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    New England Biolabs dpn i
    DpnI
    DpnI 5 000 units
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    Average 99 stars, based on 163 article reviews
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    Images

    1) Product Images from "Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli"

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    Journal: Frontiers in Public Health

    doi: 10.3389/fpubh.2016.00131

    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).
    Figure Legend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Techniques Used: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.
    Figure Legend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .
    Figure Legend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Techniques Used: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation

    2) Product Images from "Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells"

    Article Title: Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

    Journal: American Journal of Human Genetics

    doi:

    STRIP assay and replication of the pFX/SV40 templates ( dark gray rings ) by primate proteins within COS-1 cells. At 48 h posttransfection, the episomally replicated DNA was extracted and digested by Dpn I to remove the unreplicated ( dark gray rings ) and partially replicated ( dark gray rings with small light gray rings on top ) templates. The Dpn I-resistant primate-replicated templates ( light gray rings ) were transformed into E. coli , and individual colonies, each an individual product of primate replication, were cultured. The resulting DNA was restriction digested and analyzed on 4% polyacrylamide gels for CGG length changes.
    Figure Legend Snippet: STRIP assay and replication of the pFX/SV40 templates ( dark gray rings ) by primate proteins within COS-1 cells. At 48 h posttransfection, the episomally replicated DNA was extracted and digested by Dpn I to remove the unreplicated ( dark gray rings ) and partially replicated ( dark gray rings with small light gray rings on top ) templates. The Dpn I-resistant primate-replicated templates ( light gray rings ) were transformed into E. coli , and individual colonies, each an individual product of primate replication, were cultured. The resulting DNA was restriction digested and analyzed on 4% polyacrylamide gels for CGG length changes.

    Techniques Used: Stripping Membranes, Transformation Assay, Cell Culture

    Replication efficiency. An aliquot of the starting mixture, composed of each pFX53 construct with unmethylated pSV40, was linearized with Alw NI. Primate-replicated DNA resulting from the cotransfection of this same starting mixture was also linearized with Alw NI, was further digested with Dpn I to remove the unreplicated parental template, and was then probed with the SV40- ori ” section).
    Figure Legend Snippet: Replication efficiency. An aliquot of the starting mixture, composed of each pFX53 construct with unmethylated pSV40, was linearized with Alw NI. Primate-replicated DNA resulting from the cotransfection of this same starting mixture was also linearized with Alw NI, was further digested with Dpn I to remove the unreplicated parental template, and was then probed with the SV40- ori ” section).

    Techniques Used: Construct, Cotransfection

    3) Product Images from "In Vivo Association of Ku with Mammalian Origins of DNA Replication"

    Article Title: In Vivo Association of Ku with Mammalian Origins of DNA Replication

    Journal: Molecular Biology of the Cell

    doi:

    Ku is associated with the ADA-associated origin of the mouse genome in Ku80 +/+ cells, but not in Ku80 −/− MEFs. Ku80 −/− cell extracts have reduced replication activity. (A) Western blot probed with 1/100th dilution of anti-Ku86, or 1/400th dilution of anti-Ku70. 1/20th of immunoprecipitation with clone162 from cross-linked or untreated Ku80 +/+ or Ku80 −/− MEFs. (B) PCR amplification with the use of primer set ADA A, which amplifies a genomic 230-bp fragment. Template DNA used was as follows. Lanes 1 and 2, total genomic DNA isolated from untreated Ku80 +/+ or Ku80 −/− cells. Lane 3, negative control to verify primer contamination; no template DNA added to PCR reaction. Lanes 4, 6, and 8, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 +/+ cells. Lanes 5, 7, and 9, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 −/− cells. (C) In vitro DNA replication assays were performed with Ku80 +/+ or Ku80 −/− cells extracts and p186 as the template DNA. The in vitro replication products were purified, digested with Dpn I, and the Dpn I-resistant bands were quantitated with the use of a phosphorimager. The amount of radioactive precursor incorporated into the DNA is expressed as a percentage relative to the Ku80 +/+ cell extract reaction (100%). The quantification was obtained from at least three different in vitro reactions. Each bar represents three experiments and 1 SD is indicated.
    Figure Legend Snippet: Ku is associated with the ADA-associated origin of the mouse genome in Ku80 +/+ cells, but not in Ku80 −/− MEFs. Ku80 −/− cell extracts have reduced replication activity. (A) Western blot probed with 1/100th dilution of anti-Ku86, or 1/400th dilution of anti-Ku70. 1/20th of immunoprecipitation with clone162 from cross-linked or untreated Ku80 +/+ or Ku80 −/− MEFs. (B) PCR amplification with the use of primer set ADA A, which amplifies a genomic 230-bp fragment. Template DNA used was as follows. Lanes 1 and 2, total genomic DNA isolated from untreated Ku80 +/+ or Ku80 −/− cells. Lane 3, negative control to verify primer contamination; no template DNA added to PCR reaction. Lanes 4, 6, and 8, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 +/+ cells. Lanes 5, 7, and 9, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 −/− cells. (C) In vitro DNA replication assays were performed with Ku80 +/+ or Ku80 −/− cells extracts and p186 as the template DNA. The in vitro replication products were purified, digested with Dpn I, and the Dpn I-resistant bands were quantitated with the use of a phosphorimager. The amount of radioactive precursor incorporated into the DNA is expressed as a percentage relative to the Ku80 +/+ cell extract reaction (100%). The quantification was obtained from at least three different in vitro reactions. Each bar represents three experiments and 1 SD is indicated.

    Techniques Used: Activity Assay, Western Blot, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Isolation, Negative Control, In Vitro, Purification

    4) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).
    Figure Legend Snippet: Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Synthesized, Staining, Activity Assay, Methylation

    5) Product Images from "Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection"

    Article Title: Fate of methylated/unmethylated H19 imprinting control region after paternal and maternal pronuclear injection

    Journal: Experimental Animals

    doi: 10.1538/expanim.17-0031

    Generation and in vitro methylation of H19 ICR transgene. (a) Genomic structure of the mouse Igf2/H19 locus. The Igf2 and H19 genes (open boxes) are −90 kb apart, and the expression of both genes depends on the shared 3′ enhancer (filled ovals). The H19 ICR is located within a 2.9-kbp Sac I/ Bam HI fragment. The black boxes in the enlarged map indicate the position of the CCCTC-binding factor (CTCF) binding sites. (b) A schematic map of the transgene containing H19 ICR (ICR-F) used in this study. pCpG-EGFP-SB contains 2.9 kb of the mouse H19 ICR (white box containing four black boxes), mCMV enhancer (gray box), hEF1 promoter (gray box), and EGFP (white box) sequences. CTCF1/2, CTCF3/4, and EGFP regions analyzed for methylation status are indicated in solid lines. Primers used for nested-PCR are shown by arrows. P, Pac I; D, Dpn I sites. (c) Confirmation of the methylation status in the H19 ICR transgene fragment used for microinjection. Unmethylated and methylated transgenes were analyzed by bisulfite sequencing analysis using primers shown in B. Methylated and unmethylated CpG motifs are shown as filled and open circles, respectively. Each horizontal row represents a single DNA template molecule. Gray bars indicate the location of the CTCF-binding sites.
    Figure Legend Snippet: Generation and in vitro methylation of H19 ICR transgene. (a) Genomic structure of the mouse Igf2/H19 locus. The Igf2 and H19 genes (open boxes) are −90 kb apart, and the expression of both genes depends on the shared 3′ enhancer (filled ovals). The H19 ICR is located within a 2.9-kbp Sac I/ Bam HI fragment. The black boxes in the enlarged map indicate the position of the CCCTC-binding factor (CTCF) binding sites. (b) A schematic map of the transgene containing H19 ICR (ICR-F) used in this study. pCpG-EGFP-SB contains 2.9 kb of the mouse H19 ICR (white box containing four black boxes), mCMV enhancer (gray box), hEF1 promoter (gray box), and EGFP (white box) sequences. CTCF1/2, CTCF3/4, and EGFP regions analyzed for methylation status are indicated in solid lines. Primers used for nested-PCR are shown by arrows. P, Pac I; D, Dpn I sites. (c) Confirmation of the methylation status in the H19 ICR transgene fragment used for microinjection. Unmethylated and methylated transgenes were analyzed by bisulfite sequencing analysis using primers shown in B. Methylated and unmethylated CpG motifs are shown as filled and open circles, respectively. Each horizontal row represents a single DNA template molecule. Gray bars indicate the location of the CTCF-binding sites.

