rnase h Search Results


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  • 99
    New England Biolabs rnase h
    R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of  BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the  BU‐1  locus in  primpol  cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired  t ‐test was used to compare differences between matched amplicons in  primpol BU‐1A (GAA)10  and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in  primpol BU‐1A (GAA)10 :Gg RNase H1 (see also   Appendix Fig S5 ) and  primpol BU‐1A (GAA)10 :hPrimPol. An unpaired  t ‐test on three biological replicates was used to compare differences to  primpol BU‐1A (GAA)10  for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced  BU‐1A  epigenetic instability in  primpol  cells. Fluctuation analysis was performed on three  primpol BU‐1A (GAA)10  clones. One‐way ANOVA was used to calculate the significance of differences in  BU‐1  instability between  primpol BU‐1A ΔG4  and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in   Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of  BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.
    Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2919 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase h
    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with <t>RNase</t> H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.
    Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7396 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rnase h
    Determination of unfolding kinetic constants of MBP and <t>RNase</t> H by pulse proteolysis
    Rnase H, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    TaKaRa rnase h
    Determination of specificity of binding of oligonucleotides designed based upon RT-ROL results to viral RNA by cleavage with RNase H. HIV-2 1-561 RNA was incubated in monomer buffer with the oligonucleotides indicated and RNase H. Digestion products were loaded onto a denaturing polyacrylamide gel and fragments were visualized by ethidium bromide staining. Lanes L: RNA size ladder (Ambion); Lane 1: complete reaction except with no oligonucleotide added; Lanes 2-8: complete reactions with oligonucleotide asROD32, asROD99, asROD172, asROD258, asROD309, asRODPBS, and asROD344 added, respectively; Lane 9: control reaction with the non-complementary sense oligonucleotide sROD228.
    Rnase H, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 501 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher e coli rnase h
    <t>RNase</t> H activities in Leishmania . (A) Zymogram for the RNase H activities in the cell extracts from L. donovani . Cells and mitochondrial enriched fraction were lysed and boiled in SDS-containing denaturation solution for 3 min before loading. Proteins equivalent to 2 × 10 7 cells were loaded per lane (TCE, ME). The separating gel was made with the RNase H substrate 32 P-poly(rA)/poly(dT). These activities were solely dependent upon Mn 2+ and addition of Mg 2+ instead did not yield any such band. The sizes of the bands shown are approximate. The major Mn 2+ -dependent RNase H activity is enriched in the mitochondrial fraction. TCE: total cellular extract; ME: mitochondrial extract. (B) Expression and purification of N-terminal His 6 -tagged L. major LRNase HIIC precursor protein and the mature LRNase HIICΔMLS in E. coli . Recombinant cells were induced with 0.2 mM IPTG. The proteins were separated in a 10% SDS-PAGE and stained with Coomasie Blue R250. Optimal induction was observed in 3 h at 37 °C. We followed up to 21 h. Lane M: size marker. Lanes 1–4 are for LRNase HIIC and lanes 5–8 are for LRNase HIICΔMLS. Lanes 1 and 5, lysates from uninduced cells; lanes 2 and 6, lysates from IPTG induced cells; lanes 3, 4, 7 and 8, fractions eluted from washed columns with buffer E (100 mM NaH 2 PO 4 , 10 mM Tris.Cl pH 4.5, and 8 M urea). (C) Western blot analysis of LRNase HIIC in L. donovani promastigotes. Left panel (i), Coomassie Brilliant Blue stained protein gel showing L. donovani total cellular protein (Cell; 50 μg) and total mitochondrial protein (Mito; 50 μg). Middle panel (ii), Western blot analysis with anti-LRNase HIIC antibodies (1:1000 dilution). The LRNase HIIC antibody detected a 49-kDa protein in the mitochondrial extract. The faint band in the total cellular extract is not visible in the photograph of the chemiluminiscence autoradiogram. Right panel (iii), Western blot with anti-ISP monoclonal antibody, showing mitochondrial enrichment. (D) Pull-down of the 49-kDa radiolabeled LRNase HIIC protein by antibody. Immunoprecipitation was done with the reagents from the SeizeX Protein A immunoprecipitation kit (Pierce Biotechnology). The size of the bands is 49-kDa. Experiment 1 and Experiment 2 are duplicates for same experiment. (E) Zymogram for the activity gel assay of the proteins isolated from L. donovani mitochondrial extracts by immunocapture. Assay was done as described above. The sizes of the bands correspond to 49-kDa.
    E Coli Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 423 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of  BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the  BU‐1  locus in  primpol  cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired  t ‐test was used to compare differences between matched amplicons in  primpol BU‐1A (GAA)10  and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in  primpol BU‐1A (GAA)10 :Gg RNase H1 (see also   Appendix Fig S5 ) and  primpol BU‐1A (GAA)10 :hPrimPol. An unpaired  t ‐test on three biological replicates was used to compare differences to  primpol BU‐1A (GAA)10  for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced  BU‐1A  epigenetic instability in  primpol  cells. Fluctuation analysis was performed on three  primpol BU‐1A (GAA)10  clones. One‐way ANOVA was used to calculate the significance of differences in  BU‐1  instability between  primpol BU‐1A ΔG4  and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in   Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of  BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: R‐loops promote ( GAA ) 10 ‐dependent epigenetic instability of BU ‐1 DRIP‐qPCR analysis reveals accumulation of R‐loops across the BU‐1 locus in primpol cells. The DRIP signal was calculated as enrichment over RNase H‐treated samples and was normalised to −0.5 kb amplicon. The mean and SD for three biological replicates is presented. An unpaired t ‐test was used to compare differences between matched amplicons in primpol BU‐1A (GAA)10 and the other cell lines indicated. **** P  ≤ 0.0001, ns = not significant. DNA:RNA hybrids in primpol BU‐1A (GAA)10 :Gg RNase H1 (see also Appendix Fig S5 ) and primpol BU‐1A (GAA)10 :hPrimPol. An unpaired t ‐test on three biological replicates was used to compare differences to primpol BU‐1A (GAA)10 for each matched amplicon. The bar represents the mean, and whiskers represent the SD. *** P  ≤ 0.001, ** P  ≤ 0.01, ns = not significant. Overexpression of chicken RNase H1 prevents (GAA) 10 ‐induced BU‐1A epigenetic instability in primpol cells. Fluctuation analysis was performed on three primpol BU‐1A (GAA)10 clones. One‐way ANOVA was used to calculate the significance of differences in BU‐1 instability between primpol BU‐1A ΔG4 and other cell lines. **** P  ≤ 0.0001, ns = not significant. Diagram of the RNase H1 hybrid binding domain (HBD)–mCherry fusion and flow cytometry expression profiles of the construct in four clones. Western blots of the same four clones are shown in Appendix Fig S6 . R‐loop stabilisation induces epigenetic instability of BU‐1 . Bu‐1a fluctuation analysis of wild‐type cells expressing HBD‐mCherry. The scatter plots pool results from at least two different clones with matched HBD expression. Mean ± SD reported. **** P  ≤ 0.0001, *** P  ≤ 0.001, ns = not significant; one‐way ANOVA.

