pvuii  (New England Biolabs)


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    Structured Review

    New England Biolabs pvuii
    Quantitative polymerase chain reaction (qPCR) targeting the rep sequence using <t>pSub201</t> plasmid undigested, linearized with HindIII, or digested with <t>PvuII</t> to create inverted terminal repeats with free ends. ( a ) The minimal differences observed between mean quantification cycles ( C q ) obtained in each experimental condition indicate that the DNA concentrations determined by spectrophotometry are similar. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a .
    Pvuii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR"

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    Journal: Molecular Therapy. Methods & Clinical Development

    doi: 10.1038/mtm.2016.19

    Quantitative polymerase chain reaction (qPCR) targeting the rep sequence using pSub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats with free ends. ( a ) The minimal differences observed between mean quantification cycles ( C q ) obtained in each experimental condition indicate that the DNA concentrations determined by spectrophotometry are similar. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a .
    Figure Legend Snippet: Quantitative polymerase chain reaction (qPCR) targeting the rep sequence using pSub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats with free ends. ( a ) The minimal differences observed between mean quantification cycles ( C q ) obtained in each experimental condition indicate that the DNA concentrations determined by spectrophotometry are similar. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a .

    Techniques Used: Real-time Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Spectrophotometry

    Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.
    Figure Legend Snippet: Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Techniques Used: Plasmid Preparation, CTG Assay, Agarose Gel Electrophoresis, Electrophoresis, Concentration Assay, Spectrophotometry

    ITR2 quantitative polymerase chain reaction (qPCR) using psub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats (ITRs) with free ends. ( a ) The differences between mean quantification cycles ( C q ) obtained in each experimental condition with an equal amount of plasmid quantified by spectrophotometry are shown. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a ).
    Figure Legend Snippet: ITR2 quantitative polymerase chain reaction (qPCR) using psub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats (ITRs) with free ends. ( a ) The differences between mean quantification cycles ( C q ) obtained in each experimental condition with an equal amount of plasmid quantified by spectrophotometry are shown. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a ).

    Techniques Used: Real-time Polymerase Chain Reaction, Plasmid Preparation, Spectrophotometry

    2) Product Images from "The mechanism of DNA replication termination in vertebrates"

    Article Title: The mechanism of DNA replication termination in vertebrates

    Journal: Nature

    doi: 10.1038/nature14887

    DNA synthesis does not stall during termination A. Cartoon depicting the assay for lacO array synthesis. B. LacR Block-IPTG release was performed on p[ lacO x12]. To measure synthesis within the array, termination intermediates were cut with AflIII and PvuII to liberate the array fragment from the vector. Cleaved products were separated by native gel electrophoresis. Different exposures of array and vector fragments are shown (see methods). C. Array synthesis, vector synthesis, and dissolution were quantified. Means±s.d. are plotted (n=3).
    Figure Legend Snippet: DNA synthesis does not stall during termination A. Cartoon depicting the assay for lacO array synthesis. B. LacR Block-IPTG release was performed on p[ lacO x12]. To measure synthesis within the array, termination intermediates were cut with AflIII and PvuII to liberate the array fragment from the vector. Cleaved products were separated by native gel electrophoresis. Different exposures of array and vector fragments are shown (see methods). C. Array synthesis, vector synthesis, and dissolution were quantified. Means±s.d. are plotted (n=3).

    Techniques Used: DNA Synthesis, Blocking Assay, Plasmid Preparation, Nucleic Acid Electrophoresis

    Sequence-specific termination can be induced at a LacR array (A) To investigate whether a LacR array blocks replication forks, a plasmid containing a tandem array of 16 lac operator ( lacO ) sequences, p[ lacO x16], was incubated with buffer or LacR and then replicated in egg extract containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with XmnI (far left cartoon) and separated according to size and shape by 2-dimensional gel electrophoresis (see schematic of 2-D gel). As replication neared completion at 4.5 minutes, mainly linear molecules were produced in the presence of buffer (orange arrowhead). In contrast, in the presence of LacR, a discrete spot appeared on the Double Y arc (blue arrowhead), demonstrating that converging replication forks accumulate at a specific locus on p[ lacO x16]. These data indicate that 16 copies of LacR block replication forks. (B–F) To test whether the double-Y structures observed in (A) arose from replication forks stalling at the outer edges of the lacO array, we tested whether LacR specifically inhibited replication of lacO sequences. To this end, p[ lacOx16 ] (C) and the parental plasmid lacking lacO repeats, p[ empty ] (B), were incubated in the presence of buffer or LacR and replicated using Xenopus egg extracts containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with AflIII and PvuII to release the 2354 bp plasmid backbone (B and C) and a 294 bp control fragment from p[ empty ] (B) or a 794 bp lacO fragment from p[ lacO x16] (C). The plasmid backbone and the respective inserts were separated on a native gel and detected by autoradiography (D). A longer exposure of the small fragments is shown, since they are less intense than the large fragments. The results in panel (D) were quantified in (E) and (F). Importantly, LacR specifically inhibited replication of the lacO -containing fragment in p[ lacO x16] (F, blue circles) but not the control fragment in p[ empty ] (E, green circles). We conclude that LacR prevents replication of the lacO array and that the double-Y’s in (A) represent forks converged on the outer edges of the array. Importantly, synthesis within the 2354 bp backbone fragment (F, orange circles) of p[ lacO x16] was not inhibited in the presence of LacR, indicating that no global structural changes occur that inhibit replication.
    Figure Legend Snippet: Sequence-specific termination can be induced at a LacR array (A) To investigate whether a LacR array blocks replication forks, a plasmid containing a tandem array of 16 lac operator ( lacO ) sequences, p[ lacO x16], was incubated with buffer or LacR and then replicated in egg extract containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with XmnI (far left cartoon) and separated according to size and shape by 2-dimensional gel electrophoresis (see schematic of 2-D gel). As replication neared completion at 4.5 minutes, mainly linear molecules were produced in the presence of buffer (orange arrowhead). In contrast, in the presence of LacR, a discrete spot appeared on the Double Y arc (blue arrowhead), demonstrating that converging replication forks accumulate at a specific locus on p[ lacO x16]. These data indicate that 16 copies of LacR block replication forks. (B–F) To test whether the double-Y structures observed in (A) arose from replication forks stalling at the outer edges of the lacO array, we tested whether LacR specifically inhibited replication of lacO sequences. To this end, p[ lacOx16 ] (C) and the parental plasmid lacking lacO repeats, p[ empty ] (B), were incubated in the presence of buffer or LacR and replicated using Xenopus egg extracts containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with AflIII and PvuII to release the 2354 bp plasmid backbone (B and C) and a 294 bp control fragment from p[ empty ] (B) or a 794 bp lacO fragment from p[ lacO x16] (C). The plasmid backbone and the respective inserts were separated on a native gel and detected by autoradiography (D). A longer exposure of the small fragments is shown, since they are less intense than the large fragments. The results in panel (D) were quantified in (E) and (F). Importantly, LacR specifically inhibited replication of the lacO -containing fragment in p[ lacO x16] (F, blue circles) but not the control fragment in p[ empty ] (E, green circles). We conclude that LacR prevents replication of the lacO array and that the double-Y’s in (A) represent forks converged on the outer edges of the array. Importantly, synthesis within the 2354 bp backbone fragment (F, orange circles) of p[ lacO x16] was not inhibited in the presence of LacR, indicating that no global structural changes occur that inhibit replication.

