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  • 99
    Thermo Fisher dctp
    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 <t>[α-</t> 32 P]dGTP (A, lanes 1 and 2; B, lanes 5 and 6) or TMnNK buffer and [α- 32 P]dGTP plus the unlabeled <t>dCTP,</t> 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.
    Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 7188 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore dctp
    Intracellular <t>5-methyl-dCTP,</t> dCTP and <t>dTTP</t> concentrations in DCTPP1 -knockdown and control BGC-823 cells. The concentrations of A. 5-methyl-dCTP, B. dCTP, and C. dTTP in DCTPP1 -knockdown and control BGC-823 cells were measured by LC-MS/MS assay. All the values shown were represented as means ± SD. (ns: not significant; **: P
    Dctp, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 861 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare α 32 p dctp
    cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 <t>P]dCTP</t> (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.
    α 32 P Dctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 10068 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    PerkinElmer α 32 p dctp
    Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of <t>dCTP</t> α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.
    α 32 P Dctp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 99/100, based on 2679 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    GE Healthcare cy5 dctp
    Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using <t>Cy5-dCTP.</t> Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m
    Cy5 Dctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 96/100, based on 2483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    GE Healthcare megaprime dna labeling system
    Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using <t>Cy5-dCTP.</t> Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m
    Megaprime Dna Labeling System, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 2850 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GE Healthcare cy3 dctp
    Rescue of DNA replication activity and nuclear localization of DNA polymerase α by recombinant RecQ4 in RecQ4-depleted extracts. (A) Sperm chromatin was incubated in mock (ΔMock)- or RecQ4 (ΔRecQ4)-depleted extracts in the absence (−) or presence of full-length RecQ4 (+low-full, 2 μg/ml), N2-RecQ4 (+low-N2, 0.8 μg/ml; +high-N2, 16 μg/ml), or N1-RecQ4 (+high-N1, 9 μg/ml) at 23°C for 40 min. The samples were treated for 2 min with 0.1% Triton X-100 in EB containing an additional 100 mM NaCl and then fixed with 3.7% formaldehyde. Nuclear localization of DNA polymerase α was visualized with rabbit anti-polymerase α antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replication was monitored as the incorporation of <t>Cy3-dCTP</t> into DNA, and DNA was visualized with Hoechst 33258 dye. (B) The fluorescent intensity of each nucleus was quantified using NIH Image software, and average intensities of over 40 nuclei were normalized by the intensities of the nuclei from mock-depleted extract.
    Cy3 Dctp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 95/100, based on 2429 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher biotin 14 dctp
    Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing <t>biotin-14-dCTP</t> (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline
    Biotin 14 Dctp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 616 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dctp  (TaKaRa)
    97
    TaKaRa dctp
    Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing <t>biotin-14-dCTP</t> (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline
    Dctp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 97/100, based on 771 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    DuPont de Nemours α 32 p dctp
    DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 <t>P]dCTP-labeled</t> cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.
    α 32 P Dctp, supplied by DuPont de Nemours, used in various techniques. Bioz Stars score: 92/100, based on 496 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene α 32 p dctp
    Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA − ] or treated with 1 μM RA for 48 h [RA + ]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3′ ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXRγ (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [α- 32 <t>P]dCTP</t> labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RARα, RARγ, RARγ2, RXRα, and RXRβ were observed. RARα and RXRβ showed two isoforms. RARβ (detected only after RA treatment) showed four isoforms. RXRγ was not detectable in untreated or RA-treated cells.
    α 32 P Dctp, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 999 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim α 32 p dctp
    T. brucei telomerase-mediated template termination synthesis with ddNTPs. Telomerase reactions containing either [α- 32 P]dGTP ( A ) or [α- 32 P]TTP ( B ) and the telomerase substrate oligonucleotides indicated were tested for the effects of ddNTP chain terminators. Control reactions lacking chain terminators (lanes 1, 5, 9, 12, and 14) were compared with reactions in which dATP was substituted by ddATP (lanes 2, 6, and 10), TTP was substituted by ddTTP (lanes 3, 7, and 11), or dGTP was substituted by ddGTP (lanes 13 and 15). Size markers (lanes M) are terminal deoxynucleotidyltransferase-labeled oligonucleotides tel 6 with [α- 32 <t>P]dCTP</t> ( A ) and tel 1 labeled with [α- 32 P]dGTP ( B ). Lanes nE and nP are mock reactions lacking extract or primer, respectively.
    α 32 P Dctp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 674 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TriLink 5 methyl dctp
    BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM <t>5-methyl-dCTP</t> were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.
    5 Methyl Dctp, supplied by TriLink, used in various techniques. Bioz Stars score: 91/100, based on 149 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    NEN Life Science α 32 p dctp
    Fig. 1. jnk-1 encodes two different transcripts. ( A ) Structure of C.elegans jnk-1 . Exons are indicated by boxes, while introns are represented as lines. Numbers refer to amino acids. RACE was performed using two different sets of primers as described in Materials and methods. In 11/15 clones (using primers based on sequences from exons 7 and 8) and in 6/10 clones (using primers from exon 12), we detected two transcripts, which we termed jnk-1α and jnk-1β containing 5′ SL1 trans ). For jnk-1α , the SL1 trans -spliced sequence was followed by exon 1, whereas for jnk-1β the SL1 trans -spliced sequence was followed by exon 2. The translation initiation site, ATG (in bold and italicized) of jnk-1α is located in exon 1 while the translation initiation site of jnk-1β is located in exon 3 (in bold and marked by a box). JNK-1α has a 91 amino acid extended N-terminal region (in gray), fused in-frame with 372 amino acid residues (in black) that are identical in JNK-1α and JNK-1β. Poly(A), polyadenylation site; SL1, trans -splicing leader sequence; E, Eco RI and H, Hin dIII restriction sites; NH 2 , N-terminus; COOH, C-terminus. ( B ) Nucleotide sequence of the 5′ regions of jnk-1α and jnk-1β transcripts. SL1 trans -splicing leader sequences (underlined and italicized) are located at the beginning of both jnk-1 transcripts. Non-translated sequences are in lower case and translated sequences are in upper case. ( C ) Southern blot analysis using full-length [α 32 <t>-P]dCTP-labeled</t> jnk-1α cDNA as a probe shows a pattern of DNA restriction compatible with that predicted from cosmid B0478.
    α 32 P Dctp, supplied by NEN Life Science, used in various techniques. Bioz Stars score: 92/100, based on 253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dntps
    Thermal denaturation transitions of duplexes 9 : 5 – 9 : 8 and H-bonding patterns expected from I, 8-oxoI, and 8-BrI (left); and RTn using <t>AMV</t> on duplexes 9 : 5 – 9 : 8 in the presence of canonical <t>dNTPs,</t> where M = equimolar mixture of all dNTPs, (right). Steady-state kinetics data is shown in the lower left corner.
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    92
    Valiant α 32 p dctp
    (a) DNA gel shift assays showing binding of VlmI-L to a 443 bp vlmA–vlmH intergenic DNA fragment. The 443 bp DNA fragment was labelled at both ends with [ α - 32 <t>P]dCTP.</t> The assays in lanes 1–6 show the behaviour when the complete 443 bp fragment was mixed with increasing amounts of VlmI-L. Lane 7 shows the behaviour when the labelled DNA fragment plus 4 μg VlmI-L was mixed with a 50-fold excess of the unlabelled 443 bp DNA fragment. Lanes 8–11 show the results of gel shift assays after the labelled 443 bp fragment was digested with Pml I, and lanes 12–15 show the results of gel shift assays after digestion of the same fragment with Sma I. The quantity of VlmI used in each assay is shown at the top of each lane. S and P show the positions of the Sma I and Pml I sites, respectively. (b) Comparison of DNA gel shifts produced by VlmI-L and VlmI-S with a radiolabelled 443 bp vlmA–vlmH intergenic DNA fragment.
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    92
    DuPont de Nemours p dctp
    Dynamics of association of the ori-β to the nuclear matrix during the cell cycle of CHO cells. ( A ) Schematic representation of the position of the ori-β probe in the non-transcribed spacer 3′ of the DHFR gene in CHO cells. A 479 bp <t>DNA</t> fragment (filled box) was amplified by PCR and used as a probe. ( B ) Relative abundance of the DHFR ori-β in the matrix-attached DNA in CHO cells. Aliquots of matrix-attached DNA isolated from CHO cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probe in vitro labeled with [ 32 <t>P]dCTP.</t> For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. The results are means of five independent experiments and the standard deviations are shown with error bars.
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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 allow DNA polymerization, priming assays were performed as described above with the following modifications: 2 μl [α-32 P]dGTP was added along with either 10 μM unlabeled dATP, dCTP, and TTP (Invitrogen) or 0.25 μM unlabeled dATP, as indicated.

