rna  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94

    Structured Review

    New England Biolabs rna
    Population genetics analysis of <t>epiRNA-associated</t> <t>RNA</t> editing events. Extent of selective constraints for epiRNA-associated editing events was evaluated as described in Materials and Methods . The distribution of SNP densities ( A ), SNP nucleotide diversity ( B ), and the frequency spectra of derived allele ( C ) in the local regions of epiRNA-associated focal editing sites were determined and quantitatively represented as boxplots or means ± SD, with the more distal regions (Upstream and Downstream) as the references. ( D ) The nucleotide diversity of epiRNAs, their flanking regions (Upstream and Downstream), and a group of 10,000 randomly selected piRNAs in piRNA clusters are shown in boxplots. Of note, the same measurements for all synonymous and nonsynonymous sites were calculated as references, with the median values denoted by the blue and red dotted lines, respectively. All P values were derived from Wilcoxon one-tailed test.
    Rna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna - by Bioz Stars, 2022-05
    94/100 stars

    Images

    1) Product Images from "Selectively Constrained RNA Editing Regulation Crosstalks with piRNA Biogenesis in Primates"

    Article Title: Selectively Constrained RNA Editing Regulation Crosstalks with piRNA Biogenesis in Primates

    Journal: Molecular Biology and Evolution

    doi: 10.1093/molbev/msv183

    Population genetics analysis of epiRNA-associated RNA editing events. Extent of selective constraints for epiRNA-associated editing events was evaluated as described in Materials and Methods . The distribution of SNP densities ( A ), SNP nucleotide diversity ( B ), and the frequency spectra of derived allele ( C ) in the local regions of epiRNA-associated focal editing sites were determined and quantitatively represented as boxplots or means ± SD, with the more distal regions (Upstream and Downstream) as the references. ( D ) The nucleotide diversity of epiRNAs, their flanking regions (Upstream and Downstream), and a group of 10,000 randomly selected piRNAs in piRNA clusters are shown in boxplots. Of note, the same measurements for all synonymous and nonsynonymous sites were calculated as references, with the median values denoted by the blue and red dotted lines, respectively. All P values were derived from Wilcoxon one-tailed test.
    Figure Legend Snippet: Population genetics analysis of epiRNA-associated RNA editing events. Extent of selective constraints for epiRNA-associated editing events was evaluated as described in Materials and Methods . The distribution of SNP densities ( A ), SNP nucleotide diversity ( B ), and the frequency spectra of derived allele ( C ) in the local regions of epiRNA-associated focal editing sites were determined and quantitatively represented as boxplots or means ± SD, with the more distal regions (Upstream and Downstream) as the references. ( D ) The nucleotide diversity of epiRNAs, their flanking regions (Upstream and Downstream), and a group of 10,000 randomly selected piRNAs in piRNA clusters are shown in boxplots. Of note, the same measurements for all synonymous and nonsynonymous sites were calculated as references, with the median values denoted by the blue and red dotted lines, respectively. All P values were derived from Wilcoxon one-tailed test.

    Techniques Used: Derivative Assay, One-tailed Test

    Experimental verification of epiRNAs. ( A , B ) For two selected epiRNA candidates, the deep sequencing raw data as well as the Sanger sequencing results corresponding to the genome, mRNA and small RNA in the macaque animal (100MGP-001) testis sample are shown. The detected editing sites are highlighted by black box (in deep sequencing) or red arrows (in Sanger sequencing).
    Figure Legend Snippet: Experimental verification of epiRNAs. ( A , B ) For two selected epiRNA candidates, the deep sequencing raw data as well as the Sanger sequencing results corresponding to the genome, mRNA and small RNA in the macaque animal (100MGP-001) testis sample are shown. The detected editing sites are highlighted by black box (in deep sequencing) or red arrows (in Sanger sequencing).

