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  • 93
    New England Biolabs ptwin1 vector dna
    Ptwin1 Vector Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 93/100, based on 43 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ptwin1 vector dna/product/New England Biolabs
    Average 93 stars, based on 43 article reviews
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    99
    Millipore dna vector
    Specific and non-specific binding of <t>DNA</t> by <t>ParB:</t> a speculative model for spreading at parS sites. ( A ) A region of a DNA molecule containing a specific binding site is shown. ( B ) Specific binding. At low concentrations, ParB binds to parS sequences via the central helix-turn-helix motifs to form a ParB 2 :DNA complex (supporting data in Figures 1 , 3 and 4 ). ( C ) Non-specific DNA binding. Elevated concentrations of ParB allow co-operative non-specific binding via a second (hypothetical) DNA binding domain (supporting data in Figures 1 and 2 ). The continued self-association of ParB (indicated with arrows) via at least two interfaces subsequently leads to formation of higher order networks and DNA condensation. This transition is not dependent on the presence of parS . ( D ) The condensed nucleoprotein network (supporting data in Figures 5 – 8 ) may contain both specific and non-specific DNA binding sites (see main text for justification) that trap loops of DNA that are anchored around parS if the parS site is present. For simplicity, the specific binding sites for most of the ParB dimers are shown unoccupied. Such structures might bridge larger distances, including between distant parS loci, through the sharing of segments of DNA, or via additional protein:protein interactions (indicated with the faded nucleoprotein complex).
    Dna Vector, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore vector dna
    Human PBMCs anchoring GR express <t>nestin</t> and the stem cell marker CD133. (A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the <t>DNA</t> stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.
    Vector Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 544 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher vector dna
    hsp40 and hsp70 influence on molecular partitioning of Httex1 as monomers, oligomers, and inclusions. The proportion of Httex1-Emerald molecules in Neuro2a cell lysate is shown as defined by gel filtration (which can quantitate monomers and oligomers) and SV analysis in 2 m sucrose at 3,000 rpm (which can quantitate inclusions). Cells were <t>transfected</t> with equal <t>DNA</t> quanta of Httex1-Emerald and hsp40 and hsp70, buffered with the GFP inv construct under the same conditions as for the flow cytometry data in Figs. 3 and 4 . Total abundance of Htt based on fluorescence yield in lysate ( n = 3, mean ± S.D. shown). For ANOVA, individual pairwise comparisons against Htt alone as control are shown for p
    Vector Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1459 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM vector dna
    Silencing of TFIIB like expression affects SL RNA synthesis in vivo. ( A ) Total RNAs prepared from RNAi-induced cells were analyzed by primer extension assays using 5′- 32 P-end labeled oligonucleotides specific for SL RNA and U2 snRNA. The lower of the two extension products for the SL RNA is a result of the hyper-methylated SL RNA cap, which prematurely terminates reverse transcription. <t>DNA</t> marker (M) sizes are indicated on the left. ( B ) Nascent RNAs were labeled with [α- 32 P]UTP in permeabilized cells in which expression of TFIIB like dsRNA was induced for the times specified. The RNAs were separated on a 6% polyacrylamide/50% urea gel and visualized by autoradiography. SL RNA and tRNA are indicated on the right and DNA marker (M) sizes on the left. ( C ) Labeled, nascent RNA was used to probe dot blots containing the complete coding regions of the SL RNA, <t>GPEET</t> procyclin, α-tubulin, heat shock protein 70 (HSP70), 18S ribosomal RNA, U2 snRNA and U6 snRNA. The vector pTZ18U served as a control. Shown are low exposures of experiments I–III for the SL RNA and a long exposure of experiment I for all other RNAs. ( D ) Quantification of the dot blot signal strengths from three independent experiments. The signal of non-induced cells was set to 100%.
    Vector Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    TaKaRa dna vector
    Production of the HA-cyclin 1 and FLAG-CDKA expressing double-knock-in strain. (A) Schematic diagram of the knock-in of <t>HA-CYC1</t> and FLAG-CDKA into the chromosomal CYC1 and CDKA loci by homologous recombination. The first line indicates the linear <t>DNA</t> vector that was transformed into the M4 strain. The second and third lines indicate the genomic structure of the parental M4 strain and resultant HA-cyclin 1 strain, respectively. The fourth line indicates the linear DNA vector that was transformed into the HA-cyclin 1 strain. The fifth and sixth lines indicate the CDKA locus of the parental HA-cyclin 1 strain and the resultant HA-cyclin 1/FLAG-CDKA strain, respectively. The arrowheads indicate the positions of the PCR primers used in (B) . (B) PCR analysis of the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains, confirming the occurrence of homologous recombination events. The M4 strain was used as a negative control. The positions of the primer sets No. 45/No. 46 and No. 47/No. 48 are shown in (A) and the exact positions and sequences are indicated in Supplementary Table S1. The predicted size of the PCR product amplified from the CYC1 locus is 6.8 kb for the HA-cyclin 1 strain and 4.0 kb for the M4 strain. The predicted size of the PCR product amplified from the CDKA locus is 4.5 kb for the HA-cyclin 1/FLAG-CDKA strain and 2.9 kb for the HA-cyclin 1 strain. (C) Immunoblotting with the anti-HA and the anti-FLAG antibodies. The predicted sizes of HA-cyclin 1 and FLAG-CDKA proteins are 48 and 40 kDa, respectively. (D) Growth curves of the D-sfGFP strain (an sfGFP expresser was used as a control), the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains.
    Dna Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 91/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    Bio-Rad dna vector
