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FUJIFILM ribonuclease inhibitor
Ribonuclease Inhibitor, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 92 stars, based on 6 article reviews
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Incubation:

Article Title: A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro
Article Snippet: .. The 100-µl reaction mixture, which contained 1× transcription buffer (Roche), 25 mM each of ATP, CTP, GTP and UTP (Roche), 2 µl of ribonuclease inhibitor (40 U/µl; Wako), 10 µg of pEUKKS3.2b and SP6 RNA polymerase (Roche), was incubated for 3 h at 37°C, after which the synthesized RNA was purified with a MicroSpin G-25 column (Amersham Pharmacia Biotech). .. The 35 S-labeled Cr-RSH precursor was synthesized by in vitro translation in the presence of [35 S]methionine (0.4 mCi/ml) with a Proteios wheat germ cell-free protein synthesis kit (Toyobo).

Synthesized:

Article Title: A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro
Article Snippet: .. The 100-µl reaction mixture, which contained 1× transcription buffer (Roche), 25 mM each of ATP, CTP, GTP and UTP (Roche), 2 µl of ribonuclease inhibitor (40 U/µl; Wako), 10 µg of pEUKKS3.2b and SP6 RNA polymerase (Roche), was incubated for 3 h at 37°C, after which the synthesized RNA was purified with a MicroSpin G-25 column (Amersham Pharmacia Biotech). .. The 35 S-labeled Cr-RSH precursor was synthesized by in vitro translation in the presence of [35 S]methionine (0.4 mCi/ml) with a Proteios wheat germ cell-free protein synthesis kit (Toyobo).

Purification:

Article Title: A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro
Article Snippet: .. The 100-µl reaction mixture, which contained 1× transcription buffer (Roche), 25 mM each of ATP, CTP, GTP and UTP (Roche), 2 µl of ribonuclease inhibitor (40 U/µl; Wako), 10 µg of pEUKKS3.2b and SP6 RNA polymerase (Roche), was incubated for 3 h at 37°C, after which the synthesized RNA was purified with a MicroSpin G-25 column (Amersham Pharmacia Biotech). .. The 35 S-labeled Cr-RSH precursor was synthesized by in vitro translation in the presence of [35 S]methionine (0.4 mCi/ml) with a Proteios wheat germ cell-free protein synthesis kit (Toyobo).

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    FUJIFILM rnase h cleavage
    Inhibition of <t>RNase</t> H activity of HIV-1 and XMRV RTs and isolated XMRV and p15-Ec HIV RNases H by RNHIs. (A) RNHIs used in this study. (B) A 20 nM concentration of RT or p15-Ec HIV RNase H or 200 nM XMRV RNase H was preincubated with increasing concentrations
    Rnase H Cleavage, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 88/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h cleavage/product/FUJIFILM
    Average 88 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    rnase h cleavage - by Bioz Stars, 2020-08
    88/100 stars
      Buy from Supplier

    92
    FUJIFILM ribonuclease a
    Inhibition of <t>RNase</t> H activity of HIV-1 and XMRV RTs and isolated XMRV and p15-Ec HIV RNases H by RNHIs. (A) RNHIs used in this study. (B) A 20 nM concentration of RT or p15-Ec HIV RNase H or 200 nM XMRV RNase H was preincubated with increasing concentrations
    Ribonuclease A, supplied by FUJIFILM, used in various techniques. Bioz Stars score: 92/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ribonuclease a/product/FUJIFILM
    Average 92 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    ribonuclease a - by Bioz Stars, 2020-08
    92/100 stars
      Buy from Supplier

    Image Search Results


    Inhibition of RNase H activity of HIV-1 and XMRV RTs and isolated XMRV and p15-Ec HIV RNases H by RNHIs. (A) RNHIs used in this study. (B) A 20 nM concentration of RT or p15-Ec HIV RNase H or 200 nM XMRV RNase H was preincubated with increasing concentrations

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Inhibition of RNase H activity of HIV-1 and XMRV RTs and isolated XMRV and p15-Ec HIV RNases H by RNHIs. (A) RNHIs used in this study. (B) A 20 nM concentration of RT or p15-Ec HIV RNase H or 200 nM XMRV RNase H was preincubated with increasing concentrations

