human placental dna Search Results


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  • 90
    New England Biolabs human placental dna
    Human Placental Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 93 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Millipore human placental dna
    Structural basis of transcription inhibition by PUM Structures of T. thermophilus transcription initiation complexes containing PUM (A) and CMPcPP (B) . Top, crystallization and refinement statistics (left) and experimental electron density and fit (right). Green, PUM; pink, RNA and CMPcPP; red, <t>DNA</t> template strand; violet sphere between <t>RNAP</t> product (P) and addition (A) sites, Mg 2+ (I); violet sphere in RNAP addition (A) site, Mg 2+ (II); gray; RNAP bridge helix; green mesh, mF o -DF c omit map (contoured at 2.5σ). Middle, stereodiagram of interactions. Green, PUM carbon atoms; pink, RNA and CMPcPP carbon atoms; gray, RNAP carbon atoms; red, blue, yellow, and orange, oxygen, nitrogen, sulfur, and phosphorous atoms; dashed lines, H-bonds; other colors, as above. Bottom, summary of interactions. Red dashed lines, H-bonds; blue arcs, van der Waals interactions. Residues numbered as in T. thermophilus RNAP and, in parentheses, E. coli RNAP. .
    Human Placental Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 359 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Millipore human placental gdna
    Structural basis of transcription inhibition by PUM Structures of T. thermophilus transcription initiation complexes containing PUM (A) and CMPcPP (B) . Top, crystallization and refinement statistics (left) and experimental electron density and fit (right). Green, PUM; pink, RNA and CMPcPP; red, <t>DNA</t> template strand; violet sphere between <t>RNAP</t> product (P) and addition (A) sites, Mg 2+ (I); violet sphere in RNAP addition (A) site, Mg 2+ (II); gray; RNAP bridge helix; green mesh, mF o -DF c omit map (contoured at 2.5σ). Middle, stereodiagram of interactions. Green, PUM carbon atoms; pink, RNA and CMPcPP carbon atoms; gray, RNAP carbon atoms; red, blue, yellow, and orange, oxygen, nitrogen, sulfur, and phosphorous atoms; dashed lines, H-bonds; other colors, as above. Bottom, summary of interactions. Red dashed lines, H-bonds; blue arcs, van der Waals interactions. Residues numbered as in T. thermophilus RNAP and, in parentheses, E. coli RNAP. .
    Human Placental Gdna, supplied by Millipore, used in various techniques. Bioz Stars score: 95/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore controls human placental genomic dna
    Analytical validation of the MS-HRMA assay for CST6 promoter methylation: A ) Specificity and reproducibility of the assay: 0%: human placental genomic <t>DNA,</t> 100% methylated control: universal methylated human DNA standard, run three times at three different days, B ) Sensitivity, Black: 0%, red: 1%, blue: 10%, green: 50%, yellow: 100% methylation.
    Controls Human Placental Genomic Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore chemicals human placental dna
    Bactericidal activity and inhibition of antibiotics by <t>DNA,</t> F-actin, <t>LPS</t> and LTA. A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of DNA/F-actin/LPS/LTA (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post t -test, and P -values were considered significant when (**) p
    Chemicals Human Placental Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore human placental control dna
    Bactericidal activity and inhibition of antibiotics by <t>DNA,</t> F-actin, <t>LPS</t> and LTA. A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of DNA/F-actin/LPS/LTA (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post t -test, and P -values were considered significant when (**) p
    Human Placental Control Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 94/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Thermo Fisher human placental dna
    Detection of the <t>JCV</t> T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from <t>DNA</t> extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.
    Human Placental Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore human placental dna d4642
    Detection of the <t>JCV</t> T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from <t>DNA</t> extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.
    Human Placental Dna D4642, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa human placental dna
    Detection of the <t>JCV</t> T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from <t>DNA</t> extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.
    Human Placental Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Meridian Life Science human placental dna
    Detection of the <t>JCV</t> T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from <t>DNA</t> extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.
    Human Placental Dna, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Roche cot 1 human placental dna
    Detection of the <t>JCV</t> T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from <t>DNA</t> extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.
    Cot 1 Human Placental Dna, supplied by Roche, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega human placental genomic dna
    AR phosphorylation status selectively promotes the recruitment of ubiquitin–proteasome components at the <t>PSA</t> promoter. WT and XPD cells were transiently transfected to overexpress either AR/WT, AR/WT together with XPD/WT or AR/S515E (as indicated at the top of each panel). ( A – D ) Expression of the PSA gene: RT–qPCR analysis was performed at indicated times after DHT (10 −7 M) treatment. The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n -fold induction relative to non-treated cells. ( E – H ) After DHT treatment, the recruitment of RNA pol II (yellow curve), TFIIH (via its XPB and cdk7 subunits, blue and green curve, respectively) and AR (red curve) were analysed by ChIP assays at the PSA proximal promoter. The results are presented as percentage of <t>DNA</t> immunoprecipitated relative to the input (% input). ( I – L ) Recruitment of the MDM2 (green curve) and CHIP (yellow curve) E3 ligases on the PSA promoter. The recruitment of the ubiquitinated AR-containing fraction (brown curve) was also analysed by ChIP/re-ChIP assays using first an anti-ubiquitin antibody and second with an anti-AR antibody. ( M – P ) Recruitment of SUG1 (red curve), β5 (light blue) and S1 (dark blue) subunits of the proteasome at the PSA promoter was analysed by ChIP assays. Note that the recruitment of S1 has not been analysed in XPD cells transiently co-transfected with plasmids encoding AR/WT and XPD/WT ( O ).
    Human Placental Genomic Dna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa human placental genomic dna
    Transfer of the Platform ACE into heterologous cell lines. ( A ) Flow karyogram of metaphase chromosomes isolated from CHR1 cells and treated with Hoechst 33258 and chromomycin A3 dyes as described in Materials and Methods. The region circled and highlighted in red consists predominantly of the Platform ACE. ( B ) FISH analysis of the ChY1 cell line. The purified Platform ACE was transferred to CHO-DG44 cells and the resultant puromycin-resistant clone ChY1 was characterized by FISH. Metaphase chromosome spreads were prepared as described and hybridized with a mixture of DIG-labeled mouse major satellite repeat probe (rhodamine) <t>PCR</t> amplimer and biotin-labeled pFK161 probe (rDNA, FITC) generated by nick translation. ( C ) Southern-blot analysis of ChY1 and ChC2 Platform ACE cell lines. Genomic <t>DNA</t> samples from wildtype cell lines LMTK − , CHO-DG44 and CHO-S, as well as the corresponding Platform ACE cell lines CHR1, ChY1 and ChC2 were digested with NcoI and were fractionated on a 1% agarose gel. These were run along side varying dilutions of NcoI-digested pSV40attPPuro DNA so as to allow for determination of copy number. The fractionated DNA was transferred to nylon membrane by capillary blotting and the resultant membrane was hybridized with a labeled att P probe fragment (upper autoradiograph), then stripped and reprobed with a PCR-labeled hamster metallothionein-1 gene fragment (lower autoradiograph). Lane 1–4: copy number controls; 5 copies (lane 1), 10 copies (lane 2), 25 copies (lane 3), 50 copies (lane 4). Lane 5: empty. Lanes 6–11: cell line DNA; LMTK − (lane 6), CHR1 (lane 7), CHO-DG44 (lane 8), ChY1 (lane 9), CHO-S (lane 10), ChC2 (lane 11). The att P probe hybridizes to the expected 1.1 kb band in the CHR1, ChY1 and ChC2 DNA samples (upper). The hamster metallothionein-1 gene (chMT-1) probe detects the single copy gene and is used as a loading control for the CHO samples (lanes 8–11, lower panel). The hamster metallothionein-1 gene probe does not hybridize to the murine LMTK − derived samples, hence the absence of signal in lanes 6 and 7 (lower). Lanes 6, 8 and 10 are derived from the parental cell lines; lanes 7, 9 and 11 are cell lines derived after Platform ACE transfer. Note that the ChC2 sample (lane 11) is overloaded when compared with the other samples.
