sp6  (New England Biolabs)


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    Name:
    SP6 RNA Polymerase
    Description:
    SP6 RNA Polymerase 10 000 units
    Catalog Number:
    m0207l
    Price:
    269
    Size:
    10 000 units
    Category:
    RNA Polymerases
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    Structured Review

    New England Biolabs sp6
    SP6 RNA Polymerase
    SP6 RNA Polymerase 10 000 units
    https://www.bioz.com/result/sp6/product/New England Biolabs
    Average 94 stars, based on 71 article reviews
    Price from $9.99 to $1999.99
    sp6 - by Bioz Stars, 2021-02
    94/100 stars

    Images

    1) Product Images from "Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells"

    Article Title: Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06092-6

    LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm
    Figure Legend Snippet: LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm

    Techniques Used: Antibody Labeling

    2) Product Images from "Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *"

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *

    Journal: The Journal of Biological Chemistry

    doi:

    Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.
    Figure Legend Snippet: Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.

    Techniques Used: IA, In Vitro, Concentration Assay, Incubation

    Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.
    Figure Legend Snippet: Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.

    Techniques Used: In Vitro, Sequencing, IA

    3) Product Images from "Secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus 1"

    Article Title: Secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus 1

    Journal: Journal of Molecular Biology

    doi: 10.1006/jmbi.1998.2186

    Construction of plasmid pRSV1. A 266 bp Avr II- Bss HII fragment encompassing the RSV frameshift region was isolated from plasmid pATV8 (which harbours a proviral clone of RSV Prague C; Schwartz et al ., 1983 ) and subcloned into the PB1 reporter gene of plasmid pPSO.1, a derivative of pPSO (Somogyi et al ., 1993) with unique Avr II and Bss HII sites. The open reading frames in pRSV1 are such that the upstream portion of the PB1 reporter gene is in frame with the gag gene and the downstream portion, the pol gene. Analysis of the secondary structure of the RNA at the RSV frameshift site by primer extension was on mRNAs prepared by linearisation of pRSV1 with Pvu II and subsequent transcription with T7 RNA polymerase. For structural analysis of end-labelled RNA, related plasmids containing a T3 promoter were prepared (see the text), linearised with Dde I and transcribed with T3 RNA polymerase. In ribosomal frameshift assays, capped mRNAs were prepared by SP6 transcription from Ava II linearised template. The predicted non-frameshifted (45 kDa) and frameshifted (70 kDa) species generated upon in vitro translation of such mRNAs in RRL are shown.
    Figure Legend Snippet: Construction of plasmid pRSV1. A 266 bp Avr II- Bss HII fragment encompassing the RSV frameshift region was isolated from plasmid pATV8 (which harbours a proviral clone of RSV Prague C; Schwartz et al ., 1983 ) and subcloned into the PB1 reporter gene of plasmid pPSO.1, a derivative of pPSO (Somogyi et al ., 1993) with unique Avr II and Bss HII sites. The open reading frames in pRSV1 are such that the upstream portion of the PB1 reporter gene is in frame with the gag gene and the downstream portion, the pol gene. Analysis of the secondary structure of the RNA at the RSV frameshift site by primer extension was on mRNAs prepared by linearisation of pRSV1 with Pvu II and subsequent transcription with T7 RNA polymerase. For structural analysis of end-labelled RNA, related plasmids containing a T3 promoter were prepared (see the text), linearised with Dde I and transcribed with T3 RNA polymerase. In ribosomal frameshift assays, capped mRNAs were prepared by SP6 transcription from Ava II linearised template. The predicted non-frameshifted (45 kDa) and frameshifted (70 kDa) species generated upon in vitro translation of such mRNAs in RRL are shown.

    Techniques Used: Plasmid Preparation, Isolation, Antiviral Assay, Generated, In Vitro

    4) Product Images from "Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells"

    Article Title: Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

    Journal: Nature Communications

    doi: 10.1038/s41467-018-06092-6

    LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm
    Figure Legend Snippet: LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm

    Techniques Used: Antibody Labeling

    5) Product Images from "Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *"

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *

    Journal: The Journal of Biological Chemistry

    doi:

    Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.
    Figure Legend Snippet: Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.

