α-32 p Search Results


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  • 99
    Thermo Fisher α 32 p
    α 32 P, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 98 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer α 32 p datp
    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and <t>dATP</t> (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
    α 32 P Datp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 980 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer dctp α 32 p
    RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 <t>P]-dCTP)</t> and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.
    Dctp α 32 P, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 131 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer α 32 p dttp
    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 <t>P]dTTP</t> end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.
    α 32 P Dttp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 241 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer α 32 p dgtp
    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 <t>P]dGTP</t> (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.
    α 32 P Dgtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    PerkinElmer α 32 p gtp
    YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 <t>P]GTP</t> and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.
    α 32 P Gtp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 96/100, based on 847 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare α 32 p
    Northern blot analysis of p29 mRNA. Total RNA isolated from partially fed adult female  H. longicornis  ticks was resolved on an 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with the α- 32 P-labeled PCR product amplified from the cloned p29 phage DNA by using the λscreen expression vector-specific primers (SP6 promoter and T7 terminator). +, native p29.
    α 32 P, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    NEN Life Science α 32 p
    Northern blot analysis of p29 mRNA. Total RNA isolated from partially fed adult female  H. longicornis  ticks was resolved on an 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with the α- 32 P-labeled PCR product amplified from the cloned p29 phage DNA by using the λscreen expression vector-specific primers (SP6 promoter and T7 terminator). +, native p29.
    α 32 P, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 86/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    PerkinElmer α 32 p
    Northern blot analysis of p29 mRNA. Total RNA isolated from partially fed adult female  H. longicornis  ticks was resolved on an 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with the α- 32 P-labeled PCR product amplified from the cloned p29 phage DNA by using the λscreen expression vector-specific primers (SP6 promoter and T7 terminator). +, native p29.
    α 32 P, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    DuPont de Nemours α 32 p
    Northern blot analysis of p29 mRNA. Total RNA isolated from partially fed adult female  H. longicornis  ticks was resolved on an 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with the α- 32 P-labeled PCR product amplified from the cloned p29 phage DNA by using the λscreen expression vector-specific primers (SP6 promoter and T7 terminator). +, native p29.
    α 32 P, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    PerkinElmer α 32 p atp
    Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 <t>P-ATP,</t> resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009
    α 32 P Atp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 95/100, based on 863 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer α 32 p ctp
    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 <t>P]CTP</t> and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.
    α 32 P Ctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 571 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    87
    DuPont de Nemours α 32 p datp
    Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 <t>P]dATP.</t> The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.
    α 32 P Datp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 87/100, based on 208 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    GE Healthcare α 32 p datp
    Formation of TP-dAMP complex catalysed by Nf and GA-1 DNA polymerases. The assays were performed as described under Materials and Methods in the presence of 1 mM (for GA-1 and φ29 DNA polymerases) and 2 mM (for Nf DNA polymerase) MnCl 2 , 5 ng of TP, 500 ng of TP-DNA, 0.1 μM [α- 32 <t>P]dATP</t> (1 μCi), and the indicated amounts of DNA polymerase. After incubation at 30°C for the indicated times, samples were analysed by SDS–PAGE and autoradiography. The position of the TP-dAMP initiation complex is indicated. Quantification was by densitometry of the band corresponding to the labelled TP-dAMP complex, detected by autoradiography.
    α 32 P Datp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 90/100, based on 2301 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    HARTMANN ANALYTIC α 32 p datp
    Formation of TP-dAMP complex catalysed by Nf and GA-1 DNA polymerases. The assays were performed as described under Materials and Methods in the presence of 1 mM (for GA-1 and φ29 DNA polymerases) and 2 mM (for Nf DNA polymerase) MnCl 2 , 5 ng of TP, 500 ng of TP-DNA, 0.1 μM [α- 32 <t>P]dATP</t> (1 μCi), and the indicated amounts of DNA polymerase. After incubation at 30°C for the indicated times, samples were analysed by SDS–PAGE and autoradiography. The position of the TP-dAMP initiation complex is indicated. Quantification was by densitometry of the band corresponding to the labelled TP-dAMP complex, detected by autoradiography.
    α 32 P Datp, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 92/100, based on 81 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ICN Pharmaceuticals α 32 p datp
    Formation of TP-dAMP complex catalysed by Nf and GA-1 DNA polymerases. The assays were performed as described under Materials and Methods in the presence of 1 mM (for GA-1 and φ29 DNA polymerases) and 2 mM (for Nf DNA polymerase) MnCl 2 , 5 ng of TP, 500 ng of TP-DNA, 0.1 μM [α- 32 <t>P]dATP</t> (1 μCi), and the indicated amounts of DNA polymerase. After incubation at 30°C for the indicated times, samples were analysed by SDS–PAGE and autoradiography. The position of the TP-dAMP initiation complex is indicated. Quantification was by densitometry of the band corresponding to the labelled TP-dAMP complex, detected by autoradiography.
    α 32 P Datp, supplied by ICN Pharmaceuticals, used in various techniques. Bioz Stars score: 88/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Valiant α 32 p datp
    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 <t>P]dATP.</t> DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .
    α 32 P Datp, supplied by Valiant, used in various techniques. Bioz Stars score: 93/100, based on 140 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    HARTMANN ANALYTIC α 32 p dct
    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 <t>P]dATP.</t> DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .
    α 32 P Dct, supplied by HARTMANN ANALYTIC, used in various techniques. Bioz Stars score: 86/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    PerkinElmer α 32 p dntp
    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 <t>P]dATP.</t> DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .
    α 32 P Dntp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 93/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    GE Healthcare dct α 32 p
    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 <t>P]dATP.</t> DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .
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    NEN Life Science α 32 p datp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    Valiant α 32 p pcp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    DuPont de Nemours α 32 p rutp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    PerkinElmer α 32 p rutp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    GE Healthcare α 32 p rctp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    GE Healthcare α 32 p dcτp
    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 <t>P]dATP-labeled</t> DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.
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    NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 <t>P]NTP</t> was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.
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    GE Healthcare α 32 p αdctp
    NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 <t>P]NTP</t> was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.
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    NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 <t>P]NTP</t> was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.
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    NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 <t>P]NTP</t> was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.
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    Thermo Fisher α 32 p datp
    NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 <t>P]NTP</t> was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.
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    Image Search Results


    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

    Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

    Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Article Snippet: To test the nucleotide specificity of in vitro HBV priming, priming assays were performed using 1 μl [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]TTP, or [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer).

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker

    Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM α- 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM dNTPs mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.

    Journal: PLoS ONE

    Article Title: Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex

    doi: 10.1371/journal.pone.0010372

    Figure Lengend Snippet: Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM α- 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM dNTPs mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.

    Article Snippet: For HIV and MuLV RT, a mixture of 200 nM 48-mer RNA template annealed to 100 nM 18-mer DNA primer , 500 µM dNTPs, 0.16 µM α-32 P-dNTPs (PerkinElmer, 3000 Ci/mmol) were incubated with RT for 1 hour at 37°C under conditions described previously (16).