    Techniques Used: In Vitro, Methylation, Expressing, Binding Assay, Nested PCR, Methylation Sequencing

    Methylation analysis of transgenic H19 ICRs at blastocyst stage. (a) Bisulfite sequencing analysis of embryos that developed to blastocyst stage after microinjection of the ICR-F into the paternal or maternal PN. The methylation status of the ICR-F was indicated as described in Fig. 2c . Genomic DNA of 5–10 blastocysts was extracted as a pool and subjected to Dpn I digestion before bisulfite treatment to eliminate the transgene fragments that were unintegrated into the genome. The result is composed of data from three pools (for the CTCF1/2 region by maternal injection using unmethylated transgene, two pools were analyzed). At least six clones were sequenced from each pool. The data from three pools were combined, because the methylation level of each pool was almost the same. (b) Ratio of the DNA methylation levels at H19 ICR (CTCF1/2 and CTCF3/4) and EGFP regions are indicated.
    Figure Legend Snippet: Methylation analysis of transgenic H19 ICRs at blastocyst stage. (a) Bisulfite sequencing analysis of embryos that developed to blastocyst stage after microinjection of the ICR-F into the paternal or maternal PN. The methylation status of the ICR-F was indicated as described in Fig. 2c . Genomic DNA of 5–10 blastocysts was extracted as a pool and subjected to Dpn I digestion before bisulfite treatment to eliminate the transgene fragments that were unintegrated into the genome. The result is composed of data from three pools (for the CTCF1/2 region by maternal injection using unmethylated transgene, two pools were analyzed). At least six clones were sequenced from each pool. The data from three pools were combined, because the methylation level of each pool was almost the same. (b) Ratio of the DNA methylation levels at H19 ICR (CTCF1/2 and CTCF3/4) and EGFP regions are indicated.

    Techniques Used: Methylation, Transgenic Assay, Methylation Sequencing, Injection, Clone Assay, DNA Methylation Assay

    6) Product Images from "DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO"

    Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

    Journal: Molecular Systems Biology

    doi: 10.1038/msb.2010.54

    The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.
    Figure Legend Snippet: The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.

    Techniques Used: Binding Assay, Methylation, Amplification, Labeling, Plasmid Preparation, Construct, Transgenic Assay, Expressing

    7) Product Images from "Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit"

    Article Title: Efficient genome replication of hepatitis B virus using adenovirus vector: a compact pregenomic RNA-expression unit

    Journal: Scientific Reports

    doi: 10.1038/srep41851

    Detection and quantification of replicating HBV genome. Cells were infected with Ax-CM103G-kS (kS) or Ax-CM103G-dP (dP) by the indicated MOIs, or transfected with plasmids possessing the same mutant HBV expression units. ( a ) Detection of the replicating HBV genome. Total DNA either from infected or transfected HepG2 cells were analysed using Southern blot analysis. AdV (◆), Kpn I-digested genome of adenovirus vector; rc, relaxed circular DNA genome of HBV; dsL, double-stranded linear DNA genome of HBV; ss, single stranded DNA of HBV; plasmid ( ¢ ), Hind III and Dpn I-digested plasmid fragments; mock, mock infection. Overexposure of blots and full-length blots are presented in Supplementary Figs S5 and S9 , respectively. ( b ) Replicating HBV genomes were quantified using qPCR. Total DNA from infected HepG2 or PXB cells were used to performed qPCR. n = 3. Error bars represent ± s.d.; mock, mock infection of the indicated cells; ** P
    Figure Legend Snippet: Detection and quantification of replicating HBV genome. Cells were infected with Ax-CM103G-kS (kS) or Ax-CM103G-dP (dP) by the indicated MOIs, or transfected with plasmids possessing the same mutant HBV expression units. ( a ) Detection of the replicating HBV genome. Total DNA either from infected or transfected HepG2 cells were analysed using Southern blot analysis. AdV (◆), Kpn I-digested genome of adenovirus vector; rc, relaxed circular DNA genome of HBV; dsL, double-stranded linear DNA genome of HBV; ss, single stranded DNA of HBV; plasmid ( ¢ ), Hind III and Dpn I-digested plasmid fragments; mock, mock infection. Overexposure of blots and full-length blots are presented in Supplementary Figs S5 and S9 , respectively. ( b ) Replicating HBV genomes were quantified using qPCR. Total DNA from infected HepG2 or PXB cells were used to performed qPCR. n = 3. Error bars represent ± s.d.; mock, mock infection of the indicated cells; ** P

    Techniques Used: Infection, Transfection, Mutagenesis, Expressing, Southern Blot, Plasmid Preparation, Real-time Polymerase Chain Reaction

    8) Product Images from "Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases"

    Article Title: Replication interference between human papillomavirus types 16 and 18 mediated by heterologous E1 helicases

    Journal: Virology Journal

    doi: 10.1186/1743-422X-11-11

    Effects of HPV16 E1 mutants on HPV18 replication. (A) Schematic representation of HPV16 E1 mutants. F, FLAG-tag; ND, N-terminal domain; DBD, DNA-binding domain; OD, oligomerization domain; HD, helicase domain. (B) C33A cells were transfected with 1 ng of the HPV18 gDNA together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV18 gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the HPV18 E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.
    Figure Legend Snippet: Effects of HPV16 E1 mutants on HPV18 replication. (A) Schematic representation of HPV16 E1 mutants. F, FLAG-tag; ND, N-terminal domain; DBD, DNA-binding domain; OD, oligomerization domain; HD, helicase domain. (B) C33A cells were transfected with 1 ng of the HPV18 gDNA together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV18 gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the HPV18 E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Techniques Used: FLAG-tag, Binding Assay, Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Replication of HPV16 or HPV18 genomes supported by homologous or heterologous E1/E2s. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication when cells were transfected 20 ng of each pF16E1 and pF16E2 (A) or 10 ng of each pF18E1 and pF18E2 (B) . Each bar represents the average of two independent experiments with the standard error of mean.
    Figure Legend Snippet: Replication of HPV16 or HPV18 genomes supported by homologous or heterologous E1/E2s. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication when cells were transfected 20 ng of each pF16E1 and pF16E2 (A) or 10 ng of each pF18E1 and pF18E2 (B) . Each bar represents the average of two independent experiments with the standard error of mean.

    Techniques Used: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    Simultaneous replication of HPV16/18 genomes in the presence of E1/E2s of both types. C33A cells were transfected with a mixture of HPV16 and HPV18 gDNAs (1 ng each) together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV16 (upper panel) and HPV18 (lower panel) gDNAs were quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of three independent experiments with the standard deviation.
    Figure Legend Snippet: Simultaneous replication of HPV16/18 genomes in the presence of E1/E2s of both types. C33A cells were transfected with a mixture of HPV16 and HPV18 gDNAs (1 ng each) together with the indicated amounts (ng) of the expression plasmids. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV16 (upper panel) and HPV18 (lower panel) gDNAs were quantified by real-time PCR and normalized to the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of three independent experiments with the standard deviation.

