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    Structured Review

    New England Biolabs dpni
    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with <t>DpnI</t> . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to <t>Dpn</t> I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Dpni, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    2) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    3) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

    4) Product Images from "iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins"

    Article Title: iDamIDseq and iDEAR: an improved method and computational pipeline to profile chromatin-binding proteins

    Journal: Development (Cambridge, England)

    doi: 10.1242/dev.139261

    Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).
    Figure Legend Snippet: Improving Dam-fusion proteins. (A) DamL122A displays low toxicity in medaka embryos compared with the unmodified protein. Medaka zygotes were injected with mRNA coding for the E. coli Dam (eD-f-G) or DamL122A fused to GFP via flexylinker (D-f-G) (see below). Embryos were scored for abnormalities at embryonic stage 25. (B) Agarose gel of isolated bacterial gDNA samples undigested (−) or digested (+) with DpnI . Dam activity depends on the flexilinker, and the type and orientation of the fused proteins. Bacterial gDNA isolated from a strain deficient in the dam/dcm systems is resistant to Dpn I digestion. This condition can be reversed in transformed bacteria only when the fusion protein generates a functional Dam. Whereas DNA from bacteria transformed with constructs coding for fusions Dam-GFP (D-G) or Dam-TF (D-TF) (OtpA from zebrafish) can be digested by Dpn I, DNA from GFP-Dam (G-D) and TF-Dam (TF-D) bacteria is resistant to Dpn I digestion. In addition, the use of flexylinker between Dam and the fusion protein (D-f-GFP and D-f-TF) generates a Dpn I digestion pattern similar to that of bacteria with a functional dam/dcm system (Top10 cells).

    Techniques Used: Injection, Agarose Gel Electrophoresis, Isolation, Activity Assay, Transformation Assay, Functional Assay, Construct

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    New England Biolabs dpn i
    Effect of linker mutations on core DNA. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and were harvested seven days post-transfection. HBV NC-associated DNA (core DNA) was extracted from cytoplasmic lysate without ( A ) or with TURBO DNase digestion ( B ), and detected by Southern blot analysis. Input plasmid DNA (but not viral replicative DNA) was removed with <t>Dpn</t> I before Southern blot analysis in A . The viral DNA signals (RC and immature DS DNA) digested by the nuclease were marked by the dotted boxes ( B ). RC, RC DNA; SS, SS DNA. C. Quantitative results from multiple experiments shown in A. SS DNA synthesis efficiency was determined by normalizing the levels of SS DNA to the pgRNA signals in Fig 4 . RC DNA synthesis efficiency was determined by normalizing the levels of RC DNA to the SS DNA signals. The efficiencies from WT was set to 1.0. *, P
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    Effect of linker mutations on core DNA. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and were harvested seven days post-transfection. HBV NC-associated DNA (core DNA) was extracted from cytoplasmic lysate without ( A ) or with TURBO DNase digestion ( B ), and detected by Southern blot analysis. Input plasmid DNA (but not viral replicative DNA) was removed with Dpn I before Southern blot analysis in A . The viral DNA signals (RC and immature DS DNA) digested by the nuclease were marked by the dotted boxes ( B ). RC, RC DNA; SS, SS DNA. C. Quantitative results from multiple experiments shown in A. SS DNA synthesis efficiency was determined by normalizing the levels of SS DNA to the pgRNA signals in Fig 4 . RC DNA synthesis efficiency was determined by normalizing the levels of RC DNA to the SS DNA signals. The efficiencies from WT was set to 1.0. *, P

    Journal: PLoS Pathogens

    Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

    doi: 10.1371/journal.ppat.1009230

    Figure Lengend Snippet: Effect of linker mutations on core DNA. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and were harvested seven days post-transfection. HBV NC-associated DNA (core DNA) was extracted from cytoplasmic lysate without ( A ) or with TURBO DNase digestion ( B ), and detected by Southern blot analysis. Input plasmid DNA (but not viral replicative DNA) was removed with Dpn I before Southern blot analysis in A . The viral DNA signals (RC and immature DS DNA) digested by the nuclease were marked by the dotted boxes ( B ). RC, RC DNA; SS, SS DNA. C. Quantitative results from multiple experiments shown in A. SS DNA synthesis efficiency was determined by normalizing the levels of SS DNA to the pgRNA signals in Fig 4 . RC DNA synthesis efficiency was determined by normalizing the levels of RC DNA to the SS DNA signals. The efficiencies from WT was set to 1.0. *, P

    Article Snippet: Where indicated, Dpn I (NEB) digestion, instead of TURBO DNase, was used to remove the transfected plasmid after DNA purification [ ].

