA and ARIA as well as for ARRET and αARRET. The black arrowheads indicate two discrete bands of ∼0.7 and 1.1 kb detected by oC and oG oligonucleotides and of so far unknown origin. The asterisks indicate major hybridization bands corresponding to ARRET and αARRET RNA species. U6 snRNA was used as loading control. Molecular weights are on the left in kb. ( C ) Total RNA from wt and Δ rap1 strains was reverse transcribed (RT) using oC (left panels) or o1 (right panels) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right samples correspond to control reactions where template was omitted. Molecular weights are on the right in bp.
    Figure Legend Snippet: ( A ) Schematic representation of S. pombe chromosome ends and telomeric transcriptome. Telomeric repeats are in black and subtelomeric sequences in white. Oligonucleotides used for RT–PCR and/or northern blotting are indicated by arrows. The sketch is not in scale. ( B ) RNA isolated from wt and Δ rap1 cells was hybridized with the indicated probes. Exposures and signal detection were performed in parallel for TERRA and ARIA as well as for ARRET and αARRET. The black arrowheads indicate two discrete bands of ∼0.7 and 1.1 kb detected by oC and oG oligonucleotides and of so far unknown origin. The asterisks indicate major hybridization bands corresponding to ARRET and αARRET RNA species. U6 snRNA was used as loading control. Molecular weights are on the left in kb. ( C ) Total RNA from wt and Δ rap1 strains was reverse transcribed (RT) using oC (left panels) or o1 (right panels) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right samples correspond to control reactions where template was omitted. Molecular weights are on the right in bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Isolation, Hybridization, Polymerase Chain Reaction, Amplification

    ( A ) Total RNA from wt and Δ rap1 strains was subjected to poly(A)+ fractionation and corresponding volumes of total and poly(A)+ RNA were hybridized to detect TERRA, ARIA, ARRET and αARRET. The asterisks indicate the major hybridization bands corresponding to ARRET and αARRET RNA species. Molecular weights are on the left in kb. ( B ) The same RNA as in A was hybridized using probes detecting the polyadenylated act1 + mRNA and the non-polyadenylated 18S rRNA. ( C ) Total and poly(A)+ RNA was RT using poly(T) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right sample corresponds to a control reaction where template was omitted. Molecular weights are on the right in bp.
    Figure Legend Snippet: ( A ) Total RNA from wt and Δ rap1 strains was subjected to poly(A)+ fractionation and corresponding volumes of total and poly(A)+ RNA were hybridized to detect TERRA, ARIA, ARRET and αARRET. The asterisks indicate the major hybridization bands corresponding to ARRET and αARRET RNA species. Molecular weights are on the left in kb. ( B ) The same RNA as in A was hybridized using probes detecting the polyadenylated act1 + mRNA and the non-polyadenylated 18S rRNA. ( C ) Total and poly(A)+ RNA was RT using poly(T) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right sample corresponds to a control reaction where template was omitted. Molecular weights are on the right in bp.

    Techniques Used: Fractionation, Hybridization, Polymerase Chain Reaction, Amplification

    21) Product Images from "The telomeric transcriptome of Schizosaccharomyces pombe"

    Article Title: The telomeric transcriptome of Schizosaccharomyces pombe

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1153

    ( A ) Chromatin isolated from wt and Δ rap1 cells was immuno-precipitated using antibodies against RNAPII C terminal domain repeats either unmodified (αtotal) or phosphorylated at Serine 2 (αpS2) or Serine 5 (αpS5). Quantitative real-time PCR was performed using primers flanking TERRA TSS (left graph) or amplifying a fragment from the highly transcribed RNAPII substrate gene act1 (right graph; positive control). Graphs show the fraction of input DNA retrieved in the different samples, after subtraction of the background signal measured for control reactions performed using only beads. Bars and error bars are averages and standard deviations from three independent experiments. ( B ) Total proteins were extracted from wt and Δ rap1 strains and analyzed by western blot with the same antibodies used for ChIP. Act1 was used as loading control.
    Figure Legend Snippet: ( A ) Chromatin isolated from wt and Δ rap1 cells was immuno-precipitated using antibodies against RNAPII C terminal domain repeats either unmodified (αtotal) or phosphorylated at Serine 2 (αpS2) or Serine 5 (αpS5). Quantitative real-time PCR was performed using primers flanking TERRA TSS (left graph) or amplifying a fragment from the highly transcribed RNAPII substrate gene act1 (right graph; positive control). Graphs show the fraction of input DNA retrieved in the different samples, after subtraction of the background signal measured for control reactions performed using only beads. Bars and error bars are averages and standard deviations from three independent experiments. ( B ) Total proteins were extracted from wt and Δ rap1 strains and analyzed by western blot with the same antibodies used for ChIP. Act1 was used as loading control.

    Techniques Used: Isolation, Real-time Polymerase Chain Reaction, Positive Control, Western Blot, Chromatin Immunoprecipitation

    ( A ) Schematic representation of S. pombe chromosome ends and telomeric transcriptome. Telomeric repeats are in black and subtelomeric sequences in white. Oligonucleotides used for RT–PCR and/or northern blotting are indicated by arrows. The sketch is not in scale. ( B ) RNA isolated from wt and Δ rap1 cells was hybridized with the indicated probes. Exposures and signal detection were performed in parallel for TERRA and ARIA as well as for ARRET and αARRET. The black arrowheads indicate two discrete bands of ∼0.7 and 1.1 kb detected by oC and oG oligonucleotides and of so far unknown origin. The asterisks indicate major hybridization bands corresponding to ARRET and αARRET RNA species. U6 snRNA was used as loading control. Molecular weights are on the left in kb. ( C ) Total RNA from wt and Δ rap1 strains was reverse transcribed (RT) using oC (left panels) or o1 (right panels) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right samples correspond to control reactions where template was omitted. Molecular weights are on the right in bp.
    Figure Legend Snippet: ( A ) Schematic representation of S. pombe chromosome ends and telomeric transcriptome. Telomeric repeats are in black and subtelomeric sequences in white. Oligonucleotides used for RT–PCR and/or northern blotting are indicated by arrows. The sketch is not in scale. ( B ) RNA isolated from wt and Δ rap1 cells was hybridized with the indicated probes. Exposures and signal detection were performed in parallel for TERRA and ARIA as well as for ARRET and αARRET. The black arrowheads indicate two discrete bands of ∼0.7 and 1.1 kb detected by oC and oG oligonucleotides and of so far unknown origin. The asterisks indicate major hybridization bands corresponding to ARRET and αARRET RNA species. U6 snRNA was used as loading control. Molecular weights are on the left in kb. ( C ) Total RNA from wt and Δ rap1 strains was reverse transcribed (RT) using oC (left panels) or o1 (right panels) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right samples correspond to control reactions where template was omitted. Molecular weights are on the right in bp.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Northern Blot, Isolation, Hybridization, Polymerase Chain Reaction, Amplification

    ( A ) Total RNA from wt and Δ rap1 strains was subjected to poly(A)+ fractionation and corresponding volumes of total and poly(A)+ RNA were hybridized to detect TERRA, ARIA, ARRET and αARRET. The asterisks indicate the major hybridization bands corresponding to ARRET and αARRET RNA species. Molecular weights are on the left in kb. ( B ) The same RNA as in A was hybridized using probes detecting the polyadenylated act1 + mRNA and the non-polyadenylated 18S rRNA. ( C ) Total and poly(A)+ RNA was RT using poly(T) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right sample corresponds to a control reaction where template was omitted. Molecular weights are on the right in bp.
    Figure Legend Snippet: ( A ) Total RNA from wt and Δ rap1 strains was subjected to poly(A)+ fractionation and corresponding volumes of total and poly(A)+ RNA were hybridized to detect TERRA, ARIA, ARRET and αARRET. The asterisks indicate the major hybridization bands corresponding to ARRET and αARRET RNA species. Molecular weights are on the left in kb. ( B ) The same RNA as in A was hybridized using probes detecting the polyadenylated act1 + mRNA and the non-polyadenylated 18S rRNA. ( C ) Total and poly(A)+ RNA was RT using poly(T) oligonucleotides and cDNA was PCR amplified using o2+o3 or o2+oC oligonucleotides. The most right sample corresponds to a control reaction where template was omitted. Molecular weights are on the right in bp.

    Techniques Used: Fractionation, Hybridization, Polymerase Chain Reaction, Amplification

    22) Product Images from "Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis"

    Article Title: Txe, an endoribonuclease of the enterococcal Axe-Txe toxin-antitoxin system, cleaves mRNA and inhibits protein synthesis

    Journal: Microbiology

    doi: 10.1099/mic.0.045492-0

    Txe-induced cleavage of  lpp  mRNA. The 5′ end of  lpp  mRNA was mapped by using the primer  lpp  21. Numbers indicate times (min) at which mRNA was harvested after the addition of IPTG. The major cleavage site is indicated by an arrow. The sequence around the major cleavage site is shown to the side, with the cleavage site again indicated by an arrow.
    Figure Legend Snippet: Txe-induced cleavage of lpp mRNA. The 5′ end of lpp mRNA was mapped by using the primer lpp 21. Numbers indicate times (min) at which mRNA was harvested after the addition of IPTG. The major cleavage site is indicated by an arrow. The sequence around the major cleavage site is shown to the side, with the cleavage site again indicated by an arrow.

    Techniques Used: Sequencing

    23) Product Images from "DNA-PK is a DNA sensor for IRF-3-dependent innate immunityDecision letterAuthor response"

    Article Title: DNA-PK is a DNA sensor for IRF-3-dependent innate immunityDecision letterAuthor response