    Techniques Used: Sequencing, Plasmid Preparation, Incubation, Nucleic Acid Electrophoresis, Produced, Blocking Assay, Autoradiography

    3) Product Images from "Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16"

    Article Title: Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-020-02702-9

    FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h
    Figure Legend Snippet: FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h

    Techniques Used: Stripping Membranes, Amplification

    Sequencing pvuII -digested LAMP amplicons. a PvuII -digested products were run on a non-denaturing gel and bands A and B were extracted. b The schematic shows the corresponding LAMP amplicon diagram for the sequenced bands A and B
    Figure Legend Snippet: Sequencing pvuII -digested LAMP amplicons. a PvuII -digested products were run on a non-denaturing gel and bands A and B were extracted. b The schematic shows the corresponding LAMP amplicon diagram for the sequenced bands A and B

    Techniques Used: Sequencing, Amplification

    FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h
    Figure Legend Snippet: FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h

    Techniques Used: Stripping Membranes, Amplification

    4) Product Images from "Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum"

    Article Title: Peptide Inhibition of Topoisomerase IB from Plasmodium falciparum

    Journal: Molecular Biology International

    doi: 10.4061/2011/854626

    Effect of peptide WRWYCRCK on restriction digestion by restriction endonucleases. Representative gel picture showing the result of incubating pUC19 plasmid with EcoRI (lanes 4–7) or PvuII (lanes 8–11) in the presence of peptide WRWYCRCK at the following concentration: 12.5 μ M, 25 μ M, or 50 μ M. The sizes, kbp, of the DNA marker (lane 1, labeled M) are shown to the left of the gel picture. 0: control lanes with no peptide added; +: control lane with 50 μ M peptide added; asterisks indicate the gel electrophoretic mobility of the digested plasmid, for EcoRI, 2.7 kbp, and for PvuII, 0.3 kbp and 2.4 kbp.
    Figure Legend Snippet: Effect of peptide WRWYCRCK on restriction digestion by restriction endonucleases. Representative gel picture showing the result of incubating pUC19 plasmid with EcoRI (lanes 4–7) or PvuII (lanes 8–11) in the presence of peptide WRWYCRCK at the following concentration: 12.5 μ M, 25 μ M, or 50 μ M. The sizes, kbp, of the DNA marker (lane 1, labeled M) are shown to the left of the gel picture. 0: control lanes with no peptide added; +: control lane with 50 μ M peptide added; asterisks indicate the gel electrophoretic mobility of the digested plasmid, for EcoRI, 2.7 kbp, and for PvuII, 0.3 kbp and 2.4 kbp.

    Techniques Used: Plasmid Preparation, Concentration Assay, Marker, Labeling

    5) Product Images from "Cleavage of a model DNA replication fork by a Type I restriction endonuclease"

    Article Title: Cleavage of a model DNA replication fork by a Type I restriction endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp214

    Preparation of a long branched DNA (eF1). ( A ) pES1 was treated with SphI, PvuII and nicking endonuclease Nb.BbvCI. A partial fragment from pGap1 was prepared by PCR with indicated primers, followed by nicking with Nt.BbvCI. ( B ) Single strands between the nicks introduced in the previous step were dissociated by heating with removal by complementary oligo DNA from the fragment containing a gap structure. ( C ) Resulting DNAs with the gap were annealed to form a branched structure (Materials and methods section). A leftward triangle indicates an EcoR124I site, 5′ CGA TGCTGTA TTC . An open circle indicates 32 P for 5′-end labeling.
    Figure Legend Snippet: Preparation of a long branched DNA (eF1). ( A ) pES1 was treated with SphI, PvuII and nicking endonuclease Nb.BbvCI. A partial fragment from pGap1 was prepared by PCR with indicated primers, followed by nicking with Nt.BbvCI. ( B ) Single strands between the nicks introduced in the previous step were dissociated by heating with removal by complementary oligo DNA from the fragment containing a gap structure. ( C ) Resulting DNAs with the gap were annealed to form a branched structure (Materials and methods section). A leftward triangle indicates an EcoR124I site, 5′ CGA TGCTGTA TTC . An open circle indicates 32 P for 5′-end labeling.

    Techniques Used: Polymerase Chain Reaction, End Labeling

    6) Product Images from "Cleavage of a model DNA replication fork by a methyl-specific endonuclease"

    Article Title: Cleavage of a model DNA replication fork by a methyl-specific endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr153

    Preparation of a long-branched DNA with methylation. The two plasmids were modified in vivo by M.FnuDII to generate 5′-m5 C GCG, a McrBC recognition sequence {(i), (ii)}. Potential unmethylated plasmids were eliminated by cleavage with BstUI (5′-CGCG). pME63 was cleaved with PvuII and then with nicking endonuclease Nb.BbvCI (iii), while pMap63 was treated with nicking endonuclease Nt.BbvCI (iv). The resulting short single strands were dissociated by heating and removed by annealing with a complementary single-strand oligo DNA. The 5′-ends of intermediate (iv) were labeled with 32 P (v), followed by BspHI cleavage for removal of one of the radio-labels (vii). The two DNAs with complementary single-strand regions {(vi), (vii)} were annealed to form a branched structure {viii, eM63(++)} as detailed in ‘Materials and Methods’ section. Open circle, 32 P label at 5′-end; filled diamond, DNA methylation.
    Figure Legend Snippet: Preparation of a long-branched DNA with methylation. The two plasmids were modified in vivo by M.FnuDII to generate 5′-m5 C GCG, a McrBC recognition sequence {(i), (ii)}. Potential unmethylated plasmids were eliminated by cleavage with BstUI (5′-CGCG). pME63 was cleaved with PvuII and then with nicking endonuclease Nb.BbvCI (iii), while pMap63 was treated with nicking endonuclease Nt.BbvCI (iv). The resulting short single strands were dissociated by heating and removed by annealing with a complementary single-strand oligo DNA. The 5′-ends of intermediate (iv) were labeled with 32 P (v), followed by BspHI cleavage for removal of one of the radio-labels (vii). The two DNAs with complementary single-strand regions {(vi), (vii)} were annealed to form a branched structure {viii, eM63(++)} as detailed in ‘Materials and Methods’ section. Open circle, 32 P label at 5′-end; filled diamond, DNA methylation.

    Techniques Used: DNA Methylation Assay, Modification, In Vivo, Sequencing, Labeling

    7) Product Images from "Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16"

    Article Title: Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-020-02702-9

    FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h
    Figure Legend Snippet: FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h

    Techniques Used: Stripping Membranes, Amplification

    Sequencing pvuII -digested LAMP amplicons. a PvuII -digested products were run on a non-denaturing gel and bands A and B were extracted. b The schematic shows the corresponding LAMP amplicon diagram for the sequenced bands A and B
    Figure Legend Snippet: Sequencing pvuII -digested LAMP amplicons. a PvuII -digested products were run on a non-denaturing gel and bands A and B were extracted. b The schematic shows the corresponding LAMP amplicon diagram for the sequenced bands A and B

    Techniques Used: Sequencing, Amplification

    FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h
    Figure Legend Snippet: FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h

    Techniques Used: Stripping Membranes, Amplification

    8) Product Images from "Mechanistic studies of the modulation of cleavage activity of topoisomerase I by DNA adducts of mono- and bi-functional PtII complexes"

    Article Title: Mechanistic studies of the modulation of cleavage activity of topoisomerase I by DNA adducts of mono- and bi-functional PtII complexes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp580

    The dependence of the DNA cleavage of the 161-bp PvuII/HindIII fragment of pBluescript SK(-) phagemid mediated by top1 in the presence of CPT on the level of DNA modification by cisplatin ( A ) and [PtCl(dien)]Cl ( B ). The relative cleavage was calculated as a ratio of the intensities of the bands corresponding to the five main cleavage sites ( Figure 3 ) induced by top1 in unplatinated and platinated DNA in the presence of CPT after removal of the platinum adducts by treatment with NaCN.
    Figure Legend Snippet: The dependence of the DNA cleavage of the 161-bp PvuII/HindIII fragment of pBluescript SK(-) phagemid mediated by top1 in the presence of CPT on the level of DNA modification by cisplatin ( A ) and [PtCl(dien)]Cl ( B ). The relative cleavage was calculated as a ratio of the intensities of the bands corresponding to the five main cleavage sites ( Figure 3 ) induced by top1 in unplatinated and platinated DNA in the presence of CPT after removal of the platinum adducts by treatment with NaCN.