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: Real-time Polymerase Chain Reaction, Amplification, Over Expression, Binding Assay, Flow Cytometry, Expressing, Construct, Clone Assay, Western Blot

    PrimPol suppresses R‐loop formation in association with  DNA  secondary structure‐forming sequences across the  DT 40 genome Representative normalised DRIP‐seq data in two genes  COL22A1 , spanning over 200 kb, and  MYC . The locations of H‐DNA and G4 motifs are shown below the gene map. Wild type in blue;  primpol  in red. The corresponding RNase H‐treated samples are dashed. See   Materials and Methods  for further details of graphic generation. Metagene analysis of DRIP peak distribution in wild‐type and  primpol  DT40 cells compared with the distribution of the indicated features in the genome. DRIP peak heights in wild type and  primpol  DT40 normalised to  Drosophila  S2 spike‐in.  n  (wild type) = 41,445;  n  ( primpol ) = 48,648. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and  primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and  primpol  cells (red). Normalised DRIP peak heights in the genes identified as associating with H‐DNA. Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and  primpol  cells (red). Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information:  P ‐values calculated with Mann–Whitney  U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd  quartile + 1.5*IQR).

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences across the DT 40 genome Representative normalised DRIP‐seq data in two genes COL22A1 , spanning over 200 kb, and MYC . The locations of H‐DNA and G4 motifs are shown below the gene map. Wild type in blue; primpol in red. The corresponding RNase H‐treated samples are dashed. See Materials and Methods for further details of graphic generation. Metagene analysis of DRIP peak distribution in wild‐type and primpol DT40 cells compared with the distribution of the indicated features in the genome. DRIP peak heights in wild type and primpol DT40 normalised to Drosophila S2 spike‐in. n (wild type) = 41,445; n ( primpol ) = 48,648. Correlation of normalised DRIP peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised DRIP peak heights in the genes identified as associating with H‐DNA. Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised DRIP peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: MANN-WHITNEY

    PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences in BOBSC iPS cells Representative normalised RNA DIP‐seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H‐DNA and G4 motifs are shown below the gene map. The corresponding RNase H‐treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Metagene analysis of RNA‐DIP peak distribution in wild‐type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. RNA DIP‐seq peak heights in wild‐type and primpol BOBSC cells normalised to a DT40 spike‐in. n (wild type) = 32,740; n ( primpol ) = 33,721. Correlation of normalised RNA DIP‐seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised RNA DIP‐seq peak heights in the genes identified as associating with H‐DNA. Normalised RNA DIP‐seq peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: PrimPol suppresses R‐loop formation in association with DNA secondary structure‐forming sequences in BOBSC iPS cells Representative normalised RNA DIP‐seq data in the SKI locus. Wild type in blue; primpol in red. The locations of H‐DNA and G4 motifs are shown below the gene map. The corresponding RNase H‐treated samples are dashed. Since so little material was recovered following RNase H treatment, all samples were pooled prior to library generation. Metagene analysis of RNA‐DIP peak distribution in wild‐type and primpol BOBSC cells compared with the distribution of the indicated features in the genome. RNA DIP‐seq peak heights in wild‐type and primpol BOBSC cells normalised to a DT40 spike‐in. n (wild type) = 32,740; n ( primpol ) = 33,721. Correlation of normalised RNA DIP‐seq peak heights in the overlapping peaks between wild type and primpol . Blue line = 1:1 correlation; red line = linear regression through data. Correlation between H‐DNA‐forming sequences and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Correlation between G4 motifs ([G 3‐5 N 1‐7 ] 4 ) and all genes (white bar), and genes with DRIP peaks in wild‐type (blue) and primpol cells (red). Normalised RNA DIP‐seq peak heights in the genes identified as associating with H‐DNA. Normalised RNA DIP‐seq peak heights in the genes identified as associating with G4 motifs ([G 3‐5 N 1‐7 ] 4 ). Data information: P ‐values calculated with Mann–Whitney U ‐test. In violin plots, bar = median; box = interquartile range (IQR); whiskers = upper and lower inner fences (1 st /3 rd quartile + 1.5*IQR).

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: DNA Immunoprecipitation Sequencing, MANN-WHITNEY

    Loss of PrimPol leads to unscheduled S phase R‐loop formation Expression of geminin‐tagged chicken RNase H1‐YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X ‐axis). RNase H1‐YFP with or without the geminin degron protein is detected on the Y ‐axis. The RNase H1‐YFP‐geminin degron is degraded in G1. In contrast, RNase H1‐YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication. Bu‐1a fluctuation analysis of two independently derived primpol BU‐1A (GAA)10 :Gg RNase H1‐YFP‐geminin degron clones. Since the expression of the RNase H1‐YFP‐geminin degron construct is not stable (unlike the RNase H1‐YFP construct without the degron), Bu‐1a expression was assessed separately in the YFP +ve and YFP −ve cells within each clone. Statistical differences calculated the Kruskal–Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** P ≤ 0.0001, *** P ≤ 0.001, ns = not significant. DRIP‐qPCR for R‐loops around the engineered +3.5 (GAA) 10 repeat in BU‐1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU‐1 DRIP signal was normalised to −0.5 kb amplicon in G1‐arrested cells ( t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol . Error bars = SD. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Workflow for the S9.6‐independent detection of newly synthesised R‐loops. See Materials and Methods for details. Validation of analysis of nascent DNA:RNA hybrid formation in BU‐1 locus. Enrichment of 4‐SU‐labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild‐type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** P ≤ 0.01, * P ≤ 0.05, ns = not significant; unpaired t ‐test. Synchronisation and 4‐SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids. Newly synthesised R‐loops in BU‐1 during S phase in wild type (black) and primpol (red). Error bars represent 1 SD of three biological repeats of the experiment. *** P ≤ 0.001, * P ≤ 0.05; unpaired t ‐test.

