rnase h Search Results


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  • 99
    New England Biolabs rnase h
    eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for <t>RNase</t> H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.
    Rnase H, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 4251 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 4251 article reviews
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    99
    Thermo Fisher rnase h
    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and <t>RNase</t> H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).
    Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9985 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript ii rnase h reverse transcriptase
    Analysis of tumor samples for expression of antigens from stable cell lines confirmed expression in the tumors. (a) Protein was extracted from primary tumor tissues, and the concentration was calculated. Tissues were homogenized by using a tissue tearer prior to processing for protein extraction. Portions (100 μg) of lysate were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Equal loading of samples was confirmed with Ponceau-S staining of the membrane in all cases. The EBNA1 and Myc-tagged Nm23-H1 or EBNA3C were analyzed as reported with the use of anti-EBV human serum and anti-Myc antibody, respectively. (b) Total RNA was isolated from tissues by using TRIZOL reagent. Tissues were homogenized by using a tissue tearer prior to processing for RNA isolation. RT was carried performed with SuperScript II <t>RNase</t> H reverse transcriptase. followed by PCR with specific primers to detect the desired transcript. The Nm23-H1-Myc transcript was amplified by using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′ designed to amplify the junction sequence between Nm23-H1 and Myc tag. The EBNA3C-Myc transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. The EBNA1 transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. Amplification products were resolved in 1.5% agarose gels.
    Superscript Ii Rnase H Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7865 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore rnase
    Characterization of the chromatin assembly reaction in yeast whole-cell extract by DNA supercoiling assays. Open circular (O), relaxed (R), and highly supercoiled (S) products are resolved by agarose gel electrophoresis. ( A ) Protein titration. Reactions were performed at 30°C for 30 min. ( B ) Time course of supercoiling with 50 μg of yeast whole-cell extract. The gradual disappearance of open circular/relaxed DNA species is accompanied by the progressive accumulation of highly supercoiled products. ( C ) <t>RNA</t> in the extract does not play a role in de novo assembly. S100 was treated with <t>RNase</t> linked to acrylic beads to digest the RNA ( Upper ). Untreated (lane 2) and treated (lane 3) extracts have similar capacities to assemble the template ( Lower ). Arrows mark the positions of the most prominent RNA species. The size (bp) of DNA markers (lane 1) is indicated on the left. ( D ) Cellular chromatin in the extract does not play a role in de novo assembly. S100 (lane 1) contains high molecular weight (HMW) DNA that can be removed by low-speed centrifugation (lane 2, DNA pellet) without significantly affecting assembly (lane 3). The template (relaxed pBluescript) was labeled with [ 32 P]dCTP by repair synthesis during assembly. ( E ) Analysis of template linking number by two-dimensional agarose gel electrophoresis. Up to 16 topoisomers can be resolved (numbered). The faintest spot (number one) was visible only upon prolonged exposure. The intense spot in the upper left corner is open circular template.
    Rnase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 13974 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii rnase h reverse transcriptase
    ) with modifications, using Invitrogen Superscript <t>III</t> RNase H- reverse transcriptase and 32 P-labeled primers for 40 min at 52°C. Lanes U to A, sequencing ladders generated by PCR with pRZ6-2 template and 32 P-labeled primers in the presence of ddATP, ddCTP, ddGTP, and ddUTP by utilizing the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH). All samples were run on an 8% polyacrylamide-8 M urea sequencing gel. The nucleotide sequence of the (+)-PSTVd is given on the left, and the arrows point to the bands corresponding to reverse transcription termination at A99.
    Superscript Iii Rnase H Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2237 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega rnase h
    Fig. 7.  Extent of RNA hybridized to switch DNA sequences in the presence and absence of DNA gyrase. The RNA species were radiolabeled internally with [α– 32 P]UTP during RNA–DNA hybrid formation. Transcription was performed with supercoiled pGD44 (lanes 1–3), pGD231 (lanes 4–6), pSG3-2 (lanes 7–9) or pGD100 (lanes 10–12). pGD44 contains 900 bp of Sμ, pGD231 contains 2.2 kb of Sγ3, pSG3-2 contains 129 bp of Sγ3 and pGD100 contains 832 bp of Sγ2b. Lanes 1, 4, 7 and 10 are plasmids transcribed in the absence of DNA gyrase; lanes 2, 5, 8 and 11 are plasmids transcribed in the presence of DNA gyrase; lanes 3, 6, 9 and 12 are plasmids transcribed in the presence of DNA gyrase and then treated with RNase H. M designates a 1 kb ladder.
    Rnase H, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 1335 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa ribonuclease h
    Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.
    Ribonuclease H, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 54 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript rnase h reverse transcriptase
    Primer extension (PE) analysis of the fimA (A) and pepO (B and C) transcripts. In lane PE, oligonucleotide was annealed to 10 μg of S. parasanguis FW213 RNA and extended using SuperScript <t>RNase</t> H − reverse transcriptase. The nucleotide sequences of pVT1198 and pVT1327 (lanes G, A, T, and C) were determined using the same oligonucleotide as a primer.
    Superscript Rnase H Reverse Transcriptase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1098 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen rnase h
    Structures of <t>RNase</t> H (D132N mutant) complexed with DNA/RNA duplexes (with the same sequences). ( a ; 1.80 Å resolution; this work). The cleavage site in the selenium-modified structure
    Rnase H, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 858 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Illumina Inc rnase h
    SOX2 interacts with the 3′-UTR of the S100A14 mRNA in vivo . (A) The diagram depicts the annealing positions of the targeting oligomer for <t>RNase</t> H digestion and the primers for reverse transcription and PCR detection of the ORF and 3′-UTR fragments. (B) FLAG-SOX2 was transiently expressed in BFTC905 cells for 48 h, followed by the CLIP assay. The matrix-bound mRNA was incubated at 37 °C with RNase H in the absence of the targeting oligomer for 30 min. The mock-treated matrix-bound mRNA was then purified and reverse-transcribed. The full-length, ORF, and 3′ fragments of the S100A14 mRNA were detected by RT-PCR. (C) After immunoprecipitation, the matrix-bound mRNA was subject to oligomer-dependent RNase H digestion at 37 °C for 30 min. The ORF and 3′-UTR fragments of the S100A14 mRNA in the supernatant and bound to the matrix were recovered, reverse transcribed, and detected by semi-quantitative PCR.
    Rnase H, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 918 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 93 stars, based on 918 article reviews
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    99
    Promega rnase h minus
    SOX2 interacts with the 3′-UTR of the S100A14 mRNA in vivo . (A) The diagram depicts the annealing positions of the targeting oligomer for <t>RNase</t> H digestion and the primers for reverse transcription and PCR detection of the ORF and 3′-UTR fragments. (B) FLAG-SOX2 was transiently expressed in BFTC905 cells for 48 h, followed by the CLIP assay. The matrix-bound mRNA was incubated at 37 °C with RNase H in the absence of the targeting oligomer for 30 min. The mock-treated matrix-bound mRNA was then purified and reverse-transcribed. The full-length, ORF, and 3′ fragments of the S100A14 mRNA were detected by RT-PCR. (C) After immunoprecipitation, the matrix-bound mRNA was subject to oligomer-dependent RNase H digestion at 37 °C for 30 min. The ORF and 3′-UTR fragments of the S100A14 mRNA in the supernatant and bound to the matrix were recovered, reverse transcribed, and detected by semi-quantitative PCR.
    Rnase H Minus, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 636 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex

    doi: 10.1093/nar/gky071

    Figure Lengend Snippet: eIF4A activity within the 48S complex. ( A ) RNase H-mapping of RNA–RNA interactions between SV-DLP U1 and 18S rRNA. The analysis was carried out in the absence or presence of 1 μM hippuristanol, with identification of the resulting RNA fragments indicated. For clarity, a schematic diagram of the ES6S and h16–18 regions of rabbit 18S rRNA with the primers used for RNase H digestion is shown. The use of oligos 4 and 9 limited the region of 18S rRNA (509–830) where the crosslinkings concentrated. Bands corresponding to crosslinking of SV DLP U1 mRNA with the ES6S region (680–1863) and h16-h18 helices (1–662) were quantified by densitometry and expressed as a ratio. Data are the mean ± SEM from four independent experiments. ( B ). Reactivity to SHAPE reagent (NMIA) is higher for unpaired nucleotides (red) and low for those involved in pairings (black). Stops corresponding to toeprints are marked with arrowheads. Quantification of toeprint ratios (17–19/23–25) in absence or presence of hippuristanol is shown from three independent experiments; data are the mean ± SEM.

    Article Snippet: Briefly, samples were annealed at 65°C for 5′ with 10 pmol of oligonucleotides covering the indicated regions of 18S rRNA and digested with 5 U of RNase H (NEB) for 15 min at 37°C.

    Techniques: Activity Assay

    H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Journal: Nature Communications

    Article Title: Chromatin conformation regulates the coordination between DNA replication and transcription

    doi: 10.1038/s41467-018-03539-8

    Figure Lengend Snippet: H1-TKO cells display impaired transcription dynamics. a Diagram of the experimental design to measure transcription elongation rates by transient inhibition of initiating RNAPIIs with DRB. Three hours after DRB incubation, the drug was washed-off to resume transcription elongation and total RNA was extracted from identical number of cells at the indicated time-points (open triangles). Global nascent transcription was evaluated by 1h-EU labelling at the indicated time points (red lines). b Time course transcription elongation measurements at the Med13l and Inpp5a genes in WT mES (upper panels) and H1-TKO mES cells (lower panels). Levels of pre-mRNA at the indicated times were determined by RT-qPCR at the positions marked in the gene maps above the graphs. Pre-mRNA values were normalized to the values of the non-DRB-treated sample. Results are shown as means ± s.d. from two independent experiments ( n = 2). c Representative images of EU staining (top) and distribution of EU nuclear intensity during DRB treatment and upon drug-release at the time points shown in the experimental scheme in a . d Representative images of S9.6 immunostaining ±RNAseA or +-RNAseH incubation (top) and distribution of S9.6 nuclear intensity (bottom) in WT and H1-TKO cells. Scale bar, 10 μm. Nuclear segmentation (white lines) was based on DAPI staining. Median values are indicated ( n c-d for S-phase distribution of S9.6 and γH2AX nuclear intensities. Differences between distributions were assessed with the Mann–Whitney rank sum test. **** P

    Article Snippet: Samples were treated either with 1 mg/ml RNAseA (Sigma), 30 U RNAseH (New England Biolabs), or both RNAseA and RNAseH, for 18 h at 37 °C before immunoprecipitation.

    Techniques: Inhibition, Incubation, Quantitative RT-PCR, Staining, Immunostaining, MANN-WHITNEY

    Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous RNase H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.

    Journal: Molecules

    Article Title: One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

    doi: 10.3390/molecules25051142

    Figure Lengend Snippet: Proof of principle on construct 1. ( A ): Denaturing polyacrylamide gel showing the cleavage of a tandem transcript and comparison of transcription from a single-repeat ssDNA template. Lane 1: Tandem transcription of the construct from the linearized plasmid. Lane 2: Simultaneous RNase H cleavage during tandem transcription leading to the main product of 20 nt. The 12* label refers to the chimeric cleavage guide consisting of 4 DNA nucleotides and 8 2′-OMe nucleotides. Lane 3: IVT from a single-repeat template encoding construct 1 showing much higher levels of longer and shorter products. For comparison, the same amount of reaction has been loaded in all lanes. ( B ): Ion-exchange HPLC chromatogram for the 1 mL IVT from A (lane 3, construct 1*) and denaturing PAGE sampling the eluted peaks. The target peak overlaps with side products and is of low intensity. ( C ): Ion-exchange HPLC chromatogram for a 1 mL reaction of cleaved tandem transcription from A (lane 2, construct 1). The main signal is pure judging from denaturing PAGE, and of significantly larger intensity than from single-repeat template.