    Techniques Used: Sequencing

    Interaction of RNA editing and piRNA biogenesis. ( A ) The plot shows the percentages of epiRNAs and mRNA degradation fragments with the nucleotide uridine at the 5′-end of the respective sequences. ( B ) Heatmaps showing the relative expression levels across seven different macaque tissues for long transcripts (left) and small RNAs (right) corresponding to the epiRNA-associated regions, with reference to the color scale on top. ( C ) The differences in ADAR expression levels and normalized piRNA tag types across four different animals are shown in linear graphs on the left. The heatmap on the right depicts the relative expression levels of piRNAs in each piRNA cluster of the four animals, organized and scaled in rows. These piRNA clusters were categorized into three groups: epiRNAs-expressing clusters (yellow), “editing-absent” epiRNA clusters (overlapping with editing sites on the long transcripts but lack detectable epiRNAs; orange), and canonical piRNA clusters (red), as indicated by the color bar on top.
    Figure Legend Snippet: Interaction of RNA editing and piRNA biogenesis. ( A ) The plot shows the percentages of epiRNAs and mRNA degradation fragments with the nucleotide uridine at the 5′-end of the respective sequences. ( B ) Heatmaps showing the relative expression levels across seven different macaque tissues for long transcripts (left) and small RNAs (right) corresponding to the epiRNA-associated regions, with reference to the color scale on top. ( C ) The differences in ADAR expression levels and normalized piRNA tag types across four different animals are shown in linear graphs on the left. The heatmap on the right depicts the relative expression levels of piRNAs in each piRNA cluster of the four animals, organized and scaled in rows. These piRNA clusters were categorized into three groups: epiRNAs-expressing clusters (yellow), “editing-absent” epiRNA clusters (overlapping with editing sites on the long transcripts but lack detectable epiRNAs; orange), and canonical piRNA clusters (red), as indicated by the color bar on top.

    Techniques Used: Expressing

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86
    New England Biolabs cid1 poly u polymerase
    Biochemical determination of inosine tail lengths. A. Scheme for analyzing inosine tails based on RNAse A resistance of purine homopolymers. Top, poly(A)+ mRNAs, bottom, non-polyadenylated RNAs. From left to right: tailing by <t>Cid1,</t> 3’ end labeling by T4 RNA ligase and 32 P-pCp, RNAse A digestion. B. Cid1 adds ~50 inosine residues onto the poly(A) tail of a model poly(A)+ mRNA. Left, labeled products without (-) and with (+) I-tailing, and without RNAse A digestion, run on a 6% denaturing polyacrylamide gel. Right, labeled products without (-) and with (+) I-tailing after RNAse A digestion, run on a 12% denaturing polyacrylamide gel. Markers are indicated as for Fig 1 . C. Cid1 adds a long heterogeneous tract of inosine residues onto the 3’ end of a model non-polyadenylated mRNA. Lanes are as for (B) above. D. Cid1 adds a long heterogeneous tract of inosine residues onto the 3’ end of the non-polyadenylated 5.8S rRNA. Lanes are as for (B) above.
    Cid1 Poly U Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cid1 poly u polymerase/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cid1 poly u polymerase - by Bioz Stars, 2022-05
    86/100 stars
      Buy from Supplier

