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 3044 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher maxiscript t7rna polymerase kit
    The C ontinuous in vitro E volution (CE) cycle. (A) Schematic of the CE cycle, which consists of three consecutive enzymatic steps of amplification [12] . Each enzymatic step constitutes a component of fitness. The first enzymatic reaction is the ligation performed by the ligase ribozyme itself. During this step the ligase catalyzes the ligation of an exogenous substrate to its 5′-end to form a ligase-substrate complex. The second step is the reverse transcription performed by the reverse transcriptase (RT). During this step, cDNA copies are made of both the ligases and ligase-substrate complexes. The third enzymatic step is forward transcription performed by the <t>T7</t> RNA polymerase (RNAP). Only the ligase-substrate complexes carry the promoter necessary for recognition and transcription by RNAP; cDNA copies of ligases (uncatalyzed) alone are not recognized and represent the end of their lineage (analog to death). (B) Secondary structure representation of the B16–19 genotype, the wild-type ligase used to seed all the evolution cycles, bound to the exogenous substrate (blue, lower case) at its 5′-end. Notice that the promoter for the T7 RNAP is located in the exogenous substrate. Mutations that emerged during the evolution cycles are shown with arrows. The unpaired 3′-end of the ligase has a mutational hotspot where most of the mutations occurred. The mutations that occurred in this region are noted with the name of the mutant ligase genotype that carries them. The U62→A mutation, present in many of the genotypes in this study, is also indicated.
    Maxiscript T7rna Polymerase Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore t7rna 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.
    T7rna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t 7 rna polymerase
    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 <t>T7</t> 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.
    T 7 Rna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 115 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 879 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega t7 rna polymerase
    RNase T 1 interferes with R-loop formation on a linearized switch substrate containing four repeats of murine Sγ3. The substrate, pDR3, was linearized with ApaL1 and then transcribed with <t>T7</t> RNA polymerase in the presence of [α- 32 P]UTP.
    T7 Rna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 8591 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher t7 rna polymerase
    Fig. 3. Effects of K86R mutant band IV protein expression. ( A ) Adenylylation assay of extracts from E.coli expressing wild-type and mutant band IV proteins (arrow); some degradation products are also seen. An adenylylatable E.coli protein (asterisk) serves as an internal control. ( B ) piLigIV-K86R drives expression in trypanosomes of the K86R mutant band IV protein and a linked phleomycin-resistance gene (Phleo R , the ble gene from Streptoalloteichus hindustanus ) from a T7 promoter with a tet operator and uses trypanosome 5′ and 3′ processing signals. It is transfected into 29-13 procyclic trypanosomes, which express <t>T7</t> RNA polymerase and the tet repressor. ( C ) Adenylylation assay of mitochondrial extract made from non-induced (lanes 1 and 3) and induced (lanes 2 and 4) K86R cells. The assays in lanes 1 and 2 used unfractionated extract and those in lanes 3 and 4 used the ∼20S peak from glycerol gradient fractionated extract. ( D ) Growth of two lines of K86R cells and control cells (the 29-13 parental line) with none or 1 µg/ml tet added at 0 h. Media was added to maintain cell densities supporting log phase growth. This semi-log plot is based on total cell numbers (cells/ml × volume), scaled to equalize minor differences in starting cell numbers. Error bars denote standard deviation for K86R-1 cells and are similar for the other cell lines. After much longer periods of induction (selection), the cultures can be overtaken by normal looking cells, possibly due to loss or inactivation of the ectopic gene. ( E ) Representative morphologies of K86R cells non-induced (first panel) and induced with tet (subsequent three panels), DAPI-stained and photographed 66 h post-induction at 100× magnification.
    T7 Rna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9704 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs t7 rna polymerase system
    A) Electrophoresis (8%) PAGE gel of the expression and purification of <t>His-LAR</t> <t>T7</t> RNA polymerase. Lanes 1) Induced insoluble fraction, 2) Induced soluble fraction, 3) Uninduced whole cell lysate, 4) Molecular weight marker, 5) Molecular weight marker,
    T7 Rna Polymerase System, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Jena Bioscience t7 rna polymerase
    A) Electrophoresis (8%) PAGE gel of the expression and purification of <t>His-LAR</t> <t>T7</t> RNA polymerase. Lanes 1) Induced insoluble fraction, 2) Induced soluble fraction, 3) Uninduced whole cell lysate, 4) Molecular weight marker, 5) Molecular weight marker,
    T7 Rna Polymerase, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 13 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: The in vitro transcription reaction was carried out with 10 μg of linearized plasmid DNA in a total volume of 100 μL containing 20 μL 5× RRL buffer (400 mM HEPES (pH 7.5), 60 mM MgCl2 , 10 mM spermidine and 200 mM DTT), NTP-Mix (3.125 mM ATP, CTP and UTP and 1.56 mM GTP), 1 U/μL RNasin (Promega, Madison, WI, USA), 2 U/μL T7 RNA polymerase (New England Biolabs) and 1 mM anti-reverse cap analogue (ARCA; 3′-O-Me- m7G(5′)ppp(5′)G; New England Biolabs).

    Techniques: Clone Assay, Construct, Agarose Gel Electrophoresis

    The C ontinuous in vitro E volution (CE) cycle. (A) Schematic of the CE cycle, which consists of three consecutive enzymatic steps of amplification [12] . Each enzymatic step constitutes a component of fitness. The first enzymatic reaction is the ligation performed by the ligase ribozyme itself. During this step the ligase catalyzes the ligation of an exogenous substrate to its 5′-end to form a ligase-substrate complex. The second step is the reverse transcription performed by the reverse transcriptase (RT). During this step, cDNA copies are made of both the ligases and ligase-substrate complexes. The third enzymatic step is forward transcription performed by the T7 RNA polymerase (RNAP). Only the ligase-substrate complexes carry the promoter necessary for recognition and transcription by RNAP; cDNA copies of ligases (uncatalyzed) alone are not recognized and represent the end of their lineage (analog to death). (B) Secondary structure representation of the B16–19 genotype, the wild-type ligase used to seed all the evolution cycles, bound to the exogenous substrate (blue, lower case) at its 5′-end. Notice that the promoter for the T7 RNAP is located in the exogenous substrate. Mutations that emerged during the evolution cycles are shown with arrows. The unpaired 3′-end of the ligase has a mutational hotspot where most of the mutations occurred. The mutations that occurred in this region are noted with the name of the mutant ligase genotype that carries them. The U62→A mutation, present in many of the genotypes in this study, is also indicated.

