ribonuclease t1 reactions Search Results


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  • 99
    Thermo Fisher ribonuclease t1 reactions
    C2c2 proteins process precursor crRNA transcripts to generate mature crRNAs a , Maximum-likelihood phylogenetic tree of C2c2 proteins. Homologs used in this study are highlighted in yellow. b , Diagram of the three Type VI CRISPR loci used in this study. Black rectangles denote repeat elements, yellow diamonds denote spacer sequences. Cas1 and Cas2 are only found in the genomic vicinity of LshC2c2. c , C2c2-mediated cleavage of pre-crRNA derived from the LbuC2c2, LseC2c2 and LshC2c2 CRISPR repeat loci. OH: alkaline hydrolysis ladder; T1: <t>RNase</t> T1 hydrolysis ladder; processing cleavage reactions were performed with 100 nM C2c2 and
    Ribonuclease T1 Reactions, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore rnase t1
    Hydroxyl radical footprinting of Cbf5–Pf9 and L7Ae–Pf9 complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 4, 11, 16) or presence of increasing concentrations of Cbf5 (lanes 5–10) or L7Ae (lanes 12–15) and subjected to hydroxyl radical cleavage. Lane 1 is undigested RNA and lanes 2 and 3 are size markers generated by alkaline hydrolysis (OH) and <t>RNase</t> T1 digestion (T1) of the free RNA, respectively. Nucleotides corresponding to secondary structure landmarks are indicated to the right. Blue and green bars indicate regions of strong Cbf5 and L7Ae protection, respectively. ( B ) Summary of protections in the context of a functional secondary structure model of Pf9 RNA. Box ACA, the pseudouridylation pocket and k-turn are boxed. Apical loop, upper and lower stems are labelled. The rRNA target of Pf9 is shown in grey lowercase letters. Cbf5 and L7Ae protections observed in A are shown as indicated in the legend. The regions shaded grey were not assessed due to the resolution limits of the gel.
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    Roche rnase t1 reactions
    LRP5 3′-UTR PG4 folds into a G4 structure in vitro . ( a ) Sequence and numbering of the wt LRP5 PG4 used in the in vitro experiments. The lowercase guanosines (g) correspond to those mutated to adenosines in the G/A-mutant version. Nucleotides that were hydrolyzed significantly more in the presence of KCl during the in-line probing are both in bold and underlined. ( b , c ) CD spectra for the LRP5 PG4 sequence using 4 µM of either the wt (b) or the G/A-mutant (c) versions performed either in the absence of salt (closed circle) or in the presence of 100 mM of either LiCl (inverted closed triangle), NaCl (open circle) or KCl (open triangle). ( d ) Autoradiogram of a 10% denaturing polyacrylamide gel of the in-line probing of the 5′-end-labeled LRP5 wt and G/A-mutant PG4 versions performed either in the absence of salt (NS), or in the presence of 100 mM of either LiCl, NaCl or KCl. Lanes L and T1 correspond to alkaline hydrolysis and <t>RNase</t> T1 mapping of the wt version, respectively. The positions of the guanosines are indicated on the left of the gel, whereas the domains of the G4 structure are indicated on the right.
    Rnase T1 Reactions, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher rnase a t1
    Hu proteins and FXR2P bind to  PSD95  mRNA.  A , Brain extracts were precipitated by specific antibodies against FMRP and HuD in the presence (+) or absence (−) of RNase A/T1 and control IgGs. Coimmunoprecipitated proteins FMRP, HuR, and other Hu members (panHu) are indicated.  B1 , Representative image of the CA3 region of hippocampus stained with anti-FMRP (red) and anti-HuR or HuD (green). Scale bar, 50 μm.  B2 , FMRP (red) and FXR2P or HuD or HuR (green) staining on primary neurons at 12 d  in vitro . White arrows indicate protein colocalization quantified by the Mander's coefficient ( n  = 122 for FXR2P;  n  = 44 for HuD;  n  = 34 for HuR). Scale bar, 12.5 μm.  C , HuD RNA-IP from WT and  Fmr1  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qRT-PCR.  D , FXR2P-IP from WT and  Fxr2  KO brain extracts detected by Western blotting.  E , Same as in  A , FMRP-IP and FXR2P-IP in the presence of RNase A/T1 detected by Western blotting.  F , FXR2P RNA-IP from WT and  Fmr1  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qPCR.  G , FMRP-IP from WT and  Fxr2  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qPCR. mRNA levels were calculated using the formula 2^ − (Ct PSD95  − Ct exogenous normalizerBC200 ) and normalized to the mRNA present in the input and the mock IP.  C ,  n  = 7 independent experiments.  F ,  n  = 7 independent experiments.  G ,  n  = 7 independent experiments. ** p
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    95
    Thermo Fisher rnase a t1 mix
    Optimization of ORF57 immunoprecipitation and RNase digestion. (A) ORF57-RNA complexes were immunoprecipitated by a rabbit anti-ORF57 antibody from cells extract of BCBL-1 cells treated with valproic acid for 24 h (input), normal rabbit IgG served as a negative control. The levels of ORF57 in input (1%) and immunoprecipitates (3%) in CLIP experiment were detected by Western blot with mouse anti-ORF57 antibody detecting both full-length ORF57 protein (upper band) and its caspase-cleavage product (lower band). (B) To determine an optimal RNase digestion condition, the immunoprecipitated ORF57 complexes were incubated with various amounts of RNase <t>A/T1</t> mix for 5 sec at room temperature and followed by proteinase K treatment and RNA extraction. The RNase A/T1 digestion efficiency was checked by RT-PCR for the remaining KSHV PAN RNA, a known ORF57 target.
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    Thermo Fisher rnase t1 mixture
    Optimization of ORF57 immunoprecipitation and RNase digestion. (A) ORF57-RNA complexes were immunoprecipitated by a rabbit anti-ORF57 antibody from cells extract of BCBL-1 cells treated with valproic acid for 24 h (input), normal rabbit IgG served as a negative control. The levels of ORF57 in input (1%) and immunoprecipitates (3%) in CLIP experiment were detected by Western blot with mouse anti-ORF57 antibody detecting both full-length ORF57 protein (upper band) and its caspase-cleavage product (lower band). (B) To determine an optimal RNase digestion condition, the immunoprecipitated ORF57 complexes were incubated with various amounts of RNase <t>A/T1</t> mix for 5 sec at room temperature and followed by proteinase K treatment and RNA extraction. The RNase A/T1 digestion efficiency was checked by RT-PCR for the remaining KSHV PAN RNA, a known ORF57 target.
    Rnase T1 Mixture, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim rnase t1
    Structural and functional probing of mccA transcript. ( A ) 32 P-5′-end labeled in vitro synthesized wild-type or mutant mccA RNA was digested with <t>RNase</t> T1 or RNase T2. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. Lanes labeled ‘control’ show material that was not treated with RNAses. Lanes labeled ‘SEQ T1’ are marker lanes, showing digestion pattern obtained with RNase T1 under denaturing conditions (cleavages at every G). In lanes labeled ‘RNase T1’ and ‘RNase T2’ transcripts were re-folded in the presence of 10 mM MgCl 2 and then digested with RNases. In lanes labeled ‘PURExpress’ wild-type mccA RNA was folded in the presence of PURExpress full system in the presence of thiostrepton. ( B ) A secondary structure of mccA RNA produced by RNAfold software ( http://rna.tbi.univie.ac.at/ ) and consistent with RNase probing results. Cleavage positions by RNase T1 and RNase T2 are shown, respectively, with black and white asterisks. Elements of the structure (loops, L, and hairpins, H) are also marked on the gel shown in panel A. A fragment of mccA tested for in vivo termination activity is indicated. ( C ) A DNA fragment coding for a hairpin structure and adjacent sequences shown in panel C was cloned in direct (pFD_mcc_dir) and inverted (pFD_mcc_inv) orientations between the galK reporter and the lac UV5 promoter of plasmid pFD100. The resulting plasmids, as well as control pFD51 plasmid lacking the promoter, were transformed into galK − E. coli cells and transformants were then tested for GalK activity on McConkey indicator agar plates. Overnight growth of cells is shown.
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    89
    Boehringer Mannheim ribonuclease t1