    Techniques Used: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase, Standard Deviation

    Effects of heterologous E1 or E2 on HPV16/18 replication. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to that of the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.
    Figure Legend Snippet: Effects of heterologous E1 or E2 on HPV16/18 replication. C33A cells were transfected with 1 ng of the HPV16 (A) or HPV18 (B) gDNAs together with the indicated amounts (ng) of the expression plasmids for E1/E2s. Three days after transfection, low molecular weight DNA was isolated by the Hirt procedure and digested with Dpn I. The Dpn I-resistant HPV gDNA was quantified by real-time PCR and normalized to that of the luciferase gene. The level of the replication was presented as the relative amount of the Dpn I-resistant DNA compared to that obtained by the replication with the homologous E1/E2 alone. Each bar represents the average of two independent experiments with the standard error of mean.

    Techniques Used: Transfection, Expressing, Molecular Weight, Isolation, Real-time Polymerase Chain Reaction, Luciferase

    9) Product Images from "Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition"

    Article Title: Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03688-w

    Helraiser circle replication and donor site repair in HEK293T cells. a Schematic of the Helraiser heteroduplex LacZ donor plasmid (pHelR(mm)-Cam-LacZ) and resulting Helraiser circle. The red cross indicates the mismatch position within the transposon sequence, and the red circle marks the position of the mismatch used in the analysis of the Helraiser circles. b Experimental design of transposon circle replication assay using heteroduplex pHelR(mm)-Cam-LacZ donor plasmid. As shown, Dpn I digestion of LMW DNA reaction products can be used to distinguish between transposition of the (+) strand and the (−) strand. c Proportion of the transposon circles containing precise LTS-to-RTS junctions before and after Dpn I digestion of electroporated LMW DNA. The data are presented as n = 3 biological replicates. d Results of the transposon circle replication assay with pHelR(mm)-Cam-LacZ plasmid. The data are presented as a mean ± s.e.m., n = 3 biological replicates. e Schematic representation of possible outcomes of the transposon circle replication assay with heteroduplex pHelR(mm)-Cam-LacZ donors. Purple line: (+) strand of transposon donor; green line: (−) strand of transposon donor; solid line: methylated DNA; dashed line: unmethylated DNA; thin black line: plasmid backbone
    Figure Legend Snippet: Helraiser circle replication and donor site repair in HEK293T cells. a Schematic of the Helraiser heteroduplex LacZ donor plasmid (pHelR(mm)-Cam-LacZ) and resulting Helraiser circle. The red cross indicates the mismatch position within the transposon sequence, and the red circle marks the position of the mismatch used in the analysis of the Helraiser circles. b Experimental design of transposon circle replication assay using heteroduplex pHelR(mm)-Cam-LacZ donor plasmid. As shown, Dpn I digestion of LMW DNA reaction products can be used to distinguish between transposition of the (+) strand and the (−) strand. c Proportion of the transposon circles containing precise LTS-to-RTS junctions before and after Dpn I digestion of electroporated LMW DNA. The data are presented as n = 3 biological replicates. d Results of the transposon circle replication assay with pHelR(mm)-Cam-LacZ plasmid. The data are presented as a mean ± s.e.m., n = 3 biological replicates. e Schematic representation of possible outcomes of the transposon circle replication assay with heteroduplex pHelR(mm)-Cam-LacZ donors. Purple line: (+) strand of transposon donor; green line: (−) strand of transposon donor; solid line: methylated DNA; dashed line: unmethylated DNA; thin black line: plasmid backbone

    Techniques Used: Plasmid Preparation, Chick Chorioallantoic Membrane Assay, Sequencing, Methylation

    10) Product Images from "The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication †"

    Article Title: The Acidic Activation Domain of the Baculovirus Transactivator IE1 Contains a Virus-Specific Domain Essential for DNA Replication †

    Journal: Journal of Virology

    doi: 10.1128/JVI.76.11.5598-5604.2002

    Dpn I-based transient-replication assays for the analysis of IE1 chimeras ability to activate replication. (a) Ld652Y cells were cotransfected with five viral replication genes ( lef-1 , -2 , and -3 , p143 , and dnapol ), ie2 , the viral non-hr origin of replication (pHdN), and different IE1 chimeras. The number to the left of the blot corresponds to the size (in kilobases) of the hybridized band of linearized pHdN. The name of the sample corresponding to each lane is shown on the top of the blot. Each sample is presented in duplicate and represents two separate transfections. The negative control lane (pBS+) contains ie1 replaced by pBS+ and lacks any replication signal, indicating that there is no background replication. The lane M is a marker lane, which is the reporter plasmid pHdN linearized with Eco RI mixed with genomic DNA cut with Dpn I. C, control blank lane. (b) Transactivation analysis of p39CAT-E3 by IE1 chimeric proteins in the presence or absence of replication factors in Ld652Y cells (as described in Materials and Methods). All transfections were repeated a minimum of two times, each in duplicate. All CAT activities obtained are reported relative to the reporter plasmid cotransfected with IE1-OpAD, which was given an arbitrary value of 100. Error bars indicate standard error.
    Figure Legend Snippet: Dpn I-based transient-replication assays for the analysis of IE1 chimeras ability to activate replication. (a) Ld652Y cells were cotransfected with five viral replication genes ( lef-1 , -2 , and -3 , p143 , and dnapol ), ie2 , the viral non-hr origin of replication (pHdN), and different IE1 chimeras. The number to the left of the blot corresponds to the size (in kilobases) of the hybridized band of linearized pHdN. The name of the sample corresponding to each lane is shown on the top of the blot. Each sample is presented in duplicate and represents two separate transfections. The negative control lane (pBS+) contains ie1 replaced by pBS+ and lacks any replication signal, indicating that there is no background replication. The lane M is a marker lane, which is the reporter plasmid pHdN linearized with Eco RI mixed with genomic DNA cut with Dpn I. C, control blank lane. (b) Transactivation analysis of p39CAT-E3 by IE1 chimeric proteins in the presence or absence of replication factors in Ld652Y cells (as described in Materials and Methods). All transfections were repeated a minimum of two times, each in duplicate. All CAT activities obtained are reported relative to the reporter plasmid cotransfected with IE1-OpAD, which was given an arbitrary value of 100. Error bars indicate standard error.

    Techniques Used: Transfection, Negative Control, Marker, Plasmid Preparation

    11) Product Images from "N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena"

    Article Title: N6-adenine DNA methylation is associated with the linker DNA of H2A.Z-containing well-positioned nucleosomes in Pol II-transcribed genes in Tetrahymena