    Human Placental Genomic Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    TaKaRa human placental cdna
    p50 protein associates with the C terminus of WRN in vitro. ( A ) Yeast two-hybrid screening. The <t>WRNCT</t> bait encoding amino acids 949-1401 (∼50 kDa) of the human WRN protein was cloned into pAS2–1 vector and used to screen a human placental <t>cDNA</t> library. The schematic representation of the WRNCT bait shows the C terminus nuclear localization signal (NLS), the conserved RecQ domain (RecQCt), and the putative nucleic acid binding domain (HRD). ( B ) Retransformation of the yeast expression plasmids to Y190 host strain. pAS2–1, bait vector alone; p50, full-length p50 in pACT2 vector in fusion with GAL4-activator domain. pLAM and pVA3 are human lamin C and murine p53, respectively, cloned into the pAS2–1 vector. ( C ) In vitro translation combined with immunoprecipitation. WRNCT and full-length p50 cloned into pcDNA3.1-His/Xpress and pcDNA 3.1-His/Myc vectors, respectively, were transcribed/translated in vitro in the presence (WRNCT) or absence (p50) of [ 35 S]methionine. Equal amounts of labeled WRNCT polypeptide were mixed with either control (lanes 1 and 3) or p50-Myc programmed nonradioactive reticulocyte lysates (lanes 2 and 4) and immunoprecipitated by anti-Myc (lanes 1 and 2) or anti-HA (lanes 3 and 4) polyclonal antibodies. The associated polypeptides were resolved by SDS-PAGE and visualized by autoradiography. The lower panel indicates 2.5% of the corresponding mixtures loaded before immunoprecipitation (input). We estimated that 4–5% of the input protein could be coprecipitated with the anti-Myc antibody used.
    Human Placental Cdna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 88/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    BioChain Institute human placental genomic dna
    p50 protein associates with the C terminus of WRN in vitro. ( A ) Yeast two-hybrid screening. The <t>WRNCT</t> bait encoding amino acids 949-1401 (∼50 kDa) of the human WRN protein was cloned into pAS2–1 vector and used to screen a human placental <t>cDNA</t> library. The schematic representation of the WRNCT bait shows the C terminus nuclear localization signal (NLS), the conserved RecQ domain (RecQCt), and the putative nucleic acid binding domain (HRD). ( B ) Retransformation of the yeast expression plasmids to Y190 host strain. pAS2–1, bait vector alone; p50, full-length p50 in pACT2 vector in fusion with GAL4-activator domain. pLAM and pVA3 are human lamin C and murine p53, respectively, cloned into the pAS2–1 vector. ( C ) In vitro translation combined with immunoprecipitation. WRNCT and full-length p50 cloned into pcDNA3.1-His/Xpress and pcDNA 3.1-His/Myc vectors, respectively, were transcribed/translated in vitro in the presence (WRNCT) or absence (p50) of [ 35 S]methionine. Equal amounts of labeled WRNCT polypeptide were mixed with either control (lanes 1 and 3) or p50-Myc programmed nonradioactive reticulocyte lysates (lanes 2 and 4) and immunoprecipitated by anti-Myc (lanes 1 and 2) or anti-HA (lanes 3 and 4) polyclonal antibodies. The associated polypeptides were resolved by SDS-PAGE and visualized by autoradiography. The lower panel indicates 2.5% of the corresponding mixtures loaded before immunoprecipitation (input). We estimated that 4–5% of the input protein could be coprecipitated with the anti-Myc antibody used.
    Human Placental Genomic Dna, supplied by BioChain Institute, used in various techniques. Bioz Stars score: 94/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Becton Dickinson human placental cdna
    p50 protein associates with the C terminus of WRN in vitro. ( A ) Yeast two-hybrid screening. The <t>WRNCT</t> bait encoding amino acids 949-1401 (∼50 kDa) of the human WRN protein was cloned into pAS2–1 vector and used to screen a human placental <t>cDNA</t> library. The schematic representation of the WRNCT bait shows the C terminus nuclear localization signal (NLS), the conserved RecQ domain (RecQCt), and the putative nucleic acid binding domain (HRD). ( B ) Retransformation of the yeast expression plasmids to Y190 host strain. pAS2–1, bait vector alone; p50, full-length p50 in pACT2 vector in fusion with GAL4-activator domain. pLAM and pVA3 are human lamin C and murine p53, respectively, cloned into the pAS2–1 vector. ( C ) In vitro translation combined with immunoprecipitation. WRNCT and full-length p50 cloned into pcDNA3.1-His/Xpress and pcDNA 3.1-His/Myc vectors, respectively, were transcribed/translated in vitro in the presence (WRNCT) or absence (p50) of [ 35 S]methionine. Equal amounts of labeled WRNCT polypeptide were mixed with either control (lanes 1 and 3) or p50-Myc programmed nonradioactive reticulocyte lysates (lanes 2 and 4) and immunoprecipitated by anti-Myc (lanes 1 and 2) or anti-HA (lanes 3 and 4) polyclonal antibodies. The associated polypeptides were resolved by SDS-PAGE and visualized by autoradiography. The lower panel indicates 2.5% of the corresponding mixtures loaded before immunoprecipitation (input). We estimated that 4–5% of the input protein could be coprecipitated with the anti-Myc antibody used.
    Human Placental Cdna, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 88/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Stratagene human placental cdna library
    Regulation of COX7RP (A) and EB9 (B) mRNA by estrogen. <t>MCF-7</t> cells were treated without (lane 1) or with (lanes 2 to 4) 10 −7 M 17β-estradiol for 6 h in the presence or absence of cycloheximide (lane 3) or actinomycin D (lane 4). Poly(A) + RNA (2 μg) was prepared from the cells, and Northern blot analysis was performed using COX7RP, EB9 <t>cDNA,</t> and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as probes. The signals of transcripts and GAPDH are indicated. Migration positions of rRNA are shown on the right. (C) Estrogen-dependent enhancer activity of the ERE present in the first intron of the COX7RP gene and in the 5′ upstream region of the EBAG9 gene. Reporter plasmids containing pGCAT, vitERE-GCAT, EB1-ERE-GCAT, and EB9-ERE-GCAT were transfected into MCF-7 cells. After culture in the absence or presence of 10 −7 M 17β-estradiol, a CAT assay was performed. The intensities of the signals were analyzed with a BAS2000 image analyzer. The results are presented as means ± standard deviations of triplicate determinations.
    Human Placental Cdna Library, supplied by Stratagene, used in various techniques. Bioz Stars score: 88/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Structural basis of transcription inhibition by PUM Structures of T. thermophilus transcription initiation complexes containing PUM (A) and CMPcPP (B) . Top, crystallization and refinement statistics (left) and experimental electron density and fit (right). Green, PUM; pink, RNA and CMPcPP; red, DNA template strand; violet sphere between RNAP product (P) and addition (A) sites, Mg 2+ (I); violet sphere in RNAP addition (A) site, Mg 2+ (II); gray; RNAP bridge helix; green mesh, mF o -DF c omit map (contoured at 2.5σ). Middle, stereodiagram of interactions. Green, PUM carbon atoms; pink, RNA and CMPcPP carbon atoms; gray, RNAP carbon atoms; red, blue, yellow, and orange, oxygen, nitrogen, sulfur, and phosphorous atoms; dashed lines, H-bonds; other colors, as above. Bottom, summary of interactions. Red dashed lines, H-bonds; blue arcs, van der Waals interactions. Residues numbered as in T. thermophilus RNAP and, in parentheses, E. coli RNAP. .

