hindiii  (New England Biolabs)


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    HindIII
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    HindIII 50 000 units
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    r0104l
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    Restriction Enzymes
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    New England Biolabs hindiii
    HindIII
    HindIII 50 000 units
    https://www.bioz.com/result/hindiii/product/New England Biolabs
    Average 99 stars, based on 786 article reviews
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    hindiii - by Bioz Stars, 2020-07
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    1) Product Images from "Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ"

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

    Journal: BMC Genomics

    doi: 10.1186/s12864-016-3331-9

    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype
    Figure Legend Snippet: Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Techniques Used: Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, In Vitro, Recombination Assay, Clone Assay, Incubation, Marker

    2) Product Images from "Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins"

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0202138

    Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).
    Figure Legend Snippet: Sites of structural changes induced by the hyper-negative supercoiling detected by Nuclease SI. (A) Experimental scheme. The red-filled circle designates 32 P. The different steps of the experiment are indicated: first (1), the digestion by the Nuclease SI; second (2), the digestion by (BamHI + BglII) or (BahmHI + HindIII); third (3), electrophoresis on a sequencing gel. (B) The enzymatic probe used to map the fine structure of the T -2 and T -6 topoisomers is Nuclease SI. Nuclease SI is at 2 mU microL -1 and DNA at 0.5 nM. After the Nuclease SI reaction, the samples are treated to remove the proteins. The DNAs are precipitated and submitted to the BamHI+HindIII double digestion to only visualize DNA fragments from one of the two radiolabeled strands. The reaction products are analyzed on two different sequencing gels (8% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 5) or the guanines and adenines (G+A lanes; lanes 2 and 6). (C) Same as 3B except that the samples are submitted to the BglII+BamHI double digestion to only visualize DNA fragments from the complementary radiolabeled strands. The reaction products are analyzed on two different sequencing gels (7% to see long DNA fragments, 12% to see short DNA fragments) as indicated. G and G+A lanes correspond to the products of the Maxam and Gilbert reactions to identify specifically the guanines (G lanes; lanes 1 and 7) or the guanines and adenines (G+A lanes; lanes 2 and 6).

    Techniques Used: Electrophoresis, Sequencing

    3) Product Images from "Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate "

    Article Title: Genomic Analysis Reveals Mycoplasma pneumoniae Repetitive Element 1-Mediated Recombination in a Clinical Isolate

    Journal: Infection and Immunity

    doi: 10.1128/IAI.01621-07

    Southern analysis of strains M129 and S1. Chromosomal DNA isolated from M129 (lanes 1, 3, 5, and 7) and S1 (lanes 2, 4, 6, and 8) was digested to completion with EcoRI and HindIII, and the fragments generated were separated on 1% agarose gels
    Figure Legend Snippet: Southern analysis of strains M129 and S1. Chromosomal DNA isolated from M129 (lanes 1, 3, 5, and 7) and S1 (lanes 2, 4, 6, and 8) was digested to completion with EcoRI and HindIII, and the fragments generated were separated on 1% agarose gels

    Techniques Used: Isolation, Generated

    4) Product Images from "Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿"

    Article Title: Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00351-10

    Gel showing a summary of Southern hybridization data from six gels of HindIII-restricted RN4220-RF transconjugant plasmid DNA, pSK41 (lane 9), and pUSA03 and pUSA02 (lane 16). Four probes each hybridized with a unique HindIII fragment in all plasmid DNA
    Figure Legend Snippet: Gel showing a summary of Southern hybridization data from six gels of HindIII-restricted RN4220-RF transconjugant plasmid DNA, pSK41 (lane 9), and pUSA03 and pUSA02 (lane 16). Four probes each hybridized with a unique HindIII fragment in all plasmid DNA

    Techniques Used: Hybridization, Plasmid Preparation

    5) Product Images from "GES Extended-Spectrum ?-Lactamases in Acinetobacter baumannii Isolates in Belgium ▿"

    Article Title: GES Extended-Spectrum ?-Lactamases in Acinetobacter baumannii Isolates in Belgium ▿

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.00871-10

    Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from
    Figure Legend Snippet: Crude plasmid extracts and banding patterns of plasmids pGES-11 and pGES-12 corestricted with HindIII and EcoRI. Lane M, 2 log DNA ladder (0.1 to 10.0 kb); lane 1, pGES-11 (from isolate 9027) extract digested with EcoRI and HindIII; lane 2, pGES-12 (from

    Techniques Used: Plasmid Preparation

    6) Product Images from "Conservative site-specific and single-copy transgenesis in human LINE-1 elements"

    Article Title: Conservative site-specific and single-copy transgenesis in human LINE-1 elements

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv1345

    att H4X targeting in human embryonic stem cell (hESCs). ( A ) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into att H4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. ( B ) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS ( pCMVssInt-C3C ), 6xHIS-tagged Int-C3CNLS ( pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H ) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). ( C ) Example of screening for att H4X × att P4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_ att H4X_F2 and att P rev (for the left junction; top left panel) and cs_ att H4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers att P rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (10 7 , 10 8 ), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP . White arrow heads indicate fragments of the expected size in the targeted clones.
    Figure Legend Snippet: att H4X targeting in human embryonic stem cell (hESCs). ( A ) Schematic diagram of pTZ-attP4X-UN-EF1α-eGFP targeting vector after integration into att H4X. Positions of relevant primers, the Southern probe targeting EGFP and HindIII and XbaI restriction sites are indicated. ( B ) Western blot showing Integrase expression in hESCs. Lysates from hESCs transfected with plasmids expressing Int-C3CNLS ( pCMVssInt-C3C ), 6xHIS-tagged Int-C3CNLS ( pCMVssInt-C3C-H, pEF-Int-C3C-H, pEFssInt-C3C-H ) and untransfected control cells were analyzed by western blotting with an anti-HIS tag antibody (top panel). Purified HIS-tagged Integrase C3 was employed as positive control. β-actin was used as loading control (bottom panel). ( C ) Example of screening for att H4X × att P4X recombination events in hESCs. PCR was performed with genomic DNA (extracted from neomycin-resistant, EGFP-positive hESC recombinants) and primers cs_ att H4X_F2 and att P rev (for the left junction; top left panel) and cs_ att H4X_R2 and pr21 (for the right junction; bottom left panel). PCR amplified products of the expected sizes (278 and 439 bp) were detected in clone #24. The right panel shows a PCR analysis to confirm site-specific recombination in clone #24 using different genomic locus-specific primers. PCR-amplified products of the expected sizes (∼1.25 kb with primers att P rev and 24G-F2, and ∼750 bp with primers pr21 and 24G-R1) were obtained and confirmed by sequencing. W, no DNA template control; ES, negative control (genomic DNA from parental hESCs); +, positive control (genomic DNA from HT1080 clone #19); M, 100 bp DNA ladder; M1, 1 kb DNA ladder; 16 to 27, genomic DNA from neomycin resistant hESC clones obtained through co-transfection of pTZ-attP4X-UN-EF1α-eGFP and pEF1α-ssInt-C3CNLS . ( D ) Southern blot analysis. Genomic DNA purified from three targeted hESC clones and parental hESC cell lines were digested with HindIII or XbaI. A probe complementary to EGFP was employed. Lanes: M1, 1 kb DNA ladder; m, DNA ladder (TeloTAGGG Telomere Length Assay kit, Roche); ES, parental DNA; 3, 24, 59, genomic DNA from targeted hESC clones; pUN4X (10 7 , 10 8 ), copies of linearized targeting vector pTZ-attP4X-UN-EF1α-eGFP . White arrow heads indicate fragments of the expected size in the targeted clones.

    Techniques Used: Plasmid Preparation, Western Blot, Expressing, Transfection, Purification, Positive Control, Polymerase Chain Reaction, Amplification, Sequencing, Negative Control, Clone Assay, Cotransfection, Southern Blot

    7) Product Images from "Analyses of germline variants associated with ovarian cancer survival identify functional candidates at the 1q22 and 19p12 outcome loci"

    Article Title: Analyses of germline variants associated with ovarian cancer survival identify functional candidates at the 1q22 and 19p12 outcome loci

    Journal: Oncotarget

    doi: 10.18632/oncotarget.18501

    Two regions containing candidate all chemo PFS candidate variants interact with the ZNF100 promoter in ovarian cancer cell lines at the 19p12 outcome locus The figure shows 3C analyses of interactions between HindIII fragments and the ZNF100 promoter region (highlighted in pink) in COV362 and OVCAR8 cells. For each cell line, interaction frequencies were normalised to those of the fragment proximal to the promoter. Interaction frequencies from three independent biological replicates are shown (error bars represent standard error of the mean). The original variant associated with any chemotherapy PFS, rs3795247, is shown with correlated candidate outcome variants ( r 2 > 0.4) in black. Roadmap Consortium chromatin state segmentation for normal ovarian tissue using a Hidden Markov Model (Chrom HMM) is shown (red = active transcription start sites, dark green = weak transcription, green = strong transcription, green/yellow = genic enhancers, yellow = enhancers, aquamarine = ZNF gene repeats and turquoise = heterochromatin). H3K4Me1 modification in normal ovarian tissue is also indicated. Putative Regulatory Elements (PREs), and their coincident candidate variants, cloned into reporter gene constructs are highlighted in blue.
    Figure Legend Snippet: Two regions containing candidate all chemo PFS candidate variants interact with the ZNF100 promoter in ovarian cancer cell lines at the 19p12 outcome locus The figure shows 3C analyses of interactions between HindIII fragments and the ZNF100 promoter region (highlighted in pink) in COV362 and OVCAR8 cells. For each cell line, interaction frequencies were normalised to those of the fragment proximal to the promoter. Interaction frequencies from three independent biological replicates are shown (error bars represent standard error of the mean). The original variant associated with any chemotherapy PFS, rs3795247, is shown with correlated candidate outcome variants ( r 2 > 0.4) in black. Roadmap Consortium chromatin state segmentation for normal ovarian tissue using a Hidden Markov Model (Chrom HMM) is shown (red = active transcription start sites, dark green = weak transcription, green = strong transcription, green/yellow = genic enhancers, yellow = enhancers, aquamarine = ZNF gene repeats and turquoise = heterochromatin). H3K4Me1 modification in normal ovarian tissue is also indicated. Putative Regulatory Elements (PREs), and their coincident candidate variants, cloned into reporter gene constructs are highlighted in blue.

