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  • 99
    Millipore dna probes
    Models of mammalian <t>mtDNA</t> replication. Two alternative mechanisms of strand-asynchronous replication have been proposed: ( A ) the strand-displacement model that depends on protein (mtSSB) to coat the displaced lagging-strand template, and ( B ) the bootlace mechanism that relies on mature transcripts. ( C ) Intermediates with the properties of conventional (concurrent) leading and lagging-strand <t>DNA</t> synthesis have also been reported in mammalian mitochondria suggesting that they also operate a strand-coupled (SC) mechanism of replication. ( D ) Restriction digestion of the intermediates of strand-asynchronous replication will not cut at sites that are single-stranded DNA (in the case of many restriction enzymes) or where there is RNA/DNA hybrid, which produces atypical structures compared to SC replication as illustrated, and these form so-called supra-Y (SY) arcs when resolved by 2D-AGE ( E ).
    Dna Probes, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Gen-Probe dna probes
    Models of mammalian <t>mtDNA</t> replication. Two alternative mechanisms of strand-asynchronous replication have been proposed: ( A ) the strand-displacement model that depends on protein (mtSSB) to coat the displaced lagging-strand template, and ( B ) the bootlace mechanism that relies on mature transcripts. ( C ) Intermediates with the properties of conventional (concurrent) leading and lagging-strand <t>DNA</t> synthesis have also been reported in mammalian mitochondria suggesting that they also operate a strand-coupled (SC) mechanism of replication. ( D ) Restriction digestion of the intermediates of strand-asynchronous replication will not cut at sites that are single-stranded DNA (in the case of many restriction enzymes) or where there is RNA/DNA hybrid, which produces atypical structures compared to SC replication as illustrated, and these form so-called supra-Y (SY) arcs when resolved by 2D-AGE ( E ).
    Dna Probes, supplied by Gen-Probe, used in various techniques. Bioz Stars score: 92/100, based on 229 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abbott Laboratories pathvysion her 2 dna probe kit
    Validation of 17q12 amplification in SDCs. Composite illustration of chromosome (Chr) 17q12 <t>DNA</t> ( A ) and corresponding <t>ERBB2</t> gene amplification by FISH ( B ) and the expression by IHC analysis ( C ) in the four SDCs with amplicons at this location (SDCs 1,
    Pathvysion Her 2 Dna Probe Kit, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 581 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Abbott Laboratories pathvysion her2 dna probe kit
    The relationship of subtypes to the <t>DNA</t> ploidy and CIN. The diploid/CIN- status was detected in 72 (64.9%) of the 111 luminal A carcinomas, 39 (41.1%) of the 95 luminal B <t>(HER2-)</t> carcinomas, 8 (11.6%) of the 69 lumminal B (HER2+) carcinomas, 2 (4.9%) of the 41 HER2 carcinomas, and 8 (17.0%) of the 26 basal-like carcinomas. In contrast, the aneuploid/CIN + status was detected in 31 (27.9%) of the 111 luminal A, 47 (49.5%) of the luminal B (HER2-), 56 (81.2%) of the 69 luminal B (HER2+), 37 (90.2%) of the 41 HER2, and 36 (86.6%) of the 47 basal-like subtype tumors. The incidence of diploid/CIN- and aneuploid/CIN+ status was different between the luminal A subtype and luminal B (HER2-), luminal B (HER2+), HER2, and basal-like subtypes (p = 0.0006, p = 5E-13, p = 5E10-12, and p = 8E10-9). In addition, the incidence of diploid/CIN- and aneuploid/CIN+ status was statistically different between the luminal B (HER2-) subtype and luminal B (HER2+), HER2, and basal-like subtypes (p = 0.00002, p = 0.000009, and p = 0.002). Black bar; diploid/CIN- tumors, gray bar; aneuploid/CIN+ tumors, white column; others.
    Pathvysion Her2 Dna Probe Kit, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 90/100, based on 591 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Abbott Laboratories dna probes
    Representative examples of conventional cytology and <t>DNA</t> <t>FISH</t> of biliary brushing specimens. ((a) and (b)) Conventional cytology. (a) Reactive changes in the context of PSC with some architectural irregularity, mild variation in nuclear size, and overall intact nuclear to cytoplasmic ratio. (b) Adenocarcinoma in a patient with underlying PSC showing hyperchromatic nuclei, marked variation in nuclear shape and size with nuclear molding, and severely disturbed nuclear to cytoplasmic ratio. In the background, necrotic debris with degenerative cells and granulocytes are observed. ((c) and (d)) Representative examples of FISH signal patterns seen in biliary strictures with probes for CEP7 [aqua], CEP17 [green], 20q [gold], and MYC [red]. (c) A normal cell (2 signals of each probe) and (d) gain of MYC ( > 2 red signals) and gain of CEP7 ( > 2 blue signals).
    Dna Probes, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 93/100, based on 308 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim dna probe
    Level of encapsidated (left) and total intracellular (right) viral <t>DNA</t> as determined by semi-quantative PCR. Total DNA (lanes 4–6) or DNase I-treated DNA samples (lanes 1–3) were isolated from cells that either did not express a ribozyme (-, lanes 1 and 4) or express ribozyme M1-I (lanes 2 and 5) or M1-F (lanes 3 and 6) and were infected with HCMV at MOI of 1. We determined the levels of viral IE1 sequence by PCR using human β-actin DNA sequence as the internal controls. The amplification by PCR was within the linear range. The <t>radiolabeled</t> PCR products were separated in 4% non-denaturing polyacrylamide gels and quantitated with a STORM840 phosphorimager.
    Dna Probe, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 221 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dna probe
    Polar sequestration enhances cell cycle robustness by maintaining the hemimethylated (HM) state of newly synthesized chromosome. ( A ) Cells expressing single copy or multicopy of e <t>yfp-ccrM</t> under the control of P xyl were grown in M2G + 0.3% xylose to exponential phase and imaged by phase contrast and epifluorescence microscopy. Expression of a higher amount of eYFP-CcrM impairs its polar localization. ( B ) Spot dilutions of strains in A . Wild-type C. crescentus NA1000 and strains harboring a single copy of P xyl -eyfp-ccrM (S), multicopies of P xyl -eyfp-ccrM (M), single copy of empty vector (pXYFPN2), and multicopies of empty vector (pBMCS2) were diluted to an OD 600 of 0.03, serially diluted, and spotted onto the PYE agar plate supplied with either 0.2% glucose or 0.3% xylose and incubated at 30 °C for 2 d before photography. Induced expression of multicopy e yfp-ccrM resulted in impaired growth. ( C ) Schematic of the Caulobacter chromosome showing position 1 and position 2 where the CYFP reporter cassettes are inserted. Chromosome methylation states are shown as a function of <t>DNA</t> replication and cell cycle progression. Fully methylated (FM, solid line) state position 1 is progressively converted to a HM (dotted line) state following the SW-to-ST cell transition and the initiation of DNA replication. Fully methylated position 2 is converted to a hemimethylated state at the end of DNA replication in the predivisional cell (PD). ( D ) Relative CYFP expressions in synchronized stalked cells treated with 0.2% glucose (eYFP-CcrM repression) or 0.3% xylose (eYFP-CcrM induction). The CYFP reporter cassettes driven by either P dnaA or P ctrA1 were inserted at 2 different positions on the chromosome of strains harboring single copy of P xyl -eyfp-ccrM or multicopies of P xyl -eyfp-ccrM . The qPCR assays were performed using primers targeting the transcribed promotor region of P dnaA or P ctrA1 and CYFP linker, so that the assayed transcriptional levels represent the expression of CYFP gene, not eYFP-CcrM. Results suggest that eYFP-CcrM polar sequestration prevents remethylation of daughter chromosome in the strain constitutively expressing eYFP-CcrM. **** P
    Dna Probe, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 244 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher dna probes
    Exclusion of RIG-I from compartments containing plus-strand and minus-strand HCV RNA. A and B) Uninfected and HCV-infected Huh7.5 cells were transfected with constructs encoding for FLAG-tagged RIG-I-K207A 2 days after HCV infection. On day 4 after infection, cells were probed with antibodies directed against the FLAG epitope (grey) and either HCV core or NS5A (green). <t>DNA</t> probes (Affymetrix) targeted to either the positive-strand or the negative-strand of the HCV RNA (red) were then hybridized to the viral RNA. DNA was stained with DAPI (blue) and cells were examined by confocal fluorescence microscopy. Boxed regions in the middle row of both panels outline the area of magnification presented in the bottom rows. Scale bars represent 5 μm.
    Dna Probes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 2169 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Stratagene dna probes
    Generation of ISP2 and ISP3 double null mutants. A. Schematic representation of the ISP2–ISP3 locus in WT L. major (upper) and the constructs for gene deletion. ORFs are shown as grey arrows and 5′ FR and 3′ FR boxes represent the FR <t>DNA</t> sequences used for gene targeting. The predicted DNA fragment sizes after restriction digest are shown. HYG , hygromycin-resistance gene; BLE , phleomycin-resistance gene. B. Southern blot of the WT L. major (lanes 1, 3 and 5) and Δ isp2/3 (lanes 2, 4 and 6) digested with AgeI or EcoRI/BamHI and probed with <t>radiolabelled</t> 5′ FR, ISP3 ORF and 3′ FR probes. C. Western blot of cell extracts from 0.5 × 10 7 purified metacyclic promastigotes of WT L. major (lane 1), Δ isp2/3 (lane 2) and Δ isp2/3 : ISP2–ISP3 (lane 3) using antibodies against ISP2, HASPB and EF1α.
    Dna Probes, supplied by Stratagene, used in various techniques. Bioz Stars score: 92/100, based on 415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Boehringer Mannheim dna hybridization probes
    Replicon localization of mob DNAs in cosmids pRmOR69 and pRmOR65. Blots of Eco RI-digested genomic DNAs were hybridized against <t>digoxigenin-labeled</t> cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild-type); 2, A. tumefaciens C58; 3, A. tumefaciens C58(pRme2011a); 4, R. meliloti GR4 (wild type); 5, R. meliloti GRM6 (cured of plasmid pRmeGR4b); 6, R. meliloti GRM10 (cured of plasmid pRmeGR4a); 7, R. meliloti GRM8 (cured of pRmeGR4a and pRmeGR4b); 8, R. meliloti GRT3 (carrying pSym1 deletion); 9, Eco RI-digested cosmid <t>DNA.</t>
    Dna Hybridization Probes, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 88/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Abbott Laboratories aneuvysion multicolor dna probe kit
    Replicon localization of mob DNAs in cosmids pRmOR69 and pRmOR65. Blots of Eco RI-digested genomic DNAs were hybridized against <t>digoxigenin-labeled</t> cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild-type); 2, A. tumefaciens C58; 3, A. tumefaciens C58(pRme2011a); 4, R. meliloti GR4 (wild type); 5, R. meliloti GRM6 (cured of plasmid pRmeGR4b); 6, R. meliloti GRM10 (cured of plasmid pRmeGR4a); 7, R. meliloti GRM8 (cured of pRmeGR4a and pRmeGR4b); 8, R. meliloti GRT3 (carrying pSym1 deletion); 9, Eco RI-digested cosmid <t>DNA.</t>
    Aneuvysion Multicolor Dna Probe Kit, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 75 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    Boehringer Mannheim digoxigenin labeled dna probes
    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the <t>digoxigenin-labeled</t> full-length DOH1 <t>DNA</t> probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.
    Digoxigenin Labeled Dna Probes, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 90/100, based on 126 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Ventana Medical inform her2 dual ish dna probe cocktail assay
    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the <t>digoxigenin-labeled</t> full-length DOH1 <t>DNA</t> probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.
    Inform Her2 Dual Ish Dna Probe Cocktail Assay, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 88/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Hologic Inc b dermatitidis dna probe
    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the <t>digoxigenin-labeled</t> full-length DOH1 <t>DNA</t> probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.
    B Dermatitidis Dna Probe, supplied by Hologic Inc, used in various techniques. Bioz Stars score: 92/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Abbott Laboratories vysis pathvysion her2 dna probe kit
    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the <t>digoxigenin-labeled</t> full-length DOH1 <t>DNA</t> probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.
    Vysis Pathvysion Her2 Dna Probe Kit, supplied by Abbott Laboratories, used in various techniques. Bioz Stars score: 85/100, based on 37 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Ventana Medical chromosome 17 cep17 probes
    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the <t>digoxigenin-labeled</t> full-length DOH1 <t>DNA</t> probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.
    Chromosome 17 Cep17 Probes, supplied by Ventana Medical, used in various techniques. Bioz Stars score: 88/100, based on 30 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Hologic Inc dna probes
    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the <t>digoxigenin-labeled</t> full-length DOH1 <t>DNA</t> probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.
    Dna Probes, supplied by Hologic Inc, used in various techniques. Bioz Stars score: 90/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Models of mammalian mtDNA replication. Two alternative mechanisms of strand-asynchronous replication have been proposed: ( A ) the strand-displacement model that depends on protein (mtSSB) to coat the displaced lagging-strand template, and ( B ) the bootlace mechanism that relies on mature transcripts. ( C ) Intermediates with the properties of conventional (concurrent) leading and lagging-strand DNA synthesis have also been reported in mammalian mitochondria suggesting that they also operate a strand-coupled (SC) mechanism of replication. ( D ) Restriction digestion of the intermediates of strand-asynchronous replication will not cut at sites that are single-stranded DNA (in the case of many restriction enzymes) or where there is RNA/DNA hybrid, which produces atypical structures compared to SC replication as illustrated, and these form so-called supra-Y (SY) arcs when resolved by 2D-AGE ( E ).

