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  • 99
    New England Biolabs endoh
    Endoh, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 3730xl dna analyzer
    3730xl Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31844 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    New England Biolabs dsrna ladder
    Dsrna Ladder, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs mspji
    Characterization of phage <t>T4</t> DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; <t>MspJI</t> (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Mspji, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs alui
    Characterization of phage <t>T4</t> DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes <t>AluI</t> (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.
    Alui, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 744 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ape1
    Assessment of the removal of rotationally and translationally positioned uracils by UDG and <t>APE1.</t> A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Ape1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 376 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs smai
    Assessment of the removal of rotationally and translationally positioned uracils by UDG and <t>APE1.</t> A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Smai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1265 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs alwni
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    Alwni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs drai
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    Drai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 388 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs cutsmart buffer
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    Cutsmart Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1840 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 pnk
    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of <t>DraI</t> and <t>AlwnI</t> enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.
    T4 Pnk, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3038 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs xmni
    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with <t>XmnI,</t> which cuts p[ lacO x16] once, or <t>Nt.BspQI,</t> which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
    Xmni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 272 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs kpni
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    Kpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2557 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs t4 polynucleotide kinase
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    T4 Polynucleotide Kinase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 22375 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rnase inhibitor
    In vitro selection process. ( A ) RNA <t>aptamer</t> library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), <t>KpnI</t> (green triangles) and PacI (red squares), as a function of selection round.
    Rnase Inhibitor, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1529 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs nt bspqi
    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with <t>XmnI,</t> which cuts p[ lacO x16] once, or <t>Nt.BspQI,</t> which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
    Nt Bspqi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 187 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bamhi
    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with <t>XmnI,</t> which cuts p[ lacO x16] once, or <t>Nt.BspQI,</t> which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.
    Bamhi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8434 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs bmti
    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of <t>pCRE-uno</t> incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the <t>PciI/BmtI</t> digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.
    Bmti, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    New England Biolabs gibson assembly mastermix kit
    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of <t>pCRE-uno</t> incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the <t>PciI/BmtI</t> digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.
    Gibson Assembly Mastermix Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of <t>pCRE-uno</t> incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the <t>PciI/BmtI</t> digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.
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    New England Biolabs m13mp18 single stranded circular dna
    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of <t>pCRE-uno</t> incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the <t>PciI/BmtI</t> digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.
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    New England Biolabs epimark nucleosome assembly kit
    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of <t>pCRE-uno</t> incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the <t>PciI/BmtI</t> digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.
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    Identification of transcription factor specificity protein (Sp)-1–binding site in the human MIOX promoter and effect of in vitro methylation on the binding with nucleoproteins. Eletrophoretic mobility shift assays using Sp-1 and differentially methylated segment (DMS) <t>oligos</t> (DMS1 includes <t>CpG</t> sites -784 and -787 and DMS2 includes -581). A: An increased binding of Sp-1 is observed under high-glucose ambience (30 mmol/L) when the Sp-1 oligo is differentially demethylated or unmethylated ( arrowhead ). No binding of methylated or demethylated oligos is observed under low-glucose (5 mmol/L) ambience. B and C: Likewise an increased binding of unmethylated DMS1 and DMS2 oligos is observed under high-glucose ambience ( arrowhead ). The binding is also seen under low-glucose ambience; nevertheless, binding with unmethylated oligos (Sp-1, DMS1, and DMS2) under high-glucose ambience is much stronger. The arrows and asterisks indicate the nonspecific bands in the autoradiograms.
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    New England Biolabs mboii
    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. <t>MboII</t> (1 unit/reaction) and <t>DNase</t> I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.
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    New England Biolabs pmal c5x vector
    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. <t>MboII</t> (1 unit/reaction) and <t>DNase</t> I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.
    Pmal C5x Vector, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 643 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs rnase b
    Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured <t>RNase</t> B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
    Rnase B, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs deep vent exo polymerase
    Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured <t>RNase</t> B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
    Deep Vent Exo Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs λ protein phosphatase
    Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured <t>RNase</t> B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
    λ Protein Phosphatase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 814 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured <t>RNase</t> B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.
    Shrimp Alkaline Phosphatase Rsap, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 230 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore ubiquitin
    The LPS-stimulated activation of Pellino 1 is prevented by inhibition of TBK1/IKK ε and reversed by phosphatase treatment ( A ) and incubated for 60 min at 30°C in 0.02 ml of 50 mM Tris/HCl (pH 7.5), 5 mM MgCl 2 , UBE1 (0.1 μ M), Ubc13–Uev1a (0.05 μ M), <t>FLAG–ubiquitin</t> (0.05 mM) and ATP (2 mM) in the presence of the non-specific protein kinase inhibitor staurosporine (75 μ ] to prevent any phosphorylation and activation of Pellino 1 during the assay by protein kinases present in the immunoprecipitates as trace contaminants. The reactions were terminated by denaturation in SDS, and immunoblotted with an anti-FLAG antibody (upper panel). Cell lysates were subjected to SDS/PAGE and immunoblotted with antibodies that recognize Pellino 1 or IRF3 phosphorylated at Ser 396 (lower two panels). ( B ) RAW264.7 macrophages were stimulated with LPS for 30 min or left unstimulated (0 min) and Pellino 1 was immunoprecipitated as in ( A ). The immunoprecipitates were then incubated for 30 min at 30°C with (+) or without (−) 100 units of phage λ gt10 phosphatase and 1 mM MnCl 2 in the presence (+) or absence (−) of 50 mM NaF. After washing three times in 50 mM Tris/HCl (pH 7.5) and 5 mM MgCl 2 , the immunoprecipitated Pellino 1 was assayed for E3 ubiquitin ligase activity as in ( A ) and cell lysates immunoblotted with the antibodies used in ( A ). ( C ) As in ( B ) except that the RAW264.7 macrophages were incubated for 1 h without (−) or with (+) 2 μ M MRT67307, before stimulation with LPS. To aid clarity, ( A – C ) do not show the whole gel, but only the region with polyubiquitylated species of > 150 kDa. ( D ) RAW264.7 macrophages were stimulated with 100 ng/ml LPS (Sigma) for 60 min and the cells were lysed in the absence of any phosphatase inhibitor. Cell lysate (20 μ g protein) was then incubated for 30 min at 30°C with (+) or without (−) 300 units of phage λ gt10 phosphatase in the presence of 1 mM MnCl 2 . The reactions were terminated in SDS, subjected to SDS/PAGE and immunoblotted with antibodies that recognize Pellino 1 or actin. The experiment was carried out three times with similar results.
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    New England Biolabs hpa ii
    Effect of MBD1 isoforms on methylated and unmethylated promoters. (A) PCR-amplified <t>DNA</t> fragments from human imprinted SNRPN and tumor suppressor p16 genes were used for a band shift analysis and subcloned upstream of a luciferase cDNA in a pGL3-Basic vector. The PCR fragments and pGL3 constructs were methylated in vitro using <t>Hpa</t> II, Hha I, and Sss I (CpG) methyltransferases. The methyl-CpG sites modified by these enzymes are shown by vertical lines. (B) Band shift of methylated DNA complexed with the methyl-CpG binding domain of MBD1. Unmethylated (−) and methylated fragments containing SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. In the upper and lower panels, the amount of the protein incubated with DNA fragments was 0.5 and 1.0 μg, respectively. (C and D) Regulation of Sp1-activated transcription by MBD1v1 and -v3 in Drosophila SL2 cells ( SNRPN [C] or p16 [D]). Unmethylated (M-) or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with Sp1-expressing plasmid pPacSp1 (0.5 μg), MBD1-expressing plasmids (pAc5.1-MBD1v1 and pAc5.1-MBD1v3) (0 to 1.0 μg), and insertless plasmid pAc5.1/V5-His (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pAc5.1-pRL (0.1 μg). (E) Detection of endogenous MBD1 by an antibody raised against the recombinant MBD1. MBD1 was found to be approximately 80 kDa in HeLa and A549 cells but not in SL2 and CHO-K1 cells.
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    Image Search Results