    Structural and functional probing of mccA transcript. ( A ) 32 P-5′-end labeled in vitro synthesized wild-type or mutant mccA RNA was digested with <t>RNase</t> T1 or RNase T2. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. Lanes labeled ‘control’ show material that was not treated with RNAses. Lanes labeled ‘SEQ T1’ are marker lanes, showing digestion pattern obtained with RNase T1 under denaturing conditions (cleavages at every G). In lanes labeled ‘RNase T1’ and ‘RNase T2’ transcripts were re-folded in the presence of 10 mM MgCl 2 and then digested with RNases. In lanes labeled ‘PURExpress’ wild-type mccA RNA was folded in the presence of PURExpress full system in the presence of thiostrepton. ( B ) A secondary structure of mccA RNA produced by RNAfold software ( http://rna.tbi.univie.ac.at/ ) and consistent with RNase probing results. Cleavage positions by RNase T1 and RNase T2 are shown, respectively, with black and white asterisks. Elements of the structure (loops, L, and hairpins, H) are also marked on the gel shown in panel A. A fragment of mccA tested for in vivo termination activity is indicated. ( C ) A DNA fragment coding for a hairpin structure and adjacent sequences shown in panel C was cloned in direct (pFD_mcc_dir) and inverted (pFD_mcc_inv) orientations between the galK reporter and the lac UV5 promoter of plasmid pFD100. The resulting plasmids, as well as control pFD51 plasmid lacking the promoter, were transformed into galK − E. coli cells and transformants were then tested for GalK activity on McConkey indicator agar plates. Overnight growth of cells is shown.
    Ribonuclease T1, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 89/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Thermo Fisher rnase cocktail enzyme mix
    Structural and functional probing of mccA transcript. ( A ) 32 P-5′-end labeled in vitro synthesized wild-type or mutant mccA RNA was digested with <t>RNase</t> T1 or RNase T2. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. Lanes labeled ‘control’ show material that was not treated with RNAses. Lanes labeled ‘SEQ T1’ are marker lanes, showing digestion pattern obtained with RNase T1 under denaturing conditions (cleavages at every G). In lanes labeled ‘RNase T1’ and ‘RNase T2’ transcripts were re-folded in the presence of 10 mM MgCl 2 and then digested with RNases. In lanes labeled ‘PURExpress’ wild-type mccA RNA was folded in the presence of PURExpress full system in the presence of thiostrepton. ( B ) A secondary structure of mccA RNA produced by RNAfold software ( http://rna.tbi.univie.ac.at/ ) and consistent with RNase probing results. Cleavage positions by RNase T1 and RNase T2 are shown, respectively, with black and white asterisks. Elements of the structure (loops, L, and hairpins, H) are also marked on the gel shown in panel A. A fragment of mccA tested for in vivo termination activity is indicated. ( C ) A DNA fragment coding for a hairpin structure and adjacent sequences shown in panel C was cloned in direct (pFD_mcc_dir) and inverted (pFD_mcc_inv) orientations between the galK reporter and the lac UV5 promoter of plasmid pFD100. The resulting plasmids, as well as control pFD51 plasmid lacking the promoter, were transformed into galK − E. coli cells and transformants were then tested for GalK activity on McConkey indicator agar plates. Overnight growth of cells is shown.
    Rnase Cocktail Enzyme Mix, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 482 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t1 ribonuclease structure mapping
    Structural and functional probing of mccA transcript. ( A ) 32 P-5′-end labeled in vitro synthesized wild-type or mutant mccA RNA was digested with <t>RNase</t> T1 or RNase T2. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. Lanes labeled ‘control’ show material that was not treated with RNAses. Lanes labeled ‘SEQ T1’ are marker lanes, showing digestion pattern obtained with RNase T1 under denaturing conditions (cleavages at every G). In lanes labeled ‘RNase T1’ and ‘RNase T2’ transcripts were re-folded in the presence of 10 mM MgCl 2 and then digested with RNases. In lanes labeled ‘PURExpress’ wild-type mccA RNA was folded in the presence of PURExpress full system in the presence of thiostrepton. ( B ) A secondary structure of mccA RNA produced by RNAfold software ( http://rna.tbi.univie.ac.at/ ) and consistent with RNase probing results. Cleavage positions by RNase T1 and RNase T2 are shown, respectively, with black and white asterisks. Elements of the structure (loops, L, and hairpins, H) are also marked on the gel shown in panel A. A fragment of mccA tested for in vivo termination activity is indicated. ( C ) A DNA fragment coding for a hairpin structure and adjacent sequences shown in panel C was cloned in direct (pFD_mcc_dir) and inverted (pFD_mcc_inv) orientations between the galK reporter and the lac UV5 promoter of plasmid pFD100. The resulting plasmids, as well as control pFD51 plasmid lacking the promoter, were transformed into galK − E. coli cells and transformants were then tested for GalK activity on McConkey indicator agar plates. Overnight growth of cells is shown.
    T1 Ribonuclease Structure Mapping, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 18 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen rnase
    snaR RT-PCR clones. ( A ) Clones of snaR identified from sequencing of RT-PCR products ( 21 ) can be grouped into two subsets. Homology within each subset is denoted by an asterisk and non-homologous nucleotides are in bold font. Differences between subsets are denoted by gray shading. Consensus sequences are given below each set of clones. The majority of clones are derived from asynchronous NF90b cell line extract. Clones with ‘m’ or ‘a’ appended to their name were immunoprecipitated from NF90b G2/M phase extract or from NF90a extract, respectively. ( B ) The genomic sequence of snaR-A and -B. Genomic nucleotides matching consensus sequence are highlighted in yellow, those differing from consensus sequence are in green. 3′-Oligo(A) and oligo(T) tracts are denoted in red and blue, respectively. Dashed line denotes sequence complementary to probe H, solid line denotes predicted <t>RNase</t> H digestion product. ( C ) Sequence alignment of snaR-A with two potential piRNAs (bold). Alu <t>RNA</t> homology is denoted in yellow. Sequence homologous to the PolIII B box motif is underlined.
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    Thermo Fisher rnase treatment
    snaR RT-PCR clones. ( A ) Clones of snaR identified from sequencing of RT-PCR products ( 21 ) can be grouped into two subsets. Homology within each subset is denoted by an asterisk and non-homologous nucleotides are in bold font. Differences between subsets are denoted by gray shading. Consensus sequences are given below each set of clones. The majority of clones are derived from asynchronous NF90b cell line extract. Clones with ‘m’ or ‘a’ appended to their name were immunoprecipitated from NF90b G2/M phase extract or from NF90a extract, respectively. ( B ) The genomic sequence of snaR-A and -B. Genomic nucleotides matching consensus sequence are highlighted in yellow, those differing from consensus sequence are in green. 3′-Oligo(A) and oligo(T) tracts are denoted in red and blue, respectively. Dashed line denotes sequence complementary to probe H, solid line denotes predicted <t>RNase</t> H digestion product. ( C ) Sequence alignment of snaR-A with two potential piRNAs (bold). Alu <t>RNA</t> homology is denoted in yellow. Sequence homologous to the PolIII B box motif is underlined.
    Rnase Treatment, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1007 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Roche rnase mix
    snaR RT-PCR clones. ( A ) Clones of snaR identified from sequencing of RT-PCR products ( 21 ) can be grouped into two subsets. Homology within each subset is denoted by an asterisk and non-homologous nucleotides are in bold font. Differences between subsets are denoted by gray shading. Consensus sequences are given below each set of clones. The majority of clones are derived from asynchronous NF90b cell line extract. Clones with ‘m’ or ‘a’ appended to their name were immunoprecipitated from NF90b G2/M phase extract or from NF90a extract, respectively. ( B ) The genomic sequence of snaR-A and -B. Genomic nucleotides matching consensus sequence are highlighted in yellow, those differing from consensus sequence are in green. 3′-Oligo(A) and oligo(T) tracts are denoted in red and blue, respectively. Dashed line denotes sequence complementary to probe H, solid line denotes predicted <t>RNase</t> H digestion product. ( C ) Sequence alignment of snaR-A with two potential piRNAs (bold). Alu <t>RNA</t> homology is denoted in yellow. Sequence homologous to the PolIII B box motif is underlined.
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    Image Search Results