    Techniques: Sequencing, Binding Assay, Concentration Assay, DNA Synthesis

    RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Bacillus subtilis RarA modulates replication restart

    doi: 10.1093/nar/gky541

    Figure Lengend Snippet: RarA has no effect on ongoing DNA replication. ( A ) Scheme of the experimental design. The B. subtilis replisome was assembled on the DNA in the absence of RarA and in the presence of limiting ATPγS and then DNA replication was started by dNTP (including [α- 32 P]-dCTP) and ATP addition. After 20 s of initiating the reaction, 100 nM RarA was added or not, and reactions were continued for the indicated times. ( B ) Quantification of leading strand synthesis (mean ± SEM of > 3 independent experiments). ( C ) The leading strand DNA products obtained in one of these assays are visualized by denaturing gel electrophoresis and autoradiography.

    Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer.

    Techniques: Nucleic Acid Electrophoresis, Autoradiography

    RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Bacillus subtilis RarA modulates replication restart

    doi: 10.1093/nar/gky541

    Figure Lengend Snippet: RarA does not inhibit SPP1 DNA replication. Quantification of leading ( A ) and lagging ( B ) strand synthesis obtained in standard SPP1 rolling circle DNA replication assays in the absence or in the presence of 100 nM RarA. Reaction mixes contained the SPP1 replisome, which is composed by SPP1 preprimosomal proteins (G 38 P and G 39 P) and DNA helicase G 40 P, and host proteins (DnaG, τ-complex, β, PolC and DnaE). The SPP1 replisome works with both SSB proteins (SsbA or G 36 P) and the effect of RarA on reactions having either viral G 36 P or host SbsA was tested. An enzyme mix consisting of all proteins except the SSB was generated, and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and the indicated SSB (none, 30 nM G 36 P, or, 90 nM SsbA). Then reactions were placed at 37°C and incubated for 10 min. Leading strand synthesis was quantified by [α- 32 P]-dCTP incorporation and lagging strand synthesis by [α- 32 P]-dGTP incorporation. The results are expressed as the mean ± SEM of > 3 independent experiments.

    Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer.

    Techniques: Generated, Incubation

    SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.

    Journal: Nucleic Acids Research

    Article Title: Bacillus subtilis RarA modulates replication restart

    doi: 10.1093/nar/gky541

    Figure Lengend Snippet: SsbA-dependent RarA-mediated inhibition of B. subtilis PriA-dependent DNA replication. ( A ) Total DNA synthesis obtained in the presence of increasing RarA concentrations (15 min, 37°C). Reaction mixes contained all replisome components (preprimosomal proteins [PriA, DnaB, DnaD, DnaI), DnaC, DnaG, SsbA, τ-complex, β, PolC, DnaE), the indicated RarA concentration, template DNA, rNTPs, dNTPs and [α- 32 P]-dCTP and [α- 32 P]-dGTP. An enzyme mix consisting of all proteins except SsbA was generated and added to a substrate mix composed of template DNA, rNTPs, dNTPs, and SsbA. Then, samples were placed at 37°C. ( B ) Visualization of products obtained in the presence of 100 nM RarA or RarAK51A (15 min, 37°C). In the presence of [α- 32 P]-dCTP very large DNA fragments derived from rolling circle leading strand DNA synthesis is observed. A parallel reaction in the presence of [α- 32 P]-dGTP renders visible the small Okazaki fragments due to lagging strand DNA synthesis. Quantification of leading ( C ) and lagging strand ( D ) synthesis in the absence/presence of 100nM RarA and the indicated SsbA concentrations (15 min, 37°C). The quantification of the results is expressed as the mean ± SEM of six independent experiments. On the right part, a representative alkaline gel visualized by autoradiography showing the products of the DNA synthesis obtained in the presence or absence of RarA and SsbA.

    Article Snippet: The radioactive nucleotides, [γ-32 P]-ATP, [α-32 P]-dCTP and [α-32 P]-dGTP, were from Perkin Elmer.

    Techniques: Inhibition, DNA Synthesis, Concentration Assay, Generated, Derivative Assay, Autoradiography

    Gene expression of inflammatory mediators in the lungs after hemorrhagic shock and resuscitation (HSR). Lungs from the HSR group rats treated with vehicle or biliverdin (BV) were excised at 3 h after resuscitation and the levels of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) mRNA were determined by Northern blot analysis. (Left) The autoradiographic signals of RNA blot hybridized with [α- 32 P] deoxycytidine triphosphate-labeled TNF-α ( A ) or iNOS ( B ) cDNA. (Right) Concentrations of TNF-α and iNOS mRNA were expressed as arbitrary units. Data are presented as means ± standard deviation and were statistically evaluated using analysis of variance followed by Tukey–Kramer honestly significant difference test (n = 3 per group). * p

    Journal: PLoS ONE

    Article Title: Effects of Biliverdin Administration on Acute Lung Injury Induced by Hemorrhagic Shock and Resuscitation in Rats

    doi: 10.1371/journal.pone.0063606

    Figure Lengend Snippet: Gene expression of inflammatory mediators in the lungs after hemorrhagic shock and resuscitation (HSR). Lungs from the HSR group rats treated with vehicle or biliverdin (BV) were excised at 3 h after resuscitation and the levels of tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) mRNA were determined by Northern blot analysis. (Left) The autoradiographic signals of RNA blot hybridized with [α- 32 P] deoxycytidine triphosphate-labeled TNF-α ( A ) or iNOS ( B ) cDNA. (Right) Concentrations of TNF-α and iNOS mRNA were expressed as arbitrary units. Data are presented as means ± standard deviation and were statistically evaluated using analysis of variance followed by Tukey–Kramer honestly significant difference test (n = 3 per group). * p

    Article Snippet: Preparation of cDNA Template cDNAs for tumor necrosis factor (TNF)-α and inducible nitric oxide synthase (iNOS) were prepared as described previously., All probes used for Northern blot analysis were [α-32 P] deoxycytidine triphosphate (dCTP) (PerkinElmer Japan Co. Yokohama, Japan)-labeled cDNA prepared using a random primer DNA labeling system (GE Healthcare Japan Co. Tokyo, Japan) according to the manufacturer’s instructions.

    Techniques: Expressing, Northern Blot, Northern blot, Labeling, Standard Deviation

    Taq polymerase I614K introduces rCTP in PCR products. DNA is amplified by I614K Taq pol mutant in the presence of radiolabeled α 32 P-dCTP or α 32 P-rCTP, and then it is separated onto an agarose gel, after purification through drop-dialysis and ethanol precipitation. The radioactive signal visible in the rCTP lane confirms that PCR products contain rNMPs.