    Journal: PLoS ONE

    Article Title: Partitioning the Fitness Components of RNA Populations Evolving In Vitro

    doi: 10.1371/journal.pone.0084454

    Figure Lengend Snippet: The C ontinuous in vitro E volution (CE) cycle. (A) Schematic of the CE cycle, which consists of three consecutive enzymatic steps of amplification [12] . Each enzymatic step constitutes a component of fitness. The first enzymatic reaction is the ligation performed by the ligase ribozyme itself. During this step the ligase catalyzes the ligation of an exogenous substrate to its 5′-end to form a ligase-substrate complex. The second step is the reverse transcription performed by the reverse transcriptase (RT). During this step, cDNA copies are made of both the ligases and ligase-substrate complexes. The third enzymatic step is forward transcription performed by the T7 RNA polymerase (RNAP). Only the ligase-substrate complexes carry the promoter necessary for recognition and transcription by RNAP; cDNA copies of ligases (uncatalyzed) alone are not recognized and represent the end of their lineage (analog to death). (B) Secondary structure representation of the B16–19 genotype, the wild-type ligase used to seed all the evolution cycles, bound to the exogenous substrate (blue, lower case) at its 5′-end. Notice that the promoter for the T7 RNAP is located in the exogenous substrate. Mutations that emerged during the evolution cycles are shown with arrows. The unpaired 3′-end of the ligase has a mutational hotspot where most of the mutations occurred. The mutations that occurred in this region are noted with the name of the mutant ligase genotype that carries them. The U62→A mutation, present in many of the genotypes in this study, is also indicated.

    Article Snippet: Once the timer reached minute 24, 2.4 U/µL of enzyme T7 RNA polymerase (Ambion) were added to the reaction vessel.

    Techniques: In Vitro, Amplification, Ligation, Mutagenesis

    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

    Steps and principle of allele-specific extension on primer arrays. Multiplex PCRs yielding amplicons tailed with 5′-T7 RNA polymerase promoter sequence are performed. The PCR products are added directly to the primer array, along with the reaction mixture, which contains both T7 RNA polymerase and rNTPs to generate RNA targets from the PCR products and RT and labeled dNTPs for the actual allele-specific genotyping reaction, which is illustrated in the inset on the right column. For genotyping a pair of allele-specific detection primers for each mutant or polymorphic site immobilized through their 5′ end are used. The two primers differ at their 3′ end, which is complementary to either of the variant alleles. The RT extends the immobilized detection primers with labeled dNTPs in a template-dependent manner. After the reaction, fluorescence scanning of the arrays and quantitation of the fluorescent signals are carried out using a commercial confocal scanner and software. The fluorescent signals from each primer pair are compared, and the signal ratios fall into distinct categories defining the genotype at each site. The timescale for the reaction steps is illustrated beside the left column of the figure.

    Journal: Genome Research

    Article Title: A System for Specific, High-throughput Genotyping by Allele-specific Primer Extension on Microarrays

    doi:

    Figure Lengend Snippet: Steps and principle of allele-specific extension on primer arrays. Multiplex PCRs yielding amplicons tailed with 5′-T7 RNA polymerase promoter sequence are performed. The PCR products are added directly to the primer array, along with the reaction mixture, which contains both T7 RNA polymerase and rNTPs to generate RNA targets from the PCR products and RT and labeled dNTPs for the actual allele-specific genotyping reaction, which is illustrated in the inset on the right column. For genotyping a pair of allele-specific detection primers for each mutant or polymorphic site immobilized through their 5′ end are used. The two primers differ at their 3′ end, which is complementary to either of the variant alleles. The RT extends the immobilized detection primers with labeled dNTPs in a template-dependent manner. After the reaction, fluorescence scanning of the arrays and quantitation of the fluorescent signals are carried out using a commercial confocal scanner and software. The fluorescent signals from each primer pair are compared, and the signal ratios fall into distinct categories defining the genotype at each site. The timescale for the reaction steps is illustrated beside the left column of the figure.

    Article Snippet: For the simultaneous template preparation and allele-specific extension reactions, 0.5 μl of the total 60 μl of pooled multiplex PCR product, 6 m m of the four rNTPs, 6 μ m dATP, dGTP, CY5-dUTP, and CY5-dCTP (Amersham-Pharmacia Biotech), 6 μ m of the four ddNTPs, 6 U of MMLV RT (Epicentre Technologies), 0.5 μl of T7-RNA polymerase solution (Ampliscribe Kit) were combined in a total volume of 6 μl of T7 Ampliscribe reaction buffer supplemented with 0.4 m trehalose (T-5251, Sigma, St. Louis, Missouri) and 15% glycerol (w/v).

    Techniques: Multiplex Assay, Sequencing, Polymerase Chain Reaction, Labeling, Mutagenesis, Variant Assay, Fluorescence, Quantitation Assay, Software

    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

    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

    Interaction of HIV-1 Rev and REBP in mammalian cells. A total of 10 6 BSC40 cells infected with the vaccinia vector vTF7-3, expressing the T7 RNA polymerase, at 10 PFU/cell were subsequently transfected with 3 μg of plasmids expressing HA-REBP, HIV-1 wt Rev, or the functionally inactive NES mutant RevΔ81s, as shown. At 16 h posttransfection, [ 35 S]cysteine-[ 35 S]methionine-labeled proteins in clarified cell lysates were immunoprecipitated with either monoclonal anti-HA antibody or polyclonal Rev antiserum, as indicated. One-eighth and one-half of the total immunoprecipitates obtained with the HA and Rev antibodies, respectively, were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of protein size markers are indicated at left.