    C2c2 proteins process precursor crRNA transcripts to generate mature crRNAs a , Maximum-likelihood phylogenetic tree of C2c2 proteins. Homologs used in this study are highlighted in yellow. b , Diagram of the three Type VI CRISPR loci used in this study. Black rectangles denote repeat elements, yellow diamonds denote spacer sequences. Cas1 and Cas2 are only found in the genomic vicinity of LshC2c2. c , C2c2-mediated cleavage of pre-crRNA derived from the LbuC2c2, LseC2c2 and LshC2c2 CRISPR repeat loci. OH: alkaline hydrolysis ladder; T1: RNase T1 hydrolysis ladder; processing cleavage reactions were performed with 100 nM C2c2 and

    Journal: Nature

    Article Title: Two Distinct RNase Activities of CRISPR-C2c2 Enable Guide RNA Processing and RNA Detection

    doi: 10.1038/nature19802

    Figure Lengend Snippet: C2c2 proteins process precursor crRNA transcripts to generate mature crRNAs a , Maximum-likelihood phylogenetic tree of C2c2 proteins. Homologs used in this study are highlighted in yellow. b , Diagram of the three Type VI CRISPR loci used in this study. Black rectangles denote repeat elements, yellow diamonds denote spacer sequences. Cas1 and Cas2 are only found in the genomic vicinity of LshC2c2. c , C2c2-mediated cleavage of pre-crRNA derived from the LbuC2c2, LseC2c2 and LshC2c2 CRISPR repeat loci. OH: alkaline hydrolysis ladder; T1: RNase T1 hydrolysis ladder; processing cleavage reactions were performed with 100 nM C2c2 and

    Article Snippet: Reactions were cooled to ambient temperature, and then 1 U of RNase T1 (Ambion) was added to reaction.

    Techniques: CRISPR, Derivative Assay

    RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Journal: Nucleic Acids Research

    Article Title: Group II intron in Bacillus cereus has an unusual 3? extension and splices 56 nucleotides downstream of the predicted site

    doi: 10.1093/nar/gkm031

    Figure Lengend Snippet: RNase T1/A protection assay ( A ) and radioactive RT-PCR ( B ) showing that the extra 56-nt element 3′ of the B.c .I4 intron is part of the intron RNA and not part of the exons. In A , lanes 1, 2 and 3 show positive controls based on mouse RNA, and lanes 4, 5 and 6 show the results based on B. cereus RNA. Lane 1: digested antisense mouse β-actin RNA probe hybridized with mouse liver RNA; lane 2: same probe as in lane 1, undigested; lane 3: same probe as in lane 1, digested, without mouse liver RNA; lane 4: undigested B.c .I4-3′exon junction probe hybridized to B. cereus ATCC 10987 total RNA; lane 5: same probe as in lane 4, digested, without RNA sample; lane 6: same probe as in lane 4, digested, with RNA sample. A schematic of the experiment illustrating the location of the probe and the expected products is shown on the right. The black area represents the extra 56-nt element. In B , lanes 1, 2 and 3: RT-PCR conducted with exon-specific primers I4B_right (radiolabeled) and I4A_left ( Table 1 ) using as template total RNA sample isolated from B. cereus ATCC 10987 at 3, 4 and 6 h of growth, respectively. Lane 4: γ [32-P] ATP 5′-end-labeled pBR322 DNA digested with MspI (New England Biolabs), as marker.

