t7 rna polymerase Search Results


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  • 99
    New England Biolabs t7 rna polymerase
    Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the <t>T7</t> RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).
    T7 Rna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 3659 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t7 rna polymerase
    Efficacy of ribozyme RZ101 against rat caspase-3. A , Expected cleavage fragments with RZ101. B , In vitro cleavage of rat caspase-3 RNA by RZ101. Ribozyme or caspase-3 RNA were generated by in vitro transcription using <t>T7</t> RNA polymerase. Caspase-3 RNA was incubated with either ribozyme or water for 1 hr at 37°C and then analyzed by electrophoresis and silver staining. Specific cleavage fragments migrated at their expected positions. C , Quantitative RT-PCR of endogenous caspase-3 in rat PC12 cells transfected with RZ101 or β-galactosidase. After 24 hr of serum deprivation, total mRNA was amplified for caspase-3 (323 bp) and cyclophilin (330 bp). D , After staining with ethidium bromide, the bands were quantified by densitometry. The ratio of caspase-3 to cyclophilin band volumes were calculated, and data were expressed as a percent value of the β-galactosidase-transfected control result. RZ101 produced a 13 ± 1% reduction in signal. Given a 20% transfection rate, the calculated reduction in caspase-3 mRNA in RZ101-transfected cells alone is ∼65%. E , Western blot for endogenous caspase-3 in PC12 cells transiently transfected with β-galactosidase or RZ101. For each condition, 10, 5, and 2.5 μg of protein dilutions were analyzed. RZ101 effectively downregulates caspase-3 protein expression.
    T7 Rna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 12384 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega t7 rna polymerase
    A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with <t>T7</t> RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.
    T7 Rna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 10162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene t7 rna polymerase
    Linear DNA templates in cell-free protein synthesis based on CHO cell lysate. Linear IRES-luciferase and IRES-Mel-EPO templates tested during cell-free protein synthesis reactions in the presence of 14 C leucine. Different concentrations of linear DNA product and <t>T7</t> RNA polymerase (Pol) were added to the individual reactions. Protein yield was quantified by hot TCA precipitation followed by scintillation measurement. Error bars show standard deviations calculated from triplicates.
    T7 Rna Polymerase, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 1076 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa t7 rna polymerase
    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, <t>T7</t> 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.
    T7 Rna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 1043 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore t7 rna polymerase
    Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by <t>T7</t> RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.
    T7 Rna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 621 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim t7 rna polymerase
    Construction of artificial defective RNAs of MBGV. The minigenomes were inserted in transcription vector 2,0 (gray) between the <t>T7</t> RNA polymerase promoter and the hepatitis delta virus ribozyme (hatched). For in vitro transcription, plasmids were linearized by using the Sal I restriction site (right side). (A) Diagram of negative-sense minigenomic cDNA 215, consisting of 439 nt of the 5′ trailer (white) adjacent to the T7 RNA polymerase promoter, 668 nt of the CAT gene in a negative-sense orientation (black), and 106 nt of the 3′ leader (white) adjacent to the ribozyme. Above the scheme are indicated the boundary between the T7 RNA polymerase promoter sequence (underlined) and the 5′ end of the minigenome (negative-sense orientation) (left side) and the boundary between the ribozyme sequence (underlined) and the 3′ end of the minigenome (right side). The CAT gene is flanked by Not I and Nde I restriction sites. (B) Diagram of positive-sense minigenomic cDNA 2.1-CAT, consisting of 106 nt of the 3′ leader (white) adjacent to the T7 RNA polymerase promoter, the CAT gene in a positive-sense orientation (black), and 439 nt of the 5′ trailer adjacent to the ribozyme sequence (white). The boundaries between the T7 RNA polymerase promoter and the ribozyme sequence (underlined), respectively, and the MBGV-specific sequences are indicated. MBGV-specific sequences are shown in the plus-strand orientation. The CAT gene is flanked by Not I restriction sites. (C) Diagram of the cDNA coding for the copy-back-type negative-stranded minigenome cb-CAT. The minigenome consists of 439 nt of the 5′ trailer (white) adjacent to the T7 RNA polymerase promoter, the CAT gene in a negative-sense orientation (black), and, adjacent to the ribozyme sequence, 105 nt complementary to the last 105 nt of the trailer (designated as c-trailer; white), which serves as the leader region. The boundaries between the T7 RNA polymerase promoter and the ribozyme sequence (underlined), respectively, and the MBGV-specific sequences are indicated. MBGV-specific sequences are shown in the negative-sense orientation. The CAT gene is flanked by Not I and Nde ). Transcription start and stop sites are indicated only for negative-stranded minigenomes. The cleavage site of the ribozyme is symbolized by a pair of scissors.
    T7 Rna Polymerase, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 640 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Agilent technologies t7 rna polymerase
    Identification of Proteins Able to Bind GAA Exon 2 by Pull-Down and Western Blot Analysis (A) Schematic representation of GAA 147–286, 357–500, and 427–572 exonic regions used as templates for <t>T7</t> RNA transcription in vitro. Putative silencers are represented by red boxes, while the putative enhancer is in green. (B) Pull-down analysis of three in vitro transcribed RNAs with HeLa nuclear extract (NE) analyzed on SDS-10% polyacrylamide gels and visualized by colloidal Coomassie staining. Proteins differentially precipitated by each RNA (letters a–e) were excised from the gel and analyzed by mass spectrometry. Colloidal Coomassie gel staining gives a representative picture of three independent experiments. Beads alone were used as a control (Beads). (C) Western blot analysis, after pull-down assay, using specific antibodies against hnRNPA1/A2, hnRNPH, hnRNPQ, and hnRNPR confirmed the identity of each protein. The nuclear extract sample corresponds to 1/20th of the total amount of protein used for the pull-down assay. The western blot images are representative pictures of three independent experiments. Beads alone were used as a control.
    T7 Rna Polymerase, supplied by Agilent technologies, used in various techniques. Bioz Stars score: 93/100, based on 676 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t7 rnap
    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage <t>T7</t> RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.
    T7 Rnap, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 175 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Enzo Biochem t7 rna polymerase
    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage <t>T7</t> RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.
    T7 Rna Polymerase, supplied by Enzo Biochem, used in various techniques. Bioz Stars score: 92/100, based on 285 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Promega t7 rna polymerase kit
    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage <t>T7</t> RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.
    T7 Rna Polymerase Kit, supplied by Promega, used in various techniques. Bioz Stars score: 89/100, based on 99 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    GE Healthcare t7 rna polymerase
    Specificity of recognition of cap structures by Flu polymerases. (A) Analysis of 5′-terminal cap structures of RNAs. <t>T7</t> RNA polymerase-synthesized RNAs were treated with nuclease P 1 and analyzed by TLC (PEI-CEL, 0.65 M LiCl), and radioactive nucleotides were detected by autoradiography. (B and C) In vitro capped RNA cleavage (B) and RNA elongation (C) reactions were performed with 600 ng of FluA (lanes 2, 5, and 8) or FluB (lanes 3, 6, and 9) vRNP using 2 fmol of variously methylated capped RNAs (m 7 GpppGm-RNA, lanes 1 to 3; m 7 GpppG-RNA, lanes 4 to 6; GpppG-RNA, lanes 7 to 9). RNA products were analyzed by 15% PAGE containing 8 M urea. The input capped RNAs (33 nt), the cleaved capped RNA products, and the elongated products are indicated as a closed triangle, open triangles, and a black bar, respectively, at the right. (D and E) Ratios of cleaved RNA products (D) and RNA transcripts (E) to total input primer RNAs. (F) Cap-binding activity for variously methylated capped RNAs. UV cross-linking was performed using 50, 100, and 200 ng of FluA (upper panel) and FluB (lower panel) vRNP and 50 fmol of variously methylated capped RNAs (GpppG-RNA, lanes 1 to 3; m 7 GpppG-RNA, lanes 4 to 6; m 7 GpppGm-RNA, lanes 7 to 9).
    T7 Rna Polymerase, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 94/100, based on 795 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Toyobo t7 rna polymerase
    RNA transcription reaction of acetaldehyde-treated plasmids. a In the absence of DNA damage, the <t>T7</t> RNA polymerase generates RNA transcripts from DNA templates. After purifying RNA, real-time reverse transcription-PCR (qRT-PCR) is performed, and the PCR products are analyzed. If acetaldehyde damages DNA, the resulting lesions inhibit RNA synthesis, as T7 RNA polymerase cannot synthesize transcripts from damaged templates, and qRT-PCR products will not be detected. Amplification plot of qRT-PCR analysis of RNA transcripts of UV-irradiated ( b ) or of acetaldehyde (AA)-treated ( c ) DNA templates. UV-irradiated ( d ) or acetaldehyde-treated ( e ) pBSII was incubated with T7 RNA polymerase, and transcription was quantified by qRT-PCR
    T7 Rna Polymerase, supplied by Toyobo, used in various techniques. Bioz Stars score: 92/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher megascript t7 rna polymerase kit
    RNA transcription reaction of acetaldehyde-treated plasmids. a In the absence of DNA damage, the <t>T7</t> RNA polymerase generates RNA transcripts from DNA templates. After purifying RNA, real-time reverse transcription-PCR (qRT-PCR) is performed, and the PCR products are analyzed. If acetaldehyde damages DNA, the resulting lesions inhibit RNA synthesis, as T7 RNA polymerase cannot synthesize transcripts from damaged templates, and qRT-PCR products will not be detected. Amplification plot of qRT-PCR analysis of RNA transcripts of UV-irradiated ( b ) or of acetaldehyde (AA)-treated ( c ) DNA templates. UV-irradiated ( d ) or acetaldehyde-treated ( e ) pBSII was incubated with T7 RNA polymerase, and transcription was quantified by qRT-PCR
    Megascript T7 Rna Polymerase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 88 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs hiscribe t7 high yield rna synthesis kit
    RNA transcription reaction of acetaldehyde-treated plasmids. a In the absence of DNA damage, the <t>T7</t> RNA polymerase generates RNA transcripts from DNA templates. After purifying RNA, real-time reverse transcription-PCR (qRT-PCR) is performed, and the PCR products are analyzed. If acetaldehyde damages DNA, the resulting lesions inhibit RNA synthesis, as T7 RNA polymerase cannot synthesize transcripts from damaged templates, and qRT-PCR products will not be detected. Amplification plot of qRT-PCR analysis of RNA transcripts of UV-irradiated ( b ) or of acetaldehyde (AA)-treated ( c ) DNA templates. UV-irradiated ( d ) or acetaldehyde-treated ( e ) pBSII was incubated with T7 RNA polymerase, and transcription was quantified by qRT-PCR
    Hiscribe T7 High Yield Rna Synthesis Kit, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1935 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).