    Journal: Journal of Virology

    Article Title: A Nuclear Kinesin-Like Protein Interacts with and Stimulates the Activity of the Leucine-Rich Nuclear Export Signal of the Human Immunodeficiency Virus Type 1 Rev Protein

    doi: 10.1128/JVI.77.13.7236-7243.2003

    Figure Lengend Snippet: Interaction of HIV-1 Rev and REBP in mammalian cells. A total of 10 6 BSC40 cells infected with the vaccinia vector vTF7-3, expressing the T7 RNA polymerase, at 10 PFU/cell were subsequently transfected with 3 μg of plasmids expressing HA-REBP, HIV-1 wt Rev, or the functionally inactive NES mutant RevΔ81s, as shown. At 16 h posttransfection, [ 35 S]cysteine-[ 35 S]methionine-labeled proteins in clarified cell lysates were immunoprecipitated with either monoclonal anti-HA antibody or polyclonal Rev antiserum, as indicated. One-eighth and one-half of the total immunoprecipitates obtained with the HA and Rev antibodies, respectively, were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of protein size markers are indicated at left.

    Article Snippet: Plasmid pCMV-T7HA contains a HinDIII- Bam HI fragment specifying the influenza virus hemagglutinin (HA) epitope YPYDVPDYA cloned downstream of the cytomegalovirus immediate-early promoter (CMV-IE) and T7 RNA polymerase promoter in the plasmid pBC/CMV/IL-2, which is replication competent in COS cells. pCMV-REBP was constructed by cloning a Bam HI- Xho I fragment, comprising residues 2 to 665 of the REBP ORF, in-frame downstream of the HA epitope, thus placing HA-REBP expression under the control of the CMV-IE promoter and interleukin-2 (IL-2) poly(A) addition signal sequences. pcDNA3-Rev wt and pcDNA3-RevΔ81s contain the HIV-1 Rev wt and Δ81s NES mutant ORFs ( ) with a Kozak translation initiation consensus cloned downstream of the CMV-IE and T7 RNA polymerase promoters in the mammalian expression vector pCDNA3 (Invitrogen).

    Techniques: Infection, Plasmid Preparation, Expressing, Transfection, Mutagenesis, Labeling, Immunoprecipitation, Polyacrylamide Gel Electrophoresis

    ), together with the predicted signal peptide cleavage site (arrow). Two mutants, A 259 R and C 260 R were generated in segment 6 ORF. ( A ) T7 RNA transcripts expressing CM2 ORF, segment 6 ORF (wt), A 259 R segment 6 ORF or C 260 R ORF were translated in vitro using rabbit reticulocyte lysate in the presence and absence of canine pancreatic microsomes. ( B ) Polypeptides from the indicated in vitro translation reactions from ( A ) were immunoprecipitated with α-CM2 serum and an aliquot was treated with PNG at 37°C for 18 h. ( C ) Plasmids encoding CM2 ORF, segment 6 ORF (wt), A 259 R segment 6 ORF or C 260 R ORF were expressed by using the vac-T7 expression system in HeLa-T4 cells and polypeptides were immunoprecipitated using α-CM2 serum. An aliquot was treated with PNG.

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

    Article Title: Influenza C virus CM2 integral membrane glycoprotein is produced from a polypeptide precursor by cleavage of an internal signal sequence

    doi:

    Figure Lengend Snippet: ), together with the predicted signal peptide cleavage site (arrow). Two mutants, A 259 R and C 260 R were generated in segment 6 ORF. ( A ) T7 RNA transcripts expressing CM2 ORF, segment 6 ORF (wt), A 259 R segment 6 ORF or C 260 R ORF were translated in vitro using rabbit reticulocyte lysate in the presence and absence of canine pancreatic microsomes. ( B ) Polypeptides from the indicated in vitro translation reactions from ( A ) were immunoprecipitated with α-CM2 serum and an aliquot was treated with PNG at 37°C for 18 h. ( C ) Plasmids encoding CM2 ORF, segment 6 ORF (wt), A 259 R segment 6 ORF or C 260 R ORF were expressed by using the vac-T7 expression system in HeLa-T4 cells and polypeptides were immunoprecipitated using α-CM2 serum. An aliquot was treated with PNG.

    Article Snippet: Bacteriophage T7 RNA polymerase transcripts were synthesized from Hin dIII linearized plasmids, by using the T7 mMessage mMachine kit (Ambion).

    Techniques: Generated, Expressing, In Vitro, Immunoprecipitation

    Fate of mtDBP when T7 RNA polymerase transcribes through mtDBP–DNA complex, as determined by combining RNA transcription with gel mobility shift assays. (Top) Diagrammatic representation of the experimental strategy. (Bottom) Autoradiogram of a 6% polyacrylamide gel showing the results of gel shift assay with the 32 P-labelled 46mer. Reactions were performed as described in Materials and Methods. DNA template:protein ratios were 1:6 and 1:13 in lanes 1–3 and 4–7, respectively. A transcription assay in the presence of [α- 32 P]UTP was performed in parallel (data not shown).

    Journal: Nucleic Acids Research

    Article Title: Contrahelicase activity of the mitochondrial transcription termination factor mtDBP

    doi: 10.1093/nar/gki693

    Figure Lengend Snippet: Fate of mtDBP when T7 RNA polymerase transcribes through mtDBP–DNA complex, as determined by combining RNA transcription with gel mobility shift assays. (Top) Diagrammatic representation of the experimental strategy. (Bottom) Autoradiogram of a 6% polyacrylamide gel showing the results of gel shift assay with the 32 P-labelled 46mer. Reactions were performed as described in Materials and Methods. DNA template:protein ratios were 1:6 and 1:13 in lanes 1–3 and 4–7, respectively. A transcription assay in the presence of [α- 32 P]UTP was performed in parallel (data not shown).

    Article Snippet: Template DNA (100 fmol) was incubated with recombinant mtDBP at 25°C for 15 min in a 20 μl mixture containing T7 RNA polymerase buffer [40 mM Tris–HCl, pH 8.0, 25 mM NaCl, 8 mM MgCl2 and 2 mM spermidine-(HCl)3 ], 28 U of RNaseOUT (Invitrogen), 0.5 mM each of ATP, CTP and GTP, and 0.05 mM UTP.