    Production of the HA-cyclin 1 and FLAG-CDKA expressing double-knock-in strain. (A) Schematic diagram of the knock-in of <t>HA-CYC1</t> and FLAG-CDKA into the chromosomal CYC1 and CDKA loci by homologous recombination. The first line indicates the linear <t>DNA</t> vector that was transformed into the M4 strain. The second and third lines indicate the genomic structure of the parental M4 strain and resultant HA-cyclin 1 strain, respectively. The fourth line indicates the linear DNA vector that was transformed into the HA-cyclin 1 strain. The fifth and sixth lines indicate the CDKA locus of the parental HA-cyclin 1 strain and the resultant HA-cyclin 1/FLAG-CDKA strain, respectively. The arrowheads indicate the positions of the PCR primers used in (B) . (B) PCR analysis of the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains, confirming the occurrence of homologous recombination events. The M4 strain was used as a negative control. The positions of the primer sets No. 45/No. 46 and No. 47/No. 48 are shown in (A) and the exact positions and sequences are indicated in Supplementary Table S1. The predicted size of the PCR product amplified from the CYC1 locus is 6.8 kb for the HA-cyclin 1 strain and 4.0 kb for the M4 strain. The predicted size of the PCR product amplified from the CDKA locus is 4.5 kb for the HA-cyclin 1/FLAG-CDKA strain and 2.9 kb for the HA-cyclin 1 strain. (C) Immunoblotting with the anti-HA and the anti-FLAG antibodies. The predicted sizes of HA-cyclin 1 and FLAG-CDKA proteins are 48 and 40 kDa, respectively. (D) Growth curves of the D-sfGFP strain (an sfGFP expresser was used as a control), the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains.
    Dna Vector, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 89/100, based on 124 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Addgene inc vector dna
    Production of the HA-cyclin 1 and FLAG-CDKA expressing double-knock-in strain. (A) Schematic diagram of the knock-in of <t>HA-CYC1</t> and FLAG-CDKA into the chromosomal CYC1 and CDKA loci by homologous recombination. The first line indicates the linear <t>DNA</t> vector that was transformed into the M4 strain. The second and third lines indicate the genomic structure of the parental M4 strain and resultant HA-cyclin 1 strain, respectively. The fourth line indicates the linear DNA vector that was transformed into the HA-cyclin 1 strain. The fifth and sixth lines indicate the CDKA locus of the parental HA-cyclin 1 strain and the resultant HA-cyclin 1/FLAG-CDKA strain, respectively. The arrowheads indicate the positions of the PCR primers used in (B) . (B) PCR analysis of the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains, confirming the occurrence of homologous recombination events. The M4 strain was used as a negative control. The positions of the primer sets No. 45/No. 46 and No. 47/No. 48 are shown in (A) and the exact positions and sequences are indicated in Supplementary Table S1. The predicted size of the PCR product amplified from the CYC1 locus is 6.8 kb for the HA-cyclin 1 strain and 4.0 kb for the M4 strain. The predicted size of the PCR product amplified from the CDKA locus is 4.5 kb for the HA-cyclin 1/FLAG-CDKA strain and 2.9 kb for the HA-cyclin 1 strain. (C) Immunoblotting with the anti-HA and the anti-FLAG antibodies. The predicted sizes of HA-cyclin 1 and FLAG-CDKA proteins are 48 and 40 kDa, respectively. (D) Growth curves of the D-sfGFP strain (an sfGFP expresser was used as a control), the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains.
    Vector Dna, supplied by Addgene inc, used in various techniques. Bioz Stars score: 92/100, based on 67 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    LGC Genomics GmbH vector dna
    Production of the HA-cyclin 1 and FLAG-CDKA expressing double-knock-in strain. (A) Schematic diagram of the knock-in of <t>HA-CYC1</t> and FLAG-CDKA into the chromosomal CYC1 and CDKA loci by homologous recombination. The first line indicates the linear <t>DNA</t> vector that was transformed into the M4 strain. The second and third lines indicate the genomic structure of the parental M4 strain and resultant HA-cyclin 1 strain, respectively. The fourth line indicates the linear DNA vector that was transformed into the HA-cyclin 1 strain. The fifth and sixth lines indicate the CDKA locus of the parental HA-cyclin 1 strain and the resultant HA-cyclin 1/FLAG-CDKA strain, respectively. The arrowheads indicate the positions of the PCR primers used in (B) . (B) PCR analysis of the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains, confirming the occurrence of homologous recombination events. The M4 strain was used as a negative control. The positions of the primer sets No. 45/No. 46 and No. 47/No. 48 are shown in (A) and the exact positions and sequences are indicated in Supplementary Table S1. The predicted size of the PCR product amplified from the CYC1 locus is 6.8 kb for the HA-cyclin 1 strain and 4.0 kb for the M4 strain. The predicted size of the PCR product amplified from the CDKA locus is 4.5 kb for the HA-cyclin 1/FLAG-CDKA strain and 2.9 kb for the HA-cyclin 1 strain. (C) Immunoblotting with the anti-HA and the anti-FLAG antibodies. The predicted sizes of HA-cyclin 1 and FLAG-CDKA proteins are 48 and 40 kDa, respectively. (D) Growth curves of the D-sfGFP strain (an sfGFP expresser was used as a control), the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains.
    Vector Dna, supplied by LGC Genomics GmbH, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vector dna/product/LGC Genomics GmbH
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    95
    New England Biolabs ptxb1 vector dna
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Ptxb1 Vector Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Thermo Fisher pcdna3 vector dna
    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the <t>pTXB1</t> vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .
    Pcdna3 Vector Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Specific and non-specific binding of DNA by ParB: a speculative model for spreading at parS sites. ( A ) A region of a DNA molecule containing a specific binding site is shown. ( B ) Specific binding. At low concentrations, ParB binds to parS sequences via the central helix-turn-helix motifs to form a ParB 2 :DNA complex (supporting data in Figures 1 , 3 and 4 ). ( C ) Non-specific DNA binding. Elevated concentrations of ParB allow co-operative non-specific binding via a second (hypothetical) DNA binding domain (supporting data in Figures 1 and 2 ). The continued self-association of ParB (indicated with arrows) via at least two interfaces subsequently leads to formation of higher order networks and DNA condensation. This transition is not dependent on the presence of parS . ( D ) The condensed nucleoprotein network (supporting data in Figures 5 – 8 ) may contain both specific and non-specific DNA binding sites (see main text for justification) that trap loops of DNA that are anchored around parS if the parS site is present. For simplicity, the specific binding sites for most of the ParB dimers are shown unoccupied. Such structures might bridge larger distances, including between distant parS loci, through the sharing of segments of DNA, or via additional protein:protein interactions (indicated with the faded nucleoprotein complex).

