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GE Healthcare rnasea
Rnasea, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 23 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 99 stars, based on 23 article reviews
Price from $9.99 to $1999.99
rnasea - by Bioz Stars, 2020-09
99/100 stars

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Agarose Gel Electrophoresis:

Article Title: Oct4/Sox2 Binding Sites Contribute to Maintaining Hypomethylation of the Maternal Igf2/H19 Imprinting Control Region
Article Snippet: .. Digested DNA was separated by agarose gel electrophoresis followed by blotting to Hybond-XL (Amersham Biosciences). .. Probes were radiolabeled with the Takara Random Primer DNA Labeling Kit and hybridized to membranes in either Rapid-Hybe Buffer (GE Healthcare) supplemented with 25 µg/ml salmon sperm (SS) DNA at 65°C for 2 hrs or 3% Poly(acrylic acid) pH8.0, 6× SSPE, 1% SDS, 0.1% BSA, 25 ug/ml SS DNA at 65°C overnight.

Article Title: Efficient disruption of Zebrafish genes using a Gal4-containing gene trap
Article Snippet: .. Genomic DNA (5 μg) was digested with HpaI and NdeI, subjected to 1% agarose gel electrophoresis and transferred onto Amersham Hybond™-XL (GE Healthcare, code RPN303S) membrane. .. Hybridization was carried out with 32 P-labelled eGFP probe (ca 700 bp) that was obtained by PCR amplification using original gene trap vector as a template.

Article Title: Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast
Article Snippet: .. RNA that had been extracted by the hot-phenol procedure ( ) was separated by agarose-gel electrophoresis and transferred to the Hybond XL (Amersham Pharmacia). .. The reporters were detected by Northern blotting using an oligonucleotide oRP121 (5′-AATTCCCCCCCCCCCCCCCCCCA-3′).

Southern Blot:

Article Title: Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum
Article Snippet: .. For Southern blot analysis, total genomic DNA was extracted from ssku80 mutant strain 20 and the wild type, digested with either Bam HI or Eco RV and transferred to a Hybond‐XL (Amersham Biosciences, UK) nylon membrane. .. Hybridization was performed at 42 °C under low‐stringency conditions in the presence of ULTRAhyb solution (Ambion, Austin, TX).

Synthesized:

Article Title: Telomere G-overhang Length Measurement: The DSN method
Article Snippet: .. 10M NaOH and 0.5M EDTA 2×loading dye: 100mM NaOH; 2mM EDTA; 5% Ficoll (type 400); 0.05% bromophenol blue (Bio-Rad, Inc) Alkali electrophoresis buffer: 50mM NaOH; 1mM EDTA UltraPure™ Agarose (Invitrogen, Inc) Telomeric DNA markers: synthesized 36, 54 and 96-mer oligonucleotides of repetitive telomere sequence were used as low MW DNA markers; high MW telomeric markers were made as described by Chai et al ( ). ( see ) Amersham Hybond™-XL (GE Healthcare, Inc) 10×SSC (transfer buffer), made from 20×SSC: 3M NaCl; 0.3M sodium citrate, pH 7.0 3MM Whatman Paper .. 10 pmol/µl GTU4 oligonucleotide dissolved in TE buffer: 5′-GGGUUAGGGUUAGGGUUAGGGAAA-3′ 100 pmol/µl T3 C3 +9 oligonucleotide (T3 C3 is complementary to the 3′ end of GTU4 above, and +9 refers to 9nt of telomeric repeats) dissolved in TE buffer: 5′-TTTCCCTAACCCTAA-3′ 1 M NaCl 10× buffer M: 100 mM Tris·Cl (pH 7.5); 100 mM MgCl2 ; 500 mM NaCl; 10 mM dithioerythritol 2 M Tris·Cl, pH 7.4 to 7.6 10 mg/ml BSA (Ambion, Inc) 1.25 mM dAdT: 1.25 mM each of dATP and dTTP [α-32 P]dCTP (3000 Ci/mmol) (PerkinElmer) 5U/µl Klenow large fragment of E. coli DNA polymerase I (NEB) 1 U/µl uracil deglycosylase (UDG) (Invitrogen) PCR machine

Mutagenesis:

Article Title: Efficient gene replacement and direct hyphal transformation in Sclerotinia sclerotiorum
Article Snippet: .. For Southern blot analysis, total genomic DNA was extracted from ssku80 mutant strain 20 and the wild type, digested with either Bam HI or Eco RV and transferred to a Hybond‐XL (Amersham Biosciences, UK) nylon membrane. .. Hybridization was performed at 42 °C under low‐stringency conditions in the presence of ULTRAhyb solution (Ambion, Austin, TX).

Electrophoresis:

Article Title: Telomere G-overhang Length Measurement: The DSN method
Article Snippet: .. 10M NaOH and 0.5M EDTA 2×loading dye: 100mM NaOH; 2mM EDTA; 5% Ficoll (type 400); 0.05% bromophenol blue (Bio-Rad, Inc) Alkali electrophoresis buffer: 50mM NaOH; 1mM EDTA UltraPure™ Agarose (Invitrogen, Inc) Telomeric DNA markers: synthesized 36, 54 and 96-mer oligonucleotides of repetitive telomere sequence were used as low MW DNA markers; high MW telomeric markers were made as described by Chai et al ( ). ( see ) Amersham Hybond™-XL (GE Healthcare, Inc) 10×SSC (transfer buffer), made from 20×SSC: 3M NaCl; 0.3M sodium citrate, pH 7.0 3MM Whatman Paper .. 10 pmol/µl GTU4 oligonucleotide dissolved in TE buffer: 5′-GGGUUAGGGUUAGGGUUAGGGAAA-3′ 100 pmol/µl T3 C3 +9 oligonucleotide (T3 C3 is complementary to the 3′ end of GTU4 above, and +9 refers to 9nt of telomeric repeats) dissolved in TE buffer: 5′-TTTCCCTAACCCTAA-3′ 1 M NaCl 10× buffer M: 100 mM Tris·Cl (pH 7.5); 100 mM MgCl2 ; 500 mM NaCl; 10 mM dithioerythritol 2 M Tris·Cl, pH 7.4 to 7.6 10 mg/ml BSA (Ambion, Inc) 1.25 mM dAdT: 1.25 mM each of dATP and dTTP [α-32 P]dCTP (3000 Ci/mmol) (PerkinElmer) 5U/µl Klenow large fragment of E. coli DNA polymerase I (NEB) 1 U/µl uracil deglycosylase (UDG) (Invitrogen) PCR machine

Article Title: Upf1 potentially serves as a RING-related E3 ubiquitin ligase via its association with Upf3 in yeast
Article Snippet: .. RNA that had been extracted by the hot-phenol procedure ( ) was separated by agarose-gel electrophoresis and transferred to the Hybond XL (Amersham Pharmacia). .. The reporters were detected by Northern blotting using an oligonucleotide oRP121 (5′-AATTCCCCCCCCCCCCCCCCCCA-3′).