    Techniques: Mobility Shift, Electrophoretic Mobility Shift Assay

    Line 2 is related to 1Kb ladder (Fermentas, cat. No.SM0311) and 2600bp band in line 1 shows amplification of T7 RNA polymerase gene.

    Journal: Iranian Journal of Cancer Prevention

    Article Title: Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines

    doi:

    Figure Lengend Snippet: Line 2 is related to 1Kb ladder (Fermentas, cat. No.SM0311) and 2600bp band in line 1 shows amplification of T7 RNA polymerase gene.

    Article Snippet: The PCR amplification of T7 RNA polymerase gene was carried out by using specific primers with Platinum-pfx kit (Invitrogen).

    Techniques: Amplification

    T7 RNA polymerase assay in HEK-293 cells (a) and T7-BHK cells (b) as a control was positive by using pFT7A vector.

    Journal: Iranian Journal of Cancer Prevention

    Article Title: Construction of an eGFP Expression Plasmid under Control of T7 Promoter and IRES Sequence for Assay of T7 RNA Polymerase Activity in Mammalian Cell Lines

    doi:

    Figure Lengend Snippet: T7 RNA polymerase assay in HEK-293 cells (a) and T7-BHK cells (b) as a control was positive by using pFT7A vector.

    Article Snippet: The PCR amplification of T7 RNA polymerase gene was carried out by using specific primers with Platinum-pfx kit (Invitrogen).

    Techniques: Plasmid Preparation

    Design and Genome Editing Activity of VEsiCas (A) EGFP disruption assay with VSV-G/SpCas9 vesicles produced in HEK293T cells. Shown are percentages of EGFP knockout HEK293-EGFP cells generated by transfection of SpCas9 (SpCas9 plasmid) together with EGFP targeting (sg EGFP5 ) or control (sgCtr) sgRNA or transduction with VSV-G/SpCas9 vesicles carrying U6-transcribed sgRNA. Where indicated, HEK293-EGFP cells were pre-transfected with sg EGFP5 or sgCtr (+ pre-sgRNA) prior to VSV-G/SpCas9 vesicle treatment. Data are presented as mean ± SEM for n = 2 independent experiments. (B) Schematic of VEsiCas production in BSR-T7/5 cells. T7 RNA polymerase, expressed in the cytosol, regulates cytosolic sgRNA expression by means of the T7 promoter. Vesicles decorated with VSV-G, expressed by BSR-T7/5 producer cells, bud incorporating SpCas9 complexed with sgRNA to form VEsiCas. In target cells, VEsiCas release active SpCas9-sgRNA complexes that enter the nuclei through two nuclear localization sequences introduced in SpCas9. (C) Genome activity of VEsiCas produced in BSR-T7/5 on HEK293-EGFP cells. Shown are percentages of non-fluorescent HEK293-EGFP cells following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sg EGFP5 or sgCtr) or treatment with VEsiCas carrying sgRNAs (sg EGFP5 or sgCtr) either with or without pre-transfection with sgRNAs, as indicated. Data are presented as mean ± SEM for n = 2 independent experiments. (D and E) VEsiCas-mediated editing of the CXCR4 (D) and VEGFA site3 (E) genomic loci. Percentages of indel formation in HEK293T cells were measured through TIDE analysis following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sg CXCR4 , sg VEGFA site3 , or sgCtr) or after three sequential treatments with VEsiCas carrying sgRNAs (sg CXCR4 , sg VEGFA site3 , or sgCtr). Data are presented as mean ± SEM for n = 2 independent experiments.

    Journal: Molecular Therapy. Nucleic Acids

    Article Title: VSV-G-Enveloped Vesicles for Traceless Delivery of CRISPR-Cas9

    doi: 10.1016/j.omtn.2018.05.010

    Figure Lengend Snippet: Design and Genome Editing Activity of VEsiCas (A) EGFP disruption assay with VSV-G/SpCas9 vesicles produced in HEK293T cells. Shown are percentages of EGFP knockout HEK293-EGFP cells generated by transfection of SpCas9 (SpCas9 plasmid) together with EGFP targeting (sg EGFP5 ) or control (sgCtr) sgRNA or transduction with VSV-G/SpCas9 vesicles carrying U6-transcribed sgRNA. Where indicated, HEK293-EGFP cells were pre-transfected with sg EGFP5 or sgCtr (+ pre-sgRNA) prior to VSV-G/SpCas9 vesicle treatment. Data are presented as mean ± SEM for n = 2 independent experiments. (B) Schematic of VEsiCas production in BSR-T7/5 cells. T7 RNA polymerase, expressed in the cytosol, regulates cytosolic sgRNA expression by means of the T7 promoter. Vesicles decorated with VSV-G, expressed by BSR-T7/5 producer cells, bud incorporating SpCas9 complexed with sgRNA to form VEsiCas. In target cells, VEsiCas release active SpCas9-sgRNA complexes that enter the nuclei through two nuclear localization sequences introduced in SpCas9. (C) Genome activity of VEsiCas produced in BSR-T7/5 on HEK293-EGFP cells. Shown are percentages of non-fluorescent HEK293-EGFP cells following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sg EGFP5 or sgCtr) or treatment with VEsiCas carrying sgRNAs (sg EGFP5 or sgCtr) either with or without pre-transfection with sgRNAs, as indicated. Data are presented as mean ± SEM for n = 2 independent experiments. (D and E) VEsiCas-mediated editing of the CXCR4 (D) and VEGFA site3 (E) genomic loci. Percentages of indel formation in HEK293T cells were measured through TIDE analysis following transfection of SpCas9 (SpCas9 plasmid) together with sgRNAs (sg CXCR4 , sg VEGFA site3 , or sgCtr) or after three sequential treatments with VEsiCas carrying sgRNAs (sg CXCR4 , sg VEGFA site3 , or sgCtr). Data are presented as mean ± SEM for n = 2 independent experiments.

