hpaii  (New England Biolabs)


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    Name:
    HpaII
    Description:
    HpaII 10 000 units
    Catalog Number:
    r0171l
    Price:
    269
    Size:
    10 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs hpaii
    HpaII
    HpaII 10 000 units
    https://www.bioz.com/result/hpaii/product/New England Biolabs
    Average 95 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    hpaii - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain"

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain

    Journal: Molecular Pain

    doi: 10.1186/1744-8069-10-51

    Repression of MOR promoter-driven transcription by CpG methylation. (A, B) Three different luciferase (LUC) constructs (pL450, pLup and pL1.3 k) were mock methylated (control) or in vitro methylated with HapI, HpaII (partial) or SssI (full) methylase and transfected into SH-SY5Y cells. The results are given as luciferase activity normalized against cotransfected pCH110 β-galactosidase activity. The data shown are the means of three independent experiments with at least two different plasmid preparations. The control with none methylase was set at 1 for quantifications. # P
    Figure Legend Snippet: Repression of MOR promoter-driven transcription by CpG methylation. (A, B) Three different luciferase (LUC) constructs (pL450, pLup and pL1.3 k) were mock methylated (control) or in vitro methylated with HapI, HpaII (partial) or SssI (full) methylase and transfected into SH-SY5Y cells. The results are given as luciferase activity normalized against cotransfected pCH110 β-galactosidase activity. The data shown are the means of three independent experiments with at least two different plasmid preparations. The control with none methylase was set at 1 for quantifications. # P

    Techniques Used: CpG Methylation Assay, Luciferase, Construct, Methylation, In Vitro, Transfection, Activity Assay, Plasmid Preparation

    2) Product Images from "CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons"

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

    Journal: Molecular Autism

    doi: 10.1186/s13229-016-0105-9

    CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “ Methods ” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a . The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by
    Figure Legend Snippet: CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “ Methods ” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a . The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by

    Techniques Used: Methylation, Derivative Assay, Mass Spectrometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Methylation Assay

    Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “ Methods ” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3 : Figure S3 for representative images during neuronal differentiation of 37D cells
    Figure Legend Snippet: Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “ Methods ” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3 : Figure S3 for representative images during neuronal differentiation of 37D cells

    Techniques Used: Methylation, Polymerase Chain Reaction

    Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a – c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a , b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols . c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately ( i ) or in a ~1:1 mixture ( ii ) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel ( iii ) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation
    Figure Legend Snippet: Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a – c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a , b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols . c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately ( i ) or in a ~1:1 mixture ( ii ) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel ( iii ) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation

    Techniques Used: Methylation, DNA Methylation Assay, Standard Deviation

    3) Product Images from "Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function"

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.05.021

    Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.
    Figure Legend Snippet: Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Recombinant, Produced, Thin Layer Chromatography, Knock-In, Knock-Out, Staining, Northern Blot, Western Blot, Expressing, Marker, Methylation, Southern Blot, DNA Methylation Assay, Immunoprecipitation, End Labeling, Sequencing, Immunofluorescence

    Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.
    Figure Legend Snippet: Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.

    Techniques Used: Activity Assay, Mutagenesis, Staining, Northern Blot, Southern Blot, Methylation, DNA Methylation Assay, Immunoprecipitation, End Labeling, Labeling, Immunofluorescence

    4) Product Images from "Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation"

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-12148-2

    Methylation sensitive comet assay (MS-CA). The observed comet tails represent double-strand DNA breaks, introduced by the isoschizomeric enzymes MspI and HpaII; in the presence of an electric field, the HpaII- and MspI-digested DNA migrates faster, compared to the significantly slower migration of the undigested DNA. Digestion of the control (Chlamy) and treated (vitamin B 12 and MTX) samples resulted in a tail DNA increase for MTX and decrease for vitamin B 12 , relative to the intact DNA in the comet head of the untreated controls – indicative of decreased DNA methylation levels in MTX-treated animals and increased DNA methylation in vitamin B 12 -treated animals.
    Figure Legend Snippet: Methylation sensitive comet assay (MS-CA). The observed comet tails represent double-strand DNA breaks, introduced by the isoschizomeric enzymes MspI and HpaII; in the presence of an electric field, the HpaII- and MspI-digested DNA migrates faster, compared to the significantly slower migration of the undigested DNA. Digestion of the control (Chlamy) and treated (vitamin B 12 and MTX) samples resulted in a tail DNA increase for MTX and decrease for vitamin B 12 , relative to the intact DNA in the comet head of the untreated controls – indicative of decreased DNA methylation levels in MTX-treated animals and increased DNA methylation in vitamin B 12 -treated animals.

    Techniques Used: Methylation, Single Cell Gel Electrophoresis, Mass Spectrometry, Migration, DNA Methylation Assay

    5) Product Images from "DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation"

    Article Title: DNA methyltransferase 1 and DNA methylation patterning contribute to germinal center B-cell differentiation

    Journal: Blood

    doi: 10.1182/blood-2011-06-357996

    Hypomethylation preferentially affects regulatory regions of the genome in GC B cells. (A) Four genes from the GC B-cell signature were selected for validation by MassArray. The results are represented as heatmaps in which the columns correspond to individual samples, while rows represent individual CpGs with color reflecting methylation value. P values are from moderated t test comparing methylation values from all tested CpGs between GC B cells and NBs. The location of MassArray amplicons (blue) and HELP probesets (red) relative to the transcriptional start site (TSS) of each gene (black) is illustrated below each heatmap. (B) The relative transcript abundance of the same 4 genes was measured by QPCR in 3 additional NB and GC B-cell specimens. The y-axis depicts fold expression difference in GC B-cells versus NBs calculated using ddCT method. All 4 genes are expressed at higher levels in GC B-cells, concordant with their hypomethylation. (C) LUMA assays performed on 3 NB and 2 GC B-cell specimens show a mild increase in the abundance of hypomethylated HpaII sites. The y-axis depicts relative signal of Hpa II vs Msp I signals. (D) Liquid chromatography-mass spectrometry was performed in 3 NB and 2 GC B-cell specimens. The y-axis depicts the percentage of methylcytosine versus total cytosines in each specimen.
    Figure Legend Snippet: Hypomethylation preferentially affects regulatory regions of the genome in GC B cells. (A) Four genes from the GC B-cell signature were selected for validation by MassArray. The results are represented as heatmaps in which the columns correspond to individual samples, while rows represent individual CpGs with color reflecting methylation value. P values are from moderated t test comparing methylation values from all tested CpGs between GC B cells and NBs. The location of MassArray amplicons (blue) and HELP probesets (red) relative to the transcriptional start site (TSS) of each gene (black) is illustrated below each heatmap. (B) The relative transcript abundance of the same 4 genes was measured by QPCR in 3 additional NB and GC B-cell specimens. The y-axis depicts fold expression difference in GC B-cells versus NBs calculated using ddCT method. All 4 genes are expressed at higher levels in GC B-cells, concordant with their hypomethylation. (C) LUMA assays performed on 3 NB and 2 GC B-cell specimens show a mild increase in the abundance of hypomethylated HpaII sites. The y-axis depicts relative signal of Hpa II vs Msp I signals. (D) Liquid chromatography-mass spectrometry was performed in 3 NB and 2 GC B-cell specimens. The y-axis depicts the percentage of methylcytosine versus total cytosines in each specimen.

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Expressing, Liquid Chromatography, Mass Spectrometry

    6) Product Images from "CpG-island promoters drive transcription of human telomeres"

    Article Title: CpG-island promoters drive transcription of human telomeres

    Journal: RNA

    doi: 10.1261/rna.1748309

    Cytosine methylation at 61-29-37 repeats. ( A,B ) Genomic DNA extracted from the indicated cell lines was digested with the methylation-sensitive HpaII restriction enzyme or with its methylation-insensitive isoschizomer MspI. Digested DNA was electrophoresed, blotted, and hybridized with radioactive DNA probes detecting 61-29-37 repeats. ( B ) DNMT1 −/− and DNMT3b − /− are HCT116-derived clonal cell lines knocked-out for DNA methyltransferases 1 and 3b, respectively. Double KO (DKO–) are HCT116-derived clonal cell lines knocked-out for both methyltransferases. Standard molecular weights are shown on the left of each blot in kilobases (kb).
    Figure Legend Snippet: Cytosine methylation at 61-29-37 repeats. ( A,B ) Genomic DNA extracted from the indicated cell lines was digested with the methylation-sensitive HpaII restriction enzyme or with its methylation-insensitive isoschizomer MspI. Digested DNA was electrophoresed, blotted, and hybridized with radioactive DNA probes detecting 61-29-37 repeats. ( B ) DNMT1 −/− and DNMT3b − /− are HCT116-derived clonal cell lines knocked-out for DNA methyltransferases 1 and 3b, respectively. Double KO (DKO–) are HCT116-derived clonal cell lines knocked-out for both methyltransferases. Standard molecular weights are shown on the left of each blot in kilobases (kb).

    Techniques Used: Methylation, Derivative Assay

    7) Product Images from "JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis"

    Article Title: JMJ24 targets CHROMOMETHYLASE3 for proteasomal degradation in Arabidopsis

    Journal: Genes & Development

    doi: 10.1101/gad.274647.115

    JMJ24 regulates CHG methylation and H3K9me2 through CMT3. ( A , B ) Southern blot of MspI- and HpaII-digested genomic DNAs probed with an Athila LTR ( A ) or a 5s rDNA repeat ( B ). See Supplemental Figure S4A for the full scanning of B . ( C , D ) DNA methylation levels on FWA ( C ) and QQS ( D ) determined by sequencing of bisulfite-treated genomic DNAs. ( E – G ) H3K9me2 accumulation on FWA ( E ), QQS ( F ), or SDC ( G ) promoters in different mutant backgrounds. ( H , I ) QQS ( H ) and SDC ( I ) transcripts levels in different mutant backgrounds. ( jmj24;cmt3 ) jmj24 and cmt3 double mutant; ( JMJ24/jmj24 ) jmj24 mutant complemented with wild-type JMJ24; ( JMJ24m/jmj24 ) jmj24 mutant complemented with JMJ24(H244A, C263S).
    Figure Legend Snippet: JMJ24 regulates CHG methylation and H3K9me2 through CMT3. ( A , B ) Southern blot of MspI- and HpaII-digested genomic DNAs probed with an Athila LTR ( A ) or a 5s rDNA repeat ( B ). See Supplemental Figure S4A for the full scanning of B . ( C , D ) DNA methylation levels on FWA ( C ) and QQS ( D ) determined by sequencing of bisulfite-treated genomic DNAs. ( E – G ) H3K9me2 accumulation on FWA ( E ), QQS ( F ), or SDC ( G ) promoters in different mutant backgrounds. ( H , I ) QQS ( H ) and SDC ( I ) transcripts levels in different mutant backgrounds. ( jmj24;cmt3 ) jmj24 and cmt3 double mutant; ( JMJ24/jmj24 ) jmj24 mutant complemented with wild-type JMJ24; ( JMJ24m/jmj24 ) jmj24 mutant complemented with JMJ24(H244A, C263S).

    Techniques Used: Methylation, Southern Blot, DNA Methylation Assay, Sequencing, Mutagenesis

    8) Product Images from "Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer"

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032921

    Wnt7a is methylated in NSCLC cell lines and tissues. A) B2B and HBEC cells were treated with 10 uM NNK for 2 hours and Wnt7a expression was measured by QPCR compared to an untreated control. Error bars are the SEM of triplicate experiments. Percent methylation in B2B and HBEC cells was analyzed using the Methyl Profiler system after 2 hr 10 uM NNK treatment compared to untreated cells. B) Mean percent methylation of the Wnt7a promoter was measured by pyrosequencing in NSCLC tissue and compared to matched normal adjacent lung tissue by paired t-test. Mean percent methylation of B2B, H157, and H1299 cell lines was measured by pyrosequencing. C) A Wnt7a promoter luciferase was modified by SssI, HpaII, and HhaI methylating enzymes and transiently transfected into B2B and HBEC cell lines. Fold-change in luciferase activity was measured compared to unmodified Wnt7a promoter luciferase activity. Error bars are the SE of triplicate experiments.
    Figure Legend Snippet: Wnt7a is methylated in NSCLC cell lines and tissues. A) B2B and HBEC cells were treated with 10 uM NNK for 2 hours and Wnt7a expression was measured by QPCR compared to an untreated control. Error bars are the SEM of triplicate experiments. Percent methylation in B2B and HBEC cells was analyzed using the Methyl Profiler system after 2 hr 10 uM NNK treatment compared to untreated cells. B) Mean percent methylation of the Wnt7a promoter was measured by pyrosequencing in NSCLC tissue and compared to matched normal adjacent lung tissue by paired t-test. Mean percent methylation of B2B, H157, and H1299 cell lines was measured by pyrosequencing. C) A Wnt7a promoter luciferase was modified by SssI, HpaII, and HhaI methylating enzymes and transiently transfected into B2B and HBEC cell lines. Fold-change in luciferase activity was measured compared to unmodified Wnt7a promoter luciferase activity. Error bars are the SE of triplicate experiments.

    Techniques Used: Methylation, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Modification, Transfection, Activity Assay

    9) Product Images from "Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma"

    Article Title: Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI98747

    AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.
    Figure Legend Snippet: AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.