    Journal: Viruses

    Article Title: A Reverse Genetics System for Zika Virus Based on a Simple Molecular Cloning Strategy

    doi: 10.3390/v10070368

    Figure Lengend Snippet: Construction and stability of synthetic full length Zika virus (synZIKV) cDNA clones. ( A ) Schematic representation of the synZIKV MR766 construct and the four fragments used to assemble the genome. The 5′ and 3′UTRs are indicated with bold black lines, the promoter for the T7 RNA polymerase with a black arrow. Restriction sites used for the assembly of the fragments are indicated. An enlargement of fragment #1 is shown below with putative CEPs (score > 0.85) indicated by red arrow heads. CEP 1 was not mutated (indicated with the pink arrow head). ( B ) Same as in panel ( A ) but for synZIKV-H/PF/2013. ( C ) Restriction patterns of pFK-synZIKV constructs obtained after digest with EcoRI (MR766) or XmnI (H/PF/2013) and agarose gel electrophoresis. Plasmids were analysed directly after assembly (original prep) and after five passages (P5) in E. coli (five DNA clones of P5 are shown).

    Article Snippet: After incubation at 37 °C for 2.5 h, 1 U/μL T7 RNA polymerase was added followed by additional 2.5 h incubation at 37 °C.

    Techniques: Clone Assay, Construct, Agarose Gel Electrophoresis

    Efficacy of ribozyme RZ101 against rat caspase-3. A , Expected cleavage fragments with RZ101. B , In vitro cleavage of rat caspase-3 RNA by RZ101. Ribozyme or caspase-3 RNA were generated by in vitro transcription using T7 RNA polymerase. Caspase-3 RNA was incubated with either ribozyme or water for 1 hr at 37°C and then analyzed by electrophoresis and silver staining. Specific cleavage fragments migrated at their expected positions. C , Quantitative RT-PCR of endogenous caspase-3 in rat PC12 cells transfected with RZ101 or β-galactosidase. After 24 hr of serum deprivation, total mRNA was amplified for caspase-3 (323 bp) and cyclophilin (330 bp). D , After staining with ethidium bromide, the bands were quantified by densitometry. The ratio of caspase-3 to cyclophilin band volumes were calculated, and data were expressed as a percent value of the β-galactosidase-transfected control result. RZ101 produced a 13 ± 1% reduction in signal. Given a 20% transfection rate, the calculated reduction in caspase-3 mRNA in RZ101-transfected cells alone is ∼65%. E , Western blot for endogenous caspase-3 in PC12 cells transiently transfected with β-galactosidase or RZ101. For each condition, 10, 5, and 2.5 μg of protein dilutions were analyzed. RZ101 effectively downregulates caspase-3 protein expression.

    Journal: The Journal of Neuroscience

    Article Title: Ribozyme-Mediated Inhibition of Caspase-3 Protects Cerebellar Granule Cells from Apoptosis Induced by Serum–Potassium Deprivation

    doi: 10.1523/JNEUROSCI.20-01-00179.2000

    Figure Lengend Snippet: Efficacy of ribozyme RZ101 against rat caspase-3. A , Expected cleavage fragments with RZ101. B , In vitro cleavage of rat caspase-3 RNA by RZ101. Ribozyme or caspase-3 RNA were generated by in vitro transcription using T7 RNA polymerase. Caspase-3 RNA was incubated with either ribozyme or water for 1 hr at 37°C and then analyzed by electrophoresis and silver staining. Specific cleavage fragments migrated at their expected positions. C , Quantitative RT-PCR of endogenous caspase-3 in rat PC12 cells transfected with RZ101 or β-galactosidase. After 24 hr of serum deprivation, total mRNA was amplified for caspase-3 (323 bp) and cyclophilin (330 bp). D , After staining with ethidium bromide, the bands were quantified by densitometry. The ratio of caspase-3 to cyclophilin band volumes were calculated, and data were expressed as a percent value of the β-galactosidase-transfected control result. RZ101 produced a 13 ± 1% reduction in signal. Given a 20% transfection rate, the calculated reduction in caspase-3 mRNA in RZ101-transfected cells alone is ∼65%. E , Western blot for endogenous caspase-3 in PC12 cells transiently transfected with β-galactosidase or RZ101. For each condition, 10, 5, and 2.5 μg of protein dilutions were analyzed. RZ101 effectively downregulates caspase-3 protein expression.

    Article Snippet: RNA transcripts were generated using T7 RNA polymerase (Ambion, Austin, TX) and purified by gel filtration chromatography (Clontech, Cambridge, UK).

    Techniques: In Vitro, Generated, Incubation, Electrophoresis, Silver Staining, Quantitative RT-PCR, Transfection, Amplification, Staining, Produced, Western Blot, Expressing

    Strategies for construction and in vitro growth characterization of Flc-LOM. (A) Circular map of pFlc-LOM showing the relative positions of the LOM cDNA, ampicillin resistance gene, and P15A origin of replication. Locations of relevant restriction sites and the T7 promoter are indicated. The T7 RNA promoter was located immediately upstream of the 5' end of the genome, and a Srf I site was at the 3' end. The geno me sequences are underlined. (B) Using the classical swine fever virus-specific monoclonal antibody LOM01, an indirect immunoperoxidase assay of Flc-LOM was performed in PK-15 cells 3 days after infection with a multiplicity of infection (MOI) of 1 (a and b, × 200). The Flc-LOM biotype was determined using the exaltation of Newcastle disease virus method. Serial dilutions from 10 1.0 to 10 6.0 of Flc-LOM were added to primary swine testicular cells. After 4 days, the supernatant was removed and the cells were infected with Newcastle disease virus (10 4.0 PFU/mL). A cytopathic effect was observed 3 days post-infection and photographed using an inverted microscope (c and d, × 200). (C) Replication kinetics of the Flc-LOM and LOM viruses. PK-15 cells seeded in T25 flasks were infected at a MOI of 1. The data are from three independent experiments. Virus titers are shown as log 10 10 4 median TCID 50 /mL. ST: swine testicular.

