pbr322 Search Results


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  • 96
    New England Biolabs pbr322
    Topology-dependent binding of Topoisomerase IV to <t>pBR322.</t> (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Pbr322, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 664 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    pbr322  (ATCC)
    95
    ATCC pbr322
    Topology-dependent binding of Topoisomerase IV to <t>pBR322.</t> (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Pbr322, supplied by ATCC, used in various techniques. Bioz Stars score: 95/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher pbr322 dna bsuri haeiii marker
    Topology-dependent binding of Topoisomerase IV to <t>pBR322.</t> (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).
    Pbr322 Dna Bsuri Haeiii Marker, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher pbr322
    Effect of ureas on topo II-α Line 1: Etoposide (100 μM) + <t>pBR322;</t> line 2: pBR322 only; line 3: pBR322 + topo II-α; line 4: pBR322 + topo II-α + 23 (10 μM); line 5: pBR322 + topo II-α + 24 (10 μM); line 6: pBR322 + topo II-α + 21 (10 μM); line 7: pBR322 + topo II-α + 22 (10 μM); line 8: pBR322 + topo II-α + 20 (10 μM); line 9: pBR322 + topo II-α + 25 (10 μM).
    Pbr322, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 360 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Thermo Fisher pbr322 plasmid dna
    GSH potentiates kinamycin F-induced <t>DNA</t> nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of <t>pBR322</t> plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.
    Pbr322 Plasmid Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 95/100, based on 76 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher plasmids pbr322
    Agarose gel (1%) electrophoresis of heat-treated and unheated plasmid DNAs from Cylindrospermum stagnale studied together with heat-treated and unheated bacterial plasmids pUC18 and <t>pBR322</t> and linear λ DNA. Heat-treated samples of DNA were obtained by heating the samples in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 °C followed by quick cooling. The arrows indicate Covalently Closed Circular (CCC) DNA. Lane M - λ DNA- Hin dIII and ϕ x174 DNA- Hae III digest Mix (Finnzymes); Lane 1 – Unheated Cylindrospermum stagnale plasmids ; Lane 2 – Heat-treated Cylindrospermum stagnale plasmids; Lane 3 – Unheated pUC18 plasmid; Lane 4 – Heat-treated pUC18 plasmid; Lane 5 – pBR322 plasmid; Lane 6 – Heat treated pBR322 plasmid; Lane 7 – Unheated λ DNA; Lane 8 – Heat treated λ DNA.
    Plasmids Pbr322, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Millipore pbr322
    A: Representative image of <t>pBR322</t> on an agarose gel following treatment with a radionuclide. Here, pBR322 was incubated with 1 MBq 111 In-chloride ( 111 In-Cl 3 or as external radiation ( 111 In (external)) for 72 h in the presence or absence of DMSO or cold indium chloride (InCl 3 ). B and C: Fraction of supercoiled (undamaged) plasmid, as measured from gels such as A. Plasmids were incubated with either 111 InCl 3 (B) or 67 GaCl 3 (C). Data points are average ± standard deviation (SD; n = 2–3). Relaxed bands represent single strand breaks; linear bands are double strand breaks.
    Pbr322, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 66 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene pbr322
    The gene disruption method. ( A ) Map of the plasmid EZTN:tet r -bs r . The EZTN:tet r -bs r plasmid generates a transposable blasticidin resistance (bs r ) cassette that can be used to create Dictyostelium gene disruptants. In order to create EZTN:tet r -bs r , the bacterial tet r cassette, isolated from <t>pBR322,</t> and the Dictyostelium Actin15Bsr cassette, isolated from Actin15ΔBamBsr, were cloned into the EZ::TNpMOD2 vector (Epicentre, USA) as indicated. The 19 bp Tn 5 transposon recognition sequences are represented as triangles and their sequence is shown below the map. The plasmid has PvuII sites flanking the transposable element and the transposable blasticidin resistance cassette is usually generated by PvuII digestion of the construct. ( B ) Diagrammatic representation of the scheme for gene disruption. A fragment of genomic DNA is isolated by PCR and inserted, by topoisomerase cloning, into pCR2.1-TOPO, a vector that confers resistance to ampicillin and kanamycin. In vitro transposition is then performed using transposon DNA prepared as in (A). The resultant DNA molecules are recovered by transformation into E.coli using a triple drug selection, with ampicillin, kanamycin and tetracycline.
    Pbr322, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATUM pbr322 plasmid dna
    Electrophoresis mobility shift assays for cisplatin and heterometallic Ti–Au compounds 5a – d (see the Experimental Section for details). <t>DNA</t> refers to untreated plasmid <t>pBR322.</t> a , b , c , and d correspond to metal/DNA bp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.
    Pbr322 Plasmid Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 69 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Promega pbr322
    Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), <t>pBR322</t> (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.
    Pbr322, supplied by Promega, used in various techniques. Bioz Stars score: 92/100, based on 143 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa pbr322 plasmid dna
    The results of validation experiments. ( a ) Chemical structure of NC. ( b ) Effect of NC on TopI mediated <t>DNA</t> relaxation indifferent concentration. Lane 1, supercoiled <t>pBR322</t> plasmid DNA; lane 2, DNA + TopI; lane 3–8, DNA + TopI + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopI + 50 μM camptothecin. ( c ) Inhibition of TopII relaxation activity. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopII; lane 3–8, DNA + TopII + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopII + 50 μM etoposide. ( d ) The effect of adrenaline (5 μg/kg) on arterial blood pressure. ( e ) NC (0.1 mg/kg) caused reversal of the adrenaline pressor response. ( f ) The effect of NC (0.01, 0.025, 0.1 mg/kg) on the arterial blood pressure response to adrenaline.
    Pbr322 Plasmid Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 34 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    ICN Biomedicals pbr322 plasmid dna
    Electrophoretograms applying to the incubated mixtures of <t>pBR322</t> plasmid <t>DNA</t> and varying concentrations of QH4, QH7, QH8 and cisplatin followed by digestion with BamH1. Lane B 1 applied to the untreated pBR322 plasmid DNA and undigested with BamH1, lane B 2 applied to untreated but digestion with BamH1. Lanes 1 to 8: apply to pBR322 plasmid DNA interacted with increasing concentrations of the compounds followed by BamH1 digestion. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12, lane 2 : 0.23, lane 3: 0.47, lane 4: 0.94, lane 5:1.88, lane 6: 3.75, lane 7: 7.50 and lane 8: 15.00.
    Pbr322 Plasmid Dna, supplied by ICN Biomedicals, used in various techniques. Bioz Stars score: 89/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    ATCC pbr322 vector
    End-point PCRs for detection of the MMTV env g ene in the samples. A . First nested PCR round resulting in a 660-bp MMTV env g ene fragment, exemplifying the expected results defining positive detection. B . Second nested PCR round resulting in a 250-bp fragment, exemplifying the expected results defining positive detection. C . Three samples that exemplify positive and negative detection of the 250-bp PCR product. MW: 100-bp molecular weight ladder; Neg: non-template negative control; C3H: strain with MMTV env g ene inserted into <t>pBR322</t> plasmid, used as a positive control; T: tumor; and NB: unaffected tissue.
    Pbr322 Vector, supplied by ATCC, used in various techniques. Bioz Stars score: 86/100, based on 144 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore pbr322 dna
    Mechanistic electrophoretic pattern of <t>pBR322</t> <t>DNA</t> (100 ng) by ( A ) complex 1 and ( B ) complex 2 (0.5–2.5 μM) in 50 mM of Tris-HCl/NaCl buffer pH 7.4 after 45 min of incubation at various concentrations. Lane 1: DNA alone (control); Lane 2: DNA + DAPI + 1 ; Lane 3: DNA + MG + 1 ; Lane 4: DNA + DMSO + 1 ; Lane 5: DNA + EtOH + 1 ; Lane 6: DNA + NaN 3 + 1 ; Lane 7: DNA + SOD+ 1 ; Lane 8: DNA + DAPI + 2 ; Lane 9: DNA + MG + 2 ; Lane 10: DNA + DMSO + 2 ; Lane 11: DNA + EtOH + 2 ; Lane 12: DNA + NaN 3 + 2 ; Lane 13: DNA + SOD + 2 .
    Pbr322 Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    89
    TopoGEN Inc pbr322 dna
    Effects of SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ) on their ability to inhibit the topoisomerase I-induced relaxation of <t>pBR322</t> <t>DNA.</t> NC, nicked circular DNA; RLX, relaxed DNA; SC, supercoiled DNA; “sn38”, “2-nitro-” and “4-nitro-” denote the tested drugs SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ), respectively; numbers indicate the final concentration [µM] of the tested drug in the assay mixture applied to the lanes above; all lanes except the lane “no top1” contained topoisomerase 1; no drug was added to the lane “Ctr”.
    Pbr322 Dna, supplied by TopoGEN Inc, used in various techniques. Bioz Stars score: 89/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Toyobo pbr322
    Effects of SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ) on their ability to inhibit the topoisomerase I-induced relaxation of <t>pBR322</t> <t>DNA.</t> NC, nicked circular DNA; RLX, relaxed DNA; SC, supercoiled DNA; “sn38”, “2-nitro-” and “4-nitro-” denote the tested drugs SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ), respectively; numbers indicate the final concentration [µM] of the tested drug in the assay mixture applied to the lanes above; all lanes except the lane “no top1” contained topoisomerase 1; no drug was added to the lane “Ctr”.
    Pbr322, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 41 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Journal: Nucleic Acids Research