    Journal: Nucleic Acids Research

    Article Title: Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

    doi: 10.1093/nar/gky852

    Figure Lengend Snippet: Models of mammalian mtDNA replication. Two alternative mechanisms of strand-asynchronous replication have been proposed: ( A ) the strand-displacement model that depends on protein (mtSSB) to coat the displaced lagging-strand template, and ( B ) the bootlace mechanism that relies on mature transcripts. ( C ) Intermediates with the properties of conventional (concurrent) leading and lagging-strand DNA synthesis have also been reported in mammalian mitochondria suggesting that they also operate a strand-coupled (SC) mechanism of replication. ( D ) Restriction digestion of the intermediates of strand-asynchronous replication will not cut at sites that are single-stranded DNA (in the case of many restriction enzymes) or where there is RNA/DNA hybrid, which produces atypical structures compared to SC replication as illustrated, and these form so-called supra-Y (SY) arcs when resolved by 2D-AGE ( E ).

    Article Snippet: Radiolabeled DNA probes were generated from fragments of human mtDNA PCR-amplified using pairs of oligonucleotide primers (Sigma) (listed in supplementary information), based on the reference sequence ( ).

    Techniques: DNA Synthesis

    Elevated expression of wild-type or mutant Twinkle increases the abundance of fully-duplex DNA replication intermediates across the major arc of mtDNA. Mitochondrial DNA was analyzed by 2D-AGE from control HEK293T cells, or cells induced to express HA-tagged wild-type or mutant (D311A) Twinkle, after digestion with 1) Dra1, 2) BsaKHA1 or 3) Hpa1. Individual fragments were detected via hybridization to probes h2, h3 or h4 (see schematic map and Supplementary data). Y - replication fork arc, 1n – unit length fragment, 2n - twice the unit length fragment where the Y arc terminates. Standard Y arcs do not distinguish the direction of replication fork movement, and so forks could be entering from either end of all the fragments shown, as illustrated to the right of the map for the Dra1 fragment.

    Journal: Nucleic Acids Research

    Article Title: Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

    doi: 10.1093/nar/gky852

    Figure Lengend Snippet: Elevated expression of wild-type or mutant Twinkle increases the abundance of fully-duplex DNA replication intermediates across the major arc of mtDNA. Mitochondrial DNA was analyzed by 2D-AGE from control HEK293T cells, or cells induced to express HA-tagged wild-type or mutant (D311A) Twinkle, after digestion with 1) Dra1, 2) BsaKHA1 or 3) Hpa1. Individual fragments were detected via hybridization to probes h2, h3 or h4 (see schematic map and Supplementary data). Y - replication fork arc, 1n – unit length fragment, 2n - twice the unit length fragment where the Y arc terminates. Standard Y arcs do not distinguish the direction of replication fork movement, and so forks could be entering from either end of all the fragments shown, as illustrated to the right of the map for the Dra1 fragment.

    Article Snippet: Radiolabeled DNA probes were generated from fragments of human mtDNA PCR-amplified using pairs of oligonucleotide primers (Sigma) (listed in supplementary information), based on the reference sequence ( ).

    Techniques: Expressing, Mutagenesis, Hybridization

    The proportion of replication forks travelling towards, rather than away from, the unidirectional origin Ori-H is markedly increased in cells expressing transgenic wild-type Twinkle. Panels 1–4 Restriction digested HEK293T cell DNA separated by 1D-AGE and then subjected to an additional in-gel digestion step with another restriction enzyme, prior to standard 2D-AGE and gel processing. Fork direction is defined as clockwise (c), or anti-clockwise (ac) as indicated on the schematic map (bottom-right). Assignments of the arcs associated with fragment 1 of control cells (no transgene) and cells expressing wild-type Twinkle (Twk) are shown below the gel images; SC – strand coupled, SA – strand asynchronous replication. Twk and Twk-mut2 (D311A) transgenes were induced with 3 ng/mL doxycycline for 72 h. Immediately below each gel image ( 1–4 ) the relevant region of mtDNA is represented as a horizontal line, broken at the in-gel digestion site. Arrows above the line indicate the direction of fork travel, the thickness and length of the arrows correspond approximately to the proportion of forks travelling in a particular direction. Black arrows – forks travelling clockwise (towards Ori-H); gray arrows – forks travelling anti-clockwise (away from Ori-H). Black boxes indicate the position of the probes, h2, h4, h5 and h6.

    Journal: Nucleic Acids Research

    Article Title: Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

    doi: 10.1093/nar/gky852

    Figure Lengend Snippet: The proportion of replication forks travelling towards, rather than away from, the unidirectional origin Ori-H is markedly increased in cells expressing transgenic wild-type Twinkle. Panels 1–4 Restriction digested HEK293T cell DNA separated by 1D-AGE and then subjected to an additional in-gel digestion step with another restriction enzyme, prior to standard 2D-AGE and gel processing. Fork direction is defined as clockwise (c), or anti-clockwise (ac) as indicated on the schematic map (bottom-right). Assignments of the arcs associated with fragment 1 of control cells (no transgene) and cells expressing wild-type Twinkle (Twk) are shown below the gel images; SC – strand coupled, SA – strand asynchronous replication. Twk and Twk-mut2 (D311A) transgenes were induced with 3 ng/mL doxycycline for 72 h. Immediately below each gel image ( 1–4 ) the relevant region of mtDNA is represented as a horizontal line, broken at the in-gel digestion site. Arrows above the line indicate the direction of fork travel, the thickness and length of the arrows correspond approximately to the proportion of forks travelling in a particular direction. Black arrows – forks travelling clockwise (towards Ori-H); gray arrows – forks travelling anti-clockwise (away from Ori-H). Black boxes indicate the position of the probes, h2, h4, h5 and h6.