    Journal: Journal of Veterinary Science

    Article Title: Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain

    doi: 10.4142/jvs.2012.13.1.81

    Figure Lengend Snippet: Strategies for construction and in vitro growth characterization of Flc-LOM. (A) Circular map of pFlc-LOM showing the relative positions of the LOM cDNA, ampicillin resistance gene, and P15A origin of replication. Locations of relevant restriction sites and the T7 promoter are indicated. The T7 RNA promoter was located immediately upstream of the 5' end of the genome, and a Srf I site was at the 3' end. The geno me sequences are underlined. (B) Using the classical swine fever virus-specific monoclonal antibody LOM01, an indirect immunoperoxidase assay of Flc-LOM was performed in PK-15 cells 3 days after infection with a multiplicity of infection (MOI) of 1 (a and b, × 200). The Flc-LOM biotype was determined using the exaltation of Newcastle disease virus method. Serial dilutions from 10 1.0 to 10 6.0 of Flc-LOM were added to primary swine testicular cells. After 4 days, the supernatant was removed and the cells were infected with Newcastle disease virus (10 4.0 PFU/mL). A cytopathic effect was observed 3 days post-infection and photographed using an inverted microscope (c and d, × 200). (C) Replication kinetics of the Flc-LOM and LOM viruses. PK-15 cells seeded in T25 flasks were infected at a MOI of 1. The data are from three independent experiments. Virus titers are shown as log 10 10 4 median TCID 50 /mL. ST: swine testicular.

    Article Snippet: Authentic viral RNA was obtained by transcription from the T7 RNA polymerase promoter using a T7 MEGAscript kit (Ambion, USA).

    Techniques: In Vitro, Indirect Immunoperoxidase Assay, Infection, Inverted Microscopy

    Test of translational independence of the second cistron in the dicistronic reporter construct. Phosphorimages of SDS–PAGE gels containing proteins labeled with [ 35 S]methionine during in vitro translation. Rabbit reticulocyte lysates were programmed with 20 ng/µl of dicistronic RNA transcribed in vitro by T7 polymerase as indicated. Rluc translation is controlled by either the EMCV IRES ( A ), or ΔEMCV ( B ), Fluc translation is controlled by the three indicated sequences. Results identical to these were obtained using 10 ng/µl of RNA in separate reactions.

    Journal: Nucleic Acids Research

    Article Title: The dicistronic RNA from the mouse LINE-1 retrotransposon contains an internal ribosome entry site upstream of each ORF: implications for retrotransposition

    doi: 10.1093/nar/gkj490

    Figure Lengend Snippet: Test of translational independence of the second cistron in the dicistronic reporter construct. Phosphorimages of SDS–PAGE gels containing proteins labeled with [ 35 S]methionine during in vitro translation. Rabbit reticulocyte lysates were programmed with 20 ng/µl of dicistronic RNA transcribed in vitro by T7 polymerase as indicated. Rluc translation is controlled by either the EMCV IRES ( A ), or ΔEMCV ( B ), Fluc translation is controlled by the three indicated sequences. Results identical to these were obtained using 10 ng/µl of RNA in separate reactions.

    Article Snippet: In vitro translation RNA was transcribed in vitro using T7 polymerase (MAXIscript, Ambion, Inc.) with templates made from dicistronic luciferase constructs after linearization with XbaI.

    Techniques: Construct, SDS Page, Labeling, In Vitro

    PEDV transfer vectors. (A) The pPEDV transfer vector contains the 5′-proximal 605 nt fused to the 3′ approximately 8 kilobases of the PEDV genome. All other vectors are derivatives thereof. The red triangle indicates the T7 promoter in the transfer vectors from which synthetic RNAs were made in vitro using T7 RNA polymerase. (B) Nucleotide sequences of junctions in the PEDV transfer vectors. Encircled numbers correspond to the numbered positions in the vector maps as indicated in Fig. 2A. (upper panel) The stop codon of ORF1b is underlined, the start codon of S is in blue, the transcription regulatory sequences (XUA(A/G)AC; [4] ) are in orange and the Bam HI site is indicated in purple. (lower panel) The stop codon of the S gene is underlined, the start codon of the ORF3 gene is in blue, the start codon of E gene is in red, the transcription regulatory sequences are in orange and the unique Pml I and Eco RV sites are indicated in purple and green, respectively.

    Journal: PLoS ONE

    Article Title: Manipulation of the Porcine Epidemic Diarrhea Virus Genome Using Targeted RNA Recombination

    doi: 10.1371/journal.pone.0069997

    Figure Lengend Snippet: PEDV transfer vectors. (A) The pPEDV transfer vector contains the 5′-proximal 605 nt fused to the 3′ approximately 8 kilobases of the PEDV genome. All other vectors are derivatives thereof. The red triangle indicates the T7 promoter in the transfer vectors from which synthetic RNAs were made in vitro using T7 RNA polymerase. (B) Nucleotide sequences of junctions in the PEDV transfer vectors. Encircled numbers correspond to the numbered positions in the vector maps as indicated in Fig. 2A. (upper panel) The stop codon of ORF1b is underlined, the start codon of S is in blue, the transcription regulatory sequences (XUA(A/G)AC; [4] ) are in orange and the Bam HI site is indicated in purple. (lower panel) The stop codon of the S gene is underlined, the start codon of the ORF3 gene is in blue, the start codon of E gene is in red, the transcription regulatory sequences are in orange and the unique Pml I and Eco RV sites are indicated in purple and green, respectively.

    Article Snippet: Capped runoff transcripts were synthesized from Pac I-linearized pPEDV, pPEDV-Rluc, pPEDV-ΔORF3, pPEDV-ΔORF3/Rluc, or pPEDV-ΔORF3/GFP, respectively, with a T7 RNA polymerase kit (Ambion) as specified by the manufacturer.

    Techniques: Plasmid Preparation, In Vitro

    A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.

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

    Article Title: Regulation of protein translation through mRNA structure influences MHC class I loading and T cell recognition

    doi: 10.1073/pnas.0801968105

    Figure Lengend Snippet: A codon-modified GAr sequence influences EBNA1 synthesis. ( A ) IVT assay of pcDNA3 expression constructs encoding EBNA1 (E1) (lane 1), E1ΔGA (lane 2), E1-GAr(100N) (lane 3), E1-GAr(100M) (lane 4), E1-GAr(200N) (lane 5), E1-GAr(200M) (lane 6), E1-GAr(300N) (lane 7), E1-GAr(300M) (lane 8), E1-GAr(400N) (lane 9), E1-GAr(400M) (lane 10), E1-GAr(500N) (lane 11), or E1-GAr(500M) (lane 12). The constructs were transcribed and translated in vitro with T7 RNA polymerase by using a coupled transcription/translation reticulocyte lysate system. 35 S-methionine-labeled proteins were visualized by autoradiography. ( B and C ) Band intensities from the IVT assay were quantified by densitometric analysis using Imagequant software (Molecular Dynamics) and graphed to demonstrate absolute intensities ( B ) or relative fold increase of EBNA1 encoded by codon-modified GAr domains compared with EBNA1 encoded by native GAr domains ( C ). ( D ) Western blot of EBV-negative HEK293 cells transfected with expression constructs encoding E1-GFP (lane 1), E1ΔGA-GFP (lane 2), E1-GAr(100N)-GFP (lane 3), E1-GAr(100M)-GFP (lane 4), E1-GAr(200N)-GFP (lane 5), E1-GAr(200M)-GFP (lane 6), E1-GAr(300N)-GFP (lane 7), E1-GAr(300M)-GFP (lane 8), E1-GAr(400N)-GFP (lane 9), E1-GAr(400M)-GFP (lane 10), E1-GAr(500N)-GFP (lane 11), or E1-GAr(500M)-GFP (lane 12) with a GFP antibody ( Upper ) or a monoclonal actin antibody ( Lower ). Molecular weight markers M r (kDa) are indicated on the left. ( E ) Band intensities after immunoblotting were quantified as described for B . Representative data from one of four experiments are presented here.