    Techniques Used: IA, In Vitro, Concentration Assay, Incubation

    Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.
    Figure Legend Snippet: Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.

    Techniques Used: In Vitro, Sequencing, IA

    6) Product Images from "Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *"

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *

    Journal: The Journal of Biological Chemistry

    doi:

    Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.
    Figure Legend Snippet: Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.

    Techniques Used: IA, In Vitro, Concentration Assay, Incubation

    Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.
    Figure Legend Snippet: Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.

    Techniques Used: In Vitro, Sequencing, IA

    7) Product Images from "Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting"

    Article Title: Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gki299

    Analysis of the Edr frameshift signal by site-directed mutagenesis. ( A ) A series of mutations were introduced into the Edr frameshift region to modify the proposed slippery sequence (GGGAAAC, in bold) or pseudoknot. ( B ). Wild-type pKT0/ Edr or mutant derivatives were digested with HindIII, transcribed with SP6 RNA polymerase and transcripts translated in RRL at a concentration of ∼50 μg/ml. Products were labelled with [ 35 S]methionine, separated on 15% SDS/polyacrylamide gels and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents 14 C protein markers (Amersham Pharmacia Biotech). ( C ) Summary of the mutations made and the resulting frameshift efficiencies. In constructs pKT0/ m5 , m8 and m11 , both arms of the relevant stem region were mutated such that the stems should reform (double/revertant).
    Figure Legend Snippet: Analysis of the Edr frameshift signal by site-directed mutagenesis. ( A ) A series of mutations were introduced into the Edr frameshift region to modify the proposed slippery sequence (GGGAAAC, in bold) or pseudoknot. ( B ). Wild-type pKT0/ Edr or mutant derivatives were digested with HindIII, transcribed with SP6 RNA polymerase and transcripts translated in RRL at a concentration of ∼50 μg/ml. Products were labelled with [ 35 S]methionine, separated on 15% SDS/polyacrylamide gels and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents 14 C protein markers (Amersham Pharmacia Biotech). ( C ) Summary of the mutations made and the resulting frameshift efficiencies. In constructs pKT0/ m5 , m8 and m11 , both arms of the relevant stem region were mutated such that the stems should reform (double/revertant).

    Techniques Used: Mutagenesis, Sequencing, Concentration Assay, Autoradiography, Construct

    Deletion analysis of the Edr frameshift signal. ( A ) Three independent in-frame deletions were created in pKT0/ Edr , Δ90, Δ63 and Δ24, to investigate the requirement for sequence information downstream of the putative Edr slippery sequence GGGAAAC (boxed). Each deletion (of 90, 63 or 24 nt) was to a common 3′ site (immediately downstream of the NdeI site, which was removed), leaving varying lengths of 5′ sequence. The nucleotides implicated as forming the stimulatory RNA in a previous study ( 20 ) are in bold. ( B ) The three deletion mutants were digested with HindIII, transcribed with SP6 RNA polymerase and transcripts translated in RRL, either undiluted (1; final concentration ∼50 μg/ml) or diluted 1/3 (1/3; about 15 μg/ml). Products were labelled with [ 35 S]methionine, separated on a 15% SDS/polyacrylamide gel and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents 14 C protein markers (Amersham Pharmacia Biotech). The species indicated with an asterisk is discussed in the text.
    Figure Legend Snippet: Deletion analysis of the Edr frameshift signal. ( A ) Three independent in-frame deletions were created in pKT0/ Edr , Δ90, Δ63 and Δ24, to investigate the requirement for sequence information downstream of the putative Edr slippery sequence GGGAAAC (boxed). Each deletion (of 90, 63 or 24 nt) was to a common 3′ site (immediately downstream of the NdeI site, which was removed), leaving varying lengths of 5′ sequence. The nucleotides implicated as forming the stimulatory RNA in a previous study ( 20 ) are in bold. ( B ) The three deletion mutants were digested with HindIII, transcribed with SP6 RNA polymerase and transcripts translated in RRL, either undiluted (1; final concentration ∼50 μg/ml) or diluted 1/3 (1/3; about 15 μg/ml). Products were labelled with [ 35 S]methionine, separated on a 15% SDS/polyacrylamide gel and detected by autoradiography. The non-frameshifted (stop) and frameshifted (FS) species are marked with arrows. M represents 14 C protein markers (Amersham Pharmacia Biotech). The species indicated with an asterisk is discussed in the text.