    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Journal: mBio

    Article Title: Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    doi: 10.1128/mBio.00648-15

    Figure Lengend Snippet: Characterization of phage T4 DNA modification. (A) Phage T4(glc-HMC), T4(HMC), and T4(C) DNA left untreated (−) or treated with (+) restriction enzymes AluI (top), which cleaves unmodified DNA; MspJI (middle), which cleaves HMC-containing DNA; or T4 glucosyltransferase (bottom), which increases the mobility of HMC-containing DNA by the addition of glucose groups. The arrows indicate the mobility shift due to glucose attachment. (B) Analysis of phage T4 DNA modification by single-molecule sequencing. Results are summarized for each genome by mapping IPD ratios at each base for each of the T4 strains studied. The coloration of each base is shown by the key at the bottom left. The T4 nucleotide sequence runs from top to bottom for each of the four genomes. The distance each colored point is displaced from the center indicates the IPD ratio (scale at bottom; leftward for the reverse strand, rightward for the forward strand). Examples of interpulse distances (indicative of modification) are shown to the right for a short segment of the T4 genome. Bars indicate the magnitude of the IPD ratio (upward for the forward strand and downward for the reverse strand). A 5′ GATC 3′ site of DAM methylation is highlighted in yellow. (C) Violin plot showing IPD ratios of A residues at 5′ GATC 3′ sequences.

    Article Snippet: One microgram of T4(C), T4(HMC), or T4(glc-HMC) was digested with AluI (R0137s; NEB), MspJI (R0661S; NEB), or T4 phage β-glucosyltransferase (M0357S; NEB) in accordance with NEB-specified protocols.

    Techniques: Modification, Mobility Shift, Sequencing, Methylation

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques: Incubation

    UDG and APE1 Digestion

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: UDG and APE1 Digestion

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques: Activity Assay, Incubation

    UDG and APE1 Digestion

    Journal: The Journal of Biological Chemistry

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    doi: 10.1074/jbc.M112.441444

    Figure Lengend Snippet: UDG and APE1 Digestion

    Article Snippet: After confirmation of formaldehyde cross-linking efficiency by electrophoretic mobility shift assays (EMSA) (data not shown), cross-linked and noncross-linked NCPs were incubated with UDG and APE1 for different times (0–40 min).

    Techniques:

    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Journal: PLoS ONE

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

    doi: 10.1371/journal.pone.0000669

    Figure Lengend Snippet: Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Article Snippet: AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Techniques: Produced, DNA Synthesis, Transmission Electron Microscopy, Incubation, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Synthesized

    Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Journal: PLoS ONE

    Article Title: HIV-1 Protease and Reverse Transcriptase Control the Architecture of Their Nucleocapsid Partner

    doi: 10.1371/journal.pone.0000669

    Figure Lengend Snippet: Extrusion of dsDNA produced by RT from ssDNA-NCp7 co-aggregates. Progression of DNA synthesis analyzed by TEM after 2 min. (A), 10 min. (B) and 40 min. (C) from reactions with ssDNA (5 nM), RT (50 nM) and NCp7 (3.4 µM). Disaggregation after 10 min. (B) appeared both at the periphery and within the aggregates. A few individual molecules were visible close to the aggregate after 40 min. (C). (D) A typical DNA product visualized by TEM after 40 min. of DNA synthesis with subsequent incubation for 15 min. at 70°C in the presence of 0.4 M NaCl. (E) Band shift analysis of DNA flap synthesis within the dsDNA produced by RT after 40 min. at 37°C with 50 nM RT, with or without 3.4 µM NCp7. An excess of DraI and AlwnI enzymes that digest this dsDNA into two fragments (1800 and 1500 bp) was added. The DraI-AlwnI digestion products of the plasmid DNA are shown as a control on the left. When the HIV-1 central DNA flap is fully synthesized (i. e. with NCp7), the 1800 bp fragment is shifted to a slower migrating band. Magnification is identical for panels A,B,C. The scale bars correspond to 250 nm.

    Article Snippet: AlwNI , DraI and Mo-MuLV RT enzymes were purchased from New England Biolabs (Ipswich, MA).

    Techniques: Produced, DNA Synthesis, Transmission Electron Microscopy, Incubation, Electrophoretic Mobility Shift Assay, Plasmid Preparation, Synthesized

    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Article Snippet: To analyze topoisomers ( ) 0.25 ng/µl of radiolabelled DNA was incubated in 1X Buffer A and 1X Buffer B (Topogen) with 0.2 U/µl Human Topo II-α (Topogen) at 37°C for 15 minutes, or in CutSmart Buffer with 0.4 U/µl XmnI or 0.04 U/µl Nt.BspQI (New England Biolabs) for 1 hour.