    Article Snippet: EBNA1/pcDNA3 expression constructs were linearized with XbaI and 1 μg of template transcribed with T7 RNA polymerase by using a Riboprobe in vitro transcription system (Promega) supplemented with 50 μCi [α-32 P]UTP (Amersham Biosciences).

    Techniques: Modification, Sequencing, Expressing, Construct, In Vitro, Labeling, Autoradiography, Software, Western Blot, Transfection, Molecular Weight

    AV reovirus possesses a truncated σ 1 protein. ( A ) WT and AV reovirus were purified from L929 murine fibroblasts and examined for the presence of σ 1. Through immunoblotting, membranes were probed with reovirus antiserum (left panel) and polyclonal reovirus σ 1 N-terminus antiserum (right panel). The N-terminal antibody was raised against σ 1 amino acids 1–158 ( Duncan and Lee, 1994 ). ( B ) Attenuated reovirus was collected from infected HT1080 and PI HTR1 supernatants by high-speed ultracentrifugation and examined by immunoblotting using reovirus antiserum (left panel) and polyclonal σ 1 N-terminus antiserum (right panel). Purified WT reovirus was used as a point of reference. ( C ) In vitro transcription of the AV reovirus S1 gene by T7 polymerase produced a σ 1 RNA transcript that was in vitro translated using a rabbit reticulocyte system. Translation of a luciferase transcript and a minus RNA reaction were used as positive and negative controls, respectively. The samples were analysed by 12% SDS–PAGE and subsequent autoradiography.

    Journal: British Journal of Cancer

    Article Title: Attenuated reovirus displays oncolysis with reduced host toxicity

    doi: 10.1038/sj.bjc.6606053

    Figure Lengend Snippet: AV reovirus possesses a truncated σ 1 protein. ( A ) WT and AV reovirus were purified from L929 murine fibroblasts and examined for the presence of σ 1. Through immunoblotting, membranes were probed with reovirus antiserum (left panel) and polyclonal reovirus σ 1 N-terminus antiserum (right panel). The N-terminal antibody was raised against σ 1 amino acids 1–158 ( Duncan and Lee, 1994 ). ( B ) Attenuated reovirus was collected from infected HT1080 and PI HTR1 supernatants by high-speed ultracentrifugation and examined by immunoblotting using reovirus antiserum (left panel) and polyclonal σ 1 N-terminus antiserum (right panel). Purified WT reovirus was used as a point of reference. ( C ) In vitro transcription of the AV reovirus S1 gene by T7 polymerase produced a σ 1 RNA transcript that was in vitro translated using a rabbit reticulocyte system. Translation of a luciferase transcript and a minus RNA reaction were used as positive and negative controls, respectively. The samples were analysed by 12% SDS–PAGE and subsequent autoradiography.

    Article Snippet: In vitro transcription was performed using a 50 μ l reaction containing 5 μ g of AV reovirus S1 template by the RiboMAX large-scale RNA production systems for T7 polymerase (Promega, Nepean, ON, Canada).

    Techniques: Purification, Infection, In Vitro, Produced, Luciferase, SDS Page, Autoradiography

    Fig. 1. Altered DNA mobility upon in vitro transcription of murine class switch sequences Sμ, Sγ3 and Sγ2b. ( A ) Diagram of switch sequences on plasmids showing the direction of physiological transcription. The bent arrows indicate the direction of transcription by either T3 or T7 RNA polymerase. ( B ) Supercoiled plasmid DNA containing either a 900 bp fragment of Sμ (pGD44), a 2.2 kb fragment of Sγ3 (pGD231), a 267 bp fragment of Sγ3 (pSG3-5), a 129 bp fragment of Sγ3 (pSG3-2), an 832 bp fragment of Sγ2b (pGD100) or a 564 bp Hin dIII fragment from λ phage (pTWEL5) was transcribed, treated with RNase A, run out on a 1% agarose gel and post-stained with ethidium bromide as described in Materials and methods. Lanes 1, 4, 7, 10, 13 and 16 are non-transcribed plasmids; lanes 2, 5, 8, 11, 14 and 17 are plasmids transcribed with T7 RNA polymerase; lanes 3, 6, 9, 12, 15 and 18 are plasmids transcribed with T7 RNA polymerase and treated with RNase H. A 1 kb ladder (Gibco-BRL) was used as a molecular weight marker (M). The positions of supercoiled (SC) and nicked circular (NC) forms of the plasmids are indicated. ( C ) Radioactive image of the gel shown in (B).

    Journal: The EMBO Journal

    Article Title: Transcription-dependent R-loop formation at mammalian class switch sequences

    doi: 10.1093/emboj/19.5.1055

    Figure Lengend Snippet: Fig. 1. Altered DNA mobility upon in vitro transcription of murine class switch sequences Sμ, Sγ3 and Sγ2b. ( A ) Diagram of switch sequences on plasmids showing the direction of physiological transcription. The bent arrows indicate the direction of transcription by either T3 or T7 RNA polymerase. ( B ) Supercoiled plasmid DNA containing either a 900 bp fragment of Sμ (pGD44), a 2.2 kb fragment of Sγ3 (pGD231), a 267 bp fragment of Sγ3 (pSG3-5), a 129 bp fragment of Sγ3 (pSG3-2), an 832 bp fragment of Sγ2b (pGD100) or a 564 bp Hin dIII fragment from λ phage (pTWEL5) was transcribed, treated with RNase A, run out on a 1% agarose gel and post-stained with ethidium bromide as described in Materials and methods. Lanes 1, 4, 7, 10, 13 and 16 are non-transcribed plasmids; lanes 2, 5, 8, 11, 14 and 17 are plasmids transcribed with T7 RNA polymerase; lanes 3, 6, 9, 12, 15 and 18 are plasmids transcribed with T7 RNA polymerase and treated with RNase H. A 1 kb ladder (Gibco-BRL) was used as a molecular weight marker (M). The positions of supercoiled (SC) and nicked circular (NC) forms of the plasmids are indicated. ( C ) Radioactive image of the gel shown in (B).

    Article Snippet: T3 and T7 RNA polymerases, and RNase H were purchased from Promega (Madison, WI).

    Techniques: In Vitro, Plasmid Preparation, Agarose Gel Electrophoresis, Staining, Molecular Weight, Marker

    Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

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

    Article Title: Inhibition of HPV-16 E6/E7 immortalization of normal keratinocytes by hairpin ribozymes

    doi:

    Figure Lengend Snippet: Cis-expression of R434 ribozyme inhibits HPV-16E6/E7 in vitro translation. ( A ) Map of HPV-16E6/E7 cis-expression constructs with R434 and R434i ribozymes. PCR-amplified fragments containing the entire HPV-16 E6/E7 genes (nucleotides 97–868) linked to R434 (pCR16E6/E7RZ) or R434i (pCR16E6/E7RZi) ribozymes were cloned in the pCR3.1 vector. The pCR16HH plasmid contains only the HPV-16E6/E7 genes. The relative positions of the Sty I sites used for cloning the ribozymes and the vector poly(A) signal are shown. ( B ) The protein products produced by plasmids pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi were examined by in vitro translation reactions using T7 RNA polymerase and rabbit reticulocyte lysates in the presence of [ 35 S]methionine. ←, the position of E6 and E7 proteins. Luc, luciferase protein reaction control.