Sequencing:

Article Title: Telomere G-overhang Length Measurement: The DSN method
Article Snippet: .. 10M NaOH and 0.5M EDTA 2×loading dye: 100mM NaOH; 2mM EDTA; 5% Ficoll (type 400); 0.05% bromophenol blue (Bio-Rad, Inc) Alkali electrophoresis buffer: 50mM NaOH; 1mM EDTA UltraPure™ Agarose (Invitrogen, Inc) Telomeric DNA markers: synthesized 36, 54 and 96-mer oligonucleotides of repetitive telomere sequence were used as low MW DNA markers; high MW telomeric markers were made as described by Chai et al ( ). ( see ) Amersham Hybond™-XL (GE Healthcare, Inc) 10×SSC (transfer buffer), made from 20×SSC: 3M NaCl; 0.3M sodium citrate, pH 7.0 3MM Whatman Paper .. 10 pmol/µl GTU4 oligonucleotide dissolved in TE buffer: 5′-GGGUUAGGGUUAGGGUUAGGGAAA-3′ 100 pmol/µl T3 C3 +9 oligonucleotide (T3 C3 is complementary to the 3′ end of GTU4 above, and +9 refers to 9nt of telomeric repeats) dissolved in TE buffer: 5′-TTTCCCTAACCCTAA-3′ 1 M NaCl 10× buffer M: 100 mM Tris·Cl (pH 7.5); 100 mM MgCl2 ; 500 mM NaCl; 10 mM dithioerythritol 2 M Tris·Cl, pH 7.4 to 7.6 10 mg/ml BSA (Ambion, Inc) 1.25 mM dAdT: 1.25 mM each of dATP and dTTP [α-32 P]dCTP (3000 Ci/mmol) (PerkinElmer) 5U/µl Klenow large fragment of E. coli DNA polymerase I (NEB) 1 U/µl uracil deglycosylase (UDG) (Invitrogen) PCR machine