    Journal: Cell

    Article Title: Antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase

    doi: 10.1016/j.cell.2017.05.042

    Figure Lengend Snippet: Structural basis of transcription inhibition by PUM Structures of T. thermophilus transcription initiation complexes containing PUM (A) and CMPcPP (B) . Top, crystallization and refinement statistics (left) and experimental electron density and fit (right). Green, PUM; pink, RNA and CMPcPP; red, DNA template strand; violet sphere between RNAP product (P) and addition (A) sites, Mg 2+ (I); violet sphere in RNAP addition (A) site, Mg 2+ (II); gray; RNAP bridge helix; green mesh, mF o -DF c omit map (contoured at 2.5σ). Middle, stereodiagram of interactions. Green, PUM carbon atoms; pink, RNA and CMPcPP carbon atoms; gray, RNAP carbon atoms; red, blue, yellow, and orange, oxygen, nitrogen, sulfur, and phosphorous atoms; dashed lines, H-bonds; other colors, as above. Bottom, summary of interactions. Red dashed lines, H-bonds; blue arcs, van der Waals interactions. Residues numbered as in T. thermophilus RNAP and, in parentheses, E. coli RNAP. .

    Article Snippet: For experiments in assessing promoter-independent transcription by E. coli RNAP and HeLa nuclear extract (human RNAP I/II/II), reaction mixtures contained (20 μl): 0–100 μM PUM, 100 nM E. coli RNAP core enzyme or 8 U HeLaScribe Nuclear Extract (Promega), 1 μg human placental DNA (Sigma-Aldrich; catalog number D7011), 400 μM ATP, 400 μM GTP, and 400 μM CTP, and 1.56, 25, or 400 μM [α32 P]UTP (0.1–1 Bq/fmol; PerkinElmer), in 40 mM Tris-HCl (pH 8.0), 7 mM HEPES-NaOH, 70 mM ammonium sulfate, 30 mM KCl, 12 mM MgCl2 , 5 mM dithiothreitol, 0.1 mM EDTA, and 12% glycerol.

    Techniques: Inhibition, Crystallization Assay

    Inference of contamination origin in commercial human genomic DNAs. Phylogenetic analysis of 301-bp gag sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), CFS-WPI-1130(GQ483508), CFS-WPI-1138(GQ483509), CFS-WPI-1169(GQ483510), CFS-WPI-CI-1303(JF907633), CFS-WPI-CI-1313(JF907643), CFS-WPI-CI-1314T(JF907644), CFS-WPI-CI-1307(JF907638), CFS-WPI-CI-1310(JF907641), CFS-WPI-CI-1327(JF907636), preXMRV2(FR871850), PC-VP35(DQ241301),and PC-VP62(DQ399707). Accession numbers for the new gag sequences generated in our study are JN629081-JN629087. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.

    Journal: PLoS ONE

    Article Title: Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs

    doi: 10.1371/journal.pone.0029050

    Figure Lengend Snippet: Inference of contamination origin in commercial human genomic DNAs. Phylogenetic analysis of 301-bp gag sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), CFS-WPI-1130(GQ483508), CFS-WPI-1138(GQ483509), CFS-WPI-1169(GQ483510), CFS-WPI-CI-1303(JF907633), CFS-WPI-CI-1313(JF907643), CFS-WPI-CI-1314T(JF907644), CFS-WPI-CI-1307(JF907638), CFS-WPI-CI-1310(JF907641), CFS-WPI-CI-1327(JF907636), preXMRV2(FR871850), PC-VP35(DQ241301),and PC-VP62(DQ399707). Accession numbers for the new gag sequences generated in our study are JN629081-JN629087. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.

    Article Snippet: In contrast, two human placental DNAs from Sigma-Aldrich both tested negative for XMRV/MLV and mouse IAP and mtDNA sequences ( ).

    Techniques: Sequencing, Generated

    Inference of contamination origin in commercial RT-PCR reagents and human genomic DNAs. Phylogenetic analysis of 168-bp polymerase ( pol ) sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in red (RT contaminants) and blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include identical sequences from CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), preXMRV2(FR871850), PC-VP35(DQ241301), PC-VP42(DQ241302), PC-VP62(DQ399707), 22Rv1/CWR-R1(FN692043) The 168-bp pol sequences are available from the authors upon request. GenBank does not accept sequences less than 200-bp in length. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.

    Journal: PLoS ONE

    Article Title: Detection of Murine Leukemia Virus or Mouse DNA in Commercial RT-PCR Reagents and Human DNAs

    doi: 10.1371/journal.pone.0029050

    Figure Lengend Snippet: Inference of contamination origin in commercial RT-PCR reagents and human genomic DNAs. Phylogenetic analysis of 168-bp polymerase ( pol ) sequences. Bootstrap values ≥60 are shown. New sequences from the current study are shown in red (RT contaminants) and blue (human genomic DNA contaminants). Sequence names in purple are those reported as contaminants from other human studies. GenBank accession numbers for prototypical murine leukemia virus (MLV) reference sequences are provided in parentheses. XMRV clades are collapsed for presentation only and include identical sequences from CFS-WPI-1106(GQ497344), CFS-WPI-1178(GQ497343), preXMRV2(FR871850), PC-VP35(DQ241301), PC-VP42(DQ241302), PC-VP62(DQ399707), 22Rv1/CWR-R1(FN692043) The 168-bp pol sequences are available from the authors upon request. GenBank does not accept sequences less than 200-bp in length. Sequences coded as XMRV VP and WPI are from prostate cancer (PC) and CFS patients, respectively. BD; blood donor sequences. Viral host receptor tropism is indicated by blue (xenotropic), purple (polytropic), and yellow (ecotropic) spheres. PMLV, polytropic MLV; XMLV, xenotropic MLV.