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Inhibition, Activity Assay, Isolation, Concentration Assay

    Interactions of RNases H with RNA-DNA substrates. (A) Molecular model showing potential interactions of XMRV ΔC RNase H (cyan cartoon) with an RNA-DNA substrate. The molecular model was built using the crystallographic coordinates of unliganded

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Interactions of RNases H with RNA-DNA substrates. (A) Molecular model showing potential interactions of XMRV ΔC RNase H (cyan cartoon) with an RNA-DNA substrate. The molecular model was built using the crystallographic coordinates of unliganded

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques:

    Crystal structure of the isolated XMRV ΔC RNase H domain. (A) 2 F o -F c electron density map of XMRV ΔC RNase H, displayed at σ = 2.0. (B) Stereo view of the isolated XMRV ΔC RNase H domain structure in cartoon representation.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Crystal structure of the isolated XMRV ΔC RNase H domain. (A) 2 F o -F c electron density map of XMRV ΔC RNase H, displayed at σ = 2.0. (B) Stereo view of the isolated XMRV ΔC RNase H domain structure in cartoon representation.

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Isolation

    Inhibition of RNase H activity.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Inhibition of RNase H activity.

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Inhibition, Activity Assay

    Dependence of RNase H activity on metal and time for HIV-1, MoMLV, and XMRV RTs. (A) A 50 nM concentration of Cy3-T r35 /P d22 was incubated with 20 nM HIV-1 RT or MoMLV RT or 100 nM XMRV RT for the indicated times. Lengths of cleavage products are indicated.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Dependence of RNase H activity on metal and time for HIV-1, MoMLV, and XMRV RTs. (A) A 50 nM concentration of Cy3-T r35 /P d22 was incubated with 20 nM HIV-1 RT or MoMLV RT or 100 nM XMRV RT for the indicated times. Lengths of cleavage products are indicated.

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Activity Assay, Concentration Assay, Incubation

    Comparison of the RNase H active sites of isolated XMRV RNase H ΔC and HIV-1 RT-dsDNA. (A) The active site of XMRV RNase H ΔC. One Mg 2+ ion (green sphere) is coordinated by the conserved catalytic residues Asp524, Glu562, and Asp583 (shown

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Comparison of the RNase H active sites of isolated XMRV RNase H ΔC and HIV-1 RT-dsDNA. (A) The active site of XMRV RNase H ΔC. One Mg 2+ ion (green sphere) is coordinated by the conserved catalytic residues Asp524, Glu562, and Asp583 (shown

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Isolation

    Effect of preincubation conditions on inhibition of RNase H. We used HIV-1 RT (A), XMRV RT in the presence of Mg 2+ (B), XMRV RT in the presence of Mn 2+ (C), XMRV RNase H in the presence of Mg 2+ (D), and XMRV RNase H in the presence of Mn 2+ (E). Each reaction

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Effect of preincubation conditions on inhibition of RNase H. We used HIV-1 RT (A), XMRV RT in the presence of Mg 2+ (B), XMRV RT in the presence of Mn 2+ (C), XMRV RNase H in the presence of Mg 2+ (D), and XMRV RNase H in the presence of Mn 2+ (E). Each reaction

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Inhibition

    Sequence alignment of XMRV, MoMLV, HIV-1, and B. halodurans ( Bh ) RNases H. All deleted residues from the XMRV ΔC RNase H structure are underlined in green. Residues contacting the RNA template are highlighted in red, residues contacting the DNA

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: Structural and Inhibition Studies of the RNase H Function of Xenotropic Murine Leukemia Virus-Related Virus Reverse Transcriptase

    doi: 10.1128/AAC.06000-11

    Figure Lengend Snippet: Sequence alignment of XMRV, MoMLV, HIV-1, and B. halodurans ( Bh ) RNases H. All deleted residues from the XMRV ΔC RNase H structure are underlined in green. Residues contacting the RNA template are highlighted in red, residues contacting the DNA

    Article Snippet: RNase H cleavage products were resolved on 15% polyacrylamide–7 M urea gels, which were scanned using a PhosphorImager (FujiFilm FLA 5000).