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  • 89
    GE Healthcare rnase h2
    <t>RNase</t> H activity in cell extracts and FLAG-tag purified fractions from RNASEH2A expressing and untransduced HeLa cell lines. The 5′-end 32 P-labeled 20-bp RNA/DNA hybrid ( A ) and DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid ( B ) were cleaved with increasing amounts of the total cell extracts and FLAG-tag purified fractions from RNASEH2A expressing cells (H2A) and untransduced cells (mock) at 37°C for 15 min. The 500 μl cell extracts from 10 7 cells yielded 200 μl of FLAG-purified samples as described in Materials and methods section. Ten picomoles of substrates were treated with 1 μl of the samples in 10 μl of reaction mixtures. Protein samples were diluted in Dilution Buffer. Lanes 1, 4, 7 and 10 contained 0.002 μl equivalents of the undiluted sample, lanes 2, 5, 8 and 11 contained 0.02 μl equivalents and lanes 3, 6, 9 and 12 contained 0.2 μl equivalents. After digestion the reactions were electrophoresed in a 20% TBE-urea PAGE and the gel analyzed on a phosphoimager. Note (B) the mobilities of the DNA 12 product of DNA 12 –RNA 1 –DNA 27 /DNA 40 migrates faster than the RNA size markers due to inherent differences in migration in the gels between RNA and DNA. In (A), major cleavage sites of 20-bp RNA/DNA hybrid with RNase H1 and <t>RNase</t> H2 are indicated with blue and red arrows, respectively. The main cleavage product of RNase H2 is indicated by a thick red arrow. Molecular size markers are indicated as M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I) (measuring the sites of cleavage from the 5′- label of the 20-mer RNA) and D (products of digestion of 32 P uniformly labeled poly-rA/poly-dT by mouse RNase H1) (measuring the sizes of products that have uniform sequences). ( C ) Uniformly 32 P-labeled poly-rA/poly-dT (1 μM) was cleaved with increasing amount of the total cell extracts and FLAG-tag purified fractions. Amounts of samples in lanes 1–12 are equivalent to those of (A). The ratios of cleavage products were determined by measuring the acid-soluble radioactivity. ( D ) SDS–PAGE of the purified RNase H2 from HeLa RNASEH2A cells with the two-step affinity immunopurification. HeLa RNASEH2A cells were extracted and subjected to anti-FLAG and anti-HA two-step purification. The purified sample was analyzed by SDS–PAGE stained with silver staining. The fragment indicated with A1, A2 and A3 were identified by mass spectrometry, as described in text.
    Rnase H2, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase h2/product/GE Healthcare
    Average 89 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
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    85
    GE Healthcare bs rnase multimers
    SEC chromatograms and PAGE under non denaturing conditions of <t>BS-RNase</t> aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution. ( A ) SEC pattern obtained with a Sephadex G100 column. Elution with ammonium acetate 0.1 M, pH 5.65, flow rate of 0.4 ml/min. ( B ) SEC chromatogram of BS-RNase <t>multimers</t> superimposed with that of RNase A oligomers: both patterns were obtained with a Superdex 75 10/300 GL column. Elution with 0.2 M NaPi, pH 6.7, flow rate 0.1 ml/min. ( C ) Enlarged Superdex 75 SEC pattern of BS-RNase aggregates; in the inset, 7.5% non denaturing PAGE of the two BS-tetramers, run-time 110 min. ( D ) Additional purification of the two BS-RNase tetramers: their mixture was concentrated to 25 µl in 0.4 M NaPi, and re-chromatographed in the Superdex 75 column equilibrated with the same buffer (dashed+dotted line). Then, TT 1 and TT 2 fractions were further purified: once for TT 1 , continuous line; twice for TT 2 , dotted and dashed lines, respectively. In the right part of the panel are reported the models of two N-swapped BS-RNase tetramers proposed by Adinolfi et al. [13] : they cannot be associated to both tetramers. The various BS-RNase species are: D, native dimer; TT 1 and TT 2 , two tetrameric conformers, H (1 and 2), hexamers; L.O., larger oligomers. Concerning RNase A, grey italics labels: M , native monomer, N D , N-terminal-swapped dimer, C D , C-terminal-swapped dimer; T , trimers; NCN TT : double N+C-swapped tetramer; CNC TT : double C+N-swapped tetramer; P* : pentamers; H* : hexamers. The asterisk* is present to mention that P and H positions are derived from data obtained in [21] .
    Bs Rnase Multimers, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 85 stars, based on 1 article reviews
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    85
    GE Healthcare rnase e ctd constructs rhlb subassemblies
    Subassemblies of <t>RNase</t> E <t>CTD</t> constructs with <t>RhlB,</t> interact with 70S ribosome. ( A ) Surface plasmon resonance analysis. Equal amount of subassemblies of RNase E CTD constructs/RhlB or NTD were injected over immobilized 70S ribosome and their association/dissociation curves compared. The dissociation curves for the subassemblies indicate a stable interaction between analytes and ligands (with weaker stability for RNase E CTDΔRBD/RhlB). The interaction between RNase E NTD and 70S ribosome is considered to be weak or non-specific. ( B ) Co-sedimentation analysis of RNase E CTDΔRBDΔAR2/RhlB complex and 70S ribosome. The CTDΔRBDΔAR2/RhlB complex migrated with 70S ribosome to 23–24% sucrose (lower gel), whereas the subassembly itself distributed at 10–20% sucrose (upper gel).
    Rnase E Ctd Constructs Rhlb Subassemblies, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase e ctd constructs rhlb subassemblies/product/GE Healthcare
    Average 85 stars, based on 1 article reviews
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    89
    GE Healthcare rnase e
    Stereo views of the crystal structure of the catalytic domain of E. coli <t>RNase</t> E and the predicted structure of its homolog RNase G. ( A
    Rnase E, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 89/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rnase e/product/GE Healthcare
    Average 89 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    RNase H activity in cell extracts and FLAG-tag purified fractions from RNASEH2A expressing and untransduced HeLa cell lines. The 5′-end 32 P-labeled 20-bp RNA/DNA hybrid ( A ) and DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid ( B ) were cleaved with increasing amounts of the total cell extracts and FLAG-tag purified fractions from RNASEH2A expressing cells (H2A) and untransduced cells (mock) at 37°C for 15 min. The 500 μl cell extracts from 10 7 cells yielded 200 μl of FLAG-purified samples as described in Materials and methods section. Ten picomoles of substrates were treated with 1 μl of the samples in 10 μl of reaction mixtures. Protein samples were diluted in Dilution Buffer. Lanes 1, 4, 7 and 10 contained 0.002 μl equivalents of the undiluted sample, lanes 2, 5, 8 and 11 contained 0.02 μl equivalents and lanes 3, 6, 9 and 12 contained 0.2 μl equivalents. After digestion the reactions were electrophoresed in a 20% TBE-urea PAGE and the gel analyzed on a phosphoimager. Note (B) the mobilities of the DNA 12 product of DNA 12 –RNA 1 –DNA 27 /DNA 40 migrates faster than the RNA size markers due to inherent differences in migration in the gels between RNA and DNA. In (A), major cleavage sites of 20-bp RNA/DNA hybrid with RNase H1 and RNase H2 are indicated with blue and red arrows, respectively. The main cleavage product of RNase H2 is indicated by a thick red arrow. Molecular size markers are indicated as M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I) (measuring the sites of cleavage from the 5′- label of the 20-mer RNA) and D (products of digestion of 32 P uniformly labeled poly-rA/poly-dT by mouse RNase H1) (measuring the sizes of products that have uniform sequences). ( C ) Uniformly 32 P-labeled poly-rA/poly-dT (1 μM) was cleaved with increasing amount of the total cell extracts and FLAG-tag purified fractions. Amounts of samples in lanes 1–12 are equivalent to those of (A). The ratios of cleavage products were determined by measuring the acid-soluble radioactivity. ( D ) SDS–PAGE of the purified RNase H2 from HeLa RNASEH2A cells with the two-step affinity immunopurification. HeLa RNASEH2A cells were extracted and subjected to anti-FLAG and anti-HA two-step purification. The purified sample was analyzed by SDS–PAGE stained with silver staining. The fragment indicated with A1, A2 and A3 were identified by mass spectrometry, as described in text.

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: RNase H activity in cell extracts and FLAG-tag purified fractions from RNASEH2A expressing and untransduced HeLa cell lines. The 5′-end 32 P-labeled 20-bp RNA/DNA hybrid ( A ) and DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid ( B ) were cleaved with increasing amounts of the total cell extracts and FLAG-tag purified fractions from RNASEH2A expressing cells (H2A) and untransduced cells (mock) at 37°C for 15 min. The 500 μl cell extracts from 10 7 cells yielded 200 μl of FLAG-purified samples as described in Materials and methods section. Ten picomoles of substrates were treated with 1 μl of the samples in 10 μl of reaction mixtures. Protein samples were diluted in Dilution Buffer. Lanes 1, 4, 7 and 10 contained 0.002 μl equivalents of the undiluted sample, lanes 2, 5, 8 and 11 contained 0.02 μl equivalents and lanes 3, 6, 9 and 12 contained 0.2 μl equivalents. After digestion the reactions were electrophoresed in a 20% TBE-urea PAGE and the gel analyzed on a phosphoimager. Note (B) the mobilities of the DNA 12 product of DNA 12 –RNA 1 –DNA 27 /DNA 40 migrates faster than the RNA size markers due to inherent differences in migration in the gels between RNA and DNA. In (A), major cleavage sites of 20-bp RNA/DNA hybrid with RNase H1 and RNase H2 are indicated with blue and red arrows, respectively. The main cleavage product of RNase H2 is indicated by a thick red arrow. Molecular size markers are indicated as M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I) (measuring the sites of cleavage from the 5′- label of the 20-mer RNA) and D (products of digestion of 32 P uniformly labeled poly-rA/poly-dT by mouse RNase H1) (measuring the sizes of products that have uniform sequences). ( C ) Uniformly 32 P-labeled poly-rA/poly-dT (1 μM) was cleaved with increasing amount of the total cell extracts and FLAG-tag purified fractions. Amounts of samples in lanes 1–12 are equivalent to those of (A). The ratios of cleavage products were determined by measuring the acid-soluble radioactivity. ( D ) SDS–PAGE of the purified RNase H2 from HeLa RNASEH2A cells with the two-step affinity immunopurification. HeLa RNASEH2A cells were extracted and subjected to anti-FLAG and anti-HA two-step purification. The purified sample was analyzed by SDS–PAGE stained with silver staining. The fragment indicated with A1, A2 and A3 were identified by mass spectrometry, as described in text.