    94
    New England Biolabs rna polymerase core
    RNAP bypass by mRNA takeover is reduced as the complexity of CO replication-transcription collisions increases. ( A ) Replication reaction mixtures on template CO100 containing [α- 32 P]GMP-labeled 19mer-RNAP (lanes 1–4), 100mer RNAP (5–8), and RNAP arrays (9–12) were incubated for the indicated times with or without E. coli replication proteins except <t>DnaA</t> (rep. pro.) and with or without DnaA. The products were analyzed by electrophoresis through a composite 5%/20% 7 M urea polyacrylamide gel. ( B ) Fraction of the labeled <t>RNA</t> extended by mRNA takeover on the gel shown in panel A. ( C ) mRNA takeover (%) relative to the extent of replication measured by [α- 32 P]dAMP incorporation in independent replication reactions using the same RNAP template preparations as in panel A. Replication efficiency averaged 34 ± 12% (1 min)—52 ± 15% (8 min) for the 19mer; 36 ± 21% (1 min)—51 ± 25% (8 min) for the 100mer; and 30 ± 8% (1 min)—45 ± 12% (8 min) for the RNAP array ( n = 4, mean ± standard deviation). ( D ) Assay protocol to estimate RNAP displacement by active DNA replication. ( E ) Ratios of β′ and α RNAP subunit signal intensities from western blots (replicated/unreplicated) in reactions before and after gel filtration in high salt compared to the RNAP subunit ratio predicted for active RNAP dissociation by replisome bypass ( n = 3, mean ± standard deviation). A more detailed analysis of one of the experimental repeats is shown in Supplementary Figure S3 . Gray ovals, RNAP.
    Rna Polymerase Core, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna polymerase core/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna polymerase core - by Bioz Stars, 2022-05
    94/100 stars
      Buy from Supplier

    Image Search Results


    Biochemical determination of inosine tail lengths. A. Scheme for analyzing inosine tails based on RNAse A resistance of purine homopolymers. Top, poly(A)+ mRNAs, bottom, non-polyadenylated RNAs. From left to right: tailing by Cid1, 3’ end labeling by T4 RNA ligase and 32 P-pCp, RNAse A digestion. B. Cid1 adds ~50 inosine residues onto the poly(A) tail of a model poly(A)+ mRNA. Left, labeled products without (-) and with (+) I-tailing, and without RNAse A digestion, run on a 6% denaturing polyacrylamide gel. Right, labeled products without (-) and with (+) I-tailing after RNAse A digestion, run on a 12% denaturing polyacrylamide gel. Markers are indicated as for Fig 1 . C. Cid1 adds a long heterogeneous tract of inosine residues onto the 3’ end of a model non-polyadenylated mRNA. Lanes are as for (B) above. D. Cid1 adds a long heterogeneous tract of inosine residues onto the 3’ end of the non-polyadenylated 5.8S rRNA. Lanes are as for (B) above.

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Biochemical determination of inosine tail lengths. A. Scheme for analyzing inosine tails based on RNAse A resistance of purine homopolymers. Top, poly(A)+ mRNAs, bottom, non-polyadenylated RNAs. From left to right: tailing by Cid1, 3’ end labeling by T4 RNA ligase and 32 P-pCp, RNAse A digestion. B. Cid1 adds ~50 inosine residues onto the poly(A) tail of a model poly(A)+ mRNA. Left, labeled products without (-) and with (+) I-tailing, and without RNAse A digestion, run on a 6% denaturing polyacrylamide gel. Right, labeled products without (-) and with (+) I-tailing after RNAse A digestion, run on a 12% denaturing polyacrylamide gel. Markers are indicated as for Fig 1 . C. Cid1 adds a long heterogeneous tract of inosine residues onto the 3’ end of a model non-polyadenylated mRNA. Lanes are as for (B) above. D. Cid1 adds a long heterogeneous tract of inosine residues onto the 3’ end of the non-polyadenylated 5.8S rRNA. Lanes are as for (B) above.

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques: End Labeling, Labeling

    Capture and analysis of RNA polymerase II (RNAPII) associated nascent transcripts by Cid1 I-tailing. A. Protocol for isolating RNAPII-associated transcripts from chromatin (see methods). B. A model for co-transcriptional splicing. Introns bounded by 5’ splice sites (green) and 3’ splice sites (gold) are removed creating exon-exon junctions (blue) in nascent transcripts as RNAPII (magenta) moves down the gene. C. genome browser views of nanopore reads mapping to ACT1 (top), RPS4B (middle), and FBA1 (bottom). The 3’ ends of reads from nascent RNAs are distributed along the gene, and splicing usually occurs before RNAPII has moved 100 nt beyond the 3’ splice site.

