Journal: Applied and Environmental Microbiology

Article Title: Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response

doi: 10.1128/AEM.69.1.679-682.2003

Figure Lengend Snippet: PFGE analysis to show the relatedness of E. pyrifoliae strains Ep2/97 and Ep4/97. Genomic DNA of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.

Article Snippet: To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (Accu Taq ; Sigma).

Techniques: Agarose Gel Electrophoresis, Marker

Journal: Extremophiles

Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

doi: 10.1007/s00792-014-0646-9

Figure Lengend Snippet: a Purified polD was separated by 4–20 % SDS-PAGE and stained with Coomassie blue. Lane 1 is a Protein Ladder (10–250 kDa) and Lane 2 is polD. b – f Nucleotide incorporation by polD into a primed M13mp18 substrate was assayed as described in “ Materials and Methods ”. b PolD temperature optimum. Nucleotide incorporation by polD (10 nM) was measured at various temperatures for 30 min. c Heat stability. PolB (10 nM) and polD (10 nM) were incubated at 95 °C in 1× ThermoPol buffer for the indicated times. Nucleotide incorporation by heat-treated polB and polD was then assayed at 65 °C. The fraction of activity remaining was plotted versus incubation time at 95 °C and fit to an exponential equation; polB filled squares ; polD filled circles . d PolD Mg 2+ optimum. Nucleotide incorporation by polD (10 nM) was assayed in 1× ThermoPol II buffer containing various Mg 2+ concentrations (0.5–32 mM). e polD extension from a DNA or RNA primer. Nucleotide incorporation by polD (10 nM) was measured using either DNA- or RNA-primed M13 substrates. DNA-primed M13 substrate filled circles ; RNA-primed M13 substrate filled squares . f PCNA stimulates polD. PolD (22 nM) synthesis is stimulated by PCNA and RFC ( filled circles ) compared to a reaction lacking PCNA and RFC ( open circles )

Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

Techniques: Purification, SDS Page, Staining, Incubation, Activity Assay

Journal: Extremophiles

Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

doi: 10.1007/s00792-014-0646-9

Figure Lengend Snippet: Aphidicolin inhibits polB but not polD synthesis. To test if aphidicolin inhibits polB and polD, DNA synthesis was measured as described in “ Materials and Methods ”. a DNA synthesis by polB (10 nM) ( filled squares ) and polD (10 nM) ( filled circles ) was measured in the presence of increasing concentrations of aphidicolin (0–400 μM) and plotted as a percentage of activity in a reaction without aphidicolin. b , c DNA synthesis was also measured over a time course. After 4 min of incubation, aphidicolin (final concentration of 200 μM; open shape ) or dH 2 O ( filled shape ) was added and the reaction was allowed to proceed

Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

Techniques: DNA Synthesis, Activity Assay, Incubation, Concentration Assay

Journal: Extremophiles

Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

doi: 10.1007/s00792-014-0646-9

Figure Lengend Snippet: Comparison of error rates and single nucleotide substitutions. a PolD (95 × 10 −5 ) and polD exo- (125 × 10 −5 ) error rates are higher than polB (20 × 10 −5 ). b Transitions ( gray ) are the majority of single base substitutions during synthesis by polD, polD exo- and Taq DNA polymerases. PolD and polD exo- synthesis also yields transversions ( white ) and frameshift deletions ( black ) at lower frequencies

Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

Techniques:

Journal: Extremophiles

Article Title: Characterization of Family D DNA polymerase from Thermococcus sp. 9?N

doi: 10.1007/s00792-014-0646-9

Figure Lengend Snippet: Characterization of polD 3′–5′ exonuclease activity. a FAM-labeled primer/template DNA was incubated in 1× ThermoPol buffer. Various concentrations (10, 5, 2.5, 1.25 nM) of polD wild type ( Lanes 2–5 ), polD/D507A ( Lanes 6–9 ), polD/H554A ( Lanes 10–13 ) or polD/D507A/H554A ( Lanes 14–17 ) were added. Reactions were incubated at 65 °C for 10 min. Reactions omitting polD were run in parallel ( Lane 1 ). Reactions were separated by 15 % TBE-UREA polyacrylamide gel electrophoresis and visualized using a phosphorimager. b DNA polymerase (1 nM) was incubated with a FAM-labeled primer-template (15 nM) in 1× ThermoPol buffer. Exonuclease product was quantified over time and fit to a linear slope to derive rates as described in “ Materials and Methods ”. PolB 3′–5′ exonuclease activity ( filled circle ; 21 fmol/min) was almost two-fold higher than polD ( open circle ; 12 fmol/min). PolD exonuclease-deficient mutants (polD/D507, open square ; polD/H554A, open triangle ; and polD/D507A/H554A, cross hatch ) lacked detectible 3′–5′ exonuclease activity

Article Snippet: To test the effect of aphidicolin on polB and polD, the DNA polymerase assay described above was repeated in the presence or absence of aphidicolin (Sigma).

Techniques: Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis

Journal: Frontiers in Microbiology

Article Title: The Heme Metabolite Carbon Monoxide Facilitates KSHV Infection by Inhibiting TLR4 Signaling in Endothelial Cells

doi: 10.3389/fmicb.2017.00568

Figure Lengend Snippet: TLR4 inhibition via CO or CLI-095 does not affect virus binding, entry or genome delivery to the nucleus. qPCR for KSHV genome copies in (A) the cytoplasmic fraction and (B) the nuclear fraction of iLEC pretreated with 3 μM CLI-095 (pale gray) for 2 h or 10 μM CORM-2 (light gray) for 10 min and then infected with KSHV-BAC16. DMSO (dark gray) was used as a vehicle control. KSHV genome copies are represented as number of genomes per 100 ng DNA. Data are expressed as the mean ± SEM ( n = 3). (C) Immunofluorescent analysis of iLEC infected with KSHV-BAC16 for 30, 60, or 120 min. Expression of ORF65 (green), Actin (red), and DAPI (blue) are shown. Single left panel: Uninfected cells (mock). Top three panels: DMSO-treated iLEC. Middle three panels: CLI-095 treated iLEC. Bottom three panels: CORM-2-treated iLEC. Images were captured at 60× magnification (scale bar = 30 μm). (D) qPCR for KSHV genome copies in iLEC pretreated with 3 μM CLI-095 for 2 h or 10 μM CORM-2 for 10 min and then infected with KSHV-BAC16 for up to 8 h in the presence or absence of 400 μM Foscarnet (+FOS). DMSO was used as a vehicle control. KSHV genome copies (2–8 hpi) are represented as number of genomes per 100 ng DNA. Data are expressed as the mean ± SEM ( n = 3). A one-way ANOVA test was used for statistical analysis. ∗ p -value ≤ 0.05, ∗∗ p -value ≤ 0.01.

Article Snippet: The viral DNA polymerase inhibitor Foscarnet (FOS, Sigma) was used at a concentration of 400 μM.

Techniques: Inhibition, Binding Assay, Real-time Polymerase Chain Reaction, Infection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: SMAD2 Inactivation Inhibits CLDN6 Methylation to Suppress Migration and Invasion of Breast Cancer Cells

doi: 10.3390/ijms18091863

Figure Lengend Snippet: DNA methyltransferase 1 (DNMT1)-mediated upregulation of CLDN6 expression by SB431542. ( A , B ) Real-time polymerase chain reaction (RT-PCR) and immunoblot analysis of DNMT1 and CLDN6, and densitometric analysis of relative gene expression levels after normalization to loading controls GAPDH and β-actin are presented. ( C ) DNMT1 activity assays. ( D ) Methylation-specific PCR (MSP) analysis of CpG island of CLDN6 promoter using bisulfite-treated genomic DNA isolated from MCF-7 and SKBR-3 cells. ( E ) CpG island methylation within the CLDN6 promoter region was measured by bisulfite sequencing in SKBR-3 cells. “Me” stands for methylated, and “U” stands for unmethylated. ( F ) Chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assay to detect the binding of DNMT1 to the promoter of CLDN6 (** p