    Article Snippet: E.coli RNase H shows unimpaired enzymatic activity within buffer systems of the T7 buffer and the commercial RNase H reaction buffer (NEB).

    Techniques: Construct, Plasmid Preparation, High Performance Liquid Chromatography, Polyacrylamide Gel Electrophoresis, Sampling

    Schematic representation of the reported protocol. ( A ): (left) Tandem transcription from a linearized plasmid template with T7 RNA polymerase (T7RNAP) and (right) successive cleavage of the transcript to target length RNA by RNase H, directed by a chimeric DNA guide. ( B ): Detailed schematic of the tandem template, which starts with the viral T7RNAP promoter, an initiation sequence. The target sequence (dark blue, example here is 20 nt length) is repeated n number of times. The repeats flanked by a 5′- and 3′-spacer sequences consisting of the last eight and first four nucleotides respectively to allow for removal of the initiation and restriction sequences from the first and last repeat unit. ( C ): Hybridization of the tandem transcript (red) and the chimeric cleavage guides (green). RNase H cleaves the RNA opposite the DNA 5′-end. The 2′-OMe RNA flanks increase specificity by enhancing the binding affinity of cleavage guide to the target RNA.

    Journal: Molecules

    Article Title: One-Pot Production of RNA in High Yield and Purity Through Cleaving Tandem Transcripts

    doi: 10.3390/molecules25051142

    Figure Lengend Snippet: Schematic representation of the reported protocol. ( A ): (left) Tandem transcription from a linearized plasmid template with T7 RNA polymerase (T7RNAP) and (right) successive cleavage of the transcript to target length RNA by RNase H, directed by a chimeric DNA guide. ( B ): Detailed schematic of the tandem template, which starts with the viral T7RNAP promoter, an initiation sequence. The target sequence (dark blue, example here is 20 nt length) is repeated n number of times. The repeats flanked by a 5′- and 3′-spacer sequences consisting of the last eight and first four nucleotides respectively to allow for removal of the initiation and restriction sequences from the first and last repeat unit. ( C ): Hybridization of the tandem transcript (red) and the chimeric cleavage guides (green). RNase H cleaves the RNA opposite the DNA 5′-end. The 2′-OMe RNA flanks increase specificity by enhancing the binding affinity of cleavage guide to the target RNA.

    Article Snippet: E.coli RNase H shows unimpaired enzymatic activity within buffer systems of the T7 buffer and the commercial RNase H reaction buffer (NEB).

    Techniques: Plasmid Preparation, Sequencing, Hybridization, Binding Assay

    Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and RNase H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).

    Journal: Nucleic Acids Research

    Article Title: Differential usage of transcriptional start sites and polyadenylation sites in FMR1 premutation alleles †

    doi: 10.1093/nar/gkr100

    Figure Lengend Snippet: Polyadenylation state of Fmr1 mRNA variants in the CGG KI mouse brain. ( A ) Left panel: schematic view of the polyadenylation assay (PAT). According to di Penta et al . ( 42 ), the poly(A) tails are tagged by incubating the RNA with a (T) 12 -tag oligonucleotide, blocked at the 3′-end, in the presence of dNTPs and Klenow enzyme to fill in the complementary tag sequence. The RNA is then denatured and annealed to a DNA primer, identical to the tag, to start a reverse transcription (RT). The cDNA is then amplified using a gene-specific forward primer and the reverse tag oligo. Right panel: cartoon of a polyadenylation profile obtained with a PAT assay. The PCR of a polyadenylated mRNA gives rise to a smear while the same mRNA deadenylated with oligodT and RNase H prior to poly(A) tagging is used as a negative control and gives a sharp band. ( B ) Upper panel: β-actin mRNA in WT (lane 1) and CGG KI (lane 3). Deadenylated RNA is shown as negative control (lane 2) and the deadenylated form is indicated by black arrows. Lower panel: dispersion graph representing the distribution of the β-actin polyadenylated transcripts in WT (black line) and CGG KI (grey line). The signal intensity along the lane has been plotted against the poly(A) tail length, estimated from the molecular markers loaded on the same gel. ( C ) PAT for all three poly(A) Fmr1 mRNA variants. Because of close proximity, the transcripts containing sites V and VI cannot be discriminated and therefore they are not taken into exam (upper panel). Black arrows points to the deadenylated form. The polyadenylation of transcripts using site IV from WT (lane 1) and CGG KI (lane 3) has been independently acquired and highlighted in the box below. Deadenylated RNA treated as mentioned above, is shown as negative control (lane 2). Right panel: dispersion graph for Fmr1 variants using site IV in WT (black line) and CGG KI (grey line). ( D ) Left panel: PAT for Fmr1 variants using site VI in WT (lane 1) and CGG KI (lane 3) brain. Deadenylated RNA as above is used as negative control (lane 2). Right panel: Dispersion graph representing the distribution of the polyadenylated transcripts using site VI in WT (black line) and CGG KI (grey line).

    Article Snippet: As a negative control, RNAs were deadenylated by incubating with Oligo(dT)12–18 (Invitrogen) and RNase H (Fermentas) at 37°C for 90 min, purified by phenol-chloroform and subjected to the same tagging reaction.

    Techniques: Sequencing, Amplification, Polymerase Chain Reaction, Negative Control

    RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

    Journal: PLoS ONE

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

    doi: 10.1371/journal.pone.0033115

    Figure Lengend Snippet: RNase treatment does not modify MV size, but reduces RNA content of MVs. A) Representative MV size analyses by direct measurement with NTA, showing no difference among MVs treated or not with RNase. B) Representative Bioanalyzer profile, showing the size distribution of total RNA extracted from MVs treated or not with RNAse. The first peak (left side of each panel) represents an internal standard. The two peaks in Sample 1 (black arrows) represent 18 S (left) and 28 S (right) ribosomal RNA, only partially detectable in MVs. The red arrows showed the reduction of 18 and 28 S fragment inside RNAse-treated MVs. C) Histogram showing the expression level of SUMO-1 , POLR2 and Act B transcripts in MVs treated or not with RNase, express as 2 -δCt , as described in material and methods.