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Activity Assay, FLAG-tag, Purification, Expressing, Labeling, Polyacrylamide Gel Electrophoresis, Migration, Radioactivity, SDS Page, Immu-Puri, Staining, Silver Staining, Mass Spectrometry

    Cleavage of poly-rA/poly-dT substrate with human RNase H2 and E. coli RNase HII. ( A ) The uniformly 32 P-labeled poly-rA/poly-dT (1 μM) was digested with human RNase H2 (0.25 fmol) and E. coli RNase HII (76 fmol) at 37°C for the times indicated for each lane. Samples were analyzed by 20% TBE-urea PAGE as described in Materials and methods section. ( B ) Graphical representations of degradation of the substrate at the time points 0 (black), 2 (red), 6 (purple) and 15 min (blue) are shown. ( C ) Processivity values are from at least three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: Cleavage of poly-rA/poly-dT substrate with human RNase H2 and E. coli RNase HII. ( A ) The uniformly 32 P-labeled poly-rA/poly-dT (1 μM) was digested with human RNase H2 (0.25 fmol) and E. coli RNase HII (76 fmol) at 37°C for the times indicated for each lane. Samples were analyzed by 20% TBE-urea PAGE as described in Materials and methods section. ( B ) Graphical representations of degradation of the substrate at the time points 0 (black), 2 (red), 6 (purple) and 15 min (blue) are shown. ( C ) Processivity values are from at least three independent experiments.

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis

    Relative specific activity and processivity. ( A ) Schematic representation of the recombinant human RNase H2 three subunits. Numbers represent the position of the amino acid residues relative to the N-terminal methionine after the pET15b-derived His-tag. AGS-related mutations examined in this study are shown above each subunit. Conserved catalytic residues (D34, E35, D141 and D169) are shown below RNASEH2A. The region shown in black (K294 to F301) in RNASEH2B represents the PIP-box. ( B, C ) A bar graph of relative specific activity and processivity: (B) Relative specific activity (red) and processivity (green) of human RNase H2 wild type and AGS-related mutants and E. coli RNase HII (EcHII) was analyzed using uniformly 32 P-labeled poly-rA/poly-dT as a substrate. (C) Relative specific activity of human RNase H2 wild-type and AGS-related mutants was analyzed by 32 P-labeled DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid. The relative values were normalized to wild type RNase H2 (100%). The error bars represent the standard deviation of at least three independent measurements.

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: Relative specific activity and processivity. ( A ) Schematic representation of the recombinant human RNase H2 three subunits. Numbers represent the position of the amino acid residues relative to the N-terminal methionine after the pET15b-derived His-tag. AGS-related mutations examined in this study are shown above each subunit. Conserved catalytic residues (D34, E35, D141 and D169) are shown below RNASEH2A. The region shown in black (K294 to F301) in RNASEH2B represents the PIP-box. ( B, C ) A bar graph of relative specific activity and processivity: (B) Relative specific activity (red) and processivity (green) of human RNase H2 wild type and AGS-related mutants and E. coli RNase HII (EcHII) was analyzed using uniformly 32 P-labeled poly-rA/poly-dT as a substrate. (C) Relative specific activity of human RNase H2 wild-type and AGS-related mutants was analyzed by 32 P-labeled DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid. The relative values were normalized to wild type RNase H2 (100%). The error bars represent the standard deviation of at least three independent measurements.

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Activity Assay, Recombinant, Derivative Assay, Labeling, Standard Deviation

    Comparison of cleavage pattern of short substrates with human RNase H2 and E. coli RNase HII. The 5′-end 32 P-labeled DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid ( A ) and 20-bp RNA/DNA hybrid ( B ) were cleaved with human RNase H2 and E. coli RNase HII at 37°C for 15 min. The reaction volume was 10 μl and the substrate concentration was 1 μM. Amounts of proteins for human RNase H2 were 1.2 fmol (lanes 2 and 14), 12 fmol (lanes 3 and 15), 120 fmol (lanes 4 and 16), 1.2 pmol (lanes 5 and 17), 12 pmol (lanes 6 and 18). The amounts of protein for E. coli RNase HII were 7.6 fmol (lanes 8 and 20), 76 fmol (lanes 9 and 21), 760 fmol (lanes 10 and 22), 7.6 pmol (lanes 11 and 23), 76 pmol (lanes 12 and 24). Lanes 1, 7, 13 and 19 contained no enzymes. The digested products were analyzed by 20% TBE-urea PAGE. Molecular size markers are indicated as M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I) and D (products of digestion of 32 P-labeled poly-rA/poly-dT by mouse RNase H1).

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: Comparison of cleavage pattern of short substrates with human RNase H2 and E. coli RNase HII. The 5′-end 32 P-labeled DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid ( A ) and 20-bp RNA/DNA hybrid ( B ) were cleaved with human RNase H2 and E. coli RNase HII at 37°C for 15 min. The reaction volume was 10 μl and the substrate concentration was 1 μM. Amounts of proteins for human RNase H2 were 1.2 fmol (lanes 2 and 14), 12 fmol (lanes 3 and 15), 120 fmol (lanes 4 and 16), 1.2 pmol (lanes 5 and 17), 12 pmol (lanes 6 and 18). The amounts of protein for E. coli RNase HII were 7.6 fmol (lanes 8 and 20), 76 fmol (lanes 9 and 21), 760 fmol (lanes 10 and 22), 7.6 pmol (lanes 11 and 23), 76 pmol (lanes 12 and 24). Lanes 1, 7, 13 and 19 contained no enzymes. The digested products were analyzed by 20% TBE-urea PAGE. Molecular size markers are indicated as M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I) and D (products of digestion of 32 P-labeled poly-rA/poly-dT by mouse RNase H1).