    Techniques Used: Cycling Probe Technology, Modification

    ( A ) Autoradiograms of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of DNA synthesis by VentR(exo−) DNA polymerase on the 161-bp PvuII/HindIII fragment of pBluescript SK(−) phagemid and subsequently modified by cisplatin or [PtCl(dien)]Cl. The gels contained the linear amplification products of control, nonplatinated DNA, and DNA treated with cisplatin or [PtCl(dien)]Cl at r b = 0.005 or 0.008. Lanes are as follows: (control) unmodified template; lanes, cisPt1 and dienPt1, DNA modified by cisplatin or [PtCl(dien)]Cl, respectively, at r b = 0.005; lanes, cisPt2 and dienPt2, DNA modified by cisplatin or [PtCl(dien)]Cl, respectively, at r b = 0.008; lanes C, G, T, A, chain-terminated marker DNAs (note that these dideoxy sequencing lanes give the sequence complementary to the template strand). The numbers correspond to the nucleotide sequence numbering of part B. Left panel, mapping of the Pt adducts formed on the top strand of the 161-bp fragment; right panel, mapping of the Pt adducts formed on the bottom strand of the 161-bp fragment. ( B ) Schematic diagram showing a portion of the sequence used to monitor inhibition of DNA synthesis on the template containing adducts of cisplatin or [PtCl(dien)]Cl. Solid circles indicate major stop signals from part A, lane cisPt2, open circles indicate major stop signals from part A, lane dienPt2. The numbering of the nucleotides in this scheme corresponds to the cleavage sites of top1 ( Figure 4 ). Nucleotides 37, 70, 92, 97 and 119 correspond respectively to nucleotides 569, 602, 624, 629 and 741 on the pBluescript SK(−) phagemid nucleotide sequence map.
    Figure Legend Snippet: ( A ) Autoradiograms of 6% polyacrylamide/8 M urea sequencing gel showing inhibition of DNA synthesis by VentR(exo−) DNA polymerase on the 161-bp PvuII/HindIII fragment of pBluescript SK(−) phagemid and subsequently modified by cisplatin or [PtCl(dien)]Cl. The gels contained the linear amplification products of control, nonplatinated DNA, and DNA treated with cisplatin or [PtCl(dien)]Cl at r b = 0.005 or 0.008. Lanes are as follows: (control) unmodified template; lanes, cisPt1 and dienPt1, DNA modified by cisplatin or [PtCl(dien)]Cl, respectively, at r b = 0.005; lanes, cisPt2 and dienPt2, DNA modified by cisplatin or [PtCl(dien)]Cl, respectively, at r b = 0.008; lanes C, G, T, A, chain-terminated marker DNAs (note that these dideoxy sequencing lanes give the sequence complementary to the template strand). The numbers correspond to the nucleotide sequence numbering of part B. Left panel, mapping of the Pt adducts formed on the top strand of the 161-bp fragment; right panel, mapping of the Pt adducts formed on the bottom strand of the 161-bp fragment. ( B ) Schematic diagram showing a portion of the sequence used to monitor inhibition of DNA synthesis on the template containing adducts of cisplatin or [PtCl(dien)]Cl. Solid circles indicate major stop signals from part A, lane cisPt2, open circles indicate major stop signals from part A, lane dienPt2. The numbering of the nucleotides in this scheme corresponds to the cleavage sites of top1 ( Figure 4 ). Nucleotides 37, 70, 92, 97 and 119 correspond respectively to nucleotides 569, 602, 624, 629 and 741 on the pBluescript SK(−) phagemid nucleotide sequence map.

    Techniques Used: Sequencing, Inhibition, DNA Synthesis, Modification, Amplification, Marker

    9) Product Images from "DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis"

    Article Title: DNA Methylation Impacts Gene Expression and Ensures Hypoxic Survival of Mycobacterium tuberculosis

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003419

    MamA is a DNA methyltransferase that protects lppC from endonucleolytic cleavage. (A) Southern blotting strategy to assess the status of a PvuII site near the 3′ end of lppC . Genomic DNA was digested with PvuII and analyzed by Southern blot with a probe hybridizing as shown. Fully cleaved DNA generates a 1.8 kb product, while protected DNA produces a 2.1 kb product. (B) DNA from the vaccine strain M. bovis BCG and from M. tuberculosis strains of the Euro-American lineage (Erdmann and CDC1551) is partially protected from PvuII cleavage while DNA from a Beijing lineage strain (HN878) is not. (C) Genetic deletion of mamA abrogates protection of lppC , while deletion of hsdM does not affect protection. (D) Protection is restored by complementation of a ΔmamA strain with an ectopic copy of mamA , but not by empty vector or mamA E270A . (E) Sequence context of the assayed PvuII site. Underlined bases are predicted to block PvuII if methylated.
    Figure Legend Snippet: MamA is a DNA methyltransferase that protects lppC from endonucleolytic cleavage. (A) Southern blotting strategy to assess the status of a PvuII site near the 3′ end of lppC . Genomic DNA was digested with PvuII and analyzed by Southern blot with a probe hybridizing as shown. Fully cleaved DNA generates a 1.8 kb product, while protected DNA produces a 2.1 kb product. (B) DNA from the vaccine strain M. bovis BCG and from M. tuberculosis strains of the Euro-American lineage (Erdmann and CDC1551) is partially protected from PvuII cleavage while DNA from a Beijing lineage strain (HN878) is not. (C) Genetic deletion of mamA abrogates protection of lppC , while deletion of hsdM does not affect protection. (D) Protection is restored by complementation of a ΔmamA strain with an ectopic copy of mamA , but not by empty vector or mamA E270A . (E) Sequence context of the assayed PvuII site. Underlined bases are predicted to block PvuII if methylated.

    Techniques Used: Southern Blot, Plasmid Preparation, Sequencing, Blocking Assay, Methylation

    10) Product Images from "The Transcriptional Enhancer of the Pea Plastocyanin Gene Associates with the Nuclear Matrix and Regulates Gene Expression through Histone Acetylation"

    Article Title: The Transcriptional Enhancer of the Pea Plastocyanin Gene Associates with the Nuclear Matrix and Regulates Gene Expression through Histone Acetylation

    Journal: The Plant Cell

    doi: 10.1105/tpc.011825

    Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with PvuII, MfeI, and SacI to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.
    Figure Legend Snippet: Association of the PetE Enhancer Region with Nuclear Matrices in Isolated Nuclei. Nuclei were isolated from E-P-GUS or P-GUS seedlings, extracted with LIS, and digested with PvuII, MfeI, and SacI to separate the enhancer, the uidA coding region, and the nos 3′ region into individual fragments. The nuclear matrices were collected by centrifugation, and DNA was isolated from the pellet (P) and supernatant (S) fractions and analyzed by semiquantitative PCR. Total represents a sample of the digestion mixture taken before centrifugation. The enhancer and promoter regions of PetE were amplified with primer pairs y6 and y13, the uidA coding region was amplified with primer pairs G1 and G2, and the PetE promoter region was amplified with primer pairs c2 and y13.

    Techniques Used: Isolation, Centrifugation, Polymerase Chain Reaction, Amplification

    11) Product Images from "The roles of hox 13 genes in newt limb development and regeneration"

    Article Title: The roles of hox 13 genes in newt limb development and regeneration

    Journal: bioRxiv

    doi: 10.1101/789180

    Genotype analysis using amplicons. (A and B) Wt and M indicate data from a wild type animal and size marker, respectively. (A) Genotypes of acd crispants were analyzed using restriction enzyme for amplicons. Digestion by PvuII was examined in PCR products of acd crispants. Data of representative 7 animals is shown. Numbers above gel photos represent individual identification numbers. “-” and “+” indicate no treatment and treatment with PvuII, respectively. Arrows and arrowheads show positions of undigested and digested amplicons, respectively. (B) Amplicons of ad crispants were analyzed by gel electrophoresis. Data of representative 15 animals is shown. Numbers above gel photos represent individual identification numbers. Arrows indicate positions of an amplicon from a wild type animal.
    Figure Legend Snippet: Genotype analysis using amplicons. (A and B) Wt and M indicate data from a wild type animal and size marker, respectively. (A) Genotypes of acd crispants were analyzed using restriction enzyme for amplicons. Digestion by PvuII was examined in PCR products of acd crispants. Data of representative 7 animals is shown. Numbers above gel photos represent individual identification numbers. “-” and “+” indicate no treatment and treatment with PvuII, respectively. Arrows and arrowheads show positions of undigested and digested amplicons, respectively. (B) Amplicons of ad crispants were analyzed by gel electrophoresis. Data of representative 15 animals is shown. Numbers above gel photos represent individual identification numbers. Arrows indicate positions of an amplicon from a wild type animal.