    Article Snippet: Radiolabeled DNA probes were generated from fragments of human mtDNA PCR-amplified using pairs of oligonucleotide primers (Sigma) (listed in supplementary information), based on the reference sequence ( ).

    Techniques: Expressing, Transgenic Assay

    Transgenic wild-type Twinkle causes mtDNA depletion and depresses transcript levels. Whole cellular DNA and RNA were isolated from control HEK293T cells or cells expressing Twinkle-HA or a mutated Twinkle-HA (D311A). ( A ) Relative mtDNA copy number was calculated from the abundance of the cytochrome b gene of mtDNA relative to the single copy nuclear gene APP1, using real-time PCR quantification. ( B ) The abundance of five mitochondrial transcripts, cytochrome b , cytochrome c oxidase II, NADH dehydrogenase 1, 16S and 12S rRNA determined by RT-q-PCR. ( C )).

    Journal: Nucleic Acids Research

    Article Title: Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

    doi: 10.1093/nar/gky852

    Figure Lengend Snippet: Transgenic wild-type Twinkle causes mtDNA depletion and depresses transcript levels. Whole cellular DNA and RNA were isolated from control HEK293T cells or cells expressing Twinkle-HA or a mutated Twinkle-HA (D311A). ( A ) Relative mtDNA copy number was calculated from the abundance of the cytochrome b gene of mtDNA relative to the single copy nuclear gene APP1, using real-time PCR quantification. ( B ) The abundance of five mitochondrial transcripts, cytochrome b , cytochrome c oxidase II, NADH dehydrogenase 1, 16S and 12S rRNA determined by RT-q-PCR. ( C )).

    Article Snippet: Radiolabeled DNA probes were generated from fragments of human mtDNA PCR-amplified using pairs of oligonucleotide primers (Sigma) (listed in supplementary information), based on the reference sequence ( ).

    Techniques: Transgenic Assay, Isolation, Expressing, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction

    Elevated Twinkle expression induces biphasic θ replication in mtDNA. 2D-AGE analysis of HEK293T cell DNA from cells expressing no transgene (control); transgenic wild-type Twinkle (Twk) or Twinkle D311A (Twk-mut2), both with HA tags. Whole cell DNA was digested with ( A ) Pvu2 that cuts mtDNA at a single site in the minor arc (nt 2650), or ( B ) Bgl1 (sites at np 6034 and 6964). SSN, single-strand-nuclease. Interpretations of the mtRIs are shown beneath the gel images, b – bubble structures, dY – double Y arc. The strong dY is indicative of slow-replication in the minor arc (as per the map to the left of B ) for further details).

    Journal: Nucleic Acids Research

    Article Title: Transcript availability dictates the balance between strand-asynchronous and strand-coupled mitochondrial DNA replication

    doi: 10.1093/nar/gky852

    Figure Lengend Snippet: Elevated Twinkle expression induces biphasic θ replication in mtDNA. 2D-AGE analysis of HEK293T cell DNA from cells expressing no transgene (control); transgenic wild-type Twinkle (Twk) or Twinkle D311A (Twk-mut2), both with HA tags. Whole cell DNA was digested with ( A ) Pvu2 that cuts mtDNA at a single site in the minor arc (nt 2650), or ( B ) Bgl1 (sites at np 6034 and 6964). SSN, single-strand-nuclease. Interpretations of the mtRIs are shown beneath the gel images, b – bubble structures, dY – double Y arc. The strong dY is indicative of slow-replication in the minor arc (as per the map to the left of B ) for further details).

    Article Snippet: Radiolabeled DNA probes were generated from fragments of human mtDNA PCR-amplified using pairs of oligonucleotide primers (Sigma) (listed in supplementary information), based on the reference sequence ( ).

    Techniques: Expressing, Transgenic Assay

    Analysis of TRL-78 transcripts. ( A ) Northern hybridizations. mtRNAs from the indicated strains were separated in a 1.8% agarose gel, which was stained with ethidium bromide and blotted to Hybond-N. The blot was hybridized with a 32 P-labeled 84-bp Bam HI– Eco RI fragment corresponding to the cloned TRL-78 from p7-2. The left shows the ethidium bromide-stained gel and the right shows an autoradiogram of the blot. The positions of the small (19S) and large (25S) rRNAs, mt tRNAs and transcripts of various mitochondrial plasmids are shown to the left: VS, Varkud small plasmid (56); VS/V1-2, a deletion derivative of the Varkud small plasmid (57); ΔV1-2, a 300-nt deletion derivative of pV1-2 (S.Mohr and A.M.Lambowitz, in preparation). ( B and C ) Northern hybridizations in which mtRNAs from Varkud (V) and V1-2 were separated in a denaturing 5% polyacrylamide gel and electroblotted to Hybond-N. (B) The blot was hybridized with 5′-end-labeled DNA oligonucleotide V1-2 3′, which is complementary to the 3′-end of TRL-64 and TRL-78. (C) The blot was hybridized with 5′-end-labeled oligonucleotide tRNA Trp , which is complementary to the 3′-end of tRNA Trp . To avoid smearing, the gels in (B) and (C) were loaded with one-twentieth of the RNA loaded in (A). ( D ) Ethidium bromide staining of V1-2 mtRNAs separated in a denaturing 5% polyacrylamide gel. The gel was used for densitometric scanning as described in the text. In (B)–(D) the numbers to the left indicate the sizes and positions of molecular weight markers ( 32 P-labeled Hin fI– Ban I fragments of pBS+) and the numbers to the right indicate the identities and/or sizes of the RNAs detected by the probe.

    Journal: Nucleic Acids Research

    Article Title: Characterization of an unusual tRNA-like sequence found inserted in a Neurospora retroplasmid

    doi:

    Figure Lengend Snippet: Analysis of TRL-78 transcripts. ( A ) Northern hybridizations. mtRNAs from the indicated strains were separated in a 1.8% agarose gel, which was stained with ethidium bromide and blotted to Hybond-N. The blot was hybridized with a 32 P-labeled 84-bp Bam HI– Eco RI fragment corresponding to the cloned TRL-78 from p7-2. The left shows the ethidium bromide-stained gel and the right shows an autoradiogram of the blot. The positions of the small (19S) and large (25S) rRNAs, mt tRNAs and transcripts of various mitochondrial plasmids are shown to the left: VS, Varkud small plasmid (56); VS/V1-2, a deletion derivative of the Varkud small plasmid (57); ΔV1-2, a 300-nt deletion derivative of pV1-2 (S.Mohr and A.M.Lambowitz, in preparation). ( B and C ) Northern hybridizations in which mtRNAs from Varkud (V) and V1-2 were separated in a denaturing 5% polyacrylamide gel and electroblotted to Hybond-N. (B) The blot was hybridized with 5′-end-labeled DNA oligonucleotide V1-2 3′, which is complementary to the 3′-end of TRL-64 and TRL-78. (C) The blot was hybridized with 5′-end-labeled oligonucleotide tRNA Trp , which is complementary to the 3′-end of tRNA Trp . To avoid smearing, the gels in (B) and (C) were loaded with one-twentieth of the RNA loaded in (A). ( D ) Ethidium bromide staining of V1-2 mtRNAs separated in a denaturing 5% polyacrylamide gel. The gel was used for densitometric scanning as described in the text. In (B)–(D) the numbers to the left indicate the sizes and positions of molecular weight markers ( 32 P-labeled Hin fI– Ban I fragments of pBS+) and the numbers to the right indicate the identities and/or sizes of the RNAs detected by the probe.

    Article Snippet: After staining with ethidium bromide, the gel was blotted to Hybond-N (Amersham-Pharmacia) and the blots were hybridized overnight at 37°C with 32 P-labeled DNA probes ( > 5 × 104 c.p.m.) in 50% formamide, 5× SSC (1× SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.2% SDS and 100 mg/ml sheared, denatured salmon sperm DNA (Sigma).

    Techniques: Northern Blot, Agarose Gel Electrophoresis, Staining, Labeling, Clone Assay, Plasmid Preparation, Molecular Weight

    Validation of 17q12 amplification in SDCs. Composite illustration of chromosome (Chr) 17q12 DNA ( A ) and corresponding ERBB2 gene amplification by FISH ( B ) and the expression by IHC analysis ( C ) in the four SDCs with amplicons at this location (SDCs 1,

    Journal: The American Journal of Pathology

    Article Title: Detailed Genome-Wide SNP Analysis of Major Salivary Carcinomas Localizes Subtype-Specific Chromosome Sites and Oncogenes of Potential Clinical Significance

    doi: 10.1016/j.ajpath.2013.02.020

    Figure Lengend Snippet: Validation of 17q12 amplification in SDCs. Composite illustration of chromosome (Chr) 17q12 DNA ( A ) and corresponding ERBB2 gene amplification by FISH ( B ) and the expression by IHC analysis ( C ) in the four SDCs with amplicons at this location (SDCs 1,

    Article Snippet: We performed fluorescence in situ hybridization (FISH) analysis on touch preparations from fresh tumor fragments using standard, commercially available, spectrum orange– and spectrum green–labeled probes for ERBB2 amplification (PathVysion HER-2 DNA probe kit; Abbott Laboratories, Des Plaines, IL).