    Article Snippet: Ribonuclease protection assay RNase T1/A protection assay (RPA) was performed using the Ambion RPA III kit following the manufacturer's protocol.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Isolation, Labeling, Marker

    Expression of oppA mRNA transcripts in cultured B. burgdorferi . Expression of the oppA genes was measured by RT PCR (A) and by RPA (B). For RT PCR, RNA was isolated from B. burgdorferi grown in BSK H medium at 37°C. cDNA was generated from total RNA, using gene-specific primers. For each sample, quantitative RT PCR for oppA-I to -V was performed. Determination of copy numbers was calculated by comparison to individual standard curves generated for each primer set. Panel A shows the results of three separate experiments. For RPA, total RNA was hybridized to biotinylated probes specific for each oppA gene and then digested with RNase A-RNase T 1 . Samples were subjected to electrophoresis in a polyacrylamide gel and transferred to nylon membranes. Detection of biotinylated probes was done using chemiluminescence, and quantitation was performed by scanning densitometry. Data shown are individual results from three separate experiments.

    Journal: Journal of Bacteriology

    Article Title: Effects of Environmental Changes on Expression of the Oligopeptide Permease (opp) Genes of Borrelia burgdorferi

    doi: 10.1128/JB.184.22.6198-6206.2002

    Figure Lengend Snippet: Expression of oppA mRNA transcripts in cultured B. burgdorferi . Expression of the oppA genes was measured by RT PCR (A) and by RPA (B). For RT PCR, RNA was isolated from B. burgdorferi grown in BSK H medium at 37°C. cDNA was generated from total RNA, using gene-specific primers. For each sample, quantitative RT PCR for oppA-I to -V was performed. Determination of copy numbers was calculated by comparison to individual standard curves generated for each primer set. Panel A shows the results of three separate experiments. For RPA, total RNA was hybridized to biotinylated probes specific for each oppA gene and then digested with RNase A-RNase T 1 . Samples were subjected to electrophoresis in a polyacrylamide gel and transferred to nylon membranes. Detection of biotinylated probes was done using chemiluminescence, and quantitation was performed by scanning densitometry. Data shown are individual results from three separate experiments.

    Article Snippet: Hybridized RNA was digested with RNase A-RNase T1 at 37°C for 30 min and inactivated according to the manufacturer's instructions (MAXIscript; Ambion).

    Techniques: Expressing, Cell Culture, Reverse Transcription Polymerase Chain Reaction, Recombinase Polymerase Amplification, Isolation, Generated, Quantitative RT-PCR, Electrophoresis, Quantitation Assay

    Hydroxyl radical footprinting of Cbf5–Pf9 and L7Ae–Pf9 complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 4, 11, 16) or presence of increasing concentrations of Cbf5 (lanes 5–10) or L7Ae (lanes 12–15) and subjected to hydroxyl radical cleavage. Lane 1 is undigested RNA and lanes 2 and 3 are size markers generated by alkaline hydrolysis (OH) and RNase T1 digestion (T1) of the free RNA, respectively. Nucleotides corresponding to secondary structure landmarks are indicated to the right. Blue and green bars indicate regions of strong Cbf5 and L7Ae protection, respectively. ( B ) Summary of protections in the context of a functional secondary structure model of Pf9 RNA. Box ACA, the pseudouridylation pocket and k-turn are boxed. Apical loop, upper and lower stems are labelled. The rRNA target of Pf9 is shown in grey lowercase letters. Cbf5 and L7Ae protections observed in A are shown as indicated in the legend. The regions shaded grey were not assessed due to the resolution limits of the gel.

    Journal: Nucleic Acids Research

    Article Title: Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

    doi: 10.1093/nar/gkm673

    Figure Lengend Snippet: Hydroxyl radical footprinting of Cbf5–Pf9 and L7Ae–Pf9 complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 4, 11, 16) or presence of increasing concentrations of Cbf5 (lanes 5–10) or L7Ae (lanes 12–15) and subjected to hydroxyl radical cleavage. Lane 1 is undigested RNA and lanes 2 and 3 are size markers generated by alkaline hydrolysis (OH) and RNase T1 digestion (T1) of the free RNA, respectively. Nucleotides corresponding to secondary structure landmarks are indicated to the right. Blue and green bars indicate regions of strong Cbf5 and L7Ae protection, respectively. ( B ) Summary of protections in the context of a functional secondary structure model of Pf9 RNA. Box ACA, the pseudouridylation pocket and k-turn are boxed. Apical loop, upper and lower stems are labelled. The rRNA target of Pf9 is shown in grey lowercase letters. Cbf5 and L7Ae protections observed in A are shown as indicated in the legend. The regions shaded grey were not assessed due to the resolution limits of the gel.

    Article Snippet: For ribonuclease cleavage, the reactions were initiated by addition of 0.1 or 0.2 U RNase T1 (Sigma), or 1 or 2 ng RNase A (Sigma) and incubated for 15 min at 37°C.

    Techniques: Footprinting, Incubation, Generated, Functional Assay

    Lead-induced cleavage footprinting of Pf9 RNA, and Cbf5–Pf9 and L7Ae–Pf9 sub-complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 5, 12, 16) or presence of increasing concentrations of Cbf5 (lanes 6–11) or L7Ae (lanes 13–15) and subjected to lead (II)-induced cleavage. Lane 1 is undigested RNA and lanes 3, 2 and 4 are size markers generated by alkaline hydrolysis (OH), and RNase T1 digestion under non-denaturing (T1) and denaturing conditions (ΔT1), respectively. Blue and green bars indicate regions of strong Cbf5 and L7Ae protection. Yellow bars indicate cleavage enhancements observed with L7Ae. Red arrowheads indicate unexpected cleavages in the upper stem of the guide RNA in the absence of protein. ( B ) Summary of cleavage protections and enhancements in the context of Pf9 RNA secondary structure model (as in Figure 1 ).

    Journal: Nucleic Acids Research

    Article Title: Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

    doi: 10.1093/nar/gkm673

    Figure Lengend Snippet: Lead-induced cleavage footprinting of Pf9 RNA, and Cbf5–Pf9 and L7Ae–Pf9 sub-complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 5, 12, 16) or presence of increasing concentrations of Cbf5 (lanes 6–11) or L7Ae (lanes 13–15) and subjected to lead (II)-induced cleavage. Lane 1 is undigested RNA and lanes 3, 2 and 4 are size markers generated by alkaline hydrolysis (OH), and RNase T1 digestion under non-denaturing (T1) and denaturing conditions (ΔT1), respectively. Blue and green bars indicate regions of strong Cbf5 and L7Ae protection. Yellow bars indicate cleavage enhancements observed with L7Ae. Red arrowheads indicate unexpected cleavages in the upper stem of the guide RNA in the absence of protein. ( B ) Summary of cleavage protections and enhancements in the context of Pf9 RNA secondary structure model (as in Figure 1 ).

