restriction endonucleases ecorv Search Results


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  • 99
    New England Biolabs restriction endonucleases ecorv
    Signature of Aberrant DNA Methylation in MYC -Induced T cell Lymphomas (A) Schematic representation of transgenes in MYC -induced T cell lymphoma model used in this study. (B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy <t>NotI</t> fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events. (C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP EGFP mice are also shown (see text and Figure 2 ). (D) Southern blot analysis of control and tumor cells using the 2C51 <t>NotI-EcoRV</t> DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites. (E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides. (F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y -axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.
    Restriction Endonucleases Ecorv, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 247 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher restriction endonucleases eco32i
    Signature of Aberrant DNA Methylation in MYC -Induced T cell Lymphomas (A) Schematic representation of transgenes in MYC -induced T cell lymphoma model used in this study. (B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy <t>NotI</t> fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events. (C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP EGFP mice are also shown (see text and Figure 2 ). (D) Southern blot analysis of control and tumor cells using the 2C51 <t>NotI-EcoRV</t> DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites. (E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides. (F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y -axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.
    Restriction Endonucleases Eco32i, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    TaKaRa restriction endonuclease ecorv
    Schematic of the recombinant plasmid pCMV6-truncated-rVegfr2. Restriction digestion of the two <t>EcoRV</t> restriction sites in the plasmid pCMV6-rVegfr2, which encodes the extracellular reserved VEGFR2 Ig 1–3 and 5 regions. The recombinant plasmid pCMV6-truncated-rVegfr2 was reconstructed after self-ligation of the plasmid vector with <t>T4</t> DNA ligase. rVegfr2: rat-Vegf-receptor2.
    Restriction Endonuclease Ecorv, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher ecorv restriction endonuclease
    Schematic of the recombinant plasmid pCMV6-truncated-rVegfr2. Restriction digestion of the two <t>EcoRV</t> restriction sites in the plasmid pCMV6-rVegfr2, which encodes the extracellular reserved VEGFR2 Ig 1–3 and 5 regions. The recombinant plasmid pCMV6-truncated-rVegfr2 was reconstructed after self-ligation of the plasmid vector with <t>T4</t> DNA ligase. rVegfr2: rat-Vegf-receptor2.
    Ecorv Restriction Endonuclease, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ecorv restriction endonuclease
    pH dependence of the <t>EcoRV</t> specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site <t>DNA</t> fragment, and the nonspecific oligonucleotide competitor
    Ecorv Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HiMedia Laboratories ecorv restriction endonuclease
    pH dependence of the <t>EcoRV</t> specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site <t>DNA</t> fragment, and the nonspecific oligonucleotide competitor
    Ecorv Restriction Endonuclease, supplied by HiMedia Laboratories, used in various techniques. Bioz Stars score: 91/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega ecorv restriction endonucleases
    pH dependence of the <t>EcoRV</t> specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site <t>DNA</t> fragment, and the nonspecific oligonucleotide competitor
    Ecorv Restriction Endonucleases, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs ecorv hf restriction endonuclease
    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with <t>EcoRV</t> and <t>SpeI</t> that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
    Ecorv Hf Restriction Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 128 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Promega restriction endonuclease eco rv
    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with <t>EcoRV</t> and <t>SpeI</t> that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
    Restriction Endonuclease Eco Rv, supplied by Promega, used in various techniques. Bioz Stars score: 85/100, based on 61 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Fisher Scientific restriction enzyme ecorv
    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with <t>EcoRV</t> and <t>SpeI</t> that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.
    Restriction Enzyme Ecorv, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 88/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TaKaRa ecorv
    Conserved Domains in Karma . (A) Karma structure. Open boxes depict the two open reading frames of Karma (ORF1 and ORF2). ORF2 contains the endonuclease ( en ) and reverse transcriptase ( rt ) domains. Black boxes depict Cys-rich motifs. Vertical lines above the boxes depict the sites of HpaII and MspI cleavage, and vertical lines below the boxes depict the sites of <t>EcoRV</t> cleavage. Hatched boxes depict probes A (1317 bp) and B (1498 bp) used in <t>DNA</t> gel blot analysis. Bar = 1 kb. (B) Alignment of the conserved reverse transcriptase domain. Gray boxes indicate residues conserved in at least seven of the eight sequences depicted. I to VII denote the seven domains characteristic of reverse transcriptases. Retrotransposon sequences are from rice Karma , maize cin4 , lily del2 , Arabidopsis Ta11-1 , Cannabis LINE-CS, human LINE-1, frog Tx1 , and fruit fly R2Dm .
    Ecorv, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 477 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TaKaRa ecorv eco ri digested pires puro3 vector
    Conserved Domains in Karma . (A) Karma structure. Open boxes depict the two open reading frames of Karma (ORF1 and ORF2). ORF2 contains the endonuclease ( en ) and reverse transcriptase ( rt ) domains. Black boxes depict Cys-rich motifs. Vertical lines above the boxes depict the sites of HpaII and MspI cleavage, and vertical lines below the boxes depict the sites of <t>EcoRV</t> cleavage. Hatched boxes depict probes A (1317 bp) and B (1498 bp) used in <t>DNA</t> gel blot analysis. Bar = 1 kb. (B) Alignment of the conserved reverse transcriptase domain. Gray boxes indicate residues conserved in at least seven of the eight sequences depicted. I to VII denote the seven domains characteristic of reverse transcriptases. Retrotransposon sequences are from rice Karma , maize cin4 , lily del2 , Arabidopsis Ta11-1 , Cannabis LINE-CS, human LINE-1, frog Tx1 , and fruit fly R2Dm .
    Ecorv Eco Ri Digested Pires Puro3 Vector, supplied by TaKaRa, used in various techniques. Bioz Stars score: 85/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Signature of Aberrant DNA Methylation in MYC -Induced T cell Lymphomas (A) Schematic representation of transgenes in MYC -induced T cell lymphoma model used in this study. (B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy NotI fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events. (C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP EGFP mice are also shown (see text and Figure 2 ). (D) Southern blot analysis of control and tumor cells using the 2C51 NotI-EcoRV DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites. (E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides. (F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y -axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.