    Techniques: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation

    In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: In vitro selection process. ( A ) RNA aptamer library format, random region and tetraloop highlighted in black. ( B ) Fraction of RNA recovered from selections against BamHI (blue circles), KpnI (green triangles) and PacI (red squares), as a function of selection round.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: In Vitro, Selection

    Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Quantitation of KpnI binding affinity by electrophoretic gel mobility shift assay for ( A ) aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; and ( D ) aptamer 30. Replicate data are shown as black and gray points.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Quantitation Assay, Binding Assay, Mobility Shift

    Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Titration of 50 nM KpnI with high affinity radiolabeled aptamer 20 in the concentration range 100 pM–500 nM.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Titration, Concentration Assay

    Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example of anti-KpnI aptamer 20 binding to KpnI as measured by quantitative electrophoretic gel mobility shift assay. A total of 13 nM radiolabeled RNA aptamer titrated with increasing concentrations of KpnI as indicated.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Binding Assay, Mobility Shift

    Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Example gels showing results of qualitative screens for aptamer activity. ( A ) Electrophoretic gel mobility shift binding screen. Radiolabeled aptamer candidates (4 nM) were exposed to their corresponding protein targets, in this case KpnI (20 nM; even lanes). ( B ) Restriction inhibition screen. Fluorescent DNA probe (20 nM, see panel C ) was exposed to REase that had been incubated with a high concentration of the indicated RNA aptamers (40 μM). The examples shown are RNA aptamers against KpnI.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Activity Assay, Mobility Shift, Binding Assay, Inhibition, Incubation, Concentration Assay

    Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: Competition gel shi ft assay showing inhibition of KpnI binding to fluorescent DNA probe in the presence of 100 pM–10 μM anti-KpnI RNA aptamers. ( A ) Aptamer 20, ( B ) aptamer 24.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Binding Assay

    ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: ( A ) In-line probing ( 36 ) of high-affinity anti-KpnI aptamers 20, 24, 29, 30. (UN: unmodified samples (lane 1, 6, 11 and 16); T1: RNAse T1 digestions (lane 2, 7, 12 and 17); OH: alkaline hydrolysis reactions (lane 3, 8, 13 and 18); IN: In-line attack reactions after 24 h (lane 4, 9, 14 and 19) and 48 h (lane 5, 10, 15 and 20). ( B ) Predicted secondary structure of aptamer 20 and validation by in-line probing shown as color-coded heat map (red, high in-line attack rate; white, intermediate in-line attack rate; blue, low in-line attack rate). Black box highlights the nucleotides that were randomized in the original library.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques:

    KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Journal: Nucleic Acids Research

    Article Title: RNA aptamer inhibitors of a restriction endonuclease

    doi: 10.1093/nar/gkv702

    Figure Lengend Snippet: KpnI inhibition by anti-KpnI RNA aptamers. KpnI in the presence of 20 nM fluorescent DNA probe was incubated with anti-KpnI aptamers in the concentration range 10 pM–100 μM. ( A ) Aptamer 20; ( B ) aptamer 24; ( C ) aptamer 29; ( D ) aptamer 30.

    Article Snippet: Binding reactions contain radiolabeled aptamer (13 nM) and the indicated concentration of KpnI in New England Biolabs 1.1 buffer (10 mM Bis-Tris-Propane-HCl, pH 7, 10 mM MgCl2 , 100 μg/ml bovine serum albumin (BSA)) for 30 min at 37°C.

    Techniques: Inhibition, Incubation, Concentration Assay

    Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Topo II-dependent decatenation of p[lacOx16] (A) The autoradiograph in primary Figure 1G is reproduced with cartoons indicating the structures of the replication and termination intermediates n-n, n-sc, sc-sc, n, and sc (see main text for definitions). The order of appearance of the different catenanes matches previous work 5 (n-n, then n-sc, then sc-sc). (B–D) To determine the role of Topo II during termination within a lacO array, termination was monitored in mock- or Topo II-depleted extracts. To confirm immunodepletion of Topo II, Mock and Topo II-depleted NPE was blotted with MCM7 and Topo II antibodies (B). p[ lacO x16] was incubated with LacR, then replicated in either mock- or Topo II-depleted egg extracts in the presence of [α- 32 P]dATP, and termination was induced with IPTG (at 7’). Untreated DNA intermediates were separated by native gel electrophoresis (C). In the mock-depleted extract, nicked and supercoiled monomers were readily produced (as in (A), albeit with slower kinetics due to nonspecific inhibition of the extracts by the immunodepletion procedure), while in the Topo II-depleted extracts, a discrete species was produced. DNA from the last time point in each reaction (lanes 4 and 8 in (C)) was purified and treated with XmnI, which cuts p[ lacO x16] once, or Nt.BspQI, which nicks p[ lacO x16] once, or recombinant Topo II, and then separated by native gel electrophoresis (D). Cleavage of the mock- and TopoII-depleted products with XmnI yielded the expected linear 3.15 kb band (lanes 2 and 6), demonstrating that in both extracts, all products were fully dissolved topoisomers of each other. Relaxation of the mock-depleted products by nicking with Nt.BspQI yielded a discrete band corresponding to nicked plasmid (lane 3), while the TopoII-depleted products were converted to a ladder of discrete topoisomers (lane 7), which we infer represent catenated dimers of different linking numbers, since the mobility difference cannot be due to differences in supercoiling. Importantly, the mobility shift following Nt.BspQI treatment (lane 5 vs lane 7) demonstrated that the Topo II-depleted products (lane 5) were covalently closed and thus in the absence of Topo II ligation of the daughter strands still occurred. Treatment of the mock- and Topo II-depleted products with recombinant human Topo II produced the same relaxed monomeric species (lanes 4 and 8), further confirming that the Topo II-depleted products contained catenanes. Collectively, these observations demonstrate that termination within a lacO array in Topo II depleted extracts produces highly catenated supercoiled-supercoiled dimers, as seen in cells lacking Topo II 16 , 17 . These data confirm that Topo II is responsible for decatenation and argue that termination within a lacO array reflects physiological termination. (E) n-n, n-sc, sc-sc, n, and sc products were also detected when plasmid lacking lacO sequences (pBlueScript) was replicated in the absence of LacR without the use of Cyclin A to synchronize replication. Therefore, these intermediates arise in the course of unperturbed DNA replication in Xenopus egg extracts.