    Article Snippet: For ribonuclease cleavage, the reactions were initiated by addition of 0.1 or 0.2 U RNase T1 (Sigma), or 1 or 2 ng RNase A (Sigma) and incubated for 15 min at 37°C.

    Techniques: Footprinting, Incubation, Generated

    Single-stranded nuclease footprinting of Pf9. ( A ) 5′-end labelled Pf9 was digested with indicated concentrations of RNase A (lanes 3, 4) or RNase T1 (lanes 5, 6). The cleavage products were separated on a denaturing 20% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Strong cleavages at nucleotides in the upper stem region are indicated with red arrowheads. ( B ) Summary of Pf9 RNA cleavages by single-stranded nucleases in the context of the predicted secondary structure of Pf9 RNA (as in Figure 1 ). Upper stem region is boxed in red.

    Journal: Nucleic Acids Research

    Article Title: Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

    doi: 10.1093/nar/gkm673

    Figure Lengend Snippet: Single-stranded nuclease footprinting of Pf9. ( A ) 5′-end labelled Pf9 was digested with indicated concentrations of RNase A (lanes 3, 4) or RNase T1 (lanes 5, 6). The cleavage products were separated on a denaturing 20% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Strong cleavages at nucleotides in the upper stem region are indicated with red arrowheads. ( B ) Summary of Pf9 RNA cleavages by single-stranded nucleases in the context of the predicted secondary structure of Pf9 RNA (as in Figure 1 ). Upper stem region is boxed in red.

    Article Snippet: For ribonuclease cleavage, the reactions were initiated by addition of 0.1 or 0.2 U RNase T1 (Sigma), or 1 or 2 ng RNase A (Sigma) and incubated for 15 min at 37°C.

    Techniques: Footprinting, Acrylamide Gel Assay, Marker, Generated

    Hydroxyl radical footprinting of Cbf5–L7Ae–Pf9 complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 1, 7, 13) or presence of either 1 μM L7Ae (lane 2) with increasing concentrations of Cbf5 (lanes 3–6), or 2 μM Cbf5 (lane 8) with increasing concentrations of L7Ae (lanes 9–12), and subjected to hydroxyl radical cleavage. Lanes 14 and 15 are size markers generated by alkaline hydrolysis (OH) and RNase T1 digestion (T1). Turquoise and purple bars indicate new sites of protection observed in the presence of both proteins and of Cbf5 protections lost upon addition of L7Ae, respectively. ( B ) Summary of changes in protection in the context of Pf9 RNA secondary structure model (as in Figure 1 ).

    Journal: Nucleic Acids Research

    Article Title: Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

    doi: 10.1093/nar/gkm673

    Figure Lengend Snippet: Hydroxyl radical footprinting of Cbf5–L7Ae–Pf9 complexes. ( A ) 5′-end labelled Pf9 was incubated in the absence (lanes 1, 7, 13) or presence of either 1 μM L7Ae (lane 2) with increasing concentrations of Cbf5 (lanes 3–6), or 2 μM Cbf5 (lane 8) with increasing concentrations of L7Ae (lanes 9–12), and subjected to hydroxyl radical cleavage. Lanes 14 and 15 are size markers generated by alkaline hydrolysis (OH) and RNase T1 digestion (T1). Turquoise and purple bars indicate new sites of protection observed in the presence of both proteins and of Cbf5 protections lost upon addition of L7Ae, respectively. ( B ) Summary of changes in protection in the context of Pf9 RNA secondary structure model (as in Figure 1 ).

    Article Snippet: For ribonuclease cleavage, the reactions were initiated by addition of 0.1 or 0.2 U RNase T1 (Sigma), or 1 or 2 ng RNase A (Sigma) and incubated for 15 min at 37°C.

    Techniques: Footprinting, Incubation, Generated

    Single-stranded nuclease footprinting of L7Ae–Pf9. ( A ) 5′-end labelled Pf9 was incubated alone (lanes 3, 5) or with 1 μM L7Ae (lanes 4, 6) and digested with RNase T1 (lanes 3, 4) or RNase A (lanes 5, 6). The cleavage products were separated on a denaturing 15% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Red arrowheads indicate cleavages in the upper stem of the guide RNA in the absence of protein. Green and yellow bars indicate strong L7Ae protections and cleavage enhancements, respectively. ( B ) Summary of L7Ae cleavage protections and enhancements in the context of the predicted secondary structure of Pf9 (as in Figure 1 ).

    Journal: Nucleic Acids Research

    Article Title: Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

    doi: 10.1093/nar/gkm673

    Figure Lengend Snippet: Single-stranded nuclease footprinting of L7Ae–Pf9. ( A ) 5′-end labelled Pf9 was incubated alone (lanes 3, 5) or with 1 μM L7Ae (lanes 4, 6) and digested with RNase T1 (lanes 3, 4) or RNase A (lanes 5, 6). The cleavage products were separated on a denaturing 15% acrylamide gel. Lane 1 is undigested RNA and lane 2 is a size marker generated by alkaline hydrolysis (OH). Red arrowheads indicate cleavages in the upper stem of the guide RNA in the absence of protein. Green and yellow bars indicate strong L7Ae protections and cleavage enhancements, respectively. ( B ) Summary of L7Ae cleavage protections and enhancements in the context of the predicted secondary structure of Pf9 (as in Figure 1 ).

    Article Snippet: For ribonuclease cleavage, the reactions were initiated by addition of 0.1 or 0.2 U RNase T1 (Sigma), or 1 or 2 ng RNase A (Sigma) and incubated for 15 min at 37°C.

    Techniques: Footprinting, Incubation, Acrylamide Gel Assay, Marker, Generated

    LRP5 3′-UTR PG4 folds into a G4 structure in vitro . ( a ) Sequence and numbering of the wt LRP5 PG4 used in the in vitro experiments. The lowercase guanosines (g) correspond to those mutated to adenosines in the G/A-mutant version. Nucleotides that were hydrolyzed significantly more in the presence of KCl during the in-line probing are both in bold and underlined. ( b , c ) CD spectra for the LRP5 PG4 sequence using 4 µM of either the wt (b) or the G/A-mutant (c) versions performed either in the absence of salt (closed circle) or in the presence of 100 mM of either LiCl (inverted closed triangle), NaCl (open circle) or KCl (open triangle). ( d ) Autoradiogram of a 10% denaturing polyacrylamide gel of the in-line probing of the 5′-end-labeled LRP5 wt and G/A-mutant PG4 versions performed either in the absence of salt (NS), or in the presence of 100 mM of either LiCl, NaCl or KCl. Lanes L and T1 correspond to alkaline hydrolysis and RNase T1 mapping of the wt version, respectively. The positions of the guanosines are indicated on the left of the gel, whereas the domains of the G4 structure are indicated on the right.