    Journal: eLife

    doi: 10.7554/eLife.00047.012

    The innate immune response to DNA requires DNA-PK in fibroblasts. ( A ) ISD DNA of different lengths was transfected into MEFs and the transcription of Cxcl10 was assayed by qPCR 6 hr later. ( B ) Double stranded oligonucleotides (bio-ISD), concatenated ISD DNA (bio-concatamers), genomic vaccinia virus DNA (bio-VACV), genomic E. coli DNA (bio- E. coli ), poly (dA:dT) or the RNA analogue poly (I:C) were biotinylated and transfected into HEK293 cells. Following affinity purification of proteins from cytoplasmic extracts using streptavidin agarose, the bound proteins were analysed by SDS-PAGE and immunoblotting. AP; affinity purification. ( C ) Primary MEFs of the indicated genotype were transfected with 10 μg/ml of the same (non-biotinylated) nucleic acids as in ( A ) followed by qRT-PCR analysis measuring induction of Cxcl10 mRNA 6 hr later. ( D ) Wild type and Prkdc −/− transformed MEFs were transfected with DNA (10 μg/ml, left panel) or stimulated with LPS (100 ng/ml, right panel) and the level of transcription of cxcl10 was measured at the indicated times post stimulation. ( E ) Levels of Cxcl10 and Ifnβ were measured by ELISA from the supernatants of primary wild type and Prkdc −/− MEFs at passage 1, 24 hr after transfection with DNA or poly (I:C). ( F ),( G ) Primary wild type and Prkdc −/− MEFs at passage 1 were transfected with DNA or poly (I:C) and the level of induction of ( F ) Ifnb and Il-6 and ( G ) ccl4 and ccl5 mRNA was measured by qRT-PCR 6 hr later. ( H ) MEFs expressing Ku80 or lacking Ku80 were transfected with DNA and the transcription of Cxcl10 or Il6 was measured by qPCR 6 hr later. ( I ) Primary murine skin fibroblasts (MSF) from wild type adult mice or those lacking both Ku genes were transfected with DNA or poly (I:C) and the level of Ifnb induction was measured 6 hr later by qRT-PCR. ( J ) Xrcc5 +/+ / Trp53 −/− and Xrcc5 −/− / Trp53 −/− MEFs were transfected with an expression plasmid encoding Ku80 or an empty vector (EV) control and Cxcl10 production was measured 24 hr later by ELISA. *** p
    Figure Legend Snippet: The innate immune response to DNA requires DNA-PK in fibroblasts. ( A ) ISD DNA of different lengths was transfected into MEFs and the transcription of Cxcl10 was assayed by qPCR 6 hr later. ( B ) Double stranded oligonucleotides (bio-ISD), concatenated ISD DNA (bio-concatamers), genomic vaccinia virus DNA (bio-VACV), genomic E. coli DNA (bio- E. coli ), poly (dA:dT) or the RNA analogue poly (I:C) were biotinylated and transfected into HEK293 cells. Following affinity purification of proteins from cytoplasmic extracts using streptavidin agarose, the bound proteins were analysed by SDS-PAGE and immunoblotting. AP; affinity purification. ( C ) Primary MEFs of the indicated genotype were transfected with 10 μg/ml of the same (non-biotinylated) nucleic acids as in ( A ) followed by qRT-PCR analysis measuring induction of Cxcl10 mRNA 6 hr later. ( D ) Wild type and Prkdc −/− transformed MEFs were transfected with DNA (10 μg/ml, left panel) or stimulated with LPS (100 ng/ml, right panel) and the level of transcription of cxcl10 was measured at the indicated times post stimulation. ( E ) Levels of Cxcl10 and Ifnβ were measured by ELISA from the supernatants of primary wild type and Prkdc −/− MEFs at passage 1, 24 hr after transfection with DNA or poly (I:C). ( F ),( G ) Primary wild type and Prkdc −/− MEFs at passage 1 were transfected with DNA or poly (I:C) and the level of induction of ( F ) Ifnb and Il-6 and ( G ) ccl4 and ccl5 mRNA was measured by qRT-PCR 6 hr later. ( H ) MEFs expressing Ku80 or lacking Ku80 were transfected with DNA and the transcription of Cxcl10 or Il6 was measured by qPCR 6 hr later. ( I ) Primary murine skin fibroblasts (MSF) from wild type adult mice or those lacking both Ku genes were transfected with DNA or poly (I:C) and the level of Ifnb induction was measured 6 hr later by qRT-PCR. ( J ) Xrcc5 +/+ / Trp53 −/− and Xrcc5 −/− / Trp53 −/− MEFs were transfected with an expression plasmid encoding Ku80 or an empty vector (EV) control and Cxcl10 production was measured 24 hr later by ELISA. *** p

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction, Affinity Purification, SDS Page, Quantitative RT-PCR, Transformation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Mouse Assay, Plasmid Preparation

    24) Product Images from "The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of “off Target” Gene (glnE) Expression"

    Article Title: The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of “off Target” Gene (glnE) Expression

    Journal: Antibiotics (Basel, Switzerland)

    doi: 10.3390/antibiotics4020188

    The nucleotide sequence upstream of glnE and MtrR-binding sites. The 301 bp sequence of the DNA upstream of glnE and the first two codons (encoding M and S, respectively) is shown with the annotated −10 and −35 hexamer sequences of the glnE promoter identified by a line under the sequences. The putative extended −10 element is shown in blue. The alternative −35 hexamer is shown by the dashed line above the sequence. The boxed regions represent predicted MtrR binding sites that were identified based on sequence similarity to that of a site upstream of mtrCDE [ 8 , 10 ] or rpoH [ 7 ]. The grey box represents a sequence with 53% identity to the region upstream of rpoH [ 7 ] while the yellow, black, and red boxes represent sequences with 55%, 67%, and 52%, respectively, identity to regions upstream of mtrCDE [ 8 , 10 ]. The MtrR-binding sites identified by DNase I protection ( Figure 3 ) are noted by the solid line above the sense strand or below the anti-sense strand; the two sites on the anti-sense strand are denoted as A′ and B′ with the DNase I hypersensitive site in A′ shown by an *. The adjacent seven nucleotide imperfect inverted element is shown in green and red.
    Figure Legend Snippet: The nucleotide sequence upstream of glnE and MtrR-binding sites. The 301 bp sequence of the DNA upstream of glnE and the first two codons (encoding M and S, respectively) is shown with the annotated −10 and −35 hexamer sequences of the glnE promoter identified by a line under the sequences. The putative extended −10 element is shown in blue. The alternative −35 hexamer is shown by the dashed line above the sequence. The boxed regions represent predicted MtrR binding sites that were identified based on sequence similarity to that of a site upstream of mtrCDE [ 8 , 10 ] or rpoH [ 7 ]. The grey box represents a sequence with 53% identity to the region upstream of rpoH [ 7 ] while the yellow, black, and red boxes represent sequences with 55%, 67%, and 52%, respectively, identity to regions upstream of mtrCDE [ 8 , 10 ]. The MtrR-binding sites identified by DNase I protection ( Figure 3 ) are noted by the solid line above the sense strand or below the anti-sense strand; the two sites on the anti-sense strand are denoted as A′ and B′ with the DNase I hypersensitive site in A′ shown by an *. The adjacent seven nucleotide imperfect inverted element is shown in green and red.

    Techniques Used: Sequencing, Binding Assay

    Identification of the MtrR-binding site in the glnE upstream DNA. ( A ) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; ( B ) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.
    Figure Legend Snippet: Identification of the MtrR-binding site in the glnE upstream DNA. ( A ) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; ( B ) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.

    Techniques Used: Binding Assay, Sequencing, Purification, Labeling

    25) Product Images from "A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR"

    Article Title: A LexA-related protein regulates redox-sensitive expression of the cyanobacterial RNA helicase, crhR

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl426

    LexA-related protein-binding analysis. EMSA using recombinant LexA (rLexA) were performed to confirm interactions between LexA and the crhR gene. ( A ) rLexA concentration curve. Increasing concentrations of rLexA were incubated with 32 P-labeled KC+5. As controls, rLexA was also incubated with Synechocystis (lane 8) and E.coli (lane 9) soluble protein extracts. ( B ) DNA competition assays. rLexA (100 nM) was incubated with 32 P-labeled KC+5 and the indicated fold excess of either specific competitor DNA (unlabeled KC+5; lanes 1–5) or non-specific competitor DNA (internal lexA fragment; lanes 6–10).
    Figure Legend Snippet: LexA-related protein-binding analysis. EMSA using recombinant LexA (rLexA) were performed to confirm interactions between LexA and the crhR gene. ( A ) rLexA concentration curve. Increasing concentrations of rLexA were incubated with 32 P-labeled KC+5. As controls, rLexA was also incubated with Synechocystis (lane 8) and E.coli (lane 9) soluble protein extracts. ( B ) DNA competition assays. rLexA (100 nM) was incubated with 32 P-labeled KC+5 and the indicated fold excess of either specific competitor DNA (unlabeled KC+5; lanes 1–5) or non-specific competitor DNA (internal lexA fragment; lanes 6–10).

    Techniques Used: Protein Binding, Recombinant, Concentration Assay, Incubation, Labeling

    ( A ) crhR nested deletion series. DNA was deleted by directional digestion from the SacI site using Exonuclease III. Deleted clones are designated by their start site relative to the transcriptional start indicated as +1. DNA fragments corresponding to each deletion were generated by PCR using the M13 forward (FP) and GWO-45 primers, expect KC+125 and KC+219 which were produced by restriction digestion (KC+125: SpeI/BssS1; KC+219: XmnI/BssS1). Plasmid and crhR insert sequences are indicated by thick and thin solid lines, respectively. Scale 50 bp = 1 cm. ( B and C ) EMSA identification of the protein-binding region in the crhR gene. ( B ) Localization of the protein-binding region. 32 P-end-labeled DNA targets were incubated either alone (−) or with 30 μg Synechocystis soluble protein extract (+). ( C ) Competition assays. KC-179 32 P-end-labeled target DNA, containing the entire crhR promoter, was incubated with no protein or 30 μg Synechocystis soluble protein extract. Increasing amounts (0–3.0 pmol) of either specific competitor DNA (unlabeled KC-179 fragment; upper panel) or non-specific competitor DNA (unlabeled 262 bp EcoRV / PvuII fragment of pBluescript KS+; lower panel) were included in the binding reaction to determine the specificity of the protein–DNA interaction.
    Figure Legend Snippet: ( A ) crhR nested deletion series. DNA was deleted by directional digestion from the SacI site using Exonuclease III. Deleted clones are designated by their start site relative to the transcriptional start indicated as +1. DNA fragments corresponding to each deletion were generated by PCR using the M13 forward (FP) and GWO-45 primers, expect KC+125 and KC+219 which were produced by restriction digestion (KC+125: SpeI/BssS1; KC+219: XmnI/BssS1). Plasmid and crhR insert sequences are indicated by thick and thin solid lines, respectively. Scale 50 bp = 1 cm. ( B and C ) EMSA identification of the protein-binding region in the crhR gene. ( B ) Localization of the protein-binding region. 32 P-end-labeled DNA targets were incubated either alone (−) or with 30 μg Synechocystis soluble protein extract (+). ( C ) Competition assays. KC-179 32 P-end-labeled target DNA, containing the entire crhR promoter, was incubated with no protein or 30 μg Synechocystis soluble protein extract. Increasing amounts (0–3.0 pmol) of either specific competitor DNA (unlabeled KC-179 fragment; upper panel) or non-specific competitor DNA (unlabeled 262 bp EcoRV / PvuII fragment of pBluescript KS+; lower panel) were included in the binding reaction to determine the specificity of the protein–DNA interaction.

    Techniques Used: Clone Assay, Generated, Polymerase Chain Reaction, Produced, Plasmid Preparation, Protein Binding, Labeling, Incubation, Binding Assay

    26) Product Images from "Nucleic acid-binding specificity of human FUS protein"

    Article Title: Nucleic acid-binding specificity of human FUS protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv679

    FUS has mutually exclusive binding sites for single-stranded RNA, DNA and dsRNA. ( A ) ssRNA and ssDNA bind FUS mutually exclusively. Increasing amounts of unlabeled ssRNA or ssDNA were mixed with 20 nM radiolabeled ssRNA and the mixtures were incubated with 350 nM FUS. ( B ) ssRNA and dsRNA compete for binding to FUS. The same binding competition assay was performed as in (A) except various amounts of unlabeled ssRNA were mixed with 20 nM radiolabeled dsRNA.
    Figure Legend Snippet: FUS has mutually exclusive binding sites for single-stranded RNA, DNA and dsRNA. ( A ) ssRNA and ssDNA bind FUS mutually exclusively. Increasing amounts of unlabeled ssRNA or ssDNA were mixed with 20 nM radiolabeled ssRNA and the mixtures were incubated with 350 nM FUS. ( B ) ssRNA and dsRNA compete for binding to FUS. The same binding competition assay was performed as in (A) except various amounts of unlabeled ssRNA were mixed with 20 nM radiolabeled dsRNA.