    Techniques Used: Variant Assay, Modification, Clone Assay, Construct

    8) Product Images from "Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells"

    Article Title: Tumor treating fields (TTFields) delay DNA damage repair following radiation treatment of glioma cells

    Journal: Radiation Oncology (London, England)

    doi: 10.1186/s13014-017-0941-6

    TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c - d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown
    Figure Legend Snippet: TTFields Influence DNA Damage Repair by Homologous Recombination in Glioma Cells. a pDNA-PKcs (pS2056) and total DNA-PK were compared between U-118 MG cells either untreated or treated with RT or TTFields alone or their combination at indicated time points post RT (4 Gy). Lamin B was used as loading control. b U-118 MG cells were transfected with an intact pGL2-Luc vector or vector that was linearized with either HindIII or EcoRI. Luc activity was measured in cells prior and post 24 h TTFields treatment. c - d U-118 MG cells were irradiated with 4 Gy and immediately treated with TTFields for 1 h, 2 h, or 24 h. c Rad 51 foci formation was analyzed by immunofluorescence at 24 h post treatment. Rad 51 foci (Red) and DAPI (blue) stained nuclei are shown. Scale bar - 5 μm. d The average Rad51 foci in cells with more than 5 foci are shown

    Techniques Used: Homologous Recombination, Transfection, Plasmid Preparation, Activity Assay, Irradiation, Immunofluorescence, Staining

    9) Product Images from "Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt ▿"

    Article Title: Initiation of Epstein-Barr Virus Lytic Replication Requires Transcription and the Formation of a Stable RNA-DNA Hybrid Molecule at OriLyt ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02175-10

    RNase H1 impairs OriLyt-dependent plasmid replication. Wild-type pBluescript OriLyt plasmids were cotransfected with BZLF1 into ZKO-293 cells with an RNase H1 expression plasmid or control vector. Plasmids were recovered from cells, digested with (A and B) or without (C and D) HindIII and DpnI enzymes, and analyzed by Southern blotting. Three identical, independent experiments were conducted in each case. One representative experiment is shown for each (A and C). Relative plasmid amounts were quantified using quantitative Southern blotting (B and D). In the cases where enzymes were used, results were calculated as the DpnI-resistant signal over the DpnI-digested signal and were normalized to the value obtained for vector controls (B). In cases where no enzymes were used, results were calculated as replicating (top band) plasmid over supercoiled (bottom band) signal and normalized to the value obtained for vector controls (D). Data averages from all three identical, independent experiments are shown in each case, with error bars representing the standard deviations. Statistical significance was calculated using a two-tailed, unpaired t test. (E) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.
    Figure Legend Snippet: RNase H1 impairs OriLyt-dependent plasmid replication. Wild-type pBluescript OriLyt plasmids were cotransfected with BZLF1 into ZKO-293 cells with an RNase H1 expression plasmid or control vector. Plasmids were recovered from cells, digested with (A and B) or without (C and D) HindIII and DpnI enzymes, and analyzed by Southern blotting. Three identical, independent experiments were conducted in each case. One representative experiment is shown for each (A and C). Relative plasmid amounts were quantified using quantitative Southern blotting (B and D). In the cases where enzymes were used, results were calculated as the DpnI-resistant signal over the DpnI-digested signal and were normalized to the value obtained for vector controls (B). In cases where no enzymes were used, results were calculated as replicating (top band) plasmid over supercoiled (bottom band) signal and normalized to the value obtained for vector controls (D). Data averages from all three identical, independent experiments are shown in each case, with error bars representing the standard deviations. Statistical significance was calculated using a two-tailed, unpaired t test. (E) Western blot assays were used to monitor Zta and BALF2 protein expression levels in all transfected cells.

    Techniques Used: Plasmid Preparation, Expressing, Southern Blot, Two Tailed Test, Western Blot, Transfection

    10) Product Images from "Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop"

    Article Title: Imprinting at the PLAGL1 domain is contained within a 70-kb CTCF/cohesin-mediated non-allelic chromatin loop

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks1355

    Chromatin interactions with the CTCF sites upstream of the PLAGL1 -DMR. ( A ) The position of the HindIII sites used for 3C analysis, and annotated ChIA-PET data showing CTCF and POL2 interactions. ( B ) The looping profile in brain (black), placental TCL1 cell line (red) and normal leucocytes (blue) using a constant primer 5 kb upstream of the PLAGL1 -DMR, showing strong interactions with the 3′ UTR and alternative promoters of PLAGL1 . The x-axis shows the position of the primers used. ( C ) The interaction profile in expressing tissues, brain (black) and placental TCL1 cell line (red), using a constant primer between the P3 and P4 promoters. ( D ) Heterozygous SNP rs2064661 in constant fragment 5 kb upstream of the PLAGL1 -DMR allowed for allele-specific chromatin interactions to be assessed in adult brain. Sequence traces reveal biallelic higher-order chromatin interactions between the constant fragment and various contact points throughout the PLAGL1 domain.
    Figure Legend Snippet: Chromatin interactions with the CTCF sites upstream of the PLAGL1 -DMR. ( A ) The position of the HindIII sites used for 3C analysis, and annotated ChIA-PET data showing CTCF and POL2 interactions. ( B ) The looping profile in brain (black), placental TCL1 cell line (red) and normal leucocytes (blue) using a constant primer 5 kb upstream of the PLAGL1 -DMR, showing strong interactions with the 3′ UTR and alternative promoters of PLAGL1 . The x-axis shows the position of the primers used. ( C ) The interaction profile in expressing tissues, brain (black) and placental TCL1 cell line (red), using a constant primer between the P3 and P4 promoters. ( D ) Heterozygous SNP rs2064661 in constant fragment 5 kb upstream of the PLAGL1 -DMR allowed for allele-specific chromatin interactions to be assessed in adult brain. Sequence traces reveal biallelic higher-order chromatin interactions between the constant fragment and various contact points throughout the PLAGL1 domain.

    Techniques Used: ChIA Pet Assay, Expressing, Sequencing

    11) Product Images from "Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses"

    Article Title: Simple and Cost-Effective Restriction Endonuclease Analysis of Human Adenoviruses

    Journal: BioMed Research International

    doi: 10.1155/2014/363790

    Picture of the REA. (a) REA pattern for BamHI and SmaI (fast digest). (b) REA pattern for BglII and HindIII . M shows Lambda DNA-HindIII digest marker. Numbers 1, 3, 4, and 37 show HAdV-1, -3, -4, and -37, respectively.
    Figure Legend Snippet: Picture of the REA. (a) REA pattern for BamHI and SmaI (fast digest). (b) REA pattern for BglII and HindIII . M shows Lambda DNA-HindIII digest marker. Numbers 1, 3, 4, and 37 show HAdV-1, -3, -4, and -37, respectively.

    Techniques Used: Lambda DNA Preparation, Marker

    12) Product Images from "Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals"

    Article Title: Transcription-Dependent Gene Looping of the HIV-1 Provirus Is Dictated by Recognition of Pre-mRNA Processing Signals

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2007.11.030

    Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control PCR panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 qRT-PCR at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).
    Figure Legend Snippet: Integrated U1 HIV-1 Proviruses Form Quantitatively Different Looping Conformations (A) Representation of integrated provirus and flanking chromosomal sequence with restriction enzyme sites and primers for BanI and HindIII 3C analysis. Numbers denote distance from 5′ (−) or 3′ (+) proviral ends. Arrows indicate primer direction and name; black/gray arrows refer to primers that detect LTR and the MSD, respectively. HIV-1 long-terminal repeat (LTR) regions (U3, R, and U5), MSD, and polyadenylation sites (pA) are indicated. (B) int-ChrX 3C. Unstimulated (−TPA), cells after 5 hr TPA (+TPA), and control PCR panel (control). Positive lanes (+) signify internal HIV-1 PCR controls on U1 gDNA (control panel) and chromatin (for −/+TPA; see Experimental Procedures ). Common PCR primers are shown above the figure, with the second primer shown above each lane. Graphs below represent quantified percentages of 3C product observed compared to PCR control, standardized between − and +TPA samples (using internal PCR controls; see + lane). (C) Quantitative analysis of Tat- or TPA-induced int-ChrX and int-Chr2 loop structures. (a) HIV-1 qRT-PCR at 0, 3, and 5 hr post-TPA treatment with nuc1 primers ( Figure 1 A) standardized to 18S rRNA transcription. (b) q3C HindIII-digested U1 chromatin analysis; primers used to detect the “loop”: interaction (primers 22/23 for int-Chr2 and X2/X3 for int-ChrX) compared to the adjacent amplified fragment (primers 22/H7 or X2/H7). (D) (a) HIV-1 qRT-PCR treated with 0 (10 μg GFP control), 5, or 10 μg of GFP-tagged Tat protein. (b) q3C HindIII analysis of U1 chromatin treated with 10 μg Tat-GFP or GFP. Error bars represent SEM from n = 6 samples from two separate chromatin preparations (except 22/23 and X2/X3 in Tat induction analysis where n = 9, from three separate chromatin preparations).

    Techniques Used: Sequencing, Polymerase Chain Reaction, Quantitative RT-PCR, Amplification

    13) Product Images from "Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes "

    Article Title: Enhancer Complexes Located Downstream of Both Human Immunoglobulin C? Genes

    Journal: The Journal of Experimental Medicine

    doi:

    Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands
    Figure Legend Snippet: Mapping of DNase I hypersensitive sites in the regions 3′ of the human Cα genes. ( A ) DNase I hypersensitive sites lie downstream from the human Cα genes in the HS Sultan plasmacytoma. DNA samples prepared from DNase I–digested nuclei isolated from K562 promyeloid and HS Sultan myeloma cells were digested with BglII, electrophoresed, blotted, and hybridized with probe a (αm, Fig.1). No DNase I hypersensitive sites are seen in the K562 samples. In contrast, at least seven DNase I hypersensitive sites are observed in samples from HS Sultan plasmacytoma cells. The size of each DNase I–generated band corresponds to its distance from the BglII sites located ∼1 kb 5′ of each α membrane exon ( αm ). This mapping strategy does not distinguish between sites in the α1 versus α2 loci; sites are labeled according to their subsequent assignment (see B and C , and sequence analyses). Due to their large size, bands resulting from DNase I cutting at the α1 and α2 HS4 sites are not resolved in this analysis. ( B ) HS4 sites are accessible to nuclease in both α1 and α2 loci. HS Sultan nuclei were digested with DNase I or SspI restriction enzyme (both the α1 and α2 HS4 sequences contain an SspI site). Purified DNA was digested with EcoRI and hybridized with probe b′, yielding two closely spaced DNase I HS bands, whose sizes correspond to the expected distance between the HS4 enhancers and the downstream EcoRI sites. Furthermore, there are two similarly positioned bands in the samples from SspI-digested nuclei, indicating that both the α1 and α2 HS4 sites are accessible to SspI. ( C ) Assignment of DNase I hypersensitive sites to the 3′ Cα2 region. HS Sultan DNA samples were digested with HindIII and hybridized with probe g (α2 HS12, Fig. 1 ). Because DNAse I–generated bands from the α1 region which hybridize to this probe are expected to be larger than the 12-kb α2 HindIII fragment, all bands

    Techniques Used: Isolation, Generated, Labeling, Sequencing, Purification

    14) Product Images from "Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1"

    Article Title: Productive Replication of Human Papillomavirus 31 Requires DNA Repair Factor Nbs1

    Journal: Journal of Virology

    doi: 10.1128/JVI.00517-14

    Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T 0 and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T 0 and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.
    Figure Legend Snippet: Nbs1 knockdown disrupts MRN complex formation. (A) Whole-cell lysates were harvested from HFK, CIN612, and CIN612 9E cells stably expressing shScramble or shNBS1 at T 0 and after 72 h of differentiation in high-calcium medium (Ca). Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. GAPDH was used as a loading control. (B) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from HFK and CIN612 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. (C) Total, nuclear (Nuc), and cytoplasmic (Cyto) lysates were harvested from stable CIN612 9E shScramble and CIN612 9E shNBS1 cells. Immunoblotting was performed using Mre11, Rad50, and Nbs1 antibodies. For panels B and C, lamin A/C and tubulin were used to confirm nuclear and cytoplasmic fractionation, respectively. (D) DNA was harvested from CIN612 9E cells at T 0 and after 72 h of differentiation in high-calcium medium with dimethyl sulfoxide (DMSO) as a vehicle control or 50 μM Mre11 inhibitor Mirin. Southern blot analysis was performed to analyze viral genome amplification of DNA digested with BamHI (nonviral genome cutter; upper panel) or HindIII (cuts viral genome once; lower panel). All results are representative of observations of two or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, Fractionation, Southern Blot, Amplification