    Journal: Biophysical Journal

    Article Title: The Incorporation of Ribonucleotides Induces Structural and Conformational Changes in DNA

    doi: 10.1016/j.bpj.2017.07.013

    Figure Lengend Snippet: Taq polymerase I614K introduces rCTP in PCR products. DNA is amplified by I614K Taq pol mutant in the presence of radiolabeled α 32 P-dCTP or α 32 P-rCTP, and then it is separated onto an agarose gel, after purification through drop-dialysis and ethanol precipitation. The radioactive signal visible in the rCTP lane confirms that PCR products contain rNMPs.

    Article Snippet: PCR with radiolabeled nucleotides PCR reactions were performed adding α 32 P-dCTP or α 32 P-rCTP (PerkinElmer, Waltham, MA) in addition to the nonradioactive dCTP or rCTP, respecting the final concentrations described above for RC-DNAs.

    Techniques: Polymerase Chain Reaction, Amplification, Mutagenesis, Agarose Gel Electrophoresis, Purification, Ethanol Precipitation

    RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Journal: Molecular and Cellular Biology

    Article Title: A Novel Retinoic Acid-Responsive Element Regulates Retinoic Acid-Induced BLR1 Expression

    doi: 10.1128/MCB.24.6.2423-2443.2004

    Figure Lengend Snippet: RA-induced nuclear extracts protect sequences in the distal region of the BLR1 promoter. A dsDNA fragment of 250 bp (spanning 217 bp [−1096 to −879] in the BLR1 promoter plus 25 bp at the 5′ end from the plasmid backbone sequence in the pBLR1-Luc promoter-reporter construct and 8 nt from the incorporated Eco RI and Pst I site) was prepared by PCR. After digestion with Eco RI and Pst I, the amplified fragment was [α- 32 P]dATP and [α- 32 P]dTTP end labeled at the 3′ recessed end with the Klenow fragment of Escherichia coli DNA polymerase I and used in the DNase I footprinting assay with nuclear extracts from HL-60 cells that were either left untreated (RA − ) or treated (RA + ) with all- trans -RA for 48 h. A DNA sequencing ladder (10 bp) was end labeled (using T4 polynucleotide kinase) with [γ- 32 P]ATP, heat denatured, and corun with the DNase I-treated samples as a size marker. The nucleotide sequence of the DNase I-protected site was determined by alignment of the protected region with the sequencing ladder. An approximately 17-bp region (−1071 to −1055) with the indicated sequence was specifically protected from DNase I digestion in the nuclear extracts from RA-treated cells. No footprint was visible with nuclear extracts from untreated cells. An autoradiograph of the DNA footprint is shown. The sizes of the denatured DNA sequence markers that were corun with the samples are indicated with arrows on the left side of the right panel. The 5′ and 3′ ends of the DNA probe used in the footprinting assay are indicated by arrows pointing up and down. The nucleotide sequence of the DNA footprint is shown on the right. Numbers indicate the positions of start and end points of the protection region relative to +1, the transcriptional initiation site.

    Article Snippet: Rabbit polyclonal antibodies for each of RARα, RXRα, Oct1, Oct2, NTATc1, NFATc2, NFATc3, NFATc4, NFATc5, CREB1, and CREB2 and normal anti-rabbit immunoglobulin G (IgG) were purchased from Santa Cruz Biotechnology Inc. [α-32 P]dCTP, [α-32 P]dATP, [α-32 P]dTTP, and [γ-32 P]ATP were obtained from Perkin Elmer Life Sciences.

    Techniques: Plasmid Preparation, Sequencing, Construct, Polymerase Chain Reaction, Amplification, Labeling, Footprinting, DNA Sequencing, Marker, Autoradiography

    Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of DP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified DP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between DP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B). The beads, which contained the primed DP, were processed for SDS-PAGE to visualize the labeled DP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of TMgNK buffer and [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled dCTP, TTP, and dATP (A, lanes 3 and 4; B, lanes 7 and 8). (C) [α- 32 P]dGTP stock was mock (lane 4) or apyrase treated (lane 5). The DP priming product obtained in TMgNK buffer and [α- 32 P]dGTP was either mock treated (lane 2) or Tdp2 treated (lane 3), which released dGMP from the DP-dGMP phosphotyrosyl linkage. Samples were resolved on a urea–20% polyacrylamide gel. The positions of 32 P-labeled 10-nucleotide marker (Invitrogen) (B) and DNA oligomers (dTG, dTGA, and dTGAA in panels B and C) are indicated, as are the positions of dGTP and dGMP. (D) HPLC analysis of dGTP and dGMP. (Panel 1) UV ( A 260 ) detection showing retention times of unlabeled dGMP and dGTP. (Panel 2) Detection of 32 P radioactivity from mock-treated DP priming products (−Tdp2), showing the absence of dGMP and the presence of residual dGTP substrate input. (Panel 3) Detection of 32 P radioactivity from Tdp2-treated DP priming products (+Tdp2), showing the presence of dGMP released by Tdp2 from DP and again some residual dGTP substrate input. The positions of dGMP and dGTP are indicated.

    Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker, High Performance Liquid Chromatography, Radioactivity

    Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Detection of in vitro protein priming by purified HP. Priming reactions were performed by incubating immunoaffinity-purified HP with TMgNK buffer and [α- 32 P]dGTP (A to C ) or another labeled nucleotide as indicated (D and E). After priming, the beads were washed, and the labeled HP was resolved on an SDS–12.5% polyacrylamide gel. A priming reaction was also performed with the DHBV MiniRT2 (DP) in TMnNK buffer and resolved on the same gel for comparison (A, lane 1). Labeled HP and DP priming products were detected by autoradiography after SDS-PAGE. (A) In vitro priming reactions with WT (lanes 3 and 4) or mutant (lanes 5 and 6) HP with (lanes 4 to 6) or without Hε (lane 3) coexpression in cells. GFP + Hε (lane 2) represents priming using the control purification product from cells cotransfected with GFP and the Hε-expressing plasmid. (B) After protein priming, primed HP was untreated (−; lane 1) or treated with DNase I (D; lane 2) or pronase (P; lane 3) before analysis by SDS-PAGE. (C) The purified HP was mock treated (lane 1) or RNase treated (lane 2) before being used in protein priming. Labeled HP was detected by autoradiography after SDS-PAGE (top), and HP protein levels were measured by Western blotting using the anti-FLAG (α-Flag) antibody (bottom). (D) HP purified either with (lanes 5 to 8) or without (lanes 1 to 4) the coexpressed Hε was assayed for priming activity in the presence of [α- 32 P]dGTP (G; lanes 2 and 6), [α- 32 P]TTP (T; lanes 1 and 5), [α- 32 P]dCTP (C; lanes 3 and 7), or [α- 32 P]dATP (A; lanes 4 and 8). Priming signals were quantified via phosphorimaging, normalized to the highest signal (dGTP priming, set as 100%), and denoted below the lane numbers (as a percentage of dGTP signal). The labeled HP and DP priming products are indicated. (E) Shown on the top is a schematic diagram of the mutant Hε RNAs, with the last 4 nucleotides of the internal bulge and part of the upper stem, including its bottom A-U base pair. In Hε-B6G (left), the last (6th) bulge residue (i.e., B6) was changed (from rC in the WT) to rG and in Hε-B6A (right), the same residue was changed to rA. The mutated residues are highlighted in bold. Shown at the bottom are priming products obtained with the mutant Hε RNAs. The Hε-B6G (lanes 1 and 2) or -B6A (lanes 3 and 4) mutant was coexpressed with HP, and the purified HP-Hε complex was assayed for protein priming in vitro in the presence of the indicated 32 P-labeled nucleotide. The labeled HP priming products are indicated, as is the position of the protein molecular mass marker (in kDa).

    Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

    Techniques: In Vitro, Purification, Labeling, Autoradiography, SDS Page, Mutagenesis, Expressing, Plasmid Preparation, Western Blot, Activity Assay, Marker

    Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Differentiation of priming initiation from DNA polymerization by S1 nuclease digestion. (A) Protein priming was conducted with DP bound to M2 affinity beads in TMnNK buffer, in the presence of [α- 32 P]dGTP and unlabeled dCTP, dATP, and TTP. Priming products were either mock treated (−; lanes 5 and 6) or S1 treated (+; lanes 7 and 8), followed by mock treatment (−; lanes 5 and 7) or Tdp2 treatment (+; lanes 6 and 8), as described in Materials and Methods. Released nucleotides or DNAs were resolved by urea-PAGE and detected by autoradiography. The 10-nucleotide marker, the dTG, dTGA, and dTGAA DNA oligomers, and dGMP positions are indicated, as is the priming initiation product (I; i.e., the single dGMP residue released by Tdp2 from DP) or polymerization products (P; DNA polymerization from the first dGMP residue). (B) Protein priming was performed with DP in TMnNK buffer with [α- 32 P]dGTP (lanes 1 and 2) or with unlabeled dGTP (unlabled dNTP denoted by parentheses) followed by the addition of [α- 32 P]TTP to extend the unlabeled DP-dGMP initiation product (lanes 3 and 4). The priming products were then mock treated (−; lanes 1 and 3) or treated with S1 nuclease (+; lanes 2 and 4), resolved by SDS-PAGE, and detected by autoradiography. (C) Priming was performed with DP (lanes 1 and 2) or HP (lanes 3 to 6) in TMgNK buffer with [α- 32 P]dGTP (lanes 1 to 4) or with unlabeled dGTP first followed by addition of [α- 32 P]dATP to extend the unlabeled HP-dGMP initiation product (lanes 5 and 6). The priming products were either mock treated (−; lanes 1, 3, and 5) or S1 treated (+; lanes 2, 4, and 6), resolved by SDS-PAGE, and detected by autoradiography. (D) The percent decreases in DP and HP priming signals as a result of S1 nuclease treatment are represented. Mock-treated DP initiation reaction in the presence of [α- 32 P]dGTP alone, with either TMnNK or TMgNK buffer, was set as 100%, and the other reaction conditions, as explained in panels B and C, were normalized to this. The decrease in priming signal due to proteolytic degradation (unrelated to S1 nuclease cleavage of internucleotide linkages) was subtracted from the calculations. (E) DP or HP was incubated with or without S1 nuclease as described above. Protease degradation was monitored by Western blotting using the M2 anti-Flag antibody. HC, antibody heavy chain. The symbol * in panels B, C, and E represents DP and HP degradation products caused by contaminating protease activity in S1. Note that only some proteolytic degradation products detected by the Western blot (E) appeared to match the 32 P-labeled degradation products (B and C) since the labeled products must have contained the priming site(s), whereas the Western blot detected only fragments containing the N-terminal FLAG tag. Also, some labeled degradation products might be present at such low levels that they were undetectable by Western blotting. Note also that the appearance of the proteolytic degradation products was accompanied by the decrease of the full-length HP or DP in panels B, C, and E. (F) The diagram depicts the cleavage of the internucleotide linkages, but not the HP-dGMP linkage, by S1.

    Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

    Techniques: Polyacrylamide Gel Electrophoresis, Autoradiography, Marker, SDS Page, Incubation, Western Blot, Activity Assay, Labeling, FLAG-tag

    Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Journal: Journal of Virology

    Article Title: In Vitro Epsilon RNA-Dependent Protein Priming Activity of Human Hepatitis B Virus Polymerase

    doi: 10.1128/JVI.07137-11

    Figure Lengend Snippet: Analysis of HP protein priming products by Tdp2 cleavage of the phosphotyrosyl bond between DNA and protein. Purified HP bound to M2 antibody affinity beads was assayed for protein priming. Free nucleotides were then removed with extensive washing, and priming products were mock treated (−) or treated with Tdp2 (+) to cleave the phosphotyrosyl-DNA linkages between HP and the linked nucleotides or DNA oligomers. The supernatant, which contained the released nucleotides/DNA, was collected and resolved on a urea–20% polyacrylamide gel (B to D). The beads, which contained the primed HP, were processed for SDS-PAGE to visualize the labeled HP (A). Radiolabeled proteins and nucleotides/DNA were detected by autoradiography. Priming was done in the presence of [α- 32 P]dGTP (A, lanes 1 and 2; B, lanes 3 and 4), [α- 32 P]dATP (A, lanes 3 and 4; B, lanes 5 and 6), [α- 32 P]dGTP plus [α- 32 P]dATP (A, lanes 5 and 6; B, lanes 1 and 2; D, lanes 1 and 2), [α- 32 P]dGTP plus [α- 32 P]dTTP (D, lanes 3 and 4), [α- 32 P]dGTP plus unlabeled dATP (C, lanes 3 and 4), or the other three unlabeled dNTPs (C, lanes 5 and 6; denoted as N). Unlabeled dNTPs are denoted with parentheses in panel C. The positions of the 32 P-labeled 10-nucleotide marker (Invitrogen) (C) and DNA oligomers (dGA, dGAA, and dGAAA in panels B to D and dTG, dTGA, and dTGAA in panel C) are indicated, as are the positions of dGTP and dGMP. (E) The top diagram depicts the HP priming product, i.e., the dGAA DNA oligomer that is covalently attached to HP via Y63 and templated by the last three nucleotides (rUUC) of the internal bulge of Hε. Part of the upper stem of Hε, with its bottom A-U base pair, is also shown. The phosphotyrosyl protein-DNA linkage is specifically cleaved by Tdp2 as shown. The bottom diagram depicts DNA strand elongation following primer transfer, whereby the HP-dGAA complex is translocated from Hε to DR1, and the dGAA oligomer is further extended, potentially up to dGAAAAA in the presence of only dGTP and dATP. The putative dGAAAA or dGAAAAA product released by Tdp2 from HP is also denoted by “GAAAA(?)” in panel D.