    Article Snippet: Total RNA was isolated from MVs, treated or not with RNase, using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s protocol.

    Techniques: Expressing, Activated Clotting Time Assay

    MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.

    Journal: PLoS ONE

    Article Title: Microvesicles Derived from Mesenchymal Stem Cells Enhance Survival in a Lethal Model of Acute Kidney Injury

    doi: 10.1371/journal.pone.0033115

    Figure Lengend Snippet: MV infusion protects SCID mice with cisplatin-induced AKI from tubular injury. Representative micrographs of renal histology of healthy SCID mice and of SCID mice treated with cisplatin and injected with vehicle alone or with MV pre-treated with RNase or with different regiments of MVs (single or multiple injections) and sacrificed at different time points (day 4, 14 and 21). Original Magnification: ×200. The typical aspect of intra-tubular casts, tubular necrosis and tubular atrophy are respectively shown by asterisks, arrows and head arrows.

    Article Snippet: Total RNA was isolated from MVs, treated or not with RNase, using the mirVana RNA isolation kit (Ambion) according to the manufacturer’s protocol.

    Techniques: Mouse Assay, Injection

    Analysis of tumor samples for expression of antigens from stable cell lines confirmed expression in the tumors. (a) Protein was extracted from primary tumor tissues, and the concentration was calculated. Tissues were homogenized by using a tissue tearer prior to processing for protein extraction. Portions (100 μg) of lysate were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Equal loading of samples was confirmed with Ponceau-S staining of the membrane in all cases. The EBNA1 and Myc-tagged Nm23-H1 or EBNA3C were analyzed as reported with the use of anti-EBV human serum and anti-Myc antibody, respectively. (b) Total RNA was isolated from tissues by using TRIZOL reagent. Tissues were homogenized by using a tissue tearer prior to processing for RNA isolation. RT was carried performed with SuperScript II RNase H reverse transcriptase. followed by PCR with specific primers to detect the desired transcript. The Nm23-H1-Myc transcript was amplified by using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′ designed to amplify the junction sequence between Nm23-H1 and Myc tag. The EBNA3C-Myc transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. The EBNA1 transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. Amplification products were resolved in 1.5% agarose gels.

    Journal: Journal of Virology

    Article Title: Epstein-Barr Virus Latent Nuclear Antigens Can Induce Metastasis in a Nude Mouse Model ▿

    doi: 10.1128/JVI.00886-07

    Figure Lengend Snippet: Analysis of tumor samples for expression of antigens from stable cell lines confirmed expression in the tumors. (a) Protein was extracted from primary tumor tissues, and the concentration was calculated. Tissues were homogenized by using a tissue tearer prior to processing for protein extraction. Portions (100 μg) of lysate were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane. Equal loading of samples was confirmed with Ponceau-S staining of the membrane in all cases. The EBNA1 and Myc-tagged Nm23-H1 or EBNA3C were analyzed as reported with the use of anti-EBV human serum and anti-Myc antibody, respectively. (b) Total RNA was isolated from tissues by using TRIZOL reagent. Tissues were homogenized by using a tissue tearer prior to processing for RNA isolation. RT was carried performed with SuperScript II RNase H reverse transcriptase. followed by PCR with specific primers to detect the desired transcript. The Nm23-H1-Myc transcript was amplified by using the forward primer 5′-GATTACACGAGCTGTGCTCA-3′ and the reverse primer 5′-TTCGCTAGCCAAGTCTTCTT-3′ designed to amplify the junction sequence between Nm23-H1 and Myc tag. The EBNA3C-Myc transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. The EBNA1 transcript was amplified by using the forward primer 5′-CGGGATCCGGAAGGAACCATGGCCA-3′ and the reverse primer 5′-GAATTCTCCTGTCATTTCATAGATCCA-3′. Amplification products were resolved in 1.5% agarose gels.

    Article Snippet: RT was carried out with SuperScript II RNase H-reverse transcriptase (Life Technologies, Gaithersburg, MD).

    Techniques: Expressing, Stable Transfection, Concentration Assay, Protein Extraction, Polyacrylamide Gel Electrophoresis, Staining, Isolation, Polymerase Chain Reaction, Amplification, Sequencing

    Characterization of the chromatin assembly reaction in yeast whole-cell extract by DNA supercoiling assays. Open circular (O), relaxed (R), and highly supercoiled (S) products are resolved by agarose gel electrophoresis. ( A ) Protein titration. Reactions were performed at 30°C for 30 min. ( B ) Time course of supercoiling with 50 μg of yeast whole-cell extract. The gradual disappearance of open circular/relaxed DNA species is accompanied by the progressive accumulation of highly supercoiled products. ( C ) RNA in the extract does not play a role in de novo assembly. S100 was treated with RNase linked to acrylic beads to digest the RNA ( Upper ). Untreated (lane 2) and treated (lane 3) extracts have similar capacities to assemble the template ( Lower ). Arrows mark the positions of the most prominent RNA species. The size (bp) of DNA markers (lane 1) is indicated on the left. ( D ) Cellular chromatin in the extract does not play a role in de novo assembly. S100 (lane 1) contains high molecular weight (HMW) DNA that can be removed by low-speed centrifugation (lane 2, DNA pellet) without significantly affecting assembly (lane 3). The template (relaxed pBluescript) was labeled with [ 32 P]dCTP by repair synthesis during assembly. ( E ) Analysis of template linking number by two-dimensional agarose gel electrophoresis. Up to 16 topoisomers can be resolved (numbered). The faintest spot (number one) was visible only upon prolonged exposure. The intense spot in the upper left corner is open circular template.