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx883

    6mA is preferentially associated with RNA Polymerase II-transcribed genes. ( A ) Comparison of 6mA methylation levels on bulk genomic DNA (MAC, blue), Polymerase II transcribed gene regions (Pol II, green) and rDNA (Pol I, red). ( B ) No 6mA methylation is localized on RNA Polymerase III transcribed genes. Pol III genes are listed in Supplementary Table S2 . ( C ) qPCR validation of nine selected GATC sites located on genes transcribed by different RNA polymerases (Pol I, II and III). Genomic DNA of SB210 was digested at 37°C with Dpn I for 30 min or overnight or with Dpn II overnight. qPCR was performed with primers flanking GATC sites or with internal control primers ( Supplementary Table S3 ). Y -axis represents methylation level that is reflected by normalized Ct value difference between digested and undigested samples. H1-H3: highly methylated sites. I1: intermediately methylated sites. N1-N5: unmethylated sites. ( D ) Methylation levels of 6mA distributed in the 1 kb region downstream the TSS of genes with different expression levels. Genes are ranked from high to low by their expression levels and divided into 10 quantiles (Q1-Q10). Each 6mA is shown as a green dot. Median of methylation level in each group of genes is marked with a red line. Inter-quartile ranges (IQR) are plotted as brown boxes. Confidence intervals are marked with blue lines. Ten groups of genes with different expression levels are divided into four homogeneous subsets according to ANOVA analysis (significance of each subset compared with others are 0.040, 0.016, 1.000 and 1.000, respectively, under condition of α = 0.01; see Supplementary Table S4 ). ( E ) Correlation matrix of different attributes of genes: (i) 6mA amount in 1 kb region downstream TSS, (ii) relative distance of 6mA to nucleosome dyad in this region, (iii) methylation level of 6mA in this region and (iv) gene expression levels (log 10 ). Correlation coefficients and correlation color dots were shown in the higher triangle of the correlation matrix ( P
    Figure Legend Snippet: 6mA is preferentially associated with RNA Polymerase II-transcribed genes. ( A ) Comparison of 6mA methylation levels on bulk genomic DNA (MAC, blue), Polymerase II transcribed gene regions (Pol II, green) and rDNA (Pol I, red). ( B ) No 6mA methylation is localized on RNA Polymerase III transcribed genes. Pol III genes are listed in Supplementary Table S2 . ( C ) qPCR validation of nine selected GATC sites located on genes transcribed by different RNA polymerases (Pol I, II and III). Genomic DNA of SB210 was digested at 37°C with Dpn I for 30 min or overnight or with Dpn II overnight. qPCR was performed with primers flanking GATC sites or with internal control primers ( Supplementary Table S3 ). Y -axis represents methylation level that is reflected by normalized Ct value difference between digested and undigested samples. H1-H3: highly methylated sites. I1: intermediately methylated sites. N1-N5: unmethylated sites. ( D ) Methylation levels of 6mA distributed in the 1 kb region downstream the TSS of genes with different expression levels. Genes are ranked from high to low by their expression levels and divided into 10 quantiles (Q1-Q10). Each 6mA is shown as a green dot. Median of methylation level in each group of genes is marked with a red line. Inter-quartile ranges (IQR) are plotted as brown boxes. Confidence intervals are marked with blue lines. Ten groups of genes with different expression levels are divided into four homogeneous subsets according to ANOVA analysis (significance of each subset compared with others are 0.040, 0.016, 1.000 and 1.000, respectively, under condition of α = 0.01; see Supplementary Table S4 ). ( E ) Correlation matrix of different attributes of genes: (i) 6mA amount in 1 kb region downstream TSS, (ii) relative distance of 6mA to nucleosome dyad in this region, (iii) methylation level of 6mA in this region and (iv) gene expression levels (log 10 ). Correlation coefficients and correlation color dots were shown in the higher triangle of the correlation matrix ( P

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Expressing

    12) Product Images from "Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans"

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-11-465

    Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).
    Figure Legend Snippet: Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).

    Techniques Used: Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Synthesized, Staining, Activity Assay, Methylation

    13) Product Images from "Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase"

    Article Title: Deaminase-Independent Inhibition of Parvoviruses by the APOBEC3A Cytidine Deaminase

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1000439

    A3A inhibits AAV2 and MVM DNA replication. (A) Inhibition of wild-type AAV replication. U2OS cells were transfected with plasmids for APOBEC3 proteins and then infected with AAV and adenovirus. HA-tagged APOBEC3 (red) and AAV Rep proteins (green) were detected by immunofluorescence using specific antibodies. (B) Inhibition of wild-type MVM replication. Southern blot detection of low molecular weight DNA extracted from A9 cells cotransfected with an infectious MVM clone together with APOBEC3A expression plasmids. The DNA was digested with Dpn -I, separated by gel electrophoresis and hybridized with a radiolabeled MVM probe. Left line is a marker (M). Replicative intermediates of ssDNA (SS), monomer (M), and dimer (D) are indicated to the right. Panel below shows immunoblots for APOBEC3 and the NS1 protein of MVM.
    Figure Legend Snippet: A3A inhibits AAV2 and MVM DNA replication. (A) Inhibition of wild-type AAV replication. U2OS cells were transfected with plasmids for APOBEC3 proteins and then infected with AAV and adenovirus. HA-tagged APOBEC3 (red) and AAV Rep proteins (green) were detected by immunofluorescence using specific antibodies. (B) Inhibition of wild-type MVM replication. Southern blot detection of low molecular weight DNA extracted from A9 cells cotransfected with an infectious MVM clone together with APOBEC3A expression plasmids. The DNA was digested with Dpn -I, separated by gel electrophoresis and hybridized with a radiolabeled MVM probe. Left line is a marker (M). Replicative intermediates of ssDNA (SS), monomer (M), and dimer (D) are indicated to the right. Panel below shows immunoblots for APOBEC3 and the NS1 protein of MVM.

    Techniques Used: Inhibition, Transfection, Infection, Immunofluorescence, Southern Blot, Molecular Weight, Expressing, Nucleic Acid Electrophoresis, Marker, Western Blot

    A3A mutants inhibit AAV DNA replication. (A) Titration of wild-type and mutant A3A expression vectors in rAAVLuc production assays. Production of rAAVLuc was assessed by transduction of target cells and quantitation of luciferase activity. Presented is the average of four independent experiments normalized to vector alone control (mock). The panels below show immunoblots to detect HA-tagged wild-type and mutant A3A proteins in transfected 293T cell lysates. (B) Southern blot detection of low molecular weight DNA extracted from 293T cells transfected for rAAVLuc production in the presence of mock (1 µg) A3G (1 µg), A3A (1, 0.1, 0.01 and 0.001 µg) and mutant A3A expression vectors (1 µg). The DNA was digested with Dpn -I, separated by gel electrophoresis, and hybridized with a radiolabeled luciferase probe.
    Figure Legend Snippet: A3A mutants inhibit AAV DNA replication. (A) Titration of wild-type and mutant A3A expression vectors in rAAVLuc production assays. Production of rAAVLuc was assessed by transduction of target cells and quantitation of luciferase activity. Presented is the average of four independent experiments normalized to vector alone control (mock). The panels below show immunoblots to detect HA-tagged wild-type and mutant A3A proteins in transfected 293T cell lysates. (B) Southern blot detection of low molecular weight DNA extracted from 293T cells transfected for rAAVLuc production in the presence of mock (1 µg) A3G (1 µg), A3A (1, 0.1, 0.01 and 0.001 µg) and mutant A3A expression vectors (1 µg). The DNA was digested with Dpn -I, separated by gel electrophoresis, and hybridized with a radiolabeled luciferase probe.

    Techniques Used: Titration, Mutagenesis, Expressing, Transduction, Quantitation Assay, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, Transfection, Southern Blot, Molecular Weight, Nucleic Acid Electrophoresis

    14) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Lytic Origin (ori-Lyt)-Dependent DNA Replication: Identification of the ori-Lyt and Association of K8 bZip Protein with the Origin"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Lytic Origin (ori-Lyt)-Dependent DNA Replication: Identification of the ori-Lyt and Association of K8 bZip Protein with the Origin

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.10.5578-5588.2003

    Mapping of boundaries of KSHV ori-Lyt ). (B) Each mutant plasmid was assayed in BCBL-1 cells for its ability to support lytic-phase DNA replication. KSHV lytic replication is induced by expression of ORF50 (Rta). Extrachromosomal DNAs were prepared using the Hirt extraction method and used for the assay. Replicated DNAs were distinguished from input DNAs by Dpn I digestion and detected by Southern blotting with 32 P-labeled pBluescript plasmid. The replication rate of each mutant relative to that of pOri-A was calculated by measuring intensities of the replicated and the input DNA bands in a phosphorimager. Each number is the average of three independent experiments.
    Figure Legend Snippet: Mapping of boundaries of KSHV ori-Lyt ). (B) Each mutant plasmid was assayed in BCBL-1 cells for its ability to support lytic-phase DNA replication. KSHV lytic replication is induced by expression of ORF50 (Rta). Extrachromosomal DNAs were prepared using the Hirt extraction method and used for the assay. Replicated DNAs were distinguished from input DNAs by Dpn I digestion and detected by Southern blotting with 32 P-labeled pBluescript plasmid. The replication rate of each mutant relative to that of pOri-A was calculated by measuring intensities of the replicated and the input DNA bands in a phosphorimager. Each number is the average of three independent experiments.