    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 allow DNA polymerization, priming assays were performed as described above with the following modifications: 2 μl [α-32 P]dGTP was added along with either 10 μM unlabeled dATP, dCTP, and TTP (Invitrogen) or 0.25 μM unlabeled dATP, as indicated.

    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 allow DNA polymerization, priming assays were performed as described above with the following modifications: 2 μl [α-32 P]dGTP was added along with either 10 μM unlabeled dATP, dCTP, and TTP (Invitrogen) or 0.25 μM unlabeled dATP, as indicated.

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

    Intracellular 5-methyl-dCTP, dCTP and dTTP concentrations in DCTPP1 -knockdown and control BGC-823 cells. The concentrations of A. 5-methyl-dCTP, B. dCTP, and C. dTTP in DCTPP1 -knockdown and control BGC-823 cells were measured by LC-MS/MS assay. All the values shown were represented as means ± SD. (ns: not significant; **: P

    Journal: Oncotarget

    Article Title: DCTPP1 attenuates the sensitivity of human gastric cancer cells to 5-fluorouracil by up-regulating MDR1 expression epigenetically

    doi: 10.18632/oncotarget.11864

    Figure Lengend Snippet: Intracellular 5-methyl-dCTP, dCTP and dTTP concentrations in DCTPP1 -knockdown and control BGC-823 cells. The concentrations of A. 5-methyl-dCTP, B. dCTP, and C. dTTP in DCTPP1 -knockdown and control BGC-823 cells were measured by LC-MS/MS assay. All the values shown were represented as means ± SD. (ns: not significant; **: P

    Article Snippet: Rabbit anti-Phospho-BRCA1 Ab (Cat# 9009), rabbit anti-Phospho-H2A.X Ab (Cat# 2197), rabbit anti-caspase-3 (8G10) mAb (Cat# 9665), rabbit anti-cleaved caspase-3 (Asp 175) mAb (Cat# 9664), rabbit anti-Bax pAb (Cat#2772), rabbit anti-Bcl-2 (50E3) mAb (Cat# 2870), HRP-linked anti-rabbit IgG Ab (Cat# 7074) and HRP-linked anti-mouse IgG Ab (Cat# 7076) were purchased from Cell Signaling Technology (Beverly, MA, USA). dCTP, dTTP and 5-FU were purchased from Sigma and 5-methyl-dCTP was purchased from New England BioLabs (Ipswich, MA, USA).

    Techniques: Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

    cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 P]dCTP (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.

    Journal: Plant Physiology

    Article Title: Antisense Suppression of a (+)-δ-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1-Cadinene Synthase Gene in Cotton Prevents the Induction of This Defense Response Gene during Bacterial Blight Infection But Not Its Constitutive Expression 1 [w]

    doi: 10.1104/pp.104.056010

    Figure Lengend Snippet: cdn 1-C4 is part of a large multigene family in Gossypium species. Southern hybridization of Eco RI-digested (left section) and Hin dIII-digested (right section) cotton genomic DNA probed with the Eco RI insert of the cdn 1-C4 clone labeled with [ α - 32 P]dCTP (10 μ Ci μ L −1 ). DNA was electrophoresed in an agarose gel and capillary blotted onto Hybond-N + . Lane 1, G. hirsutum cv Coker 315; lane 2, G. hirsutum cv Sicala V2; lane 3, G. hirsutum cv DP16 glanded; lane 4, G. hirsutum cv DP16 glandless; lane 5, G. arboreum ; lane 6, G. sturtianum . Molecular weight markers are shown in kbs. There are no internal Eco RI sites in the cdn 1-C4 gene; however, there are three internal Hin dIII sites.

    Article Snippet: A 0.75-kb Sal I/ Eco RI fragment of CSZ-7 was labeled with [ α -32 P]dCTP (10 μ Ci μ L−1 ; New Megaprime Random Labeling kit, Amersham Biosciences, Uppsala).

    Techniques: Hybridization, Labeling, Agarose Gel Electrophoresis, Molecular Weight

    TPX2-Δ nuclei form fully functional NEs. (A) TPX2-Δ nuclei import a nuclear substrate. Mock-depleted (a–d) or TPX2-Δ (e–h) nuclear assembly reactions were supplemented with rhodamine-labeled nucleoplasmin to assay nuclear import. To distinguish import from nonspecific association of nucleoplasmin with the nuclei, some reactions (c, d, g, and h) were supplemented with the inhibitor of nuclear import, wheat germ agglutinin (WGA). Nuclei were visualized by DNA stain (a, c, e, e′, and g) or rhodamine fluorescence (b, d, f, f′, and h). (e′ and f′) Higher magnification views of e and f, respectively. (B) TPX2-Δ nuclei exclude a nonnuclear substance. Mock-depleted (a and b) or TPX2-Δ (c and d) nuclear assembly reactions were spiked with rhodamine-labeled 155-kD dextran, and a small amount of the reaction was spotted onto a coverslip. Nuclei were visualized by Hoechst staining (a and c) and the dextran by fluorescence (b and d). (C) TPX2-Δ nuclei replicate their DNA. Autoradiographs of two representative replication assays are shown. [ 32 P]dCTP was added to control (C), mock-depleted (M), or TPX2-Δ (T) extracts supplemented with ∼100 sperm chromatin/microliter, and the reactions were incubated for 3 h at room temperature to allow DNA replication. Reactions were then stopped, separated on a 0.8% agarose gel, and visualized by autoradiography. Two types of control extracts were used, with similar results: (a) duplicate reactions were treated with the inhibitor of replication, aphidicolin (AC); (b) [ 32 P]dCTP was added to mitotic extracts. (D) Quantitation of DNA replication in four independent experiments, normalized to the amount of label incorporated in the mock-depleted controls. Error bars indicate standard deviation. Bars: (A [a–f, g, and h] and B) 25 μm; (A, e' and f') 10 μm.