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: Specific and non-specific binding of DNA by ParB: a speculative model for spreading at parS sites. ( A ) A region of a DNA molecule containing a specific binding site is shown. ( B ) Specific binding. At low concentrations, ParB binds to parS sequences via the central helix-turn-helix motifs to form a ParB 2 :DNA complex (supporting data in Figures 1 , 3 and 4 ). ( C ) Non-specific DNA binding. Elevated concentrations of ParB allow co-operative non-specific binding via a second (hypothetical) DNA binding domain (supporting data in Figures 1 and 2 ). The continued self-association of ParB (indicated with arrows) via at least two interfaces subsequently leads to formation of higher order networks and DNA condensation. This transition is not dependent on the presence of parS . ( D ) The condensed nucleoprotein network (supporting data in Figures 5 – 8 ) may contain both specific and non-specific DNA binding sites (see main text for justification) that trap loops of DNA that are anchored around parS if the parS site is present. For simplicity, the specific binding sites for most of the ParB dimers are shown unoccupied. Such structures might bridge larger distances, including between distant parS loci, through the sharing of segments of DNA, or via additional protein:protein interactions (indicated with the faded nucleoprotein complex).

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Binding Assay

    Specific binding of ParB to the parS sequence. Electrophoretic mobility shift assay of ParB binding to a radiolabelled 147-bp substrate in a magnesium acetate containing gel-running buffer. ( A ) Titration of ParB on DNA containing a single parS site in the centre. ( B ) ParB titration on an equivalent substrate that is lacking a parS site (see Supplementary Table S1 for details). The species assigned as specific and non-specific complexes are labelled. The lower panels show the quantification of the gels revealing a highly sigmoidal pattern for non-specific binding. These data were fit to Equation ( 1 ) to yield the values shown.

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: Specific binding of ParB to the parS sequence. Electrophoretic mobility shift assay of ParB binding to a radiolabelled 147-bp substrate in a magnesium acetate containing gel-running buffer. ( A ) Titration of ParB on DNA containing a single parS site in the centre. ( B ) ParB titration on an equivalent substrate that is lacking a parS site (see Supplementary Table S1 for details). The species assigned as specific and non-specific complexes are labelled. The lower panels show the quantification of the gels revealing a highly sigmoidal pattern for non-specific binding. These data were fit to Equation ( 1 ) to yield the values shown.

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Binding Assay, Sequencing, Electrophoretic Mobility Shift Assay, Titration

    Specific binding of parS to ParB protects the helix-turn-helix region from proteolysis. ParB (2-μM dimer) was progressively digested into a large and a small fragment by trypsin, with approximate weights of 26 and 15 kDa, respectively, as determined by a comparison to molecular weight markers. N-terminal sequencing of the excised bands revealed the N-terminal sequences of these fragments to be MAKX and KXIN, respectively. The N-terminus of the large fragment is M1, with the C-terminus lying within the linker region between the central and C-terminal domains of ParB. The N-terminus of the small fragment is K7, which lies within the Box I motif, and the C-terminus is within the helix-turn-helix motif (K132 or K143). The lower panel shows a cartoon representation of the primary structure indicating the major degradation products. In the presence of parS DNA (20 μM), the degradation of the large fragment to the small fragment (and therefore cleavage near the helix-turn-helix motif) is substantially reduced, whereas an equivalent non-specific DNA does not have this effect.

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: Specific binding of parS to ParB protects the helix-turn-helix region from proteolysis. ParB (2-μM dimer) was progressively digested into a large and a small fragment by trypsin, with approximate weights of 26 and 15 kDa, respectively, as determined by a comparison to molecular weight markers. N-terminal sequencing of the excised bands revealed the N-terminal sequences of these fragments to be MAKX and KXIN, respectively. The N-terminus of the large fragment is M1, with the C-terminus lying within the linker region between the central and C-terminal domains of ParB. The N-terminus of the small fragment is K7, which lies within the Box I motif, and the C-terminus is within the helix-turn-helix motif (K132 or K143). The lower panel shows a cartoon representation of the primary structure indicating the major degradation products. In the presence of parS DNA (20 μM), the degradation of the large fragment to the small fragment (and therefore cleavage near the helix-turn-helix motif) is substantially reduced, whereas an equivalent non-specific DNA does not have this effect.

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Binding Assay, Molecular Weight, Sequencing

    Condensation of DNA by ParB monitored by magnetic tweezers. ( A ) Experimental configuration used to measure condensation dynamics mediated by ParB proteins with magnetic tweezers. ( B ) Schematic representation of the parS DNA substrate. ( C ) Condensation assay. At 4-pN stretching force, 1-μM ParB 2 was injected in the fluid cell and incubated for 2 min. Following incubation, the force was reduced to 0.34 pN. In the absence of protein this leads to the change in extension represented in the grey trace. However, in the presence of ParB we observed a progressive decrease of the extension until reaching a final extension near the surface. Raw data were acquired at 60 Hz (red) and filtered down to 2.4 Hz (black).

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: Condensation of DNA by ParB monitored by magnetic tweezers. ( A ) Experimental configuration used to measure condensation dynamics mediated by ParB proteins with magnetic tweezers. ( B ) Schematic representation of the parS DNA substrate. ( C ) Condensation assay. At 4-pN stretching force, 1-μM ParB 2 was injected in the fluid cell and incubated for 2 min. Following incubation, the force was reduced to 0.34 pN. In the absence of protein this leads to the change in extension represented in the grey trace. However, in the presence of ParB we observed a progressive decrease of the extension until reaching a final extension near the surface. Raw data were acquired at 60 Hz (red) and filtered down to 2.4 Hz (black).

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Injection, Incubation

    The stoichiometry of the ParB– parS complex. ( A ) Binding of ParB 2 (9 μM) to 24-bp Hex-labelled DNA (10 μM) analysed by SEC-MALS. Only DNA-containing species were observed by monitoring the (normalized) absorbance at 535 nm. With a parS containing DNA substrate (solid line) the major complex has a calculated Mw of 81.6 ± 1.9 kDa, consistent with a single ParB dimer bound to DNA. A lower abundance species is also seen with a calculated Mw of 112.1 ± 3. In contrast, ParB is unable to bind a non-specific substrate (dotted line). In that case, the DNA is found in a late eluting peak, for which no weight could be assigned due to poor light scattering. ( B ) Native-mass spectrometry of ParB binding to a 100-bp substrate containing a single parS sequence predominantly showed a single dimer bound to the DNA, as well as free DNA. The peak assignments are indicated using cartoons on the graph. Binding of ParB to non-specific DNA was not observed.