    Journal: Nucleic Acids Research

    Article Title: Exploring mRNA 3?-UTR G-quadruplexes: evidence of roles in both alternative polyadenylation and mRNA shortening

    doi: 10.1093/nar/gkt265

    Figure Lengend Snippet: LRP5 3′-UTR PG4 folds into a G4 structure in vitro . ( a ) Sequence and numbering of the wt LRP5 PG4 used in the in vitro experiments. The lowercase guanosines (g) correspond to those mutated to adenosines in the G/A-mutant version. Nucleotides that were hydrolyzed significantly more in the presence of KCl during the in-line probing are both in bold and underlined. ( b , c ) CD spectra for the LRP5 PG4 sequence using 4 µM of either the wt (b) or the G/A-mutant (c) versions performed either in the absence of salt (closed circle) or in the presence of 100 mM of either LiCl (inverted closed triangle), NaCl (open circle) or KCl (open triangle). ( d ) Autoradiogram of a 10% denaturing polyacrylamide gel of the in-line probing of the 5′-end-labeled LRP5 wt and G/A-mutant PG4 versions performed either in the absence of salt (NS), or in the presence of 100 mM of either LiCl, NaCl or KCl. Lanes L and T1 correspond to alkaline hydrolysis and RNase T1 mapping of the wt version, respectively. The positions of the guanosines are indicated on the left of the gel, whereas the domains of the G4 structure are indicated on the right.

    Article Snippet: The reactions were incubated for 2 min at 37°C in the presence of 0.6 U of RNase T1 (Roche Diagnostic), and they were then quenched by the addition of 20 µl of formamide loading buffer.

    Techniques: In Vitro, Sequencing, Mutagenesis, Labeling

    Hu proteins and FXR2P bind to  PSD95  mRNA.  A , Brain extracts were precipitated by specific antibodies against FMRP and HuD in the presence (+) or absence (−) of RNase A/T1 and control IgGs. Coimmunoprecipitated proteins FMRP, HuR, and other Hu members (panHu) are indicated.  B1 , Representative image of the CA3 region of hippocampus stained with anti-FMRP (red) and anti-HuR or HuD (green). Scale bar, 50 μm.  B2 , FMRP (red) and FXR2P or HuD or HuR (green) staining on primary neurons at 12 d  in vitro . White arrows indicate protein colocalization quantified by the Mander's coefficient ( n  = 122 for FXR2P;  n  = 44 for HuD;  n  = 34 for HuR). Scale bar, 12.5 μm.  C , HuD RNA-IP from WT and  Fmr1  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qRT-PCR.  D , FXR2P-IP from WT and  Fxr2  KO brain extracts detected by Western blotting.  E , Same as in  A , FMRP-IP and FXR2P-IP in the presence of RNase A/T1 detected by Western blotting.  F , FXR2P RNA-IP from WT and  Fmr1  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qPCR.  G , FMRP-IP from WT and  Fxr2  KO hippocampal extracts.  PSD95  and  Cyp46  mRNAs were amplified by qPCR. mRNA levels were calculated using the formula 2^ − (Ct PSD95  − Ct exogenous normalizerBC200 ) and normalized to the mRNA present in the input and the mock IP.  C ,  n  = 7 independent experiments.  F ,  n  = 7 independent experiments.  G ,  n  = 7 independent experiments. ** p

    Journal: The Journal of Neuroscience

    Article Title: FXR2P Exerts a Positive Translational Control and Is Required for the Activity-Dependent Increase of PSD95 Expression

    doi: 10.1523/JNEUROSCI.4800-14.2015

    Figure Lengend Snippet: Hu proteins and FXR2P bind to PSD95 mRNA. A , Brain extracts were precipitated by specific antibodies against FMRP and HuD in the presence (+) or absence (−) of RNase A/T1 and control IgGs. Coimmunoprecipitated proteins FMRP, HuR, and other Hu members (panHu) are indicated. B1 , Representative image of the CA3 region of hippocampus stained with anti-FMRP (red) and anti-HuR or HuD (green). Scale bar, 50 μm. B2 , FMRP (red) and FXR2P or HuD or HuR (green) staining on primary neurons at 12 d in vitro . White arrows indicate protein colocalization quantified by the Mander's coefficient ( n = 122 for FXR2P; n = 44 for HuD; n = 34 for HuR). Scale bar, 12.5 μm. C , HuD RNA-IP from WT and Fmr1 KO hippocampal extracts. PSD95 and Cyp46 mRNAs were amplified by qRT-PCR. D , FXR2P-IP from WT and Fxr2 KO brain extracts detected by Western blotting. E , Same as in A , FMRP-IP and FXR2P-IP in the presence of RNase A/T1 detected by Western blotting. F , FXR2P RNA-IP from WT and Fmr1 KO hippocampal extracts. PSD95 and Cyp46 mRNAs were amplified by qPCR. G , FMRP-IP from WT and Fxr2 KO hippocampal extracts. PSD95 and Cyp46 mRNAs were amplified by qPCR. mRNA levels were calculated using the formula 2^ − (Ct PSD95 − Ct exogenous normalizerBC200 ) and normalized to the mRNA present in the input and the mock IP. C , n = 7 independent experiments. F , n = 7 independent experiments. G , n = 7 independent experiments. ** p

    Article Snippet: Brain extracts were incubated with 2 μg of specific antibodies at 4°C overnight in the absence or presence of a mix of RNase A/T1 (10 μl/ml) (Fermentas).

    Techniques: Staining, In Vitro, Amplification, Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction

    Optimization of ORF57 immunoprecipitation and RNase digestion. (A) ORF57-RNA complexes were immunoprecipitated by a rabbit anti-ORF57 antibody from cells extract of BCBL-1 cells treated with valproic acid for 24 h (input), normal rabbit IgG served as a negative control. The levels of ORF57 in input (1%) and immunoprecipitates (3%) in CLIP experiment were detected by Western blot with mouse anti-ORF57 antibody detecting both full-length ORF57 protein (upper band) and its caspase-cleavage product (lower band). (B) To determine an optimal RNase digestion condition, the immunoprecipitated ORF57 complexes were incubated with various amounts of RNase A/T1 mix for 5 sec at room temperature and followed by proteinase K treatment and RNA extraction. The RNase A/T1 digestion efficiency was checked by RT-PCR for the remaining KSHV PAN RNA, a known ORF57 target.