    Article Snippet: HPV-16 E6/E7 proteins were produced using the T7 RNA polymerase promoter from pCR16HH, pCR16E6/E7RZ, and pCR16E6/E7RZi and using an in vitro transcription–translation system (rabbit reticulocyte TnT, Promega) and [35 S]methionine (Amersham).

    Techniques: Expressing, In Vitro, Construct, Polymerase Chain Reaction, Amplification, Clone Assay, Plasmid Preparation, Produced, Luciferase

    Linear DNA templates in cell-free protein synthesis based on CHO cell lysate. Linear IRES-luciferase and IRES-Mel-EPO templates tested during cell-free protein synthesis reactions in the presence of 14 C leucine. Different concentrations of linear DNA product and T7 RNA polymerase (Pol) were added to the individual reactions. Protein yield was quantified by hot TCA precipitation followed by scintillation measurement. Error bars show standard deviations calculated from triplicates.

    Journal: PLoS ONE

    Article Title: Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    doi: 10.1371/journal.pone.0163670

    Figure Lengend Snippet: Linear DNA templates in cell-free protein synthesis based on CHO cell lysate. Linear IRES-luciferase and IRES-Mel-EPO templates tested during cell-free protein synthesis reactions in the presence of 14 C leucine. Different concentrations of linear DNA product and T7 RNA polymerase (Pol) were added to the individual reactions. Protein yield was quantified by hot TCA precipitation followed by scintillation measurement. Error bars show standard deviations calculated from triplicates.

    Article Snippet: It was shown before that T7 RNA polymerase and other macromolecules are positively affected by molecular crowding, thereby influencing biochemical kinetics by volume exclusion effects [ ].

    Techniques: Luciferase, TCA Precipitation

    Influence of PEG on protein production in cell-free systems based on translationally active CHO lysate. Two concentrations (1%, 2%) of different PEG molecules (3350, 5000, 20000) were analyzed in cell-free protein synthesis reactions using pIX3.0-CRPV(GCT)-eYFP plasmid template. Translation reactions without the addition of PEG but with supplementation of 3 U/μl (increased concentration) and 1 U/μl (standard concentration) T7 RNA polymerase served as control reactions A . Fluorescence signals of synthesized eYFP proteins were detected by fluorescence imaging on μ-Ibidi slides using the Typhoon Trio Plus Imager. B . Quantification of fluorescence signals was accomplished by using Image Quant TL Array analysis software. Error bars show standard deviations calculated from triplicate analysis.

    Journal: PLoS ONE

    Article Title: Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    doi: 10.1371/journal.pone.0163670

    Figure Lengend Snippet: Influence of PEG on protein production in cell-free systems based on translationally active CHO lysate. Two concentrations (1%, 2%) of different PEG molecules (3350, 5000, 20000) were analyzed in cell-free protein synthesis reactions using pIX3.0-CRPV(GCT)-eYFP plasmid template. Translation reactions without the addition of PEG but with supplementation of 3 U/μl (increased concentration) and 1 U/μl (standard concentration) T7 RNA polymerase served as control reactions A . Fluorescence signals of synthesized eYFP proteins were detected by fluorescence imaging on μ-Ibidi slides using the Typhoon Trio Plus Imager. B . Quantification of fluorescence signals was accomplished by using Image Quant TL Array analysis software. Error bars show standard deviations calculated from triplicate analysis.

    Article Snippet: It was shown before that T7 RNA polymerase and other macromolecules are positively affected by molecular crowding, thereby influencing biochemical kinetics by volume exclusion effects [ ].

    Techniques: Plasmid Preparation, Concentration Assay, Fluorescence, Synthesized, Imaging, Software

    Enrichment of membrane proteins in microsomal fractions of CHO lysate using a repetitive vesicle addressing procedure. During the first synthesis step (Cycle 1) Mel-EPO and OPMR1 protein was produced according to the optimized conditions (3 U/μl T7 RNA polymerase). After completing the cell-free reaction, translation mixture was separated into microsomal fraction and supernatant by centrifugation (15 min, 4°C and 16000xg) in a standard table top centrifuge. Supernatant was removed and microsomal fraction was resuspended using freshly prepared translation mixture containing CHO cell lysate without microsomal structures to initiate the second cell-free translation cycle (Cycle 2). This procedure was repeated again, after finishing the second translation step to obtain a third step of addressing the microsomal fraction (Cycle 3). Samples of translation mixture (TM), supernatant (S) and microsomal fraction (MF) were collected after each cycle for further analysis. Protein yields were quantified by hot TCA precipitation of 14 C leucine labeled cell-free produced proteins followed by scintillation measurement. Error bars show standard deviations calculated from triplicate analysis. Molecular weight and modifications of proteins were visualized by SDS-PAGE separation and autoradiography.

    Journal: PLoS ONE

    Article Title: Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    doi: 10.1371/journal.pone.0163670

    Figure Lengend Snippet: Enrichment of membrane proteins in microsomal fractions of CHO lysate using a repetitive vesicle addressing procedure. During the first synthesis step (Cycle 1) Mel-EPO and OPMR1 protein was produced according to the optimized conditions (3 U/μl T7 RNA polymerase). After completing the cell-free reaction, translation mixture was separated into microsomal fraction and supernatant by centrifugation (15 min, 4°C and 16000xg) in a standard table top centrifuge. Supernatant was removed and microsomal fraction was resuspended using freshly prepared translation mixture containing CHO cell lysate without microsomal structures to initiate the second cell-free translation cycle (Cycle 2). This procedure was repeated again, after finishing the second translation step to obtain a third step of addressing the microsomal fraction (Cycle 3). Samples of translation mixture (TM), supernatant (S) and microsomal fraction (MF) were collected after each cycle for further analysis. Protein yields were quantified by hot TCA precipitation of 14 C leucine labeled cell-free produced proteins followed by scintillation measurement. Error bars show standard deviations calculated from triplicate analysis. Molecular weight and modifications of proteins were visualized by SDS-PAGE separation and autoradiography.

    Article Snippet: It was shown before that T7 RNA polymerase and other macromolecules are positively affected by molecular crowding, thereby influencing biochemical kinetics by volume exclusion effects [ ].

    Techniques: Produced, Centrifugation, TCA Precipitation, Labeling, Molecular Weight, SDS Page, Autoradiography

    Evaluation of plasmid and T7 RNA polymerase concentration applied for CHO lysate based cell-free synthesis. A. Analysis of plasmid concentration of pIX3.0-EMCV-Luc and pT7CFE1-Luc used for cell-free protein synthesis. Protein yields of active luciferase were determined by standard luciferase assay B. Dependence of protein yield on plasmid and T7 RNA polymerase concentration during cell-free synthesis based on the template pcDNA3.1-CRPV(GCT)-Luc. Protein yields of de novo synthesized luciferase were detected and calculated by quantification of 14 C leucine labeled proteins and scintillation measurement. Luciferase assay was used for investigation of the amount of functional protein. Error bars represent standard deviations calculated from triplicate analysis. C Investigation of protein yield using pIX2.0-CRPV(GCT)-Mel-EPO DNA template. Variation of template DNA and T7 RNA polymerase concentration. Radio labeled proteins were analyzed by TCA precipitation followed by scintillation measurement (upper part C). Additionally, de novo synthesized proteins were visualized by autoradiography (lower part C).

    Journal: PLoS ONE

    Article Title: Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of “Difficult-to-Express” Proteins and Future Perspectives

    doi: 10.1371/journal.pone.0163670

    Figure Lengend Snippet: Evaluation of plasmid and T7 RNA polymerase concentration applied for CHO lysate based cell-free synthesis. A. Analysis of plasmid concentration of pIX3.0-EMCV-Luc and pT7CFE1-Luc used for cell-free protein synthesis. Protein yields of active luciferase were determined by standard luciferase assay B. Dependence of protein yield on plasmid and T7 RNA polymerase concentration during cell-free synthesis based on the template pcDNA3.1-CRPV(GCT)-Luc. Protein yields of de novo synthesized luciferase were detected and calculated by quantification of 14 C leucine labeled proteins and scintillation measurement. Luciferase assay was used for investigation of the amount of functional protein. Error bars represent standard deviations calculated from triplicate analysis. C Investigation of protein yield using pIX2.0-CRPV(GCT)-Mel-EPO DNA template. Variation of template DNA and T7 RNA polymerase concentration. Radio labeled proteins were analyzed by TCA precipitation followed by scintillation measurement (upper part C). Additionally, de novo synthesized proteins were visualized by autoradiography (lower part C).