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: The stoichiometry of the ParB– parS complex. ( A ) Binding of ParB 2 (9 μM) to 24-bp Hex-labelled DNA (10 μM) analysed by SEC-MALS. Only DNA-containing species were observed by monitoring the (normalized) absorbance at 535 nm. With a parS containing DNA substrate (solid line) the major complex has a calculated Mw of 81.6 ± 1.9 kDa, consistent with a single ParB dimer bound to DNA. A lower abundance species is also seen with a calculated Mw of 112.1 ± 3. In contrast, ParB is unable to bind a non-specific substrate (dotted line). In that case, the DNA is found in a late eluting peak, for which no weight could be assigned due to poor light scattering. ( B ) Native-mass spectrometry of ParB binding to a 100-bp substrate containing a single parS sequence predominantly showed a single dimer bound to the DNA, as well as free DNA. The peak assignments are indicated using cartoons on the graph. Binding of ParB to non-specific DNA was not observed.

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Binding Assay, Size-exclusion Chromatography, Mass Spectrometry, Sequencing

    The ParB– parS complex does not recruit additional ParB molecules to neighbouring non-specific DNA. The binding of ParB to a 147-bp DNA labelled with Cy3 (Supplementary Table S1) results in an increase in fluorescence intensity. ( A ) Titration of ParB dimer on DNA containing a parS site in its centre. ( B ) Titration on an equivalent substrate that is lacking the parS site. These data were fit to Equation ( 1 ) to yield the values shown. The error bars represent the standard errors from three independent experiments.

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: The ParB– parS complex does not recruit additional ParB molecules to neighbouring non-specific DNA. The binding of ParB to a 147-bp DNA labelled with Cy3 (Supplementary Table S1) results in an increase in fluorescence intensity. ( A ) Titration of ParB dimer on DNA containing a parS site in its centre. ( B ) Titration on an equivalent substrate that is lacking the parS site. These data were fit to Equation ( 1 ) to yield the values shown. The error bars represent the standard errors from three independent experiments.

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Binding Assay, Fluorescence, Titration

    ParB-dependent condensation of DNA is reversible. ( A ) Decondensation of DNA by force. Characteristic force-induced decondensation traces for parS substrates are characterized by multiple small steps and a gradual increase of extension. ( B ) Decondensation of DNA by parS competitor DNA. Following condensation by reduction of force, a 5-μM parS competitor DNA was injected into the flow cell resulting in a process of decondensation characterized by large discrete steps. Decondensation stopped at the extension expected for 0.34 pN applied force in the absence of protein (indicated by the grey dashed line). The lack of protein bound to DNA was checked by raising the force up to 4 pN and reduction down to 0 pN; no (de)condensation effects were observed. ( C ) Condensation force dependency on ParB concentration. A maximum condensation force of 2.1 pN was measured at saturating protein concentration for both parS and non-specific DNA substrates. Errors are the standard deviation of measurements on different molecules ( N ≥ 5 molecules). ( D ) Mean force-extension curve of DNA molecules in the presence of 1-μM ParB 2 (circles). The solid line is included as a guide for the eye. Data in squares are the control experiment in the absence of protein and the solid line is a fit to the worm-like chain model. Errors are the standard deviation of measurements on different molecules ( N ≥ 15 molecules).

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: ParB-dependent condensation of DNA is reversible. ( A ) Decondensation of DNA by force. Characteristic force-induced decondensation traces for parS substrates are characterized by multiple small steps and a gradual increase of extension. ( B ) Decondensation of DNA by parS competitor DNA. Following condensation by reduction of force, a 5-μM parS competitor DNA was injected into the flow cell resulting in a process of decondensation characterized by large discrete steps. Decondensation stopped at the extension expected for 0.34 pN applied force in the absence of protein (indicated by the grey dashed line). The lack of protein bound to DNA was checked by raising the force up to 4 pN and reduction down to 0 pN; no (de)condensation effects were observed. ( C ) Condensation force dependency on ParB concentration. A maximum condensation force of 2.1 pN was measured at saturating protein concentration for both parS and non-specific DNA substrates. Errors are the standard deviation of measurements on different molecules ( N ≥ 5 molecules). ( D ) Mean force-extension curve of DNA molecules in the presence of 1-μM ParB 2 (circles). The solid line is included as a guide for the eye. Data in squares are the control experiment in the absence of protein and the solid line is a fit to the worm-like chain model. Errors are the standard deviation of measurements on different molecules ( N ≥ 15 molecules).

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Injection, Flow Cytometry, Concentration Assay, Protein Concentration, Standard Deviation

    ParB stabilizes crossovers and writhe formed by DNA braiding and bridging. ( A ) Cartoon of the experiment to braid DNA segments in trans . The application of one turn (clockwise or anti-clockwise) to doubly tethered beads promotes the cross-over of both DNAs, leading to a change of the extension (Δ z ). Subsequent untwisting to zero rotation immediately recovers the original extension. ( B ) Time trace of an experiment with two doubly tethered beads recorded simultaneously on bare DNA. ( C ) In the presence of ParB the cross-over is stabilized, and the extension does not recover after untwisting to zero rotation or even when one additional turn in the direction opposite to the cross-over is applied. ( D ) Injection of 5-μM parS DNA competitor oligonucleotide promotes the recovery of the full extension following ParB-mediated stabilization of a braid. ( E ) Cartoon of the experiment to bridge DNA segments in cis . Single torsionally constrained DNA molecules ( 1 ) are positively supercoiled at 4-pN force by applying 60 turns ( 2 ). Then, 1-μM ParB 2 is injected into the fluid cell ( 3 ). After full exchange of buffer, all of the turns are released ( 4 ). ( F ) DNA extension is displayed as a function of turns to highlight the hysteresis observed due to bridging of different regions of supercoiled DNA after introduction of ParB. The numbers indicate the different stages of the experiment as per the cartoon in part (E).