    Techniques: Transfection, Expressing, Construct, Southern Blot, Plasmid Preparation, DNA Synthesis

    Analysis of CCC DNA from TT146/147AA in the presence and absence of the L protein. The full-length HBV replicon, with WT or TT146/147AA mutant HBc, or their L - derivative was transfected into HepG2 cells. Transfected cells were harvested seven days post-transfection. A. HBV NC-associated DNA (core DNA) was released by SDS-proteinase K digestion from cytoplasmic lysates and detected by Southern blot analysis. B. PF DNA was extracted by Hirt extraction and digested with Dpn I (lane 1–4) or Dpn I plus exonuclease I and III (lane 5–8). RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. Quantitative results from multiple experiments. Left, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized values from the WT were set to 1.0. **, P

    Journal: PLoS Pathogens

    Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

    doi: 10.1371/journal.ppat.1009230

    Figure Lengend Snippet: Analysis of CCC DNA from TT146/147AA in the presence and absence of the L protein. The full-length HBV replicon, with WT or TT146/147AA mutant HBc, or their L - derivative was transfected into HepG2 cells. Transfected cells were harvested seven days post-transfection. A. HBV NC-associated DNA (core DNA) was released by SDS-proteinase K digestion from cytoplasmic lysates and detected by Southern blot analysis. B. PF DNA was extracted by Hirt extraction and digested with Dpn I (lane 1–4) or Dpn I plus exonuclease I and III (lane 5–8). RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. Quantitative results from multiple experiments. Left, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized values from the WT were set to 1.0. **, P

    Article Snippet: Where indicated, Dpn I (NEB) digestion, instead of TURBO DNase, was used to remove the transfected plasmid after DNA purification [ ].

    Techniques: Countercurrent Chromatography, Mutagenesis, Transfection, Southern Blot

    Effects of linker mutations on CCC DNA formation. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and HBV PF DNA was extracted from the transfected cells seven days after transfection. The extracted DNA was digested with Dpn I to degrade input plasmids ( A ), or Dpn I plus the exonuclease I and III to removal all DNA except closed circular DNA ( B ), before agarose gel electrophoresis and Southern blot analysis. Novel PF DNA smears detected from certain mutants are marked with the white asterisks to the left of the relevant lanes (S141D, S141R, L143A, TT146/147DD). PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. CCC DNA and PF-RC DNA signals of each mutant were compared with WT (top two panels). CCC DNA and PF-RC DNA are normalized to core RC DNA (middle two panels), and CCC DNA is normalized PF-RC DNA (bottom). All values from the WT were set to 1.0. *, P

    Journal: PLoS Pathogens

    Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

    doi: 10.1371/journal.ppat.1009230

    Figure Lengend Snippet: Effects of linker mutations on CCC DNA formation. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and HBV PF DNA was extracted from the transfected cells seven days after transfection. The extracted DNA was digested with Dpn I to degrade input plasmids ( A ), or Dpn I plus the exonuclease I and III to removal all DNA except closed circular DNA ( B ), before agarose gel electrophoresis and Southern blot analysis. Novel PF DNA smears detected from certain mutants are marked with the white asterisks to the left of the relevant lanes (S141D, S141R, L143A, TT146/147DD). PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. CCC DNA and PF-RC DNA signals of each mutant were compared with WT (top two panels). CCC DNA and PF-RC DNA are normalized to core RC DNA (middle two panels), and CCC DNA is normalized PF-RC DNA (bottom). All values from the WT were set to 1.0. *, P

    Article Snippet: Where indicated, Dpn I (NEB) digestion, instead of TURBO DNase, was used to remove the transfected plasmid after DNA purification [ ].

