Journal: Nucleic Acids Research

Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

doi: 10.1093/nar/gks316

Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

Article Snippet: Termination of purified DPE activity with dideoxyCTP DPE reactions were prepared as described above with a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM dideoxyCTP (ddCTP) (Affymetrix cat# 77332).

Techniques: Purification, Polymerase Chain Reaction, Construct

Journal: Nucleic Acids Research

Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

doi: 10.1093/nar/gks316

Figure Lengend Snippet: Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

Article Snippet: Termination of purified DPE activity with dideoxyCTP DPE reactions were prepared as described above with a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM dideoxyCTP (ddCTP) (Affymetrix cat# 77332).

Techniques: Polymerase Chain Reaction, Lysis, Derivative Assay, Activity Assay, Real-time Polymerase Chain Reaction, Generated

Journal: The Plant Cell

Article Title: Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis

doi:

Figure Lengend Snippet: cDNA Microarray Analysis of Gene Expression after Mechanical Wounding. A fluorescently labeled cDNA probe was prepared from mRNA isolated from control Arabidopsis leaves by reverse transcription in the presence of Cy3-dCTP. A second probe, labeled with Cy5-dCTP, was prepared from leaves that were mechanically wounded (60 min). After the simultaneous hybridization of both probes with a cDNA microarray containing 150 defense-related Arabidopsis ESTs and scanning of the array, a pseudocolor image was generated. Genes induced or repressed after mechanical wounding are represented as red or green signals, respectively. Genes expressed at approximately equal levels between treatments appear as yellow spots. The intensity of each spot corresponds to the absolute amount of expression of each gene. The actual size of the array is 8 × 8 mm. Control genes are in the first row of top left, top right, and bottom left quadrants.

Article Snippet: Each reaction was performed in a 30-μL volume containing 2 μg of mRNA, 2 μg of oligo(dT) 21-mer, 500 μM each for dATP, dGTP, and dTTP, 200 μM dCTP, 100 μM Cy3-dCTP or Cy5-dCTP, 30 units of RNase inhibitor (Life Technologies, Basel, Switzerland), 10 μM DTT, and 400 units of SuperScriptII reverse transcriptase (Life Technologies) in SuperScript buffer (Life Technologies).

Techniques: Microarray, Expressing, Labeling, Isolation, Hybridization, Generated

Journal: Nucleic Acids Research

Article Title: An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

doi:

Figure Lengend Snippet: Hybridization consistency on the 9K microarray using TSA amplification. In each graph, Cy5 signals are shown on the ordinate and Cy3 signals on the abscissa on a log 10 scale after non-linear normalization. Each dot represents the hybridization intensity of each gene. While there was more variability in hybridizations obtained from the same sample hybridized to different arrays ( B ) relative to the same array ( A ), both showed high reproducibility.

Article Snippet: The labeling reaction contained 50 µg total RNA, 500 µM dUTP, 500 µM dATP, 500 µM dTTP, 100 µM dCTP, 1 mM Cy3–dCTP/Cy5–dCTP, 400 U Superscript II reverse transcriptase (Gibco BRL), 1 mM DTT and 1× reverse transcriptase buffer supplied by the manufacturer (NEN).

Techniques: Hybridization, Microarray, Amplification

Journal: Nucleic Acids Research

Article Title: An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

doi:

Figure Lengend Snippet: Scatter plots of experiments with total RNA isolated from mouse neural stem cell cultures (neurospheres, NS). The same RNA sample was used for cDNA synthesis and labeled with either fluorescein-12-dCTP or biotin-11-dCTP. Both cDNAs were co-hybridized on the mouse 9k microarray (homotypic hybridization) and developed according to the TSA protocol provided by the manufacturer (MICROMAX; NEN). ( A ) The plot (on a log 10 scale) of Cy5 signal versus background from a representative homotypic hybridization of NS RNA on the mouse 9k array. The plots for Cy3 were similar. The points above the solid line have a signal higher than 2-fold background. The letter E indicates the signal produced by negative control spots. Notable is the clear separation between signal and background apparent in the two clusters of spots that are observed when the data are plotted in this manner. ( B ) A representative plot of the log 10 ratio (Cy3/Cy5) versus the average log 10 signal from both Cy3 and Cy5 channels for each spot in homotypic hybridization. It can be seen that the likelihood of false positive ratios decreases with signal strength. ( C ) Scatter plots of the signal generated from NS versus NS hybridizations from two experiments where the dyes were reversed. In this case, rather than labeling the same RNA, each NS culture, NS7 and NS8, was grown in separate flasks and processed separately. The clear non-linearity (curvature) produced by differences in dye incorporation and signal at different cDNA abundances can be observed.