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    doi: 10.1093/nar/gku1381

    Figure Lengend Snippet: Topology-dependent binding of Topoisomerase IV to pBR322. (A) Unbound and Topo IV-bound DNA visualized by SYBR Green staining after electrophoresis in a 1% agarose gel. The sharpest central parts of the gel lanes were quantified to maximize the resolution (blue and red boxes). (B) Free (red) and Topo IV-bound (blue) DNA from densitometry scans of the gel in A plotted as intensity per pixel. The free DNA distribution has relatively more DNA at lower linking numbers, whereas the bound DNA distribution has relatively more DNA at higher linking numbers. (C) Relative K a as a function of the plasmid linking number Δ L k . K a ratios were calculated for DNA topoisomers bound by Topo IV (Equation 3 ), normalized to the affinity for the topoisomer Δ L k = −1 (green dots; error bars represent the standard error of four measurements) and fit to a line ( K a ratio = 1.12 + 0.15(−Δ L k ); χ 2 = 1.49). Inset, relative K a as a function of the plasmid linking number Δ L k for Topo IV with an active site mutation Y120F (error bars represent the standard error of four measurements).

    Article Snippet: Tfam binding reactions contained 10 nM Tfam and 10 nM pBR322 in 150 mM NaCl, 10 mM Tris pH 7.5, 10 nM MgCl2 and 1 mM DTT based on the buffer conditions used in ( ).

    Techniques: Binding Assay, SYBR Green Assay, Staining, Electrophoresis, Agarose Gel Electrophoresis, Plasmid Preparation, Mutagenesis

    (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Journal: Nucleic Acids Research

    Article Title: A robust assay to measure DNA topology-dependent protein binding affinity

    doi: 10.1093/nar/gku1381

    Figure Lengend Snippet: (A) Relative binding affinities ( K a ) normalized to the affinity for topoisomer Δ L k = 0 (ntop1, light green; top1mt, dark green; RecQ, blue; EcoRV, yellow) or to the affinity for topoisomer Δ L k = −1 (Tfam, red; pink). The undifferentiated band was assigned a value of Δ L k = −23 as an estimate of the expected value of the unresolvable band containing all topoisomers with Δ L k values below −10. Other Gaussians fit to bands not clearly separable as individual topoisomers were assigned intermediate values. Pink circles represent the results of a Tfam binding experiment using supercoiled pBR322, and were normalized to the empirically determined relative K a value for Δ L k = −23. The data points to which the data were normalized are ringed by black circles and error bars represent the standard error of at least four experiments. The gel images to the right of each protein contain unenhanced images of agarose gels containing unbound (left column) and bound (right column) pBR322 topoisomer distributions. In each case the topmost band contains nicked DNA, followed by topoisomers in order of decreasing Δ L k . The bottommost image contains supercoiled DNA for the higher topoisomer range Tfam binding experiment and was electrophoresed in the presence of 3.5 μg/ml chloroquine. (B) Table of relative binding affinities for nicked plasmids ( K aN / K a0 ) and for highly supercoiled (Δ L k = −23) plasmids ( K aS / K a0 ).

    Article Snippet: Tfam binding reactions contained 10 nM Tfam and 10 nM pBR322 in 150 mM NaCl, 10 mM Tris pH 7.5, 10 nM MgCl2 and 1 mM DTT based on the buffer conditions used in ( ).

    Techniques: Binding Assay

    The DNA-binding properties of β-Casein 197. a ProtParam analysis of predicted binding residues of β-casein 197, predicted binding residues was labeled with a red “+”. b Gel retardation assays. Binding was assayed by the migration of pDNA. Various concentrations of peptides were incubated with 5 µL of 25 µg mL −1 pBR322 vector from E. coli at 37 °C for 1 h, and then the reaction mixtures were applied to 0.7% agarose gel electrophoresis. Lane M DNA marker DL 10,000; Lane 1 5 µL pDNA as control; Lanes 2–6 mixture of pDNA and various concentrations of peptides (12.5, 25, 50, 100 and 500 µg mL −1 )

    Journal: AMB Express

    Article Title: Investigation into the antimicrobial action and mechanism of a novel endogenous peptide β-casein 197 from human milk

    doi: 10.1186/s13568-017-0409-y

    Figure Lengend Snippet: The DNA-binding properties of β-Casein 197. a ProtParam analysis of predicted binding residues of β-casein 197, predicted binding residues was labeled with a red “+”. b Gel retardation assays. Binding was assayed by the migration of pDNA. Various concentrations of peptides were incubated with 5 µL of 25 µg mL −1 pBR322 vector from E. coli at 37 °C for 1 h, and then the reaction mixtures were applied to 0.7% agarose gel electrophoresis. Lane M DNA marker DL 10,000; Lane 1 5 µL pDNA as control; Lanes 2–6 mixture of pDNA and various concentrations of peptides (12.5, 25, 50, 100 and 500 µg mL −1 )

    Article Snippet: DNA binding assay Gel-retardation experiments were performed using 5 µL of a 25 µg mL−1 pBR322 vector from E. coli (BioLabs, New England).

    Techniques: Binding Assay, Labeling, Electrophoretic Mobility Shift Assay, Migration, Incubation, Plasmid Preparation, Agarose Gel Electrophoresis, Marker

    GM1-ELISA to measure binding of LT 192 and LT 192 -Gly:Pro-STb proteins to GM1. Total proteins extracted from equivalent amounts of cells (determined by OD readings) of each strain (3030-2 [K88ac + LT + STb + ], 8017 [1836-2/pBR322], 8035 [1836-2 LT], 8221 [1836-2 LT 192 ], 8145 [1836-2 LT-Gly:Pro-STb], and 8488 [1836-2 LT 192 -Gly:Pro-STb]) were tested in GM1 binding using anti-CT as the primary antibody (1:5,000) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000) as the secondary antibody. Optical densities, measured at 405 nm, showed significant differences for 3030-2, 8035, and 8221 strains ( P

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies ▿

    doi: 10.1128/CVI.00095-10

    Figure Lengend Snippet: GM1-ELISA to measure binding of LT 192 and LT 192 -Gly:Pro-STb proteins to GM1. Total proteins extracted from equivalent amounts of cells (determined by OD readings) of each strain (3030-2 [K88ac + LT + STb + ], 8017 [1836-2/pBR322], 8035 [1836-2 LT], 8221 [1836-2 LT 192 ], 8145 [1836-2 LT-Gly:Pro-STb], and 8488 [1836-2 LT 192 -Gly:Pro-STb]) were tested in GM1 binding using anti-CT as the primary antibody (1:5,000) and horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5,000) as the secondary antibody. Optical densities, measured at 405 nm, showed significant differences for 3030-2, 8035, and 8221 strains ( P

    Article Snippet: Final PCR products (inserts) and vector pBR322 were digested with NheI and EagI restriction enzymes (New England Biolabs, Ipswich, MA) and ligated with T4 DNA ligase (Invitrogen) under standard conditions ( ).

    Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

    Detection of toxic activity in a porcine ligated-gut-loop assay. Individual ligated intestinal loops from the ileum and jejunum sections were inoculated with 2 × 10 9 CFU of overnight culture from strain 3030-2 (K88 + LT + STb + , a positive control), 8488 (1836-2 LT 192 -Gly:Pro-STb), 8221(1836-2 LT 192 ), 8017 (1836-2/pBR322, a negative control), or 8816 (1836-2 STb). Fluid accumulation was measured after 8 h postinoculation; the data are presented in the inserted table. Statistical analysis indicated that the fluid accumulation in loops incubated with strains 8488 and 8221 was not significantly different from that in loops incubated with the negative control 8017 strain ( P > 0.05), whereas fluid accumulated in the loops incubated with 8816 and 3030-2 was significantly different ( P

    Journal: Clinical and Vaccine Immunology : CVI

    Article Title: Genetic Fusions of Heat-Labile Toxoid (LT) and Heat-Stable Toxin b (STb) of Porcine Enterotoxigenic Escherichia coli Elicit Protective Anti-LT and Anti-STb Antibodies ▿

    doi: 10.1128/CVI.00095-10

    Figure Lengend Snippet: Detection of toxic activity in a porcine ligated-gut-loop assay. Individual ligated intestinal loops from the ileum and jejunum sections were inoculated with 2 × 10 9 CFU of overnight culture from strain 3030-2 (K88 + LT + STb + , a positive control), 8488 (1836-2 LT 192 -Gly:Pro-STb), 8221(1836-2 LT 192 ), 8017 (1836-2/pBR322, a negative control), or 8816 (1836-2 STb). Fluid accumulation was measured after 8 h postinoculation; the data are presented in the inserted table. Statistical analysis indicated that the fluid accumulation in loops incubated with strains 8488 and 8221 was not significantly different from that in loops incubated with the negative control 8017 strain ( P > 0.05), whereas fluid accumulated in the loops incubated with 8816 and 3030-2 was significantly different ( P

    Article Snippet: Final PCR products (inserts) and vector pBR322 were digested with NheI and EagI restriction enzymes (New England Biolabs, Ipswich, MA) and ligated with T4 DNA ligase (Invitrogen) under standard conditions ( ).

    Techniques: Activity Assay, Positive Control, Negative Control, Incubation

    Effect of ureas on topo II-α Line 1: Etoposide (100 μM) + pBR322; line 2: pBR322 only; line 3: pBR322 + topo II-α; line 4: pBR322 + topo II-α + 23 (10 μM); line 5: pBR322 + topo II-α + 24 (10 μM); line 6: pBR322 + topo II-α + 21 (10 μM); line 7: pBR322 + topo II-α + 22 (10 μM); line 8: pBR322 + topo II-α + 20 (10 μM); line 9: pBR322 + topo II-α + 25 (10 μM).

    Journal: Molecules

    Article Title: Solvent-Free Synthesis, DNA-Topoisomerase II Activity and Molecular Docking Study of New Asymmetrically N,N'-Substituted Ureas

    doi: 10.3390/molecules171112882

    Figure Lengend Snippet: Effect of ureas on topo II-α Line 1: Etoposide (100 μM) + pBR322; line 2: pBR322 only; line 3: pBR322 + topo II-α; line 4: pBR322 + topo II-α + 23 (10 μM); line 5: pBR322 + topo II-α + 24 (10 μM); line 6: pBR322 + topo II-α + 21 (10 μM); line 7: pBR322 + topo II-α + 22 (10 μM); line 8: pBR322 + topo II-α + 20 (10 μM); line 9: pBR322 + topo II-α + 25 (10 μM).

    Article Snippet: In the topo II-α assay, two units were utilised to relax 0.125 μg/mL pBR322 (Gibco).

    Techniques:

    GSH potentiates kinamycin F-induced DNA nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

    Journal: Free radical biology & medicine

    Article Title: Mechanism of the cytotoxicity of the diazoparaquinone antitumor antibiotic kinamycin F

    doi: 10.1016/j.freeradbiomed.2007.07.005

    Figure Lengend Snippet: GSH potentiates kinamycin F-induced DNA nicking. (A) This fluorescent image of the ethidium bromide-stained gel shows that when GSH (5 mM) was added to reaction mixtures containing various amounts of kinamycin F, the amount of DNA nicking was increased. In the absence of GSH a slight increase in nicking was seen at 400 μM kinamycin F. (B) In this image it is shown that deferoxamine (100 μM) and DMSO (0.5% (v/v)) pre-treatment of the DNA reduced the amount of DNA nicking induced by kinamycin F (200 μM) and GSH (5 mM). (C) In this image it is shown that catalase (50 μg/ml) decreased the amount of DNA nicking, but boiled catalase (b, 50 μg/ml) did not. Catalase was added immediately prior to the kinamycin F (200 μM) and GSH (5 mM). All lanes contained 80 ng of pBR322 plasmid DNA, except for those with linear DNA which contained 80 ng of linear pBR322 plasmid DNA. Linear DNA was present in lane 7 and lanes 4 and 11 in (A) and (B), respectively. NC, nicked circular DNA; LIN, linear DNA; SC, supercoiled DNA. All incubations for the DNA nicking experiments were for 5 h at 37°C.

    Article Snippet: Thioglo-1 was from EMD Biosciences (Mississauga, Canada), kDNA was obtained from TopoGEN (Columbus, OH) and pBR322 plasmid DNA was obtained from MBI Fermentas (Burlington, ON, Canada).

    Techniques: Staining, Plasmid Preparation

    Effect of dovitinib on the relaxation activity and cellular poisoning of topoisomerase I. (A) Effect of dovitinib and camptothecin on the ability of topoisomerase I to relax supercoiled pBR322 DNA. This fluorescent image of the ethidium bromide-stained

    Journal: Biochemical pharmacology

    Article Title: The anticancer multi-kinase inhibitor dovitinib also targets topoisomerase I and topoisomerase II

    doi: 10.1016/j.bcp.2012.09.023

    Figure Lengend Snippet: Effect of dovitinib on the relaxation activity and cellular poisoning of topoisomerase I. (A) Effect of dovitinib and camptothecin on the ability of topoisomerase I to relax supercoiled pBR322 DNA. This fluorescent image of the ethidium bromide-stained

    Article Snippet: The pBR322 DNA was from MBI Fermentas (Burlington, Canada).

    Techniques: Activity Assay, Staining

    G226 inhibited the activity of Topo II. G226 inhibited Topo II-mediated supercoiled pBR322 relaxation (A) and Topo II-mediated kDNA decatenation (B) but had no effect on Topo I-mediated supercoiled pBR322 relaxation (C).

    Journal: Acta Pharmacologica Sinica

    Article Title: G226, a new epipolythiodioxopiperazine derivative, triggers DNA damage and apoptosis in human cancer cells in vitro via ROS generation

    doi: 10.1038/aps.2014.105

    Figure Lengend Snippet: G226 inhibited the activity of Topo II. G226 inhibited Topo II-mediated supercoiled pBR322 relaxation (A) and Topo II-mediated kDNA decatenation (B) but had no effect on Topo I-mediated supercoiled pBR322 relaxation (C).

    Article Snippet: The reaction buffer contained 50 mmol/L Tris-HCl (pH 8.0), 150 mmol/L NaCl, 10 mmol/L MgCl2 , 2 mmol/L ATP, 0.5 mmol/L dithiothreitol (DTT), 12.5 μg/mL supercoiled pBR322 DNA (Thermo Scientific, Beijing, China) or 20 μg/mL kDNA (TopoGEN, Port Orange, FL, USA), 1 unit of Topo II α (Topogen), and the indicated concentrations of compounds in a total volume of 20 μL.

    Techniques: Activity Assay

    Agarose gel (1%) electrophoresis of heat-treated and unheated plasmid DNAs from Cylindrospermum stagnale studied together with heat-treated and unheated bacterial plasmids pUC18 and pBR322 and linear λ DNA. Heat-treated samples of DNA were obtained by heating the samples in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 °C followed by quick cooling. The arrows indicate Covalently Closed Circular (CCC) DNA. Lane M - λ DNA- Hin dIII and ϕ x174 DNA- Hae III digest Mix (Finnzymes); Lane 1 – Unheated Cylindrospermum stagnale plasmids ; Lane 2 – Heat-treated Cylindrospermum stagnale plasmids; Lane 3 – Unheated pUC18 plasmid; Lane 4 – Heat-treated pUC18 plasmid; Lane 5 – pBR322 plasmid; Lane 6 – Heat treated pBR322 plasmid; Lane 7 – Unheated λ DNA; Lane 8 – Heat treated λ DNA.

    Journal: Saudi Journal of Biological Sciences

    Article Title: Isolation and characterization of two novel plasmids pCYM01 and pCYM02 of Cylindrospermum stagnale

    doi: 10.1016/j.sjbs.2019.11.017

    Figure Lengend Snippet: Agarose gel (1%) electrophoresis of heat-treated and unheated plasmid DNAs from Cylindrospermum stagnale studied together with heat-treated and unheated bacterial plasmids pUC18 and pBR322 and linear λ DNA. Heat-treated samples of DNA were obtained by heating the samples in the presence of 0.1% (w/v) sarkosyl for 2 min at 100 °C followed by quick cooling. The arrows indicate Covalently Closed Circular (CCC) DNA. Lane M - λ DNA- Hin dIII and ϕ x174 DNA- Hae III digest Mix (Finnzymes); Lane 1 – Unheated Cylindrospermum stagnale plasmids ; Lane 2 – Heat-treated Cylindrospermum stagnale plasmids; Lane 3 – Unheated pUC18 plasmid; Lane 4 – Heat-treated pUC18 plasmid; Lane 5 – pBR322 plasmid; Lane 6 – Heat treated pBR322 plasmid; Lane 7 – Unheated λ DNA; Lane 8 – Heat treated λ DNA.