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

    Article Title: Chromatin assembly in a yeast whole-cell extract

    doi:

    Figure Lengend Snippet: Characterization of the chromatin assembly reaction in yeast whole-cell extract by DNA supercoiling assays. Open circular (O), relaxed (R), and highly supercoiled (S) products are resolved by agarose gel electrophoresis. ( A ) Protein titration. Reactions were performed at 30°C for 30 min. ( B ) Time course of supercoiling with 50 μg of yeast whole-cell extract. The gradual disappearance of open circular/relaxed DNA species is accompanied by the progressive accumulation of highly supercoiled products. ( C ) RNA in the extract does not play a role in de novo assembly. S100 was treated with RNase linked to acrylic beads to digest the RNA ( Upper ). Untreated (lane 2) and treated (lane 3) extracts have similar capacities to assemble the template ( Lower ). Arrows mark the positions of the most prominent RNA species. The size (bp) of DNA markers (lane 1) is indicated on the left. ( D ) Cellular chromatin in the extract does not play a role in de novo assembly. S100 (lane 1) contains high molecular weight (HMW) DNA that can be removed by low-speed centrifugation (lane 2, DNA pellet) without significantly affecting assembly (lane 3). The template (relaxed pBluescript) was labeled with [ 32 P]dCTP by repair synthesis during assembly. ( E ) Analysis of template linking number by two-dimensional agarose gel electrophoresis. Up to 16 topoisomers can be resolved (numbered). The faintest spot (number one) was visible only upon prolonged exposure. The intense spot in the upper left corner is open circular template.

    Article Snippet: To digest RNA, 50 μg of extract was treated with 0.5 unit of RNase cross-linked to acrylic beads (Sigma R-7005, resuspended at 0.25 units/μl of YDBI) for 30 min at 37°C ( ).

    Techniques: Agarose Gel Electrophoresis, Titration, Molecular Weight, Centrifugation, Labeling

    Fig. 3. CBTF 122 and CBTF 98 bind RNA in vitro and are associated with non-translating mRNAs in cleavage stage embryos. ( A ) Northwestern blot analysis of total soluble proteins from two stage VI oocytes (VI) or two embryos at the stages shown. Proteins were separated by SDS–PAGE and transferred to nitrocellulose. The blot was probed first with 32 P-labelled L1 5′-UTR (northwestern) and subsequently probed with affinity-purified anti-CBTF 122/98 antibody (western). ( B ) Cytoplasmic proteins from cleavage-stage embryos were incubated in the absence or presence of RNase A and T1 as indicated and then sedimented through a 20–60% nycodenz gradient. Protein precipitated from the indicated fractions was analysed by western blotting using affinity-purified anti-CBTF 122/98 antibody or anti-mRNP3+4 antibody. ( C ) Fractions 12 and 13 of the (–)RNase gradient were mixed and aliquots were incubated in the absence or presence of RNase A and T1. The RNase-treated or untreated aliquots were incubated with anti-mRNP3+4 antiserum (α-mRNP3+4) and protein A beads as shown. Western blots of the supernatant (S) and pellet (P) fractions were probed with affinity-purified anti-p122/p98 (upper panel) or anti-mRNP3+4 (lower panel) antibodies.

    Journal: The EMBO Journal

    Article Title: RNA-dependent cytoplasmic anchoring of a transcription factor subunit during Xenopus development

    doi: 10.1093/emboj/19.14.3683

    Figure Lengend Snippet: Fig. 3. CBTF 122 and CBTF 98 bind RNA in vitro and are associated with non-translating mRNAs in cleavage stage embryos. ( A ) Northwestern blot analysis of total soluble proteins from two stage VI oocytes (VI) or two embryos at the stages shown. Proteins were separated by SDS–PAGE and transferred to nitrocellulose. The blot was probed first with 32 P-labelled L1 5′-UTR (northwestern) and subsequently probed with affinity-purified anti-CBTF 122/98 antibody (western). ( B ) Cytoplasmic proteins from cleavage-stage embryos were incubated in the absence or presence of RNase A and T1 as indicated and then sedimented through a 20–60% nycodenz gradient. Protein precipitated from the indicated fractions was analysed by western blotting using affinity-purified anti-CBTF 122/98 antibody or anti-mRNP3+4 antibody. ( C ) Fractions 12 and 13 of the (–)RNase gradient were mixed and aliquots were incubated in the absence or presence of RNase A and T1. The RNase-treated or untreated aliquots were incubated with anti-mRNP3+4 antiserum (α-mRNP3+4) and protein A beads as shown. Western blots of the supernatant (S) and pellet (P) fractions were probed with affinity-purified anti-p122/p98 (upper panel) or anti-mRNP3+4 (lower panel) antibodies.

    Article Snippet: For RNase treatment, nuclear and embryonic extracts, and nycodenz gradient fractions were incubated in the presence of 100 µg/ml RNase A (Sigma) and 1500 U/ml RNase T1 (Amersham) at 37°C for 30 min either before centrifugation or before immunoprecipitation.

    Techniques: In Vitro, SDS Page, Affinity Purification, Western Blot, Incubation

    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:

    ) with modifications, using Invitrogen Superscript III RNase H- reverse transcriptase and 32 P-labeled primers for 40 min at 52°C. Lanes U to A, sequencing ladders generated by PCR with pRZ6-2 template and 32 P-labeled primers in the presence of ddATP, ddCTP, ddGTP, and ddUTP by utilizing the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH). All samples were run on an 8% polyacrylamide-8 M urea sequencing gel. The nucleotide sequence of the (+)-PSTVd is given on the left, and the arrows point to the bands corresponding to reverse transcription termination at A99.

    Journal: Journal of Virology

    Article Title: Evidence for the Existence of the Loop E Motif of Potato Spindle Tuber Viroid In Vivo ▿

    doi: 10.1128/JVI.01781-06

    Figure Lengend Snippet: ) with modifications, using Invitrogen Superscript III RNase H- reverse transcriptase and 32 P-labeled primers for 40 min at 52°C. Lanes U to A, sequencing ladders generated by PCR with pRZ6-2 template and 32 P-labeled primers in the presence of ddATP, ddCTP, ddGTP, and ddUTP by utilizing the Thermo Sequenase cycle sequencing kit (USB, Cleveland, OH). All samples were run on an 8% polyacrylamide-8 M urea sequencing gel. The nucleotide sequence of the (+)-PSTVd is given on the left, and the arrows point to the bands corresponding to reverse transcription termination at A99.