    Phosphorylation of ATM and Chk2 is maintained with Nbs1 knockdown upon differentiation. (A) Whole-cell lysates were harvested from HFKs and CIN612 9E cells, as well as CIN612 9E cells stably expressing shScramble or shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated ATM (Ser1981) (pATM), total ATM, and Nbs1. Tubulin was used as a loading control. Protein levels were quantified using ImageJ, with phosphorylated protein levels normalized first to total levels and then to tubulin. Levels for this representative experiment are graphed as fold change compared to the T 0 HFK sample, which is set to 1. (B) Whole-cell lysates were harvested from HFK, CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated Chk2 (Thr68) (pChk2), total Chk2, and Nbs1. GAPDH was used as a loading control. Protein levels were quantified using ImageJ as indicated above. Shown is a representative experiment where levels are graphed as fold change compared to the T 0 HFK sample, which is set at 1. (C) DNA was harvested from CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and after 72 h of differentiation in high-calcium medium and linearized by digestion with HindIII. HPV episomes were visualized via Southern blot analysis. Results shown are representative observations of four or more independent experiments.
    Figure Legend Snippet: Phosphorylation of ATM and Chk2 is maintained with Nbs1 knockdown upon differentiation. (A) Whole-cell lysates were harvested from HFKs and CIN612 9E cells, as well as CIN612 9E cells stably expressing shScramble or shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated ATM (Ser1981) (pATM), total ATM, and Nbs1. Tubulin was used as a loading control. Protein levels were quantified using ImageJ, with phosphorylated protein levels normalized first to total levels and then to tubulin. Levels for this representative experiment are graphed as fold change compared to the T 0 HFK sample, which is set to 1. (B) Whole-cell lysates were harvested from HFK, CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and 72 h after differentiation in high-calcium medium. Immunoblotting was performed using antibodies to phosphorylated Chk2 (Thr68) (pChk2), total Chk2, and Nbs1. GAPDH was used as a loading control. Protein levels were quantified using ImageJ as indicated above. Shown is a representative experiment where levels are graphed as fold change compared to the T 0 HFK sample, which is set at 1. (C) DNA was harvested from CIN612 9E, CIN612 9E shScramble, and CIN612 9E shNBS1 cells at T 0 and after 72 h of differentiation in high-calcium medium and linearized by digestion with HindIII. HPV episomes were visualized via Southern blot analysis. Results shown are representative observations of four or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, Southern Blot

    Nbs1 is necessary for productive viral replication. (A) DNA was harvested from CIN612 9E cells stably expressing a Scramble shRNA or Nbs1 shRNA at T 0 (undifferentiated) or after 48 and 96 h of differentiation in high-calcium medium. Southern blot analysis was performed to analyze viral genome amplification. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble, which was set to 1. Densitometry was performed using ImageJ. Ca, calcium. (B) Total protein was harvested from CIN612 9E shScramble and shNbs1 cells at T 0 or after 48 and 96 h of differentiation in high-calcium medium. Western blot analysis was performed using antibodies to Nbs1 and involucrin, and GAPDH as a loading control. (C) DNA was harvested from human foreskin keratinocytes stably maintaining HPV31 genomes (HFK-31) at T 0 or after 24 and 48 h of differentiation in methylcellulose (MC) and analyzed by Southern blotting for amplification of viral genomes. DNA samples were digested with BamHI (does not cut the viral genome; upper panel) or with HindIII to linearize viral genomes. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble (set to 1). Densitometry was performed using ImageJ. (D) Western blot analysis was performed on lysates harvested from HFK-31 cells at T 0 or after 24 and 48 h of differentiation in methylcellulose using antibodies to Nbs1 and involucrin. GAPDH was used as a loading control. All results are representative of observations of four or more independent experiments.
    Figure Legend Snippet: Nbs1 is necessary for productive viral replication. (A) DNA was harvested from CIN612 9E cells stably expressing a Scramble shRNA or Nbs1 shRNA at T 0 (undifferentiated) or after 48 and 96 h of differentiation in high-calcium medium. Southern blot analysis was performed to analyze viral genome amplification. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble, which was set to 1. Densitometry was performed using ImageJ. Ca, calcium. (B) Total protein was harvested from CIN612 9E shScramble and shNbs1 cells at T 0 or after 48 and 96 h of differentiation in high-calcium medium. Western blot analysis was performed using antibodies to Nbs1 and involucrin, and GAPDH as a loading control. (C) DNA was harvested from human foreskin keratinocytes stably maintaining HPV31 genomes (HFK-31) at T 0 or after 24 and 48 h of differentiation in methylcellulose (MC) and analyzed by Southern blotting for amplification of viral genomes. DNA samples were digested with BamHI (does not cut the viral genome; upper panel) or with HindIII to linearize viral genomes. The bar graph represents quantification of the episome copy number present at each time point, relative to T 0 shScramble (set to 1). Densitometry was performed using ImageJ. (D) Western blot analysis was performed on lysates harvested from HFK-31 cells at T 0 or after 24 and 48 h of differentiation in methylcellulose using antibodies to Nbs1 and involucrin. GAPDH was used as a loading control. All results are representative of observations of four or more independent experiments.

    Techniques Used: Stable Transfection, Expressing, shRNA, Southern Blot, Amplification, Western Blot

    15) Product Images from "A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila"

    Article Title: A view through a chromatin loop: insights into the ecdysone activation of early genes in Drosophila

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku754

    Long-distance interactions at the E75 gene in imaginal discs. 3C analysis at the E75 locus was performed in imaginal discs using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar imaginal discs. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘
    Figure Legend Snippet: Long-distance interactions at the E75 gene in imaginal discs. 3C analysis at the E75 locus was performed in imaginal discs using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar imaginal discs. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘

    Techniques Used: Binding Assay, Derivative Assay

    Long-distance 3C interactions at the E75 gene. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel), or E75C (lower panel) and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘
    Figure Legend Snippet: Long-distance 3C interactions at the E75 gene. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel), or E75C (lower panel) and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘

    Techniques Used:

    Long-distance 3C interactions at the Broad locus. Schematic of Broad is shown at the top. Horizontal arrow indicates the TSS, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y -axis) between the fixed HindIII anchor at the proximal promoter and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Locations of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle bracket (‘ > ’) indicates the location and direction of the anchor primer. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter, either in the presence or absence of ecdysone. Coordinates are given along the x -axis relative to the proximal TSS. Asterisk indicates significant difference between ecdysone and control samples (Paired Student's t -test, P
    Figure Legend Snippet: Long-distance 3C interactions at the Broad locus. Schematic of Broad is shown at the top. Horizontal arrow indicates the TSS, vertical bars indicate HindIII sites and numbered arrows designate EcREs. Cross-linking frequencies ( y -axis) between the fixed HindIII anchor at the proximal promoter and the rest of the locus were measured in S2 cells in the absence or presence of ecdysone as indicated. Locations of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle bracket (‘ > ’) indicates the location and direction of the anchor primer. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter, either in the presence or absence of ecdysone. Coordinates are given along the x -axis relative to the proximal TSS. Asterisk indicates significant difference between ecdysone and control samples (Paired Student's t -test, P

    Techniques Used:

    Effect of EcR knockdown on pre-existing promoter–EcRE interactions at the E75 and Broad loci. S2 cells were incubated for 3 days with or without dsRNA targeting EcR , then incubated with or without 1×10 −6 M ecdysone as indicated. Each sample was divided and used in qRT-PCR and 3C analyses. HindIII-based 3C analysis was performed, and interactions were measured between the E75 (A) or Broad (B) promoters and the EcRE(s) indicated ( x- axis). Cross-linking frequencies ( y- axis) are shown relative to the control sample. Bars indicate mean ± SEM from three independent experiments. Inset: total RNA was extracted and transcript abundance was measured by qRT-PCR for E75B , E75A , E75C and Broad core. Expression was normalized to the rp49 transcript and is shown relative to the control sample ( y -axis). Bars indicate mean ± SEM from three independent experiments. Number above each bar indicates fold change relative to control sample.
    Figure Legend Snippet: Effect of EcR knockdown on pre-existing promoter–EcRE interactions at the E75 and Broad loci. S2 cells were incubated for 3 days with or without dsRNA targeting EcR , then incubated with or without 1×10 −6 M ecdysone as indicated. Each sample was divided and used in qRT-PCR and 3C analyses. HindIII-based 3C analysis was performed, and interactions were measured between the E75 (A) or Broad (B) promoters and the EcRE(s) indicated ( x- axis). Cross-linking frequencies ( y- axis) are shown relative to the control sample. Bars indicate mean ± SEM from three independent experiments. Inset: total RNA was extracted and transcript abundance was measured by qRT-PCR for E75B , E75A , E75C and Broad core. Expression was normalized to the rp49 transcript and is shown relative to the control sample ( y -axis). Bars indicate mean ± SEM from three independent experiments. Number above each bar indicates fold change relative to control sample.

    Techniques Used: Incubation, Quantitative RT-PCR, Expressing

    Long-distance interactions at the E75 gene in fat body. 3C analysis at the E75 locus was performed in the fat body using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar or mid-third instar fat bodies as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘
    Figure Legend Snippet: Long-distance interactions at the E75 gene in fat body. 3C analysis at the E75 locus was performed in the fat body using HindIII. Schematic of the E75 locus is shown at the top. Horizontal arrows indicate TSSs and vertical bars indicate HindIII sites. EcR binding peaks (A–L) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for E75B (upper panel), E75A (middle panel) or E75C (lower panel) and the rest of the locus were measured in late third instar or mid-third instar fat bodies as indicated. Location of fixed anchor sites for E75B , E75A and E75C are marked by red, blue and green bars, respectively, and test sites are marked by gray bars. Black angle brackets (‘

    Techniques Used: Binding Assay, Derivative Assay

    Long-distance interactions at the Broad locus in larval tissues. Schematic of Broad is shown at the top. Horizontal arrow indicates TSS and vertical bars indicate HindIII sites. EcR binding peaks (A–E) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for Broad and the rest of the locus were measured in late third instar imaginal discs (upper panel) and in late and mid-third instar fat bodies (lower panel). Location of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle brackets (‘ > ’) indicate the location and direction of anchor primers. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter. Coordinates are given along the x- axis relative to the proximal TSS. Asterisk indicates significant difference between late and mid-third instar fat bodies (Unpaired Student's t -test, P
    Figure Legend Snippet: Long-distance interactions at the Broad locus in larval tissues. Schematic of Broad is shown at the top. Horizontal arrow indicates TSS and vertical bars indicate HindIII sites. EcR binding peaks (A–E) from whole larvae at 0 APF, derived from the modENCODE database, are denoted by red boxes at the top of the diagram. Cross-linking frequencies ( y- axis) between the fixed HindIII anchor for Broad and the rest of the locus were measured in late third instar imaginal discs (upper panel) and in late and mid-third instar fat bodies (lower panel). Location of the fixed anchor site and test sites are marked by blue and gray bars, respectively. Black angle brackets (‘ > ’) indicate the location and direction of anchor primers. Blue brackets indicate the location and direction of test primers in fragments that interact with the Broad promoter. Coordinates are given along the x- axis relative to the proximal TSS. Asterisk indicates significant difference between late and mid-third instar fat bodies (Unpaired Student's t -test, P

    Techniques Used: Binding Assay, Derivative Assay

    16) Product Images from "Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates"

    Article Title: Genetic Environments of the rmtA Gene in Pseudomonas aeruginosa Clinical Isolates

    Journal: Antimicrobial Agents and Chemotherapy

    doi: 10.1128/AAC.48.6.2069-2074.2004

    Comparison of the genetic organizations of AR-2 and AR-11. Double-headed striped arrows indicate the position of the rmtA locus and that of the region common to both sequenced areas. Inserts of pBCRMTH2, pBCRMTE2, and pBCRMTE11 are indicated by horizontal lines. Rectangles filled with wavy lines, sequences similar to part of Tn 5041 . Solid arrowheads in the 15.8-kbp EcoRI fragment, terminal inverted repeats. mer , the mercury resistance operon, includes merR . Sequence 1, transposase gene-like sequence; sequence 2, Na + /H + antiporter-like sequence; orfA , probable tRNA ribosyltransferase gene; orfQ ′, part of orfQ ; orfA ′, part of orfA ; IR, probable inverted repeat. Restriction sites: H, HindIII; E, EcoRI. Sequences 1 and 2 encode no complete proteins due to several frameshifts and deletions.
    Figure Legend Snippet: Comparison of the genetic organizations of AR-2 and AR-11. Double-headed striped arrows indicate the position of the rmtA locus and that of the region common to both sequenced areas. Inserts of pBCRMTH2, pBCRMTE2, and pBCRMTE11 are indicated by horizontal lines. Rectangles filled with wavy lines, sequences similar to part of Tn 5041 . Solid arrowheads in the 15.8-kbp EcoRI fragment, terminal inverted repeats. mer , the mercury resistance operon, includes merR . Sequence 1, transposase gene-like sequence; sequence 2, Na + /H + antiporter-like sequence; orfA , probable tRNA ribosyltransferase gene; orfQ ′, part of orfQ ; orfA ′, part of orfA ; IR, probable inverted repeat. Restriction sites: H, HindIII; E, EcoRI. Sequences 1 and 2 encode no complete proteins due to several frameshifts and deletions.