    Article Snippet: It was shown before that T7 RNA polymerase and other macromolecules are positively affected by molecular crowding, thereby influencing biochemical kinetics by volume exclusion effects [ ].

    Techniques: Plasmid Preparation, Concentration Assay, Luciferase, Synthesized, Labeling, Functional Assay, TCA Precipitation, Autoradiography

    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

    Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    Journal: Scientific Reports

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

    doi: 10.1038/srep10404

    Figure Lengend Snippet: Schematic representation of the transcription- and translation-coupled DNA replication system. Circular DNA encoding phi29 DNA polymerase under control of the T7 promoter is incubated with the reconstituted translation system including T7 RNA polymerase. mRNA is transcribed from the DNA, and phi29 DNA polymerase is translated. The polymerase attaches to the circular DNA and initiates the polymerization of a long single-stranded RNA in a rolling-circle manner. The polymerase further synthesizes the complementary strand to produce double-stranded DNA, which is a long repeat of the circular DNA sequence. The next round of transcription and translation occurs from the double-stranded DNA.

    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, Sequencing

    Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by T7 RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.

    Journal: Nucleic Acids Research

    Article Title: Identification and characterization of two forms of mouse MUTYH proteins encoded by alternatively spliced transcripts

    doi: 10.1093/nar/gkh214

    Figure Lengend Snippet: Two forms of mMUTYH protein encoded by the alternatively spliced transcripts. ( A ) In vitro translation of type b and type c Mutyh mRNAs. RNAs synthesized from pT7Blue plasmids carrying type b and type c Mutyh cDNA or human MUTYH ) by T7 RNA polymerase were translated using rabbit reticulocyte lysate, and translation products were subjected to a western blot analysis with anti-hMUTYH antibody. No template, during in vitro translation, template RNA was omitted. An arrow indicates 50 kDa mMUTYHα encoded by type b mRNA, and an arrowhead indicates the 47 kDa mMUTYHβ encoded by type c mRNA, respectively. ( B ) The detection of two forms of MUTYH in mouse ES cells. Whole cell extracts prepared from wild-type ES cell line, CCE28 cells, MUTYH-null YDK15 cells, and YDKα or YDKβ cells to which an expression construct for type b or type c cDNA was stably introduced, respectively, were subjected to western blot analysis with anti-hMUTYH antibody. An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. ( C ) The detection of two forms of mMUTYH protein in mouse thymocytes. Thymocyte extracts prepared from two independent wild-type (lanes 1, 2) and MUTYH-null mice ( Mutyh –/– ; lanes 3, 4) were subjected to western blot analysis with anti-hMUTYH antibody (top panel), or with anti-mMUTYHβN (middle panel). An arrow indicates mMUTYHα and an arrowhead indicates mMUTYHβ, respectively. Stained filter with Coomassie Brilliant Blue is shown (bottom) for loading control.

    Article Snippet: The transcripts were synthesized from BamHI linearized plasmids by T7 RNA polymerase at 30°C for 15 min using Single Tube Protein System 2 (Novagen).

    Techniques: In Vitro, Synthesized, Western Blot, Expressing, Construct, Stable Transfection, Mouse Assay, Staining

    The mutations 3/MA, 4A/A48-T, and 5B/DG, which are critical for efficient NS2-3-independent formation of CSFV, affect neither polyprotein processing nor RNA replication. (A) (Top) Scheme of the bicistronic reporter replicon CSFV Bici RLuc IRES-Ubi-NS3-3′, coding for N pro and Renilla luciferase in the first ORF and the viral replicase proteins NS3-5B in the second ORF. Ubiquitin (Ubi) was inserted upstream of NS3 to generate the authentic N terminus of NS3. Full-length replicon RNA can be generated by in vitro transcription with SP6 RNA polymerase (SP6 promoter [gray arrow]). Since a T7 promoter (black arrow) was inserted upstream of the second ORF, it could be transcribed (dashed line) using T7 RNA polymerase (T7 pol ) to analyze polyprotein processing in a replication-independent manner. (Bottom) Schematic drawing of the experimental setup. WB, Western blotting. (B) Huh7-T7 cells were infected with modified vaccinia virus Ankara (MVA) encoding T7 pol to increase the T7 pol amounts. At 1 h postinfection, cells were transfected with plasmid DNA of the indicated pCSFV Bici RLuc IRES-Ubi-NS3-3′ variants, followed by T7-mediated expression for 18 h. Subsequently, the cells were lysed in sample buffer and protein samples were separated by SDS-PAGE followed by Western blot analyses. Proteins were visualized using the indicated protein-specific primary antibodies. Mock, untransfected cells (negative control); 3/S163-A, inactivated NS3 protease (negative control for polyprotein processing); 5B/GAA, RNA replication-deficient mutant (negative control for RNA replication); WT, wild type. The mutations 3/MA, 4A/A48-T, and 5B/DG are indicated. Western blots of one representative experiment are depicted. Molecular mass markers (in kilodaltons [kDa]) are indicated on the left. Protein products are depicted on the right. (C) (Top) Scheme of CSFV RLuc IRES-Ubi-NS3-3′ highlighting the SP6 RNA polymerase promoter and the respective RNA transcript (dashed line) that was used to generate full-length replicon RNA. (Bottom) Experimental setup used to analyze RNA replication efficiencies. (D) SK6 cells were electroporated with 1 μg of wild-type (WT) or the indicated mutated replicon RNA. At given time points p.e., cells were harvested, followed by lysis and determination of luciferase activities as relative light units (RLUs). Mean values and standard deviations from three experiments are depicted.

    Journal: Journal of Virology

    Article Title: Determination of Critical Requirements for Classical Swine Fever Virus NS2-3-Independent Virion Formation

    doi: 10.1128/JVI.00679-19

    Figure Lengend Snippet: The mutations 3/MA, 4A/A48-T, and 5B/DG, which are critical for efficient NS2-3-independent formation of CSFV, affect neither polyprotein processing nor RNA replication. (A) (Top) Scheme of the bicistronic reporter replicon CSFV Bici RLuc IRES-Ubi-NS3-3′, coding for N pro and Renilla luciferase in the first ORF and the viral replicase proteins NS3-5B in the second ORF. Ubiquitin (Ubi) was inserted upstream of NS3 to generate the authentic N terminus of NS3. Full-length replicon RNA can be generated by in vitro transcription with SP6 RNA polymerase (SP6 promoter [gray arrow]). Since a T7 promoter (black arrow) was inserted upstream of the second ORF, it could be transcribed (dashed line) using T7 RNA polymerase (T7 pol ) to analyze polyprotein processing in a replication-independent manner. (Bottom) Schematic drawing of the experimental setup. WB, Western blotting. (B) Huh7-T7 cells were infected with modified vaccinia virus Ankara (MVA) encoding T7 pol to increase the T7 pol amounts. At 1 h postinfection, cells were transfected with plasmid DNA of the indicated pCSFV Bici RLuc IRES-Ubi-NS3-3′ variants, followed by T7-mediated expression for 18 h. Subsequently, the cells were lysed in sample buffer and protein samples were separated by SDS-PAGE followed by Western blot analyses. Proteins were visualized using the indicated protein-specific primary antibodies. Mock, untransfected cells (negative control); 3/S163-A, inactivated NS3 protease (negative control for polyprotein processing); 5B/GAA, RNA replication-deficient mutant (negative control for RNA replication); WT, wild type. The mutations 3/MA, 4A/A48-T, and 5B/DG are indicated. Western blots of one representative experiment are depicted. Molecular mass markers (in kilodaltons [kDa]) are indicated on the left. Protein products are depicted on the right. (C) (Top) Scheme of CSFV RLuc IRES-Ubi-NS3-3′ highlighting the SP6 RNA polymerase promoter and the respective RNA transcript (dashed line) that was used to generate full-length replicon RNA. (Bottom) Experimental setup used to analyze RNA replication efficiencies. (D) SK6 cells were electroporated with 1 μg of wild-type (WT) or the indicated mutated replicon RNA. At given time points p.e., cells were harvested, followed by lysis and determination of luciferase activities as relative light units (RLUs). Mean values and standard deviations from three experiments are depicted.