    Article Snippet: One microliter of [α-32 P]dGTP (10 mCi/ml [3,000 Ci/mmol]; PerkinElmer) was then added, and the reaction mixtures were incubated at 25°C for 4 h with shaking.

    Techniques: Purification, SDS Page, Labeling, Autoradiography, Marker

    YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: YgdH binds (p)ppGpp antagonistically with magnesium. (A) Competition assay of purified YgdH protein (20 μM) binding a 1:1 mixture of ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA. Representative DRaCALA spots and quantifications (average values for bound fractions and standards errors of the means [SEM]) of binding signals are shown. (B) Thin-layer chromatography (TLC) of DRaCALA binding reactions determined by using 1.5 M K 2 HPO 4 (pH 3.4) as the mobile phase. Binding reactions performed with purified MutT, Der, or YgdH were run in parallel with standards of [α- 32 P]GTP and a mixture of [α- 32 P]ppGpp and [α- 32 P]pppGpp (2 nM [each]). (C) Binding curves and K d determinations for YgdH interacting with α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]) without or with 1.5 mM MgCl 2 . The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Magnesium (0 to 10.15 mM) IC 50 determinations of binding of [α- 32 P]ppGpp (2 nM) to YgdH (50 μM). IC 50 values are shown. (E) Competition assay of YgdH (50 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of 100 μM cold competitors [including (p)ppGpp and the substrates of YgdH (GMP, AMP, and IMP)] without or with 1.5 mM magnesium.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Purification, Binding Assay, Thin Layer Chromatography, Labeling

    Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: Translational GTPases are conserved targets of (p)ppGpp. (A) Competition assay of RF3 and Der (20 μM) and LepA (10 μM) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B and C) Binding curves and K d determination of RF3 (B) and Der (C) binding of α- 32 P-labeled ppGpp, pppGpp, GTP, or GDP (2 nM [each]). At least three replicates were performed. The apparent K d values corresponding to each protein-ligand interaction are shown. (D) Dissociation curves for Der (50 μM) and [α- 32 P]ppGpp (2 nM) in the presence of either ppGpp or GTP (100 μM) (cold). (E and F) Binding curves and K d determination for DerG1 (E) and SaDer (F) binding α- 32 P-labeled ppGpp, pppGpp, or GTP (2 nM [each]). At least three replicates were performed. The apparent K d values are shown for each protein-ligand interaction.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Binding Assay, Labeling

    HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: HypB specifically binds (p)ppGpp with physiological affinity. (A) Competition assay of HypB (20 μM) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (B) Binding curves and K d determination for HypB binding α- 32 P-labeled ppGpp, pppGpp, and GTP (2 nM [each]). Three replicates were performed, and the apparent K d values are indicated.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Competitive Binding Assay, Binding Assay, Labeling

    In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 P]ppGpp (10 nM) determined using purified MutT and NudG (1 μM) in the presence of cold competitors (100 μM) or excess EDTA (25 mM). Samples were incubated at 30°C for 10 min (or 1 h; see Fig. S6D ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: In vitro cleavage of ppGpp by MutT, NudG, NadR, and TrmE. (A) Competition assay of whole-cell lysates containing overexpressed MutT and NudG binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). (B) Competition assay of purified NadR (left) and TrmE (right) (20 μM [each]) binding α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the presence of cold competitors (100 μM). (C) DRaCALA spots of purified proteins (10 μM) binding a mixture of α- 32 P-labeled ppGpp and pppGpp (2 nM [each]) in the absence or presence of EDTA (25 mM). (D) TLC assessment of cleavage products from the binding reactions described for panel C. A mixture of ppGpp and pppGpp was run as the standard, and both molecules are indicated. (E) Quantification of (p)ppGpp percentage determined as described for panel D. (F) Competition assay of whole-cell lysates containing overproduced MutT and NudG binding [α- 32 P](p)ppGpp (2 nM) in the presence of cold competitors and their native substrates (100 μM [each]). Representative DRaCALA spots are shown. 8OdG, 8-oxo-dGTP; 8OG, 8-oxo-GTP; 2OdA, 2-hydroxyl-dATP; 2OA, 2-hydroxyl-ATP. (G) TLC assessment of cleavage products of [α- 32 P]ppGpp (10 nM) determined using purified MutT and NudG (1 μM) in the presence of cold competitors (100 μM) or excess EDTA (25 mM). Samples were incubated at 30°C for 10 min (or 1 h; see Fig. S6D ), and reactions were stopped by addition of excess EDTA (25 mM). pGp and ppGpp are indicated.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: In Vitro, Competitive Binding Assay, Binding Assay, Purification, Labeling, Thin Layer Chromatography, Incubation

    GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d values determined as previously described ( 37 ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

    Journal: mBio

    Article Title: Novel (p)ppGpp Binding and Metabolizing Proteins of Escherichia coli

    doi: 10.1128/mBio.02188-17

    Figure Lengend Snippet: GTP biosynthesis and salvage pathways are targeted by (p)ppGpp. (A) Schematic of purine biosynthesis pathways with (p)ppGpp targets highlighted by colored boxes. Green indicates E. coli targets identified here; blue indicates specific Bacillus / Staphylococcus targets; red indicates E. coli targets reported previously but not confirmed in this study; gray indicates a target found in E. coli , Bacillus , and Staphylococcus . G, guanine; X, xanthine; H, hypoxanthine; A, adenine; PRPP, phosphoribosyl pyrophosphate; Gln, glutamine. (B) Binding curves and apparent K d values for E. coli Gpt, Hpt, and Apt binding pppGpp and ppGpp (2 nM [each]). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted and the curve-fitted and K d values determined as previously described ( 37 ). The apparent K d values corresponding to each protein-ligand interaction are shown. (C) Competition assay of Gpt, Hpt, and Apt (20 μM [each]) binding [α- 32 P]ppGpp (2 nM) in the presence of cold competitors (100 μM). The average values for bound fractions and standard errors of the means (SEM) determined for at least three replicates were plotted. Representative DRaCALA spots are shown above the respective diagrams.

    Article Snippet: 32 P-labeled pppGpp was synthesized from [α-32 P]GTP (PerkinElmer) by incubating 125 nM [α-32 P]GTP with 4 μM purified RelSeq (1–285)-His protein ( ) in buffer S (containing 25 mM Tris [pH 9.0], 100 mM NaCl, 15 mM MgCl2 , and 8 mM ATP) at 37°C for 1 h. The sample was subsequently incubated for 5 min at 95°C to stop synthesis, and the denatured RelSeq (1–285)-His protein was removed by centrifugation at 13,400 rpm for 10 min at 4°C.