    Techniques Used: Sequencing

    17) Product Images from "The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness"

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    Journal: Viruses

    doi: 10.3390/v10070343

    MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.
    Figure Legend Snippet: MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.

    Techniques Used: Modification, Agarose Gel Electrophoresis, Incubation, Staining

    18) Product Images from "Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage"

    Article Title: Rescue of Infectious Recombinant Hazara Nairovirus from cDNA Reveals the Nucleocapsid Protein DQVD Caspase Cleavage Motif Performs an Essential Role other than Cleavage

    Journal: Journal of Virology

    doi: 10.1128/JVI.00616-19

    Confirmation of cDNA origin via recovery of mutant rHAZV. (A) Table outlining the change (underlined) to the cDNA sequence of the HAZV-N ORF and the resulting amino acid sequence. (B) Detection of HAZV-N by Western blotting posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf). Supernatant samples collected from transfected BSR-T7 cells at 96 h posttransfection were used to infect monolayers of SW13 cells. Following a 48-h infection, lysates were collected and analyzed by Western blotting for N expression. Recovery of rHAZV containing a HindIII restriction site (rHAZV-G723T) was carried out alongside complete control recovery of rHAZV. Detection of GAPDH abundance was included as a loading control. (C) Schematic showing the location of the inserted HindIII restriction site in the S segment ORF cDNA. UTR, untranslated region. (D) Restriction digest of double-stranded DNA fragments following RNA extraction of rHAZV- and rHAZV-G723T-containing supernatants, first-strand synthesis, and PCR amplification of viral genetic material.
    Figure Legend Snippet: Confirmation of cDNA origin via recovery of mutant rHAZV. (A) Table outlining the change (underlined) to the cDNA sequence of the HAZV-N ORF and the resulting amino acid sequence. (B) Detection of HAZV-N by Western blotting posttransfection (p.Tr) of BSR-T7 cells and 48 h postinfection (p.Inf). Supernatant samples collected from transfected BSR-T7 cells at 96 h posttransfection were used to infect monolayers of SW13 cells. Following a 48-h infection, lysates were collected and analyzed by Western blotting for N expression. Recovery of rHAZV containing a HindIII restriction site (rHAZV-G723T) was carried out alongside complete control recovery of rHAZV. Detection of GAPDH abundance was included as a loading control. (C) Schematic showing the location of the inserted HindIII restriction site in the S segment ORF cDNA. UTR, untranslated region. (D) Restriction digest of double-stranded DNA fragments following RNA extraction of rHAZV- and rHAZV-G723T-containing supernatants, first-strand synthesis, and PCR amplification of viral genetic material.

    Techniques Used: Mutagenesis, Sequencing, Western Blot, Transfection, Infection, Expressing, RNA Extraction, Polymerase Chain Reaction, Amplification

    19) Product Images from "A simple biophysical model emulates budding yeast chromosome condensation"

    Article Title: A simple biophysical model emulates budding yeast chromosome condensation

    Journal: eLife

    doi: 10.7554/eLife.05565

    Experimental and computational intrachromosomal interaction frequency maps. ( A ) Close-up of the chromosomal viewpoints selected for 4C analysis. Condensin localization along part of the chromosome 5 right arm is shown together with genomic HindIII recognition sites and the four 4C view points that do (1 and 4) or do not (2 and 3) contain a condensin binding site. ( B ) Experimental 4C interaction maps of the four regions, in both interphase and mitosis. Shown is also a 4C map of region 4 in mitosis after condensin has been depleted from the nucleus using the brn1-aa allele. The y-axis shows sequencing read counts normalized to the total number of mapped reads in each sample. The percentage of interactions that extend farther than 100 kb from the viewpoint is indicated. ( C ) Averaged computational intrachromosomal interaction maps of 6 viewpoints within 50 kb from the chromosome ends, on or between condensin binding sites, generated using both the Type I and Type II model and sampled over 1000 time points and 30 simulations in interphase and mitosis (condensin interaction dissociation rates 10 −3 and 10 −4 , respectively). The y-axis shows interaction frequencies of the viewpoints normalized to all interactions. ( D ) Percentage of interactions that extend beyond 100 kb from the viewpoint under the indicated conditions. The mean of the four experimental fragments, or of the simulated distributions, is shown together with the standard deviation. *p
    Figure Legend Snippet: Experimental and computational intrachromosomal interaction frequency maps. ( A ) Close-up of the chromosomal viewpoints selected for 4C analysis. Condensin localization along part of the chromosome 5 right arm is shown together with genomic HindIII recognition sites and the four 4C view points that do (1 and 4) or do not (2 and 3) contain a condensin binding site. ( B ) Experimental 4C interaction maps of the four regions, in both interphase and mitosis. Shown is also a 4C map of region 4 in mitosis after condensin has been depleted from the nucleus using the brn1-aa allele. The y-axis shows sequencing read counts normalized to the total number of mapped reads in each sample. The percentage of interactions that extend farther than 100 kb from the viewpoint is indicated. ( C ) Averaged computational intrachromosomal interaction maps of 6 viewpoints within 50 kb from the chromosome ends, on or between condensin binding sites, generated using both the Type I and Type II model and sampled over 1000 time points and 30 simulations in interphase and mitosis (condensin interaction dissociation rates 10 −3 and 10 −4 , respectively). The y-axis shows interaction frequencies of the viewpoints normalized to all interactions. ( D ) Percentage of interactions that extend beyond 100 kb from the viewpoint under the indicated conditions. The mean of the four experimental fragments, or of the simulated distributions, is shown together with the standard deviation. *p

    Techniques Used: Binding Assay, Sequencing, Generated, Standard Deviation

    20) Product Images from "Characterization of the Human SNM1A and SNM1B/Apollo DNA Repair Exonucleases *"

    Article Title: Characterization of the Human SNM1A and SNM1B/Apollo DNA Repair Exonucleases *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.367243

    hSNM1A and hSNM1B are both able to hydrolyze dsDNA past a blockage in the hydrolyzed as well as in the complementary strand, but only hSNM1A hydrolyzes plasmid-based gapped and linearized DNA, a reaction that is only inhibited by the presence of a large excess (more than 25-fold) of oligonucleotide substrate. A , hydrolysis of plasmid substrate, either intact, gapped, or linearized, by hSNM1A or hSNM1B is shown. Lane M , GeneRuler 1kb DNA ladder, plasmid substrate (pUC18-shortGAP46, 325 ng; 16 n m free ends) in the absence of enzyme ( lanes 1–3 ), in the presence of 21 n m hSNM1A ( lanes 4–6 ), or in the presence of 219 n m hSNM1B ( lanes 7–9 ). Plasmid substrate was either left untreated ( lanes 1 , 4 , and 7 ), gapped with NbBbvCI ( lanes 2 , 5 , and 8 ), or linearized with HindIII ( lanes 3 , 6 , and 9 ). B , hydrolysis of plasmid substrate either gapped or linearized by hSNM1A in the presence of competitor dsDNA. Lane M , GeneRuler 1-kb DNA ladder, plasmid substrate (pUC18-shortGAP46, 325 ng; 16 n m free ends) that has been gapped by NbBbvCI ( lanes 1–9 ) or linearized by HindIII ( lanes 10–18 ) and treated with by 21 n m hSNM1A in the presence of an increasing concentration of 21mer dsDNA. C , shown are time courses (0, 5, 30, 60, 120 min) of hydrolysis of cross-linked substrate ( lanes 1–10 ) and a non-cross-linked control ( lanes 11–20 ) by 0.001 μg (2.0 n m ) hSNM1A ( lanes 1–5 and 11–15 ) or 0.195 μg (427 n m ) hSNM1B ( lanes 6–10 and 16–20 ). nt , nucleotides.
    Figure Legend Snippet: hSNM1A and hSNM1B are both able to hydrolyze dsDNA past a blockage in the hydrolyzed as well as in the complementary strand, but only hSNM1A hydrolyzes plasmid-based gapped and linearized DNA, a reaction that is only inhibited by the presence of a large excess (more than 25-fold) of oligonucleotide substrate. A , hydrolysis of plasmid substrate, either intact, gapped, or linearized, by hSNM1A or hSNM1B is shown. Lane M , GeneRuler 1kb DNA ladder, plasmid substrate (pUC18-shortGAP46, 325 ng; 16 n m free ends) in the absence of enzyme ( lanes 1–3 ), in the presence of 21 n m hSNM1A ( lanes 4–6 ), or in the presence of 219 n m hSNM1B ( lanes 7–9 ). Plasmid substrate was either left untreated ( lanes 1 , 4 , and 7 ), gapped with NbBbvCI ( lanes 2 , 5 , and 8 ), or linearized with HindIII ( lanes 3 , 6 , and 9 ). B , hydrolysis of plasmid substrate either gapped or linearized by hSNM1A in the presence of competitor dsDNA. Lane M , GeneRuler 1-kb DNA ladder, plasmid substrate (pUC18-shortGAP46, 325 ng; 16 n m free ends) that has been gapped by NbBbvCI ( lanes 1–9 ) or linearized by HindIII ( lanes 10–18 ) and treated with by 21 n m hSNM1A in the presence of an increasing concentration of 21mer dsDNA. C , shown are time courses (0, 5, 30, 60, 120 min) of hydrolysis of cross-linked substrate ( lanes 1–10 ) and a non-cross-linked control ( lanes 11–20 ) by 0.001 μg (2.0 n m ) hSNM1A ( lanes 1–5 and 11–15 ) or 0.195 μg (427 n m ) hSNM1B ( lanes 6–10 and 16–20 ). nt , nucleotides.

    Techniques Used: Plasmid Preparation, Concentration Assay

    21) Product Images from "Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides *"

    Article Title: Cloning and Characterization of an Armillaria gallica cDNA Encoding Protoilludene Synthase, Which Catalyzes the First Committed Step in the Synthesis of Antimicrobial Melleolides *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.165845

    Determination of A. gallica protoilludene synthase gene copy number by Southern blot hybridization. One clear band is visible in the BamHI and EcoRI lanes (neither enzyme has a target site in the genomic clone), whereas two bands are visible in the HindIII lane (which has two target sites in the clone, 85 bp apart).
    Figure Legend Snippet: Determination of A. gallica protoilludene synthase gene copy number by Southern blot hybridization. One clear band is visible in the BamHI and EcoRI lanes (neither enzyme has a target site in the genomic clone), whereas two bands are visible in the HindIII lane (which has two target sites in the clone, 85 bp apart).

    Techniques Used: Southern Blot, Hybridization

    22) Product Images from "Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100 ▿ HD100 ▿ §"

    Article Title: Identification of Genes Essential for Prey-Independent Growth of Bdellovibrio bacteriovorus HD100 ▿ HD100 ▿ §

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.01343-10

    BamHI-HindIII fragment containing a Cm r cassette integrated into the chromosome of B. bacteriovorus HD100 used for construction of knockout plasmid pNR16. The fragment was obtained by PCR amplification using primers Bd29 and Bd30 from chromosomal DNA
    Figure Legend Snippet: BamHI-HindIII fragment containing a Cm r cassette integrated into the chromosome of B. bacteriovorus HD100 used for construction of knockout plasmid pNR16. The fragment was obtained by PCR amplification using primers Bd29 and Bd30 from chromosomal DNA

    Techniques Used: Knock-Out, Plasmid Preparation, Polymerase Chain Reaction, Amplification

    23) Product Images from "Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A"

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A

    Journal: mBio

    doi: 10.1128/mBio.01298-18

    Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.
    Figure Legend Snippet: Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.