    Article Snippet: The following plasmids pBR322 and pUC18, and λDNA were obtained from ThermoFisher Scientific, treated in the same way and used as controls.

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Plasmid Preparation, Countercurrent Chromatography

    Comparison of digital PCR quantification with 16S DNA Free master mix (DF) for linearlized and supercoiled pBR322 plasmid sample (O99) using Assay I and Assay II (statistically insignificant for Linear and Supercoil DNA, p > 0.05).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: Comparison of digital PCR quantification with 16S DNA Free master mix (DF) for linearlized and supercoiled pBR322 plasmid sample (O99) using Assay I and Assay II (statistically insignificant for Linear and Supercoil DNA, p > 0.05).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Digital PCR, Plasmid Preparation

    PCR amplification curves of linearized and supercoil pBR322 plasmid with ( a ) Gene Expression master mix (GE), ( b ) Environment master mix (EN) and ( c ) 16S DNA Free master mix (DF).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: PCR amplification curves of linearized and supercoil pBR322 plasmid with ( a ) Gene Expression master mix (GE), ( b ) Environment master mix (EN) and ( c ) 16S DNA Free master mix (DF).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Expressing

    Supercoiled pBR322 plasmid DNA concentration optimization for digital PCR by using Assay I and Assay II labeling with HEX (A1 × 2, B1 × 2 and C1 × 2 are the two times dilution of sample A1, B1 and C1, statistically insignificant for sample A1, B1 and C1 between two assays and two dilutions, p > 0.05).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: Supercoiled pBR322 plasmid DNA concentration optimization for digital PCR by using Assay I and Assay II labeling with HEX (A1 × 2, B1 × 2 and C1 × 2 are the two times dilution of sample A1, B1 and C1, statistically insignificant for sample A1, B1 and C1 between two assays and two dilutions, p > 0.05).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Plasmid Preparation, Concentration Assay, Digital PCR, Labeling

    Digital PCR hit map of serial dilution of pBR322 (sample B) by using Assay I labeling with HEX (Panel 1–18, three replicates of each dilution from SD1 to SD6, panel 19–22, four replicates of dilution SD7, panel 23–24, NTC).

    Journal: Scientific Reports

    Article Title: Accurate quantification of supercoiled DNA by digital PCR

    doi: 10.1038/srep24230

    Figure Lengend Snippet: Digital PCR hit map of serial dilution of pBR322 (sample B) by using Assay I labeling with HEX (Panel 1–18, three replicates of each dilution from SD1 to SD6, panel 19–22, four replicates of dilution SD7, panel 23–24, NTC).

    Article Snippet: Two PCR assays (Assay I and Assay II) were designed for the quantification of pBR322 plasmid using Primer Express 3.0.1 (Applied Biosystems).

    Techniques: Digital PCR, Serial Dilution, Labeling

    Complementation of the lpp mutant of Y. pestis CO92 with the htrA gene restores intracellular survival in macrophages. Intracellular survival of WT with pBR322, Δ lpp mutant with pBR322, and the Δ lpp with pBR322 htrA was determined by infecting RAW 264.7 murine macrophages with an MOI of 1 for 45 minutes, followed by a 60-minute gentamicin wash, and plating the surviving intracellular bacteria at 4, 8, and 12 hours. The Δ lpp mutant with pBR322 htrA has a significant increase in percent survival compared to the Δ lpp mutant with pBR322 alone, as determined by ANOVA and Holm-Sidak method.

    Journal: Comparative and Functional Genomics

    Article Title: Comparative Global Gene Expression Profiles of Wild-Type Yersinia pestis CO92 and Its Braun Lipoprotein Mutant at Flea and Human Body Temperatures

    doi: 10.1155/2010/342168

    Figure Lengend Snippet: Complementation of the lpp mutant of Y. pestis CO92 with the htrA gene restores intracellular survival in macrophages. Intracellular survival of WT with pBR322, Δ lpp mutant with pBR322, and the Δ lpp with pBR322 htrA was determined by infecting RAW 264.7 murine macrophages with an MOI of 1 for 45 minutes, followed by a 60-minute gentamicin wash, and plating the surviving intracellular bacteria at 4, 8, and 12 hours. The Δ lpp mutant with pBR322 htrA has a significant increase in percent survival compared to the Δ lpp mutant with pBR322 alone, as determined by ANOVA and Holm-Sidak method.

    Article Snippet: The PCR product was purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA) and digested using the restriction endonucleases Bam HI and Sal I (New England BioLabs, Ipswich, MA) to prepare the fragment for ligation into pBR322 vector (Fermentas, Glen Burnie, MD) at the appropriate restriction enzyme sites.

    Techniques: Mutagenesis

    A: Representative image of pBR322 on an agarose gel following treatment with a radionuclide. Here, pBR322 was incubated with 1 MBq 111 In-chloride ( 111 In-Cl 3 or as external radiation ( 111 In (external)) for 72 h in the presence or absence of DMSO or cold indium chloride (InCl 3 ). B and C: Fraction of supercoiled (undamaged) plasmid, as measured from gels such as A. Plasmids were incubated with either 111 InCl 3 (B) or 67 GaCl 3 (C). Data points are average ± standard deviation (SD; n = 2–3). Relaxed bands represent single strand breaks; linear bands are double strand breaks.

    Journal: Nuclear Medicine and Biology

    Article Title: Re-assessing gallium-67 as a therapeutic radionuclide

    doi: 10.1016/j.nucmedbio.2016.10.008

    Figure Lengend Snippet: A: Representative image of pBR322 on an agarose gel following treatment with a radionuclide. Here, pBR322 was incubated with 1 MBq 111 In-chloride ( 111 In-Cl 3 or as external radiation ( 111 In (external)) for 72 h in the presence or absence of DMSO or cold indium chloride (InCl 3 ). B and C: Fraction of supercoiled (undamaged) plasmid, as measured from gels such as A. Plasmids were incubated with either 111 InCl 3 (B) or 67 GaCl 3 (C). Data points are average ± standard deviation (SD; n = 2–3). Relaxed bands represent single strand breaks; linear bands are double strand breaks.

    Article Snippet: 2.2 Cell-free DNA damage by 111 In and 67 Ga 125 ng pBR322 (10 μL supplied in 10 mM Tris–HCl (pH 8.0) with 1 mM ethylenediaminetetraacetic acid (EDTA), Sigma) was mixed with 1 MBq 111 In-chloride, 67 Ga-chloride or 67 Ga-citrate and incubated up to 72 h at 4 °C (final EDTA concentration 0.1 mM).

    Techniques: Agarose Gel Electrophoresis, Incubation, Plasmid Preparation, Standard Deviation

    HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 enhances ATP hydrolysis by topoisomerase IIα. ( A ) HMGB1 reduces inhibition of catalytic activity of topo IIα by ICRF-193. kDNA (0.2 μg) was decatenated with topo IIα (2 nM) in the absence or presence of 10 μM ICRF-193. Some decatenation reactions contained HMGB1 (0.5, 1.5 and 3 μM, left to right). Decatenation was carried out as detailed in the Materials and Methods section. Due to the absence of ethidium bromide in the agarose gel in the course of electrophoresis, the decatenated minicircles migrated as single bands comprising both nicked and closed-circular DNA (designated as oc and rel in Figures 3 and 4 ). A representative ethidium bromide-stained 1% agarose gel (presented as a negative) is shown. ( B ) HMGB1 enhances the rate of ATP hydrolysis by topo IIα. ATP hydrolysis by topo IIα (5 nM) was studied in reactions containing 1 mM cold ATP and [γ- 32 P]ATP and negatively supercoiled plasmid pBR322 (50 nM). The ATP hydrolysis (as determined by a time-dependent release of free PO 4 ) was measured by thin layer chromatography ( 24 ) and quantified by PhosporImaging. Data represent the average of two independent experiments.