    Article Snippet: To analyze topoisomers ( ) 0.25 ng/µl of radiolabelled DNA was incubated in 1X Buffer A and 1X Buffer B (Topogen) with 0.2 U/µl Human Topo II-α (Topogen) at 37°C for 15 minutes, or in CutSmart Buffer with 0.4 U/µl XmnI or 0.04 U/µl Nt.BspQI (New England Biolabs) for 1 hour.

    Techniques: Autoradiography, Incubation, Nucleic Acid Electrophoresis, Produced, Inhibition, Purification, Recombinant, Plasmid Preparation, Mobility Shift, Ligation

    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of pCRE-uno incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the PciI/BmtI digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter

    doi: 10.1093/nar/gkx718

    Figure Lengend Snippet: Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of pCRE-uno incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the PciI/BmtI digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.

    Article Snippet: Electrophoretic mobility shift assays Vectors pCRE-uno and pCRE-zero were digested with PciI and BmtI (both NEB) to produce the CRE-less 4228 bp fragment and the 180 bp fragment with one (pCRE-uno) or no CRE (pCRE-zero) and cleaned up.

    Techniques: Methylation, Binding Assay, Expressing, Sequencing, Inhibition, Agarose Gel Electrophoresis, Incubation, Migration, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Transfection, FACS

    Impacts of one or three 5-hmC in the minimal CRE promoter on the EGFP gene expression and comparison with the respective effects of 5-mC. ( A ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one or three 5-hmC. Representative fluorescence scatter plots and the correspondent overlaid fluorescence distribution plots of HeLa cells 24 h after transfection (on the left) as well as mean EGFP expression values determined relative to the construct containing unmodified oligonucleotide (‘C’). Mean of nine independent experiments ± SD; P -values calculated by the Student's t -test. ( B ) CREB binding to CRE containing one 5-mC or 5-hmC in the central CG-dinucleotide detected by EMSA of the pCRE-uno PciI/BmtI fragment. ( C ) EGFP expression in HeLa cells transfected with pCRE-uno containing either one (1×) or three (3×) of the indicated CpG modifications in the promoter fragment (relative to pCRE-uno without CpG modifications). Lines represent independent transfections.

    Journal: Nucleic Acids Research

    Article Title: Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter

    doi: 10.1093/nar/gkx718

    Figure Lengend Snippet: Impacts of one or three 5-hmC in the minimal CRE promoter on the EGFP gene expression and comparison with the respective effects of 5-mC. ( A ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one or three 5-hmC. Representative fluorescence scatter plots and the correspondent overlaid fluorescence distribution plots of HeLa cells 24 h after transfection (on the left) as well as mean EGFP expression values determined relative to the construct containing unmodified oligonucleotide (‘C’). Mean of nine independent experiments ± SD; P -values calculated by the Student's t -test. ( B ) CREB binding to CRE containing one 5-mC or 5-hmC in the central CG-dinucleotide detected by EMSA of the pCRE-uno PciI/BmtI fragment. ( C ) EGFP expression in HeLa cells transfected with pCRE-uno containing either one (1×) or three (3×) of the indicated CpG modifications in the promoter fragment (relative to pCRE-uno without CpG modifications). Lines represent independent transfections.

    Article Snippet: Electrophoretic mobility shift assays Vectors pCRE-uno and pCRE-zero were digested with PciI and BmtI (both NEB) to produce the CRE-less 4228 bp fragment and the 180 bp fragment with one (pCRE-uno) or no CRE (pCRE-zero) and cleaned up.

    Techniques: Expressing, Transfection, Fluorescence, Construct, Binding Assay

    Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of pCRE-uno incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the PciI/BmtI digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter

    doi: 10.1093/nar/gkx718

    Figure Lengend Snippet: Impact of CpG hemi-methylation in the minimal CRE promoter on the CREB binding and the reporter EGFP gene expression. ( A ) Artificial CRE-uno promoter: transcription start (broken arrow), CRE sequence (bold), AatII site (underlined), Nb.BsrDI nicking positions (vertical arrows), and positions of 5-mC/5-hmC/5-fC/5-caC in the incorporated synthetic oligonucleotides (asterisks). ( B ) Inhibition of the AatII restriction endonuclease by the M.SssI methylation of the central CG-dinucleotide in CRE. Agarose gel electrophoresis of pCRE-uno incubated with AatII. Arrows indicate migration positions of the linearized vector (4408 bp) and of the covalently closed (cc) and nicked (nc) forms of circular vector DNA. ( C ) Verification of the incorporation of synthetic oligonucleotides containing one or three 5-mC (1 × 5mC, 3 × 5mC) by inhibition of the AatII cleavage. ( D ) Scheme of the PciI/BmtI digest of the pCRE-uno vector and electrophoretic mobility shift assay (EMSA) of the PciI/BmtI fragments under the indicated CREB:DNA molar ratios. The shorter (180 bp) fragment contains CRE with no or one 5-mC in the central CG-dinucleotide. The right panel shows control CRE-less vector (pCRE-zero) incubated with CREB in parallel. ( E ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one (in the central CG-dinucleotide of CRE) or three 5mC. Representative FACS data and relative EGFP expression values (mean ± SD, n = 7). Expression in the absence of an EGFP-coding vector (DsRed) denotes the lower detection limit. CRE-less vector (pCRE-zero) was included in two experiments as a reference for the basal expression level. P -values calculated by the Student's t -test.

    Article Snippet: Electrophoretic mobility shift assays Vectors pCRE-uno and pCRE-zero were digested with PciI and BmtI (both NEB) to produce the CRE-less 4228 bp fragment and the 180 bp fragment with one (pCRE-uno) or no CRE (pCRE-zero) and cleaned up.