    Techniques Used: Mutagenesis, Southern Blot, Western Blot, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Expressing, Marker

    24) Product Images from "The Arabidopsis thaliana Dihydroxyacetone Phosphate Reductase Gene SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 Is Required for Glycerolipid Metabolism and for the Activation of Systemic Acquired Resistance W⃞"

    Article Title: The Arabidopsis thaliana Dihydroxyacetone Phosphate Reductase Gene SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 Is Required for Glycerolipid Metabolism and for the Activation of Systemic Acquired Resistance W⃞

    Journal: The Plant Cell

    doi: 10.1105/tpc.016907

    Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.
    Figure Legend Snippet: Cloning of SFD1 . (A) Alignment of the SFD1 sequence with the consensus sequence from COG0240 and Pfam0210.8. Basic Local Alignment Search Tool (BLASTp) analysis of the predicted SFD1 protein sequence revealed homology to DHAP reductases/G3P dehydrogenases in the COG2040 and Pfam0210.8 protein family data sets. The alignment of SFD1 amino acids 89 to 419 to the consensus sequence from COG2040 and Pfam0210.8 is shown. Solid and dashed lines above the SFD1 sequence mark the predicted NAD + and substrate binding domains, respectively. The arrow indicates the Ala 381 that is mutated to yield Thr 381 in sfd1-2 . Amino acids that are identical to SFD1 are shown in red and those that are similar are shown in blue. (B) Complementation of the sfd1-1 and sfd1-2 mutants by SFD1 . Comparison of the morphology of 4-week-old, soil-grown wild-type sfd1-1 , ssi2 npr1 , and sfd1-1 ssi2 npr1 plants and the sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. The photographs were taken from the same distance. (C) Restoration of the ssi2 -conferred cell death by SFD1 . Leaves from 4-week-old, soil-grown ssi2 npr1 , sfd1-1 ssi2 npr1 , sfd1-1/SFD1 ssi2 npr1 , and sfd1-2/SFD1 ssi2 npr1 plants were stained with trypan blue. sfd1-1/SFD1 ssi2 npr1 and sfd1-2/SFD1 ssi2 npr1 plants are sfd1-1 ssi2 npr1 and sfd1-2 ssi2 npr1 plants transformed with a 5-kb HindIII fragment containing the genomic SFD1 clone. Trypan blue–stained leaves from the transgenic plants and from the ssi2 npr1 plants show intensely stained dead cells (yellow arrows). All of the photographs were taken at the same magnification. (D) Restoration of the ssi2 -conferred constitutive PR1 expression by SFD1 . PR1 expression was monitored in 4-week-old, soil-grown sfd1-1 ssi2 npr1 ( sfd1-1 ) and sfd1-2 ssi2 npr1 ( sfd1-2 ) plants transformed with pBI121 (Vector) or with pBI121 containing a 5-kb HindIII genomic fragment spanning SFD1 (SFD1). Blots were hybridized with a radiolabeled PR1 probe. Gel loading was monitored by photographing the ethidium bromide (EtBr)–stained gel before transfer to the membrane.

    Techniques Used: Clone Assay, Sequencing, Binding Assay, Transformation Assay, Staining, Transgenic Assay, Expressing, Plasmid Preparation

    25) Product Images from "Enhancement of UV Light Sensitivity of a Vibrio parahaemolyticus O3:K6 Pandemic Strain Due to Natural Lysogenization by a Telomeric Phage ▿"

    Article Title: Enhancement of UV Light Sensitivity of a Vibrio parahaemolyticus O3:K6 Pandemic Strain Due to Natural Lysogenization by a Telomeric Phage ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01995-08

    Restriction maps of plasmid and bacteriophage DNA obtained from V. parahaemolyticus strain PMC 58.5. N, H, and S correspond to NheI, HindIII, and SphI restriction enzymes sites, respectively. A hypothetical circular map is shown in the middle, with the possible ends shown with a red arrow and the restriction sites with a black line. b, bacteriophage; p, plasmid.
    Figure Legend Snippet: Restriction maps of plasmid and bacteriophage DNA obtained from V. parahaemolyticus strain PMC 58.5. N, H, and S correspond to NheI, HindIII, and SphI restriction enzymes sites, respectively. A hypothetical circular map is shown in the middle, with the possible ends shown with a red arrow and the restriction sites with a black line. b, bacteriophage; p, plasmid.

    Techniques Used: Plasmid Preparation

    26) Product Images from "Sequence Analysis and Organization of the Neodiprion abietis Nucleopolyhedrovirus Genome"

    Article Title: Sequence Analysis and Organization of the Neodiprion abietis Nucleopolyhedrovirus Genome

    Journal:

    doi: 10.1128/JVI.00187-06

    Linear sequence map and HindIII physical map of the circular NeabNPV genome. The transcriptional direction of each ORF is represented by an arrow labeled with the ORF number. Known baculovirus predicted homologues are presented below the ORF numbers.
    Figure Legend Snippet: Linear sequence map and HindIII physical map of the circular NeabNPV genome. The transcriptional direction of each ORF is represented by an arrow labeled with the ORF number. Known baculovirus predicted homologues are presented below the ORF numbers.

    Techniques Used: Sequencing, Labeling

    27) Product Images from "A Lamina-Associated Domain Border Governs Nuclear Lamina Interactions, Transcription, and Recombination of the Tcrb Locus"

    Article Title: A Lamina-Associated Domain Border Governs Nuclear Lamina Interactions, Transcription, and Recombination of the Tcrb Locus

    Journal: Cell reports

    doi: 10.1016/j.celrep.2018.10.052

    Looping of the RC to RC-Proximal Chromatin Is Suppressed by usRC (A) Scale schematic of the Tcrb locus indicating positions of 3C viewpoint and test fragments analyzed. (B) 3C analysis of long-distance interactions between test HindIII fragments and the E β -containing HindIII viewpoint fragment. Results for different ligation products were normalized to their abundance in a digested and religated BAC standard and were expressed relative to the frequency of ligation with an E β nearest neighbor fragment. The data represent the mean and SE of five to six independent experiments. *p
    Figure Legend Snippet: Looping of the RC to RC-Proximal Chromatin Is Suppressed by usRC (A) Scale schematic of the Tcrb locus indicating positions of 3C viewpoint and test fragments analyzed. (B) 3C analysis of long-distance interactions between test HindIII fragments and the E β -containing HindIII viewpoint fragment. Results for different ligation products were normalized to their abundance in a digested and religated BAC standard and were expressed relative to the frequency of ligation with an E β nearest neighbor fragment. The data represent the mean and SE of five to six independent experiments. *p

    Techniques Used: Ligation, BAC Assay

    28) Product Images from "Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance"

    Article Title: Kaposi’s Sarcoma-Associated Herpesvirus LANA-Adjacent Regions with Distinct Functions in Episome Segregation or Maintenance

    Journal: Journal of Virology

    doi: 10.1128/JVI.02158-18

    Deletions within LANA residues 33 to 273 do not reduce DNA replication. LANA or each LANA mutant was cotransfected with a plasmid containing 8 TR copies (p8TR-gB) into 293T cells. Twenty-four or 72 h after transfection, DNA was extracted by the method of Hirt. (A, B, D, and E) HindIII-digested Hirt DNA harvested at 24 h (A and D) or Hirt DNA digested with HindIII and DpnI at 72 h (B and E) was assessed by Southern blotting after incubation with 32 P-radiolabeled TR probe. (C and F) Western blotting for LANA or tubulin 24 h after 293T cell transfection. Brightness and contrast were uniformly adjusted using Adobe Photoshop. The results are representative of at least 3 independent experiments. The arrows in panels A and D indicate linear p8TR-gB plasmid; the arrows in panels B and E indicate replicated DNA.
    Figure Legend Snippet: Deletions within LANA residues 33 to 273 do not reduce DNA replication. LANA or each LANA mutant was cotransfected with a plasmid containing 8 TR copies (p8TR-gB) into 293T cells. Twenty-four or 72 h after transfection, DNA was extracted by the method of Hirt. (A, B, D, and E) HindIII-digested Hirt DNA harvested at 24 h (A and D) or Hirt DNA digested with HindIII and DpnI at 72 h (B and E) was assessed by Southern blotting after incubation with 32 P-radiolabeled TR probe. (C and F) Western blotting for LANA or tubulin 24 h after 293T cell transfection. Brightness and contrast were uniformly adjusted using Adobe Photoshop. The results are representative of at least 3 independent experiments. The arrows in panels A and D indicate linear p8TR-gB plasmid; the arrows in panels B and E indicate replicated DNA.

    Techniques Used: Mutagenesis, Plasmid Preparation, Transfection, Southern Blot, Incubation, Western Blot

    29) Product Images from "The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media"

    Article Title: The Moraxella catarrhalis phase-variable DNA methyltransferase ModM3 is an epigenetic regulator that affects bacterial survival in an in vivo model of otitis media

    Journal: BMC Microbiology

    doi: 10.1186/s12866-019-1660-y

    Southern blotting confirms the ModM3 methylation target sequence 5′-AC m6 ATC-3′. a Schematic representation of the restriction-inhibition assay used to confirm the ModM3 methylation site. The location of the Southern blot probe, and the BtsCI and ModM3 recognition sites are shown. The central BtsCI site overlaps the ModM3 recognition sequence and is sensitive to overlapping N6-methyladenine methylation. b Restriction-inhibition assay of modM3 ON, modM3 OFF and ∆ modM3 genomic DNA using the methyl-sensitive restriction endonuclease BtsCI. The restriction endonuclease HindIII is not sensitive to methylation and is included as a control for digestion. c Southern blot of BtsCI digested genomic DNA isolated from modM3 ON, modM3 OFF, and ∆ modM3 strains. Methylated DNA in the ModM3 ON strain is protected from BtsCI digestion resulting in a 1.5 kb band. All BtsCI sites are cleaved in the modM3 OFF and ∆ modM3 strains as ModM3 methylation is absent, resulting in a 0.5 kb band. d qRT-PCR indicating the relative abundance of methylated, undigested genomic DNA in modM3 ON and modM3 OFF relative to ∆ modM3 following digestion with BtsCI (Ct values of 18.32, 21.97, 24.77, respectively, normalised to copB reference)
    Figure Legend Snippet: Southern blotting confirms the ModM3 methylation target sequence 5′-AC m6 ATC-3′. a Schematic representation of the restriction-inhibition assay used to confirm the ModM3 methylation site. The location of the Southern blot probe, and the BtsCI and ModM3 recognition sites are shown. The central BtsCI site overlaps the ModM3 recognition sequence and is sensitive to overlapping N6-methyladenine methylation. b Restriction-inhibition assay of modM3 ON, modM3 OFF and ∆ modM3 genomic DNA using the methyl-sensitive restriction endonuclease BtsCI. The restriction endonuclease HindIII is not sensitive to methylation and is included as a control for digestion. c Southern blot of BtsCI digested genomic DNA isolated from modM3 ON, modM3 OFF, and ∆ modM3 strains. Methylated DNA in the ModM3 ON strain is protected from BtsCI digestion resulting in a 1.5 kb band. All BtsCI sites are cleaved in the modM3 OFF and ∆ modM3 strains as ModM3 methylation is absent, resulting in a 0.5 kb band. d qRT-PCR indicating the relative abundance of methylated, undigested genomic DNA in modM3 ON and modM3 OFF relative to ∆ modM3 following digestion with BtsCI (Ct values of 18.32, 21.97, 24.77, respectively, normalised to copB reference)