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Capture and analysis of RNA polymerase II (RNAPII) associated nascent transcripts by Cid1 I-tailing. A. Protocol for isolating RNAPII-associated transcripts from chromatin (see methods). B. A model for co-transcriptional splicing. Introns bounded by 5’ splice sites (green) and 3’ splice sites (gold) are removed creating exon-exon junctions (blue) in nascent transcripts as RNAPII (magenta) moves down the gene. C. genome browser views of nanopore reads mapping to ACT1 (top), RPS4B (middle), and FBA1 (bottom). The 3’ ends of reads from nascent RNAs are distributed along the gene, and splicing usually occurs before RNAPII has moved 100 nt beyond the 3’ splice site.

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques:

    Quantitative evaluation of yeast mRNA detection after I-tailing. A. Strongly correlated abundance measurements for > 5000 yeast mRNAs using either standard poly(A)+ capture or I-tailing by Cid1 and capture with the oligoC adapter. B. Estimation of median tail lengths by Nanopolish of poly(A)+ mRNAs using direct capture of poly(A)+ with the standard oligo(dT) adapter (black) or after tailing and capture with the oligoC adapter (blue). The version of Nanopolish used here has only been trained to distinguish and measure poly(A) tails (see text).

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Quantitative evaluation of yeast mRNA detection after I-tailing. A. Strongly correlated abundance measurements for > 5000 yeast mRNAs using either standard poly(A)+ capture or I-tailing by Cid1 and capture with the oligoC adapter. B. Estimation of median tail lengths by Nanopolish of poly(A)+ mRNAs using direct capture of poly(A)+ with the standard oligo(dT) adapter (black) or after tailing and capture with the oligoC adapter (blue). The version of Nanopolish used here has only been trained to distinguish and measure poly(A) tails (see text).

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques:

    Ability of Cid1 to use modified UTP analogs for tailing. Cid1 tailing reactions were set up with the indicated analog either without (-, 1mM) or with (+, 0.5 mM each) competing unmodified UTP, using the MYL6(A+) model mRNA as a substrate. The 6% denaturing polyacrylamide gel was stained with SYBR Gold and imaged on a Typhoon Imager.

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Ability of Cid1 to use modified UTP analogs for tailing. Cid1 tailing reactions were set up with the indicated analog either without (-, 1mM) or with (+, 0.5 mM each) competing unmodified UTP, using the MYL6(A+) model mRNA as a substrate. The 6% denaturing polyacrylamide gel was stained with SYBR Gold and imaged on a Typhoon Imager.

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques: Modification, Staining

    Nanopore ionic current signals for poly(I)-tailed RNA molecules. A. Schematic for Nanopore adaptation of poly(I)-tailed RNA molecules. This step employs a poly(dC)10 oligomer that anneals to the terminal 10 nucleotides of the added inosine tail. B. Ionic current traces produced by the GLuc200 model mRNA (IV, blue) with or without a poly(A) tail (II, green) and with or without a ligated 30 nt segment of inosine (III, purple). Adaptor sequence trace is shown in gray (I). RNA molecules enter the pore 3’ end first, so the trace records transit through the pore in the 3’ to 5’ direction. C. Ionic current traces produced by translocation of native yeast TDH3 mRNA or 25S rRNA, with or without I-tailing by Cid1. Segments are colored as in ( B ).

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Nanopore ionic current signals for poly(I)-tailed RNA molecules. A. Schematic for Nanopore adaptation of poly(I)-tailed RNA molecules. This step employs a poly(dC)10 oligomer that anneals to the terminal 10 nucleotides of the added inosine tail. B. Ionic current traces produced by the GLuc200 model mRNA (IV, blue) with or without a poly(A) tail (II, green) and with or without a ligated 30 nt segment of inosine (III, purple). Adaptor sequence trace is shown in gray (I). RNA molecules enter the pore 3’ end first, so the trace records transit through the pore in the 3’ to 5’ direction. C. Ionic current traces produced by translocation of native yeast TDH3 mRNA or 25S rRNA, with or without I-tailing by Cid1. Segments are colored as in ( B ).

