exonuclease iii Search Results


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  • 99
    New England Biolabs exonuclease iii
    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq <t>(three</t> experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1118 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher exonuclease iii
    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq <t>(three</t> experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
    Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 293 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Promega exonuclease iii
    Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic <t>DNA</t> from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of <t>three</t> independent experiments. n = 4 replicates per single experiment. Student's t -test.
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    93
    TaKaRa exonuclease iii
    Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic <t>DNA</t> from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of <t>three</t> independent experiments. n = 4 replicates per single experiment. Student's t -test.
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    92
    Boehringer Mannheim exonuclease iii
    Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with <t>Exo</t> <t>III</t> (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.
    Exonuclease Iii, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega exonuclease iii digestion
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    Exonuclease Iii Digestion, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 95 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    TaKaRa e coli exonuclease iii
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    E Coli Exonuclease Iii, supplied by TaKaRa, used in various techniques. Bioz Stars score: 96/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Pacific Biosciences exonuclease iii
    Repression of <t>HIF-1β</t> promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease <t>III</t> digestion. Relative positions of each clone and major TSSs
    Exonuclease Iii, supplied by Pacific Biosciences, used in various techniques. Bioz Stars score: 92/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene exonuclease iii
    VPS35 , VPS30, and <t>VPS29</t> are highly conserved in eukaryotes. ( A ) Schematic view of homology between Vps35p and proteins found in the C. elegans and M. musculus proteomes. The numbers between the proteins are percent identities between Vps35p and the other two proteins. In the lower section, the two most highly conserved domains (designated domains I and <t>III)</t> are aligned to show the regions of greatest homology between the yeast, mouse, nematode, and also human homologues. The black boxes indicate completely conserved residues. ( B ) Alignments between Vps29p and homologues present in the proteomes of C. elegans and H. sapiens , and also alignments between Vps30p and homologues present in the proteomes of C. elegans and H. sapiens . Black shaded regions indicate areas that are completely conserved.
    Exonuclease Iii, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 59 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher lambda exonuclease iii
    VPS35 , VPS30, and <t>VPS29</t> are highly conserved in eukaryotes. ( A ) Schematic view of homology between Vps35p and proteins found in the C. elegans and M. musculus proteomes. The numbers between the proteins are percent identities between Vps35p and the other two proteins. In the lower section, the two most highly conserved domains (designated domains I and <t>III)</t> are aligned to show the regions of greatest homology between the yeast, mouse, nematode, and also human homologues. The black boxes indicate completely conserved residues. ( B ) Alignments between Vps29p and homologues present in the proteomes of C. elegans and H. sapiens , and also alignments between Vps30p and homologues present in the proteomes of C. elegans and H. sapiens . Black shaded regions indicate areas that are completely conserved.
    Lambda Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 21 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    80
    Stratagene exonuclease iii mung bean nuclease deletion kit
    Analysis of polysomal mRNA. Data from spermidine- and arginine-grown cultures are shown on the left and right, respectively, and the tops of the gradients are to the right. (A) A 254 profile of 10 to 40% sucrose gradients. (B) Northern blot analysis of <t>spe-1</t> mRNA from the aga strain (IC3). (C) Northern blot analysis of spe-1 mRNA from transformant DMH3, lacking the 5′-UTR sequences between the Afl <t>III</t> and Nru I sites. (D) Northern blot analysis of tub mRNA of strain IC3 shown in panel B ( tub mRNA from DMH3 was similar). Polysomal fractions are aligned below their approximate positions in the gradient.
    Exonuclease Iii Mung Bean Nuclease Deletion Kit, supplied by Stratagene, used in various techniques. Bioz Stars score: 80/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher superscript iii cdna kit
    Analysis of polysomal mRNA. Data from spermidine- and arginine-grown cultures are shown on the left and right, respectively, and the tops of the gradients are to the right. (A) A 254 profile of 10 to 40% sucrose gradients. (B) Northern blot analysis of <t>spe-1</t> mRNA from the aga strain (IC3). (C) Northern blot analysis of spe-1 mRNA from transformant DMH3, lacking the 5′-UTR sequences between the Afl <t>III</t> and Nru I sites. (D) Northern blot analysis of tub mRNA of strain IC3 shown in panel B ( tub mRNA from DMH3 was similar). Polysomal fractions are aligned below their approximate positions in the gradient.
    Superscript Iii Cdna Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 329 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega exonuclease iii solution
    Analysis of polysomal mRNA. Data from spermidine- and arginine-grown cultures are shown on the left and right, respectively, and the tops of the gradients are to the right. (A) A 254 profile of 10 to 40% sucrose gradients. (B) Northern blot analysis of <t>spe-1</t> mRNA from the aga strain (IC3). (C) Northern blot analysis of spe-1 mRNA from transformant DMH3, lacking the 5′-UTR sequences between the Afl <t>III</t> and Nru I sites. (D) Northern blot analysis of tub mRNA of strain IC3 shown in panel B ( tub mRNA from DMH3 was similar). Polysomal fractions are aligned below their approximate positions in the gradient.
    Exonuclease Iii Solution, 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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    Image Search Results