    Techniques Used: Mutagenesis, Plasmid Preparation, Expressing, Southern Blot, Labeling

    (A) Schematic presentation of the duplicated ori-Lyt region in the KSHV genome. The homology between the two duplicated sequences is indicated. The positions of the inserts in the plasmids pOri-A and pOri-B are shown below the ori-Lyt regions. (B) Replication of KSHV ori-Lyt -containing plasmids in cotransfected BCBL-1 cells receiving ori-Lyt plasmids plus pCR3.1-ORF50. KSHV lytic replication was induced by expression of ORF50 (Rta). Total DNAs were isolated from the transfected cells and used in the replication assay. Replicated DNAs were distinguished from input DNAs by Dpn I digestion and detected by Southern blotting with 32 P-labeled pBluescript plasmid.
    Figure Legend Snippet: (A) Schematic presentation of the duplicated ori-Lyt region in the KSHV genome. The homology between the two duplicated sequences is indicated. The positions of the inserts in the plasmids pOri-A and pOri-B are shown below the ori-Lyt regions. (B) Replication of KSHV ori-Lyt -containing plasmids in cotransfected BCBL-1 cells receiving ori-Lyt plasmids plus pCR3.1-ORF50. KSHV lytic replication was induced by expression of ORF50 (Rta). Total DNAs were isolated from the transfected cells and used in the replication assay. Replicated DNAs were distinguished from input DNAs by Dpn I digestion and detected by Southern blotting with 32 P-labeled pBluescript plasmid.

    Techniques Used: Expressing, Isolation, Transfection, Southern Blot, Labeling, Plasmid Preparation

    15) Product Images from "Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition"

    Article Title: Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition

    Journal: Nature Communications

    doi: 10.1038/s41467-018-03688-w

    Helraiser circle replication and donor site repair in HEK293T cells. a Schematic of the Helraiser heteroduplex LacZ donor plasmid (pHelR(mm)-Cam-LacZ) and resulting Helraiser circle. The red cross indicates the mismatch position within the transposon sequence, and the red circle marks the position of the mismatch used in the analysis of the Helraiser circles. b Experimental design of transposon circle replication assay using heteroduplex pHelR(mm)-Cam-LacZ donor plasmid. As shown, Dpn I digestion of LMW DNA reaction products can be used to distinguish between transposition of the (+) strand and the (−) strand. c Proportion of the transposon circles containing precise LTS-to-RTS junctions before and after Dpn I digestion of electroporated LMW DNA. The data are presented as n = 3 biological replicates. d Results of the transposon circle replication assay with pHelR(mm)-Cam-LacZ plasmid. The data are presented as a mean ± s.e.m., n = 3 biological replicates. e Schematic representation of possible outcomes of the transposon circle replication assay with heteroduplex pHelR(mm)-Cam-LacZ donors. Purple line: (+) strand of transposon donor; green line: (−) strand of transposon donor; solid line: methylated DNA; dashed line: unmethylated DNA; thin black line: plasmid backbone
    Figure Legend Snippet: Helraiser circle replication and donor site repair in HEK293T cells. a Schematic of the Helraiser heteroduplex LacZ donor plasmid (pHelR(mm)-Cam-LacZ) and resulting Helraiser circle. The red cross indicates the mismatch position within the transposon sequence, and the red circle marks the position of the mismatch used in the analysis of the Helraiser circles. b Experimental design of transposon circle replication assay using heteroduplex pHelR(mm)-Cam-LacZ donor plasmid. As shown, Dpn I digestion of LMW DNA reaction products can be used to distinguish between transposition of the (+) strand and the (−) strand. c Proportion of the transposon circles containing precise LTS-to-RTS junctions before and after Dpn I digestion of electroporated LMW DNA. The data are presented as n = 3 biological replicates. d Results of the transposon circle replication assay with pHelR(mm)-Cam-LacZ plasmid. The data are presented as a mean ± s.e.m., n = 3 biological replicates. e Schematic representation of possible outcomes of the transposon circle replication assay with heteroduplex pHelR(mm)-Cam-LacZ donors. Purple line: (+) strand of transposon donor; green line: (−) strand of transposon donor; solid line: methylated DNA; dashed line: unmethylated DNA; thin black line: plasmid backbone

    Techniques Used: Plasmid Preparation, Chick Chorioallantoic Membrane Assay, Sequencing, Methylation

    16) Product Images from "Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner"

    Article Title: Generation of Covalently Closed Circular DNA of Hepatitis B Viruses via Intracellular Recycling Is Regulated in a Virus Specific Manner

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001082

    Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.
    Figure Legend Snippet: Polymerase linkage status of cytoplasmic versus nuclear viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Full-length rcDNA. DNA was extracted from cytoplasmic lysate (cyto) or gradient-purified cell nuclei (nuc) by phenol extraction with or without prior PK treatment (+/− PK) and subsequently incubated with Dpn I. For HBV small amounts of partial Dpn I digestion products extended up to close to the position of ssDNA (Pla). Note the comparably strong signal for nuclear, but not cytoplasmic, HBV rcDNA even without PK treatment. ( B ) Nuclease resistant DNAs. Cytoplasmic lysate and gradient-purified nuclei were treated with MN. Subsequently, DNA was prepared by phenol extraction with or without prior PK digestion. All samples not treated with PK produced only weak if at all detectable signals.

    Techniques Used: Transfection, Purification, Incubation, Proximity Ligation Assay, Produced

    Intracellular distribution and nuclease sensitivity of viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. ( B ) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.
    Figure Legend Snippet: Intracellular distribution and nuclease sensitivity of viral DNAs. HepG2 cells were transfected with vectors for surface-deficient DHBV (left panels) or HBV (right panels). ( A ) Relative nuclear distribution. DNA was extracted, after prior PK treatment, from total cells or from gradient-purified nuclei and subsequently digested with DpnI plus PsD. Serially diluted samples were loaded on the gel. Loading volumes are indicated in percent of the total sample volume, obtained from one well of a 6-well plate. One of three experiments used for quantification (see text for details) is shown. ( B ) Direct comparison of MN resistant versus total nuclear viral DNA. DNA was prepared from total cell extract treated with MN plus PK (MN), or from gradient-purified nuclei; equal aliquots of the nuclei were treated with MN plus PK before DNA extraction (MN), or with only PK followed by incubation with Dpn I plus PsD (total nuclear DNA). A six times longer exposure for the nuclear HBV samples is shown to better reveal weak signals. Quantification indicated that only ∼10% of the nuclear full-length HBV versus ≥80% of the nuclear DHBV DNA were MN resistant.