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques: Produced, Sequencing, Translocation Assay

    Nucleotide-adding activity of S. pombe Cid1 and S. cerevisiae Poly(A) polymerase (PAP) on various model RNA substrates. RNA was incubated with enzyme and rNTPs as indicated and run on denaturing polyacrylamide gels, stained with SYBR Gold and imaged on a Typhoon scanner. Markers are DNA with the indicated chain lengths for the equivalently migrating RNA, using the ~1.04X greater mass to charge ratio of RNA per residue. For example, a 500 nt DNA marks the approximate migration of a 482 residue RNA. A. Cid1 adds long tails of A, U, or I, but much shorter tails of C or G to A24. B. PAP adds long tails of A, a 35-50 nt stretch of G, but only short stretches of C, U, or I to A24. C. Cid1 efficiently adds long stretches of A or U, but mostly just 50 residues of I to a model poly(A)+ mRNA, whereas it inefficiently adds A, U, or I to a non-polyadenylated RNA. D. PAP efficiently adds A to either polyadenylated or non-polyadenylated RNA (lanes 3 and 6) and adds a short stretch of I to poly(A)+ RNA, but only inefficiently adds U or I to non-polyadenylated RNA.

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Nucleotide-adding activity of S. pombe Cid1 and S. cerevisiae Poly(A) polymerase (PAP) on various model RNA substrates. RNA was incubated with enzyme and rNTPs as indicated and run on denaturing polyacrylamide gels, stained with SYBR Gold and imaged on a Typhoon scanner. Markers are DNA with the indicated chain lengths for the equivalently migrating RNA, using the ~1.04X greater mass to charge ratio of RNA per residue. For example, a 500 nt DNA marks the approximate migration of a 482 residue RNA. A. Cid1 adds long tails of A, U, or I, but much shorter tails of C or G to A24. B. PAP adds long tails of A, a 35-50 nt stretch of G, but only short stretches of C, U, or I to A24. C. Cid1 efficiently adds long stretches of A or U, but mostly just 50 residues of I to a model poly(A)+ mRNA, whereas it inefficiently adds A, U, or I to a non-polyadenylated RNA. D. PAP efficiently adds A to either polyadenylated or non-polyadenylated RNA (lanes 3 and 6) and adds a short stretch of I to poly(A)+ RNA, but only inefficiently adds U or I to non-polyadenylated RNA.

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques: Activity Assay, Incubation, Staining, Migration

    Detection of non-polyadenylated RNAs. A. I-tailing of total yeast RNA. Yeast RNA treated with (+) or without (-) Cid1 and ITP for I-tailing were run on a 6% denaturing polyacrylamide gel and stained with SYBR Gold. Slight shift up of 18 and 25S rRNAs indicates addition of poly(I). Disappearance of 5.8S rRNA is consistent with its acquisition of a long heterogeneous tail (See Fig 2 ). B. genome browser view of the rRNA genes of S. cerevisiae showing detection of rRNA by different library preparation methods. Top line is poly(A) selected RNA using the direct oligoT capture method, second line is poly(A) selected and I-tailed RNA captured with the oligoC adapter, third line is total RNA I-tailed and captured with the oligoC adapter, fourth line is Ribominus rRNA depleted total RNA I-tailed and captured with the oligoC adapter. C. Scatter plot showing robust detection of non-polyadenylated noncoding RNAs by I-tailing and capture with the oligoC adapter.