    Journal: Current protocols in microbiology

    Article Title: CLIP-seq to identify KSHV ORF57-binding RNA in host B cells

    doi: 10.1002/cpmc.3

    Figure Lengend Snippet: Optimization of ORF57 immunoprecipitation and RNase digestion. (A) ORF57-RNA complexes were immunoprecipitated by a rabbit anti-ORF57 antibody from cells extract of BCBL-1 cells treated with valproic acid for 24 h (input), normal rabbit IgG served as a negative control. The levels of ORF57 in input (1%) and immunoprecipitates (3%) in CLIP experiment were detected by Western blot with mouse anti-ORF57 antibody detecting both full-length ORF57 protein (upper band) and its caspase-cleavage product (lower band). (B) To determine an optimal RNase digestion condition, the immunoprecipitated ORF57 complexes were incubated with various amounts of RNase A/T1 mix for 5 sec at room temperature and followed by proteinase K treatment and RNA extraction. The RNase A/T1 digestion efficiency was checked by RT-PCR for the remaining KSHV PAN RNA, a known ORF57 target.

    Article Snippet: Protein A beads (EMD Millipore, cat. no. 16–125) RNase A/T1 mix (2 mg/ml of RNase A and 5000 U/ml of RNase T1, ThermoFisher Scientific, cat. no. EN0551) Recombinant Shrimp Alkaline Phosphatase (rSAP, 1000U/ml, New England Biolabs, cat. no. M0371S) Proteinase K (600 mAU/ml, EMD Millipore, cat. no. 71049) Proteinase K buffer (1× IP buffer supplemented with 1% SDS) Phase Lock Gel Light, 1.5 ml tubes (VWR, cat. no. 10052–164) 3M sodium acetate, pH 5.2 (Quality Biological, cat. no. 351-035-721EA) 70–75% (v/v) ethanol 100% (v/v) ethanol Agilent RNA 6000 Pico Kit (Agilent Technologies, cat. no. 5067–1513) Universal miRNA Cloning Linker (New England BioLabs, cat. no. S1315S) 50% PEG8000 (New England BioLabs, from the T4 RNA Ligase II kit) RNaseOUT (ThermoFisher Scientific, cat. no. 10777–019) T4 RNA Ligase 2, truncated KQ (200,000 units/ml, New England Biolabs, cat. no. M0373L) Agencourt RNAClean beads (Beckman Coulter, cat. no. A29168) Agencourt AMPure XP - PCR Purification beads (Beckman Coulter, cat. no. ) dNTP Mix, 10 mM each (Bioline, cat. no. BIO-39044) SuperScript III First-Strand Synthesis System (ThermoFisher Scientific, cat. no. 18080–051) CircLigase ssDNA ligase (Epicentre, cat. no. CL4111K) Phusion DNA polymerase kit (New England BioLabs, cat. no. M0530S) Reverse transcription primer (IDT custom synthesis) [5’-(Phos)-AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGC-(SpC18)-CACTCA-(SpC18)-TTCAGACGTGTGCTCTTCCGATCTATTGATGGTGCCTACAG-3’] riboPCR_F primer (5’-AATGATACGGCGACCACCGAGATCTACAC-3’, IDT custom synthesis) Indexed primers (5’-CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGA-CGTGTGCTCTTCCG-3’) (‘NNNNNN’ denotes the index, of which each index has a unique sequence.

    Techniques: Immunoprecipitation, Negative Control, Cross-linking Immunoprecipitation, Western Blot, Incubation, Size-exclusion Chromatography, RNA Extraction, Reverse Transcription Polymerase Chain Reaction

    Structural and functional probing of mccA transcript. ( A ) 32 P-5′-end labeled in vitro synthesized wild-type or mutant mccA RNA was digested with RNase T1 or RNase T2. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. Lanes labeled ‘control’ show material that was not treated with RNAses. Lanes labeled ‘SEQ T1’ are marker lanes, showing digestion pattern obtained with RNase T1 under denaturing conditions (cleavages at every G). In lanes labeled ‘RNase T1’ and ‘RNase T2’ transcripts were re-folded in the presence of 10 mM MgCl 2 and then digested with RNases. In lanes labeled ‘PURExpress’ wild-type mccA RNA was folded in the presence of PURExpress full system in the presence of thiostrepton. ( B ) A secondary structure of mccA RNA produced by RNAfold software ( http://rna.tbi.univie.ac.at/ ) and consistent with RNase probing results. Cleavage positions by RNase T1 and RNase T2 are shown, respectively, with black and white asterisks. Elements of the structure (loops, L, and hairpins, H) are also marked on the gel shown in panel A. A fragment of mccA tested for in vivo termination activity is indicated. ( C ) A DNA fragment coding for a hairpin structure and adjacent sequences shown in panel C was cloned in direct (pFD_mcc_dir) and inverted (pFD_mcc_inv) orientations between the galK reporter and the lac UV5 promoter of plasmid pFD100. The resulting plasmids, as well as control pFD51 plasmid lacking the promoter, were transformed into galK − E. coli cells and transformants were then tested for GalK activity on McConkey indicator agar plates. Overnight growth of cells is shown.

    Journal: Nucleic Acids Research

    Article Title: Ribosome-controlled transcription termination is essential for the production of antibiotic microcin C

    doi: 10.1093/nar/gku880

    Figure Lengend Snippet: Structural and functional probing of mccA transcript. ( A ) 32 P-5′-end labeled in vitro synthesized wild-type or mutant mccA RNA was digested with RNase T1 or RNase T2. Reaction products were resolved by denaturing PAGE and revealed by autoradiography. Lanes labeled ‘control’ show material that was not treated with RNAses. Lanes labeled ‘SEQ T1’ are marker lanes, showing digestion pattern obtained with RNase T1 under denaturing conditions (cleavages at every G). In lanes labeled ‘RNase T1’ and ‘RNase T2’ transcripts were re-folded in the presence of 10 mM MgCl 2 and then digested with RNases. In lanes labeled ‘PURExpress’ wild-type mccA RNA was folded in the presence of PURExpress full system in the presence of thiostrepton. ( B ) A secondary structure of mccA RNA produced by RNAfold software ( http://rna.tbi.univie.ac.at/ ) and consistent with RNase probing results. Cleavage positions by RNase T1 and RNase T2 are shown, respectively, with black and white asterisks. Elements of the structure (loops, L, and hairpins, H) are also marked on the gel shown in panel A. A fragment of mccA tested for in vivo termination activity is indicated. ( C ) A DNA fragment coding for a hairpin structure and adjacent sequences shown in panel C was cloned in direct (pFD_mcc_dir) and inverted (pFD_mcc_inv) orientations between the galK reporter and the lac UV5 promoter of plasmid pFD100. The resulting plasmids, as well as control pFD51 plasmid lacking the promoter, were transformed into galK − E. coli cells and transformants were then tested for GalK activity on McConkey indicator agar plates. Overnight growth of cells is shown.

    Article Snippet: Reactions were next supplied with 1 μl of RNase T1 (0.01 u/μl) or RNase T2 (0.01 u/μl) (Boehringer-Mannheim) and incubated for 5 min at 37°C.