    Techniques: Methylation, Binding Assay, Expressing, Sequencing, Inhibition, Agarose Gel Electrophoresis, Incubation, Migration, Plasmid Preparation, Electrophoretic Mobility Shift Assay, Transfection, FACS

    Impacts of one or three 5-hmC in the minimal CRE promoter on the EGFP gene expression and comparison with the respective effects of 5-mC. ( A ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one or three 5-hmC. Representative fluorescence scatter plots and the correspondent overlaid fluorescence distribution plots of HeLa cells 24 h after transfection (on the left) as well as mean EGFP expression values determined relative to the construct containing unmodified oligonucleotide (‘C’). Mean of nine independent experiments ± SD; P -values calculated by the Student's t -test. ( B ) CREB binding to CRE containing one 5-mC or 5-hmC in the central CG-dinucleotide detected by EMSA of the pCRE-uno PciI/BmtI fragment. ( C ) EGFP expression in HeLa cells transfected with pCRE-uno containing either one (1×) or three (3×) of the indicated CpG modifications in the promoter fragment (relative to pCRE-uno without CpG modifications). Lines represent independent transfections.

    Journal: Nucleic Acids Research

    Article Title: Functional impacts of 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxycytosine at a single hemi-modified CpG dinucleotide in a gene promoter

    doi: 10.1093/nar/gkx718

    Figure Lengend Snippet: Impacts of one or three 5-hmC in the minimal CRE promoter on the EGFP gene expression and comparison with the respective effects of 5-mC. ( A ) EGFP expression in HeLa cells transfected with pCRE-uno containing none, one or three 5-hmC. Representative fluorescence scatter plots and the correspondent overlaid fluorescence distribution plots of HeLa cells 24 h after transfection (on the left) as well as mean EGFP expression values determined relative to the construct containing unmodified oligonucleotide (‘C’). Mean of nine independent experiments ± SD; P -values calculated by the Student's t -test. ( B ) CREB binding to CRE containing one 5-mC or 5-hmC in the central CG-dinucleotide detected by EMSA of the pCRE-uno PciI/BmtI fragment. ( C ) EGFP expression in HeLa cells transfected with pCRE-uno containing either one (1×) or three (3×) of the indicated CpG modifications in the promoter fragment (relative to pCRE-uno without CpG modifications). Lines represent independent transfections.

    Article Snippet: Electrophoretic mobility shift assays Vectors pCRE-uno and pCRE-zero were digested with PciI and BmtI (both NEB) to produce the CRE-less 4228 bp fragment and the 180 bp fragment with one (pCRE-uno) or no CRE (pCRE-zero) and cleaned up.

    Techniques: Expressing, Transfection, Fluorescence, Construct, Binding Assay

    Identification of transcription factor specificity protein (Sp)-1–binding site in the human MIOX promoter and effect of in vitro methylation on the binding with nucleoproteins. Eletrophoretic mobility shift assays using Sp-1 and differentially methylated segment (DMS) oligos (DMS1 includes CpG sites -784 and -787 and DMS2 includes -581). A: An increased binding of Sp-1 is observed under high-glucose ambience (30 mmol/L) when the Sp-1 oligo is differentially demethylated or unmethylated ( arrowhead ). No binding of methylated or demethylated oligos is observed under low-glucose (5 mmol/L) ambience. B and C: Likewise an increased binding of unmethylated DMS1 and DMS2 oligos is observed under high-glucose ambience ( arrowhead ). The binding is also seen under low-glucose ambience; nevertheless, binding with unmethylated oligos (Sp-1, DMS1, and DMS2) under high-glucose ambience is much stronger. The arrows and asterisks indicate the nonspecific bands in the autoradiograms.

    Journal: The American Journal of Pathology

    Article Title: High Glucose–Induced Hypomethylation Promotes Binding of Sp-1 to Myo-Inositol Oxygenase

    doi: 10.1016/j.ajpath.2016.12.011

    Figure Lengend Snippet: Identification of transcription factor specificity protein (Sp)-1–binding site in the human MIOX promoter and effect of in vitro methylation on the binding with nucleoproteins. Eletrophoretic mobility shift assays using Sp-1 and differentially methylated segment (DMS) oligos (DMS1 includes CpG sites -784 and -787 and DMS2 includes -581). A: An increased binding of Sp-1 is observed under high-glucose ambience (30 mmol/L) when the Sp-1 oligo is differentially demethylated or unmethylated ( arrowhead ). No binding of methylated or demethylated oligos is observed under low-glucose (5 mmol/L) ambience. B and C: Likewise an increased binding of unmethylated DMS1 and DMS2 oligos is observed under high-glucose ambience ( arrowhead ). The binding is also seen under low-glucose ambience; nevertheless, binding with unmethylated oligos (Sp-1, DMS1, and DMS2) under high-glucose ambience is much stronger. The arrows and asterisks indicate the nonspecific bands in the autoradiograms.

    Article Snippet: For preparation of methylated oligos, CpG methytransferase ( M. SssI ; catalog number MO226S; New England Biolabs, Ipswich, MA) was used.

    Techniques: Binding Assay, In Vitro, Methylation, Mobility Shift

    ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Journal: Nucleic Acids Research

    Article Title: Multipronged regulatory functions of a novel endonuclease (TieA) from Helicobacter pylori

    doi: 10.1093/nar/gkw730

    Figure Lengend Snippet: ( A ) Binding of TieA to dsDNA: electrophoretic mobility shift assays were carried out by incubating different concentrations of TieA (0.1, 0.5, 1 and 2 μg) with 0.5 nM 32 P-labeled DNA substrates. Samples were subjected to electrophoresis on native PAGE and visualized by autoradiography as mentioned in materials and methods section. ( B ) TieA binds to DNA non-specifically: electrophoretic mobility shift assays were carried out by incubating 1 μg of TieA with mutated oligos 1–5 (see Supplementary Table S1). ( C ) Nuclease activity of TieA: different concentrations of TieA (0.01, 0.1, 0.2, 0.5, 1 and 2 μg corresponding to lanes 7-12, respectively) were incubated with 1 μg of pUC19 DNA for 1 h at 37 °C. The reaction was stopped by addition of 10 mM EDTA and samples were deprotonized by adding proteinase K (10 μg/sample) in presence of 0.05% SDS for 15 min at 65°C. The digested products were separated on 1.2% agarose gel. Rv3131 (0.5 μg) was used as a negative control in lane 6. MboII (1 unit/reaction) and DNase I (1 unit/reaction) served as positive controls in lanes 3 and 5, respectively. Lane 4 represents heat inactivated TieA. ( D ) TieA cleaves both pUC19 (circular) and Lambda DNA (linear): pUC19 and Lambda DNA were incubated with TieA (lanes 5, 6, 14 and 15) for 1 h at 37°C and processed as described above. MboII (lanes 3 and 12) and DNase I (lanes 4 and 13) were used as positive controls. Rv3131 protein was used as a negative control (lanes 7 and 16). Ca 2+ –Mg 2+ dependent nuclease activity of TieA was confirmed by pre-incubating pUC19/Lambda DNA with either SDS (0.05%) or EDTA (10 mM) for 10 min (lanes 8, 9, 17 and 18) and later 1 μg of TieA was added and further processed as described above. Data are representative of three independent experiments. HI: heat inactivated.