    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Journal: Nature Communications

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    doi: 10.1038/ncomms15335

    Figure Lengend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Techniques: Mutagenesis, Two Tailed Test

    Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Overexpression of PRL-3 promotes telomere dysfunction. ( A ) Validation of PRL-3 stable overexpression. WI38 fibroblasts were infected with control or PRL-3-expressing letivirus. Expression vectors pcDNA3-myc-PRL-3 (for HCT116 cells), pcDNA3.1-myc-PRL-3 (for LoVo cells) and the respective control plasmids were transfected into cells, followed by selection and pooling of stable colonies. Cell lysates were examined by western blot with antibodies to PRL-3, TRF2 and RAP1. ( B ) Effects of PRL-3 stable overexpression on γH2AX, pCHK1 and p53 levels. Indicated cells were treated with ETP (20 μM) or DMSO (1:1000) for 4 h. ( C ) Effects of PRL-3 stable overexpression on TIF formation. WI38 cells were analyzed by IF-FISH staining of pATM (green) and telomere (red). Left, representative staining. Arrows, foci of TIFs. Scale bar, 5 μm. Right, quantification of cells with ≥5 TIFs. Mean ± SD of two independent experiments. n > 60 metaphase per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on dysfunctional telomere repair pathways. Upper, representative CO-FISH staining of WI38 cells. Metaphase cells were stained with probes specific for leading (red) and lagging (green) strands and counterstained with DAPI (blue). Yellow arrow, a typical T-SCE. White arrow, a chromosome–chromosome fusion. Red arrowhead, a MTS. Scale bar, 2.5 μm. Lower, quantification of abnormalities. Mean ± SD of two independent experiments. n > 1300 chromosomes per single experiment. Student's t -test. ( E ) Southern blot analysis of PRL-3 stable overexpression-induced telomere deprotection. Genomic DNA from indicated cells were resolved on agarose gel, transferred to nitrocellulose membrane and probed with biotin-labeled telomere probe. ( F ) qPCR analysis of PRL-3 stable overexpression-induced telomere deprotection. Relative telomere to single copy gene (T/S) ratio of control cells was set as 1. Mean ± SD of three independent experiments. n = 4 replicates per single experiment. Student's t -test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Over Expression, Infection, Expressing, Transfection, Selection, Western Blot, Fluorescence In Situ Hybridization, Staining, Southern Blot, Agarose Gel Electrophoresis, Labeling, Real-time Polymerase Chain Reaction

    Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Overexpression of PRL-3 promotes chromosomal instability and senescence. ( A ) Effects of PRL-3 stable overexpression on APB and MN formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. Student's t -test. n > 1500 cells scored per sample for MN or n > 60 anaphase cells scored per sample for APB. ( B ) Effects of PRL-3 stable overexpression on BrdU incorporation. Indicated cells were treated with double-thymidine block, released into fresh medium containing 10 μM BrdU and incubated for 45 min. Cells were fixed, immunostained with anti-BrdU (green), and counterstained with DAPI (blue). Left, representative staining of BrdU. Scale bar, 15 μm. Right, quantification of BrdU-positive cells. Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on senescence. Indicated cells were treated with DMSO (1:1000) or Ku55933 (5 μM) for 24 h, followed by β-galactosidase staining. Left, representative staining. Right, quantification of β-galactosidase positive cells. Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 stable overexpression on H3K9me3 levels. Indicated cells were fixed, immunostained with anti-H3K9me3 (red), and counterstained with DAPI (blue). ( E ) Effects of reconstituted PRL-3 on telomere length, DNA damage and senescence in PRL-3 stable knockdown cells. HCT116 control and PRL-3 stable knockdown cells were co-transfected with indicated amount of pcDNA3 and pcDNA3-PRL-3 plasmids. The total amount of plasmids for each sample was adjusted to 4 μg. After 72 h, protein lysates were subjected to western blot of PRL-3, γH2AX, H3K9me3 (lower). Genomic DNA was used for qPCR analysis of telomere length (upper). Protein bands were scanned and relative OD was calculated by normalizing to GAPDH. T/S ratio of HCT116 control cells transfected with pcDNA3 was set as 1. Pearson χ2 test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Over Expression, Staining, BrdU Incorporation Assay, Blocking Assay, Incubation, Transfection, Western Blot, Real-time Polymerase Chain Reaction

    Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Silencing of PRL-3 promotes DDR and senescence. ( A ) Efficiencies of PRL-3 silencing in HCT116 (knockdown by two shRNAs using lentivirus system, left) and SW480 (knockout by CRISPR/Cas9 system, right) cells and its effects on indicated protein levels. WT, wild-type. KO, knockout. ( B ) Effects of PRL-3 silencing on phosphorylations of H2AX and CHK1. Samples treated with 20 μM etoposide (ETP) for 4 h were used as positive controls. ( C ) Effects of PRL-3 silencing on TIF formation. Indicated HCT116 cells were subjected to IF-FISH staining. Upper, representative staining. Arrows, colocalizations between γH2AX and telomere (TIFs). Scale bar, 5 μm. Lower, quantification of cells with ≥5 TIF. Mean ± SD of two independent experiments. n > 200 cells per single experiment. Student's t -test. ( D ) Effects of PRL-3 silencing on anaphase bridges (APB) and micronuclei (MN) formation. Indicated cells were treated with aphidicolin (0.2 μM) or DMSO (1:1000) for 24 h, followed by DAPI staining. Mean ± SD of two independent experiments. n > 1000 cells scored per sample for MN and n > 50 anaphase cells scored per sample for APB. Student's t -test. Representative images of APB (red arrow) and MN (white arrow) of HCT116 cells stained with DAPI were shown. ( E ) ChIP analysis of RAP1 and TRF2's binding to telomeric or Alu DNA in HCT116 and S480 cells silenced for PRL-3. Upper, representative blots after ChIP with indicated antibodies or IgG. Input, 2% DNA. Lower, quantification of relative OD. Relative OD was calculated by normalizing to that of input and relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( F ) PRL-3 silencing induced ROS-dependent cellular senescence and DNA damage response. Indica ted HCT116 cells were treated with NAC (10 mM), GSH (10 mM) or DMSO (1:1000) for 24 h. Part of cells were fixed and processed for β-galactosidase staining, others were analyzed by western blot. Upper, representative β-galactosidase staining of cells treated with DMSO. Middle, quantification of β-galactosidase positive cells. Mean ± SD of two independent experiments. n > 400 cells per single experiment. Student's t -test. Lower, western blot of γH2AX.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Knock-Out, CRISPR, Fluorescence In Situ Hybridization, Staining, Chromatin Immunoprecipitation, Binding Assay, Western Blot

    RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: RAP1 and TRF2-dependent recruitment of PRL-3 to telomere. ( A ) In situ PLA analysis of PRL-3's associations with RAP1 and TRF2. HCT116 cells were pre-extracted, fixed, inmunostained with indicated pairs of antibodies and probed with Duolink in situ PLA reagent. Binding foci were in red and dashed lines indicated outline of nucleus (determined by DAPI counter staining). Scale bar, 10 μm. ( B ) TRF2- and RAP1-dependent recruitment of PRL-3 to telomeric DNA in vitro . Purified myc-TRF2 (150 ng), His-RAP1 (120 ng), and His-PRL-3 (30 ng) were co-incubated with 1 μg biotin-labeled telomere (lanes 1–4) or Alu (lanes 5–8) probe as indicated and subjected to pull-down analysis with Streptavidin agarose. Precipitates were analyzed by western blot with antibodies to TRF2, RAP1 and PRL-3. ( C and D ) TRF2 and RAP1-dependent recruitment of PRL-3 to telomere in cells. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h, pre-extracted, fixed and subjected to IF-FISH staining. (C) Representative PRL-3 association with telomere. Scale bar, 10 μm. Areas in white squares were enlarged. (D) Quantification of cells with ≥5 associations between PRL-3 foci and telomere. Mean ± SD of three independent experiments. n > 100 cells per single experiment. Student's t -test. ( E ) Knockdown efficiencies of RAP1 and TRF2. HCT116 cells were transfected with 50 nM siRNAs against RAP1 or TRF2 for 48 h. Lysates were analyzed by western blot with indicated antibodies. ( F ) ChIP analysis of PRL-3 binding to telomeric and Alu DNA. HCT116 cells were transfected with 50 nM indicated siRNAs for 48 h and processed for ChIP using anti-PRL-3 or pre-immune IgG. Upper, representative blots of hybridization with probe to telomere or Alu. Input, 2% DNA. Lower, quantification of relative optical densities (OD). Relative OD was calculated by normalizing to OD of Input and relative OD of control siRNA-transfected sample was set as 100%. Mean ± SD of three independent experiments. Student's t -test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: In Situ, Proximity Ligation Assay, Binding Assay, Staining, In Vitro, Purification, Incubation, Labeling, Western Blot, Transfection, Fluorescence In Situ Hybridization, Chromatin Immunoprecipitation, Hybridization

    PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: PRL-3 relocates RAP1 and TRF2 from telomeric DNA. ( A ) Effects of PRL-3 stable overexpression on the chromatin abundance of RAP1, TRF2 and TRF1. Nuclei from HCT116 cells were homogenized in buffer containing indicated concentrations of NaCl. Chromatin-enriched fractions were analyzed by western blot. Left, representative blots. Right, relative levels of TRF2, RAP1 and TRF1. Protein band were scanned and relative OD was calculated by normalizing to OD of H2B. The relative OD of sample prepared with 150 mM NaCl was set as 100%. Mean ± SD of three independent experiments. ANOVA. ( B ) Effects of PRL-3 stable overexpression on bindings of RAP1 and TRF2 to telomeric and Alu DNA. Indicated cells were crosslinked, immunoprecipitated with antibodies to RAP1, TRF2 or pre-immune IgG, and precipitated DNA was analyzed by ChIP. Upper, representative blots. Lower, quantification of relative OD, which was calculated by normalizing to that of Input. Relative OD of control was set as 100%. Mean ± SD of three independent experiments. Student's t -test. ( C ) Effects of PRL-3 stable overexpression on telomere associations of RAP1 and TRF2 in WI38 cells. Left, representative IF-FISH staining of telomere (red) and RAP1 or TRF2 (green). Arrows, foci of co-localization. Scale bar, 10 μm. Right, quantification of cells with ≥5 associations between RAP1 or TRF2 foci and telomere. Mean ± SD of two independent experiments. n > 80 cells per single experiment. Student's t -test. ( D ) EMSA analysis of PRL-3, RAP1 and TRF2's associations with telomeric DNA. Indicated concentrations of purified FLAG-TRF2, His-RAP1, myc-PRL-3 were co-incubated with Biotin-labeled telomere probe (20 nM). To induce super-shift, 0.1 μg anti-PRL-3 (lane 5), anti-TRF2 (lanes 6 and 18) and IgG (lane 7) were used. Note that anti-PRL-3 and anti-TRF2-induced super-shifts of Complex II partially co-migrated with Complex I (lanes 5 and 6).

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Over Expression, Western Blot, Immunoprecipitation, Chromatin Immunoprecipitation, Fluorescence In Situ Hybridization, Staining, Purification, Incubation, Labeling

    Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

    Journal: Nucleic Acids Research

    Article Title: PRL-3 promotes telomere deprotection and chromosomal instability

    doi: 10.1093/nar/gkx392

    Figure Lengend Snippet: Disrupting PRL-3-RAP1 complex or expressing ectopic TRF2 attenuates PRL-3 overexpression-promoted telomere deprotection, DNA damage, chromosomal instability and senescence. ( A ) HCT116 control and PRL-3 overexpressing cells were transfected with 0.5 μg of pEGFP-N1-Myb or pEGFP-N1 plasmid for 72 h, and indicated proteins were analyzed by western blot. ( B ) qPCR analysis of telomere length of cells in (A). T/S ratio of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test. ( C ) Quantification of micronuclei of cells in (A). Mean ± SD of two independent experiments. n > 500 cells per single experiment. Student's t -test. ( D ) Quantification of β-galactosidase-positive cells in (A). Mean ± SD of two independent experiments. n > 300 cells per single experiment. Student's t -test. ( E ) Relative migration of cells in (A). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells transfected with pEGFP-N1 was set as 1. Mean ± SD of two independent experiments. Three replicates per single experiment. Student's t -test. ( F ) HCT116 control and PRL-3 overexpressing cells were infected with control (Lv-con) or TRF2-expressing lentivirus (Lv-TRF2) for 120 h, and lysates were subjected to western blot. ( G ) qPCR analysis of telomere length of cells in (F). T/S ratio of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. 3 replicates per single experiment. Student's t -test. ( H ) Quantification of micronuclei of cells in (F). Mean ± SD of three independent experiments. n > 500 cells per single experiment. Student's t -test. ( I ) Quantification of β-galactosidase-positive cells in (F). Mean ± SD of three independent experiments. n > 300 cells per single experiment. Student's t -test. ( J ) Relative migration of cells of (F). Cells were allowed to migrate through transwell chambers for 24 h. Value of HCT116 control cells infected with Lv-con was set as 1. Mean ± SD of three independent experiments. Three replicates per single experiment. Student's t -test.