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: ParB stabilizes crossovers and writhe formed by DNA braiding and bridging. ( A ) Cartoon of the experiment to braid DNA segments in trans . The application of one turn (clockwise or anti-clockwise) to doubly tethered beads promotes the cross-over of both DNAs, leading to a change of the extension (Δ z ). Subsequent untwisting to zero rotation immediately recovers the original extension. ( B ) Time trace of an experiment with two doubly tethered beads recorded simultaneously on bare DNA. ( C ) In the presence of ParB the cross-over is stabilized, and the extension does not recover after untwisting to zero rotation or even when one additional turn in the direction opposite to the cross-over is applied. ( D ) Injection of 5-μM parS DNA competitor oligonucleotide promotes the recovery of the full extension following ParB-mediated stabilization of a braid. ( E ) Cartoon of the experiment to bridge DNA segments in cis . Single torsionally constrained DNA molecules ( 1 ) are positively supercoiled at 4-pN force by applying 60 turns ( 2 ). Then, 1-μM ParB 2 is injected into the fluid cell ( 3 ). After full exchange of buffer, all of the turns are released ( 4 ). ( F ) DNA extension is displayed as a function of turns to highlight the hysteresis observed due to bridging of different regions of supercoiled DNA after introduction of ParB. The numbers indicate the different stages of the experiment as per the cartoon in part (E).

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques: Injection

    ParB-mediated DNA condensation parameters. ( A ) Mean condensation curves for parS (black) and non-specific (red) DNA substrates ( N > 20). ( B ) Distribution of condensation times for parS (black) and non-specific (red) DNA substrates. ( C ) Distribution of final extensions after condensation for parS (black) and non-specific (red) DNA substrates. ( D ) Scatter plot of initial and final extensions for lambda-based substrates (black), parS -based substrates (blue) and pSP73-based substrates (green). All of the data shown were obtained from condensation curves at 0.34 pN.

    Journal: Nucleic Acids Research

    Article Title: Specific and non-specific interactions of ParB with DNA: implications for chromosome segregation

    doi: 10.1093/nar/gku1295

    Figure Lengend Snippet: ParB-mediated DNA condensation parameters. ( A ) Mean condensation curves for parS (black) and non-specific (red) DNA substrates ( N > 20). ( B ) Distribution of condensation times for parS (black) and non-specific (red) DNA substrates. ( C ) Distribution of final extensions after condensation for parS (black) and non-specific (red) DNA substrates. ( D ) Scatter plot of initial and final extensions for lambda-based substrates (black), parS -based substrates (blue) and pSP73-based substrates (green). All of the data shown were obtained from condensation curves at 0.34 pN.

    Article Snippet: DNA vector and substrate preparation The B. subtilis parB gene, which contains a single parS site (the sequence in the coding strand is 5′-TGTTCCACGTGAAACA), was amplified from B. subtilis 168 genomic DNA and cloned into the vectors pET28a(+) (Novagen) and pSP73 (Promega).

    Techniques:

    Human PBMCs anchoring GR express nestin and the stem cell marker CD133. (A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.

    Journal: PLoS ONE

    Article Title: Nestin Modulates Glucocorticoid Receptor Function by Cytoplasmic Anchoring

    doi: 10.1371/journal.pone.0006084

    Figure Lengend Snippet: Human PBMCs anchoring GR express nestin and the stem cell marker CD133. (A–D) Confocal images of PBMCs isolated from cord blood; (A1–A3) cells were attached to glass coverslips, fixed with acetone, and double labelled with antibodies to GR and Nes. The proportion of Nes positive cells is less than 1%. (B) Nes positive cells are negative for the hematopoietic lineage marker CD34.(C) Nes positive cells express the stem cell marker CD133 (merged image). (D) Merged image of cells extracted with a non-ionic detergent, fixed with paraformaldehyde, double labelled with antibodies to GR and Nes and incubated with the DNA stain DRAQ5 [79]. Cytoplasmic GR in Nes expressing cells resists extraction with a non-ionic detergent, whereas Nes negative cells retain GR only in the nucleus (arrow). Bars in B, C, and D = 5 µm.

    Article Snippet: Silencing of GR and nestin The lentiviral control vector DNA containing a scrambled sequence and the vector DNA for silencing human GR and human nestin were obtained from Sigma, Germany.

    Techniques: Marker, Isolation, Incubation, Staining, Expressing

    KSHV LANA-1 decreases BRD4 S -induced cyclin E promoter activity. Luciferase based reporter assay in HEK 293T cells. (A) HEK 293T cells in six-well plates were transfected with 50 ng of reporter plasmid (human cyclin E promoter) and 500 ng of expression plasmid for the myc-tagged BRD4 S ) expression constructs and lysed at confluence 48 h later. The total amount of DNA per transfection was adjusted to 1.05 μg using salmon sperm DNA. For details see Material and Methods. Absolute luciferase activities are shown with standard deviations (error bars). Additionally, mean relative activities are given as numbers on top of each column. (B) HEK 293T cells were cotransfected with 50 ng of reporter plasmid (human cyclin E promoter) and constant amounts of 500 ng of either empty vector pcDNA3 (mock) or BRD4 S ) expression constructs, and lysed at confluence 48 h later. Mean luciferase activities in the absence of BRD4 S expression were set at 1 for each amount (50 ng, 200 ng, or 500 ng) of the ORF 73 constructs or pcDNA3 (left three bars in panels 1, 2, 3, and 4). The relative activities in the presence of BRD4 S are shown in the three right bars in panels 1, 2, 3, and 4. Mean relative luciferase activities are depicted and given as numbers on top of each column. Standard deviations (error bars) are shown. +, presence of; −, absence of.

    Journal: Journal of Virology

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus LANA-1 Interacts with the Short Variant of BRD4 and Releases Cells from a BRD4- and BRD2/RING3-Induced G1 Cell Cycle Arrest ▿

    doi: 10.1128/JVI.00804-06

    Figure Lengend Snippet: KSHV LANA-1 decreases BRD4 S -induced cyclin E promoter activity. Luciferase based reporter assay in HEK 293T cells. (A) HEK 293T cells in six-well plates were transfected with 50 ng of reporter plasmid (human cyclin E promoter) and 500 ng of expression plasmid for the myc-tagged BRD4 S ) expression constructs and lysed at confluence 48 h later. The total amount of DNA per transfection was adjusted to 1.05 μg using salmon sperm DNA. For details see Material and Methods. Absolute luciferase activities are shown with standard deviations (error bars). Additionally, mean relative activities are given as numbers on top of each column. (B) HEK 293T cells were cotransfected with 50 ng of reporter plasmid (human cyclin E promoter) and constant amounts of 500 ng of either empty vector pcDNA3 (mock) or BRD4 S ) expression constructs, and lysed at confluence 48 h later. Mean luciferase activities in the absence of BRD4 S expression were set at 1 for each amount (50 ng, 200 ng, or 500 ng) of the ORF 73 constructs or pcDNA3 (left three bars in panels 1, 2, 3, and 4). The relative activities in the presence of BRD4 S are shown in the three right bars in panels 1, 2, 3, and 4. Mean relative luciferase activities are depicted and given as numbers on top of each column. Standard deviations (error bars) are shown. +, presence of; −, absence of.