    Article Snippet: To select cells that retain the T7 RNA polymerase construct, the medium was supplemented with 1 mg/mL G418 (Gibco-Life Technologies).

    Techniques: Activity Assay, Produced, Knock-Out, Generated, Transfection, Plasmid Preparation, Transduction, Expressing

    (A) Components of a T7 RNA polymerase-based BUN-minireplicon system in mosquito cells. The L, N, S, NSs, or firefly luciferase (FFLuc) ORF was cloned into pT7AcCat to produce the pT7Ac constructs used in this study. 5′Ac and 3′Ac, 5′ and 3′ translational enhancers of the baculovirus p10 gene; φ10, T7 RNA polymerase promoter. (B) Induction of firefly luciferase activity in mosquito cells expressing T7 RNA polymerase (C6-IBT7/3) or in parental cells (C6/36). Cells were either transfected with 100 ng of pT7AcLuc (bar 1) or of pT7AcCat (bar 2) or were not transfected (bar 3). (C) Analysis of various IRESs and translational enhancer elements in C6-IBT7/3 mosquito cells expressing T7 RNA polymerase. Monocistronic reporter constructs containing either the RhPV IRES in the sense (pSP72Δ1luc) or antisense (pSP72 Δ1asluc) orientation, the baculovirus p10 gene 5′ and 3′ translational enhancers (pT7AcLuc), or the EMCV IRES (pTM1-FF-Luc) were used. C6-IBT7/3 cells were transfected with 1 μg of the indicated plasmid. Firefly luciferase activities (expressed here as fold induction) were determined at 24 h posttransfection and normalized against an internal standard (the φ10 promoter-containing construct expressing Renilla luciferase). (D) Dual-reporter constructs containing either the RhPV IRES (pGEMCATΔ1luc), the EMCV IRES (pGEMCATEMCV-Luc), or no IRES element (PGEMCATluc), as indicated. C6-IBT7/3 cells were transfected with 1 μg of the indicated plasmid. Firefly luciferase activities (expressed here as fold induction) were determined at 24 h posttransfection and normalized against an internal standard (the φ10 promoter-containing construct expressing Renilla luciferase).

    Journal: Journal of Virology

    Article Title: A Bunyamwera Virus Minireplicon System in Mosquito Cells

    doi: 10.1128/JVI.78.11.5679-5685.2004

    Figure Lengend Snippet: (A) Components of a T7 RNA polymerase-based BUN-minireplicon system in mosquito cells. The L, N, S, NSs, or firefly luciferase (FFLuc) ORF was cloned into pT7AcCat to produce the pT7Ac constructs used in this study. 5′Ac and 3′Ac, 5′ and 3′ translational enhancers of the baculovirus p10 gene; φ10, T7 RNA polymerase promoter. (B) Induction of firefly luciferase activity in mosquito cells expressing T7 RNA polymerase (C6-IBT7/3) or in parental cells (C6/36). Cells were either transfected with 100 ng of pT7AcLuc (bar 1) or of pT7AcCat (bar 2) or were not transfected (bar 3). (C) Analysis of various IRESs and translational enhancer elements in C6-IBT7/3 mosquito cells expressing T7 RNA polymerase. Monocistronic reporter constructs containing either the RhPV IRES in the sense (pSP72Δ1luc) or antisense (pSP72 Δ1asluc) orientation, the baculovirus p10 gene 5′ and 3′ translational enhancers (pT7AcLuc), or the EMCV IRES (pTM1-FF-Luc) were used. C6-IBT7/3 cells were transfected with 1 μg of the indicated plasmid. Firefly luciferase activities (expressed here as fold induction) were determined at 24 h posttransfection and normalized against an internal standard (the φ10 promoter-containing construct expressing Renilla luciferase). (D) Dual-reporter constructs containing either the RhPV IRES (pGEMCATΔ1luc), the EMCV IRES (pGEMCATEMCV-Luc), or no IRES element (PGEMCATluc), as indicated. C6-IBT7/3 cells were transfected with 1 μg of the indicated plasmid. Firefly luciferase activities (expressed here as fold induction) were determined at 24 h posttransfection and normalized against an internal standard (the φ10 promoter-containing construct expressing Renilla luciferase).

    Article Snippet: The T7 RNA polymerase ORF was amplified by PCR from pSFV-T7 , and the amplified product was first digested with HindIII and XbaI and then inserted into the HindIII/XbaI-digested expression vector pIB/V5-His (Invitrogen).

    Techniques: Luciferase, Clone Assay, Construct, Activity Assay, Expressing, Transfection, Plasmid Preparation

    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

    RNase T 1 interferes with R-loop formation on a linearized switch substrate containing four repeats of murine Sγ3. The substrate, pDR3, was linearized with ApaL1 and then transcribed with T7 RNA polymerase in the presence of [α- 32 P]UTP.

    Journal:

    Article Title: Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences ▿Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences ▿ †

    doi: 10.1128/MCB.01251-07

    Figure Lengend Snippet: RNase T 1 interferes with R-loop formation on a linearized switch substrate containing four repeats of murine Sγ3. The substrate, pDR3, was linearized with ApaL1 and then transcribed with T7 RNA polymerase in the presence of [α- 32 P]UTP.

    Article Snippet: Moreover, the amount of R-loop formation is similar or higher for all of our buffers containing Li+ , Na+ , K+ , or Cs+ (Fig. , lanes 4 to 7 and lanes 12 to 15 and data not shown) relative to the manufacturer's buffer for T7 RNA polymerase (Fig. , lanes 2 and 10), which is prepared using Na+ (and has the same composition as our Na+ -based transcription buffer).

    Techniques:

    Cell–cell fusion assay using parental PM1 cells (PM1-P) and clones derived from the control transduced PM1 cells (PM1-RAI3–5) and from PM1 cells transduced with ST6-encoding retrovirus (PM1–6-G). PM1 cells were infected with a vaccinia recombinant encoding T7 RNA polymerase. To assess background luciferase activity, parental PM1 cells were infected with vaccinia virus encoding β-galactosidase instead (PM1-P-lacZ). Reporter gene activity upon coculturing of PM1 cells with 293T cells expressing HIV-1 env and transfected with reporter plasmid is shown as relative light units (RLU) on the y -axis. Pairs of individual experiments are shown.