    Journal: bioRxiv

    Article Title: Synthesis of modified nucleotide polymers by the poly(U) polymerase Cid1: Application to direct RNA sequencing on nanopores

    doi: 10.1101/2021.07.06.451372

    Figure Lengend Snippet: Detection of non-polyadenylated RNAs. A. I-tailing of total yeast RNA. Yeast RNA treated with (+) or without (-) Cid1 and ITP for I-tailing were run on a 6% denaturing polyacrylamide gel and stained with SYBR Gold. Slight shift up of 18 and 25S rRNAs indicates addition of poly(I). Disappearance of 5.8S rRNA is consistent with its acquisition of a long heterogeneous tail (See Fig 2 ). B. genome browser view of the rRNA genes of S. cerevisiae showing detection of rRNA by different library preparation methods. Top line is poly(A) selected RNA using the direct oligoT capture method, second line is poly(A) selected and I-tailed RNA captured with the oligoC adapter, third line is total RNA I-tailed and captured with the oligoC adapter, fourth line is Ribominus rRNA depleted total RNA I-tailed and captured with the oligoC adapter. C. Scatter plot showing robust detection of non-polyadenylated noncoding RNAs by I-tailing and capture with the oligoC adapter.

    Article Snippet: Cid1 poly(U) polymerase can efficiently add inosine to the 3’ ends of RNA To find new ways to add modified nucleotides to the 3’ ends of RNA, we tested commercial preparations of two well-studied enzymes, the S. pombe poly(U) polymerase Cid1 , and S. cerevisiae poly(A) polymerase (PAP) ( ).

    Techniques: Staining

    RNAP bypass by mRNA takeover is reduced as the complexity of CO replication-transcription collisions increases. ( A ) Replication reaction mixtures on template CO100 containing [α- 32 P]GMP-labeled 19mer-RNAP (lanes 1–4), 100mer RNAP (5–8), and RNAP arrays (9–12) were incubated for the indicated times with or without E. coli replication proteins except DnaA (rep. pro.) and with or without DnaA. The products were analyzed by electrophoresis through a composite 5%/20% 7 M urea polyacrylamide gel. ( B ) Fraction of the labeled RNA extended by mRNA takeover on the gel shown in panel A. ( C ) mRNA takeover (%) relative to the extent of replication measured by [α- 32 P]dAMP incorporation in independent replication reactions using the same RNAP template preparations as in panel A. Replication efficiency averaged 34 ± 12% (1 min)—52 ± 15% (8 min) for the 19mer; 36 ± 21% (1 min)—51 ± 25% (8 min) for the 100mer; and 30 ± 8% (1 min)—45 ± 12% (8 min) for the RNAP array ( n = 4, mean ± standard deviation). ( D ) Assay protocol to estimate RNAP displacement by active DNA replication. ( E ) Ratios of β′ and α RNAP subunit signal intensities from western blots (replicated/unreplicated) in reactions before and after gel filtration in high salt compared to the RNAP subunit ratio predicted for active RNAP dissociation by replisome bypass ( n = 3, mean ± standard deviation). A more detailed analysis of one of the experimental repeats is shown in Supplementary Figure S3 . Gray ovals, RNAP.

    Journal: Nucleic Acids Research

    Article Title: Bypass of complex co-directional replication-transcription collisions by replisome skipping

    doi: 10.1093/nar/gkab760

    Figure Lengend Snippet: RNAP bypass by mRNA takeover is reduced as the complexity of CO replication-transcription collisions increases. ( A ) Replication reaction mixtures on template CO100 containing [α- 32 P]GMP-labeled 19mer-RNAP (lanes 1–4), 100mer RNAP (5–8), and RNAP arrays (9–12) were incubated for the indicated times with or without E. coli replication proteins except DnaA (rep. pro.) and with or without DnaA. The products were analyzed by electrophoresis through a composite 5%/20% 7 M urea polyacrylamide gel. ( B ) Fraction of the labeled RNA extended by mRNA takeover on the gel shown in panel A. ( C ) mRNA takeover (%) relative to the extent of replication measured by [α- 32 P]dAMP incorporation in independent replication reactions using the same RNAP template preparations as in panel A. Replication efficiency averaged 34 ± 12% (1 min)—52 ± 15% (8 min) for the 19mer; 36 ± 21% (1 min)—51 ± 25% (8 min) for the 100mer; and 30 ± 8% (1 min)—45 ± 12% (8 min) for the RNAP array ( n = 4, mean ± standard deviation). ( D ) Assay protocol to estimate RNAP displacement by active DNA replication. ( E ) Ratios of β′ and α RNAP subunit signal intensities from western blots (replicated/unreplicated) in reactions before and after gel filtration in high salt compared to the RNAP subunit ratio predicted for active RNAP dissociation by replisome bypass ( n = 3, mean ± standard deviation). A more detailed analysis of one of the experimental repeats is shown in Supplementary Figure S3 . Gray ovals, RNAP.