    Article Snippet: In contrast, two human placental DNAs from Sigma-Aldrich both tested negative for XMRV/MLV and mouse IAP and mtDNA sequences ( ).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing

    Analytical validation of the MS-HRMA assay for CST6 promoter methylation: A ) Specificity and reproducibility of the assay: 0%: human placental genomic DNA, 100% methylated control: universal methylated human DNA standard, run three times at three different days, B ) Sensitivity, Black: 0%, red: 1%, blue: 10%, green: 50%, yellow: 100% methylation.

    Journal: BMC Cancer

    Article Title: A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples

    doi: 10.1186/1471-2407-12-486

    Figure Lengend Snippet: Analytical validation of the MS-HRMA assay for CST6 promoter methylation: A ) Specificity and reproducibility of the assay: 0%: human placental genomic DNA, 100% methylated control: universal methylated human DNA standard, run three times at three different days, B ) Sensitivity, Black: 0%, red: 1%, blue: 10%, green: 50%, yellow: 100% methylation.

    Article Snippet: Controls Human placental genomic DNA (gDNA; Sigma-Aldrich) and Universal Methylated Human DNA Standard (ZYMO Research Co., Orange, CA), were used as fully unmethylated and fully methylated controls respectively.

    Techniques: Mass Spectrometry, Methylation

    The primers of MS-HRM and MSP assays for CST6 promoter methylation. The MS-HRM primers are shown in red. The outer MSP primers are framed, while the inner ones are shown in purple. The region from −162 up to +188 is depicted (+1 is the Transcriptional start site). Note that this sequence is produced after bisulfite conversion of genomic DNA. All CpGs are considered to be methylated, and therefore are unaffected during the conversion process.

    Journal: BMC Cancer

    Article Title: A closed-tube methylation-sensitive high resolution melting assay (MS-HRMA) for the semi-quantitative determination of CST6 promoter methylation in clinical samples

    doi: 10.1186/1471-2407-12-486

    Figure Lengend Snippet: The primers of MS-HRM and MSP assays for CST6 promoter methylation. The MS-HRM primers are shown in red. The outer MSP primers are framed, while the inner ones are shown in purple. The region from −162 up to +188 is depicted (+1 is the Transcriptional start site). Note that this sequence is produced after bisulfite conversion of genomic DNA. All CpGs are considered to be methylated, and therefore are unaffected during the conversion process.

    Article Snippet: Controls Human placental genomic DNA (gDNA; Sigma-Aldrich) and Universal Methylated Human DNA Standard (ZYMO Research Co., Orange, CA), were used as fully unmethylated and fully methylated controls respectively.

    Techniques: Mass Spectrometry, Methylation, Sequencing, Produced

    Bactericidal activity and inhibition of antibiotics by DNA, F-actin, LPS and LTA. A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of DNA/F-actin/LPS/LTA (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post t -test, and P -values were considered significant when (**) p

    Journal: PLoS ONE

    Article Title: Activity and Interactions of Liposomal Antibiotics in Presence of Polyanions and Sputum of Patients with Cystic Fibrosis

    doi: 10.1371/journal.pone.0005724

    Figure Lengend Snippet: Bactericidal activity and inhibition of antibiotics by DNA, F-actin, LPS and LTA. A) Bactericidal concentrations of free tobramycin (F-TOB) and liposomal tobramycin (L-TOB) were incubated in presence of LPS/LTA (1 to 1000 mg/L). B) Bactericidal concentrations of free polymyxin B (F-PMB) and liposomal polymyxin B (L-PMB) were incubated in presence of DNA/F-actin/LPS/LTA (125 to 1000 mg/L). Growth controls are represented at 0 h (empty bar), and 3 h (dark bar). Comparisons between free and liposomal formulations were made by ANOVA one-way post t -test, and P -values were considered significant when (**) p

    Article Snippet: F-actin and other chemicals Human placental DNA, G-actin, Escherichia coli (O111:B4) lipopolysaccharide (LPS), and Staphylococcus aureus lipoteichoic acid (LTA) were purchased from Sigma Chemicals Co (St. Louis, MO, USA).

    Techniques: Activity Assay, Inhibition, Incubation

    Detection of the JCV T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from DNA extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.

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

    Article Title: JC virus DNA is present in the mucosa of the human colon and in colorectal cancers

    doi:

    Figure Lengend Snippet: Detection of the JCV T Ag sequence in human colorectal tissues. The 520-bp fragment (spanning JCV nucleotides 4481–5000, by using the primers JCT1 and JCT2) was amplified from DNA extracted from matched samples of normal colon (N) and colorectal cancers (T). ( Upper ) An EtdBr-stained 2% agarose gel demonstrating the 520-bp amplicons. ( Lower ) A Southern blot of the gel hybridized with a radiolabeled 416-bp probe for the JCV T antigen sequence obtained from the plasmid pMITC digested with Hin dIII. B indicates a PCR control lane (blank template plus all other reagents), and M refers to the molecular weight marker lane.

    Article Snippet: JCV-containing plasmids were serially diluted in 1-μg aliquots of human placental DNA (HumanCOT, GIBCO) and subjected to PCR amplification of the 120-bp target described above, by using primers JCT2 and JCN2, denaturing at 94°C for 2 min, annealing and extending at 62°C for 60 sec over 45 cycles by using Taq polymerase (GIBCO) and standard buffers (50 mM KCl, 10 mM Tris⋅HCl, and 2.0 mM MgCl2 ).

    Techniques: Sequencing, Amplification, Staining, Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Polymerase Chain Reaction, Molecular Weight, Marker

    Semiquantitative amplification of JCV T Ag sequence from colorectal tissue samples. ( Upper ) The 120-bp PCR product (spanning JCV nucleotides 4881–5000) obtained by amplifying the pMad1 (JCV sequence) plasmid. Concentrations of the plasmid DNA initially were determined by A 260 , and then diluted in 1 μg of human placental DNA (GIBCO), which provided an estimate of the minimal amount of template required to detect viral sequences (assuming similar efficiencies of amplification of plasmid DNA and authentic viral DNA). By using serial dilutions of template DNA to determine the point of extinction of the PCR product, the viral content in tumor specimens was estimated to be ≈0.01 viral copies/human genome equivalent. ( Lower ) An EtdBr-stained 2% agarose gel, and immediately below it, the corresponding Southern blot hybridized with the radiolabeled oligoprobe ESPB to confirm the sequence. Paired samples of template DNA from normal colon (N) and tumor specimens (T) from individual patients were diluted in parallel before amplification to determine the point at which the viral sequence could no longer be detected. Extinction of the PCR product from the normal samples, although still detectable from tumor samples, indicates that the tumor specimens contained a greater amount of amplifiable JCV than the matched normal colonic epithelium.