Article Snippet: Antibodies included anti-DNMT1 (1:500, Cell Signaling Technology), anti-IgG (Millipore), and anti-DNA polymerase II (Millipore); anti-IgG was the negative control and anti-DNA polymerase II was the positive control.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Methylation, Polymerase Chain Reaction, Isolation, Methylation Sequencing, Chromatin Immunoprecipitation, Binding Assay

Journal: Antioxidants & Redox Signaling

Article Title: Identification of a Redox-Modulatory Interaction Between Uncoupling Protein 3 and Thioredoxin 2 in the Mitochondrial Intermembrane Space

doi: 10.1089/ars.2011.3888

Figure Lengend Snippet: Submitochondrial localization of the UCP3/Trx2 interaction. (A) Mitochondria isolated from HeLa cells transfected with UCP3 and Trx2 were suspended in RIPA buffer+SDS or lysis buffer+Triton X-100, respectively. Mitochondrial pellets and supernatants were subjected to SDS-PAGE followed by immunoblotting (IB) with anti-myc, anti-V5, anti-COX4, and anti-DNA polymerase γ (DPγ) antibodies. PPT, mitochondrial pellet after centrifugation; SDS, RIPA buffer; SUP, supernatant after centrifugation; Triton X-100; lysis buffer. (B) . Mitochondrial pellets and supernatants were resuspended in SDS-PAGE buffer, and then loaded onto an SDS-PAGE gel. Immunoblots show the presence of the intermembrane space (IMS) resident Smac and the matrix resident HSP60 in mitochondria that were untreated or treated with proteinase K (Pro K) (lanes 2–7) and increasing concentrations of digitonin (DIG) (lanes 3–7, 0.1 to 0.7 mg/ml DIG). (C) N (VN)- and C (VC)- terminal fragments of Venus fluorescent proteins were fused to the C-terminus of UCP3 (resides 1–308) (UCP3-VN) or ΔCUCP3 (residues1–234) (ΔCUCP3-VN), and the C-terminus of Trx2, respectively. The fragments of fluorescent proteins of belonging to UCP3-VN and ΔCUCP3-VN were localized to the mitochondrial IMS and matrix, respectively. (D) HeLa cells were transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC. Cell extracts (100 μg) were immunoprecipitated (IP) with anti-myc antibodies and analyzed by IB to detect V5 (UCP3) and myc (Trx2). β-Actin was used as an internal standard. (E) Fluorescent images of HeLa cells transfected with UCP3-VN, Trx2-VC, ΔCUCP3-VN and Trx2-VC, and UCP3-VN and Trx2-VC as indicated in each panel. (F) ).

Article Snippet: The proteins were transferred to a nitrocellulose membrane and probed with primary antibody according to the manufacturer's instructions; anti-V5, anti-UCP3, anti-aconitase 2, anti-β-actin (Abcam, Cambridge, MA), anti-cytochrome c (BD Biosciences, Franklin Lakes, NJ), anti-myc, anti-phospho-p38, anti-p38 (Cell Signaling), anti-COX4 (Clontech, Mountain View, CA), anti-DNA polymerase γ (Neo Markers, Fremont, CA), anti-T7 (Novagen, Gibbstown, NJ), and anti-thioredoxin 2 (Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used.