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Inhibition, Activity Assay, Agarose Gel Electrophoresis, Electrophoresis, Staining, Plasmid Preparation, Thin Layer Chromatography

    HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 promotes intermolecular association of DNA. ( A ) Macromolecular crowding favors intermolecular ligase-mediated DNA end-joining by HMGB1. Linearized plasmid pTZ19R (∼15 nM) was pre-incubated with 0.5 μM (lanes 3 and 6) or 1.5 μM (lanes 4 and 7) HMGB1, and then treated with 0.2 U of T4 DNA ligase in the presence (lanes 6 and 7) or absence (lanes 3 and 4) of 5% polyethyleneglycol (PEG). L2, dimers; L3 trimers or higher multimers. Linear, linearized plasmid pBR322; circular, closed-circular plasmid pBR322. ( B ) HMGB1 promotes topo IIα-catalyzed interlocking of DNA into multimers (catenanes) in the presence of PEG. Supercoiled plasmid pTZ19R (∼15 nM, lane 1) was pre-incubated with HMGB1 (4.5 μM) in the absence or presence of PEG (as indicated), and treated with topo IIα (∼7 nM). ( C ) Both relaxed and supercoiled plasmid DNAs form multimers with HMGB1 and topo IIα. Relaxed or supercoiled plasmids pTZ19R (∼15 nM) were pre-incubated with 0.5 μM (lanes 3 and 7), 1.5 μM (lanes 4 and 8) and 4.5 μM HMGB1 (lanes 5 and 9) in the presence of 5% PEG, followed by treatment with topo IIα (∼7 nM). ( D ) DNA multimers formed by topo IIα and HMGB1 are catenanes. Reactions from (C) (lane 4) were deproteinized and treated with increasing amounts of topo IIα (10 and 20 nM, left to right) for 30 min at 37°C. Deproteinized samples in (A–D) were separated on 1% agarose gels, and the resolved DNA samples were visualized by ethidium bromide staining as detailed in Materials and Methods section. The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA; FIII, linearized plasmid DNA ( Hin dIII).

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Plasmid Preparation, Incubation, Staining

    Stimulation of topo IIα activity by HMGB1 is not due to the effect of HMGB1 on DNA topology. ( A ) HMGB1 protein and structure of HMGB1 domains A and B indicating positions of the mutated intercalating amino acid residues (in red frames). ( B ) Purity of HMGB1 and mutant as revealed by electrophoresis on an SDS–18% polyacrylamide gel and Coomassie blue R-250 staining. Lane 1, wild-type HMGB1; lane 2, HMGB1 mutant (F38A/F103A/I122A). A, alanine; F, phenylalanine; I, isoleucine. ( C ) Alanine mutagenesis of intercalating residues of HMGB1 abrogated the ability of HMGB1 to introduce supercoils into DNA by topoisomerase I. Closed-circular pBR322 plasmid DNA (∼9 nM) was incubated with wheat germ topoisomerase I in the absence or presence of HMGB1 or mutant (1, 2, 3 and 6 μM, left to right). FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. (D ) Both wild-type HMGB1 and HMGB1 mutant can enhance decatenation of kDNA by human topo IIα. Decatenation of kDNA was carried out as in Figure 3 A. HMGB1 and mutant were at 1 μM, topo IIα was 7.5 and 15 nM. ( E ) HMGB1 does not stimulate decatenation of kDNA by E. coli topoisomerase IV. Decatenation of kDNA by E. coli topoisomerase IV (0.15 and 0.6 U) was carried out as detailed in the Materials and Methods section using HMGB1 or mutant (1 and 4 μM, left to right). The DNA samples in (C–E) were separated on 0.8% agarose gels without (C) or with (D–E) ethidium bromide in the course of electrophoresis as detailed in the Materials and Methods section. The gels are presented as negatives. WT, wild-type HMGB1; Mut, triple-mutant (F38A/F103A/I122A) of HMGB1.

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: Stimulation of topo IIα activity by HMGB1 is not due to the effect of HMGB1 on DNA topology. ( A ) HMGB1 protein and structure of HMGB1 domains A and B indicating positions of the mutated intercalating amino acid residues (in red frames). ( B ) Purity of HMGB1 and mutant as revealed by electrophoresis on an SDS–18% polyacrylamide gel and Coomassie blue R-250 staining. Lane 1, wild-type HMGB1; lane 2, HMGB1 mutant (F38A/F103A/I122A). A, alanine; F, phenylalanine; I, isoleucine. ( C ) Alanine mutagenesis of intercalating residues of HMGB1 abrogated the ability of HMGB1 to introduce supercoils into DNA by topoisomerase I. Closed-circular pBR322 plasmid DNA (∼9 nM) was incubated with wheat germ topoisomerase I in the absence or presence of HMGB1 or mutant (1, 2, 3 and 6 μM, left to right). FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. (D ) Both wild-type HMGB1 and HMGB1 mutant can enhance decatenation of kDNA by human topo IIα. Decatenation of kDNA was carried out as in Figure 3 A. HMGB1 and mutant were at 1 μM, topo IIα was 7.5 and 15 nM. ( E ) HMGB1 does not stimulate decatenation of kDNA by E. coli topoisomerase IV. Decatenation of kDNA by E. coli topoisomerase IV (0.15 and 0.6 U) was carried out as detailed in the Materials and Methods section using HMGB1 or mutant (1 and 4 μM, left to right). The DNA samples in (C–E) were separated on 0.8% agarose gels without (C) or with (D–E) ethidium bromide in the course of electrophoresis as detailed in the Materials and Methods section. The gels are presented as negatives. WT, wild-type HMGB1; Mut, triple-mutant (F38A/F103A/I122A) of HMGB1.

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Activity Assay, Mutagenesis, Electrophoresis, Staining, Introduce, Plasmid Preparation, Incubation

    HMGB1 stimulates decatenation of kinetoplast DNA and relaxation of supercoiled DNA by topoisomerase IIα. ( A ) Decatenation of kinetoplast DNA (kDNA) by topo IIα is stimulated by HMGB1. kDNA (0.2 μg) was incubated with increasing concentrations of topo IIα (as indicated) in the absence or presence of HMGB1 (1 μM). ( B ) Domain structure of HMGB1, HMGB1-ΔC, HMGB1 lacking the acidic C-tail (depicted as oval); A, domain A; B, domain B. ( C ) HMGB1 stimulates decatenation of kDNA by topo IIα via the HMG-box domains. kDNA (0.2 μg) was incubated with topo IIα (2 nM) in the presence or absence of increasing amounts of HMGB1, HMGB1-ΔC, domain A or domain B (as indicated). ΔC, HMGB1-ΔC. ( D ) HMGB1 stimulates relaxation of negatively supercoiled DNA by HMGB1. Relaxation of negatively supercoiled pBR322 plasmid DNA (∼8 nM) by different concentrations of topo IIα (as indicated) in the presence or absence of recombinant HMGB1 (1 μM), 37°C for 45 min. Decatenation and relaxation assays were carried out in the absence of PEG. Deproteinized DNA samples were resolved on 1% agarose gels containing ethidium bromide (A and C) or without ethidium bromide (panel D). The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. oc, nicked closed-circular DNA minicircle; rel, relaxed closed-circular DNA minicircle.

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 stimulates decatenation of kinetoplast DNA and relaxation of supercoiled DNA by topoisomerase IIα. ( A ) Decatenation of kinetoplast DNA (kDNA) by topo IIα is stimulated by HMGB1. kDNA (0.2 μg) was incubated with increasing concentrations of topo IIα (as indicated) in the absence or presence of HMGB1 (1 μM). ( B ) Domain structure of HMGB1, HMGB1-ΔC, HMGB1 lacking the acidic C-tail (depicted as oval); A, domain A; B, domain B. ( C ) HMGB1 stimulates decatenation of kDNA by topo IIα via the HMG-box domains. kDNA (0.2 μg) was incubated with topo IIα (2 nM) in the presence or absence of increasing amounts of HMGB1, HMGB1-ΔC, domain A or domain B (as indicated). ΔC, HMGB1-ΔC. ( D ) HMGB1 stimulates relaxation of negatively supercoiled DNA by HMGB1. Relaxation of negatively supercoiled pBR322 plasmid DNA (∼8 nM) by different concentrations of topo IIα (as indicated) in the presence or absence of recombinant HMGB1 (1 μM), 37°C for 45 min. Decatenation and relaxation assays were carried out in the absence of PEG. Deproteinized DNA samples were resolved on 1% agarose gels containing ethidium bromide (A and C) or without ethidium bromide (panel D). The gels are presented as negatives. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. oc, nicked closed-circular DNA minicircle; rel, relaxed closed-circular DNA minicircle.

