Journal: Cell Division

Article Title: IMP/GTP balance modulates cytoophidium assembly and IMPDH activity

doi: 10.1186/s13008-018-0038-0

Figure Lengend Snippet: IMPDH-based cytoophidia in iPSCs respond to GTP levels and proliferation arrest. a iPSCs were labelled with anti-IMPDH2 antibody and EdU. b Cytoophidia disassembled completely in 12 h of 2 mM thymidine treatment. Once thymidine was removed and dCTP was added, cytoophidia reassembled in 12 h. c Quantitative results of conditions in b . d Cytoophidia disassembled when cells were treated with 1 mM guanosine for 4 h. After removal of guanosine, cytoophidia reassembled in 12 h. e With 1 mM GTP supplementation, cytoophidia disassembled in 4 h and reassembled in 4 h after removal of GTP. f Quantitative results of conditions in d and e indicating the proportion of cells with cytoophidium. g Proportion of cells labelled by EdU after 4 h of guanosine or GTP treatment. Mean (± SEM) is presented in c , f and g from at least 200 cells counted for each time point of the treatments in at least two independent experiments

Article Snippet: DON (Sigma #D2141), ribavirin (Abcam #ab120660), dCTP (Invitrogen #10217016) and GTP (Sigma #G8877) were dissolved in water.

Techniques:

Journal: Viruses

Article Title: The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

doi: 10.3390/v7052641

Figure Lengend Snippet: Southern blot analysis of transgenic Nicotiana benthamiana plants agroinoculated with SLCMV-[Attur2] DNA A and DNA B partial dimers. DNA (1 µg) from control uninfected (C), control agroinoculated (C-I), and the three agroinoculated transgenic plants (E3, E6, E7) was loaded in the respective lanes. Bi: SLCMV-[Attur2] full-length DNA A (50 pg) was used as a positive control. [α- 32 P]dCTP-labeled full-length SLCMV-[Attur2] DNA A was used as the probe. Single-stranded (ss), supercoiled (sc), open circular (oc) and linear (lin) forms of viral DNA are marked. E: empty lane. The bottom panel represents equal loading of plant DNA (1 µg) in all lanes.

Article Snippet: DNA was labeled with [α-32 P]dCTP using the Megaprime DNA labeling system (GE Healthcare UK Ltd., Little Chalfont, UK) to prepare probes.

Techniques: Southern Blot, Transgenic Assay, Positive Control, Labeling

Journal: Viruses

Article Title: The Agrobacterium tumefaciens Ti Plasmid Virulence Gene virE2 Reduces Sri Lankan Cassava Mosaic Virus Infection in Transgenic Nicotiana benthamiana Plants

doi: 10.3390/v7052641

Figure Lengend Snippet: ( a ) Southern blot analysis of virE2 transgenic Nicotiana benthamiana plants with the hpt probe. Total DNA (10 µg) from transgenic (E1-E7) as well as control (Nb C) plants was digested with Eco RI. Agrobacterium tumefaciens (pCAM- virE2 ) DNA (0.25 ng) digested with Eco RI (At) was used as a positive control. The hpt gene (50 ng) labeled with [α- 32 P]dCTP was used as the probe; ( b ) Internal T-DNA fragment and junction fragment analyses of virE2 transgenic plants by Southern blotting. Total DNA (10 µg) from transgenic (E1-E7) and control (Nb C) plants was digested with Eco RV. Total A. tumefaciens (pCAM- virE2 ) DNA (0.25 ng) digested with Eco RV served as a positive control (At). The virE2 gene (50 ng) labelled with [α- 32 P]dCTP was used as the probe.

Article Snippet: DNA was labeled with [α-32 P]dCTP using the Megaprime DNA labeling system (GE Healthcare UK Ltd., Little Chalfont, UK) to prepare probes.