    Techniques Used: Southern Blot, Methylation, Sequencing, Inhibition, Isolation, Quantitative RT-PCR

    30) Product Images from "Allele-specific control of replication timing and genome organization during development"

    Article Title: Allele-specific control of replication timing and genome organization during development

    Journal: Genome Research

    doi: 10.1101/gr.232561.117

    RT asynchrony correlates with genome organization. ( A ) RT synchronous and asynchronous genomic regions contain similar SNP densities. ( B ) Genome organization, chromatin accessibility, and gene expression of RT synchronous regions that replicate either early or late during S-phase. ( C ) Spearman correlation values of RT and distinct genomic features per genome. ( D . ( E ) Spearman correlation values of RT asynchrony and changes in Hi-C compartments, Hi-C interaction counts and distances, differences in chromatin accessibility, and SNP density. ( F ) Two exemplary chromosome regions showing the RT asynchrony associated with changes in Hi-C compartments, differential expression, and distinct promoter-enhancer interactions. Two replicates of each cell line are shown in each plot of RT profiles. Allele-specific RT, total nuclear RNA-seq, and ATAC-seq were determined based on the SNPs shown in red. Allele-specific Hi-C and PC-Hi-C data were obtained using HindIII fragments containing SNPs for each genome (HindIII track). Differentially expressed genes measured by total nuclear RNA-seq are shown at the bottom , color- coded according to the allele showing the highest expression value ( musculus = black, light blue = castaneus ). These two exemplary loci illustrate the strongest differences in RT associated with a measurable difference in gene expression. This association with transcription differences was not seen for most asynchronous regions (see text for details).
    Figure Legend Snippet: RT asynchrony correlates with genome organization. ( A ) RT synchronous and asynchronous genomic regions contain similar SNP densities. ( B ) Genome organization, chromatin accessibility, and gene expression of RT synchronous regions that replicate either early or late during S-phase. ( C ) Spearman correlation values of RT and distinct genomic features per genome. ( D . ( E ) Spearman correlation values of RT asynchrony and changes in Hi-C compartments, Hi-C interaction counts and distances, differences in chromatin accessibility, and SNP density. ( F ) Two exemplary chromosome regions showing the RT asynchrony associated with changes in Hi-C compartments, differential expression, and distinct promoter-enhancer interactions. Two replicates of each cell line are shown in each plot of RT profiles. Allele-specific RT, total nuclear RNA-seq, and ATAC-seq were determined based on the SNPs shown in red. Allele-specific Hi-C and PC-Hi-C data were obtained using HindIII fragments containing SNPs for each genome (HindIII track). Differentially expressed genes measured by total nuclear RNA-seq are shown at the bottom , color- coded according to the allele showing the highest expression value ( musculus = black, light blue = castaneus ). These two exemplary loci illustrate the strongest differences in RT associated with a measurable difference in gene expression. This association with transcription differences was not seen for most asynchronous regions (see text for details).

    Techniques Used: Expressing, Hi-C, RNA Sequencing Assay

    31) Product Images from "Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum"

    Article Title: Whole-Genome Shotgun Optical Mapping of Rhodospirillum rubrum

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.9.5511-5522.2005

    Use of optical maps in confirming assembly and order of sequence contigs. In silico maps of the sequence contigs were made and aligned against the whole-genome optical maps located in the center of each diagram. A) The high-resolution HindIII map enabled alignment of the small (∼80 kb) sequence contig (contig 83) between contigs 85c and 86c. Aside from the redundant alignment of contigs 87 and 90, no gaps remained in alignment between the optical map and sequence. B) The NheI map gave the clearest representation of the redundant alignment, shown in red in the optical map, of contigs 87 and 90. The alignment of contig 87 had a better match to the optical map than do the rightmost eight fragments in contig 90 (shown in the red square). C) Removing the rightmost eight fragments from contig 90 to make contigs 90a and 90b and reversing the orientation of 90b permitted alignment to the gap between contig 87 and contig 85c. Alignments were made with MapViewer Software (OpGen, Inc, Madison WI.).
    Figure Legend Snippet: Use of optical maps in confirming assembly and order of sequence contigs. In silico maps of the sequence contigs were made and aligned against the whole-genome optical maps located in the center of each diagram. A) The high-resolution HindIII map enabled alignment of the small (∼80 kb) sequence contig (contig 83) between contigs 85c and 86c. Aside from the redundant alignment of contigs 87 and 90, no gaps remained in alignment between the optical map and sequence. B) The NheI map gave the clearest representation of the redundant alignment, shown in red in the optical map, of contigs 87 and 90. The alignment of contig 87 had a better match to the optical map than do the rightmost eight fragments in contig 90 (shown in the red square). C) Removing the rightmost eight fragments from contig 90 to make contigs 90a and 90b and reversing the orientation of 90b permitted alignment to the gap between contig 87 and contig 85c. Alignments were made with MapViewer Software (OpGen, Inc, Madison WI.).

    Techniques Used: Sequencing, In Silico, Software

    Comparisons of the XbaI, NheI, and HindIII optical maps to sequence data. (A, B, and C) Plots of optical map fragment sizes versus the DNA sequence-based map fragment sizes from the finished sequence for XbaI (A), NheI (B), and HindIII (C). The error bars represent the standard deviation of optical map fragment size about the mean. (D, E, F) Plots of the relative fragment sizing error [100 × (optical map fragment size - corresponding DNA sequence-based map fragment size)/corresponding DNA sequence-based map fragment size] versus DNA sequence-based map fragment size for XbaI ( D), NheI (E), and HindIII (F).
    Figure Legend Snippet: Comparisons of the XbaI, NheI, and HindIII optical maps to sequence data. (A, B, and C) Plots of optical map fragment sizes versus the DNA sequence-based map fragment sizes from the finished sequence for XbaI (A), NheI (B), and HindIII (C). The error bars represent the standard deviation of optical map fragment size about the mean. (D, E, F) Plots of the relative fragment sizing error [100 × (optical map fragment size - corresponding DNA sequence-based map fragment size)/corresponding DNA sequence-based map fragment size] versus DNA sequence-based map fragment size for XbaI ( D), NheI (E), and HindIII (F).

    Techniques Used: Sequencing, Standard Deviation

    32) Product Images from "T-cell receptor α enhancer is inactivated in αβ T lymphocytes"

    Article Title: T-cell receptor α enhancer is inactivated in αβ T lymphocytes

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

    doi: 10.1073/pnas.1406551112

    . The results represent the mean ± SEM of duplicate qPCR from three independent experiments. Two-tailed Student’s t tests were used to determine the statistical significance between the values of the qPCRs obtained after ligation of HindIII fragments from DP thymocytes vs. T lymphocytes (** P
    Figure Legend Snippet: . The results represent the mean ± SEM of duplicate qPCR from three independent experiments. Two-tailed Student’s t tests were used to determine the statistical significance between the values of the qPCRs obtained after ligation of HindIII fragments from DP thymocytes vs. T lymphocytes (** P

    Techniques Used: Real-time Polymerase Chain Reaction, Two Tailed Test, Ligation

    33) Product Images from "The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †"

    Article Title: The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿The NF90/NF45 Complex Participates in DNA Break Repair via Nonhomologous End Joining ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.05849-11

    In vitro NHEJ assay using DNA substrate with noncohesive ends. (A) Schematic showing the derivation of the 5.7-kb fragment with noncohesive BstXI termini, A and B. Corresponding PCR primers a and b contain HindIII and XbaI sites, respectively. Possible
    Figure Legend Snippet: In vitro NHEJ assay using DNA substrate with noncohesive ends. (A) Schematic showing the derivation of the 5.7-kb fragment with noncohesive BstXI termini, A and B. Corresponding PCR primers a and b contain HindIII and XbaI sites, respectively. Possible

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

    34) Product Images from "Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry"

    Article Title: Significance of the Bacteriophage Treatment Schedule in Reducing Salmonella Colonization of Poultry

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01257-12

    DNA restriction patterns of the bacteriophages UAB_Phi20 (lanes 3 to 5), UAB_Phi87 (lanes 6 to 8), and UAB_Phi78 (lanes 9 to 11) with the restriction enzymes EcoRI (lanes 3, 6, and 9), EcoRV (lanes 4, 7, and 10), and HindIII (lanes 5, 8, and 11). Lanes
    Figure Legend Snippet: DNA restriction patterns of the bacteriophages UAB_Phi20 (lanes 3 to 5), UAB_Phi87 (lanes 6 to 8), and UAB_Phi78 (lanes 9 to 11) with the restriction enzymes EcoRI (lanes 3, 6, and 9), EcoRV (lanes 4, 7, and 10), and HindIII (lanes 5, 8, and 11). Lanes

    Techniques Used:

    35) Product Images from "Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31"

    Article Title: Homologous Recombination Repair Factors Rad51 and BRCA1 Are Necessary for Productive Replication of Human Papillomavirus 31

    Journal: Journal of Virology

    doi: 10.1128/JVI.02495-15

    Knockdown of Rad51 and BRCA1 decreases HPV31 genome amplification upon differentiation. (A) HPV31-positive CIN612 cells were transduced with lentivirus containing control scramble shRNA sequences (scScram) or two shRNA sequences for Rad51 (shRad51) or BRCA1 (shBRCA1) to analyze efficiency of knockdown. Western blot analysis was performed using antibodies to Rad51 and BRCA1. GAPDH served as a loading control. (B) CIN612 cells were transduced with shScram, shRad51 #1, or shBRCA1 #1 and grown as a monolayer for 96 h. DNA was harvested at the indicated times, digested with BamHI (noncutter) or HindIII (which linearizes the viral genome), and examined by Southern blotting for changes in viral episome levels using the HPV31 genome as a probe. (C and D) CIN612 cells were left untreated (UT) or transiently transduced with lentivirus particles containing a control shRNA (shScram), Rad51 shRNA #1, or BRCA1 shRNA #1 for 48 h. At this time, either DNA and protein were harvested as a T 0 (undifferentiated) sample or cells were suspended in methylcellulose to induce differentiation for 24 and 48 h. DNA harvested at each time point was analyzed by Southern blotting using the HPV31 genome as a probe. Whole-cell lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate cellular differentiation (involucrin) and reduced Rad51 or BRCA1 protein in shRNA-transduced cells. GAPDH was used as a loading control. Fold change in episome copy number was determined by performing densitometry of episomal bands from four independent experiments using ImageJ software. Shown is the fold change relative to T 0 shScram, which is set to 1. Error bars represent means ± standard errors. *, P ≤ 0.05. WB, Western blot.
    Figure Legend Snippet: Knockdown of Rad51 and BRCA1 decreases HPV31 genome amplification upon differentiation. (A) HPV31-positive CIN612 cells were transduced with lentivirus containing control scramble shRNA sequences (scScram) or two shRNA sequences for Rad51 (shRad51) or BRCA1 (shBRCA1) to analyze efficiency of knockdown. Western blot analysis was performed using antibodies to Rad51 and BRCA1. GAPDH served as a loading control. (B) CIN612 cells were transduced with shScram, shRad51 #1, or shBRCA1 #1 and grown as a monolayer for 96 h. DNA was harvested at the indicated times, digested with BamHI (noncutter) or HindIII (which linearizes the viral genome), and examined by Southern blotting for changes in viral episome levels using the HPV31 genome as a probe. (C and D) CIN612 cells were left untreated (UT) or transiently transduced with lentivirus particles containing a control shRNA (shScram), Rad51 shRNA #1, or BRCA1 shRNA #1 for 48 h. At this time, either DNA and protein were harvested as a T 0 (undifferentiated) sample or cells were suspended in methylcellulose to induce differentiation for 24 and 48 h. DNA harvested at each time point was analyzed by Southern blotting using the HPV31 genome as a probe. Whole-cell lysates harvested at the indicated time points were analyzed by immunoblotting to demonstrate cellular differentiation (involucrin) and reduced Rad51 or BRCA1 protein in shRNA-transduced cells. GAPDH was used as a loading control. Fold change in episome copy number was determined by performing densitometry of episomal bands from four independent experiments using ImageJ software. Shown is the fold change relative to T 0 shScram, which is set to 1. Error bars represent means ± standard errors. *, P ≤ 0.05. WB, Western blot.