    Techniques Used: Marker, Polymerase Chain Reaction, Nucleic Acid Electrophoresis, Amplification

    12) Product Images from "Identification of Somatic Mitochondrial DNA Mutations, Heteroplasmy, and Increased Levels of Catenanes in Tumor Specimens Obtained from Three Endometrial Cancer Patients"

    Article Title: Identification of Somatic Mitochondrial DNA Mutations, Heteroplasmy, and Increased Levels of Catenanes in Tumor Specimens Obtained from Three Endometrial Cancer Patients

    Journal: Life

    doi: 10.3390/life12040562

    mtDNA copy number is altered in patient 2 and 3 tumors relative to peri-normal controls. BamHI (B) or BamHI and PvuII (B/P) digested tissue-extracted DNA samples were analyzed using Southern blotting and non-radioactive probe hybridization. The blots were simultaneously probed with the digoxigenin (DIG)-labeled 18S nDNA probe (2.2 kb band) and the mtDNA-specific probe (16.6 kb band). As previously described, bands were quantitated using the open-source image-processing package Fiji [ 32 , 34 ]. As the patient 1 A14260G SNP removes the BamHI cut site, PvuII and BamHI double digests were used to cut both the 18S and mtDNA loci. On the left-hand side blot, patient 1 samples digested with only BamHI are shown alongside patient 2 and 3 samples, but as the mtDNA banding patterns are different from the other samples, they were not used to quantitate mtDNA content. As the BamHI/PvuII digested patient 1 peri-normal DNA extract contained the highest average mtDNA to nDNA values, this sample was set to 100%, and the others were normalized to it. N, peri-normal; T, tumor; P1, patient 1; P2, patient 2; P3, patient 3. Significant differences between normal and tumor mtDNA contents were determined using t -tests, n = 4 for each sample set (see the graph below the blots; a representative blot is shown for each patient with n = 2 lanes shown for each sample, N and T); ****, p
    Figure Legend Snippet: mtDNA copy number is altered in patient 2 and 3 tumors relative to peri-normal controls. BamHI (B) or BamHI and PvuII (B/P) digested tissue-extracted DNA samples were analyzed using Southern blotting and non-radioactive probe hybridization. The blots were simultaneously probed with the digoxigenin (DIG)-labeled 18S nDNA probe (2.2 kb band) and the mtDNA-specific probe (16.6 kb band). As previously described, bands were quantitated using the open-source image-processing package Fiji [ 32 , 34 ]. As the patient 1 A14260G SNP removes the BamHI cut site, PvuII and BamHI double digests were used to cut both the 18S and mtDNA loci. On the left-hand side blot, patient 1 samples digested with only BamHI are shown alongside patient 2 and 3 samples, but as the mtDNA banding patterns are different from the other samples, they were not used to quantitate mtDNA content. As the BamHI/PvuII digested patient 1 peri-normal DNA extract contained the highest average mtDNA to nDNA values, this sample was set to 100%, and the others were normalized to it. N, peri-normal; T, tumor; P1, patient 1; P2, patient 2; P3, patient 3. Significant differences between normal and tumor mtDNA contents were determined using t -tests, n = 4 for each sample set (see the graph below the blots; a representative blot is shown for each patient with n = 2 lanes shown for each sample, N and T); ****, p

    Techniques Used: Southern Blot, Hybridization, Labeling

    13) Product Images from "Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity"

    Article Title: Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity

    Journal: BMC Biochemistry

    doi: 10.1186/s12858-015-0041-x

    PCR-based lacZα -complementation system used to determine the fidelity of HIV RT. (A) An overview of the procedure used to assess polymerase fidelity is presented. RNA is represented by broken lines and DNA is represented by solid line. Primers have arrowheads at the 3′ end. The ~760 nt template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3′ and 5′ ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. The filled box at the bottom of the figure is the 115 base region of the lacZ α gene that was scored in the assay. Details for specific steps are provided under Materials and Methods. (B) Plasmid pBSM13ΔPvuII 1146 , is shown. Relevant sites on the plasmid are indicated and numbering is based on the parent plasmid (pBSM13+ (Stratagene)). (C) The nt and amino acid sequence for the 115 base region of the lacZ α gene that was scored in the assay is shown. Both strands of the DNA plasmid are shown since HIV RT synthesis was performed in both directions (see Figure 2A). A line is drawn above the 92 nts that are in the detectable area for substitution mutations while frameshifts can be detected over the entire 115 nt region. Based on a previous cataloging of mutations in this gene [ 51 ], the assay can detect 116 different substitutions (33.6% of the 345 possible substitutions in the 115 nt sequence) and 100% of the frameshift mutations.
    Figure Legend Snippet: PCR-based lacZα -complementation system used to determine the fidelity of HIV RT. (A) An overview of the procedure used to assess polymerase fidelity is presented. RNA is represented by broken lines and DNA is represented by solid line. Primers have arrowheads at the 3′ end. The ~760 nt template RNA used as the initial template for HIV RT RNA-directed DNA synthesis is shown at the top with the 3′ and 5′ ends indicated. The positions of PvuII and EcoRI restriction sites are indicated for reference to the vector. The filled box at the bottom of the figure is the 115 base region of the lacZ α gene that was scored in the assay. Details for specific steps are provided under Materials and Methods. (B) Plasmid pBSM13ΔPvuII 1146 , is shown. Relevant sites on the plasmid are indicated and numbering is based on the parent plasmid (pBSM13+ (Stratagene)). (C) The nt and amino acid sequence for the 115 base region of the lacZ α gene that was scored in the assay is shown. Both strands of the DNA plasmid are shown since HIV RT synthesis was performed in both directions (see Figure 2A). A line is drawn above the 92 nts that are in the detectable area for substitution mutations while frameshifts can be detected over the entire 115 nt region. Based on a previous cataloging of mutations in this gene [ 51 ], the assay can detect 116 different substitutions (33.6% of the 345 possible substitutions in the 115 nt sequence) and 100% of the frameshift mutations.

    Techniques Used: Polymerase Chain Reaction, DNA Synthesis, Plasmid Preparation, Sequencing

    14) Product Images from "Defining characteristics of Tn5 Transposase non-specific DNA binding"

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl179

    Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.
    Figure Legend Snippet: Single ES substrates of differing lengths are cleaved with variable rate constants. ( A ) A partial restriction map of the plasmid (pWSR6103) used to create substrates for in vitro transposition reactions is shown. The Tnp ES is represented as a black arrow. This plasmid was digested with PflMI and either PvuII, BglI, NarI, NdeI, AatII or XmnI to create substrates varying in size from 485 to 1183 bp. Each restriction fragment contained 395 bp of transposon (Tn) DNA and varying lengths of donor backbone (dbb) DNA as shown. The location of the transposon ES in each substrate is marked with a black arrow. ( B ) A schematic of the in vitro transposition reactions with single-ended substrates is shown. Each substrate DNA was incubated (together with non-specific DNA remaining from the restriction digest) with Tnp and MgAc at 37°C. Time points were taken from 0 to 8 h. Following PEC formation, the substrate was cleaved into two products, the dbb and Tn DNA. In this figure, the single ended substrate DNA is shown as two parallel lines containing a transposon ES (gray box). The cleavage site is marked with +1. The non-specific DNA remaining from the restriction digest is shown as linear double stranded DNA. Both product DNAs are appropriately labeled and other reaction components are described as in Figure 4 . ( C ) Each time point was run on an appropriate agarose gel to separate the full-length, unreacted substrate from the dbb and Tn DNA products. In this representative gel of the 555 bp substrate, time points are shown in lanes 3–13 and DNA size markers are shown in lanes 1 and 2. The substrate, dbb and Tn DNAs are represented as in (B). ( D ) The percentage of substrates cleaved was determined for each time point as described in the Materials and Methods. The mean percentage cleaved at each time point was calculated from at least three independent experiments and was then plotted (together with error bars representing the standard error) versus time and the data were fit to a one-phase exponential equation. The plot shown here represents data for the 555 bp substrate. In vitro transposition reactions and analysis were performed in this fashion for each of the six single end substrates. ( E ) k obs,cleavage and the standard error (SE) of this value were calculated from the fits described in (D). These are shown for each of the six substrates tested. ( F ) To better visualize the effect of substrate length on k obs,cleavage , k obs,cleavage was plotted versus substrate length for each substrate. The error bars represent the standard error of k obs,cleavage for each substrate.

    Techniques Used: Plasmid Preparation, In Vitro, Incubation, Labeling, Agarose Gel Electrophoresis

    15) Product Images from "Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization"

    Article Title: Micromanipulation of prophase I chromosomes from mouse spermatocytes reveals high stiffness and gel-like chromatin organization

    Journal: Communications Biology

    doi: 10.1038/s42003-020-01265-w

    Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched ( a ), and chromosomes following enzyme treatments ( b – d ). Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 µm. b Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG˅CTG). c Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG˅CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). d Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). e Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes ( N = 10) and a 1 ± 4% weakening of meiotic chromosomes ( N = 10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes ( N = 4) and 70 ± 9% reduction in stiffness for meiotic chromosomes ( N = 4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes ( N = 4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 µm.
    Figure Legend Snippet: Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched ( a ), and chromosomes following enzyme treatments ( b – d ). Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 µm. b Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG˅CTG). c Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG˅CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). d Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). e Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes ( N = 10) and a 1 ± 4% weakening of meiotic chromosomes ( N = 10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes ( N = 4) and 70 ± 9% reduction in stiffness for meiotic chromosomes ( N = 4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes ( N = 4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 µm.