    Techniques: Sequencing

    Biochemical characterization of HCV RNase III cleavage product end-groups. S 1 was internally labeled at low specific radioactivity and cleaved with RNase III. P2 purified from the gel was subjected to different specific enzymatic reactions to determine chemical groups of the ( A ): 3′ end. Lane 1, P2 labeled with [ 32 P]pCp and T4 RNA ligase; lane 2, P2 alone; lane 3, control bands resulting from a standard reaction of RNase III on S 1 . ( B ) 5′ End. Lane 1, P2 central product band labeled with T4 polynucleotide kinase and [γ- 32 P]ATP after treatment with alkaline phosphatase; lane 2, P2 was identically treated as before without prior incubation with alkaline phosphatase; lane 3, P2 untreated. ( C ) 3′ and 5′ ends by P2 product circularization: lane 1, P2 alone; lane 2, P2 circularization with T4 RNA ligase. The band that corresponds to circularized P 2 migrates more slowly than the starting material; lane3, standard RNase III reaction. ( D ) Coincident mobility of the 5′-terminal RNase III and nuclease P1 cleavage products of substrate S 1 5′ Cap labeled S 1 incubated with: lane 1, RNase III (the positions of the products P1 (27 nt) and P1′ (33 nt) are indicated by arrows); lane 2, OH − hydrolysis; lane 3, nuclease P1 treatment.

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Biochemical characterization of HCV RNase III cleavage product end-groups. S 1 was internally labeled at low specific radioactivity and cleaved with RNase III. P2 purified from the gel was subjected to different specific enzymatic reactions to determine chemical groups of the ( A ): 3′ end. Lane 1, P2 labeled with [ 32 P]pCp and T4 RNA ligase; lane 2, P2 alone; lane 3, control bands resulting from a standard reaction of RNase III on S 1 . ( B ) 5′ End. Lane 1, P2 central product band labeled with T4 polynucleotide kinase and [γ- 32 P]ATP after treatment with alkaline phosphatase; lane 2, P2 was identically treated as before without prior incubation with alkaline phosphatase; lane 3, P2 untreated. ( C ) 3′ and 5′ ends by P2 product circularization: lane 1, P2 alone; lane 2, P2 circularization with T4 RNA ligase. The band that corresponds to circularized P 2 migrates more slowly than the starting material; lane3, standard RNase III reaction. ( D ) Coincident mobility of the 5′-terminal RNase III and nuclease P1 cleavage products of substrate S 1 5′ Cap labeled S 1 incubated with: lane 1, RNase III (the positions of the products P1 (27 nt) and P1′ (33 nt) are indicated by arrows); lane 2, OH − hydrolysis; lane 3, nuclease P1 treatment.

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Labeling, Radioactivity, Purification, Incubation

    Competition assays. (A and B) dsRNA competes with and inhibits the RNase III-specific cleavage of HCV RNA. Cleavage by RNase III of S 1 transcript labeled at ( A ) 5′ end; ( B ) 3′ end, in the presence of increasing concentrations of competitor dsRNA. (A) Lanes 1 and 2 correspond to 5′ end-labeled RNA alone (incubated on ice) and incubated with buffer, respectively; lane 3, standard reaction with RNase III; lanes 2–9, cleavage reactions using a constant concentration of S 1 (10 ng) and increasing concentrations of dsRNA (20, 40, 60, 80, 100 and 120 ng, respectively). (B) Identical to (A) but using 3′ end-labeled S 1 . ( C ) Reverse competition of RNase III cleavage of T7 R1.1 RNA with HCV RNA. A constant amount of a 60 nt fragment of T7 R1.1 RNA (internally labeled) was cleaved in the presence of increasing concentrations of HCV RNA S 1 without label. Lane 1 and 2, RNA T7 R1.1 alone or incubated with buffer, respectively; lane 3, standard RNase reaction, but with final concentration of RNase III of 0.0001 U/µl; lane 4, standard reaction with RNase III (0.0005 U/µl); lanes 5–9, RNase III cleavage of a constant concentration of T7 R1.1 RNA (1.8 nM) and increasing concentrations of S 1 RNA (0.45, 0.9, 1.8, 3.6 and 7.2 nM respectively); lane 9, inhibition of T7 R1.1 RNA cleavage with 300 ng of Penicillium chrysogenum dsRNA (positive control).