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

    Article Title: JC virus DNA is present in the mucosa of the human colon and in colorectal cancers

    doi:

    Figure Lengend Snippet: Semiquantitative amplification of JCV T Ag sequence from colorectal tissue samples. ( Upper ) The 120-bp PCR product (spanning JCV nucleotides 4881–5000) obtained by amplifying the pMad1 (JCV sequence) plasmid. Concentrations of the plasmid DNA initially were determined by A 260 , and then diluted in 1 μg of human placental DNA (GIBCO), which provided an estimate of the minimal amount of template required to detect viral sequences (assuming similar efficiencies of amplification of plasmid DNA and authentic viral DNA). By using serial dilutions of template DNA to determine the point of extinction of the PCR product, the viral content in tumor specimens was estimated to be ≈0.01 viral copies/human genome equivalent. ( Lower ) An EtdBr-stained 2% agarose gel, and immediately below it, the corresponding Southern blot hybridized with the radiolabeled oligoprobe ESPB to confirm the sequence. Paired samples of template DNA from normal colon (N) and tumor specimens (T) from individual patients were diluted in parallel before amplification to determine the point at which the viral sequence could no longer be detected. Extinction of the PCR product from the normal samples, although still detectable from tumor samples, indicates that the tumor specimens contained a greater amount of amplifiable JCV than the matched normal colonic epithelium.

    Article Snippet: JCV-containing plasmids were serially diluted in 1-μg aliquots of human placental DNA (HumanCOT, GIBCO) and subjected to PCR amplification of the 120-bp target described above, by using primers JCT2 and JCN2, denaturing at 94°C for 2 min, annealing and extending at 62°C for 60 sec over 45 cycles by using Taq polymerase (GIBCO) and standard buffers (50 mM KCl, 10 mM Tris⋅HCl, and 2.0 mM MgCl2 ).

    Techniques: Amplification, Sequencing, Polymerase Chain Reaction, Plasmid Preparation, Staining, Agarose Gel Electrophoresis, Southern Blot

    AR phosphorylation status selectively promotes the recruitment of ubiquitin–proteasome components at the PSA promoter. WT and XPD cells were transiently transfected to overexpress either AR/WT, AR/WT together with XPD/WT or AR/S515E (as indicated at the top of each panel). ( A – D ) Expression of the PSA gene: RT–qPCR analysis was performed at indicated times after DHT (10 −7 M) treatment. The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n -fold induction relative to non-treated cells. ( E – H ) After DHT treatment, the recruitment of RNA pol II (yellow curve), TFIIH (via its XPB and cdk7 subunits, blue and green curve, respectively) and AR (red curve) were analysed by ChIP assays at the PSA proximal promoter. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). ( I – L ) Recruitment of the MDM2 (green curve) and CHIP (yellow curve) E3 ligases on the PSA promoter. The recruitment of the ubiquitinated AR-containing fraction (brown curve) was also analysed by ChIP/re-ChIP assays using first an anti-ubiquitin antibody and second with an anti-AR antibody. ( M – P ) Recruitment of SUG1 (red curve), β5 (light blue) and S1 (dark blue) subunits of the proteasome at the PSA promoter was analysed by ChIP assays. Note that the recruitment of S1 has not been analysed in XPD cells transiently co-transfected with plasmids encoding AR/WT and XPD/WT ( O ).

    Journal: The EMBO Journal

    Article Title: The phosphorylation of the androgen receptor by TFIIH directs the ubiquitin/proteasome process

    doi: 10.1038/emboj.2010.337

    Figure Lengend Snippet: AR phosphorylation status selectively promotes the recruitment of ubiquitin–proteasome components at the PSA promoter. WT and XPD cells were transiently transfected to overexpress either AR/WT, AR/WT together with XPD/WT or AR/S515E (as indicated at the top of each panel). ( A – D ) Expression of the PSA gene: RT–qPCR analysis was performed at indicated times after DHT (10 −7 M) treatment. The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n -fold induction relative to non-treated cells. ( E – H ) After DHT treatment, the recruitment of RNA pol II (yellow curve), TFIIH (via its XPB and cdk7 subunits, blue and green curve, respectively) and AR (red curve) were analysed by ChIP assays at the PSA proximal promoter. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). ( I – L ) Recruitment of the MDM2 (green curve) and CHIP (yellow curve) E3 ligases on the PSA promoter. The recruitment of the ubiquitinated AR-containing fraction (brown curve) was also analysed by ChIP/re-ChIP assays using first an anti-ubiquitin antibody and second with an anti-AR antibody. ( M – P ) Recruitment of SUG1 (red curve), β5 (light blue) and S1 (dark blue) subunits of the proteasome at the PSA promoter was analysed by ChIP assays. Note that the recruitment of S1 has not been analysed in XPD cells transiently co-transfected with plasmids encoding AR/WT and XPD/WT ( O ).

    Article Snippet: For luciferase reporter assays, the full-length PSA promoter (−5025 to +25) was amplified from human placental genomic DNA and cloned into pGL3.basic (Promega) using the Gateway technology (Invitrogen) giving rise to pGL3.PSA-Luc.

    Techniques: Transfection, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Immunoprecipitation

    Transfer of the Platform ACE into heterologous cell lines. ( A ) Flow karyogram of metaphase chromosomes isolated from CHR1 cells and treated with Hoechst 33258 and chromomycin A3 dyes as described in Materials and Methods. The region circled and highlighted in red consists predominantly of the Platform ACE. ( B ) FISH analysis of the ChY1 cell line. The purified Platform ACE was transferred to CHO-DG44 cells and the resultant puromycin-resistant clone ChY1 was characterized by FISH. Metaphase chromosome spreads were prepared as described and hybridized with a mixture of DIG-labeled mouse major satellite repeat probe (rhodamine) PCR amplimer and biotin-labeled pFK161 probe (rDNA, FITC) generated by nick translation. ( C ) Southern-blot analysis of ChY1 and ChC2 Platform ACE cell lines. Genomic DNA samples from wildtype cell lines LMTK − , CHO-DG44 and CHO-S, as well as the corresponding Platform ACE cell lines CHR1, ChY1 and ChC2 were digested with NcoI and were fractionated on a 1% agarose gel. These were run along side varying dilutions of NcoI-digested pSV40attPPuro DNA so as to allow for determination of copy number. The fractionated DNA was transferred to nylon membrane by capillary blotting and the resultant membrane was hybridized with a labeled att P probe fragment (upper autoradiograph), then stripped and reprobed with a PCR-labeled hamster metallothionein-1 gene fragment (lower autoradiograph). Lane 1–4: copy number controls; 5 copies (lane 1), 10 copies (lane 2), 25 copies (lane 3), 50 copies (lane 4). Lane 5: empty. Lanes 6–11: cell line DNA; LMTK − (lane 6), CHR1 (lane 7), CHO-DG44 (lane 8), ChY1 (lane 9), CHO-S (lane 10), ChC2 (lane 11). The att P probe hybridizes to the expected 1.1 kb band in the CHR1, ChY1 and ChC2 DNA samples (upper). The hamster metallothionein-1 gene (chMT-1) probe detects the single copy gene and is used as a loading control for the CHO samples (lanes 8–11, lower panel). The hamster metallothionein-1 gene probe does not hybridize to the murine LMTK − derived samples, hence the absence of signal in lanes 6 and 7 (lower). Lanes 6, 8 and 10 are derived from the parental cell lines; lanes 7, 9 and 11 are cell lines derived after Platform ACE transfer. Note that the ChC2 sample (lane 11) is overloaded when compared with the other samples.