    Journal: PLoS Genetics

    Article Title: CpG Island Methylation in a Mouse Model of Lymphoma Is Driven by the Genetic Configuration of Tumor Cells

    doi: 10.1371/journal.pgen.0030167

    Figure Lengend Snippet: Signature of Aberrant DNA Methylation in MYC -Induced T cell Lymphomas (A) Schematic representation of transgenes in MYC -induced T cell lymphoma model used in this study. (B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy NotI fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events. (C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP EGFP mice are also shown (see text and Figure 2 ). (D) Southern blot analysis of control and tumor cells using the 2C51 NotI-EcoRV DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites. (E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides. (F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y -axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.

    Article Snippet: A total of 10 μg of genomic DNA isolated from normal thymocytes or thymic tumors was digested with NotI and EcoRV (New England Biolabs).

    Techniques: DNA Methylation Assay, RLGS, Methylation, Isolation, Mouse Assay, Southern Blot, Amplification, Methylation Sequencing, Sequencing, Expressing, Quantitative RT-PCR

    The images of EcoR I and Rsa I digestion on ds-DNA array. (A): the image of the ds-DNA array which created by insertion with Cy3-dUTP (a) digested by EcoR I under 1h(b) and 12h(c); (B): the image of ds-DNA array (a) digested by Rsa I under 1 h(b) and 12 h(c) digestion. The four different concentrations of Oligo II (C, 1, 2, 3 and 4 below the two images indicate control, 80 µM, 40 µM, 20 µM and 10 µM Oligo II, respectively.) were spotted onto the array.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Investigation of DNA-protein Sequence-Specific Interactions with a ds-DNA Array

    doi: 10.3390/10020417

    Figure Lengend Snippet: The images of EcoR I and Rsa I digestion on ds-DNA array. (A): the image of the ds-DNA array which created by insertion with Cy3-dUTP (a) digested by EcoR I under 1h(b) and 12h(c); (B): the image of ds-DNA array (a) digested by Rsa I under 1 h(b) and 12 h(c) digestion. The four different concentrations of Oligo II (C, 1, 2, 3 and 4 below the two images indicate control, 80 µM, 40 µM, 20 µM and 10 µM Oligo II, respectively.) were spotted onto the array.