    Journal: The EMBO Journal

    Article Title: R‐loop formation during S phase is restricted by PrimPol‐mediated repriming

    doi: 10.15252/embj.201899793

    Figure Lengend Snippet: Loss of PrimPol leads to unscheduled S phase R‐loop formation Expression of geminin‐tagged chicken RNase H1‐YFP. Phases of the cell cycle were determined by staining DNA content in live cells by Hoechst 33342 ( X ‐axis). RNase H1‐YFP with or without the geminin degron protein is detected on the Y ‐axis. The RNase H1‐YFP‐geminin degron is degraded in G1. In contrast, RNase H1‐YFP levels remain stable irrespective of the phase of the cell cycle. 2n and 4n indicate the chromosome number before and after DNA replication. Bu‐1a fluctuation analysis of two independently derived primpol BU‐1A (GAA)10 :Gg RNase H1‐YFP‐geminin degron clones. Since the expression of the RNase H1‐YFP‐geminin degron construct is not stable (unlike the RNase H1‐YFP construct without the degron), Bu‐1a expression was assessed separately in the YFP +ve and YFP −ve cells within each clone. Statistical differences calculated the Kruskal–Wallis test. For all panels, at least 36 individual clones were analysed; mean ± SD reported. **** P ≤ 0.0001, *** P ≤ 0.001, ns = not significant. DRIP‐qPCR for R‐loops around the engineered +3.5 (GAA) 10 repeat in BU‐1 in different phases of the cell cycle. The location of the qPCR amplicons is indicated in the map at the top of the panel. The BU‐1 DRIP signal was normalised to −0.5 kb amplicon in G1‐arrested cells ( t = 0 h). See Fig EV4 for representative cell cycle synchronisation profiles. Black: wild type; red: primpol . Error bars = SD. **** P ≤ 0.0001, *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. Workflow for the S9.6‐independent detection of newly synthesised R‐loops. See Materials and Methods for details. Validation of analysis of nascent DNA:RNA hybrid formation in BU‐1 locus. Enrichment of 4‐SU‐labelled RNA moiety of DNA:RNA hybrids was calculated relative to input in three independent asynchronous wild‐type (black) or primpol (red) cells, with or without exogenous RNase H treatment. Error bars = SD. ** P ≤ 0.01, * P ≤ 0.05, ns = not significant; unpaired t ‐test. Synchronisation and 4‐SU pulse labelling scheme to identify nascently formed DNA:RNA hybrids. Newly synthesised R‐loops in BU‐1 during S phase in wild type (black) and primpol (red). Error bars represent 1 SD of three biological repeats of the experiment. *** P ≤ 0.001, * P ≤ 0.05; unpaired t ‐test.

    Article Snippet: The specificity of the pull‐down was tested with RNase H and RNase III treatments prior to immunoprecipitation: one‐third of the digested material was treated with 25 U of RNase H (NEB, M0297), or with 10 U of RNase III (Ambion, AM2290) in appropriate buffers overnight at 37°C, with the subsequent steps performed as described above.

    Techniques: Expressing, Staining, Derivative Assay, Clone Assay, Construct, Real-time Polymerase Chain Reaction, Amplification

    RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Journal: PLoS ONE

    Article Title: RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

    doi: 10.1371/journal.pone.0128686

    Figure Lengend Snippet: RNase H-mediated degradation of pre-formed ASO:RNA duplexes and in vitro -synthesized RNAs targeted by ASOs. (A) Schematic of the experimental setup for panels B and C. Applicable for some ASOs: Y, 8-oxo-dG residue; +, LNA sugar base. (B, C) Cleavage of pre-formed ASO:target RNA duplexes by RNase H. (B) Five femtomoles of 33 P-labeled substrate was treated with RNase H for the indicated times. The reaction products were collected, denatured by heating at 95°C for 2 min and analyzed by PAGE in native 15% gels. Arrows at right point to the substrate (S) and major cleavage product(s) (P). Results from one of three independent reproducible experiments are shown. (C) Kinetics of RNase H cleavage of different ASO:RNA duplexes. The amounts of radioactivity remaining in the uncleaved substrate were quantified using a Typhoon Trio instrument. Quantifications were performed for each gel. The obtained values were normalized to the radioactivity present in the substrate before adding RNase H (set to 100%). Each point corresponds to the average of three independent experiments. Error bars indicate the standard deviation. (D) Cleavage of FR3131 RNA by RNase H in the presence of different ASOs. The RNA and ASOs were mixed and incubated at 37°C for 10 min; then, RNase H was added to the reaction mixture. RNA samples were collected at the indicated time points and analyzed by electrophoresis in native 0.8% TAE agarose gels. The results from one of three independent reproducible experiments are shown. S: substrate; P1 and P2: cleavage products.

    Article Snippet: This target RNA was pre-incubated with D4676, DM4676, LD4676 or LDM4676 for 10 min at 37°C; next, RNase H was added to the reaction mixture.

    Techniques: Allele-specific Oligonucleotide, In Vitro, Synthesized, Labeling, Polyacrylamide Gel Electrophoresis, Radioactivity, Standard Deviation, Incubation, Electrophoresis

    Determination of unfolding kinetic constants of MBP and RNase H by pulse proteolysis

    Journal: Protein Science : A Publication of the Protein Society

    Article Title: Investigating protein unfolding kinetics by pulse proteolysis

    doi: 10.1002/pro.29

    Figure Lengend Snippet: Determination of unfolding kinetic constants of MBP and RNase H by pulse proteolysis

    Article Snippet: E.coli MBP and RNase H were prepared as described previously., Thermolysin (EC 3.4.24.27) from B. thermoproteolyticus rokko (Sigma Chemical, St. Louis, MO) was prepared in 2.5 M NaCl containing 10 m M CaCl2 .

    Techniques:

    Alignment of the predicted amino acid sequences of the pro-pol open reading frame in WDSV, WEHV1, and WEHV2. The seven conserved domains in RT are shown with brackets. LPQG and YMDD are indicated by four asterisks. The five conserved regions of RNase H are outlined in blocks labeled I through V. Identity with respect to WDSV (-), gaps (.), and differences between WEHV1 and WEHV2 are in bold-faced type. Amino acid changes between WEHV2 subclones are indicated in parentheses.