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

    Article Title: Functional deletion of the CCR5 receptor by intracellular immunization produces cells that are refractory to CCR5-dependent HIV-1 infection and cell fusion

    doi:

    Figure Lengend Snippet: Cell–cell fusion assay using parental PM1 cells (PM1-P) and clones derived from the control transduced PM1 cells (PM1-RAI3–5) and from PM1 cells transduced with ST6-encoding retrovirus (PM1–6-G). PM1 cells were infected with a vaccinia recombinant encoding T7 RNA polymerase. To assess background luciferase activity, parental PM1 cells were infected with vaccinia virus encoding β-galactosidase instead (PM1-P-lacZ). Reporter gene activity upon coculturing of PM1 cells with 293T cells expressing HIV-1 env and transfected with reporter plasmid is shown as relative light units (RLU) on the y -axis. Pairs of individual experiments are shown.

    Article Snippet: A reporter plasmid containing the luciferase gene under the control of the T7 RNA polymerase promoter was purchased from Promega, and plasmid pcDNA3.1/Zeo was purchased from Invitrogen.

    Techniques: Cell-Cell Fusion Assay, Clone Assay, Derivative Assay, Transduction, Infection, Recombinant, Luciferase, Activity Assay, Expressing, Transfection, Plasmid Preparation

    The N-terminal half of p50 cdc37 mediates association with the catalytic domain of Raf-1 but is impaired for Hsp90 interaction and accumulation to Raf-1. (A) Plasmids pSG5-p50 cdc37 and pSG5-p50 cdc37 ΔC were transcribed and translated in vitro, using T7 RNA polymerase and a reticulocyte lysate system (Promega); 5 μl of each reaction mixture was either analyzed directly (input lanes) or assayed in vitro for binding to either GST or bacterially purified GST–ΔN-Raf-1(Δ26-309) and visualized by SDS-PAGE and fluorography. Comparable results were obtained with full-length GST–Raf-1 (not shown). (B) Cos-1 cells transfected with pSG5-FLAG vector, pSG5-FLAG-p50 cdc37 , and pSG5-FLAG-p50 cdc37 ΔC were [ 35 S]methionine labeled, and anti-FLAG IPs in NP-40 LB of each transfected sample were analyzed by SDS-PAGE and fluorography (lanes 1 to 3, respectively). Proteins at the sizes predicted for overexpressed FLAG-p50 cdc37 proteins or associated endogenous Hsp90 are also indicated. (C) Two micrograms of pEBG-GST-Raf-1 was cotransfected with 5 μg of pSG5-FLAG vector (lane 1), pSG5-FLAG-p50 cdc37 (lanes 2 and 3), or pSG5-FLAG-p50 cdc37 ΔC (lanes 4 and 5) at 5 or 15 μg as indicated. After 48 h in DMEM-FBS, all five cultures were harvested and lysed in NP-40 LB, and GST–Raf-1 was GSH-Sepharose purified and tested for associated p50 cdc37 or Hsp90 proteins with rabbit anti-p50 cdc37 or rat anti-Hsp90 antibody. A control anti-GST immunoblot was also included to detect overexpressed GST–Raf-1 (top panel). (D) Diagram indicating regions of interaction between p50 cdc37 , Raf-1, and Hsp90. The N-terminal half of p50 cdc37 (gray area) which corresponds to p50 cdc37 ), higher-order complexes of p50 cdc37 –Raf-1–Hsp90 can also be envisioned.

    Journal: Molecular and Cellular Biology

    Article Title: p50cdc37 Acting in Concert with Hsp90 Is Required for Raf-1 Function †

    doi:

    Figure Lengend Snippet: The N-terminal half of p50 cdc37 mediates association with the catalytic domain of Raf-1 but is impaired for Hsp90 interaction and accumulation to Raf-1. (A) Plasmids pSG5-p50 cdc37 and pSG5-p50 cdc37 ΔC were transcribed and translated in vitro, using T7 RNA polymerase and a reticulocyte lysate system (Promega); 5 μl of each reaction mixture was either analyzed directly (input lanes) or assayed in vitro for binding to either GST or bacterially purified GST–ΔN-Raf-1(Δ26-309) and visualized by SDS-PAGE and fluorography. Comparable results were obtained with full-length GST–Raf-1 (not shown). (B) Cos-1 cells transfected with pSG5-FLAG vector, pSG5-FLAG-p50 cdc37 , and pSG5-FLAG-p50 cdc37 ΔC were [ 35 S]methionine labeled, and anti-FLAG IPs in NP-40 LB of each transfected sample were analyzed by SDS-PAGE and fluorography (lanes 1 to 3, respectively). Proteins at the sizes predicted for overexpressed FLAG-p50 cdc37 proteins or associated endogenous Hsp90 are also indicated. (C) Two micrograms of pEBG-GST-Raf-1 was cotransfected with 5 μg of pSG5-FLAG vector (lane 1), pSG5-FLAG-p50 cdc37 (lanes 2 and 3), or pSG5-FLAG-p50 cdc37 ΔC (lanes 4 and 5) at 5 or 15 μg as indicated. After 48 h in DMEM-FBS, all five cultures were harvested and lysed in NP-40 LB, and GST–Raf-1 was GSH-Sepharose purified and tested for associated p50 cdc37 or Hsp90 proteins with rabbit anti-p50 cdc37 or rat anti-Hsp90 antibody. A control anti-GST immunoblot was also included to detect overexpressed GST–Raf-1 (top panel). (D) Diagram indicating regions of interaction between p50 cdc37 , Raf-1, and Hsp90. The N-terminal half of p50 cdc37 (gray area) which corresponds to p50 cdc37 ), higher-order complexes of p50 cdc37 –Raf-1–Hsp90 can also be envisioned.