    Article Snippet: Endonucleases (EcoR I, Rsa I) and EcoR I methylase were obtained from New England BioLabs.

    Techniques: DNA Array

    The fluorescence intensity variation of EcoR I and Rsa I digestion on the same array after methylation. EM indicated methylation enzyme and ER indicated EcoR I.

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Investigation of DNA-protein Sequence-Specific Interactions with a ds-DNA Array

    doi: 10.3390/10020417

    Figure Lengend Snippet: The fluorescence intensity variation of EcoR I and Rsa I digestion on the same array after methylation. EM indicated methylation enzyme and ER indicated EcoR I.

    Article Snippet: Endonucleases (EcoR I, Rsa I) and EcoR I methylase were obtained from New England BioLabs.

    Techniques: Fluorescence, Methylation

    The images of array one digested by EcoR I and Rsa I after methylation. (A): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by EcoR I (c); (B): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by Rsa I (c). (C and E below the images indicate Control probe and Oligo I, respectively)

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Investigation of DNA-protein Sequence-Specific Interactions with a ds-DNA Array

    doi: 10.3390/10020417

    Figure Lengend Snippet: The images of array one digested by EcoR I and Rsa I after methylation. (A): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by EcoR I (c); (B): Images of array one which created by inserted Cy3-dUTP (a) was treated by EcoR I methylation enzyme (b) and then by Rsa I (c). (C and E below the images indicate Control probe and Oligo I, respectively)

    Article Snippet: Endonucleases (EcoR I, Rsa I) and EcoR I methylase were obtained from New England BioLabs.

    Techniques: Methylation

    Selection of putative CaMV35S : AtGA20ox transgenic kenaf plants in MS media containing hygromycin B and the confirmation through PCR amplification and Southern blot analysis. Growth of UT plants in the MS medium without hygromycin B (a) and with hygromycin B (45 μg mL −1 ) (b). Growing of putative transformants in the MS media with hygromycin B (45 μg mL −1 ) (c and d). Detection of the transgene encoding AtGA20ox under duplicate 35S promoter in transgenic G4 and V36 kenaf and UT plants either amplified by PCR using GA-R and 35S-F primers (e and f) or southern blot analysis (g and h). Lane M is ladder, lanes 1–3, 5, 7 (e) and 1–3, 5, 13 (f) are putative transformed G4 and V36 plants respectively showing two amplifications 1.3 kb and 1.6 kb, except for line G4-1 (lane 1 in e) which produced only 1.3 kb amplification product; lane UT is the DNA of untransformed plants; lane −Ve is negative control used PCR product without DNA; lane +Ve is the plasmid DNA of expression clone pEXP32-AtGA20ox. Southern blot analysis was performed with genomic DNA (6 μg–30 μg) digested with EcoR V, separated on 0.8% agarose gel using biotin labelled ORF of AtGA20ox as probe: lane M is marker; lanes 1–3, 5, 7 are putative G4 transformants; lane UT is the DNA of G4 UT and lane +Ve is plasmid DNA (g). In Figure h, lanes 1, 3, 13 are the putative V36 transformants, lane UT is the DNA from V36 UT plant and lane +Ve is plasmid DNA of pEXP32-AtGA20ox digested with EcoR V.

    Journal: Breeding Science

    Article Title: Overexpression of Arabidopsis thaliana gibberellic acid 20 oxidase (AtGA20ox) gene enhance the vegetative growth and fiber quality in kenaf (Hibiscus cannabinus L.) plants

    doi: 10.1270/jsbbs.65.177

    Figure Lengend Snippet: Selection of putative CaMV35S : AtGA20ox transgenic kenaf plants in MS media containing hygromycin B and the confirmation through PCR amplification and Southern blot analysis. Growth of UT plants in the MS medium without hygromycin B (a) and with hygromycin B (45 μg mL −1 ) (b). Growing of putative transformants in the MS media with hygromycin B (45 μg mL −1 ) (c and d). Detection of the transgene encoding AtGA20ox under duplicate 35S promoter in transgenic G4 and V36 kenaf and UT plants either amplified by PCR using GA-R and 35S-F primers (e and f) or southern blot analysis (g and h). Lane M is ladder, lanes 1–3, 5, 7 (e) and 1–3, 5, 13 (f) are putative transformed G4 and V36 plants respectively showing two amplifications 1.3 kb and 1.6 kb, except for line G4-1 (lane 1 in e) which produced only 1.3 kb amplification product; lane UT is the DNA of untransformed plants; lane −Ve is negative control used PCR product without DNA; lane +Ve is the plasmid DNA of expression clone pEXP32-AtGA20ox. Southern blot analysis was performed with genomic DNA (6 μg–30 μg) digested with EcoR V, separated on 0.8% agarose gel using biotin labelled ORF of AtGA20ox as probe: lane M is marker; lanes 1–3, 5, 7 are putative G4 transformants; lane UT is the DNA of G4 UT and lane +Ve is plasmid DNA (g). In Figure h, lanes 1, 3, 13 are the putative V36 transformants, lane UT is the DNA from V36 UT plant and lane +Ve is plasmid DNA of pEXP32-AtGA20ox digested with EcoR V.

    Article Snippet: DNA from putative transgenic and UT plants was digested with EcoR V (NEB, UK).

    Techniques: Selection, Transgenic Assay, Mass Spectrometry, Polymerase Chain Reaction, Amplification, Southern Blot, Transformation Assay, Produced, Negative Control, Plasmid Preparation, Expressing, Agarose Gel Electrophoresis, Marker

    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Journal: International Journal of Molecular Sciences

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    doi: 10.3390/ijms17101690

    Figure Lengend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Article Snippet: The cDNA was digested with the restriction enzymes EcoR I and Mse I (New England Biolabs (NEB), Beijing, China) at 37 °C overnight.

    Techniques: DNA Methylation Assay, Methylation

    Schematic of the recombinant plasmid pCMV6-truncated-rVegfr2. Restriction digestion of the two EcoRV restriction sites in the plasmid pCMV6-rVegfr2, which encodes the extracellular reserved VEGFR2 Ig 1–3 and 5 regions. The recombinant plasmid pCMV6-truncated-rVegfr2 was reconstructed after self-ligation of the plasmid vector with T4 DNA ligase. rVegfr2: rat-Vegf-receptor2.

    Journal: Cell & Bioscience

    Article Title: Expression and characterization of a soluble VEGF receptor 2 protein

    doi: 10.1186/2045-3701-4-14

    Figure Lengend Snippet: Schematic of the recombinant plasmid pCMV6-truncated-rVegfr2. Restriction digestion of the two EcoRV restriction sites in the plasmid pCMV6-rVegfr2, which encodes the extracellular reserved VEGFR2 Ig 1–3 and 5 regions. The recombinant plasmid pCMV6-truncated-rVegfr2 was reconstructed after self-ligation of the plasmid vector with T4 DNA ligase. rVegfr2: rat-Vegf-receptor2.

    Article Snippet: The restriction endonuclease EcoRV and T4 DNA Ligase were obtained from Takara China (TAKARA Biotechnology Co., LTD., Dalian, Shangdong China). pCMV6-rVegfr2 was constructed by ORIGENE (ORIGENE, China).

    Techniques: Recombinant, Plasmid Preparation, Ligation

    pH dependence of the EcoRV specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: pH dependence of the EcoRV specific-nonspecific free binding energy difference. The pH dependence of ln(K nsp-sp ) is shown for the range 5.5 to 8.0. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay

    Kinetics of the EcoRV-DNA complex formation. The kinetics of DNA-protein complex formation was measured using the self-cleavage assay at different conditions of pH: pH 6.3 (▲); pH 7.6 (■). The binding of the EcoRV proceeds in at least

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: Kinetics of the EcoRV-DNA complex formation. The kinetics of DNA-protein complex formation was measured using the self-cleavage assay at different conditions of pH: pH 6.3 (▲); pH 7.6 (■). The binding of the EcoRV proceeds in at least

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Cleavage Assay, Binding Assay

    A direct comparison of EcoRV-DNA binding analyzed by the gel mobility shift assay and by the self-cleavage assay. (A ) A gel image is shown illustrating a direct comparison of the EcoRV-DNA binding by the gel mobility shift assay (left), and by the self-cleavage

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: A direct comparison of EcoRV-DNA binding analyzed by the gel mobility shift assay and by the self-cleavage assay. (A ) A gel image is shown illustrating a direct comparison of the EcoRV-DNA binding by the gel mobility shift assay (left), and by the self-cleavage

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Mobility Shift, Cleavage Assay

    Equilibrium competition between specific and nonspecific DNA sequences for the EcoRV binding. Mixtures of EcoRV, the 310 bp DNA fragment with a specific recognition site, and nonspecific oligonucleotide competitor were incubated at 20 °C overnight

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: Equilibrium competition between specific and nonspecific DNA sequences for the EcoRV binding. Mixtures of EcoRV, the 310 bp DNA fragment with a specific recognition site, and nonspecific oligonucleotide competitor were incubated at 20 °C overnight

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Incubation

    The dependence of the EcoRV specific-nonspecific binding free energy difference, ln(K nsp-sp ) in the units of kT, on solute osmolal concentration is shown for four neutral osmolytes. Mixtures of the specific site DNA fragment, nonspecific oligonucleotide,

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: The dependence of the EcoRV specific-nonspecific binding free energy difference, ln(K nsp-sp ) in the units of kT, on solute osmolal concentration is shown for four neutral osmolytes. Mixtures of the specific site DNA fragment, nonspecific oligonucleotide,

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Concentration Assay

    The pH dependence of Knsp-sp for EcoRV-DNA binding

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: The pH dependence of Knsp-sp for EcoRV-DNA binding

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay

    The dependence of the EcoRV specific-nonspecific binding free energy difference on triethylene glycol concentration is shown for different pH values. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Journal: The FEBS journal

    Article Title: Solution parameters modulating DNA binding specificity of the restriction endonuclease EcoRV

    doi: 10.1111/j.1742-4658.2011.08198.x

    Figure Lengend Snippet: The dependence of the EcoRV specific-nonspecific binding free energy difference on triethylene glycol concentration is shown for different pH values. Mixtures of EcoRV, the 310 bp specific site DNA fragment, and the nonspecific oligonucleotide competitor

    Article Snippet: DNA binding and cleavage experiments were performed with highly purified EcoRV restriction endonuclease (described below) or with commercial EcoRV sample purchased from New England Biolabs.

    Techniques: Binding Assay, Concentration Assay

    Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Journal: EMBO Molecular Medicine

    Article Title: A single epidermal stem cell strategy for safe ex vivo gene therapy