    Techniques: Amplification, Fluorescence In Situ Hybridization, Expressing, Immunohistochemistry

    The relationship of subtypes to the DNA ploidy and CIN. The diploid/CIN- status was detected in 72 (64.9%) of the 111 luminal A carcinomas, 39 (41.1%) of the 95 luminal B (HER2-) carcinomas, 8 (11.6%) of the 69 lumminal B (HER2+) carcinomas, 2 (4.9%) of the 41 HER2 carcinomas, and 8 (17.0%) of the 26 basal-like carcinomas. In contrast, the aneuploid/CIN + status was detected in 31 (27.9%) of the 111 luminal A, 47 (49.5%) of the luminal B (HER2-), 56 (81.2%) of the 69 luminal B (HER2+), 37 (90.2%) of the 41 HER2, and 36 (86.6%) of the 47 basal-like subtype tumors. The incidence of diploid/CIN- and aneuploid/CIN+ status was different between the luminal A subtype and luminal B (HER2-), luminal B (HER2+), HER2, and basal-like subtypes (p = 0.0006, p = 5E-13, p = 5E10-12, and p = 8E10-9). In addition, the incidence of diploid/CIN- and aneuploid/CIN+ status was statistically different between the luminal B (HER2-) subtype and luminal B (HER2+), HER2, and basal-like subtypes (p = 0.00002, p = 0.000009, and p = 0.002). Black bar; diploid/CIN- tumors, gray bar; aneuploid/CIN+ tumors, white column; others.

    Journal: BMC Research Notes

    Article Title: Luminal A and luminal B (HER2 negative) subtypes of breast cancer consist of a mixture of tumors with different genotype

    doi: 10.1186/1756-0500-5-376

    Figure Lengend Snippet: The relationship of subtypes to the DNA ploidy and CIN. The diploid/CIN- status was detected in 72 (64.9%) of the 111 luminal A carcinomas, 39 (41.1%) of the 95 luminal B (HER2-) carcinomas, 8 (11.6%) of the 69 lumminal B (HER2+) carcinomas, 2 (4.9%) of the 41 HER2 carcinomas, and 8 (17.0%) of the 26 basal-like carcinomas. In contrast, the aneuploid/CIN + status was detected in 31 (27.9%) of the 111 luminal A, 47 (49.5%) of the luminal B (HER2-), 56 (81.2%) of the 69 luminal B (HER2+), 37 (90.2%) of the 41 HER2, and 36 (86.6%) of the 47 basal-like subtype tumors. The incidence of diploid/CIN- and aneuploid/CIN+ status was different between the luminal A subtype and luminal B (HER2-), luminal B (HER2+), HER2, and basal-like subtypes (p = 0.0006, p = 5E-13, p = 5E10-12, and p = 8E10-9). In addition, the incidence of diploid/CIN- and aneuploid/CIN+ status was statistically different between the luminal B (HER2-) subtype and luminal B (HER2+), HER2, and basal-like subtypes (p = 0.00002, p = 0.000009, and p = 0.002). Black bar; diploid/CIN- tumors, gray bar; aneuploid/CIN+ tumors, white column; others.

    Article Snippet: HER2 amplification was tested on the smear preparations for IHC equivocal cases using a PathVysion HER2 DNA Probe Kit (Abbott Laboratories) according to the manufacturer’s instructions as described previously.

    Techniques:

    Representative examples of conventional cytology and DNA FISH of biliary brushing specimens. ((a) and (b)) Conventional cytology. (a) Reactive changes in the context of PSC with some architectural irregularity, mild variation in nuclear size, and overall intact nuclear to cytoplasmic ratio. (b) Adenocarcinoma in a patient with underlying PSC showing hyperchromatic nuclei, marked variation in nuclear shape and size with nuclear molding, and severely disturbed nuclear to cytoplasmic ratio. In the background, necrotic debris with degenerative cells and granulocytes are observed. ((c) and (d)) Representative examples of FISH signal patterns seen in biliary strictures with probes for CEP7 [aqua], CEP17 [green], 20q [gold], and MYC [red]. (c) A normal cell (2 signals of each probe) and (d) gain of MYC ( > 2 red signals) and gain of CEP7 ( > 2 blue signals).

    Journal: Gastroenterology Research and Practice

    Article Title: Genetic Abnormalities in Biliary Brush Samples for Distinguishing Cholangiocarcinoma from Benign Strictures in Primary Sclerosing Cholangitis

    doi: 10.1155/2016/4381513

    Figure Lengend Snippet: Representative examples of conventional cytology and DNA FISH of biliary brushing specimens. ((a) and (b)) Conventional cytology. (a) Reactive changes in the context of PSC with some architectural irregularity, mild variation in nuclear size, and overall intact nuclear to cytoplasmic ratio. (b) Adenocarcinoma in a patient with underlying PSC showing hyperchromatic nuclei, marked variation in nuclear shape and size with nuclear molding, and severely disturbed nuclear to cytoplasmic ratio. In the background, necrotic debris with degenerative cells and granulocytes are observed. ((c) and (d)) Representative examples of FISH signal patterns seen in biliary strictures with probes for CEP7 [aqua], CEP17 [green], 20q [gold], and MYC [red]. (c) A normal cell (2 signals of each probe) and (d) gain of MYC ( > 2 red signals) and gain of CEP7 ( > 2 blue signals).

    Article Snippet: FISH was performed as previously described using seven different DNA probes including the centromeric probes CEP7 and CEP17 and the locus-specific probes to CDKN2A , TP53 , ERBB2 , 20q , and MYC (Abbott Molecular, Abbott Park, Illinois) [ ].

    Techniques: Fluorescence In Situ Hybridization

    Level of encapsidated (left) and total intracellular (right) viral DNA as determined by semi-quantative PCR. Total DNA (lanes 4–6) or DNase I-treated DNA samples (lanes 1–3) were isolated from cells that either did not express a ribozyme (-, lanes 1 and 4) or express ribozyme M1-I (lanes 2 and 5) or M1-F (lanes 3 and 6) and were infected with HCMV at MOI of 1. We determined the levels of viral IE1 sequence by PCR using human β-actin DNA sequence as the internal controls. The amplification by PCR was within the linear range. The radiolabeled PCR products were separated in 4% non-denaturing polyacrylamide gels and quantitated with a STORM840 phosphorimager.

    Journal: Nucleic Acids Research

    Article Title: RNase P ribozyme inhibits cytomegalovirus replication by blocking the expression of viral capsid proteins

    doi: 10.1093/nar/gkh660

    Figure Lengend Snippet: Level of encapsidated (left) and total intracellular (right) viral DNA as determined by semi-quantative PCR. Total DNA (lanes 4–6) or DNase I-treated DNA samples (lanes 1–3) were isolated from cells that either did not express a ribozyme (-, lanes 1 and 4) or express ribozyme M1-I (lanes 2 and 5) or M1-F (lanes 3 and 6) and were infected with HCMV at MOI of 1. We determined the levels of viral IE1 sequence by PCR using human β-actin DNA sequence as the internal controls. The amplification by PCR was within the linear range. The radiolabeled PCR products were separated in 4% non-denaturing polyacrylamide gels and quantitated with a STORM840 phosphorimager.

    Article Snippet: The radiolabeled DNA probe used to detect M1GS RNAs was synthesized from plasmid pFL117, by using a random primed labeling kit (Boehringer Manheim, Co., Indianapolis, IN).

    Techniques: Polymerase Chain Reaction, Isolation, Infection, Sequencing, Amplification

    Southern blot analysis of selected wzx mutants. Chromosomal DNA from representative A L (S2 and X10) and A + (S7 and X24) wzx mutants from the wzx s and wzx x series and a representative A − (X14) wzx x mutant was digested with Hin dIII and separated on a 0.8% agarose gel, transferred to a nylon membrane, and probed with a dUTP-digoxigenin-labelled Bam HI- Bgl II fragment corresponding to the insert of pFV162-26. All mutants show an increase in the size of the Hin dIII fragment of approximately 0.9 kb, corresponding to the size of the Gm r cassette. No gross rearrangements that could be responsible for the varied A-band LPS phenotypes of these mutants were found in this region.

    Journal: Journal of Bacteriology

    Article Title: Effect of wzx (rfbX) Mutations on A-Band and B-Band Lipopolysaccharide Biosynthesis in Pseudomonas aeruginosa O5

    doi:

    Figure Lengend Snippet: Southern blot analysis of selected wzx mutants. Chromosomal DNA from representative A L (S2 and X10) and A + (S7 and X24) wzx mutants from the wzx s and wzx x series and a representative A − (X14) wzx x mutant was digested with Hin dIII and separated on a 0.8% agarose gel, transferred to a nylon membrane, and probed with a dUTP-digoxigenin-labelled Bam HI- Bgl II fragment corresponding to the insert of pFV162-26. All mutants show an increase in the size of the Hin dIII fragment of approximately 0.9 kb, corresponding to the size of the Gm r cassette. No gross rearrangements that could be responsible for the varied A-band LPS phenotypes of these mutants were found in this region.

    Article Snippet: For detection of specific fragments, probe DNA was labelled with digoxigenin-dUTP (Boehringer Mannheim, Laval, Quebec, Canada), and hybridization and detection were performed according to the manufacturer’s directions.

