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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 157 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 157 article reviews
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    99
    Millipore dna oligonucleotide probe
    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP <t>(DNA</t> duplex probe) + Circle DNA + hairpin probes <t>(GHP)</t> + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.
    Dna Oligonucleotide Probe, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 16 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Hologic Inc deoxyribonucleic acid probe assay
    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP <t>(DNA</t> duplex probe) + Circle DNA + hairpin probes <t>(GHP)</t> + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.
    Deoxyribonucleic Acid Probe Assay, supplied by Hologic Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/deoxyribonucleic acid probe assay/product/Hologic Inc
    Average 93 stars, based on 1 article reviews
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    85
    GE Healthcare complementary deoxyribonucleic acid probe
    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP <t>(DNA</t> duplex probe) + Circle DNA + hairpin probes <t>(GHP)</t> + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.
    Complementary Deoxyribonucleic Acid Probe, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 85/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/complementary deoxyribonucleic acid probe/product/GE Healthcare
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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 212 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna probe/product/Boehringer Mannheim
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    90
    Evrogen 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 Evrogen, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna probe/product/Evrogen
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    92
    Gen-Probe 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 Gen-Probe, used in various techniques. Bioz Stars score: 92/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna probe/product/Gen-Probe
    Average 92 stars, based on 196 article reviews
    Price from $9.99 to $1999.99
    dna probe - by Bioz Stars, 2020-05
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    Image Search Results


    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

    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    Journal: Nanomaterials

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    doi: 10.3390/nano6100190

    Figure Lengend Snippet: Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    Article Snippet: Chemicals All the HPLC-purified oligonucleotide sequences were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) and listed below: the anti-BPA aptamer with the sequence of 5′-CC GGT GGG TGG TCA GGT GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC-3′ (denoted DNA1 ); the 20-base complementary fragment (denoted P1 ), 5′-CCC ACC TGA CCA CCC ACC GG-3′; Circular DNA template (P-circle), 5′-P-GTC AGG T GGG C TTG ATA ACT ACC CGT GAG GAA CCA ATT ATC CCA ACT TAT TCG TGA CTC CAG TGA AGC TAA ATG C CGG TG G GTG-3′; the hairpin DNA probe (GHP), 5′-GGG TTG GGC GGG ATG GGT TAC CTC AGT GCT TAT TCA ACC CTA TTA-3′; Protoporphyrin IX zinc(II) (ZnPPIX) was purchased from Sigma-Aldrich (Shanghai, China) and used without further purification.

    Techniques: Fluorescence

    ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.

    Journal: Nanomaterials

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    doi: 10.3390/nano6100190

    Figure Lengend Snippet: ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.

    Article Snippet: Chemicals All the HPLC-purified oligonucleotide sequences were synthesized by Shanghai Sangon Biotechnology Co., Ltd. (Shanghai, China) and listed below: the anti-BPA aptamer with the sequence of 5′-CC GGT GGG TGG TCA GGT GGG ATA GCG TTC CGC GTA TGG CCC AGC GCA TCA CGG GTT CGC ACC-3′ (denoted DNA1 ); the 20-base complementary fragment (denoted P1 ), 5′-CCC ACC TGA CCA CCC ACC GG-3′; Circular DNA template (P-circle), 5′-P-GTC AGG T GGG C TTG ATA ACT ACC CGT GAG GAA CCA ATT ATC CCA ACT TAT TCG TGA CTC CAG TGA AGC TAA ATG C CGG TG G GTG-3′; the hairpin DNA probe (GHP), 5′-GGG TTG GGC GGG ATG GGT TAC CTC AGT GCT TAT TCA ACC CTA TTA-3′; Protoporphyrin IX zinc(II) (ZnPPIX) was purchased from Sigma-Aldrich (Shanghai, China) and used without further purification.

    Techniques: Agarose Gel Electrophoresis, Electrophoresis, Microscopy, Amplification

    Schematic showing the principle of the triggered self-assembly amplification for DNA detection on GFET. The GFET was functionalized by hairpin probe DNA H1 through the PBASE linker. The target DNA (T) opens the hairpin probe to form the complex H1·T. T is then displaced by helper DNA H2 through the toehold-mediated strand displacement reaction, leading to the formation of the H1·H2 complex and enabling target recycling. Hybridization chain reaction (HCR) was triggered by H1·H2 in the presence of two additional helper DNAs, H3 and H4. Amplified HCR products are then detected through a shift of the GFET Dirac voltage.

    Journal: Nano Letters

    Article Title: Detection of Sub-fM DNA with Target Recycling and Self-Assembly Amplification on Graphene Field-Effect Biosensors

    doi: 10.1021/acs.nanolett.8b00572

    Figure Lengend Snippet: Schematic showing the principle of the triggered self-assembly amplification for DNA detection on GFET. The GFET was functionalized by hairpin probe DNA H1 through the PBASE linker. The target DNA (T) opens the hairpin probe to form the complex H1·T. T is then displaced by helper DNA H2 through the toehold-mediated strand displacement reaction, leading to the formation of the H1·H2 complex and enabling target recycling. Hybridization chain reaction (HCR) was triggered by H1·H2 in the presence of two additional helper DNAs, H3 and H4. Amplified HCR products are then detected through a shift of the GFET Dirac voltage.

    Article Snippet: 1-Pyrenebutyric acid N -Hydroxysuccinimide Ester (PBASE) Functionalization with Hairpin Probe DNA Immobilization and Testing against Target or Control Solutions GFET sensors were soaked in a 0.2 mM PBASE (Sigma-Aldrich) in DMF for 20 h and then washed thoroughly with DMF, IPA, and DI water for 3 min each.

    Techniques: Amplification, 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