    Journal: Journal of Virology

    Article Title: Two Closely Related but Distinct Retroviruses Are Associated with Walleye Discrete Epidermal Hyperplasia

    doi:

    Figure Lengend Snippet: Alignment of the predicted amino acid sequences of the pro-pol open reading frame in WDSV, WEHV1, and WEHV2. The seven conserved domains in RT are shown with brackets. LPQG and YMDD are indicated by four asterisks. The five conserved regions of RNase H are outlined in blocks labeled I through V. Identity with respect to WDSV (-), gaps (.), and differences between WEHV1 and WEHV2 are in bold-faced type. Amino acid changes between WEHV2 subclones are indicated in parentheses.

    Article Snippet: After RNase H digestion, the cDNA was purified by centrifugation and filtered through Millipore Ultrafree-MC 30,000 NMWL filters.

    Techniques: Labeling

    Dose-response curve showing inhibition of RNase H activity by penicillin and streptomycin. The 100% of signal corresponds to the activity of 10 U/mL of RNase H (20 min at 25°C). Fluorescence values were collected after 40 min in the second step. Error bars represent the standard deviation (SD) calculated from triplicate experiments.

    Journal: Scientific Reports

    Article Title: A hybrid chimeric system for versatile and ultra-sensitive RNase detection

    doi: 10.1038/srep09558

    Figure Lengend Snippet: Dose-response curve showing inhibition of RNase H activity by penicillin and streptomycin. The 100% of signal corresponds to the activity of 10 U/mL of RNase H (20 min at 25°C). Fluorescence values were collected after 40 min in the second step. Error bars represent the standard deviation (SD) calculated from triplicate experiments.

    Article Snippet: Proteinase K and all DNase/RNase-free reagents used to prepare the RNase H buffer (50 mM Tris-HCl, 100 mM KCl, 9 mM MgCl2 , 1 mM DTT, 20 μg/mL BSA, pH 7.5) were purchased from Sigma-Aldrich.

    Techniques: Inhibition, Activity Assay, Fluorescence, Standard Deviation

    Characterization of biostability of LS OMe MBs (green) compared to DS MBs (blue). ( a ) The response of LS OMe MB1 to its target and DNase I was tested. ( b ) Signal enhancement and stem melting temperature of each LS OMe MB were calculated. ( c ) Selectivity of LS OMe MB1 was tested in the same way as LS MBs using mock target sequences. ( d – e ) RNase H sensitivity of LS OMe MB1 (d) and DS MB1 (e) was experimented. First, each MB was incubated with its RNA target, then RNase H was added to the mixture and the fluorescence change was monitored. In the case of DS MB (e), DNA target was added to ensure the DS MB1 had conformational change after the RNA targets were digested. ( f ) Cell lysate sensitivity of LS OMe MB 1 (green) and DS MB 1 (blue) were tested.

    Journal: Nucleic Acids Research

    Article Title: Superior structure stability and selectivity of hairpin nucleic acid probes with an l-DNA stem

    doi: 10.1093/nar/gkm771

    Figure Lengend Snippet: Characterization of biostability of LS OMe MBs (green) compared to DS MBs (blue). ( a ) The response of LS OMe MB1 to its target and DNase I was tested. ( b ) Signal enhancement and stem melting temperature of each LS OMe MB were calculated. ( c ) Selectivity of LS OMe MB1 was tested in the same way as LS MBs using mock target sequences. ( d – e ) RNase H sensitivity of LS OMe MB1 (d) and DS MB1 (e) was experimented. First, each MB was incubated with its RNA target, then RNase H was added to the mixture and the fluorescence change was monitored. In the case of DS MB (e), DNA target was added to ensure the DS MB1 had conformational change after the RNA targets were digested. ( f ) Cell lysate sensitivity of LS OMe MB 1 (green) and DS MB 1 (blue) were tested.

    Article Snippet: RNase H sensitivity To test the vulnerability of MB-RNA duplexes to ribonuclease H from Sigma–Aldrich, Inc. (St Louis, MO) digestion, 65 nM of MBs and RNA targets were incubated in the MB buffer while the fluorescence intensity was monitored.

    Techniques: Incubation, Fluorescence

    Determination of specificity of binding of oligonucleotides designed based upon RT-ROL results to viral RNA by cleavage with RNase H. HIV-2 1-561 RNA was incubated in monomer buffer with the oligonucleotides indicated and RNase H. Digestion products were loaded onto a denaturing polyacrylamide gel and fragments were visualized by ethidium bromide staining. Lanes L: RNA size ladder (Ambion); Lane 1: complete reaction except with no oligonucleotide added; Lanes 2-8: complete reactions with oligonucleotide asROD32, asROD99, asROD172, asROD258, asROD309, asRODPBS, and asROD344 added, respectively; Lane 9: control reaction with the non-complementary sense oligonucleotide sROD228.

    Journal: Antisense & nucleic acid drug development

    Article Title: Elucidation and characterization of oligonucleotide-accessible sites on HIV-2 leader region RNA

    doi: 10.1089/108729003764097331

    Figure Lengend Snippet: Determination of specificity of binding of oligonucleotides designed based upon RT-ROL results to viral RNA by cleavage with RNase H. HIV-2 1-561 RNA was incubated in monomer buffer with the oligonucleotides indicated and RNase H. Digestion products were loaded onto a denaturing polyacrylamide gel and fragments were visualized by ethidium bromide staining. Lanes L: RNA size ladder (Ambion); Lane 1: complete reaction except with no oligonucleotide added; Lanes 2-8: complete reactions with oligonucleotide asROD32, asROD99, asROD172, asROD258, asROD309, asRODPBS, and asROD344 added, respectively; Lane 9: control reaction with the non-complementary sense oligonucleotide sROD228.

    Article Snippet: The selection and identification of sites using this method relies on the recognition and cleavage of DNA:RNA heteroduplexes by RNase H, then using primer extension from an interior site on the RNA to detect the exact sites of cleavage.

    Techniques: Binding Assay, Incubation, Staining

    Overall scheme of the rapid determination of viral RNA sequence method. *By adding RNase H; WGA, whole genome amplification; †With specially designed primer sets as shown in   Figure 2 .

    Journal: Emerging Infectious Diseases

    Article Title: Rapid Genome Sequencing of RNA Viruses

    doi: 10.3201/eid1302.061032

    Figure Lengend Snippet: Overall scheme of the rapid determination of viral RNA sequence method. *By adding RNase H; WGA, whole genome amplification; †With specially designed primer sets as shown in Figure 2 .