    Journal: The Journal of Cell Biology

    Article Title: TPX2 is required for postmitotic nuclear assembly in cell-free Xenopus laevis egg extracts

    doi: 10.1083/jcb.200512107

    Figure Lengend Snippet: TPX2-Δ nuclei form fully functional NEs. (A) TPX2-Δ nuclei import a nuclear substrate. Mock-depleted (a–d) or TPX2-Δ (e–h) nuclear assembly reactions were supplemented with rhodamine-labeled nucleoplasmin to assay nuclear import. To distinguish import from nonspecific association of nucleoplasmin with the nuclei, some reactions (c, d, g, and h) were supplemented with the inhibitor of nuclear import, wheat germ agglutinin (WGA). Nuclei were visualized by DNA stain (a, c, e, e′, and g) or rhodamine fluorescence (b, d, f, f′, and h). (e′ and f′) Higher magnification views of e and f, respectively. (B) TPX2-Δ nuclei exclude a nonnuclear substance. Mock-depleted (a and b) or TPX2-Δ (c and d) nuclear assembly reactions were spiked with rhodamine-labeled 155-kD dextran, and a small amount of the reaction was spotted onto a coverslip. Nuclei were visualized by Hoechst staining (a and c) and the dextran by fluorescence (b and d). (C) TPX2-Δ nuclei replicate their DNA. Autoradiographs of two representative replication assays are shown. [ 32 P]dCTP was added to control (C), mock-depleted (M), or TPX2-Δ (T) extracts supplemented with ∼100 sperm chromatin/microliter, and the reactions were incubated for 3 h at room temperature to allow DNA replication. Reactions were then stopped, separated on a 0.8% agarose gel, and visualized by autoradiography. Two types of control extracts were used, with similar results: (a) duplicate reactions were treated with the inhibitor of replication, aphidicolin (AC); (b) [ 32 P]dCTP was added to mitotic extracts. (D) Quantitation of DNA replication in four independent experiments, normalized to the amount of label incorporated in the mock-depleted controls. Error bars indicate standard deviation. Bars: (A [a–f, g, and h] and B) 25 μm; (A, e' and f') 10 μm.

    Article Snippet: DNA replication assays DNA replication was measured in seven independent experiments by incorporation of [32 P]dCTP (3,000 Ci/mmol; Redivue [GE Healthcare]) as described by with the following modifications: 20 μl of extract (TPX2-Δ, mock-depleted, or negative control extract) was supplemented with 1,000 sperm/microliter and 10 μCi of [32 P]dCTP and was incubated for 3 h at 22°C.

    Techniques: Functional Assay, Labeling, Whole Genome Amplification, Staining, Fluorescence, Incubation, Agarose Gel Electrophoresis, Autoradiography, Quantitation Assay, Standard Deviation

    Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV ( A ) or the Ser42Ala mutant ( B ) was incubated with DNA duplexes (T A P*, T T P*, T G P* and T C P*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z -axis for different template nucleotides (dA, dT, dG and dC; y -axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x -axis) for the two proteins (wtPolIV and Ser42Ala; x -axis).

    Journal: Nucleic Acids Research

    Article Title: A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli

    doi: 10.1093/nar/gkt146

    Figure Lengend Snippet: Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV ( A ) or the Ser42Ala mutant ( B ) was incubated with DNA duplexes (T A P*, T T P*, T G P* and T C P*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z -axis for different template nucleotides (dA, dT, dG and dC; y -axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x -axis) for the two proteins (wtPolIV and Ser42Ala; x -axis).

    Article Snippet: In the case of chain terminated DNA, dCTP (GE Amersham) was added to a final concentration of 5 mM.

    Techniques: Mutagenesis, Primer Extension Assay, Incubation

    Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of dCTP α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.

    Journal: Nucleic Acids Research

    Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource

    doi: 10.1093/nar/gkx900

    Figure Lengend Snippet: Analysis of minigene induction and splicing. ( A ) Schematic of primer target locations for analysis of minigene-derived RNA. ( B ) Quantitative RT-PCR (qRT-PCR) with primers directed against Tnp1 exon 2 and the Pfn3 ORF following 4 h of treatment with Dox or vehicle. Minigene expression was normalized to the average of three reference genes ( ALDO, GAPDH and RPS16 ). Data shown are from three independent, biological replicates; error bars depict mean ± SEM. ( C ) Phosphorimaging analysis of 4 h induced RT-PCR products generated with intron-flanking Tnp1 primers or 5′ and 3′-directed Pfn3 ORF primers in the presence of dCTP α 32 P. Percent-spliced-in (PSI) was determined as the signal intensity of the spliced product versus the sum of spliced and unspliced products. Amplification of β-tubulin ( TUBB ) was performed in a multiplex reaction as an internal control for loading ( bottom ). ( D ) 3′-end RT-PCR to assess minigene polyadenylation. Distinct products reflect use of either the endogenous polyadenylation sites-encoded within the Tnp1 and Pfn3 minigenes or the vector-encoded site located within the BGH cassette. β-tubulin ( TUBB ) served as a loading control.

    Article Snippet: For splicing analysis, template cDNA was amplified by Taq polymerase (NEB) using Tnp1 -specific intron-flanking primers, along with control primers against the reference gene TUBB , in a multiplexed PCR reaction containing 33 nM α-32 P-dCTP (3000 Ci/mmol, Perkin Elmer).

    Techniques: Derivative Assay, Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, Generated, Amplification, Multiplex Assay, Plasmid Preparation

    Effects of inhibitors of BER, ATM, ATR and DNA-PK on survival after exposure of cells to HmdUrd, FdUrd, FU and FUrd. ( A ) Inhibition of APE1 cleavage of methoxyamine-modified AP-sites. A double-stranded oligonucleotide containing an AP-site was pre-treated for 20 min with various concentrations of MX, and then incubated with recombinant APE1. The upper bands observed after denaturing PAGE represent uncleaved 19-mer substrate, and the lower bands represent cleaved products. Bottom row numbers represent quantification of AP-site cleavage (lower gel band) as a percentage of control for various concentrations of MX (top row). ( B ) BER assay of repair of cccDNA substrate containing a single 5-hmU:G base pair in the presence of increasing concentrations of the PARP-1 inhibitor 4-AN. Nuclear extracts from the SW480 cells were pre-incubated with various concentration of 4-AN. Bands represent incorporation of [α 33 P]dCTP at the position of HmU. Bottom row numbers represent quantification of band signal as a percentage of control for various concentrations of 4-AN (top row). ( C ) SW480 and HeLa cell survival measured by the MTT assay after four days of continuous exposure to varying concentrations of HmdUrd, FdUrd, FU or FUrd in the presence or absence (black) of either 50 mM MX (orange), 20 µM 4-AN (green), 2 mM vanillin (blue), 2 mM caffeine (red), or 10 µM ATM kinase inhibitor (violet). The curves are normalized to untreated cells in the presence of the indicated molecular inhibitors. The data represent the mean ± SD of at least two parallel experiments.

    Journal: Nucleic Acids Research

    Article Title: UNG-initiated base excision repair is the major repair route for 5-fluorouracil in DNA, but 5-fluorouracil cytotoxicity depends mainly on RNA incorporation

    doi: 10.1093/nar/gkr563

    Figure Lengend Snippet: Effects of inhibitors of BER, ATM, ATR and DNA-PK on survival after exposure of cells to HmdUrd, FdUrd, FU and FUrd. ( A ) Inhibition of APE1 cleavage of methoxyamine-modified AP-sites. A double-stranded oligonucleotide containing an AP-site was pre-treated for 20 min with various concentrations of MX, and then incubated with recombinant APE1. The upper bands observed after denaturing PAGE represent uncleaved 19-mer substrate, and the lower bands represent cleaved products. Bottom row numbers represent quantification of AP-site cleavage (lower gel band) as a percentage of control for various concentrations of MX (top row). ( B ) BER assay of repair of cccDNA substrate containing a single 5-hmU:G base pair in the presence of increasing concentrations of the PARP-1 inhibitor 4-AN. Nuclear extracts from the SW480 cells were pre-incubated with various concentration of 4-AN. Bands represent incorporation of [α 33 P]dCTP at the position of HmU. Bottom row numbers represent quantification of band signal as a percentage of control for various concentrations of 4-AN (top row). ( C ) SW480 and HeLa cell survival measured by the MTT assay after four days of continuous exposure to varying concentrations of HmdUrd, FdUrd, FU or FUrd in the presence or absence (black) of either 50 mM MX (orange), 20 µM 4-AN (green), 2 mM vanillin (blue), 2 mM caffeine (red), or 10 µM ATM kinase inhibitor (violet). The curves are normalized to untreated cells in the presence of the indicated molecular inhibitors. The data represent the mean ± SD of at least two parallel experiments.