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Incubation, Plasmid Preparation, Recombinant

    HMGB1 stimulates DNA cleavage activity of topoisomerase IIα. ( A ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the absence of etoposide. DNA cleavage reactions contained negatively supercoiled plasmid pBR322 (∼4 nM), human topo IIα (∼8 nM) and increasing concentrations of HMGB1 (1, 2 and 3 μM, left to right). ATP or a non-hydrolyzable ATP analog (AMP-PNP) were at 1 mM. Arrowhead indicates position of linearized DNA. ( B ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the presence of etoposide. The molar concentrations of topo IIα and plasmid pBR322 were identical as in panel A. HMGB1 protein was at 1 μM and etoposide at 50 μM. ( C ) Effect of HMGB1 on DNA cleavage by topo IIα in the presence of increasing concentrations of etoposide. Negatively supercoiled plasmid DNA (4 nM ) was pre-incubated on ice either with buffer (control) or HMGB1 (1 μM) in the presence of 0–100 μM etoposide. Cleavage reactions were initiated by addition of topo IIα (8 nM) and incubation was carried out at 37°C for 15 min. The cleavage complexes in A–C reactions were trapped by 1% SDS, followed by digestion with proteinase K as detailed in Materials and Methods section. Deproteinized DNA samples were resolved on 1% agarose gels and subsequently stained with ethidium bromide. FIII, Hin dIII-digested plasmid pBR322 (last right lanes in A and B) indicates mobility of linearized plasmid DNA. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. The gels are presented as negatives. The percentage of cleaved plasmid DNA was quantified from three independent cleavage experiments, each in duplicates, by ImageQuant TL (GE Healthcare). The average SD for the data was

    Journal: Nucleic Acids Research

    Article Title: HMGB1 interacts with human topoisomerase II? and stimulates its catalytic activity

    doi: 10.1093/nar/gkm525

    Figure Lengend Snippet: HMGB1 stimulates DNA cleavage activity of topoisomerase IIα. ( A ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the absence of etoposide. DNA cleavage reactions contained negatively supercoiled plasmid pBR322 (∼4 nM), human topo IIα (∼8 nM) and increasing concentrations of HMGB1 (1, 2 and 3 μM, left to right). ATP or a non-hydrolyzable ATP analog (AMP-PNP) were at 1 mM. Arrowhead indicates position of linearized DNA. ( B ) Effect of HMGB1 on DNA cleavage activity of topo IIα in the presence of etoposide. The molar concentrations of topo IIα and plasmid pBR322 were identical as in panel A. HMGB1 protein was at 1 μM and etoposide at 50 μM. ( C ) Effect of HMGB1 on DNA cleavage by topo IIα in the presence of increasing concentrations of etoposide. Negatively supercoiled plasmid DNA (4 nM ) was pre-incubated on ice either with buffer (control) or HMGB1 (1 μM) in the presence of 0–100 μM etoposide. Cleavage reactions were initiated by addition of topo IIα (8 nM) and incubation was carried out at 37°C for 15 min. The cleavage complexes in A–C reactions were trapped by 1% SDS, followed by digestion with proteinase K as detailed in Materials and Methods section. Deproteinized DNA samples were resolved on 1% agarose gels and subsequently stained with ethidium bromide. FIII, Hin dIII-digested plasmid pBR322 (last right lanes in A and B) indicates mobility of linearized plasmid DNA. FI, supercoiled plasmid DNA; FII, relaxed closed-circular plasmid DNA. The gels are presented as negatives. The percentage of cleaved plasmid DNA was quantified from three independent cleavage experiments, each in duplicates, by ImageQuant TL (GE Healthcare). The average SD for the data was

    Article Snippet: ‘DNA cleavage assay’ was carried out by incubation of plasmid pBR322 (∼4 nM) with topo IIα in DNA cleavage buffer (10 mM Tris-HCl pH 8, 50 mM KCl, 50 mM NaCl, 5 mM MgCl2 , 2.5% glycerol, 0.1 mM EDTA, containing either 1 mM ATP or 1 mM non-hydrolyzable analog ANP-PNP, Sigma) in a total volume of 20 μl at 37°C for 20 min.

    Techniques: Activity Assay, Plasmid Preparation, Incubation, Staining

    The gene disruption method. ( A ) Map of the plasmid EZTN:tet r -bs r . The EZTN:tet r -bs r plasmid generates a transposable blasticidin resistance (bs r ) cassette that can be used to create Dictyostelium gene disruptants. In order to create EZTN:tet r -bs r , the bacterial tet r cassette, isolated from pBR322, and the Dictyostelium Actin15Bsr cassette, isolated from Actin15ΔBamBsr, were cloned into the EZ::TNpMOD2 vector (Epicentre, USA) as indicated. The 19 bp Tn 5 transposon recognition sequences are represented as triangles and their sequence is shown below the map. The plasmid has PvuII sites flanking the transposable element and the transposable blasticidin resistance cassette is usually generated by PvuII digestion of the construct. ( B ) Diagrammatic representation of the scheme for gene disruption. A fragment of genomic DNA is isolated by PCR and inserted, by topoisomerase cloning, into pCR2.1-TOPO, a vector that confers resistance to ampicillin and kanamycin. In vitro transposition is then performed using transposon DNA prepared as in (A). The resultant DNA molecules are recovered by transformation into E.coli using a triple drug selection, with ampicillin, kanamycin and tetracycline.

    Journal: Nucleic Acids Research

    Article Title: Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

    doi: 10.1093/nar/gng095

    Figure Lengend Snippet: The gene disruption method. ( A ) Map of the plasmid EZTN:tet r -bs r . The EZTN:tet r -bs r plasmid generates a transposable blasticidin resistance (bs r ) cassette that can be used to create Dictyostelium gene disruptants. In order to create EZTN:tet r -bs r , the bacterial tet r cassette, isolated from pBR322, and the Dictyostelium Actin15Bsr cassette, isolated from Actin15ΔBamBsr, were cloned into the EZ::TNpMOD2 vector (Epicentre, USA) as indicated. The 19 bp Tn 5 transposon recognition sequences are represented as triangles and their sequence is shown below the map. The plasmid has PvuII sites flanking the transposable element and the transposable blasticidin resistance cassette is usually generated by PvuII digestion of the construct. ( B ) Diagrammatic representation of the scheme for gene disruption. A fragment of genomic DNA is isolated by PCR and inserted, by topoisomerase cloning, into pCR2.1-TOPO, a vector that confers resistance to ampicillin and kanamycin. In vitro transposition is then performed using transposon DNA prepared as in (A). The resultant DNA molecules are recovered by transformation into E.coli using a triple drug selection, with ampicillin, kanamycin and tetracycline.

    Article Snippet: We first generated a transposable bsr cassette containing the Dictyostelium blasticidin S (bsr ) resistance cassette from Actin15ΔBamBsr ( ) and the bacterial tetracycline resistance (tetr ) cassette from pBR322 (Stratagene), linked in tandem. pBR322 was digested with AvaI and, after blunt-ending the AvaI site, it was digested with EcoRI to produce an AvaI (blunt)–EcoRI fragment containing tetr .

    Techniques: Plasmid Preparation, Isolation, Clone Assay, Sequencing, Generated, Construct, Polymerase Chain Reaction, In Vitro, Transformation Assay, Selection

    Electrophoresis mobility shift assays for cisplatin and heterometallic Ti–Au compounds 5a – d (see the Experimental Section for details). DNA refers to untreated plasmid pBR322. a , b , c , and d correspond to metal/DNA bp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Journal: Organometallics

    Article Title: Titanocene–Gold Complexes Containing N-Heterocyclic Carbene Ligands Inhibit Growth of Prostate, Renal, and Colon Cancers in Vitro

    doi: 10.1021/acs.organomet.6b00051

    Figure Lengend Snippet: Electrophoresis mobility shift assays for cisplatin and heterometallic Ti–Au compounds 5a – d (see the Experimental Section for details). DNA refers to untreated plasmid pBR322. a , b , c , and d correspond to metal/DNA bp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Article Snippet: Interactions of the New Compounds with Plasmid pBR322 (Gel Electrophoresis Mobility Shift Assay) Ten microliter aliquots of pBR322 plasmid DNA (20 μg/mL) in buffer (5 mM Tris/HCl, 50 mM NaClO4 , pH 7.39) were incubated with different concentrations of the compounds ( 4a – d , 5a – d , and cisplatin as control) (in the range 0.25 and 4.0 metal complex to DNA bp) at 37 °C for 20 h in the dark.