    Techniques: Binding Assay, Competitive Binding Assay

    Northern blot analysis of p29 mRNA. Total RNA isolated from partially fed adult female  H. longicornis  ticks was resolved on an 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with the α- 32 P-labeled PCR product amplified from the cloned p29 phage DNA by using the λscreen expression vector-specific primers (SP6 promoter and T7 terminator). +, native p29.

    Journal: Infection and Immunity

    Article Title: Molecular Characterization of a Haemaphysalis longicornis Tick Salivary Gland-Associated 29-Kilodalton Protein and Its Effect as a Vaccine against Tick Infestation in Rabbits

    doi:

    Figure Lengend Snippet: Northern blot analysis of p29 mRNA. Total RNA isolated from partially fed adult female H. longicornis ticks was resolved on an 1% agarose gel containing formaldehyde and transferred to a nylon membrane (Hybond N+; Amersham). The membrane was hybridized with the α- 32 P-labeled PCR product amplified from the cloned p29 phage DNA by using the λscreen expression vector-specific primers (SP6 promoter and T7 terminator). +, native p29.

    Article Snippet: The membranes were dried at room temperature for about 30 min and subsequently fixed in an oven at 120°C for 20 min. Three of the longest cDNAs obtained from immunoscreening were radiolabeled with α-32 P by using a Multiprime DNA labeling kit (Amersham) and used as a probe.

    Techniques: Northern Blot, Isolation, Agarose Gel Electrophoresis, Labeling, Polymerase Chain Reaction, Amplification, Clone Assay, Expressing, Plasmid Preparation

    Southern blot analysis of tick genomic DNA. Tick genomic DNA was extracted and digested with indicated restriction enzymes followed by electrophoresis on a 0.8% agarose gel. Following electrophoresis, the separated DNA was transferred to a nylon membrane (Hybond N+; Amersham). The membrane was probed with the α- 32 P-labeled PCR product amplified from cloned p29 phage DNA by using the vector-specific primers (SP6 promoter and T7 terminator).

    Journal: Infection and Immunity

    Article Title: Molecular Characterization of a Haemaphysalis longicornis Tick Salivary Gland-Associated 29-Kilodalton Protein and Its Effect as a Vaccine against Tick Infestation in Rabbits

    doi:

    Figure Lengend Snippet: Southern blot analysis of tick genomic DNA. Tick genomic DNA was extracted and digested with indicated restriction enzymes followed by electrophoresis on a 0.8% agarose gel. Following electrophoresis, the separated DNA was transferred to a nylon membrane (Hybond N+; Amersham). The membrane was probed with the α- 32 P-labeled PCR product amplified from cloned p29 phage DNA by using the vector-specific primers (SP6 promoter and T7 terminator).

    Article Snippet: The membranes were dried at room temperature for about 30 min and subsequently fixed in an oven at 120°C for 20 min. Three of the longest cDNAs obtained from immunoscreening were radiolabeled with α-32 P by using a Multiprime DNA labeling kit (Amersham) and used as a probe.

    Techniques: Southern Blot, Electrophoresis, Agarose Gel Electrophoresis, Labeling, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation

    Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009

    Journal: eLife

    Article Title: Single-molecule tracking of the transcription cycle by sub-second RNA detection

    doi: 10.7554/eLife.01775

    Figure Lengend Snippet: Labeling and biochemical characterization of T7 RNAP activity. ( A ) Synthesis of Cy5-conjugated HaloTag Ligand ( S3 ) used to label RNAP from Cy5 acid S1 and HaloTag ligand-amine S2 . See ‘Materials and methods’ for details. ( B ) SDS-PAGE gel images of the unlabeled (‘RNAP’: no HaloTag; ‘Halo-RNAP’: with HaloTag) and the Cy5-labeled Halo-RNAP (‘Cy5-Halo-RNAP’) T7 RNAP. All RNAPs have a 6(His) tag at the N-terminus used for purification. Top: image stained with Coomassie Brilliant Blue. Bottom: the same gel scanned for Cy5 fluorescence (prior to Coomassie staining). ( C ) In vitro transcription activity of RNAP. The concentrations of RNAP derivatives were as indicated. The DNA template (a PCR fragment spanning from −75 to +295 of the concensus T7 RNAP promoter), was used at 10 nM. RNA products were labeled by incorporation of α 32 P-ATP, resolved on a denaturing polyacrylamide gel, and imaged by autoradiography. The major bands at ∼295 nt are the expected run-off products. DOI: http://dx.doi.org/10.7554/eLife.01775.009

    Article Snippet: In ensemble measurements, 10 nM DNA template (same PCR fragment as used in single-molecule assays), 0.17 μCi/μl α-32 P-ATP (Perkin Elmer, Waltham, MA) and different concentrations of RNAP were also included in the reaction.

    Techniques: Labeling, Activity Assay, SDS Page, Purification, Staining, Fluorescence, In Vitro, Polymerase Chain Reaction, Autoradiography

    Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.

    Journal: The Journal of Biological Chemistry

    Article Title: Further Insights into the Roles of GTP and the C Terminus of the Hepatitis C Virus Polymerase in the Initiation of RNA Synthesis *

    doi: 10.1074/jbc.M110.151316

    Figure Lengend Snippet: Single round RNA synthesis by H77 and J4 NS5b. HCV RdRp and template RNA were preincubated for 30 min at 25 °C in the reaction mixture without NTP or with GTP or with the 2 initiating oligonucleotides. Heparin ( M r 4000–6000, 200 μg/ml) was then added followed by [α- 32 P]CTP and NTP needed to start the elongation. The reaction mixture was further incubated at 25 °C for 0, 5, 10, 20, and 60 min. The 32 P RNA products were quantified after TCA precipitation and counted in a Wallac Counter. A , reactions were performed with G1-C and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : empty diamonds , preincubation without NTP; filled squares , preincubation with GTP; filled triangles , preincubation with CTP and GTP). B , reactions were performed with G3-U and H77_NS5b ( upper panel ) or J4_NS5b ( lower panel : filled squares , preincubation with GTP; filled circles , preincubation with ATP and CTP). Data were the mean of 3–6 independent experiments ± S.D.

    Article Snippet: GTP was added at different concentrations before or at the same time as 0.5 m m ATP, 3′-dUTP, and 10 or 100 μ m CTP with 4 μCi of [α-32 P]CTP (3000 Ci·mmol−1 , PerkinElmer Life Sciences).

    Techniques: Incubation, TCA Precipitation

    Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

    doi:

    Figure Lengend Snippet: Inhibitory effect of select nucleoside analogs on WHV Pol-dependent viral plus-strand DNA synthesis. (A) Partially purified virions from the serum of a woodchuck with chronic WHV infection were used to conduct endogenous Pol reactions with various amounts of guanosine-TPs or cytosine-TPs, as indicated at the top, and constant concentrations of cold dNTPs and [α- 32 P]dATP. The characteristic double-stranded linear (ds) and relaxed circular (rc) DNA species were isolated, resolved on 1% agarose gels, and imaged by autoradiography. Substr., substrate. (B) Inhibition curves generated by phosphorimaging analysis of the gels in panel A.