    Techniques: Countercurrent Chromatography, Transfection, Expressing, Construct, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis

    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.

    Journal: Molecular Systems Biology

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

    doi: 10.1038/msb.2010.54

    Figure Lengend 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.

    Article Snippet: Briefly, genomic DNA was isolated using a DNAeasy kit (Qiagen) and then digested overnight with Dpn I (New England Biolabs (NEB)) to cut at GAm TC sites.

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

    Detection of shortened HBV genomes in the cytoplasm of LMH cells. Transfection was done with a plasmid coding for splice-deficient virus with the natural HBV core promoter driving transcription of pgRNA and the surface protein coding sequence mutated. ( A ) Viral DNA from cytoplasm of LMH cells was compared with viral DNA from HepG2 cell nuclei and HepG2 cytoplasm. Preparation of viral DNA involved nuclease treatment of cytoplasmic lysates and purified cell nuclei. Numbers below indicate relative amounts of DNA loaded in the respective lanes. Hybridisation was done with a probe encompassing both strands of the whole HBV genome. The arrow points to a prominent DNA species obtained both in cytoplasm of LMH cells and nucleus of HepG2 cells. ( B ) Viral DNA from transfected LMH cells was denatured (+/− denat) by heating before gel loading, as indicated. Hybridisation was done with 32 P-labelled oligonucleotides detecting either the 3′-end or the 5′-end of plus-strand DNA at genome positions 2682 and 17 (see map of the HBV genome in Figure 1D ). Positions of the 3.2 kb and 2.0 kb marker fragments are indicated. The arrow points to viral genomes migrating between these marker fragments. ( C ) Viral DNA was prepared from cytoplasm of transfected LMH cells with or without micrococcal nuclease (+/− MN) treatment and with or without protease (+/− ProtK) digestion done before phenol extraction. The purified DNA was digested with restriction enzyme Dpn I, which selectively cuts transfected plasmid (Pla) into multiple short fragments. Southern blot hybridisation was done with a probe covering both strands of the whole HBV genome. This probe also detects several Dpn I fragments of transfected plasmid DNA. ( D ) Viral DNA was prepared from cytoplasm of HepG2 and LMH cells with or without micrococcal nuclease (+/− MN) treatment. Purified DNA was subsequently digested with restriction enzyme Dpn I and denatured by heating before gel electrophoresis. Southern blot hybridisation was done with a mixture of 32 P-labeled oligonucleotide complementary to the 3′-end of plus-strand DNA at genome position 2307, 2340, 2385, 2439 and 2682. These five oligonucleotides were combined to enhance sensitivity. The vertical lane on the right side indicates shortened plus-strand molecules. The probe detects a 0.8 kb Dpn I fragment of the transfected plasmid (Pla) if micrococcal nuclease treatment is not done.

    Journal: PLoS ONE

    Article Title: Human Hepatitis B Virus Production in Avian Cells Is Characterized by Enhanced RNA Splicing and the Presence of Capsids Containing Shortened Genomes