    Techniques Used: Binding Assay, Incubation, Competitive Binding Assay

    27) Product Images from "Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function"

    Article Title: Mutations in the nucleotide binding and hydrolysis domains of Helicobacter pylori MutS2 lead to altered biochemical activities and inactivation of its in vivo function

    Journal: BMC Microbiology

    doi: 10.1186/s12866-016-0629-3

    a Schematic representation of HpMutS2 variants used in this study. Walker-A and Walker-B motifs of HpMutS2 were identified by multiple sequence alignment of MutS2 proteins from different bacteria using Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The mutations in the walker-A and Walker-B motifs were introduced using site directed mutagenesis. The mutated amino acids are highlighted in red. All the proteins were C-terminally His 6 -tagged. The LDLK motif and Smr domain are conserved nuclease sites of HpMutS2. b Effect of DNA substrates on ATPase activity of HpMutS2. HpMutS2 (45 nM) was incubated with increasing concentrations of ATP [0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1000 (μM)]. DNA substrates (1 μM) were added separately to the reaction mixtures. After incubation at 37 °C for 30 min the reactions were stopped by EDTA (50 mM) and the products were separated by TLC. ATP [γ- 32 P] was used as tracer to monitor the product formation. Reaction velocities were calculated by quantifying the proportion of products formed to un-reacted substrate divided by incubation time
    Figure Legend Snippet: a Schematic representation of HpMutS2 variants used in this study. Walker-A and Walker-B motifs of HpMutS2 were identified by multiple sequence alignment of MutS2 proteins from different bacteria using Clustal Omega ( http://www.ebi.ac.uk/Tools/msa/clustalo/ ). The mutations in the walker-A and Walker-B motifs were introduced using site directed mutagenesis. The mutated amino acids are highlighted in red. All the proteins were C-terminally His 6 -tagged. The LDLK motif and Smr domain are conserved nuclease sites of HpMutS2. b Effect of DNA substrates on ATPase activity of HpMutS2. HpMutS2 (45 nM) was incubated with increasing concentrations of ATP [0, 10, 20, 40, 60, 80, 100, 200, 400, 600, 800, and 1000 (μM)]. DNA substrates (1 μM) were added separately to the reaction mixtures. After incubation at 37 °C for 30 min the reactions were stopped by EDTA (50 mM) and the products were separated by TLC. ATP [γ- 32 P] was used as tracer to monitor the product formation. Reaction velocities were calculated by quantifying the proportion of products formed to un-reacted substrate divided by incubation time

    Techniques Used: Sequencing, Mutagenesis, Activity Assay, Incubation, Thin Layer Chromatography

    28) Product Images from "RNA aptamer inhibitors of a restriction endonuclease"

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv702

    Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.
    Figure Legend Snippet: Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Techniques Used: Activity Assay, Mobility Shift, Binding Assay, Inhibition, Incubation, Concentration Assay

    Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.
    Figure Legend Snippet: Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Techniques Used: Inhibition, Binding Assay

    KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.
    Figure Legend Snippet: KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Techniques Used: Inhibition, Incubation, Concentration Assay

    29) Product Images from "Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function"

    Article Title: Secondary Structure across the Bacterial Transcriptome Reveals Versatile Roles in mRNA Regulation and Function

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1005613

    Stronger SD sequence has a higher propensity to form secondary structure which does not correlate with the translation efficiency. (A) SD strength does not correlate with translation efficiency (i.e. the total RPFs per coding mRNA) of a gene. SD hybridization energies fall into four major distributions: strong SD, MHE
    Figure Legend Snippet: Stronger SD sequence has a higher propensity to form secondary structure which does not correlate with the translation efficiency. (A) SD strength does not correlate with translation efficiency (i.e. the total RPFs per coding mRNA) of a gene. SD hybridization energies fall into four major distributions: strong SD, MHE

    Techniques Used: Sequencing, Hybridization

    30) Product Images from "Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma"

    Article Title: Regulation of HK2 expression through alterations in CpG methylation of the HK2 promoter during progression of hepatocellular carcinoma

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9723

    Identification of −234/−230 HRE, a key region in the HK2 promoter regulated by methylation ( A ) The promoter activity of HK2 promoter-deleted luciferase constructs was evaluated in HCC cell lines under normoxic and hypoxic conditions. Luciferase activity of all test constructs was normalized to that of the null construct. The relative luciferase activity was plotted as the percentage of the -1921 construct under normoxic or hypoxic conditions. ( B ) The promoter activity of HK2 promoter-deleted luciferase constructs under normoxic or hypoxic conditions using Hep3B cells (rectangle; putative HRE, circle; putative Sp1-binding site). ( C ) The HK2 promoter site-specific mutant luciferase constructs for putative HIF-1α and Sp1 binding sites were constructed as described in the upper panel. The promoter activity of each mutant construct under normoxic or hypoxic conditions is shown as the luciferase activity relative to the −175 construct (red closed circle; putative Sp1-binding site, green open rectangle; putative HRE). ( D ) The luciferase activity of the −965W and −965M under normoxic (N) or hypoxic (H) conditions. ( E ) The interaction between the −234/−230 HRE and HIF-1α was evaluated by ChIP assay. Sp1 was used as a positive control. The −302/−114 region and a non-relevant region (480/792) designed as shown in the upper panel were amplified by PCR. The specific interaction was plotted as the percentage of the input in the lower panel. ( F ) EMSA experiment involving the −234/−230 HRE and the mutant version (HREm) on the human HK2 promoter. The oligonucleotides shown in the upper panel were labeled and incubated with nuclear extracts from Hep3B cells. NS, non-specific bands.
    Figure Legend Snippet: Identification of −234/−230 HRE, a key region in the HK2 promoter regulated by methylation ( A ) The promoter activity of HK2 promoter-deleted luciferase constructs was evaluated in HCC cell lines under normoxic and hypoxic conditions. Luciferase activity of all test constructs was normalized to that of the null construct. The relative luciferase activity was plotted as the percentage of the -1921 construct under normoxic or hypoxic conditions. ( B ) The promoter activity of HK2 promoter-deleted luciferase constructs under normoxic or hypoxic conditions using Hep3B cells (rectangle; putative HRE, circle; putative Sp1-binding site). ( C ) The HK2 promoter site-specific mutant luciferase constructs for putative HIF-1α and Sp1 binding sites were constructed as described in the upper panel. The promoter activity of each mutant construct under normoxic or hypoxic conditions is shown as the luciferase activity relative to the −175 construct (red closed circle; putative Sp1-binding site, green open rectangle; putative HRE). ( D ) The luciferase activity of the −965W and −965M under normoxic (N) or hypoxic (H) conditions. ( E ) The interaction between the −234/−230 HRE and HIF-1α was evaluated by ChIP assay. Sp1 was used as a positive control. The −302/−114 region and a non-relevant region (480/792) designed as shown in the upper panel were amplified by PCR. The specific interaction was plotted as the percentage of the input in the lower panel. ( F ) EMSA experiment involving the −234/−230 HRE and the mutant version (HREm) on the human HK2 promoter. The oligonucleotides shown in the upper panel were labeled and incubated with nuclear extracts from Hep3B cells. NS, non-specific bands.

    Techniques Used: Methylation, Activity Assay, Luciferase, Construct, Binding Assay, Mutagenesis, Chromatin Immunoprecipitation, Positive Control, Amplification, Polymerase Chain Reaction, Labeling, Incubation

    The induction of HK2 expression in HK2negative SNU449 cells by treatment with 5-Aza-CdR and hypoxia ( A ) Hypoxia-mediated HK2 expression. ( B ) The suppression of HK2 expression by HIF-1α silencing. ( C ) The methylation status of the HK2 promoter CpGs plotted for the 5-Aza-CdR-treated SNU475 cells and SNU449 cells. The difference in methylation frequency between 5-Aza-CdR-treated cells and untreated cells is shown in each lower panel. ( D ) The induction of HK2 expression in SNU475 and SNU449 cells by treatment with 5-Aza-CdR for 2 d, followed by hypoxic stimuli for 1 d. In all experiments, the expression of HIF-1α and HK2 were evaluated by immunoblot. ( E ) The interaction between the −234/−230 HRE and HIF-1α following 5-Aza-CdR treatment was evaluated using a ChIP assay.
    Figure Legend Snippet: The induction of HK2 expression in HK2negative SNU449 cells by treatment with 5-Aza-CdR and hypoxia ( A ) Hypoxia-mediated HK2 expression. ( B ) The suppression of HK2 expression by HIF-1α silencing. ( C ) The methylation status of the HK2 promoter CpGs plotted for the 5-Aza-CdR-treated SNU475 cells and SNU449 cells. The difference in methylation frequency between 5-Aza-CdR-treated cells and untreated cells is shown in each lower panel. ( D ) The induction of HK2 expression in SNU475 and SNU449 cells by treatment with 5-Aza-CdR for 2 d, followed by hypoxic stimuli for 1 d. In all experiments, the expression of HIF-1α and HK2 were evaluated by immunoblot. ( E ) The interaction between the −234/−230 HRE and HIF-1α following 5-Aza-CdR treatment was evaluated using a ChIP assay.

    Techniques Used: Expressing, Methylation, Chromatin Immunoprecipitation

    31) Product Images from "The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis"

    Article Title: The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis

    Journal: eLife

    doi: 10.7554/eLife.00048

    Sequencing read coverage of the 3′; end nucleotide positions in tBip1 mRNA from derived from mRNA enriched by subtractive hybridization against rRNA (Ribominus kit, Invitrogen kit). Cells were treated with DTT (2 mM, 1 hr). DOI: http://dx.doi.org/10.7554/eLife.00048.014
    Figure Legend Snippet: Sequencing read coverage of the 3′; end nucleotide positions in tBip1 mRNA from derived from mRNA enriched by subtractive hybridization against rRNA (Ribominus kit, Invitrogen kit). Cells were treated with DTT (2 mM, 1 hr). DOI: http://dx.doi.org/10.7554/eLife.00048.014

    Techniques Used: Sequencing, Derivative Assay, Hybridization

    32) Product Images from "Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication"

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Journal: Cancers

    doi: 10.3390/cancers6031464

    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.
    Figure Legend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

    33) Product Images from "Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A"

    Article Title: Regulation of the alternative splicing of tau exon 10 by SC35 and Dyrk1A

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr195

    SC35 promotes tau exon 10 inclusion. ( A ) SC35 promoted tau exon 10 inclusion dose dependently. The pCI/SI9–SI10 mini-tau-gene was co-transfected with different amount of pCEP4-SC35 into HEK-239T cells. Total RNA was subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection. ( B ) SC35 promoted tau exon 10 inclusion cell-type independently. pCI/SI9–SI10 was co-transfected with pCEP4/SC35 into various cell lines indicated under each panel. Tau exon 10 splicing was measured by RT–PCR after 36 h transfection. ( C and D ) siRNA of SC35 suppressed tau exon 10 inclusion. pCI/SI9–SI10 was co-trasnfected with siRNA of SC35 into N2a cells for 48 h, and then the splicing products of tau exon 10 of mini gene (C) and endogenous mouse tau (D) were measured by RT–PCR. The same amount of scramble siRNA was used for controls. The data are presented as mean ± SD. ** P
    Figure Legend Snippet: SC35 promotes tau exon 10 inclusion. ( A ) SC35 promoted tau exon 10 inclusion dose dependently. The pCI/SI9–SI10 mini-tau-gene was co-transfected with different amount of pCEP4-SC35 into HEK-239T cells. Total RNA was subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection. ( B ) SC35 promoted tau exon 10 inclusion cell-type independently. pCI/SI9–SI10 was co-transfected with pCEP4/SC35 into various cell lines indicated under each panel. Tau exon 10 splicing was measured by RT–PCR after 36 h transfection. ( C and D ) siRNA of SC35 suppressed tau exon 10 inclusion. pCI/SI9–SI10 was co-trasnfected with siRNA of SC35 into N2a cells for 48 h, and then the splicing products of tau exon 10 of mini gene (C) and endogenous mouse tau (D) were measured by RT–PCR. The same amount of scramble siRNA was used for controls. The data are presented as mean ± SD. ** P

    Techniques Used: Transfection, Reverse Transcription Polymerase Chain Reaction

    SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P ATP. The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.
    Figure Legend Snippet: SC35 promotes tau exon 10 inclusion through SC35-like enhancer. ( A ) SC35 was immunopurified by anti-HA. SC35 tagged with HA was overexpressed in HEK-293T cells and immunopurified with anti-HA-crosslinked protein G beads. Different elution fractions (left panel) were subjected to western blot analysis with anti-HA. Immunoprecipitated SC35 without elution was dephosphorylated with alkaline phosphatase and determined by anti-HA and 1H4, an antibody to phosphorylated SR proteins. E1, elution fraction 1; E2, elution fraction 2; E3, elution fraction 3; ALP, alkaline phosphatase. ( B ) SC35 bound to RNA of tau exon 10. Immunopurified SC35 (E1 and E2) was incubated with tau pre-mRNA (tau-RNA) or SC35-like enhancer deleted tau pre-mRNA (tau-RNA ΔSC35 like ) pre-labeled with γ- 32 P ATP. The incubation products were subjected to native-gel electrophoresis. After drying, the gel was analyzed with phosphoimaging device (BAS-1500, Fujifilm). ( C ) Schematic of mutations of mini-tau-gene on SC35-like enhancer of tau exon 10. ( D ) Mutations of SC35-like enhancer affected SC35 promoted tau exon 10 inclusion. Different mutants of mini-tau-gene, pCI/SI9–SI10, at SC35 like enhancer were transfected alone or together with pCEP4/SC35. RT–PCR was carried out to measure tau exon 10 splicing after 36 h transfection.