    Techniques Used: Sequencing, Concentration Assay, Autoradiography

    Construction of plasmid pKT0/ Edr . A 1230 bp DNA fragment (631–1861) encompassing the Edr frameshift region was amplified by PCR from plasmid pSP64T/ Edr ( 20 ) and cloned into NcoI/HindIII digested plasmid pKT0 ( 28 ). The 5′ and 3′ portions of the cloned Edr segment are shown in lower case, numbered according to the mRNA sequence (A of natural AUG start site is base 452; accession no. AJ006464). The AUG for the expression of Edr sequences in pKT0/ Edr is derived from the vector (upper case, underlined). In ribosomal frameshifting assays, capped mRNAs were prepared by SP6 transcription of NdeI-linearized templates (unless otherwise stated). The predicted size of the non-frameshifted (stop) and frameshifted (fs) products generated from the translation of this mRNA in RRL is 29 and 32 kDa, respectively. For structural analysis of the Edr stimulatory RNA, a T3 promoter was introduced into the Edr sequence (at position 1357) to generate plasmid pKT0/ Edr /T3. Linearization of this plasmid with NdeI and subsequent transcription with T3 RNA polymerase yields a transcript of 152 nt.
    Figure Legend Snippet: Construction of plasmid pKT0/ Edr . A 1230 bp DNA fragment (631–1861) encompassing the Edr frameshift region was amplified by PCR from plasmid pSP64T/ Edr ( 20 ) and cloned into NcoI/HindIII digested plasmid pKT0 ( 28 ). The 5′ and 3′ portions of the cloned Edr segment are shown in lower case, numbered according to the mRNA sequence (A of natural AUG start site is base 452; accession no. AJ006464). The AUG for the expression of Edr sequences in pKT0/ Edr is derived from the vector (upper case, underlined). In ribosomal frameshifting assays, capped mRNAs were prepared by SP6 transcription of NdeI-linearized templates (unless otherwise stated). The predicted size of the non-frameshifted (stop) and frameshifted (fs) products generated from the translation of this mRNA in RRL is 29 and 32 kDa, respectively. For structural analysis of the Edr stimulatory RNA, a T3 promoter was introduced into the Edr sequence (at position 1357) to generate plasmid pKT0/ Edr /T3. Linearization of this plasmid with NdeI and subsequent transcription with T3 RNA polymerase yields a transcript of 152 nt.

    Techniques Used: Plasmid Preparation, Amplification, Polymerase Chain Reaction, Clone Assay, Sequencing, Expressing, Derivative Assay, Generated

    8) Product Images from "Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection"

    Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection

    Journal: Viruses

    doi: 10.3390/v10080422

    A trio of functional ZIKV cDNAs was created for the rescue of three molecularly cloned genetically divergent strains: rMR-766, rP6-740, and rPRVABC-59. ( A ) Construction of three full-length ZIKV cDNAs as BACs for MR-766, P6-740, and PRVABC-59. In all three cases, each genomic RNA (top panel) was first subcloned into five overlapping cDNAs (middle panel), which were then joined at four shared restriction sites as indicated to assemble its full-length cDNA without introducing any point mutations for cloning (bottom panel). Presented below the three full-length cDNAs are the sequences corresponding to the 5′ and 3′ termini conserved in all three ZIKVs (black lowercase), an SP6 promoter placed just upstream of the viral genome (magenta uppercase), and a run-off site positioned immediately downstream of the viral genome ( Psr I or Bar I, blue uppercase). Marked below the sequences are the transcription start (white arrowhead) and run-off (black arrowhead) sites; ( B ) Functionality of the three full-length ZIKV cDNAs. After linearization with Psr I or Bar I, as appropriate, each full-length cDNA was used as a template for in vitro transcription with SP6 RNA polymerase in the presence of the dinucleotide cap analog m 7 GpppA. Capped RNA transcripts were transfected into Vero cells to determine the number of infectious centers (plaques) counterstained with crystal violet at 5 days after transfection (RNA infectivity). At 36 h post-transfection, culture supernatants from RNA-transfected cells were harvested to estimate the level of virus production by plaque assays on Vero cells (Virus yield). Means and standard deviations from three independent experiments are shown; ( C ) Plaque morphology. The average plaque sizes were estimated by measuring 20 representative plaques.
    Figure Legend Snippet: A trio of functional ZIKV cDNAs was created for the rescue of three molecularly cloned genetically divergent strains: rMR-766, rP6-740, and rPRVABC-59. ( A ) Construction of three full-length ZIKV cDNAs as BACs for MR-766, P6-740, and PRVABC-59. In all three cases, each genomic RNA (top panel) was first subcloned into five overlapping cDNAs (middle panel), which were then joined at four shared restriction sites as indicated to assemble its full-length cDNA without introducing any point mutations for cloning (bottom panel). Presented below the three full-length cDNAs are the sequences corresponding to the 5′ and 3′ termini conserved in all three ZIKVs (black lowercase), an SP6 promoter placed just upstream of the viral genome (magenta uppercase), and a run-off site positioned immediately downstream of the viral genome ( Psr I or Bar I, blue uppercase). Marked below the sequences are the transcription start (white arrowhead) and run-off (black arrowhead) sites; ( B ) Functionality of the three full-length ZIKV cDNAs. After linearization with Psr I or Bar I, as appropriate, each full-length cDNA was used as a template for in vitro transcription with SP6 RNA polymerase in the presence of the dinucleotide cap analog m 7 GpppA. Capped RNA transcripts were transfected into Vero cells to determine the number of infectious centers (plaques) counterstained with crystal violet at 5 days after transfection (RNA infectivity). At 36 h post-transfection, culture supernatants from RNA-transfected cells were harvested to estimate the level of virus production by plaque assays on Vero cells (Virus yield). Means and standard deviations from three independent experiments are shown; ( C ) Plaque morphology. The average plaque sizes were estimated by measuring 20 representative plaques.

    Techniques Used: Functional Assay, Clone Assay, In Vitro, Transfection, Infection

    9) Product Images from "A molecular clone of Chronic Bee Paralysis Virus (CBPV) causes mortality in honey bee pupae (Apis mellifera)"

    Article Title: A molecular clone of Chronic Bee Paralysis Virus (CBPV) causes mortality in honey bee pupae (Apis mellifera)

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52822-1

    Synthetic RNA of the molecular CBPV clone. Plasmid DNAs were linearized with Sca I (RNA1) or Sac II (RNA2) and transcribed using SP6 polymerase. The synthetic RNA was injected into honey bee pupae to start the cycle of infection. Note that a DNA ladder is used for size estimation (left side).
    Figure Legend Snippet: Synthetic RNA of the molecular CBPV clone. Plasmid DNAs were linearized with Sca I (RNA1) or Sac II (RNA2) and transcribed using SP6 polymerase. The synthetic RNA was injected into honey bee pupae to start the cycle of infection. Note that a DNA ladder is used for size estimation (left side).

    Techniques Used: Plasmid Preparation, Injection, Infection

    10) Product Images from "The role of RNA pseudoknot stem 1 length in the promotion of efficient −1 ribosomal frameshifting 1"

    Article Title: The role of RNA pseudoknot stem 1 length in the promotion of efficient −1 ribosomal frameshifting 1