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Labeling, Concentration Assay, Polyacrylamide Gel Electrophoresis

    Comparison of enzymatic characteristics of human RNase H2 purified from HeLa cells and E. coli . ( A ) SDS–PAGE of human RNase H2 expressed in E. coli . Whole-cell extracts of overnight culture of E. coli MIC1066 transformed with pET15b (lane 1), pET-hH2ABC (lane 2) and pET-hH2BC2 (lane 3), and purified human RNase H2 A/B/C complex (lane 4) and B/C complex (lane 5) were analyzed by 10–20% gradient SDS–PAGE (Bio-Rad) and the gel was stained with CBB. The molecular weight marker is Rainbow Marker from Amersham Bioscience. The proteins corresponding to RNASEH2A, RNASEH2B and RNASEH2C are indicated as A, B and C, respectively. ( B ) Cleavage of 20-bp substrate with human RNase H2 purified from HeLa cells and E. coli . The 5′-end 32 P-labeled 20-bp RNA/DNA hybrid (10 pmol) was hydrolyzed with human RNase H2 purified from HeLa cells (as in lane 8 of Figure 1A) and E. coli (12 fmol) at 37°C for the time (min) indicated above each lane in 10 μl reaction mixtures. Molecular size marker is indicated with M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I). The digested products were analyzed by 20% TBE-urea PAGE. Prominent cleavage sites were indicated with red arrows. The thick red arrow indicates the main cleavage site. Dependence of pH ( C ), salt concentration ( D ) and Mg 2+ and Mn 2+ ions concentration ( E ) of human RNase H2 purified from HeLa cells (filled) and E. coli (open) were analyzed using uniformly 32 P-labeled poly-rA/poly-dT substrate. MgCl 2 and MnSO 4 concentrations were indicated by circle and triangle in ( E ), respectively.

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: Comparison of enzymatic characteristics of human RNase H2 purified from HeLa cells and E. coli . ( A ) SDS–PAGE of human RNase H2 expressed in E. coli . Whole-cell extracts of overnight culture of E. coli MIC1066 transformed with pET15b (lane 1), pET-hH2ABC (lane 2) and pET-hH2BC2 (lane 3), and purified human RNase H2 A/B/C complex (lane 4) and B/C complex (lane 5) were analyzed by 10–20% gradient SDS–PAGE (Bio-Rad) and the gel was stained with CBB. The molecular weight marker is Rainbow Marker from Amersham Bioscience. The proteins corresponding to RNASEH2A, RNASEH2B and RNASEH2C are indicated as A, B and C, respectively. ( B ) Cleavage of 20-bp substrate with human RNase H2 purified from HeLa cells and E. coli . The 5′-end 32 P-labeled 20-bp RNA/DNA hybrid (10 pmol) was hydrolyzed with human RNase H2 purified from HeLa cells (as in lane 8 of Figure 1A) and E. coli (12 fmol) at 37°C for the time (min) indicated above each lane in 10 μl reaction mixtures. Molecular size marker is indicated with M (products of digestion of 32 P-labeled 20-mer RNA by Phosphodiesterase I). The digested products were analyzed by 20% TBE-urea PAGE. Prominent cleavage sites were indicated with red arrows. The thick red arrow indicates the main cleavage site. Dependence of pH ( C ), salt concentration ( D ) and Mg 2+ and Mn 2+ ions concentration ( E ) of human RNase H2 purified from HeLa cells (filled) and E. coli (open) were analyzed using uniformly 32 P-labeled poly-rA/poly-dT substrate. MgCl 2 and MnSO 4 concentrations were indicated by circle and triangle in ( E ), respectively.

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Purification, SDS Page, Transformation Assay, Positron Emission Tomography, Staining, Molecular Weight, Marker, Labeling, Polyacrylamide Gel Electrophoresis, Concentration Assay

    Physical interaction of RNase H2 with PCNA. ( A ) Physical interaction between RNase H2 and PCNA was analyzed using gel filtration column chromatography. Mixture of RNase H2 and PCNA (top), PCNA alone (middle) and RNase H2 alone (bottom) were analyzed on the gel filtration column. The indicated fractions were subjected to 10–20% gradient SDS–PAGE and the proteins were visualized by CBB. A representative result is shown in this figure. A, B, C, and PCNA mark the migration of RNASEH2A, RNASEH2B, RNASEH2C and PCNA, respectively. ( B ) PCNA interacts with C-terminal tail of RNASEH2B: Bacterial lysate containing untagged PCNA was incubated with bacterial lysates containing His-tagged wild-type RNase H2 A/B/C complex, B/C complex, mutant A/B/C complex with two F to A mutations in PIP-box (RNase H2 FA ) and mutant A/B/C complex with PIP-box deleted (RNase H2 ΔPIP ). Pulled-down samples were analyzed by SDS–PAGE with CBB stain and western blotting with anti-PCNA antibody. ( C ) AGS-related mutations do not affect physical interaction between RNase H2 and PCNA: Bacterial lysate containing untagged PCNA was incubated with bacterial lysates containing His-tagged wild-type RNase H2 and mutant RNase H2 with AGS-related mutations. Pulled-down samples were analyzed by western blotting with anti-PCNA antibody.

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: Physical interaction of RNase H2 with PCNA. ( A ) Physical interaction between RNase H2 and PCNA was analyzed using gel filtration column chromatography. Mixture of RNase H2 and PCNA (top), PCNA alone (middle) and RNase H2 alone (bottom) were analyzed on the gel filtration column. The indicated fractions were subjected to 10–20% gradient SDS–PAGE and the proteins were visualized by CBB. A representative result is shown in this figure. A, B, C, and PCNA mark the migration of RNASEH2A, RNASEH2B, RNASEH2C and PCNA, respectively. ( B ) PCNA interacts with C-terminal tail of RNASEH2B: Bacterial lysate containing untagged PCNA was incubated with bacterial lysates containing His-tagged wild-type RNase H2 A/B/C complex, B/C complex, mutant A/B/C complex with two F to A mutations in PIP-box (RNase H2 FA ) and mutant A/B/C complex with PIP-box deleted (RNase H2 ΔPIP ). Pulled-down samples were analyzed by SDS–PAGE with CBB stain and western blotting with anti-PCNA antibody. ( C ) AGS-related mutations do not affect physical interaction between RNase H2 and PCNA: Bacterial lysate containing untagged PCNA was incubated with bacterial lysates containing His-tagged wild-type RNase H2 and mutant RNase H2 with AGS-related mutations. Pulled-down samples were analyzed by western blotting with anti-PCNA antibody.