    Techniques Used: Transfection, Purification, DNA Extraction, Incubation

    Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA. Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. ( A ) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. ( B ) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently ( Figure S5B ), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).
    Figure Legend Snippet: Site-specific discontinuity confirms the rcDNA nature of a substantial fraction of nuclear HBV DNA. Extensive nicking of HBV cccDNA might pretend an artifactually high ratio of rcDNA to cccDNA in the nucleus. However, nicking should occur at random whereas RC-DNA is distinctly discontinuous where the minus-strand and plus-strand DNA start. ( A ) Scheme of HBV rcDNA discontinuities. Restriction site positions are indicated with the first and last nucleotide of the recognition sequences. The Apa LI site is located immediately upstream of the plus-strand start. DNA in which the plus-strand is not sufficiently extended can not be cut. DR1 and DR2, direct repeats 1 and 2; wavy line at DR2, RNA primer at plus-strand 5′ end. ( B ) About one third of the rcDNA signal is resistant to Apa LI but not Nco I or Fsp I digestion. Cytoplasmic (treated with MN) and nuclear (treated with Dpn I) DNA preparations (both after prior PK digestion) were incubated with the indicated restriction enzymes. Consistently ( Figure S5B ), ∼35% of the rcDNA signal from the cytoplasm as well as the nucleus remained upon incubation with Apa LI (arrowheads) but not Nco I or Fsp I. Activity of Apa LI in the reactions is documented by the absence of a plasmid-derived Dpn I fragment containing internal sites for Apa LI and Fsp I but not Nco I. All samples were run on the same gel but a six-times longer exposure is shown for the nuclear samples; lane 5L on the longer exposure corresponds to lane 5 on the left panel. M, marker fragments of the indicated sizes (in kb).

    Techniques Used: Incubation, Activity Assay, Plasmid Preparation, Derivative Assay, Marker

    DHBV replication in avian cells and HBV replication in human cells. ( A ) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. ( B ) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.
    Figure Legend Snippet: DHBV replication in avian cells and HBV replication in human cells. ( A ) Chicken LMH cells transfected with wild-type and surface-deficient DHBV. ( B ) Human HepG2 cells transfected with wild-type and surface-deficient HBV. DNAs were extracted, after prior PK digestion, from cytoplasmic lysates and the nuclear fractions. One aliquot of the cytoplasmic lysates was treated with micrococcal nuclease (MN) before DNA extraction and analyzed without further treatment. All other samples were incubated, post extraction, with either Dpn I alone (Dpn), or Dpn I plus Plasmid safe DNAse (Dpn+PsD). Nomenclature of the various DNA species: RC, relaxed circular; DL, double strand linear; SS, single strand; CCC, covalently closed circular DNA; Pla, Dpn I restriction fragments of transfected plasmid DNA.

    Techniques Used: Transfection, DNA Extraction, Incubation, Plasmid Preparation, Countercurrent Chromatography, Proximity Ligation Assay

    Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility. ( A ) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. ( B ) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. ( C ) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.
    Figure Legend Snippet: Restriction mapping of nuclear HBV rcDNA suggests genome region-selective MN accessibility. ( A ) Cytoplasmic versus nuclear MN-resistant viral DNAs. DNAs were isolated from HepG2 cells transfected with the surface-deficient HBV vector after prior MN plus PK treatment, and incubated with restriction enzymes Spe I and Nco I (ø, no restriction). Amounts equivalent to 10% of the total cytoplasmic fraction and 90% of the total nuclear fraction were loaded. ( B ) Total nuclear rcDNA versus MN resistant nuclear DNA. Viral DNA from isolated nuclei was prepared by either the PK plus Dpn I plus PsD procedure (total nuclear rcDNA), or after prior MN plus PK treatment (MN resistant nuclear DNA), and incubated with Nsi I, Eco RI or Bsp EI. Asterisks denote newly formed distinct fragments; lane ø on the right is a longer exposure of lane ø on the left. ( C ) Restriction map of HBV. The restriction sites probed in A and B are indicated. DR1 and DR2, direct repeats 1 and 2; P, covalently linked polymerase; wiggly red line, RNA primer at (+)-DNA 5′ end. Together, the patterns can consistently be explained if MN treatment removed a defined region from the rcDNA that approximately encompasses position 3000 to 500.

    Techniques Used: Isolation, Transfection, Plasmid Preparation, Incubation

    Core protein association of nuclear DHBV and HBV DNA. Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. ( A ) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. ( B ) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7 .
    Figure Legend Snippet: Core protein association of nuclear DHBV and HBV DNA. Vectors for surface-deficient DHBV and HBV genomes were transfected into Huh7 cells. IPs were performed in cytoplasmic extracts and extracts of purified nuclei containing 0.75× RIPA buffer, using antibodies against DHBV core protein (αDc) or HBV core protein (αHBc); in the mock IPs αDc was replaced by αHBc and vice versa. Immunopellets were treated with MN or not as indicated, and extracted after prior PK digestion. Purified DNAs from not MN-treated samples were digested with Dpn I. ø, extract directly treated with MN. ( A ) DHBV. M1, marker for cccDNA and rcDNA; M2, marker for single-stranded DNA; * and **, positions of cccDNA and ssDNA, respectively. ( B ) HBV. The rightmost panel shows the nuclear samples from an analogous experiment in HepG2 cells; the cytoplasmic samples are shown in Figure S7 .

    Techniques Used: Transfection, Purification, Marker

    17) Product Images from "A tool kit for rapid cloning and expression of recombinant antibodies"

    Article Title: A tool kit for rapid cloning and expression of recombinant antibodies

    Journal: Scientific Reports

    doi: 10.1038/srep05885

    Schematic representation of PIPE cloning strategy for swapping antibody variable regions. pVITRO1-IgE/κ vector is PCR linearized by V H and V K flanking primer pairs in two independent PCR reactions, resulting in two vector fragments subsequently treated with Dpn I. Simultaneously, the CSPG4 specific V H and V K are PCR amplified for generation of vector fragment terminal end-homology. The Dpn I-treated vector fragments are mixed with unpurified V H and V K , the single-stranded DNA fragments anneal directionally across the complementary sequences and nicks and gaps are repaired in vivo after transformation, generating pVITRO1-CSPG4-IgE/κ expression vector.
    Figure Legend Snippet: Schematic representation of PIPE cloning strategy for swapping antibody variable regions. pVITRO1-IgE/κ vector is PCR linearized by V H and V K flanking primer pairs in two independent PCR reactions, resulting in two vector fragments subsequently treated with Dpn I. Simultaneously, the CSPG4 specific V H and V K are PCR amplified for generation of vector fragment terminal end-homology. The Dpn I-treated vector fragments are mixed with unpurified V H and V K , the single-stranded DNA fragments anneal directionally across the complementary sequences and nicks and gaps are repaired in vivo after transformation, generating pVITRO1-CSPG4-IgE/κ expression vector.

    Techniques Used: Clone Assay, Plasmid Preparation, Polymerase Chain Reaction, Amplification, In Vivo, Transformation Assay, Expressing

    18) Product Images from "DNA Replication and Postreplication Mismatch Repair in Cell-Free Extracts from Cultured Human Neuroblastoma and Fibroblast Cells"

    Article Title: DNA Replication and Postreplication Mismatch Repair in Cell-Free Extracts from Cultured Human Neuroblastoma and Fibroblast Cells