    Article Snippet: In accordance with the Invitrogen manual, cDNA was synthesized, by using random hexamers (Takara Bio Inc., Kyoto, Japan) and Superscript III (Invitrogen, Carlsbad, CA, USA) lacking RNase H activity, at 50°C for 1 h. Then 60 U of RNase H (Takara Bio Inc.) added before synthesis of second-strand cDNA at 50°C for 1 h. In accordance with the manual, a whole genome amplification system (WGA; Sigma-Aldrich, Saint Louis, MO, USA), which was developed for amplification of genomic DNA, was used to amplify viral double-stranded cDNA.

    Techniques: Sequencing, Whole Genome Amplification

    Back-spliced CRM1 RNA in the maize centromere. (A) Procedure for anti-CENH3 RIP and subsequent high-throughput sequencing and cDNA library screening. (B) BLAST results of the back-spliced CRM1 reads from the anti-CENH3 RIP-seq, input-seq data, and the 354-nt RNA from the anti-CENH3 RIP cDNA library. The arrow shows the back-splicing site. (C) The location of the 607-bp combined sequence in CRM1, and the back-spliced form of the 354-nt RNA. The red line represents the 607-bp sequence. (D) Distribution of the 354-bp sequence on CRM1. The first track of each panel represents the centromeric region indicated by CENH3 enrichment, and the peak height represents the RPM value (0–1). The other 4 tracks represent the distributions of the 354-bp, 269-bp, 85-bp, and CRM1 sequences along a specific region of cen5. The lower panel shows a detailed version of the information displayed in the upper panel. The arrows inside the rectangular bars represent the directions of the sequences. (E) The public raw genome sequencing data (including Pacbio [65×] and Illumina [100×] reads) and 4 anti-CENH3 ChIP-seq datasets from B73 (including 1 generated in this study and 3 from public resources), together with 1 input-seq dataset, were mapped to the assumed 354-bp DNA. Only one read from anti-CENH3 ChIP-seq dataset was matched to the region containing the back-spliced junction site (purple line). All the other reads show no covering the back-spliced junction site. The data underlying this figure can be found in the GEO with accession numbers GSE124242, SRR3018834, SRR2000635, SRR2000640, SRR2000648, SRX1472849, and SRX1452310 and on Github ( https://github.com/sxx-ying/maize-centromere-circRNA ). CB, chromatin binding; CENH3, centromeric H3 variant; ChIP-seq, chromatin immunoprecipitation following high-throughput sequencing; Chr, chromosome; CRM, centromeric retrotransposon; Gag, gag protein; GEO, Gene Expression Omnibus; input-seq, input sequencing; nt, nucleotides; PR, protease; RIP, RNA immunoprecipitation; RIP-seq, RIP sequencing; RNH, RNase H; RPM, reads per million; RT, reverse transcriptase.

    Journal: PLoS Biology

    Article Title: Back-spliced RNA from retrotransposon binds to centromere and regulates centromeric chromatin loops in maize

    doi: 10.1371/journal.pbio.3000582

    Figure Lengend Snippet: Back-spliced CRM1 RNA in the maize centromere. (A) Procedure for anti-CENH3 RIP and subsequent high-throughput sequencing and cDNA library screening. (B) BLAST results of the back-spliced CRM1 reads from the anti-CENH3 RIP-seq, input-seq data, and the 354-nt RNA from the anti-CENH3 RIP cDNA library. The arrow shows the back-splicing site. (C) The location of the 607-bp combined sequence in CRM1, and the back-spliced form of the 354-nt RNA. The red line represents the 607-bp sequence. (D) Distribution of the 354-bp sequence on CRM1. The first track of each panel represents the centromeric region indicated by CENH3 enrichment, and the peak height represents the RPM value (0–1). The other 4 tracks represent the distributions of the 354-bp, 269-bp, 85-bp, and CRM1 sequences along a specific region of cen5. The lower panel shows a detailed version of the information displayed in the upper panel. The arrows inside the rectangular bars represent the directions of the sequences. (E) The public raw genome sequencing data (including Pacbio [65×] and Illumina [100×] reads) and 4 anti-CENH3 ChIP-seq datasets from B73 (including 1 generated in this study and 3 from public resources), together with 1 input-seq dataset, were mapped to the assumed 354-bp DNA. Only one read from anti-CENH3 ChIP-seq dataset was matched to the region containing the back-spliced junction site (purple line). All the other reads show no covering the back-spliced junction site. The data underlying this figure can be found in the GEO with accession numbers GSE124242, SRR3018834, SRR2000635, SRR2000640, SRR2000648, SRX1472849, and SRX1452310 and on Github ( https://github.com/sxx-ying/maize-centromere-circRNA ). CB, chromatin binding; CENH3, centromeric H3 variant; ChIP-seq, chromatin immunoprecipitation following high-throughput sequencing; Chr, chromosome; CRM, centromeric retrotransposon; Gag, gag protein; GEO, Gene Expression Omnibus; input-seq, input sequencing; nt, nucleotides; PR, protease; RIP, RNA immunoprecipitation; RIP-seq, RIP sequencing; RNH, RNase H; RPM, reads per million; RT, reverse transcriptase.

    Article Snippet: RNase H treatment To confirm that circular RNAs can form RNA:DNA hybrids, 4 μg of chromatin-binding RNAs was treated with 120 U of RNase H (Takara, Category Number 2151) at 37°C for 3 h. The RNA was then purified using a phenol-chloroform extraction, and Superscript III reverse transcriptase was used for reverse transcription.