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Filtration, Column Chromatography, SDS Page, Migration, Incubation, Mutagenesis, Staining, Western Blot

    Cleavage of oligomeric substrates with RNase H2 in the presence of PCNA. The 5′-end 32 P-labeled DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid (i), DNA 39 –RNA 1 –DNA 40 /DNA 80 hybrid (ii) and 20-bp RNA/DNA hybrid (ii) were cleaved with human RNase H2 (3 fmol for DNA 12 –RNA 1 –DNA 27 /DNA 40 and DNA 39 –RNA 1 –DNA 40 /DNA 80 hybrids, 1.2 fmol for 20-bp RNA/DNA hybird) at 37°C for 15 min. The reaction volume was 10 μl. Substrates amounts were 150 fmol. PCNA quantities were 300 fmol (lanes 4, 12 and20), 1 pmol (lanes 5, 13 and 21), 3 pmol (lanes 6, 14 and 22), 10 pmol (lanes 7, 15 and 23), 30 pmol (lanes 2, 8, 10, 15, 18 and 24). Lanes 1, 9 and 17 contained no enzyme. Reactions were analyzed by 20% TBE-urea PAGE.

    Journal: Nucleic Acids Research

    Article Title: Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex

    doi: 10.1093/nar/gkn913

    Figure Lengend Snippet: Cleavage of oligomeric substrates with RNase H2 in the presence of PCNA. The 5′-end 32 P-labeled DNA 12 –RNA 1 –DNA 27 /DNA 40 hybrid (i), DNA 39 –RNA 1 –DNA 40 /DNA 80 hybrid (ii) and 20-bp RNA/DNA hybrid (ii) were cleaved with human RNase H2 (3 fmol for DNA 12 –RNA 1 –DNA 27 /DNA 40 and DNA 39 –RNA 1 –DNA 40 /DNA 80 hybrids, 1.2 fmol for 20-bp RNA/DNA hybird) at 37°C for 15 min. The reaction volume was 10 μl. Substrates amounts were 150 fmol. PCNA quantities were 300 fmol (lanes 4, 12 and20), 1 pmol (lanes 5, 13 and 21), 3 pmol (lanes 6, 14 and 22), 10 pmol (lanes 7, 15 and 23), 30 pmol (lanes 2, 8, 10, 15, 18 and 24). Lanes 1, 9 and 17 contained no enzyme. Reactions were analyzed by 20% TBE-urea PAGE.

    Article Snippet: Analysis of physical interaction of RNase H2 with PCNA by gel filtration column chromatography Gel filtrations of RNase H2, PCNA and their complex were performed at 4°C using a column (20 × 900 mm) packed with Sephacryl S-200 (GE Healthcare).

    Techniques: Labeling, Polyacrylamide Gel Electrophoresis

    SEC chromatograms and PAGE under non denaturing conditions of BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution. ( A ) SEC pattern obtained with a Sephadex G100 column. Elution with ammonium acetate 0.1 M, pH 5.65, flow rate of 0.4 ml/min. ( B ) SEC chromatogram of BS-RNase multimers superimposed with that of RNase A oligomers: both patterns were obtained with a Superdex 75 10/300 GL column. Elution with 0.2 M NaPi, pH 6.7, flow rate 0.1 ml/min. ( C ) Enlarged Superdex 75 SEC pattern of BS-RNase aggregates; in the inset, 7.5% non denaturing PAGE of the two BS-tetramers, run-time 110 min. ( D ) Additional purification of the two BS-RNase tetramers: their mixture was concentrated to 25 µl in 0.4 M NaPi, and re-chromatographed in the Superdex 75 column equilibrated with the same buffer (dashed+dotted line). Then, TT 1 and TT 2 fractions were further purified: once for TT 1 , continuous line; twice for TT 2 , dotted and dashed lines, respectively. In the right part of the panel are reported the models of two N-swapped BS-RNase tetramers proposed by Adinolfi et al. [13] : they cannot be associated to both tetramers. The various BS-RNase species are: D, native dimer; TT 1 and TT 2 , two tetrameric conformers, H (1 and 2), hexamers; L.O., larger oligomers. Concerning RNase A, grey italics labels: M , native monomer, N D , N-terminal-swapped dimer, C D , C-terminal-swapped dimer; T , trimers; NCN TT : double N+C-swapped tetramer; CNC TT : double C+N-swapped tetramer; P* : pentamers; H* : hexamers. The asterisk* is present to mention that P and H positions are derived from data obtained in [21] .

    Journal: PLoS ONE

    Article Title: Double Domain Swapping in Bovine Seminal RNase: Formation of Distinct N- and C-swapped Tetramers and Multimers with Increasing Biological Activities

    doi: 10.1371/journal.pone.0046804

    Figure Lengend Snippet: SEC chromatograms and PAGE under non denaturing conditions of BS-RNase aggregates obtained by lyophilising the protein from a 40% (v/v) acetic acid solution. ( A ) SEC pattern obtained with a Sephadex G100 column. Elution with ammonium acetate 0.1 M, pH 5.65, flow rate of 0.4 ml/min. ( B ) SEC chromatogram of BS-RNase multimers superimposed with that of RNase A oligomers: both patterns were obtained with a Superdex 75 10/300 GL column. Elution with 0.2 M NaPi, pH 6.7, flow rate 0.1 ml/min. ( C ) Enlarged Superdex 75 SEC pattern of BS-RNase aggregates; in the inset, 7.5% non denaturing PAGE of the two BS-tetramers, run-time 110 min. ( D ) Additional purification of the two BS-RNase tetramers: their mixture was concentrated to 25 µl in 0.4 M NaPi, and re-chromatographed in the Superdex 75 column equilibrated with the same buffer (dashed+dotted line). Then, TT 1 and TT 2 fractions were further purified: once for TT 1 , continuous line; twice for TT 2 , dotted and dashed lines, respectively. In the right part of the panel are reported the models of two N-swapped BS-RNase tetramers proposed by Adinolfi et al. [13] : they cannot be associated to both tetramers. The various BS-RNase species are: D, native dimer; TT 1 and TT 2 , two tetrameric conformers, H (1 and 2), hexamers; L.O., larger oligomers. Concerning RNase A, grey italics labels: M , native monomer, N D , N-terminal-swapped dimer, C D , C-terminal-swapped dimer; T , trimers; NCN TT : double N+C-swapped tetramer; CNC TT : double C+N-swapped tetramer; P* : pentamers; H* : hexamers. The asterisk* is present to mention that P and H positions are derived from data obtained in [21] .