    Journal: The Journal of Neuroscience

    doi: 10.1523/JNEUROSCI.17-22-08711.1997

    Origin-dependent replication using cell-free extracts from human fibroblasts and SY5Y neuroblastoma cells. DNA synthesis was performed as described in Materials and Methods (4 hr, 37°C) in the presence or absence of T-antigen. DNA was purified and subjected to electrophoresis in a 1% agarose gel, followed by ethidium bromide staining (0.2 mg/ml) and autoradiography. DNA standards were run in adjacent lanes. The positions of the different forms of replicated DNA are indicated at the left . HeLa extract was used as a positive control for replication that was both Tag- and plasmid-dependent. To verify that semiconservative replication was occurring, the product DNA was treated with Dpn I, which only cuts fully methylated 5′-GATC sequences. Hemimethylated product DNAs resulting from semiconservative replication, dependent on the presence of T-antigen in the reaction, were refractory to cleavage (data not shown). Extract protein concentration in all the reactions involving WI38 cells was 2 mg/ml, in reactions with SY5Y was 1 mg/ml, and in reactions with HeLa was 3 mg/ml. The bands at the top are caused by radioactive material remaining in the wells.
    Figure Legend Snippet: Origin-dependent replication using cell-free extracts from human fibroblasts and SY5Y neuroblastoma cells. DNA synthesis was performed as described in Materials and Methods (4 hr, 37°C) in the presence or absence of T-antigen. DNA was purified and subjected to electrophoresis in a 1% agarose gel, followed by ethidium bromide staining (0.2 mg/ml) and autoradiography. DNA standards were run in adjacent lanes. The positions of the different forms of replicated DNA are indicated at the left . HeLa extract was used as a positive control for replication that was both Tag- and plasmid-dependent. To verify that semiconservative replication was occurring, the product DNA was treated with Dpn I, which only cuts fully methylated 5′-GATC sequences. Hemimethylated product DNAs resulting from semiconservative replication, dependent on the presence of T-antigen in the reaction, were refractory to cleavage (data not shown). Extract protein concentration in all the reactions involving WI38 cells was 2 mg/ml, in reactions with SY5Y was 1 mg/ml, and in reactions with HeLa was 3 mg/ml. The bands at the top are caused by radioactive material remaining in the wells.

    Techniques Used: DNA Synthesis, Purification, Electrophoresis, Agarose Gel Electrophoresis, Staining, Autoradiography, Positive Control, Plasmid Preparation, Methylation, Protein Concentration

    Related Articles

    Methylation:

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli
    Article Snippet: .. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites. ..

    Isolation:

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins
    Article Snippet: .. Bacterial genomic DNA was isolated from 3 ml LBamp cultures from individual colonies using the DNeasy Tissue kit (Qiagen, 69504). gDNA (1 µg) was digested with 10 units of Dpn I (NEB, R0176S) for 1 h at 37°C. ..

    Ligation:

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans
    Article Snippet: .. Ligation to Linker A was carried out in a 50 μl reaction using the following mix: 10 μl Dpn I product, 1.5 μl of 0.05 mM Linker A, 11.0 μl dH2 O, 25.0 μl 2× Quick Ligase Buffer, 2.5 μl Quick Ligase (NEB #M2200). .. To increase the number of ligated molecules, we added a second ligation step using the following mix: 10 μl Quick Ligase product, 7 μl dH2 O, 2 μl 10× ligase buffer, 1 μl T4 DNA ligase (2,000 U/μl; NEB #M0202).

    Construct:

    Article Title: Secondary structure formation and DNA instability at fragile site FRA16B
    Article Snippet: .. To determine replication efficiency of the constructs ( B), SV40-replicated DNAs were digested with HindIII and NdeI (New England Biolabs) to linearize the plasmids, and with DpnI (New England Biolabs) to remove unreplicated parental templates. ..

    other:

    Article Title: Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication ▿
    Article Snippet: HindIII, DpnI, and λ DNA were purchased from New England Biolabs.

    Plasmid Preparation:

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli
    Article Snippet: .. Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites. ..

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    New England Biolabs dpn i
    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with <t>Dpn</t> I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).
    Dpn I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genetic and growth characteristics displayed by dam- complemented mutant strains of UPEC relative to wild-type . (A) Dam methylation pattern in UPEC CFT073 strain subsequent to digestion with Dpn I (lane 1) and Mbo I (lane 2). The 1 kb plus DNA ladder (MW) is also shown. (B) Growth curve (CFU/milliliter versus time) for dam complement UPEC strains of CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam . (C) Micrographs for wild-type (WT) and dam mutant (Δ dam ) UPEC strains, illustrating the morphological occurrence of shortened- and filamentous rods, respectively. (D) Semi-quantitative RT-PCR for mdh, rec A, and arc A expression at cycles 23, 25, and 30 for CFT073 (lanes 1–3), CFT073 Δ dam (lanes 4–6), CFT073 + pGEMdam (lanes 7–9), CFT073 Δ dam + pGEMdam (lanes 10–12). The 100 bp molecular marker MW (Promega, WI, USA) and negative control are shown (lane 13).

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Methylation, Quantitative RT-PCR, Expressing, Marker, Negative Control

    Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Phenotypic influence of Dam on P fimbriae . (A) PCR screening for pap EF in UPEC strains cC119 (lane 4), CFT073 (lane 5), and cU155 (lane 6). The 100-bp molecular weight marker (Invitrogen), negative control and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 1 and 2, respectively. (B) PCR screening for pap I– pap B intergenic regulatory region in UPEC strains from UPEC strains cC119 (lane 2), CFT073 (lane 3), and cU155 (lane 4). The 1-kb plus molecular marker (Invitrogen, CA, USA), negative control, and positive control ( E. coli strain Lo qnr A + / pap EF + ) are represented as MW, 2 and 5, respectively. (C) Schematic representation of pSAMS1 recombinant plasmid containing cloned pap IB insert within pCRII–TOPOII vector. (D) Dam methylation patterns for pap I-B regulatory region. Sau 3AI (lane 2), Mbo I (lane 3), and Dpn I (lane 4) digests of pSAMS2 isolated from cC119 are shown. MW represents the 1 kb Plus molecular marker (Invitrogen). An undigested pap IB fragment (lane 5) is also represented. (E) Semi-quantitative (sq) RT-PCR for pap I expression in cC119 (lane 1), cC119 Δ dam (lane 2), CFT073 wild-type (lane 3) and CFT073 Δ dam (lane 4). The 1 kb Plus molecular marker (Invitrogen) and amplified chromosomal DNA for UPEC strains cC119 and CFT073 are shown in lanes MW, 5 and 6, respectively.

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Negative Control, Positive Control, Recombinant, Plasmid Preparation, Clone Assay, Methylation, Isolation, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification

    Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Journal: Frontiers in Public Health

    Article Title: Epigenetic Influence of Dam Methylation on Gene Expression and Attachment in Uropathogenic Escherichia coli

    doi: 10.3389/fpubh.2016.00131

    Figure Lengend Snippet: Genotypic and growth characteristics displayed by parental and dam- mutant strains of UPEC . (A) Schematic diagram of gene disruption strategy for chromosomal insertion of chloramphenicol resistance gene from pKD3 into dam gene within UPEC chromosome subsequent to λ red recombineering with pKM208. (B) Amplified dam fragment from wild type UPEC strains CFT073 (lane 1) and cured parental strains C119 (lane 2) to produce 1071 bp amplicon. MW is 1 kb DNA ladder (Bioneer Corporation, Republic of Korea) and −ve is negative control. (C) PCR screening of UPEC candidates for dam mutation observed as 1323 bp products using primers UR427 and UR428. MW is a 1 kb Plus DNA ladder (Invitrogen, USA). (D) Dam methylation pattern in UPEC CFT073 wild type (lanes 1, 2, 8, 9, 14, 15), C119 wild type (lanes 3, 4, 10, 11, 16, 17), and E. coli K-12 substrain MG1655 (5, 12, 18) strains subsequent to digestion with Mbo I, Sau 3AI, and Dpn I. The negative control (7, 13, 19) and 1 kb Plus DNA ladder (MW) are also shown. (E) Dam methylation pattern in UPEC dam mutants CFT073 (lanes 1, 2, 3, 8, 9, 10, 15, 16, 17) and C119 wild-type (lanes 4, 5, 6, 11, 12, 13, 18, 19) subsequent to digestion with Sau 3AI, Mbo I, and Dpn I. The negative control (lanes 7, 14) and 1 kb Plus DNA ladder (MW) are also shown. (F) Growth curve (CFU/milliliter versus time) for UPEC strains CFT073, CFT073 Δ dam , cC119, and cC119 Δ dam .