Techniques: Isolation, Transfection, Lysis, SDS Page, Centrifugation, Western Blot, Immunoprecipitation

Journal: BMC Biotechnology

Article Title: Development of a gene synthesis platform for the efficient large scale production of small genes encoding animal toxins

doi: 10.1186/s12896-016-0316-3

Figure Lengend Snippet: Performance of four thermostable DNA polymerases for the synthesis of gene B using PCA-DTF. Lane 1: negative control reaction performed at the same conditions, without addition of primers; lane 2: KOD Hot Start DNA polymerase; lane 3: Q5 Hot Start High Fidelity DNA polymerase; lane 4: Pfu Turbo DNA polymerase and lane 5: Taq DNA polymerase. M2: NZYladder I

Article Snippet: Four DNA polymerases were selected for these studies: KOD Hot Start DNA polymerase (EMD-Millipore), Q5® Hot Star High Fidelity DNA polymerase (New England Biolabs), Pfu Turbo DNA polymerase (Agilent Technologies) and Taq DNA polymerase (Sigma-Aldrich).

Techniques: Negative Control

Journal: Nature Communications

Article Title: 53BP1 can limit sister-chromatid rupture and rearrangements driven by a distinct ultrafine DNA bridging-breakage process

doi: 10.1038/s41467-018-03098-y

Figure Lengend Snippet: The formation of sister DNA entanglements in 53BP1-depleted HeLa cells is dependent on RAD51. a Quantitation of 53BP1-depleted HeLa (left) and U2OS (right) anaphase cells forming FANCD2-negative UFBs. Numbers of anaphase counted: HeLa = 135, B2 = 112, D4 = 105; U2OS = 91, B18 = 90 from three independent experiments. b Maximum z-projection high-resolution image showing multiple short FANCD2-negative PICH-coated UFBs (arrows), linking the separating chromatin and lagging chromosomes in B2 (53BP1 hypo ) cells. Inset shows that PICH stained UFBs (green) are not associated with FANCD2 foci (red). c Maximum z-projection high-resolution image showing the association of FANCD2 foci (red) on PICH-UFBs (green) in aphidicolin-treated HeLa B2 (53BP1 hypo ) cells. Inset shows enlarged view of PICH-coated UFBs are positive of FANCD2 foci at their termini. d Representative images showing γH2AX present on chromatin bridges and lagging chromatin pairs in HeLa 53BP1 hypo cells. Left: maximum z-projection image showing γH2AX (red) at the junction (arrows) of the differentially labelled sister-chromatid bridges (EdU; green). Right: a pair of lagging sister chromatin intertwined by a PICH-UFB (red) and positive of γH2AX (blue) at their termini. Bottom Right: panels showing single-plane images of the intertwining lagging sister chromatin. Blue arrows indicate γH2AX present at the tips of the chromatin. e HeLa B2 (53BP1 hypo ) cell showing the presence of γH2AX signals (red) at the termini of chromatin that were tethered by PICH-coated UFBs (green). f HeLa B2 (53BP1 hypo ) cells were transfected with control or Rad51 siRNA oligos, followed by IF analysis using anti-Rad51. Nuclei are outlined (grey). g RAD51 knockdown caused the formation of FANCD2-assoicated (red) PICH-UFBs (green) in HeLa B2 (53BP1 hypo ) cells. h Quantitation of HeLa B2 (53BP1 hypo ) anaphase cells with FANCD2-positive UFBs following RAD51 knockdown. i Quantitation of HeLa B2 (53BP1 hypo ) anaphase cells with FANCD2-negative UFBs following RAD51 knockdown. Numbers of anaphase cells scored: B2 + control siRNA = 350, B2 + RAD51 siRNA = 220 from three independent experiments. j A model showing the potential roles of 53BP1 and RAD51 in the formation of FANCD2-negative sister DNA bridges in the 53BP1-depleted cells. Error bars represent s.d of three independent experiments. Statistical significance was determined by T -test (* p

Article Snippet: As indicated, the cells were treated with the DNA polymerase inhibitor aphidicolin (Sigma, A0781; 0.3 μM).

Techniques: Quantitation Assay, Staining, Transfection