e coli rnase h  (Thermo Fisher)


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    Name:
    Ambion RNase H
    Description:
    Ambion RNase H (Ribonuclease H) is isolated from an E. coli strain that over-expresses the gene. RNase H specifically degrades the RNA in RNA:DNA hybrids to produce 3' -hydroxyl and 5' -phosphate terminated products. It is supplied in one tube containing 1,000 U (10 U/ µL). The enzyme will not degrade DNA or unhybridized RNA. RNase H is an integral part of most RNA amplification and NASBA protocols. It can also be used to degrade specific RNAs when the complementary DNA oligo is hybridized, such as poly(A) tail removal from mRNA hybridized to oligo(dT). Ribonuclease H is rigorously tested for contaminating nonspecific endonuclease, exonuclease, RNase, and protease activity.Unit Definition:One unit of Ribonuclease H is the amount of enzyme required to increase fluorescence 1.5 RFUs per sec at 37°C using 20 pmol of RNaseAlert probe coupled to 1,000 pmol of a complementary oligonucleotide as substrate.
    Catalog Number:
    AM2293
    Price:
    None
    Applications:
    General Real-Time PCR Reagents|PCR & Real-Time PCR|Real Time PCR (qPCR)|Reverse Transcription
    Size:
    1 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher e coli rnase h
    ( A ) Schematic drawing of 1–570 RNA, indicating a subset of 20-base ODNs, each complementary to an HCV IRES region beginning with the residue number shown; the (−) sign indicates complementary to the viral sequence. ( B ) Cleavage reaction of 1–570 RNA with  E. coli  RNase III in the presence of a set of ODNs complementary to the viral sequences: 22(−), 371(−), 425(−), 473(−) and 492(−). Cleavage reactions were performed at 0.0005 U/µl of RNase III. Lanes 1 and 2: 1–570 RNA alone incubated on ice and incubated with buffer, respectively. Lane 3: control cleavage reaction without oligonucleotide. Lanes 4–6: cleavage reaction in the presence of increasing concentrations of ODN 22(−): (lane 4: 15 nM; lane 5: 150 nM; lane 6: 1500 nM). The same for lanes 7–9, 10–12, 13–15 and 16–18 for ODN 371(−), 425(−), 473(−) and 492(−), respectively. RNA molecular weight markers of 1–502 and 1–466 nt in length are indicated by a line on the left of the gel fragments. ( C ) Analysis of  E. coli  RNase H digestion products of 1–570 RNA annealed with complementary ODNs. Lane 1 is RNA incubated on ice, lane 2 incubated in buffer and lane 3, buffer with 0.5 units of  E. coli  RNase H. Lanes 4 to 18: the annealing reaction was performed with a set of 20-mer ODNs, 22(−), 371(−), 425(−), 473(−) and 492(−), at increasing concentrations of 15, 150 and 1500 nM, as indicated at the top of the gel, and treated with 0.5 U/µl of RNase H. Lane 19 is a commercial radiolabeled ladder of RNA fragments of 100, 200, 300, 400, 500, 750 and 1000 bases in length, which will be referred to as ‘MW marker’ in the remaining figure legends. Only fragments 100–500 bases in size appear in this gel image. ( D ) Table summarizing the changes in reactivity of 1–570 RNA to RNase III in the presence of complementary ODNs. The ‘oligonucleotide’ column indicates the positions where DNA oligonucleotides hybridize to RNA. The ‘X’ column shows the relative activation of RNase III cleavage at the X site (including fragments X and P1X) in 1–570 RNA by the presence of ODNs. The ‘accessibility’ column indicates the relative sensitivities of 1–570 RNA to DNA-mediated RNase H cleavage.
    Ambion RNase H (Ribonuclease H) is isolated from an E. coli strain that over-expresses the gene. RNase H specifically degrades the RNA in RNA:DNA hybrids to produce 3' -hydroxyl and 5' -phosphate terminated products. It is supplied in one tube containing 1,000 U (10 U/ µL). The enzyme will not degrade DNA or unhybridized RNA. RNase H is an integral part of most RNA amplification and NASBA protocols. It can also be used to degrade specific RNAs when the complementary DNA oligo is hybridized, such as poly(A) tail removal from mRNA hybridized to oligo(dT). Ribonuclease H is rigorously tested for contaminating nonspecific endonuclease, exonuclease, RNase, and protease activity.Unit Definition:One unit of Ribonuclease H is the amount of enzyme required to increase fluorescence 1.5 RFUs per sec at 37°C using 20 pmol of RNaseAlert probe coupled to 1,000 pmol of a complementary oligonucleotide as substrate.
    https://www.bioz.com/result/e coli rnase h/product/Thermo Fisher
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    e coli rnase h - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "In vitro characterization of a miR-122-sensitive double-helical switch element in the 5? region of hepatitis C virus RNA"

    Article Title: In vitro characterization of a miR-122-sensitive double-helical switch element in the 5? region of hepatitis C virus RNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp553

    ( A ) Schematic drawing of 1–570 RNA, indicating a subset of 20-base ODNs, each complementary to an HCV IRES region beginning with the residue number shown; the (−) sign indicates complementary to the viral sequence. ( B ) Cleavage reaction of 1–570 RNA with  E. coli  RNase III in the presence of a set of ODNs complementary to the viral sequences: 22(−), 371(−), 425(−), 473(−) and 492(−). Cleavage reactions were performed at 0.0005 U/µl of RNase III. Lanes 1 and 2: 1–570 RNA alone incubated on ice and incubated with buffer, respectively. Lane 3: control cleavage reaction without oligonucleotide. Lanes 4–6: cleavage reaction in the presence of increasing concentrations of ODN 22(−): (lane 4: 15 nM; lane 5: 150 nM; lane 6: 1500 nM). The same for lanes 7–9, 10–12, 13–15 and 16–18 for ODN 371(−), 425(−), 473(−) and 492(−), respectively. RNA molecular weight markers of 1–502 and 1–466 nt in length are indicated by a line on the left of the gel fragments. ( C ) Analysis of  E. coli  RNase H digestion products of 1–570 RNA annealed with complementary ODNs. Lane 1 is RNA incubated on ice, lane 2 incubated in buffer and lane 3, buffer with 0.5 units of  E. coli  RNase H. Lanes 4 to 18: the annealing reaction was performed with a set of 20-mer ODNs, 22(−), 371(−), 425(−), 473(−) and 492(−), at increasing concentrations of 15, 150 and 1500 nM, as indicated at the top of the gel, and treated with 0.5 U/µl of RNase H. Lane 19 is a commercial radiolabeled ladder of RNA fragments of 100, 200, 300, 400, 500, 750 and 1000 bases in length, which will be referred to as ‘MW marker’ in the remaining figure legends. Only fragments 100–500 bases in size appear in this gel image. ( D ) Table summarizing the changes in reactivity of 1–570 RNA to RNase III in the presence of complementary ODNs. The ‘oligonucleotide’ column indicates the positions where DNA oligonucleotides hybridize to RNA. The ‘X’ column shows the relative activation of RNase III cleavage at the X site (including fragments X and P1X) in 1–570 RNA by the presence of ODNs. The ‘accessibility’ column indicates the relative sensitivities of 1–570 RNA to DNA-mediated RNase H cleavage.
    Figure Legend Snippet: ( A ) Schematic drawing of 1–570 RNA, indicating a subset of 20-base ODNs, each complementary to an HCV IRES region beginning with the residue number shown; the (−) sign indicates complementary to the viral sequence. ( B ) Cleavage reaction of 1–570 RNA with E. coli RNase III in the presence of a set of ODNs complementary to the viral sequences: 22(−), 371(−), 425(−), 473(−) and 492(−). Cleavage reactions were performed at 0.0005 U/µl of RNase III. Lanes 1 and 2: 1–570 RNA alone incubated on ice and incubated with buffer, respectively. Lane 3: control cleavage reaction without oligonucleotide. Lanes 4–6: cleavage reaction in the presence of increasing concentrations of ODN 22(−): (lane 4: 15 nM; lane 5: 150 nM; lane 6: 1500 nM). The same for lanes 7–9, 10–12, 13–15 and 16–18 for ODN 371(−), 425(−), 473(−) and 492(−), respectively. RNA molecular weight markers of 1–502 and 1–466 nt in length are indicated by a line on the left of the gel fragments. ( C ) Analysis of E. coli RNase H digestion products of 1–570 RNA annealed with complementary ODNs. Lane 1 is RNA incubated on ice, lane 2 incubated in buffer and lane 3, buffer with 0.5 units of E. coli RNase H. Lanes 4 to 18: the annealing reaction was performed with a set of 20-mer ODNs, 22(−), 371(−), 425(−), 473(−) and 492(−), at increasing concentrations of 15, 150 and 1500 nM, as indicated at the top of the gel, and treated with 0.5 U/µl of RNase H. Lane 19 is a commercial radiolabeled ladder of RNA fragments of 100, 200, 300, 400, 500, 750 and 1000 bases in length, which will be referred to as ‘MW marker’ in the remaining figure legends. Only fragments 100–500 bases in size appear in this gel image. ( D ) Table summarizing the changes in reactivity of 1–570 RNA to RNase III in the presence of complementary ODNs. The ‘oligonucleotide’ column indicates the positions where DNA oligonucleotides hybridize to RNA. The ‘X’ column shows the relative activation of RNase III cleavage at the X site (including fragments X and P1X) in 1–570 RNA by the presence of ODNs. The ‘accessibility’ column indicates the relative sensitivities of 1–570 RNA to DNA-mediated RNase H cleavage.