    Article Snippet: Different full-length and deletion forms of p50cdc37 were transcribed and translated in vitro from the pSG5 expression constructs in the presence of 20 μCi of [35 S]methionine (EXPRESS protein labeling mix; NEN), using the coupled rabbit reticulocyte lysate and T7 RNA polymerase system (Promega).

    Techniques: In Vitro, Binding Assay, Purification, SDS Page, Transfection, Plasmid Preparation, Labeling

    Spermine attenuates the inhibition of transcription by actinomycin D in vitro. ( A ) The effect of ACTD on T7 RNA polymerase activity in the presence of various concentrations of spermine (SPM). ( B ) Quantification of the percentage of RNA polymerase activity relative to the control treated with or without ACTD in the presence of various concentrations of spermine (SPM) (0, 0.1, and 0.2 mM). The data represent the mean values ±SDs from three separate experiments.

    Journal: PLoS ONE

    Article Title: Spermine Attenuates the Action of the DNA Intercalator, Actinomycin D, on DNA Binding and the Inhibition of Transcription and DNA Replication

    doi: 10.1371/journal.pone.0047101

    Figure Lengend Snippet: Spermine attenuates the inhibition of transcription by actinomycin D in vitro. ( A ) The effect of ACTD on T7 RNA polymerase activity in the presence of various concentrations of spermine (SPM). ( B ) Quantification of the percentage of RNA polymerase activity relative to the control treated with or without ACTD in the presence of various concentrations of spermine (SPM) (0, 0.1, and 0.2 mM). The data represent the mean values ±SDs from three separate experiments.

    Article Snippet: RNA polymerase assays To analyze in vitro transcription, a T7 RNA polymerase assay (Promega) was used.

    Techniques: Inhibition, In Vitro, Activity Assay

    Fig. 3. Effects of K86R mutant band IV protein expression. ( A ) Adenylylation assay of extracts from E.coli expressing wild-type and mutant band IV proteins (arrow); some degradation products are also seen. An adenylylatable E.coli protein (asterisk) serves as an internal control. ( B ) piLigIV-K86R drives expression in trypanosomes of the K86R mutant band IV protein and a linked phleomycin-resistance gene (Phleo R , the ble gene from Streptoalloteichus hindustanus ) from a T7 promoter with a tet operator and uses trypanosome 5′ and 3′ processing signals. It is transfected into 29-13 procyclic trypanosomes, which express T7 RNA polymerase and the tet repressor. ( C ) Adenylylation assay of mitochondrial extract made from non-induced (lanes 1 and 3) and induced (lanes 2 and 4) K86R cells. The assays in lanes 1 and 2 used unfractionated extract and those in lanes 3 and 4 used the ∼20S peak from glycerol gradient fractionated extract. ( D ) Growth of two lines of K86R cells and control cells (the 29-13 parental line) with none or 1 µg/ml tet added at 0 h. Media was added to maintain cell densities supporting log phase growth. This semi-log plot is based on total cell numbers (cells/ml × volume), scaled to equalize minor differences in starting cell numbers. Error bars denote standard deviation for K86R-1 cells and are similar for the other cell lines. After much longer periods of induction (selection), the cultures can be overtaken by normal looking cells, possibly due to loss or inactivation of the ectopic gene. ( E ) Representative morphologies of K86R cells non-induced (first panel) and induced with tet (subsequent three panels), DAPI-stained and photographed 66 h post-induction at 100× magnification.

    Journal: The EMBO Journal

    Article Title: Roles for ligases in the RNA editing complex of Trypanosoma brucei: band IV is needed for U-deletion and RNA repair

    doi: 10.1093/emboj/20.17.4694

    Figure Lengend Snippet: Fig. 3. Effects of K86R mutant band IV protein expression. ( A ) Adenylylation assay of extracts from E.coli expressing wild-type and mutant band IV proteins (arrow); some degradation products are also seen. An adenylylatable E.coli protein (asterisk) serves as an internal control. ( B ) piLigIV-K86R drives expression in trypanosomes of the K86R mutant band IV protein and a linked phleomycin-resistance gene (Phleo R , the ble gene from Streptoalloteichus hindustanus ) from a T7 promoter with a tet operator and uses trypanosome 5′ and 3′ processing signals. It is transfected into 29-13 procyclic trypanosomes, which express T7 RNA polymerase and the tet repressor. ( C ) Adenylylation assay of mitochondrial extract made from non-induced (lanes 1 and 3) and induced (lanes 2 and 4) K86R cells. The assays in lanes 1 and 2 used unfractionated extract and those in lanes 3 and 4 used the ∼20S peak from glycerol gradient fractionated extract. ( D ) Growth of two lines of K86R cells and control cells (the 29-13 parental line) with none or 1 µg/ml tet added at 0 h. Media was added to maintain cell densities supporting log phase growth. This semi-log plot is based on total cell numbers (cells/ml × volume), scaled to equalize minor differences in starting cell numbers. Error bars denote standard deviation for K86R-1 cells and are similar for the other cell lines. After much longer periods of induction (selection), the cultures can be overtaken by normal looking cells, possibly due to loss or inactivation of the ectopic gene. ( E ) Representative morphologies of K86R cells non-induced (first panel) and induced with tet (subsequent three panels), DAPI-stained and photographed 66 h post-induction at 100× magnification.

    Article Snippet: Cell line 29-13, a 427 derivative that expresses the T7 RNA polymerase and the tetracycline repressor protein ( ) was grown in this medium with 15 µg/ml G418 (Gibco-BRL) and 50 µg/ml hygromycin (Sigma), as were transfectants bearing piLigIV-K86R.