    Article Snippet: Total amounts of transfected DNA were adjusted by using the respective empty vector DNA or salmon sperm DNA (Sigma).

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

    hsp40 and hsp70 influence on molecular partitioning of Httex1 as monomers, oligomers, and inclusions. The proportion of Httex1-Emerald molecules in Neuro2a cell lysate is shown as defined by gel filtration (which can quantitate monomers and oligomers) and SV analysis in 2 m sucrose at 3,000 rpm (which can quantitate inclusions). Cells were transfected with equal DNA quanta of Httex1-Emerald and hsp40 and hsp70, buffered with the GFP inv construct under the same conditions as for the flow cytometry data in Figs. 3 and 4 . Total abundance of Htt based on fluorescence yield in lysate ( n = 3, mean ± S.D. shown). For ANOVA, individual pairwise comparisons against Htt alone as control are shown for p

    Journal: The Journal of Biological Chemistry

    Article Title: A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70 *

    doi: 10.1074/jbc.M113.486944

    Figure Lengend Snippet: hsp40 and hsp70 influence on molecular partitioning of Httex1 as monomers, oligomers, and inclusions. The proportion of Httex1-Emerald molecules in Neuro2a cell lysate is shown as defined by gel filtration (which can quantitate monomers and oligomers) and SV analysis in 2 m sucrose at 3,000 rpm (which can quantitate inclusions). Cells were transfected with equal DNA quanta of Httex1-Emerald and hsp40 and hsp70, buffered with the GFP inv construct under the same conditions as for the flow cytometry data in Figs. 3 and 4 . Total abundance of Htt based on fluorescence yield in lysate ( n = 3, mean ± S.D. shown). For ANOVA, individual pairwise comparisons against Htt alone as control are shown for p

    Article Snippet: The following day, the cells in each well were transfected with 2 μl of Lipofectamine 2000/0.8 μg of vector DNA according to the manufacturer's instructions (Invitrogen).

    Techniques: Filtration, Transfection, Construct, Flow Cytometry, Cytometry, Fluorescence

    hsp40 and hsp70 increase total abundance of Httex1 molecules in the cell population. The total amount of Httex1-Emerald molecules (based on Emerald fluorescence intensities) in Neuro2a cell lysate is shown as defined by gel filtration (which can quantitate monomers and oligomers) and SV analysis in 2 m sucrose at 3,000 rpm (which can quantitate inclusions). Cells were transfected with equal DNA quanta of Httex1-Emerald and hsp40 and hsp70, buffered with the GFP inv construct under the same conditions as for the flow cytometry data in Figs. 3 and 4 . n = 3, mean ± S.D. shown. For ANOVA, individual pairwise comparisons against Htt alone as control are shown for p

    Journal: The Journal of Biological Chemistry

    Article Title: A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70 *

    doi: 10.1074/jbc.M113.486944

    Figure Lengend Snippet: hsp40 and hsp70 increase total abundance of Httex1 molecules in the cell population. The total amount of Httex1-Emerald molecules (based on Emerald fluorescence intensities) in Neuro2a cell lysate is shown as defined by gel filtration (which can quantitate monomers and oligomers) and SV analysis in 2 m sucrose at 3,000 rpm (which can quantitate inclusions). Cells were transfected with equal DNA quanta of Httex1-Emerald and hsp40 and hsp70, buffered with the GFP inv construct under the same conditions as for the flow cytometry data in Figs. 3 and 4 . n = 3, mean ± S.D. shown. For ANOVA, individual pairwise comparisons against Htt alone as control are shown for p

    Article Snippet: The following day, the cells in each well were transfected with 2 μl of Lipofectamine 2000/0.8 μg of vector DNA according to the manufacturer's instructions (Invitrogen).

    Techniques: Fluorescence, Filtration, Transfection, Construct, Flow Cytometry, Cytometry

    Aggregation “kinetics” of Httex1 in cells and effect of overexpressing hsp40 and hsp70. Flow cytometry data of Neuro2a cells transfected with Httex1 TC9 -Cerulean in the mKate2-F IRES vector (pTIREX) is binned into Htt expression level and shown as proportion of cells with inclusions (i population) from total number of cells in each bin. The i population was calculated by gating the pulse width and height values of Cerulean (Pacific-Blue channel) as described previously ( 19 ). The Httex1 TC9 -Cerulean IRES vector was coexpressed with a nonfluorescent Y66L mutant of GFP (GFP inv ) as a “blank” (alone) or with DNAJB1 plus GFP inv (Hsp40), HSPA1A plus GFP inv (Hsp70), or DNAJB1 plus HSPA1A (Hsp40 and Hsp70). GFP inv was used to buffer DNA levels so to enable the same quanta of DNA of Httex1 TC9 -Cerulean (1/3 quantum), and each chaperone (1/3 quantum) was used in transfections for each treatment. Data shows mean (central lines) ± S.D. (outer boundaries); n = 3.