    Techniques Used: Sequencing, Incubation, Molecular Weight, Marker, Activation Assay

    2) Product Images from "RNase H sequence preferences influence antisense oligonucleotide efficiency"

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1073

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Flow Cytometry, 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 ).
    Figure Legend 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 ).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Refining, Sequencing

    3) Product Images from "Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT"

    Article Title: Comparison of Second-Strand Transfer Requirements and RNase H Cleavages Catalyzed by Human Immunodeficiency Virus Type 1 Reverse Transcriptase (RT) and E478Q RT

    Journal:

    doi:

    Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.
    Figure Legend Snippet: Second-strand transfer assay with model substrates. This illustrates the model strand transfer assay with the truncated substrate possessing only 12 (Δ6), 9 (Δ9), and 6 (Δ12) positions of the RNA sequence. Step 1 illustrates the input substrate for each truncated substrate, along with their respective input RNA-DNA sizes. Step 2 illustrates the polymerization reaction which can occur in the presence of RT and dNTPs and the size of the polymerization product for each substrate, 52-mer (Δ6), 49-mer (Δ9), and 46-mer (Δ12). DNA polymerization creates the RNA-DNA hybrid, which is a substrate for the RNase H domain (step 3). Once the RNA has been removed between the terminal ribo-A and ribo-C, the acceptor molecule can enter and produce a strand transfer product, 70-mer (step 4). In each step, the RNA portion is indicated in bold and the 5′ radiolabel is indicated by an asterisk. The size of the strand transfer product (70-mer) would be the same for each truncated substrate.

    Techniques Used: Sequencing

    Model of WT RT and E478Q RT polymerase-dependent and polymerase-independent RNase H activities. The possible binding orientations for HIV-1 RT and E478Q RT are shown. (A to C) WT RT and E478Q RT are shown positioned on the various substrates, 50-mer substrate (32-mer DNA, 18-mer RNA) (A), 44-mer substrate (32-mer DNA, 12-mer RNA) (B), and 19-mer (7-mer DNA, 12-mer RNA) (C). The RNA portion is indicated by the thick line, and the DNA is indicated by the solid black line. The position of RNase H cleavage is indicated by a nick in the RNA strand. The WT and E478Q RT can be distinguished by the presence of an R (WT) or E/Q (E478Q RT) in the RNase H domain. Additionally, the thumb and polymerase domains are indicated by T and P, respectively. The 5′ phosphate is indicated, as well as the size of the RNA on each model substrate. (D) Models of substrates bound in the polymerase active site (left) and the RNase H active site (right) in HIV-1 RT. The electrostatic potential mapped on the molecular surface rendering of the HIV-1 RT (GRASP ) is shown with the template-primer as bound in the structure reported by Huang et al. (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.
    Figure Legend Snippet: Model of WT RT and E478Q RT polymerase-dependent and polymerase-independent RNase H activities. The possible binding orientations for HIV-1 RT and E478Q RT are shown. (A to C) WT RT and E478Q RT are shown positioned on the various substrates, 50-mer substrate (32-mer DNA, 18-mer RNA) (A), 44-mer substrate (32-mer DNA, 12-mer RNA) (B), and 19-mer (7-mer DNA, 12-mer RNA) (C). The RNA portion is indicated by the thick line, and the DNA is indicated by the solid black line. The position of RNase H cleavage is indicated by a nick in the RNA strand. The WT and E478Q RT can be distinguished by the presence of an R (WT) or E/Q (E478Q RT) in the RNase H domain. Additionally, the thumb and polymerase domains are indicated by T and P, respectively. The 5′ phosphate is indicated, as well as the size of the RNA on each model substrate. (D) Models of substrates bound in the polymerase active site (left) and the RNase H active site (right) in HIV-1 RT. The electrostatic potential mapped on the molecular surface rendering of the HIV-1 RT (GRASP ) is shown with the template-primer as bound in the structure reported by Huang et al. (1rtd), and substrates are shown as stick models. Positively charged amino acids are shown in blue, and negatively charged amino acids are shown in red. In the right-hand panel, the substrate found in the 1rtd structure has been truncated to include only 12 bp of template-primer extending from the RNase H active site. This truncated substrate makes very limited interactions with the thumb. T, thumb; F, fingers; RH, RNase H active site; Pol, polymerase active site.

    Techniques Used: Binding Assay

    RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.
    Figure Legend Snippet: RNase H cleavage analysis of truncated DNA substrates. (A) The substrates utilized are illustrated and are labeled A through F. The RNA portions are indicated in bold, and an asterisk indicates the radiolabel. The substrates were prepared as described in Materials and Methods. (B) Substrates B to F assayed with HIV-1 RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow. (C) Substrates B to F assayed with E478Q RT. Reactions were performed as described in Materials and Methods. Time course reactions are shown, and time points are indicated above each lane, along with the substrate utilized. The RNase H cleavage product is designated and indicated by an arrow.

    Techniques Used: Labeling

    4) Product Images from "Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells"

    Article Title: Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5?-Terminal Region of p53 mRNA In Vitro and in the Living Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0078863

    RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA. The 5′-end-[ 32 P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′- O Me or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H 2 O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the  Firefly  luciferase sequence.
    Figure Legend Snippet: RNase H assay in rabbit reticulocyte lysate in the presence of antisense oligomers no. 1 and 7b targeted to ΔNp53utr RNA. The 5′-end-[ 32 P]-labelled ΔNp53utr-Luc RNA was incubated in RRL and subsequently antisense oligomers no. 1 and no. 7b in their unmodified (UM) and modified (2′- O Me or GAP) form were added to the mixture. After 10 and 30 min incubation at 30°C, RNA was isolated and resolved on 8% polyacrylamide gel in denaturing conditions. The ΔNp53utr-Luc RNA was also subjected to limited hydrolysis by RNase T1 in denaturing conditions to determine the positions of RNase H cleavages. Lanes (−) and H 2 O indicate control reactions in the absence of antisense nucleotide in RRL and water, respectively. Lane (+) denotes the reaction in the presence of a control antisense oligonucleotide which is complementary to the Firefly luciferase sequence.