    Article Snippet: For standard EPAs , WHV virions or immunocomplexed HBV capsids were resuspended in 50 μl of EPA buffer (50 mM Tris hydrochloride [pH 7.4], 75 mM NH4 Cl, 1 mM EDTA, 20 mM MgCl2 , 0.1 mM β-mercaptoethanol, 0.5% Tween 20) supplemented with 50 μM (or in some reactions 12.5 μM) unlabeled dNTPs (dGTP, dCTP, and TTP) and 33 nM [α-32 P]dATP (3,000 Ci/mmol; NEN-Dupont, Boston, Mass.).

    Techniques: DNA Synthesis, Purification, Infection, Isolation, Autoradiography, Inhibition, Generated

    Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α- 32 P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the 35 S-labeled DHBV Pol (lane 35 S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: In Vitro Inhibition of Hepadnavirus Polymerases by the Triphosphates of BMS-200475 and Lobucavir

    doi:

    Figure Lengend Snippet: Effect of guanosine versus cytosine analogs on DHBV priming. In vitro-translated DHBV Pol was incubated with 250 nM [α- 32 P]dGTP; 220 nM unlabeled dCTP, dATP, and TTP; and increasing concentrations of the indicated analog-TPs. (A) The radiolabeled Pol-oligonucleotide adducts were analyzed by conventional SDS-PAGE and autoradiography. The migration positions of the 35 S-labeled DHBV Pol (lane 35 S) are indicated at the sides. Substr., substrate. (B) Titration curves were generated by phosphorimaging.

    Article Snippet: For standard EPAs , WHV virions or immunocomplexed HBV capsids were resuspended in 50 μl of EPA buffer (50 mM Tris hydrochloride [pH 7.4], 75 mM NH4 Cl, 1 mM EDTA, 20 mM MgCl2 , 0.1 mM β-mercaptoethanol, 0.5% Tween 20) supplemented with 50 μM (or in some reactions 12.5 μM) unlabeled dNTPs (dGTP, dCTP, and TTP) and 33 nM [α-32 P]dATP (3,000 Ci/mmol; NEN-Dupont, Boston, Mass.).

    Techniques: In Vitro, Incubation, SDS Page, Autoradiography, Migration, Labeling, Titration, Generated

    Formation of TP-dAMP complex catalysed by Nf and GA-1 DNA polymerases. The assays were performed as described under Materials and Methods in the presence of 1 mM (for GA-1 and φ29 DNA polymerases) and 2 mM (for Nf DNA polymerase) MnCl 2 , 5 ng of TP, 500 ng of TP-DNA, 0.1 μM [α- 32 P]dATP (1 μCi), and the indicated amounts of DNA polymerase. After incubation at 30°C for the indicated times, samples were analysed by SDS–PAGE and autoradiography. The position of the TP-dAMP initiation complex is indicated. Quantification was by densitometry of the band corresponding to the labelled TP-dAMP complex, detected by autoradiography.

    Journal: Nucleic Acids Research

    Article Title: Functional characterization of highly processive protein-primed DNA polymerases from phages Nf and GA-1, endowed with a potent strand displacement capacity

    doi: 10.1093/nar/gkl769

    Figure Lengend Snippet: Formation of TP-dAMP complex catalysed by Nf and GA-1 DNA polymerases. The assays were performed as described under Materials and Methods in the presence of 1 mM (for GA-1 and φ29 DNA polymerases) and 2 mM (for Nf DNA polymerase) MnCl 2 , 5 ng of TP, 500 ng of TP-DNA, 0.1 μM [α- 32 P]dATP (1 μCi), and the indicated amounts of DNA polymerase. After incubation at 30°C for the indicated times, samples were analysed by SDS–PAGE and autoradiography. The position of the TP-dAMP initiation complex is indicated. Quantification was by densitometry of the band corresponding to the labelled TP-dAMP complex, detected by autoradiography.

    Article Snippet: Nucleotides and DNAs Unlabelled nucleotides, as well as [α-32 P]dATP [3000 Ci/mmol (1 Ci = 37 GBq)] and [γ-32 P]ATP (3000 Ci/mmol) were obtained from Amersham Pharmacia.

    Techniques: Incubation, SDS Page, Autoradiography

    External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 P]dATP. DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .

    Journal: Nucleic Acids Research

    Article Title: Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding

    doi: 10.1093/nar/gkr345

    Figure Lengend Snippet: External mutations on the surface of MCM disrupt unwinding and protection of the 5′-tail. ( A ) Alignment of proposed exterior surface residues on MCM that interact with ssDNA using CLUSTAL W2 ( http://www.ebi.ac.uk/Tools/clustalw2 ). Aligned are MCM exterior surface residues proposed to bind ssDNA from Sulfolobus solfataricus ( Sso ), Methanothermobacter thermoautotrophicus ( Mth ), Xenopus laevis MCM2 (xMCM2) and human MCM2 (hMCM2). ( B ) DNA unwinding assays comparing wild-type and mutant MCM activities at 700 nM hexamer. Fork DNA with 30 base 3′- and 5′-tails were examined for unwinding at 60°C for 30 min as described in ‘Materials and Methods’ section. ( C ) Quantification of fraction unwound in (B) for WT at 700 nM and the three mutants at four separate concentrations (350, 700, 1400 and 2800 nM) from at least three independent experiments. ( D ) Nuclease assays were performed in the presence and absence of Sso MCM with different length 5′-tails as described in ‘Materials and Methods’ section. DNA was labeled at the 3′-end with [α- 32 P]dATP. DNA markers (M) are shown in lane 1. The length of the 5′-tail was varied from 20, 30, 40, 50 and 80 bases. The duplex region (36 bases) and 3′-tail (30 bases) were identical for lanes 2–9. The duplex region for lanes 10–11 were 20 bases and 3′-tail were 30 bases. ( E ) Quantification of the fraction protected from at least three independent mung bean nuclease assays comparing WT Sso MCM to mutants (K232A, R440A and K323A/R440A) with 30, 50 or 80 base 5′-tails and shown and reported in Supplementary Table S2 .

    Article Snippet: [γ-32 P]ATP and [α-32 P]dATP were purchased from MP Biomedicals and used with PNK/Optikinase or TdT to 32 P label the 5′- or 3′-ends of DNA, respectively.

    Techniques: Mutagenesis, Labeling

    Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 P]dATP-labeled DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.