    doi: 10.1371/journal.pone.0037248

    Figure Lengend Snippet: Detection of shortened HBV genomes in the cytoplasm of LMH cells. Transfection was done with a plasmid coding for splice-deficient virus with the natural HBV core promoter driving transcription of pgRNA and the surface protein coding sequence mutated. ( A ) Viral DNA from cytoplasm of LMH cells was compared with viral DNA from HepG2 cell nuclei and HepG2 cytoplasm. Preparation of viral DNA involved nuclease treatment of cytoplasmic lysates and purified cell nuclei. Numbers below indicate relative amounts of DNA loaded in the respective lanes. Hybridisation was done with a probe encompassing both strands of the whole HBV genome. The arrow points to a prominent DNA species obtained both in cytoplasm of LMH cells and nucleus of HepG2 cells. ( B ) Viral DNA from transfected LMH cells was denatured (+/− denat) by heating before gel loading, as indicated. Hybridisation was done with 32 P-labelled oligonucleotides detecting either the 3′-end or the 5′-end of plus-strand DNA at genome positions 2682 and 17 (see map of the HBV genome in Figure 1D ). Positions of the 3.2 kb and 2.0 kb marker fragments are indicated. The arrow points to viral genomes migrating between these marker fragments. ( C ) Viral DNA was prepared from cytoplasm of transfected LMH cells with or without micrococcal nuclease (+/− MN) treatment and with or without protease (+/− ProtK) digestion done before phenol extraction. The purified DNA was digested with restriction enzyme Dpn I, which selectively cuts transfected plasmid (Pla) into multiple short fragments. Southern blot hybridisation was done with a probe covering both strands of the whole HBV genome. This probe also detects several Dpn I fragments of transfected plasmid DNA. ( D ) Viral DNA was prepared from cytoplasm of HepG2 and LMH cells with or without micrococcal nuclease (+/− MN) treatment. Purified DNA was subsequently digested with restriction enzyme Dpn I and denatured by heating before gel electrophoresis. Southern blot hybridisation was done with a mixture of 32 P-labeled oligonucleotide complementary to the 3′-end of plus-strand DNA at genome position 2307, 2340, 2385, 2439 and 2682. These five oligonucleotides were combined to enhance sensitivity. The vertical lane on the right side indicates shortened plus-strand molecules. The probe detects a 0.8 kb Dpn I fragment of the transfected plasmid (Pla) if micrococcal nuclease treatment is not done.

    Article Snippet: The DNA samples were treated with Plasmid safe DNase (Epicentre Biotechnologies), or digested with restriction enzyme Dpn I (New England Biolabs) or denatured by heating at 95°C for 5 minutes, as indicated in the respective experiments.

    Techniques: Transfection, Plasmid Preparation, Sequencing, Purification, Hybridization, Marker, Proximity Ligation Assay, Southern Blot, Nucleic Acid Electrophoresis, Labeling

    Identification and characterization of the Dnmt1-binding regions in early mouse embryos by DamID and DELP sequencing. ( A ) Structure of Dnmt1–Dam (DnmtDam) fusion protein. V5 is an epitope for immunostaining. Refer to Supplementary Figure S4 for the regulatory region (R). ( B ) DamID and DELP-seq ( Dpn I fragment enrichment by ligation-mediated PCR sequencing) procedures. Mouse zygotes were injected with the DamID toolkit and were cultured until the four- and eight-cell stages. The embryos were treated with Ponasterone A for 24 h to induce the expression of DnmtDam for the methylation of nearby Dpn I sites (5′-GATC-3′). Genomic DNA from the four/eight-cell embryos was subjected to DELP-seq. ( C ) Nuclear localization of DnmtDam in mouse embryos. Two- and four-cell nuclei were stained with the anti-V5 antibody (DnmtDam, green). The nuclei were stained for histone H3-lysine 9 acetylation (H3K9ac, red) and were counterstained with DAPI (blue). ( D ) Collection of DamID embryo samples and DELP-seq statistics. The table shows the number of cleavage embryos used for DamID/DELP-seq and the number of sequencing reads and mapped reads. The mapping efficiencies are shown in parentheses. ( E ) A genome browser snapshot for DELP-seq results ( Xist locus). The relative enrichment of DnmtDam over Dam is depicted as red bars, while Dam-enriched loci are colored in gray. CGIs (green) and genes (blue) in this region are also displayed. The genomic coordinates are indicated below. ( F ) Genomic distributions of DnmtDam or Dam peaks. The proportion of DnmtDam or Dam peaks over genomic features, including proximal (from TSS/−3 kb to TSS) and distal promoters (from TSS/−10 kb to TSS/3 kb), gene bodies and intergenic regions. TSS and TES refer to transcription start and end sites, respectively. ( G ) Read coverages of DnmtDam (red) and Dam (blue) over genes and CGIs. The genes and CGIs of various lengths were scaled to fit into a fixed width surrounded by TSS/TES or S/E (start/end), respectively. For comprehensive analysis, the analytic regions were extended 5 kb from both the TSS and TES for genes and the S and E for CGIs. ( H ) Transcriptional profiles of genes having DnmtDam or Dam peaks at promoters. Consulting two publicly available transcriptomic datasets (GSE18920 and GSE66582), the expression of genes with an increasing occurrence of DnmtDam or Dam peaks at the promoter regions (TSS/±3 kb) was profiled. The x -axes indicate the number of corresponding peaks at the promoters and the y -axes represent the expression levels of the associated genes. The number of genes with a specified number of peaks is denoted on each box plot. For statistical analysis, one-way ANOVA tests were performed.

    Journal: Nucleic Acids Research

    Article Title: Dnmt1 binds and represses genomic retroelements via DNA methylation in mouse early embryos

    doi: 10.1093/nar/gkaa584

    Figure Lengend Snippet: Identification and characterization of the Dnmt1-binding regions in early mouse embryos by DamID and DELP sequencing. ( A ) Structure of Dnmt1–Dam (DnmtDam) fusion protein. V5 is an epitope for immunostaining. Refer to Supplementary Figure S4 for the regulatory region (R). ( B ) DamID and DELP-seq ( Dpn I fragment enrichment by ligation-mediated PCR sequencing) procedures. Mouse zygotes were injected with the DamID toolkit and were cultured until the four- and eight-cell stages. The embryos were treated with Ponasterone A for 24 h to induce the expression of DnmtDam for the methylation of nearby Dpn I sites (5′-GATC-3′). Genomic DNA from the four/eight-cell embryos was subjected to DELP-seq. ( C ) Nuclear localization of DnmtDam in mouse embryos. Two- and four-cell nuclei were stained with the anti-V5 antibody (DnmtDam, green). The nuclei were stained for histone H3-lysine 9 acetylation (H3K9ac, red) and were counterstained with DAPI (blue). ( D ) Collection of DamID embryo samples and DELP-seq statistics. The table shows the number of cleavage embryos used for DamID/DELP-seq and the number of sequencing reads and mapped reads. The mapping efficiencies are shown in parentheses. ( E ) A genome browser snapshot for DELP-seq results ( Xist locus). The relative enrichment of DnmtDam over Dam is depicted as red bars, while Dam-enriched loci are colored in gray. CGIs (green) and genes (blue) in this region are also displayed. The genomic coordinates are indicated below. ( F ) Genomic distributions of DnmtDam or Dam peaks. The proportion of DnmtDam or Dam peaks over genomic features, including proximal (from TSS/−3 kb to TSS) and distal promoters (from TSS/−10 kb to TSS/3 kb), gene bodies and intergenic regions. TSS and TES refer to transcription start and end sites, respectively. ( G ) Read coverages of DnmtDam (red) and Dam (blue) over genes and CGIs. The genes and CGIs of various lengths were scaled to fit into a fixed width surrounded by TSS/TES or S/E (start/end), respectively. For comprehensive analysis, the analytic regions were extended 5 kb from both the TSS and TES for genes and the S and E for CGIs. ( H ) Transcriptional profiles of genes having DnmtDam or Dam peaks at promoters. Consulting two publicly available transcriptomic datasets (GSE18920 and GSE66582), the expression of genes with an increasing occurrence of DnmtDam or Dam peaks at the promoter regions (TSS/±3 kb) was profiled. The x -axes indicate the number of corresponding peaks at the promoters and the y -axes represent the expression levels of the associated genes. The number of genes with a specified number of peaks is denoted on each box plot. For statistical analysis, one-way ANOVA tests were performed.

    Article Snippet: Genomic DNAs were isolated from the embryos by incubating them in an embryo lysis buffer (10 mM Tris–HCl, pH 8.0, 0.1 M EDTA, 0.5% SDS, 20 mg/ml RNAse A and 0.4 mg/ml proteinase K) for 3 h at 56°C ( ) and serially digested by Dpn I (NEB) and Fat I (NEB).

    Techniques: Binding Assay, Sequencing, Immunostaining, Ligation, Polymerase Chain Reaction, Injection, Cell Culture, Expressing, Methylation, Staining