    Article Snippet: The reverse transcription was carried out following the protocols described by Baumstark et al. ( ) with modifications, using primer R1 (5′-AAACCCTGTTTCGGCGGGAATTAC-3′, complementary to position 154-179 on the PSTVd genome) (Fig. ) and the SuperScript III RNase H- reverse transcriptase (Invitrogen, Carlsbad, CA) at 52°C for 40 min. As shown in Fig. , reverse transcription terminated predominantly at A99 when the cross-linked PSTVd RNA was used as the template (lane cI), compared to the situation when the non-cross-linked circular PSTVd RNA was used as the template (lane c).

    Techniques: Labeling, Sequencing, Generated, Polymerase Chain Reaction

    Fig. 7.  Extent of RNA hybridized to switch DNA sequences in the presence and absence of DNA gyrase. The RNA species were radiolabeled internally with [α– 32 P]UTP during RNA–DNA hybrid formation. Transcription was performed with supercoiled pGD44 (lanes 1–3), pGD231 (lanes 4–6), pSG3-2 (lanes 7–9) or pGD100 (lanes 10–12). pGD44 contains 900 bp of Sμ, pGD231 contains 2.2 kb of Sγ3, pSG3-2 contains 129 bp of Sγ3 and pGD100 contains 832 bp of Sγ2b. Lanes 1, 4, 7 and 10 are plasmids transcribed in the absence of DNA gyrase; lanes 2, 5, 8 and 11 are plasmids transcribed in the presence of DNA gyrase; lanes 3, 6, 9 and 12 are plasmids transcribed in the presence of DNA gyrase and then treated with RNase H. M designates a 1 kb ladder.

    Journal: The EMBO Journal

    Article Title: Transcription-dependent R-loop formation at mammalian class switch sequences

    doi: 10.1093/emboj/19.5.1055

    Figure Lengend Snippet: Fig. 7. Extent of RNA hybridized to switch DNA sequences in the presence and absence of DNA gyrase. The RNA species were radiolabeled internally with [α– 32 P]UTP during RNA–DNA hybrid formation. Transcription was performed with supercoiled pGD44 (lanes 1–3), pGD231 (lanes 4–6), pSG3-2 (lanes 7–9) or pGD100 (lanes 10–12). pGD44 contains 900 bp of Sμ, pGD231 contains 2.2 kb of Sγ3, pSG3-2 contains 129 bp of Sγ3 and pGD100 contains 832 bp of Sγ2b. Lanes 1, 4, 7 and 10 are plasmids transcribed in the absence of DNA gyrase; lanes 2, 5, 8 and 11 are plasmids transcribed in the presence of DNA gyrase; lanes 3, 6, 9 and 12 are plasmids transcribed in the presence of DNA gyrase and then treated with RNase H. M designates a 1 kb ladder.

    Article Snippet: T3 and T7 RNA polymerases, and RNase H were purchased from Promega (Madison, WI).

    Techniques:

    Fig. 1.  Altered DNA mobility upon  in vitro  transcription of murine class switch sequences Sμ, Sγ3 and Sγ2b. ( A ) Diagram of switch sequences on plasmids showing the direction of physiological transcription. The bent arrows indicate the direction of transcription by either T3 or T7 RNA polymerase. ( B ) Supercoiled plasmid DNA containing either a 900 bp fragment of Sμ (pGD44), a 2.2 kb fragment of Sγ3 (pGD231), a 267 bp fragment of Sγ3 (pSG3-5), a 129 bp fragment of Sγ3 (pSG3-2), an 832 bp fragment of Sγ2b (pGD100) or a 564 bp  Hin dIII fragment from λ phage (pTWEL5) was transcribed, treated with RNase A, run out on a 1% agarose gel and post-stained with ethidium bromide as described in Materials and methods. Lanes 1, 4, 7, 10, 13 and 16 are non-transcribed plasmids; lanes 2, 5, 8, 11, 14 and 17 are plasmids transcribed with T7 RNA polymerase; lanes 3, 6, 9, 12, 15 and 18 are plasmids transcribed with T7 RNA polymerase and treated with RNase H. A 1 kb ladder (Gibco-BRL) was used as a molecular weight marker (M). The positions of supercoiled (SC) and nicked circular (NC) forms of the plasmids are indicated. ( C ) Radioactive image of the gel shown in (B).

    Journal: The EMBO Journal

    Article Title: Transcription-dependent R-loop formation at mammalian class switch sequences

    doi: 10.1093/emboj/19.5.1055

    Figure Lengend Snippet: Fig. 1. Altered DNA mobility upon in vitro transcription of murine class switch sequences Sμ, Sγ3 and Sγ2b. ( A ) Diagram of switch sequences on plasmids showing the direction of physiological transcription. The bent arrows indicate the direction of transcription by either T3 or T7 RNA polymerase. ( B ) Supercoiled plasmid DNA containing either a 900 bp fragment of Sμ (pGD44), a 2.2 kb fragment of Sγ3 (pGD231), a 267 bp fragment of Sγ3 (pSG3-5), a 129 bp fragment of Sγ3 (pSG3-2), an 832 bp fragment of Sγ2b (pGD100) or a 564 bp Hin dIII fragment from λ phage (pTWEL5) was transcribed, treated with RNase A, run out on a 1% agarose gel and post-stained with ethidium bromide as described in Materials and methods. Lanes 1, 4, 7, 10, 13 and 16 are non-transcribed plasmids; lanes 2, 5, 8, 11, 14 and 17 are plasmids transcribed with T7 RNA polymerase; lanes 3, 6, 9, 12, 15 and 18 are plasmids transcribed with T7 RNA polymerase and treated with RNase H. A 1 kb ladder (Gibco-BRL) was used as a molecular weight marker (M). The positions of supercoiled (SC) and nicked circular (NC) forms of the plasmids are indicated. ( C ) Radioactive image of the gel shown in (B).

    Article Snippet: T3 and T7 RNA polymerases, and RNase H were purchased from Promega (Madison, WI).

    Techniques: In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker

    Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.