    Techniques Used: Transferring, Isolation, Sequencing

    Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched ( a ), and chromosomes following enzyme treatments ( b – d ). Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 µm. b Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG˅CTG). c Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG˅CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). d Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). e Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes ( N = 10) and a 1 ± 4% weakening of meiotic chromosomes ( N = 10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes ( N = 4) and 70 ± 9% reduction in stiffness for meiotic chromosomes ( N = 4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes ( N = 4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 µm.
    Figure Legend Snippet: Mitotic and meiotic chromosomes have a contiguous DNA connection, which is dissolved by 4 bp restriction enzymes, but only weakened by 6 bp restriction enzymes. Image pairs show pipette positions untreated (native isolated) chromosomes when relaxed and stretched ( a ), and chromosomes following enzyme treatments ( b – d ). Vertical blue lines mark positions of force pipettes. Force pipette deflection by pulling (horizontal blue lines) indicates mechanical connection; no movement (no horizontal blue line) indicates no mechanical connection. Red notches mark positions of stiff pipettes. Bars are 5 µm. b Both mitotic and meiotic chromosomes were weakened, but not fully digested after treatment with PvuII (cut sequence CAG˅CTG). c Both mitotic and meiotic chromosomes lost connectivity after treatment with AluI (cut sequence AG˅CT; for 1 of 4 trials meiotic chromosomes were not fully digested by AluI). d Both mitotic and meiotic chromosomes lost connectivity when treated with MNase (cleaves all DNA sequences). e Quantification of chromosome stretching elasticity after no treatment or after being treated with PvuII, AluI, and MNase. No treatment caused a 13 ± 4% weakening of mitotic chromosomes ( N = 10) and a 1 ± 4% weakening of meiotic chromosomes ( N = 10). PvuII treatment caused a 70 ± 8% reduction in stiffness for MEF chromosomes ( N = 4) and 70 ± 9% reduction in stiffness for meiotic chromosomes ( N = 4). One of four AluI treatments of meiotic chromosomes caused a 90% reduction in stiffness (rather than fully digesting), while AluI treatment digested 4 of 4 mitotic chromosomes. All MNase treatments caused full digestion of mitotic and meiotic chromosomes ( N = 4 in both cases). All averages are reported as mean value ± SEM. Bars are 5 µm.

    Techniques Used: Transferring, Isolation, Sequencing

    16) Product Images from "Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16"

    Article Title: Method for the elucidation of LAMP products captured on lateral flow strips in a point of care test for HPV 16

    Journal: Analytical and Bioanalytical Chemistry

    doi: 10.1007/s00216-020-02702-9

    FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h
    Figure Legend Snippet: FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h

    Techniques Used: Stripping Membranes, Amplification

    Sequencing pvuII -digested LAMP amplicons. a PvuII -digested products were run on a non-denaturing gel and bands A and B were extracted. b The schematic shows the corresponding LAMP amplicon diagram for the sequenced bands A and B
    Figure Legend Snippet: Sequencing pvuII -digested LAMP amplicons. a PvuII -digested products were run on a non-denaturing gel and bands A and B were extracted. b The schematic shows the corresponding LAMP amplicon diagram for the sequenced bands A and B

    Techniques Used: Sequencing, Amplification

    FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h
    Figure Legend Snippet: FAM band comparisons of pvuII -digested and undigested lateral flow strip elutions. Three different representations of the LAMP amplicon products are shown. Gel a and b are LAMP amplicons digested with pvuII (independent of LFS). Gels c , d , f , and g are eluted products from the LFS itself. Furthermore, gels c and d contain uncleaved LFS-LAMP products ( pvuII− ), whereas gels f and g are cleaved LFS-LAMP products ( pvuII+ ). The FAM bands 94–114 (band C) bp and 71–80 bp (b and D) are highlighted as the HPV 16 FAM amplicons. Gel b also shows the previously sequenced bands of 146 (band A) and 92 (band B). The LFS strips that DNA was eluted from are shown in LFS images e and h of Fig. 4. The gels in panels a and b are pvuII -digested LAMP products; the lateral flow strips results are equivalent to the LFS shown in panel h

    Techniques Used: Stripping Membranes, Amplification

    17) Product Images from "Negative Regulation of the Androgen Receptor Gene Through a Primate-Specific Androgen Response Element Present in the 5′ UTR"

    Article Title: Negative Regulation of the Androgen Receptor Gene Through a Primate-Specific Androgen Response Element Present in the 5′ UTR

    Journal: Hormones & Cancer

    doi: 10.1007/s12672-014-0185-y

    ChIP analysis confirms binding of hAR to 5′ UTR ARE . a Line diagram (not to scale) of the hAR 5′ UTR showing the recognition sites of the restriction endonucleases NheI and PvuII used to digest chromatin and plasmid, plus the forward (F) and reverse (R) primers ( solid arrows ) used for ChIP semiquantitative PCR. Oligonucleotides Gen-R and Vect-R are specific for the genomic and plasmid vector sequences, respectively, and the bent arrow indicates the transcriptional start site. b , c and d Representative agarose gels of PCR amplified immunoprecipitated DNA. b LNCaP cells were treated with either vehicle or 10 nM DHT (shown above gel) and ChIP was performed using PG21 anti-hAR antibody. Precipitated genomic DNA was amplified using primers for the ARE in the hAR 5′ UTR ( ARE ), or in the PSA upstream promoter ( PSA-ARE-III ) with DHT treated cells. Lanes : IP input sample, Ig preimmune rabbit IgG, Ab antibody. Charts display values expressed as percentage of input DNA and represent means ± S.D, * p
    Figure Legend Snippet: ChIP analysis confirms binding of hAR to 5′ UTR ARE . a Line diagram (not to scale) of the hAR 5′ UTR showing the recognition sites of the restriction endonucleases NheI and PvuII used to digest chromatin and plasmid, plus the forward (F) and reverse (R) primers ( solid arrows ) used for ChIP semiquantitative PCR. Oligonucleotides Gen-R and Vect-R are specific for the genomic and plasmid vector sequences, respectively, and the bent arrow indicates the transcriptional start site. b , c and d Representative agarose gels of PCR amplified immunoprecipitated DNA. b LNCaP cells were treated with either vehicle or 10 nM DHT (shown above gel) and ChIP was performed using PG21 anti-hAR antibody. Precipitated genomic DNA was amplified using primers for the ARE in the hAR 5′ UTR ( ARE ), or in the PSA upstream promoter ( PSA-ARE-III ) with DHT treated cells. Lanes : IP input sample, Ig preimmune rabbit IgG, Ab antibody. Charts display values expressed as percentage of input DNA and represent means ± S.D, * p

    Techniques Used: Chromatin Immunoprecipitation, Binding Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, Immunoprecipitation

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    New England Biolabs pvuii restriction enzymes
    <t>PvuII</t> R-M system control region. (A) Genetic structure. The three genes specify a <t>DNA</t> m ethyltransferase ( pvuIIM ), r estriction endonuclease ( pvuIIR ) and c ontroller (activator/repressor, pvuIIC ). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) ( 25 ). The two pvuIIM promoters are also shown (leftward bent arrows). The four vertical rectangles represent the C-boxes, which are binding sites for C.PvuII. ( B) The pvuIICR regulatory region sequence showing C-boxes and promoter elements. The nearly palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or O L (operator left) and 2AB or O R (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated by bent arrows. (C) C-box sequence Logos. The Logos represent the subset of C-box regions associated with the subset of C proteins having HRTY in the recognition helix [21 cases, ( 28 )]. The Logo ( 55 ) was generated by the server at http://weblogo.berkeley.edu . The C-box int ra -operator spacers are boxed, and the central TGTA int er -operator spacer is underlined.
    Pvuii Restriction Enzymes, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PvuII R-M system control region. (A) Genetic structure. The three genes specify a DNA m ethyltransferase ( pvuIIM ), r estriction endonuclease ( pvuIIR ) and c ontroller (activator/repressor, pvuIIC ). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) ( 25 ). The two pvuIIM promoters are also shown (leftward bent arrows). The four vertical rectangles represent the C-boxes, which are binding sites for C.PvuII. ( B) The pvuIICR regulatory region sequence showing C-boxes and promoter elements. The nearly palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or O L (operator left) and 2AB or O R (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated by bent arrows. (C) C-box sequence Logos. The Logos represent the subset of C-box regions associated with the subset of C proteins having HRTY in the recognition helix [21 cases, ( 28 )]. The Logo ( 55 ) was generated by the server at http://weblogo.berkeley.edu . The C-box int ra -operator spacers are boxed, and the central TGTA int er -operator spacer is underlined.