    Techniques Used: Western Blot, Immunoprecipitation, ALP Assay, Incubation, Labeling, Nucleic Acid Electrophoresis, Transfection, Reverse Transcription Polymerase Chain Reaction

    SC35 acts on exon 10 of tau pre-mRNA to promote tau exon 10 inclusion. ( A ) Tau pre-mRNA could be immunoprecipitated by SC35. pCI/SI9–SI10 was co-transfected with pCEP4/SC35-HA into HEK-293T cells. SC35 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated pre-mRNA of tau with SC35 was determined by RT–PCR with random primer or oligo-dT for generating cDNA and with two sets of primers specific to introns 9 and 10 as indicated for amplifying the cDNA derived from tau pre-mRNA. The RT–PCR product was separated by agarose electrophoresis and quantitated by densitometry and presented in B from two separated experiments. ( C ) Schematic of SC35 deletion mutants. ( D ) Tau pre-mRNA was immunoprecipitated by deletion mutants of SC35 differentially. Different deletion mutants of SC35 showed in panel C tagged with HA were overexpressed in pCI/SI9–SI10 transfected HEK-293T cells. RNA-IP was carried out with anti-HA antibody and co-immunoprecipitated pre-mRNA of tau was measured by RT–PCR as in panel A. Total pre-mRNA of tau, Input, was also measured by RT–PCR with same primers. The immunoprecipitated deletion mutations of SC35 were examined by western blot using anti-HA antibody (lower panel). ( E ) Deletion mutations of SC35 promoted tau exon 10 inclusion differentially. pCI/SI9–SI10 was co-transfected with different deletion mutants of SC35 into HEK-293T. Total RNA was extracted and subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection.
    Figure Legend Snippet: SC35 acts on exon 10 of tau pre-mRNA to promote tau exon 10 inclusion. ( A ) Tau pre-mRNA could be immunoprecipitated by SC35. pCI/SI9–SI10 was co-transfected with pCEP4/SC35-HA into HEK-293T cells. SC35 was immunoprecipitated with anti-HA antibody. Co-immunoprecipitated pre-mRNA of tau with SC35 was determined by RT–PCR with random primer or oligo-dT for generating cDNA and with two sets of primers specific to introns 9 and 10 as indicated for amplifying the cDNA derived from tau pre-mRNA. The RT–PCR product was separated by agarose electrophoresis and quantitated by densitometry and presented in B from two separated experiments. ( C ) Schematic of SC35 deletion mutants. ( D ) Tau pre-mRNA was immunoprecipitated by deletion mutants of SC35 differentially. Different deletion mutants of SC35 showed in panel C tagged with HA were overexpressed in pCI/SI9–SI10 transfected HEK-293T cells. RNA-IP was carried out with anti-HA antibody and co-immunoprecipitated pre-mRNA of tau was measured by RT–PCR as in panel A. Total pre-mRNA of tau, Input, was also measured by RT–PCR with same primers. The immunoprecipitated deletion mutations of SC35 were examined by western blot using anti-HA antibody (lower panel). ( E ) Deletion mutations of SC35 promoted tau exon 10 inclusion differentially. pCI/SI9–SI10 was co-transfected with different deletion mutants of SC35 into HEK-293T. Total RNA was extracted and subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection.

    Techniques Used: Immunoprecipitation, Transfection, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Electrophoresis, Western Blot

    Dyrk1A phosphorylates SC35 and suppresses SC35 promoted tau exon 10 inclusion. (A ) autoradiography of SC35 phosphorylation by Dyrk1A in vitro . Recombinant GST-SC35 was incubated with various concentrations of Dyrk1A indicated above each lane for 30 min at 30°C and separated by SDS–PAGE and visualized with Coomassie blue staining (lower panel). The last lane is Dyrk1A alone, without GST-SC35. After drying the gel, the 32 P incorporated into SC35 was measured by using a phosphorimaging device (BAS-1500, Fuji) (upper panel). ( B ) The incorporated 32 P into SC35 was by different concentration of Dyrk1A. ( C ) Dyrk1A, but not Dyrk1A K188R, inhibited tau exon 10 inclusion promoted by SC35. pcDNA/Dyrk1A or pcDNA/Dyrk1A K188R was transfected only or together with SC35 into HEK-293T. Total RNA was subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection. ( D ) siRNA of Dyrk1A enhanced SC35-promoted tau exon 10 inclusion. pCEP4/SC35 was co-transfected with various concentration of siRNA of Dyrk1A into pCI/SI9–SI10 transfected HEK-293FT cells for 48 h, and the products of tau exon 10 splicing were measured by RT–PCR. ( E ) siRNA of Dyrk1A promoted 4R-tau expression. N2a or SH-SY5Y cells were transfected with Dyrk1A siRNA for 48 h, and then 3R-tau and 4R-tau were measured by RT–PCR. The same amount of scramble siRNA was used for controls. * P
    Figure Legend Snippet: Dyrk1A phosphorylates SC35 and suppresses SC35 promoted tau exon 10 inclusion. (A ) autoradiography of SC35 phosphorylation by Dyrk1A in vitro . Recombinant GST-SC35 was incubated with various concentrations of Dyrk1A indicated above each lane for 30 min at 30°C and separated by SDS–PAGE and visualized with Coomassie blue staining (lower panel). The last lane is Dyrk1A alone, without GST-SC35. After drying the gel, the 32 P incorporated into SC35 was measured by using a phosphorimaging device (BAS-1500, Fuji) (upper panel). ( B ) The incorporated 32 P into SC35 was by different concentration of Dyrk1A. ( C ) Dyrk1A, but not Dyrk1A K188R, inhibited tau exon 10 inclusion promoted by SC35. pcDNA/Dyrk1A or pcDNA/Dyrk1A K188R was transfected only or together with SC35 into HEK-293T. Total RNA was subjected to RT–PCR for measurement of tau exon 10 splicing after 36 h transfection. ( D ) siRNA of Dyrk1A enhanced SC35-promoted tau exon 10 inclusion. pCEP4/SC35 was co-transfected with various concentration of siRNA of Dyrk1A into pCI/SI9–SI10 transfected HEK-293FT cells for 48 h, and the products of tau exon 10 splicing were measured by RT–PCR. ( E ) siRNA of Dyrk1A promoted 4R-tau expression. N2a or SH-SY5Y cells were transfected with Dyrk1A siRNA for 48 h, and then 3R-tau and 4R-tau were measured by RT–PCR. The same amount of scramble siRNA was used for controls. * P

    Techniques Used: Autoradiography, In Vitro, Recombinant, Incubation, SDS Page, Staining, Concentration Assay, Transfection, Reverse Transcription Polymerase Chain Reaction, Expressing

    34) Product Images from "Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins"

    Article Title: Polynucleotide phosphorylase exonuclease and polymerase activities on single-stranded DNA ends are modulated by RecN, SsbA and RecA proteins

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr635

    RecA stimulates phosphorolysis of ssDNA with a 3′-ddTMP. The 60-nt long 5′-end labeled [ 32 P]-oligo-F (0.25 nM, lanes 1–4) or the 61-nt long 5′-end labeled [ 32 P]-oligo-F’ with a ddTMP at the 3′-end (0.25 nM, lanes 5–11) was pre-incubated with PNPase Bsu (0.5, 1 and 5 nM) for 5 min in buffer A. Then RecA (20 nM) was added and the reaction incubated for 30 min. The samples were separated as in Figure 2 .
    Figure Legend Snippet: RecA stimulates phosphorolysis of ssDNA with a 3′-ddTMP. The 60-nt long 5′-end labeled [ 32 P]-oligo-F (0.25 nM, lanes 1–4) or the 61-nt long 5′-end labeled [ 32 P]-oligo-F’ with a ddTMP at the 3′-end (0.25 nM, lanes 5–11) was pre-incubated with PNPase Bsu (0.5, 1 and 5 nM) for 5 min in buffer A. Then RecA (20 nM) was added and the reaction incubated for 30 min. The samples were separated as in Figure 2 .

    Techniques Used: Labeling, Incubation

    35) Product Images from "Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2"

    Article Title: Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2

    Journal: Journal of Molecular Biology

    doi: 10.1016/j.jmb.2015.11.014

    RNA sequences in the MS2 virion that contact the capsid proteins. (a) Outline of the CLIP-Seq protocol used to map RNA residues that contact the MS2 capsid proteins. (b) PAGE analysis of a time course of RNA cleavage using 10 mM ZnCl 2 with poly(A:U) RNA. (c) SDS-PAGE gel of immune-precipitated MS2 CP containing covalently linked RNA treated with zinc ions. Irradiated and control samples were transferred to a nitrocellulose membrane and subsequently visualized by a Western blot with an anti-MS2 CP primary antibody (left-hand panel). The regions of the same gel containing cross-linked RNA were identified in parallel samples by prior 5′ end labeling of the RNA fragments with polynucleotide kinase (right-hand panel). The smear likely represents cross-linked CP–RNA. The white lines identify the regions excised from the membrane and processed for deep sequencing (see Materials and Methods ). (d) Amount of input RNA recovered with CP after purification. (e) Abundances of cDNA fragments that co-purified with the MS2 CP. The sequences for the cDNAs identified by Illumina DNA sequencing were aligned with the MS2 genome, and histograms were produced denoting the frequency of particular sequences within the datasets for irradiated (green) and control (blue) samples. The peak that corresponds to the MS2 operator hairpin is identified by an inverted blue triangle. A schematic of the MS2 genome is shown below the graph to allow identification the approximate locations of the most significant peaks. Lines in blue highlight those sites matching previous PS predictions by Dykeman et al. [41] ; those in orange, similarly for Bleckley et al. [66] ; and green, for matches to both predictions. Black lines indicate the 10 peaks not predicted previously.
    Figure Legend Snippet: RNA sequences in the MS2 virion that contact the capsid proteins. (a) Outline of the CLIP-Seq protocol used to map RNA residues that contact the MS2 capsid proteins. (b) PAGE analysis of a time course of RNA cleavage using 10 mM ZnCl 2 with poly(A:U) RNA. (c) SDS-PAGE gel of immune-precipitated MS2 CP containing covalently linked RNA treated with zinc ions. Irradiated and control samples were transferred to a nitrocellulose membrane and subsequently visualized by a Western blot with an anti-MS2 CP primary antibody (left-hand panel). The regions of the same gel containing cross-linked RNA were identified in parallel samples by prior 5′ end labeling of the RNA fragments with polynucleotide kinase (right-hand panel). The smear likely represents cross-linked CP–RNA. The white lines identify the regions excised from the membrane and processed for deep sequencing (see Materials and Methods ). (d) Amount of input RNA recovered with CP after purification. (e) Abundances of cDNA fragments that co-purified with the MS2 CP. The sequences for the cDNAs identified by Illumina DNA sequencing were aligned with the MS2 genome, and histograms were produced denoting the frequency of particular sequences within the datasets for irradiated (green) and control (blue) samples. The peak that corresponds to the MS2 operator hairpin is identified by an inverted blue triangle. A schematic of the MS2 genome is shown below the graph to allow identification the approximate locations of the most significant peaks. Lines in blue highlight those sites matching previous PS predictions by Dykeman et al. [41] ; those in orange, similarly for Bleckley et al. [66] ; and green, for matches to both predictions. Black lines indicate the 10 peaks not predicted previously.