    Techniques Used: Amplification, Transduction, shRNA, Western Blot, Southern Blot, Cell Differentiation, Software

    Productive replication is abrogated in HFK-31 cells treated with an inhibitor of Rad51 activity, which reduces HR activity. (A) DNA was harvested from HFK-31 cells at T 0 as well as after differentiation in high-calcium medium for 72 h in the presence of DMSO (DM) or increasing concentrations of the Rad51 inhibitor B02. DMSO was added at a volume equal to that of the 30 μM concentration of B02. DNA was digested with BamHI (noncutter) or HindIII to linearize the viral genome and then analyzed by Southern blotting to examine episome copy number using the HPV31 genome as a probe. Western blot analysis was performed to examine the effect of B02 treatment on Rad51 levels and differentiation (involucrin). (B) Fold change in episome copy number in response to B02 treatment was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Graphed is the average fold change relative to DMSO (DM)–72-h Ca, which is set to 1. Error bars represent means ± standard errors. Statistics were assayed using a two-tailed t test. *, P
    Figure Legend Snippet: Productive replication is abrogated in HFK-31 cells treated with an inhibitor of Rad51 activity, which reduces HR activity. (A) DNA was harvested from HFK-31 cells at T 0 as well as after differentiation in high-calcium medium for 72 h in the presence of DMSO (DM) or increasing concentrations of the Rad51 inhibitor B02. DMSO was added at a volume equal to that of the 30 μM concentration of B02. DNA was digested with BamHI (noncutter) or HindIII to linearize the viral genome and then analyzed by Southern blotting to examine episome copy number using the HPV31 genome as a probe. Western blot analysis was performed to examine the effect of B02 treatment on Rad51 levels and differentiation (involucrin). (B) Fold change in episome copy number in response to B02 treatment was determined by performing densitometry of episomal bands from three independent experiments using ImageJ software. Graphed is the average fold change relative to DMSO (DM)–72-h Ca, which is set to 1. Error bars represent means ± standard errors. Statistics were assayed using a two-tailed t test. *, P

    Techniques Used: Activity Assay, Concentration Assay, Southern Blot, Western Blot, Software, Two Tailed Test

    36) Product Images from "SNP haplotyping technique for evaluation of MGP 5′ UTR power in osteoblast cells"

    Article Title: SNP haplotyping technique for evaluation of MGP 5′ UTR power in osteoblast cells

    Journal: SpringerPlus

    doi: 10.1186/s40064-016-2329-8

    pGL3 Basic plasmid and constructs on agarose gel. I ARMS products; the ARMS–PCR reactions were performed in two microtubes and the products (1624 bp) were run on the agarose gel (2 %) electrophoresis ( Lanes A , B and Ladder). II Constructs containing three polymorphic sites were digested using restriction enzymes (REs) and were run on the agarose gel (3.5 %) electrophoresis ( A amplified product, 568 bp, B digestion with SspI, 555 bp, C digestion with SspI and HindIII, 540 bp). III pGL3 Basic plasmid was digested using restriction enzymes (SmaI and HindIII) and was run on the agarose gel (2 %) electrophoresis ( A intact plasmid, B digested plasmid with REs). IV The REs-digested constructs (539 bp) were inserted and ligated into pGL3 Basic plasmid and were compared on the agarose gel (2 %) ( A construct-inserted plasmid, B intact plasmid)
    Figure Legend Snippet: pGL3 Basic plasmid and constructs on agarose gel. I ARMS products; the ARMS–PCR reactions were performed in two microtubes and the products (1624 bp) were run on the agarose gel (2 %) electrophoresis ( Lanes A , B and Ladder). II Constructs containing three polymorphic sites were digested using restriction enzymes (REs) and were run on the agarose gel (3.5 %) electrophoresis ( A amplified product, 568 bp, B digestion with SspI, 555 bp, C digestion with SspI and HindIII, 540 bp). III pGL3 Basic plasmid was digested using restriction enzymes (SmaI and HindIII) and was run on the agarose gel (2 %) electrophoresis ( A intact plasmid, B digested plasmid with REs). IV The REs-digested constructs (539 bp) were inserted and ligated into pGL3 Basic plasmid and were compared on the agarose gel (2 %) ( A construct-inserted plasmid, B intact plasmid)

    Techniques Used: Plasmid Preparation, Construct, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Electrophoresis, Amplification

    37) Product Images from "Force regulated dynamics of RPA on a DNA fork"

    Article Title: Force regulated dynamics of RPA on a DNA fork

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw187

    DNA melting capacity of human RPA depends on Mg 2+ concentration. ( A ) yRPA does not melt dsDNA. Lambda/HindIII DNA (lane 1) was incubated with 2.2 μM yRPA (corresponding to 300% saturation) with various magnesium acetate concentrations (lanes 2–9, 0–10 mM as indicated) for 30 min at 30°C and subsequently analyzed on a 1% agarose gel. Throughout the range of magnesium concentrations tested, no melting occurred ( cf . heat denatured substrate in lane 10). ( B ) Experiment as in panel a but with 2.2 μM hRPA (corresponds to 375% DNA saturation) incubated for 30 min at 37°C. In contrast to yRPA, hRPA melts dsDNA at Mg 2+ concentrations below 3 mM (lanes 5–9).
    Figure Legend Snippet: DNA melting capacity of human RPA depends on Mg 2+ concentration. ( A ) yRPA does not melt dsDNA. Lambda/HindIII DNA (lane 1) was incubated with 2.2 μM yRPA (corresponding to 300% saturation) with various magnesium acetate concentrations (lanes 2–9, 0–10 mM as indicated) for 30 min at 30°C and subsequently analyzed on a 1% agarose gel. Throughout the range of magnesium concentrations tested, no melting occurred ( cf . heat denatured substrate in lane 10). ( B ) Experiment as in panel a but with 2.2 μM hRPA (corresponds to 375% DNA saturation) incubated for 30 min at 37°C. In contrast to yRPA, hRPA melts dsDNA at Mg 2+ concentrations below 3 mM (lanes 5–9).

    Techniques Used: Recombinase Polymerase Amplification, Concentration Assay, Incubation, Agarose Gel Electrophoresis

    38) Product Images from "C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents"

    Article Title: C3-symmetric opioid scaffolds are pH-responsive DNA condensation agents

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1097

    Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.
    Figure Legend Snippet: Experimental design for the Bioanalyzer 2100 to identify site-specific endonuclease inhibition by opioid compounds, HindIII, EcoRI, BamHI and SalI.

    Techniques Used: Inhibition

    ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.
    Figure Legend Snippet: ( A ) Electrograms generated using the Bioanalyzer 2100 of 742 bp dsDNA fragment with treatment by endonucleases BamHI, HindIII, SalI and EcoRI. Electrograms of the 742 bp fragment were pre-incubated for 5 h with either ( B ) MC3 , ( C ) HC3 and ( D ) OC3 , followed by exposure over night to the type II restriction endonuclease.

    Techniques Used: Generated, Incubation

    Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .
    Figure Legend Snippet: Atomic force microscopy (AFM) images of MC3 -treated supercoiled and HindIII linearized pUC19 DNA; ( A–D ) supercoiled pUC19 with 8, 9, 10 and 20 μM MC3 ; ( E–H ) linear pUC19 with 5, 10, 20 and 50 μM MC3 .

    Techniques Used: Microscopy

    39) Product Images from "Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A"

    Article Title: Acinetobacter baumannii Gastrointestinal Colonization Is Facilitated by Secretory IgA Which Is Reductively Dissociated by Bacterial Thioredoxin A

    Journal: mBio

    doi: 10.1128/mBio.01298-18

    Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.
    Figure Legend Snippet: Generation of a trxA deletion mutant. A schematic representation of the WT A. baumannii Ci79 and Δ trxA mutant genome surrounding the trxA locus with predicted fragment sizes detected by probe following either HindIII (white arrowhead) or XbaI (black arrowhead) digestion as well as probe target region (dotted box) (A). Southern blot (B) and Western blot (C) analyses of genomic and protein extracts, respectively, from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii were subsequently performed. PCR amplification of the trxA gene locus was performed using DNA from WT Ci79, ΔtrxA , and ΔtrxA c A. baumannii isolates. The PCR amplicons were subsequently digested with SalI and subjected to agarose gel electrophoresis (C). Total bacterial proteins were separated by electrophoresis and visualized in a Coomassie blue-stained polyacrylamide gel (D, left panel) or probed with anti-TrxA antibody to detect TrxA expression (D, right panel). Molecular marker sizes for DNA (bp; B and C) and protein (kDa; D) are provided.

    Techniques Used: Mutagenesis, Southern Blot, Western Blot, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Electrophoresis, Staining, Expressing, Marker

    40) Product Images from "The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness"

    Article Title: The Bacteriophage T4 MotB Protein, a DNA-Binding Protein, Improves Phage Fitness

    Journal: Viruses

    doi: 10.3390/v10070343

    MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.
    Figure Legend Snippet: MotB and H-NS bind to both unmodified λ and GHme-C modified T4 DNA. Agarose gel shows the DNA (500 ng) λ or T4 DNA pretreated with HindIII and SspI restriction nucleases (lanes 1 and 5, respectively), after incubation with 60 pmol MotB (lanes 2 and 6), 60 pmol H-NS (lanes 3 and 7), or both (lanes 4 and 8). DNA was visualized by ethidium bromide staining and UV illumination.

    Techniques Used: Modification, Agarose Gel Electrophoresis, Incubation, Staining

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ
    Article Snippet: .. For floxCol12a1 screening, PCR products were digested with HindIII (NEB). .. in vitro Cre-recombination 200 ng of PCR products of cloned floxCol12a1 alleles from floxed mice or genomic Col12a1 PCR products of wildtype mice were incubated with Cre recombinase (NEB) and analyzed by 2% agarose gel electrophoresis according to the manufacture’s instruction.

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: .. The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG. .. The D277A point mutation inactivating the hDNA2 nuclease was introduced with oligonucleotide pair 5'-GGCCTGAAGGGAAAGATCGCTGTCACAGTTGGAGTGAAG-3' and 5'-CTTCACTCCAACTGTGACAGCGATCTTTCCCTTCAGGCC-3' whereas the K654R point mutation abolishing the hDNA2 helicase was introduced with oligonucleotide pair 5'-GGCATGCCGGGAACTGGCAGGACAACCACTATCTGCACA-3' and 5'-TGTGCAGATAGTGGTTGTCCTGCCAGTTCCCGGCATGCC-3' using the QuikChange XL Site-directed mutagenesis kit (Agilent, Santa Clara, CA) according to manufacturer's recommendations.

    Transformation Assay:

    Article Title: Single-stranded DNA and RNA origami
    Article Snippet: .. In the next step, the two digested fragments were ligated with Eco RI and Hind III digested pGEM-7zf (−) vector, and transformed into E. coli cells (NEB stable competent cells). .. This was a three-fragment ligation to form a circular piece of DNA.

    other:

    Article Title: Probing hyper-negatively supercoiled mini-circles with nucleases and DNA binding proteins
    Article Snippet: Materials Escherichia coli topoisomerase I (Ec TopoI), T4 polynucleotide kinase (PNK), calf intestinal phosphatase, T4 DNA ligase, DNAse I, BamHI, BglII and HindIII were from New England Biolabs.