    Journal: Nucleic Acids Research

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system

    doi: 10.1093/nar/gkn1010

    Figure Lengend Snippet: PvuII R-M system control region. (A) Genetic structure. The three genes specify a DNA m ethyltransferase ( pvuIIM ), r estriction endonuclease ( pvuIIR ) and c ontroller (activator/repressor, pvuIIC ). The two transcription starts for pvuIICR are identified by rightward bent arrows: from the C-independent weak promoter (thin) and C-dependent strong promoter (thick) ( 25 ). The two pvuIIM promoters are also shown (leftward bent arrows). The four vertical rectangles represent the C-boxes, which are binding sites for C.PvuII. ( B) The pvuIICR regulatory region sequence showing C-boxes and promoter elements. The nearly palindromic operators each contain a pair of C-boxes, designated as boxes 1AB or O L (operator left) and 2AB or O R (operator right). Conserved elements of the stronger, C-dependent promoter are indicated by heavy rectangles, while thinner rectangles indicate the weak C-independent promoter. Transcript starts are indicated by bent arrows. (C) C-box sequence Logos. The Logos represent the subset of C-box regions associated with the subset of C proteins having HRTY in the recognition helix [21 cases, ( 28 )]. The Logo ( 55 ) was generated by the server at http://weblogo.berkeley.edu . The C-box int ra -operator spacers are boxed, and the central TGTA int er -operator spacer is underlined.

    Article Snippet: Equal amounts of DNA (400 ng) digested with 10 u of PvuII restriction enzymes (NEB) were run on 1% agarose TAE gels to compare the methylation status of the substrate plasmid.

    Techniques: Binding Assay, Sequencing, Generated

    In vitro interaction of C.PvuII protein with wild-type and altered C-box regions. ( A ) EMSA reactions were processed as outlined in ‘Materials and methods’ section and shown in Supplementary Data (Figure S4) . Data are shown as % of unshifted DNA versus increasing concentration of added C.PvuII. ‘WT’ refers to the native PvuII C-boxes and spacers. The others all have symmetrized spacers with varied intra-operator spacers. ( B ) EMSA competition assays were performed using 200 nM of C.PvuII and 20 nM of biotin-labeled WT C-box 126-mer (as described in ‘Materials and methods’ section). Competition reactions contained increasing amounts of unlabeled 126-mer DNA fragments (from 1- to 10-fold molar excess). The competitor DNAs contained intra-operator spacers: CAA/CAA (white circles), CAA/CAT (white squares), CAT/CAA (black squares), CAT/CAT (black circles) or no C-boxes (triangles, negative control). Following EMSA and electroblotting, the shifted bands for each reaction were visualized and quantified via chemiluminescent detection of the biotinylated DNA as described in ‘Materials and methods’ section. For clarity symbols are the same as in Figure 3.

    Journal: Nucleic Acids Research

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system

    doi: 10.1093/nar/gkn1010

    Figure Lengend Snippet: In vitro interaction of C.PvuII protein with wild-type and altered C-box regions. ( A ) EMSA reactions were processed as outlined in ‘Materials and methods’ section and shown in Supplementary Data (Figure S4) . Data are shown as % of unshifted DNA versus increasing concentration of added C.PvuII. ‘WT’ refers to the native PvuII C-boxes and spacers. The others all have symmetrized spacers with varied intra-operator spacers. ( B ) EMSA competition assays were performed using 200 nM of C.PvuII and 20 nM of biotin-labeled WT C-box 126-mer (as described in ‘Materials and methods’ section). Competition reactions contained increasing amounts of unlabeled 126-mer DNA fragments (from 1- to 10-fold molar excess). The competitor DNAs contained intra-operator spacers: CAA/CAA (white circles), CAA/CAT (white squares), CAT/CAA (black squares), CAT/CAT (black circles) or no C-boxes (triangles, negative control). Following EMSA and electroblotting, the shifted bands for each reaction were visualized and quantified via chemiluminescent detection of the biotinylated DNA as described in ‘Materials and methods’ section. For clarity symbols are the same as in Figure 3.

    Article Snippet: Equal amounts of DNA (400 ng) digested with 10 u of PvuII restriction enzymes (NEB) were run on 1% agarose TAE gels to compare the methylation status of the substrate plasmid.

    Techniques: In Vitro, Concentration Assay, Labeling, Cellular Antioxidant Activity Assay, Negative Control

    DNA bending by C.PvuII. WT C-boxes ( A ) and O L spacer variant with CAA ( B ). With the indicated restriction enzymes 150-nt DNA fragments (60-ng each) were released, and contain the C-box in the center (EcoRV) or close to either end (MluI or BamHI). C.PvuII (200 ng) was added and EMSA was carried out (as in Figure 3). ( C ) Flexure displacement analysis was as described ( 52 ). The net DNA bending average angle was calculated.

    Journal: Nucleic Acids Research

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system

    doi: 10.1093/nar/gkn1010

    Figure Lengend Snippet: DNA bending by C.PvuII. WT C-boxes ( A ) and O L spacer variant with CAA ( B ). With the indicated restriction enzymes 150-nt DNA fragments (60-ng each) were released, and contain the C-box in the center (EcoRV) or close to either end (MluI or BamHI). C.PvuII (200 ng) was added and EMSA was carried out (as in Figure 3). ( C ) Flexure displacement analysis was as described ( 52 ). The net DNA bending average angle was calculated.

    Article Snippet: Equal amounts of DNA (400 ng) digested with 10 u of PvuII restriction enzymes (NEB) were run on 1% agarose TAE gels to compare the methylation status of the substrate plasmid.

    Techniques: Variant Assay, Cellular Antioxidant Activity Assay

    Testing the two alternative symmetry patterns in the C-box region. ( A ) Two alternative symmetry patterns. The top portion shows the ‘AGTC’ consensus [in blue, ( 33 )] comprising the C-box elements themselves, the actual sequence from PvuII R-M system, and the ‘TATA’ consensus [in yellow, ( 41 )]. The centers of symmetry are shown. The numbers at the right indicate the extent of match to each consensus, and whether a characteristic repression complex forms in vitro at ≤500 nM C.PvuII. The lower portion shows several previously tested C.PvuII operator variants ( 28 ). Magenta shading indicates mutations introduced in each case, in the context of symmetrized C-boxes (underlined positions). ( B ) Binding effects of ‘TATA’ consensus alteration. A series of 126-bp dsDNA-binding targets were prepared by PCR amplification, and included in each binding reaction at 20 nM. The DNAs contained WT or variant C boxes flanked on either side by 50 bp of native PvuII sequence. EMSA reactions with concentrations of C.PvuII increasing from 0–500 nM were processed as outlined in ‘Materials and methods’ section. PvuSym indicates wild-type spacers in the context of symmetrized C-box sequences (see sequence in A). Other variants are in the same sequence context, but with different intra-operator spacers as indicated (O L /O R ). Reactions were resolved on 10% native polyacrylamide gels, and DNA was visualized by staining with ethidium bromide. Numbers at right indicate the expected numbers of bound C.PvuII dimers. ( C ) In vivo titration of intra-operator spacers variants with C.PvuII. Cells, carrying pvuIIC under the control of P BAD , were grown in minimal media with 0.2% glucose and the indicated concentration of arabinose, as described before ( 28 ). Expression from variants (labeled as in B) was measured via transcriptional fusion of the variant C-boxes/P pvuIICR to reporter gene lacZ . Each point represents β-galactosidase specific activity, determined by linear regression of the plot of LacZ activity (modified Miller units) versus optical density of the culture, as previously ( 49 ). Each point represents the slope of a regression from at least 3 points; in all cases R 2 was > 0.97. The beginning of the curve (0), indicates values obtained in glucose with no arabinose. Black diamonds represent LacZ activity from cells with the WT symmetrized PvuII C-boxes with CAT/CAA intra-operator spacers (pIM8). The ‘TATA’ consensus variant (TAT/TAT) is shown as blue circles, and the ‘TATA’-disrupted variant (CGC/CGC) is shown as red diamonds. As negative control vector plasmid with no pvuIIC was used (open circles).