    Techniques Used: Activity Assay, Mass Spectrometry, In Vitro, Incubation, IF-P, Genome Wide, Methylation, HELP Assay

    10) Product Images from "The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition"

    Article Title: The high mobility group A2 protein epigenetically silences the Cdh1 gene during epithelial-to-mesenchymal transition

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku1293

    The Cdh1 promoter is epigenetically silenced in HMGA2-overexpressing NMuMG cells. ( A ) An illustration of the mouse Cdh1 promoter, position −230 to +200 base pairs relative to the transcription start site (+1), containing E-boxes (yellow) and CpG dinucleotides (black lines). The blue underline indicates the proximal region (position −108 to +3) examined in ChIP-qPCR assays in this study. ( B ) HMGA2 binding to proximal region of the mouse Cdh1 promoter was analysed by ChIP assays with non-specific IgG or HA antibody in NM-Mock and NM-Hmga2 cells. Precipitated DNA was analysed by qPCR and data are graphed as explained in the methods. ( C ) ChIP-qPCR assays were performed to examine levels of histone H3 and its lysine modifications (active marks, K4me3 and K9ac; repressive marks, K9me3 and K27me3) on the proximal region of the mouse Cdh1 promoter in NM-Mock and NM-Hmga2 cells. ( D ) HpaII–MspI digestion–methylation assay using primers which span the proximal region of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells. The PCR product was subjected to agarose gel electrophoresis and a band observed after HpaII-digestion indicates that the amplified DNA is methylated (asterisk). ( E ) The DNA methylation status of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells was analysed by bisulphite sequencing of the promoter region shown in panel A, where CpG sites are denoted by circles, and five independent clones of each cell line are shown here. White and black circles represent unmethylated and methylated CpG sites respectively. ( F ) Expression of HMGA2 and CDH1 in human breast cancer cell lines classified as basal A (red), basal B (grey) and luminal (blue) subtypes. Expression values derived from microarray analysis of gene expression are shown in logarithmic (log 2 ) scale.
    Figure Legend Snippet: The Cdh1 promoter is epigenetically silenced in HMGA2-overexpressing NMuMG cells. ( A ) An illustration of the mouse Cdh1 promoter, position −230 to +200 base pairs relative to the transcription start site (+1), containing E-boxes (yellow) and CpG dinucleotides (black lines). The blue underline indicates the proximal region (position −108 to +3) examined in ChIP-qPCR assays in this study. ( B ) HMGA2 binding to proximal region of the mouse Cdh1 promoter was analysed by ChIP assays with non-specific IgG or HA antibody in NM-Mock and NM-Hmga2 cells. Precipitated DNA was analysed by qPCR and data are graphed as explained in the methods. ( C ) ChIP-qPCR assays were performed to examine levels of histone H3 and its lysine modifications (active marks, K4me3 and K9ac; repressive marks, K9me3 and K27me3) on the proximal region of the mouse Cdh1 promoter in NM-Mock and NM-Hmga2 cells. ( D ) HpaII–MspI digestion–methylation assay using primers which span the proximal region of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells. The PCR product was subjected to agarose gel electrophoresis and a band observed after HpaII-digestion indicates that the amplified DNA is methylated (asterisk). ( E ) The DNA methylation status of the Cdh1 promoter in NM-Mock and NM-Hmga2 cells was analysed by bisulphite sequencing of the promoter region shown in panel A, where CpG sites are denoted by circles, and five independent clones of each cell line are shown here. White and black circles represent unmethylated and methylated CpG sites respectively. ( F ) Expression of HMGA2 and CDH1 in human breast cancer cell lines classified as basal A (red), basal B (grey) and luminal (blue) subtypes. Expression values derived from microarray analysis of gene expression are shown in logarithmic (log 2 ) scale.

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Methylation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Amplification, DNA Methylation Assay, Bisulfite Sequencing, Clone Assay, Expressing, Derivative Assay, Microarray

    DNMT3A upregulation by TGFβ in NMuMG cells. ( A ) Immunoblots for E-cadherin and GAPDH in NMuMG cells unstimulated or stimulated with TGFβ1 (5 ng/ml) for 24 or 48 h, in the presence of vehicle or 10 μM 5-aza. ( B ) Phase contrast microscopy of cells as described in (A). Scale bar: 50 μm. ( C ) Luciferase reporter assay of human Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( D ) HpaII–MspI digestion–methylation assay of Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( E ) Dnmt mRNA levels after TGFβ1 treatment of NMuMG cells for the indicated time periods, were normalized to Gapdh mRNA expression. Expression values of the 0-h time point are normalized to 1. ( F ) Dnmt3a mRNA expression in NMuMG cells pre-treated for 30 min with or without TGFβ type I receptor kinase inhibitor SB505124 (10 μM), in the absence or presence of TGFβ1 for 24 h. DMSO was used as a vehicle. qPCR values were normalized to that of Gapdh and the expression values of DMSO-treated, unstimulated cells were normalized to 1. ( G ) Phase contrast images (left panel) and immunoblot analysis (right panel) of DNMT3A and GAPDH protein levels in NMuMG cells transfected with si Control or si Dnmt3a , untreated or treated with TGFβ1 for 24 h. Scale bar : 50 μm. ( H ) E-cadherin, DNMT1, DNMT3A and α-tubulin protein levels in NMuMG cells transfected with si Control , si Dnmt1 or si Dnmt3a , in the absence or presence of TGFβ1 for 48 h.
    Figure Legend Snippet: DNMT3A upregulation by TGFβ in NMuMG cells. ( A ) Immunoblots for E-cadherin and GAPDH in NMuMG cells unstimulated or stimulated with TGFβ1 (5 ng/ml) for 24 or 48 h, in the presence of vehicle or 10 μM 5-aza. ( B ) Phase contrast microscopy of cells as described in (A). Scale bar: 50 μm. ( C ) Luciferase reporter assay of human Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( D ) HpaII–MspI digestion–methylation assay of Cdh1 promoter in NMuMG cells stimulated with TGFβ1 for the indicated time period. ( E ) Dnmt mRNA levels after TGFβ1 treatment of NMuMG cells for the indicated time periods, were normalized to Gapdh mRNA expression. Expression values of the 0-h time point are normalized to 1. ( F ) Dnmt3a mRNA expression in NMuMG cells pre-treated for 30 min with or without TGFβ type I receptor kinase inhibitor SB505124 (10 μM), in the absence or presence of TGFβ1 for 24 h. DMSO was used as a vehicle. qPCR values were normalized to that of Gapdh and the expression values of DMSO-treated, unstimulated cells were normalized to 1. ( G ) Phase contrast images (left panel) and immunoblot analysis (right panel) of DNMT3A and GAPDH protein levels in NMuMG cells transfected with si Control or si Dnmt3a , untreated or treated with TGFβ1 for 24 h. Scale bar : 50 μm. ( H ) E-cadherin, DNMT1, DNMT3A and α-tubulin protein levels in NMuMG cells transfected with si Control , si Dnmt1 or si Dnmt3a , in the absence or presence of TGFβ1 for 48 h.

    Techniques Used: Western Blot, Microscopy, Luciferase, Reporter Assay, Methylation, Expressing, Real-time Polymerase Chain Reaction, Transfection

    11) Product Images from "Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion"

    Article Title: Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2013.01.032

    Evidence that 5hmC Levels Increase at ICRs in Somatic Cells after Fusion with EGCs (A) Detection of 5hmC ( I ) and unmodified cytosine ( II ) in human heterokaryon samples. Genomic DNA was divided and was either treated with T4-β-glucosyltransferase, which binds glucose groups selectively at 5hmC sites (red asterisk) and creates 5hgmC (open hexagon, left), or left untreated (H 2 O). Samples were digested with MspI (which does not digest 5hgmC) or left undigested (H 2 O), and the abundance of locus-specific DNA in each was compared by qPCR. In strategy II , unmodified (C) and modified CpG (5mC and 5hmC) levels were evaluated by HpaII digestion (right); DNA samples were treated with HpaII (which does not cut 5mC and 5hmC), left undigested (H 2 O), or treated with MspI (which cuts both and provides a positive control). The abundance of locus-specific DNA within each of these samples was estimated by qPCR and used to calculate the percentage of HpaII resistance. (B) Levels of 5hmC at OCT4 in hB cells before (0 hr), and 48 hr and 72 hr after fusion with mouse EGCs (black bars) or ESCs (white bars) are shown as the mean and SE of three to five independent experiments. (C) HpaII digestion analysis of OCT4 in hB lymphocytes before (0 hr) and 48 hr and 72 hr after fusion with EGCs (closed circles) or ESCs (open circles) are shown. Red bars mark the position of primer-amplified PCR products derived from the promoter (right) and downstream of the TSS (left), and values represent the mean and SE of three to five independent experiments. ∗∗ , p value
    Figure Legend Snippet: Evidence that 5hmC Levels Increase at ICRs in Somatic Cells after Fusion with EGCs (A) Detection of 5hmC ( I ) and unmodified cytosine ( II ) in human heterokaryon samples. Genomic DNA was divided and was either treated with T4-β-glucosyltransferase, which binds glucose groups selectively at 5hmC sites (red asterisk) and creates 5hgmC (open hexagon, left), or left untreated (H 2 O). Samples were digested with MspI (which does not digest 5hgmC) or left undigested (H 2 O), and the abundance of locus-specific DNA in each was compared by qPCR. In strategy II , unmodified (C) and modified CpG (5mC and 5hmC) levels were evaluated by HpaII digestion (right); DNA samples were treated with HpaII (which does not cut 5mC and 5hmC), left undigested (H 2 O), or treated with MspI (which cuts both and provides a positive control). The abundance of locus-specific DNA within each of these samples was estimated by qPCR and used to calculate the percentage of HpaII resistance. (B) Levels of 5hmC at OCT4 in hB cells before (0 hr), and 48 hr and 72 hr after fusion with mouse EGCs (black bars) or ESCs (white bars) are shown as the mean and SE of three to five independent experiments. (C) HpaII digestion analysis of OCT4 in hB lymphocytes before (0 hr) and 48 hr and 72 hr after fusion with EGCs (closed circles) or ESCs (open circles) are shown. Red bars mark the position of primer-amplified PCR products derived from the promoter (right) and downstream of the TSS (left), and values represent the mean and SE of three to five independent experiments. ∗∗ , p value

    Techniques Used: Real-time Polymerase Chain Reaction, Modification, Positive Control, Amplification, Polymerase Chain Reaction, Derivative Assay

    12) Product Images from "Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation"

    Article Title: Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnj022

    COMPARE-MS overview and rationale. Genomic DNA is digested with AluI with or without the methylation-sensitive restriction enzyme HpaII. After digestion, either the MBD2-MBD captured methylated DNA or all digested DNA are subjected to real-time PCR at a gene-specific locus. We hypothesized that enrichment of methylated DNA by methylation-sensitive restriction enzyme digestion alone or by MBD2-MBD capture of methylated DNA alone may result in false positives associated with incomplete digestion or nonspecific capture, respectively, while the combination of the two approaches (COMPARE-MS) would maintain sensitivity while minimizing false-positive results.
    Figure Legend Snippet: COMPARE-MS overview and rationale. Genomic DNA is digested with AluI with or without the methylation-sensitive restriction enzyme HpaII. After digestion, either the MBD2-MBD captured methylated DNA or all digested DNA are subjected to real-time PCR at a gene-specific locus. We hypothesized that enrichment of methylated DNA by methylation-sensitive restriction enzyme digestion alone or by MBD2-MBD capture of methylated DNA alone may result in false positives associated with incomplete digestion or nonspecific capture, respectively, while the combination of the two approaches (COMPARE-MS) would maintain sensitivity while minimizing false-positive results.

    Techniques Used: Mass Spectrometry, Methylation, Real-time Polymerase Chain Reaction, DNA Methylation Assay

    13) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    Analysis of the methylation status of the repeat region in DNA from carriers of normal (N), premutation (PM), and full mutation (FM) FMR1 . The resultant products were resolved on a 1% agarose gel. The + sign indicates those reactions that were predigested with HpaII in addition to HindIII. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-Kb molecular weight ladders, respectively. A: Water (lane 1), DNA from a normal embryonic stem cell (ESC) line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). B: Analysis of DNA from a fragile X syndrome ESC WCMC37, MC37B, a subclone isolated from this cell line, and from two female (F) FM carriers F23856 and F23690. M, male.
    Figure Legend Snippet: Analysis of the methylation status of the repeat region in DNA from carriers of normal (N), premutation (PM), and full mutation (FM) FMR1 . The resultant products were resolved on a 1% agarose gel. The + sign indicates those reactions that were predigested with HpaII in addition to HindIII. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-Kb molecular weight ladders, respectively. A: Water (lane 1), DNA from a normal embryonic stem cell (ESC) line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). B: Analysis of DNA from a fragile X syndrome ESC WCMC37, MC37B, a subclone isolated from this cell line, and from two female (F) FM carriers F23856 and F23690. M, male.

    Techniques Used: Methylation, Mutagenesis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Isolation

    14) Product Images from "DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells"

    Article Title: DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky947

    DNMT3L deficiency in mESCs results in hypomethylation at specific heterochromatin regions. ( A ) Mouse breeding strategies to generate mESC lines with different Dnmt3l genotypes. ( B and C ) Western blot analysis of the mESC lines (2 independent clones per genotype) for expression of DNMT3L (B) and the pluripotency factors OCT4 and SOX2 (C), with β-ACTIN serving as a loading control. ( D and E ) Southern blot analysis of the mESC lines (2-3 independent clones per genotype) for DNA methylation at the major satellite repeats (D) and the minor satellite repeats (E) after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite repeats and HpaII for minor satellite repeats). DKO, Dnmt3a/3b double KO mESC line.
    Figure Legend Snippet: DNMT3L deficiency in mESCs results in hypomethylation at specific heterochromatin regions. ( A ) Mouse breeding strategies to generate mESC lines with different Dnmt3l genotypes. ( B and C ) Western blot analysis of the mESC lines (2 independent clones per genotype) for expression of DNMT3L (B) and the pluripotency factors OCT4 and SOX2 (C), with β-ACTIN serving as a loading control. ( D and E ) Southern blot analysis of the mESC lines (2-3 independent clones per genotype) for DNA methylation at the major satellite repeats (D) and the minor satellite repeats (E) after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite repeats and HpaII for minor satellite repeats). DKO, Dnmt3a/3b double KO mESC line.