    Journal: Nucleic Acids Research

    Article Title: A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

    doi: 10.1093/nar/gnh169

    Figure Lengend Snippet: Transfer of the Platform ACE into heterologous cell lines. ( A ) Flow karyogram of metaphase chromosomes isolated from CHR1 cells and treated with Hoechst 33258 and chromomycin A3 dyes as described in Materials and Methods. The region circled and highlighted in red consists predominantly of the Platform ACE. ( B ) FISH analysis of the ChY1 cell line. The purified Platform ACE was transferred to CHO-DG44 cells and the resultant puromycin-resistant clone ChY1 was characterized by FISH. Metaphase chromosome spreads were prepared as described and hybridized with a mixture of DIG-labeled mouse major satellite repeat probe (rhodamine) PCR amplimer and biotin-labeled pFK161 probe (rDNA, FITC) generated by nick translation. ( C ) Southern-blot analysis of ChY1 and ChC2 Platform ACE cell lines. Genomic DNA samples from wildtype cell lines LMTK − , CHO-DG44 and CHO-S, as well as the corresponding Platform ACE cell lines CHR1, ChY1 and ChC2 were digested with NcoI and were fractionated on a 1% agarose gel. These were run along side varying dilutions of NcoI-digested pSV40attPPuro DNA so as to allow for determination of copy number. The fractionated DNA was transferred to nylon membrane by capillary blotting and the resultant membrane was hybridized with a labeled att P probe fragment (upper autoradiograph), then stripped and reprobed with a PCR-labeled hamster metallothionein-1 gene fragment (lower autoradiograph). Lane 1–4: copy number controls; 5 copies (lane 1), 10 copies (lane 2), 25 copies (lane 3), 50 copies (lane 4). Lane 5: empty. Lanes 6–11: cell line DNA; LMTK − (lane 6), CHR1 (lane 7), CHO-DG44 (lane 8), ChY1 (lane 9), CHO-S (lane 10), ChC2 (lane 11). The att P probe hybridizes to the expected 1.1 kb band in the CHR1, ChY1 and ChC2 DNA samples (upper). The hamster metallothionein-1 gene (chMT-1) probe detects the single copy gene and is used as a loading control for the CHO samples (lanes 8–11, lower panel). The hamster metallothionein-1 gene probe does not hybridize to the murine LMTK − derived samples, hence the absence of signal in lanes 6 and 7 (lower). Lanes 6, 8 and 10 are derived from the parental cell lines; lanes 7, 9 and 11 are cell lines derived after Platform ACE transfer. Note that the ChC2 sample (lane 11) is overloaded when compared with the other samples.

    Article Snippet: For the construction of pZeo-EPO (Figure B), the 2.1 kb genomic erythropoietin (EPO) coding sequence was generated by PCR from a human placental genomic DNA library (Clontech) using the EPO primer set (see Supplementary Material, Primer Table.pdf).

    Techniques: Flow Cytometry, Isolation, Fluorescence In Situ Hybridization, Purification, Labeling, Polymerase Chain Reaction, Generated, Nick Translation, Southern Blot, Agarose Gel Electrophoresis, Autoradiography, Derivative Assay

    Targeted integration of hrGFP onto Platform ACE. ATV pBLAS-GFP was co-transfected with the ACE Integrase expression vector pCXlamIntROK into ChY1 cells. Correct integration of the pBLAS-GFP into the Platform ACE acceptor site(s) would result in activation of the blast R gene. Transfected cells were placed in selective medium (blasticidin supplemented) and drug-resistant colonies were analyzed as described below (panels A–C). A huEPO ATV, pZeo-EPO was co-transfected with the ACE Integrase expression vector pCXlamIntROK into the Y1F5 clone, obtained from the above transfection. Correct integration of pZeo-EPO into the Platform ACE acceptor site(s) would result in activation of the zeo R gene. Transfected cells were placed in selective medium (Bleocin supplemented) and drug-resistant colonies were analyzed as described below (panels D–G). ( A ) DNA was prepared from blasticidin-resistant clones and analyzed by PCR for the presence of an att R site, indicative of targeted integration of the pBLAS-GFP onto the att P acceptor site(s) of the Platform ACE. The blasticidin primers (See Supplementary Material, Primer Table.pdf) will amplify a 961 bp fragment spanning the att R junction of a targeted integration. PCR reactions were analyzed by agarose gel electrophoresis: Analyzed clones (lanes 1–10), ChY1 negative control (lane 11), positive control (lane 12). On the basis of positive PCR signal, 9 out of the 10 clones analyzed had undergone targeted integration of the pBLAS-GFP ATV onto the Platform ACE. Clone Y1F5 (lane 9) was selected for double loading. ( B ) Clone Y1F5 (panel A, lane 9) was harvested and analyzed by flow cytometry for GFP fluorescence as described in Materials and Methods. As can be seen, 98% of cells are expressing GFP. ( C ) Metaphase chromosome spreads prepared from Clone Y1F5 were hybridized with a mixture of DIG-labeled mouse major satellite DNA repeat PCR amplimer probe (rhodamine) and biotin-labeled hrGFP targeting vector, pBLAS-GFP, (FITC) generated by nick translation. Note that the Platform ACE is labeled with both probes and, by contrast, neither probe labeled the native CHO chromosomes. ( D ) DNA was prepared from Bleocin-resistant clones and analyzed by PCR for the presence of an att R site, indicative of targeted integration of the pZeo-EPO onto the att P acceptor site(s) of the Platform ACE. The zeomycin primers (see Supplementary Material, Primer Table.pdf) will amplify a 697 bp fragment spanning the att R junction of a targeted integration event. PCR reactions were analyzed by agarose gel electrophoresis: Analyzed clones (lanes 1–24), positive control (lane 25), negative control (lane 26). On the basis of positive PCR signal, 20 out of 24 clones analyzed had undergone targeted integration of the pZeo-EPO ATV onto the Platform ACE. Clone 14-1-36P (lane 16) was selected for further analysis. ( E ) Clone 14-1-36P (panel D, lane 16) was harvested and analyzed by flow cytometry for GFP fluorescence as described in Materials and Methods. As can be seen, after a second round of gene loading > 98% of cells still expressed GFP. ( F ) Metaphase chromosome spreads prepared from Clone 14-1-36P were hybridized with a mixture of DIG-labeled mouse major satellite repeat probe (rhodamine) PCR amplimer and biotin-labeled huEPO targeting vector, pZeo-EPO, (FITC) generated by nick translation. Note that the Platform ACE is labeled with both probes and, by contrast, neither probe labeled the native CHO chromosomes. ( G ) The mean huEPO productivity was determined for selected clones as described in Materials and Methods.