    Techniques: Next-Generation Sequencing, cDNA Library Assay, Sequencing, Chromatin Immunoprecipitation, Generated, Binding Assay, Variant Assay, Expressing, Immunoprecipitation

    QKI5 binds to the pri-124-1 transcript in vitro and in vivo . (A) A schematic representation of co-transfection with wild-type (124_WT) or mutant (124_MUT) pri-124-1 constructs and either wild-type (QKI5) or mutant (QKI ΔKH and QKI5 V157E ) QKI5 constructs. The nucleotide positions of the QRE and stem loop are indicated in the cloned pri-124-1 sequence used in this study. (B) q-PCR shows the changes in the levels of pri-124-1 (red bar) and miR-124 (green bar) upon transfection of the indicated combinations into 293T cells as shown in A . (C) A schematic representation of the primers used in RNA-IP-PCR analysis. (D) RNA-IP-PCR assays for pri-124-1 performed on anti-QKI5 immunoprecipitates (QKI5) from K562 and HEL cell lysates. An unrelated IgG served as the negative control (IgG). RT: no reverse-transcribed PCR. QKI5 RNA-IP assays for the irrelevant pri-23a are shown in the right panel. (E) q-PCR analysis of pri-124-1 and pri-23a associated with QKI5 evaluated by RNA-IP assays in K562 cells. The RNA-IP-q-PCR results are shown as fold enrichment compared with input. (F) A schematic representation of the DNA oligonucleotides used in the RNase H protection assays. The antisense DNA oligonucleotides A-C span different regions of 124-QRE RNAs, whereas Ctr is an unrelated control oligonucleotide. (G , H) RNase H protection assays. RNase H cleavage results of reaction with either 124-QRE RNAs or QKI5-protected 124-QRE RNAs targeted by antisense DNA oligonucleotides A-C (lanes 2-4 and 7-9) or by an unrelated control oligonucleotide (Ctr, lanes 5 and 10; G ). Additional control reactions were performed in the absence of oligonucleotide (input, lanes 1 and 6). Arrow indicates a reduced cutting site (G) . The quantitative data were shown in H . The relative protection ratio was defined by the percentage of undigested 124-QRE in reactions with QKI5 protein to that without QKI5, and the relative digestion ratio was defined by the percentage of digested segments in reactions with QKI5 protein to that without QKI5. The data were quantified using ImageJ software. (I) In vitro association of QKI5 with the pri-124-1 QRE as identified by an RNA-EMSA assay in which 5′-biotin-labeled miR-124-QRE probes were incubated with different concentration of purified Flag-QKI5 (the left panel). A reported 5′-biotin-labeled p27-QRE probe was used as the positive control (the right panel). A mutant probe was used as the control, and the unlabeled miR-124-QRE probe was used in the competitive assays. (J) A schematic representation of the RNA pull-down assays using MS2-tagged pri-124-1 affinity purification. (K) Immunoblot of endogenous QKI5 in RNA pull-down assays from 293T cells transfected with either MS2-tagged wild-type pri-124-1 (124_WT-MS2) or MS2-tagged QRE mutant pri-124-1 (124_MUT-MS2) constructs. An unrelated protein GAPDH was used as the control. (L) The competitive association of non-MS2-tagged pri-124-1 with endogenous QKI5 in RNA pull-down assays performed on 293T cells co-transfected with 124_WT-MS2 and increased dosages of non-tagged 124_WT. Error bars reflect SEM from three biological replicates if not stated otherwise. Significance was determined by t -test with * P

    Journal: Cell Research

    Article Title: The RNA-binding protein QKI5 regulates primary miR-124-1 processing via a distal RNA motif during erythropoiesis

    doi: 10.1038/cr.2017.26

    Figure Lengend Snippet: QKI5 binds to the pri-124-1 transcript in vitro and in vivo . (A) A schematic representation of co-transfection with wild-type (124_WT) or mutant (124_MUT) pri-124-1 constructs and either wild-type (QKI5) or mutant (QKI ΔKH and QKI5 V157E ) QKI5 constructs. The nucleotide positions of the QRE and stem loop are indicated in the cloned pri-124-1 sequence used in this study. (B) q-PCR shows the changes in the levels of pri-124-1 (red bar) and miR-124 (green bar) upon transfection of the indicated combinations into 293T cells as shown in A . (C) A schematic representation of the primers used in RNA-IP-PCR analysis. (D) RNA-IP-PCR assays for pri-124-1 performed on anti-QKI5 immunoprecipitates (QKI5) from K562 and HEL cell lysates. An unrelated IgG served as the negative control (IgG). RT: no reverse-transcribed PCR. QKI5 RNA-IP assays for the irrelevant pri-23a are shown in the right panel. (E) q-PCR analysis of pri-124-1 and pri-23a associated with QKI5 evaluated by RNA-IP assays in K562 cells. The RNA-IP-q-PCR results are shown as fold enrichment compared with input. (F) A schematic representation of the DNA oligonucleotides used in the RNase H protection assays. The antisense DNA oligonucleotides A-C span different regions of 124-QRE RNAs, whereas Ctr is an unrelated control oligonucleotide. (G , H) RNase H protection assays. RNase H cleavage results of reaction with either 124-QRE RNAs or QKI5-protected 124-QRE RNAs targeted by antisense DNA oligonucleotides A-C (lanes 2-4 and 7-9) or by an unrelated control oligonucleotide (Ctr, lanes 5 and 10; G ). Additional control reactions were performed in the absence of oligonucleotide (input, lanes 1 and 6). Arrow indicates a reduced cutting site (G) . The quantitative data were shown in H . The relative protection ratio was defined by the percentage of undigested 124-QRE in reactions with QKI5 protein to that without QKI5, and the relative digestion ratio was defined by the percentage of digested segments in reactions with QKI5 protein to that without QKI5. The data were quantified using ImageJ software. (I) In vitro association of QKI5 with the pri-124-1 QRE as identified by an RNA-EMSA assay in which 5′-biotin-labeled miR-124-QRE probes were incubated with different concentration of purified Flag-QKI5 (the left panel). A reported 5′-biotin-labeled p27-QRE probe was used as the positive control (the right panel). A mutant probe was used as the control, and the unlabeled miR-124-QRE probe was used in the competitive assays. (J) A schematic representation of the RNA pull-down assays using MS2-tagged pri-124-1 affinity purification. (K) Immunoblot of endogenous QKI5 in RNA pull-down assays from 293T cells transfected with either MS2-tagged wild-type pri-124-1 (124_WT-MS2) or MS2-tagged QRE mutant pri-124-1 (124_MUT-MS2) constructs. An unrelated protein GAPDH was used as the control. (L) The competitive association of non-MS2-tagged pri-124-1 with endogenous QKI5 in RNA pull-down assays performed on 293T cells co-transfected with 124_WT-MS2 and increased dosages of non-tagged 124_WT. Error bars reflect SEM from three biological replicates if not stated otherwise. Significance was determined by t -test with * P

    Article Snippet: Briefly, in 25 μl reaction, 32 P-labeled 124-QRE transcripts of 1 × 105 cpm, 10 μg/ml DNA oligonucleotide, 12 mM HEPES, pH 8.0, 60 mM KCl, 3 mM MgCl2 , 1 mM DTT, 20 U of RNasin (Invitrogen) and 1 U of E. coli RNase H (TaKaRa, 2150A) were incubated with 10 ng/μl Flag-QKI5 protein purified from 293T cells by anti-Flag M2 magnetic beads (Sigma) or not.