Article Snippet: The labeling reaction contained 50 µg total RNA, 500 µM dUTP, 500 µM dATP, 500 µM dTTP, 100 µM dCTP, 1 mM Cy3–dCTP/Cy5–dCTP, 400 U Superscript II reverse transcriptase (Gibco BRL), 1 mM DTT and 1× reverse transcriptase buffer supplied by the manufacturer (NEN).

Techniques: Isolation, Labeling, Microarray, Hybridization, Produced, Negative Control, Generated

Journal: Biochemistry

Article Title: The Mechanism and Dynamics of Translesion DNA Synthesis Catalyzed by the Escherichia coli Klenow fragment

doi: 10.1021/bi800324r

Figure Lengend Snippet: (A) Structures of 2' deoxynucleoside triphosphates used or referred to in this study are dATP, dCTP, dGTP, dTTP, Ind-TP, 5-FITP, 5-AITP, 5-NITP, 5-PhITP, 5-CE-ITP, 5-CH-ITP, and 5-NapITP. For convenience, dR is used to represent the 2'-deoxyribose 5'-triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes T or the presence of a tetrahydrofuran moiety that mimics an abasic site.

Article Snippet: Oligonucleotides, including those containing a tetrahydrofuran moiety mimicking an abasic site, were synthesized by Operon Technologies (Alameda, CA). dATP, dCTP, dGTP, and dTTP were obtained from Sigma in greater than 99% purity.

Techniques:

Journal: Biochemistry

Article Title: Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase by Raman crystallography

doi: 10.1021/bi400524h

Figure Lengend Snippet: The Raman spectra of deoxycytidine triphosphate. A) Raman spectrum of 100 mM 2′-deoxycytidine- 13 C 9 , 15 N 3 , (labeled deoxycytidine) 5′-triphosphate (d*CTP) in solution, pH 7.0, in a hanging drop. B) Raman spectrum of 100 mM 2′-deoxycytidine

Article Snippet: 2′-Deoxycytidine 5′-triphosphate (dCTP), thymidine 5′-triphosphate, and 15 N and 13 C labeled dCTP (d*CTP) were purchased from Sigma-Aldrich.

Techniques: Labeling

Journal: Applied and Environmental Microbiology

Article Title: High-Throughput Plasmid Content Analysis of Borrelia burgdorferi B31 by Using Luminex Multiplex Technology ▿ B31 by Using Luminex Multiplex Technology ▿ †

doi: 10.1128/AEM.01877-10

Figure Lengend Snippet: Overview of Luminex procedure for determining plasmid content. (1) Plasmid-specific regions are amplified by multiplex PCR, using genomic DNA or B. burgdorferi culture as the template. Unincorporated primers and dNTPs in amplified PCR products are removed by treatment with exonuclease I and alkaline phosphatase. (2) Primers that contain an xTAG sequence are utilized in an asymmetric PCR that incorporates biotin-dCTP. (3) Biotinylated products are hybridized with xTAG microspheres that are coupled to antitag sequences and detected by binding of streptavidin-R-phycoerythrin. MFI, mean fluorescence intensity.

Article Snippet: The ASPE reaction mixture consisted of 10.1 μl of nuclease-free water, 2 μl of 10× PCR buffer (20 mM Tris-HCl, pH 8.4, 50 mM KCl), 1 μl of pooled tag-ASPE primers (8 primers per pool; 500 nM [each]), 1 μl of 20× dNTPs (without dCTP; 1 μM [each]), 0.5 μl 50 mM MgCl2 , 0.25 μl 0.4 mM biotin-dCTP (Invitrogen), 0.15 μl of Platinum GenoTYPE Tsp DNA polymerase (Invitrogen), and 5 μl of ExoSapIT-treated PCR product.