    Techniques: Southern Blot, Mutagenesis, Agarose Gel Electrophoresis

    Polar sequestration enhances cell cycle robustness by maintaining the hemimethylated (HM) state of newly synthesized chromosome. ( A ) Cells expressing single copy or multicopy of e yfp-ccrM under the control of P xyl were grown in M2G + 0.3% xylose to exponential phase and imaged by phase contrast and epifluorescence microscopy. Expression of a higher amount of eYFP-CcrM impairs its polar localization. ( B ) Spot dilutions of strains in A . Wild-type C. crescentus NA1000 and strains harboring a single copy of P xyl -eyfp-ccrM (S), multicopies of P xyl -eyfp-ccrM (M), single copy of empty vector (pXYFPN2), and multicopies of empty vector (pBMCS2) were diluted to an OD 600 of 0.03, serially diluted, and spotted onto the PYE agar plate supplied with either 0.2% glucose or 0.3% xylose and incubated at 30 °C for 2 d before photography. Induced expression of multicopy e yfp-ccrM resulted in impaired growth. ( C ) Schematic of the Caulobacter chromosome showing position 1 and position 2 where the CYFP reporter cassettes are inserted. Chromosome methylation states are shown as a function of DNA replication and cell cycle progression. Fully methylated (FM, solid line) state position 1 is progressively converted to a HM (dotted line) state following the SW-to-ST cell transition and the initiation of DNA replication. Fully methylated position 2 is converted to a hemimethylated state at the end of DNA replication in the predivisional cell (PD). ( D ) Relative CYFP expressions in synchronized stalked cells treated with 0.2% glucose (eYFP-CcrM repression) or 0.3% xylose (eYFP-CcrM induction). The CYFP reporter cassettes driven by either P dnaA or P ctrA1 were inserted at 2 different positions on the chromosome of strains harboring single copy of P xyl -eyfp-ccrM or multicopies of P xyl -eyfp-ccrM . The qPCR assays were performed using primers targeting the transcribed promotor region of P dnaA or P ctrA1 and CYFP linker, so that the assayed transcriptional levels represent the expression of CYFP gene, not eYFP-CcrM. Results suggest that eYFP-CcrM polar sequestration prevents remethylation of daughter chromosome in the strain constitutively expressing eYFP-CcrM. **** P

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

    Article Title: Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

    doi: 10.1073/pnas.1906119116

    Figure Lengend Snippet: Polar sequestration enhances cell cycle robustness by maintaining the hemimethylated (HM) state of newly synthesized chromosome. ( A ) Cells expressing single copy or multicopy of e yfp-ccrM under the control of P xyl were grown in M2G + 0.3% xylose to exponential phase and imaged by phase contrast and epifluorescence microscopy. Expression of a higher amount of eYFP-CcrM impairs its polar localization. ( B ) Spot dilutions of strains in A . Wild-type C. crescentus NA1000 and strains harboring a single copy of P xyl -eyfp-ccrM (S), multicopies of P xyl -eyfp-ccrM (M), single copy of empty vector (pXYFPN2), and multicopies of empty vector (pBMCS2) were diluted to an OD 600 of 0.03, serially diluted, and spotted onto the PYE agar plate supplied with either 0.2% glucose or 0.3% xylose and incubated at 30 °C for 2 d before photography. Induced expression of multicopy e yfp-ccrM resulted in impaired growth. ( C ) Schematic of the Caulobacter chromosome showing position 1 and position 2 where the CYFP reporter cassettes are inserted. Chromosome methylation states are shown as a function of DNA replication and cell cycle progression. Fully methylated (FM, solid line) state position 1 is progressively converted to a HM (dotted line) state following the SW-to-ST cell transition and the initiation of DNA replication. Fully methylated position 2 is converted to a hemimethylated state at the end of DNA replication in the predivisional cell (PD). ( D ) Relative CYFP expressions in synchronized stalked cells treated with 0.2% glucose (eYFP-CcrM repression) or 0.3% xylose (eYFP-CcrM induction). The CYFP reporter cassettes driven by either P dnaA or P ctrA1 were inserted at 2 different positions on the chromosome of strains harboring single copy of P xyl -eyfp-ccrM or multicopies of P xyl -eyfp-ccrM . The qPCR assays were performed using primers targeting the transcribed promotor region of P dnaA or P ctrA1 and CYFP linker, so that the assayed transcriptional levels represent the expression of CYFP gene, not eYFP-CcrM. Results suggest that eYFP-CcrM polar sequestration prevents remethylation of daughter chromosome in the strain constitutively expressing eYFP-CcrM. **** P

    Article Snippet: DNA‐binding capacity of CcrM was evaluated by incubation of purified CcrM with 20 nM of indicated DNA probe in the presence of 200 µM sinefungin (Sigma) in EMSA buffer (50 mM Hepes pH 7.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) for 30 min at room temperature and subjected to electrophoresis in 4 to 15% Mini-PROTEAN TGX precast protein gels (Bio-Rad) at constant 80 V for 3 h at 4 °C in 1× Tris glycine native gel buffer (25 mM Tris base, 192 mM glycine).

    Techniques: Synthesized, Expressing, Epifluorescence Microscopy, Plasmid Preparation, Incubation, Methylation, Real-time Polymerase Chain Reaction

    Cell-pole sequestration separates CcrM from chromosomal DNA and the Lon protease. ( A ) Cells are shown with CcrM-eYFP diffraction-limited data overlaid with its centroid location (blue signal with yellow cross) and single-molecule localizations of PAmCherry-PopZ (red scatterplot). PAmCherry-PopZ is concentrated at the poles; localizations with more than 30 neighbors within a 100-nm radius are shown in filled red scatters with black outlines. The rest of the PAmCherry-PopZ that are typically nonpolar are shown in empty red circles. ( Inset ) The spatial correlation of CcrM-eYFP and PAmCherry-PopZ across 207 cells exhibited an average Pearson’s correlation coefficient of 0.46. (Scale bar, 600 nm.) This is a montage of different fields of view. ( B ) PAmCherry-PopZ localizations in 25-nm bins (yellow-red color scale) along with all Lon-eYFP localizations (pink scatterplot). The spatial correlation of PAmCherry-PopZ and Lon-eYFP across 270 cells (532 values) exhibited an average Pearson’s correlation coefficient of −0.74. (Scale bar, 600 nm.) This is a montage of a different field of views. ( C ) Distributions of the distance between the centroid of the CcrM-eYFP cluster to the center of PAmCherry-PopZ (blue) and the averaged distribution of distances between Lon-eYFP to the center of PAmCherry-PopZ (pink). The average distance of ParB-eYFP to PopZ was marked to show the PopZ-cytosol interface (red). The area that PopZ covers is marked by the gray shading, assuming symmetry along the cellular axis. A cartoon ( Right ) shows the physical separation between CcrM (blue) and Lon (pink). The cytosolic boundary of the PopZ domain is marked by red lines.

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

    Article Title: Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

    doi: 10.1073/pnas.1906119116

    Figure Lengend Snippet: Cell-pole sequestration separates CcrM from chromosomal DNA and the Lon protease. ( A ) Cells are shown with CcrM-eYFP diffraction-limited data overlaid with its centroid location (blue signal with yellow cross) and single-molecule localizations of PAmCherry-PopZ (red scatterplot). PAmCherry-PopZ is concentrated at the poles; localizations with more than 30 neighbors within a 100-nm radius are shown in filled red scatters with black outlines. The rest of the PAmCherry-PopZ that are typically nonpolar are shown in empty red circles. ( Inset ) The spatial correlation of CcrM-eYFP and PAmCherry-PopZ across 207 cells exhibited an average Pearson’s correlation coefficient of 0.46. (Scale bar, 600 nm.) This is a montage of different fields of view. ( B ) PAmCherry-PopZ localizations in 25-nm bins (yellow-red color scale) along with all Lon-eYFP localizations (pink scatterplot). The spatial correlation of PAmCherry-PopZ and Lon-eYFP across 270 cells (532 values) exhibited an average Pearson’s correlation coefficient of −0.74. (Scale bar, 600 nm.) This is a montage of a different field of views. ( C ) Distributions of the distance between the centroid of the CcrM-eYFP cluster to the center of PAmCherry-PopZ (blue) and the averaged distribution of distances between Lon-eYFP to the center of PAmCherry-PopZ (pink). The average distance of ParB-eYFP to PopZ was marked to show the PopZ-cytosol interface (red). The area that PopZ covers is marked by the gray shading, assuming symmetry along the cellular axis. A cartoon ( Right ) shows the physical separation between CcrM (blue) and Lon (pink). The cytosolic boundary of the PopZ domain is marked by red lines.

    Article Snippet: DNA‐binding capacity of CcrM was evaluated by incubation of purified CcrM with 20 nM of indicated DNA probe in the presence of 200 µM sinefungin (Sigma) in EMSA buffer (50 mM Hepes pH 7.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) for 30 min at room temperature and subjected to electrophoresis in 4 to 15% Mini-PROTEAN TGX precast protein gels (Bio-Rad) at constant 80 V for 3 h at 4 °C in 1× Tris glycine native gel buffer (25 mM Tris base, 192 mM glycine).