    Article Snippet: Excision activities by purified proteins were measured using recombinant human His-tagged UNG2, SMUG1, or TDG, 0.1 pmol oligonucleotide substrate in UDG buffer ( ) containing 50 mM NaCl and 0.1 pmol recombinant hAPE1 ( ) after incubation at 37°C for 30 min. BER incorporation assays were carried out in the same buffer as BER/MMR assays, supplemented with 3 µCi dCTP or dTTP (3000 Ci/mmol, Perkin-Elmer) essentially as described ( ).

    Techniques: Inhibition, Modification, Incubation, Recombinant, Polyacrylamide Gel Electrophoresis, Concentration Assay, MTT Assay

    Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Transcription- and translation-coupled DNA (TTcDR) replication. To perform the TTcDR reaction, circular plasmid DNA encoding phi29 DNA polymerase was incubated with the translation system optimized in a previous study 11 , including dNTPs, yeast ppiase, T7 RNA polymerase, and [ 32 P]-dCTP, for 12 h at 30 °C. An aliquot of the mixture after incubation was used in 1% agarose gel electrophoresis and autoradiography. The arrowhead indicates the product of the TTcDR reaction. Lane 1: lambda-BstPI marker. Lane 2: TTcDR reaction without plasmid DNA. Lane 3: TTcDR reaction with plasmid DNA. Lane 4: DNA polymerization with a purified phi29 in phi29 standard buffer.

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Autoradiography, Marker, Purification

    Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Journal: Scientific Reports

    Article Title: A transcription and translation-coupled DNA replication system using rolling-circle replication

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Characterization of the optimized TTcDR reaction. A ) Cleavage of the TTcDR product by restriction enzymes. After the TTcDR reaction was conducted for 12 h at 30 °C, the indicated restriction enzymes were added and incubated for 1 h at 37 °C. An aliquot was used for 1% agarose gel electrophoresis and autoradiography. The sample treated with Pst I was purified using a DNA column (Life Technologies) before electrophoresis. B ) Time-course data for the translation of DNA polymerase during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 35 S]-methionine at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 10% SDS-PAGE and autoradiography. The error bars indicate the standard error (n = 4). C ) Time-course data for DNA replication during the TTcDR reaction. The circular DNA (1 ng/μl) was incubated with the optimized TTcDR mixture containing [ 32 P]-dCTP at 30 °C with or without chloramphenicol (25 μg/μl). After the indicated time, an aliquot was used for 1% agarose gel electrophoresis and autoradiography. The error bars indicate the standard error (n = 4).

    Article Snippet: Assay of the TTcDR reaction The optimized composition of the TTcDR system was as follows: template plasmid DNA (1 ng/μl), dNTPs (0.3 mM each, Takara), [32 -P] dCTP (3.3 μM, PerkinElmer), magnesium acetate (7.9 mM, Wako), potassium glutamate (70 mM, Wako), spermidine (0.375 mM, Nakarai), dithiothreitol (6 mM, Nakarai), ATP (0.375 mM, GE Healthcare), GTP (0.25 mM, GE Healthcare), CTP (0.125 mM, GE Healthcare), UTP (0.125 mM, GE Healthcare), creatine phosphate (25 mM, Nakarai), E. coli tRNA mixture (0.518 μg/μl, Roche), 10-formyl-5,6,7,8-tetrahydrofolic acid (10 ng/μl), Cys (0.3 mM, Wako), Tyr (0.3 mM, Wako), the other 18 amino acids except for Cys and Tyr (0.36 mM, Wako), 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (100 mM, pH 7.6, Sigma), ribosomes (1 μM), IF1 (25 μM), IF2 (1 μM), IF3 (4.9 μM), EF-G (1.1 μM), EF-Tu (80 μM), EF-Ts (3.3 μM), RF1 (0.05 μM), RF2 (0.05 μM), RF3 (0.17 μM), RRF (3.9 μM), AlaRS (730 nM), ArgRS (30 nM), AsnRS (420 nM), AspRS (120 nM), CysRS (20 nM), GlnRS (60 nM), GluRS (230 nM), GlyRS (90 nM), HisRS (90 nM), IleRS (370 nM), LeuRS (40 nM), LysRS (120 nM), MetRS (110 nM), PheRS (130 nM), ProRS (170 nM), SerRS (80 nM), ThrRS (80 nM), TrpRS (30 nM), TyrRS (150 nM), ValRS (20 nM), MTF (590 nM), creatine kinase (0.25 μM), myokinase (1.4 μM), nucleoside-diphosphate kinase (20 nM), pyrophosphatase (40 nM), yeast inorganic pyrophosphatase (0.2 mU/μl, New England BioLabs (NEB)), ribonuclease inhibitor (0.1 U/μl; Promega), and T7 RNA polymerase (0.42 U/μl; Takara).

    Techniques: Incubation, Agarose Gel Electrophoresis, Autoradiography, Purification, Electrophoresis, SDS Page

    Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Identification of Four Entamoeba histolytica Organellar DNA Polymerases of the Family B and Cellular Localization of the Ehodp1 Gene and EhODP1 Protein

    doi: 10.1155/2010/734898

    Figure Lengend Snippet: Ehodp1 gene localization in nuclei and cytoplasmic DNA-containing structures by in situ PCR. Trophozoites of E. histolytica clone A were fixed, permeabilized and used to amplify a specific DNA fragment of the Ehodp1 gene by IS -PCR using Cy5-dCTP. Then, cells were RNase-treated and stained with PI and observed through a laser confocal microscope. (a)–(b) Amplification of Ehodp1 by IS -PCR. (c) Negative control of IS -PCR carried out without Taq DNA polymerase. (d) Negative control of IS -PCR performed without Ehodp1 specific oligonucleotides. (PI) Cells stained with propidium iodide (red channel). (Cy5) Ehodp1 amplification products labeled with Cy5-dCTP (blue channel). (M) Merging of red and blue fluorescent signals. Squares show an image amplification of a nucleus and a cytoplasmic DNA-containing structure. (MN) Merging of fluorescent signals superimposed on the corresponding cellular images obtained by Nomarsky microscopy. Nucleus (n). Arrows indicate cytoplasmic DNA-containing structures. Bar scale corresponds to 8 μ m

    Article Snippet: For IS -PCR, samples were covered with 50 μ L of reaction mixture containing 200 μ M dNTPs, 400 μ M of each odp1-f and odp1-r primers (described in Materials and Methods, ), 4.5 mM MgCl2 , 10 U of AmpliTaq DNA Polymerase IS (Perkin-Elmer) and 0.1 μ L of Cy5-dCTP (Amersham).

    Techniques: In Situ, Polymerase Chain Reaction, Staining, Microscopy, Amplification, Negative Control, Labeling

    Rescue of DNA replication activity and nuclear localization of DNA polymerase α by recombinant RecQ4 in RecQ4-depleted extracts. (A) Sperm chromatin was incubated in mock (ΔMock)- or RecQ4 (ΔRecQ4)-depleted extracts in the absence (−) or presence of full-length RecQ4 (+low-full, 2 μg/ml), N2-RecQ4 (+low-N2, 0.8 μg/ml; +high-N2, 16 μg/ml), or N1-RecQ4 (+high-N1, 9 μg/ml) at 23°C for 40 min. The samples were treated for 2 min with 0.1% Triton X-100 in EB containing an additional 100 mM NaCl and then fixed with 3.7% formaldehyde. Nuclear localization of DNA polymerase α was visualized with rabbit anti-polymerase α antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replication was monitored as the incorporation of Cy3-dCTP into DNA, and DNA was visualized with Hoechst 33258 dye. (B) The fluorescent intensity of each nucleus was quantified using NIH Image software, and average intensities of over 40 nuclei were normalized by the intensities of the nuclei from mock-depleted extract.