Techniques: Luminex, Plasmid Preparation, Amplification, Multiplex Assay, Polymerase Chain Reaction, Sequencing, Binding Assay, Fluorescence

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

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0063606

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

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

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

Journal: Nucleic Acids Research

Article Title: Bacillus subtilis RarA modulates replication restart

doi: 10.1093/nar/gky541

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

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

Techniques: Nucleic Acid Electrophoresis, Autoradiography

Journal: Nucleic Acids Research

Article Title: Bacillus subtilis RarA modulates replication restart

doi: 10.1093/nar/gky541

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

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

Techniques: Generated, Incubation

Journal: Nucleic Acids Research

Article Title: Bacillus subtilis RarA modulates replication restart

doi: 10.1093/nar/gky541

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

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

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

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

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

Journal: Plants

Article Title: Innovative RNAi Strategies and Tactics to Tackle Plum Pox Virus (PPV) Genome in Prunus domestica-Plum

doi: 10.3390/plants8120565

Figure Lengend Snippet: ( A ) Autoradiogram showing the accumulated RNAi detected in leaves of 3 different plants (Lanes 1 to 3) of each clone (amisiCPRNAplum-3, -4, and -7) challenged with PPV. 20 µg of total RNA was fractionated onto PAGE. Transferred onto membrane, RNAi was probed with a PPVCP probe labeled with α- 32P-dCTP. Lane MW represents the miRNA molecular weight marker (indicated by the arrow in the right margin) hybridized with a γ-32P-ATP miRNA probe. Ethidium bromide staining of rRNA was used as loading control (bottom panels). ( B ): Agarose gel showing the DNA methylation analysis of the virus CP transgene introduced in amisiCPRNAplums. Genomic DNA was extracted from leaves of 2 different plants (Lanes 1 and 2) of each clone (amisiCPRNAplum-3, -4) and 3 plants of the amisiCPRNAplum-7 challenged with PPV. After over-digestion of DNA with either the isoschizomer MboI (not shown) or the BFCuI restriction enzymes, the methylated status of the PPVCP cistron engineered in these clones was verified by PCR. With the use of a couple of primers (340FWD and 660REV, Table 1 ) flanking the two GATC sites potentially methylated, an amplicon of 425 bp (arrow in the right margin) indicates the expected band amplified, as got from the uncut DNA control (lane P, uncut pHellsgate-amisiCPRNA plasmid). Lane 1Kb DNA ladder (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) represents the molecular weight markers in Kbp.

Article Snippet: Electro-transferred to Hybond-NX membrane (GE Healthcare), the amisiCPRNA was hybridized with α-32P- dCTP (Perkin Elmer, Waltham, MA, USA) radiolabeled CP probe ( B, A).

Techniques: Polyacrylamide Gel Electrophoresis, Labeling, Molecular Weight, Marker, Staining, Agarose Gel Electrophoresis, DNA Methylation Assay, Methylation, Clone Assay, Polymerase Chain Reaction, Amplification, Plasmid Preparation

Journal: Plants

Article Title: Innovative RNAi Strategies and Tactics to Tackle Plum Pox Virus (PPV) Genome in Prunus domestica-Plum

doi: 10.3390/plants8120565

Figure Lengend Snippet: ( A ) Scheme of the gene construct harbored by the recombinant pHellsgate- amisiCPRNA and pHellsgate-amiCPRNA. ( B ) Autoradiograms showing results of Northern-blot done with total RNA extracted from young leaves of the 10 clones (lanes 1 to 10) obtained from plum transformation with pHellsgate-amisiCPRNA. The membrane was hybridized with α-32P-dCTP PPV CP amplicon probe ( Table 1 ). ( C ): Results of Northern blot analysis with total RNA extracted from young leaves of 6 plums including 5 clones obtained from plum transformation with pHellsgate-amiCPRNA, the conventional plum BO70146 used as control ( C ) and a set of synthetic single-stranded RNA oligonucleotides 17, 21, and 25 residues long (MW) as the molecular weight markers (New England Biolabs, Ipswich, MA, USA). After separation onto 16% PAGE. RNA was transferred onto membrane and hybridized with a mixture of probes including miRNA 157, 159, 171, and a miRNA molecular weight marker probe labeled with γ- 32P-ATP ( Table 1 ). The numbers (upper lanes) represent the clones studied. Arrow (right margin) indicates the expected bands detected.