    Techniques Used: Rnase H Assay, Incubation, Modification, Isolation, Luciferase, Sequencing

    Mapping of the accessibility of the 5′-terminal region of ΔNp53utr-Luc transcript to oligonucleotide hybridization. Semi-random libraries of DNA 6-mers and RNase H hydrolysis of RNA-DNA hybrids were used to search for sites accessible to hybridization in the 5′-terminal region of ΔNp53utr-Luc transcript. ( A ) The cleavage sites were identified by reverse transcription with the 5′-end-[ 32 P]-labelled DNA primer that was bound to nucleotides 232–257 of the ΔNp53utr-Luc sequence. The cDNA products were analyzed on 8% polyacrylamide gels. Selected cytosine and guanosine residues are marked on the left and the short and long run of the gel is shown. Lane (−) denotes the control reaction in the absence of semi-random oligonucleotide libraries. A, T, C, G – sequencing reaction with adenosine, thymidine, cytosine and guanosine dideoxy terminating nucleotides, respectively. ( B ) The cleavage sites occurring in the presence of libraries  a ,  c ,  g ,  t  displayed on the RNA secondary structure model. Continuous grey lines along the RNA sequence show the most probable positions of oligonucleotide hybridization on the ΔNp53utr-Luc transcript. The designed specific antisense oligonucleotides are marked with black lines and numbered respectively.
    Figure Legend Snippet: Mapping of the accessibility of the 5′-terminal region of ΔNp53utr-Luc transcript to oligonucleotide hybridization. Semi-random libraries of DNA 6-mers and RNase H hydrolysis of RNA-DNA hybrids were used to search for sites accessible to hybridization in the 5′-terminal region of ΔNp53utr-Luc transcript. ( A ) The cleavage sites were identified by reverse transcription with the 5′-end-[ 32 P]-labelled DNA primer that was bound to nucleotides 232–257 of the ΔNp53utr-Luc sequence. The cDNA products were analyzed on 8% polyacrylamide gels. Selected cytosine and guanosine residues are marked on the left and the short and long run of the gel is shown. Lane (−) denotes the control reaction in the absence of semi-random oligonucleotide libraries. A, T, C, G – sequencing reaction with adenosine, thymidine, cytosine and guanosine dideoxy terminating nucleotides, respectively. ( B ) The cleavage sites occurring in the presence of libraries a , c , g , t displayed on the RNA secondary structure model. Continuous grey lines along the RNA sequence show the most probable positions of oligonucleotide hybridization on the ΔNp53utr-Luc transcript. The designed specific antisense oligonucleotides are marked with black lines and numbered respectively.

    Techniques Used: Hybridization, Sequencing

    5) Product Images from "Primary microRNA processing is functionally coupled to RNAP II transcription in vitro"

    Article Title: Primary microRNA processing is functionally coupled to RNAP II transcription in vitro

    Journal: Scientific Reports

    doi: 10.1038/srep11992

    Identification of RNA species generated in the RNAP II txn/processing system. ( a ) Predicted secondary structure of let-7a pri-miRNA showing the binding sites of the X, Y and Z DNA oligos used for oligonucleotide-directed RNAse H cleavage. ( b ) Schematic of let-7a pri-miRNA showing RNAse H cleavage sites in the 5′ flanking region, 3′ flanking region, and pre-let-7a loop using the X, Y and Z DNA oligos. ( c ) No oligo (lane 1) or the indicated oligos (lanes 2–7) were used for RNAse H cleavage of total RNA isolated from the txn/pri-miRNA processing system (lanes 2–4) or carried out directly in the txn/pri-miRNA processing system after completion of the reaction (at the 10 min time point). The RNAse H cleavage products or pri-miRNA processing products are indicated by an asterisk to the left of the gel lanes and are described in panel  d  from top to bottom for each gel lane in panel  c .
    Figure Legend Snippet: Identification of RNA species generated in the RNAP II txn/processing system. ( a ) Predicted secondary structure of let-7a pri-miRNA showing the binding sites of the X, Y and Z DNA oligos used for oligonucleotide-directed RNAse H cleavage. ( b ) Schematic of let-7a pri-miRNA showing RNAse H cleavage sites in the 5′ flanking region, 3′ flanking region, and pre-let-7a loop using the X, Y and Z DNA oligos. ( c ) No oligo (lane 1) or the indicated oligos (lanes 2–7) were used for RNAse H cleavage of total RNA isolated from the txn/pri-miRNA processing system (lanes 2–4) or carried out directly in the txn/pri-miRNA processing system after completion of the reaction (at the 10 min time point). The RNAse H cleavage products or pri-miRNA processing products are indicated by an asterisk to the left of the gel lanes and are described in panel d from top to bottom for each gel lane in panel c .

    Techniques Used: Generated, Binding Assay, Isolation

    6) Product Images from "R-loops cause replication impairment and genome instability during meiosis"

    Article Title: R-loops cause replication impairment and genome instability during meiosis

    Journal:

    doi: 10.1038/embor.2012.119

    Replication is impaired in C. elegans thoc-2 germlines and partially alleviated by RNase H microinjection. ( A ) Quantification of Cy3-dUTP incorporation 24 and 48 h after microinjection in the germline regions of mitosis (MT), transition zone (TZ),
    Figure Legend Snippet: Replication is impaired in C. elegans thoc-2 germlines and partially alleviated by RNase H microinjection. ( A ) Quantification of Cy3-dUTP incorporation 24 and 48 h after microinjection in the germline regions of mitosis (MT), transition zone (TZ),

    Techniques Used:

    7) Product Images from "Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma"

    Article Title: Protocol for Nearly Full-Length Sequencing of HIV-1 RNA from Plasma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001420

    Nearly full-length RT-PCR method. Viral RNA was reverse-transcribed by priming with UNINEF 7′ or with VIF-VPUoutR1 using SuperScript TM  III RNase H −  RT. The locations of all the primers used for cDNA synthesis and nested PCR were depicted in this diagram with the map of the complete HIV-1 genome. Two different strategies were employed to amplify the nearly full-length genome: one amplified the 2.6-kb ( gag-pol ) and 7.0-kb ( pol-nef ) fragments with the overlap of 797-bp, and the other amplified three overlapping fragments of 2.6-kb ( gag-pol ), 3.7-kb ( pol-vpu ) and 3.3-kb ( env-nef ) with the 797-bp and 321-bp over lap regions, respectively.
    Figure Legend Snippet: Nearly full-length RT-PCR method. Viral RNA was reverse-transcribed by priming with UNINEF 7′ or with VIF-VPUoutR1 using SuperScript TM III RNase H − RT. The locations of all the primers used for cDNA synthesis and nested PCR were depicted in this diagram with the map of the complete HIV-1 genome. Two different strategies were employed to amplify the nearly full-length genome: one amplified the 2.6-kb ( gag-pol ) and 7.0-kb ( pol-nef ) fragments with the overlap of 797-bp, and the other amplified three overlapping fragments of 2.6-kb ( gag-pol ), 3.7-kb ( pol-vpu ) and 3.3-kb ( env-nef ) with the 797-bp and 321-bp over lap regions, respectively.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Nested PCR, Amplification

    8) Product Images from "RNase H sequence preferences influence antisense oligonucleotide efficiency"

    Article Title: RNase H sequence preferences influence antisense oligonucleotide efficiency

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1073

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Functional Assay, Flow Cytometry, 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 ).
    Figure Legend 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 ).

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: Refining, Sequencing

    9) Product Images from "Synthetic in vitro transcriptional oscillators"