    Journal: Journal of Molecular Biology

    doi: 10.1006/jmbi.1999.2688

    The frameshift reporter construct pKA1. (a) Plasmid pKA1 was derived from pFScass 6 (Brierley et al ., 1992) by site-directed mutagenesis (see Materials and Methods). pKA1 contains a truncated version of the minimal IBV pseudoknot (white box) cloned into a reporter gene, the influenza PB2 gene (shaded boxes). Linearisation of the plasmid with Bam H1 and in vitro transcription using SP6 RNA polymerase yields an mRNA (2.4kb) that, when translated in RRL, is predicted to produce a 19 kDa non-frameshift product corresponding to ribosomes that terminate at the UGA termination codon (located immediately downstream of the slippery sequence UUUAAAC, shaded), and a 22 kDa −1 frameshift product. The 0-frame and −2/+1 frames are also open (to some extent) in this construct. Ribosomes which enter these frames produce 28 kDa and 85 kDa products respectively. A bacteriophage T7 promoter is present just upstream of the frameshift region; this promoter is employed to generate short, pseudoknot-containing transcripts from Bst NI-digested templates for secondary structure analysis. (b) The wild-type (wt) IBV, the minimal IBV and the pKA1 pseudoknots (PK). The minimal IBV frameshift signal present in pFScass 6 is based on the wild-type IBV frameshift signal and is fully functional in frameshifting (Brierley et al ., 1992) . It differs from the wild-type in a number of ways; a termination codon (UGA) is present immediately downstream of the slippery sequence (UUUAAAC, boxed) to terminate zero frame ribosomes, loop 2 of the pseudoknot contains 8 rather than 32 nt, the G·A mismatched pair in stem 1 of the wild-type pseudoknot is replaced by a U·G pair, the G nucleotide of loop 1 is replaced by a C nucleotide and, finally, the minimal pseudoknot has no stop codons. Plasmid pKA1 was derived from pFScass 6 by deletion mutagenesis (see Materials and Methods). The type and number of bases in the loops and stem 2 remained unaltered. The predicted stability of stem 1 of each construct is shown (calculated according to the rules by Turner et al ., 1988 , using a loop length of 8 nt; see the text).
    Figure Legend Snippet: The frameshift reporter construct pKA1. (a) Plasmid pKA1 was derived from pFScass 6 (Brierley et al ., 1992) by site-directed mutagenesis (see Materials and Methods). pKA1 contains a truncated version of the minimal IBV pseudoknot (white box) cloned into a reporter gene, the influenza PB2 gene (shaded boxes). Linearisation of the plasmid with Bam H1 and in vitro transcription using SP6 RNA polymerase yields an mRNA (2.4kb) that, when translated in RRL, is predicted to produce a 19 kDa non-frameshift product corresponding to ribosomes that terminate at the UGA termination codon (located immediately downstream of the slippery sequence UUUAAAC, shaded), and a 22 kDa −1 frameshift product. The 0-frame and −2/+1 frames are also open (to some extent) in this construct. Ribosomes which enter these frames produce 28 kDa and 85 kDa products respectively. A bacteriophage T7 promoter is present just upstream of the frameshift region; this promoter is employed to generate short, pseudoknot-containing transcripts from Bst NI-digested templates for secondary structure analysis. (b) The wild-type (wt) IBV, the minimal IBV and the pKA1 pseudoknots (PK). The minimal IBV frameshift signal present in pFScass 6 is based on the wild-type IBV frameshift signal and is fully functional in frameshifting (Brierley et al ., 1992) . It differs from the wild-type in a number of ways; a termination codon (UGA) is present immediately downstream of the slippery sequence (UUUAAAC, boxed) to terminate zero frame ribosomes, loop 2 of the pseudoknot contains 8 rather than 32 nt, the G·A mismatched pair in stem 1 of the wild-type pseudoknot is replaced by a U·G pair, the G nucleotide of loop 1 is replaced by a C nucleotide and, finally, the minimal pseudoknot has no stop codons. Plasmid pKA1 was derived from pFScass 6 by deletion mutagenesis (see Materials and Methods). The type and number of bases in the loops and stem 2 remained unaltered. The predicted stability of stem 1 of each construct is shown (calculated according to the rules by Turner et al ., 1988 , using a loop length of 8 nt; see the text).