    Techniques:

    ). Cell division generates 2 types of progeny cells: a motile swarmer cell and a sessile stalked cell. In swarmer cells, CcrM is completely proteolyzed by Lon, which is stimulated on a DNA-based platform, so by the time it differentiates into a stalked cell and DNA replication is initiated, CcrM is cleared from the cell. In progeny stalked cells, however, immediate chromosomal replication does not provide adequate time for proteolysis of remaining CcrM inherited from the predivisional cells. Instead of clearance by protein degradation, CcrM is sequestered to the new cell pole away from the chromosome, concurrent with the immediate initiation of chromosome replication. Sequestered CcrM has minimum contact with chromosome DNA and Lon, therefore preventing remethylation of newly synthesized chromosomal DNA. The fully methylated chromosome is indicated by a solid red line, and the hemimethylated chromosome is indicated by a dashed red line.

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

    Article Title: Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

    doi: 10.1073/pnas.1906119116

    Figure Lengend Snippet: ). Cell division generates 2 types of progeny cells: a motile swarmer cell and a sessile stalked cell. In swarmer cells, CcrM is completely proteolyzed by Lon, which is stimulated on a DNA-based platform, so by the time it differentiates into a stalked cell and DNA replication is initiated, CcrM is cleared from the cell. In progeny stalked cells, however, immediate chromosomal replication does not provide adequate time for proteolysis of remaining CcrM inherited from the predivisional cells. Instead of clearance by protein degradation, CcrM is sequestered to the new cell pole away from the chromosome, concurrent with the immediate initiation of chromosome replication. Sequestered CcrM has minimum contact with chromosome DNA and Lon, therefore preventing remethylation of newly synthesized chromosomal DNA. The fully methylated chromosome is indicated by a solid red line, and the hemimethylated chromosome is indicated by a dashed red line.

    Article Snippet: DNA‐binding capacity of CcrM was evaluated by incubation of purified CcrM with 20 nM of indicated DNA probe in the presence of 200 µM sinefungin (Sigma) in EMSA buffer (50 mM Hepes pH 7.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) for 30 min at room temperature and subjected to electrophoresis in 4 to 15% Mini-PROTEAN TGX precast protein gels (Bio-Rad) at constant 80 V for 3 h at 4 °C in 1× Tris glycine native gel buffer (25 mM Tris base, 192 mM glycine).

    Techniques: Synthesized, Methylation

    DNA binding facilities CcrM-Lon recognition. ( A ) Schematic view of DNA probe designs according to genome locus. Probe 2 is the same as probe 1 except with the mutation of GATTC to AATAC. Probe 3 contains the upstream sequence of pliA . P1-P2 and P3-P4 are primer pairs to amplify probe 1 (or probe 2) and probe 3, respectively. CcrM methylation sites are shown circled “M.” ( B ) The direct binding of purified LonS674A to CcrM or probe 1 was measured in vitro by microscale thermophoresis. LonS674A was fluorescently labeled with Atto-488 dye. The concentration of LonS674A 6 was held constant at 20 nM while CcrM or probe 1 was titrated in 2-fold serial dilutions against it. The purified proteins were incubated at room temperature for 10 min before the binding assay. The data report the fraction of LonS674A 6 for description of curve fits. ( C ) A schematic view showing affinities between CcrM, Lon, and DNA. CcrM and Lon have higher affinities to DNA than that of direct interaction.

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

    Article Title: Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

    doi: 10.1073/pnas.1906119116

    Figure Lengend Snippet: DNA binding facilities CcrM-Lon recognition. ( A ) Schematic view of DNA probe designs according to genome locus. Probe 2 is the same as probe 1 except with the mutation of GATTC to AATAC. Probe 3 contains the upstream sequence of pliA . P1-P2 and P3-P4 are primer pairs to amplify probe 1 (or probe 2) and probe 3, respectively. CcrM methylation sites are shown circled “M.” ( B ) The direct binding of purified LonS674A to CcrM or probe 1 was measured in vitro by microscale thermophoresis. LonS674A was fluorescently labeled with Atto-488 dye. The concentration of LonS674A 6 was held constant at 20 nM while CcrM or probe 1 was titrated in 2-fold serial dilutions against it. The purified proteins were incubated at room temperature for 10 min before the binding assay. The data report the fraction of LonS674A 6 for description of curve fits. ( C ) A schematic view showing affinities between CcrM, Lon, and DNA. CcrM and Lon have higher affinities to DNA than that of direct interaction.

    Article Snippet: DNA‐binding capacity of CcrM was evaluated by incubation of purified CcrM with 20 nM of indicated DNA probe in the presence of 200 µM sinefungin (Sigma) in EMSA buffer (50 mM Hepes pH 7.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) for 30 min at room temperature and subjected to electrophoresis in 4 to 15% Mini-PROTEAN TGX precast protein gels (Bio-Rad) at constant 80 V for 3 h at 4 °C in 1× Tris glycine native gel buffer (25 mM Tris base, 192 mM glycine).

    Techniques: Binding Assay, Mutagenesis, Sequencing, Methylation, Purification, In Vitro, Microscale Thermophoresis, Labeling, Concentration Assay, Incubation

    DNA serves as a platform in stimulating CcrM proteolysis by Lon. ( A ) In vitro degradation assays showing the stimulatory effect of DNA on CcrM degradation by Lon. CcrM (1 µM) was incubated with Lon 6 . The means ± SDs ( n = 3) are plotted. ( B for description of curve fits. ( C ) A proposed model of DNA-facilitated CcrM degradation by Lon. The Top shows the presence of CcrM, Lon, and DNA fragments in a mixed reaction. A zoomed-in schematic view shows the 3 steps of CcrM degradation by Lon on DNA: first, binding of CcrM and Lon to DNA fragment due to individual high affinity; second, enhanced-intermolecular collision frequency driven by CcrM processivity; third, substrate unfolding and proteolysis. ( D ) In vivo degradation assays showing the proteolysis of eYFP-CcrM by LonQM and the proteolysis of eYFP-CcrMS315A by Lon in isolated populations of swarmer, stalked, and predivisional (PD) cells in which CcrM and its variant are constitutively expressed. Cells expressing eYFP-CcrM or eYFP- CcrMS315A controlled by P xyl were grown in M2G and induced with 0.3% xylose, synchronized, and harvested at 0 (swarmer cells), 45 (stalked cells), and 120 (predivisional cells) mps. Samples were treated with chloramphenicol (200 µg/mL) to shut off protein synthesis. Relative protein levels were monitored by immunoblots using anti-CcrM antibody ( Top ). Band intensities were quantified ( Bottom ) and error bars represent SDs ( n = 4).

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

    Article Title: Asymmetric division yields progeny cells with distinct modes of regulating cell cycle-dependent chromosome methylation

    doi: 10.1073/pnas.1906119116

    Figure Lengend Snippet: DNA serves as a platform in stimulating CcrM proteolysis by Lon. ( A ) In vitro degradation assays showing the stimulatory effect of DNA on CcrM degradation by Lon. CcrM (1 µM) was incubated with Lon 6 . The means ± SDs ( n = 3) are plotted. ( B for description of curve fits. ( C ) A proposed model of DNA-facilitated CcrM degradation by Lon. The Top shows the presence of CcrM, Lon, and DNA fragments in a mixed reaction. A zoomed-in schematic view shows the 3 steps of CcrM degradation by Lon on DNA: first, binding of CcrM and Lon to DNA fragment due to individual high affinity; second, enhanced-intermolecular collision frequency driven by CcrM processivity; third, substrate unfolding and proteolysis. ( D ) In vivo degradation assays showing the proteolysis of eYFP-CcrM by LonQM and the proteolysis of eYFP-CcrMS315A by Lon in isolated populations of swarmer, stalked, and predivisional (PD) cells in which CcrM and its variant are constitutively expressed. Cells expressing eYFP-CcrM or eYFP- CcrMS315A controlled by P xyl were grown in M2G and induced with 0.3% xylose, synchronized, and harvested at 0 (swarmer cells), 45 (stalked cells), and 120 (predivisional cells) mps. Samples were treated with chloramphenicol (200 µg/mL) to shut off protein synthesis. Relative protein levels were monitored by immunoblots using anti-CcrM antibody ( Top ). Band intensities were quantified ( Bottom ) and error bars represent SDs ( n = 4).

    Article Snippet: DNA‐binding capacity of CcrM was evaluated by incubation of purified CcrM with 20 nM of indicated DNA probe in the presence of 200 µM sinefungin (Sigma) in EMSA buffer (50 mM Hepes pH 7.0, 200 mM NaCl, 1 mM EDTA, 1 mM DTT) for 30 min at room temperature and subjected to electrophoresis in 4 to 15% Mini-PROTEAN TGX precast protein gels (Bio-Rad) at constant 80 V for 3 h at 4 °C in 1× Tris glycine native gel buffer (25 mM Tris base, 192 mM glycine).

    Techniques: In Vitro, Incubation, Binding Assay, In Vivo, Isolation, Variant Assay, Expressing, Western Blot

    Exclusion of RIG-I from compartments containing plus-strand and minus-strand HCV RNA. A and B) Uninfected and HCV-infected Huh7.5 cells were transfected with constructs encoding for FLAG-tagged RIG-I-K207A 2 days after HCV infection. On day 4 after infection, cells were probed with antibodies directed against the FLAG epitope (grey) and either HCV core or NS5A (green). DNA probes (Affymetrix) targeted to either the positive-strand or the negative-strand of the HCV RNA (red) were then hybridized to the viral RNA. DNA was stained with DAPI (blue) and cells were examined by confocal fluorescence microscopy. Boxed regions in the middle row of both panels outline the area of magnification presented in the bottom rows. Scale bars represent 5 μm.