    Journal: Molecular and Cellular Biology

    Article Title: The N-Terminal Noncatalytic Region of Xenopus RecQ4 Is Required for Chromatin Binding of DNA Polymerase ? in the Initiation of DNA Replication

    doi: 10.1128/MCB.02267-05

    Figure Lengend Snippet: Rescue of DNA replication activity and nuclear localization of DNA polymerase α by recombinant RecQ4 in RecQ4-depleted extracts. (A) Sperm chromatin was incubated in mock (ΔMock)- or RecQ4 (ΔRecQ4)-depleted extracts in the absence (−) or presence of full-length RecQ4 (+low-full, 2 μg/ml), N2-RecQ4 (+low-N2, 0.8 μg/ml; +high-N2, 16 μg/ml), or N1-RecQ4 (+high-N1, 9 μg/ml) at 23°C for 40 min. The samples were treated for 2 min with 0.1% Triton X-100 in EB containing an additional 100 mM NaCl and then fixed with 3.7% formaldehyde. Nuclear localization of DNA polymerase α was visualized with rabbit anti-polymerase α antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replication was monitored as the incorporation of Cy3-dCTP into DNA, and DNA was visualized with Hoechst 33258 dye. (B) The fluorescent intensity of each nucleus was quantified using NIH Image software, and average intensities of over 40 nuclei were normalized by the intensities of the nuclei from mock-depleted extract.

    Article Snippet: Sperm nuclei (4,000/μl) were incubated in the egg extract containing 10 μM Cy3-dCTP (Amersham Biosciences) for appropriate times at 23°C.

    Techniques: Activity Assay, Recombinant, Incubation, Labeling, Software

    Effect of RecQ4 depletion from the egg extract on DNA replication and chromatin binding of DNA polymerase α. (A) Depletion of RecQ4 from the extracts. The egg extracts were treated with preimmune or anti-RecQ4 antibodies conjugated to protein A beads. Untreated egg extract and mock- and RecQ4-depleted extracts (1 μl) were resolved by SDS-PAGE and immunoblotted with the antibodies indicated in the figure. (B) Immunofluorescent detection of nuclear RecQ4. Xenopus sperm chromatin was incubated in mock- and RecQ4-depleted extracts for 40 min at 23°C. The samples were treated with 0.1% NP-40 and then fixed with 3.7% formaldehyde in EB. Nuclear localization of RecQ4 was visualized with rabbit anti-RecQ4 antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replications were monitored as the incorporation of Cy3-dCTP into DNA, and DNA was visualized with Hoechst 33258 dye. Fluorescence images were captured with the OpenLab imaging program (Improvision). (C) Chromatin binding of Cut5 in the absence of RecQ4. Xenopus sperm chromatin was incubated in mock- or RecQ4-depleted extracts for 40 min at 23°C. Isolated chromatin fractions were resolved by SDS-PAGE and immunoblotted with the antibodies indicated. (D) Chromatin binding of RecQ4 in the absence of Cut5. Xenopus sperm chromatin was incubated in mock- or Cut5-depleted extracts with (+) or without (−) recombinant Cut5 for 40 min at 23°C.

    Journal: Molecular and Cellular Biology

    Article Title: The N-Terminal Noncatalytic Region of Xenopus RecQ4 Is Required for Chromatin Binding of DNA Polymerase ? in the Initiation of DNA Replication

    doi: 10.1128/MCB.02267-05

    Figure Lengend Snippet: Effect of RecQ4 depletion from the egg extract on DNA replication and chromatin binding of DNA polymerase α. (A) Depletion of RecQ4 from the extracts. The egg extracts were treated with preimmune or anti-RecQ4 antibodies conjugated to protein A beads. Untreated egg extract and mock- and RecQ4-depleted extracts (1 μl) were resolved by SDS-PAGE and immunoblotted with the antibodies indicated in the figure. (B) Immunofluorescent detection of nuclear RecQ4. Xenopus sperm chromatin was incubated in mock- and RecQ4-depleted extracts for 40 min at 23°C. The samples were treated with 0.1% NP-40 and then fixed with 3.7% formaldehyde in EB. Nuclear localization of RecQ4 was visualized with rabbit anti-RecQ4 antibody followed by Alexa 488-labeled anti-rabbit immunoglobulin G. DNA replications were monitored as the incorporation of Cy3-dCTP into DNA, and DNA was visualized with Hoechst 33258 dye. Fluorescence images were captured with the OpenLab imaging program (Improvision). (C) Chromatin binding of Cut5 in the absence of RecQ4. Xenopus sperm chromatin was incubated in mock- or RecQ4-depleted extracts for 40 min at 23°C. Isolated chromatin fractions were resolved by SDS-PAGE and immunoblotted with the antibodies indicated. (D) Chromatin binding of RecQ4 in the absence of Cut5. Xenopus sperm chromatin was incubated in mock- or Cut5-depleted extracts with (+) or without (−) recombinant Cut5 for 40 min at 23°C.

    Article Snippet: Sperm nuclei (4,000/μl) were incubated in the egg extract containing 10 μM Cy3-dCTP (Amersham Biosciences) for appropriate times at 23°C.

    Techniques: Binding Assay, SDS Page, Incubation, Labeling, Fluorescence, Imaging, Isolation, Recombinant

    Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline

    Journal: BMC Genomics

    Article Title: A streamlined tethered chromosome conformation capture protocol

    doi: 10.1186/s12864-016-2596-3

    Figure Lengend Snippet: Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline

    Article Snippet: Restriction enzyme (DpnII or AvaII) generated 5’ overhangs were filled in by adding 63 μl water, 1 μl 1 M MgCl, 10 μl 10× NEBuffer 2, 0.7 μl 10 mM dATP, 0.7 μl 10 mM dTTP, 0.7 μl 10 mM 2’-Deoxyguanosine-5’-O-(1-thiotriphosphate), sodium salt, Sp-isomer (Axxora, San Diego, CA, USA), 15 μl 0.4 mM Biotin-14-dCTP (Life Technologies), 4 μl 10 % Triton X-100 and 5 μl 5 U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB).

    Techniques: Sequencing, Magnetic Beads, Purification, Ligation, Generated, Polymerase Chain Reaction, Amplification

    DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 P]dCTP-labeled cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

    doi:

    Figure Lengend Snippet: DDA of cDNAs derived from mRNAs from etiolated and illuminated maize leaves and Northern blot analyses of these mRNAs. ( A ) Part of a gel from a DDA done with primers T12MG and AP12 (GATCTAACCG) is shown. Total RNA preparations from 10-day-old etiolated maize leaves (D) and greening leaves illuminated with white light (W) or red light (R) for 8 hr or 24 hr were subjected to differential display reverse transcription-PCR. Band No. 40 represents the light-regulated cDNA segment of L29. ( B ) Confirmation of the differential expression pattern of L29. Twenty-five μg of total RNA from etiolated leaves (D) or greening leaves illuminated with blue light (B) or red light (R) for 8 hr or 24 hr was fractionated electrophoretically in a 1% formaldehyde agarose gel, transferred, and probed with [α- 32 P]dCTP-labeled cDNA of reamplified fragment No. 40. A single band of about 0.8 kbp was detected in both blue light- and red light-treated samples but not in the etiolated samples. Equal loading was confirmed by 23S rRNA hybridization.