    Journal: Sensors (Basel, Switzerland)

    Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition

    doi: 10.3390/s18082441

    Figure Lengend Snippet: Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.

    Article Snippet: Ribonuclease H (RNase H) was obtained from Takara Biotechnology Co., Ltd. (DaLian, China).

    Techniques: Fluorescence

    Primer extension (PE) analysis of the fimA (A) and pepO (B and C) transcripts. In lane PE, oligonucleotide was annealed to 10 μg of S. parasanguis FW213 RNA and extended using SuperScript RNase H − reverse transcriptase. The nucleotide sequences of pVT1198 and pVT1327 (lanes G, A, T, and C) were determined using the same oligonucleotide as a primer.

    Journal: Infection and Immunity

    Article Title: The Divergently Transcribed Streptococcus parasanguis Virulence-Associated fimA Operon Encoding an Mn2+-Responsive Metal Transporter and pepO Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated

    doi: 10.1128/IAI.70.10.5706-5714.2002

    Figure Lengend Snippet: Primer extension (PE) analysis of the fimA (A) and pepO (B and C) transcripts. In lane PE, oligonucleotide was annealed to 10 μg of S. parasanguis FW213 RNA and extended using SuperScript RNase H − reverse transcriptase. The nucleotide sequences of pVT1198 and pVT1327 (lanes G, A, T, and C) were determined using the same oligonucleotide as a primer.

    Article Snippet: The labeled oligonucleotide was hybridized with 10 μg of S. parasanguis total RNA, and extension was performed using SuperScript RNase H− reverse transcriptase (Gibco BRL) for 1 h at 42°C.

    Techniques:

    Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Sequence preferences of Escherichia coli, Homo sapiens and HIV-1 RNase H ( A ) The heatmaps display the changes in nucleotide composition at different positions for the R7 construct (left) and the R4b construct (right) after cleavage with the three different RNase H enzymes. The intensity of the red and blue color indicates the k rel of having given nucleotide at a given position fixed relative to the average hydrolysis rate of the randomized pool. The barplots below the heatmaps show the overall information content at each position and the sequence logos are based on the 1% most downregulated pentamers. Note that only the randomized parts of the probed duplexes is displayed. ( B ) Cleavage of sequences predicted to be preferred (‘P’), avoided (‘A’) and neutral (‘N’) with respect to cleavage with human RNase H1 compared to the cleavage of a reference substrate. With respect to the reference substrate, the k rel of the preferred substrate is 3.7, of the avoided is 0.26 and of the neutral it is 1.4. ( C ) The design of the dumbbell substrate mimics. The gray box indicates the region having either the preferred (‘P’) or avoided (‘A’) sequence. ( D ) The cleavage of a reference substrate in the presence of increasing concentrations of a preferred or avoided dumbbell substrate mimic.

    Article Snippet: Nevertheless, only one of the modified nucleotides directly interacts with the RNase H1 enzyme ( ) ( ) and reassuringly, for the human and E. coli RNase H, we obtained similar results with all three constructs having different modification patterns (Figure and ), suggesting that the different modifications in the substrates did not distort the results.

    Techniques: Sequencing, Construct

    Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Functional significance of predicted HIV-1 RNase H cleavage sites. ( A ) Predicted RNase H cleavage efficiency of the HIV-1 genome, shown as log 2 (fold change) (log 2 FC). ( B ) Schematic of the HIV reverse transcription. White scissors at the black circle indicate specific areas zoomed-in in subsequent panels. ( C ) Comparison of distances (in nucleotides) between well-cleaved sites in the HIV-1 genome and in the randomized HIV-1 genomes. The red rhombi shows the observed count of distances between positions predicted to be efficiently cleaved in HIV-1 genome that fall into the indicated distance intervals. The violin plots show the density of the distributions that resulted from the same analysis, but repeated 10 000× on HIV-1 genome sequences that were randomized with preserving the local dinucleotide content; Predicted cleavage efficiency of ( D ) the sequence surrounding the 3′PPT, ( E ) of the terminal 18 nt of the tRNA-Lys3 primer and ( F ) it is reverse complement (primer binding site). ( G ) Predicted cleavage efficiency of the best-cleaved site in the terminal 18 nt of the different human tRNAs (plus CCA) and of the corresponding reverse complement. The tRNA-Lys3 is indicated in red.

    Article Snippet: Nevertheless, only one of the modified nucleotides directly interacts with the RNase H1 enzyme ( ) ( ) and reassuringly, for the human and E. coli RNase H, we obtained similar results with all three constructs having different modification patterns (Figure and ), suggesting that the different modifications in the substrates did not distort the results.

    Techniques: Functional Assay, Preserving, Sequencing, Binding Assay

    RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT ( 30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 ( 31 ).

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: RNase H Sequence Preferences correlate with gapmer efficiency. ( A ) Correlation between the log 2 fold changes of different hexamers observed for the R4b construct in the experiment and the corresponding log 2 fold changes as predicted by a single nucleotide model prepared from the data obtained in the R4a experiment. ( B ) As in (A), but with prediction with a dinucleotide model. ( C ) Prediction of RNase H1 mediated downregulation of the different 11-mers present in RNA sequence used for the RNase H RNA–DNA heteroduplex crystal structure [PDB: 2QK9]. Each bar corresponds to the cleavage site of a potential binding mode of RNase H1. The filled bar corresponds to the RNase H1 binding mode observed in the crystal structure and is also indicated in the drawing below the plot. ( D ) Correlation between the change of target RNA level for MAPT ( 30 ) after treatment with 1518 different gapmers and the corresponding downregulation predicted by the dinucleotide model for the different binding modes of RNase H1 on each gapmer target duplex. The error bars show the 99% confidence intervals. The drawing below the plot indicates the RNase H1 binding mode associated with the best-observed correlation. ( E ) The same analysis as in (D), but with 1581 different gapmers targeted against ANGPTL3 ( 31 ).