Techniques: Southern Blot, Transgenic Assay, Positive Control, Labeling

Journal: PLoS Genetics

Article Title: The presence of rNTPs decreases the speed of mitochondrial DNA replication

doi: 10.1371/journal.pgen.1007315

Figure Lengend Snippet: The influence of exonuclease activity on rNMP incorporation by Pol γ. ( A ) Schematic view of reaction set up in Fig 3B. ( B ) Comparison of rNMP incorporation frequencies of wild type (WT) and exonuclease deficient (exo - ) Pol γ. In vitro replication of a primed 7.3 kb ssDNA template was performed with 10 μM dNTPs in the presence (+) or absence (-) of rNTPs. Reaction products were followed by addition of [α- 32 P]-dCTP. Samples were alkaline-treated (“+ NaOH” lanes 5–8) or untreated control (“-NaOH” lanes 1–4) for 2 h at 55°C and run on a denaturing alkaline gel. Fig 3B is a representative picture of three independent experiments. ( C ) Distribution plot of the percentage of total signal intensity from NaOH-treated samples in Fig 3B. The curves for WT (black and grey) and exo - (brown and orange) Pol γ overlap, which indicates a similar incorporation frequency. ( D ) Southern blot analysis against the 16S rDNA region of mtDNA isolated from the liver of WT PolgA (n = 2) and exonuclease-deficient PolgA D257A (n = 3; “exo - ”) mice. SacI-linearized mtDNA was treated with alkaline hydrolysis (“A”) and run on an alkaline gel alongside untreated control samples (“C”). ( E ) Distribution plot of the DNA fragments in control and alkaline-treated samples from Fig 3D. The comparable distribution of DNA fragment size after alkaline-treatment is consistent with a comparable rNMP incorporation frequency in liver mtDNA of WT PolgA and PolgA D257A mice.

Article Snippet: To follow the reaction, [α-32 P]-dCTP (Perkin Elmer) was added.

Techniques: Activity Assay, In Vitro, Southern Blot, Isolation, Mouse Assay

Journal: PLoS Genetics

Article Title: The presence of rNTPs decreases the speed of mitochondrial DNA replication

doi: 10.1371/journal.pgen.1007315

Figure Lengend Snippet: rNMP incorporation frequency of Pol γ and the mtDNA replisome on long DNA templates. ( A ) Schematic diagram of the 7.3 kb M13 ssDNA substrate used to compare the incorporation of rNMPs by Pol γ and yeast Pol δ in the primer extension assay in Fig 2B. The newly synthesized DNA was labelled by addition of [α- 32 P]-dCTP to the reaction. ( B ) Analysis of the rNMP incorporation frequency of Pol γ and yeast Pol δ at “normal” (“N”, S phase) concentrations, “low” (“L”, concentration during the rest of the cell cycle and in non-dividing cells), or S . cerevisiae (“Sc”) dNTP concentrations, in the absence or presence of rNTPs. In all reactions, the DNA template was coated with the relevant single-stranded DNA-binding proteins (lanes 1–6 with RPA; lanes 7–14 with mtSSB) to avoid stalling of DNA synthesis due to formation of DNA secondary structures. Untreated (“-NaOH”) or alkaline (“+NaOH”) treated reaction products were analysed on an agarose gel under denaturing conditions. To estimate rNMP incorporation frequencies from the presented gel, the median length of alkali stable products was determined and used to calculate the frequencies as described in Materials and Methods. Numbers on the left-hand side of the gel indicate positions of DNA marker bands and the full-length starting product (7.3) in kb. The gel is a representative picture of four independent experiments. ( C ) Schematic diagram of the primed mini-circle substrate with a 40 nt 5′ overhang used in Fig 2D. ( D ) Analysis of the rNMP incorporation frequency by the mitochondrial replisome consisting of mtSSB, Twinkle and Pol γ (AB 2 ) on a primed mini-circle substrate with a 5′ overhang for Twinkle loading. Reactions were carried out at normal (“N), low (“L”), and “ S . cerevisiae ” (“Sc”) dNTP concentrations in the presence or absence of rNTPs. Untreated (“-NaOH”) and alkaline (“+NaOH”) treated reaction products were analysed on a denaturing (alkaline) agarose gel. The gel is a representative picture of two independent experiments.

Article Snippet: To follow the reaction, [α-32 P]-dCTP (Perkin Elmer) was added.

Techniques: Primer Extension Assay, Synthesized, Concentration Assay, DNA Binding Assay, Recombinase Polymerase Amplification, DNA Synthesis, Agarose Gel Electrophoresis, Marker