Article Snippet: Electro-transferred to Hybond-NX membrane (GE Healthcare), the amisiCPRNA was hybridized with α-32P- dCTP (Perkin Elmer, Waltham, MA, USA) radiolabeled CP probe ( B, A).

Techniques: Construct, Recombinant, Northern Blot, Clone Assay, Transformation Assay, Amplification, Molecular Weight, Polyacrylamide Gel Electrophoresis, Marker, Labeling

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

Journal: Scientific Reports

Article Title: Structural basis for polymerase η–promoted resistance to the anticancer nucleoside analog cytarabine

doi: 10.1038/s41598-018-30796-w

Figure Lengend Snippet: Polη-catalyzed extension from AraC 3′-terminated primers. ( A ) Extension of 32 P 5′-end-labeled 23-nt primer with the unmodified C or AraC residues at the 3′-end bound to the 5′-…CCTAA G A…-3′ 39-nt template in the presence of dATP, dGTP, and dTTP. Lane 1, unmodified DNA substrate; Lane 2, extension from the unmodified C; Lane 3, extension from AraC. Lanes 1, 2 and 3 were run on the same gel, but lane 1 was cropped from a different part of the gel. Thus, lane 1 is separated with white space from lanes 2 and 3. The original gel is shown in Supplementary Fig. 1 . ( B ) Extension with the 5′-…GGGGG G A…-3′ 39-nt template. Lane 1, unmodified DNA substrate; Lane 2, extension from the unmodified C in the presence of dATP, dGTP, dTTP and dCTP; Lane 3, extension from the AraC in the presence of dATP, dGTP, dTTP and AraCTP. Lanes 1–3 were run together on the same gel. The original gel is shown in Supplementary Fig. 2 . All extension reactions were conducted at 37 °C for 10 min.

Article Snippet: Primer extension was assayed at 37 °C for 10 min in the presence of 25 μM of dATP, dGTP, and dTTP for G1 template and 50 μM each of dATP, dGTP, dTTP and dCTP (Roche Biochemicals, Indianapolis) for extension from C and 50 μM each of dATP, dGTP, dTTP and araCTP for extension from AraC on G6 template DNA.

Techniques: Labeling

Journal: Leukemia

Article Title: AZA-MS: a novel multiparameter mass spectrometry method to determine the intracellular dynamics of azacitidine therapy in vivo

doi: 10.1038/leu.2017.340

Figure Lengend Snippet: Development of a quantitative high-resolution mass spectrometry to directly measure intracellular 5-AZA-CdR and AZA. ( a ) Chemical structures of cytidine, deoxycytidine, AZA and 5-AZA-CdR. ( b ) Schematic depicting intracellular metabolism of AZA. Following cellular uptake and phosphorylation, ~80% of AZA gets incorporated into RNA by RNA polymerases. The remaining fraction is converted into 5-AZA-CdR by ribonucleotide reductase and incorporated into DNA by DNA polymerases. ( c ) Representative high-resolution Orbitrap mass spectra at RT of 0.98 min, showing clear baseline separation between 5-AZA-CdR, 15N-dC and 13C-dC (left, with respective m/z values) despite their identical chromatographic retention times (right). ( d ) Schematic depicting the optimised method incorporating steps to improve sensitivity of detection of intracellular 5-AZA-CdR and AZA by LC–MS.

