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    Structured Review

    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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    1) Product Images from "Tuning the relative affinities for activating and repressing operators of a temporally regulated restriction-modification system"

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1010

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

    Techniques Used: 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).
    Figure Legend 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).

    Techniques Used: 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).
    Figure Legend 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).

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

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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
    https://www.bioz.com/result/pvuii restriction enzymes/product/New England Biolabs
    Average 94 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pvuii restriction enzymes - by Bioz Stars, 2022-07
    94/100 stars
      Buy from Supplier

    95
    New England Biolabs pvuii restriction enzyme
    ATM activity and F-actin polymerization are required for chromosomal stability after induction of DSBs <t>HTori-3</t> cells were treated for 4 h with ATM kinase inhibitor KU55933 or DNA-PK inhibitor NU7441 immediately after electroporation with <t>PvuII</t> restriction endonuclease. (A) Graph showing the effect of ATM and DNA-PK inhibition on the formation of RET/PTC rearrangements in HTori-3 cells with and without PvuII-induced DNA DSBs. (B) Graph showing that F-actin polymerization deficiency after introduction of G13R-mutated actin increased the frequency of RET/PTC rearrangements in HTori-3 cells treated with PvuII endonuclease. Data are presented as means with 95%CI; * , P
    Pvuii Restriction Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/pvuii restriction enzyme/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    pvuii restriction enzyme - by Bioz Stars, 2022-07
    95/100 stars
      Buy from Supplier

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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

    Homologous recombination between vector and insert generated by restriction endonucleases. (A) The pNatMX was cleaved with the PvuII endonuclease generating the natMX fragment of 1469 bp. The pUC19 was prepared by digestion with the EcoRI and HindIII restriction enzymes, resulting in a 2639 bp linear plasmid. Homologous recombination between the natMX and pUC19 fragments generated the pUC19Nat plasmid. (B) Agarose gel electrophoresis after gel purification of the fragments natMX and pUC19. (C) The counting of colonies after transformation of the vector alone and co-transformation of the pUC19 plus the fragment natMX. (D) Colony PCR screening confirmed 100% positive cloning events. Abbreviations are as described in Fig. 2 .

    Journal: PLoS ONE

    Article Title: Optimal Cloning of PCR Fragments by Homologous Recombination in Escherichia coli

    doi: 10.1371/journal.pone.0119221

    Figure Lengend Snippet: Homologous recombination between vector and insert generated by restriction endonucleases. (A) The pNatMX was cleaved with the PvuII endonuclease generating the natMX fragment of 1469 bp. The pUC19 was prepared by digestion with the EcoRI and HindIII restriction enzymes, resulting in a 2639 bp linear plasmid. Homologous recombination between the natMX and pUC19 fragments generated the pUC19Nat plasmid. (B) Agarose gel electrophoresis after gel purification of the fragments natMX and pUC19. (C) The counting of colonies after transformation of the vector alone and co-transformation of the pUC19 plus the fragment natMX. (D) Colony PCR screening confirmed 100% positive cloning events. Abbreviations are as described in Fig. 2 .

    Article Snippet: Restriction endonucleases PvuII, EcoRI and HindIII were purchased from New England Biolabs (Ipswich, MA, USA) and used according to manufacturer’s instructions.

    Techniques: Homologous Recombination, Plasmid Preparation, Generated, Agarose Gel Electrophoresis, Gel Purification, Transformation Assay, Polymerase Chain Reaction, Clone Assay

    ATM activity and F-actin polymerization are required for chromosomal stability after induction of DSBs HTori-3 cells were treated for 4 h with ATM kinase inhibitor KU55933 or DNA-PK inhibitor NU7441 immediately after electroporation with PvuII restriction endonuclease. (A) Graph showing the effect of ATM and DNA-PK inhibition on the formation of RET/PTC rearrangements in HTori-3 cells with and without PvuII-induced DNA DSBs. (B) Graph showing that F-actin polymerization deficiency after introduction of G13R-mutated actin increased the frequency of RET/PTC rearrangements in HTori-3 cells treated with PvuII endonuclease. Data are presented as means with 95%CI; * , P

    Journal: Oncotarget

    Article Title: Nuclear myosin/actin-motored contact between homologous chromosomes is initiated by ATM kinase and homology-directed repair proteins at double-strand DNA breaks to suppress chromosome rearrangements

    doi: 10.18632/oncotarget.24434

    Figure Lengend Snippet: ATM activity and F-actin polymerization are required for chromosomal stability after induction of DSBs HTori-3 cells were treated for 4 h with ATM kinase inhibitor KU55933 or DNA-PK inhibitor NU7441 immediately after electroporation with PvuII restriction endonuclease. (A) Graph showing the effect of ATM and DNA-PK inhibition on the formation of RET/PTC rearrangements in HTori-3 cells with and without PvuII-induced DNA DSBs. (B) Graph showing that F-actin polymerization deficiency after introduction of G13R-mutated actin increased the frequency of RET/PTC rearrangements in HTori-3 cells treated with PvuII endonuclease. Data are presented as means with 95%CI; * , P

    Article Snippet: For each experiment 2×106 of HTori-3 cells were electroporated with 25U of PvuII restriction enzyme (New England Bio Labs, Ipswick, MA, USA) at 50 mC charge (400 V and 125 mF) using Gene Pulser Xcell Electroporator (Bio-Rad) as previously described [ ].

    Techniques: Activity Assay, Electroporation, Inhibition

    ATM activity and F-actin polymerization are required for chromosomal stability after induction of DSBs HTori-3 cells were treated for 4 h with ATM kinase inhibitor KU55933 or DNA-PK inhibitor NU7441 immediately after electroporation with PvuII restriction endonuclease. (A) Graph showing the effect of ATM and DNA-PK inhibition on the formation of RET/PTC rearrangements in HTori-3 cells with and without PvuII-induced DNA DSBs. (B) Graph showing that F-actin polymerization deficiency after introduction of G13R-mutated actin increased the frequency of RET/PTC rearrangements in HTori-3 cells treated with PvuII endonuclease. Data are presented as means with 95%CI; * , P

    Journal: Oncotarget

    Article Title: Nuclear myosin/actin-motored contact between homologous chromosomes is initiated by ATM kinase and homology-directed repair proteins at double-strand DNA breaks to suppress chromosome rearrangements

    doi: 10.18632/oncotarget.24434

    Figure Lengend Snippet: ATM activity and F-actin polymerization are required for chromosomal stability after induction of DSBs HTori-3 cells were treated for 4 h with ATM kinase inhibitor KU55933 or DNA-PK inhibitor NU7441 immediately after electroporation with PvuII restriction endonuclease. (A) Graph showing the effect of ATM and DNA-PK inhibition on the formation of RET/PTC rearrangements in HTori-3 cells with and without PvuII-induced DNA DSBs. (B) Graph showing that F-actin polymerization deficiency after introduction of G13R-mutated actin increased the frequency of RET/PTC rearrangements in HTori-3 cells treated with PvuII endonuclease. Data are presented as means with 95%CI; * , P

    Article Snippet: For each experiment 2×106 of HTori-3 cells were electroporated with 25U of PvuII restriction enzyme (New England Bio Labs, Ipswick, MA, USA) at 50 mC charge (400 V and 125 mF) using Gene Pulser Xcell Electroporator (Bio-Rad) as previously described [ ].

    Techniques: Activity Assay, Electroporation, Inhibition