    Journal: Antimicrobial Agents and Chemotherapy

    Article Title: The Candida dubliniensis CdCDR1 Gene Is Not Essential for Fluconazole Resistance

    doi: 10.1128/AAC.46.9.2829-2841.2002

    Figure Lengend Snippet: Northern blot showing expression levels of CdCDR1 , CdMDR1 , and CdTEF3 mRNAs in matched pairs of C. dubliniensis clinical isolates and in vitro-generated derivatives exhibiting reduced susceptibility to fluconazole. (A) Total RNA was extracted from C. dubliniensis isolates and derivatives grown to the mid-exponential phase in YEPD broth cultures and analyzed by Northern hybridization analysis with [α- 32 P]dATP-labeled DNA probes homologous to CdCDR1 , CdMDR1 , and the constitutively expressed internal control CdTEF3 gene (see Materials and Methods). (B) Graphical representation of CdCDR1 and CdMDR1 mRNA expression levels. Hybridization signals were analyzed by scanning densitometry and normalized against levels of CdTEF3 expression.

    Article Snippet: RNA was hybridized at 42°C with DNA probes homologous to CdCDR1 , CdMDR1 , and CdTEF3 labeled with [α-32 P]dATP (6,000 Ci/mmol, 220 TBq/mmol; NEN Life Sciences, Boston, Mass.) by random primer labeling as described by Moran et al. ( ).

    Techniques: Northern Blot, Expressing, In Vitro, Generated, Hybridization, Labeling

    NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 P]NTP was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.

    Journal: Journal of Virology

    Article Title: The Helicase-Like Domain of Plant Potexvirus Replicase Participates in Formation of RNA 5? Cap Structure by Exhibiting RNA 5?-Triphosphatase Activity

    doi: 10.1128/JVI.75.24.12114-12120.2001

    Figure Lengend Snippet: NTPase activity of the helicase-like domain analyzed by TLC (A) ATPase; (B) UTPase; (C) CTPase; (D) GTPase. [α- 32 P]NTP was incubated with various enzyme preparations (lanes 1, wild-type helicase-like domain; lanes 2, motif I mutant (GKS→GAA); lanes 3, blank), and the reaction products were analyzed by TLC as described in Materials and Methods. Standard nucleotides (arrowheads) were run along with the radiolabeled samples in a same TLC sheet, visualized under UV at 254 nm.

    Article Snippet: Unless otherwise stated, the standard NTPase reaction was performed at 37°C for 30 min in a 10-μl solution containing 50 mM Tris (pH 7.4), 5 mM MgCl2 , 5 mM DTT, 200 μM NTP, 5 μCi of [α-32 P]NTP (Amersham; 5,000 Ci/mmol), 40 U of RNase inhibitor (HPR I; Takara), and 30 ng of the purified enzymes.

    Techniques: Activity Assay, Thin Layer Chromatography, Incubation, Mutagenesis

    RfaH N effects on Rho-dependent termination. A. Transcript generated on a linear pIA267 DNA template; transcription start site (+1), ops , Rho-dependent RNA release sites and transcript end are indicated. B. Halted, [α- 32 P]-GMP-labelled TECs were formed at 40 nM with E. coli RNAP. Rho, NusG, RfaH or RfaH N were added at indicated concentrations, followed by addition of NTPs and rifapentin. The reactions were incubated for 15 min at 37°C, quenched, and analysed on a 6% denaturing gel. A representative gel and four selected traces for Rho alone (gray), full-length RfaH at 300 nM (red), RfaH N at 60 nM (blue) and NusG at 40 nM (green) are shown.

    Journal: Molecular Microbiology

    Article Title: Functional regions of the N-terminal domain of the antiterminator RfaH

    doi: 10.1111/j.1365-2958.2010.07056.x

    Figure Lengend Snippet: RfaH N effects on Rho-dependent termination. A. Transcript generated on a linear pIA267 DNA template; transcription start site (+1), ops , Rho-dependent RNA release sites and transcript end are indicated. B. Halted, [α- 32 P]-GMP-labelled TECs were formed at 40 nM with E. coli RNAP. Rho, NusG, RfaH or RfaH N were added at indicated concentrations, followed by addition of NTPs and rifapentin. The reactions were incubated for 15 min at 37°C, quenched, and analysed on a 6% denaturing gel. A representative gel and four selected traces for Rho alone (gray), full-length RfaH at 300 nM (red), RfaH N at 60 nM (blue) and NusG at 40 nM (green) are shown.

    Article Snippet: Proteins and reagents Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA), NTPs and [α-32 P]-NTPs were from GE Healthcare (Piscataway, NJ, USA), restriction and modification enzymes – from NEB (Ipswich, MA, USA), PCR reagents – from Roche (Indianapolis, IN, USA), other chemicals – from Sigma (St Louis, MO, USA) and Fisher (Pittsburgh, PA, USA).

    Techniques: Generated, Incubation

    RfaH N effects on intrinsic termination. Transcript generated on a linear pIA416 DNA template; transcription start site (+1), the ops element (boxed), T hly terminator structure, terminated and run-off RNA products are shown on top. Halted [α- 32 P]-CMP-labelled G37 TECs were formed at 60 nM with E. coli RNAP and challenged with NTPs (10 µM UTP, 200 µM ATP, CTP, GTP) and rifapentin at 25 µg ml −1 in the absence or in the presence of full-length RfaH or RfaH N . The reactions were incubated for 15 min at 37°C, quenched, and analysed on a 6% denaturing gel along with the [γ- 32 P]-ATP-labelled pBR322 Msp I digest as a molecular weight standard (the sizes of fragments are indicated. Termination efficiency (219-nt long RNA as a fraction of total RNA) was determined in three independent experiments.

    Journal: Molecular Microbiology

    Article Title: Functional regions of the N-terminal domain of the antiterminator RfaH

    doi: 10.1111/j.1365-2958.2010.07056.x

    Figure Lengend Snippet: RfaH N effects on intrinsic termination. Transcript generated on a linear pIA416 DNA template; transcription start site (+1), the ops element (boxed), T hly terminator structure, terminated and run-off RNA products are shown on top. Halted [α- 32 P]-CMP-labelled G37 TECs were formed at 60 nM with E. coli RNAP and challenged with NTPs (10 µM UTP, 200 µM ATP, CTP, GTP) and rifapentin at 25 µg ml −1 in the absence or in the presence of full-length RfaH or RfaH N . The reactions were incubated for 15 min at 37°C, quenched, and analysed on a 6% denaturing gel along with the [γ- 32 P]-ATP-labelled pBR322 Msp I digest as a molecular weight standard (the sizes of fragments are indicated. Termination efficiency (219-nt long RNA as a fraction of total RNA) was determined in three independent experiments.

    Article Snippet: Proteins and reagents Oligonucleotides were obtained from Integrated DNA Technologies (Coralville, IA, USA), NTPs and [α-32 P]-NTPs were from GE Healthcare (Piscataway, NJ, USA), restriction and modification enzymes – from NEB (Ipswich, MA, USA), PCR reagents – from Roche (Indianapolis, IN, USA), other chemicals – from Sigma (St Louis, MO, USA) and Fisher (Pittsburgh, PA, USA).

    Techniques: Generated, Incubation, Molecular Weight