Article Snippet: Calibration standard mixes containing all analytes (AZA, 5-AZA-CdR, C, dC, mC and mdC) were prepared by dilution in CE buffer of the following chemicals: 5-azacytidine and decitabine (Selleckchem, Houston, TX, USA), cytidine triphosphate and deoxycytidine triphosphate (Promega, Madison, WI, USA) and 5-methylcytidine and 5-methyl-2′-deoxycytidine (MP Biomedicals, Irvine, CA, USA).

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Infectivity analysis of SLCMV-[SeM] partial dimers in N. benthamiana plants. ( a ) Symptoms displayed by N. benthamiana plants agroinoculated with the SLCMV-[SeM] partial dimers. Bottom half shows individual leaves of the tested plants. ( b ) Southern blot analysis using SLCMV-[SeM] DNA A (without common region, ΔCR) labeled with [α- 32 P]dCTP as the probe. The plasmid pBS-SLCMV-Ma-A digested with PstI (50 pg and 250 pg) was used as the positive control. ( c ) Southern blot analysis using [α- 32 P]dCTP-labeled SLCMV-[SeM] DNA B (ΔCR) as the probe. The plasmid pBS-SLCMV-Ma-B digested with BamHI (50 pg and 250 pg) was used as the positive control. ( b , c ) DNA (1 μg) from uninfected plant (C), plant mock infected with the Agrobacterium tumefaciens strain Ach5 (Mock), plant agroinoculated with partial dimers of DNA A alone (A), DNA B alone (B) and two plants independently co-agroinoculated with the partial dimers of DNA A + DNA B (A + B) were loaded in the respective lanes. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom.

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Infection, Southern Blot, Labeling, Plasmid Preparation, Positive Control, Staining, Molecular Weight

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Sri Lankan cassava mosaic virus (SLCMV) DNA A and small interfering RNA (siRNA) analysis in symptomatic and symptom-recovered cassava plants. ( a ) Southern blot analysis of field-infected cassava plants maintained in the greenhouse. DNA (1 μg) samples from a virus-free, axenic (tissue culture-grown) cassava plant (C), and field-infected cassava (cultivar Sengutchi) plants collected from Malappuram (M) and Thiruvananthapuram (T) were analyzed. The field-infected cassava plants were established in the Madurai Kamaraj University (MKU) greenhouse and the DNA was extracted during first year (M1 and T1), second year (M2 and T2) and third year (M3 and T3) of establishment in the greenhouse ( Figure 1 ). pBS-SLCMV-Ma-A plasmid (50 pg and 250 pg) digested with PstI was used as a positive control. [α- 32 P]dCTP-labeled full-length SLCMV-[SeM] DNA A was used as the probe. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom panel. ( b ) Diagnostic multiplex PCR using SLCMV- and ICMV-specific primers to analyze mixed infection in the plant sample T1 (field-infected SLCMV-SeT1, symptomatic plant) and TR (plant which initially recovered and subsequently showed re-emergence of CMD symptoms in the Basel greenhouse) ( Figure 1 ). Genomic DNA and RCA-amplified DNA from T1 and TR were used as templates for polymerase chain reaction (PCR) analysis. Amplified fragments of SLCMV (615bp) and Indian cassava mosaic virus (ICMV) (386bp) are marked. M, Molecular weight marker. ( c ) Small RNA Northern blot analysis of field-infected cassava plants. RNA (10 μg) from field-infected cassava plants collected from Malappuram (M3) and Thiruvananthapuram (T3) were analyzed. The field-infected cassava plants were established in the MKU greenhouse and RNA was extracted after 25 months of initial establishment in the greenhouse (M3 and T3). [α- 32 P]dCTP-labeled SLCMV-[SeM] AC1 (1 kb) gene fragment was used as the probe. The position of siRNA (21 nt) is marked. The bottom panel shows ethidium bromide stained tRNA, as a loading control. ( d ) The proportion of the plant genome (cassava)- and the viral genome (DNA A + DNA B)-derived small RNAs in a total population of 20–25nt small RNAs accumulating in the Sengutchi cassava plants “SeM-SLCMV”, “SeT-recovered” and “SeT-ICMV” ( Figure 1 ).