    Techniques Used: Cross-linking Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Western Blot, End Labeling, Sequencing, Purification, DNA Sequencing, Produced

    36) Product Images from "Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2"

    Article Title: Direct Evidence for Packaging Signal-Mediated Assembly of Bacteriophage MS2

    Journal: Journal of Molecular Biology

    doi: 10.1016/j.jmb.2015.11.014

    RNA sequences in the MS2 virion that contact the capsid proteins. (a) Outline of the CLIP-Seq protocol used to map RNA residues that contact the MS2 capsid proteins. (b) PAGE analysis of a time course of RNA cleavage using 10 mM ZnCl 2 with poly(A:U) RNA. (c) SDS-PAGE gel of immune-precipitated MS2 CP containing covalently linked RNA treated with zinc ions. Irradiated and control samples were transferred to a nitrocellulose membrane and subsequently visualized by a Western blot with an anti-MS2 CP primary antibody (left-hand panel). The regions of the same gel containing cross-linked RNA were identified in parallel samples by prior 5′ end labeling of the RNA fragments with polynucleotide kinase (right-hand panel). The smear likely represents cross-linked CP–RNA. The white lines identify the regions excised from the membrane and processed for deep sequencing (see Materials and Methods ). (d) Amount of input RNA recovered with CP after purification. (e) Abundances of cDNA fragments that co-purified with the MS2 CP. The sequences for the cDNAs identified by Illumina DNA sequencing were aligned with the MS2 genome, and histograms were produced denoting the frequency of particular sequences within the datasets for irradiated (green) and control (blue) samples. The peak that corresponds to the MS2 operator hairpin is identified by an inverted blue triangle. A schematic of the MS2 genome is shown below the graph to allow identification the approximate locations of the most significant peaks. Lines in blue highlight those sites matching previous PS predictions by Dykeman et al. [41] ; those in orange, similarly for Bleckley et al. [66] ; and green, for matches to both predictions. Black lines indicate the 10 peaks not predicted previously.
    Figure Legend Snippet: RNA sequences in the MS2 virion that contact the capsid proteins. (a) Outline of the CLIP-Seq protocol used to map RNA residues that contact the MS2 capsid proteins. (b) PAGE analysis of a time course of RNA cleavage using 10 mM ZnCl 2 with poly(A:U) RNA. (c) SDS-PAGE gel of immune-precipitated MS2 CP containing covalently linked RNA treated with zinc ions. Irradiated and control samples were transferred to a nitrocellulose membrane and subsequently visualized by a Western blot with an anti-MS2 CP primary antibody (left-hand panel). The regions of the same gel containing cross-linked RNA were identified in parallel samples by prior 5′ end labeling of the RNA fragments with polynucleotide kinase (right-hand panel). The smear likely represents cross-linked CP–RNA. The white lines identify the regions excised from the membrane and processed for deep sequencing (see Materials and Methods ). (d) Amount of input RNA recovered with CP after purification. (e) Abundances of cDNA fragments that co-purified with the MS2 CP. The sequences for the cDNAs identified by Illumina DNA sequencing were aligned with the MS2 genome, and histograms were produced denoting the frequency of particular sequences within the datasets for irradiated (green) and control (blue) samples. The peak that corresponds to the MS2 operator hairpin is identified by an inverted blue triangle. A schematic of the MS2 genome is shown below the graph to allow identification the approximate locations of the most significant peaks. Lines in blue highlight those sites matching previous PS predictions by Dykeman et al. [41] ; those in orange, similarly for Bleckley et al. [66] ; and green, for matches to both predictions. Black lines indicate the 10 peaks not predicted previously.

    Techniques Used: Cross-linking Immunoprecipitation, Polyacrylamide Gel Electrophoresis, SDS Page, Irradiation, Western Blot, End Labeling, Sequencing, Purification, DNA Sequencing, Produced

    Identification of different classes of PS sites. (a) Shared lead ion cleavage positions detected in both the transcripts and the MS2 virion are highlighted in magenta on the RNA secondary structure [73] . Proposed hairpins are indicated as I–XIV. (b) Altered lead-ion-induced cleavage sites are indicative of altered RNA structure following genome packaging. The lead-ion-induced cleavage sites detected only in transcripts, that is, which are protected in the virion, are highlighted in red. Those that cleave only in the virion are in blue. Comparison of these positions with MiSeq reads, shown here with a green line spanning 40 nt around the peak nucleotide (*), and previously predicted PSs [41] within the MS2 genome, shown here as orange lines and annotated as SL − 3 to SL + 5, allows the identification of PSs that have a propensity to fold prior to capsid assembly or that must refold during assembly.
    Figure Legend Snippet: Identification of different classes of PS sites. (a) Shared lead ion cleavage positions detected in both the transcripts and the MS2 virion are highlighted in magenta on the RNA secondary structure [73] . Proposed hairpins are indicated as I–XIV. (b) Altered lead-ion-induced cleavage sites are indicative of altered RNA structure following genome packaging. The lead-ion-induced cleavage sites detected only in transcripts, that is, which are protected in the virion, are highlighted in red. Those that cleave only in the virion are in blue. Comparison of these positions with MiSeq reads, shown here with a green line spanning 40 nt around the peak nucleotide (*), and previously predicted PSs [41] within the MS2 genome, shown here as orange lines and annotated as SL − 3 to SL + 5, allows the identification of PSs that have a propensity to fold prior to capsid assembly or that must refold during assembly.

    Techniques Used:

    Roles of the MP. (a) Sequence of the MP with the peptides identified via RCAP highlighted in red. The Jpred3-predicted secondary structure elements of the MP are shown using the same format as for the CP ( Fig. 2 c). Predicted RNA-binding residues (RNABindRPlus) are highlighted in orange. Peptides that are highly conserved across both Leviviridae and Alloleviviridae phage MPs are boxed (motif 1, amino acids 193–210, 51% average identity over 18 amino acids; motif 2, amino acids 279–308, 56% average identity over 30 amino acids; motif 3, amino acids 385–393, average 52% identity over 9 amino acids). These regions may therefore be part of the pilin-binding site. (b) Sequence homology of Leviviridae MP sequences. Filled bars represent regions homologous to MS2 MP and open boxes represent non-homologous regions. These bars cover the full length of each protein, without indicating the short gaps required to accommodate the alignment. Most proteins in this comparison are ~ 400 amino acids long. Colors represent the homology score using the BLOSUM62 matrix. Potential RNA-binding sites predicted by RNABindRPlus are represented by asterisks.
    Figure Legend Snippet: Roles of the MP. (a) Sequence of the MP with the peptides identified via RCAP highlighted in red. The Jpred3-predicted secondary structure elements of the MP are shown using the same format as for the CP ( Fig. 2 c). Predicted RNA-binding residues (RNABindRPlus) are highlighted in orange. Peptides that are highly conserved across both Leviviridae and Alloleviviridae phage MPs are boxed (motif 1, amino acids 193–210, 51% average identity over 18 amino acids; motif 2, amino acids 279–308, 56% average identity over 30 amino acids; motif 3, amino acids 385–393, average 52% identity over 9 amino acids). These regions may therefore be part of the pilin-binding site. (b) Sequence homology of Leviviridae MP sequences. Filled bars represent regions homologous to MS2 MP and open boxes represent non-homologous regions. These bars cover the full length of each protein, without indicating the short gaps required to accommodate the alignment. Most proteins in this comparison are ~ 400 amino acids long. Colors represent the homology score using the BLOSUM62 matrix. Potential RNA-binding sites predicted by RNABindRPlus are represented by asterisks.

    Techniques Used: Sequencing, RNA Binding Assay, Binding Assay

    Components of the MS2 virion and its lifecycle. (a) Ribbon representations of the two quasi-equivalent MS2 CP dimers: The symmetric dimer (C/C) is colored pink, while the asymmetric (A/B) is blue/green. Binding the TR high-affinity PS, sequence and secondary structure triggers conformer switching of C/C to A/B [26] . In principle, 60 such conformer switching events are required to create the T = 3 capsid shown in (b). (b) Structure of the MS2 virion. Left, surface view of the icosahedrally averaged X-ray structure of the T = 3 MS2 virion [27] , [28] . Right, cutaway schematic of the equivalent EM reconstitution at 9 Å resolution [29] , showing the extensive contacts between the genome, density radially colored pink to blue as the radius increases and the overlying protein shell. X-ray coordinates for these images were taken from PDB ID 1ZDH. (c) Schematic of the phage lifecycle. Virions initially bind to the sides of the bacterial pilus via the MP. MPs are an essential single-copy structural component of RNA phages. By a mechanism that remains largely obscure, the RNA–MP complex but not the remaining capsid shell enters the cell. MP also binds to its own PSs located toward either end of the MS2 genome [30] . Recent asymmetric reconstruction of the MS2–pilus complex suggests that MP replaces a CP dimer of the C/C conformer in an otherwise entirely icosahedral protein shell [31] , from which position it is ideally placed to contact the cellular receptor and escort the RNA into the cytoplasm. Note that the presence of the asymmetric MP component could not be detected in averaged X-ray and EM density maps. Once internalized, the MP is cleaved into two separate fragments by protease, allowing translation and replication to start. Temporal control of phage gene expression then regulates the production of progeny genomes and structural proteins that assemble prior to the action of the phage lysis protein.
    Figure Legend Snippet: Components of the MS2 virion and its lifecycle. (a) Ribbon representations of the two quasi-equivalent MS2 CP dimers: The symmetric dimer (C/C) is colored pink, while the asymmetric (A/B) is blue/green. Binding the TR high-affinity PS, sequence and secondary structure triggers conformer switching of C/C to A/B [26] . In principle, 60 such conformer switching events are required to create the T = 3 capsid shown in (b). (b) Structure of the MS2 virion. Left, surface view of the icosahedrally averaged X-ray structure of the T = 3 MS2 virion [27] , [28] . Right, cutaway schematic of the equivalent EM reconstitution at 9 Å resolution [29] , showing the extensive contacts between the genome, density radially colored pink to blue as the radius increases and the overlying protein shell. X-ray coordinates for these images were taken from PDB ID 1ZDH. (c) Schematic of the phage lifecycle. Virions initially bind to the sides of the bacterial pilus via the MP. MPs are an essential single-copy structural component of RNA phages. By a mechanism that remains largely obscure, the RNA–MP complex but not the remaining capsid shell enters the cell. MP also binds to its own PSs located toward either end of the MS2 genome [30] . Recent asymmetric reconstruction of the MS2–pilus complex suggests that MP replaces a CP dimer of the C/C conformer in an otherwise entirely icosahedral protein shell [31] , from which position it is ideally placed to contact the cellular receptor and escort the RNA into the cytoplasm. Note that the presence of the asymmetric MP component could not be detected in averaged X-ray and EM density maps. Once internalized, the MP is cleaved into two separate fragments by protease, allowing translation and replication to start. Temporal control of phage gene expression then regulates the production of progeny genomes and structural proteins that assemble prior to the action of the phage lysis protein.