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Competition assays. (A and B) dsRNA competes with and inhibits the RNase III-specific cleavage of HCV RNA. Cleavage by RNase III of S 1 transcript labeled at ( A ) 5′ end; ( B ) 3′ end, in the presence of increasing concentrations of competitor dsRNA. (A) Lanes 1 and 2 correspond to 5′ end-labeled RNA alone (incubated on ice) and incubated with buffer, respectively; lane 3, standard reaction with RNase III; lanes 2–9, cleavage reactions using a constant concentration of S 1 (10 ng) and increasing concentrations of dsRNA (20, 40, 60, 80, 100 and 120 ng, respectively). (B) Identical to (A) but using 3′ end-labeled S 1 . ( C ) Reverse competition of RNase III cleavage of T7 R1.1 RNA with HCV RNA. A constant amount of a 60 nt fragment of T7 R1.1 RNA (internally labeled) was cleaved in the presence of increasing concentrations of HCV RNA S 1 without label. Lane 1 and 2, RNA T7 R1.1 alone or incubated with buffer, respectively; lane 3, standard RNase reaction, but with final concentration of RNase III of 0.0001 U/µl; lane 4, standard reaction with RNase III (0.0005 U/µl); lanes 5–9, RNase III cleavage of a constant concentration of T7 R1.1 RNA (1.8 nM) and increasing concentrations of S 1 RNA (0.45, 0.9, 1.8, 3.6 and 7.2 nM respectively); lane 9, inhibition of T7 R1.1 RNA cleavage with 300 ng of Penicillium chrysogenum dsRNA (positive control).

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Labeling, Incubation, Concentration Assay, Inhibition, Positive Control

    Localization of RNase III cleavage sites on HCV transcripts in the viral genome. ( A – C ) Autoradiograms of E.coli RNase III cleavage of internally labeled, 5′ end-labeled and 3′ end-labeled S 1 transcripts, respectively. Lane 1, the transcript alone incubated on ice; lane 2, transcript incubated in under ‘working conditions’; lane 3, RNase III cleavage reaction. The arrows indicate the substrate band (S 1 ) and the product bands–partial products (P1–P2) and (P2–P3) and total products (P1, P2 and P3). ( D ) Diagram of the cleavage sites of HCV RNA by E.coli RNase III. S 1 transcript (1–570) is represented with a line. RNase III cleavage sites in the positions 27, 33 and 439 respectively, are indicated by three arrows. Below are represented the final and partial cleavage products bands observed on a 4% polyacrylamide electrophoresis gels, (P1, P1′, P2 and P3) and (P1–P2, P2–P3), respectively.

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Localization of RNase III cleavage sites on HCV transcripts in the viral genome. ( A – C ) Autoradiograms of E.coli RNase III cleavage of internally labeled, 5′ end-labeled and 3′ end-labeled S 1 transcripts, respectively. Lane 1, the transcript alone incubated on ice; lane 2, transcript incubated in under ‘working conditions’; lane 3, RNase III cleavage reaction. The arrows indicate the substrate band (S 1 ) and the product bands–partial products (P1–P2) and (P2–P3) and total products (P1, P2 and P3). ( D ) Diagram of the cleavage sites of HCV RNA by E.coli RNase III. S 1 transcript (1–570) is represented with a line. RNase III cleavage sites in the positions 27, 33 and 439 respectively, are indicated by three arrows. Below are represented the final and partial cleavage products bands observed on a 4% polyacrylamide electrophoresis gels, (P1, P1′, P2 and P3) and (P1–P2, P2–P3), respectively.