    Article Snippet: Replication proteins were purified as described previously: RNA polymerase core ( ); σ70 ( ); DnaA and HU ( ); DnaB, DnaC, and DnaG ( ); DnaN (β clamp) ( ); Pol III* ( ); SSB ( ); Tus ( ); DNA Gyrase ( ); UvrD, gift of T. Lohman.

    Techniques: Labeling, Incubation, Electrophoresis, Standard Deviation, Western Blot, Filtration

    Inhibition of viral infection in benfooxythiamine (BOT)-treated cells in combination with 2-deoxy- d -glucose (2DG). Caco-2 cells were pre-treated with different concentration of BOT for 24 h. Then, the 2DG at concentration 5mM or 10 mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. ( A ) Representative images illustrating immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. ( B ) Quantification of illustrating immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Values represent the mean ± SD of the three independent experiments. p -values were determined with a two-sided unpaired t -test. *** p ≤ 0.001 ( C ) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. SARS-CoV-2/FFM7 RNA copy numbers used the RNA-polymerase (RdRp) gene by qRT-PCR of RdRp gene. Values represent mean ± SD of the three independent experiments. p -values were determined with a two-sided unpaired t -test. ns: not significant; * p ≤ 0.05; ** p ≤ 0.01. Effects of BOT in combination with 2DG on cell viability are provided in Supplementary Figure S1 . ( D ) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com (accessed on 18 August 2021).

    Journal: Metabolites

    Article Title: Targeting the Pentose Phosphate Pathway for SARS-CoV-2 Therapy

    doi: 10.3390/metabo11100699

    Figure Lengend Snippet: Inhibition of viral infection in benfooxythiamine (BOT)-treated cells in combination with 2-deoxy- d -glucose (2DG). Caco-2 cells were pre-treated with different concentration of BOT for 24 h. Then, the 2DG at concentration 5mM or 10 mM was added and cells were infected with SARS-CoV-2/FFM7 at MOI 0.01. ( A ) Representative images illustrating immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. ( B ) Quantification of illustrating immunohistochemistry staining of SARS-CoV-2 spike protein in SARS-CoV-2/FFM7 infected Caco-2 cells treated with BOT in combination with 2DG. Values represent the mean ± SD of the three independent experiments. p -values were determined with a two-sided unpaired t -test. *** p ≤ 0.001 ( C ) Quantification of viral genomes in supernatant of SARS-CoV-2 infected Caco-2 cells treated with BOT in combination with 2DG or BOT alone. SARS-CoV-2/FFM7 RNA copy numbers used the RNA-polymerase (RdRp) gene by qRT-PCR of RdRp gene. Values represent mean ± SD of the three independent experiments. p -values were determined with a two-sided unpaired t -test. ns: not significant; * p ≤ 0.05; ** p ≤ 0.01. Effects of BOT in combination with 2DG on cell viability are provided in Supplementary Figure S1 . ( D ) Simplified scheme of glycolysis and pentose phosphate pathway. The targets for 2DG and BOT are depicted in red. The scheme was created with BioRender.com (accessed on 18 August 2021).