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Small Interfering RNA, Southern Blot, Infection, Plasmid Preparation, Positive Control, Labeling, Staining, Molecular Weight, Diagnostic Assay, Multiplex Assay, Polymerase Chain Reaction, Amplification, Marker, Northern Blot, Derivative Assay

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Pseudo-recombination in N. benthamiana with SLCMV-[SeM] DNA A and ICMV-[SeT4] DNA B. ( a ) Symptoms in N. benthamiana mock inoculated plants (Mock) and plants agroinoculated with the partial dimers of SLCMV-[SeM] DNA A + DNA B, ICMV-[SeT4] DNA A + DNA B and SLCMV-[SeM] DNA A + ICMV-[SeT4] DNA B. ( b ) Southern blot analysis using SLCMV-[SeM] DNA A (ΔCR) labeled with [α- 32 P]dCTP as the probe. The plasmid pBS-SLCMV-Ma-A digested with PstI (50 pg and 250 pg) was used as the positive control. ( c ) Southern blot analysis using [α- 32 P]dCTP-labeled SLCMV-[SeM] DNA B (ΔCR) as the probe. The plasmid pBS-SLCMV-Ma-B digested with BamHI (250 pg) was used as the positive control. ( b , c ) DNA (1 μg) from uninfected N. benthamiana plant (C), plant mock infected with the A. tumefaciens strain Ach5 (Mock), plant co-agroinoculated with the partial dimers of SLCMV-[SeM] DNA A + DNA B (SA + SB), plant co-agroinoculated with the partial dimers of ICMV-[SeT4] DNA A + DNA B (IA + IB) and plants co-agroinoculated with the partial dimers of SLCMV-[SeM] DNA A + ICMV-[SeT4] DNA B (SA + IB) were loaded in the respective lanes. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom.

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Southern Blot, Labeling, Plasmid Preparation, Positive Control, Infection, IA, Staining, Molecular Weight

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Infectivity analysis of ICMV-[SeT4] in N. benthamiana plants. ( a ) Symptoms displayed by N. benthamiana plants agroinoculated with the partial dimers of ICMV-[SeT4]. Bottom half shows individual leaves of the tested plants. ( b ) Southern blot analysis using ICMV-[SeT4] DNA A (ΔCR) labeled with [α- 32 P]dCTP as the probe. The plasmid pBS-ICMV-Tv-A digested with PstI (250 pg) was used as the positive control. ( c ) Southern blot analysis using [α- 32 P]dCTP-labeled ICMV-[SeT4] DNA B (ΔCR) as the probe. The plasmid pBS-ICMV-Tv-B digested with BamHI (50 pg and 250 pg) was used as the positive control. ( b , c ) DNA (1 μg) from uninfected plant (C), plant mock infected with the A. tumefaciens strain Ach5 (Mock), plant agroinoculated with the partial dimers of DNA A alone (A), DNA B alone (B) and two plants independently co-agroinoculated with the partial dimers of DNA A + DNA B (A + B) were loaded in the respective lanes. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom.

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Infection, Southern Blot, Labeling, Plasmid Preparation, Positive Control, Staining, Molecular Weight

Journal: Cell Stress & Chaperones

Article Title: Heat shock protein 70 stimulation of the deoxyribonucleic acid base excision repair enzyme polymerase ?

doi:

Figure Lengend Snippet: Stimulation of repair of an oligonucleotide by heat shock protein 70 (Hsp70). Gapped 30-mer duplexes were incubated with 5 μCi [α 32 P]deoxycytidine triphosphate, β pol supplemented with 0.5 μg Hsp70 (lanes 1–3) or with 0.5 μg bovine serum albumin (lanes 4–6), or no stimulatory protein (lanes 7–9). Each reaction (20 μL) included 400 ng of gapped duplex, (20 pmol containing ∼0.3 pmol AP sites), 2 μL of β pol at a 1/50 dilution (lanes 1, 4, and 7), 1/200 dilution (lanes 2, 5, and 8), or buffer only (lanes 3, 6, and 9). Reactions at 1/50 dilution of β pol contained 0.16 units, and reactions at a 1/200 dilution of β pol contained 0.04 units of the enzyme. The denatured samples were analyzed on a 20% acrylamide denaturing gel, as described in Experimental Procedures and in the text. the upper panel represents an autoradiogram from a 3-hour exposure of the gel, and the lower panel represents an 18-hour exposure. Hsp70 (0.5 μg) is 7.0 pmol. BSA (0.5 μg) is ∼7.4 pmol