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Labeling, Incubation, Electrophoresis

    The 5′ and 3′ cleavages in HCV RNA occur in a single structural motif. Kinetic analysis of E.coli RNAse III cleavage of HCV RNA in the presence or absence of a complementary RNA oligonucleotide. Left panel: autoradiogram of RNase III cleavage of internally labeled S 1 HCV RNA transcript in the presence (+) or in the absence (−) of a synthetic RNA oligonucleotide complementary to HCV nt 23–43 (5′-GGAGUGAUCUAUGGUGGAGUG-3′). HCV RNA substrate (0.6 nM final concentration) was pre-heated at 90°C for 1 min, before the addition of reaction buffer [10 mM HEPES–KOH, pH 7.5, 10 mM Mg(OAc) 2 and 100 mM NH 4 (OAc)] and left to cool down to room temperature. Cleavage reactions were performed with 20 U RNAsin, and 0.0005 U/µl (final concentration) of E.coli RNase III, in the presence of 1 µg/µl of yeast tRNA, and carried out at 37°C in a volume of 10 µl for 1 h. These optimal conditions were used in all of the experiments. In (+) reactions the synthetic complementary RNA oligonucleotide was added to a final concentration of 15 nM. Lane 13 (‘M’) contains RNA molecular weight markers composed of transcripts with a length of 82, 99, 110, 400 and 570 (S 1 ) nt. Lanes starting from the left represent sequentially 10, 20, 30, 40, 50 and 60 min of incubation with RNase III without (−) or with (+) complementary RNA oligonucleotide. Cleavage products were separated on 4% denaturing polyacrylamide gels and visualized by autoradiography. The arrows on the left indicate the products of the RNase III cleavage directed by the complementary oligonucleotide and on the right; the products of RNase III alone (P1+P2, P2 and P3) are indicated. Quantitation was performed with a Radioisotopic Image Analyzer. Right panel: Graphic representation of the time course processing of S 1 by E.coli RNAse III in the absence of the complementary RNA oligonucleotide.

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: The 5′ and 3′ cleavages in HCV RNA occur in a single structural motif. Kinetic analysis of E.coli RNAse III cleavage of HCV RNA in the presence or absence of a complementary RNA oligonucleotide. Left panel: autoradiogram of RNase III cleavage of internally labeled S 1 HCV RNA transcript in the presence (+) or in the absence (−) of a synthetic RNA oligonucleotide complementary to HCV nt 23–43 (5′-GGAGUGAUCUAUGGUGGAGUG-3′). HCV RNA substrate (0.6 nM final concentration) was pre-heated at 90°C for 1 min, before the addition of reaction buffer [10 mM HEPES–KOH, pH 7.5, 10 mM Mg(OAc) 2 and 100 mM NH 4 (OAc)] and left to cool down to room temperature. Cleavage reactions were performed with 20 U RNAsin, and 0.0005 U/µl (final concentration) of E.coli RNase III, in the presence of 1 µg/µl of yeast tRNA, and carried out at 37°C in a volume of 10 µl for 1 h. These optimal conditions were used in all of the experiments. In (+) reactions the synthetic complementary RNA oligonucleotide was added to a final concentration of 15 nM. Lane 13 (‘M’) contains RNA molecular weight markers composed of transcripts with a length of 82, 99, 110, 400 and 570 (S 1 ) nt. Lanes starting from the left represent sequentially 10, 20, 30, 40, 50 and 60 min of incubation with RNase III without (−) or with (+) complementary RNA oligonucleotide. Cleavage products were separated on 4% denaturing polyacrylamide gels and visualized by autoradiography. The arrows on the left indicate the products of the RNase III cleavage directed by the complementary oligonucleotide and on the right; the products of RNase III alone (P1+P2, P2 and P3) are indicated. Quantitation was performed with a Radioisotopic Image Analyzer. Right panel: Graphic representation of the time course processing of S 1 by E.coli RNAse III in the absence of the complementary RNA oligonucleotide.

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Labeling, Concentration Assay, Molecular Weight, Incubation, Autoradiography, Quantitation Assay

    Diagram including the proposed structural motif which contains both RNase III cleavage domains and encloses the HCV IRES. On the left is shown the interaction between nt 24 and 38 of the 5′ (UTR) with the nt 428–442 of the HCV Core–coding sequence. The arrows represent the RNase III cleavage sites. The ‘loop’ is shown within a box on the right of the figure. Symbol ‘P’ refers to the cleavage position of human RNase P, which is another RNA structure dependent nuclease. After the annealing, both RNase III and RNase P cleavage sites became in close proximity.

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Diagram including the proposed structural motif which contains both RNase III cleavage domains and encloses the HCV IRES. On the left is shown the interaction between nt 24 and 38 of the 5′ (UTR) with the nt 428–442 of the HCV Core–coding sequence. The arrows represent the RNase III cleavage sites. The ‘loop’ is shown within a box on the right of the figure. Symbol ‘P’ refers to the cleavage position of human RNase P, which is another RNA structure dependent nuclease. After the annealing, both RNase III and RNase P cleavage sites became in close proximity.

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Sequencing

    Determination of the cleavage sites in the HCV transcript. ( A ) To determine the cleavage sites near the 5′ end, a ‘Cap’ labeled S 1 was prepared. Lane 1, S 1 RNA transcript alone incubated on ice; lane 2, S 1 incubated with RNase III (see conditions in Figure 2 ). Arrows indicate the most prominent bands (P1, P1′); lane 3, alkaline hydrolysis reaction; lane 4, S 1 RNase P1 reaction; lane 5, RNA molecular weight markers composed of transcripts with a length of 21, 40, 69, 76, 77, 109 and 135 nt; lane 6, S 1 incubated under ‘working’ conditions buffer without enzyme. Samples were electrophoresed on a 20% denaturing polyacrylamide gel. The RNase III cleavage products (with expected 3′ OH end groups) run between two alkaline hydrolysis products (mixtures of RNA fragments with 3′P and 2′P end groups), and have been numbered according to the band running faster. Further experiments based on fingerprinting of internally labeled RNA are in progress to confirm the cleavage position presented here. ( B ) For determination of the cleavage site near the 3′ end, the S 2 (1–465) transcript was labeled with 32 pCp at its 3′ end with the T4 RNA ligase, cleaved with RNase III, and the most 3′-proximal product P3 * (indicated by an arrow) gel purified on a 20% denaturing polyacrylamide gel. Lane 1, S 2 transcript on ice; lane 2, S 2 treated with RNase III (standard conditions); lane 3 and 4, partial hydrolysis of P3 * with OH − after incubations of 4 and 8 min, respectively; lane 5, P3 * alone; lanes 6 and 7, partial digestions with nuclease T1 at 0.01 and 0.02 µg/µl final concentrations, respectively. The product bands of RNase T1 identified as A, B, C, D, E, at the left of the electrophoresis gel are described in ( D ). ( C ) Representation of the 3′ end RNA sequence of S 2 containing the P3 * fragment. RNase T1 cleavages after every G-residue are marked. A gap of 8 nt between the B and C product bands in the gel could be clearly positioned between positions U 442 and G 450 , the largest RNase T1 digestion product of the P3 * fragment. This gap serves as a reference to localize the cleavage site two positions upstream, between bases G 439 and A 440 .

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Determination of the cleavage sites in the HCV transcript. ( A ) To determine the cleavage sites near the 5′ end, a ‘Cap’ labeled S 1 was prepared. Lane 1, S 1 RNA transcript alone incubated on ice; lane 2, S 1 incubated with RNase III (see conditions in Figure 2 ). Arrows indicate the most prominent bands (P1, P1′); lane 3, alkaline hydrolysis reaction; lane 4, S 1 RNase P1 reaction; lane 5, RNA molecular weight markers composed of transcripts with a length of 21, 40, 69, 76, 77, 109 and 135 nt; lane 6, S 1 incubated under ‘working’ conditions buffer without enzyme. Samples were electrophoresed on a 20% denaturing polyacrylamide gel. The RNase III cleavage products (with expected 3′ OH end groups) run between two alkaline hydrolysis products (mixtures of RNA fragments with 3′P and 2′P end groups), and have been numbered according to the band running faster. Further experiments based on fingerprinting of internally labeled RNA are in progress to confirm the cleavage position presented here. ( B ) For determination of the cleavage site near the 3′ end, the S 2 (1–465) transcript was labeled with 32 pCp at its 3′ end with the T4 RNA ligase, cleaved with RNase III, and the most 3′-proximal product P3 * (indicated by an arrow) gel purified on a 20% denaturing polyacrylamide gel. Lane 1, S 2 transcript on ice; lane 2, S 2 treated with RNase III (standard conditions); lane 3 and 4, partial hydrolysis of P3 * with OH − after incubations of 4 and 8 min, respectively; lane 5, P3 * alone; lanes 6 and 7, partial digestions with nuclease T1 at 0.01 and 0.02 µg/µl final concentrations, respectively. The product bands of RNase T1 identified as A, B, C, D, E, at the left of the electrophoresis gel are described in ( D ). ( C ) Representation of the 3′ end RNA sequence of S 2 containing the P3 * fragment. RNase T1 cleavages after every G-residue are marked. A gap of 8 nt between the B and C product bands in the gel could be clearly positioned between positions U 442 and G 450 , the largest RNase T1 digestion product of the P3 * fragment. This gap serves as a reference to localize the cleavage site two positions upstream, between bases G 439 and A 440 .