    Journal: Nucleic Acids Research

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system

    doi: 10.1093/nar/gkn1010

    Figure Lengend Snippet: Testing the two alternative symmetry patterns in the C-box region. ( A ) Two alternative symmetry patterns. The top portion shows the ‘AGTC’ consensus [in blue, ( 33 )] comprising the C-box elements themselves, the actual sequence from PvuII R-M system, and the ‘TATA’ consensus [in yellow, ( 41 )]. The centers of symmetry are shown. The numbers at the right indicate the extent of match to each consensus, and whether a characteristic repression complex forms in vitro at ≤500 nM C.PvuII. The lower portion shows several previously tested C.PvuII operator variants ( 28 ). Magenta shading indicates mutations introduced in each case, in the context of symmetrized C-boxes (underlined positions). ( B ) Binding effects of ‘TATA’ consensus alteration. A series of 126-bp dsDNA-binding targets were prepared by PCR amplification, and included in each binding reaction at 20 nM. The DNAs contained WT or variant C boxes flanked on either side by 50 bp of native PvuII sequence. EMSA reactions with concentrations of C.PvuII increasing from 0–500 nM were processed as outlined in ‘Materials and methods’ section. PvuSym indicates wild-type spacers in the context of symmetrized C-box sequences (see sequence in A). Other variants are in the same sequence context, but with different intra-operator spacers as indicated (O L /O R ). Reactions were resolved on 10% native polyacrylamide gels, and DNA was visualized by staining with ethidium bromide. Numbers at right indicate the expected numbers of bound C.PvuII dimers. ( C ) In vivo titration of intra-operator spacers variants with C.PvuII. Cells, carrying pvuIIC under the control of P BAD , were grown in minimal media with 0.2% glucose and the indicated concentration of arabinose, as described before ( 28 ). Expression from variants (labeled as in B) was measured via transcriptional fusion of the variant C-boxes/P pvuIICR to reporter gene lacZ . Each point represents β-galactosidase specific activity, determined by linear regression of the plot of LacZ activity (modified Miller units) versus optical density of the culture, as previously ( 49 ). Each point represents the slope of a regression from at least 3 points; in all cases R 2 was > 0.97. The beginning of the curve (0), indicates values obtained in glucose with no arabinose. Black diamonds represent LacZ activity from cells with the WT symmetrized PvuII C-boxes with CAT/CAA intra-operator spacers (pIM8). The ‘TATA’ consensus variant (TAT/TAT) is shown as blue circles, and the ‘TATA’-disrupted variant (CGC/CGC) is shown as red diamonds. As negative control vector plasmid with no pvuIIC was used (open circles).

    Article Snippet: Equal amounts of DNA (400 ng) digested with 10 u of PvuII restriction enzymes (NEB) were run on 1% agarose TAE gels to compare the methylation status of the substrate plasmid.

    Techniques: Sequencing, In Vitro, Binding Assay, Polymerase Chain Reaction, Amplification, Variant Assay, Staining, In Vivo, Titration, Concentration Assay, Expressing, Labeling, Activity Assay, Modification, Cellular Antioxidant Activity Assay, Negative Control, Plasmid Preparation

    Methylation status of plasmid isolated from cells expressing varied levels of WT M.PvuII or its variants. The complementary strands for the symmetrized C-box region of the PvuII R-M system are shown at the top. The upper strand specifies M.PvuII. The two changes that symmetrize the C-boxes are shown in red, along with the resultant changes in the MTase sequence (N4S/S10R). Substitution of the O L spacer, from CAT to CAA, would result in an additional change (M8L). Escherichia coli TOP10 cells carried two plasmids: pUC × 7PvuII (with seven PvuII sites) and plasmids expressing either WT pvuIIM (pBadMTwt-kan) or one of its variants (MTase N4S/S10R–pBadMT2-kan; MTase N4S/S10R/M8L–pBadMT2-kan) under the arabinose inducible promoter P BAD . After induction with a range of arabinose concentrations, cells were pelleted and plasmid DNA was isolated. The extent of methylation was assessed by digestion with PvuII restriction enzyme. Results ranged from full cleavage (no protection as in control lane 1; asterisk shows the position of highest bands) to no cleavage (complete methylation as in control lane 2). Digests were resolved on 1% agarose gel. Lanes 4 to 8 for each MTase represent equivalent increasing arabinose concentration from 0.01 to 0.2%. The topmost band in lanes 4–8 for each MTase is the pBAD plasmid linearized with XhoI prior to digestion with PvuII indicated by arrow; pUC × 7PvuII lacks a XhoI site. M lane shows DNA markers (1 kB Plus ladder; Invitrogen).

    Journal: Nucleic Acids Research

    Article Title: Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system

    doi: 10.1093/nar/gkn1010

    Figure Lengend Snippet: Methylation status of plasmid isolated from cells expressing varied levels of WT M.PvuII or its variants. The complementary strands for the symmetrized C-box region of the PvuII R-M system are shown at the top. The upper strand specifies M.PvuII. The two changes that symmetrize the C-boxes are shown in red, along with the resultant changes in the MTase sequence (N4S/S10R). Substitution of the O L spacer, from CAT to CAA, would result in an additional change (M8L). Escherichia coli TOP10 cells carried two plasmids: pUC × 7PvuII (with seven PvuII sites) and plasmids expressing either WT pvuIIM (pBadMTwt-kan) or one of its variants (MTase N4S/S10R–pBadMT2-kan; MTase N4S/S10R/M8L–pBadMT2-kan) under the arabinose inducible promoter P BAD . After induction with a range of arabinose concentrations, cells were pelleted and plasmid DNA was isolated. The extent of methylation was assessed by digestion with PvuII restriction enzyme. Results ranged from full cleavage (no protection as in control lane 1; asterisk shows the position of highest bands) to no cleavage (complete methylation as in control lane 2). Digests were resolved on 1% agarose gel. Lanes 4 to 8 for each MTase represent equivalent increasing arabinose concentration from 0.01 to 0.2%. The topmost band in lanes 4–8 for each MTase is the pBAD plasmid linearized with XhoI prior to digestion with PvuII indicated by arrow; pUC × 7PvuII lacks a XhoI site. M lane shows DNA markers (1 kB Plus ladder; Invitrogen).

    Article Snippet: Equal amounts of DNA (400 ng) digested with 10 u of PvuII restriction enzymes (NEB) were run on 1% agarose TAE gels to compare the methylation status of the substrate plasmid.

    Techniques: Methylation, Plasmid Preparation, Isolation, Expressing, Sequencing, Cellular Antioxidant Activity Assay, Agarose Gel Electrophoresis, Concentration Assay

    Quantitative polymerase chain reaction (qPCR) targeting the rep sequence using pSub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats with free ends. ( a ) The minimal differences observed between mean quantification cycles ( C q ) obtained in each experimental condition indicate that the DNA concentrations determined by spectrophotometry are similar. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a .

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    doi: 10.1038/mtm.2016.19

    Figure Lengend Snippet: Quantitative polymerase chain reaction (qPCR) targeting the rep sequence using pSub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats with free ends. ( a ) The minimal differences observed between mean quantification cycles ( C q ) obtained in each experimental condition indicate that the DNA concentrations determined by spectrophotometry are similar. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a .

    Article Snippet: For the preparation of the linearized plasmids, psub201 was digested with either HindIII or PvuII (New England BioLabs, Ipswich, MA) under the conditions determined by the manufacturer.

    Techniques: Real-time Polymerase Chain Reaction, Sequencing, Plasmid Preparation, Spectrophotometry

    Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    doi: 10.1038/mtm.2016.19

    Figure Lengend Snippet: Generation of free ends for the plasmid inverted terminal repeats (ITRs). ( a ) Schematic representation of the plasmid psub201 and the PvuII and HindIII restriction sites. ( b ) Magnification of the plasmid DNA sequences close to the PvuII digestion sites. pEMBL8(+) plasmid backbone (lower case); AAV2-sub201 viral genome (upper case) and PvuII site (CAG/CTG): underlined. ( c ) Separation of undigested and digested plasmid DNA on a 1% agarose gel; supercoiled and linear DNA ladder were used as electrophoresis standards. ( d ) Plasmid DNA purity and concentration measured by spectrophotometry.

    Article Snippet: For the preparation of the linearized plasmids, psub201 was digested with either HindIII or PvuII (New England BioLabs, Ipswich, MA) under the conditions determined by the manufacturer.

    Techniques: Plasmid Preparation, CTG Assay, Agarose Gel Electrophoresis, Electrophoresis, Concentration Assay, Spectrophotometry

    ITR2 quantitative polymerase chain reaction (qPCR) using psub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats (ITRs) with free ends. ( a ) The differences between mean quantification cycles ( C q ) obtained in each experimental condition with an equal amount of plasmid quantified by spectrophotometry are shown. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a ).

    Journal: Molecular Therapy. Methods & Clinical Development

    Article Title: Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR

    doi: 10.1038/mtm.2016.19

    Figure Lengend Snippet: ITR2 quantitative polymerase chain reaction (qPCR) using psub201 plasmid undigested, linearized with HindIII, or digested with PvuII to create inverted terminal repeats (ITRs) with free ends. ( a ) The differences between mean quantification cycles ( C q ) obtained in each experimental condition with an equal amount of plasmid quantified by spectrophotometry are shown. ( b ) r-square, slope, and intersection values of the qPCR tests shown in a ).