    Techniques Used: Western Blot, Clone Assay, Expressing, Southern Blot, DNA Methylation Assay

    15) Product Images from "Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture"

    Article Title: Myc-induced anchorage of the rDNA IGS region to nucleolar matrix modulates growth-stimulated changes in higher-order rDNA architecture

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gku183

    Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.
    Figure Legend Snippet: Growth-dependent and c-Myc-dependent attachment of rDNA to the nucleolar matrix. (A) Matrix attachment of the rDNA IGS is induced upon growth stimulation of HeLa cells. The left panel represents quantitative real-time PCR showing the relative levels of matrix-attached rDNA throughout the rDNA repeat after digestion with DNase I for starved HeLa cells before (−S) or after (+S) re-feeding with serum-containing medium. The right panel shows the corresponding changes in the level of pre-rRNA measured by quantitative real-time PCR. (B) Growth-induced attachment of the rDNA IGS to nuclear matrix requires c-Myc in rat fibroblasts. Levels of matrix attachment after restriction digestion (see Figure 2B ) are shown for starved TGR-1 cells re-fed with serum in the absence or presence of actinomycin D (0.1 μg/ml) or the Myc inhibitor, 10058-F4 (80 μM). Growing HO1519 ( myc −/− ) cells were treated and analyzed in parallel. Pre-rRNA levels corresponding to the different cells and treatments are shown (right panel). (C) Activation of Myc in serum-starved cells is sufficient to induce rDNA IGS matrix attachment. The relative levels of matrix-attached rDNA after restriction digestion (see Figure 2B ) at the indicated rDNA regions in Rat1MycER cells, which express a Myc-ER fusion protein, before (−4-HT) and after (+4-HT) activation of Myc-ER by addition of 4-hydroxytamoxifen in the absence or presence of c-Myc inhibitor 10058-F4. Pre-rRNA levels corresponding to the different treatments are shown (right panel). The cutting efficiencies of restriction enzymes on samples in (B) and (C) are shown in Supplementary Figure S2D and E. (D) rRNA genes that associate with nucleolar matrix are hypomethylated in the promoter region. A diagram of the rat rDNA promoter region shows the primer set (forward primer, F; reverse primer, R), described in the Materials and Methods section, used to detect the methylation status of the CpG residue at −145 bp from the transcription start site, while the primer set R0 is for normalization between samples (upper panel; see the Materials and Methods section). The quantitative real-time PCR signal for genomic DNA from serum-starved (−Serum) and growing (+ Serum) TGR-1 cells after cleavage with HpaII or MspI, prior to (left panel) or after separation of non-matrix-associated DNA (middle panel) and matrix-associated DNA (right panel). The values in (A–D) are the means and standard deviations of results from three independent experiments.

    Techniques Used: Real-time Polymerase Chain Reaction, Activation Assay, Methylation

    16) Product Images from "Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease"

    Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194036

    Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).
    Figure Legend Snippet: Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

    Techniques Used: Methylation, DNA Methylation Assay, Microarray

    17) Product Images from "How to Isolate a Plant's Hypomethylome in One Shot"

    Article Title: How to Isolate a Plant's Hypomethylome in One Shot

    Journal: BioMed Research International

    doi: 10.1155/2015/570568

    Complementary identification of genomic regions in rice due to restriction site locations. A detailed representation of the mapping results is shown for both enzymes, AciI and HpaII. The identified regions around the displayed gene differ due to the lack of recognition sites for the other enzyme. On the right, an example for overlapping but expanded regions is given.
    Figure Legend Snippet: Complementary identification of genomic regions in rice due to restriction site locations. A detailed representation of the mapping results is shown for both enzymes, AciI and HpaII. The identified regions around the displayed gene differ due to the lack of recognition sites for the other enzyme. On the right, an example for overlapping but expanded regions is given.

    Techniques Used:

    Length distribution of genomic regions identified for AciI, HpaII, and the combined dataset in rice. The length distribution of hypomethylated regions identified with the three datasets up to the maximal length is shown as well as a closer view to the region between 0 and 2.000 bp, where an increase in length is visible for the combined dataset. Additionally, the amount of regions, the average and maximum length, and the average reads per region are given.
    Figure Legend Snippet: Length distribution of genomic regions identified for AciI, HpaII, and the combined dataset in rice. The length distribution of hypomethylated regions identified with the three datasets up to the maximal length is shown as well as a closer view to the region between 0 and 2.000 bp, where an increase in length is visible for the combined dataset. Additionally, the amount of regions, the average and maximum length, and the average reads per region are given.

    Techniques Used:

    Genes and transposable elements identified in the rice genome with the methyl filtration technique. The regions comprised of at least five reads (left), and all regions (middle) show a clear depletion of transposable elements for AciI, Bsh1236I, and HpaII. On the right a representation of genes and transposable elements is given showing potential methylation sites within their gene space. All values are shown in percent based on the annotated 39.954 genes and 15.847 transposable elements.
    Figure Legend Snippet: Genes and transposable elements identified in the rice genome with the methyl filtration technique. The regions comprised of at least five reads (left), and all regions (middle) show a clear depletion of transposable elements for AciI, Bsh1236I, and HpaII. On the right a representation of genes and transposable elements is given showing potential methylation sites within their gene space. All values are shown in percent based on the annotated 39.954 genes and 15.847 transposable elements.

    Techniques Used: Filtration, Methylation

    18) Product Images from "Characterization of Dnmt3b:thymine-DNA glycosylase interaction and stimulation of thymine glycosylase-mediated repair by DNA methyltransferase(s) and RNA"

    Article Title: Characterization of Dnmt3b:thymine-DNA glycosylase interaction and stimulation of thymine glycosylase-mediated repair by DNA methyltransferase(s) and RNA

    Journal: Journal of molecular biology

    doi: 10.1016/j.jmb.2008.02.049

    Evidence for the existence of an endogenous RNA component essential to T·G mismatch repair. (a) T·GC ODN was treated with RNase A (Lanes 1, 2), J1 nuclear extract (Lanes 3, 4) or aliquots of a J1 nuclear extract were treated with DNase-free RNase A for 30 min (Lanes 5, 6), with RNase A blocked with an excess of RNase A inhibitor (Lanes 7, 8), or treated with RNase A for 30 min followed by addition of RNase A inhibitor for 30 min and then supplemented with 10 μg of J1 total RNA (Lanes 9, 10). The untreated or treated aliquots were used in T·GC repair assays. The expected 26 nt repair product is indicated by the arrowhead. Both the untreated extract and the extract treated with blocked RNase A were competent for repair. The extract treated with RNase A alone was not able to repair the ODN. However, when extracts treated with RNase A followed by RNase A inhibitor were supplemented with J1 total RNA (lanes 9, 10), they regained the ability to correctly repair the T·GC ODN. Naked ODN incubated with RNase A for 16 hs at 37°C was not detectably degraded (lanes 1, 2). M, MspI. H, HpaII. (b) J1 nuclear extracts treated sequentially with RNase A and RNase inhibitor were supplemented with J1 total RNA, E. coli tRNA or RNase-treated salmon sperm genomic DNA as indicated. Reactions were split in half and either digested with HpaII or not digested. Addition of tRNA or genomic DNA resulted in non-specific cleavage of the ODN phosphodiester backbone (lanes 6, 8 respectively). M, sizing markers. H, HpaII. Ø, no digestion.
    Figure Legend Snippet: Evidence for the existence of an endogenous RNA component essential to T·G mismatch repair. (a) T·GC ODN was treated with RNase A (Lanes 1, 2), J1 nuclear extract (Lanes 3, 4) or aliquots of a J1 nuclear extract were treated with DNase-free RNase A for 30 min (Lanes 5, 6), with RNase A blocked with an excess of RNase A inhibitor (Lanes 7, 8), or treated with RNase A for 30 min followed by addition of RNase A inhibitor for 30 min and then supplemented with 10 μg of J1 total RNA (Lanes 9, 10). The untreated or treated aliquots were used in T·GC repair assays. The expected 26 nt repair product is indicated by the arrowhead. Both the untreated extract and the extract treated with blocked RNase A were competent for repair. The extract treated with RNase A alone was not able to repair the ODN. However, when extracts treated with RNase A followed by RNase A inhibitor were supplemented with J1 total RNA (lanes 9, 10), they regained the ability to correctly repair the T·GC ODN. Naked ODN incubated with RNase A for 16 hs at 37°C was not detectably degraded (lanes 1, 2). M, MspI. H, HpaII. (b) J1 nuclear extracts treated sequentially with RNase A and RNase inhibitor were supplemented with J1 total RNA, E. coli tRNA or RNase-treated salmon sperm genomic DNA as indicated. Reactions were split in half and either digested with HpaII or not digested. Addition of tRNA or genomic DNA resulted in non-specific cleavage of the ODN phosphodiester backbone (lanes 6, 8 respectively). M, sizing markers. H, HpaII. Ø, no digestion.

    Techniques Used: Incubation

    DNA methyltransferases stimulate base excision repair of T·G mismatches. A: Design and testing of oligodeoxyribonucleotides (ODNs) used in a mismatch repair assay. (a) Sequence of the double-stranded ODN. The sense strand contains either a normal CCGG site or a mutated CTGG site. The antisense strand contains either C or 5mC within an MspI/HpaII recognition site. Positions of variable nucleotides y and x are shown in bold. The MspI/HpaII recognition sequence is indicated with a bar. The asterisk indicates the position of the 32 P label. (b) Predicted sensitivity to MspI or HpaII digestion of the control C·GC and C·G m C ODNs compared to the mismatched T·GC and T·G m C ODNs. (c) MspI digestion of the control ODNs results in the expected 26 nt long radiolabeled fragment from both controls, while a 26 nt long fragment is only observed after HpaII digestion of the unmethylated control. In contrast, T·G mismatch ODNs are refractory to digestion by either enzyme. M, MspI. H, HpaII. B: De novo DNA methyltrasnferases stimulate repair of T·G mismatches. (a) Evaluation of Dnmt and Tdg protein levels in J1 and Dnmt null ES cells. Nuclear extracts prepared from each cell line were immunoblotted using antibodies specific for Dnmt1, Dnmt3a, Dnmt3b and Tdg to verify the expression of Tdg and the absence of specific Dnmt expression in each cell line relative to wild-type J1. Immunoblot for Pcna served as a loading control. (b) Predicted T·GC ODN sensitivity to digestion by MspI or HpaII depending on extent of repair/DNA methylation. (c) Typical results from one of three independent experiments in which the T·GC ODN was incubated with wild-type J1 or Dnmt null ES cell nuclear extracts followed by digestion with either MspI or HpaII. The expected 26 nt repair product produced as a result of digestion is indicated by the arrowhead. In the absence of either Dnmt3b and/or Dnmt3a there is a reduction in repair efficiency when compared to wild-type nuclear extracts (refer to text for discussion of Dnmt1 null extracts). As a control (last lane), T·GC ODN was used in a repair assay but was not digested. Lack of a 26 nt fragment indicates the ODN is being repaired and not merely nicked 5′ to the mismatched thymine. (d) Quantitation of differences in the extent of mismatch repair by extracts from normal ES cells (J1) and ES cells nullizygous for the indicated Dnmts. Error bars represent the mean (± SD) of three independent experiments. [*] P
    Figure Legend Snippet: DNA methyltransferases stimulate base excision repair of T·G mismatches. A: Design and testing of oligodeoxyribonucleotides (ODNs) used in a mismatch repair assay. (a) Sequence of the double-stranded ODN. The sense strand contains either a normal CCGG site or a mutated CTGG site. The antisense strand contains either C or 5mC within an MspI/HpaII recognition site. Positions of variable nucleotides y and x are shown in bold. The MspI/HpaII recognition sequence is indicated with a bar. The asterisk indicates the position of the 32 P label. (b) Predicted sensitivity to MspI or HpaII digestion of the control C·GC and C·G m C ODNs compared to the mismatched T·GC and T·G m C ODNs. (c) MspI digestion of the control ODNs results in the expected 26 nt long radiolabeled fragment from both controls, while a 26 nt long fragment is only observed after HpaII digestion of the unmethylated control. In contrast, T·G mismatch ODNs are refractory to digestion by either enzyme. M, MspI. H, HpaII. B: De novo DNA methyltrasnferases stimulate repair of T·G mismatches. (a) Evaluation of Dnmt and Tdg protein levels in J1 and Dnmt null ES cells. Nuclear extracts prepared from each cell line were immunoblotted using antibodies specific for Dnmt1, Dnmt3a, Dnmt3b and Tdg to verify the expression of Tdg and the absence of specific Dnmt expression in each cell line relative to wild-type J1. Immunoblot for Pcna served as a loading control. (b) Predicted T·GC ODN sensitivity to digestion by MspI or HpaII depending on extent of repair/DNA methylation. (c) Typical results from one of three independent experiments in which the T·GC ODN was incubated with wild-type J1 or Dnmt null ES cell nuclear extracts followed by digestion with either MspI or HpaII. The expected 26 nt repair product produced as a result of digestion is indicated by the arrowhead. In the absence of either Dnmt3b and/or Dnmt3a there is a reduction in repair efficiency when compared to wild-type nuclear extracts (refer to text for discussion of Dnmt1 null extracts). As a control (last lane), T·GC ODN was used in a repair assay but was not digested. Lack of a 26 nt fragment indicates the ODN is being repaired and not merely nicked 5′ to the mismatched thymine. (d) Quantitation of differences in the extent of mismatch repair by extracts from normal ES cells (J1) and ES cells nullizygous for the indicated Dnmts. Error bars represent the mean (± SD) of three independent experiments. [*] P