    Article Snippet: The amount of viral RNA was detected by primers targeting the RNA-dependent RNA polymerase (RdRp): RdRP_SARSr-F2 (GTGARATGGTCATGTGTGGCGG) and dRP_SARSr-R1(CARATGTTAAASACACTATTAGCATA) using the Luna Universal One-Step RT-qPCR Kit (New England Biolabs) and a CFX96 Real-Time System C1000 Touch Thermal Cycler.

    Techniques: Inhibition, Infection, Concentration Assay, Immunohistochemistry, Staining, Quantitative RT-PCR

    Immunofluorescence of differentiated HBE cells infected with HRV-C651 or HRV-C15. Immunofluorescence with the rabbit polyclonal anti-HRV-C VP1 protein-antibody only ( A ) and mAbJ2 antibody ( D ). At 24 h post-inoculation with HRV-C651 or HRV-C15, the HBE cells were fixed, followed by incubation with VP1 protein-antibody ( B and C ) and mAb12 antibody detecting double-strand RNA ( E and F ). The red region indicates VP1 protein ( B and C ) or the double-stranded RNA of HRV-C ( E and F ). Confocal images were taken with a magnification of 200×. Nuclei were stained with DAPI

    Journal: Virology Journal

    Article Title: Comparison of immune response to human rhinovirus C and respiratory syncytial virus in highly differentiated human airway epithelial cells

    doi: 10.1186/s12985-022-01805-2

    Figure Lengend Snippet: Immunofluorescence of differentiated HBE cells infected with HRV-C651 or HRV-C15. Immunofluorescence with the rabbit polyclonal anti-HRV-C VP1 protein-antibody only ( A ) and mAbJ2 antibody ( D ). At 24 h post-inoculation with HRV-C651 or HRV-C15, the HBE cells were fixed, followed by incubation with VP1 protein-antibody ( B and C ) and mAb12 antibody detecting double-strand RNA ( E and F ). The red region indicates VP1 protein ( B and C ) or the double-stranded RNA of HRV-C ( E and F ). Confocal images were taken with a magnification of 200×. Nuclei were stained with DAPI

    Article Snippet: The two plasmids, Lz651 and PC15, containing HRV-C viral genome were digested to linearize using Endonuclease Cal I (NEB), followed by HRV-C RNA transcription in vitro with a MEGAscript® T7 Transcription Kit (Ambion) in accordance with the manufacturer’s instructions.

    Techniques: Immunofluorescence, Infection, Incubation, Staining

    Four HRV-C strains and two RSV strains grew successfully in ALI HBE cells ( A and B ). Virus RNA load from infected tissue supernatants was measured by real-time RT-PCR collected at the apical surface of ALI HBE cells infected with HRV-C strains (HRV-C 651, HRV-C15, HRV-C 79, or HRV-C 101) and two RSV strains (RSV1 or RSV2). Error bars represent the standard deviation calculated from biological replicates (n = 3)

    Journal: Virology Journal

    Article Title: Comparison of immune response to human rhinovirus C and respiratory syncytial virus in highly differentiated human airway epithelial cells

    doi: 10.1186/s12985-022-01805-2

    Figure Lengend Snippet: Four HRV-C strains and two RSV strains grew successfully in ALI HBE cells ( A and B ). Virus RNA load from infected tissue supernatants was measured by real-time RT-PCR collected at the apical surface of ALI HBE cells infected with HRV-C strains (HRV-C 651, HRV-C15, HRV-C 79, or HRV-C 101) and two RSV strains (RSV1 or RSV2). Error bars represent the standard deviation calculated from biological replicates (n = 3)

    Article Snippet: The two plasmids, Lz651 and PC15, containing HRV-C viral genome were digested to linearize using Endonuclease Cal I (NEB), followed by HRV-C RNA transcription in vitro with a MEGAscript® T7 Transcription Kit (Ambion) in accordance with the manufacturer’s instructions.

    Techniques: Infection, Quantitative RT-PCR, Standard Deviation