    Techniques Used: Construct, Plasmid Preparation, Derivative Assay, Mutagenesis, Clone Assay, In Vitro, Sequencing, Functional Assay

    Related Articles

    Transfection:

    Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
    Article Snippet: .. Transcription and Transfection Infectious transcripts were synthesized from Psr I/Bar I-linearized BAC plasmid DNA with SP6 RNA polymerase as described [ ] in reactions containing m7 GpppA (New England Biolabs, Ipswich, MA, USA). .. RNA integrity was examined by agarose gel electrophoresis.

    In Vitro:

    Article Title: Characterization of the frameshift signal of Edr, a mammalian example of programmed -1 ribosomal frameshifting
    Article Snippet: .. In vitro transcription reactions employing the bacteriophage SP6 RNA polymerase were carried out essentially as described by Melton et al . ( ) and included the synthetic cap structure 7meGpppG (New England Biolabs) to generate capped mRNA. ..

    Article Title: Secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus 1
    Article Snippet: .. In vitro transcription reactions employing the bacteriophage SP6 RNA polymerase were carried out essentially as described by and included the synthetic cap structure me7 GpppG (New England Biolabs) to generate capped mRNA. .. Product RNA was recovered by a single extraction with phenol:chloroform (50:50, v/v) followed by ethanol precipitation in the presence of 2 M ammonium acetate.

    Synthesized:

    Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
    Article Snippet: .. Transcription and Transfection Infectious transcripts were synthesized from Psr I/Bar I-linearized BAC plasmid DNA with SP6 RNA polymerase as described [ ] in reactions containing m7 GpppA (New England Biolabs, Ipswich, MA, USA). .. RNA integrity was examined by agarose gel electrophoresis.

    other:

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *
    Article Snippet: SP6 RNA polymerase is a single large polypeptide chain of ≈98 kDa.

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *
    Article Snippet: The effect of Br-UTP on elongation by SP6 RNA polymerase ( ) is also consistent with prior observations that an RNA-DNA hybrid is important for lateral stability of phage polymerase on DNA and that bacteriophage T7 RNA polymerase terminates at sites at which the RNA is U-rich but which lacks a discernible stem-loop secondary structure ( , – ).

    BAC Assay:

    Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
    Article Snippet: .. Transcription and Transfection Infectious transcripts were synthesized from Psr I/Bar I-linearized BAC plasmid DNA with SP6 RNA polymerase as described [ ] in reactions containing m7 GpppA (New England Biolabs, Ipswich, MA, USA). .. RNA integrity was examined by agarose gel electrophoresis.

    Plasmid Preparation:

    Article Title: Functional Genomics and Immunologic Tools: The Impact of Viral and Host Genetic Variations on the Outcome of Zika Virus Infection
    Article Snippet: .. Transcription and Transfection Infectious transcripts were synthesized from Psr I/Bar I-linearized BAC plasmid DNA with SP6 RNA polymerase as described [ ] in reactions containing m7 GpppA (New England Biolabs, Ipswich, MA, USA). .. RNA integrity was examined by agarose gel electrophoresis.

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    New England Biolabs sp6
    LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for <t>SP6-9</t> . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm
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    LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm

    Journal: Nature Communications

    Article Title: Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

    doi: 10.1038/s41467-018-06092-6

    Figure Lengend Snippet: LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm

    Article Snippet: T7 (Roche) and SP6 (NEB) RNA polymerases were used for transcription of each strand.

    Techniques: Antibody Labeling

    Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.

    Journal: The Journal of Biological Chemistry

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *

    doi:

    Figure Lengend Snippet: Transcription through site Ia by phage SP6 RNA polymerase A, pGEMTerm DNA cut with Bst EII ( lane 1 ), Pst I ( lane 2 ), or Nsi I ( lanes 3 – 5 ) were transcribed in vitro with SP6 RNA polymerase in the presence of 2.4 mM each ATP, UTP, and GTP, and the indicated concentration of CTP for 2 h ( lanes 1 – 5 ) or 30 min ( lanes 6 – 9 ). In lane 9 , UTP was replaced with 2 mM 5-Br-UTP. After incubation, samples were made of 100 μ g/ml in heparin ( lanes 7 and 8 ) and 1 mM in CTP ( lane 8 ) and incubated for an additional 15 min. The addition of heparin results in a new slower migrating band, the identity of which is unknown. Lane M, reference RNAs of 260, 380, 420, and 540 bases. B, reactions containing 2.4 mM each ATP and GTP, 10 μ M CTP, and either 1 mM ( lane 1 ), 750 μ M ( lane 2 ), 500 μ M ( lane 3 ), 250 μ M ( lane 4 ), or no UTP ( lane 5 ) were incubated for 30 min at 37 °C. The indicated concentrations of 5-Br-UTP were added to each to bring the total uridine nucleotide concentration to 1 mM. Percent readthrough (100 × (phosphorimager units in the run-off RNA)/(units in run-off RNA + transcript Ia + transcript II)), was plotted versus Br-UTP concentration.

    Article Snippet: One reaction contained 10 units of SP6 RNA polymerase (New England Biolabs, Beverly, MA or Life Technologies, Inc.), 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 28 units of RNasin™ (Promega, Madison, WI), 0.5 μ g of linearized pGEMTerm, 10 μ Ci [ α -32 P]CTP (400 Ci/mmol), 2.4 mM each of ATP, UTP, GTP, and varying concentrations of CTP.

    Techniques: IA, In Vitro, Concentration Assay, Incubation

    Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.

    Journal: The Journal of Biological Chemistry

    Article Title: Recognition of a Human Arrest Site Is Conserved between RNA Polymerase II and Prokaryotic RNA Polymerases *

    doi:

    Figure Lengend Snippet: Templates used for in vitro transcription A, pAdTerm-2 contains the core adenovirus major late promoter (−50 to +10) and a 285-base pair segment of the human histone H3.3 gene. The sequence around arrest site Ia ( overlined ) is shown. Numbering refers to base positions in the transcript relative to the transcription start (+1). The phosphodiester bond cleaved by RNA polymerase II in arrested Ia complexes is indicated ( downward arrowhead ). Approximate sizes of the expected transcripts are shown. Bold ). B, p rrn -Ia contains DNA sequence from the P 2 promoter of the E. coli rrn B operon inserted upstream of the human H3.3 sequences in pGEMTerm (see below). Approximate lengths of transcripts initiating from P 2 are indicated on the right. C, pGEMTerm contains the human H3.3 sequences inserted into pGEM2 downstream of an SP6 RNA polymerase promoter. Approximate transcript lengths are indicated on the right.

    Article Snippet: One reaction contained 10 units of SP6 RNA polymerase (New England Biolabs, Beverly, MA or Life Technologies, Inc.), 40 mM Tris-HCl (pH 7.9), 6 mM MgCl2 , 2 mM spermidine, 10 mM dithiothreitol, 28 units of RNasin™ (Promega, Madison, WI), 0.5 μ g of linearized pGEMTerm, 10 μ Ci [ α -32 P]CTP (400 Ci/mmol), 2.4 mM each of ATP, UTP, GTP, and varying concentrations of CTP.

    Techniques: In Vitro, Sequencing, IA

    Construction of plasmid pRSV1. A 266 bp Avr II- Bss HII fragment encompassing the RSV frameshift region was isolated from plasmid pATV8 (which harbours a proviral clone of RSV Prague C; Schwartz et al ., 1983 ) and subcloned into the PB1 reporter gene of plasmid pPSO.1, a derivative of pPSO (Somogyi et al ., 1993) with unique Avr II and Bss HII sites. The open reading frames in pRSV1 are such that the upstream portion of the PB1 reporter gene is in frame with the gag gene and the downstream portion, the pol gene. Analysis of the secondary structure of the RNA at the RSV frameshift site by primer extension was on mRNAs prepared by linearisation of pRSV1 with Pvu II and subsequent transcription with T7 RNA polymerase. For structural analysis of end-labelled RNA, related plasmids containing a T3 promoter were prepared (see the text), linearised with Dde I and transcribed with T3 RNA polymerase. In ribosomal frameshift assays, capped mRNAs were prepared by SP6 transcription from Ava II linearised template. The predicted non-frameshifted (45 kDa) and frameshifted (70 kDa) species generated upon in vitro translation of such mRNAs in RRL are shown.