    Article Snippet: Specific probes were generated by labeling reamplified or cloned cDNA fragments with [α-32 P]dCTP (Dupont/NEN) with a random primer DNA labeling kit (Life Technologies).

    Techniques: Derivative Assay, Northern Blot, Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Labeling, Hybridization

    Time course of L29 mRNA accumulation in leaves on illumination of etiolated maize seedlings. Total RNA was extracted from leaves of 10-day-old etiolated (D) or greening plants illuminated with white light for the number of hours indicated and fractionated electrophoretically in a 1% formaldehyde agarose gel (25 μg/lane). After transfer to Gene Screen filter (DuPont/NEN), the filter was hybridized with [α- 32 P] dCTP-labeled maize L29 full-length cDNA, and 23S rRNA probe was used to assess loading differences.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Subpopulations of chloroplast ribosomes change during photoregulated development of Zea mays leaves: Ribosomal proteins L2, L21, and L29

    doi:

    Figure Lengend Snippet: Time course of L29 mRNA accumulation in leaves on illumination of etiolated maize seedlings. Total RNA was extracted from leaves of 10-day-old etiolated (D) or greening plants illuminated with white light for the number of hours indicated and fractionated electrophoretically in a 1% formaldehyde agarose gel (25 μg/lane). After transfer to Gene Screen filter (DuPont/NEN), the filter was hybridized with [α- 32 P] dCTP-labeled maize L29 full-length cDNA, and 23S rRNA probe was used to assess loading differences.

    Article Snippet: Specific probes were generated by labeling reamplified or cloned cDNA fragments with [α-32 P]dCTP (Dupont/NEN) with a random primer DNA labeling kit (Life Technologies).

    Techniques: Agarose Gel Electrophoresis, Labeling

    Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA − ] or treated with 1 μM RA for 48 h [RA + ]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3′ ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXRγ (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [α- 32 P]dCTP labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RARα, RARγ, RARγ2, RXRα, and RXRβ were observed. RARα and RXRβ showed two isoforms. RARβ (detected only after RA treatment) showed four isoforms. RXRγ was not detectable in untreated or RA-treated cells.

    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: Expression of RARs and RXRs in HL-60 cells. Total RNA (isolated from HL-60 cells that were left untreated [RA − ] or treated with 1 μM RA for 48 h [RA + ]) was analyzed by Northern blotting. RAR and RXR subtype-specific primers located in the 3′ ends of their corresponding cDNAs were used so that for each receptor subtype, all possible isoforms derived from alternative in-frame translational start codons would be detected. Except for the primer for RXRγ (which was amplified from a plasmid), all other specific primers were prepared by RT-PCR from HL-60 cells. The probes were [α- 32 P]dCTP labeled. Ethidium bromide-stained 28S and 18S rRNA bands are shown to serve as size markers. Constitutively expressed transcripts of RARα, RARγ, RARγ2, RXRα, and RXRβ were observed. RARα and RXRβ showed two isoforms. RARβ (detected only after RA treatment) showed four isoforms. RXRγ was not detectable in untreated or RA-treated cells.

    Article Snippet: The PCR-amplified fragments were gel purified and labeled with [α-32 P]dCTP by using a Prime-It RmT random primer labeling kit (Stratagene), and the labeled probes were purified with a NucTrap probe purification column (Stratagene) for detection of mRNA transcripts of blr1 , RARs, and RXRs by hybridization at 50°C overnight after a 2-h prehybridization at 42°C for blr1 and 45°C for RARs and RXRs.

    Techniques: Expressing, Isolation, Northern Blot, Derivative Assay, Amplification, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Labeling, Staining

    T. brucei telomerase-mediated template termination synthesis with ddNTPs. Telomerase reactions containing either [α- 32 P]dGTP ( A ) or [α- 32 P]TTP ( B ) and the telomerase substrate oligonucleotides indicated were tested for the effects of ddNTP chain terminators. Control reactions lacking chain terminators (lanes 1, 5, 9, 12, and 14) were compared with reactions in which dATP was substituted by ddATP (lanes 2, 6, and 10), TTP was substituted by ddTTP (lanes 3, 7, and 11), or dGTP was substituted by ddGTP (lanes 13 and 15). Size markers (lanes M) are terminal deoxynucleotidyltransferase-labeled oligonucleotides tel 6 with [α- 32 P]dCTP ( A ) and tel 1 labeled with [α- 32 P]dGTP ( B ). Lanes nE and nP are mock reactions lacking extract or primer, respectively.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei telomerase-mediated template termination synthesis with ddNTPs. Telomerase reactions containing either [α- 32 P]dGTP ( A ) or [α- 32 P]TTP ( B ) and the telomerase substrate oligonucleotides indicated were tested for the effects of ddNTP chain terminators. Control reactions lacking chain terminators (lanes 1, 5, 9, 12, and 14) were compared with reactions in which dATP was substituted by ddATP (lanes 2, 6, and 10), TTP was substituted by ddTTP (lanes 3, 7, and 11), or dGTP was substituted by ddGTP (lanes 13 and 15). Size markers (lanes M) are terminal deoxynucleotidyltransferase-labeled oligonucleotides tel 6 with [α- 32 P]dCTP ( A ) and tel 1 labeled with [α- 32 P]dGTP ( B ). Lanes nE and nP are mock reactions lacking extract or primer, respectively.

    Article Snippet: Ten microliters of the telomerase reaction was added to a 50-μl final volume PCR mix containing 1× modified TRAP buffer, 50 μM each dNTP, 20 pmol of TS primer, 20 pmol of CX-ext primer, 0.1 μCi/μl [α-32 P]dGTP or [α-32 P]dCTP and 1 unit of Taq polymerase (Boehringer Mannheim).

    Techniques: Labeling

    T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: Telomerase in kinetoplastid parasitic protozoa

    doi:

    Figure Lengend Snippet: T. brucei activity monitored directly by telomerase primer-extension assay. Reactions were performed with DEAE fraction and primer tel 2. Lane 1, standard reaction; lane 2, extract pretreated with 100 ng of RNase A; lane 3, extract incubated with RNasin before addition of RNase A; lane 4, RNase A treatment after telomerase reaction (+); lane 5, nP, no input primer, lane 6, nE, extract substituted by reaction buffer; lane M, terminal deoxynucleotidyltransferase used to label tel 6 with [α- 32 P]dCTP (19 indicates the position of the primer plus 1-nt molecular weight marker).

    Article Snippet: Ten microliters of the telomerase reaction was added to a 50-μl final volume PCR mix containing 1× modified TRAP buffer, 50 μM each dNTP, 20 pmol of TS primer, 20 pmol of CX-ext primer, 0.1 μCi/μl [α-32 P]dGTP or [α-32 P]dCTP and 1 unit of Taq polymerase (Boehringer Mannheim).

    Techniques: Activity Assay, Primer Extension Assay, Incubation, Molecular Weight, Marker

    BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM 5-methyl-dCTP were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: BAC clone sequencing based on the MspJI-based DNA fragmentation approach. ( a ) Amplified BAC DNA directly from glycerol stocks, using the Φ29 enzyme. In the amplification solution, 30 μM 5-methyl-dCTP were included. ( b ) Resequencing results of the BAC clones. Read coverage depth for each clone was visualised on the Tablet software.

    Article Snippet: The reaction mixture, consisting of 29 μl REPLI-g Reaction Buffer, 1 μl REPLI-g Mini DNA Polymerase and 1 μl 5-methyl-dCTP (750 μM), was added into 9 μl of denatured sample, and the sample was incubated at 30°C for 16 hours.