    Techniques Used: Sequencing, Expressing, DNA Methylation Assay, Incubation, Produced, Quantitation Assay

    Treatment of nuclear extracts with RNase A leads to random cleavage of the T·G m C ODN. (a) Diagram depicting an experimental protocol devised to examine methylation of repaired T·G m C ODN in normal and Dnmt null ES cell lines. Following repair of the T·G m C ODN using RNase A-treated ES cell nuclear extracts, the ODN was purified and heat denatured. It was then annealed to a 5-fold excess of cold antisense CG strand. This will generate an unmethylated or hemimethylated ODN depending on the methylation status of the repaired cytosine. MspI will digest either ODN but HpaII digestion will be inhibited if the repaired cytosine is methylated. (b) Results of the experiment diagrammed in (a). The T·G m C ODN appears to be degraded by each RNase-treated nuclear extract in a consistent pattern irrespective of methyltransferase expression. As a control, the C·GC ODN was incubated with RNase-treated J1 nuclear extract. The expected 26 nt digestion product is indicated by an arrow.
    Figure Legend Snippet: Treatment of nuclear extracts with RNase A leads to random cleavage of the T·G m C ODN. (a) Diagram depicting an experimental protocol devised to examine methylation of repaired T·G m C ODN in normal and Dnmt null ES cell lines. Following repair of the T·G m C ODN using RNase A-treated ES cell nuclear extracts, the ODN was purified and heat denatured. It was then annealed to a 5-fold excess of cold antisense CG strand. This will generate an unmethylated or hemimethylated ODN depending on the methylation status of the repaired cytosine. MspI will digest either ODN but HpaII digestion will be inhibited if the repaired cytosine is methylated. (b) Results of the experiment diagrammed in (a). The T·G m C ODN appears to be degraded by each RNase-treated nuclear extract in a consistent pattern irrespective of methyltransferase expression. As a control, the C·GC ODN was incubated with RNase-treated J1 nuclear extract. The expected 26 nt digestion product is indicated by an arrow.

    Techniques Used: Methylation, Purification, Expressing, Incubation

    19) Product Images from "Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia"

    Article Title: Microsphere-Based Multiplex Analysis of DNA Methylation in Acute Myeloid Leukemia

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2013.10.010

    The MELP assay accurately reflects HELP-derived data. A: Comparison of HELP-derived HpaII/MspI ratios ( x axis) to MELP-derived ratios ( y axis) at the 18 loci used in the methylation classifier for AML ( r = 0.63 to 0.92, P
    Figure Legend Snippet: The MELP assay accurately reflects HELP-derived data. A: Comparison of HELP-derived HpaII/MspI ratios ( x axis) to MELP-derived ratios ( y axis) at the 18 loci used in the methylation classifier for AML ( r = 0.63 to 0.92, P

    Techniques Used: Derivative Assay, Methylation

    A: Schematic of the HELP assay. Genomic DNA is digested with either MspI (methylation insensitive) or HpaII (methylation sensitive). The resulting fragments are ligated to linkers and PCR amplified with linker-specific primers. Amplicons are fluorescently
    Figure Legend Snippet: A: Schematic of the HELP assay. Genomic DNA is digested with either MspI (methylation insensitive) or HpaII (methylation sensitive). The resulting fragments are ligated to linkers and PCR amplified with linker-specific primers. Amplicons are fluorescently

    Techniques Used: HELP Assay, Methylation, Polymerase Chain Reaction, Amplification

    20) Product Images from "An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation"

    Article Title: An Integrative Genomic and Epigenomic Approach for the Study of Transcriptional Regulation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0001882

    Promoter DNA methylation shows genome-wide inverse correlation with gene expression and H3K9 acetylation: Panel A: Smoothed histogram of gene-by-gene correlations between log(HpaII/MspI) values and gene expression, showing a positive correlation between the two measures for the majority of genes, which translates into a negative biological correlation ( i.e. higher promoter methylation correlates with lower gene expression). Panel B: Smoothed histogram of gene-by-gene correlations between log(HpaII/MspI) values and H3K9 acetylation, showing a positive correlation between the two measures for many of the genes, which translates into a negative biological correlation ( i.e. higher promoter methylation correlates with lower promoter H3K9 acetylation). Panel C: Graphical representation of the data from all three platforms for one of the cases (AML.2) as custom tracks in the UCSC genome browser [40] . Four representative genes are shown here to illustrate the correlation between the three platforms. H3K9 acetylation data (in blue) is represented as the ratio of the signal between the H3K9 acetyl channel and the input channel; DNA methylation (in red) is represented as log(HpaII/MspI), so that a negative deflection corresponds to a methylated HpaII fragment while a positive one corresponds to a hypomethylated fragment; finally, gene expression data (in green) is represented as median-centered log 2 of RMA-normalized intensities.
    Figure Legend Snippet: Promoter DNA methylation shows genome-wide inverse correlation with gene expression and H3K9 acetylation: Panel A: Smoothed histogram of gene-by-gene correlations between log(HpaII/MspI) values and gene expression, showing a positive correlation between the two measures for the majority of genes, which translates into a negative biological correlation ( i.e. higher promoter methylation correlates with lower gene expression). Panel B: Smoothed histogram of gene-by-gene correlations between log(HpaII/MspI) values and H3K9 acetylation, showing a positive correlation between the two measures for many of the genes, which translates into a negative biological correlation ( i.e. higher promoter methylation correlates with lower promoter H3K9 acetylation). Panel C: Graphical representation of the data from all three platforms for one of the cases (AML.2) as custom tracks in the UCSC genome browser [40] . Four representative genes are shown here to illustrate the correlation between the three platforms. H3K9 acetylation data (in blue) is represented as the ratio of the signal between the H3K9 acetyl channel and the input channel; DNA methylation (in red) is represented as log(HpaII/MspI), so that a negative deflection corresponds to a methylated HpaII fragment while a positive one corresponds to a hypomethylated fragment; finally, gene expression data (in green) is represented as median-centered log 2 of RMA-normalized intensities.

    Techniques Used: DNA Methylation Assay, Genome Wide, Expressing, Methylation

    21) Product Images from "In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays"

    Article Title: In Silico Enhanced Restriction Enzyme Based Methylation Analysis of the Human Glioblastoma Genome Using Agilent 244K CpG Island Microarrays

    Journal: Frontiers in Neuroscience

    doi: 10.3389/neuro.15.005.2009

    Schematic diagram of in silico annotated genomic DNA and MSRE method . (A) Using in silico simulation, genomic DNA can be separated into MSRE sensitive and insensitive BfaI fragments (flanked by gray boxes) based on the presence or absence of internal HpaII/MspI sites (CCGG) (blue squares). Microarray probes binding to sensitive fragments are represented with green rectangles. Probes binding to insensitive fragments are represented by blue rectangles. Sensitive fragments can have multiple CCGG sites that can be fully methylated, partially methylated, or completely unmethylated in tandem. Methylated CCGG sites (CC m GG) are indicated with a red dot placed above the blue square. (B) Genomic DNA is fragmented with BfaI, ligated to H12/H24 linkers, digested with HpaII and MspI in parallel, amplified, differentially labeled and co-hybridized to Agilent CpG island arrays. Uncleaved fragments will have high intensities compared to cleaved fragments since only uncleaved fragments are amplified. Fully methylated fragments are cut by MspI but not HpaII, resulting in amplification of HpaII digested fragments only with resultant M -value [log 2 (HpaII/MspI)] > 0. Completely unmethylated or partially methylated (not all CCGGs methylated in tandem) fragments are cut by both MspI and HpaII. With the absence of amplification, these fragments resulting in low signal intensities in both channels (HpaII and MspI ∼ 0) with M = 0. Insensitive fragments should not be cut by either enzyme, resulting in M = 0.
    Figure Legend Snippet: Schematic diagram of in silico annotated genomic DNA and MSRE method . (A) Using in silico simulation, genomic DNA can be separated into MSRE sensitive and insensitive BfaI fragments (flanked by gray boxes) based on the presence or absence of internal HpaII/MspI sites (CCGG) (blue squares). Microarray probes binding to sensitive fragments are represented with green rectangles. Probes binding to insensitive fragments are represented by blue rectangles. Sensitive fragments can have multiple CCGG sites that can be fully methylated, partially methylated, or completely unmethylated in tandem. Methylated CCGG sites (CC m GG) are indicated with a red dot placed above the blue square. (B) Genomic DNA is fragmented with BfaI, ligated to H12/H24 linkers, digested with HpaII and MspI in parallel, amplified, differentially labeled and co-hybridized to Agilent CpG island arrays. Uncleaved fragments will have high intensities compared to cleaved fragments since only uncleaved fragments are amplified. Fully methylated fragments are cut by MspI but not HpaII, resulting in amplification of HpaII digested fragments only with resultant M -value [log 2 (HpaII/MspI)] > 0. Completely unmethylated or partially methylated (not all CCGGs methylated in tandem) fragments are cut by both MspI and HpaII. With the absence of amplification, these fragments resulting in low signal intensities in both channels (HpaII and MspI ∼ 0) with M = 0. Insensitive fragments should not be cut by either enzyme, resulting in M = 0.

    Techniques Used: In Silico, Microarray, Binding Assay, Methylation, Amplification, Labeling

    22) Product Images from "Early Life Stress Inhibits Expression of Ribosomal RNA in the Developing Hippocampus"

    Article Title: Early Life Stress Inhibits Expression of Ribosomal RNA in the Developing Hippocampus

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0115283

    PND14 pups exposed to BDS show increased methylation rate at CG_6 in a manner that is highly correlated with a decrease in rRNA levels. A (top) Nucleotide sequence of primer rRNA-2F (arrow) is shown with HpaII restriction site at CG_6 is highlighted in grey, (bottom), schematic diagram of Q-PCR primers used to assess DNA methylation at CG_6. Note that no product will be generated if CG_6 is not methylated and the DNA is digested with HpaII. Exposure to BDS decreases rRNA levels (B) while increasing DNA methylation at CG_6 in 14-day old pups (C). D. Levels of methylation at CG_6 are inversely correlated with rRNA levels in the developing hippocampus. Unpaired Student t tests (B, C), Pearson correlation (D). Error bars represent mean ± SEM, *p
    Figure Legend Snippet: PND14 pups exposed to BDS show increased methylation rate at CG_6 in a manner that is highly correlated with a decrease in rRNA levels. A (top) Nucleotide sequence of primer rRNA-2F (arrow) is shown with HpaII restriction site at CG_6 is highlighted in grey, (bottom), schematic diagram of Q-PCR primers used to assess DNA methylation at CG_6. Note that no product will be generated if CG_6 is not methylated and the DNA is digested with HpaII. Exposure to BDS decreases rRNA levels (B) while increasing DNA methylation at CG_6 in 14-day old pups (C). D. Levels of methylation at CG_6 are inversely correlated with rRNA levels in the developing hippocampus. Unpaired Student t tests (B, C), Pearson correlation (D). Error bars represent mean ± SEM, *p

    Techniques Used: Methylation, Sequencing, Polymerase Chain Reaction, DNA Methylation Assay, Generated

    23) Product Images from "Biochemical reconstitution of TET1–TDG–BER-dependent active DNA demethylation reveals a highly coordinated mechanism"

    Article Title: Biochemical reconstitution of TET1–TDG–BER-dependent active DNA demethylation reveals a highly coordinated mechanism