    Article Snippet: The plasmid pCITE-2a, containing the internal ribosomal entry site (IRES) of encephalomyocarditis virus (EMCV) downstream of the T7 RNA polymerase promoter, was obtained from Novagen (Madison, WI, USA).

    Techniques: Luciferase, Generated, In Vitro, Western Blot, Infection, Modification, Transfection, Plasmid Preparation, Expressing, SDS Page, Negative Control, Mutagenesis, Lysis

    Construction of artificial defective RNAs of MBGV. The minigenomes were inserted in transcription vector 2,0 (gray) between the T7 RNA polymerase promoter and the hepatitis delta virus ribozyme (hatched). For in vitro transcription, plasmids were linearized by using the Sal I restriction site (right side). (A) Diagram of negative-sense minigenomic cDNA 215, consisting of 439 nt of the 5′ trailer (white) adjacent to the T7 RNA polymerase promoter, 668 nt of the CAT gene in a negative-sense orientation (black), and 106 nt of the 3′ leader (white) adjacent to the ribozyme. Above the scheme are indicated the boundary between the T7 RNA polymerase promoter sequence (underlined) and the 5′ end of the minigenome (negative-sense orientation) (left side) and the boundary between the ribozyme sequence (underlined) and the 3′ end of the minigenome (right side). The CAT gene is flanked by Not I and Nde I restriction sites. (B) Diagram of positive-sense minigenomic cDNA 2.1-CAT, consisting of 106 nt of the 3′ leader (white) adjacent to the T7 RNA polymerase promoter, the CAT gene in a positive-sense orientation (black), and 439 nt of the 5′ trailer adjacent to the ribozyme sequence (white). The boundaries between the T7 RNA polymerase promoter and the ribozyme sequence (underlined), respectively, and the MBGV-specific sequences are indicated. MBGV-specific sequences are shown in the plus-strand orientation. The CAT gene is flanked by Not I restriction sites. (C) Diagram of the cDNA coding for the copy-back-type negative-stranded minigenome cb-CAT. The minigenome consists of 439 nt of the 5′ trailer (white) adjacent to the T7 RNA polymerase promoter, the CAT gene in a negative-sense orientation (black), and, adjacent to the ribozyme sequence, 105 nt complementary to the last 105 nt of the trailer (designated as c-trailer; white), which serves as the leader region. The boundaries between the T7 RNA polymerase promoter and the ribozyme sequence (underlined), respectively, and the MBGV-specific sequences are indicated. MBGV-specific sequences are shown in the negative-sense orientation. The CAT gene is flanked by Not I and Nde ). Transcription start and stop sites are indicated only for negative-stranded minigenomes. The cleavage site of the ribozyme is symbolized by a pair of scissors.

    Journal: Journal of Virology

    Article Title: Three of the Four Nucleocapsid Proteins of Marburg Virus, NP, VP35, and L, Are Sufficient To Mediate Replication and Transcription of Marburg Virus-Specific Monocistronic Minigenomes

    doi:

    Figure Lengend Snippet: Construction of artificial defective RNAs of MBGV. The minigenomes were inserted in transcription vector 2,0 (gray) between the T7 RNA polymerase promoter and the hepatitis delta virus ribozyme (hatched). For in vitro transcription, plasmids were linearized by using the Sal I restriction site (right side). (A) Diagram of negative-sense minigenomic cDNA 215, consisting of 439 nt of the 5′ trailer (white) adjacent to the T7 RNA polymerase promoter, 668 nt of the CAT gene in a negative-sense orientation (black), and 106 nt of the 3′ leader (white) adjacent to the ribozyme. Above the scheme are indicated the boundary between the T7 RNA polymerase promoter sequence (underlined) and the 5′ end of the minigenome (negative-sense orientation) (left side) and the boundary between the ribozyme sequence (underlined) and the 3′ end of the minigenome (right side). The CAT gene is flanked by Not I and Nde I restriction sites. (B) Diagram of positive-sense minigenomic cDNA 2.1-CAT, consisting of 106 nt of the 3′ leader (white) adjacent to the T7 RNA polymerase promoter, the CAT gene in a positive-sense orientation (black), and 439 nt of the 5′ trailer adjacent to the ribozyme sequence (white). The boundaries between the T7 RNA polymerase promoter and the ribozyme sequence (underlined), respectively, and the MBGV-specific sequences are indicated. MBGV-specific sequences are shown in the plus-strand orientation. The CAT gene is flanked by Not I restriction sites. (C) Diagram of the cDNA coding for the copy-back-type negative-stranded minigenome cb-CAT. The minigenome consists of 439 nt of the 5′ trailer (white) adjacent to the T7 RNA polymerase promoter, the CAT gene in a negative-sense orientation (black), and, adjacent to the ribozyme sequence, 105 nt complementary to the last 105 nt of the trailer (designated as c-trailer; white), which serves as the leader region. The boundaries between the T7 RNA polymerase promoter and the ribozyme sequence (underlined), respectively, and the MBGV-specific sequences are indicated. MBGV-specific sequences are shown in the negative-sense orientation. The CAT gene is flanked by Not I and Nde ). Transcription start and stop sites are indicated only for negative-stranded minigenomes. The cleavage site of the ribozyme is symbolized by a pair of scissors.

    Article Snippet: For preparation of positive-stranded riboprobes, 1 μg of the positive-sense minigenomic DNA 2.1-CAT was digested with Hin dIII and transcribed in vitro, using T7 RNA polymerase and the Dig RNA Labeling Kit (Boehringer Mannheim).

    Techniques: Plasmid Preparation, In Vitro, Sequencing

    Identification of Proteins Able to Bind GAA Exon 2 by Pull-Down and Western Blot Analysis (A) Schematic representation of GAA 147–286, 357–500, and 427–572 exonic regions used as templates for T7 RNA transcription in vitro. Putative silencers are represented by red boxes, while the putative enhancer is in green. (B) Pull-down analysis of three in vitro transcribed RNAs with HeLa nuclear extract (NE) analyzed on SDS-10% polyacrylamide gels and visualized by colloidal Coomassie staining. Proteins differentially precipitated by each RNA (letters a–e) were excised from the gel and analyzed by mass spectrometry. Colloidal Coomassie gel staining gives a representative picture of three independent experiments. Beads alone were used as a control (Beads). (C) Western blot analysis, after pull-down assay, using specific antibodies against hnRNPA1/A2, hnRNPH, hnRNPQ, and hnRNPR confirmed the identity of each protein. The nuclear extract sample corresponds to 1/20th of the total amount of protein used for the pull-down assay. The western blot images are representative pictures of three independent experiments. Beads alone were used as a control.

    Journal: Molecular Therapy

    Article Title: Glycogen Reduction in Myotubes of Late-Onset Pompe Disease Patients Using Antisense Technology

    doi: 10.1016/j.ymthe.2017.05.019

    Figure Lengend Snippet: Identification of Proteins Able to Bind GAA Exon 2 by Pull-Down and Western Blot Analysis (A) Schematic representation of GAA 147–286, 357–500, and 427–572 exonic regions used as templates for T7 RNA transcription in vitro. Putative silencers are represented by red boxes, while the putative enhancer is in green. (B) Pull-down analysis of three in vitro transcribed RNAs with HeLa nuclear extract (NE) analyzed on SDS-10% polyacrylamide gels and visualized by colloidal Coomassie staining. Proteins differentially precipitated by each RNA (letters a–e) were excised from the gel and analyzed by mass spectrometry. Colloidal Coomassie gel staining gives a representative picture of three independent experiments. Beads alone were used as a control (Beads). (C) Western blot analysis, after pull-down assay, using specific antibodies against hnRNPA1/A2, hnRNPH, hnRNPQ, and hnRNPR confirmed the identity of each protein. The nuclear extract sample corresponds to 1/20th of the total amount of protein used for the pull-down assay. The western blot images are representative pictures of three independent experiments. Beads alone were used as a control.