    Article Title: Synthetic in vitro transcriptional oscillators

    Journal: Molecular Systems Biology

    doi: 10.1038/msb.2010.119

    Schematics for  in vitro  transcriptional oscillators. ( A ) Reaction diagram for the two-switch negative-feedback oscillator (Design I). On the top left is a block diagram, wherein arrowheads indicate activation or production and circular ends indicate inhibition. The block diagram corresponds to the detailed diagram as highlighted by gray shading: T21A1 (ON-state switch Sw21) and T21 (OFF-state switch Sw21) are summarized by the Sw21 block; RNA inhibitor rI2, together with its threshold, DNA activator A2, and their complex, A2rI2, is summarized by the rI2 block; and similarly for the Sw12 and rA1 blocks. The sequence domains are color coded to indicate identical or complementary sequences; for the switch templates, the dark blue sequence domain inside the rectangle indicates the T7 RNAP promoter sequence with arrows pointing in the direction of transcription, with transcription domains indicated by light blue dashed circles. For fluorescence monitoring, OFF-state switches are labeled with fluorophores, T21 with Texas Red (red circle) and T12 with TAMRA (green circle), and both activators A1 and A2 are labeled with Iowa Black RQ quenchers (black circle). Four types of hybridization reactions are indicated by arrows: activation (magenta), inhibition (orange), annihilation (brown), and release (blue). Hybridization reactions are reversible; the arrowhead corresponds to the thermodynamically favorable direction, and the reverse reactions are expected to be so slow as to be negligible ( Wetmur, 1991 ;  Yurke and Mills, 2003 ;  Zhang and Winfree, 2009 ). Transcription by RNAP is shown as black dashed arrows and degradation of RNA within RNA-DNA hybrids by RNase H is shown as black dotted arrows. DNA and RNA sequences of single-stranded species and complexes for all three oscillators are shown in  Supplementary Figure S1 . ( B ) List of hybridization and enzyme reactions. See  Supplementary information  section 1.4 for details. ( C ) Theoretical end states of hybridization reactions in the absence of enzymes. As the input RNA inhibitor rI2 concentration increases, initially the free DNA activator A2 is consumed without affecting switch state. When all free A2 is consumed (i.e., [rI2]=[A2 tot ]–[T12 tot ]), rI2 displaces A2 from the T12A2 complex in stoichiometric amounts until all A2 is consumed (i.e., [rI2]=[A2 tot ]), resulting in a piecewise linear graph. Similarly, the response of switch Sw21 to rA1 input is a piecewise linear graph. See  Supplementary information  section 1.1 for details. ( D ) Block diagrams for the amplified negative-feedback oscillator (Design II) and the three-switch ring oscillator (Design III). See  Supplementary Figures S2 and S3  for detailed reaction diagrams.
    Figure Legend Snippet: Schematics for in vitro transcriptional oscillators. ( A ) Reaction diagram for the two-switch negative-feedback oscillator (Design I). On the top left is a block diagram, wherein arrowheads indicate activation or production and circular ends indicate inhibition. The block diagram corresponds to the detailed diagram as highlighted by gray shading: T21A1 (ON-state switch Sw21) and T21 (OFF-state switch Sw21) are summarized by the Sw21 block; RNA inhibitor rI2, together with its threshold, DNA activator A2, and their complex, A2rI2, is summarized by the rI2 block; and similarly for the Sw12 and rA1 blocks. The sequence domains are color coded to indicate identical or complementary sequences; for the switch templates, the dark blue sequence domain inside the rectangle indicates the T7 RNAP promoter sequence with arrows pointing in the direction of transcription, with transcription domains indicated by light blue dashed circles. For fluorescence monitoring, OFF-state switches are labeled with fluorophores, T21 with Texas Red (red circle) and T12 with TAMRA (green circle), and both activators A1 and A2 are labeled with Iowa Black RQ quenchers (black circle). Four types of hybridization reactions are indicated by arrows: activation (magenta), inhibition (orange), annihilation (brown), and release (blue). Hybridization reactions are reversible; the arrowhead corresponds to the thermodynamically favorable direction, and the reverse reactions are expected to be so slow as to be negligible ( Wetmur, 1991 ; Yurke and Mills, 2003 ; Zhang and Winfree, 2009 ). Transcription by RNAP is shown as black dashed arrows and degradation of RNA within RNA-DNA hybrids by RNase H is shown as black dotted arrows. DNA and RNA sequences of single-stranded species and complexes for all three oscillators are shown in Supplementary Figure S1 . ( B ) List of hybridization and enzyme reactions. See Supplementary information section 1.4 for details. ( C ) Theoretical end states of hybridization reactions in the absence of enzymes. As the input RNA inhibitor rI2 concentration increases, initially the free DNA activator A2 is consumed without affecting switch state. When all free A2 is consumed (i.e., [rI2]=[A2 tot ]–[T12 tot ]), rI2 displaces A2 from the T12A2 complex in stoichiometric amounts until all A2 is consumed (i.e., [rI2]=[A2 tot ]), resulting in a piecewise linear graph. Similarly, the response of switch Sw21 to rA1 input is a piecewise linear graph. See Supplementary information section 1.1 for details. ( D ) Block diagrams for the amplified negative-feedback oscillator (Design II) and the three-switch ring oscillator (Design III). See Supplementary Figures S2 and S3 for detailed reaction diagrams.

    Techniques Used: In Vitro, Blocking Assay, Activation Assay, Inhibition, Sequencing, Fluorescence, Labeling, Hybridization, Concentration Assay, Amplification

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    Polyacrylamide Gel Electrophoresis:

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    Autoradiography:

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    High Performance Liquid Chromatography:

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    Concentration Assay:

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    Article Snippet: RNA complementary to the cDNA was removed by E. coli RNase H (10 U; Ambion) and remaining RNAs were digested with 20 U of RNase Cocktail (Ambion) by incubating at 37°C for 20 minutes. .. PCR was performed for the sense and antisense transcripts independently.

    Article Title: 2?-Fluoro-4?-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity
    Article Snippet: The activity of E. coli RNase HI (USB Corporation, Cleveland, OH) was tested with antisense oligonucleotides under conditions recommended by the manufacturer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM MgCl2 , 0.25 mM EDTA, 0.25 mM DTT). .. The antisense and 5′-32 P labeled sense strands were combined in a 2:1 ratio and annealed by heating to 90°C followed by slow cooling to room temperature.

    Northern Blot:

    Article Title: The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific
    Article Snippet: Poly(A)- RNA was prepared in vitro by treating RNA samples with oligo(dT) and RNase H ( ). .. RNA (12 μg) was mixed with 0.5 μg of oligo(dT)15 (Promega) in annealing buffer (20 mM Tris, pH 8.0/5 mM MgCl2 /2 mM DTT/0.006% BSA) and then digested with Escherichia coli RNase H (Ambion) at 37°C for 30 min. RNA was phenol extracted, ethanol precipitated, electrophoresed in a 1.5% agarose gel, and analyzed by Northern blot hybridizations. .. Northern Blots.

    Hemagglutination Assay:

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature. .. For IP using mAbs, 30 µL protein A/G beads were first incubated with 5 µg mAbs for 1 h at room temperature followed by blocking with 4% BSA for 1 h. The mAb-coated beads were then incubated with the precleared ribosome samples at 4 °C for 1 h, followed by washing with polysome lysis buffer for three times.

    Sedimentation:

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Precipitated RNA was used for cDNA library construction and high-throughput sequencing described below. .. Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion. .. Total RNA was extracted using TRIzol reagent.

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Strand-specific RNA sequencing reveals extensive regulated long antisense transcripts that are conserved across yeast species
    Article Snippet: Paragraph title: Strand-specific RT-PCR ... RNA complementary to the cDNA was removed by E. coli RNase H (10 U; Ambion) and remaining RNAs were digested with 20 U of RNase Cocktail (Ambion) by incubating at 37°C for 20 minutes.

    Binding Assay:

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature. .. For IP using mAbs, 30 µL protein A/G beads were first incubated with 5 µg mAbs for 1 h at room temperature followed by blocking with 4% BSA for 1 h. The mAb-coated beads were then incubated with the precleared ribosome samples at 4 °C for 1 h, followed by washing with polysome lysis buffer for three times.

    Cleavage Assay:

    Article Title: In vitro characterization of a miR-122-sensitive double-helical switch element in the 5? region of hepatitis C virus RNA
    Article Snippet: Fold increase of P1X was measured as: P1X product at 60 min in the desired conditions divided by P1X product in the control lane at 60 min, run in the same gel. .. The salt and buffer conditions used for E. coli RNase H (Ambion) digestion were the same as those used in the RNase III cleavage assay. .. HCV RNA substrate was pretreated identically.

    Nucleic Acid Electrophoresis:

    Article Title: The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis
    Article Snippet: ∼50 A260 units of clarified lysate was treated with 750 U of E. coli RNase I (Ambion) for 1 hr on ice to minimize 80S degradation. .. RNA from monosome or polysome fractions was isolated using the hot acid phenol method.

    Isolation:

    Article Title: The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis
    Article Snippet: Paragraph title: Ribosome footprint isolation for polysome profile and deep-sequencing library ... ∼50 A260 units of clarified lysate was treated with 750 U of E. coli RNase I (Ambion) for 1 hr on ice to minimize 80S degradation.