    Techniques Used: Binding Assay, Sequencing, Expressing, Lysis

    Lead acetate probing of the MS2 ssRNA genome. (a) Outline of the lead probing experiment. The time-dependent cleavage of the genome within MS2 virions or sub-genomic fragments was performed in separate reactions at room temperature. Following Pb 2 + incubation, we quenched the reactions with EDTA and we precipitated the RNAs at high salt concentration. Following addition of EDTA, we phenol/chloroform extracted the MS2 phage sample to remove virion proteins. (b) Schematic showing the RNA fragments footprinted and the region from 1419 to 2190 probed. The arrows indicate the sites of hybridisation of the primers; blue bar, the location of the TR site. (c) An autoradiograph of a 6% (w/v) PAGE assay of primer extension products obtained by reverse transcription of the 5′ RNA treated with Pb 2 + . The TR operator is highlighted by the blue bar. Samples were treated with 0.4 mM Pb 2 + in assembly buffer for 5, 10, 30 and 60 min. A control reaction lacking Pb 2 + is indicated by “÷”. Arrows highlight sites cleaved specifically by lead-ion-induced hydrolysis positions. Dideoxy sequencing ladders (G and C), a hydrolysis ladder (OH) and size standards (L) analyzed in adjacent lanes allowed identification of the nucleotide sequence and hence the sites of cleavage.
    Figure Legend Snippet: Lead acetate probing of the MS2 ssRNA genome. (a) Outline of the lead probing experiment. The time-dependent cleavage of the genome within MS2 virions or sub-genomic fragments was performed in separate reactions at room temperature. Following Pb 2 + incubation, we quenched the reactions with EDTA and we precipitated the RNAs at high salt concentration. Following addition of EDTA, we phenol/chloroform extracted the MS2 phage sample to remove virion proteins. (b) Schematic showing the RNA fragments footprinted and the region from 1419 to 2190 probed. The arrows indicate the sites of hybridisation of the primers; blue bar, the location of the TR site. (c) An autoradiograph of a 6% (w/v) PAGE assay of primer extension products obtained by reverse transcription of the 5′ RNA treated with Pb 2 + . The TR operator is highlighted by the blue bar. Samples were treated with 0.4 mM Pb 2 + in assembly buffer for 5, 10, 30 and 60 min. A control reaction lacking Pb 2 + is indicated by “÷”. Arrows highlight sites cleaved specifically by lead-ion-induced hydrolysis positions. Dideoxy sequencing ladders (G and C), a hydrolysis ladder (OH) and size standards (L) analyzed in adjacent lanes allowed identification of the nucleotide sequence and hence the sites of cleavage.

    Techniques Used: Incubation, Concentration Assay, Hybridization, Autoradiography, Polyacrylamide Gel Electrophoresis, Sequencing

    RCAP of the MS2 virion. (a) MALDI-ToF spectra of trypsin-generated peptides co-purified with the MS2 RNA ( Materials and Methods ) from samples mock-treated or cross-linked with formaldehyde. (b) Residue numbers of CP peptides recovered in experiments similar to those shown in (a) for the different protease treatments. “+” indicates the intensity of each peptide segment. (c) MS2 CP peptides in contact with the genome in the virion. Peptides assigned following RCAP are shown in red and comprise the entire region from amino acid 32 to amino acid 105, which form the four β-strands, C, D, E and F, facing the interior of the virion. The secondary structure elements (PDB ID 2MS2; Fig. 1 a) are represented by green arrows, blue bars and a gold bar for β-strands, α-helices and a 3 10 helix, respectively. Residues contacting the TR PS are shown with an asterisk above ( Fig. 1 b).
    Figure Legend Snippet: RCAP of the MS2 virion. (a) MALDI-ToF spectra of trypsin-generated peptides co-purified with the MS2 RNA ( Materials and Methods ) from samples mock-treated or cross-linked with formaldehyde. (b) Residue numbers of CP peptides recovered in experiments similar to those shown in (a) for the different protease treatments. “+” indicates the intensity of each peptide segment. (c) MS2 CP peptides in contact with the genome in the virion. Peptides assigned following RCAP are shown in red and comprise the entire region from amino acid 32 to amino acid 105, which form the four β-strands, C, D, E and F, facing the interior of the virion. The secondary structure elements (PDB ID 2MS2; Fig. 1 a) are represented by green arrows, blue bars and a gold bar for β-strands, α-helices and a 3 10 helix, respectively. Residues contacting the TR PS are shown with an asterisk above ( Fig. 1 b).

    Techniques Used: Generated, Purification

    37) Product Images from "Comparative analysis of LIN28-RNA binding sites identified at single nucleotide resolution"

    Article Title: Comparative analysis of LIN28-RNA binding sites identified at single nucleotide resolution

    Journal: RNA Biology

    doi: 10.1080/15476286.2017.1356566

    The LIN28A ZKD can crosslink RNA in vitro . (A) Surface representation of the LIN28A ZKD in complex with the GGAG fragment of preE M -let-7f. G20 and G21, the 2 major points of mutation in the crosslinked preE M -let-7f fragment are highlighted in red. (B) Cartoon representations of the LIN28A ZKD with G20 and G21 interacting with the side chains of residues K159, H162 and M170. (C) Gel shift binding assays with the radiolabeled pre-let-7 fragment UAGGAGAU, mixed with increasing concentrations of LIN28A-ZKD (0, 56, 225, 900 nM, 1.8, 3.6, 5.4 and 7.2 μM). (D) Corresponding SDS-PAGE gel shows a crosslinked complex band following UV irradiation.
    Figure Legend Snippet: The LIN28A ZKD can crosslink RNA in vitro . (A) Surface representation of the LIN28A ZKD in complex with the GGAG fragment of preE M -let-7f. G20 and G21, the 2 major points of mutation in the crosslinked preE M -let-7f fragment are highlighted in red. (B) Cartoon representations of the LIN28A ZKD with G20 and G21 interacting with the side chains of residues K159, H162 and M170. (C) Gel shift binding assays with the radiolabeled pre-let-7 fragment UAGGAGAU, mixed with increasing concentrations of LIN28A-ZKD (0, 56, 225, 900 nM, 1.8, 3.6, 5.4 and 7.2 μM). (D) Corresponding SDS-PAGE gel shows a crosslinked complex band following UV irradiation.

    Techniques Used: In Vitro, Mutagenesis, Electrophoretic Mobility Shift Assay, Binding Assay, SDS Page, Irradiation

    38) Product Images from "Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense"

    Article Title: Frame-Insensitive Expression Cloning of Fluorescent Protein from Scolionema suvaense

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms19020371

    Frame-insensitive expression of eGFP with pRSET-TriEX vector. ( a ) The genes encoding eGFP with different reading frames were inserted in pRSET-TriEX. ( b ) pRSET B was used as a negative control. The labels A, B, and C indicate the frames of eGFP CDS.
    Figure Legend Snippet: Frame-insensitive expression of eGFP with pRSET-TriEX vector. ( a ) The genes encoding eGFP with different reading frames were inserted in pRSET-TriEX. ( b ) pRSET B was used as a negative control. The labels A, B, and C indicate the frames of eGFP CDS.

    Techniques Used: Expressing, Plasmid Preparation, Negative Control

    Structure of frame-insensitive bacterial expression cloning vector, pRSET-TriEX. ( a ) Vector map of pRSET-TriEX. ( b ) Detailed information around multiple cloning site (MCS). The DNA sequence (dT) 14  and restriction enzyme sites (SmaI, ClaI, and SalI) are inserted after original start codon of pRSET B.
    Figure Legend Snippet: Structure of frame-insensitive bacterial expression cloning vector, pRSET-TriEX. ( a ) Vector map of pRSET-TriEX. ( b ) Detailed information around multiple cloning site (MCS). The DNA sequence (dT) 14 and restriction enzyme sites (SmaI, ClaI, and SalI) are inserted after original start codon of pRSET B.

    Techniques Used: Expressing, Clone Assay, Plasmid Preparation, Sequencing

    39) Product Images from "Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress"

    Article Title: Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006133

    VgrR-VgrS is a two-component signaling system. (A) VgrS has autokinase activity. Inverted membrane vesicles of full-length VgrS and VgrS H186A recombinant proteins (5 μM) were incubated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. (B) VgrS transferred phosphoryl group to VgrR. VgrS (5 μM) was autophosphorylated as in (A) for 2 min. Twenty μM VgrR or 80 μM VgrR D51A proteins were added into the reaction, respectively. In both (A) and (B), the reaction was stopped by loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins (lower panels). Each experiment was repeated three times. (C) vgrR and vgrS constitute a bicistronic operon. RT-PCR was used to amplify cDNA. RT: amplification using cDNA transcribed from total RNA as template.–RT: negative control lacking reverse transcriptase during cDNA synthesis. DNA: amplification using bacterial DNA as template. Amplification of vgrS cDNA was used as a positive control. Location of the primers is shown in Fig 1A (upper panel). The assay was repeated three times. (D) Mapping the transcription initiation site (TIS) of the vgrR-vgrS operon. Primer extension using total RNA of X . campestris pv. campestris as the template. The G-A-T-C lanes show the dideoxy chain termination sequencing reaction in the same promoter region (note that the top of the ladder is blur for high GC content of the template). 1 and 2: vgrR mRNA was reverse transcribed at 42°C and 52°C, respectively, 3: △vgrR mRNA template (with primer binding site being deleted) was reverse transcribed at 42°C as negative control. TIS sites (P1 and P2) are shown as asterisks. NS: non-specific bands. The experiment was repeated independently twice. (E) Nucleotide sequence of the 5′ region upstream of vgrR . (F) GUS activity assay of promoters. GUS activity assays were conducted among all the recombinant bacterial strains with transcriptional fusions of P1-GUS, P2-GUS and P1+P2-GUS, respectively. Bacterial strains were induced in iron depleted (MMX) or iron replete (MMX plus Fe 3+ ) conditions. Vertical bars represent the standard deviations (n = 3).
    Figure Legend Snippet: VgrR-VgrS is a two-component signaling system. (A) VgrS has autokinase activity. Inverted membrane vesicles of full-length VgrS and VgrS H186A recombinant proteins (5 μM) were incubated with 100 μM ATP containing 10 μCi [γ- 32 P]ATP. (B) VgrS transferred phosphoryl group to VgrR. VgrS (5 μM) was autophosphorylated as in (A) for 2 min. Twenty μM VgrR or 80 μM VgrR D51A proteins were added into the reaction, respectively. In both (A) and (B), the reaction was stopped by loading buffer before SDS-PAGE separation and autoradiography. The gel was stained by Coomassie brilliant blue (CBB) to check the amount of proteins (lower panels). Each experiment was repeated three times. (C) vgrR and vgrS constitute a bicistronic operon. RT-PCR was used to amplify cDNA. RT: amplification using cDNA transcribed from total RNA as template.–RT: negative control lacking reverse transcriptase during cDNA synthesis. DNA: amplification using bacterial DNA as template. Amplification of vgrS cDNA was used as a positive control. Location of the primers is shown in Fig 1A (upper panel). The assay was repeated three times. (D) Mapping the transcription initiation site (TIS) of the vgrR-vgrS operon. Primer extension using total RNA of X . campestris pv. campestris as the template. The G-A-T-C lanes show the dideoxy chain termination sequencing reaction in the same promoter region (note that the top of the ladder is blur for high GC content of the template). 1 and 2: vgrR mRNA was reverse transcribed at 42°C and 52°C, respectively, 3: △vgrR mRNA template (with primer binding site being deleted) was reverse transcribed at 42°C as negative control. TIS sites (P1 and P2) are shown as asterisks. NS: non-specific bands. The experiment was repeated independently twice. (E) Nucleotide sequence of the 5′ region upstream of vgrR . (F) GUS activity assay of promoters. GUS activity assays were conducted among all the recombinant bacterial strains with transcriptional fusions of P1-GUS, P2-GUS and P1+P2-GUS, respectively. Bacterial strains were induced in iron depleted (MMX) or iron replete (MMX plus Fe 3+ ) conditions. Vertical bars represent the standard deviations (n = 3).