    Article Snippet: BS-RNase multimers were chromatographed also through a Source 15S HR10/10 or Mono-S cation-exchange columns (GE-Healthcare): elution was performed with a 0.09–0.20 M NaPi gradient, pH 6.7 ; flow rate was between 0.4 and 1.2 ml/min.

    Techniques: Size-exclusion Chromatography, Polyacrylamide Gel Electrophoresis, Flow Cytometry, Purification, Derivative Assay

    Subassemblies of RNase E CTD constructs with RhlB, interact with 70S ribosome. ( A ) Surface plasmon resonance analysis. Equal amount of subassemblies of RNase E CTD constructs/RhlB or NTD were injected over immobilized 70S ribosome and their association/dissociation curves compared. The dissociation curves for the subassemblies indicate a stable interaction between analytes and ligands (with weaker stability for RNase E CTDΔRBD/RhlB). The interaction between RNase E NTD and 70S ribosome is considered to be weak or non-specific. ( B ) Co-sedimentation analysis of RNase E CTDΔRBDΔAR2/RhlB complex and 70S ribosome. The CTDΔRBDΔAR2/RhlB complex migrated with 70S ribosome to 23–24% sucrose (lower gel), whereas the subassembly itself distributed at 10–20% sucrose (upper gel).

    Journal: Nucleic Acids Research

    Article Title: Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

    doi: 10.1093/nar/gks739

    Figure Lengend Snippet: Subassemblies of RNase E CTD constructs with RhlB, interact with 70S ribosome. ( A ) Surface plasmon resonance analysis. Equal amount of subassemblies of RNase E CTD constructs/RhlB or NTD were injected over immobilized 70S ribosome and their association/dissociation curves compared. The dissociation curves for the subassemblies indicate a stable interaction between analytes and ligands (with weaker stability for RNase E CTDΔRBD/RhlB). The interaction between RNase E NTD and 70S ribosome is considered to be weak or non-specific. ( B ) Co-sedimentation analysis of RNase E CTDΔRBDΔAR2/RhlB complex and 70S ribosome. The CTDΔRBDΔAR2/RhlB complex migrated with 70S ribosome to 23–24% sucrose (lower gel), whereas the subassembly itself distributed at 10–20% sucrose (upper gel).

    Article Snippet: Reconstitution of RNase E CTD constructs/RhlB subassemblies was based on a previous protocol ( ) and the subassemblies were further purified by size exclusion chromatography [Sephacryl S-200 HR column (GE Healthcare) with 20 mM sodium potassium phosphate buffer, pH 7.7, 150 mM NaCl].

    Techniques: Construct, SPR Assay, Injection, Sedimentation

    Organization of the RNA degradosome and its component constructs. ( A ) Schematic representation of the RNA degradosome. RNase E CTD organizes the degradosome and is punctuated by small recognition elements: segment A is the membrane-binding amphipathic helix corresponding to RNase E residues 565–585; segment B is the RhlB binding site; an enolase dimer binds segment C and a PNPase trimer binds segment D. RBD and AR2 are the two RNA binding sites of RNase E CTD. ( B ) Recombinant constructs used in this study. The hexagon represents the hexa-histidine tag and the grey lines in the RNase E-(628-843) diagram represent the flanking non-RNase E sequences derived from its expression vector (a 13-residue head and a 20-residue tail).

    Journal: Nucleic Acids Research

    Article Title: Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

    doi: 10.1093/nar/gks739

    Figure Lengend Snippet: Organization of the RNA degradosome and its component constructs. ( A ) Schematic representation of the RNA degradosome. RNase E CTD organizes the degradosome and is punctuated by small recognition elements: segment A is the membrane-binding amphipathic helix corresponding to RNase E residues 565–585; segment B is the RhlB binding site; an enolase dimer binds segment C and a PNPase trimer binds segment D. RBD and AR2 are the two RNA binding sites of RNase E CTD. ( B ) Recombinant constructs used in this study. The hexagon represents the hexa-histidine tag and the grey lines in the RNase E-(628-843) diagram represent the flanking non-RNase E sequences derived from its expression vector (a 13-residue head and a 20-residue tail).

    Article Snippet: Reconstitution of RNase E CTD constructs/RhlB subassemblies was based on a previous protocol ( ) and the subassemblies were further purified by size exclusion chromatography [Sephacryl S-200 HR column (GE Healthcare) with 20 mM sodium potassium phosphate buffer, pH 7.7, 150 mM NaCl].

    Techniques: Construct, Binding Assay, RNA Binding Assay, Recombinant, Derivative Assay, Expressing, Plasmid Preparation

    RNase E CTD constructs containing at least one RNA-binding site interact with 70S ribosome and its subunits, 50S and 30S. ( A ) Interactions between RNase E CTD constructs and 70S ribosome in vitro . (i) Electrophoretic mobility shift assay of RNase E CTD constructs and 70S ribosome. Prior to electrophoresis, protein samples were mixed at 8:1 (CTD constructs:70S ribosome) molar ratio. Protein samples were stained with Coomassie Blue. (ii) Gradient co-sedimentation of RNase E CTD construct (a), CTDΔRBD (b), CTDΔAR2 (c) and CTDΔRBDΔAR2 (d) with (lower gel) and without (upper gel) 70S ribosome. Before layering protein samples on a 10–50% sucrose gradient, the samples of CTD constructs with 70S ribosome were mixed at a 16:1 molar ratio. The fractionated samples were analysed using 4–12% SDS–PAGE and the protein samples were stained with Coomassie Blue. (iii) Data for dissociation constants of RNase E CTD constructs and 70S ribosome were estimated by kinetic analyses. Biotinylated ribosomes were immobilized on a SA chip while RNase E CTD constructs were injected as analytes. ( B ) Native gel electrophoresis of RNase E constructs and ribosome subunits (50S and 30S). Prior to electrophoresis, RNase E constructs were mixed with 50S and 30S ribosomal subunits, respectively at 1:1 molar ratio, except the RNase E-(628–843) peptide was mixed with the subunits at 1.3:1 peptide:subunit ratio (asterisk). The protein samples were stained with Coomassie Blue.