    Techniques: In Vitro, In Vivo, Cotransfection, Mutagenesis, Construct, Clone Assay, Sequencing, Polymerase Chain Reaction, Transfection, Negative Control, Software, Labeling, Incubation, Concentration Assay, Purification, Positive Control, Affinity Purification

    RNase H activities in Leishmania . (A) Zymogram for the RNase H activities in the cell extracts from L. donovani . Cells and mitochondrial enriched fraction were lysed and boiled in SDS-containing denaturation solution for 3 min before loading. Proteins equivalent to 2 × 10 7 cells were loaded per lane (TCE, ME). The separating gel was made with the RNase H substrate 32 P-poly(rA)/poly(dT). These activities were solely dependent upon Mn 2+ and addition of Mg 2+ instead did not yield any such band. The sizes of the bands shown are approximate. The major Mn 2+ -dependent RNase H activity is enriched in the mitochondrial fraction. TCE: total cellular extract; ME: mitochondrial extract. (B) Expression and purification of N-terminal His 6 -tagged L. major LRNase HIIC precursor protein and the mature LRNase HIICΔMLS in E. coli . Recombinant cells were induced with 0.2 mM IPTG. The proteins were separated in a 10% SDS-PAGE and stained with Coomasie Blue R250. Optimal induction was observed in 3 h at 37 °C. We followed up to 21 h. Lane M: size marker. Lanes 1–4 are for LRNase HIIC and lanes 5–8 are for LRNase HIICΔMLS. Lanes 1 and 5, lysates from uninduced cells; lanes 2 and 6, lysates from IPTG induced cells; lanes 3, 4, 7 and 8, fractions eluted from washed columns with buffer E (100 mM NaH 2 PO 4 , 10 mM Tris.Cl pH 4.5, and 8 M urea). (C) Western blot analysis of LRNase HIIC in L. donovani promastigotes. Left panel (i), Coomassie Brilliant Blue stained protein gel showing L. donovani total cellular protein (Cell; 50 μg) and total mitochondrial protein (Mito; 50 μg). Middle panel (ii), Western blot analysis with anti-LRNase HIIC antibodies (1:1000 dilution). The LRNase HIIC antibody detected a 49-kDa protein in the mitochondrial extract. The faint band in the total cellular extract is not visible in the photograph of the chemiluminiscence autoradiogram. Right panel (iii), Western blot with anti-ISP monoclonal antibody, showing mitochondrial enrichment. (D) Pull-down of the 49-kDa radiolabeled LRNase HIIC protein by antibody. Immunoprecipitation was done with the reagents from the SeizeX Protein A immunoprecipitation kit (Pierce Biotechnology). The size of the bands is 49-kDa. Experiment 1 and Experiment 2 are duplicates for same experiment. (E) Zymogram for the activity gel assay of the proteins isolated from L. donovani mitochondrial extracts by immunocapture. Assay was done as described above. The sizes of the bands correspond to 49-kDa.

    Journal: Molecular and biochemical parasitology

    Article Title: A type II ribonuclease H from Leishmania mitochondria: An enzyme essential for the growth of the parasite

    doi: 10.1016/j.molbiopara.2005.05.009

    Figure Lengend Snippet: RNase H activities in Leishmania . (A) Zymogram for the RNase H activities in the cell extracts from L. donovani . Cells and mitochondrial enriched fraction were lysed and boiled in SDS-containing denaturation solution for 3 min before loading. Proteins equivalent to 2 × 10 7 cells were loaded per lane (TCE, ME). The separating gel was made with the RNase H substrate 32 P-poly(rA)/poly(dT). These activities were solely dependent upon Mn 2+ and addition of Mg 2+ instead did not yield any such band. The sizes of the bands shown are approximate. The major Mn 2+ -dependent RNase H activity is enriched in the mitochondrial fraction. TCE: total cellular extract; ME: mitochondrial extract. (B) Expression and purification of N-terminal His 6 -tagged L. major LRNase HIIC precursor protein and the mature LRNase HIICΔMLS in E. coli . Recombinant cells were induced with 0.2 mM IPTG. The proteins were separated in a 10% SDS-PAGE and stained with Coomasie Blue R250. Optimal induction was observed in 3 h at 37 °C. We followed up to 21 h. Lane M: size marker. Lanes 1–4 are for LRNase HIIC and lanes 5–8 are for LRNase HIICΔMLS. Lanes 1 and 5, lysates from uninduced cells; lanes 2 and 6, lysates from IPTG induced cells; lanes 3, 4, 7 and 8, fractions eluted from washed columns with buffer E (100 mM NaH 2 PO 4 , 10 mM Tris.Cl pH 4.5, and 8 M urea). (C) Western blot analysis of LRNase HIIC in L. donovani promastigotes. Left panel (i), Coomassie Brilliant Blue stained protein gel showing L. donovani total cellular protein (Cell; 50 μg) and total mitochondrial protein (Mito; 50 μg). Middle panel (ii), Western blot analysis with anti-LRNase HIIC antibodies (1:1000 dilution). The LRNase HIIC antibody detected a 49-kDa protein in the mitochondrial extract. The faint band in the total cellular extract is not visible in the photograph of the chemiluminiscence autoradiogram. Right panel (iii), Western blot with anti-ISP monoclonal antibody, showing mitochondrial enrichment. (D) Pull-down of the 49-kDa radiolabeled LRNase HIIC protein by antibody. Immunoprecipitation was done with the reagents from the SeizeX Protein A immunoprecipitation kit (Pierce Biotechnology). The size of the bands is 49-kDa. Experiment 1 and Experiment 2 are duplicates for same experiment. (E) Zymogram for the activity gel assay of the proteins isolated from L. donovani mitochondrial extracts by immunocapture. Assay was done as described above. The sizes of the bands correspond to 49-kDa.

    Article Snippet: Bovine pancreatic RNase A (Type 1A, Sigma Chemical Co.) and E. coli RNase H (Invitrogen) were used as negative and positive control, respectively, to verify the efficacy and specificity of the substrate.