    Agarose Gel Electrophoresis:

    Article Title: Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿
    Article Snippet: .. HindIII (New England Biolabs, Beverly, MA)-restricted plasmid fragments were separated on a 0.75% agarose gel in Tris-borate-EDTA (TBE) buffer at 80 V for 5 h. The Trackit 1 Kb DNA ladder (Invitrogen, Carlsbad, CA) was used for sizing the linear fragments. .. Digoxigenin-labeled DNA probes were generated by PCR using the oligonucleotide primers shown in Table and plasmid DNA isolated from the following control organisms: S. aureus FPR3757 ( ) for tra E , tra I , rep A , and mup A , and S. aureus HIP11714 MI-1 (vancomycin resistant S. aureus [VRSA-1]) ( ) for aac 6 ′- aph 2 ″ and dfr A .

    Plasmid Preparation:

    Article Title: Single-stranded DNA and RNA origami
    Article Snippet: .. In the next step, the two digested fragments were ligated with Eco RI and Hind III digested pGEM-7zf (−) vector, and transformed into E. coli cells (NEB stable competent cells). .. This was a three-fragment ligation to form a circular piece of DNA.

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
    Article Snippet: .. The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG. .. The D277A point mutation inactivating the hDNA2 nuclease was introduced with oligonucleotide pair 5'-GGCCTGAAGGGAAAGATCGCTGTCACAGTTGGAGTGAAG-3' and 5'-CTTCACTCCAACTGTGACAGCGATCTTTCCCTTCAGGCC-3' whereas the K654R point mutation abolishing the hDNA2 helicase was introduced with oligonucleotide pair 5'-GGCATGCCGGGAACTGGCAGGACAACCACTATCTGCACA-3' and 5'-TGTGCAGATAGTGGTTGTCCTGCCAGTTCCCGGCATGCC-3' using the QuikChange XL Site-directed mutagenesis kit (Agilent, Santa Clara, CA) according to manufacturer's recommendations.

    Article Title: Emergence of Resistance among USA300 Methicillin-Resistant Staphylococcus aureus Isolates Causing Invasive Disease in the United States ▿
    Article Snippet: .. HindIII (New England Biolabs, Beverly, MA)-restricted plasmid fragments were separated on a 0.75% agarose gel in Tris-borate-EDTA (TBE) buffer at 80 V for 5 h. The Trackit 1 Kb DNA ladder (Invitrogen, Carlsbad, CA) was used for sizing the linear fragments. .. Digoxigenin-labeled DNA probes were generated by PCR using the oligonucleotide primers shown in Table and plasmid DNA isolated from the following control organisms: S. aureus FPR3757 ( ) for tra E , tra I , rep A , and mup A , and S. aureus HIP11714 MI-1 (vancomycin resistant S. aureus [VRSA-1]) ( ) for aac 6 ′- aph 2 ″ and dfr A .

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    New England Biolabs hin diii
    Southern blots of Hin <t>dIII</t> and Hin dIII-VDE digests of <t>DNA</t> from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015
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    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from spo11 strains with inserts at HIS4 (top) and at URA3 (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.015

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Spo11-initiated events at the two insert loci. ( A ) Spo11-catalyzed DSBs are more frequent at HIS4 that at URA3 . Left—Southern blots of Eco RI digests of DNA from vde∆ strains, probed with pBR322 sequences, showing Spo11-DSBs in the Parent 2 insert (see Figure 1 ) in resection/repair-deficient sae2∆ mutant strains. Right—location of DSBs and probe and DSB frequencies (average of 7 and 8 hr samples from a single experiment; error bars represent range). Spo11-DSBs in the Parent 1 inserts at HIS4 and URA3 were at different locations within the insert, but displayed similar ratios between the two loci (data not shown). ( B ) Southern blots of Hin dIII digests of DNA from vde∆ strains, to detect total Spo11-initiated crossovers. ( C ) Southern blots of Hin dIII-VDE double digests of the same samples, to determine the background contribution of Spo11-initiated COs in subsequent experiments measuring VDE-initiated COs, which will be VDE-resistant due to conversion of the VRS site to VRS103 . Probes were as shown in Figure 1 . ( D ) Quantification of data in panels B (total COs; filled circles) and C (VDE-resistant COs; open circles). Data are from a single experiment. DOI: http://dx.doi.org/10.7554/eLife.19669.004

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques: Mutagenesis

    70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: 70–80% of VDE-DSBs are repaired. ( A ) Fraction of inserts remaining, calculated using Hin dIII digests (see Figure 1 ). For the arg4-VRS103 insert, the ratio (Parent 2 + CO2)/ (0.5 x LC) was calculated at 9 hr, and was then normalized to the 0 hr value. For the arg4-VRS insert, a similar calculation was made: (Parent 1 + NCO + CO1)/(0.5 x LC) ( B ) Relative recovery of interhomolog recombination products, calculated using Hin dIII-VDE double digests (see Figure 1 ). The sum of CO (average of CO1 and CO2) and NCO frequencies was divided by the frequency of total DSBs, as calculated in Figure 2A . Data are the average of two independent experiments; error bars represent range. DOI: http://dx.doi.org/10.7554/eLife.19669.006

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Probes and gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. DOI: http://dx.doi.org/10.7554/eLife.19669.009

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Journal: eLife

    Article Title: Local chromosome context is a major determinant of crossover pathway biochemistry during budding yeast meiosis

    doi: 10.7554/eLife.19669

    Figure Lengend Snippet: Southern blots of Hin dIII and Hin dIII-VDE digests of DNA from HIS4 insert-containing strains (top) and from URA3 insert-contaning strains (bottom). Gel labels are as in Figure 1 ; JM—joint molecule recombination intermediates. In the gel with Hin DIII digests of samples from a pch2∆ mm4-mn yen1∆ slx1∆ strain with inserts at URA3 , the 9 hr sample was originally loaded between the 4 and 5 hr samples; this image was cut and spliced as indicated by vertical lines for presentation purposes. DOI: http://dx.doi.org/10.7554/eLife.19669.012

    Article Snippet: Recombination products were detected on Southern blots containing genomic DNA digested with Hin dIII and VDE (P I-Sce I, New England Biolabs), using specific buffer for P I-Sce I.

    Techniques:

    hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Journal: eLife

    Article Title: Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

    doi: 10.7554/eLife.18574

    Figure Lengend Snippet: hDNA2 D277A unwinds plasmid- and oligonucleotide-based DNA substrates. ( A ) Representative 1% agarose gel showing hDNA2 D277A helicase activity on a λDNA/HindIII substrate in a time-course experiment with 346 nM hRPA. Heat, heat-denatured DNA substrate. ( B ) Representative 1% agarose gel showing that nuclease- and helicase-deficient hDNA2 D277A K654R (lanes 2–6) and helicase-deficient hDNA2 K654R (lane 8) do not exhibit helicase activity. Lane 7, DNA unwinding by nuclease-deficient DNA2 D277A. Reactions contained 215 nM hRPA. ( C – E ) Representative 10% polyacrylamide gels showing the helicase activity of hDNA2 D277A with ( C ) 5’ overhang, ( D ) 3’ overhang and with ( E ) dsDNA substrates. Reactions contained 7.5 nM RPA. Heat, heat-denatured DNA substrate. ( F ) Representative 1% agarose gels showing DNA unwinding of a 2.7 kbp-long substrate by either hDNA2 D277A (left part, at 37°C) or yDna2 E675A (right part, at 30°C) in a kinetic experiment with 215 nM human RPA or 267 nM yeast RPA respectively. ( G ) Quantitation of experiments such as shown in F. Averages shown, n = 2; error bars, SEM. DOI: http://dx.doi.org/10.7554/eLife.18574.007

    Article Snippet: The PCR products were digested with BamHI and HindIII restriction endonucleases (New England Biolabs, Ipswich, MA) and ligated into a pFastBac1 vector (Invitrogen, Carlsbad, CA) generating pFB-His-hDNA2-FLAG.

    Techniques: Plasmid Preparation, Agarose Gel Electrophoresis, Activity Assay, Recombinase Polymerase Amplification, Quantitation Assay

    Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Journal: BMC Genomics

    Article Title: Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ

    doi: 10.1186/s12864-016-3331-9

    Figure Lengend Snippet: Generation of floxed mice by the enhanced PITCh system. a Targeting strategy for the generation of flox Col12a1 mice by the enhanced PITCh system. Purple highlights indicate microhomologies between endogenous Col12a1 locus and PITCh-donor. Blue characters indicate CRISPR target sequences. Red characters indicate protospacer adjacent motif (PAM) sequences. Yellow lightnings indicate DSB sites. b Schematic diagram of pronuclear injection of Cas9 protein, Col12a1 -left, -right, and gRNA-s1 crRNAs, tracrRNA, PITCh-donor, and Exo1 mRNA. The red, purple, and blue boxes indicate the insert, Col12a1 microhomologies, and gRNA-s1 target sequences, respectively. c PCR screenings of newborns. d PCR-RFLP (restriction fragment length polymorphism) screenings of floxed newborn mice. e Summary of flox Col12a1 mouse production by the enhanced PITCh system. f Sequences of boundaries between Col12a1 and LoxPs. Blue, green, and red characters indicate microhomologies, HindIII sites, and LoxPs, respectively. g in vitro Cre-recombination assay. Cloned PCR products of flox alleles from three flox Col12a1 mice and genomic PCR of wildtype were incubated with or without Cre-recombinase. LF: left forward primer, RR: right reverse primer, MH: microhomology, M: molecular marker, and WT: wildtype

    Article Snippet: For floxCol12a1 screening, PCR products were digested with HindIII (NEB).

    Techniques: Mouse Assay, CRISPR, Injection, Polymerase Chain Reaction, In Vitro, Recombination Assay, Clone Assay, Incubation, Marker

    Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).

    Journal: Science (New York, N.Y.)

    Article Title: Single-stranded DNA and RNA origami

    doi: 10.1126/science.aao2648

    Figure Lengend Snippet: Schematic of ssOrigami synthesis and replication by in vitro PCR and by in vivo cloning of ssOrigami genes. ( A ) One-step PCR with two double-stranded gBlock templates containing 30-bp sequence overlap (yellow sections) and two modified primers (phosphorothioate modification on green primer and phosphorylation modification on red primer). ( B ) Double-stranded PCR product with modified 5′ ends. (C) ssDNA product after lambda exonuclease digestion. Phosphorothioate modification protects the forward strand from being digested. ( D ) Folded ssOrigami structure. Note that the folded ssOrigami product can be directly used as a template for its PCR replication. ( E ) Double-stranded gBlock DNA fragments with restriction enzyme sites designed at both ends. ( F ) Ligation of two half fragments into linearized pGEM-7zf (−) vector to form the full-length ssOrigami gene. ( G ) The ligation products were transformed into E. coli NEB stable competent cells. ( H ) Full-length ssOrigami genes were amplified as plasmid DNA in E. coli NEB stable cells. ( I ) The harvested genes were treated by the nicking endonuclease Nb.BbvCI and the restriction endonuclease Hind III. ( J . ( K and L ) Schematic (K) and AFM images [(K), zoomed-in; (L), large field of view] of the 5 × 5 ssOrigami structures produced by the PCR synthesis [first cycle in (A) to (D)]. ( M ) AFM image of 5 × 5 ssOrigami structures produced by PCR replication method [the second cycle in (A) to (D), that is, the re-PCR product]. ( N ) AFM image of 5 × 5 rhombus ssOrigami produced by in vivo cloning method. Detailed experimental information is shown in sections S6 (in vitro PCR) and S7 (in vivo cloning).

    Article Snippet: In the next step, the two digested fragments were ligated with Eco RI and Hind III digested pGEM-7zf (−) vector, and transformed into E. coli cells (NEB stable competent cells).

    Techniques: In Vitro, Polymerase Chain Reaction, In Vivo, Clone Assay, Sequencing, Modification, Ligation, Plasmid Preparation, Transformation Assay, Amplification, Produced