    Article Title: The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific
    Article Snippet: Total cytoplasmic RNA was isolated at various times from mock- and HSV-1(F)-infected cells. .. RNA (12 μg) was mixed with 0.5 μg of oligo(dT)15 (Promega) in annealing buffer (20 mM Tris, pH 8.0/5 mM MgCl2 /2 mM DTT/0.006% BSA) and then digested with Escherichia coli RNase H (Ambion) at 37°C for 30 min. RNA was phenol extracted, ethanol precipitated, electrophoresed in a 1.5% agarose gel, and analyzed by Northern blot hybridizations.

    Size-exclusion Chromatography:

    Article Title: A New Method for Stranded Whole Transcriptome RNA-seq
    Article Snippet: Starting with 10 μL 3′-5′ adapter ligated RNA (sec. 2.6), 2.5 μL RS-TS-PCR1A primer ( ) was added, the mixture was heat denatured for 2 min at 70°C, and then quenched on ice. .. Reverse transcription was carried out by adding 2.5 μL Superscript RT III/RNaseOut enzyme mix plus 13.5 μL 2X Superscript reaction buffer (Life Technologies, Grand Island, NY 14072, cat. no. 18080-400) and incubating the reaction at 50°C for 60 min. Once the reverse transcription reaction was completed, the RNA was digested with 1 μL/10 U RNase H (Life Technologies, Grand Island, NY 14072, cat. no. AM2293) for 20 min at 37°C followed by PCR1 amplification.

    Labeling:

    Article Title: Synthetic in vitro transcriptional oscillators
    Article Snippet: T21-nt was labeled with a Texas Red fluorophore at the 5′ end, T12-nt was labeled with a TAMRA fluorophore at the 5′ end, A1 and A2 were labeled with Iowa Black-RQ quenchers at the 3′ end. .. The T7 RNA polymerase (enzyme mix), transcription buffer, and NTP as part of the T7 Megashortscript kit and E. coli RNase H were purchased from Ambion.

    Article Title: Functional characterization of the SOFA delta ribozyme
    Article Snippet: Trace amounts of 5′ end labeled SOFA-δRz-303 (~10,000 c.p.m; < 0.1 pmol) in the presence of 50 pmol of either the unlabeled small substrate (44 nt) or yeast tRNA as carrier (Roche Diagnostic) were preincubated in a volume of 8 μL containing 25 mM Tris-HCl (pH 7.5), 25 mM KCl, 12 mM MgCl2 , 0.13 mM EDTA, and 0.13 mM DTT for 10 min at 25°C. .. Finally, Escherichia coli RNase H (2 U, Ambion) was added to the mixtures and the samples incubated for 10 min at 37°C.

    Purification:

    Article Title: Synthetic in vitro transcriptional oscillators
    Article Snippet: All DNA oligonucleotides were synthesized and PAGE or HPLC purified by Integrated DNA Technologies. .. The T7 RNA polymerase (enzyme mix), transcription buffer, and NTP as part of the T7 Megashortscript kit and E. coli RNase H were purchased from Ambion.

    Article Title: Nuclear RNA Sequencing of the Mouse Erythroid Cell Transcriptome
    Article Snippet: Purified RNA was treated with 10 U of DNaseI (Roche) for 20 min at 28°C. .. Second strand synthesis was performed using E. coli RNase H (Ambion) and E. coli DNA Polymerase I (NEB) as described in Sambrook and Russell 2001 .

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: Paragraph title: Ribosome Purification. ... To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature.

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion. .. Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion.

    Article Title: Quantitative profiling of initiating ribosomes in vivo
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added to stop digestion. .. Total RNA extraction was performed using TRIzol reagent.

    Article Title: 2?-Fluoro-4?-thioarabino-modified oligonucleotides: conformational switches linked to siRNA activity
    Article Snippet: The activity of E. coli RNase HI (USB Corporation, Cleveland, OH) was tested with antisense oligonucleotides under conditions recommended by the manufacturer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM MgCl2 , 0.25 mM EDTA, 0.25 mM DTT). .. The activity of E. coli RNase HI (USB Corporation, Cleveland, OH) was tested with antisense oligonucleotides under conditions recommended by the manufacturer (50 mM Tris-HCl, pH 7.5, 50 mM KCl, 25 mM MgCl2 , 0.25 mM EDTA, 0.25 mM DTT).

    Sequencing:

    Article Title: Synthetic in vitro transcriptional oscillators
    Article Snippet: The sequence of all DNA molecules and expected RNA transcript sequences were chosen to minimize the occurrence of alternative secondary structures, checked by the Vienna group's DNA and RNA folding program ( ). .. The T7 RNA polymerase (enzyme mix), transcription buffer, and NTP as part of the T7 Megashortscript kit and E. coli RNase H were purchased from Ambion.

    Article Title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine
    Article Snippet: Briefly, equal aliquots of the ribosome fractions separated by sucrose gradient were pooled and treated with E. coli RNase I (Ambion) to digest regions of mRNAs not protected by ribosomes. cDNA libraries were constructed from the ribosome-protected mRNA fragments by poly(A) tailing and reverse transcription using barcode-containing oligonucleotides (one for each treatment condition). .. For deep sequencing, the cDNA library was amplified by PCR using the Phusion High-Fidelity polymerase and primers containing Illumina cluster generation sequences.

    Article Title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine
    Article Snippet: Briefly, equal aliquots of the ribosome fractions separated by sucrose gradient were pooled and treated with E. coli RNase I (Ambion) to digest regions of mRNAs not protected by ribosomes. cDNA libraries were constructed from the ribosome-protected mRNA fragments by poly(A) tailing and reverse transcription using barcode-containing oligonucleotides (one for each treatment condition). .. For deep sequencing, the cDNA library was amplified by PCR using the Phusion High-Fidelity polymerase and primers containing Illumina cluster generation sequences.

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion. .. Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion.

    Blocking Assay:

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature. .. For FKBP binding assay, 20 µg recombinant HA-FKBP proteins purified from E. coli (BL21) were first immobilized on protein A/G beads using anti-HA antibody.

    De-Phosphorylation Assay:

    Article Title: Quantitative profiling of initiating ribosomes in vivo
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added to stop digestion. .. Purified RNA samples were dephosphorylated in a 15 μL reaction containing 1× T4 polynucleotide kinase buffer, 10 U SUPERase_In, and 20 U T4 polynucleotide kinase (NEB).

    cDNA Library Assay:

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion. .. Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion.

    Article Title: Quantitative profiling of initiating ribosomes in vivo
    Article Snippet: Paragraph title: cDNA library construction of ribosome-protected mRNA fragments ... To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added to stop digestion.

    RNA Extraction:

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature. .. After blocking with 4% BSA for 1 h, the beads were then incubated with the precleared ribosome samples at 4 °C for 1 h in the absence or presence of 1 µM rapalog.

    Article Title: Quantitative profiling of initiating ribosomes in vivo
    Article Snippet: The pooled polysome samples were treated with E. coli RNase I (Ambion, 750 U per 100 A260 units) at 4 °C for 1 h to convert the polysome into monosome. .. The pooled polysome samples were treated with E. coli RNase I (Ambion, 750 U per 100 A260 units) at 4 °C for 1 h to convert the polysome into monosome.

    Recombinant:

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature. .. For IP using mAbs, 30 µL protein A/G beads were first incubated with 5 µg mAbs for 1 h at room temperature followed by blocking with 4% BSA for 1 h. The mAb-coated beads were then incubated with the precleared ribosome samples at 4 °C for 1 h, followed by washing with polysome lysis buffer for three times.

    Agarose Gel Electrophoresis:

    Article Title: The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific
    Article Snippet: Poly(A)- RNA was prepared in vitro by treating RNA samples with oligo(dT) and RNase H ( ). .. RNA (12 μg) was mixed with 0.5 μg of oligo(dT)15 (Promega) in annealing buffer (20 mM Tris, pH 8.0/5 mM MgCl2 /2 mM DTT/0.006% BSA) and then digested with Escherichia coli RNase H (Ambion) at 37°C for 30 min. RNA was phenol extracted, ethanol precipitated, electrophoresed in a 1.5% agarose gel, and analyzed by Northern blot hybridizations. .. Northern Blots.