    Techniques: Electrophoresis, Mobility Shift, Plasmid Preparation

    Electrophoresis mobility shift assays for cisplatin and compounds 1 – 5 (see Experimental Section for details). DNA refers to untreated plasmid pBR322. A, B, C, and D correspond to metal/DNAbp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Journal: Journal of Medicinal Chemistry

    Article Title: Cyclometalated Iminophosphorane Gold(III) and Platinum(II) Complexes. A Highly Permeable Cationic Platinum(II) Compound with Promising Anticancer Properties

    doi: 10.1021/acs.jmedchem.5b00427

    Figure Lengend Snippet: Electrophoresis mobility shift assays for cisplatin and compounds 1 – 5 (see Experimental Section for details). DNA refers to untreated plasmid pBR322. A, B, C, and D correspond to metal/DNAbp ratios of 0.25, 0.5, 1.0, and 2.0, respectively.

    Article Snippet: Interaction of Compounds 1 – 5 and Cisplatin with Plasmid (pBR322) DNA by Electrophoresis (Mobility Shift Assay) First, 10 μL aliquots of pBR322 plasmid DNA (20 μg/mL) in buffer (5 mM Tris-HCl, 50 mM NaClO4 , pH 7.39) were incubated with different concentrations of the compounds 1 – 5 (in the range 0.25–2.0 metal complex:DNAbp) at 37 °C for 20 h in the dark.

    Techniques: Electrophoresis, Mobility Shift, Plasmid Preparation

    Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.

    Journal: Retrovirology

    Article Title: BLV-CoCoMo-qPCR: Quantitation of bovine leukemia virus proviral load using the CoCoMo algorithm

    doi: 10.1186/1742-4690-7-91

    Figure Lengend Snippet: Evaluation of the specificity of the BLV-CoCoMo-qPCR primers . (A) Real-time PCR using the CoCoMo 6 and CoCoMo 81 primers from the BLV-CoCoMo-qPCR was performed using 0.3 ng of the following infectious molecular clones: BLV (pBLV-IF, lane 2); HTLV-1 (pK30, lane 3); HIV-1 (pNL4-3, lane 4); SIV (pSIVmac239/WT, lane 5); MMTV (hybrid MMTV, lane 6); M-MLV (pL-4, lane 7); and the plasmids pUC18 (lane 8), pUC19 (lane 9), pBR322 (lane 10), and pBluescript SK(+) (lane 11). PCR products were subjected to 3% agarose gel electrophoresis. Lane 1, DNA marker Φ × 174- Hae III digest. A PCR product 168 bp in length is indicated by an arrow. (B) The number of BLV provirus copies in 1 μg of DNA from each DNA sample is indicated by lowercase. Values represent the mean ± standard deviation (SD) of the results of three independent experiments.

    Article Snippet: Additional clones used included a BLV infectious clone, pBLV-IF [ ]; a HTLV-1 infectious clone, pK30 [ ]; a HIV-1 infectious clone, pNL4-3 [ ]; a SIV infectious clone, SIVmac239/WT [ ]; a hybrid MMTV provirus plasmid [ ]; a M-MLV infectious clone, pL-4 [ ] and plasmids including pUC18 (Takara Bio Inc., Tokyo, Japan), pUC19 (Takara Bio Inc.), and pBR322 (Promega).

    Techniques: Real-time Polymerase Chain Reaction, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker, Standard Deviation

    The results of validation experiments. ( a ) Chemical structure of NC. ( b ) Effect of NC on TopI mediated DNA relaxation indifferent concentration. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopI; lane 3–8, DNA + TopI + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopI + 50 μM camptothecin. ( c ) Inhibition of TopII relaxation activity. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopII; lane 3–8, DNA + TopII + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopII + 50 μM etoposide. ( d ) The effect of adrenaline (5 μg/kg) on arterial blood pressure. ( e ) NC (0.1 mg/kg) caused reversal of the adrenaline pressor response. ( f ) The effect of NC (0.01, 0.025, 0.1 mg/kg) on the arterial blood pressure response to adrenaline.

    Journal: Scientific Reports

    Article Title: The gene expression profiles in response to 102 traditional Chinese medicine (TCM) components: a general template for research on TCMs

    doi: 10.1038/s41598-017-00535-8

    Figure Lengend Snippet: The results of validation experiments. ( a ) Chemical structure of NC. ( b ) Effect of NC on TopI mediated DNA relaxation indifferent concentration. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopI; lane 3–8, DNA + TopI + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopI + 50 μM camptothecin. ( c ) Inhibition of TopII relaxation activity. Lane 1, supercoiled pBR322 plasmid DNA; lane 2, DNA + TopII; lane 3–8, DNA + TopII + NC (0.1, 1, 5, 10, 20, 50 μM); lane 9, DNA + TopII + 50 μM etoposide. ( d ) The effect of adrenaline (5 μg/kg) on arterial blood pressure. ( e ) NC (0.1 mg/kg) caused reversal of the adrenaline pressor response. ( f ) The effect of NC (0.01, 0.025, 0.1 mg/kg) on the arterial blood pressure response to adrenaline.

    Article Snippet: Compound concentrations, pBR322 plasmid DNA (0.25 μg) and 1 unit of TopI (TaKaRa Biotechnology Co., Ltd., Dalian) were combined in a final volume of 20 μL buffer (35 mM pH 8.0 Tris-HCl, 72 mM KCl, 5 mM MgCl2 , 5 mM dithiothreitol, 5 mM spermidine, 0.1% bovine serum albumin).

    Techniques: Concentration Assay, Plasmid Preparation, Inhibition, Activity Assay

    Electrophoretograms applying to the incubated mixtures of pBR322 plasmid DNA and varying concentrations of QH4, QH7, QH8 and cisplatin followed by digestion with BamH1. Lane B 1 applied to the untreated pBR322 plasmid DNA and undigested with BamH1, lane B 2 applied to untreated but digestion with BamH1. Lanes 1 to 8: apply to pBR322 plasmid DNA interacted with increasing concentrations of the compounds followed by BamH1 digestion. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12, lane 2 : 0.23, lane 3: 0.47, lane 4: 0.94, lane 5:1.88, lane 6: 3.75, lane 7: 7.50 and lane 8: 15.00.

    Journal: Journal of Biomedical Science

    Article Title: Synthesis and activity of three new trinuclear platinums with cis-geometry for terminal metal centres

    doi: 10.1186/1423-0127-21-41

    Figure Lengend Snippet: Electrophoretograms applying to the incubated mixtures of pBR322 plasmid DNA and varying concentrations of QH4, QH7, QH8 and cisplatin followed by digestion with BamH1. Lane B 1 applied to the untreated pBR322 plasmid DNA and undigested with BamH1, lane B 2 applied to untreated but digestion with BamH1. Lanes 1 to 8: apply to pBR322 plasmid DNA interacted with increasing concentrations of the compounds followed by BamH1 digestion. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12, lane 2 : 0.23, lane 3: 0.47, lane 4: 0.94, lane 5:1.88, lane 6: 3.75, lane 7: 7.50 and lane 8: 15.00.

    Article Snippet: Exactly, 1.5 μL of supplied pBR322 plasmid DNA in solution was added to solutions of the compounds at different concentrations ranging from 0.55 μM to 70 μM for QH4, QH8 and cisplatin.

    Techniques: Incubation, Plasmid Preparation

    Electrophotograms applying to the interaction of pBR322 plasmid DNA with increasing concentrations of QH4, QH7, QH8 and cisplatin. Lane B applied to untreated pBR322 plasmid DNA to serve as a control, lanes 1 to 8 applied to plasmid DNA interacted with increasing concentrations of the compounds and cisplatin. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12 μM, lane 2 : 0.23 μM, lane 3: 0.47 μM, lane 4: 0.94 μM, lane 5:1.88 μM, lane 6: 3.75 μM, lane 7: 7.50 μM and lane 8: 15.00 μM.

    Journal: Journal of Biomedical Science

    Article Title: Synthesis and activity of three new trinuclear platinums with cis-geometry for terminal metal centres

    doi: 10.1186/1423-0127-21-41

    Figure Lengend Snippet: Electrophotograms applying to the interaction of pBR322 plasmid DNA with increasing concentrations of QH4, QH7, QH8 and cisplatin. Lane B applied to untreated pBR322 plasmid DNA to serve as a control, lanes 1 to 8 applied to plasmid DNA interacted with increasing concentrations of the compounds and cisplatin. The concentrations for QH4, QH8 and cisplatin were: lane 1: 0.55 μM, lane 2: 1.09 μM, lane 3: 2.19 μM, lane 4: 4.38 μM, lane 5: 8.75 μM, lane 6: 17.50 μM, lane 7: 35.00 μM, and lane 8: 70.00 μM. The concentrations for QH7 is as follows lane 1: 0.12 μM, lane 2 : 0.23 μM, lane 3: 0.47 μM, lane 4: 0.94 μM, lane 5:1.88 μM, lane 6: 3.75 μM, lane 7: 7.50 μM and lane 8: 15.00 μM.

