Journal: Nucleic Acids Research

Article Title: Protein–protein interaction analysis by C-terminally specific fluorescence labeling and fluorescence cross-correlation spectroscopy

doi: 10.1093/nar/gkl477

Figure Lengend Snippet: Materials for fluorescence labeling. ( A ) The structure of a fluorescent puromycin derivative. A fluorophore (Cy5 or RhG) and iminobiotin were chemically conjugated to puromycin through a linker. ( B ) DNA construction for fluorescence labeling of proteins. Template DNA consists of SP6 promoter, Omega sequence and an open reading frame (ORF) with a T7·tag at the N-terminus and a polyhistidine tag at the C-terminus, followed by a XhoI restriction enzyme site.

Article Snippet: The purified DNA was transcribed in an SP6 large-scale RNA production system (Promega).

Techniques: Fluorescence, Labeling, Sequencing

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: BTV core-derived ssRNAs (lane 2), single S8 T7 ssRNA (lane 3) and all ten segments T7 ssRNAs (lane 4) were used for the in vitro polymerase assay as described in and synthesized dsRNA were analyzed by PAGE. The radiolabeled dsRNAs were detected by autoradiography using Storage Phospher screen and image analyzer, Typhoon Trio. The end-labeled BTV1 genome segments were used as markers (lane 1). Genome segments are indicated.

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Derivative Assay, In Vitro, Synthesized, Autoradiography, Labeling

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: A. Schematic representation of modified S9 transcripts . B. ssRNA templates were synthesized using T7 polymerase. Lane 1, ssRNA markers; lane 2, luciferase gene (about 1800 nucleotides); lane 3, PAC gene (about 609 nucleotides); lane 4, EGFP gene (about 729 nucleotides); lane 5, chimeric S9-EGFP, EGFP277/657, (1389 nucleotides) and lane 6, wild type BTV10 S9 (1049 nucleotides). The numbers on the left indicate the lengths of the markers in nucleotides. C. dsRNA synthesis from luciferase gene (lane 2), PAC gene (lane 3), EGFP gene (lane 4) and chimeric S9-EGFP, EGFP277/657, ssRNA template (lane 5) was compared with wild type BTV10 S9 (lane 6). The radiolabeled bands were detected by autoradiography. The arrows indicate the bands with the correct size. The end-labeled BTV1 genome segments were used as markers (lane 1). Genome segments are indicated on the left.

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Modification, Synthesized, Luciferase, Autoradiography, Labeling

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: The recovery of BTV from RNA generated in vitro was compared using uncapped and capped ssRNA in the second transfection. BSR cells were transfected first with 6 capped ssRNAs (S1, S3, S4, S6, S8 & S9) and subsequently with all capped ssRNAs (column 1), all uncapped ssRNAs (column 2) or uncapped S9 together with remaining 9 capped ssRNAs (column 3). Each ssRNA was at 50 ng per transfection. The recovery of virus was shown as the total number of plaques per well (Mean ± SD).

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Generated, In Vitro, Transfection, Virus

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: Each 20 pmol of cap analogues indicated at the bottom of each column, was added to the reaction mixture with 0.5 µg of uncapped (upper panel) or capped (lower panel) T7 S9 ssRNA and 70 µg of VP1. The radioactive intensity of each sample was standardized by control sample (ctrl), which was added water instead of cap analogue. The efficiency of dsRNA synthesis of each sample was shown as a fold of control (Mean ± SD). Asterisks are showing significant difference (**P<0.01; *P<0.05).

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Analogues, Control

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: Each 0.5 µg of uncapped (Uncapped, column 1), anti-reverse capped (ARCA, column 2), non-methylated capped (GpppG, column 3) or 5′ extended T7 S9 ssRNAs (GpG, column 4; ApG, column 5) was added to the reaction mixture. Schematic representation of each 5′-modified T7 S9 ssRNA was shown on the upper panel. The radioactive intensity of each sample was standardized by uncapped T7 S9 ssRNA sample (lower panel). The efficiency of dsRNA synthesis of each sample was shown as a fold of uncapped T7 S9 ssRNA sample (Mean ± SD). The radiolabeled bands were detected by autoradiography. The intensity of each band was counted using ImageJ software. Asterisks are showing significant difference (**P<0.01; *P<0.05).

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Methylation, Modification, Autoradiography, Software

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: Each 20 pmol of dinucleotide indicated at the bottom of each column was added to the reaction mixture containing 0.5 µg of either uncapped (upper panel) or capped (lower panel) T7 S9 ssRNA template. The radioactive intensity of each sample was standardized by control sample (ctrl, column 1), which was added water instead of dinucleotide. The efficiency of dsRNA synthesis of each sample was shown as a fold of control (Mean ± SD). Asterisks show significant difference (**P<0.01; *P<0.05).

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Control

Journal: PLoS ONE

Article Title: Bluetongue Virus VP1 Polymerase Activity In Vitro : Template Dependency, Dinucleotide Priming and Cap Dependency

doi: 10.1371/journal.pone.0027702

Figure Lengend Snippet: A. The effect of biotin-labeled ApG (Bio-ApG) on BTV polymerase was compared with non-labeled ApG (ApG). Each 20 pmol of ApG or Bio-ApG was added to the reaction mixture containing [α- 32 P] CTP. B. Priming activity by dinucleotide, ApG, was detected using biotin-labeled ApG and non-radioactive polymerase assay as described in . As markers, BTV genomic segments was used and stained with 0.02% (w/v) methylene blue after transferring to the nylon membrane (lane 1). Non-labeled ApG (lane 2) or biotin-labeled ApG (lane 3) was added the reaction mixture containing non-labeled rNTP with uncapped T7 S9 ssRNA. Biotin-labeled dsRNAs were detected using Streptavidin e-alkaline phosphatase conjugate as described in . Genome segments are indicated on the left.

Article Snippet: For synthesis of luciferase ssRNA, control linear DNA from RiboMAX Large Scale RNA Production System-T7 kit (Promega) was used.

Techniques: Labeling, Activity Assay, Staining, Transferring, Membrane