    Article Snippet: The BrdU/BrdC-substituted DNA was digested with 10 U/μl Exonuclease III (Promega) in 50 mM Tris–HCl pH 8.0, 5 mM MgCl2 and 5 mM DTT for 10 min at room temperature.

    Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, Western Blot, Real-time Polymerase Chain Reaction, Migration, Infection

    Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with Exo III (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.

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

    Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

    doi:

    Figure Lengend Snippet: Diagram of experimental design and intrinsic terminator RNA secondary structures. ( A ) Transcription was initiated at the promoter (bent rightward arrow) and the ternary complexes were walked proximal to the termination site (T) on double-stranded PCR-generated templates that were biotinylated (shaded circle containing the letter B) at one of the 5′ ends. Next, the stalled elongation complexes (shaded ellipse) were digested with Exo III (shaded circles, missing a piece) and then all four NTPs were added, allowing the stalled elongation complex to resume transcript elongation. 5′ and 3′ ends of the DNA are as indicated. ( B ) The RNA secondary structures of the three intrinsic terminators, λ tR2, T 7 Te, and rrn BT1, are shown. The underlined bases indicate the positions of transcript release and the length of the RNA product [λ tR2 and rrn , T. Kerppola and M.J.C., unpublished results) and T 7 )]. The RNA secondary structures are drawn to conform to the classical model of transcript termination, with a G+C-rich stem and U-rich region immediately downstream. It should be noted, however, that for the λ tR2 and the rrn BT1 terminators, the hairpin can be extended to include upstream adenosines and downstream uridines.

    Article Snippet: The following reagents were purchased from the sources noted: α-32 P-labeled nucleoside triphosphates (NEN or Amersham); HPLC grade nucleoside triphosphates and deoxynucleoside triphosphates (Pharmacia); Hybond N+ nylon membrane (Amersham); dinucleotide ApU, BSA, yeast Torula RNA, polymin P, and rifampicin (Sigma); M-280 streptavidin Dynabeads (Dynal, Oslo); Seq-Light (Tropix, Bedford MA); exonuclease III (Exo III) (Boehringer Mannheim); AmpliTaq (Perkin–Elmer); Ni2+ -NTA agarose (Qiagen, Chatsworth CA); phosphocellulose (Whatman); X-Omat scientific imaging film (Kodak); and 5′ biotin phosphoramidite (Glen Research, Sterling, VA).

    Techniques: Polymerase Chain Reaction, Generated

    Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. ( A ) Stalled C 47 complexes (lanes 1 and 5) in TGK-B 40 M 1 were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C 47 ). ( B ) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.

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

    Article Title: Escherichia coli RNA polymerase terminates transcription efficiently at rho-independent terminators on single-stranded DNA templates

    doi:

    Figure Lengend Snippet: Assaying for intrinsic termination at the λ tR2 terminator on both ss- and dsDNA templates. ( A ) Stalled C 47 complexes (lanes 1 and 5) in TGK-B 40 M 1 were chased with 500 μM ATP, 500 μM GTP, 500 μM CTP, and 500 μM UTP for 10 min at 42°C in the presence of rifampicin at 20 μg/ml (lanes 2–4 and 6–8). Lanes 2 and 6 are aliquots of the chase reactions before the pellet was separated from the supernatant fluid, lanes 3 and 7 are the RNAs associated with the Dynabeads (P), and lanes 4 and 8 are the RNAs released into the supernatant fluid (S). The λ tR2 terminator is at positions +104 to +105 relative to the transcription start site, whereas the end of the DNA template or run-off occurs at position +177. Lanes: 1–4, no Exo III; 5–8, digested with Exo III (2,000 units/ml) for 5 min at 30°C. Quantitation of the total counts within a lane between the C 47 ). ( B ) Exo III-digested DNA was analyzed on a 6% denaturing PAGE gel and detected by chemiluminescence. The nontemplate strand was end-labeled with on the 5′ end with a biotin moiety. Lanes: 1, no Exo III; 2, Exo III at 2,000 units/ml for 5 min at 30°C. The initial DNA template, 315 nucleotides long was reduced to approximately 204 nucleotides after Exo III digestion.