    Journal: PLoS Pathogens

    Article Title: The Hepatitis C Virus-Induced Membranous Web and Associated Nuclear Transport Machinery Limit Access of Pattern Recognition Receptors to Viral Replication Sites

    doi: 10.1371/journal.ppat.1005428

    Figure Lengend Snippet: Exclusion of RIG-I from compartments containing plus-strand and minus-strand HCV RNA. A and B) Uninfected and HCV-infected Huh7.5 cells were transfected with constructs encoding for FLAG-tagged RIG-I-K207A 2 days after HCV infection. On day 4 after infection, cells were probed with antibodies directed against the FLAG epitope (grey) and either HCV core or NS5A (green). DNA probes (Affymetrix) targeted to either the positive-strand or the negative-strand of the HCV RNA (red) were then hybridized to the viral RNA. DNA was stained with DAPI (blue) and cells were examined by confocal fluorescence microscopy. Boxed regions in the middle row of both panels outline the area of magnification presented in the bottom rows. Scale bars represent 5 μm.

    Article Snippet: DNA probes (Affymetrix) complementary to either positive-strand (panel A, red) or negative-strand HCV RNA (panel B, red) were then hybridized to the samples using the manufacturers protocol.

    Techniques: Infection, Transfection, Construct, FLAG-tag, Staining, Fluorescence, Microscopy

    Generation of ISP2 and ISP3 double null mutants. A. Schematic representation of the ISP2–ISP3 locus in WT L. major (upper) and the constructs for gene deletion. ORFs are shown as grey arrows and 5′ FR and 3′ FR boxes represent the FR DNA sequences used for gene targeting. The predicted DNA fragment sizes after restriction digest are shown. HYG , hygromycin-resistance gene; BLE , phleomycin-resistance gene. B. Southern blot of the WT L. major (lanes 1, 3 and 5) and Δ isp2/3 (lanes 2, 4 and 6) digested with AgeI or EcoRI/BamHI and probed with radiolabelled 5′ FR, ISP3 ORF and 3′ FR probes. C. Western blot of cell extracts from 0.5 × 10 7 purified metacyclic promastigotes of WT L. major (lane 1), Δ isp2/3 (lane 2) and Δ isp2/3 : ISP2–ISP3 (lane 3) using antibodies against ISP2, HASPB and EF1α.

    Journal: Cellular Microbiology

    Article Title: Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major

    doi: 10.1111/j.1462-5822.2008.01243.x

    Figure Lengend Snippet: Generation of ISP2 and ISP3 double null mutants. A. Schematic representation of the ISP2–ISP3 locus in WT L. major (upper) and the constructs for gene deletion. ORFs are shown as grey arrows and 5′ FR and 3′ FR boxes represent the FR DNA sequences used for gene targeting. The predicted DNA fragment sizes after restriction digest are shown. HYG , hygromycin-resistance gene; BLE , phleomycin-resistance gene. B. Southern blot of the WT L. major (lanes 1, 3 and 5) and Δ isp2/3 (lanes 2, 4 and 6) digested with AgeI or EcoRI/BamHI and probed with radiolabelled 5′ FR, ISP3 ORF and 3′ FR probes. C. Western blot of cell extracts from 0.5 × 10 7 purified metacyclic promastigotes of WT L. major (lane 1), Δ isp2/3 (lane 2) and Δ isp2/3 : ISP2–ISP3 (lane 3) using antibodies against ISP2, HASPB and EF1α.

    Article Snippet: The DNA probes were radiolabelled with a random priming kit (Stratagene) and the blots were hybridized at 65°C overnight.

    Techniques: Construct, Southern Blot, Western Blot, Purification

    Detection of a cis -encoded antisense small RNA transcribed from the 5′ ureB noncoding strand by Northern analysis. (A) ureAB gene structure and the DNA fragments that were used as templates for RNA probe synthesis by in vitro transcription. The strand-specific oligonucleotide probes are also shown. The bent arrows denote promoters. The ovals represent HP0166 binding sites. (B) The total RNAs were extracted from wild-type H. pylori strain 43504. RNAs were separated in a 10% polyacrylamide-urea gel and then transferred to a Zeta-GT membrane. Sense RNA probes (lanes S) for 5′ ureA , IGR ureAB , 5′ ureB , full ureB , 3′ ureB , and IGR ureBI were used for Northern blot analysis. As a control, antisense RNA probes (lanes AS) were also used for Northern blot analysis. (C) For further characterization of the ∼290-nt antisense sRNA complementary to the 5′ region of ureB , total RNAs isolated from wild-type H. pylori strains 43504 and 26695 were separated in a 6% polyacrylamide-urea gel and then transferred to a Zeta-GB membrane. Six strand-specific oligonucleotide sense probes (1-1S, 1-2S, 1-3S, 2-1S, 2-3S, and 3-1S, each with a size of 51 to 55 nt) corresponding to different regions of the 5′- ureB probe were used in Northern blot analysis.

    Journal: Journal of Bacteriology

    Article Title: A cis-Encoded Antisense Small RNA Regulated by the HP0165-HP0166 Two-Component System Controls Expression of ureB in Helicobacter pylori ▿

    doi: 10.1128/JB.00800-10

    Figure Lengend Snippet: Detection of a cis -encoded antisense small RNA transcribed from the 5′ ureB noncoding strand by Northern analysis. (A) ureAB gene structure and the DNA fragments that were used as templates for RNA probe synthesis by in vitro transcription. The strand-specific oligonucleotide probes are also shown. The bent arrows denote promoters. The ovals represent HP0166 binding sites. (B) The total RNAs were extracted from wild-type H. pylori strain 43504. RNAs were separated in a 10% polyacrylamide-urea gel and then transferred to a Zeta-GT membrane. Sense RNA probes (lanes S) for 5′ ureA , IGR ureAB , 5′ ureB , full ureB , 3′ ureB , and IGR ureBI were used for Northern blot analysis. As a control, antisense RNA probes (lanes AS) were also used for Northern blot analysis. (C) For further characterization of the ∼290-nt antisense sRNA complementary to the 5′ region of ureB , total RNAs isolated from wild-type H. pylori strains 43504 and 26695 were separated in a 6% polyacrylamide-urea gel and then transferred to a Zeta-GB membrane. Six strand-specific oligonucleotide sense probes (1-1S, 1-2S, 1-3S, 2-1S, 2-3S, and 3-1S, each with a size of 51 to 55 nt) corresponding to different regions of the 5′- ureB probe were used in Northern blot analysis.

    Article Snippet: For preparation of the DNA probe, the DNA fragment for 5′- ureA (510 bp) generated by PCR as described above was radiolabeled with [α-32 P]dCTP using a Prim-It II random primer labeling kit (Stratagene, La Jolla, CA).

    Techniques: Northern Blot, In Vitro, Binding Assay, Isolation

    Replicon localization of mob DNAs in cosmids pRmOR69 and pRmOR65. Blots of Eco RI-digested genomic DNAs were hybridized against digoxigenin-labeled cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild-type); 2, A. tumefaciens C58; 3, A. tumefaciens C58(pRme2011a); 4, R. meliloti GR4 (wild type); 5, R. meliloti GRM6 (cured of plasmid pRmeGR4b); 6, R. meliloti GRM10 (cured of plasmid pRmeGR4a); 7, R. meliloti GRM8 (cured of pRmeGR4a and pRmeGR4b); 8, R. meliloti GRT3 (carrying pSym1 deletion); 9, Eco RI-digested cosmid DNA.

    Journal: Journal of Bacteriology

    Article Title: Cloning and Identification of Conjugative Transfer Origins in the Rhizobium meliloti Genome

    doi:

    Figure Lengend Snippet: Replicon localization of mob DNAs in cosmids pRmOR69 and pRmOR65. Blots of Eco RI-digested genomic DNAs were hybridized against digoxigenin-labeled cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild-type); 2, A. tumefaciens C58; 3, A. tumefaciens C58(pRme2011a); 4, R. meliloti GR4 (wild type); 5, R. meliloti GRM6 (cured of plasmid pRmeGR4b); 6, R. meliloti GRM10 (cured of plasmid pRmeGR4a); 7, R. meliloti GRM8 (cured of pRmeGR4a and pRmeGR4b); 8, R. meliloti GRT3 (carrying pSym1 deletion); 9, Eco RI-digested cosmid DNA.

    Article Snippet: DNA hybridization probes were digoxigenin-labeled according to standard protocols (Boehringer, Mannheim, Germany).

    Techniques: Labeling, Molecular Weight, Marker, Plasmid Preparation, Cosmid DNA

    Replicon localization of mob DNAs in cosmids pRmOR106, -1012, -1026, -1034, and -1030. Blots of Eco RI-digested genomic DNAs were hybridized against digoxigenin-labeled cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild type); 2, A. tumefaciens C58; 3, A. tumefaciens C58(pRme2011a); 4, R. meliloti GR4 (wild type); 5, R. meliloti GRM6 (cured of plasmid pRmeGR4b); 6, R. meliloti GRM10 (cured of plasmid pRmeGR4a); 7, R. meliloti GRM8 (cured of pRmeGR4a and pRmeGR4b); 8, R. meliloti GRT3 (carrying pSym1 deletion); 9, Eco RI-digested cosmid DNA.