    Techniques: BAC Assay, Sequencing, Amplification, Clone Assay, Software

    Sequencing of the fungal endophyte genome with the MspJI-based DNA fragmentation method. Two independent experiments (rep1 and rep2) were performed using the single endophyte strain. ( a ) WGA of the endophyte genome with the Φ29 enzyme in the presence of 5-methyl-dCTP (X μM). ( b ) MspJI digestion of the endophyte genome-derived amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from WGA products.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: Sequencing of the fungal endophyte genome with the MspJI-based DNA fragmentation method. Two independent experiments (rep1 and rep2) were performed using the single endophyte strain. ( a ) WGA of the endophyte genome with the Φ29 enzyme in the presence of 5-methyl-dCTP (X μM). ( b ) MspJI digestion of the endophyte genome-derived amplicons. ( c ) Flowchart of the MspJI- and physical shearing-based library prep procedures from WGA products.

    Article Snippet: The reaction mixture, consisting of 29 μl REPLI-g Reaction Buffer, 1 μl REPLI-g Mini DNA Polymerase and 1 μl 5-methyl-dCTP (750 μM), was added into 9 μl of denatured sample, and the sample was incubated at 30°C for 16 hours.

    Techniques: Sequencing, Whole Genome Amplification, Derivative Assay

    MspJI-enzymatic digestion of 5 m C-containing PCR and Φ29 products. ( a ) DNA fragments amplified with the locus-specific PCR primers for the Agro _gc50 sequence under the presence of 5-methyl-dCTP (0, 2, 4 or 8 μM). ( b ) MspJI-digested DNA fragments derived from PCR products with each locus-specific primers and Agro gDNA as DNA template. Molar concentration denotes the 5-methyl-dCTP-concentration in PCR solution. ( c ) MspJI-enzymatic digestion of Φ29 enzyme-amplified DNA with randomly incorporated 5 m C from a range of DNA templates. 0, 10, 15 and 20 μM denote final concentrations of 5-methyl-dCTP in the REPLI-g WGA mixture.

    Journal: BMC Biotechnology

    Article Title: A simple method for semi-random DNA amplicon fragmentation using the methylation-dependent restriction enzyme MspJI

    doi: 10.1186/s12896-015-0139-7

    Figure Lengend Snippet: MspJI-enzymatic digestion of 5 m C-containing PCR and Φ29 products. ( a ) DNA fragments amplified with the locus-specific PCR primers for the Agro _gc50 sequence under the presence of 5-methyl-dCTP (0, 2, 4 or 8 μM). ( b ) MspJI-digested DNA fragments derived from PCR products with each locus-specific primers and Agro gDNA as DNA template. Molar concentration denotes the 5-methyl-dCTP-concentration in PCR solution. ( c ) MspJI-enzymatic digestion of Φ29 enzyme-amplified DNA with randomly incorporated 5 m C from a range of DNA templates. 0, 10, 15 and 20 μM denote final concentrations of 5-methyl-dCTP in the REPLI-g WGA mixture.

    Article Snippet: The reaction mixture, consisting of 29 μl REPLI-g Reaction Buffer, 1 μl REPLI-g Mini DNA Polymerase and 1 μl 5-methyl-dCTP (750 μM), was added into 9 μl of denatured sample, and the sample was incubated at 30°C for 16 hours.

    Techniques: Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Concentration Assay, Whole Genome Amplification

    Fig. 1. jnk-1 encodes two different transcripts. ( A ) Structure of C.elegans jnk-1 . Exons are indicated by boxes, while introns are represented as lines. Numbers refer to amino acids. RACE was performed using two different sets of primers as described in Materials and methods. In 11/15 clones (using primers based on sequences from exons 7 and 8) and in 6/10 clones (using primers from exon 12), we detected two transcripts, which we termed jnk-1α and jnk-1β containing 5′ SL1 trans ). For jnk-1α , the SL1 trans -spliced sequence was followed by exon 1, whereas for jnk-1β the SL1 trans -spliced sequence was followed by exon 2. The translation initiation site, ATG (in bold and italicized) of jnk-1α is located in exon 1 while the translation initiation site of jnk-1β is located in exon 3 (in bold and marked by a box). JNK-1α has a 91 amino acid extended N-terminal region (in gray), fused in-frame with 372 amino acid residues (in black) that are identical in JNK-1α and JNK-1β. Poly(A), polyadenylation site; SL1, trans -splicing leader sequence; E, Eco RI and H, Hin dIII restriction sites; NH 2 , N-terminus; COOH, C-terminus. ( B ) Nucleotide sequence of the 5′ regions of jnk-1α and jnk-1β transcripts. SL1 trans -splicing leader sequences (underlined and italicized) are located at the beginning of both jnk-1 transcripts. Non-translated sequences are in lower case and translated sequences are in upper case. ( C ) Southern blot analysis using full-length [α 32 -P]dCTP-labeled jnk-1α cDNA as a probe shows a pattern of DNA restriction compatible with that predicted from cosmid B0478.

    Journal: The EMBO Journal

    Article Title: jkk-1 and mek-1 regulate body movement coordination and response to heavy metals through jnk-1 in Caenorhabditis elegans

    doi: 10.1093/emboj/20.18.5114

    Figure Lengend Snippet: Fig. 1. jnk-1 encodes two different transcripts. ( A ) Structure of C.elegans jnk-1 . Exons are indicated by boxes, while introns are represented as lines. Numbers refer to amino acids. RACE was performed using two different sets of primers as described in Materials and methods. In 11/15 clones (using primers based on sequences from exons 7 and 8) and in 6/10 clones (using primers from exon 12), we detected two transcripts, which we termed jnk-1α and jnk-1β containing 5′ SL1 trans ). For jnk-1α , the SL1 trans -spliced sequence was followed by exon 1, whereas for jnk-1β the SL1 trans -spliced sequence was followed by exon 2. The translation initiation site, ATG (in bold and italicized) of jnk-1α is located in exon 1 while the translation initiation site of jnk-1β is located in exon 3 (in bold and marked by a box). JNK-1α has a 91 amino acid extended N-terminal region (in gray), fused in-frame with 372 amino acid residues (in black) that are identical in JNK-1α and JNK-1β. Poly(A), polyadenylation site; SL1, trans -splicing leader sequence; E, Eco RI and H, Hin dIII restriction sites; NH 2 , N-terminus; COOH, C-terminus. ( B ) Nucleotide sequence of the 5′ regions of jnk-1α and jnk-1β transcripts. SL1 trans -splicing leader sequences (underlined and italicized) are located at the beginning of both jnk-1 transcripts. Non-translated sequences are in lower case and translated sequences are in upper case. ( C ) Southern blot analysis using full-length [α 32 -P]dCTP-labeled jnk-1α cDNA as a probe shows a pattern of DNA restriction compatible with that predicted from cosmid B0478.

    Article Snippet: The blots were pre-hybridized with ExpressHyb hybridization solution (Clontech) for 1 h, and then probed with random-primed (Prime-it II, Stratagene), [α-32 P]dCTP (NEN Life Science)-labeled full-length jnk-1β cDNA at 68°C for 1 h. The blots were washed under stringent conditions (65°C and 0.1% SSC/0.1% SDS) and exposed for 48 h to Kodak XAR-5 film at –70°C.

    Techniques: Clone Assay, Sequencing, Southern Blot, Labeling

    Thermal denaturation transitions of duplexes 9 : 5 – 9 : 8 and H-bonding patterns expected from I, 8-oxoI, and 8-BrI (left); and RTn using AMV on duplexes 9 : 5 – 9 : 8 in the presence of canonical dNTPs, where M = equimolar mixture of all dNTPs, (right). Steady-state kinetics data is shown in the lower left corner.