    Article Snippet: The following reagents were purchased from the sources noted: α-32 P-labeled nucleoside triphosphates (NEN or Amersham); HPLC grade nucleoside triphosphates and deoxynucleoside triphosphates (Pharmacia); Hybond N+ nylon membrane (Amersham); dinucleotide ApU, BSA, yeast Torula RNA, polymin P, and rifampicin (Sigma); M-280 streptavidin Dynabeads (Dynal, Oslo); Seq-Light (Tropix, Bedford MA); exonuclease III (Exo III) (Boehringer Mannheim); AmpliTaq (Perkin–Elmer); Ni2+ -NTA agarose (Qiagen, Chatsworth CA); phosphocellulose (Whatman); X-Omat scientific imaging film (Kodak); and 5′ biotin phosphoramidite (Glen Research, Sterling, VA).

    Techniques: Quantitation Assay, Polyacrylamide Gel Electrophoresis, Labeling

    Repression of HIF-1β promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease III digestion. Relative positions of each clone and major TSSs

    Journal: Journal of immunology (Baltimore, Md. : 1950)

    Article Title: IFN-? Attenuates Hypoxia-Inducible Factor (HIF) Activity in Intestinal Epithelial Cells through Transcriptional Repression of HIF-1?

    doi: 10.4049/jimmunol.1001442

    Figure Lengend Snippet: Repression of HIF-1β promoter activity by IFN-γ. A , Cloned HIF-1β promoter and series of truncated promoter-luciferase reporter constructs generated using exonuclease III digestion. Relative positions of each clone and major TSSs

    Article Snippet: Sequential truncations of the cloned HIF-1β promoter sequence were generated by exonuclease III digestion (Erase-a-Base; Promega).

    Techniques: Activity Assay, Clone Assay, Luciferase, Construct, Generated

    VPS35 , VPS30, and VPS29 are highly conserved in eukaryotes. ( A ) Schematic view of homology between Vps35p and proteins found in the C. elegans and M. musculus proteomes. The numbers between the proteins are percent identities between Vps35p and the other two proteins. In the lower section, the two most highly conserved domains (designated domains I and III) are aligned to show the regions of greatest homology between the yeast, mouse, nematode, and also human homologues. The black boxes indicate completely conserved residues. ( B ) Alignments between Vps29p and homologues present in the proteomes of C. elegans and H. sapiens , and also alignments between Vps30p and homologues present in the proteomes of C. elegans and H. sapiens . Black shaded regions indicate areas that are completely conserved.

    Journal: The Journal of Cell Biology

    Article Title: Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

    doi:

    Figure Lengend Snippet: VPS35 , VPS30, and VPS29 are highly conserved in eukaryotes. ( A ) Schematic view of homology between Vps35p and proteins found in the C. elegans and M. musculus proteomes. The numbers between the proteins are percent identities between Vps35p and the other two proteins. In the lower section, the two most highly conserved domains (designated domains I and III) are aligned to show the regions of greatest homology between the yeast, mouse, nematode, and also human homologues. The black boxes indicate completely conserved residues. ( B ) Alignments between Vps29p and homologues present in the proteomes of C. elegans and H. sapiens , and also alignments between Vps30p and homologues present in the proteomes of C. elegans and H. sapiens . Black shaded regions indicate areas that are completely conserved.

    Article Snippet: Constructs for sequencing the VPS29 gene were made by treating pEMY29-1 with exonuclease III and mung bean nuclease as described in the Stratagene BlueScript manual.

    Techniques:

    Analysis of polysomal mRNA. Data from spermidine- and arginine-grown cultures are shown on the left and right, respectively, and the tops of the gradients are to the right. (A) A 254 profile of 10 to 40% sucrose gradients. (B) Northern blot analysis of spe-1 mRNA from the aga strain (IC3). (C) Northern blot analysis of spe-1 mRNA from transformant DMH3, lacking the 5′-UTR sequences between the Afl III and Nru I sites. (D) Northern blot analysis of tub mRNA of strain IC3 shown in panel B ( tub mRNA from DMH3 was similar). Polysomal fractions are aligned below their approximate positions in the gradient.