    Journal: The Journal of Biological Chemistry

    Article Title: A Platform to View Huntingtin Exon 1 Aggregation Flux in the Cell Reveals Divergent Influences from Chaperones hsp40 and hsp70 *

    doi: 10.1074/jbc.M113.486944

    Figure Lengend Snippet: Aggregation “kinetics” of Httex1 in cells and effect of overexpressing hsp40 and hsp70. Flow cytometry data of Neuro2a cells transfected with Httex1 TC9 -Cerulean in the mKate2-F IRES vector (pTIREX) is binned into Htt expression level and shown as proportion of cells with inclusions (i population) from total number of cells in each bin. The i population was calculated by gating the pulse width and height values of Cerulean (Pacific-Blue channel) as described previously ( 19 ). The Httex1 TC9 -Cerulean IRES vector was coexpressed with a nonfluorescent Y66L mutant of GFP (GFP inv ) as a “blank” (alone) or with DNAJB1 plus GFP inv (Hsp40), HSPA1A plus GFP inv (Hsp70), or DNAJB1 plus HSPA1A (Hsp40 and Hsp70). GFP inv was used to buffer DNA levels so to enable the same quanta of DNA of Httex1 TC9 -Cerulean (1/3 quantum), and each chaperone (1/3 quantum) was used in transfections for each treatment. Data shows mean (central lines) ± S.D. (outer boundaries); n = 3.

    Article Snippet: The following day, the cells in each well were transfected with 2 μl of Lipofectamine 2000/0.8 μg of vector DNA according to the manufacturer's instructions (Invitrogen).

    Techniques: Flow Cytometry, Cytometry, Transfection, Plasmid Preparation, Expressing, Mutagenesis

    Silencing of TFIIB like expression affects SL RNA synthesis in vivo. ( A ) Total RNAs prepared from RNAi-induced cells were analyzed by primer extension assays using 5′- 32 P-end labeled oligonucleotides specific for SL RNA and U2 snRNA. The lower of the two extension products for the SL RNA is a result of the hyper-methylated SL RNA cap, which prematurely terminates reverse transcription. DNA marker (M) sizes are indicated on the left. ( B ) Nascent RNAs were labeled with [α- 32 P]UTP in permeabilized cells in which expression of TFIIB like dsRNA was induced for the times specified. The RNAs were separated on a 6% polyacrylamide/50% urea gel and visualized by autoradiography. SL RNA and tRNA are indicated on the right and DNA marker (M) sizes on the left. ( C ) Labeled, nascent RNA was used to probe dot blots containing the complete coding regions of the SL RNA, GPEET procyclin, α-tubulin, heat shock protein 70 (HSP70), 18S ribosomal RNA, U2 snRNA and U6 snRNA. The vector pTZ18U served as a control. Shown are low exposures of experiments I–III for the SL RNA and a long exposure of experiment I for all other RNAs. ( D ) Quantification of the dot blot signal strengths from three independent experiments. The signal of non-induced cells was set to 100%.

    Journal: Nucleic Acids Research

    Article Title: A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei

    doi: 10.1093/nar/gkl090

    Figure Lengend Snippet: Silencing of TFIIB like expression affects SL RNA synthesis in vivo. ( A ) Total RNAs prepared from RNAi-induced cells were analyzed by primer extension assays using 5′- 32 P-end labeled oligonucleotides specific for SL RNA and U2 snRNA. The lower of the two extension products for the SL RNA is a result of the hyper-methylated SL RNA cap, which prematurely terminates reverse transcription. DNA marker (M) sizes are indicated on the left. ( B ) Nascent RNAs were labeled with [α- 32 P]UTP in permeabilized cells in which expression of TFIIB like dsRNA was induced for the times specified. The RNAs were separated on a 6% polyacrylamide/50% urea gel and visualized by autoradiography. SL RNA and tRNA are indicated on the right and DNA marker (M) sizes on the left. ( C ) Labeled, nascent RNA was used to probe dot blots containing the complete coding regions of the SL RNA, GPEET procyclin, α-tubulin, heat shock protein 70 (HSP70), 18S ribosomal RNA, U2 snRNA and U6 snRNA. The vector pTZ18U served as a control. Shown are low exposures of experiments I–III for the SL RNA and a long exposure of experiment I for all other RNAs. ( D ) Quantification of the dot blot signal strengths from three independent experiments. The signal of non-induced cells was set to 100%.

    Article Snippet: Briefly, reactions of 40 µl containing 8 µl of extract, 20 mM potassium l -glutamate, 20 mM KCl, 3 mM MgCl2 , 20 mM HEPES–KOH, pH 7.7, 0.5 mM of each nucleoside triphosphate (NTP), 20 mM creatine phosphate, 0.48 mg/ml of creatine kinase, 2.5% polyethylene glycol, 0.2 mM EDTA, 0.5 mM EGTA, 4 mM DTT, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 12.5 µg/ml vector DNA, 20 µg/ml GPEET-trm template and 7.5 µg/ml SLins19 template were incubated for 1 h at 27°C.

    Techniques: Expressing, In Vivo, Labeling, Methylation, Marker, Autoradiography, Plasmid Preparation, Dot Blot

    TFIIB like specifically binds to the SLRNA promoter. Promoter pull-down assays were carried out with TbH8 extract and immobilized DNAs of GPEET, SLRNA, RRNA and VSG expression site ( VSG ES ) promoters. The numbers indicate the end positions of the DNA fragments relative to the transcription initiation sites. In addition to these promoter DNAs, a 222 bp nonspecific DNA and the complete intergenic region of HSP70 genes 2 and 3 ( H23 ) were analyzed. Proteins which bound to the DNAs were analyzed by immunoblotting using the PTP-specific PAP reagent and a polyclonal antibody against the SNAPc subunit SNAP3.

    Journal: Nucleic Acids Research

    Article Title: A TFIIB-like protein is indispensable for spliced leader RNA gene transcription in Trypanosoma brucei

    doi: 10.1093/nar/gkl090

    Figure Lengend Snippet: TFIIB like specifically binds to the SLRNA promoter. Promoter pull-down assays were carried out with TbH8 extract and immobilized DNAs of GPEET, SLRNA, RRNA and VSG expression site ( VSG ES ) promoters. The numbers indicate the end positions of the DNA fragments relative to the transcription initiation sites. In addition to these promoter DNAs, a 222 bp nonspecific DNA and the complete intergenic region of HSP70 genes 2 and 3 ( H23 ) were analyzed. Proteins which bound to the DNAs were analyzed by immunoblotting using the PTP-specific PAP reagent and a polyclonal antibody against the SNAPc subunit SNAP3.