    Journal: Nucleic Acids Research

    Article Title: A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

    doi: 10.1093/nar/gnh169

    Figure Lengend Snippet: Targeted integration of hrGFP onto Platform ACE. ATV pBLAS-GFP was co-transfected with the ACE Integrase expression vector pCXlamIntROK into ChY1 cells. Correct integration of the pBLAS-GFP into the Platform ACE acceptor site(s) would result in activation of the blast R gene. Transfected cells were placed in selective medium (blasticidin supplemented) and drug-resistant colonies were analyzed as described below (panels A–C). A huEPO ATV, pZeo-EPO was co-transfected with the ACE Integrase expression vector pCXlamIntROK into the Y1F5 clone, obtained from the above transfection. Correct integration of pZeo-EPO into the Platform ACE acceptor site(s) would result in activation of the zeo R gene. Transfected cells were placed in selective medium (Bleocin supplemented) and drug-resistant colonies were analyzed as described below (panels D–G). ( A ) DNA was prepared from blasticidin-resistant clones and analyzed by PCR for the presence of an att R site, indicative of targeted integration of the pBLAS-GFP onto the att P acceptor site(s) of the Platform ACE. The blasticidin primers (See Supplementary Material, Primer Table.pdf) will amplify a 961 bp fragment spanning the att R junction of a targeted integration. PCR reactions were analyzed by agarose gel electrophoresis: Analyzed clones (lanes 1–10), ChY1 negative control (lane 11), positive control (lane 12). On the basis of positive PCR signal, 9 out of the 10 clones analyzed had undergone targeted integration of the pBLAS-GFP ATV onto the Platform ACE. Clone Y1F5 (lane 9) was selected for double loading. ( B ) Clone Y1F5 (panel A, lane 9) was harvested and analyzed by flow cytometry for GFP fluorescence as described in Materials and Methods. As can be seen, 98% of cells are expressing GFP. ( C ) Metaphase chromosome spreads prepared from Clone Y1F5 were hybridized with a mixture of DIG-labeled mouse major satellite DNA repeat PCR amplimer probe (rhodamine) and biotin-labeled hrGFP targeting vector, pBLAS-GFP, (FITC) generated by nick translation. Note that the Platform ACE is labeled with both probes and, by contrast, neither probe labeled the native CHO chromosomes. ( D ) DNA was prepared from Bleocin-resistant clones and analyzed by PCR for the presence of an att R site, indicative of targeted integration of the pZeo-EPO onto the att P acceptor site(s) of the Platform ACE. The zeomycin primers (see Supplementary Material, Primer Table.pdf) will amplify a 697 bp fragment spanning the att R junction of a targeted integration event. PCR reactions were analyzed by agarose gel electrophoresis: Analyzed clones (lanes 1–24), positive control (lane 25), negative control (lane 26). On the basis of positive PCR signal, 20 out of 24 clones analyzed had undergone targeted integration of the pZeo-EPO ATV onto the Platform ACE. Clone 14-1-36P (lane 16) was selected for further analysis. ( E ) Clone 14-1-36P (panel D, lane 16) was harvested and analyzed by flow cytometry for GFP fluorescence as described in Materials and Methods. As can be seen, after a second round of gene loading > 98% of cells still expressed GFP. ( F ) Metaphase chromosome spreads prepared from Clone 14-1-36P were hybridized with a mixture of DIG-labeled mouse major satellite repeat probe (rhodamine) PCR amplimer and biotin-labeled huEPO targeting vector, pZeo-EPO, (FITC) generated by nick translation. Note that the Platform ACE is labeled with both probes and, by contrast, neither probe labeled the native CHO chromosomes. ( G ) The mean huEPO productivity was determined for selected clones as described in Materials and Methods.

    Article Snippet: For the construction of pZeo-EPO (Figure B), the 2.1 kb genomic erythropoietin (EPO) coding sequence was generated by PCR from a human placental genomic DNA library (Clontech) using the EPO primer set (see Supplementary Material, Primer Table.pdf).

    Techniques: Transfection, Expressing, Plasmid Preparation, Activation Assay, Clone Assay, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Negative Control, Positive Control, Flow Cytometry, Cytometry, Fluorescence, Labeling, Generated, Nick Translation

    FISH analyses of CHR1 Platform ACE Cell Line. Mitotic spreads of the CHR1 Platform ACE cell line (derived from murine LMTK − cells). The arrows indicate single Platform ACEs. ( A ) Mixture of probes: biotin-labeled att P (FITC signal) PCR amplimer and DIG-labeled mouse major satellite DNA repeat PCR amplimer (rhodamine signal). att P labeling was further enhanced by TSA treatment. ( B ) Probe is a biotin-labeled SV40attP PCR amplimer (FITC signal). ( C ) Mixture of probes: biotin-labeled pFK161 (rDNA; FITC signal) was generated by nick translation and DIG-labeled mouse major satellite DNA repeat (rhodamine signal) PCR amplimer. ( D ) Mixture of probes: a biotin-labeled telomere peptide nucleic acid probe (FITC signal), commercially prepared (Dako) and DIG-labeled pSV40attPPuro (rhodamine signal) generated by nick translation. Note that telomere probe signal is apparent at the ends of both short and long arms of the Platform ACE. ( E ) Mixture of probes: biotin-labeled pSV40attPPuro probe (FITC signal) and a DIG-labeled pFK161 (rDNA DNA fragment) probe (rhodamine signal), both probes were generated by nick translation. High-resolution FISH performed on spreads obtained from nuclei treated with Hoechst dye to allow for partial de-condensation of metaphase chromosomes. Note that while both probes label the long arm of the Platform ACE selectively they are not entirely coincident in all regions. ( F ) Mixture of probes: biotin-labeled mouse minor satellite DNA repeat (FITC signal) PCR amplimer and a DIG-labeled pSV40attPpuro probe (rhodamine signal) generated by nick translation.

    Journal: Nucleic Acids Research

    Article Title: A mammalian artificial chromosome engineering system (ACE System) applicable to biopharmaceutical protein production, transgenesis and gene-based cell therapy

    doi: 10.1093/nar/gnh169

    Figure Lengend Snippet: FISH analyses of CHR1 Platform ACE Cell Line. Mitotic spreads of the CHR1 Platform ACE cell line (derived from murine LMTK − cells). The arrows indicate single Platform ACEs. ( A ) Mixture of probes: biotin-labeled att P (FITC signal) PCR amplimer and DIG-labeled mouse major satellite DNA repeat PCR amplimer (rhodamine signal). att P labeling was further enhanced by TSA treatment. ( B ) Probe is a biotin-labeled SV40attP PCR amplimer (FITC signal). ( C ) Mixture of probes: biotin-labeled pFK161 (rDNA; FITC signal) was generated by nick translation and DIG-labeled mouse major satellite DNA repeat (rhodamine signal) PCR amplimer. ( D ) Mixture of probes: a biotin-labeled telomere peptide nucleic acid probe (FITC signal), commercially prepared (Dako) and DIG-labeled pSV40attPPuro (rhodamine signal) generated by nick translation. Note that telomere probe signal is apparent at the ends of both short and long arms of the Platform ACE. ( E ) Mixture of probes: biotin-labeled pSV40attPPuro probe (FITC signal) and a DIG-labeled pFK161 (rDNA DNA fragment) probe (rhodamine signal), both probes were generated by nick translation. High-resolution FISH performed on spreads obtained from nuclei treated with Hoechst dye to allow for partial de-condensation of metaphase chromosomes. Note that while both probes label the long arm of the Platform ACE selectively they are not entirely coincident in all regions. ( F ) Mixture of probes: biotin-labeled mouse minor satellite DNA repeat (FITC signal) PCR amplimer and a DIG-labeled pSV40attPpuro probe (rhodamine signal) generated by nick translation.

