lambda exonuclease  (New England Biolabs)


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    Lambda Exonuclease
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    Lambda Exonuclease 5 000 units
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    New England Biolabs lambda exonuclease
    Lambda Exonuclease
    Lambda Exonuclease 5 000 units
    https://www.bioz.com/result/lambda exonuclease/product/New England Biolabs
    Average 99 stars, based on 19714 article reviews
    Price from $9.99 to $1999.99
    lambda exonuclease - by Bioz Stars, 2020-07
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    Images

    1) Product Images from "A comprehensive assay for targeted multiplex amplification of human DNA sequences"

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences

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

    doi: 10.1073/pnas.0803240105

    Construction of single-strand probes. ( A ) The bacteriophage lambda DNA (gray) was used as a template, and the primers containing the common amplification primers as adaptors on the 5′ end were used to PCR amplify the spacer that is common to all of the probes. This common spacer with the amplification sequences was the further template for building the double-stranded probe precursor. The left primer with the target sequence (red) had an adaptor with a BsaI site (blue) The right primer with the target sequences (red) has an adaptor (green) with the MlyI site. ( B ) The construct was digested with BsaI (red triangle) cutting 5 bases inwards from the recognition sequence. The digestion creates a phosphorylated 5′end and a recessed 3′ end. The phosphate group is removed by shrimp alkaline phosphatase digestion. A subsequent digestion with MlyI (black triangle) cuts 5 bases inwards from the right-hand recognition sequence to create a blunt end with a 5′ phosphate. The lambda exonuclease digests the lower strand from the 5′ phosphorylated end created by the Mly I digestion leaving the upper strand intact. ( C ) The dHPLC profile of the double-stranded template has two peaks (blue) at ≈4.5 min. After exonuclease digestion (red) there is only one peak at 4.5 min, and one large early peak that represent the digested products. ( D ) The LPP is hybridized to genomic DNA (gray) and gap-filled (orange) using the genomic DNA as template. Ligation occurs when the polymerase reaches the 5′ end of the probe (yellow circle) to form a circular molecule. The amplification sequences (yellow) in the circle are then used to amplify the targeted exons. Digestion with AscI and ClaI (sites are included in the amplification primers) separate the primers from the genomic target sequences.
    Figure Legend Snippet: Construction of single-strand probes. ( A ) The bacteriophage lambda DNA (gray) was used as a template, and the primers containing the common amplification primers as adaptors on the 5′ end were used to PCR amplify the spacer that is common to all of the probes. This common spacer with the amplification sequences was the further template for building the double-stranded probe precursor. The left primer with the target sequence (red) had an adaptor with a BsaI site (blue) The right primer with the target sequences (red) has an adaptor (green) with the MlyI site. ( B ) The construct was digested with BsaI (red triangle) cutting 5 bases inwards from the recognition sequence. The digestion creates a phosphorylated 5′end and a recessed 3′ end. The phosphate group is removed by shrimp alkaline phosphatase digestion. A subsequent digestion with MlyI (black triangle) cuts 5 bases inwards from the right-hand recognition sequence to create a blunt end with a 5′ phosphate. The lambda exonuclease digests the lower strand from the 5′ phosphorylated end created by the Mly I digestion leaving the upper strand intact. ( C ) The dHPLC profile of the double-stranded template has two peaks (blue) at ≈4.5 min. After exonuclease digestion (red) there is only one peak at 4.5 min, and one large early peak that represent the digested products. ( D ) The LPP is hybridized to genomic DNA (gray) and gap-filled (orange) using the genomic DNA as template. Ligation occurs when the polymerase reaches the 5′ end of the probe (yellow circle) to form a circular molecule. The amplification sequences (yellow) in the circle are then used to amplify the targeted exons. Digestion with AscI and ClaI (sites are included in the amplification primers) separate the primers from the genomic target sequences.

    Techniques Used: Lambda DNA Preparation, Amplification, Polymerase Chain Reaction, Sequencing, Construct, Ligation

    2) Product Images from "Structures of the human pre-catalytic spliceosome and its precursor spliceosome"

    Article Title: Structures of the human pre-catalytic spliceosome and its precursor spliceosome

    Journal: Cell Research

    doi: 10.1038/s41422-018-0094-7

    Cryo-EM structures of the human precursor pre-catalytic spliceosome (the pre-B complex) and the pre-catalytic spliceosome (the B complex). a Structures of the pre-B complex (left panel) and the B complex (right panel). The protein and RNA components are color-coded and tabulated below the images. The pre-B complex, but not the B complex, contains Sad1, Prp28, and U1 snRNP. Components of U1 snRNP (colored grey) remain unassigned due to the low resolution of the EM density map in this region. Compared to pre-B, the B complex has eight additional protein components: CypH, FBP21, MFAP1, Prp38, Smu1, Snu23, Snu66, and UBL5. Although the U4/U6.U5 tri-snRNP is the core in both complexes, it undergoes pronounced structural rearrangement. b
    Figure Legend Snippet: Cryo-EM structures of the human precursor pre-catalytic spliceosome (the pre-B complex) and the pre-catalytic spliceosome (the B complex). a Structures of the pre-B complex (left panel) and the B complex (right panel). The protein and RNA components are color-coded and tabulated below the images. The pre-B complex, but not the B complex, contains Sad1, Prp28, and U1 snRNP. Components of U1 snRNP (colored grey) remain unassigned due to the low resolution of the EM density map in this region. Compared to pre-B, the B complex has eight additional protein components: CypH, FBP21, MFAP1, Prp38, Smu1, Snu23, Snu66, and UBL5. Although the U4/U6.U5 tri-snRNP is the core in both complexes, it undergoes pronounced structural rearrangement. b

    Techniques Used:

    3) Product Images from "A novel phenotype-genotype relationship with a TGFBI exon 14 mutation in a pedigree with a unique corneal dystrophy of Bowman’s layer"

    Article Title: A novel phenotype-genotype relationship with a TGFBI exon 14 mutation in a pedigree with a unique corneal dystrophy of Bowman’s layer

    Journal: Molecular Vision

    doi:

    Electropherogram of TGFBI exon 14 in an affected individual. This revealed a missense transversion A→C at nucleotide 1877, resulting in a substitution of histidine by proline (C A T→C C T, H626P).
    Figure Legend Snippet: Electropherogram of TGFBI exon 14 in an affected individual. This revealed a missense transversion A→C at nucleotide 1877, resulting in a substitution of histidine by proline (C A T→C C T, H626P).

    Techniques Used:

    4) Product Images from "A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs"

    Article Title: A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100677

    Cas9-HR 8 again shows decreased toxicity and increased HDR rates in an independent assay. (A) Left, diagram showing the Puromycin RT template, and guides Int-G2 and G3. CMV promoter: yellow, PuroR CDS: orange, SV40 polyA sequence: light blue, guide targets: red, surrounding genomic sequence: white. Right, graph showing cellular toxicity of Cas9-HRs 4,8, Cas9, PuroR RT and untransfected controls (Con) in A549 cells. Both Cas9-HR 4 and 8 show significant reductions in toxicity relative to Cas9 (two independent replicates with 8 individual transfections per replicate, p
    Figure Legend Snippet: Cas9-HR 8 again shows decreased toxicity and increased HDR rates in an independent assay. (A) Left, diagram showing the Puromycin RT template, and guides Int-G2 and G3. CMV promoter: yellow, PuroR CDS: orange, SV40 polyA sequence: light blue, guide targets: red, surrounding genomic sequence: white. Right, graph showing cellular toxicity of Cas9-HRs 4,8, Cas9, PuroR RT and untransfected controls (Con) in A549 cells. Both Cas9-HR 4 and 8 show significant reductions in toxicity relative to Cas9 (two independent replicates with 8 individual transfections per replicate, p

    Techniques Used: Sequencing, Transfection

    Enzymatically active Cas9-HRs can be purified from E. Coli . (A) SDS-PAGE gel showing successful purification of Cas9-HR 3. Lanes 1-7 Cas9-HR 3, Lanes 8-15 Cas9. Lanes 1,8 whole lysate; 2,9 soluble fraction; 3,10 insoluble fraction; 4,11 His-purification; 5,12 Sepharose wash; 6,13 Sepharose Elution 1; 7,14 Sepharose Elution 2. (B) Diagram of HBB genomic region, with primers in red showing the amplified fragment (956bp). (C) Agarose Gel image of exonuclease activity of purified Cas9-HRs 3,4,8, Cas9, Solvent (Cas9-HR buffer), and DNA (Con). Reduction in band intensity of Cas9-HR 3 and 8 indicates exonuclease activity.
    Figure Legend Snippet: Enzymatically active Cas9-HRs can be purified from E. Coli . (A) SDS-PAGE gel showing successful purification of Cas9-HR 3. Lanes 1-7 Cas9-HR 3, Lanes 8-15 Cas9. Lanes 1,8 whole lysate; 2,9 soluble fraction; 3,10 insoluble fraction; 4,11 His-purification; 5,12 Sepharose wash; 6,13 Sepharose Elution 1; 7,14 Sepharose Elution 2. (B) Diagram of HBB genomic region, with primers in red showing the amplified fragment (956bp). (C) Agarose Gel image of exonuclease activity of purified Cas9-HRs 3,4,8, Cas9, Solvent (Cas9-HR buffer), and DNA (Con). Reduction in band intensity of Cas9-HR 3 and 8 indicates exonuclease activity.

    Techniques Used: Purification, SDS Page, Amplification, Agarose Gel Electrophoresis, Activity Assay

    Initial Construction and Characterization of Cas9-HRs. (A) Diagram of the px330 plasmid used as the expression vector for Cas9 and Cas9-HRs. (B) Diagrams showing fusions of Cas9-HRs 1-9, with Cas9 in red, NLS sequences in green, hExo1 in teal and linkers being black lines. Sequences of linkers used are available in Table S1. (C) Example of a 96 well seeding pattern for standard A549 toxicity assays used throughout the paper. All experiments contained at least two independent replicates. (D) Cas9-HRs reduce cellular toxicity in A549 cells. The target site on Chromosome 12 is depicted above graph showing cellular toxicity for Cas9-HRs 1-8 (1-8), Cas9, Cas9+hExo1, and untransfected controls (Con). Fluorescence values were normalized to untransfected control values, two independent experiments each with 8 individual transfections. All Cas9-HRs and GFP show significant reductions in cellular toxicity relative to Cas9 or Cas9+hExo1 (p
    Figure Legend Snippet: Initial Construction and Characterization of Cas9-HRs. (A) Diagram of the px330 plasmid used as the expression vector for Cas9 and Cas9-HRs. (B) Diagrams showing fusions of Cas9-HRs 1-9, with Cas9 in red, NLS sequences in green, hExo1 in teal and linkers being black lines. Sequences of linkers used are available in Table S1. (C) Example of a 96 well seeding pattern for standard A549 toxicity assays used throughout the paper. All experiments contained at least two independent replicates. (D) Cas9-HRs reduce cellular toxicity in A549 cells. The target site on Chromosome 12 is depicted above graph showing cellular toxicity for Cas9-HRs 1-8 (1-8), Cas9, Cas9+hExo1, and untransfected controls (Con). Fluorescence values were normalized to untransfected control values, two independent experiments each with 8 individual transfections. All Cas9-HRs and GFP show significant reductions in cellular toxicity relative to Cas9 or Cas9+hExo1 (p

    Techniques Used: Plasmid Preparation, Expressing, Fluorescence, Transfection

    Cas9-HR 8 decreases cellular toxicity and increases HDR. (A) Diagram of the H2B-mNeon repair template, as well as showing the location of hH2B-G4 guide. Blue: H2B coding sequence, Green: mNeon, Red: silent mutations introduced in RT sequence, black lines: surrounding genomic sequence. (B) Graph of cellular toxicity of Cas9-HRs 4,5,6,8, Cas9 and untransfected controls (Con) targeting H2B-G4 in A549 cells. Only Cas9-HR shows a significant reduction in toxicity compared to Cas9 (two replicates with 8 individual transfections, p
    Figure Legend Snippet: Cas9-HR 8 decreases cellular toxicity and increases HDR. (A) Diagram of the H2B-mNeon repair template, as well as showing the location of hH2B-G4 guide. Blue: H2B coding sequence, Green: mNeon, Red: silent mutations introduced in RT sequence, black lines: surrounding genomic sequence. (B) Graph of cellular toxicity of Cas9-HRs 4,5,6,8, Cas9 and untransfected controls (Con) targeting H2B-G4 in A549 cells. Only Cas9-HR shows a significant reduction in toxicity compared to Cas9 (two replicates with 8 individual transfections, p

    Techniques Used: Sequencing, Transfection

    5) Product Images from "A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs"

    Article Title: A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100677

    Cas9-HR 8 again shows decreased toxicity and increased HDR rates in an independent assay. (A) Left, diagram showing the Puromycin RT template, and guides Int-G2 and G3. CMV promoter: yellow, PuroR CDS: orange, SV40 polyA sequence: light blue, guide targets: red, surrounding genomic sequence: white. Right, graph showing cellular toxicity of Cas9-HRs 4,8, Cas9, PuroR RT and untransfected controls (Con) in A549 cells. Both Cas9-HR 4 and 8 show significant reductions in toxicity relative to Cas9 (two independent replicates with 8 individual transfections per replicate, p
    Figure Legend Snippet: Cas9-HR 8 again shows decreased toxicity and increased HDR rates in an independent assay. (A) Left, diagram showing the Puromycin RT template, and guides Int-G2 and G3. CMV promoter: yellow, PuroR CDS: orange, SV40 polyA sequence: light blue, guide targets: red, surrounding genomic sequence: white. Right, graph showing cellular toxicity of Cas9-HRs 4,8, Cas9, PuroR RT and untransfected controls (Con) in A549 cells. Both Cas9-HR 4 and 8 show significant reductions in toxicity relative to Cas9 (two independent replicates with 8 individual transfections per replicate, p

    Techniques Used: Sequencing, Transfection

    Enzymatically active Cas9-HRs can be purified from E. Coli . (A) SDS-PAGE gel showing successful purification of Cas9-HR 3. Lanes 1-7 Cas9-HR 3, Lanes 8-15 Cas9. Lanes 1,8 whole lysate; 2,9 soluble fraction; 3,10 insoluble fraction; 4,11 His-purification; 5,12 Sepharose wash; 6,13 Sepharose Elution 1; 7,14 Sepharose Elution 2. (B) Diagram of HBB genomic region, with primers in red showing the amplified fragment (956bp). (C) Agarose Gel image of exonuclease activity of purified Cas9-HRs 3,4,8, Cas9, Solvent (Cas9-HR buffer), and DNA (Con). Reduction in band intensity of Cas9-HR 3 and 8 indicates exonuclease activity.
    Figure Legend Snippet: Enzymatically active Cas9-HRs can be purified from E. Coli . (A) SDS-PAGE gel showing successful purification of Cas9-HR 3. Lanes 1-7 Cas9-HR 3, Lanes 8-15 Cas9. Lanes 1,8 whole lysate; 2,9 soluble fraction; 3,10 insoluble fraction; 4,11 His-purification; 5,12 Sepharose wash; 6,13 Sepharose Elution 1; 7,14 Sepharose Elution 2. (B) Diagram of HBB genomic region, with primers in red showing the amplified fragment (956bp). (C) Agarose Gel image of exonuclease activity of purified Cas9-HRs 3,4,8, Cas9, Solvent (Cas9-HR buffer), and DNA (Con). Reduction in band intensity of Cas9-HR 3 and 8 indicates exonuclease activity.

    Techniques Used: Purification, SDS Page, Amplification, Agarose Gel Electrophoresis, Activity Assay

    Initial Construction and Characterization of Cas9-HRs. (A) Diagram of the px330 plasmid used as the expression vector for Cas9 and Cas9-HRs. (B) Diagrams showing fusions of Cas9-HRs 1-9, with Cas9 in red, NLS sequences in green, hExo1 in teal and linkers being black lines. Sequences of linkers used are available in Table S1. (C) Example of a 96 well seeding pattern for standard A549 toxicity assays used throughout the paper. All experiments contained at least two independent replicates. (D) Cas9-HRs reduce cellular toxicity in A549 cells. The target site on Chromosome 12 is depicted above graph showing cellular toxicity for Cas9-HRs 1-8 (1-8), Cas9, Cas9+hExo1, and untransfected controls (Con). Fluorescence values were normalized to untransfected control values, two independent experiments each with 8 individual transfections. All Cas9-HRs and GFP show significant reductions in cellular toxicity relative to Cas9 or Cas9+hExo1 (p
    Figure Legend Snippet: Initial Construction and Characterization of Cas9-HRs. (A) Diagram of the px330 plasmid used as the expression vector for Cas9 and Cas9-HRs. (B) Diagrams showing fusions of Cas9-HRs 1-9, with Cas9 in red, NLS sequences in green, hExo1 in teal and linkers being black lines. Sequences of linkers used are available in Table S1. (C) Example of a 96 well seeding pattern for standard A549 toxicity assays used throughout the paper. All experiments contained at least two independent replicates. (D) Cas9-HRs reduce cellular toxicity in A549 cells. The target site on Chromosome 12 is depicted above graph showing cellular toxicity for Cas9-HRs 1-8 (1-8), Cas9, Cas9+hExo1, and untransfected controls (Con). Fluorescence values were normalized to untransfected control values, two independent experiments each with 8 individual transfections. All Cas9-HRs and GFP show significant reductions in cellular toxicity relative to Cas9 or Cas9+hExo1 (p

    Techniques Used: Plasmid Preparation, Expressing, Fluorescence, Transfection

    Cas9-HR 8 decreases cellular toxicity and increases HDR. (A) Diagram of the H2B-mNeon repair template, as well as showing the location of hH2B-G4 guide. Blue: H2B coding sequence, Green: mNeon, Red: silent mutations introduced in RT sequence, black lines: surrounding genomic sequence. (B) Graph of cellular toxicity of Cas9-HRs 4,5,6,8, Cas9 and untransfected controls (Con) targeting H2B-G4 in A549 cells. Only Cas9-HR shows a significant reduction in toxicity compared to Cas9 (two replicates with 8 individual transfections, p
    Figure Legend Snippet: Cas9-HR 8 decreases cellular toxicity and increases HDR. (A) Diagram of the H2B-mNeon repair template, as well as showing the location of hH2B-G4 guide. Blue: H2B coding sequence, Green: mNeon, Red: silent mutations introduced in RT sequence, black lines: surrounding genomic sequence. (B) Graph of cellular toxicity of Cas9-HRs 4,5,6,8, Cas9 and untransfected controls (Con) targeting H2B-G4 in A549 cells. Only Cas9-HR shows a significant reduction in toxicity compared to Cas9 (two replicates with 8 individual transfections, p

    Techniques Used: Sequencing, Transfection

    6) Product Images from "Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration"

    Article Title: Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

    Journal: Genome Research

    doi: 10.1101/gr.219089.116

    Targeted mutagenesis in the  Vegfa/VEGFA  gene via Cas9 ribonucleoproteins (RNPs). ( A ) The target sequence in the  Vegfa/VEGFA  locus. The PAM sequence and the sgRNA target sequence are shown in red and blue, respectively. ( B )  Vegfa -specific Cas9 RNP-driven mutations in NIH3T3 and ARPE-19 cells detected by the T7 endonuclease I (T7E1) assay. Arrows indicate the expected positions of DNA bands cleaved by T7E1. ( C ) Mutation frequencies measured by targeted deep sequencing. Error bars indicate SEM ( n  = 3). One-way ANOVA and Tukey post-hoc tests: (*)  P
    Figure Legend Snippet: Targeted mutagenesis in the Vegfa/VEGFA gene via Cas9 ribonucleoproteins (RNPs). ( A ) The target sequence in the Vegfa/VEGFA locus. The PAM sequence and the sgRNA target sequence are shown in red and blue, respectively. ( B ) Vegfa -specific Cas9 RNP-driven mutations in NIH3T3 and ARPE-19 cells detected by the T7 endonuclease I (T7E1) assay. Arrows indicate the expected positions of DNA bands cleaved by T7E1. ( C ) Mutation frequencies measured by targeted deep sequencing. Error bars indicate SEM ( n = 3). One-way ANOVA and Tukey post-hoc tests: (*) P

    Techniques Used: Mutagenesis, Sequencing

    7) Product Images from "Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase"

    Article Title: Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase

    Journal: Cancer cell

    doi: 10.1016/j.ccr.2010.08.012

    Knockdown of Fbx4 stabilizes nuclear cyclin D1 resulting in increased PRMT5-dependent histone methylation
    Figure Legend Snippet: Knockdown of Fbx4 stabilizes nuclear cyclin D1 resulting in increased PRMT5-dependent histone methylation

    Techniques Used: Methylation

    8) Product Images from "Structures of the human pre-catalytic spliceosome and its precursor spliceosome"

    Article Title: Structures of the human pre-catalytic spliceosome and its precursor spliceosome

    Journal: Cell Research

    doi: 10.1038/s41422-018-0094-7

    Cryo-EM structures of the human precursor pre-catalytic spliceosome (the pre-B complex) and the pre-catalytic spliceosome (the B complex). a Structures of the pre-B complex (left panel) and the B complex (right panel). The protein and RNA components are color-coded and tabulated below the images. The pre-B complex, but not the B complex, contains Sad1, Prp28, and U1 snRNP. Components of U1 snRNP (colored grey) remain unassigned due to the low resolution of the EM density map in this region. Compared to pre-B, the B complex has eight additional protein components: CypH, FBP21, MFAP1, Prp38, Smu1, Snu23, Snu66, and UBL5. Although the U4/U6.U5 tri-snRNP is the core in both complexes, it undergoes pronounced structural rearrangement. b Structures of the RNA elements in the pre-B complex (left panel) and the B complex (right panel). The pre-mRNA, U2, U5, and U6 snRNAs are colored red, marine, orange, and green, respectively. This color scheme is preserved throughout this manuscript. In the pre-B complex, the BPS is already recognized by complementary sequences of U2 snRNA through duplex formation, and the 3’-end sequences of U6 snRNA forms helix II with the 5’-end sequences of U2 snRNA. The 5’-exon of the pre-mRNA is anchored to loop I of U5 snRNA in the B complex, but not the pre-B complex. All structural images were created using PyMol 68
    Figure Legend Snippet: Cryo-EM structures of the human precursor pre-catalytic spliceosome (the pre-B complex) and the pre-catalytic spliceosome (the B complex). a Structures of the pre-B complex (left panel) and the B complex (right panel). The protein and RNA components are color-coded and tabulated below the images. The pre-B complex, but not the B complex, contains Sad1, Prp28, and U1 snRNP. Components of U1 snRNP (colored grey) remain unassigned due to the low resolution of the EM density map in this region. Compared to pre-B, the B complex has eight additional protein components: CypH, FBP21, MFAP1, Prp38, Smu1, Snu23, Snu66, and UBL5. Although the U4/U6.U5 tri-snRNP is the core in both complexes, it undergoes pronounced structural rearrangement. b Structures of the RNA elements in the pre-B complex (left panel) and the B complex (right panel). The pre-mRNA, U2, U5, and U6 snRNAs are colored red, marine, orange, and green, respectively. This color scheme is preserved throughout this manuscript. In the pre-B complex, the BPS is already recognized by complementary sequences of U2 snRNA through duplex formation, and the 3’-end sequences of U6 snRNA forms helix II with the 5’-end sequences of U2 snRNA. The 5’-exon of the pre-mRNA is anchored to loop I of U5 snRNA in the B complex, but not the pre-B complex. All structural images were created using PyMol 68

    Techniques Used:

    9) Product Images from "YY1 Acts as a Transcriptional Activator of Hoxa5 Gene Expression in Mouse Organogenesis"

    Article Title: YY1 Acts as a Transcriptional Activator of Hoxa5 Gene Expression in Mouse Organogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093989

    Analysis of the lung phenotype in Yy1 flox/flox Dermo1 +/Cre mutants. (A)Ratios of genotypes of litters obtained from matings between Yy1 flox/+ Dermo1 +/Cre and Yy1 flox/flox mice. (B–E) Comparative lung histology of E18.5 Yy1 flox/+ Dermo1 +/+ , Yy1 flox/+ Dermo1 +/Cre , Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- embryos. Yy1 flox/+ Dermo1 +/+ and Yy1 flox/+ Dermo1 +/Cre specimens presented a normal lung structure, whereas lungs from Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- embryos were collapsed. (F–M) Characterization of the respiratory epithelium of E18.5 Yy1 flox/flox Dermo1 +/Cre embryos. (F–I) Detection of club cells by CC10 immunostaining showed decreased labelling in lungs from Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- specimens. (J–M) Immunostaining with T1α, a marker of type I pneumocytes, was reduced in lungs from Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- embryos. (N–P) YY1 immunostaining showed ubiquitous YY1 expression in lung epithelial and mesenchymal compartments in E15.5 control embryos, but an important decreased staining in lung mesenchyme from Yy1 flox/flox Dermo1 +/Cre specimens. e, epithelium; m, mesenchyme. Scale bar: 200 μm. (Q–S) qRT-PCR analysis for Scgb1a1 , T1α , Yy1 , Hoxa5 and Hoxa4 expression in lungs from E18.5 Yy1 flox/+ Dermo1 +/+ and Yy1 flox/flox Dermo1 +/Cre embryos. Expression levels were significantly diminished for all genes tested in Yy1 flox/flox Dermo1 +/Cre specimens. Values are expressed as means ± SEM. * p
    Figure Legend Snippet: Analysis of the lung phenotype in Yy1 flox/flox Dermo1 +/Cre mutants. (A)Ratios of genotypes of litters obtained from matings between Yy1 flox/+ Dermo1 +/Cre and Yy1 flox/flox mice. (B–E) Comparative lung histology of E18.5 Yy1 flox/+ Dermo1 +/+ , Yy1 flox/+ Dermo1 +/Cre , Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- embryos. Yy1 flox/+ Dermo1 +/+ and Yy1 flox/+ Dermo1 +/Cre specimens presented a normal lung structure, whereas lungs from Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- embryos were collapsed. (F–M) Characterization of the respiratory epithelium of E18.5 Yy1 flox/flox Dermo1 +/Cre embryos. (F–I) Detection of club cells by CC10 immunostaining showed decreased labelling in lungs from Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- specimens. (J–M) Immunostaining with T1α, a marker of type I pneumocytes, was reduced in lungs from Yy1 flox/flox Dermo1 +/Cre and Hoxa5 -/- embryos. (N–P) YY1 immunostaining showed ubiquitous YY1 expression in lung epithelial and mesenchymal compartments in E15.5 control embryos, but an important decreased staining in lung mesenchyme from Yy1 flox/flox Dermo1 +/Cre specimens. e, epithelium; m, mesenchyme. Scale bar: 200 μm. (Q–S) qRT-PCR analysis for Scgb1a1 , T1α , Yy1 , Hoxa5 and Hoxa4 expression in lungs from E18.5 Yy1 flox/+ Dermo1 +/+ and Yy1 flox/flox Dermo1 +/Cre embryos. Expression levels were significantly diminished for all genes tested in Yy1 flox/flox Dermo1 +/Cre specimens. Values are expressed as means ± SEM. * p

    Techniques Used: Mouse Assay, Immunostaining, Marker, Expressing, Staining, Quantitative RT-PCR

    Characterization of the 163-bp Nco I- Sac I DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 163-bp Nco I- Sac I regulatory region. Sequence of the 163-bp Nco I- Sac I DNA fragment is indicated. Oligos G1, G2, G3, G4 and Oligo-18(G3) are underlined. Boxed nucleotides correspond to YY1 binding sites. Symbols @ indicate point mutations into YY1 binding sites (see Table 1C for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 433-bp Mfe I- Sac I radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(G3) fragment via YY1 binding sites (lanes 8, 15–16). (C) EMSA with WCE and Oligo G3 probe showed binding that was competed by an excess of cold Oligo G3, Oligo-18(G3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo G2), when several mutations were distributed along the YY1 binding sites in Oligo G3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor.
    Figure Legend Snippet: Characterization of the 163-bp Nco I- Sac I DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 163-bp Nco I- Sac I regulatory region. Sequence of the 163-bp Nco I- Sac I DNA fragment is indicated. Oligos G1, G2, G3, G4 and Oligo-18(G3) are underlined. Boxed nucleotides correspond to YY1 binding sites. Symbols @ indicate point mutations into YY1 binding sites (see Table 1C for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 433-bp Mfe I- Sac I radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(G3) fragment via YY1 binding sites (lanes 8, 15–16). (C) EMSA with WCE and Oligo G3 probe showed binding that was competed by an excess of cold Oligo G3, Oligo-18(G3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo G2), when several mutations were distributed along the YY1 binding sites in Oligo G3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor.

    Techniques Used: Sequencing, Binding Assay, Protein Binding

    Characterization of the 259-bp Xba I- Bss HII DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 259-bp Xba I- Bss HII regulatory region. Sequence of the 259-bp Xba I -Bss HII DNA fragment is indicated. Fragments A, B and C used as competitor in EMSA are underlined. Fragment C was further subdivided into Oligos C1, C2, C3 and Oligo RARE and Oligo-18(C3). Boxed nucleotides correspond to RARE-DR5 sequence and YY1 binding sites. Symbols * and @ indicate point mutations into RARE and YY1 binding sites, respectively (see Table 1B for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 259-bp Xba I- Bss HII radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(C3) fragment via YY1 binding sites (lanes 9, 12, 18–19). No binding with the RARE site was observed (lanes 4-6). EMSA with in vitro -translated YY1 protein and the 259-bp Xba I- Bss HII probe showed specific binding that was competed by Oligo C3 (lanes 21–24). (C) The binding of WCE with YY1 consensus binding site and the loss of binding when the YY1 antibody was added confirmed the presence of YY1 protein in WCE (lanes 1–5). (D) EMSA with WCE and Oligo C3 radiolabelled probe showed binding that was competed by an excess of cold Oligo C3, Oligo-18(C3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo C2), when several mutations were distributed along the YY1 binding sites in Oligo C3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor; r. lysate, reticulocyte lysate.
    Figure Legend Snippet: Characterization of the 259-bp Xba I- Bss HII DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 259-bp Xba I- Bss HII regulatory region. Sequence of the 259-bp Xba I -Bss HII DNA fragment is indicated. Fragments A, B and C used as competitor in EMSA are underlined. Fragment C was further subdivided into Oligos C1, C2, C3 and Oligo RARE and Oligo-18(C3). Boxed nucleotides correspond to RARE-DR5 sequence and YY1 binding sites. Symbols * and @ indicate point mutations into RARE and YY1 binding sites, respectively (see Table 1B for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 259-bp Xba I- Bss HII radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(C3) fragment via YY1 binding sites (lanes 9, 12, 18–19). No binding with the RARE site was observed (lanes 4-6). EMSA with in vitro -translated YY1 protein and the 259-bp Xba I- Bss HII probe showed specific binding that was competed by Oligo C3 (lanes 21–24). (C) The binding of WCE with YY1 consensus binding site and the loss of binding when the YY1 antibody was added confirmed the presence of YY1 protein in WCE (lanes 1–5). (D) EMSA with WCE and Oligo C3 radiolabelled probe showed binding that was competed by an excess of cold Oligo C3, Oligo-18(C3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo C2), when several mutations were distributed along the YY1 binding sites in Oligo C3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor; r. lysate, reticulocyte lysate.

    Techniques Used: Sequencing, Binding Assay, Protein Binding, In Vitro

    Characterization of the RARE and YY1 binding sites in E13.5 F0 transgenic mouse embryos. (A) Schematic representation of the 1.5-kb Xba I- Xba I DNA fragment in the Hoxa4-Hoxa6 genomic region. (B) Diagram of the Hoxa5/lacZ constructs used to generate E13.5 F0 transgenic embryos and summary of transgenic expression analyses. The asterisks in constructs 17–18 and the @ symbols in constructs 19–22 correspond to mutations in the RARE and YY1 binding sites, respectively. (C–H) Carcass of representative E13.5 transgenic embryos and the associated organs (I–N) stained for β-galactosidase activity showed the effects of the mutations on the expression pattern. Open arrowhead points the anterior limit of transgene expression in the neural tube. i, intestine; l, lung; nt, neural tube; pv, prevertebrae; s, stomach .
    Figure Legend Snippet: Characterization of the RARE and YY1 binding sites in E13.5 F0 transgenic mouse embryos. (A) Schematic representation of the 1.5-kb Xba I- Xba I DNA fragment in the Hoxa4-Hoxa6 genomic region. (B) Diagram of the Hoxa5/lacZ constructs used to generate E13.5 F0 transgenic embryos and summary of transgenic expression analyses. The asterisks in constructs 17–18 and the @ symbols in constructs 19–22 correspond to mutations in the RARE and YY1 binding sites, respectively. (C–H) Carcass of representative E13.5 transgenic embryos and the associated organs (I–N) stained for β-galactosidase activity showed the effects of the mutations on the expression pattern. Open arrowhead points the anterior limit of transgene expression in the neural tube. i, intestine; l, lung; nt, neural tube; pv, prevertebrae; s, stomach .

    Techniques Used: Binding Assay, Transgenic Assay, Construct, Expressing, Staining, Activity Assay

    In vivo detection of YY1 protein binding to Oligo C3 and Oligo G3 regions by ChIP analysis. (A) Schematic representation and position relative to the Hoxa5 TSS of the two YY1 binding sites in the Hoxa4-Hoxa5 intergenic region. The black circles represent the Oligo C3 and Oligo G3 sequences identified by EMSA. The position of the qPCR fragments corresponding to Oligo C3, Oligo G3 and a Hox control locus located 15-kb downstream the Hoxa5 TSS that does not contain YY1 binding sites (ctl locus) is indicated. (B) ChIP analysis of the Hoxa4-Hoxa5 intergenic region in lungs from E13.5 mouse embryos. Chromatin was immunoprecipitated with rabbit IgG (negative control), anti-YY1 and anti-histone H3 (for chromatin integrity control) antibodies. Recruitment of YY1 and histone H3 on Oligo C3 and Oligo G3 sequences, an YY1 negative control ( Rcor3 ), an YY1 positive control ( Sfrs10 ), and the Hox control locus was evaluated by qPCR and is indicated as the percentage of input. The data are mean ± SEM of three independent experiments. * p
    Figure Legend Snippet: In vivo detection of YY1 protein binding to Oligo C3 and Oligo G3 regions by ChIP analysis. (A) Schematic representation and position relative to the Hoxa5 TSS of the two YY1 binding sites in the Hoxa4-Hoxa5 intergenic region. The black circles represent the Oligo C3 and Oligo G3 sequences identified by EMSA. The position of the qPCR fragments corresponding to Oligo C3, Oligo G3 and a Hox control locus located 15-kb downstream the Hoxa5 TSS that does not contain YY1 binding sites (ctl locus) is indicated. (B) ChIP analysis of the Hoxa4-Hoxa5 intergenic region in lungs from E13.5 mouse embryos. Chromatin was immunoprecipitated with rabbit IgG (negative control), anti-YY1 and anti-histone H3 (for chromatin integrity control) antibodies. Recruitment of YY1 and histone H3 on Oligo C3 and Oligo G3 sequences, an YY1 negative control ( Rcor3 ), an YY1 positive control ( Sfrs10 ), and the Hox control locus was evaluated by qPCR and is indicated as the percentage of input. The data are mean ± SEM of three independent experiments. * p

    Techniques Used: In Vivo, Protein Binding, Chromatin Immunoprecipitation, Binding Assay, Real-time Polymerase Chain Reaction, CTL Assay, Immunoprecipitation, Negative Control, Positive Control

    10) Product Images from "Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase"

    Article Title: Nuclear Cyclin D1/CDK4 Kinase Regulates CUL4 Expression and Triggers Neoplastic Growth via Activation of the PRMT5 Methyltransferase

    Journal: Cancer cell

    doi: 10.1016/j.ccr.2010.08.012

    Cyclin D1T286A/CDK4 increases PRMT5 methyltransferase activity through MEP50
    Figure Legend Snippet: Cyclin D1T286A/CDK4 increases PRMT5 methyltransferase activity through MEP50

    Techniques Used: Activity Assay

    Knockdown of Fbx4 stabilizes nuclear cyclin D1 resulting in increased PRMT5-dependent histone methylation
    Figure Legend Snippet: Knockdown of Fbx4 stabilizes nuclear cyclin D1 resulting in increased PRMT5-dependent histone methylation

    Techniques Used: Methylation

    PRMT5/MEP50 mediates cyclin D1T286A/CDK4-dependent CUL4 repression and CDT1 stabilization
    Figure Legend Snippet: PRMT5/MEP50 mediates cyclin D1T286A/CDK4-dependent CUL4 repression and CDT1 stabilization

    Techniques Used:

    Cyclin D1T286A/CDK4 activity increases PRMT5 methyltransferase activity in vivo
    Figure Legend Snippet: Cyclin D1T286A/CDK4 activity increases PRMT5 methyltransferase activity in vivo

    Techniques Used: Activity Assay, In Vivo

    PRMT5 knockdown inhibits cyclin D1T286A-dependent DNA re-replication, cell transformation and increases death of tumor cells
    Figure Legend Snippet: PRMT5 knockdown inhibits cyclin D1T286A-dependent DNA re-replication, cell transformation and increases death of tumor cells

    Techniques Used: Transformation Assay

    Identification of cyclin D1T286A/PRMT5/MEP50 complexes
    Figure Legend Snippet: Identification of cyclin D1T286A/PRMT5/MEP50 complexes

    Techniques Used:

    11) Product Images from "UBR2 of the N-End Rule Pathway Is Required for Chromosome Stability via Histone Ubiquitylation in Spermatocytes and Somatic Cells"

    Article Title: UBR2 of the N-End Rule Pathway Is Required for Chromosome Stability via Histone Ubiquitylation in Spermatocytes and Somatic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037414

    UBR2 is involved in ubiquitylation of chromatin-associated proteins in DNA-damaged somatic cells. ( A ) MEFs were treated with 0.1 µg/ml mitomycin C (MMC) or irradiated with UV at 20 J/m 2 . After 24 hrs later, cells were immunostained for UBR2 (red) and gH2AX (green). ( B ) HeLa and U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs and immunostained for UBR2. ( C ) Immunoblotting analysis of +/+ and UBR2 −/− MEFs treated with mitomycin C, UV, or MG132. ( D ) UBR2 is enriched in the nucleus of MEFs treated with 5 µM MG132. ( E ) Control and UBR2 knockdown U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs, followed by immunostaining with FK2 antibody.
    Figure Legend Snippet: UBR2 is involved in ubiquitylation of chromatin-associated proteins in DNA-damaged somatic cells. ( A ) MEFs were treated with 0.1 µg/ml mitomycin C (MMC) or irradiated with UV at 20 J/m 2 . After 24 hrs later, cells were immunostained for UBR2 (red) and gH2AX (green). ( B ) HeLa and U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs and immunostained for UBR2. ( C ) Immunoblotting analysis of +/+ and UBR2 −/− MEFs treated with mitomycin C, UV, or MG132. ( D ) UBR2 is enriched in the nucleus of MEFs treated with 5 µM MG132. ( E ) Control and UBR2 knockdown U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs, followed by immunostaining with FK2 antibody.

    Techniques Used: Irradiation, Immunostaining

    Defective DSB repair in UBR2 −/− spermatocytes. ( A ) Prolonged retention of the γH2AX staining in pachytene chromosomes of UBR2 −/− spermatocytes. Wild type and UBR2 −/− spermatocytes were stained with γH2AX (green) and SCP3 (red). Scale bars: 10 µm. ( B ) The near absence of MLH1 (green) foci in UBR2 −/− spermatocytes at pachytene. ( C ) Asynapsis in UBR2 −/− spermatocytes at pachytene as indicated by unpaired autosomes segregated near the X-Y pair. Control and UBR2 −/− chromosomes were stained for RNA polymerase II, SCP3, or BRCA1.
    Figure Legend Snippet: Defective DSB repair in UBR2 −/− spermatocytes. ( A ) Prolonged retention of the γH2AX staining in pachytene chromosomes of UBR2 −/− spermatocytes. Wild type and UBR2 −/− spermatocytes were stained with γH2AX (green) and SCP3 (red). Scale bars: 10 µm. ( B ) The near absence of MLH1 (green) foci in UBR2 −/− spermatocytes at pachytene. ( C ) Asynapsis in UBR2 −/− spermatocytes at pachytene as indicated by unpaired autosomes segregated near the X-Y pair. Control and UBR2 −/− chromosomes were stained for RNA polymerase II, SCP3, or BRCA1.

    Techniques Used: Staining

    The localization of polyubiquitin conjugates on meiotic chromosomes in comparison with UBR2. Surface-spread meiotic chromosomes were coimmunostained with FK1 antibody (red) which specifically recognizes polyubiquitin conjugates (poly-Ub) and an antibody to UBR2 (green). ( A ) Zygotene. ( B ) Early pachytene. ( C ) Mid-pachytene. ( D ) Mid-late pachytene. Different from the FK2 staining, polyubiquitin conjugates are enriched in sex chromosomes until mid-pachytene. At mid-pachytene, both polyubiquitin and UBR2 signals are drastically induced in the majority of chromosomal regions except that the chromatin domain of sex chromosomes is relatively devoid of the UBR2 staining. Scale bar: 10 µm.
    Figure Legend Snippet: The localization of polyubiquitin conjugates on meiotic chromosomes in comparison with UBR2. Surface-spread meiotic chromosomes were coimmunostained with FK1 antibody (red) which specifically recognizes polyubiquitin conjugates (poly-Ub) and an antibody to UBR2 (green). ( A ) Zygotene. ( B ) Early pachytene. ( C ) Mid-pachytene. ( D ) Mid-late pachytene. Different from the FK2 staining, polyubiquitin conjugates are enriched in sex chromosomes until mid-pachytene. At mid-pachytene, both polyubiquitin and UBR2 signals are drastically induced in the majority of chromosomal regions except that the chromatin domain of sex chromosomes is relatively devoid of the UBR2 staining. Scale bar: 10 µm.

    Techniques Used: Staining

    Neonatal lethality in UBR2 −/− newborn pups associated with defects in lung expansion and neural development. ( A ) The majority of UBR2 −/− mice in the C57 genetic background die neonatally. Shown is gross morphology of neonatal pups enriched in the C57 genetic background at P1. Surviving UBR2 −/− neonates weighed slightly less than their +/+ and UBR2 +/− littermates of the same gender. No gross morphological differences were observed between UBR2 −/− and control mice during embryogenesis and after birth. Arrowheads mark stomachs with or without milk, which indicate the feeding from mother. ( B ) The lungs of UBR2 −/− newborn pups are not properly expanded. Shown are H E-stained cross sections from +/+ and UBR2 −/− lungs at P0. Arrowheads indicate alveoli. ( C ) In situ hybridization of UBR2 mRNA on cross sections of +/+ and UBR2 −/− brains at P1. ( D ) Dilated ventricles in UBR2 −/− brain (arrowhead). Shown are Nissl-stained cross sections of +/+ and UBR2 −/− brains at P1. ( E ) Defective gliogenesis and neuronal differentiation in hippocampus of UBR2 −/− brains at P1. Cross sections of a mildly affected UBR2 −/− brain, together with its littermate control, were subjected to Nissl or DAPI staining, or immunofluorescent staining of GFAP or NeuN.
    Figure Legend Snippet: Neonatal lethality in UBR2 −/− newborn pups associated with defects in lung expansion and neural development. ( A ) The majority of UBR2 −/− mice in the C57 genetic background die neonatally. Shown is gross morphology of neonatal pups enriched in the C57 genetic background at P1. Surviving UBR2 −/− neonates weighed slightly less than their +/+ and UBR2 +/− littermates of the same gender. No gross morphological differences were observed between UBR2 −/− and control mice during embryogenesis and after birth. Arrowheads mark stomachs with or without milk, which indicate the feeding from mother. ( B ) The lungs of UBR2 −/− newborn pups are not properly expanded. Shown are H E-stained cross sections from +/+ and UBR2 −/− lungs at P0. Arrowheads indicate alveoli. ( C ) In situ hybridization of UBR2 mRNA on cross sections of +/+ and UBR2 −/− brains at P1. ( D ) Dilated ventricles in UBR2 −/− brain (arrowhead). Shown are Nissl-stained cross sections of +/+ and UBR2 −/− brains at P1. ( E ) Defective gliogenesis and neuronal differentiation in hippocampus of UBR2 −/− brains at P1. Cross sections of a mildly affected UBR2 −/− brain, together with its littermate control, were subjected to Nissl or DAPI staining, or immunofluorescent staining of GFAP or NeuN.

    Techniques Used: Mouse Assay, Staining, In Situ Hybridization

    Total ubiquitylation activities are significantly reduced in UBR2 −/− spermatocytes at pachytene. Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. ( A , B ) Leptotene. ( C , D ) Zygotene. ( E , F ) Early pachytene. ( G , H ) Mid-pachytene. ( I , J ) Late pachytene. Compared with controls, the FK2 staining in UBR2 −/− chromosomes is relatively weak in the XY body at mid-pachytene and throughout the entire chromosomal regions at mid-late pachytene. Scale bar: 10 µm.
    Figure Legend Snippet: Total ubiquitylation activities are significantly reduced in UBR2 −/− spermatocytes at pachytene. Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. ( A , B ) Leptotene. ( C , D ) Zygotene. ( E , F ) Early pachytene. ( G , H ) Mid-pachytene. ( I , J ) Late pachytene. Compared with controls, the FK2 staining in UBR2 −/− chromosomes is relatively weak in the XY body at mid-pachytene and throughout the entire chromosomal regions at mid-late pachytene. Scale bar: 10 µm.

    Techniques Used: Staining

    The localization of Ub conjugates on meiotic chromosomes in comparison with UBR2. Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to UBR2 (green). ( A ) Leptotene. ( B ) Mid−/late zygotene. ( C ) Early pachytene. ( D ) Mid-pachytene. ( E ) Mid-late pachytene. ( F , G ) The comparison of FK2 and UBR2 signals near the telomere region. Both Ub and UBR2 signals are enriched in specific regions of meiotic chromosomes until mid-pachytene, with significant colocalization along unsynapsed axial regions. At mid-pachytene, both signals are drastically induced in the majority of chromosomal regions except that the chromatin domain of sex chromosomes is relatively devoid of the UBR2 staining. The spermatocytes were staged based on the localization profile of UBR2 and the topology of the sex chromosomes. Scale bar: 10 µm (A–E), 5 µm (F, G).
    Figure Legend Snippet: The localization of Ub conjugates on meiotic chromosomes in comparison with UBR2. Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to UBR2 (green). ( A ) Leptotene. ( B ) Mid−/late zygotene. ( C ) Early pachytene. ( D ) Mid-pachytene. ( E ) Mid-late pachytene. ( F , G ) The comparison of FK2 and UBR2 signals near the telomere region. Both Ub and UBR2 signals are enriched in specific regions of meiotic chromosomes until mid-pachytene, with significant colocalization along unsynapsed axial regions. At mid-pachytene, both signals are drastically induced in the majority of chromosomal regions except that the chromatin domain of sex chromosomes is relatively devoid of the UBR2 staining. The spermatocytes were staged based on the localization profile of UBR2 and the topology of the sex chromosomes. Scale bar: 10 µm (A–E), 5 µm (F, G).

    Techniques Used: Staining

    UBR2 mediates monoubiquitylation and polyubiquitylation of H2A and H2B but not H3 and H4. ( A ) In vitro ubiquitylation assay (20 µL) with 1 µg of histone H2A, H2B, H1, or H3. The reaction contains 100 ng E3-F (or E3-V) prepared from rat testes, 30 ng UbcH2, and Ub activating reagents, including 1 µg flag-Ub and 100 ng E1. E3-F and E3-V represent protein mixtures that have been captured by Phe-peptide and Val-peptide, respectively. ( B ) In vitro binding assay in which UBR2 (as a mixture with UBR1) immobilized on Phe-peptide-beads was mixed with histone H2A, H2B, or H3 in the presence of HR6B, E1, and Ub activating reagents, followed by immunoblotting of histones retained by X-peptide (X = Phe or Val). ( C ) The screening of E2s which can support E3-F-mediated ubiquitylation of H2A. In vitro ubiquitylation assays were performed as (A) with different E2s indicated above. In this screening, UbcH2 and UbcH5b showed reproducibly the E2 activity in H2A ubiquitylation. ( D – F ) Allosteric modulation, an additional E2, and synthetic ligands for UBR2. ( D ) The interaction between UBR2 and HR6B is cooperatively accelerated by H2A and Arg-Ala. UBR2 (0.2 µg) from 10 mg rat testes extracts were immobilized with Phe-peptide conjugated with beads. Precipitated E3-peptide beads were mixed with 60 ng HR6B, 1 µg H2A, and/or 2 mM Arg-Ala, followed by immunoblotting analysis. ( E ) The HR6B-H2A interaction is cooperatively facilitated by UBR2 and Arg-Ala. GST-pulldown assays were done with 200 ng GST-HR6B, 200 ng UBR2, 1 µg H2A, and/or 2 mM Arg-Ala. ( F ) UBR2-dependent H2A ubiquitylation is synergistically activated by type-1 and type-2 N-end rule ligands.
    Figure Legend Snippet: UBR2 mediates monoubiquitylation and polyubiquitylation of H2A and H2B but not H3 and H4. ( A ) In vitro ubiquitylation assay (20 µL) with 1 µg of histone H2A, H2B, H1, or H3. The reaction contains 100 ng E3-F (or E3-V) prepared from rat testes, 30 ng UbcH2, and Ub activating reagents, including 1 µg flag-Ub and 100 ng E1. E3-F and E3-V represent protein mixtures that have been captured by Phe-peptide and Val-peptide, respectively. ( B ) In vitro binding assay in which UBR2 (as a mixture with UBR1) immobilized on Phe-peptide-beads was mixed with histone H2A, H2B, or H3 in the presence of HR6B, E1, and Ub activating reagents, followed by immunoblotting of histones retained by X-peptide (X = Phe or Val). ( C ) The screening of E2s which can support E3-F-mediated ubiquitylation of H2A. In vitro ubiquitylation assays were performed as (A) with different E2s indicated above. In this screening, UbcH2 and UbcH5b showed reproducibly the E2 activity in H2A ubiquitylation. ( D – F ) Allosteric modulation, an additional E2, and synthetic ligands for UBR2. ( D ) The interaction between UBR2 and HR6B is cooperatively accelerated by H2A and Arg-Ala. UBR2 (0.2 µg) from 10 mg rat testes extracts were immobilized with Phe-peptide conjugated with beads. Precipitated E3-peptide beads were mixed with 60 ng HR6B, 1 µg H2A, and/or 2 mM Arg-Ala, followed by immunoblotting analysis. ( E ) The HR6B-H2A interaction is cooperatively facilitated by UBR2 and Arg-Ala. GST-pulldown assays were done with 200 ng GST-HR6B, 200 ng UBR2, 1 µg H2A, and/or 2 mM Arg-Ala. ( F ) UBR2-dependent H2A ubiquitylation is synergistically activated by type-1 and type-2 N-end rule ligands.

    Techniques Used: In Vitro, Ubiquitin Assay, Binding Assay, Activity Assay

    Pachytene arrest of UBR2 −/− spermatocytes at stage IV. ( A – D ) Testis sections from 8-week (A–C) and 3-week (D) old UBR2 −/− tubules. ( A ) Tubules that did not yet reach epithelial stage IV. A large number of spermatocytes (thick arrow) are present, indicating that the spermatogonial compartment keeps forming spermatocytes. Arrowhead, diplotene spermatocyte; thin arrow, round spermatid. The predecessors of these cells survived the stage IV arrest. ( B ) Tubules in epithelial stage IV as evidenced by the presence of large, G2 phase intermediate (In) spermatogonia (blue arrow) about to or dividing into B spermatogonia (yellow arrowhead). There is massive apoptosis of spermatocytes (asterisks). ( C ) Tubules after stage IV. A variable number of spermatocytes survive the passage through stage IV. The left tubule shows only one spermatocyte (arrowhead) and a few round spermatids (thin arrow) that stem from spermatocytes that survived stage IV one epithelial cycle earlier. The tubule on the right shows more spermatocytes (arrowhead) and round spermatids (black arrow) and even a few elongated spermatids (yellow arrow). ( D ) Stage IV arrest at the age of 3 weeks. The lower tubule shows massive apoptosis (asterisk). The upper tubule is after stage IV and shows two surviving spermatocytes (arrowhead), indicating that the arrest was already present before three weeks. ( E ) Surface-spread chromosomes of 781 control and 691 UBR2 −/− spermatocytes isolated from mice at P17 were stained with SCP3 and staged based on the morphology of SCP3-positive chromosomes. ( F ) Surface-spread chromosomes of 344 +/+ and 161 UBR2 −/− pachytene spermatocytes were substaged.
    Figure Legend Snippet: Pachytene arrest of UBR2 −/− spermatocytes at stage IV. ( A – D ) Testis sections from 8-week (A–C) and 3-week (D) old UBR2 −/− tubules. ( A ) Tubules that did not yet reach epithelial stage IV. A large number of spermatocytes (thick arrow) are present, indicating that the spermatogonial compartment keeps forming spermatocytes. Arrowhead, diplotene spermatocyte; thin arrow, round spermatid. The predecessors of these cells survived the stage IV arrest. ( B ) Tubules in epithelial stage IV as evidenced by the presence of large, G2 phase intermediate (In) spermatogonia (blue arrow) about to or dividing into B spermatogonia (yellow arrowhead). There is massive apoptosis of spermatocytes (asterisks). ( C ) Tubules after stage IV. A variable number of spermatocytes survive the passage through stage IV. The left tubule shows only one spermatocyte (arrowhead) and a few round spermatids (thin arrow) that stem from spermatocytes that survived stage IV one epithelial cycle earlier. The tubule on the right shows more spermatocytes (arrowhead) and round spermatids (black arrow) and even a few elongated spermatids (yellow arrow). ( D ) Stage IV arrest at the age of 3 weeks. The lower tubule shows massive apoptosis (asterisk). The upper tubule is after stage IV and shows two surviving spermatocytes (arrowhead), indicating that the arrest was already present before three weeks. ( E ) Surface-spread chromosomes of 781 control and 691 UBR2 −/− spermatocytes isolated from mice at P17 were stained with SCP3 and staged based on the morphology of SCP3-positive chromosomes. ( F ) Surface-spread chromosomes of 344 +/+ and 161 UBR2 −/− pachytene spermatocytes were substaged.

    Techniques Used: Isolation, Mouse Assay, Staining

    Chromosome instability and hypersensitivity to DNA damage of UBR2-deficient somatic cells. ( A ) UBR2-knockdown induces hyperproliferation in HeLa cells. ( B ) Metaphase chromosomes of UBR2 −/− MEFs show increased chromosomal aberrations, including breaks and fragmentations, compared with control cells. Arrowhead, break; Arrow, fragmentation ( C ) Quantitation of chromosomal abnormalities (breaks and fragments) observed in metaphase chromosomes from +/+ and UBR2 −/− MEFs. ( D ) UBR2 −/− MEFs are hypersensitive to hydroxyurea, or methyl methanesulfonate (Sigma).
    Figure Legend Snippet: Chromosome instability and hypersensitivity to DNA damage of UBR2-deficient somatic cells. ( A ) UBR2-knockdown induces hyperproliferation in HeLa cells. ( B ) Metaphase chromosomes of UBR2 −/− MEFs show increased chromosomal aberrations, including breaks and fragmentations, compared with control cells. Arrowhead, break; Arrow, fragmentation ( C ) Quantitation of chromosomal abnormalities (breaks and fragments) observed in metaphase chromosomes from +/+ and UBR2 −/− MEFs. ( D ) UBR2 −/− MEFs are hypersensitive to hydroxyurea, or methyl methanesulfonate (Sigma).

    Techniques Used: Quantitation Assay

    Genome-wide polyubiquitylation activities on meiotic chromosomes are reduced in UBR2 −/− spermatocytes. Surface-spread meiotic chromosomes were coimmunostained with FK1 antibody (red) which specifically recognizes polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. ( A ) Zygotene. ( B ) Early pachytene. ( C ) Mid-pachytene. Arrowhead indicates the sex chromosome. Note that polyubiquitin signals are virtually nondetectible in UBR2 −/− chromosomes. Scale bar: 10 µm.
    Figure Legend Snippet: Genome-wide polyubiquitylation activities on meiotic chromosomes are reduced in UBR2 −/− spermatocytes. Surface-spread meiotic chromosomes were coimmunostained with FK1 antibody (red) which specifically recognizes polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. ( A ) Zygotene. ( B ) Early pachytene. ( C ) Mid-pachytene. Arrowhead indicates the sex chromosome. Note that polyubiquitin signals are virtually nondetectible in UBR2 −/− chromosomes. Scale bar: 10 µm.

    Techniques Used: Genome Wide

    UBR2 is associated with chromatin during cell cycle of somatic cells. ( A ) UBR2 is enriched in the nucleus of MEFs. MEFs were stained for UBR2 without (top) and with a peptide that has been used to raise the antibody. ( B ) UBR2 is associated with chromatin in MEFs. Control and UBR2 −/− cells were separated into cytosolic, nuclear soluble, and chromatin-bound fractions in the presence or absence of iodoacetamide, followed by immunoblotting for proteins indicated. ( C ) Chromatin association of UBR2 is cell cycle-dependent. HeLa cells were synchronized at the G1-S border using the double thymidine block, released from G1-S arrest, and subjected to time-course fractionation and immunoblotting. Cell cycle stages were verified using flow cytometry and based on behaviors of cell cycle regulators, including down-regulation of chromatin-associated cyclin A and CDC6.
    Figure Legend Snippet: UBR2 is associated with chromatin during cell cycle of somatic cells. ( A ) UBR2 is enriched in the nucleus of MEFs. MEFs were stained for UBR2 without (top) and with a peptide that has been used to raise the antibody. ( B ) UBR2 is associated with chromatin in MEFs. Control and UBR2 −/− cells were separated into cytosolic, nuclear soluble, and chromatin-bound fractions in the presence or absence of iodoacetamide, followed by immunoblotting for proteins indicated. ( C ) Chromatin association of UBR2 is cell cycle-dependent. HeLa cells were synchronized at the G1-S border using the double thymidine block, released from G1-S arrest, and subjected to time-course fractionation and immunoblotting. Cell cycle stages were verified using flow cytometry and based on behaviors of cell cycle regulators, including down-regulation of chromatin-associated cyclin A and CDC6.

    Techniques Used: Staining, Blocking Assay, Fractionation, Flow Cytometry, Cytometry

    12) Product Images from "A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs"

    Article Title: A Novel Set of Cas9 Fusion Proteins to stimulate Homologous Recombination: Cas9-HRs

    Journal: bioRxiv

    doi: 10.1101/2020.05.17.100677

    Enzymatically active Cas9-HRs can be purified from E. Coli . (A) SDS-PAGE gel showing successful purification of Cas9-HR 3. Lanes 1-7 Cas9-HR 3, Lanes 8-15 Cas9. Lanes 1,8 whole lysate; 2,9 soluble fraction; 3,10 insoluble fraction; 4,11 His-purification; 5,12 Sepharose wash; 6,13 Sepharose Elution 1; 7,14 Sepharose Elution 2. (B) Diagram of HBB genomic region, with primers in red showing the amplified fragment (956bp). (C) Agarose Gel image of exonuclease activity of purified Cas9-HRs 3,4,8, Cas9, Solvent (Cas9-HR buffer), and DNA (Con). Reduction in band intensity of Cas9-HR 3 and 8 indicates exonuclease activity.
    Figure Legend Snippet: Enzymatically active Cas9-HRs can be purified from E. Coli . (A) SDS-PAGE gel showing successful purification of Cas9-HR 3. Lanes 1-7 Cas9-HR 3, Lanes 8-15 Cas9. Lanes 1,8 whole lysate; 2,9 soluble fraction; 3,10 insoluble fraction; 4,11 His-purification; 5,12 Sepharose wash; 6,13 Sepharose Elution 1; 7,14 Sepharose Elution 2. (B) Diagram of HBB genomic region, with primers in red showing the amplified fragment (956bp). (C) Agarose Gel image of exonuclease activity of purified Cas9-HRs 3,4,8, Cas9, Solvent (Cas9-HR buffer), and DNA (Con). Reduction in band intensity of Cas9-HR 3 and 8 indicates exonuclease activity.

    Techniques Used: Purification, SDS Page, Amplification, Agarose Gel Electrophoresis, Activity Assay

    13) Product Images from "YY1 Acts as a Transcriptional Activator of Hoxa5 Gene Expression in Mouse Organogenesis"

    Article Title: YY1 Acts as a Transcriptional Activator of Hoxa5 Gene Expression in Mouse Organogenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093989

    Characterization of the 163-bp Nco I- Sac I DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 163-bp Nco I- Sac I regulatory region. Sequence of the 163-bp Nco I- Sac I DNA fragment is indicated. Oligos G1, G2, G3, G4 and Oligo-18(G3) are underlined. Boxed nucleotides correspond to YY1 binding sites. Symbols @ indicate point mutations into YY1 binding sites (see Table 1C for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 433-bp Mfe I- Sac I radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(G3) fragment via YY1 binding sites (lanes 8, 15–16). (C) EMSA with WCE and Oligo G3 probe showed binding that was competed by an excess of cold Oligo G3, Oligo-18(G3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo G2), when several mutations were distributed along the YY1 binding sites in Oligo G3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor.
    Figure Legend Snippet: Characterization of the 163-bp Nco I- Sac I DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 163-bp Nco I- Sac I regulatory region. Sequence of the 163-bp Nco I- Sac I DNA fragment is indicated. Oligos G1, G2, G3, G4 and Oligo-18(G3) are underlined. Boxed nucleotides correspond to YY1 binding sites. Symbols @ indicate point mutations into YY1 binding sites (see Table 1C for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 433-bp Mfe I- Sac I radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(G3) fragment via YY1 binding sites (lanes 8, 15–16). (C) EMSA with WCE and Oligo G3 probe showed binding that was competed by an excess of cold Oligo G3, Oligo-18(G3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo G2), when several mutations were distributed along the YY1 binding sites in Oligo G3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor.

    Techniques Used: Sequencing, Binding Assay, Protein Binding

    Characterization of the 259-bp Xba I- Bss HII DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 259-bp Xba I- Bss HII regulatory region. Sequence of the 259-bp Xba I -Bss HII DNA fragment is indicated. Fragments A, B and C used as competitor in EMSA are underlined. Fragment C was further subdivided into Oligos C1, C2, C3 and Oligo RARE and Oligo-18(C3). Boxed nucleotides correspond to RARE-DR5 sequence and YY1 binding sites. Symbols * and @ indicate point mutations into RARE and YY1 binding sites, respectively (see Table 1B for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 259-bp Xba I- Bss HII radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(C3) fragment via YY1 binding sites (lanes 9, 12, 18–19). No binding with the RARE site was observed (lanes 4-6). EMSA with in vitro -translated YY1 protein and the 259-bp Xba I- Bss HII probe showed specific binding that was competed by Oligo C3 (lanes 21–24). (C) The binding of WCE with YY1 consensus binding site and the loss of binding when the YY1 antibody was added confirmed the presence of YY1 protein in WCE (lanes 1–5). (D) EMSA with WCE and Oligo C3 radiolabelled probe showed binding that was competed by an excess of cold Oligo C3, Oligo-18(C3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo C2), when several mutations were distributed along the YY1 binding sites in Oligo C3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor; r. lysate, reticulocyte lysate.
    Figure Legend Snippet: Characterization of the 259-bp Xba I- Bss HII DNA fragment by EMSA. (A) Restriction map of the 1.5-kb Xba I- Xba I sequence extending from +9.3-kb to +10.8-kb in the 3′ half of the Hoxa4 - Hoxa5 intergenic region. The box denotes the location of the 259-bp Xba I- Bss HII regulatory region. Sequence of the 259-bp Xba I -Bss HII DNA fragment is indicated. Fragments A, B and C used as competitor in EMSA are underlined. Fragment C was further subdivided into Oligos C1, C2, C3 and Oligo RARE and Oligo-18(C3). Boxed nucleotides correspond to RARE-DR5 sequence and YY1 binding sites. Symbols * and @ indicate point mutations into RARE and YY1 binding sites, respectively (see Table 1B for nucleotide sequences). (B) EMSA with WCE from lung/stomach/intestine of E13.5 embryos and the 259-bp Xba I- Bss HII radiolabelled probe in presence of unlabelled competitors in 100-fold excess showed that protein binding occurred with the Oligo-18(C3) fragment via YY1 binding sites (lanes 9, 12, 18–19). No binding with the RARE site was observed (lanes 4-6). EMSA with in vitro -translated YY1 protein and the 259-bp Xba I- Bss HII probe showed specific binding that was competed by Oligo C3 (lanes 21–24). (C) The binding of WCE with YY1 consensus binding site and the loss of binding when the YY1 antibody was added confirmed the presence of YY1 protein in WCE (lanes 1–5). (D) EMSA with WCE and Oligo C3 radiolabelled probe showed binding that was competed by an excess of cold Oligo C3, Oligo-18(C3) sequence, the YY1 consensus sequence, localized mutations in YY1 sites, and the addition of the YY1-specific antibody (lanes 1–4, 6–9). No competition occurred when a non-specific probe was used (Oligo C2), when several mutations were distributed along the YY1 binding sites in Oligo C3 or when the CDX2 control antibody was used (lanes 5, 10–11). Arrows and brackets indicate the bands corresponding to YY1 binding. COMP, competitor; r. lysate, reticulocyte lysate.

    Techniques Used: Sequencing, Binding Assay, Protein Binding, In Vitro

    14) Product Images from "KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1"

    Article Title: KDM2A integrates DNA and histone modification signals through a CXXC/PHD module and direct interaction with HP1

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw979

    Mapping of nucleosome binding sites within KDM2A. ( A ) Schematic representation of the assembly of modified nucleosomes and nucleosome pull-downs. Modified histone H3.1 was prepared by native chemical ligation and assembled into nucleosomes together with purified core histones H2A, H2B and H4 and nucleosomal 601-DNA. Nucleosomes were immobilized on streptavidin beads via the biotinylated DNA and incubated with protein extracts or recombinant proteins to identify modification-binding factors. ( B ) Recruitment of KDM2A to H3K9me3-modified nucleosomes is stimulated by HP1 and counteracted by CpG-methylation. HeLa S3 nuclear extracts were incubated with immobilized modified nucleosomes as indicated. Binding reactions were supplemented with recombinant purified HP1α or GST as a control. 4% of the nuclear extract input and 20% of the pull-downs were separated by SDS-PAGE and nucleosome-bound KDM2A and HP1α were detected by immunoblot. Equal loading was confirmed by Coomassie stain and modification of histone H3 was verified by immunoblot against H3 tri-methyl lysine marks. Both, the full length and the short isoform of KDM2A (KDM2A SF ) show increased binding to H3K9me3-modified nucleosomes, which is strongly stimulated by addition of HP1α and reduced by CpG-methylation. ( C ) Mapping of nucleosome binding domains in KDM2A. Unmodified or H3K9me3-modified nucleosomes were immobilized on streptavidin beads and incubated with 293T whole cell extracts overexpressing wild-type (WT) FLAG-GFP-tagged KDM2A or point/deletion mutants as indicated. All binding reactions were supplemented with recombinant purified HP1α. Forty percent of each input and pull-down were separated by SDS-PAGE and binding of KDM2A was detected by immunoblot against the FLAG tag. The S603D point mutation disrupts binding of the CXXC-ZnF to CpG dinucleotides ( 37 ) and the PHD C > A mutant contains C620A and C623A point mutations disrupting the structural integrity of the PHD domain. In the ΔF-box mutant amino acids 893 to 933 and in the ΔLRR mutant amino acids 1000 to 1118 are deleted. Loading controls can be found in Supplementary Figure S2A . ( D ) Mapping of a nucleosome interaction module within KDM2A. Nucleosome binding reactions were carried out as above using FLAG-GFP-tagged full length (FL) KDM2A or N- or C-terminal deletion mutants including amino acids as indicated. Loading controls can be found in Supplementary Figure S2B . ( E ) Schematic representation of the domain structure of KDM2A and the point and deletion mutants used in Figure 1C and D .
    Figure Legend Snippet: Mapping of nucleosome binding sites within KDM2A. ( A ) Schematic representation of the assembly of modified nucleosomes and nucleosome pull-downs. Modified histone H3.1 was prepared by native chemical ligation and assembled into nucleosomes together with purified core histones H2A, H2B and H4 and nucleosomal 601-DNA. Nucleosomes were immobilized on streptavidin beads via the biotinylated DNA and incubated with protein extracts or recombinant proteins to identify modification-binding factors. ( B ) Recruitment of KDM2A to H3K9me3-modified nucleosomes is stimulated by HP1 and counteracted by CpG-methylation. HeLa S3 nuclear extracts were incubated with immobilized modified nucleosomes as indicated. Binding reactions were supplemented with recombinant purified HP1α or GST as a control. 4% of the nuclear extract input and 20% of the pull-downs were separated by SDS-PAGE and nucleosome-bound KDM2A and HP1α were detected by immunoblot. Equal loading was confirmed by Coomassie stain and modification of histone H3 was verified by immunoblot against H3 tri-methyl lysine marks. Both, the full length and the short isoform of KDM2A (KDM2A SF ) show increased binding to H3K9me3-modified nucleosomes, which is strongly stimulated by addition of HP1α and reduced by CpG-methylation. ( C ) Mapping of nucleosome binding domains in KDM2A. Unmodified or H3K9me3-modified nucleosomes were immobilized on streptavidin beads and incubated with 293T whole cell extracts overexpressing wild-type (WT) FLAG-GFP-tagged KDM2A or point/deletion mutants as indicated. All binding reactions were supplemented with recombinant purified HP1α. Forty percent of each input and pull-down were separated by SDS-PAGE and binding of KDM2A was detected by immunoblot against the FLAG tag. The S603D point mutation disrupts binding of the CXXC-ZnF to CpG dinucleotides ( 37 ) and the PHD C > A mutant contains C620A and C623A point mutations disrupting the structural integrity of the PHD domain. In the ΔF-box mutant amino acids 893 to 933 and in the ΔLRR mutant amino acids 1000 to 1118 are deleted. Loading controls can be found in Supplementary Figure S2A . ( D ) Mapping of a nucleosome interaction module within KDM2A. Nucleosome binding reactions were carried out as above using FLAG-GFP-tagged full length (FL) KDM2A or N- or C-terminal deletion mutants including amino acids as indicated. Loading controls can be found in Supplementary Figure S2B . ( E ) Schematic representation of the domain structure of KDM2A and the point and deletion mutants used in Figure 1C and D .

    Techniques Used: Binding Assay, Modification, Ligation, Purification, Incubation, Recombinant, CpG Methylation Assay, SDS Page, Staining, FLAG-tag, Mutagenesis

    HP1α recruits KDM2A to H3K9me3-modified nucleosomes independently of DNA binding. ( A ) HP1-mediated recruitment of KDM2A to H3K9me3-modified nucleosomes independently of the ZnF and PHD domains. Unmodified or H3K9me3-modified nucleosomes were immobilized on streptavidin beads and incubated with WT HP1α and WT full length FLAG-GFP-tagged KDM2A or mutants in the ZnF (S603D) or PHD (PHD C > A) domains or the LxVxL motif (V801A/L803A) as indicated. 15% of the KDM2A inputs and pull-downs and 3% of the HP1α input were separated by SDS-PAGE and nucleosome-bound KDM2A and HP1α were detected by immunoblot. Equal loading was confirmed by immunoblot against Histone H3 and H3K9me3 modification of histone H3 was verified by immunoblot against the H3 tri-methyl lysine 9 mark. The apparent stronger binding of the V801A/L803A mutant is due to the difference in the input compared to the WT pull-downs. ( B ) Recruitment of KDM2A to H3K9me3-modified nucleosomes via HP1 is mediated through its LxVxL motif. Unmodified or H3K9me3-modified nucleosomes containing either unmethylated or CpG-methylated 601-DNA were immobilized on streptavidin beads and incubated with WT HP1α and WT full length FLAG-GFP-tagged KDM2A or the V801A/L803A mutant as indicated. Binding of KDM2A and HP1α to the nucleosomes was detected as described in Figure 3A .
    Figure Legend Snippet: HP1α recruits KDM2A to H3K9me3-modified nucleosomes independently of DNA binding. ( A ) HP1-mediated recruitment of KDM2A to H3K9me3-modified nucleosomes independently of the ZnF and PHD domains. Unmodified or H3K9me3-modified nucleosomes were immobilized on streptavidin beads and incubated with WT HP1α and WT full length FLAG-GFP-tagged KDM2A or mutants in the ZnF (S603D) or PHD (PHD C > A) domains or the LxVxL motif (V801A/L803A) as indicated. 15% of the KDM2A inputs and pull-downs and 3% of the HP1α input were separated by SDS-PAGE and nucleosome-bound KDM2A and HP1α were detected by immunoblot. Equal loading was confirmed by immunoblot against Histone H3 and H3K9me3 modification of histone H3 was verified by immunoblot against the H3 tri-methyl lysine 9 mark. The apparent stronger binding of the V801A/L803A mutant is due to the difference in the input compared to the WT pull-downs. ( B ) Recruitment of KDM2A to H3K9me3-modified nucleosomes via HP1 is mediated through its LxVxL motif. Unmodified or H3K9me3-modified nucleosomes containing either unmethylated or CpG-methylated 601-DNA were immobilized on streptavidin beads and incubated with WT HP1α and WT full length FLAG-GFP-tagged KDM2A or the V801A/L803A mutant as indicated. Binding of KDM2A and HP1α to the nucleosomes was detected as described in Figure 3A .

    Techniques Used: Modification, Binding Assay, Incubation, SDS Page, Mutagenesis, Methylation

    Model for KDM2A-mediated establishment of heterochromatin. The CXXC-Znf enables KDM2A to bind chromatin containing no pre-existing DNA and histone modifications (top). Binding of HP1 to the KDM2A LTVTL motif establishes an alternative to the HP1 recruitment mechanism via the H3K9me3-modification deposited by H3K9 lysine methyltransferases (KMTs) that does not require the K3K9me3 mark. Subsequent deposition of the H3K9me3-modification by H3K9 KMTs leads to reinforced HP1 binding and a stabilization of the KDM2A/HP1/nucleosome interaction. The ability of HP1 to recruit DNMTs leads to deposition of CpG methylation (black pinheads) which results in loss of KDM2A leaving behind DNA-methylated, H3K9me3-modified and HP1-decorated heterochromatin (bottom).
    Figure Legend Snippet: Model for KDM2A-mediated establishment of heterochromatin. The CXXC-Znf enables KDM2A to bind chromatin containing no pre-existing DNA and histone modifications (top). Binding of HP1 to the KDM2A LTVTL motif establishes an alternative to the HP1 recruitment mechanism via the H3K9me3-modification deposited by H3K9 lysine methyltransferases (KMTs) that does not require the K3K9me3 mark. Subsequent deposition of the H3K9me3-modification by H3K9 KMTs leads to reinforced HP1 binding and a stabilization of the KDM2A/HP1/nucleosome interaction. The ability of HP1 to recruit DNMTs leads to deposition of CpG methylation (black pinheads) which results in loss of KDM2A leaving behind DNA-methylated, H3K9me3-modified and HP1-decorated heterochromatin (bottom).

    Techniques Used: Binding Assay, Modification, CpG Methylation Assay, Methylation

    15) Product Images from "UBR2 of the N-End Rule Pathway Is Required for Chromosome Stability via Histone Ubiquitylation in Spermatocytes and Somatic Cells"

    Article Title: UBR2 of the N-End Rule Pathway Is Required for Chromosome Stability via Histone Ubiquitylation in Spermatocytes and Somatic Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0037414

    UBR2 is involved in ubiquitylation of chromatin-associated proteins in DNA-damaged somatic cells. ( A ) MEFs were treated with 0.1 µg/ml mitomycin C (MMC) or irradiated with UV at 20 J/m 2 . After 24 hrs later, cells were immunostained for UBR2 (red) and gH2AX (green). ( B ) HeLa and U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs and immunostained for UBR2. ( C ) Immunoblotting analysis of +/+ and UBR2 −/− MEFs treated with mitomycin C, UV, or MG132. ( D ) UBR2 is enriched in the nucleus of MEFs treated with 5 µM MG132. ( E ) Control and UBR2 knockdown U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs, followed by immunostaining with FK2 antibody.
    Figure Legend Snippet: UBR2 is involved in ubiquitylation of chromatin-associated proteins in DNA-damaged somatic cells. ( A ) MEFs were treated with 0.1 µg/ml mitomycin C (MMC) or irradiated with UV at 20 J/m 2 . After 24 hrs later, cells were immunostained for UBR2 (red) and gH2AX (green). ( B ) HeLa and U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs and immunostained for UBR2. ( C ) Immunoblotting analysis of +/+ and UBR2 −/− MEFs treated with mitomycin C, UV, or MG132. ( D ) UBR2 is enriched in the nucleus of MEFs treated with 5 µM MG132. ( E ) Control and UBR2 knockdown U2OS cells were treated with 0.1 µg/ml mitomycin C for 24 hrs, followed by immunostaining with FK2 antibody.

    Techniques Used: Irradiation, Immunostaining

    Total ubiquitylation activities are significantly reduced in UBR2 −/− spermatocytes at pachytene. Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. ( A , B ) Leptotene. ( C , D ) Zygotene. ( E , F ) Early pachytene. ( G , H ) Mid-pachytene. ( I , J ) Late pachytene. Compared with controls, the FK2 staining in UBR2 −/− chromosomes is relatively weak in the XY body at mid-pachytene and throughout the entire chromosomal regions at mid-late pachytene. Scale bar: 10 µm.
    Figure Legend Snippet: Total ubiquitylation activities are significantly reduced in UBR2 −/− spermatocytes at pachytene. Surface-spread meiotic chromosomes were coimmunostained with FK2 antibody (red) which recognizes both monoubiquitin and polyubiquitin conjugates and an antibody to SCP3 (green), a component of the synaptonemal complex. ( A , B ) Leptotene. ( C , D ) Zygotene. ( E , F ) Early pachytene. ( G , H ) Mid-pachytene. ( I , J ) Late pachytene. Compared with controls, the FK2 staining in UBR2 −/− chromosomes is relatively weak in the XY body at mid-pachytene and throughout the entire chromosomal regions at mid-late pachytene. Scale bar: 10 µm.

    Techniques Used: Staining

    UBR2 mediates monoubiquitylation and polyubiquitylation of H2A and H2B but not H3 and H4. ( A ) In vitro ubiquitylation assay (20 µL) with 1 µg of histone H2A, H2B, H1, or H3. The reaction contains 100 ng E3-F (or E3-V) prepared from rat testes, 30 ng UbcH2, and Ub activating reagents, including 1 µg flag-Ub and 100 ng E1. E3-F and E3-V represent protein mixtures that have been captured by Phe-peptide and Val-peptide, respectively. ( B ) In vitro binding assay in which UBR2 (as a mixture with UBR1) immobilized on Phe-peptide-beads was mixed with histone H2A, H2B, or H3 in the presence of HR6B, E1, and Ub activating reagents, followed by immunoblotting of histones retained by X-peptide (X = Phe or Val). ( C ) The screening of E2s which can support E3-F-mediated ubiquitylation of H2A. In vitro ubiquitylation assays were performed as (A) with different E2s indicated above. In this screening, UbcH2 and UbcH5b showed reproducibly the E2 activity in H2A ubiquitylation. ( D – F ) Allosteric modulation, an additional E2, and synthetic ligands for UBR2. ( D ) The interaction between UBR2 and HR6B is cooperatively accelerated by H2A and Arg-Ala. UBR2 (0.2 µg) from 10 mg rat testes extracts were immobilized with Phe-peptide conjugated with beads. Precipitated E3-peptide beads were mixed with 60 ng HR6B, 1 µg H2A, and/or 2 mM Arg-Ala, followed by immunoblotting analysis. ( E ) The HR6B-H2A interaction is cooperatively facilitated by UBR2 and Arg-Ala. GST-pulldown assays were done with 200 ng GST-HR6B, 200 ng UBR2, 1 µg H2A, and/or 2 mM Arg-Ala. ( F ) UBR2-dependent H2A ubiquitylation is synergistically activated by type-1 and type-2 N-end rule ligands.
    Figure Legend Snippet: UBR2 mediates monoubiquitylation and polyubiquitylation of H2A and H2B but not H3 and H4. ( A ) In vitro ubiquitylation assay (20 µL) with 1 µg of histone H2A, H2B, H1, or H3. The reaction contains 100 ng E3-F (or E3-V) prepared from rat testes, 30 ng UbcH2, and Ub activating reagents, including 1 µg flag-Ub and 100 ng E1. E3-F and E3-V represent protein mixtures that have been captured by Phe-peptide and Val-peptide, respectively. ( B ) In vitro binding assay in which UBR2 (as a mixture with UBR1) immobilized on Phe-peptide-beads was mixed with histone H2A, H2B, or H3 in the presence of HR6B, E1, and Ub activating reagents, followed by immunoblotting of histones retained by X-peptide (X = Phe or Val). ( C ) The screening of E2s which can support E3-F-mediated ubiquitylation of H2A. In vitro ubiquitylation assays were performed as (A) with different E2s indicated above. In this screening, UbcH2 and UbcH5b showed reproducibly the E2 activity in H2A ubiquitylation. ( D – F ) Allosteric modulation, an additional E2, and synthetic ligands for UBR2. ( D ) The interaction between UBR2 and HR6B is cooperatively accelerated by H2A and Arg-Ala. UBR2 (0.2 µg) from 10 mg rat testes extracts were immobilized with Phe-peptide conjugated with beads. Precipitated E3-peptide beads were mixed with 60 ng HR6B, 1 µg H2A, and/or 2 mM Arg-Ala, followed by immunoblotting analysis. ( E ) The HR6B-H2A interaction is cooperatively facilitated by UBR2 and Arg-Ala. GST-pulldown assays were done with 200 ng GST-HR6B, 200 ng UBR2, 1 µg H2A, and/or 2 mM Arg-Ala. ( F ) UBR2-dependent H2A ubiquitylation is synergistically activated by type-1 and type-2 N-end rule ligands.

    Techniques Used: In Vitro, Ubiquitin Assay, Binding Assay, Activity Assay

    16) Product Images from "Inhibition of chromatin remodeling by Polycomb Group protein Posterior Sex Combs is mechanistically distinct from nucleosome binding 1"

    Article Title: Inhibition of chromatin remodeling by Polycomb Group protein Posterior Sex Combs is mechanistically distinct from nucleosome binding 1

    Journal: Biochemistry

    doi: 10.1021/bi100532a

    PSC inhibits remodeling of mononucleosomes poorly Restriction enzyme accessibility (REA) assays were carried out with PSC at the indicated concentrations and hSwi/Snf. A,B) Representative gels of REAs with 6N (A) and 2N (B) templates. (N refers to nucleosomes, so 6N is a 6-nucleosome template; all of these templates are composed of repeats of the 5S nucleosome positioning sequence.). C) Summary of REAs with 6N and 2N templates. D, E) Representative gel of REA with mononucleosomes assembled on a 157-bp (10-1N) 5S template at 1nM (D) or 5nM (E). Note that PstI was used for digestion in the experiment with 1nM nucleosomes, while HhaI was used with 5nM nucleosomes. Both enzymes have single digestion sites in the template, but produce slightly different digestion patterns (HhaI digests the template into 77 and 80 bp fragments, which are not resolved, while PstI digests it into 99 and 52 bp fragments). Both enzymes were used in these assays and they produce very similar results. D) Summary of REAs with mononucleosome template. Error bars in are SEM.
    Figure Legend Snippet: PSC inhibits remodeling of mononucleosomes poorly Restriction enzyme accessibility (REA) assays were carried out with PSC at the indicated concentrations and hSwi/Snf. A,B) Representative gels of REAs with 6N (A) and 2N (B) templates. (N refers to nucleosomes, so 6N is a 6-nucleosome template; all of these templates are composed of repeats of the 5S nucleosome positioning sequence.). C) Summary of REAs with 6N and 2N templates. D, E) Representative gel of REA with mononucleosomes assembled on a 157-bp (10-1N) 5S template at 1nM (D) or 5nM (E). Note that PstI was used for digestion in the experiment with 1nM nucleosomes, while HhaI was used with 5nM nucleosomes. Both enzymes have single digestion sites in the template, but produce slightly different digestion patterns (HhaI digests the template into 77 and 80 bp fragments, which are not resolved, while PstI digests it into 99 and 52 bp fragments). Both enzymes were used in these assays and they produce very similar results. D) Summary of REAs with mononucleosome template. Error bars in are SEM.

    Techniques Used: Sequencing

    Related Articles

    Binding Assay:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. ..

    In Vitro:

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Ligation:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Ethanol Precipitation:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Purification:

    Article Title: Nucleotidyl transferase assisted DNA labeling with different click chemistries
    Article Snippet: .. Splinted ligation was performed by first annealing tailed DNA2 with DNA5/6 and DNA7 by heating to 90°C for 30 s and cooling to room temperature for 5 min, adding all other components after this step [final concentrations: 10 μM DNA2, 22.5 μM DNA5/6, 25 μM blocked and phosphorylated DNA7, 50 μM ATP, 50 mM Tris-HCl (pH 7.4), 10 mM MgCl2 , 1.5 U/μl T4 DNA ligase] incubating at 37°C for 4 h and heating to 80°C for 10 min. DNA5/6 and DNA7 were optionally removed from reaction mixtures to obtain pure, ligated/extended ssDNA by adding λ-exonuclease (0.25 U/μl for primer extension or 0.5 U/μl for ligation; New England Biolabs) directly into the reaction mixture and incubating at 37°C for 1 h, followed by 80°C for 10 min. DNA was purified by ethanol precipitation in the presence of 0.3 M sodium acetate (pH 5.5). .. Purified DNA (ds or ss) was subjected to CuAAC (1 μM DNA, 500 μM biotin azide, 500 μM CuSO4 , 2.5 mM THPTA, 5 mM sodium ascorbate) at 50°C for 2 h and reactions were purified by ethanol precipitation.

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Incubation:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    other:

    Article Title: Human Heart Mitochondrial DNA Is Organized in Complex Catenated Networks Containing Abundant Four-way Junctions and Replication Forks *
    Article Snippet: Subsequent treatments with topoisomerase IV (John Innes Enterprises), topoisomerase I, T7 endonuclease I, or λ-exonuclease (all New England Biolabs) used the manufacturers' recommended conditions.

    Nuclease Assay:

    Article Title: The hSNM1 protein is a DNA 5?-exonuclease
    Article Snippet: .. In vitro nuclease assay The assay was similar to an assay for yeast SNM1 ( ) Briefly, 0.5 pmol of radiolabeled substrate was combined with indicated amounts of purified protein (see the figure legends) in 15 μl of 1× Buffer F (50 mM Tris-acetate pH 7.2, 10 mM Mg acetate, 75 mM Potassium acetate, 1 mM DTT) supplemented with 100 μg/ml BSA, and incubated at 37°C for 20 min. For control reactions, 10 units of Rec-Jf or λ-exonuclease (for double-stranded substrate) (New England Biolabs, Ipswich, MA, USA) were used as recommended by the supplier. ..

    Polymerase Chain Reaction:

    Article Title: A comprehensive assay for targeted multiplex amplification of human DNA sequences
    Article Snippet: .. The digested PCR product was treated with 0.1 units lambda exonuclease (New England Biolabs) at 37°C for 15 min in the same restriction enzyme buffer. .. The probes were phosphorylated with 5 units of T4 polynucleotide kinase (NEB) in 50 mM Tris·HCl, pH7.9; 10 mM MgCl2 . dHPLC analysis was used to monitor the efficiency of lambda exonuclease digestion.

    Affinity Purification:

    Article Title: Rap1 and Cdc13 have complementary roles in preventing exonucleolytic degradation of telomere 5′ ends
    Article Snippet: .. For the binding assay, 10 fmol probe in presence of 1.5 µg competitor mix (0.5 µg each of sheared E.coli DNA (~250 bp), salmon sperm DNA and yeast t-RNA) in 1x λ-exonuclease buffer (New England Biolabs; 67 mM Glycine-KOH, pH 9.4, 2.5 MgCl2 and 50 µg/µl BSA) supplemented with 8% glycerol was mixed with varying concentrations of affinity purified Cdc13 (~0.8–4.8 μg), Rap1 (~0.07–7 μg), Rap1-DBD or DBD-mutants (~0.1–1.6 μg), in a total of 15 µl reaction. ..

    Recombinant:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. The substrates were then incubated with lambda exonuclease (0.025 unit/μl; NEB, MA) or purified recombinant Xenopus Exo1 (0.25ng/μl) at 22ºC. .. To assay the effect of the MRN complexes on the nucleases, the reactions also contained recombinant wild-type or mutant MRN proteins at 16 ng/μl.

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