    Journal: Nature Communications

    doi: 10.1038/ncomms10806

    Processing of differentially modified CpGs by TET1 CD –TDG or TDG. ( a ) Base release from fully methylated CpGs by His6–TET1 CD –His6–TDG. His6–TET1 CD –His6–TDG (50 nM) was incubated with labelled 60mer substrates (25 nM) containing a single 5mC modification on the fluorescent labelled top (5′-Texas Red, T) or bottom strand (5′-fluorescein, F) or a fully methylated CpG with labels on both strands. Product formation was monitored and quantified by denaturing gel electrophoresis and fluorescent scanning (Texas Red, R-channel and fluorescein, F-channel); positions of the 60mer substrate DNA and the resulting base incision products of both strands are indicated. *Unlabelled DNA strand. ( b ) Release of 5caC from differentially modified CpGs by His6–TDG. 60mer DNA substrates (25 nM) containing 5caC opposite C, 5mC or 5hmC in a CpG dinucleotide or in single-stranded (ss) DNA were incubated with His6–TDG (25 nM) and analysed by denaturing gel electrophoresis. Shown are mean percentages of product formation with s.d. ( n =3). ( c ) 5caC release from a symmetrically modified CpG dinucleotide by His6–TDG. A substrate (25 nM) containing 5caC on both strands within a CpG dinucleotide and labels of both strands was incubated with His6–TDG (25 nM) for indicated time, analysed by denaturing gel electrophoresis and visualized by fluorescent scanning of both labels. Shown are mean percentages of product formation with s.d. ( n =3). ( d ) TDG and APE1 only generate DNA DSBs at symmetrically modified CpGs. Base release assay using TDG and APE1 on a labelled 59-bp substrate containing either a single 5caC or a symmetrically 5caC-modified base pair within a HpaII recognition site (CCGG). Reactions were analysed by native PAGE. Substrate DNA and product fragment are indicated. ( e ) Full reconstitution of TDG–BER on a symmetrically modified 5caC substrate. A labelled 59-bp substrate (25 nM) containing a symmetrically 5caC-modified base pair within a HpaII recognition site (CCGG) was incubated with TDG (40 nM) and BER factors (200 nM APE1, 40 nM POLβ and 40 nM XRCC1-LIG3). Recovered DNA was digested with HpaII endonuclease and analysed by native PAGE. Substrate DNA and product fragments are indicated; ssDNA, free single-stranded DNA.
    Figure Legend Snippet: Processing of differentially modified CpGs by TET1 CD –TDG or TDG. ( a ) Base release from fully methylated CpGs by His6–TET1 CD –His6–TDG. His6–TET1 CD –His6–TDG (50 nM) was incubated with labelled 60mer substrates (25 nM) containing a single 5mC modification on the fluorescent labelled top (5′-Texas Red, T) or bottom strand (5′-fluorescein, F) or a fully methylated CpG with labels on both strands. Product formation was monitored and quantified by denaturing gel electrophoresis and fluorescent scanning (Texas Red, R-channel and fluorescein, F-channel); positions of the 60mer substrate DNA and the resulting base incision products of both strands are indicated. *Unlabelled DNA strand. ( b ) Release of 5caC from differentially modified CpGs by His6–TDG. 60mer DNA substrates (25 nM) containing 5caC opposite C, 5mC or 5hmC in a CpG dinucleotide or in single-stranded (ss) DNA were incubated with His6–TDG (25 nM) and analysed by denaturing gel electrophoresis. Shown are mean percentages of product formation with s.d. ( n =3). ( c ) 5caC release from a symmetrically modified CpG dinucleotide by His6–TDG. A substrate (25 nM) containing 5caC on both strands within a CpG dinucleotide and labels of both strands was incubated with His6–TDG (25 nM) for indicated time, analysed by denaturing gel electrophoresis and visualized by fluorescent scanning of both labels. Shown are mean percentages of product formation with s.d. ( n =3). ( d ) TDG and APE1 only generate DNA DSBs at symmetrically modified CpGs. Base release assay using TDG and APE1 on a labelled 59-bp substrate containing either a single 5caC or a symmetrically 5caC-modified base pair within a HpaII recognition site (CCGG). Reactions were analysed by native PAGE. Substrate DNA and product fragment are indicated. ( e ) Full reconstitution of TDG–BER on a symmetrically modified 5caC substrate. A labelled 59-bp substrate (25 nM) containing a symmetrically 5caC-modified base pair within a HpaII recognition site (CCGG) was incubated with TDG (40 nM) and BER factors (200 nM APE1, 40 nM POLβ and 40 nM XRCC1-LIG3). Recovered DNA was digested with HpaII endonuclease and analysed by native PAGE. Substrate DNA and product fragments are indicated; ssDNA, free single-stranded DNA.

    Techniques Used: Modification, Methylation, Incubation, Nucleic Acid Electrophoresis, Release Assay, Clear Native PAGE

    Full reconstitution of TET–TDG–BER-mediated DNA demethylation. ( a ) Intermediate steps of the oxidative DNA demethylation reaction were reconstituted and visualized by denaturing gel electrophoresis. Labelled 60mer substrate DNA containing one G·5mC base pair was incubated sequentially with TET–TDG–BER enzymes at concentrations indicated. Reaction products were separated by denaturing gel electrophoresis and visualized by fluorescent scanning; sizes of the 60mer substrate DNA and reaction products are indicated. ( b ) Complete DNA demethylation by the reconstituted TET–TDG–BER system analysed by the generation of a HpaII-sensitive restriction site. Reconstituted DNA demethylation was done with a 5′-labelled 59-bp substrate containing one G·5mC base pair within a HpaII recognition site (CCGG). Recovered DNA was digested with methylation-sensitive HpaII endonuclease and analysed by native PAGE; positions of the 59-bp substrate DNA and product fragment are indicated.
    Figure Legend Snippet: Full reconstitution of TET–TDG–BER-mediated DNA demethylation. ( a ) Intermediate steps of the oxidative DNA demethylation reaction were reconstituted and visualized by denaturing gel electrophoresis. Labelled 60mer substrate DNA containing one G·5mC base pair was incubated sequentially with TET–TDG–BER enzymes at concentrations indicated. Reaction products were separated by denaturing gel electrophoresis and visualized by fluorescent scanning; sizes of the 60mer substrate DNA and reaction products are indicated. ( b ) Complete DNA demethylation by the reconstituted TET–TDG–BER system analysed by the generation of a HpaII-sensitive restriction site. Reconstituted DNA demethylation was done with a 5′-labelled 59-bp substrate containing one G·5mC base pair within a HpaII recognition site (CCGG). Recovered DNA was digested with methylation-sensitive HpaII endonuclease and analysed by native PAGE; positions of the 59-bp substrate DNA and product fragment are indicated.

    Techniques Used: Nucleic Acid Electrophoresis, Incubation, Methylation, Clear Native PAGE

    DNA demethylation blocks G·T repair and can induce mutations. ( a ) Enzymatic activity of TDG on G·5caC- and G·T-containing substrates. Release of 5caC and T by His6–TDG (25 nM) was monitored over time on 5′-labelled 60-bp substrates (25 nM) containing either a G·5caC or G·T base pair. Reactions were stopped at indicated time, separated by denaturing gel electrophoresis, visualized with fluorescent scanning and quantified. Shown are mean percentages of product formation with s.d. ( n =3) ( b ) Base release from a substrate containing a G·5caC next to a G·T mismatch. Substrate preference of TDG (25 nM) was evaluated on a 59-bp DNA fragment (25 nM) containing 5caC on the labelled top strand (5′-Texas Red) and T on the labelled bottom strand (5′-fluorescein) within the same CpG context as illustrated. Reactions were stopped after indicated time, separated by denaturing gel electrophoresis, and both strands visualized by fluorescent scanning and quantified. Shown are mean percentages of product formation with s.d. ( n =3). ( c ) Full reconstitution of TDG–BER on a G·5caC/G·T-containing substrate. A labelled 59-bp substrate containing a G·5caC next to a G·T mismatch was incubated with His6–TDG and BER factors. Correct repair of the 5caC and the introduction of an A opposite of T was monitored by MscI digestion and analysed by native PAGE and fluorescent scanning. Unmodified (CG/CG) substrate DNA digested with HpaII was used as size marker; positions of the substrate DNA and product fragments are indicated. ssDNA, free single-stranded DNA. ( d ) Mechanistic model of TET–TDG–BER-mediated DNA demethylation. In the presence of all the necessary factors, DNA demethylation at fully methylated CpGs occurs in a coordinated and sequential manner to correctly re-establish the unmodified state (regular BER). Lack of coordination, for example, in the absence of downstream BER factors, repair-mediated DNA demethylation can lead to the induction of DNA DSBs (incomplete BER). Coincident oxidation and hydrolytic deamination at fully methylated CpG sites can lead to increased C to T transitions caused by the sequential repair mechanism (coincident deamination).
    Figure Legend Snippet: DNA demethylation blocks G·T repair and can induce mutations. ( a ) Enzymatic activity of TDG on G·5caC- and G·T-containing substrates. Release of 5caC and T by His6–TDG (25 nM) was monitored over time on 5′-labelled 60-bp substrates (25 nM) containing either a G·5caC or G·T base pair. Reactions were stopped at indicated time, separated by denaturing gel electrophoresis, visualized with fluorescent scanning and quantified. Shown are mean percentages of product formation with s.d. ( n =3) ( b ) Base release from a substrate containing a G·5caC next to a G·T mismatch. Substrate preference of TDG (25 nM) was evaluated on a 59-bp DNA fragment (25 nM) containing 5caC on the labelled top strand (5′-Texas Red) and T on the labelled bottom strand (5′-fluorescein) within the same CpG context as illustrated. Reactions were stopped after indicated time, separated by denaturing gel electrophoresis, and both strands visualized by fluorescent scanning and quantified. Shown are mean percentages of product formation with s.d. ( n =3). ( c ) Full reconstitution of TDG–BER on a G·5caC/G·T-containing substrate. A labelled 59-bp substrate containing a G·5caC next to a G·T mismatch was incubated with His6–TDG and BER factors. Correct repair of the 5caC and the introduction of an A opposite of T was monitored by MscI digestion and analysed by native PAGE and fluorescent scanning. Unmodified (CG/CG) substrate DNA digested with HpaII was used as size marker; positions of the substrate DNA and product fragments are indicated. ssDNA, free single-stranded DNA. ( d ) Mechanistic model of TET–TDG–BER-mediated DNA demethylation. In the presence of all the necessary factors, DNA demethylation at fully methylated CpGs occurs in a coordinated and sequential manner to correctly re-establish the unmodified state (regular BER). Lack of coordination, for example, in the absence of downstream BER factors, repair-mediated DNA demethylation can lead to the induction of DNA DSBs (incomplete BER). Coincident oxidation and hydrolytic deamination at fully methylated CpG sites can lead to increased C to T transitions caused by the sequential repair mechanism (coincident deamination).

    Techniques Used: Activity Assay, Nucleic Acid Electrophoresis, Incubation, Clear Native PAGE, Marker, Methylation

    24) Product Images from "Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression "

    Article Title: Host Cell Detection of Noncoding Stuffer DNA Contained in Helper-Dependent Adenovirus Vectors Leads to Epigenetic Repression of Transgene Expression

    Journal: Journal of Virology

    doi: 10.1128/JVI.00796-09

    hdAd-prok DNA is unmethylated. (A) hdAd-prok plasmid DNA or DNA from purified hdAd-prok virions was digested with MspI (M) or HpaII (H). The digested DNA was resolved on an agarose gel and visualized by ethidium bromide staining. (B) A549 cells were infected
    Figure Legend Snippet: hdAd-prok DNA is unmethylated. (A) hdAd-prok plasmid DNA or DNA from purified hdAd-prok virions was digested with MspI (M) or HpaII (H). The digested DNA was resolved on an agarose gel and visualized by ethidium bromide staining. (B) A549 cells were infected

    Techniques Used: Plasmid Preparation, Purification, Agarose Gel Electrophoresis, Staining, Infection

    25) Product Images from "Oligonucleotide treatment causes flax β-glucanase up-regulation via changes in gene-body methylation"

    Article Title: Oligonucleotide treatment causes flax β-glucanase up-regulation via changes in gene-body methylation

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-014-0261-z

    Methylation of crucial sites of β-1,3-glucanase gene. The analysis of the methylation of two sites in exons and one site in the intron of flax treated with OLIGOs (B1, B2, B3 and B4) at 24 h and 48 h after exposure to OLIGOs in comparison with control, non-treated flax (C) and in EMO-βGlu flax (EB) in comparison with control flax from field (Cf) was determined by digesting genomic DNA with restriction enzymes HpaII-MspI with subsequent semi-quantitative PCR reaction, the separation of PCR product on agarose gel and the quantification of the bands by densitometry. Data represent the mean ± standard deviations from three independent experiments. The significance of the differences between the means was determined using Student’s t test (*P
    Figure Legend Snippet: Methylation of crucial sites of β-1,3-glucanase gene. The analysis of the methylation of two sites in exons and one site in the intron of flax treated with OLIGOs (B1, B2, B3 and B4) at 24 h and 48 h after exposure to OLIGOs in comparison with control, non-treated flax (C) and in EMO-βGlu flax (EB) in comparison with control flax from field (Cf) was determined by digesting genomic DNA with restriction enzymes HpaII-MspI with subsequent semi-quantitative PCR reaction, the separation of PCR product on agarose gel and the quantification of the bands by densitometry. Data represent the mean ± standard deviations from three independent experiments. The significance of the differences between the means was determined using Student’s t test (*P

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    26) Product Images from "Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease"

    Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194036

    Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).
    Figure Legend Snippet: Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

    Techniques Used: Methylation, DNA Methylation Assay, Microarray

    27) Product Images from "Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii"

    Article Title: Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030077

    DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4 (A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1 A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1 D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.
    Figure Legend Snippet: DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4 (A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1 A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1 D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.

    Techniques Used: Sequencing, Isolation, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Southern Blot, Marker, Construct, Transformation Assay

    28) Product Images from "Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells"

    Article Title: Promoter targeted small RNAs induce long-term transcriptional gene silencing in human cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp127

    Role of DNA methylation in long-term transcriptional silencing. ( A ) Increased DNA methylation at the UbC promoter was observed several days post Tet induction of UBC167 shRNA. Genomic DNA was collected and pooled from duplicate samples of 293Trex-UBC167 cells treated without Tet (Day 0) and after 1, 2, 3 or 10 days of Tet treatment. Proportion of densely, intermediately and unmethylated DNA was calculated using method described ( 31 ). Methyl-sensitive restriction enzymes: HpaII and HhaI, methyl-dependent restriction enzyme: McrBC. qPCR was performed using promoter-specific primers. ( B ) Silencing of UbC is observed after Tet treatment. 293Trex-UBC167 cells were treated with Tet for 10 days. Cellular mRNA was isolated at various time points and UbC expression relative to GAPDH determined. The averages with the standard errors of the means are shown as determined by qRT-PCR along with P -values for paired T -tests from each treatment relative to the control. ( C ) DNA from H3K27me3 ChIP ( Figureure S3 ) was treated with bisulfite and analyzed by methylation-specific PCR using primers specific for methylated or unmethylated DNA species overlapping the UBC167 target site.
    Figure Legend Snippet: Role of DNA methylation in long-term transcriptional silencing. ( A ) Increased DNA methylation at the UbC promoter was observed several days post Tet induction of UBC167 shRNA. Genomic DNA was collected and pooled from duplicate samples of 293Trex-UBC167 cells treated without Tet (Day 0) and after 1, 2, 3 or 10 days of Tet treatment. Proportion of densely, intermediately and unmethylated DNA was calculated using method described ( 31 ). Methyl-sensitive restriction enzymes: HpaII and HhaI, methyl-dependent restriction enzyme: McrBC. qPCR was performed using promoter-specific primers. ( B ) Silencing of UbC is observed after Tet treatment. 293Trex-UBC167 cells were treated with Tet for 10 days. Cellular mRNA was isolated at various time points and UbC expression relative to GAPDH determined. The averages with the standard errors of the means are shown as determined by qRT-PCR along with P -values for paired T -tests from each treatment relative to the control. ( C ) DNA from H3K27me3 ChIP ( Figureure S3 ) was treated with bisulfite and analyzed by methylation-specific PCR using primers specific for methylated or unmethylated DNA species overlapping the UBC167 target site.

