methylation-sensitive enzymes hpaii and mspi  (Pyrosequencing Inc)

Journal: Epigenetics & Chromatin

Article Title: Independent functions of DNMT1 and USP7 at replication foci

doi: 10.1186/s13072-018-0179-z

Figure Lengend Snippet: Ablation of USP7 does not affect steady-state level of DNMT1 or global DNA methylation. a MEFs that lack USP7 after Cre-mediated excision of a Floxed allele of Usp7 show normal levels of DNMT1 (lane 2) but no detectable USP7 protein. Biological replicates are shown as Experiments 1 and 2. b Normal expression of DNMT1 and UHRF1 in human H1299 lung carcinoma cells containing an inducible shRNA against USP7 mRNA. Ablation of USP7 has no detectable effect on expression of other proteins. c Normal steady-state expression of USP7 in the absence of DNMT1 in ES cells. d Normal DNA methylation in MEFs that lack USP7. McrBC cleaves methylated DNA; HpaII cleaves unmethylated DNA at CCGG sites; MspI cleaves CCGG sites whether methylated or unmethylated. DNA from wild-type and Usp7 − / − MEFs show similar patterns of resistance to both McrBC and HpaII. e Removal of USP7 by an inducible shRNA against USP7 from H1299 human lung carcinoma cells does not affect DNA methylation levels as assessed by resistance of DNA to HpaII. f LUMA analysis shows that DNA methylation is not measurably affected by removal of USP7. n = 2 (biological replicates). Error bars show standard deviations, center value is mean, and p values were calculated using the two-tailed t test

Article Snippet: Methylation levels were quantified by LUminometric Methylation Assay (LUMA) that uses methylation-sensitive enzymes HpaII and MspI followed by Pyrosequencing which quantitates the number of HpaII cleavage events [ ].

Techniques: DNA Methylation Assay, Expressing, shRNA, Methylation, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: Telomere-binding protein regulates the chromosome ends through the interaction with histone deacetylases in Arabidopsis thaliana

doi: 10.1093/nar/gkw067

Figure Lengend Snippet: DNA methylation is involved in telomere homeostasis in Arabidopsis thaliana . ( A ) Southern blot analysis showing the pattern of DNA methylation of telomeric repeats in cmt3, met1, axe1–5 (left panel), hdt4–1 and attrb2–1 mutants (right panel). DNA blots of methylation-sensitive restriction enzyme-digested gDNA probed with [TTTAGGG] 70 repeat sequences. M, MspI; H, HpaII; B, BstNI; E, EcoRII. Arrowheads represent the different signals detected by probe compared to wild-type. Small and large white bar indicate the high and low molecular weight ranges of the gDNAs, respectively, affected by restriction enzyme digestion. ( B ) Chop-PCR in subtelomeric and ITSs regions using McrBC endonuclease. 4R-300, -600, -800 and -2100 indicate the different regions on the subtelomeric regions closed to telomere in the right arm of chromosome IV. The 60S RP gene was used as a reference for quantitative comparisons. −, +; McrBC-untreated/ -treated.

Article Snippet: A total of 10 μg of genomic DNA was digested with the methylation sensitive restriction enzymes, 20 U of MspI, HpaII, BstNI and EcoRII (Fermentas, Thermo Scientific) and then separated by electrophoresis in a 0.8% agarose gel.

Techniques: DNA Methylation Assay, Southern Blot, Methylation, Molecular Weight