    Article Snippet: Essentially, 0.5 μg of chromosomal and plasmid DNA was digested for 1.5 h at 37°C with 2 U Sau 3AI (Promega, WI, USA), 10 U Dpn I (New England Biolabs, MA, USA), or 2.5 U Mbo I. Sau 3AI cleaves DNA at GATC sites regardless of methylation state, Dpn I cleaves GATC sites that have a methylated adenine residue, and Mbo I cleaves unmethylated GATC sites.

    Techniques: Mutagenesis, Amplification, Negative Control, Polymerase Chain Reaction, Methylation

    STRIP assay and replication of the pFX/SV40 templates ( dark gray rings ) by primate proteins within COS-1 cells. At 48 h posttransfection, the episomally replicated DNA was extracted and digested by Dpn I to remove the unreplicated ( dark gray rings ) and partially replicated ( dark gray rings with small light gray rings on top ) templates. The Dpn I-resistant primate-replicated templates ( light gray rings ) were transformed into E. coli , and individual colonies, each an individual product of primate replication, were cultured. The resulting DNA was restriction digested and analyzed on 4% polyacrylamide gels for CGG length changes.

    Journal: American Journal of Human Genetics

    Article Title: Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

    doi:

    Figure Lengend Snippet: STRIP assay and replication of the pFX/SV40 templates ( dark gray rings ) by primate proteins within COS-1 cells. At 48 h posttransfection, the episomally replicated DNA was extracted and digested by Dpn I to remove the unreplicated ( dark gray rings ) and partially replicated ( dark gray rings with small light gray rings on top ) templates. The Dpn I-resistant primate-replicated templates ( light gray rings ) were transformed into E. coli , and individual colonies, each an individual product of primate replication, were cultured. The resulting DNA was restriction digested and analyzed on 4% polyacrylamide gels for CGG length changes.

    Article Snippet: Episomal DNAs were digested with Dpn I (New England Biolabs) to eliminate unreplicated parental templates.

    Techniques: Stripping Membranes, Transformation Assay, Cell Culture

    Replication efficiency. An aliquot of the starting mixture, composed of each pFX53 construct with unmethylated pSV40, was linearized with Alw NI. Primate-replicated DNA resulting from the cotransfection of this same starting mixture was also linearized with Alw NI, was further digested with Dpn I to remove the unreplicated parental template, and was then probed with the SV40- ori ” section).

    Journal: American Journal of Human Genetics

    Article Title: Role of Replication and CpG Methylation in Fragile X Syndrome CGG Deletions in Primate Cells

    doi:

    Figure Lengend Snippet: Replication efficiency. An aliquot of the starting mixture, composed of each pFX53 construct with unmethylated pSV40, was linearized with Alw NI. Primate-replicated DNA resulting from the cotransfection of this same starting mixture was also linearized with Alw NI, was further digested with Dpn I to remove the unreplicated parental template, and was then probed with the SV40- ori ” section).

    Article Snippet: Episomal DNAs were digested with Dpn I (New England Biolabs) to eliminate unreplicated parental templates.

    Techniques: Construct, Cotransfection

    Ku is associated with the ADA-associated origin of the mouse genome in Ku80 +/+ cells, but not in Ku80 −/− MEFs. Ku80 −/− cell extracts have reduced replication activity. (A) Western blot probed with 1/100th dilution of anti-Ku86, or 1/400th dilution of anti-Ku70. 1/20th of immunoprecipitation with clone162 from cross-linked or untreated Ku80 +/+ or Ku80 −/− MEFs. (B) PCR amplification with the use of primer set ADA A, which amplifies a genomic 230-bp fragment. Template DNA used was as follows. Lanes 1 and 2, total genomic DNA isolated from untreated Ku80 +/+ or Ku80 −/− cells. Lane 3, negative control to verify primer contamination; no template DNA added to PCR reaction. Lanes 4, 6, and 8, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 +/+ cells. Lanes 5, 7, and 9, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 −/− cells. (C) In vitro DNA replication assays were performed with Ku80 +/+ or Ku80 −/− cells extracts and p186 as the template DNA. The in vitro replication products were purified, digested with Dpn I, and the Dpn I-resistant bands were quantitated with the use of a phosphorimager. The amount of radioactive precursor incorporated into the DNA is expressed as a percentage relative to the Ku80 +/+ cell extract reaction (100%). The quantification was obtained from at least three different in vitro reactions. Each bar represents three experiments and 1 SD is indicated.

    Journal: Molecular Biology of the Cell

    Article Title: In Vivo Association of Ku with Mammalian Origins of DNA Replication

    doi:

    Figure Lengend Snippet: Ku is associated with the ADA-associated origin of the mouse genome in Ku80 +/+ cells, but not in Ku80 −/− MEFs. Ku80 −/− cell extracts have reduced replication activity. (A) Western blot probed with 1/100th dilution of anti-Ku86, or 1/400th dilution of anti-Ku70. 1/20th of immunoprecipitation with clone162 from cross-linked or untreated Ku80 +/+ or Ku80 −/− MEFs. (B) PCR amplification with the use of primer set ADA A, which amplifies a genomic 230-bp fragment. Template DNA used was as follows. Lanes 1 and 2, total genomic DNA isolated from untreated Ku80 +/+ or Ku80 −/− cells. Lane 3, negative control to verify primer contamination; no template DNA added to PCR reaction. Lanes 4, 6, and 8, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 +/+ cells. Lanes 5, 7, and 9, Ku70, Ku86, or clone162 immunoprecipitate from Ku80 −/− cells. (C) In vitro DNA replication assays were performed with Ku80 +/+ or Ku80 −/− cells extracts and p186 as the template DNA. The in vitro replication products were purified, digested with Dpn I, and the Dpn I-resistant bands were quantitated with the use of a phosphorimager. The amount of radioactive precursor incorporated into the DNA is expressed as a percentage relative to the Ku80 +/+ cell extract reaction (100%). The quantification was obtained from at least three different in vitro reactions. Each bar represents three experiments and 1 SD is indicated.

    Article Snippet: Samples were digested with 0.8 U of Dpn I ( New England Biolabs ) for 45 min at 37°C in the presence of 1× NEB 4 buffer and 100 mM NaCl.

    Techniques: Activity Assay, Western Blot, Immunoprecipitation, Polymerase Chain Reaction, Amplification, Isolation, Negative Control, In Vitro, Purification

    Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).

    Journal: BMC Genomics

    Article Title: Distributed probing of chromatin structure in vivo reveals pervasive chromatin accessibility for expressed and non-expressed genes during tissue differentiation in C. elegans

    doi: 10.1186/1471-2164-11-465

    Figure Lengend Snippet: Copy number determination for lines PD3994 and PD5122 . Panels a, c, e, g, and i are agarose gel images of Dpn I , Mbo I , and Sau3A I digested DNA from PD3994, PD5122, and N2 (control) animals. Below each agarose gel is the corresponding Southern blot (b, d, f, h, j). pPD98.38 is a plasmid from which the probe (808 bp of the C. elegans 5S rDNA/SL1) was synthesized. The slight smearing seen in ethidium bromide stained gels for N2 (a and e) likely resulted from non specific activity of Dpn I . Compared to fully methylated GATC, Dpn I can cut non-methylated GATC 1,000 fold slower and hemimethylated GATC 60 fold slower (Derek Robinson, New England Biolabs, personal communication).

    Article Snippet: Ligation to Linker A was carried out in a 50 μl reaction using the following mix: 10 μl Dpn I product, 1.5 μl of 0.05 mM Linker A, 11.0 μl dH2 O, 25.0 μl 2× Quick Ligase Buffer, 2.5 μl Quick Ligase (NEB #M2200).

    Techniques: Agarose Gel Electrophoresis, Southern Blot, Plasmid Preparation, Synthesized, Staining, Activity Assay, Methylation