    Techniques Used: DNA Methylation Assay, shRNA, Real-time Polymerase Chain Reaction, Isolation, Expressing, Quantitative RT-PCR, Chromatin Immunoprecipitation, Methylation, Polymerase Chain Reaction

    29) Product Images from "Mutations in CDCA7 and HELLS cause immunodeficiency–centromeric instability–facial anomalies syndrome"

    Article Title: Mutations in CDCA7 and HELLS cause immunodeficiency–centromeric instability–facial anomalies syndrome

    Journal: Nature Communications

    doi: 10.1038/ncomms8870

    Mutations in HELLS in five ICF4 patients. ( a ) Schematic representation of HELLS, with the identified mutations in red. ( b ) Sanger sequencing confirmation of HELLS mutations in family E. Only c.370+2T > A was identified in maternal DNA, indicating different allelic origins of both mutations, or de novo occurrence of the second mutation. ( c ) RT–PCR analysis of HELLS mRNA on treatment of patient-derived fibroblasts with cycloheximide (C) revealed that c.370+2T > A leads to complete skipping of exon 5 and disruption of the open reading frame. Ethanol-treated samples (E) served as controls, alternative splicing was confirmed using Sanger's sequencing in two independent experiments for both samples. ( d ) Sanger sequencing confirmation of a homozygous out-of-frame deletion in HELLS in family F. Both parents as well as unaffected sibling 2.1 are heterozygous for the deletion allele; unaffected sibling 2.3 is homozygous for the wt allele. ( e ) Sanger sequencing confirmation of a homozygous in-frame deletion in HELLS in family G. Both parents are heterozygous for the deletion allele. ( f ) Sanger sequencing confirmation of nonsense mutations in HELLS in family H. Different allelic origin was confirmed in parental DNA. ( g ) Southern blot analysis of minor satellite DNA methylation in Dnmt3b −/− and siRNA-treated wt MEFs after digesting DNA with MspI or its methylation-sensitive isoschizomer HpaII revealed CpG hypomethylation on knockdown of Zbtb24, Cdca7 and Hells. Molecular weights of the 2-Log DNA size marker are in kilobasepairs.
    Figure Legend Snippet: Mutations in HELLS in five ICF4 patients. ( a ) Schematic representation of HELLS, with the identified mutations in red. ( b ) Sanger sequencing confirmation of HELLS mutations in family E. Only c.370+2T > A was identified in maternal DNA, indicating different allelic origins of both mutations, or de novo occurrence of the second mutation. ( c ) RT–PCR analysis of HELLS mRNA on treatment of patient-derived fibroblasts with cycloheximide (C) revealed that c.370+2T > A leads to complete skipping of exon 5 and disruption of the open reading frame. Ethanol-treated samples (E) served as controls, alternative splicing was confirmed using Sanger's sequencing in two independent experiments for both samples. ( d ) Sanger sequencing confirmation of a homozygous out-of-frame deletion in HELLS in family F. Both parents as well as unaffected sibling 2.1 are heterozygous for the deletion allele; unaffected sibling 2.3 is homozygous for the wt allele. ( e ) Sanger sequencing confirmation of a homozygous in-frame deletion in HELLS in family G. Both parents are heterozygous for the deletion allele. ( f ) Sanger sequencing confirmation of nonsense mutations in HELLS in family H. Different allelic origin was confirmed in parental DNA. ( g ) Southern blot analysis of minor satellite DNA methylation in Dnmt3b −/− and siRNA-treated wt MEFs after digesting DNA with MspI or its methylation-sensitive isoschizomer HpaII revealed CpG hypomethylation on knockdown of Zbtb24, Cdca7 and Hells. Molecular weights of the 2-Log DNA size marker are in kilobasepairs.

    Techniques Used: Sequencing, Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Derivative Assay, Southern Blot, DNA Methylation Assay, Methylation, Marker

    30) Product Images from "Copper bis-Dipyridoquinoxaline Is a Potent DNA Intercalator that Induces Superoxide-Mediated Cleavage via the Minor Groove"

    Article Title: Copper bis-Dipyridoquinoxaline Is a Potent DNA Intercalator that Induces Superoxide-Mediated Cleavage via the Minor Groove

    Journal: Molecules

    doi: 10.3390/molecules24234301

    ( A ) Cartoon representation of enzyme restriction sites. ( B ) Control experiment with isoschizomers HpaII and MspI in the presence and absence of HpaII-MT. ( C ) A quantity of 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the absence of reductant. ( D ) 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the presence of reductant Na- L -asc.
    Figure Legend Snippet: ( A ) Cartoon representation of enzyme restriction sites. ( B ) Control experiment with isoschizomers HpaII and MspI in the presence and absence of HpaII-MT. ( C ) A quantity of 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the absence of reductant. ( D ) 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the presence of reductant Na- L -asc.

    Techniques Used: Sequencing, Methylation

    31) Product Images from "The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma"

    Article Title: The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma

    Journal: JCI Insight

    doi: 10.1172/jci.insight.120422

    Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).
    Figure Legend Snippet: Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).

    Techniques Used: DNA Methylation Assay, Methylation, Marker

    32) Product Images from "Parp1 Localizes within the Dnmt1 Promoter and Protects Its Unmethylated State by Its Enzymatic Activity"

    Article Title: Parp1 Localizes within the Dnmt1 Promoter and Protects Its Unmethylated State by Its Enzymatic Activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0004717

    Dnmt1 down-regulation dependent on PARG over-expression leads to a widespread genome hypomethylation. A, Endogenous DNA methyltransferase activity (dnmt) of nuclear extract from cultures at 24 and 72 hours of puromycin selection transfected with either pCS2 (white bar) or pCS2-Myc-PARG (black bar) vectors. The DNA methyltransferase activity of pCS2 samples was considered as 1.0. B, Methyl-accepting ability assay was carried out on genomic DNA purified from cells transfected with either pCS2 (white bars) or pCS2-Myc-PARG (black bars) vectors at 24 and 72 hours of puromycin selection. Results are displayed as number of picomoles of labelled S-Adenosyl methionine incorporated per microgram of DNA. DNA obtained from cells treated with 5-AZA was used as positive control for genome hypomethylation (black bar). Data reported in A and B are mean±S.E. of three experiments, each performed in triplicate. C, Analysis of Southern blot against minor satellite DNA repeats performed on genomic DNA purified from cells transfected with either pCS2 or pCS2-Myc-PARG vectors at 24 and 72 hours of puromycin selection and digested with HpaII or MspI restriction enzymes. DNA obtained from cells treated with 5-AZA was used as positive control for genome hypomethylation.
    Figure Legend Snippet: Dnmt1 down-regulation dependent on PARG over-expression leads to a widespread genome hypomethylation. A, Endogenous DNA methyltransferase activity (dnmt) of nuclear extract from cultures at 24 and 72 hours of puromycin selection transfected with either pCS2 (white bar) or pCS2-Myc-PARG (black bar) vectors. The DNA methyltransferase activity of pCS2 samples was considered as 1.0. B, Methyl-accepting ability assay was carried out on genomic DNA purified from cells transfected with either pCS2 (white bars) or pCS2-Myc-PARG (black bars) vectors at 24 and 72 hours of puromycin selection. Results are displayed as number of picomoles of labelled S-Adenosyl methionine incorporated per microgram of DNA. DNA obtained from cells treated with 5-AZA was used as positive control for genome hypomethylation (black bar). Data reported in A and B are mean±S.E. of three experiments, each performed in triplicate. C, Analysis of Southern blot against minor satellite DNA repeats performed on genomic DNA purified from cells transfected with either pCS2 or pCS2-Myc-PARG vectors at 24 and 72 hours of puromycin selection and digested with HpaII or MspI restriction enzymes. DNA obtained from cells treated with 5-AZA was used as positive control for genome hypomethylation.

    Techniques Used: Over Expression, Activity Assay, Selection, Transfection, Purification, Positive Control, Southern Blot

    33) Product Images from "Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB"

    Article Title: Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146064

    Enrichment workflow using HpaII. (A) Biotinylated HpaII enzyme is conjugated to streptavidin coated magnetic beads. A DNA mixture can then be added to the conjugated beads and following incubation the mixture is segregated into fractions that are bound (containing majority of Y . pestis ) or unbound (containing majority of human) to the beads. (B) Adding salt to the binding buffer enhances segregation of human (blue bars) from Y . pestis (green bars).
    Figure Legend Snippet: Enrichment workflow using HpaII. (A) Biotinylated HpaII enzyme is conjugated to streptavidin coated magnetic beads. A DNA mixture can then be added to the conjugated beads and following incubation the mixture is segregated into fractions that are bound (containing majority of Y . pestis ) or unbound (containing majority of human) to the beads. (B) Adding salt to the binding buffer enhances segregation of human (blue bars) from Y . pestis (green bars).

    Techniques Used: Magnetic Beads, Incubation, Binding Assay

    Genomic sequencing coverage of M . tuberculosis improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of M . tuberculosis .
    Figure Legend Snippet: Genomic sequencing coverage of M . tuberculosis improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of M . tuberculosis .

    Techniques Used: Genomic Sequencing

    HpaII mediated enrichment of DNA from a pooled sputum sample improves microbe sequencing detection and coverage. (A) The percent of microbial ID reads (blue) increases and human ID reads (red) decrease with enrichment. (B) Normalized microbial Order sequence Identification reads are plotted for bound and input samples. Greater than 95% of identified microbes have increased sequenced reads. Many microbes (red points) are only detectable after enrichment. (C) Comparison of ratio of microbial sequence ID reads in sputum input and sputum bound samples. (D) Genomic sequencing coverage of bacteria such as P . aeruginosa improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of P . aeruginosa .
    Figure Legend Snippet: HpaII mediated enrichment of DNA from a pooled sputum sample improves microbe sequencing detection and coverage. (A) The percent of microbial ID reads (blue) increases and human ID reads (red) decrease with enrichment. (B) Normalized microbial Order sequence Identification reads are plotted for bound and input samples. Greater than 95% of identified microbes have increased sequenced reads. Many microbes (red points) are only detectable after enrichment. (C) Comparison of ratio of microbial sequence ID reads in sputum input and sputum bound samples. (D) Genomic sequencing coverage of bacteria such as P . aeruginosa improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of P . aeruginosa .

    Techniques Used: Sequencing, Genomic Sequencing

    34) Product Images from "DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells"

    Article Title: DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky947

    DNMT3L deficiency in mESCs results in hypomethylation at specific heterochromatin regions. ( A ) Mouse breeding strategies to generate mESC lines with different Dnmt3l genotypes. ( B and C ) Western blot analysis of the mESC lines (2 independent clones per genotype) for expression of DNMT3L (B) and the pluripotency factors OCT4 and SOX2 (C), with β-ACTIN serving as a loading control. ( D and E ) Southern blot analysis of the mESC lines (2-3 independent clones per genotype) for DNA methylation at the major satellite repeats (D) and the minor satellite repeats (E) after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite repeats and HpaII for minor satellite repeats). DKO, Dnmt3a/3b double KO mESC line.
    Figure Legend Snippet: DNMT3L deficiency in mESCs results in hypomethylation at specific heterochromatin regions. ( A ) Mouse breeding strategies to generate mESC lines with different Dnmt3l genotypes. ( B and C ) Western blot analysis of the mESC lines (2 independent clones per genotype) for expression of DNMT3L (B) and the pluripotency factors OCT4 and SOX2 (C), with β-ACTIN serving as a loading control. ( D and E ) Southern blot analysis of the mESC lines (2-3 independent clones per genotype) for DNA methylation at the major satellite repeats (D) and the minor satellite repeats (E) after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite repeats and HpaII for minor satellite repeats). DKO, Dnmt3a/3b double KO mESC line.

    Techniques Used: Western Blot, Clone Assay, Expressing, Southern Blot, DNA Methylation Assay

    35) Product Images from "Investigation of the Role of DNA Methylation in the Expression of ERBB2 in Human Myocardium"

    Article Title: Investigation of the Role of DNA Methylation in the Expression of ERBB2 in Human Myocardium

    Journal: Gene

    doi: 10.1016/j.gene.2017.07.058

    Reporter gene activities of human ERBB2 promoter constructs (A) Luciferase activities of progressive ERBB2 deletion constructs in A549 cells (see text for details). Normalized luciferase activities were expressed relative to the values from the −499 ERBB2 -pCpGL construct, which was assigned an arbitrary value of 100. (B) Effect of DNA methylation status on the luciferase activity of the −499 ERBB2 -pCpGL construct. CpG sites methylated by HpaII or SssI methyltransferase are indicated. Data represent the mean ± standard deviation of three independent experiments. *** p
    Figure Legend Snippet: Reporter gene activities of human ERBB2 promoter constructs (A) Luciferase activities of progressive ERBB2 deletion constructs in A549 cells (see text for details). Normalized luciferase activities were expressed relative to the values from the −499 ERBB2 -pCpGL construct, which was assigned an arbitrary value of 100. (B) Effect of DNA methylation status on the luciferase activity of the −499 ERBB2 -pCpGL construct. CpG sites methylated by HpaII or SssI methyltransferase are indicated. Data represent the mean ± standard deviation of three independent experiments. *** p

    Techniques Used: Construct, Luciferase, DNA Methylation Assay, Activity Assay, Methylation, Standard Deviation

    36) Product Images from "VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization"

    Article Title: VIM1, a methylcytosine-binding protein required for centromeric heterochromatinization

    Journal: Genes & Development

    doi: 10.1101/gad.1512007

    Reduced cytosine methylation of centromeric repeats in Bor-4. ( A ) Genomic DNA samples from the indicated genotypes were digested with the isoschizomers HpaII or MspI, and DNA blot hybridization with a 180-bp centromere repeat probe (CEN) was performed. ( B ) The filter shown in A was rehybridized with a 5S rRNA probe (5S rRNA). ( C ) A DNA blot hybridization pattern with the CEN probe after HpaII digestion demonstrates that centromeric repeat arrays hypomethylated in Bor-4 were fully remethylated in F1 hybrids resulting from reciprocal crosses between strains Bor-4 and Ler. The lane labeled Ler + Bor-4 contains a 1:1 mixture of Ler and Bor-4 HpaII-digested genomic DNA and shows the hybridization pattern expected if no remethylation occurred in the F1 hybrids.
    Figure Legend Snippet: Reduced cytosine methylation of centromeric repeats in Bor-4. ( A ) Genomic DNA samples from the indicated genotypes were digested with the isoschizomers HpaII or MspI, and DNA blot hybridization with a 180-bp centromere repeat probe (CEN) was performed. ( B ) The filter shown in A was rehybridized with a 5S rRNA probe (5S rRNA). ( C ) A DNA blot hybridization pattern with the CEN probe after HpaII digestion demonstrates that centromeric repeat arrays hypomethylated in Bor-4 were fully remethylated in F1 hybrids resulting from reciprocal crosses between strains Bor-4 and Ler. The lane labeled Ler + Bor-4 contains a 1:1 mixture of Ler and Bor-4 HpaII-digested genomic DNA and shows the hybridization pattern expected if no remethylation occurred in the F1 hybrids.

    Techniques Used: Methylation, Hybridization, Labeling

    37) Product Images from "Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿"

    Article Title: Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02797-06

    Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.
    Figure Legend Snippet: Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.

    Techniques Used: Southern Blot, Molecular Weight, Infection, Methylation, CpG Methylation Assay

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    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: For 16S rRNA products amplified from cultured bacteria, a 1.5 kbp 16S product was amplified from 48 bacterial isolates using 20 ng DNA each and Taq polymerase (NEB) in a 50 μl reaction. .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Paragraph title: Methylation‐sensitive amplification polymorphism assay ... Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Paragraph title: Assessment of DNA methylation polymorphism using Methylation Sensitive Amplified Polymorphisms (MSAP) ... Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs).

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. For amplicon generation, 2 µg of genomic DNA derived from nine pediatric malignant astrocytic tumors and a pool of control DNA obtained from the white matter of three pediatric brain tissue samples were digested with Mse I (New England Biolabs).

    Article Title: A Large X-Chromosomal Deletion is Associated with Microphthalmia with Linear Skin Defects (MLS) and Amelogenesis Imperfecta (XAI)
    Article Snippet: DNA aliquots of 250 ng were incubated in a total volume of 12.5 μl with no enzyme, Dde I only, Hpa II only, or Dde I and Hpa II for 18 h at 37 °C (New England Biolabs) using the manufacturer's instructions. .. Digestion with Dde I can prevent falsely skewed X-inactivation patterns by allowing equal amplification of methylated and unmethylated DNA [ ].

    Article Title: (Epi)Genetic Analyses of Age-Related Macular Degeneration: Case-control and Discordant Twin Studies
    Article Snippet: For methylation analysis, 3 μg of genomic DNA was digested at 37 degrees Celsius for16 hours with a methyl sensitive restriction enzyme (MSRE) cocktail including Aci I (60 units), BsaH I (3.9 units), Hha I (7.5 units), Hpa II (7.5 units), and HpyCH4 IV (30 units) (New England Biolabs), in a 200 μL reaction volume with 1% BSA and 10% NEB buffer #4 (New England Biolabs) and heat inactivated for 20 minutes at 60°C. .. After this pre-treatment, the DNA enters the mapping array procedure; it is further digested with the Nsp I and Sty I restriction enzymes, and fragments of 100–1,100 bp (containing the polymorphic sites to be assessed) are PCR amplified, with the resulting amplicons then end-labeled and hybridized to the array.

    Polymerase Chain Reaction:

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: .. Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). ..

    Article Title: A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples
    Article Snippet: .. Multiple Restriction Enzyme Digestion Eighty microliters (~2 μg) of PCR products were digested with 10–20 units each, Alu I, Ava I, Dde I, Hha I, and Hpa II (New England BioLabs) as per the supplier's directives for 2.5 to 4 h. .. Gel Extraction and Subcloning MRED digestions were ethanol precipitated, resuspended in TE buffer pH 8.0, and electrophoresed on 3% agarose gels.

    Article Title: Lack of Association between the Serotonin Transporter (5-HTT) and Serotonin Receptor (5-HT2A) Gene Polymorphisms with Smoking Behavior among Malaysian Malays
    Article Snippet: .. The 342 bp PCR product was digested with Hpa II (New England Biolabs, MA, USA) for 3 h at 37°C. .. The digestion products were then resolved on a 2% agarose gel containing ethidium bromide and were visualized under UV light.

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB). ..

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. A ligation mixture in a volume of 5 μl contained 5 pmol of Eco RI adaptor and 50 pmol of Hpa II/Msp I adaptor, and 30 U of T4 DNA ligase (New England Biolabs) was added to digestion products and incubated at 37°C for 3 hr, followed by 16°C for 9 hr, and then 65°C for 20 min. Preselective polymerase chain reaction (PCR) was carried out in a total volume of 20 μl containing 2 μl of the ligation product, 0.4 μM each of Eco RI_ADAPTER (F)+A and Hpa II/Msp I_ADAPTER (F)+T preselective primers, 0.5 U of Taq DNA polymerase (Takara), 2 mM of MgCl2 , and 0.2 mM of each nucleotide.

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs). .. Amplification of DNA fragments for MSAP followed the same PCR cycling conditions used for AFLP.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. Sixteen thousand suitable fragments were polymerase chain reaction (PCR)-amplified using plasmid primer and spotted onto UltraGAPS microarrays (Corning, Acton, MA).

    Article Title: A Large X-Chromosomal Deletion is Associated with Microphthalmia with Linear Skin Defects (MLS) and Amelogenesis Imperfecta (XAI)
    Article Snippet: DNA aliquots of 250 ng were incubated in a total volume of 12.5 μl with no enzyme, Dde I only, Hpa II only, or Dde I and Hpa II for 18 h at 37 °C (New England Biolabs) using the manufacturer's instructions. .. Subsequently, regions of the AR and JARID1C (formerly SMCX ) loci were amplified by PCR in separate 25 μl reactions with 2.5 μl of the digested DNA by adding Taq Buffer (5× Taq Buffer is 83 mM Tris-HCl [pH 8.8], 850 mg/mL BSA, 83 mM (NH4 )2 SO4 , 33.5 mM MgCl2 , 34 mM EDTA, and 50mM β-mercaptoethanol), 5% dimethyl sulfoxide, 12.5 pmol of each primer, 1.5 mM dNTPs, and 0.625 U AmpliTaq (Applied Biosystems).

    Article Title: (Epi)Genetic Analyses of Age-Related Macular Degeneration: Case-control and Discordant Twin Studies
    Article Snippet: For methylation analysis, 3 μg of genomic DNA was digested at 37 degrees Celsius for16 hours with a methyl sensitive restriction enzyme (MSRE) cocktail including Aci I (60 units), BsaH I (3.9 units), Hha I (7.5 units), Hpa II (7.5 units), and HpyCH4 IV (30 units) (New England Biolabs), in a 200 μL reaction volume with 1% BSA and 10% NEB buffer #4 (New England Biolabs) and heat inactivated for 20 minutes at 60°C. .. After this pre-treatment, the DNA enters the mapping array procedure; it is further digested with the Nsp I and Sty I restriction enzymes, and fragments of 100–1,100 bp (containing the polymorphic sites to be assessed) are PCR amplified, with the resulting amplicons then end-labeled and hybridized to the array.

    Real-time Polymerase Chain Reaction:

    Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4
    Article Snippet: Cells were stimulated with 10 µM RA (Sigma, St. Louis, MO, USA) for 7 days, then harvested, DNA extracted and digested (600ng) with the methylation-sensitive enzymes HpaII or HhaI [R0171 and R0139 respectively (New England Biolabs, Ipswich, MA, USA)], see for location of restriction sites]. .. The digested DNA was then subjected to real-time PCR with primers targeting regions of the Oct4 promoter that flank the restriction sites.

    Incubation:

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. A ligation mixture in a volume of 5 μl contained 5 pmol of Eco RI adaptor and 50 pmol of Hpa II/Msp I adaptor, and 30 U of T4 DNA ligase (New England Biolabs) was added to digestion products and incubated at 37°C for 3 hr, followed by 16°C for 9 hr, and then 65°C for 20 min. Preselective polymerase chain reaction (PCR) was carried out in a total volume of 20 μl containing 2 μl of the ligation product, 0.4 μM each of Eco RI_ADAPTER (F)+A and Hpa II/Msp I_ADAPTER (F)+T preselective primers, 0.5 U of Taq DNA polymerase (Takara), 2 mM of MgCl2 , and 0.2 mM of each nucleotide.

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The reconstituted chromatin (total volume = 70 μl) was mixed with 100 μl of a preassembled MNase mixture (94 μl of Ex50 buffer, 5 μl of 100 mM CaCl2 , 1 μl of MNase [50 U/μl; Sigma]) and incubated at room temperature.

    Article Title: A Large X-Chromosomal Deletion is Associated with Microphthalmia with Linear Skin Defects (MLS) and Amelogenesis Imperfecta (XAI)
    Article Snippet: .. DNA aliquots of 250 ng were incubated in a total volume of 12.5 μl with no enzyme, Dde I only, Hpa II only, or Dde I and Hpa II for 18 h at 37 °C (New England Biolabs) using the manufacturer's instructions. .. Digestion with Dde I can prevent falsely skewed X-inactivation patterns by allowing equal amplification of methylated and unmethylated DNA [ ].

    Luciferase:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The pGL3-basic vector, pRL-TK, and dual luciferase assay system were purchased from Promega (Madison, WI). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Modification:

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Assessment of DNA methylation polymorphism using Methylation Sensitive Amplified Polymorphisms (MSAP) The MSAP protocol was followed with slight modification of the original protocol [ ]. .. Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs).

    Western Blot:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Paragraph title: DNA gel blot analysis ... Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions.

    DNA Methylation Assay:

    Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4
    Article Snippet: Paragraph title: DNA methylation assay ... Cells were stimulated with 10 µM RA (Sigma, St. Louis, MO, USA) for 7 days, then harvested, DNA extracted and digested (600ng) with the methylation-sensitive enzymes HpaII or HhaI [R0171 and R0139 respectively (New England Biolabs, Ipswich, MA, USA)], see for location of restriction sites].

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Paragraph title: Assessment of DNA methylation polymorphism using Methylation Sensitive Amplified Polymorphisms (MSAP) ... Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs).

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. CR and IGS DNA methylation levels were determined from phosphorimager (Bio-Rad, USA) files using Quantity One™ (Bio-Rad, USA) software.

    Derivative Assay:

    Article Title: Evidence for Widespread Genomic Methylation in the Migratory Locust, Locusta migratoria (Orthoptera: Acrididae)
    Article Snippet: M. musculus genomic DNA was obtained commercially (New England Biolabs) and was derived from the embryonic fibroblast cell line NIH 3T3. .. Restriction digests with Hpa II and Msp I (New England Biolabs) were carried out for 4 hr at 37°C with 2 µg of genomic DNA and 10 U of enzyme in a final volume of 50 µl.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. For amplicon generation, 2 µg of genomic DNA derived from nine pediatric malignant astrocytic tumors and a pool of control DNA obtained from the white matter of three pediatric brain tissue samples were digested with Mse I (New England Biolabs).

    Hybridization:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: Paragraph title: Differential Methylation Hybridization Analysis and CGI Identification ... CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites.

    Ligation:

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. A ligation mixture in a volume of 5 μl contained 5 pmol of Eco RI adaptor and 50 pmol of Hpa II/Msp I adaptor, and 30 U of T4 DNA ligase (New England Biolabs) was added to digestion products and incubated at 37°C for 3 hr, followed by 16°C for 9 hr, and then 65°C for 20 min. Preselective polymerase chain reaction (PCR) was carried out in a total volume of 20 μl containing 2 μl of the ligation product, 0.4 μM each of Eco RI_ADAPTER (F)+A and Hpa II/Msp I_ADAPTER (F)+T preselective primers, 0.5 U of Taq DNA polymerase (Takara), 2 mM of MgCl2 , and 0.2 mM of each nucleotide.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. After ligation of linkers H12/H24 (New England Biolabs), fragments were digested with methylation-sensitive restriction endonucleases Bst UI and Hpa II and amplified for 20 cycles with H24 as a primer.

    Cell Culture:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: Cell culture media, fetal calf serum, ITS (insulin, transferrin, and sodium selenite), Lipofectamine-2000, and Superscript one-step RT-PCR kit were obtained from Invitrogen (Carlsbad, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: For 16S rRNA products amplified from cultured bacteria, a 1.5 kbp 16S product was amplified from 48 bacterial isolates using 20 ng DNA each and Taq polymerase (NEB) in a 50 μl reaction. .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Generated:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Sequencing:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: We purchased sequence-specific oligonucleotides from Midland Certified Reagent Company (Midland, TX). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). .. Directing sequencing of MYH6 was conducted on at least 2 individuals from each sampling locality (n = 64; Additional file ; GenBank FJ638345 – FJ638410 ).