    Journal: Nucleic Acids Research

    Article Title: Recognition of the 70S ribosome and polysome by the RNA degradosome in Escherichia coli

    doi: 10.1093/nar/gks739

    Figure Lengend Snippet: RNase E CTD constructs containing at least one RNA-binding site interact with 70S ribosome and its subunits, 50S and 30S. ( A ) Interactions between RNase E CTD constructs and 70S ribosome in vitro . (i) Electrophoretic mobility shift assay of RNase E CTD constructs and 70S ribosome. Prior to electrophoresis, protein samples were mixed at 8:1 (CTD constructs:70S ribosome) molar ratio. Protein samples were stained with Coomassie Blue. (ii) Gradient co-sedimentation of RNase E CTD construct (a), CTDΔRBD (b), CTDΔAR2 (c) and CTDΔRBDΔAR2 (d) with (lower gel) and without (upper gel) 70S ribosome. Before layering protein samples on a 10–50% sucrose gradient, the samples of CTD constructs with 70S ribosome were mixed at a 16:1 molar ratio. The fractionated samples were analysed using 4–12% SDS–PAGE and the protein samples were stained with Coomassie Blue. (iii) Data for dissociation constants of RNase E CTD constructs and 70S ribosome were estimated by kinetic analyses. Biotinylated ribosomes were immobilized on a SA chip while RNase E CTD constructs were injected as analytes. ( B ) Native gel electrophoresis of RNase E constructs and ribosome subunits (50S and 30S). Prior to electrophoresis, RNase E constructs were mixed with 50S and 30S ribosomal subunits, respectively at 1:1 molar ratio, except the RNase E-(628–843) peptide was mixed with the subunits at 1.3:1 peptide:subunit ratio (asterisk). The protein samples were stained with Coomassie Blue.

    Article Snippet: Reconstitution of RNase E CTD constructs/RhlB subassemblies was based on a previous protocol ( ) and the subassemblies were further purified by size exclusion chromatography [Sephacryl S-200 HR column (GE Healthcare) with 20 mM sodium potassium phosphate buffer, pH 7.7, 150 mM NaCl].

    Techniques: Construct, RNA Binding Assay, In Vitro, Electrophoretic Mobility Shift Assay, Electrophoresis, Staining, Sedimentation, SDS Page, Chromatin Immunoprecipitation, Injection, Nucleic Acid Electrophoresis

    Stereo views of the crystal structure of the catalytic domain of E. coli RNase E and the predicted structure of its homolog RNase G. ( A

    Journal: RNA

    Article Title: Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

    doi: 10.1261/rna.2104810

    Figure Lengend Snippet: Stereo views of the crystal structure of the catalytic domain of E. coli RNase E and the predicted structure of its homolog RNase G. ( A

    Article Snippet: The membranes were then probed with either RNase E (1:2000 dilution) or RNase G (1:10,000 dilution) antibodies using the ECL Plus Western Blotting Detection Kit (GE Healthcare) as specified by the manufacturer.

    Techniques:

    Expanded view of the RNase H subdomain of the RNase E, RNase G, Rng-219, and Rng-248 proteins. The structures were prepared as described in Materials and Methods and are viewed in PyMOL. The adjacent 5′ sensor and DNase I subdomains are indicated.

    Journal: RNA

    Article Title: Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

    doi: 10.1261/rna.2104810

    Figure Lengend Snippet: Expanded view of the RNase H subdomain of the RNase E, RNase G, Rng-219, and Rng-248 proteins. The structures were prepared as described in Materials and Methods and are viewed in PyMOL. The adjacent 5′ sensor and DNase I subdomains are indicated.

    Article Snippet: The membranes were then probed with either RNase E (1:2000 dilution) or RNase G (1:10,000 dilution) antibodies using the ECL Plus Western Blotting Detection Kit (GE Healthcare) as specified by the manufacturer.

    Techniques:

    The maturation of tRNACys , tRNAHis , and tRNAPro but not tRNAAsn is completely dependent on RNase E

    Journal: RNA

    Article Title: Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

    doi: 10.1261/rna.2104810

    Figure Lengend Snippet: The maturation of tRNACys , tRNAHis , and tRNAPro but not tRNAAsn is completely dependent on RNase E

    Article Snippet: The membranes were then probed with either RNase E (1:2000 dilution) or RNase G (1:10,000 dilution) antibodies using the ECL Plus Western Blotting Detection Kit (GE Healthcare) as specified by the manufacturer.

    Techniques:

    The absence of RNase E differentially affects the decay of specific mRNAs

    Journal: RNA

    Article Title: Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

    doi: 10.1261/rna.2104810

    Figure Lengend Snippet: The absence of RNase E differentially affects the decay of specific mRNAs

    Article Snippet: The membranes were then probed with either RNase E (1:2000 dilution) or RNase G (1:10,000 dilution) antibodies using the ECL Plus Western Blotting Detection Kit (GE Healthcare) as specified by the manufacturer.

    Techniques:

    Complementation of the growth defect associated with RNase E-deficient strains is dependent on the intracellular level of the Rng-219 and Rng-248 proteins

    Journal: RNA

    Article Title: Single amino acid changes in the predicted RNase H domain of Escherichia coli RNase G lead to complementation of RNase E deletion mutants

    doi: 10.1261/rna.2104810

    Figure Lengend Snippet: Complementation of the growth defect associated with RNase E-deficient strains is dependent on the intracellular level of the Rng-219 and Rng-248 proteins

    Article Snippet: The membranes were then probed with either RNase E (1:2000 dilution) or RNase G (1:10,000 dilution) antibodies using the ECL Plus Western Blotting Detection Kit (GE Healthcare) as specified by the manufacturer.

    Techniques: