haeiii  (New England Biolabs)


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    Structured Review

    New England Biolabs haeiii
    The mutational effects in the laboratory drift correlate with sequence exchanges in <t>M.HaeIII</t> orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target <t>DNA</t> binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.
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    Images

    1) Product Images from "Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations"

    Article Title: Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations

    Journal: PLoS Computational Biology

    doi: 10.1371/journal.pcbi.1004421

    The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.
    Figure Legend Snippet: The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Techniques Used: Sequencing, Functional Assay, Binding Assay

    2) Product Images from "CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation"

    Article Title: CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms23031082

    NHEJ-mediated integration of MRSs in HEK293T cells. ( a ) Gel image showing T7EI cleavage products (red brackets) of edited BCL11A-XL 3′ UTR 1 site. ( b ) Quantification of T7EI gel bands showing similar percentages of genome editing across samples (32.6% to 44.8%). ( c ) Apparent absence of PCR-RFLP HaeIII cleavage for test samples, indicative of dsODN integration being below the detection threshold. ( d ) Top: schematic illustration of dsODN-specific amplification assay at the 3′ UTR 1 site; bottom: amplicons of ~250 and ~302 bp revealing the presence of 2 MRSs and 4 MRSs in cells, respectively. Of note, undesirable inverted integration of MRSs into 3′ UTR 1 (see greyed-out part of Figure 2 ) is not detected by this assay. MOCK: mock-nucleofected cell sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, dsODN451-2MRSs/*: cell samples nucleofected with RNPs and dsODN bearing two miR451a MRSs at the indicated picomole quantity, dsODN451-4MRSs/5: cell sample nucleofected with RNPs and 5 pmole dsODN bearing four miR451a MRSs, +: a 994-bp PCR product giving cleavage products of 572 and 422 bp after digestion with HaeIII.
    Figure Legend Snippet: NHEJ-mediated integration of MRSs in HEK293T cells. ( a ) Gel image showing T7EI cleavage products (red brackets) of edited BCL11A-XL 3′ UTR 1 site. ( b ) Quantification of T7EI gel bands showing similar percentages of genome editing across samples (32.6% to 44.8%). ( c ) Apparent absence of PCR-RFLP HaeIII cleavage for test samples, indicative of dsODN integration being below the detection threshold. ( d ) Top: schematic illustration of dsODN-specific amplification assay at the 3′ UTR 1 site; bottom: amplicons of ~250 and ~302 bp revealing the presence of 2 MRSs and 4 MRSs in cells, respectively. Of note, undesirable inverted integration of MRSs into 3′ UTR 1 (see greyed-out part of Figure 2 ) is not detected by this assay. MOCK: mock-nucleofected cell sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, dsODN451-2MRSs/*: cell samples nucleofected with RNPs and dsODN bearing two miR451a MRSs at the indicated picomole quantity, dsODN451-4MRSs/5: cell sample nucleofected with RNPs and 5 pmole dsODN bearing four miR451a MRSs, +: a 994-bp PCR product giving cleavage products of 572 and 422 bp after digestion with HaeIII.

    Techniques Used: Non-Homologous End Joining, Polymerase Chain Reaction, Amplification

    HDR-mediated integration of MRSs in HUDEP-2 cells. PCR-RFLP analysis of cells nucleofected with RNPs and ss DNA oligos was performed for the assessment of integration efficiency. Bands of 436 bp represent unmodified target sites, larger bands insertions with incomplete digestion by DdeI/HaeIII, and smaller bands cleavage products of insertions. All bands corresponding to insertions are indicated by arrowheads (band sizes of 554 bp and 277 bp for donors with 2 MRSs, and of 528 bp and 264 bp for donors with 1 MRS). Corresponding rates of DdeI/HaeIII cleavage in PCR-RFLP (%) (after subtraction of background average cleavage rates of control samples) are reported below the gels. ( a ) Analysis of cells nucleofected with RNPs and Ultramer DNA Oligos. ( b ) Analysis of cells nucleofected with Alt-R RNPs and Alt-R HDR-2MRSs DNA Oligo (left gel), shown in comparison with analysis of cells nucleofected with standard Cas9 RNPs and Ultramer ssODN451TS_2MRSs DNA Oligo (right gel). Separate gels are indicated by dashed lines. MOCK: mock-nucleofected sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, NTS: non-target strand, TS: target strand, +: PCR products bearing restriction sites for DdeI/HaeIII used as positive controls. See Section 4.2 and Supplementary Table S2 for ss DNA oligo naming.
    Figure Legend Snippet: HDR-mediated integration of MRSs in HUDEP-2 cells. PCR-RFLP analysis of cells nucleofected with RNPs and ss DNA oligos was performed for the assessment of integration efficiency. Bands of 436 bp represent unmodified target sites, larger bands insertions with incomplete digestion by DdeI/HaeIII, and smaller bands cleavage products of insertions. All bands corresponding to insertions are indicated by arrowheads (band sizes of 554 bp and 277 bp for donors with 2 MRSs, and of 528 bp and 264 bp for donors with 1 MRS). Corresponding rates of DdeI/HaeIII cleavage in PCR-RFLP (%) (after subtraction of background average cleavage rates of control samples) are reported below the gels. ( a ) Analysis of cells nucleofected with RNPs and Ultramer DNA Oligos. ( b ) Analysis of cells nucleofected with Alt-R RNPs and Alt-R HDR-2MRSs DNA Oligo (left gel), shown in comparison with analysis of cells nucleofected with standard Cas9 RNPs and Ultramer ssODN451TS_2MRSs DNA Oligo (right gel). Separate gels are indicated by dashed lines. MOCK: mock-nucleofected sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, NTS: non-target strand, TS: target strand, +: PCR products bearing restriction sites for DdeI/HaeIII used as positive controls. See Section 4.2 and Supplementary Table S2 for ss DNA oligo naming.

    Techniques Used: Polymerase Chain Reaction

    3) Product Images from "Detection of a Reproducible, Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen ▿"

    Article Title: Detection of a Reproducible, Single-Member Shift in Soil Bacterial Communities Exposed to Low Levels of Hydrogen ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02072-09

    (A and B) T-RFLP profiles generated with the RE HaeIII, from a QRS soil microcosm experiment, showing the similarity of an H 2 -treated soil T-RFLP profile (A) to an air-treated soil T-RFLP profile (B). The scale bar at the top indicates the length of the
    Figure Legend Snippet: (A and B) T-RFLP profiles generated with the RE HaeIII, from a QRS soil microcosm experiment, showing the similarity of an H 2 -treated soil T-RFLP profile (A) to an air-treated soil T-RFLP profile (B). The scale bar at the top indicates the length of the

    Techniques Used: Generated

    4) Product Images from "Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4?,6?-Diamidino-2-Phenylindole) and Tetracycline Labeling ▿Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4?,6?-Diamidino-2-Phenylindole) and Tetracycline Labeling ▿ †"

    Article Title: Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4?,6?-Diamidino-2-Phenylindole) and Tetracycline Labeling ▿Dynamics of Polyphosphate-Accumulating Bacteria in Wastewater Treatment Plant Microbial Communities Detected via DAPI (4?,6?-Diamidino-2-Phenylindole) and Tetracycline Labeling ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01540-08

    Relative abundances of T-RF after digestion with the restriction endonucleases AluI, HaeIII, and Sau3AI. The T-RF values were assigned to phylotypes according to the experimentally determined T-RF values of the respective clones. P, Pseudomonas spp.;
    Figure Legend Snippet: Relative abundances of T-RF after digestion with the restriction endonucleases AluI, HaeIII, and Sau3AI. The T-RF values were assigned to phylotypes according to the experimentally determined T-RF values of the respective clones. P, Pseudomonas spp.;

    Techniques Used: Clone Assay

    5) Product Images from "Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome"

    Article Title: Restriction Enzyme Based Enriched L1Hs Sequencing (REBELseq): A Scalable Technique for Detection of Ta Subfamily L1Hs in the Human Genome

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.119.400613

    Schematic of the construction of Ta subfamily enriched L1Hs sequencing libraries. gDNA isolated from NeuN+ nuclei was enzymatically digested with HaeIII in the presence of shrimp alkaline phosphatase (rSAP) to fragment the genome and remove 5′ phosphates from cleavage products. A single primer extension using the Ta subfamily specific L1HsACA primer extends the 3′ end of the L1 sequence into the downstream gDNA. The 3′ ‘A’ overhang from the single primer extension is ligated to a custom T-linker, and primary PCR amplifies the construct using L1HsACA and T-linker specific primers. Hemi-nested secondary PCR using the L1Hs specific L1HsG primer and T-linker primer reduces the length of the L1 sequence carried forward and adds a sequencing adapter to the L1 end. Tertiary PCR uses primers complementary to the 5′ end of library amplicons to add a barcode to the L1 end and Illumina flow cell adapters to both ends of the amplicons.
    Figure Legend Snippet: Schematic of the construction of Ta subfamily enriched L1Hs sequencing libraries. gDNA isolated from NeuN+ nuclei was enzymatically digested with HaeIII in the presence of shrimp alkaline phosphatase (rSAP) to fragment the genome and remove 5′ phosphates from cleavage products. A single primer extension using the Ta subfamily specific L1HsACA primer extends the 3′ end of the L1 sequence into the downstream gDNA. The 3′ ‘A’ overhang from the single primer extension is ligated to a custom T-linker, and primary PCR amplifies the construct using L1HsACA and T-linker specific primers. Hemi-nested secondary PCR using the L1Hs specific L1HsG primer and T-linker primer reduces the length of the L1 sequence carried forward and adds a sequencing adapter to the L1 end. Tertiary PCR uses primers complementary to the 5′ end of library amplicons to add a barcode to the L1 end and Illumina flow cell adapters to both ends of the amplicons.

    Techniques Used: Sequencing, Isolation, Polymerase Chain Reaction, Construct

    6) Product Images from "Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops"

    Article Title: Telomeric DNA in ALT Cells Is Characterized by Free Telomeric Circles and Heterogeneous t-Loops

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.24.22.9948-9957.2004

    Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ- 32 P](CCCTAA) 6 , and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.
    Figure Legend Snippet: Telomere measurement, telomerase activity, and telomere isolation in GM847, GM847-Tert, and VA13 cells. (A) Total DNA (10 μg) was digested with HinfI/HaeIII and separated by PFGE, and the telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe. The signal was detected using a PhosphorImager. (B) Telomerase activity as determined by TRAP assay. IC refers to the internal PCR control. (C) Total DNA content and relative telomeric DNA abundance in GM847 psoralen photo-cross-linked fractions following genomic DNA digestion with MboI/AluI and fractionation over an A-15m Bio-Gel column. DNA content (solid line, scale on left) as determined by optical density at 260 nm (OD 260). Telomeric signal intensity (dotted line, scale on right) determined by quantitation of slot blot shown in panel D. (D) Slot blot analysis of selected fractions from the column elution shown in panel C. DNA (50 ng) was applied to a nylon membrane, probed with [γ- 32 P](CCCTAA) 6 , and detected by PhosphorImager. Controls included buffer alone (TE), the pRST5 plasmid containing 96 (TTAGGG) repeats, Bluescript (pBS) cloning vector lacking telomeric repeats, undigested GM847 genomic DNA, and MboI/AluI-digested GM847 genomic DNA.

    Techniques Used: Activity Assay, Isolation, Hybridization, TRAP Assay, Polymerase Chain Reaction, Fractionation, Quantitation Assay, Dot Blot, Plasmid Preparation, Clone Assay

    2D PFGE of TRFs from ALT and non-ALT cells. Total DNA (20 μg) was digested with HinfI/HaeIII and then separated by 2D PFGE. Telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe and was visualized using a PhosphorImager. The black and white arrows indicate linear- and circular-form DNA, respectively.
    Figure Legend Snippet: 2D PFGE of TRFs from ALT and non-ALT cells. Total DNA (20 μg) was digested with HinfI/HaeIII and then separated by 2D PFGE. Telomeric material was detected by in-gel hybridization with a [γ- 32 P](CCCTAA) 6 probe and was visualized using a PhosphorImager. The black and white arrows indicate linear- and circular-form DNA, respectively.

    Techniques Used: Hybridization

    7) Product Images from "Methylation by a Unique ?-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725"

    Article Title: Methylation by a Unique ?-class N4-Cytosine Methyltransferase Is Required for DNA Transformation of Caldicellulosiruptor bescii DSM6725

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0043844

    Cloning, expression, purification and partial characterization of M.CbeI. (A) The region of the C. bescii chromosome containing CbeI and M.CbeI. (B) Diagram of pDCW73 used to produce a His-tagged version of M.CbeI in E. coli . (C) Purified M.CbeI displayed on a 10–20% Tris-HCl gradient gel (CriterionTM Precast Gel, Bio-Rad Laboratories, Hercules, CA) stained with coomassie blue. lane 1: protein molecular weight standards (BioRad); lane 2: 15 ng of purified M.CbeI protein. (D) lane 1, undigested unmethylated pDCW70; lane 2, undigested M.CbeI methylated pDCW70; lane 3, unmethylated pDCW70 digested with purified CbeI; lane 4, M.CbeI methylated pDCW70 digested with purified CbeI, MW: 1 kb DNA ladder (NEB). (E) DNA sequence traces of DNA methylated with M.CbeI (top panel) or M.HaeIII (bottom panel). Differences in the G residue signals between M.HaeIII and M.CbeI pUC18 are shown in the middle panel.
    Figure Legend Snippet: Cloning, expression, purification and partial characterization of M.CbeI. (A) The region of the C. bescii chromosome containing CbeI and M.CbeI. (B) Diagram of pDCW73 used to produce a His-tagged version of M.CbeI in E. coli . (C) Purified M.CbeI displayed on a 10–20% Tris-HCl gradient gel (CriterionTM Precast Gel, Bio-Rad Laboratories, Hercules, CA) stained with coomassie blue. lane 1: protein molecular weight standards (BioRad); lane 2: 15 ng of purified M.CbeI protein. (D) lane 1, undigested unmethylated pDCW70; lane 2, undigested M.CbeI methylated pDCW70; lane 3, unmethylated pDCW70 digested with purified CbeI; lane 4, M.CbeI methylated pDCW70 digested with purified CbeI, MW: 1 kb DNA ladder (NEB). (E) DNA sequence traces of DNA methylated with M.CbeI (top panel) or M.HaeIII (bottom panel). Differences in the G residue signals between M.HaeIII and M.CbeI pUC18 are shown in the middle panel.

    Techniques Used: Clone Assay, Expressing, Purification, Staining, Molecular Weight, Methylation, Sequencing

    Protection of DNA by M.CbeI or M.HaeIII from digestion in vitro . In each panel, lane 1 is unmethylated plasmid DNA (pUC18) isolated from E. coli ( dam + dcm + ), lane 2 is plasmid DNA methylated in vitro with M.HaeIII (NEB) at 37°C, lane 3 is plasmid methylated with purified M.CbeI at 78°C. (A) undigested. (B) digested with HaeIII (C) digested with purified CbeI. DNAs were subjected to electrophoresis in a 1.2% TAE-agarose gel, and then stained with ethidium bromide. M: 1kb DNA ladder (NEB).
    Figure Legend Snippet: Protection of DNA by M.CbeI or M.HaeIII from digestion in vitro . In each panel, lane 1 is unmethylated plasmid DNA (pUC18) isolated from E. coli ( dam + dcm + ), lane 2 is plasmid DNA methylated in vitro with M.HaeIII (NEB) at 37°C, lane 3 is plasmid methylated with purified M.CbeI at 78°C. (A) undigested. (B) digested with HaeIII (C) digested with purified CbeI. DNAs were subjected to electrophoresis in a 1.2% TAE-agarose gel, and then stained with ethidium bromide. M: 1kb DNA ladder (NEB).

    Techniques Used: In Vitro, Plasmid Preparation, Isolation, Methylation, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining

    8) Product Images from "Correlated Occurrence and Bypass of Frame-Shifting Insertion-Deletions (InDels) to Give Functional Proteins"

    Article Title: Correlated Occurrence and Bypass of Frame-Shifting Insertion-Deletions (InDels) to Give Functional Proteins

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1003882

    InDels frequencies in the selected (G17) and unselected (G0) repertoires. Panel A indicates the length of the homonucleotide repeat within which the InDels occurred. Panels B and C represent frequencies plotted per nucleotide position in each library. Panel D illustrates the conservation pattern in the M.HaeIII as derived by Consurf [49] – the highest the score, the higher the conservation. Also indicated are the locations of the conserved motifs shared by all DNA methyltransferases of this class (C5 methyltransferases). The serial #1–19 numbers indicate individually tested InDels (listed in Table 3 ).
    Figure Legend Snippet: InDels frequencies in the selected (G17) and unselected (G0) repertoires. Panel A indicates the length of the homonucleotide repeat within which the InDels occurred. Panels B and C represent frequencies plotted per nucleotide position in each library. Panel D illustrates the conservation pattern in the M.HaeIII as derived by Consurf [49] – the highest the score, the higher the conservation. Also indicated are the locations of the conserved motifs shared by all DNA methyltransferases of this class (C5 methyltransferases). The serial #1–19 numbers indicate individually tested InDels (listed in Table 3 ).

    Techniques Used: Derivative Assay

    9) Product Images from "Characteristics of ?-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii"

    Article Title: Characteristics of ?-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-7-25

    Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)
    Figure Legend Snippet: Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)
    Figure Legend Snippet: Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    10) Product Images from "Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple) Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple"

    Article Title: Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple) Methylation of MdMYB1 locus mediated by RdDM pathway regulates anthocyanin biosynthesis in apple

    Journal: Plant Biotechnology Journal

    doi: 10.1111/pbi.13337

    Heterologous expression of MdAGO4s or MdDRM2s rescues CHH methylation of AGO4‐binding sites in Arabidopsis ago4 and drm2 mutants. (a) Ten‐day‐old wild‐type Arabidopsis (WT), ago4 mutant, drm2 mutant and transgenic Arabidopsis (35S:: AGO4‐1/2 and 35S::DRM2‐1/2 ). DNA methylation was analysed using three methylation‐sensitive restriction endonucleases, AluI (b), HaeIII (c) and DdeI (d). Genomic DNA was extracted from 2‐week‐old seedlings, and digested genomic DNAs were amplified by PCR. Sequences lacking AluI sites (IGN5), HaeIII (AT5G27860) and DdeI (AT2G36490) were used as loading controls. Sequence and size of Arabidopsis genes are listed in Table S2 . 1–7 indicate 35S:: AGO4‐1 / ago4 , 35S:: AGO4‐2 / ago4 , ago4 , WT, drm2 , 35S:: DRM2‐1 / drm2 and 35S:: DRM2‐2 / drm2 , respectively.
    Figure Legend Snippet: Heterologous expression of MdAGO4s or MdDRM2s rescues CHH methylation of AGO4‐binding sites in Arabidopsis ago4 and drm2 mutants. (a) Ten‐day‐old wild‐type Arabidopsis (WT), ago4 mutant, drm2 mutant and transgenic Arabidopsis (35S:: AGO4‐1/2 and 35S::DRM2‐1/2 ). DNA methylation was analysed using three methylation‐sensitive restriction endonucleases, AluI (b), HaeIII (c) and DdeI (d). Genomic DNA was extracted from 2‐week‐old seedlings, and digested genomic DNAs were amplified by PCR. Sequences lacking AluI sites (IGN5), HaeIII (AT5G27860) and DdeI (AT2G36490) were used as loading controls. Sequence and size of Arabidopsis genes are listed in Table S2 . 1–7 indicate 35S:: AGO4‐1 / ago4 , 35S:: AGO4‐2 / ago4 , ago4 , WT, drm2 , 35S:: DRM2‐1 / drm2 and 35S:: DRM2‐2 / drm2 , respectively.

    Techniques Used: Expressing, Methylation, Binding Assay, Mutagenesis, Transgenic Assay, DNA Methylation Assay, Amplification, Polymerase Chain Reaction, Sequencing

    11) Product Images from "Evidence for Transgenerational Transmission of Epigenetic Tumor Susceptibility in Drosophila"

    Article Title: Evidence for Transgenerational Transmission of Epigenetic Tumor Susceptibility in Drosophila

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030151

    Increased Methylation of ftz Regulatory Region in Kr +/− Animals and Its Inheritance (A) Schematic representation of the ftz-lacZ reporter, showing the minimal ftz 5′ regulatory region previously shown to be sufficient to drive expression of a ftz-lacZ reporter transgene in ftz patterns [ 30 ]. Arrows above and below the horizontal line represent PCR primers used to amplify a 778-bp fragment, encompassing the 620-bp minimal ftz enhancer (see Figure S2 for sequence). Bent arrow indicates the start of the lacZ sequence. Vertical bars above and below the line represent positions of recognition sequences for restriction enzymes BstUI (CGCG) and HaeIII (GGCC), respectively. (B–E) Time courses of restriction digests of genomic DNA with enzymes sensitive (BstUI) or insensitive (HaeIII) to methylated DNA are shown as agarose gel pictures and quantifications. Genomic DNA was isolated from Kr 1 /CyO ftz-lacZ and +/CyO ftz-lacZ (wild-type control) adult flies (B, C), or from the F1 progeny flies of Kr 1 /CyO ftz-lacZ males crossed to hop Tum-l/+ females or wild-type females (D, E), and digested with the indicated enzymes for the indicated times (minutes). Digested DNA was amplified with PCR primers shown in (A) and run on an agarose gel. Note that the genomic DNA from Kr +/ − flies or from the F1 progeny of hop Tum-l/+ females and Kr 1 /CyO ftz-lacZ males is more resistant to BstUI digestion than the controls. (F, G) Digested genomic DNA purified from embryos was immunoprecipitated by antibodies to methylated cytosine and amplified by PCR primers shown in (A). Embryos derived from wild-type or Kr +/ − parents (F), or from Kr 1 /ftz-lacZ males crossed to wild-type (top) or Tum-l/+ (bottom) females (G) were used for DNA isolation. Note the presence of higher levels of 5-meC in the ftz-lacZ promoter in Kr +/ − embryos or in those from Tum-l/+ females crossed to Kr 1 /ftz-lacZ males.
    Figure Legend Snippet: Increased Methylation of ftz Regulatory Region in Kr +/− Animals and Its Inheritance (A) Schematic representation of the ftz-lacZ reporter, showing the minimal ftz 5′ regulatory region previously shown to be sufficient to drive expression of a ftz-lacZ reporter transgene in ftz patterns [ 30 ]. Arrows above and below the horizontal line represent PCR primers used to amplify a 778-bp fragment, encompassing the 620-bp minimal ftz enhancer (see Figure S2 for sequence). Bent arrow indicates the start of the lacZ sequence. Vertical bars above and below the line represent positions of recognition sequences for restriction enzymes BstUI (CGCG) and HaeIII (GGCC), respectively. (B–E) Time courses of restriction digests of genomic DNA with enzymes sensitive (BstUI) or insensitive (HaeIII) to methylated DNA are shown as agarose gel pictures and quantifications. Genomic DNA was isolated from Kr 1 /CyO ftz-lacZ and +/CyO ftz-lacZ (wild-type control) adult flies (B, C), or from the F1 progeny flies of Kr 1 /CyO ftz-lacZ males crossed to hop Tum-l/+ females or wild-type females (D, E), and digested with the indicated enzymes for the indicated times (minutes). Digested DNA was amplified with PCR primers shown in (A) and run on an agarose gel. Note that the genomic DNA from Kr +/ − flies or from the F1 progeny of hop Tum-l/+ females and Kr 1 /CyO ftz-lacZ males is more resistant to BstUI digestion than the controls. (F, G) Digested genomic DNA purified from embryos was immunoprecipitated by antibodies to methylated cytosine and amplified by PCR primers shown in (A). Embryos derived from wild-type or Kr +/ − parents (F), or from Kr 1 /ftz-lacZ males crossed to wild-type (top) or Tum-l/+ (bottom) females (G) were used for DNA isolation. Note the presence of higher levels of 5-meC in the ftz-lacZ promoter in Kr +/ − embryos or in those from Tum-l/+ females crossed to Kr 1 /ftz-lacZ males.

    Techniques Used: Methylation, Expressing, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Isolation, Amplification, Purification, Immunoprecipitation, Derivative Assay, DNA Extraction

    12) Product Images from "DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force"

    Article Title: DNA looping by two-site restriction endonucleases: heterogeneous probability distributions for loop size and unbinding force

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl382

    Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.
    Figure Legend Snippet: Typical DNA force-extension plots. ( A ) The two-site enzyme BsgI tested on the LBAC-B template, which has four recognition sites. The one detected loop had a measured length of 946 bp, in excellent agreement with the separation between the binding sites at positions 8342 and 9287 bp (a distance of 945 bp). ( B ) Sau3AI on the LBAC-A template, which has 55 recognition sites. ( C ) Control experiments: (i) the two-site enzyme Sau3AI was tested on bacteriophage phiX174 DNA, which contains no copies of its recognition sequence; (ii) the one-site enzyme HaeIII was tested on LBAC-B DNA containing 36 copies of its recognition sequence; (iii) the two-site enzyme SfiI was tested on LBAC-B DNA containing one copy of its recognition sequence and (iv) LBAC-B DNA, containing 26 FokI binding sites, incubated in the FokI reaction buffer, but no FokI added.

    Techniques Used: Binding Assay, Sequencing, Incubation

    13) Product Images from "FIREWACh: High-throughput Functional Detection of Transcriptional Regulatory Modules in Mammalian Cells"

    Article Title: FIREWACh: High-throughput Functional Detection of Transcriptional Regulatory Modules in Mammalian Cells

    Journal: Nature methods

    doi: 10.1038/nmeth.2885

    NFR-derived DNAs correspond to accessible chromatin regions located throughout the genome (a) Carpet plots depicting the correspondence of in silico -generated genomic DNA fragments ( n = 61,844) or Input Library NFR-DNAs ( n = 84,241) with DNaseI hypersensitive sites (HS) in ESC chromatin 9 . The DNAs in each dataset were ranked according to the expression level of their associated gene (s) in ESCs (bar to the right of each panel; red, high expression, green, low expression). The presence of a DNaseI HS site (black) was assessed for a region corresponding to the genomic interval ±1 kb of the center (green vertical line) of the DNA fragments within Library NFR- or in silico-generated random DNA fragments. (b) Venn diagram examining the relatedness of genomic regions present in the HaeIII- and RsaI-NFR DNA libraries. The total number of elements in each library is indicated at the top of each circle. (c). Genomic distribution of HaeIII- or RsaI- NFR-input library DNA populations relative to annotated transcription start sites (TSS, black arrow) determined using the GREAT analysis tool.
    Figure Legend Snippet: NFR-derived DNAs correspond to accessible chromatin regions located throughout the genome (a) Carpet plots depicting the correspondence of in silico -generated genomic DNA fragments ( n = 61,844) or Input Library NFR-DNAs ( n = 84,241) with DNaseI hypersensitive sites (HS) in ESC chromatin 9 . The DNAs in each dataset were ranked according to the expression level of their associated gene (s) in ESCs (bar to the right of each panel; red, high expression, green, low expression). The presence of a DNaseI HS site (black) was assessed for a region corresponding to the genomic interval ±1 kb of the center (green vertical line) of the DNA fragments within Library NFR- or in silico-generated random DNA fragments. (b) Venn diagram examining the relatedness of genomic regions present in the HaeIII- and RsaI-NFR DNA libraries. The total number of elements in each library is indicated at the top of each circle. (c). Genomic distribution of HaeIII- or RsaI- NFR-input library DNA populations relative to annotated transcription start sites (TSS, black arrow) determined using the GREAT analysis tool.

    Techniques Used: Derivative Assay, In Silico, Generated, Expressing

    FIREWACh Elements are associated with expressed genes and correlate with chromatin marks of active promoters and enhancers (a) Distribution of NFR-derived DNAs with respect to annotated TSSs. These datasets each represent the combined populations of HaeIII-and RsaI DNAs for Library ( top) and FIREWACh elements ( bottom). (b) Boxplot of the expression status of genes associated with the promoter-proximal DNAs (i.e 2 kb+/− of an annotated TSS) in each dataset. Median values of the distribution are denoted by a red line; whiskers show tails of the distribution and outliers are marked by red crosses. P -values were determined by nonparametric Kruskal-Wallis test. (c) Carpet plots assessing genomic loci corresponding to Promoter-proximal FIREWACh DNAs (top panel) or Distal FIREWACh DNAs (bottom panel) for the presence of histone modifications H3K4me4, H3K27ac, H3K27me3, or cohesin complex protein Nipbl. Each row corresponds to 1kb +/− from the center (green line) of a single FIREWACh DNA. Read density for the histone modification or feature indicated on the top of the column is depicted in black. FIREWACh DNAs were ranked according to the expression level of their associated gene(s) in ESCs (bar to the right of the panel; red, high expression, green, low expression).
    Figure Legend Snippet: FIREWACh Elements are associated with expressed genes and correlate with chromatin marks of active promoters and enhancers (a) Distribution of NFR-derived DNAs with respect to annotated TSSs. These datasets each represent the combined populations of HaeIII-and RsaI DNAs for Library ( top) and FIREWACh elements ( bottom). (b) Boxplot of the expression status of genes associated with the promoter-proximal DNAs (i.e 2 kb+/− of an annotated TSS) in each dataset. Median values of the distribution are denoted by a red line; whiskers show tails of the distribution and outliers are marked by red crosses. P -values were determined by nonparametric Kruskal-Wallis test. (c) Carpet plots assessing genomic loci corresponding to Promoter-proximal FIREWACh DNAs (top panel) or Distal FIREWACh DNAs (bottom panel) for the presence of histone modifications H3K4me4, H3K27ac, H3K27me3, or cohesin complex protein Nipbl. Each row corresponds to 1kb +/− from the center (green line) of a single FIREWACh DNA. Read density for the histone modification or feature indicated on the top of the column is depicted in black. FIREWACh DNAs were ranked according to the expression level of their associated gene(s) in ESCs (bar to the right of the panel; red, high expression, green, low expression).

    Techniques Used: Derivative Assay, Expressing, Modification

    14) Product Images from "The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities"

    Article Title: The ATP-dependent chromatin remodeling enzymes CHD6, CHD7, and CHD8 exhibit distinct nucleosome binding and remodeling activities

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M117.779470

    Chromatin remodeling activities of group III CHD enzyme s . A , mononucleosome sliding assay. 20 n m of dye-labeled, end-positioned nucleosome substrates (illustrated at the top ) were incubated with 0, 0.25, 1, 4, or 16 n m CHD enzymes in the presence of ATP. After competing the enzymes off with excess plasmid DNA, the reaction products were visualized by native PAGE. The white arrowheads correspond to end-positioned species; the black arrowheads correspond to middle-positioned species. B , MNase accessibility assay. Top panel , reaction schematic; CHD enzymes were tested for their ability to alter the MNase accessibility of a chromatin array in an ATP-dependent manner. Bottom left panel , SYBR-stained native PAGE of MNase-freed product DNA species. The mononucleosome band is denoted by white arrowheads. Bottom right panel , the intensity of the mononucleosome band in each reaction was quantified as a fraction of the whole lane signal. Then, for each enzyme, the ratio of −ATP to + ATP mono band intensity was calculated. Values are mean ± S.D. ( n = 3 or 5; see “Experimental Procedures”). C , HaeIII accessibility assay. Left panel , reaction schematic. CHD enzymes were tested for their ability to regulate HaeIII endonuclease accessibility of a chromatin array in an ATP-dependent manner. Right panel , SYBR-stained native PAGE of HaeIII-digested plasmid DNA species.
    Figure Legend Snippet: Chromatin remodeling activities of group III CHD enzyme s . A , mononucleosome sliding assay. 20 n m of dye-labeled, end-positioned nucleosome substrates (illustrated at the top ) were incubated with 0, 0.25, 1, 4, or 16 n m CHD enzymes in the presence of ATP. After competing the enzymes off with excess plasmid DNA, the reaction products were visualized by native PAGE. The white arrowheads correspond to end-positioned species; the black arrowheads correspond to middle-positioned species. B , MNase accessibility assay. Top panel , reaction schematic; CHD enzymes were tested for their ability to alter the MNase accessibility of a chromatin array in an ATP-dependent manner. Bottom left panel , SYBR-stained native PAGE of MNase-freed product DNA species. The mononucleosome band is denoted by white arrowheads. Bottom right panel , the intensity of the mononucleosome band in each reaction was quantified as a fraction of the whole lane signal. Then, for each enzyme, the ratio of −ATP to + ATP mono band intensity was calculated. Values are mean ± S.D. ( n = 3 or 5; see “Experimental Procedures”). C , HaeIII accessibility assay. Left panel , reaction schematic. CHD enzymes were tested for their ability to regulate HaeIII endonuclease accessibility of a chromatin array in an ATP-dependent manner. Right panel , SYBR-stained native PAGE of HaeIII-digested plasmid DNA species.

    Techniques Used: Labeling, Incubation, Plasmid Preparation, Clear Native PAGE, Staining

    15) Product Images from "Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1"

    Article Title: Editing of the TRIM5 Gene Decreases the Permissiveness of Human T Lymphocytic Cells to HIV-1

    Journal: Viruses

    doi: 10.3390/v13010024

    Tripartite-motif-containing protein 5 ( TRIM5) editing strategy and outcome. Top, the TRIM5 region targeted for mutagenesis is aligned with the reverse-complement sequence of the donor single-stranded donor DNA (ssDNA) central region. The mutated nucleotides are shown in colors, and their purpose is indicated. Substitutions leading to R332G and R335G mutations are in blue. The PAM-blocking silent mutation is in purple. The silent mutation creating an HaeIII restriction cut site is in green. Additional silent mutations in the gRNA-binding region are in grey. Bottom, sequence of TRIM5 alleles found in clonal Jurkat populations following electroporation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoproteins (RNPs) and the ssDNA and PCR-based screening. For each cell population, alleles were found in equal amounts, except for clone 38, in which the relative amounts were 44% (unedited parental (WT) allele), 24% (homology-directed repair (HDR)-edited allele) and 32% (indel-containing allele). Substituted nucleotides are in bold. Substitutions leading to R332G and R335G mutations are in blue. Indels or undesirable substitutions are in red. The number of desired mutations for each allele is shown on the right, along with the presence (y) or absence (n) of insertions and deletions.
    Figure Legend Snippet: Tripartite-motif-containing protein 5 ( TRIM5) editing strategy and outcome. Top, the TRIM5 region targeted for mutagenesis is aligned with the reverse-complement sequence of the donor single-stranded donor DNA (ssDNA) central region. The mutated nucleotides are shown in colors, and their purpose is indicated. Substitutions leading to R332G and R335G mutations are in blue. The PAM-blocking silent mutation is in purple. The silent mutation creating an HaeIII restriction cut site is in green. Additional silent mutations in the gRNA-binding region are in grey. Bottom, sequence of TRIM5 alleles found in clonal Jurkat populations following electroporation of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 ribonucleoproteins (RNPs) and the ssDNA and PCR-based screening. For each cell population, alleles were found in equal amounts, except for clone 38, in which the relative amounts were 44% (unedited parental (WT) allele), 24% (homology-directed repair (HDR)-edited allele) and 32% (indel-containing allele). Substituted nucleotides are in bold. Substitutions leading to R332G and R335G mutations are in blue. Indels or undesirable substitutions are in red. The number of desired mutations for each allele is shown on the right, along with the presence (y) or absence (n) of insertions and deletions.

    Techniques Used: Mutagenesis, Sequencing, Blocking Assay, Binding Assay, Electroporation, CRISPR, Polymerase Chain Reaction

    16) Product Images from "Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling"

    Article Title: Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr304

    H3(T118ph) increases DNA accessibility near the dyad symmetry axis. ( A and B ) PAGE analysis of unmodified and H3(T118ph) nucleosomes, respectively, digested with HhaI. The lanes are labeled with the digestion quench time in minutes. ( C ) and ( D ) Plots of the fraction of HaeIII and HhaI undigested DNA, respectively. Digestions of unmodified (squares) and H3(T118ph) (circles) nucleosomes were fit with a single exponential decay. ( E ) Plot of the site accessibility of H3(T118ph) nucleosomes, K eq-pT118 relative to the site accessibility of unmodified nucleosomes, K eq-unmod , for five separate RE sites. The error bars were determined from the standard deviation of at least three separate experiments. ( F ) The nucleosomes crystal structure ( 40 ) with the five RE sites is shown in blue and the two H3(T118) residues are shown in red.
    Figure Legend Snippet: H3(T118ph) increases DNA accessibility near the dyad symmetry axis. ( A and B ) PAGE analysis of unmodified and H3(T118ph) nucleosomes, respectively, digested with HhaI. The lanes are labeled with the digestion quench time in minutes. ( C ) and ( D ) Plots of the fraction of HaeIII and HhaI undigested DNA, respectively. Digestions of unmodified (squares) and H3(T118ph) (circles) nucleosomes were fit with a single exponential decay. ( E ) Plot of the site accessibility of H3(T118ph) nucleosomes, K eq-pT118 relative to the site accessibility of unmodified nucleosomes, K eq-unmod , for five separate RE sites. The error bars were determined from the standard deviation of at least three separate experiments. ( F ) The nucleosomes crystal structure ( 40 ) with the five RE sites is shown in blue and the two H3(T118) residues are shown in red.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Labeling, Standard Deviation

    17) Product Images from "Identification and Characterization of a Linear-Plasmid-Encoded Factor H-Binding Protein (FhbA) of the Relapsing Fever Spirochete Borrelia hermsii"

    Article Title: Identification and Characterization of a Linear-Plasmid-Encoded Factor H-Binding Protein (FhbA) of the Relapsing Fever Spirochete Borrelia hermsii

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.186.9.2612-2618.2004

    Demonstration that fhbA is a single genetic locus carried by a 220-kb linear plasmid and expressed as a monocistronic mRNA. DNAs from several TBRF isolates were fractionated in the first dimension by PFGE. The gel was rotated 90°, and then electrophoresis in the second dimension was performed, followed by ethidium bromide staining (A). The DNA was transferred onto membranes for hybridization analysis (B). To determine whether fhbA is a single genetic locus or a member of a paralogous gene family, hybridization analyses were performed with HaeIII-digested DNAs from B. hermsii isolates YOR and MAN. The DNA was fractionated in a 0.8% agarose gel and transferred onto membranes for hybridization analyses (C). To determine the size of the fhbA transcript, Northern blot analysis was performed (D.) All blots were hybridized with an internally labeled, PCR-generated fhbA probe. All methods are described in the text. DNA size markers (in kilobases) are indicated for panels A, B, and C. RNA size markers (in bases) were used for panel D.
    Figure Legend Snippet: Demonstration that fhbA is a single genetic locus carried by a 220-kb linear plasmid and expressed as a monocistronic mRNA. DNAs from several TBRF isolates were fractionated in the first dimension by PFGE. The gel was rotated 90°, and then electrophoresis in the second dimension was performed, followed by ethidium bromide staining (A). The DNA was transferred onto membranes for hybridization analysis (B). To determine whether fhbA is a single genetic locus or a member of a paralogous gene family, hybridization analyses were performed with HaeIII-digested DNAs from B. hermsii isolates YOR and MAN. The DNA was fractionated in a 0.8% agarose gel and transferred onto membranes for hybridization analyses (C). To determine the size of the fhbA transcript, Northern blot analysis was performed (D.) All blots were hybridized with an internally labeled, PCR-generated fhbA probe. All methods are described in the text. DNA size markers (in kilobases) are indicated for panels A, B, and C. RNA size markers (in bases) were used for panel D.

    Techniques Used: Plasmid Preparation, Electrophoresis, Staining, Hybridization, Agarose Gel Electrophoresis, Northern Blot, Labeling, Polymerase Chain Reaction, Generated

    18) Product Images from "Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen ▿O Fluxes of an Acidic Fen ▿ †"

    Article Title: Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen ▿O Fluxes of an Acidic Fen ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.02256-09

    Comparative TRFLP analyses of narG (A to C) and nosZ (D to F) amplified from different soil layers of the acidic fen. PCR products were digested with CfoI (A), HaeIII (B), XhoI (C), BtgI (D), NlaIV (E), and PvuI and SacI (F). Mean values of three replicates
    Figure Legend Snippet: Comparative TRFLP analyses of narG (A to C) and nosZ (D to F) amplified from different soil layers of the acidic fen. PCR products were digested with CfoI (A), HaeIII (B), XhoI (C), BtgI (D), NlaIV (E), and PvuI and SacI (F). Mean values of three replicates

    Techniques Used: Terminal Restriction Fragment Length Polymorphism, Amplification, Polymerase Chain Reaction

    19) Product Images from "Characteristics of ?-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii"

    Article Title: Characteristics of ?-lactamases and their genes (blaA and blaB) in Yersinia intermedia and Y. frederiksenii

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-7-25

    Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)
    Figure Legend Snippet: Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)
    Figure Legend Snippet: Types of restriction profiles of blaA with NciI (Lanes 1–2) and HaeIII (Lanes 3–5), and blaB with HaeIII (Lanes 6–7) and RsaI (Lanes 8–9). PCR amplification and restriction analysis were carried out for 49 strains of Y. intermedia and Y. frederiksenii . M, molecular weight DNA marker (100 bp DNA ladder)

    Techniques Used: Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    20) Product Images from "Bovine Mastitis Associated with Prototheca blaschkeae ▿"

    Article Title: Bovine Mastitis Associated with Prototheca blaschkeae ▿

    Journal:

    doi: 10.1128/JCM.00323-08

    Restriction patterns of amplified 18S rDNA fragments of four Prototheca isolates. PCR fragments were digested with HaeIII, yielding two different patterns, RP 1 and RP 2. RP 2 lacks the 400-bp fragment observed in RP 1. Lanes 50 bp, molecular weight standard
    Figure Legend Snippet: Restriction patterns of amplified 18S rDNA fragments of four Prototheca isolates. PCR fragments were digested with HaeIII, yielding two different patterns, RP 1 and RP 2. RP 2 lacks the 400-bp fragment observed in RP 1. Lanes 50 bp, molecular weight standard

    Techniques Used: Amplification, Polymerase Chain Reaction, Molecular Weight

    21) Product Images from "Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ?AQ113"

    Article Title: Characterization of the Genome of the Dairy Lactobacillus helveticus Bacteriophage ?AQ113

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00620-13

    Restriction analysis of ΦAQ113 DNA. The phage DNA was digested with BglII, HhaI, PstI, HaeIII, MspI, EcoRI, and EcoRV. Lanes M1 and M2, 1-kb Plus and λ-HindIII DNA ladders; A and B indicate that the digests were unheated and heated prior
    Figure Legend Snippet: Restriction analysis of ΦAQ113 DNA. The phage DNA was digested with BglII, HhaI, PstI, HaeIII, MspI, EcoRI, and EcoRV. Lanes M1 and M2, 1-kb Plus and λ-HindIII DNA ladders; A and B indicate that the digests were unheated and heated prior

    Techniques Used:

    22) Product Images from "OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica"

    Article Title: OxyR-dependent formation of DNA methylation patterns in OpvABOFF and OpvABON cell lineages of Salmonella enterica

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1483

    Methylation state of GATC sites in the opvAB regulatory region in wild-type and oxyR backgrounds. ( A ) Southern blot of genomic DNA obtained from wild-type and oxyR cultures and digested with HaeIII and with AccI (control) and DpnI, MboI or Sau3AI. Fragment sizes are indicated in base pairs. ( B ) Diagram of the HaeIII-AccI fragment and pattern of fragments obtained.
    Figure Legend Snippet: Methylation state of GATC sites in the opvAB regulatory region in wild-type and oxyR backgrounds. ( A ) Southern blot of genomic DNA obtained from wild-type and oxyR cultures and digested with HaeIII and with AccI (control) and DpnI, MboI or Sau3AI. Fragment sizes are indicated in base pairs. ( B ) Diagram of the HaeIII-AccI fragment and pattern of fragments obtained.

    Techniques Used: Methylation, Southern Blot

    23) Product Images from "Direct Identification of Mycobacterium haemophilum in Skin Lesions of Immunocompromised Patients by PCR-Restriction Endonuclease Analysis"

    Article Title: Direct Identification of Mycobacterium haemophilum in Skin Lesions of Immunocompromised Patients by PCR-Restriction Endonuclease Analysis

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.42.7.3336-3338.2004

    PRA patterns for reference strains and superficial lesion clinical specimens. Lanes: 1, 100-bp markers; 2, M. tuberculosis ATCC 27294; 3, M. intracellulare ATCC 13950; 4, M. kansasii ATCC 35775; 5, M. haemophilum ATCC 29548; 6, MC29362; 7, MC17735; 8, MC31558 (lanes 2 to 8 correspond to BstEII-digested amplicons); 9, 50-bp markers; 10, MC31558; 11, MC17735; 12, MC29362; 13, M. haemophilum ATCC 29548; 14, M. kansasii ATCC 35775; 15, M. intracellulare ATCC 13950; and 16, M. tuberculosis ATCC 27294 (lanes 10 to 16 correspond to HaeIII-digested amplicons). Isolates from clinical specimens MC29362, MC17735, and MC31558 were identified as M. haemophilum by matching each specimen's PRA band pattern with that of reference strain M. haemophilum ATCC 29548.
    Figure Legend Snippet: PRA patterns for reference strains and superficial lesion clinical specimens. Lanes: 1, 100-bp markers; 2, M. tuberculosis ATCC 27294; 3, M. intracellulare ATCC 13950; 4, M. kansasii ATCC 35775; 5, M. haemophilum ATCC 29548; 6, MC29362; 7, MC17735; 8, MC31558 (lanes 2 to 8 correspond to BstEII-digested amplicons); 9, 50-bp markers; 10, MC31558; 11, MC17735; 12, MC29362; 13, M. haemophilum ATCC 29548; 14, M. kansasii ATCC 35775; 15, M. intracellulare ATCC 13950; and 16, M. tuberculosis ATCC 27294 (lanes 10 to 16 correspond to HaeIII-digested amplicons). Isolates from clinical specimens MC29362, MC17735, and MC31558 were identified as M. haemophilum by matching each specimen's PRA band pattern with that of reference strain M. haemophilum ATCC 29548.

    Techniques Used:

    24) Product Images from "PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation"

    Article Title: PADI4 Haplotypes in Association with RA Mexican Patients, a New Prospect for Antigen Modulation

    Journal: Clinical and Developmental Immunology

    doi: 10.1155/2013/383681

    PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).
    Figure Legend Snippet: PADI4 SNPs enzyme digestion. The figure shows digestion of three SNPs in the PADI4 gene. (a) Shows digestion of PADI4_89, with HaeIII enzyme; lane 1 represents the A/A genotype, lane 2 A/G and 3 G/G. (b) Demonstrates PADI4_90 amplification (221 bp) in lane 1 and digested products with MscI enzyme in lanes 2 (C/C genotype), 3 (C/T genotype), and 4 (T/T genotype). (c) Shows amplification product of PADI4_92 in lane 1 (363 bp) and restriction products obtained with the enzyme MspI; lane 2 corresponds to the G/G genotype, lane 3 G/C, and lane 4 C/C. Visualized in 8% (29 : 1) polyacrylamide gel with silver staining. M: molecular weight marker (50 bp).

    Techniques Used: Amplification, Silver Staining, Molecular Weight, Marker

    25) Product Images from "The Tumor Suppressor Chromodomain Helicase DNA-binding Protein 5 (CHD5) Remodels Nucleosomes by Unwrapping *"

    Article Title: The Tumor Suppressor Chromodomain Helicase DNA-binding Protein 5 (CHD5) Remodels Nucleosomes by Unwrapping *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.568568

    CHD5 is a chromatin remodeling enzyme that increases accessibility of internal nucleosomal DNA sites. A , purification of wild-type CHD5, mut CHD5, ACF, BRG1. B , restriction enzyme accessibility assay using HaeIII and plasmid-assembled chromatin incubated
    Figure Legend Snippet: CHD5 is a chromatin remodeling enzyme that increases accessibility of internal nucleosomal DNA sites. A , purification of wild-type CHD5, mut CHD5, ACF, BRG1. B , restriction enzyme accessibility assay using HaeIII and plasmid-assembled chromatin incubated

    Techniques Used: Purification, Plasmid Preparation, Incubation

    26) Product Images from "Editing of the TRIM5 gene decreases the permissiveness of human T lymphocytic cells to HIV-1"

    Article Title: Editing of the TRIM5 gene decreases the permissiveness of human T lymphocytic cells to HIV-1

    Journal: bioRxiv

    doi: 10.1101/2020.12.07.413708

    HaeIII screening of selected Jurkat clones. The 6 clones found to be positive in the HDR editing-specific PCR test, along with 8 randomly chosen negative clones, were subjected to a PCR assay using primers that bind outside of the genomic region complementary to the HDR donor DNA, followed by digestion with HaeIII. U and D indicates bands of the expected size for the undigested PCR product and the HaeIII digestion products, respectively.
    Figure Legend Snippet: HaeIII screening of selected Jurkat clones. The 6 clones found to be positive in the HDR editing-specific PCR test, along with 8 randomly chosen negative clones, were subjected to a PCR assay using primers that bind outside of the genomic region complementary to the HDR donor DNA, followed by digestion with HaeIII. U and D indicates bands of the expected size for the undigested PCR product and the HaeIII digestion products, respectively.

    Techniques Used: Clone Assay, Polymerase Chain Reaction

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    New England Biolabs haeiii
    The mutational effects in the laboratory drift correlate with sequence exchanges in <t>M.HaeIII</t> orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target <t>DNA</t> binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.
    Haeiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    New England Biolabs haeiii restriction endonuclease enzyme
    Restriction Enzyme Digestion Profile of Amplified ITS1 Region with the <t>HaeIII</t> Restriction Enzyme M, molecular marker (50 bp); lane 1, standard L. major ; lane 2, standard L. tropica ; lanes 3 - 9, <t>PCR</t> products; lane 10, the only sample different from the normal samples and standard patterns, probably flagellates; lane 11, negative control.
    Haeiii Restriction Endonuclease Enzyme, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Journal: PLoS Computational Biology

    Article Title: Systematic Mapping of Protein Mutational Space by Prolonged Drift Reveals the Deleterious Effects of Seemingly Neutral Mutations

    doi: 10.1371/journal.pcbi.1004421

    Figure Lengend Snippet: The mutational effects in the laboratory drift correlate with sequence exchanges in M.HaeIII orthologs. a . The positional rates of evolution in M.HaeIII’s natural orthologs (‘Rate4site’, μ; red line) were plotted alongside the positional W rel values in M.HaeIII (blue line). The positional W rel values correspond to the average W rel ∑ i { W r e l i ⋅ log 2 [ 1 + 10 ⋅ f ( G 17 i ) ] } , Where i refers to all the possible single nucleotide mutations at a given residue position. Upper panel –positions 2 to 175; Lower panel –positions 176 to 330. Noted are M.HaeIII’s key functional residues, those of the cofactor binding site, the catalytic residues including the enzyme’s reaction center (Cys71, black arrow), and the target DNA binding residues. Also noted are positions of compensatory mutations that were enriched in the drift W rel > 1.1, listed in S2 Table ). b. M.HaeIII’s three-dimensional structure illustrated as a cartoon (PDB id 1dct). Residues are colored from blue to red according to their averaged W rel values (as in c ). The cofactor, SAM, is in yellow, and the enzyme’s catalytic residue (Cys71) is in green. c. The same as b for the positional diversity calculated by Rate4site (μ, as in c ) [ 65 ]. d. The distribution of PROVEAN scores for all possible single nucleotide missense mutations (n = 1,957). Shown are the distribution of mutations categorized as ‘deleterious’ ( W rel ≤0.6), and of mutations categorized as ‘nearly-neutral’, ‘neutral’ and ‘beneficial’ ( W rel > 0.6). e. The same distribution while excluding ‘nearly-neutral’ mutations.

    Article Snippet: About 106 individual transformants were obtained in each round. (ii ) Colonies grown at 37°C overnight were combined, plasmid DNA was extracted and digested with HaeIII (10–20 units, in 50 μl of NEB buffer 2, for 2 hours at 37°C), and re-purified (PCR purification kit, QIAGEN). (iii ) The recovered plasmid DNA was re-transformed for another round of enrichment.

    Techniques: Sequencing, Functional Assay, Binding Assay

    Relative gel mobility of ribozyme decreases with metal ion size (a) Native 8% PAGE of the unfolded (U) or folded (F) Tetrahymena ribozyme, in running buffer containing 3 mM metal chloride (see Methods). The unfolded ribozyme samples were in HE buffer, the folded RNA was in HE plus 1.4 mM MgCl 2 , 1.4 mM CaCl 2 or 6 mM SrCl 2 , respectively. ΦX, 5’- 32 P-labeled ΦX/ Hae III DNA. (b) Relative mobility of the folded ribozyme compared to the 271 or 310 bp DNA, as a function of counterion charge density (ζ). Similar results were obtained in two trials.

    Journal: Journal of the American Chemical Society

    Article Title: Charge density of divalent metal cations determines RNA stability

    doi: 10.1021/ja068027r

    Figure Lengend Snippet: Relative gel mobility of ribozyme decreases with metal ion size (a) Native 8% PAGE of the unfolded (U) or folded (F) Tetrahymena ribozyme, in running buffer containing 3 mM metal chloride (see Methods). The unfolded ribozyme samples were in HE buffer, the folded RNA was in HE plus 1.4 mM MgCl 2 , 1.4 mM CaCl 2 or 6 mM SrCl 2 , respectively. ΦX, 5’- 32 P-labeled ΦX/ Hae III DNA. (b) Relative mobility of the folded ribozyme compared to the 271 or 310 bp DNA, as a function of counterion charge density (ζ). Similar results were obtained in two trials.

    Article Snippet: ΦX/ Hae III DNA markers (New England Biolabs) were labeled with γ-[32 P]-ATP and T4 polynucleotide kinase.