    Article Snippet: Exactly, 1.5 μL of supplied pBR322 plasmid DNA in solution was added to solutions of the compounds at different concentrations ranging from 0.55 μM to 70 μM for QH4, QH8 and cisplatin.

    Techniques: Plasmid Preparation

    End-point PCRs for detection of the MMTV env g ene in the samples. A . First nested PCR round resulting in a 660-bp MMTV env g ene fragment, exemplifying the expected results defining positive detection. B . Second nested PCR round resulting in a 250-bp fragment, exemplifying the expected results defining positive detection. C . Three samples that exemplify positive and negative detection of the 250-bp PCR product. MW: 100-bp molecular weight ladder; Neg: non-template negative control; C3H: strain with MMTV env g ene inserted into pBR322 plasmid, used as a positive control; T: tumor; and NB: unaffected tissue.

    Journal: BMC Cancer

    Article Title: Prevalence of HMTV in breast carcinomas and unaffected tissue from Mexican women

    doi: 10.1186/1471-2407-14-942

    Figure Lengend Snippet: End-point PCRs for detection of the MMTV env g ene in the samples. A . First nested PCR round resulting in a 660-bp MMTV env g ene fragment, exemplifying the expected results defining positive detection. B . Second nested PCR round resulting in a 250-bp fragment, exemplifying the expected results defining positive detection. C . Three samples that exemplify positive and negative detection of the 250-bp PCR product. MW: 100-bp molecular weight ladder; Neg: non-template negative control; C3H: strain with MMTV env g ene inserted into pBR322 plasmid, used as a positive control; T: tumor; and NB: unaffected tissue.

    Article Snippet: The pBR322 vector (Cat 45006, ATCC) containing the MMTV env g ene from the C3H strain (GenBank AF228552) and propagated in Escherichia coli XL1 Blue [ ] was used as the positive control.

    Techniques: Nested PCR, Polymerase Chain Reaction, Molecular Weight, Negative Control, Plasmid Preparation, Positive Control

    Mechanistic electrophoretic pattern of pBR322 DNA (100 ng) by ( A ) complex 1 and ( B ) complex 2 (0.5–2.5 μM) in 50 mM of Tris-HCl/NaCl buffer pH 7.4 after 45 min of incubation at various concentrations. Lane 1: DNA alone (control); Lane 2: DNA + DAPI + 1 ; Lane 3: DNA + MG + 1 ; Lane 4: DNA + DMSO + 1 ; Lane 5: DNA + EtOH + 1 ; Lane 6: DNA + NaN 3 + 1 ; Lane 7: DNA + SOD+ 1 ; Lane 8: DNA + DAPI + 2 ; Lane 9: DNA + MG + 2 ; Lane 10: DNA + DMSO + 2 ; Lane 11: DNA + EtOH + 2 ; Lane 12: DNA + NaN 3 + 2 ; Lane 13: DNA + SOD + 2 .

    Journal: International Journal of Molecular Sciences

    Article Title: Design, Synthesis, and Biological Evaluation of Benzimidazole-Derived Biocompatible Copper(II) and Zinc(II) Complexes as Anticancer Chemotherapeutics

    doi: 10.3390/ijms19051492

    Figure Lengend Snippet: Mechanistic electrophoretic pattern of pBR322 DNA (100 ng) by ( A ) complex 1 and ( B ) complex 2 (0.5–2.5 μM) in 50 mM of Tris-HCl/NaCl buffer pH 7.4 after 45 min of incubation at various concentrations. Lane 1: DNA alone (control); Lane 2: DNA + DAPI + 1 ; Lane 3: DNA + MG + 1 ; Lane 4: DNA + DMSO + 1 ; Lane 5: DNA + EtOH + 1 ; Lane 6: DNA + NaN 3 + 1 ; Lane 7: DNA + SOD+ 1 ; Lane 8: DNA + DAPI + 2 ; Lane 9: DNA + MG + 2 ; Lane 10: DNA + DMSO + 2 ; Lane 11: DNA + EtOH + 2 ; Lane 12: DNA + NaN 3 + 2 ; Lane 13: DNA + SOD + 2 .

    Article Snippet: Material First, 2-aminobenzimidazole, 1-methyl-1H -imidazole-2-carbaldehyde, Cu(CH3 COO)2 ·H2 O, Zn(CH3 COO)2 ·2H2 O, pBR322 DNA, sodium azide, superoxide dismutase, H2 O2 , DAPI, methyl green (MG), the sodium salt of CT-DNA, and HSA essentially fatty acid-free (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Incubation

    Electrophoretic pattern of pBR322 DNA (100 ng) by ( A ) complex 1 and ( B ) complex 2 (0.5–2.5 μM) in 50 mM Tris-HCl/NaCl buffer pH, 7.4 after 45 min of incubation at various concentrations. Lane 1: DNA alone (control); Lane 2: DNA + 0.5 μM 1 ; Lane 3: DNA + 1.0 μM 1 ; Lane 4: DNA + 1.5 μM 1 ; Lane 5: DNA + 2.0 μM 1 ; Lane 6: DNA + 2.5 μM 1 ; Lane 7: DNA alone (control); Lane 8: DNA + 0.5 μM 2 ; Lane 9: DNA + 1.0 μM 2 ; Lane 10: DNA + 1.5 μM 2 ; Lane 11: DNA + 2.0 μM 2 ; Lane 12: DNA + 2.5 μM 2 .

    Journal: International Journal of Molecular Sciences

    Article Title: Design, Synthesis, and Biological Evaluation of Benzimidazole-Derived Biocompatible Copper(II) and Zinc(II) Complexes as Anticancer Chemotherapeutics

    doi: 10.3390/ijms19051492

    Figure Lengend Snippet: Electrophoretic pattern of pBR322 DNA (100 ng) by ( A ) complex 1 and ( B ) complex 2 (0.5–2.5 μM) in 50 mM Tris-HCl/NaCl buffer pH, 7.4 after 45 min of incubation at various concentrations. Lane 1: DNA alone (control); Lane 2: DNA + 0.5 μM 1 ; Lane 3: DNA + 1.0 μM 1 ; Lane 4: DNA + 1.5 μM 1 ; Lane 5: DNA + 2.0 μM 1 ; Lane 6: DNA + 2.5 μM 1 ; Lane 7: DNA alone (control); Lane 8: DNA + 0.5 μM 2 ; Lane 9: DNA + 1.0 μM 2 ; Lane 10: DNA + 1.5 μM 2 ; Lane 11: DNA + 2.0 μM 2 ; Lane 12: DNA + 2.5 μM 2 .

    Article Snippet: Material First, 2-aminobenzimidazole, 1-methyl-1H -imidazole-2-carbaldehyde, Cu(CH3 COO)2 ·H2 O, Zn(CH3 COO)2 ·2H2 O, pBR322 DNA, sodium azide, superoxide dismutase, H2 O2 , DAPI, methyl green (MG), the sodium salt of CT-DNA, and HSA essentially fatty acid-free (≥98%) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Incubation

    Effects of SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ) on their ability to inhibit the topoisomerase I-induced relaxation of pBR322 DNA. NC, nicked circular DNA; RLX, relaxed DNA; SC, supercoiled DNA; “sn38”, “2-nitro-” and “4-nitro-” denote the tested drugs SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ), respectively; numbers indicate the final concentration [µM] of the tested drug in the assay mixture applied to the lanes above; all lanes except the lane “no top1” contained topoisomerase 1; no drug was added to the lane “Ctr”.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Evaluation of Nitrobenzyl Derivatives of Camptothecin as Anti-Cancer Agents and Potential Hypoxia Targeting Prodrugs

    doi: 10.3390/molecules23082041

    Figure Lengend Snippet: Effects of SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ) on their ability to inhibit the topoisomerase I-induced relaxation of pBR322 DNA. NC, nicked circular DNA; RLX, relaxed DNA; SC, supercoiled DNA; “sn38”, “2-nitro-” and “4-nitro-” denote the tested drugs SN-38 ( 1 ), 2-nitrobenzyl-SN-38 ( 2 ) and 4-nitrobenzyl-SN-38 ( 4 ), respectively; numbers indicate the final concentration [µM] of the tested drug in the assay mixture applied to the lanes above; all lanes except the lane “no top1” contained topoisomerase 1; no drug was added to the lane “Ctr”.

    Article Snippet: The recombinant human topoisomerase 1, pBR322 DNA, and assay buffer were from TopoGEN, Inc., Buena Vista, CO, USA.

    Techniques: Concentration Assay