    Journal: Journal of Bacteriology

    Article Title: Cloning and Identification of Conjugative Transfer Origins in the Rhizobium meliloti Genome

    doi:

    Figure Lengend Snippet: Replicon localization of mob DNAs in cosmids pRmOR106, -1012, -1026, -1034, and -1030. Blots of Eco RI-digested genomic DNAs were hybridized against digoxigenin-labeled cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild type); 2, A. tumefaciens C58; 3, A. tumefaciens C58(pRme2011a); 4, R. meliloti GR4 (wild type); 5, R. meliloti GRM6 (cured of plasmid pRmeGR4b); 6, R. meliloti GRM10 (cured of plasmid pRmeGR4a); 7, R. meliloti GRM8 (cured of pRmeGR4a and pRmeGR4b); 8, R. meliloti GRT3 (carrying pSym1 deletion); 9, Eco RI-digested cosmid DNA.

    Article Snippet: DNA hybridization probes were digoxigenin-labeled according to standard protocols (Boehringer, Mannheim, Germany).

    Techniques: Labeling, Molecular Weight, Marker, Plasmid Preparation, Cosmid DNA

    Genome localization of mob DNAs in cosmids pRmOR1035, -1033, -1041, and -1042. Blots of Eco RI-digested genomic DNAs were hybridized against digoxigenin-labeled cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild type); 2, A. tumefaciens C58; 3, A. tumefaciens 104 (C58 carrying pRmeSU47b); 4, A. tumefaciens 117 (C58 carrying pRmeSU47a); 5, R. meliloti GR4 (wild type); 6, Eco RI-digested cosmid DNA.

    Journal: Journal of Bacteriology

    Article Title: Cloning and Identification of Conjugative Transfer Origins in the Rhizobium meliloti Genome

    doi:

    Figure Lengend Snippet: Genome localization of mob DNAs in cosmids pRmOR1035, -1033, -1041, and -1042. Blots of Eco RI-digested genomic DNAs were hybridized against digoxigenin-labeled cosmid probes. Lanes: M, digoxigenin-labeled molecular weight marker; 1, R. meliloti 2011 (wild type); 2, A. tumefaciens C58; 3, A. tumefaciens 104 (C58 carrying pRmeSU47b); 4, A. tumefaciens 117 (C58 carrying pRmeSU47a); 5, R. meliloti GR4 (wild type); 6, Eco RI-digested cosmid DNA.

    Article Snippet: DNA hybridization probes were digoxigenin-labeled according to standard protocols (Boehringer, Mannheim, Germany).

    Techniques: Labeling, Molecular Weight, Marker, Cosmid DNA

    RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.

    Journal: The Plant Cell

    Article Title: DOH1, a Class 1 knox Gene, Is Required for Maintenance of the Basic Plant Architecture and Floral Transition in Orchid

    doi:

    Figure Lengend Snippet: RNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. (A) Expression of DOH1 in orchid 35S :: DOH1se transformants. Total RNA was isolated from mature CLLs from wild-type plants (wt) and the independent 35S :: DOH1se transgenic lines (lines se1, se7, se17, se28, and se33). The gel blot, containing 10 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. (B) Expression of the DOH1 sense and antisense genes in 35S :: DOH1as transformants. Total RNA was isolated from 6-week-old VSAMs (length, 1.5 mm) from wild-type plants (wt) and independent 35S :: DOH1as transformants (lines as4, as10, as15, as23, as27, as31, and as38). The gel blot, containing 20 μg of total RNA in each lane, was hybridized with the digoxigenin-labeled full-length DOH1 RNA sense probe (SP) or antisense probe (AP). The rRNAs, stained by methylene blue, indicate the amount of total RNA in each lane.

    Article Snippet: Blots were hybridized overnight with the digoxigenin-labeled DNA probes at 42°C in DIG Easy Hyb buffer (Boehringer Mannheim).

    Techniques: Western Blot, Transgenic Assay, Expressing, Isolation, Labeling, Staining

    Genomic DNA Gel Blot Analysis of the DOH1 Gene in Wild-Type Plants. A DNA gel blot containing 20 μg of orchid genomic DNA digested with various restriction enzymes was hybridized at low stringency with the digoxigenin-labeled DOH1 DNA probe. The sizes of the DNA markers are given at right in kilobases. A, ApaI; E, EcoRV; P, PstI; Sa, SmaI; Sp, SspI; Su, StuI; X, XbaI.

    Journal: The Plant Cell

    Article Title: DOH1, a Class 1 knox Gene, Is Required for Maintenance of the Basic Plant Architecture and Floral Transition in Orchid

    doi:

    Figure Lengend Snippet: Genomic DNA Gel Blot Analysis of the DOH1 Gene in Wild-Type Plants. A DNA gel blot containing 20 μg of orchid genomic DNA digested with various restriction enzymes was hybridized at low stringency with the digoxigenin-labeled DOH1 DNA probe. The sizes of the DNA markers are given at right in kilobases. A, ApaI; E, EcoRV; P, PstI; Sa, SmaI; Sp, SspI; Su, StuI; X, XbaI.

    Article Snippet: Blots were hybridized overnight with the digoxigenin-labeled DNA probes at 42°C in DIG Easy Hyb buffer (Boehringer Mannheim).

    Techniques: Western Blot, Labeling

    DNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. A gel blot containing genomic DNA from independent transgenic lines (10 μg per lane) digested with SmaI was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. DNA was isolated successively from 35S :: DOH1se transformants (lines se1, se7, and se17), 35S :: DOH1as transformants (lines as4 and as10), and wild-type plants (wt). The endogenous DOH1 gene band present in all of the plants is indicated with an arrow. The sizes of the DNA markers are given at right in kilobases.

    Journal: The Plant Cell

    Article Title: DOH1, a Class 1 knox Gene, Is Required for Maintenance of the Basic Plant Architecture and Floral Transition in Orchid

    doi:

    Figure Lengend Snippet: DNA Gel Blot Analysis of the Orchid DOH1 Sense and Antisense Transgenic Plants. A gel blot containing genomic DNA from independent transgenic lines (10 μg per lane) digested with SmaI was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. DNA was isolated successively from 35S :: DOH1se transformants (lines se1, se7, and se17), 35S :: DOH1as transformants (lines as4 and as10), and wild-type plants (wt). The endogenous DOH1 gene band present in all of the plants is indicated with an arrow. The sizes of the DNA markers are given at right in kilobases.

    Article Snippet: Blots were hybridized overnight with the digoxigenin-labeled DNA probes at 42°C in DIG Easy Hyb buffer (Boehringer Mannheim).

    Techniques: Western Blot, Transgenic Assay, Labeling, Isolation

    RNA Gel Blot Analysis of the DOH1 Gene in Wild-Type Plants. (A) RNA gel blot analysis of the DOH1 gene in different orchid tissues. An RNA gel blot containing 10 μg of total RNA in each lane was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. RNA was isolated from roots (R; length, 1 cm), vegetative stems (VS; length, 0.4 cm), transitional stems (TS; length, 0.4 cm), young leaves (YL; length, 0.5 cm), old leaves (OL; length, 2 cm), VSAMs (V; 6 weeks old; length, 1.5 mm), TSAMs (T; 12 weeks old; length, 2 mm), and flowers (F; length, 1.5 cm). (B) Time course of DOH1 expression in orchid development. Lanes are labeled according to the age (in weeks) of tissues. From left to right, total RNA (25 μg per lane) was successively extracted from thin sections of protocorms (P; 0 weeks old; length, 1 mm), PLBs (PLB; 2 weeks old; length, 4 to 5 mm), VSAMs (VSAM; 4, 6, and 8 weeks old; ength, 1.5 mm), TSAMs (TSAM; 9 and 12 weeks old; length, 2 mm), and inflorescence meristems (IM; 14 weeks old; length, 3 to 5 mm). Hybridization was performed as in (A) . The rRNAs, stained by methylene blue, indicate the amount of total RNA loaded in each lane.

    Journal: The Plant Cell

    Article Title: DOH1, a Class 1 knox Gene, Is Required for Maintenance of the Basic Plant Architecture and Floral Transition in Orchid

    doi:

    Figure Lengend Snippet: RNA Gel Blot Analysis of the DOH1 Gene in Wild-Type Plants. (A) RNA gel blot analysis of the DOH1 gene in different orchid tissues. An RNA gel blot containing 10 μg of total RNA in each lane was hybridized with the digoxigenin-labeled full-length DOH1 DNA probe. RNA was isolated from roots (R; length, 1 cm), vegetative stems (VS; length, 0.4 cm), transitional stems (TS; length, 0.4 cm), young leaves (YL; length, 0.5 cm), old leaves (OL; length, 2 cm), VSAMs (V; 6 weeks old; length, 1.5 mm), TSAMs (T; 12 weeks old; length, 2 mm), and flowers (F; length, 1.5 cm). (B) Time course of DOH1 expression in orchid development. Lanes are labeled according to the age (in weeks) of tissues. From left to right, total RNA (25 μg per lane) was successively extracted from thin sections of protocorms (P; 0 weeks old; length, 1 mm), PLBs (PLB; 2 weeks old; length, 4 to 5 mm), VSAMs (VSAM; 4, 6, and 8 weeks old; ength, 1.5 mm), TSAMs (TSAM; 9 and 12 weeks old; length, 2 mm), and inflorescence meristems (IM; 14 weeks old; length, 3 to 5 mm). Hybridization was performed as in (A) . The rRNAs, stained by methylene blue, indicate the amount of total RNA loaded in each lane.

    Article Snippet: Blots were hybridized overnight with the digoxigenin-labeled DNA probes at 42°C in DIG Easy Hyb buffer (Boehringer Mannheim).

    Techniques: Western Blot, Labeling, Isolation, Expressing, Hybridization, Staining