    Article Snippet: For the preparation of the linearized plasmids, psub201 was digested with either HindIII or PvuII (New England BioLabs, Ipswich, MA) under the conditions determined by the manufacturer.

    Techniques: Real-time Polymerase Chain Reaction, Plasmid Preparation, Spectrophotometry

    DNA synthesis does not stall during termination A. Cartoon depicting the assay for lacO array synthesis. B. LacR Block-IPTG release was performed on p[ lacO x12]. To measure synthesis within the array, termination intermediates were cut with AflIII and PvuII to liberate the array fragment from the vector. Cleaved products were separated by native gel electrophoresis. Different exposures of array and vector fragments are shown (see methods). C. Array synthesis, vector synthesis, and dissolution were quantified. Means±s.d. are plotted (n=3).

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: DNA synthesis does not stall during termination A. Cartoon depicting the assay for lacO array synthesis. B. LacR Block-IPTG release was performed on p[ lacO x12]. To measure synthesis within the array, termination intermediates were cut with AflIII and PvuII to liberate the array fragment from the vector. Cleaved products were separated by native gel electrophoresis. Different exposures of array and vector fragments are shown (see methods). C. Array synthesis, vector synthesis, and dissolution were quantified. Means±s.d. are plotted (n=3).

    Article Snippet: To monitor DNA synthesis within a lacO array , 0.25–1.0 ng/µl of purified DNA was incubated in Buffer 3.1 with 0.2 units/µl PvuII and 0.2 units/µl AflIII (New England BioLabs) at 37°C for 1 hour.

    Techniques: DNA Synthesis, Blocking Assay, Plasmid Preparation, Nucleic Acid Electrophoresis

    Sequence-specific termination can be induced at a LacR array (A) To investigate whether a LacR array blocks replication forks, a plasmid containing a tandem array of 16 lac operator ( lacO ) sequences, p[ lacO x16], was incubated with buffer or LacR and then replicated in egg extract containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with XmnI (far left cartoon) and separated according to size and shape by 2-dimensional gel electrophoresis (see schematic of 2-D gel). As replication neared completion at 4.5 minutes, mainly linear molecules were produced in the presence of buffer (orange arrowhead). In contrast, in the presence of LacR, a discrete spot appeared on the Double Y arc (blue arrowhead), demonstrating that converging replication forks accumulate at a specific locus on p[ lacO x16]. These data indicate that 16 copies of LacR block replication forks. (B–F) To test whether the double-Y structures observed in (A) arose from replication forks stalling at the outer edges of the lacO array, we tested whether LacR specifically inhibited replication of lacO sequences. To this end, p[ lacOx16 ] (C) and the parental plasmid lacking lacO repeats, p[ empty ] (B), were incubated in the presence of buffer or LacR and replicated using Xenopus egg extracts containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with AflIII and PvuII to release the 2354 bp plasmid backbone (B and C) and a 294 bp control fragment from p[ empty ] (B) or a 794 bp lacO fragment from p[ lacO x16] (C). The plasmid backbone and the respective inserts were separated on a native gel and detected by autoradiography (D). A longer exposure of the small fragments is shown, since they are less intense than the large fragments. The results in panel (D) were quantified in (E) and (F). Importantly, LacR specifically inhibited replication of the lacO -containing fragment in p[ lacO x16] (F, blue circles) but not the control fragment in p[ empty ] (E, green circles). We conclude that LacR prevents replication of the lacO array and that the double-Y’s in (A) represent forks converged on the outer edges of the array. Importantly, synthesis within the 2354 bp backbone fragment (F, orange circles) of p[ lacO x16] was not inhibited in the presence of LacR, indicating that no global structural changes occur that inhibit replication.

    Journal: Nature

    Article Title: The mechanism of DNA replication termination in vertebrates

    doi: 10.1038/nature14887

    Figure Lengend Snippet: Sequence-specific termination can be induced at a LacR array (A) To investigate whether a LacR array blocks replication forks, a plasmid containing a tandem array of 16 lac operator ( lacO ) sequences, p[ lacO x16], was incubated with buffer or LacR and then replicated in egg extract containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with XmnI (far left cartoon) and separated according to size and shape by 2-dimensional gel electrophoresis (see schematic of 2-D gel). As replication neared completion at 4.5 minutes, mainly linear molecules were produced in the presence of buffer (orange arrowhead). In contrast, in the presence of LacR, a discrete spot appeared on the Double Y arc (blue arrowhead), demonstrating that converging replication forks accumulate at a specific locus on p[ lacO x16]. These data indicate that 16 copies of LacR block replication forks. (B–F) To test whether the double-Y structures observed in (A) arose from replication forks stalling at the outer edges of the lacO array, we tested whether LacR specifically inhibited replication of lacO sequences. To this end, p[ lacOx16 ] (C) and the parental plasmid lacking lacO repeats, p[ empty ] (B), were incubated in the presence of buffer or LacR and replicated using Xenopus egg extracts containing [α- 32 P]dATP. Radiolabelled replication intermediates were cleaved with AflIII and PvuII to release the 2354 bp plasmid backbone (B and C) and a 294 bp control fragment from p[ empty ] (B) or a 794 bp lacO fragment from p[ lacO x16] (C). The plasmid backbone and the respective inserts were separated on a native gel and detected by autoradiography (D). A longer exposure of the small fragments is shown, since they are less intense than the large fragments. The results in panel (D) were quantified in (E) and (F). Importantly, LacR specifically inhibited replication of the lacO -containing fragment in p[ lacO x16] (F, blue circles) but not the control fragment in p[ empty ] (E, green circles). We conclude that LacR prevents replication of the lacO array and that the double-Y’s in (A) represent forks converged on the outer edges of the array. Importantly, synthesis within the 2354 bp backbone fragment (F, orange circles) of p[ lacO x16] was not inhibited in the presence of LacR, indicating that no global structural changes occur that inhibit replication.

    Article Snippet: To monitor DNA synthesis within a lacO array , 0.25–1.0 ng/µl of purified DNA was incubated in Buffer 3.1 with 0.2 units/µl PvuII and 0.2 units/µl AflIII (New England BioLabs) at 37°C for 1 hour.

    Techniques: Sequencing, Plasmid Preparation, Incubation, Nucleic Acid Electrophoresis, Produced, Blocking Assay, Autoradiography

    Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.

    Journal: Scientific Reports

    Article Title: Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)

    doi: 10.1038/s41598-017-07231-7

    Figure Lengend Snippet: Consensus sequence of the SSUsat monomers from Spisula subtruncata . On the basis of the DNA sequences of the recovered SSUsat monomers from Spisula subtruncata genome, a consensus sequence was derived. Restriction sites for Msp I/ Hpa II, Pvu II and Taq I are underlined. Green and red arrows indicate the positions of PCR primers used for SSUsat amplification in related species.

    Article Snippet: Southern and dot blot hybridisation Genomic DNAs were digested with Hae III (Fermentas), Hpa II (Fermentas), Msp I (Promega), Pvu II (New England Biolabs) and Taq I (Roche).

    Techniques: Sequencing, Derivative Assay, Polymerase Chain Reaction, Amplification

    Southern blot hybridisation analysis of SSUsat repeats in Spisula subtruncata . Agarose gel electrophoresis of Pvu II and Hae III digested genomic DNA of Spisula subtruncata showing ladder-like multimer bands of a 315 bp monomer unit ( a ). After being Southern blotted on a nitrocellulose membrane, the electrophoresed DNA was hybridised with an SSUsat monomer probe yielding identical ladder-like multimer bands of a 315 bp monomer unit ( b ).

    Journal: Scientific Reports

    Article Title: Methylation profile of a satellite DNA constituting the intercalary G+C-rich heterochromatin of the cut trough shell Spisula subtruncata (Bivalvia, Mactridae)

    doi: 10.1038/s41598-017-07231-7

    Figure Lengend Snippet: Southern blot hybridisation analysis of SSUsat repeats in Spisula subtruncata . Agarose gel electrophoresis of Pvu II and Hae III digested genomic DNA of Spisula subtruncata showing ladder-like multimer bands of a 315 bp monomer unit ( a ). After being Southern blotted on a nitrocellulose membrane, the electrophoresed DNA was hybridised with an SSUsat monomer probe yielding identical ladder-like multimer bands of a 315 bp monomer unit ( b ).

    Article Snippet: Southern and dot blot hybridisation Genomic DNAs were digested with Hae III (Fermentas), Hpa II (Fermentas), Msp I (Promega), Pvu II (New England Biolabs) and Taq I (Roche).

    Techniques: Southern Blot, Hybridization, Agarose Gel Electrophoresis