    Journal: bioRxiv

    Article Title: Translesion Synthesis by MmLV-, AMV-, and HIV-Reverse Transcriptases Using RNA Templates Containing Inosine, Guanosine, and Their 8-oxo-7,8-Dihydropurine Derivatives

    doi: 10.1101/2020.06.10.144048

    Figure Lengend Snippet: Thermal denaturation transitions of duplexes 9 : 5 – 9 : 8 and H-bonding patterns expected from I, 8-oxoI, and 8-BrI (left); and RTn using AMV on duplexes 9 : 5 – 9 : 8 in the presence of canonical dNTPs, where M = equimolar mixture of all dNTPs, (right). Steady-state kinetics data is shown in the lower left corner.

    Article Snippet: Reactions corresponding to the HIV-RT were carried out using the buffer for AMV-RT. dNTPs (dGTP, dCTP, dATP, dTTP) were purchased from Thermo Fisher or New England Biolabs (at a concentration of 100 mM) and diluted to a final concentration of 1.7 or 0.5 mM per experiment (single tube and well loaded onto the gel). dNTP mix, typically labeled M on subsequent figures, was diluted to a final concentration of 425 μM in each dNTP per experiment.

    Techniques:

    (a) DNA gel shift assays showing binding of VlmI-L to a 443 bp vlmA–vlmH intergenic DNA fragment. The 443 bp DNA fragment was labelled at both ends with [ α - 32 P]dCTP. The assays in lanes 1–6 show the behaviour when the complete 443 bp fragment was mixed with increasing amounts of VlmI-L. Lane 7 shows the behaviour when the labelled DNA fragment plus 4 μg VlmI-L was mixed with a 50-fold excess of the unlabelled 443 bp DNA fragment. Lanes 8–11 show the results of gel shift assays after the labelled 443 bp fragment was digested with Pml I, and lanes 12–15 show the results of gel shift assays after digestion of the same fragment with Sma I. The quantity of VlmI used in each assay is shown at the top of each lane. S and P show the positions of the Sma I and Pml I sites, respectively. (b) Comparison of DNA gel shifts produced by VlmI-L and VlmI-S with a radiolabelled 443 bp vlmA–vlmH intergenic DNA fragment.

    Journal: Microbiology

    Article Title: Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

    doi: 10.1099/mic.0.033167-0

    Figure Lengend Snippet: (a) DNA gel shift assays showing binding of VlmI-L to a 443 bp vlmA–vlmH intergenic DNA fragment. The 443 bp DNA fragment was labelled at both ends with [ α - 32 P]dCTP. The assays in lanes 1–6 show the behaviour when the complete 443 bp fragment was mixed with increasing amounts of VlmI-L. Lane 7 shows the behaviour when the labelled DNA fragment plus 4 μg VlmI-L was mixed with a 50-fold excess of the unlabelled 443 bp DNA fragment. Lanes 8–11 show the results of gel shift assays after the labelled 443 bp fragment was digested with Pml I, and lanes 12–15 show the results of gel shift assays after digestion of the same fragment with Sma I. The quantity of VlmI used in each assay is shown at the top of each lane. S and P show the positions of the Sma I and Pml I sites, respectively. (b) Comparison of DNA gel shifts produced by VlmI-L and VlmI-S with a radiolabelled 443 bp vlmA–vlmH intergenic DNA fragment.

    Article Snippet: [ α -32 P]dCTP was obtained from MP Biomedicals.

    Techniques: Electrophoretic Mobility Shift Assay, Binding Assay, Produced

    (a) Diagrammatic illustration of single-crossover disruption in the vlmI gene created by plasmid pKC1139Δ vlmI , showing expected restriction fragments. (b) Southern blot analysis of Kpn I-digested genomic DNA from wild-type S. viridifaciens MG456-hF10 (WT) and S. viridifaciens MG456-hF10 ( vlmI : : pKC1139Δ vlmI ). A 478 bp internal vlmI fragment obtained by Eco RI digestion of plasmid pGEMTΔ vlmI was labelled with [ α - 32 P]dCTP and used as a probe. Kpn I sites are indicated by K .

    Journal: Microbiology

    Article Title: Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)

    doi: 10.1099/mic.0.033167-0

    Figure Lengend Snippet: (a) Diagrammatic illustration of single-crossover disruption in the vlmI gene created by plasmid pKC1139Δ vlmI , showing expected restriction fragments. (b) Southern blot analysis of Kpn I-digested genomic DNA from wild-type S. viridifaciens MG456-hF10 (WT) and S. viridifaciens MG456-hF10 ( vlmI : : pKC1139Δ vlmI ). A 478 bp internal vlmI fragment obtained by Eco RI digestion of plasmid pGEMTΔ vlmI was labelled with [ α - 32 P]dCTP and used as a probe. Kpn I sites are indicated by K .

    Article Snippet: [ α -32 P]dCTP was obtained from MP Biomedicals.

    Techniques: Plasmid Preparation, Southern Blot

    Dynamics of association of the ori-β to the nuclear matrix during the cell cycle of CHO cells. ( A ) Schematic representation of the position of the ori-β probe in the non-transcribed spacer 3′ of the DHFR gene in CHO cells. A 479 bp DNA fragment (filled box) was amplified by PCR and used as a probe. ( B ) Relative abundance of the DHFR ori-β in the matrix-attached DNA in CHO cells. Aliquots of matrix-attached DNA isolated from CHO cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probe in vitro labeled with [ 32 P]dCTP. For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. The results are means of five independent experiments and the standard deviations are shown with error bars.

    Journal: Nucleic Acids Research

    Article Title: Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

    doi:

    Figure Lengend Snippet: Dynamics of association of the ori-β to the nuclear matrix during the cell cycle of CHO cells. ( A ) Schematic representation of the position of the ori-β probe in the non-transcribed spacer 3′ of the DHFR gene in CHO cells. A 479 bp DNA fragment (filled box) was amplified by PCR and used as a probe. ( B ) Relative abundance of the DHFR ori-β in the matrix-attached DNA in CHO cells. Aliquots of matrix-attached DNA isolated from CHO cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probe in vitro labeled with [ 32 P]dCTP. For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. The results are means of five independent experiments and the standard deviations are shown with error bars.

    Article Snippet: DNA probes were labeled in vitro with [32 P]dCTP (3000 Ci/mmol; DuPont) using the RadPrime DNA Labeling System (Gibco BRL).

    Techniques: Amplification, Polymerase Chain Reaction, Isolation, In Vitro, Labeling

    Dynamics of association of the β-globin origin of replication with the nuclear matrix during the cell cycle of HeLa cells. ( A ) Schematic representation of the positions of the probes from the human β-globin locus (filled boxes). ( B ) Aliquots of matrix-attached DNA isolated from HeLa cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probes in vitro labeled with [ 32 P]dCTP. For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. Shaded columns, relative abundance of the control probe C1; filled columns, relative abundance of the β-globin replicator probe; white columns, relative abundance of the control probe C2. The results are means of five independent experiments and the standard deviations are shown with error bars.

    Journal: Nucleic Acids Research

    Article Title: Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

    doi:

    Figure Lengend Snippet: Dynamics of association of the β-globin origin of replication with the nuclear matrix during the cell cycle of HeLa cells. ( A ) Schematic representation of the positions of the probes from the human β-globin locus (filled boxes). ( B ) Aliquots of matrix-attached DNA isolated from HeLa cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probes in vitro labeled with [ 32 P]dCTP. For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. Shaded columns, relative abundance of the control probe C1; filled columns, relative abundance of the β-globin replicator probe; white columns, relative abundance of the control probe C2. The results are means of five independent experiments and the standard deviations are shown with error bars.

    Article Snippet: DNA probes were labeled in vitro with [32 P]dCTP (3000 Ci/mmol; DuPont) using the RadPrime DNA Labeling System (Gibco BRL).

    Techniques: Isolation, In Vitro, Labeling