    Article Snippet: Nevertheless, only one of the modified nucleotides directly interacts with the RNase H1 enzyme ( ) ( ) and reassuringly, for the human and E. coli RNase H, we obtained similar results with all three constructs having different modification patterns (Figure and ), suggesting that the different modifications in the substrates did not distort the results.

    Techniques: Sequencing, Construct, Binding Assay

    Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Journal: Nucleic Acids Research

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    doi: 10.1093/nar/gkx1073

    Figure Lengend Snippet: Refining the HIV-1 RNase H sequence preference model. ( A ) Distributions of the observed log 2 fold changes of RNA heptamers in R7 for human RNase H1 (right) and HIV-1 RNase H (left). ( B ) The observed log 2 fold changes after cleavage with HIV-1 RNase H for an efficiently cleaved hexamer (GCGCAA) located at different positions of R7. The position of the arrow indicates the cleavage site as aligned to the picture of scissors in the box and the arrow length represents the efficiency of cleavage. ( C ) Sequence logos of the best cleaved quartile of sets of heptamers predicted to have the same cleavage site. The arrows indicate the predicted cleavage site, with the length proportional to the observed cleavage efficiency.

    Article Snippet: Nevertheless, only one of the modified nucleotides directly interacts with the RNase H1 enzyme ( ) ( ) and reassuringly, for the human and E. coli RNase H, we obtained similar results with all three constructs having different modification patterns (Figure and ), suggesting that the different modifications in the substrates did not distort the results.

    Techniques: Refining, 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

    Structures of RNase H (D132N mutant) complexed with DNA/RNA duplexes (with the same sequences). ( a ; 1.80 Å resolution; this work). The cleavage site in the selenium-modified structure

    Journal: Acta Crystallographica Section D: Biological Crystallography

    Article Title: Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

    doi: 10.1107/S1399004713027922

    Figure Lengend Snippet: Structures of RNase H (D132N mutant) complexed with DNA/RNA duplexes (with the same sequences). ( a ; 1.80 Å resolution; this work). The cleavage site in the selenium-modified structure

    Article Snippet: Co-crystallization of the Se-DNA/RNA duplex with RNase H was achieved by screening with the The Classics Suite kit (Qiagen).

    Techniques: Mutagenesis, Modification

    ‘Pseudo-specific’ cleavage of RNA substrates by RNase H. RNase H cleavage of short native and modified RNA substrates in the presence of the short native and modified DNA guides (see sequences in Fig. 1). In the reactions, the

    Journal: Acta Crystallographica Section D: Biological Crystallography

    Article Title: Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

    doi: 10.1107/S1399004713027922

    Figure Lengend Snippet: ‘Pseudo-specific’ cleavage of RNA substrates by RNase H. RNase H cleavage of short native and modified RNA substrates in the presence of the short native and modified DNA guides (see sequences in Fig. 1). In the reactions, the

    Article Snippet: Co-crystallization of the Se-DNA/RNA duplex with RNase H was achieved by screening with the The Classics Suite kit (Qiagen).

    Techniques: Modification

    Crystal structure of RNase H complexed with the selenium-modified DNA and RNA duplex. ( a ). The protein

    Journal: Acta Crystallographica Section D: Biological Crystallography

    Article Title: Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

    doi: 10.1107/S1399004713027922

    Figure Lengend Snippet: Crystal structure of RNase H complexed with the selenium-modified DNA and RNA duplex. ( a ). The protein

    Article Snippet: Co-crystallization of the Se-DNA/RNA duplex with RNase H was achieved by screening with the The Classics Suite kit (Qiagen).

    Techniques: Modification

    SOX2 interacts with the 3′-UTR of the S100A14 mRNA in vivo . (A) The diagram depicts the annealing positions of the targeting oligomer for RNase H digestion and the primers for reverse transcription and PCR detection of the ORF and 3′-UTR fragments. (B) FLAG-SOX2 was transiently expressed in BFTC905 cells for 48 h, followed by the CLIP assay. The matrix-bound mRNA was incubated at 37 °C with RNase H in the absence of the targeting oligomer for 30 min. The mock-treated matrix-bound mRNA was then purified and reverse-transcribed. The full-length, ORF, and 3′ fragments of the S100A14 mRNA were detected by RT-PCR. (C) After immunoprecipitation, the matrix-bound mRNA was subject to oligomer-dependent RNase H digestion at 37 °C for 30 min. The ORF and 3′-UTR fragments of the S100A14 mRNA in the supernatant and bound to the matrix were recovered, reverse transcribed, and detected by semi-quantitative PCR.

    Journal: Biochemistry and Biophysics Reports

    Article Title: SOX2 suppresses the mobility of urothelial carcinoma by promoting the expression of S100A14

    doi: 10.1016/j.bbrep.2016.06.016

    Figure Lengend Snippet: SOX2 interacts with the 3′-UTR of the S100A14 mRNA in vivo . (A) The diagram depicts the annealing positions of the targeting oligomer for RNase H digestion and the primers for reverse transcription and PCR detection of the ORF and 3′-UTR fragments. (B) FLAG-SOX2 was transiently expressed in BFTC905 cells for 48 h, followed by the CLIP assay. The matrix-bound mRNA was incubated at 37 °C with RNase H in the absence of the targeting oligomer for 30 min. The mock-treated matrix-bound mRNA was then purified and reverse-transcribed. The full-length, ORF, and 3′ fragments of the S100A14 mRNA were detected by RT-PCR. (C) After immunoprecipitation, the matrix-bound mRNA was subject to oligomer-dependent RNase H digestion at 37 °C for 30 min. The ORF and 3′-UTR fragments of the S100A14 mRNA in the supernatant and bound to the matrix were recovered, reverse transcribed, and detected by semi-quantitative PCR.

    Article Snippet: Cleavage of the S100A14 mRNA by RNase H produces the ORF and 3′-UTR fragments, and the ORF and 3′-UTR fragments was detected by RT-PCR ( A).

    Techniques: In Vivo, Polymerase Chain Reaction, Cross-linking Immunoprecipitation, Incubation, Purification, Reverse Transcription Polymerase Chain Reaction, Immunoprecipitation, Real-time Polymerase Chain Reaction