    Techniques Used: Activity Assay, Recombinant, Incubation, SDS Page, Autoradiography, Staining, Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control, Positive Control, Sequencing, Binding Assay

    Iron-VgrR binding disassociates VgrR-DNA and VgrR-VgrS interactions. (A) The amount of VgrR–P tdvA binding was decreased in iron-replete conditions. ChIP-qPCR was conducted to quantify the enrichment of VgrR at the tdvA promoter in vivo when bacteria were grown under iron-replete and iron-depleted conditions, respectively. The experiment was repeated three times. Vertical bars indicate the standard deviations. (B) The presence of Fe 2+ inhibits the formation of the VgrR–P tdvA complex in vitro . Electrophoretic mobility shift assays were conducted to determine the impact of Fe 2+ on VgrR–P tdvA binding. The concentrations of Fe 2+ were gradually increased from 10 μM to 1.0 mM. The assay was repeated independently three times. (C). Fe 2+ directly binds VgrR. 2 μM VgrR and Fe 2+ or Mn 2+ was used in an MST assay. The titer of Fe 2+ ranged from 0.12 μM to 2 mM. The titer of Mn 2+ ranged from 6.1 to 100 mM. (D) Addition of ferrous iron disassociates the binding between phosphorylated VgrR and P tdvA . Electrophoretic mobility shift assays were conducted to determine the impact of Fe 2+ on VgrR–P tdvA binding. VgrR was phosphorylated by MBP-VgrS and ATP. The concentrations of Fe 2+ were gradually increased from 20 μM to 1.0 mM. The assay was repeated independently three times. (E) Ferrous iron disassociates the interaction between phosphorylated VgrR and P tdvA . 5′-FAM labelled P tdvA DNA was used in the MST assay. Different concentrations of Fe 2+ (0.61 μM -10 mM) were added to the mixture as indicated. (F) Ferrous iron disassociates the interaction between VgrR and VgrS. VgrR protein was labelled in the MST assay. If needed, VgrR was phosphorylated by acetyl phosphate. Different concentrations of VgrS membrane were added to the mixture as indicated. In (C, E and F), the experiment was repeated independently three times.
    Figure Legend Snippet: Iron-VgrR binding disassociates VgrR-DNA and VgrR-VgrS interactions. (A) The amount of VgrR–P tdvA binding was decreased in iron-replete conditions. ChIP-qPCR was conducted to quantify the enrichment of VgrR at the tdvA promoter in vivo when bacteria were grown under iron-replete and iron-depleted conditions, respectively. The experiment was repeated three times. Vertical bars indicate the standard deviations. (B) The presence of Fe 2+ inhibits the formation of the VgrR–P tdvA complex in vitro . Electrophoretic mobility shift assays were conducted to determine the impact of Fe 2+ on VgrR–P tdvA binding. The concentrations of Fe 2+ were gradually increased from 10 μM to 1.0 mM. The assay was repeated independently three times. (C). Fe 2+ directly binds VgrR. 2 μM VgrR and Fe 2+ or Mn 2+ was used in an MST assay. The titer of Fe 2+ ranged from 0.12 μM to 2 mM. The titer of Mn 2+ ranged from 6.1 to 100 mM. (D) Addition of ferrous iron disassociates the binding between phosphorylated VgrR and P tdvA . Electrophoretic mobility shift assays were conducted to determine the impact of Fe 2+ on VgrR–P tdvA binding. VgrR was phosphorylated by MBP-VgrS and ATP. The concentrations of Fe 2+ were gradually increased from 20 μM to 1.0 mM. The assay was repeated independently three times. (E) Ferrous iron disassociates the interaction between phosphorylated VgrR and P tdvA . 5′-FAM labelled P tdvA DNA was used in the MST assay. Different concentrations of Fe 2+ (0.61 μM -10 mM) were added to the mixture as indicated. (F) Ferrous iron disassociates the interaction between VgrR and VgrS. VgrR protein was labelled in the MST assay. If needed, VgrR was phosphorylated by acetyl phosphate. Different concentrations of VgrS membrane were added to the mixture as indicated. In (C, E and F), the experiment was repeated independently three times.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, In Vivo, In Vitro, Electrophoretic Mobility Shift Assay, Microscale Thermophoresis

    Dissection of the VgrR binding consensus motif. (A) Venn diagram showing the number of VgrR-regulated genes identified by ChIP-seq. (B) Functional categories of the VgrR-regulated genes identified by ChIP-seq. Details of the genes are listed in S6 and S7 Tables. (C) Deduced consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. (D) Mapping the VgrR protected DNA region in the 5′ upstream sequence of XC1241 ( tdvA ) by DNase I footprinting. The amounts of VgrR protein used in the reactions were 1: zero; 2: 0.08 μM; 3: 0.8 μM; 4: 3.2 μM; and 5: 8.0 μM. The DNA regions protected by VgrR are shown on the right of the footprinting results, with the three possible VgrR-binding motifs shown in red, green, and black, respectively. A-T-C-G lanes are the DNA ladders obtained by a dideoxy-mediated chain-termination method using the same DNA sequence as the template. (E) Electrophoretic mobility shift assay verified the DNA motif of the XC1241 promoter bound by VgrR. The DNA probes were chemically synthesized according to those shown in (D). Sequences of the promoter region of XC1241 are listed above each panel. Each DNA probe was labeled by [γ- 32 P]ATP. Triangles indicate the VgrR-DNA complexes. All experiments were repeated three times.
    Figure Legend Snippet: Dissection of the VgrR binding consensus motif. (A) Venn diagram showing the number of VgrR-regulated genes identified by ChIP-seq. (B) Functional categories of the VgrR-regulated genes identified by ChIP-seq. Details of the genes are listed in S6 and S7 Tables. (C) Deduced consensus VgrR-binding DNA motif based on ChIP-seq data. Weblogo was used to show the nucleotide composition. (D) Mapping the VgrR protected DNA region in the 5′ upstream sequence of XC1241 ( tdvA ) by DNase I footprinting. The amounts of VgrR protein used in the reactions were 1: zero; 2: 0.08 μM; 3: 0.8 μM; 4: 3.2 μM; and 5: 8.0 μM. The DNA regions protected by VgrR are shown on the right of the footprinting results, with the three possible VgrR-binding motifs shown in red, green, and black, respectively. A-T-C-G lanes are the DNA ladders obtained by a dideoxy-mediated chain-termination method using the same DNA sequence as the template. (E) Electrophoretic mobility shift assay verified the DNA motif of the XC1241 promoter bound by VgrR. The DNA probes were chemically synthesized according to those shown in (D). Sequences of the promoter region of XC1241 are listed above each panel. Each DNA probe was labeled by [γ- 32 P]ATP. Triangles indicate the VgrR-DNA complexes. All experiments were repeated three times.

    Techniques Used: Dissection, Binding Assay, Chromatin Immunoprecipitation, Functional Assay, Sequencing, Footprinting, Electrophoretic Mobility Shift Assay, Synthesized, Labeling

    40) Product Images from "pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana"

    Article Title: pKAMA-ITACHI Vectors for Highly Efficient CRISPR/Cas9-Mediated Gene Knockout in Arabidopsis thaliana

    Journal: Plant and Cell Physiology

    doi: 10.1093/pcp/pcw191

    Flowchart for isolation of Cas9-free target mutants. For construction using pKIR1.1, hybridized 23 bp oligonucleotides containing an sgRNA sequence for the target gene are inserted into Aar I-cut pKIR1.1 (Steps 1 and 2; Fig. 6 ). The pKIR series enables red fluorescence selection of seeds from T 1 plants (Steps 3 and 4; hygromycin selection is also available). In T 2 selection, seeds without red fluorescence represent Cas9-free plants (Step 5). By genotyping T 2 plants, we can obtain a null mutant in the target gene (Step 6).
    Figure Legend Snippet: Flowchart for isolation of Cas9-free target mutants. For construction using pKIR1.1, hybridized 23 bp oligonucleotides containing an sgRNA sequence for the target gene are inserted into Aar I-cut pKIR1.1 (Steps 1 and 2; Fig. 6 ). The pKIR series enables red fluorescence selection of seeds from T 1 plants (Steps 3 and 4; hygromycin selection is also available). In T 2 selection, seeds without red fluorescence represent Cas9-free plants (Step 5). By genotyping T 2 plants, we can obtain a null mutant in the target gene (Step 6).

    Techniques Used: Isolation, Sequencing, Fluorescence, Selection, Mutagenesis

    pKIR1.1 construction. (A) For pKIR1.1, U6.26p::2 × AarI:sgRNA was inserted into the Sbf I site of pKIR1.0. (B) Flowchart of pKIR1.1-based vector construction. The upper sequence is an enlarged view of the boundary (yellow) between the U6.26 promoter (pale green) and the sgRNA scaffold (orange). This boundary contains an inverted repeat of Aar I recognition sites, and Aar I digestion generates four-base overhangs. A hybridized primer with overhangs can be inserted into this site as shown. G with a red triangle is the first base for RNA transcription from the U6.26 promoter. G + (N) 19 indicates the target sequence.
    Figure Legend Snippet: pKIR1.1 construction. (A) For pKIR1.1, U6.26p::2 × AarI:sgRNA was inserted into the Sbf I site of pKIR1.0. (B) Flowchart of pKIR1.1-based vector construction. The upper sequence is an enlarged view of the boundary (yellow) between the U6.26 promoter (pale green) and the sgRNA scaffold (orange). This boundary contains an inverted repeat of Aar I recognition sites, and Aar I digestion generates four-base overhangs. A hybridized primer with overhangs can be inserted into this site as shown. G with a red triangle is the first base for RNA transcription from the U6.26 promoter. G + (N) 19 indicates the target sequence.

    Techniques Used: Plasmid Preparation, Sequencing

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    Article Snippet: .. To radioactively label NrrF for in vitro binding assays with the Hfq protein and/or the possible sdh targets, 20 pmol of in vitro-transcribed NrrF or NrrfΔ31-58 was dephosphorylated with calf intestinal phosphatase (New England Biolabs) at 37°C, purified by using a MEGAclear kit, and 5′ end labeled with 30 μCi of [γ-32 P]ATP using 10 U of T4 polynucleotide kinase. .. The unincorporated radioactive nucleotides were removed by using TE-30 Chromaspin columns, and the band of the labeled in vitro transcript of appropriate size was extracted after electrophoresis on a denaturing 6% polyacrylamide urea gel and eluted overnight at 4°C in RNA elution buffer (0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA).

    Synthesized:

    Article Title: Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression *
    Article Snippet: .. The 15-nt RNA oligos used in the in vitro RNase assay, 5′-AGUAGAAACAAGGCC-3′, were chemically synthesized, and 5′ labeled with [γ-32 P]ATP using a T4 polynucleotide kinase (New England Biolabs). .. Crystallization and Data Collection The LASV NP-C protein was mixed with the 5′-triphosphate dsRNA substrate at a 1:1 molar ratio on ice for 30 min.

    Ligation:

    Article Title: Multiple RNA–RNA tertiary interactions are dispensable for formation of a functional U2/U6 RNA catalytic core in the spliceosome
    Article Snippet: .. Yeast U6 snRNAs containing N7-deaza modifications and abasic mutations were prepared by splinted ligation ( ) with T4 DNA ligase (NEB) and a U6(19–108) DNA splint that bridges three U6 snRNA fragments, with the desired mutations located in the central fragment ( ). .. To monitor ligation and recovery, the central U6 fragments were trace-labelled with γ-[32 P] ATP (Perkin Elmer) and polynucleotide kinase (NEB).

    Labeling:

    Article Title: Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403
    Article Snippet: .. DNA probes of about 400 bp corresponding to the promoter regions of cysD , cysM , fhuR , metA , metB2 , plpA , yhcE , and yjgC were generated by PCR using specific primers (Table ) and labeled at the 5′ end with [γ-32 P]ATP by the T4 polynucleotide kinase (NEB). .. Unincorporated nucleotides were removed with the NucleoSpin PCR purification kit (Macherey Nagel).

    Article Title: Structures of Arenaviral Nucleoproteins with Triphosphate dsRNA Reveal a Unique Mechanism of Immune Suppression *
    Article Snippet: .. The 15-nt RNA oligos used in the in vitro RNase assay, 5′-AGUAGAAACAAGGCC-3′, were chemically synthesized, and 5′ labeled with [γ-32 P]ATP using a T4 polynucleotide kinase (New England Biolabs). .. Crystallization and Data Collection The LASV NP-C protein was mixed with the 5′-triphosphate dsRNA substrate at a 1:1 molar ratio on ice for 30 min.