    Techniques: Functional Assay, Labeling, In Vitro, Synthesized, Mutagenesis, Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, Produced, Software, In Vivo, Activity Assay, Clone Assay, Plasmid Preparation, Transformation Assay

    snaR RT-PCR clones. ( A ) Clones of snaR identified from sequencing of RT-PCR products ( 21 ) can be grouped into two subsets. Homology within each subset is denoted by an asterisk and non-homologous nucleotides are in bold font. Differences between subsets are denoted by gray shading. Consensus sequences are given below each set of clones. The majority of clones are derived from asynchronous NF90b cell line extract. Clones with ‘m’ or ‘a’ appended to their name were immunoprecipitated from NF90b G2/M phase extract or from NF90a extract, respectively. ( B ) The genomic sequence of snaR-A and -B. Genomic nucleotides matching consensus sequence are highlighted in yellow, those differing from consensus sequence are in green. 3′-Oligo(A) and oligo(T) tracts are denoted in red and blue, respectively. Dashed line denotes sequence complementary to probe H, solid line denotes predicted RNase H digestion product. ( C ) Sequence alignment of snaR-A with two potential piRNAs (bold). Alu RNA homology is denoted in yellow. Sequence homologous to the PolIII B box motif is underlined.

    Journal: Nucleic Acids Research

    Article Title: Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution

    doi: 10.1093/nar/gkm668

    Figure Lengend Snippet: snaR RT-PCR clones. ( A ) Clones of snaR identified from sequencing of RT-PCR products ( 21 ) can be grouped into two subsets. Homology within each subset is denoted by an asterisk and non-homologous nucleotides are in bold font. Differences between subsets are denoted by gray shading. Consensus sequences are given below each set of clones. The majority of clones are derived from asynchronous NF90b cell line extract. Clones with ‘m’ or ‘a’ appended to their name were immunoprecipitated from NF90b G2/M phase extract or from NF90a extract, respectively. ( B ) The genomic sequence of snaR-A and -B. Genomic nucleotides matching consensus sequence are highlighted in yellow, those differing from consensus sequence are in green. 3′-Oligo(A) and oligo(T) tracts are denoted in red and blue, respectively. Dashed line denotes sequence complementary to probe H, solid line denotes predicted RNase H digestion product. ( C ) Sequence alignment of snaR-A with two potential piRNAs (bold). Alu RNA homology is denoted in yellow. Sequence homologous to the PolIII B box motif is underlined.

    Article Snippet: The reaction was terminated at 70°C for 15 min and RNA was digested with RNase (15 U/μl RNase T1, 4 U/μl RNase H) for 30 min at 37°C. cDNA was twice purified through a QIAquick desalting column (Qiagen), heated at 94°C for 2 min in Tailing buffer then incubated with terminal deoxynucleotidyl transferase (New England BioLabs) for 10 min at 37°C.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Clone Assay, Sequencing, Derivative Assay, Immunoprecipitation

    snaRs are highly structured NF90-associated RNAs. ( A ) RNA immunoprecipitated from cell lines with anti-omni antibody was 3′-end labeled and resolved in a 5% acrylamide/7 M urea gel ( 21 ). Cell lines contained omni-tagged NF90a or NF90b (lanes 2 and 3) or empty vector (lane 1). Note that the cell extracts contained more NF90b than NF90a. ( B ) The most stable snaR-A and -B structures predicted by MFOLD ( 27 ). Base-pairing is represented by dots. snaR-B bases altered or deleted (open triangle) in snaR-C are circled. ( C ) Northern blot of supernatant ×10 −3 (Sup) and immunoprecipitated (IP) RNA from the cell lines probed with Probe-A (Figure S2A). 3′-pCp-labeled RNA precipitated with NF90a served as a marker (M). ( D ) RNA immunoprecipitated with NF90b was 3′-end labeled, digested with RNase H in the presence of sense (S) or antisense (AS) oligonucleotides corresponding to snaR (probe H, Figure 2 B) or 5S rRNA and resolved in a 5% acrylamide/7 M urea gel. Asterisk marks snaR-A RNase H digestion product, bromophenol blue (bpb) migration is denoted. ( E ) In vitro binding assay of T7 RNA polymerase transcribed snaR-A to equal amounts of GST fusion proteins (Figure S1) in the presence of 2000-fold molar excess yeast tRNA. NF90b [A458P,A588P] mutant is denoted by ‘GST-Mut’ and 20% input was loaded.

    Journal: Nucleic Acids Research

    Article Title: Novel rapidly evolving hominid RNAs bind nuclear factor 90 and display tissue-restricted distribution

    doi: 10.1093/nar/gkm668

    Figure Lengend Snippet: snaRs are highly structured NF90-associated RNAs. ( A ) RNA immunoprecipitated from cell lines with anti-omni antibody was 3′-end labeled and resolved in a 5% acrylamide/7 M urea gel ( 21 ). Cell lines contained omni-tagged NF90a or NF90b (lanes 2 and 3) or empty vector (lane 1). Note that the cell extracts contained more NF90b than NF90a. ( B ) The most stable snaR-A and -B structures predicted by MFOLD ( 27 ). Base-pairing is represented by dots. snaR-B bases altered or deleted (open triangle) in snaR-C are circled. ( C ) Northern blot of supernatant ×10 −3 (Sup) and immunoprecipitated (IP) RNA from the cell lines probed with Probe-A (Figure S2A). 3′-pCp-labeled RNA precipitated with NF90a served as a marker (M). ( D ) RNA immunoprecipitated with NF90b was 3′-end labeled, digested with RNase H in the presence of sense (S) or antisense (AS) oligonucleotides corresponding to snaR (probe H, Figure 2 B) or 5S rRNA and resolved in a 5% acrylamide/7 M urea gel. Asterisk marks snaR-A RNase H digestion product, bromophenol blue (bpb) migration is denoted. ( E ) In vitro binding assay of T7 RNA polymerase transcribed snaR-A to equal amounts of GST fusion proteins (Figure S1) in the presence of 2000-fold molar excess yeast tRNA. NF90b [A458P,A588P] mutant is denoted by ‘GST-Mut’ and 20% input was loaded.

    Article Snippet: The reaction was terminated at 70°C for 15 min and RNA was digested with RNase (15 U/μl RNase T1, 4 U/μl RNase H) for 30 min at 37°C. cDNA was twice purified through a QIAquick desalting column (Qiagen), heated at 94°C for 2 min in Tailing buffer then incubated with terminal deoxynucleotidyl transferase (New England BioLabs) for 10 min at 37°C.

    Techniques: Immunoprecipitation, Labeling, Plasmid Preparation, Northern Blot, Marker, Migration, In Vitro, Binding Assay, Mutagenesis