    Article Snippet: The cleavage reaction was initiated with the addition of MboII or DNase I (1 U/reaction, New England Biolabs) enzyme.

    Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Labeling, Electrophoresis, Clear Native PAGE, Autoradiography, Activity Assay, Incubation, Agarose Gel Electrophoresis, Negative Control, Lambda DNA Preparation

    Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.

    Journal: PLoS ONE

    Article Title: High-Level Expression of Endo-β-N-Acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and Its Application for the Deglycosylation of Glycoproteins

    doi: 10.1371/journal.pone.0120458

    Figure Lengend Snippet: Determining the enzyme activity of Endo H-P through mobility shift assay of RNase B. M protein molecular weight markers (the size of each band was indicated on the left); Lane 1 denatured RNase B (negative control); Lane 2 denatured RNase B treated with 1 U of commercial Endo H from NEB, USA (positive control); Lane 3–8 denatured RNase B treated with 1μl of Endo H-P diluted into 5.0%, 4.0%, 3.5%, 3.0%, 2.5% and 2.0%, respectively.

    Article Snippet: RNase B, commercial Endo H and PNGase F were purchased from NEB (USA).

    Techniques: Activity Assay, Mobility Shift, Molecular Weight, Negative Control, Positive Control

    Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B. (A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.

    Journal: PLoS ONE

    Article Title: High-Level Expression of Endo-β-N-Acetylglucosaminidase H from Streptomyces plicatus in Pichia pastoris and Its Application for the Deglycosylation of Glycoproteins

    doi: 10.1371/journal.pone.0120458

    Figure Lengend Snippet: Analyzing the characteristics of Endo H-P through mobility shift assay of RNase B. (A).Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 to Lane 6 denatured RNase B treated with concentrated Endo H-P at 25°C, 30°C, 35°C, 40°C, 45°C and 50°C, respectively; Lane 7 denatured RNase B treated with concentrated Endo H-P at 37°C, respectively;Lane 8 the negative control (RNase B without treatment); Lane 9 the positive control (overdose of Endo H-P was added to the reaction system);(B). Identifying the optimum temperature of Endo H-P with SDS-PAGE. M protein molecular weight markers (the size of each band was indicated on the left);Lane 1 the positive control (overdose of Endo H-P was added to the reaction system); Lane 2 the negative control (RNase B without treatment);Lane 3–8 denatured RNase B treated with Endo H-P at pH5.0, 5.5, 6.0, 6.5, 7.0 and 7.5, respectively.

    Article Snippet: RNase B, commercial Endo H and PNGase F were purchased from NEB (USA).

    Techniques: Mobility Shift, SDS Page, Molecular Weight, Negative Control, Positive Control

    The LPS-stimulated activation of Pellino 1 is prevented by inhibition of TBK1/IKK ε and reversed by phosphatase treatment ( A ) and incubated for 60 min at 30°C in 0.02 ml of 50 mM Tris/HCl (pH 7.5), 5 mM MgCl 2 , UBE1 (0.1 μ M), Ubc13–Uev1a (0.05 μ M), FLAG–ubiquitin (0.05 mM) and ATP (2 mM) in the presence of the non-specific protein kinase inhibitor staurosporine (75 μ ] to prevent any phosphorylation and activation of Pellino 1 during the assay by protein kinases present in the immunoprecipitates as trace contaminants. The reactions were terminated by denaturation in SDS, and immunoblotted with an anti-FLAG antibody (upper panel). Cell lysates were subjected to SDS/PAGE and immunoblotted with antibodies that recognize Pellino 1 or IRF3 phosphorylated at Ser 396 (lower two panels). ( B ) RAW264.7 macrophages were stimulated with LPS for 30 min or left unstimulated (0 min) and Pellino 1 was immunoprecipitated as in ( A ). The immunoprecipitates were then incubated for 30 min at 30°C with (+) or without (−) 100 units of phage λ gt10 phosphatase and 1 mM MnCl 2 in the presence (+) or absence (−) of 50 mM NaF. After washing three times in 50 mM Tris/HCl (pH 7.5) and 5 mM MgCl 2 , the immunoprecipitated Pellino 1 was assayed for E3 ubiquitin ligase activity as in ( A ) and cell lysates immunoblotted with the antibodies used in ( A ). ( C ) As in ( B ) except that the RAW264.7 macrophages were incubated for 1 h without (−) or with (+) 2 μ M MRT67307, before stimulation with LPS. To aid clarity, ( A – C ) do not show the whole gel, but only the region with polyubiquitylated species of > 150 kDa. ( D ) RAW264.7 macrophages were stimulated with 100 ng/ml LPS (Sigma) for 60 min and the cells were lysed in the absence of any phosphatase inhibitor. Cell lysate (20 μ g protein) was then incubated for 30 min at 30°C with (+) or without (−) 300 units of phage λ gt10 phosphatase in the presence of 1 mM MnCl 2 . The reactions were terminated in SDS, subjected to SDS/PAGE and immunoblotted with antibodies that recognize Pellino 1 or actin. The experiment was carried out three times with similar results.