Article Snippet: Human β polymerase was from Trevigen, Gaithersburg, MD, USA, [α32 P]deoxyguanosine triphosphate (dGTP) and [α32 P]deoxycytidine triphosphate (dCTP), each at 3000 Ci/mM, were from Amersham Pharmacia Biotech (Piscataway, NJ, USA).

Techniques: Incubation

Journal: Nucleic Acids Research

Article Title: Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies

doi:

Figure Lengend Snippet: Representative polony images. Lys131Glu mutant time series from left to right: ( A ) 0 h, ( B ) 51.75 h and ( C ) 97 h. Polonies were initially extended with cyanine-5-dCTP (A and C) or cyanine-5-dUTP (B). The slides were then imaged and the extended primers were removed. The procedure was then repeated with cyanine-5-dCTP used to extend (B) and cyanine-5-dUTP used to extend (A) and (C). A positive extension with cyanine-5-dUTP indicates that the wild-type nucleotide is present at position 391 of PGK1 (red), while positive extension with cyanine-5-dCTP indicates the mutant nucleotide (green) at this position.

Article Snippet: Slides were then covered with 55 µl of an extension solution consisting of 50.67 µl Klenow Extension Buffer, 1 µl Klenow fragment (50 000 U/ml; New England Biolabs), 0.83 µl single-stranded DNA binding protein (1–5 µg/µl; USB) and 2.5 µl of either cyanine-5-dATP, cyanine-5-dUTP, cyanine-5-dCTP or cyanine-5-dGTP (10 µM; PerkinElmer Life Sciences) depending upon the desired extension.

Techniques: Mutagenesis

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

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

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

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

Journal: Journal of Virology

Article Title: The Human Immunodeficiency Virus Type 1 Gag Polyprotein Has Nucleic Acid Chaperone Activity: Possible Role in Dimerization of Genomic RNA and Placement of tRNA on the Primer Binding Site

doi:

Figure Lengend Snippet: Placement of tRNA 3 Lys on HIV-1 1-331 RNA by GagΔp6. (A) 32 P-labeled tRNA 3 Lys was incubated with retroviral transcript and GagΔp6, and the reactions were analyzed as described in Materials and Methods. Lanes: 1, tRNA alone; 2, tRNA plus HaSV 34-378 plus 6 μg of GagΔp6; 3, tRNA plus HIV-1 1-331 plus 3 μg of GagΔp6; 4, tRNA plus HIV-1 1-331 plus 6 μg of GagΔp6; 5, tRNA plus HIV-1 1-331. (B) Unlabeled tRNA 3 Lys was annealed to the PBS on HIV-1 1-331 RNA by incubation as indicated. The mixtures were deproteinized as described in Materials and Methods and then tested for the presence of primer on the HIV-1 template RNA by addition of reverse transcriptase and dNTPs, including [α- 32 P]dCTP. The reactions were analyzed as described in Materials and Methods. Size of the labeled DNA product was determined ± 10 nt compared with labeled DNAs of known sizes (data not shown). Lanes: 1, tRNA plus 6 μg of GagΔp6; 2, 6 μg of GagΔp6 plus HIV-1 1-331; 3, tRNA plus HIV-1 1-331; 4, tRNA plus 6 μg of GagΔp6 plus HIV-1 1-331.

Article Snippet: After ethanol precipitation, the RNAs were redissolved in 20 μl of 75 mM KCl–5 mM MgCl2 –0.1 mM dithiothreitol–50 mM Tris (pH 7.5) containing 57 U of HIV-1 reverse transcriptase (Worthington) and 0.1 mM (each) deoxynucleoside triphosphate (dNTP), including 1 μCi of [α-32 P]dCTP (Amersham).

Techniques: Labeling, Incubation

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

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

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