    Article Snippet: The PCR products were then used for generating the pull-down target RNAs by T7 RNA polymerase (Agilent) transcription in vitro, according to the manufacturer’s instructions.

    Techniques: Western Blot, In Vitro, Staining, Mass Spectrometry, Pull Down Assay

    Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.

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

    Article Title: Single-molecule FRET studies on the cotranscriptional folding of a thiamine pyrophosphate riboswitch

    doi: 10.1073/pnas.1712983115

    Figure Lengend Snippet: Single-molecule fluorescence cotranscriptional folding assay. ( A ) Model for TPP-induced structural transition of the E. coli ThiM riboswitch. The fluorophore-labeling positions for single-molecule studies are indicated by green and red boxes (green for Cy3 or Dy547, red for Dy647). ( B ) Experimental scheme. Dy647-labeled seed RNA was incubated with a template DNA strand and phage T7 RNAP in a tube for 50 min at 37 °C. To assemble a full EC, Cy3-labeled UTP and a nontemplate DNA strand were added to the tube and incubated for 20 min. ECs were immobilized on a polymer-passivated quartz surface using streptavidin–biotin interactions. Elongation was resumed by injecting NTP, while RNA folding was observed using a single-molecule FRET microscope. ( C ) Single-molecule fluorescence images of EC ( Top ) and control (ctrl) images of nonspecifically bound Cy3-UTP ( Bottom ). The colocalized Cy3 and Cy5 spots are enclosed by circles. The percentage of acceptor spots colocalized with donor spots was estimated as 57 ± 14% from 10 measurements.

    Article Snippet: The seed RNA (800 nM) was incubated in a tube with a template DNA strand (200 nM) and T7 RNAP (40 nM; New England Biolabs) for 50 min at 37 °C.

    Techniques: Fluorescence, Labeling, Incubation, Microscopy

    Specificity of recognition of cap structures by Flu polymerases. (A) Analysis of 5′-terminal cap structures of RNAs. T7 RNA polymerase-synthesized RNAs were treated with nuclease P 1 and analyzed by TLC (PEI-CEL, 0.65 M LiCl), and radioactive nucleotides were detected by autoradiography. (B and C) In vitro capped RNA cleavage (B) and RNA elongation (C) reactions were performed with 600 ng of FluA (lanes 2, 5, and 8) or FluB (lanes 3, 6, and 9) vRNP using 2 fmol of variously methylated capped RNAs (m 7 GpppGm-RNA, lanes 1 to 3; m 7 GpppG-RNA, lanes 4 to 6; GpppG-RNA, lanes 7 to 9). RNA products were analyzed by 15% PAGE containing 8 M urea. The input capped RNAs (33 nt), the cleaved capped RNA products, and the elongated products are indicated as a closed triangle, open triangles, and a black bar, respectively, at the right. (D and E) Ratios of cleaved RNA products (D) and RNA transcripts (E) to total input primer RNAs. (F) Cap-binding activity for variously methylated capped RNAs. UV cross-linking was performed using 50, 100, and 200 ng of FluA (upper panel) and FluB (lower panel) vRNP and 50 fmol of variously methylated capped RNAs (GpppG-RNA, lanes 1 to 3; m 7 GpppG-RNA, lanes 4 to 6; m 7 GpppGm-RNA, lanes 7 to 9).

    Journal: Journal of Virology

    Article Title: Recognition of Cap Structure by Influenza B Virus RNA Polymerase Is Less Dependent on the Methyl Residue than Recognition by Influenza A Virus Polymerase ▿Recognition of Cap Structure by Influenza B Virus RNA Polymerase Is Less Dependent on the Methyl Residue than Recognition by Influenza A Virus Polymerase ▿ †

    doi: 10.1128/JVI.02375-10

    Figure Lengend Snippet: Specificity of recognition of cap structures by Flu polymerases. (A) Analysis of 5′-terminal cap structures of RNAs. T7 RNA polymerase-synthesized RNAs were treated with nuclease P 1 and analyzed by TLC (PEI-CEL, 0.65 M LiCl), and radioactive nucleotides were detected by autoradiography. (B and C) In vitro capped RNA cleavage (B) and RNA elongation (C) reactions were performed with 600 ng of FluA (lanes 2, 5, and 8) or FluB (lanes 3, 6, and 9) vRNP using 2 fmol of variously methylated capped RNAs (m 7 GpppGm-RNA, lanes 1 to 3; m 7 GpppG-RNA, lanes 4 to 6; GpppG-RNA, lanes 7 to 9). RNA products were analyzed by 15% PAGE containing 8 M urea. The input capped RNAs (33 nt), the cleaved capped RNA products, and the elongated products are indicated as a closed triangle, open triangles, and a black bar, respectively, at the right. (D and E) Ratios of cleaved RNA products (D) and RNA transcripts (E) to total input primer RNAs. (F) Cap-binding activity for variously methylated capped RNAs. UV cross-linking was performed using 50, 100, and 200 ng of FluA (upper panel) and FluB (lower panel) vRNP and 50 fmol of variously methylated capped RNAs (GpppG-RNA, lanes 1 to 3; m 7 GpppG-RNA, lanes 4 to 6; m 7 GpppGm-RNA, lanes 7 to 9).

    Article Snippet: Triphosphate-ended RNA with the 33-nucleotide sequence 5′-GAAAAAAAAAAAAAAAAAAAAAAAAAAAAUAAA-3′, designated pppG-RNA, was synthesized by using T7 RNA polymerase (Amersham Biosciences) and a synthetic DNA template.

    Techniques: Synthesized, Thin Layer Chromatography, Autoradiography, In Vitro, Methylation, Polyacrylamide Gel Electrophoresis, Binding Assay, Activity Assay

    RNA transcription reaction of acetaldehyde-treated plasmids. a In the absence of DNA damage, the T7 RNA polymerase generates RNA transcripts from DNA templates. After purifying RNA, real-time reverse transcription-PCR (qRT-PCR) is performed, and the PCR products are analyzed. If acetaldehyde damages DNA, the resulting lesions inhibit RNA synthesis, as T7 RNA polymerase cannot synthesize transcripts from damaged templates, and qRT-PCR products will not be detected. Amplification plot of qRT-PCR analysis of RNA transcripts of UV-irradiated ( b ) or of acetaldehyde (AA)-treated ( c ) DNA templates. UV-irradiated ( d ) or acetaldehyde-treated ( e ) pBSII was incubated with T7 RNA polymerase, and transcription was quantified by qRT-PCR

    Journal: Genes and Environment

    Article Title: Effects of acetaldehyde-induced DNA lesions on DNA metabolism

    doi: 10.1186/s41021-019-0142-7

    Figure Lengend Snippet: RNA transcription reaction of acetaldehyde-treated plasmids. a In the absence of DNA damage, the T7 RNA polymerase generates RNA transcripts from DNA templates. After purifying RNA, real-time reverse transcription-PCR (qRT-PCR) is performed, and the PCR products are analyzed. If acetaldehyde damages DNA, the resulting lesions inhibit RNA synthesis, as T7 RNA polymerase cannot synthesize transcripts from damaged templates, and qRT-PCR products will not be detected. Amplification plot of qRT-PCR analysis of RNA transcripts of UV-irradiated ( b ) or of acetaldehyde (AA)-treated ( c ) DNA templates. UV-irradiated ( d ) or acetaldehyde-treated ( e ) pBSII was incubated with T7 RNA polymerase, and transcription was quantified by qRT-PCR

    Article Snippet: T7 RNA polymerase and reverse transcriptase were from TOYOBO (Osaka, Japan).

    Techniques: Polymerase Chain Reaction, Quantitative RT-PCR, Amplification, Irradiation, Incubation