    Article Snippet: For the construction of pZeo-EPO (Figure B), the 2.1 kb genomic erythropoietin (EPO) coding sequence was generated by PCR from a human placental genomic DNA library (Clontech) using the EPO primer set (see Supplementary Material, Primer Table.pdf).

    Techniques: Fluorescence In Situ Hybridization, Derivative Assay, Labeling, Polymerase Chain Reaction, Generated, Nick Translation

    p50 protein associates with the C terminus of WRN in vitro. ( A ) Yeast two-hybrid screening. The WRNCT bait encoding amino acids 949-1401 (∼50 kDa) of the human WRN protein was cloned into pAS2–1 vector and used to screen a human placental cDNA library. The schematic representation of the WRNCT bait shows the C terminus nuclear localization signal (NLS), the conserved RecQ domain (RecQCt), and the putative nucleic acid binding domain (HRD). ( B ) Retransformation of the yeast expression plasmids to Y190 host strain. pAS2–1, bait vector alone; p50, full-length p50 in pACT2 vector in fusion with GAL4-activator domain. pLAM and pVA3 are human lamin C and murine p53, respectively, cloned into the pAS2–1 vector. ( C ) In vitro translation combined with immunoprecipitation. WRNCT and full-length p50 cloned into pcDNA3.1-His/Xpress and pcDNA 3.1-His/Myc vectors, respectively, were transcribed/translated in vitro in the presence (WRNCT) or absence (p50) of [ 35 S]methionine. Equal amounts of labeled WRNCT polypeptide were mixed with either control (lanes 1 and 3) or p50-Myc programmed nonradioactive reticulocyte lysates (lanes 2 and 4) and immunoprecipitated by anti-Myc (lanes 1 and 2) or anti-HA (lanes 3 and 4) polyclonal antibodies. The associated polypeptides were resolved by SDS-PAGE and visualized by autoradiography. The lower panel indicates 2.5% of the corresponding mixtures loaded before immunoprecipitation (input). We estimated that 4–5% of the input protein could be coprecipitated with the anti-Myc antibody used.

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

    Article Title: Werner protein recruits DNA polymerase ? to the nucleolus

    doi:

    Figure Lengend Snippet: p50 protein associates with the C terminus of WRN in vitro. ( A ) Yeast two-hybrid screening. The WRNCT bait encoding amino acids 949-1401 (∼50 kDa) of the human WRN protein was cloned into pAS2–1 vector and used to screen a human placental cDNA library. The schematic representation of the WRNCT bait shows the C terminus nuclear localization signal (NLS), the conserved RecQ domain (RecQCt), and the putative nucleic acid binding domain (HRD). ( B ) Retransformation of the yeast expression plasmids to Y190 host strain. pAS2–1, bait vector alone; p50, full-length p50 in pACT2 vector in fusion with GAL4-activator domain. pLAM and pVA3 are human lamin C and murine p53, respectively, cloned into the pAS2–1 vector. ( C ) In vitro translation combined with immunoprecipitation. WRNCT and full-length p50 cloned into pcDNA3.1-His/Xpress and pcDNA 3.1-His/Myc vectors, respectively, were transcribed/translated in vitro in the presence (WRNCT) or absence (p50) of [ 35 S]methionine. Equal amounts of labeled WRNCT polypeptide were mixed with either control (lanes 1 and 3) or p50-Myc programmed nonradioactive reticulocyte lysates (lanes 2 and 4) and immunoprecipitated by anti-Myc (lanes 1 and 2) or anti-HA (lanes 3 and 4) polyclonal antibodies. The associated polypeptides were resolved by SDS-PAGE and visualized by autoradiography. The lower panel indicates 2.5% of the corresponding mixtures loaded before immunoprecipitation (input). We estimated that 4–5% of the input protein could be coprecipitated with the anti-Myc antibody used.

    Article Snippet: The WRNCT bait was cotransformed intoY190 yeast cells with a human placental cDNA library fused to the GAL4 activation domain (CLONTECH HL4025AH).

    Techniques: In Vitro, Two Hybrid Screening, Clone Assay, Plasmid Preparation, cDNA Library Assay, Binding Assay, Expressing, Immunoprecipitation, Labeling, SDS Page, Autoradiography

    Regulation of COX7RP (A) and EB9 (B) mRNA by estrogen. MCF-7 cells were treated without (lane 1) or with (lanes 2 to 4) 10 −7 M 17β-estradiol for 6 h in the presence or absence of cycloheximide (lane 3) or actinomycin D (lane 4). Poly(A) + RNA (2 μg) was prepared from the cells, and Northern blot analysis was performed using COX7RP, EB9 cDNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as probes. The signals of transcripts and GAPDH are indicated. Migration positions of rRNA are shown on the right. (C) Estrogen-dependent enhancer activity of the ERE present in the first intron of the COX7RP gene and in the 5′ upstream region of the EBAG9 gene. Reporter plasmids containing pGCAT, vitERE-GCAT, EB1-ERE-GCAT, and EB9-ERE-GCAT were transfected into MCF-7 cells. After culture in the absence or presence of 10 −7 M 17β-estradiol, a CAT assay was performed. The intensities of the signals were analyzed with a BAS2000 image analyzer. The results are presented as means ± standard deviations of triplicate determinations.

    Journal: Molecular and Cellular Biology

    Article Title: Isolation of Estrogen-Responsive Genes with a CpG Island Library

    doi:

    Figure Lengend Snippet: Regulation of COX7RP (A) and EB9 (B) mRNA by estrogen. MCF-7 cells were treated without (lane 1) or with (lanes 2 to 4) 10 −7 M 17β-estradiol for 6 h in the presence or absence of cycloheximide (lane 3) or actinomycin D (lane 4). Poly(A) + RNA (2 μg) was prepared from the cells, and Northern blot analysis was performed using COX7RP, EB9 cDNA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as probes. The signals of transcripts and GAPDH are indicated. Migration positions of rRNA are shown on the right. (C) Estrogen-dependent enhancer activity of the ERE present in the first intron of the COX7RP gene and in the 5′ upstream region of the EBAG9 gene. Reporter plasmids containing pGCAT, vitERE-GCAT, EB1-ERE-GCAT, and EB9-ERE-GCAT were transfected into MCF-7 cells. After culture in the absence or presence of 10 −7 M 17β-estradiol, a CAT assay was performed. The intensities of the signals were analyzed with a BAS2000 image analyzer. The results are presented as means ± standard deviations of triplicate determinations.

    Article Snippet: An MCF-7 cDNA library was prepared from poly(A)+ RNA of MCF-7 cells with a λZAPII cDNA synthesis kit (Stratagene), and a human placental cDNA library was purchased (Stratagene).

    Techniques: Northern Blot, Migration, Activity Assay, Transfection