    Journal: The Biochemical journal

    Article Title: The role of TBK1 and IKKε in the expression and activation of Pellino 1

    doi: 10.1042/BJ20101421

    Figure Lengend Snippet: The LPS-stimulated activation of Pellino 1 is prevented by inhibition of TBK1/IKK ε and reversed by phosphatase treatment ( A ) and incubated for 60 min at 30°C in 0.02 ml of 50 mM Tris/HCl (pH 7.5), 5 mM MgCl 2 , UBE1 (0.1 μ M), Ubc13–Uev1a (0.05 μ M), FLAG–ubiquitin (0.05 mM) and ATP (2 mM) in the presence of the non-specific protein kinase inhibitor staurosporine (75 μ ] to prevent any phosphorylation and activation of Pellino 1 during the assay by protein kinases present in the immunoprecipitates as trace contaminants. The reactions were terminated by denaturation in SDS, and immunoblotted with an anti-FLAG antibody (upper panel). Cell lysates were subjected to SDS/PAGE and immunoblotted with antibodies that recognize Pellino 1 or IRF3 phosphorylated at Ser 396 (lower two panels). ( B ) RAW264.7 macrophages were stimulated with LPS for 30 min or left unstimulated (0 min) and Pellino 1 was immunoprecipitated as in ( A ). The immunoprecipitates were then incubated for 30 min at 30°C with (+) or without (−) 100 units of phage λ gt10 phosphatase and 1 mM MnCl 2 in the presence (+) or absence (−) of 50 mM NaF. After washing three times in 50 mM Tris/HCl (pH 7.5) and 5 mM MgCl 2 , the immunoprecipitated Pellino 1 was assayed for E3 ubiquitin ligase activity as in ( A ) and cell lysates immunoblotted with the antibodies used in ( A ). ( C ) As in ( B ) except that the RAW264.7 macrophages were incubated for 1 h without (−) or with (+) 2 μ M MRT67307, before stimulation with LPS. To aid clarity, ( A – C ) do not show the whole gel, but only the region with polyubiquitylated species of > 150 kDa. ( D ) RAW264.7 macrophages were stimulated with 100 ng/ml LPS (Sigma) for 60 min and the cells were lysed in the absence of any phosphatase inhibitor. Cell lysate (20 μ g protein) was then incubated for 30 min at 30°C with (+) or without (−) 300 units of phage λ gt10 phosphatase in the presence of 1 mM MnCl 2 . The reactions were terminated in SDS, subjected to SDS/PAGE and immunoblotted with antibodies that recognize Pellino 1 or actin. The experiment was carried out three times with similar results.

    Article Snippet: The protein phosphatase encoded by bacteriophage λgt10 was obtained from New England Biolabs, and ubiquitin was purchased from Sigma.

    Techniques: Activation Assay, Inhibition, Incubation, SDS Page, Immunoprecipitation, Activity Assay

    TBK1 and IKK ε phosphorylate Pellino 1 and enhance its E3 ubiquitin ligase activity ( A ) Pellino 1 (1 μ M) was incubated for 30 min at 30°C with MgATP in the presence (+) or absence (−) of 2 units/ml IRAK4 or 2 units/ml TBK1 in 50 mM Tris/HCl (pH 7.5) which had been pre-incubated in the absence (−) or presence (+) of 2 μ M MRT67307 or 10 μ M IRAK4 inhibitor. The reactions were terminated by denaturation in SDS, subjected to SDS/PAGE and the gel was stained with Coomassie Blue. The positions of unphosphorylated and phosphorylated (p) forms of Pellino 1 are indicated. ( B ) The experiment was carried out as in as ( A ), except that IKK ε was used instead of TBK1. ( C and D ) Pellino 1 (1 μ M), Ubc13–Uev1a (1 μ M), ubiquitin (0.1 mM), MgCl 2 (5 mM) and ATP (2 mM) were incubated for 30 min at 30°C without (−) or with (+) 2 units/ml TBK1 ( C ) or 2 units/ml IKK ε ( D ) and the ubiquitylation reactions initiated by the addition of E1 enzyme (0.1 μ ].

    Journal: The Biochemical journal

    Article Title: The role of TBK1 and IKKε in the expression and activation of Pellino 1

    doi: 10.1042/BJ20101421

    Figure Lengend Snippet: TBK1 and IKK ε phosphorylate Pellino 1 and enhance its E3 ubiquitin ligase activity ( A ) Pellino 1 (1 μ M) was incubated for 30 min at 30°C with MgATP in the presence (+) or absence (−) of 2 units/ml IRAK4 or 2 units/ml TBK1 in 50 mM Tris/HCl (pH 7.5) which had been pre-incubated in the absence (−) or presence (+) of 2 μ M MRT67307 or 10 μ M IRAK4 inhibitor. The reactions were terminated by denaturation in SDS, subjected to SDS/PAGE and the gel was stained with Coomassie Blue. The positions of unphosphorylated and phosphorylated (p) forms of Pellino 1 are indicated. ( B ) The experiment was carried out as in as ( A ), except that IKK ε was used instead of TBK1. ( C and D ) Pellino 1 (1 μ M), Ubc13–Uev1a (1 μ M), ubiquitin (0.1 mM), MgCl 2 (5 mM) and ATP (2 mM) were incubated for 30 min at 30°C without (−) or with (+) 2 units/ml TBK1 ( C ) or 2 units/ml IKK ε ( D ) and the ubiquitylation reactions initiated by the addition of E1 enzyme (0.1 μ ].

    Article Snippet: The protein phosphatase encoded by bacteriophage λgt10 was obtained from New England Biolabs, and ubiquitin was purchased from Sigma.

    Techniques: Activity Assay, Incubation, SDS Page, Staining

    Effect of MBD1 isoforms on methylated and unmethylated promoters. (A) PCR-amplified DNA fragments from human imprinted SNRPN and tumor suppressor p16 genes were used for a band shift analysis and subcloned upstream of a luciferase cDNA in a pGL3-Basic vector. The PCR fragments and pGL3 constructs were methylated in vitro using Hpa II, Hha I, and Sss I (CpG) methyltransferases. The methyl-CpG sites modified by these enzymes are shown by vertical lines. (B) Band shift of methylated DNA complexed with the methyl-CpG binding domain of MBD1. Unmethylated (−) and methylated fragments containing SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. In the upper and lower panels, the amount of the protein incubated with DNA fragments was 0.5 and 1.0 μg, respectively. (C and D) Regulation of Sp1-activated transcription by MBD1v1 and -v3 in Drosophila SL2 cells ( SNRPN [C] or p16 [D]). Unmethylated (M-) or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with Sp1-expressing plasmid pPacSp1 (0.5 μg), MBD1-expressing plasmids (pAc5.1-MBD1v1 and pAc5.1-MBD1v3) (0 to 1.0 μg), and insertless plasmid pAc5.1/V5-His (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pAc5.1-pRL (0.1 μg). (E) Detection of endogenous MBD1 by an antibody raised against the recombinant MBD1. MBD1 was found to be approximately 80 kDa in HeLa and A549 cells but not in SL2 and CHO-K1 cells.