    Journal: Molecular and Cellular Biology

    Article Title: Polyamine Regulation of Ornithine Decarboxylase Synthesis in Neurospora crassa

    doi:

    Figure Lengend Snippet: Analysis of polysomal mRNA. Data from spermidine- and arginine-grown cultures are shown on the left and right, respectively, and the tops of the gradients are to the right. (A) A 254 profile of 10 to 40% sucrose gradients. (B) Northern blot analysis of spe-1 mRNA from the aga strain (IC3). (C) Northern blot analysis of spe-1 mRNA from transformant DMH3, lacking the 5′-UTR sequences between the Afl III and Nru I sites. (D) Northern blot analysis of tub mRNA of strain IC3 shown in panel B ( tub mRNA from DMH3 was similar). Polysomal fractions are aligned below their approximate positions in the gradient.

    Article Snippet: This plasmid was used as starting material for 5′-to-3′ deletions from the spe-1 upstream Pst I site using an exonuclease III/mung bean nuclease deletion kit (Stratagene) as specified by the manufacturer.

    Techniques: Northern Blot

    Restriction map of the spe-1 gene and flanking sequences found in plasmids pPHL1 and pPH1. The boxed area represents the transcribed region, beginning with the right-pointing arrow. The coding sequence, interrupted by one intron, is shown in black. Abbreviations: MCS, multiple-cloning site; B, Bgl II; V, Eco V; C, Cla I; R, Eco RI; P, Pst I; S, Sac I; A, Afl III; N, Nru I; K, Kpn I; Sa, Sal I; H, Hin dIII.

    Journal: Molecular and Cellular Biology

    Article Title: Polyamine Regulation of Ornithine Decarboxylase Synthesis in Neurospora crassa

    doi:

    Figure Lengend Snippet: Restriction map of the spe-1 gene and flanking sequences found in plasmids pPHL1 and pPH1. The boxed area represents the transcribed region, beginning with the right-pointing arrow. The coding sequence, interrupted by one intron, is shown in black. Abbreviations: MCS, multiple-cloning site; B, Bgl II; V, Eco V; C, Cla I; R, Eco RI; P, Pst I; S, Sac I; A, Afl III; N, Nru I; K, Kpn I; Sa, Sal I; H, Hin dIII.

    Article Snippet: This plasmid was used as starting material for 5′-to-3′ deletions from the spe-1 upstream Pst I site using an exonuclease III/mung bean nuclease deletion kit (Stratagene) as specified by the manufacturer.

    Techniques: Sequencing, Clone Assay

    Role of the UAR on the regulation of spe-1 genes lacking the Afl III- Nru I segment of the 5′-UTR. (A) Schematic of spe-1 genes lacking 5′-UTR sequences in which the spe-1 UAR is either present (DMH3) or absent (DMH4). The positions of the Afl III and Nru I sites of the 5′-UTR are indicated. (B) Northern blot analysis of repressed (SPD) and derepressed (ARG) cultures of the transformants. Northern blots were hybridized with probes derived from spe-1 cDNA or the coding region of the tub gene.

    Journal: Molecular and Cellular Biology

    Article Title: Polyamine Regulation of Ornithine Decarboxylase Synthesis in Neurospora crassa

    doi:

    Figure Lengend Snippet: Role of the UAR on the regulation of spe-1 genes lacking the Afl III- Nru I segment of the 5′-UTR. (A) Schematic of spe-1 genes lacking 5′-UTR sequences in which the spe-1 UAR is either present (DMH3) or absent (DMH4). The positions of the Afl III and Nru I sites of the 5′-UTR are indicated. (B) Northern blot analysis of repressed (SPD) and derepressed (ARG) cultures of the transformants. Northern blots were hybridized with probes derived from spe-1 cDNA or the coding region of the tub gene.

    Article Snippet: This plasmid was used as starting material for 5′-to-3′ deletions from the spe-1 upstream Pst I site using an exonuclease III/mung bean nuclease deletion kit (Stratagene) as specified by the manufacturer.

    Techniques: Northern Blot, Derivative Assay

    Expression of various spe-1 and chimeric transcripts driven by the β-tubulin ( tub ) promoter of N. crassa . (A) Schematic diagram of tub :: spe-1 genes introduced into strain IC54, with functional regions of each gene listed across the top. The spe-1 sequences are represented by open boxes; and tub sequences are represented by shaded boxes. The positions of the Afl III and Nru I sites in the spe-1 5′-UTR are shown. (B) Northern blots of 10 μg of total RNA from repressed (SPD) or derepressed (ARG) cultures, probed with spe-1 cDNA or the coding region of tub DNA. The relative abundance of tub :: spe-1 mRNA in each transformant, normalized to tub mRNA and relative to that in DMH43/SPD, is given below the panel.