    In Vitro:

    Article Title: The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific
    Article Snippet: Poly(A)- RNA was prepared in vitro by treating RNA samples with oligo(dT) and RNase H ( ). .. RNA (12 μg) was mixed with 0.5 μg of oligo(dT)15 (Promega) in annealing buffer (20 mM Tris, pH 8.0/5 mM MgCl2 /2 mM DTT/0.006% BSA) and then digested with Escherichia coli RNase H (Ambion) at 37°C for 30 min. RNA was phenol extracted, ethanol precipitated, electrophoresed in a 1.5% agarose gel, and analyzed by Northern blot hybridizations.

    Ethanol Precipitation:

    Article Title: The herpes simplex virus 1 UL41 gene-dependent destabilization of cellular RNAs is selective and may be sequence-specific
    Article Snippet: DNase treatment (Life Technologies), phenol/chloroform extraction, and ethanol precipitation (Fisher Scientific) were carried out to remove possible DNA contamination. .. RNA (12 μg) was mixed with 0.5 μg of oligo(dT)15 (Promega) in annealing buffer (20 mM Tris, pH 8.0/5 mM MgCl2 /2 mM DTT/0.006% BSA) and then digested with Escherichia coli RNase H (Ambion) at 37°C for 30 min. RNA was phenol extracted, ethanol precipitated, electrophoresed in a 1.5% agarose gel, and analyzed by Northern blot hybridizations.

    Quantitation Assay:

    Article Title: Secondary Structure and Hybridization Accessibility of Hepatitis C Virus 3?-Terminal Sequences
    Article Snippet: ODN digestions were carried out in the presence of 1 U of E. coli RNase H (Ambion). .. ODN digestions were carried out in the presence of 1 U of E. coli RNase H (Ambion).

    Immunoprecipitation:

    Article Title: Quantitative profiling of initiating ribosomes in vivo
    Article Snippet: Paragraph title: Immunoprecipitation of ribosomes from liver polysome fractions ... The pooled polysome samples were treated with E. coli RNase I (Ambion, 750 U per 100 A260 units) at 4 °C for 1 h to convert the polysome into monosome.

    Fractionation:

    Article Title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine
    Article Snippet: Separated samples were fractionated at 0.75 mL/min through an automated fractionation system (Isco) that continually monitors A254 values. .. Briefly, equal aliquots of the ribosome fractions separated by sucrose gradient were pooled and treated with E. coli RNase I (Ambion) to digest regions of mRNAs not protected by ribosomes. cDNA libraries were constructed from the ribosome-protected mRNA fragments by poly(A) tailing and reverse transcription using barcode-containing oligonucleotides (one for each treatment condition).

    Article Title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine
    Article Snippet: Separated samples were fractionated at 0.75 mL/min through an automated fractionation system (Isco) that continually monitors A254 values. .. Briefly, equal aliquots of the ribosome fractions separated by sucrose gradient were pooled and treated with E. coli RNase I (Ambion) to digest regions of mRNAs not protected by ribosomes. cDNA libraries were constructed from the ribosome-protected mRNA fragments by poly(A) tailing and reverse transcription using barcode-containing oligonucleotides (one for each treatment condition).

    Article Title: Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation
    Article Snippet: Separated samples were fractionated at 0.375 ml/min by using a fractionation system (Isco) that continually monitored OD254 values. .. To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. SUPERase_In (50 U per 100 U RNase I) was then added to stop digestion.

    Random Hexamer Labeling:

    Article Title: Nuclear RNA Sequencing of the Mouse Erythroid Cell Transcriptome
    Article Snippet: Reverse transcription was performed using Superscript II (Invitrogen) and 10 µg random hexamer primers (Roche) per 500 ng RNA. .. Second strand synthesis was performed using E. coli RNase H (Ambion) and E. coli DNA Polymerase I (NEB) as described in Sambrook and Russell 2001 .

    High Throughput Screening Assay:

    Article Title: Dynamic m6A mRNA methylation directs translational control of heat shock response
    Article Snippet: Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion. .. Ribosome fractions separated by sucrose gradient sedimentation were pooled and digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by incubation at 4 °C for 1 h. SUPERase inhibitor (50 U per 100 U RNase I) was then added into the reaction mixture to stop digestion.

    Lysis:

    Article Title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine
    Article Snippet: For ribosome profiling without cycloheximide pretreatment, the same lysis buffer was used but cycloheximide was omitted. .. Briefly, equal aliquots of the ribosome fractions separated by sucrose gradient were pooled and treated with E. coli RNase I (Ambion) to digest regions of mRNAs not protected by ribosomes. cDNA libraries were constructed from the ribosome-protected mRNA fragments by poly(A) tailing and reverse transcription using barcode-containing oligonucleotides (one for each treatment condition).

    Article Title: Effects of single amino acid deficiency on mRNA translation are markedly different for methionine versus leucine
    Article Snippet: For ribosome profiling without cycloheximide pretreatment, the same lysis buffer was used but cycloheximide was omitted. .. Briefly, equal aliquots of the ribosome fractions separated by sucrose gradient were pooled and treated with E. coli RNase I (Ambion) to digest regions of mRNAs not protected by ribosomes. cDNA libraries were constructed from the ribosome-protected mRNA fragments by poly(A) tailing and reverse transcription using barcode-containing oligonucleotides (one for each treatment condition).

    Article Title: The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis
    Article Snippet: Frozen cells were cryogenically lysed in the presence of 3 ml of frozen polysome lysis buffer (20 mM Tris pH 8.0, 140 mM KCl, 1.5 mM MgCl2 , 100 μg/ml cycloheximide, 1% Triton) on a Retsch MM301 mixer mill, and thawed lysates were subsequently clarified by centrifugation as described. .. ∼50 A260 units of clarified lysate was treated with 750 U of E. coli RNase I (Ambion) for 1 hr on ice to minimize 80S degradation.

    Article Title: Monitoring cotranslational protein folding in mammalian cells at codon resolution
    Article Snippet: To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. Preclearance was conducted by incubating the ribosome samples with 30 µL protein A/G beads coated with 4% BSA for 1 h at room temperature. .. After blocking with 4% BSA for 1 h, the beads were then incubated with the precleared ribosome samples at 4 °C for 1 h in the absence or presence of 1 µM rapalog.

    Article Title: Ribosome profiling reveals sequence-independent post-initiation pausing as a signature of translation
    Article Snippet: After ice-cold PBS solution wash, cells were then harvested by ice-cold polysome lysis buffer (10 mM Hepes, pH 7.4, 100 mM KCl, 5 mM MgCl2 , 100 μg/ml CHX, 5 mM DTT, 20 U/ml SUPERase_In, and 2% (vol/vol) Triton X-100). .. To convert the polysome into monosome, E. coli RNase I (Ambion) was added into the pooled polysome samples (750 U per 100 A260 units) and incubated at 4 °C for 1 h. SUPERase_In (50 U per 100 U RNase I) was then added to stop digestion.

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  • 99
    Thermo Fisher e coli rnase h
    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.
    E Coli Rnase H, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/e coli rnase h/product/Thermo Fisher
    Average 99 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    e coli rnase h - by Bioz Stars, 2020-01
    99/100 stars
      Buy from Supplier

    77
    Thermo Fisher v5 tagged rnase h1
    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.
    V5 Tagged Rnase H1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 77/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/v5 tagged rnase h1/product/Thermo Fisher
    Average 77 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    v5 tagged rnase h1 - by Bioz Stars, 2020-01
    77/100 stars
      Buy from Supplier

    Image Search Results


    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: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    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: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Functional Assay, Flow Cytometry, 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: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    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: Reactions with E. coli RNase H from Thermo Scientific (cat. EN0201) were performed at 37°C in a buffer composed of 50 mM Tris–HCl pH 8.3, 75 mM KCl, 3 mM MgCl2 , 10 mM dithiothreitol (DTT), 0.4 mg/ml bovine serum albumin and 0.1 mM EDTA using the same protocol as for the reactions with human-derived enzyme, but with a final enzyme concentration 0.5 mU/μl.

    Techniques: Refining, Sequencing

    Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review   [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in  Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in  Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in   Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Both RNase H1 and P32 interact with mitochondrial DNA and pre-rRNA. ( A ) The positions of Probes and PCR primers for the human mitochondrial DNA. The DNA map was derived from published review [65] . Two oligonucleotide probes specific to 12 S and 16 S mitochondria rRNA regions are shown in Blue bars . Three sets of PCR probes corresponding to the A, B and C regions are indicated in Green arrows . ( B ) RNase H1 and P32 bind mitochondrial DNA. Cell extracts were prepared from an HA-H1 stably expressing cell line (RNase H1), control HEK cells or HEK cells transfected with the HA-P32 expression plasmid (P32). Equal amounts of each extract were used for immunoprecipitation with anti-HA beads. Nucleic acids were extracted from the precipitated samples using phenol/chloroform and subjected to PCR analysis. The probe sets for PCR were shown in Figure 6A . Genomic DNA from HEK cells that was used as a positive control. The PCR products were analyzed on 2% Agarose gels. ( C ) RNase H1 may interact with the mitochondrial rDNA region. The extracts from HA-H1 cell and control HEK cells were used for immunoprecipitation with HA-antibody. The precipitates were digested on beads with (+) or without (−) DNase I. The DNA associated with beads was then extracted and subjected to PCR analysis. The PCR products were separated in 2% agarose gel. ( D ) RNase H1 and P32 also co-immunoprecipitated with mitochondrial pre-rRNA. The same extracted nucleic acids from panel B were digested with DNase I. The RNA is used for reverse transcription with (+) or without (−) reverse transcriptase, followed by PCR amplification using different primer sets as indicated below the panels. PCR reaction using primers specific to U16 snoRNA was used as control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Polymerase Chain Reaction, Derivative Assay, Hemagglutination Assay, Stable Transfection, Expressing, Transfection, Plasmid Preparation, Immunoprecipitation, Positive Control, Agarose Gel Electrophoresis, Amplification

    Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with  32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Depletion of RNase H1 or P32 resulted in accumulation of mitochondrial pre-12S/16S rRNA. HeLa cells were treated with 2 nM or 20 nM of RNase H1-siRNA or P32 –siRNA for 24 or 48 hours. ( A ) The mRNA levels of RNase H1 and P32 were determined by qRT-PCR 24 hrs after siRNA treatment. ( B ) Protein levels of RNase H1 and P32 were analyzed by western analysis 24 hours post siRNA treatment. ( C ) Reduction of RNase H1 or P32 significantly increased the level of mitochondrial pre-rRNA. HeLa cells were treated with either RNase H1-siRNA (2 nM) or P32-siRNA (2 nM) for 24 hours. Total RNA was prepared and subjected to Northern analysis with 32 P labeled probes specific to 12S or 16S rRNAs. U3 snoRNA was detected and served as a control. The relative levels of pre-rRNA were measured from the results obtained with 12 S probe, normalized to U3, and plotted in the right panel. The error bars indicate standard error of the three replicates. (D) RT-PCR assay for the levels of pre-16 S and pre-ND3 RNAs. Total RNA prepared from HeLa cells treated for 24 hrs with corresponding siRNAs was analyzed by qRT-PCR, using primer probe sets specific to the tRNA Val -16 S rRNA junction (pre-16 S) or to the tRNA Gly -ND3 junction (pre-ND3). The error bars represent standard deviation of three replicates.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Quantitative RT-PCR, Western Blot, Northern Blot, Labeling, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Co-localization of P32 and RNase H1. ( A ) Immunofluorescence Staining of P32 and RNase H1. Upper panel: HeLa cells were stained for endogenous P32 and RNase H1 using mouse monoclonal anti-P32 antibody and rabbit anti-RNase H1 antibody, respectively, followed by FITC conjugated donkey anti-mouse ( green ) and TRITC conjugated anti-rabbit secondary antibodies ( red ). Nuclei were stained with DAP1 ( Blue ) and Mitochondria were stained with mitotracker ( white ). Lower panel: HeLa cells were infected with adenovirus expressing RNase H1. Cells were stained as described in upper panel. ( B ) Subcellular fractionation of P32 protein. The proteins from sub-cellular compartments (cytosol, mitochondrial and ER membranes, nucleus and cytoskeleton) were prepared from HEK cells using proteome cell compartment kit (Qiagen). About 10 µg protein samples from each fraction were analyzed by western for P32. The same blot was stripped and tubulin-γ was detected to serve as a control.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Immunofluorescence, Staining, Infection, Expressing, Fractionation, Western Blot

    Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing  32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d  were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Recombinant P32 binds to recombinant RNase H1, enhances its turnover rate, and reduces the binding affinity of the enzyme for the heteroduplex substrate. ( A ) Coomassie blue staining of the purified human His-H1, GST protein, and GST-P32 proteins separated by SDS-PAGE. The sizes for the standard protein markers are indicated. ( B ) RNase H1 but not P32 appears to bind the heteroduplex substrate. Gel shift assay was performed using 0.4 ug purified RNase H1, GST-P32, or GST proteins incubated at 4°C for 30 min with a non-cleavable heteroduplex containing 32 P labeled uniformly modified 2′-fluoro RNA annealed to DNA and subjected to native gel electrophoresis. ( C ) The interaction between RNase H1 and P32 appears to be equal molar. A fixed amount of GST-P32 was bound to GST affinity beads and then incubated with increasing amounts of RNase H1. Glutathione (GSH) eluted RNase H1 and P32 were quantified by Western blot as described in the Material and Methods. The amounts of bead-bound P32 and P32-associated RNase H1 were determined by loading known amounts of the respective proteins (left panel). The molecular ratio of bound RNase H1 relative to P32 was calculated and plotted in the right panel. ( D ) The effects of ionic strength on RNase H1/P32 interaction. Left panel: RNase H1 binds GST-P32 but not GST protein. GST or GST-P32 bound to anti-GST beads was incubated with RNase H1 in NaCl concentrations ranging from 0-950 mM as described in the Material and Methods. Middle panel: increasing NaCl concentration inhibits binding of RNase H1 to P32. Both unbound (flow through) and bound (affinity eluted) fractions were collected and the levels of RNase H1 and P32 evaluated by western blot. Right panel: Increasing pH reduced binding of RNase H1 to P32. ( E ) Michaelis-Menten kinetics and binding constants for RNase H1 cleavage of an RNA/DNA duplex in the presence or absence of P32. The K m , V max , and K d were determined by incubating the Apo B RNA/DNA duplex with RNase H1 plus GST (as control) or RNase H1 plus different amounts of P32 resulting in an H1:P32 ratio = 1∶1 or 1∶5. An uncleavable competitive inhibitor (2′-fluororibonucleotide/DNA) was used to determine the binding to the RNA/DNA duplex, as described in the Material and Methods. The calculated constants are indicated in the right panel. The error bars indicate the standard error from three parallel experiments.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Recombinant, Binding Assay, Staining, Purification, SDS Page, Electrophoretic Mobility Shift Assay, Incubation, Labeling, Modification, Nucleic Acid Electrophoresis, Western Blot, Concentration Assay, Flow Cytometry

    P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat  for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: P32 appears to interact with the N-terminal duplex binding domain of RNase H1. ( A ) Expression and purification of RNase H1 deletion mutants. Left panel: Schematic depiction of the different human RNase H1 deletion mutants. DL1 deletes the hybrid binding domain (amino acid positions 1–73); DL2 deletes both the hybrid binding domain and the spacer domain (amino acid 1–129). The black bars at the N-terminus of each mutant represent a His tag. Right panel: Coomassie blue staining of the purified RNase H1 deletion mutants. The sizes of the standard markers are given. ( B ) Interaction of full length RNase H1 and its deletion mutants with P32. The full length or truncated RNase H1 proteins were incubated with GST-P32 bound to GST-beads under different NaCl concentrations ranging from 150–450 mM in both the binding and washing solutions. The P32 and RNase H1 or deletion mutants were eluted and analyzed by Western blot, using P32 or RNase H1 antibodies, respectively (right panel). Western blot to RNase H1 and deletion mutants DL1 and DL2 demonstrates that the mutant proteins are recognized by the RNase H1 antibody (left panel). ( C ) Michaelis-Menten Kinetics of DL-1 mutant in the presence or absence of P32. K m , V max , and k cat for DL-1 plus GST or GST-P32 (DL-1:P32 = 1:5 in molecular ratio) were determined in 50 and 150 mM NaCl concentration with the Apo B RNA/DNA duplex as described in the Material and Methods.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Binding Assay, Expressing, Purification, Mutagenesis, Staining, Incubation, Western Blot, Concentration Assay

    Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a  32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Journal: PLoS ONE

    Article Title: Human RNase H1 Is Associated with Protein P32 and Is Involved in Mitochondrial Pre-rRNA Processing

    doi: 10.1371/journal.pone.0071006

    Figure Lengend Snippet: Human RNase H1 is associated with P32. ( A ) Western blot analysis of cell lysates and immunoprecipitated samples show Flag-tagged RNase H1 and H2 expression from cells stably transformed with RNase H1 (H1) or H2 (H2) or wild type (control) HEK cell lines. ( B ) Co-selection of RNase H1 binding proteins by immunoprecipitation. Extracts from cells expressing the Flag-H1, Flag-H2, or HA-H1 cell lines were immunoprecipitated with either anti-Flag or anti-HA antibody. Co-precipitated proteins were resolved by SDS-PAGE, and visualized by silver staining. Protein bands that were different from the co-precipitated proteins from control cells were subjected to mass spectrometry. The protein bands corresponding to the tagged RNase H1, H2 and the co-precipitated P32 proteins are indicated. The size marker was SeeBlue Plus2 Pre-Stained Standard (Invitrogen). ( C ) 2D gel electrophoresis of proteins co-precipitated with Flag-H1 or Flag-H2. About 5 mg cell lysates were prepared for immunoprecipitation with anti-flag beads from cell lines which stably express Flag-H1 or Flag-H2. The immunoprecipitates were washed four times with RIPA buffer and directly sent to Applied Biomics Inc. (San Francisco, CA) for 2D gel electrophoresis coupled with MS analysis. In brief, the co-precipitated proteins from Flag-H1 or Flag-H2 cells were labeled by fluorescent DIGE CyDyers, respectively, followed by 2D gel electrophoresis. The protein image was scanned with a fluorescence detector. The figure illustrates the proteins differentially associated with RNase H1 (green) or H2 (red). The P32 protein was confirmed with mass spectrum from the extracted gel sample. Circled spots were identified as RNase H1, H2 or P32 by mass spectrometric analysis. ( D ) Both endogenous and expressed RNase H1 are co-precipitated with the expressed P32. Left panel: western blots with P32, RNase H1, or H2 antibodies for proteins co-precipitated using anti-HA antibody from extracts of control HeLa cells or cells transfected with HA-P32 expression plasmid. Right panel: western blots for proteins co-selected using anti-HA antibody from extracts of Flag-H1, Flag-H2 stable cell lines and control cells, all of which were transfected with HA-P32 expression plasmid. ( E ) Confirmation of the specific interaction between RNase H1 and P32. RNase H cleavage activity indicates that the P32 co-immunoprecipitated material contains only RNase H1 enzyme activity. Upper panel: Cleavage patterns of human RNase H1 and H2 from IP-coupled enzyme activity assays. Immunoprecipitations were performed with either anti-flag, anti-RNase H1 or anti-H2 antibodies from extracts of Flag-H1, Flag-H2 expressing cells or control cells. The co-precipitated samples were incubated for the indicated times with a 32 P-labeled RNA/DNA-methoxyethyl (MOE) gapmer duplex and the cleavage products were separated using denaturing gel electrophoresis. The preferred cleavage sites of RNase H1 and H2 are indicated with * or #, respectively. The positions of the preferred cleavage sites in the heteroduplex are shown in the middle panel with the sequences of the RNA substrate (upper strand) and the oligonucleotide (lower strand). The bold nucleotides in the oligonucleotide strand indicate the position of the MOE substitutions. Lower panel: only the RNase H1 enzyme activity was detected in the co-precipitated material from lysates containing tagged P32. Immunoprecipitations were performed with anti-HA antibody from extracts of Flag-H1 or Flag-H2 stable cell lines or control HEK cells, which were all transfected or not transfected with HA-P32 expression plasmid. The precipitated samples were analyzed for cleavage patterns as described above. The position of the cleavage bands relative to the sequence of the cleavage products is shown on the left. A partial alkaline digestion of the same labeled RNA was used as a sequence ladder. The cleavage pattern of purified human RNase H1 is shown at the far right of the lower panel.

    Article Snippet: The full length human RNase H1, H2, and P32 cDNAs (GenBank accession numbers NM-002936, NM-006397, and NM-001212, respectively) were used to construct the plasmids with N-terminal Flag- or C-terminal HA-tag in pcDNA3.1 vector (Invitrogen) for transient expression or creation of stable cell lines.

    Techniques: Western Blot, Immunoprecipitation, Expressing, Stable Transfection, Transformation Assay, Selection, Binding Assay, Hemagglutination Assay, SDS Page, Silver Staining, Mass Spectrometry, Marker, Staining, Two-Dimensional Gel Electrophoresis, Electrophoresis, Labeling, Fluorescence, Transfection, Plasmid Preparation, Activity Assay, Incubation, Nucleic Acid Electrophoresis, Sequencing, Purification

    Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Overexpression of rnh1 relieves replication pausing. A–D , 2DNAGE of four restriction fragments of Drosophila S2 cells mtDNA, probed as indicated, in material from control cells and cells overexpressing RNase H1 in the form of epitope-tagged RNase H1-V5 (denoted OE ), both treated with 500 μ m CuSO 4 for 48 h to induce expression. E , schematic map of Drosophila mtDNA, as also shown in Fig. 8 , indicating the location of relevant restriction sites ( open circles ), mTTF-binding sites (bs1 and bs2; filled circles ), the noncoding region ( bold ), and the probes used. The open arrowhead marks the location and direction of replication initiation (see Ref. 40 ). The directions of first- and second-dimension electrophoresis in all gels are as indicated by the arrows . The images show relatively low exposures to reveal fine details of the arcs of RIs.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Over Expression, Expressing, Binding Assay, Electrophoresis

    Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular localization of epitope-tagged RNase H1. A , immunocytochemistry of cells transiently transfected with RNase H1-V5, probed for the V5 epitope tag ( red ), Cox4 ( green ), and DAPI ( blue ), showing examples of the three types of intracellular distribution of V5-tagged RNase H1: nucleus and mitochondria ( i ), mitochondria only ( ii ), and nucleus only ( iii ). B , subcellular distribution of RNase H1-V5 in 100 transfected cells as indicated (mean of three experiments, error bars denote S.D.). C , Western blots of subcellular fractions from cells transfected with RNase H1-V5, highly enriched for nuclei ( nuc ) or mitochondria ( mt ) as indicated, probed simultaneously for V5 and for the markers indicated. M , molecular mass markers.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Immunocytochemistry, Transfection, Western Blot

    Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Journal: The Journal of Biological Chemistry

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    doi: 10.1074/jbc.RA118.007015

    Figure Lengend Snippet: Subcellular targeting of RNase H1 variants. A , intracellular localization of RNase H1-V5 variants in cultures of stably transfected cells exemplified in B . M1V and M16V, N-terminal methionine variants (see Fig. S2 A ); ΔNLS, with the putative nuclear localization signal deleted (see Fig. S2 C ). C , intracellular localization of RNase H1-V5 in cells synchronized in G1 and G2 (see FACS profiles in Fig. S2 E ). All plotted values are means of three experiments. Error bars denote S.D. nuc , nuclei; mt , mitochondria.

    Article Snippet: To establish cell clones stably expressing V5-tagged RNase H1 and variants, pCoBlast (Thermo Fisher Scientific) was included in transfections.

    Techniques: Stable Transfection, Transfection, FACS