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis ▿
    Article Snippet: .. To radioactively label NrrF for in vitro binding assays with the Hfq protein and/or the possible sdh targets, 20 pmol of in vitro-transcribed NrrF or NrrfΔ31-58 was dephosphorylated with calf intestinal phosphatase (New England Biolabs) at 37°C, purified by using a MEGAclear kit, and 5′ end labeled with 30 μCi of [γ-32 P]ATP using 10 U of T4 polynucleotide kinase. .. The unincorporated radioactive nucleotides were removed by using TE-30 Chromaspin columns, and the band of the labeled in vitro transcript of appropriate size was extracted after electrophoresis on a denaturing 6% polyacrylamide urea gel and eluted overnight at 4°C in RNA elution buffer (0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA).

    Purification:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis ▿
    Article Snippet: .. To radioactively label NrrF for in vitro binding assays with the Hfq protein and/or the possible sdh targets, 20 pmol of in vitro-transcribed NrrF or NrrfΔ31-58 was dephosphorylated with calf intestinal phosphatase (New England Biolabs) at 37°C, purified by using a MEGAclear kit, and 5′ end labeled with 30 μCi of [γ-32 P]ATP using 10 U of T4 polynucleotide kinase. .. The unincorporated radioactive nucleotides were removed by using TE-30 Chromaspin columns, and the band of the labeled in vitro transcript of appropriate size was extracted after electrophoresis on a denaturing 6% polyacrylamide urea gel and eluted overnight at 4°C in RNA elution buffer (0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA).

    Generated:

    Article Title: Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403
    Article Snippet: .. DNA probes of about 400 bp corresponding to the promoter regions of cysD , cysM , fhuR , metA , metB2 , plpA , yhcE , and yjgC were generated by PCR using specific primers (Table ) and labeled at the 5′ end with [γ-32 P]ATP by the T4 polynucleotide kinase (NEB). .. Unincorporated nucleotides were removed with the NucleoSpin PCR purification kit (Macherey Nagel).

    Migration:

    Article Title: ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species
    Article Snippet: .. Helicase and branch migration assays Helicase assay substrates were 5′ end-labeled with T4 polynucleotide kinase (T4 PNK; NEB) and γ[32 P]-ATP. .. The two-strand forked helicase substrates was annealed by combining equimolar amounts of a labeled oligonucleotide with the unlabeled complement and incubating at 37°C overnight.

    Polymerase Chain Reaction:

    Article Title: Sulfur Amino Acid Metabolism and Its Control in Lactococcus lactis IL1403
    Article Snippet: .. DNA probes of about 400 bp corresponding to the promoter regions of cysD , cysM , fhuR , metA , metB2 , plpA , yhcE , and yjgC were generated by PCR using specific primers (Table ) and labeled at the 5′ end with [γ-32 P]ATP by the T4 polynucleotide kinase (NEB). .. Unincorporated nucleotides were removed with the NucleoSpin PCR purification kit (Macherey Nagel).

    Binding Assay:

    Article Title: The Hfq-Dependent Small Noncoding RNA NrrF Directly Mediates Fur-Dependent Positive Regulation of Succinate Dehydrogenase in Neisseria meningitidis ▿
    Article Snippet: .. To radioactively label NrrF for in vitro binding assays with the Hfq protein and/or the possible sdh targets, 20 pmol of in vitro-transcribed NrrF or NrrfΔ31-58 was dephosphorylated with calf intestinal phosphatase (New England Biolabs) at 37°C, purified by using a MEGAclear kit, and 5′ end labeled with 30 μCi of [γ-32 P]ATP using 10 U of T4 polynucleotide kinase. .. The unincorporated radioactive nucleotides were removed by using TE-30 Chromaspin columns, and the band of the labeled in vitro transcript of appropriate size was extracted after electrophoresis on a denaturing 6% polyacrylamide urea gel and eluted overnight at 4°C in RNA elution buffer (0.1 M sodium acetate, 0.1% SDS, 10 mM EDTA).

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    New England Biolabs t4 polynucleotide kinase
    Retroposon- and repeat-derived siRNAs have modified 5′ and 3′ termini. ( A ) A synthetic 30-nt RNA (lane 1) was sequentially treated with <t>T4</t> polynucleotide kinase (PNK, lane 2) and calf intestinal alkaline phosphatase (CIP, lane 3), separated
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1540 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Retroposon- and repeat-derived siRNAs have modified 5′ and 3′ termini. ( A ) A synthetic 30-nt RNA (lane 1) was sequentially treated with T4 polynucleotide kinase (PNK, lane 2) and calf intestinal alkaline phosphatase (CIP, lane 3), separated

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Distinct and overlapping roles for two Dicer-like proteins in the RNA interference pathways of the ancient eukaryote Trypanosoma brucei

    doi: 10.1073/pnas.0907766106

    Figure Lengend Snippet: Retroposon- and repeat-derived siRNAs have modified 5′ and 3′ termini. ( A ) A synthetic 30-nt RNA (lane 1) was sequentially treated with T4 polynucleotide kinase (PNK, lane 2) and calf intestinal alkaline phosphatase (CIP, lane 3), separated

    Article Snippet: Small RNAs were size-selected ( ) and treated with a variety of enzymes under manufacturer-recommended conditions: Calf intestinal alkaline phosphatase (CIP; Amersham) and T4 polynucleotide kinase (T4 PNK; New England Biolabs) assays were carried out for 1 h at 37 °C, and Terminator exonuclease (Epicentre) was added for 1 h at 30 °C.

    Techniques: Derivative Assay, Modification

    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Journal: Genome Research

    Article Title: Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

    doi: 10.1101/gr.121426.111

    Figure Lengend Snippet: Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Article Snippet: Total RNA was isolated using TRIzol (Invitrogen), and small RNA samples were 5′ labeled by first treating with calf alkaline phosphatase (Roche) followed by phosphorylation with T4 polynucleotide kinase (NEB) and 32 P-γ-ATP.

    Techniques: Labeling, Molecular Weight

    PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Journal: Nature Communications

    Article Title: PARP3 is a sensor of nicked nucleosomes and monoribosylates histone H2BGlu2

    doi: 10.1038/ncomms12404

    Figure Lengend Snippet: PARP3 monoribosylates H2B in damaged chromatin. ( a , left) 10μg of the chicken chromatin employed in these experiments was fractionated by SDS–PAGE and stained with Coomassie blue. (right) One microgram of soluble MNase-treated chicken chromatin or 50-mer oligonucleotide duplex (200 nM) harbouring a nick with 3′-P/5′-OH termini was mock-treated (0) or treated with 1, 0.5 or 0.25 U T4 PNK to restore 3′-OH/5′-P termini. These DNA substrates were then incubated with 100 nM hPARP3 and 12.5 μM biotin-NAD + for 30 min and biotinylated products separated by 15% SDS–PAGE and detected with streptavidin-HRP. ( b ) 1 μg chicken chromatin or the indicated recombinant histone was incubated with 100 nM hPARP3 in the presence of 300 nM 32 P-NAD + or 12.5 μM biotin-NAD and oligonucleotide harbouring either a DSB (middle) or SSB (right) and the reaction products fractionated by 15% SDS–PAGE and detected by autoradiography or streptavidin-HRP. (left) An aliquot of the chicken chromatin and recombinant histones was fractionated by SDS–PAGE and stained with Coomassie blue. ( c , left) Aliquots of recombinant histone standards were fractionated separately or together as an octamer on triton-acid urea gels and analysed by staining with Coomassie blue. (right) The products of the PARP3 ribosylation reactions conducted in b were fractionated on triton-acid urea gels and analysed by autoradiography. HRP, horseradish peroxidase.

    Article Snippet: DNA from the MNase concentration that produced the greatest SSB/DSB ratio (0.015 U) was mock-treated or treated with T4 PNK in the presence of 2 mM ATP and 10U T4 PNK enzyme (wild-type or 3′-phosphatase dead; New England Biolabs).

    Techniques: SDS Page, Staining, Incubation, TNKS1 Histone Ribosylation Assay, Recombinant, Autoradiography

    Dynamic expression of placental/decidual microRNAs and tRNA fragments. (A) Scheme of sample collection for this figure. Placenta/decidua control samples were collected at embryonic development days E12.5, E13.5, E14.5 and E18.5 (corresponding to the 3 hours, 24 hours, 48 hours, 144 hours time point control samples). (B) Principle component analysis of microRNAs and tRFs from placenta/decidua control samples at different time points by sequence-level analysis. (C) Heatmap shows dynamic expression of placental/decidual microRNAs and tRFs across embryonic development time. Each row in heatmap represents one unique sequence. Expression values were calculated by log10RPM (reads per million total mapped reads) and averaged from 6 samples at each time point and then scaled across row. For visualization purpose, only abundantly expressed miRs or tRFs (mean expression cut-off 100 RPM) that show time-dependent changes are shown in the heatmap. A complete list of dynamically expressed miRs and tRFs please refer to Table S2. (D-E) Examples of dynamically expressed microRNAs. mmu-miR-215-5p (D) is up-regulated over time and mmu-miR-146b-5p (E) is down-regulated over time. (F-H) Examples of dynamically expressed tRFs, including down-regulated 5’ halves from tRNA GluTTC and tRNA GlyCCC . (D-F) Box plots showing RPM (reads per million total mapped reads) for specific miR or tRF sequence, with each dot represents one sample (n = 6 for each time point) and middle line represents median value. (G-H) qRT-PCR validation of temporal decrease of 5’ tRNA halves in both NT (no treatment control) and T4 PNK treatment samples. Error bars represent standard deviation from two biological replicates (male and female control samples pooled for each time point).

    Journal: bioRxiv

    Article Title: tRNA-derived fragments and microRNAs in the maternal-fetal interface of a mouse maternal-immune-activation autism model

    doi: 10.1101/2019.12.20.884650

    Figure Lengend Snippet: Dynamic expression of placental/decidual microRNAs and tRNA fragments. (A) Scheme of sample collection for this figure. Placenta/decidua control samples were collected at embryonic development days E12.5, E13.5, E14.5 and E18.5 (corresponding to the 3 hours, 24 hours, 48 hours, 144 hours time point control samples). (B) Principle component analysis of microRNAs and tRFs from placenta/decidua control samples at different time points by sequence-level analysis. (C) Heatmap shows dynamic expression of placental/decidual microRNAs and tRFs across embryonic development time. Each row in heatmap represents one unique sequence. Expression values were calculated by log10RPM (reads per million total mapped reads) and averaged from 6 samples at each time point and then scaled across row. For visualization purpose, only abundantly expressed miRs or tRFs (mean expression cut-off 100 RPM) that show time-dependent changes are shown in the heatmap. A complete list of dynamically expressed miRs and tRFs please refer to Table S2. (D-E) Examples of dynamically expressed microRNAs. mmu-miR-215-5p (D) is up-regulated over time and mmu-miR-146b-5p (E) is down-regulated over time. (F-H) Examples of dynamically expressed tRFs, including down-regulated 5’ halves from tRNA GluTTC and tRNA GlyCCC . (D-F) Box plots showing RPM (reads per million total mapped reads) for specific miR or tRF sequence, with each dot represents one sample (n = 6 for each time point) and middle line represents median value. (G-H) qRT-PCR validation of temporal decrease of 5’ tRNA halves in both NT (no treatment control) and T4 PNK treatment samples. Error bars represent standard deviation from two biological replicates (male and female control samples pooled for each time point).

    Article Snippet: T4 PNK treatment and RT-PCR 1 μg total RNA was treated with T4 PNK (NEB) in the presence of 10 mM ATP at 37 °C for 40 minutes and then cleaned up by ethanol precipitation.

    Techniques: Expressing, Sequencing, Quantitative RT-PCR, Standard Deviation