    Journal: Molecular and Cellular Biology

    Article Title: Polyamine Regulation of Ornithine Decarboxylase Synthesis in Neurospora crassa

    doi:

    Figure Lengend Snippet: Expression of various spe-1 and chimeric transcripts driven by the β-tubulin ( tub ) promoter of N. crassa . (A) Schematic diagram of tub :: spe-1 genes introduced into strain IC54, with functional regions of each gene listed across the top. The spe-1 sequences are represented by open boxes; and tub sequences are represented by shaded boxes. The positions of the Afl III and Nru I sites in the spe-1 5′-UTR are shown. (B) Northern blots of 10 μg of total RNA from repressed (SPD) or derepressed (ARG) cultures, probed with spe-1 cDNA or the coding region of tub DNA. The relative abundance of tub :: spe-1 mRNA in each transformant, normalized to tub mRNA and relative to that in DMH43/SPD, is given below the panel.

    Article Snippet: This plasmid was used as starting material for 5′-to-3′ deletions from the spe-1 upstream Pst I site using an exonuclease III/mung bean nuclease deletion kit (Stratagene) as specified by the manufacturer.

    Techniques: Expressing, Functional Assay, Northern Blot

    Effects of 5′-to-3′ deletions of the spe-1 upstream region on ODC activity and derepression of spe-1 mRNA. (A) Schematic representation of the wild-type (P2) and deleted spe-1 genes integrated at the his-3 locus of strain IC2794-5. Distances from the major transcription start site, indicated by the arrow, are given in base pairs. The relative positions of the Pst I (−1000), Sac I (−167), and Afl III (+97) sites are also shown. ODC activity (in units per milligram of protein) of the transformants grown with 1 mM spermidine (SPD) or 1 mM arginine (ARG) are given to the right. (B) Northern blots of total RNA (10 μg) from repressed (left) and derepressed (right) cultures of these transformants were probed with spe-1 cDNA ( spe-1 ) or a fragment of the β-tubulin gene ( tub ), the latter as a loading control. Lanes: 1, P2; 2, PΔ1; 3, PΔ2; 4, PΔ3; 5, PΔ4; 6, PΔ5; 7, S8; 8, PΔ6; 9, PΔ7. (C) Approximately 25 μg of total RNA from the derepressed transformants was analyzed by primer extension reactions with the MH12 primer to determine the 5′ ends of their spe-1 transcripts. The molecular size marker on the right is given in nucleotides. Lanes are labeled as in panel B.

    Journal: Molecular and Cellular Biology

    Article Title: Polyamine Regulation of Ornithine Decarboxylase Synthesis in Neurospora crassa

    doi:

    Figure Lengend Snippet: Effects of 5′-to-3′ deletions of the spe-1 upstream region on ODC activity and derepression of spe-1 mRNA. (A) Schematic representation of the wild-type (P2) and deleted spe-1 genes integrated at the his-3 locus of strain IC2794-5. Distances from the major transcription start site, indicated by the arrow, are given in base pairs. The relative positions of the Pst I (−1000), Sac I (−167), and Afl III (+97) sites are also shown. ODC activity (in units per milligram of protein) of the transformants grown with 1 mM spermidine (SPD) or 1 mM arginine (ARG) are given to the right. (B) Northern blots of total RNA (10 μg) from repressed (left) and derepressed (right) cultures of these transformants were probed with spe-1 cDNA ( spe-1 ) or a fragment of the β-tubulin gene ( tub ), the latter as a loading control. Lanes: 1, P2; 2, PΔ1; 3, PΔ2; 4, PΔ3; 5, PΔ4; 6, PΔ5; 7, S8; 8, PΔ6; 9, PΔ7. (C) Approximately 25 μg of total RNA from the derepressed transformants was analyzed by primer extension reactions with the MH12 primer to determine the 5′ ends of their spe-1 transcripts. The molecular size marker on the right is given in nucleotides. Lanes are labeled as in panel B.

    Article Snippet: This plasmid was used as starting material for 5′-to-3′ deletions from the spe-1 upstream Pst I site using an exonuclease III/mung bean nuclease deletion kit (Stratagene) as specified by the manufacturer.

    Techniques: Activity Assay, Northern Blot, Marker, Labeling