    doi: 10.15252/emmm.201404353

    Figure Lengend Snippet: Isolation of genetically corrected recessive dystrophic epidermolysis bullosa (RDEB) epidermal stem cells Single cells were isolated from a mass culture (passage V) of RDEB keratinocytes infected with SIN retroviruses bearing a COL7A1 cDNA. Clonal types were determined (Barrandon Green, 1987 ) and listed in Supplementary Table S1. Growing clones were expanded for further characterisation. COLVII detection in clones by immunostaining. COLVII expression (green) was detectable in some clones (6, 17, 22, 58 and 61) and not in others (3, 24 and 54); nuclei were stained with Hoechst 33342 (blue). Dotted lines delimit the periphery of keratinocyte colonies from the surrounding irradiated 3T3-J2 feeder cells. Scale bar: 50 μm. Quantitative RT–PCR analysis of COL7A1 expression in transduced clones compared to untransduced RDEB keratinocytes. All clones shown in (A) were transduced but expressed different levels of COL7A1 transcripts. Clones 6, 17, 22, 54, 58 and 61 expressed higher levels of COL7A1 than control RDEB cells and keratinocytes obtained from healthy donors (YF29 and OR-CA, control 1 and 2, respectively). The level of COL7A1 expression in the RDEB untransduced cells was referenced as 1. Determination of proviral rearrangements in transduced clones. A Southern blot was performed using genomic DNA of RDEB cells, clones and the infected mass culture from which the clones were isolated. Genomic DNA was digested with EcoRV and SpeI that cut at the 3′ and 5′ end of the provirus (Supplementary Fig S2) and hybridised with a 907-bp COL7A1 probe radiolabelled with 32 P isotope. The upper band corresponded to the endogenous signal. The retroviral producer line Flp293A-E1aColVII1 was used as a control for the digested 9.6-kb provirus (proviral signal). Smaller bands corresponded to rearranged proviruses marked with an asterisk. Identification of stem cells producing COLVII. Western blotting revealed that only clone 6 secreted COLVII in the culture supernatant, while clone 54 and surprisingly clone 22 did not (see A). RDEB cells were used as a negative control and healthy donor cells as a positive control. The secreted matrix metalloproteinase 2 (MMP2) was used as a loading control.

    Article Snippet: Ten micrograms of DNA was codigested with SpeI/EcoRV HF (New England Biolabs) and loaded on a 0.8% agarose (Promega) gel.

    Techniques: Isolation, Infection, Clone Assay, Immunostaining, Expressing, Staining, Irradiation, Quantitative RT-PCR, Southern Blot, Western Blot, Negative Control, Positive Control

    Conserved Domains in Karma . (A) Karma structure. Open boxes depict the two open reading frames of Karma (ORF1 and ORF2). ORF2 contains the endonuclease ( en ) and reverse transcriptase ( rt ) domains. Black boxes depict Cys-rich motifs. Vertical lines above the boxes depict the sites of HpaII and MspI cleavage, and vertical lines below the boxes depict the sites of EcoRV cleavage. Hatched boxes depict probes A (1317 bp) and B (1498 bp) used in DNA gel blot analysis. Bar = 1 kb. (B) Alignment of the conserved reverse transcriptase domain. Gray boxes indicate residues conserved in at least seven of the eight sequences depicted. I to VII denote the seven domains characteristic of reverse transcriptases. Retrotransposon sequences are from rice Karma , maize cin4 , lily del2 , Arabidopsis Ta11-1 , Cannabis LINE-CS, human LINE-1, frog Tx1 , and fruit fly R2Dm .

    Journal: The Plant Cell

    Article Title: Two-Step Regulation and Continuous Retrotransposition of the Rice LINE-Type Retrotransposon Karma

    doi: 10.1105/tpc.011809

    Figure Lengend Snippet: Conserved Domains in Karma . (A) Karma structure. Open boxes depict the two open reading frames of Karma (ORF1 and ORF2). ORF2 contains the endonuclease ( en ) and reverse transcriptase ( rt ) domains. Black boxes depict Cys-rich motifs. Vertical lines above the boxes depict the sites of HpaII and MspI cleavage, and vertical lines below the boxes depict the sites of EcoRV cleavage. Hatched boxes depict probes A (1317 bp) and B (1498 bp) used in DNA gel blot analysis. Bar = 1 kb. (B) Alignment of the conserved reverse transcriptase domain. Gray boxes indicate residues conserved in at least seven of the eight sequences depicted. I to VII denote the seven domains characteristic of reverse transcriptases. Retrotransposon sequences are from rice Karma , maize cin4 , lily del2 , Arabidopsis Ta11-1 , Cannabis LINE-CS, human LINE-1, frog Tx1 , and fruit fly R2Dm .

    Article Snippet: Two micrograms of genomic DNA extracted from leaves was digested with EcoRV and self-ligated using the DNA Ligation Kit Version 2 (Takara Bio, Otsu, Japan).

    Techniques: Western Blot