    Journal: Journal of Molecular Biology

    Article Title: Secondary structure and mutational analysis of the ribosomal frameshift signal of rous sarcoma virus 1

    doi: 10.1006/jmbi.1998.2186

    Figure Lengend Snippet: Construction of plasmid pRSV1. A 266 bp Avr II- Bss HII fragment encompassing the RSV frameshift region was isolated from plasmid pATV8 (which harbours a proviral clone of RSV Prague C; Schwartz et al ., 1983 ) and subcloned into the PB1 reporter gene of plasmid pPSO.1, a derivative of pPSO (Somogyi et al ., 1993) with unique Avr II and Bss HII sites. The open reading frames in pRSV1 are such that the upstream portion of the PB1 reporter gene is in frame with the gag gene and the downstream portion, the pol gene. Analysis of the secondary structure of the RNA at the RSV frameshift site by primer extension was on mRNAs prepared by linearisation of pRSV1 with Pvu II and subsequent transcription with T7 RNA polymerase. For structural analysis of end-labelled RNA, related plasmids containing a T3 promoter were prepared (see the text), linearised with Dde I and transcribed with T3 RNA polymerase. In ribosomal frameshift assays, capped mRNAs were prepared by SP6 transcription from Ava II linearised template. The predicted non-frameshifted (45 kDa) and frameshifted (70 kDa) species generated upon in vitro translation of such mRNAs in RRL are shown.

    Article Snippet: In vitro transcription reactions employing the bacteriophage SP6 RNA polymerase were carried out essentially as described by and included the synthetic cap structure me7 GpppG (New England Biolabs) to generate capped mRNA.

    Techniques: Plasmid Preparation, Isolation, Antiviral Assay, Generated, In Vitro

    LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm

    Journal: Nature Communications

    Article Title: Conservation of epigenetic regulation by the MLL3/4 tumour suppressor in planarian pluripotent stem cells

    doi: 10.1038/s41467-018-06092-6

    Figure Lengend Snippet: LPT (RNAi) results in differentiation defects and outgrowth formation during regeneration. a A schematic showing the amputation of RNAi worms into head (H), middle (M) and tail (T) pieces in order to observe regeneration of different structures. The time-course of all the experiments on Mll3/4 knockdown animals is depicted underneath the worm schematic. A total of 9 days of dsRNA microinjection-mediated RNAi was followed by amputation on the 10 th day and subsequent observation of regeneration. b Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 7 of regeneration. Yellow arrows point towards the defects in blastema formation. c Head, middle and tail pieces following LPT (RNAi) or control GFP (RNAi) at day 11 of regeneration. Red arrows point towards outgrowths. d Homeostatic animals following LPT (RNAi) or control GFP (RNAi) at day 14 post RNAi. Red arrows point towards outgrowths. e Gut regeneration and maintenance in middle pieces following LPT (RNAi), as illustrated by RNA probe for the gene porcupine-1 at 8 days of regeneration. f Brain regeneration in middle pieces at 8 days post-amputation following LPT (RNAi), as illustrated by anti-SYNORF-1 antibody labeling the central nervous system (CNS). g Optic chiasm recovery in tail pieces at 8 days of regeneration following LPT (RNAi), as shown by anti-VC-1 antibody. h Recovery of optic cups and organized trail of optic cup precursor cells in tail pieces at 8 days of regeneration following LPT (RNAi), as demonstrated by RNA probe for SP6-9 . i Pharynx recovery in head pieces at 8 days of regeneration following LPT (RNAi), as illustrated by RNA probe for laminin . Number of animals with observable phenotype are recorded out of the total number of animals in the top right of each panel. Scale bars: 200 μm

    Article Snippet: T7 (Roche) and SP6 (NEB) RNA polymerases were used for transcription of each strand.

    Techniques: Antibody Labeling