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1

    doi:

    Figure Lengend Snippet: Effect of MBD1 isoforms on methylated and unmethylated promoters. (A) PCR-amplified DNA fragments from human imprinted SNRPN and tumor suppressor p16 genes were used for a band shift analysis and subcloned upstream of a luciferase cDNA in a pGL3-Basic vector. The PCR fragments and pGL3 constructs were methylated in vitro using Hpa II, Hha I, and Sss I (CpG) methyltransferases. The methyl-CpG sites modified by these enzymes are shown by vertical lines. (B) Band shift of methylated DNA complexed with the methyl-CpG binding domain of MBD1. Unmethylated (−) and methylated fragments containing SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. In the upper and lower panels, the amount of the protein incubated with DNA fragments was 0.5 and 1.0 μg, respectively. (C and D) Regulation of Sp1-activated transcription by MBD1v1 and -v3 in Drosophila SL2 cells ( SNRPN [C] or p16 [D]). Unmethylated (M-) or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with Sp1-expressing plasmid pPacSp1 (0.5 μg), MBD1-expressing plasmids (pAc5.1-MBD1v1 and pAc5.1-MBD1v3) (0 to 1.0 μg), and insertless plasmid pAc5.1/V5-His (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pAc5.1-pRL (0.1 μg). (E) Detection of endogenous MBD1 by an antibody raised against the recombinant MBD1. MBD1 was found to be approximately 80 kDa in HeLa and A549 cells but not in SL2 and CHO-K1 cells.

    Article Snippet: DNA fragments (516 bp long) for the promoter region of the human SNRPN gene were amplified from human genomic DNA ( ) and were methylated with Hpa II, Hha I, or Sss I methyltransferases as indicated by the manufacturer (New England Biolabs, Beverly, Mass.).

    Techniques: Methylation, Polymerase Chain Reaction, Amplification, Electrophoretic Mobility Shift Assay, Luciferase, Plasmid Preparation, Construct, In Vitro, Modification, Binding Assay, Incubation, Expressing, Activity Assay, Transfection, Recombinant

    Transcriptional regulation by MBD1 isoforms and their mutants with deletions of the methyl-CpG binding domain in mammalian CHO-K1 cells. Unmethylated or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with MBD1-expressing plasmids (pCGN-MBD1v1, pCGN-MBD1v3, pCGN-MBD1v1ΔN, and pCGN-MBD1v3ΔN) (0 to 1.0 μg) and insertless plasmid pCGN (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with 1.0 μg of pCGN (mock) was normalized to 10,000, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pRL-SV40 (0.1 μg). The combinations of pGL3-SNRPN and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (A), pGL3-SNRPN and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (B), pGL3-p16 and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (C), and pGL3-p16 and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (D) are shown.

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1

    doi:

    Figure Lengend Snippet: Transcriptional regulation by MBD1 isoforms and their mutants with deletions of the methyl-CpG binding domain in mammalian CHO-K1 cells. Unmethylated or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with MBD1-expressing plasmids (pCGN-MBD1v1, pCGN-MBD1v3, pCGN-MBD1v1ΔN, and pCGN-MBD1v3ΔN) (0 to 1.0 μg) and insertless plasmid pCGN (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with 1.0 μg of pCGN (mock) was normalized to 10,000, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pRL-SV40 (0.1 μg). The combinations of pGL3-SNRPN and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (A), pGL3-SNRPN and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (B), pGL3-p16 and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (C), and pGL3-p16 and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (D) are shown.

    Article Snippet: DNA fragments (516 bp long) for the promoter region of the human SNRPN gene were amplified from human genomic DNA ( ) and were methylated with Hpa II, Hha I, or Sss I methyltransferases as indicated by the manufacturer (New England Biolabs, Beverly, Mass.).

    Techniques: Binding Assay, Methylation, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Transfection

    Effect of MBD1v1 mutants deleted of the CXXC domains on Sp1-activated transcription in Drosophila SL2 cells. (A) Four pAc5.1/V5-His plasmids expressing deletion mutants of MBD1v1 were designated MBD1v1Δ1 to MBD1v1Δ4. (B) Each of the plasmids for MBD1v1 deletion mutants (1.0 μg) was transfected into Drosophila cells together with pPacSp1 (0.5 μg), and either unmethylated (black bars) or Hpa II (gray bars)-, Hha I (white bars)-, or Sss I (hatched bars)-methylated pGL3-SNRPN (0.5 μg). The luciferase activity of unmethylated pGL3-SNRPN in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100. Relative luciferase activities (means + standard deviations [error bars]) are given.

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1

    doi:

    Figure Lengend Snippet: Effect of MBD1v1 mutants deleted of the CXXC domains on Sp1-activated transcription in Drosophila SL2 cells. (A) Four pAc5.1/V5-His plasmids expressing deletion mutants of MBD1v1 were designated MBD1v1Δ1 to MBD1v1Δ4. (B) Each of the plasmids for MBD1v1 deletion mutants (1.0 μg) was transfected into Drosophila cells together with pPacSp1 (0.5 μg), and either unmethylated (black bars) or Hpa II (gray bars)-, Hha I (white bars)-, or Sss I (hatched bars)-methylated pGL3-SNRPN (0.5 μg). The luciferase activity of unmethylated pGL3-SNRPN in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100. Relative luciferase activities (means + standard deviations [error bars]) are given.

    Article Snippet: DNA fragments (516 bp long) for the promoter region of the human SNRPN gene were amplified from human genomic DNA ( ) and were methylated with Hpa II, Hha I, or Sss I methyltransferases as indicated by the manufacturer (New England Biolabs, Beverly, Mass.).

    Techniques: Expressing, Transfection, Methylation, Luciferase, Activity Assay