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. Msp I and Hpa II can recognize and cleave the same sequence 5′‐CCGG‐3′, but their susceptibility is different depending on the methylation state of cytosines at restriction sites (Schulz et al., ).

    Antiviral Assay:

    Article Title: A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples
    Article Snippet: .. Multiple Restriction Enzyme Digestion Eighty microliters (~2 μg) of PCR products were digested with 10–20 units each, Alu I, Ava I, Dde I, Hha I, and Hpa II (New England BioLabs) as per the supplier's directives for 2.5 to 4 h. .. Gel Extraction and Subcloning MRED digestions were ethanol precipitated, resuspended in TE buffer pH 8.0, and electrophoresed on 3% agarose gels.

    Methylation:

    Article Title: Evidence for Widespread Genomic Methylation in the Migratory Locust, Locusta migratoria (Orthoptera: Acrididae)
    Article Snippet: Paragraph title: Methylation-specific restriction enzyme assays ... Restriction digests with Hpa II and Msp I (New England Biolabs) were carried out for 4 hr at 37°C with 2 µg of genomic DNA and 10 U of enzyme in a final volume of 50 µl.

    Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4
    Article Snippet: .. Cells were stimulated with 10 µM RA (Sigma, St. Louis, MO, USA) for 7 days, then harvested, DNA extracted and digested (600ng) with the methylation-sensitive enzymes HpaII or HhaI [R0171 and R0139 respectively (New England Biolabs, Ipswich, MA, USA)], see for location of restriction sites]. .. The digested DNA was then subjected to real-time PCR with primers targeting regions of the Oct4 promoter that flank the restriction sites.

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Paragraph title: Methylation‐sensitive amplification polymorphism assay ... Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Paragraph title: Assessment of DNA methylation polymorphism using Methylation Sensitive Amplified Polymorphisms (MSAP) ... Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs).

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: Paragraph title: Differential Methylation Hybridization Analysis and CGI Identification ... CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites.

    Article Title: A Large X-Chromosomal Deletion is Associated with Microphthalmia with Linear Skin Defects (MLS) and Amelogenesis Imperfecta (XAI)
    Article Snippet: The X-inactivation pattern was evaluated by examining the methylation pattern at the AR (androgen receptor) locus [ ]. .. DNA aliquots of 250 ng were incubated in a total volume of 12.5 μl with no enzyme, Dde I only, Hpa II only, or Dde I and Hpa II for 18 h at 37 °C (New England Biolabs) using the manufacturer's instructions.

    Article Title: (Epi)Genetic Analyses of Age-Related Macular Degeneration: Case-control and Discordant Twin Studies
    Article Snippet: .. For methylation analysis, 3 μg of genomic DNA was digested at 37 degrees Celsius for16 hours with a methyl sensitive restriction enzyme (MSRE) cocktail including Aci I (60 units), BsaH I (3.9 units), Hha I (7.5 units), Hpa II (7.5 units), and HpyCH4 IV (30 units) (New England Biolabs), in a 200 μL reaction volume with 1% BSA and 10% NEB buffer #4 (New England Biolabs) and heat inactivated for 20 minutes at 60°C. ..

    Mutagenesis:

    Article Title: Lack of Association between the Serotonin Transporter (5-HTT) and Serotonin Receptor (5-HT2A) Gene Polymorphisms with Smoking Behavior among Malaysian Malays
    Article Snippet: The 342 bp PCR product was digested with Hpa II (New England Biolabs, MA, USA) for 3 h at 37°C. .. The 102T mutant remained uncut (342 bp), whereas the wild type 102C allele was digested into two bands of 216 and 126 bp; three fragments of 342, 216, and 126 bp were observed for samples containing both the wild type and mutant alleles ( ).

    Isolation:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The plasmid isolation kit and RNeasy mini-kit for total RNA isolation were obtained from Qiagen (Valencia, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: 2.2 Methylation‐sensitive amplification polymorphism assay Total genomic DNA was isolated from approximately 50 mg of siphon tissues following the proteinase K method (Waters, Dijkstra, & Wallis, ). .. Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: .. CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. Sixteen thousand suitable fragments were polymerase chain reaction (PCR)-amplified using plasmid primer and spotted onto UltraGAPS microarrays (Corning, Acton, MA).

    Purification:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: Cell culture media, fetal calf serum, ITS (insulin, transferrin, and sodium selenite), Lipofectamine-2000, and Superscript one-step RT-PCR kit were obtained from Invitrogen (Carlsbad, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Polymorphism Assay:

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Paragraph title: Methylation‐sensitive amplification polymorphism assay ... Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Chromatin Immunoprecipitation:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA). .. We obtained the LightShift Chemiluminescent EMSA kit from Pierce (Rockford, IL) and the EZ ChIP kit from Upstate Biotechnology (Temecula, CA).

    Plasmid Preparation:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The plasmid isolation kit and RNeasy mini-kit for total RNA isolation were obtained from Qiagen (Valencia, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: For PKS products amplified from sorted bacteria, two of fifty microliters of each plasmid preparation were digested with each of seven restriction enzymes: Kpn I, Bam HI, Xba I, Xho I, Hpa II, Rsa I and Sph I (NEB). .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The DNA template used for in vitro reconstitution of chromatin was the pBS HPRT 1.8-kb plasmid, which is a pBluescript-derived plasmid containing a 1.8-kb Eco RI-to- Bam HI fragment that includes the entire HPRT promoter. .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier.

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. Sixteen thousand suitable fragments were polymerase chain reaction (PCR)-amplified using plasmid primer and spotted onto UltraGAPS microarrays (Corning, Acton, MA).

    Software:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. CR and IGS DNA methylation levels were determined from phosphorimager (Bio-Rad, USA) files using Quantity One™ (Bio-Rad, USA) software.

    Agarose Gel Electrophoresis:

    Article Title: Lack of Association between the Serotonin Transporter (5-HTT) and Serotonin Receptor (5-HT2A) Gene Polymorphisms with Smoking Behavior among Malaysian Malays
    Article Snippet: The PCR products were then resolved using a 2% agarose gel and were visualized under UV light. .. The 342 bp PCR product was digested with Hpa II (New England Biolabs, MA, USA) for 3 h at 37°C.

    In Vitro:

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Sampling:

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). .. Directing sequencing of MYH6 was conducted on at least 2 individuals from each sampling locality (n = 64; Additional file ; GenBank FJ638345 – FJ638410 ).

    Alkaline Lysis:

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: The plasmid alkaline lysis minipreparation procedure was performed as described previously ( ). .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Staining:

    Article Title: Evidence for Widespread Genomic Methylation in the Migratory Locust, Locusta migratoria (Orthoptera: Acrididae)
    Article Snippet: Restriction digests with Hpa II and Msp I (New England Biolabs) were carried out for 4 hr at 37°C with 2 µg of genomic DNA and 10 U of enzyme in a final volume of 50 µl. .. Digested DNA was separated on 0.8% agarose/TAE gel and visualised using ethidium bromide staining.

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). .. Digested products were run on 2% agarose stained with ethidium bromide.

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    New England Biolabs hpaii
    Repression of MOR promoter-driven transcription by CpG methylation. (A, B) Three different luciferase (LUC) constructs (pL450, pLup and pL1.3 k) were mock methylated (control) or in vitro methylated with HapI, <t>HpaII</t> (partial) or <t>SssI</t> (full) methylase and transfected into SH-SY5Y cells. The results are given as luciferase activity normalized against cotransfected pCH110 β-galactosidase activity. The data shown are the means of three independent experiments with at least two different plasmid preparations. The control with none methylase was set at 1 for quantifications. # P
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    Repression of MOR promoter-driven transcription by CpG methylation. (A, B) Three different luciferase (LUC) constructs (pL450, pLup and pL1.3 k) were mock methylated (control) or in vitro methylated with HapI, HpaII (partial) or SssI (full) methylase and transfected into SH-SY5Y cells. The results are given as luciferase activity normalized against cotransfected pCH110 β-galactosidase activity. The data shown are the means of three independent experiments with at least two different plasmid preparations. The control with none methylase was set at 1 for quantifications. # P

    Journal: Molecular Pain

    Article Title: Increased methylation of the MOR gene proximal promoter in primary sensory neurons plays a crucial role in the decreased analgesic effect of opioids in neuropathic pain

    doi: 10.1186/1744-8069-10-51

    Figure Lengend Snippet: Repression of MOR promoter-driven transcription by CpG methylation. (A, B) Three different luciferase (LUC) constructs (pL450, pLup and pL1.3 k) were mock methylated (control) or in vitro methylated with HapI, HpaII (partial) or SssI (full) methylase and transfected into SH-SY5Y cells. The results are given as luciferase activity normalized against cotransfected pCH110 β-galactosidase activity. The data shown are the means of three independent experiments with at least two different plasmid preparations. The control with none methylase was set at 1 for quantifications. # P

    Article Snippet: Briefly, methylases SssI, HpaI and HpaII were used to methylate MOR promoter/luciferase reporter constructs following the recommendations of the manufacturer (New England Biolabs).

    Techniques: CpG Methylation Assay, Luciferase, Construct, Methylation, In Vitro, Transfection, Activity Assay, Plasmid Preparation

    CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “ Methods ” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a . The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by

    Journal: Molecular Autism

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

    doi: 10.1186/s13229-016-0105-9

    Figure Lengend Snippet: CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “ Methods ” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a . The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by

    Article Snippet: Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Techniques: Methylation, Derivative Assay, Mass Spectrometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Methylation Assay

    Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “ Methods ” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3 : Figure S3 for representative images during neuronal differentiation of 37D cells

    Journal: Molecular Autism

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

    doi: 10.1186/s13229-016-0105-9

    Figure Lengend Snippet: Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “ Methods ” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3 : Figure S3 for representative images during neuronal differentiation of 37D cells

    Article Snippet: Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Techniques: Methylation, Polymerase Chain Reaction

    Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a – c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a , b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols . c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately ( i ) or in a ~1:1 mixture ( ii ) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel ( iii ) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation

    Journal: Molecular Autism

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

    doi: 10.1186/s13229-016-0105-9

    Figure Lengend Snippet: Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a – c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a , b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols . c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately ( i ) or in a ~1:1 mixture ( ii ) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel ( iii ) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation

    Article Snippet: Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl2 , 22 mM (NH4 )2 SO4 , and 0.2 % Triton X-100.

    Techniques: Methylation, DNA Methylation Assay, Standard Deviation

    Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.

    Journal: Developmental Cell

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function

    doi: 10.1016/j.devcel.2017.05.021

    Figure Lengend Snippet: Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.

    Article Snippet: Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S).

    Techniques: Activity Assay, Binding Assay, Mutagenesis, Recombinant, Produced, Thin Layer Chromatography, Knock-In, Knock-Out, Staining, Northern Blot, Western Blot, Expressing, Marker, Methylation, Southern Blot, DNA Methylation Assay, Immunoprecipitation, End Labeling, Sequencing, Immunofluorescence

    Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.

    Journal: Developmental Cell

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function

    doi: 10.1016/j.devcel.2017.05.021

    Figure Lengend Snippet: Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.

    Article Snippet: Approximately 5 μg of genomic DNA was digested overnight at 37°C with 20 U of methylation-sensitive restriction enzyme HpaII (New England Biolabs, R0171S) or 40 U of methylation insensitive restriction enzyme MspI (New England Biolabs, R0106S).

    Techniques: Activity Assay, Mutagenesis, Staining, Northern Blot, Southern Blot, Methylation, DNA Methylation Assay, Immunoprecipitation, End Labeling, Labeling, Immunofluorescence

    Methylation sensitive comet assay (MS-CA). The observed comet tails represent double-strand DNA breaks, introduced by the isoschizomeric enzymes MspI and HpaII; in the presence of an electric field, the HpaII- and MspI-digested DNA migrates faster, compared to the significantly slower migration of the undigested DNA. Digestion of the control (Chlamy) and treated (vitamin B 12 and MTX) samples resulted in a tail DNA increase for MTX and decrease for vitamin B 12 , relative to the intact DNA in the comet head of the untreated controls – indicative of decreased DNA methylation levels in MTX-treated animals and increased DNA methylation in vitamin B 12 -treated animals.

    Journal: Scientific Reports

    Article Title: Bi-directional effects of vitamin B12 and methotrexate on Daphnia magna fitness and genomic methylation

    doi: 10.1038/s41598-017-12148-2

    Figure Lengend Snippet: Methylation sensitive comet assay (MS-CA). The observed comet tails represent double-strand DNA breaks, introduced by the isoschizomeric enzymes MspI and HpaII; in the presence of an electric field, the HpaII- and MspI-digested DNA migrates faster, compared to the significantly slower migration of the undigested DNA. Digestion of the control (Chlamy) and treated (vitamin B 12 and MTX) samples resulted in a tail DNA increase for MTX and decrease for vitamin B 12 , relative to the intact DNA in the comet head of the untreated controls – indicative of decreased DNA methylation levels in MTX-treated animals and increased DNA methylation in vitamin B 12 -treated animals.

    Article Snippet: Exposed nuclei were digested for 30 min at 37 °C in 50 µl reactions containing 1 x CutSmart buffer and 10 U HpaII, MspI (New England Biolabs, Hertfordshire, UK) or no enzyme, respectively.

    Techniques: Methylation, Single Cell Gel Electrophoresis, Mass Spectrometry, Migration, DNA Methylation Assay