    Techniques: Activity Assay, Expressing, Purification, Recombinant, SDS Page, Staining, Marker, Western Blot, Immunoprecipitation, Isolation

    Mitochondrial ribonuclease H (LRNase HIIC) is essential for the survival of  Leishmania . (A) Northern analysis of RNA isolated from  L. donovani  promastigotes treated for 72 h with the sense or the antisense oligonucleotide (25 μM). Top panel is the autoradiogram and the lower panel corresponds to the ethidium bromide stained gel showing rRNA bands. (B) Activity gel assay with the proteins (100 μg) from the mitochondrial fraction of  L. donovani  promastigotes treated for 72 h with sense or antisense oligonucleotide (25 μM). Top panel is the zymogram and lower panel is the corresponding Coommasie blue-stained gel. (C) Inhibition of the growth of  L. donovani  promastigotes by antisense gapmer oligonucleotides against LRNase HIIC. Results are mean ± S.E. ( n  = 6). Similar results were obtained with axenic amastigotes and  L. major  promastigotes (not shown). (D) Inhibition of the growth of  L. major  amastigotes inside cultured macrophages (J774A1) by antisense gapmer oligonucleotides against LRNase HIIC. Results are mean ± S.E. ( n  = 6). SSN, the sense oligo; INV, oligo with the inverted sequence; SCR, the oligo with the sequence scrambled, and ASN, the antisense oligo. The amastigotes were counted 5-days post-treatment. The counts of amastigotes in the control macrophages (no oligo treatment) were 587 ± 12 cells/100 macrophages.

    Journal: Molecular and biochemical parasitology

    Article Title: A type II ribonuclease H from Leishmania mitochondria: An enzyme essential for the growth of the parasite

    doi: 10.1016/j.molbiopara.2005.05.009

    Figure Lengend Snippet: Mitochondrial ribonuclease H (LRNase HIIC) is essential for the survival of Leishmania . (A) Northern analysis of RNA isolated from L. donovani promastigotes treated for 72 h with the sense or the antisense oligonucleotide (25 μM). Top panel is the autoradiogram and the lower panel corresponds to the ethidium bromide stained gel showing rRNA bands. (B) Activity gel assay with the proteins (100 μg) from the mitochondrial fraction of L. donovani promastigotes treated for 72 h with sense or antisense oligonucleotide (25 μM). Top panel is the zymogram and lower panel is the corresponding Coommasie blue-stained gel. (C) Inhibition of the growth of L. donovani promastigotes by antisense gapmer oligonucleotides against LRNase HIIC. Results are mean ± S.E. ( n = 6). Similar results were obtained with axenic amastigotes and L. major promastigotes (not shown). (D) Inhibition of the growth of L. major amastigotes inside cultured macrophages (J774A1) by antisense gapmer oligonucleotides against LRNase HIIC. Results are mean ± S.E. ( n = 6). SSN, the sense oligo; INV, oligo with the inverted sequence; SCR, the oligo with the sequence scrambled, and ASN, the antisense oligo. The amastigotes were counted 5-days post-treatment. The counts of amastigotes in the control macrophages (no oligo treatment) were 587 ± 12 cells/100 macrophages.

    Article Snippet: Bovine pancreatic RNase A (Type 1A, Sigma Chemical Co.) and E. coli RNase H (Invitrogen) were used as negative and positive control, respectively, to verify the efficacy and specificity of the substrate.

    Techniques: Northern Blot, Isolation, Staining, Activity Assay, Inhibition, Cell Culture, Sequencing

    Pan3 knockdown affects P-body formation and has differential effects on mRNA decay.  (A, left) Immunofluorescence microscopy results showing a significant loss of P-bodies in cells transfected with the Pan3-specific siRNA (Pan3 siRNA) but not with the control nonspecific (NS) siRNA. Rabbit anti-Dcp1a antibody was used to detect P-bodies. (A, bottom) A summary of the changes in P-body number and size after Pan3 knockdown (see Materials and methods). (A, right) Western blots showing that the Pan3 siRNA efficiently knocked down the Pan3 expression but had little effect on the expression of the three other P-body components (Dcp1a, Rck/p54, and Caf1) and the control (GAPDH). (B) Northern blots showing the effects of Pan 3 knockdown on deadenylation and decay of BBB+PTC (top), BBB+ARE (middle), or BBB (bottom) mRNA. NIH3T3 B2A2 cells were transiently cotransfected with a Tet promoter–regulated plasmid encoding a reporter mRNA as indicated and either the nonspecific siRNA or Pan3 siRNA. A plasmid encoding constitutively expressed α-globin–GAPDH mRNA was also cotransfected to provide an internal standard for transfection efficiency and sample handling (control). Times correspond to hours after tetracycline addition. Poly(A) −  RNA was prepared in vitro by treating an RNA sample from an early time point with oligo(dT) and RNase H.

    Journal: The Journal of Cell Biology

    Article Title: Deadenylation is prerequisite for P-body formation and mRNA decay in mammalian cells

    doi: 10.1083/jcb.200801196

    Figure Lengend Snippet: Pan3 knockdown affects P-body formation and has differential effects on mRNA decay. (A, left) Immunofluorescence microscopy results showing a significant loss of P-bodies in cells transfected with the Pan3-specific siRNA (Pan3 siRNA) but not with the control nonspecific (NS) siRNA. Rabbit anti-Dcp1a antibody was used to detect P-bodies. (A, bottom) A summary of the changes in P-body number and size after Pan3 knockdown (see Materials and methods). (A, right) Western blots showing that the Pan3 siRNA efficiently knocked down the Pan3 expression but had little effect on the expression of the three other P-body components (Dcp1a, Rck/p54, and Caf1) and the control (GAPDH). (B) Northern blots showing the effects of Pan 3 knockdown on deadenylation and decay of BBB+PTC (top), BBB+ARE (middle), or BBB (bottom) mRNA. NIH3T3 B2A2 cells were transiently cotransfected with a Tet promoter–regulated plasmid encoding a reporter mRNA as indicated and either the nonspecific siRNA or Pan3 siRNA. A plasmid encoding constitutively expressed α-globin–GAPDH mRNA was also cotransfected to provide an internal standard for transfection efficiency and sample handling (control). Times correspond to hours after tetracycline addition. Poly(A) − RNA was prepared in vitro by treating an RNA sample from an early time point with oligo(dT) and RNase H.

    Article Snippet: RNase H treatment of cytoplasmic mRNA after annealing to Oligo dT (Invitrogen) was used to generate poly(A)− RNA as described previously ( ).

    Techniques: Immunofluorescence, Microscopy, Transfection, Western Blot, Expressing, Northern Blot, Plasmid Preparation, In Vitro