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Labeling, Incubation, Molecular Weight, Purification, Electrophoresis, Sequencing

    Organization of the HCV genome and localization of HCV transcripts in the viral genome. Relative location of the 5′-UTR structural genes (C, Core; E1 and E2, envelope genes 1 and 2), the p7 coding region and non-structural genes (NS2 to NS5) and the 3′-UTR. S 1 (1–570), S 2 (1–466), S 3 (1–402) have been used as substrates in the E.coli RNase III assays. All transcripts initiate at the first base of the HCV genome, contain the complete 5′ UTR and expand to different sites within the Core-coding region.

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Organization of the HCV genome and localization of HCV transcripts in the viral genome. Relative location of the 5′-UTR structural genes (C, Core; E1 and E2, envelope genes 1 and 2), the p7 coding region and non-structural genes (NS2 to NS5) and the 3′-UTR. S 1 (1–570), S 2 (1–466), S 3 (1–402) have been used as substrates in the E.coli RNase III assays. All transcripts initiate at the first base of the HCV genome, contain the complete 5′ UTR and expand to different sites within the Core-coding region.

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques:

    Characterization of commercial RNase III secondary cleaving activity in comparison to the natural enzyme. Activity of the natural and commercial RNase III in buffer under working conditions (lower salt). Lane 1 is the R1.1 substrate incubated in buffer alone—it runs as band A. Lanes 2–4, natural RNase III and R1.1 substrate incubated in the presence of 9, 3 and 1 µg of poly(I–C) dsRNA competitor; lane 5, control reaction with no dsRNA; lanes 6–9 all contain substrate and 1 µl of Ambion RNase III at a 1/100 dilution; lanes 6–8 contain decreasing dsRNA competitor as in lanes 2–4, while lane 9 contains no dsRNA; lanes 10–13 are identical to lanes 6–9, except that they contain 1 µl of 1/10 Ambion RNase III. At the lower enzyme: substrate ratio, bands C and D are only faintly present, and they disappear at once when the dsRNA competitor is added.

    Journal: Nucleic Acids Research

    Article Title: RNase III cleavage demonstrates a long range RNA: RNA duplex element flanking the hepatitis C virus internal ribosome entry site

    doi: 10.1093/nar/gki822

    Figure Lengend Snippet: Characterization of commercial RNase III secondary cleaving activity in comparison to the natural enzyme. Activity of the natural and commercial RNase III in buffer under working conditions (lower salt). Lane 1 is the R1.1 substrate incubated in buffer alone—it runs as band A. Lanes 2–4, natural RNase III and R1.1 substrate incubated in the presence of 9, 3 and 1 µg of poly(I–C) dsRNA competitor; lane 5, control reaction with no dsRNA; lanes 6–9 all contain substrate and 1 µl of Ambion RNase III at a 1/100 dilution; lanes 6–8 contain decreasing dsRNA competitor as in lanes 2–4, while lane 9 contains no dsRNA; lanes 10–13 are identical to lanes 6–9, except that they contain 1 µl of 1/10 Ambion RNase III. At the lower enzyme: substrate ratio, bands C and D are only faintly present, and they disappear at once when the dsRNA competitor is added.

    Article Snippet: Quantitative data relating to RNase III cleavage kinetics were obtained using a Radioisotopic Image Analyser BAS-1800 (Fuji film).

    Techniques: Activity Assay, Incubation