    Techniques: Polyacrylamide Gel Electrophoresis, Labeling

    NHEJ-mediated integration of MRSs in HEK293T cells. ( a ) Gel image showing T7EI cleavage products (red brackets) of edited BCL11A-XL 3′ UTR 1 site. ( b ) Quantification of T7EI gel bands showing similar percentages of genome editing across samples (32.6% to 44.8%). ( c ) Apparent absence of PCR-RFLP HaeIII cleavage for test samples, indicative of dsODN integration being below the detection threshold. ( d ) Top: schematic illustration of dsODN-specific amplification assay at the 3′ UTR 1 site; bottom: amplicons of ~250 and ~302 bp revealing the presence of 2 MRSs and 4 MRSs in cells, respectively. Of note, undesirable inverted integration of MRSs into 3′ UTR 1 (see greyed-out part of Figure 2 ) is not detected by this assay. MOCK: mock-nucleofected cell sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, dsODN451-2MRSs/*: cell samples nucleofected with RNPs and dsODN bearing two miR451a MRSs at the indicated picomole quantity, dsODN451-4MRSs/5: cell sample nucleofected with RNPs and 5 pmole dsODN bearing four miR451a MRSs, +: a 994-bp PCR product giving cleavage products of 572 and 422 bp after digestion with HaeIII.

    Journal: International Journal of Molecular Sciences

    Article Title: CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

    doi: 10.3390/ijms23031082

    Figure Lengend Snippet: NHEJ-mediated integration of MRSs in HEK293T cells. ( a ) Gel image showing T7EI cleavage products (red brackets) of edited BCL11A-XL 3′ UTR 1 site. ( b ) Quantification of T7EI gel bands showing similar percentages of genome editing across samples (32.6% to 44.8%). ( c ) Apparent absence of PCR-RFLP HaeIII cleavage for test samples, indicative of dsODN integration being below the detection threshold. ( d ) Top: schematic illustration of dsODN-specific amplification assay at the 3′ UTR 1 site; bottom: amplicons of ~250 and ~302 bp revealing the presence of 2 MRSs and 4 MRSs in cells, respectively. Of note, undesirable inverted integration of MRSs into 3′ UTR 1 (see greyed-out part of Figure 2 ) is not detected by this assay. MOCK: mock-nucleofected cell sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, dsODN451-2MRSs/*: cell samples nucleofected with RNPs and dsODN bearing two miR451a MRSs at the indicated picomole quantity, dsODN451-4MRSs/5: cell sample nucleofected with RNPs and 5 pmole dsODN bearing four miR451a MRSs, +: a 994-bp PCR product giving cleavage products of 572 and 422 bp after digestion with HaeIII.

    Article Snippet: Briefly, PCR products of 300–500 bp containing the DSB-enabled tag-insertion site were digested overnight with HaeIII (for dsODNs, ssODN451TS-2MRSs, ssODN451NTS-2MRSs and Alt-R HDR-2MRSs) and DdeI (for ssODN451TS-1MRS and ssODN451NTS-1MRS) restriction enzymes (New England Biolabs, Ipswich, MA, USA) in CutSmart buffer according to the manufacturer’s instructions.

    Techniques: Non-Homologous End Joining, Polymerase Chain Reaction, Amplification

    HDR-mediated integration of MRSs in HUDEP-2 cells. PCR-RFLP analysis of cells nucleofected with RNPs and ss DNA oligos was performed for the assessment of integration efficiency. Bands of 436 bp represent unmodified target sites, larger bands insertions with incomplete digestion by DdeI/HaeIII, and smaller bands cleavage products of insertions. All bands corresponding to insertions are indicated by arrowheads (band sizes of 554 bp and 277 bp for donors with 2 MRSs, and of 528 bp and 264 bp for donors with 1 MRS). Corresponding rates of DdeI/HaeIII cleavage in PCR-RFLP (%) (after subtraction of background average cleavage rates of control samples) are reported below the gels. ( a ) Analysis of cells nucleofected with RNPs and Ultramer DNA Oligos. ( b ) Analysis of cells nucleofected with Alt-R RNPs and Alt-R HDR-2MRSs DNA Oligo (left gel), shown in comparison with analysis of cells nucleofected with standard Cas9 RNPs and Ultramer ssODN451TS_2MRSs DNA Oligo (right gel). Separate gels are indicated by dashed lines. MOCK: mock-nucleofected sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, NTS: non-target strand, TS: target strand, +: PCR products bearing restriction sites for DdeI/HaeIII used as positive controls. See Section 4.2 and Supplementary Table S2 for ss DNA oligo naming.

    Journal: International Journal of Molecular Sciences

    Article Title: CRISPR Editing Enables Consequential Tag-Activated MicroRNA-Mediated Endogene Deactivation

    doi: 10.3390/ijms23031082

    Figure Lengend Snippet: HDR-mediated integration of MRSs in HUDEP-2 cells. PCR-RFLP analysis of cells nucleofected with RNPs and ss DNA oligos was performed for the assessment of integration efficiency. Bands of 436 bp represent unmodified target sites, larger bands insertions with incomplete digestion by DdeI/HaeIII, and smaller bands cleavage products of insertions. All bands corresponding to insertions are indicated by arrowheads (band sizes of 554 bp and 277 bp for donors with 2 MRSs, and of 528 bp and 264 bp for donors with 1 MRS). Corresponding rates of DdeI/HaeIII cleavage in PCR-RFLP (%) (after subtraction of background average cleavage rates of control samples) are reported below the gels. ( a ) Analysis of cells nucleofected with RNPs and Ultramer DNA Oligos. ( b ) Analysis of cells nucleofected with Alt-R RNPs and Alt-R HDR-2MRSs DNA Oligo (left gel), shown in comparison with analysis of cells nucleofected with standard Cas9 RNPs and Ultramer ssODN451TS_2MRSs DNA Oligo (right gel). Separate gels are indicated by dashed lines. MOCK: mock-nucleofected sample, NO DONOR: cell sample nucleofected only with RNPs, MRS: miRNA recognition site, NTS: non-target strand, TS: target strand, +: PCR products bearing restriction sites for DdeI/HaeIII used as positive controls. See Section 4.2 and Supplementary Table S2 for ss DNA oligo naming.

    Article Snippet: Briefly, PCR products of 300–500 bp containing the DSB-enabled tag-insertion site were digested overnight with HaeIII (for dsODNs, ssODN451TS-2MRSs, ssODN451NTS-2MRSs and Alt-R HDR-2MRSs) and DdeI (for ssODN451TS-1MRS and ssODN451NTS-1MRS) restriction enzymes (New England Biolabs, Ipswich, MA, USA) in CutSmart buffer according to the manufacturer’s instructions.

    Techniques: Polymerase Chain Reaction

    Restriction Enzyme Digestion Profile of Amplified ITS1 Region with the HaeIII Restriction Enzyme M, molecular marker (50 bp); lane 1, standard L. major ; lane 2, standard L. tropica ; lanes 3 - 9, PCR products; lane 10, the only sample different from the normal samples and standard patterns, probably flagellates; lane 11, negative control.

    Journal: Jundishapur Journal of Microbiology

    Article Title: Study of Genetic Variation of Leishmania major Based on Internal Transcribed Spacer 1 (ITS1) in Chabahar, Iran

    doi: 10.5812/jjm.33498

    Figure Lengend Snippet: Restriction Enzyme Digestion Profile of Amplified ITS1 Region with the HaeIII Restriction Enzyme M, molecular marker (50 bp); lane 1, standard L. major ; lane 2, standard L. tropica ; lanes 3 - 9, PCR products; lane 10, the only sample different from the normal samples and standard patterns, probably flagellates; lane 11, negative control.

    Article Snippet: Restriction Fragment Length Polymorphism The PCR products from ITS1-DNA PCR were digested using the HaeIII restriction endonuclease enzyme and the Taq1 restriction enzyme, as recommended by the manufacture (New England BioLabs, Inc.).

    Techniques: Amplification, Marker, Polymerase Chain Reaction, Negative Control