    Article Snippet: Briefly, reactions of 40 µl containing 8 µl of extract, 20 mM potassium l -glutamate, 20 mM KCl, 3 mM MgCl2 , 20 mM HEPES–KOH, pH 7.7, 0.5 mM of each nucleoside triphosphate (NTP), 20 mM creatine phosphate, 0.48 mg/ml of creatine kinase, 2.5% polyethylene glycol, 0.2 mM EDTA, 0.5 mM EGTA, 4 mM DTT, 10 mg/ml leupeptin, 10 mg/ml aprotinin, 12.5 µg/ml vector DNA, 20 µg/ml GPEET-trm template and 7.5 µg/ml SLins19 template were incubated for 1 h at 27°C.

    Techniques: Expressing

    Production of the HA-cyclin 1 and FLAG-CDKA expressing double-knock-in strain. (A) Schematic diagram of the knock-in of HA-CYC1 and FLAG-CDKA into the chromosomal CYC1 and CDKA loci by homologous recombination. The first line indicates the linear DNA vector that was transformed into the M4 strain. The second and third lines indicate the genomic structure of the parental M4 strain and resultant HA-cyclin 1 strain, respectively. The fourth line indicates the linear DNA vector that was transformed into the HA-cyclin 1 strain. The fifth and sixth lines indicate the CDKA locus of the parental HA-cyclin 1 strain and the resultant HA-cyclin 1/FLAG-CDKA strain, respectively. The arrowheads indicate the positions of the PCR primers used in (B) . (B) PCR analysis of the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains, confirming the occurrence of homologous recombination events. The M4 strain was used as a negative control. The positions of the primer sets No. 45/No. 46 and No. 47/No. 48 are shown in (A) and the exact positions and sequences are indicated in Supplementary Table S1. The predicted size of the PCR product amplified from the CYC1 locus is 6.8 kb for the HA-cyclin 1 strain and 4.0 kb for the M4 strain. The predicted size of the PCR product amplified from the CDKA locus is 4.5 kb for the HA-cyclin 1/FLAG-CDKA strain and 2.9 kb for the HA-cyclin 1 strain. (C) Immunoblotting with the anti-HA and the anti-FLAG antibodies. The predicted sizes of HA-cyclin 1 and FLAG-CDKA proteins are 48 and 40 kDa, respectively. (D) Growth curves of the D-sfGFP strain (an sfGFP expresser was used as a control), the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains.

    Journal: Frontiers in Plant Science

    Article Title: Development of a Double Nuclear Gene-Targeting Method by Two-Step Transformation Based on a Newly Established Chloramphenicol-Selection System in the Red Alga Cyanidioschyzon merolae

    doi: 10.3389/fpls.2017.00343

    Figure Lengend Snippet: Production of the HA-cyclin 1 and FLAG-CDKA expressing double-knock-in strain. (A) Schematic diagram of the knock-in of HA-CYC1 and FLAG-CDKA into the chromosomal CYC1 and CDKA loci by homologous recombination. The first line indicates the linear DNA vector that was transformed into the M4 strain. The second and third lines indicate the genomic structure of the parental M4 strain and resultant HA-cyclin 1 strain, respectively. The fourth line indicates the linear DNA vector that was transformed into the HA-cyclin 1 strain. The fifth and sixth lines indicate the CDKA locus of the parental HA-cyclin 1 strain and the resultant HA-cyclin 1/FLAG-CDKA strain, respectively. The arrowheads indicate the positions of the PCR primers used in (B) . (B) PCR analysis of the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains, confirming the occurrence of homologous recombination events. The M4 strain was used as a negative control. The positions of the primer sets No. 45/No. 46 and No. 47/No. 48 are shown in (A) and the exact positions and sequences are indicated in Supplementary Table S1. The predicted size of the PCR product amplified from the CYC1 locus is 6.8 kb for the HA-cyclin 1 strain and 4.0 kb for the M4 strain. The predicted size of the PCR product amplified from the CDKA locus is 4.5 kb for the HA-cyclin 1/FLAG-CDKA strain and 2.9 kb for the HA-cyclin 1 strain. (C) Immunoblotting with the anti-HA and the anti-FLAG antibodies. The predicted sizes of HA-cyclin 1 and FLAG-CDKA proteins are 48 and 40 kDa, respectively. (D) Growth curves of the D-sfGFP strain (an sfGFP expresser was used as a control), the HA-cyclin 1 and the HA-cyclin 1/FLAG-CDKA strains.

    Article Snippet: The linear DNA vector, which consists of 1,500 bp of CYC1 upstream, 3xHA-CYC1 orf , 200-bp of CYC1 downstream, URA and 1,800-bp of CYC1 downstream, was amplified by PCR with M13 forward/reverse primers (TAKARA) and pHA-CYC1-URA as the template, and it was used for transformation of the C. merolae M4 strain.

    Techniques: Expressing, Knock-In, Homologous Recombination, Plasmid Preparation, Transformation Assay, Polymerase Chain Reaction, Negative Control, Amplification

    Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Journal: Biopolymers

    Article Title: Novel Semi-synthetic Method for Generating Full Length ?-Amyloid Peptides

    doi: 10.1002/bip.21391

    Figure Lengend Snippet: Scheme for semi-synthetic production of native sequence Aβ 1-40 . DNA encoding Aβ 1-29 with an N-terminal extension of a single amino acid, Met, is inserted into the pTXB1 vector for expression in E. coli (BL21DE3 cells). After induction with IPTG, the fusion protein is expressed at high levels. The fusion protein consists of Met-Aβ 1-29 in a thioester bond to an intein segment, and a C-terminal Chitin-Binding Domain (CBD). This allows for affinity purification of the fusion protein using Chitin beads. Met-Aβ 1-29 is cleaved from the Intein-CBD segment and eluted from the bead using MESNA in buffer, which yields Met-Aβ 1-29 -MESNA, i.e., with MESNA in a thioester linkage to Met-Aβ 1-29 . The next step is CNBr cleavage of the N-terminal Met residue; the thioester is stable to the acidic conditions under which this is performed. The recombinant Aβ 1-29 -MESNA is then linked to A30C-Aβ 30-40 by native chemical ligation. The final step is selective desulfurization, using Raney Ni, which yields full length, native sequence Aβ 1-40 .

    Article Snippet: As described above, a miniprep of DNA for the pTXB1 vector containing Aβ1-29 was used as a template for synthesis of the mutated strand by PCR.

    Techniques: Sequencing, Plasmid Preparation, Expressing, Binding Assay, Affinity Purification, Recombinant, Ligation