    Techniques: Mutagenesis, Expressing, Transfection, Standard Deviation, Selection, Staining

    A) Electrophoresis (8%) PAGE gel of the expression and purification of His-LAR T7 RNA polymerase. Lanes 1) Induced insoluble fraction, 2) Induced soluble fraction, 3) Uninduced whole cell lysate, 4) Molecular weight marker, 5) Molecular weight marker,

    Journal: Biomaterials

    Article Title: Highly Stable Aptamers Selected from a 2′-Fully Modified fGmH RNA Library for Targeting Biomaterials

    doi: 10.1016/j.biomaterials.2014.08.046

    Figure Lengend Snippet: A) Electrophoresis (8%) PAGE gel of the expression and purification of His-LAR T7 RNA polymerase. Lanes 1) Induced insoluble fraction, 2) Induced soluble fraction, 3) Uninduced whole cell lysate, 4) Molecular weight marker, 5) Molecular weight marker,

    Article Snippet: LAR T7 RNA polymerase was used to transcribe the fYrR, rGmH, and fGmH A12 variants using the optimized buffer system described above, while wild-type T7 RNA polymerase (New England Biolabs, Ipswich, MA) was used to transcribe the natural A12 RNA.

    Techniques: Electrophoresis, Polyacrylamide Gel Electrophoresis, Expressing, Purification, Molecular Weight, Marker

    Primer extension of synthetic RNA in the absence of promoter sequence. ( A ) RNA-Seq quantification of RNA products by length, for a 4 h incubation of synthetic RNA with T7 RNA polymerase, in the presence of 0.4 mM each NTP. ( B ) Most abundant RNA-Seq sequences. Extended RNA products show complementarity (blue: sequence beyond the synthetic RNA sequence, green: upstream sequence of the synthetic RNA that is complementary to the blue region). ( C ) Denaturing gel electrophoreses (20% denaturing urea) analysis of a parallel reaction using radiolabeled, synthetic RNA. Lane 1: Radiolabeled 24-base synthetic RNA. Lanes 2 and 3: Incubation of synthetic RNA with T7 RNA polymerase for 5 min and 4 h, respectively.

    Journal: Nucleic Acids Research

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses

    doi: 10.1093/nar/gky796

    Figure Lengend Snippet: Primer extension of synthetic RNA in the absence of promoter sequence. ( A ) RNA-Seq quantification of RNA products by length, for a 4 h incubation of synthetic RNA with T7 RNA polymerase, in the presence of 0.4 mM each NTP. ( B ) Most abundant RNA-Seq sequences. Extended RNA products show complementarity (blue: sequence beyond the synthetic RNA sequence, green: upstream sequence of the synthetic RNA that is complementary to the blue region). ( C ) Denaturing gel electrophoreses (20% denaturing urea) analysis of a parallel reaction using radiolabeled, synthetic RNA. Lane 1: Radiolabeled 24-base synthetic RNA. Lanes 2 and 3: Incubation of synthetic RNA with T7 RNA polymerase for 5 min and 4 h, respectively.

    Article Snippet: Reactions were carried out in a buffer containing 15 mM magnesium acetate, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25 mM potassium glutamate, 0.25 mM ethylenediaminetetraacetic acid and 0.05% Tween-20 at 37°C for 5 min. To be representative of common practices, ‘high yield’ transcription was performed using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England BioLabs), in a transcription mixture containing 2 μM each of template and nontemplate DNA, 1.5 μl of T7 RNA Polymerase Mix™ for 20 μl reaction volume, 7.5 mM each of GTP, ATP, CTP and UTP, in the T7 Reaction Buffer (New England BioLabs).

    Techniques: Sequencing, RNA Sequencing Assay, Incubation

    The ‘release and rebind’ model for the formation of longer RNA products by a cis primer extension mechanism. In the ‘On-pathway’ reaction, the promoter binding domain (pink) of T7 RNA polymerase binds promoter DNA and directs synthesis of the expected runoff RNA. In a competing ‘Off-pathway’ reaction, released RNA (R x ) rebinds to (a different) RNA polymerase and self-primes extension to longer RNA products in cis . RNAs shorter or longer than full length can function as R x . Repeated rebinding/extension can (distributively) lead to still longer products.

    Journal: Nucleic Acids Research

    Article Title: 3′ end additions by T7 RNA polymerase are RNA self-templated, distributive and diverse in character—RNA-Seq analyses

    doi: 10.1093/nar/gky796

    Figure Lengend Snippet: The ‘release and rebind’ model for the formation of longer RNA products by a cis primer extension mechanism. In the ‘On-pathway’ reaction, the promoter binding domain (pink) of T7 RNA polymerase binds promoter DNA and directs synthesis of the expected runoff RNA. In a competing ‘Off-pathway’ reaction, released RNA (R x ) rebinds to (a different) RNA polymerase and self-primes extension to longer RNA products in cis . RNAs shorter or longer than full length can function as R x . Repeated rebinding/extension can (distributively) lead to still longer products.

    Article Snippet: Reactions were carried out in a buffer containing 15 mM magnesium acetate, 30 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 25 mM potassium glutamate, 0.25 mM ethylenediaminetetraacetic acid and 0.05% Tween-20 at 37°C for 5 min. To be representative of common practices, ‘high yield’ transcription was performed using the HiScribe™ T7 High Yield RNA Synthesis Kit (New England BioLabs), in a transcription mixture containing 2 μM each of template and nontemplate DNA, 1.5 μl of T7 RNA Polymerase Mix™ for 20 μl reaction volume, 7.5 mM each of GTP, ATP, CTP and UTP, in the T7 Reaction Buffer (New England BioLabs).

    Techniques: Binding Assay

    . Panel (a): The purification scheme begins with T7 RNA polymerase transcription of a conjugated RNA composed of target RNA- glmS ribozyme-binding sequence. The binding sequence binds to a biotinylated

    Journal: Methods in molecular biology (Clifton, N.J.)

    Article Title: Native purification and labeling of RNA for single molecule fluorescence studies

    doi: 10.1007/978-1-4939-1896-6_6

    Figure Lengend Snippet: . Panel (a): The purification scheme begins with T7 RNA polymerase transcription of a conjugated RNA composed of target RNA- glmS ribozyme-binding sequence. The binding sequence binds to a biotinylated

    Article Snippet: 3 Plasmids encoding for the T7 RNA polymerase gene for protein overexpression and purification are available ( ) or the enzyme is available for purchase through New England Biolabs.

    Techniques: Purification, Binding Assay, Sequencing