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Reorganization of the Genome During Aging of Proliferative Cell Compartments
Published in Alvaro Macieira-Coelho, Molecular Basis of Aging, 2017
When the size of the monosome fragment was analyzed for all digestion times with the cells of different PDL, the size of the fragment declined with longer digestion times to a constant value of about 146 bp after 7 min of digestion, and was identical for low and high PDL cells (Figure 20). These results indicate that the structural changes detected with the micrococcal nuclease (Figure 19) are located in the linker region.
Epigenetics
Published in Sara C. Zapico, Mechanisms Linking Aging, Diseases and Biological Age Estimation, 2017
Christian Thomas, Sara C. Zapico
Histone modifications can be analyzed by ChIP in a similar way to the method used to analyze DNA methylation, i.e., coupled to qPCR, microarrays (ChIP-chip) or sequencing (ChIP-Sep). Other complementary techniques can be used to determine chromatin accessibility and nucleosome dynamics like sonication followed by sequencing (Sono-Seq) or FAIRE-Seq (formaldehyde-assisted isolation of regulatory elements followed by sequencing) (Qureshi and Mehler 2011). Likewise, Micrococcal nuclease digestion and sequencing (MNase-Seq) can be used to define maps of nucleosome locations throughout the genome (Schones et al. 2008, Kaplan et al. 2009).
Nonrandom Binding of Chemical Carcinogens in Mammalian Nuclear Subfractions
Published in Isaac Bekhor, Carol J. Mirell, C. C. Liew, Progress in Nonhistone Protein Research, 1985
It has generally been postulated that carcinogenesis is actually a mutagenic event. In fact, it has become commonplace to test the oncogenicity of various compounds by monitoring point mutations and reversions in bacterial populations.1 Direct attack on DNA has been thought to be the mechanism that could lead to neoplastic growth.13,31,40,41 The binding of ultimate carcinogens to DNA has been suggested to be the most critical event in the initiation of carcinogenesis.15,27,29,30 There have been extensive reports evaluating DNA-carcinogen adducts from numerous laboratories. In a study with mice, Eastman and Bresnick20a have shown that the degree of persistence of DNA-methylcholanthrene adducts correlates directly with susceptibility to neoplastic transformation. Arrand and Murray3 have demonstrated that the DNA of human lung epithelium (a target tissue) binds benzopyrene metabolites to a much greater extent than does nontarget fibroblast cells. In another study, Shoyab67 showed that reiterated DNA sequences preferentially bind dimethylbenzanthracene when the carcinogen was administered at low dosages. Studies by Walker et al.67 indicate random distribution of N-2-acetylaminofluorene and its N-hydroxy derivative to DNA when the chromatin is fractionated on the basis of hydrodynamic shearing or DNAase sensitivity. However, it has been reported that preferential binding does occur in micrococcal nuclease sensitive sites.32,45,67
Nuclear receptor co-repressor RIP140 regulates diurnal expression of cytochrome P450 2b10 in mouse liver
Published in Xenobiotica, 2020
Mengjing Zhao, Huan Zhao, Luomin Lin, Yi Wang, Menglin Chen, Baojian Wu
ChIP assays were performed using the SimpleChIP Enzymatic Chromatin IP kit (Cell Signaling Technology, Beverly, MA) as described previously (Zhao et al., 2019). In brief, liver samples were fixed in 1% formaldehyde at room temperature for 20 min and quenched by glycine. Cross-linked samples were digested with micrococcal nuclease followed by sonication (chromatin fragments of 150–900 bp size were obtained). Chromatin fragments were incubated with anti-RIP140, anti-Car or control rabbit IgG overnight at 4 °C. On the next day, protein G magnetic beads were added to each sample for 2 h at 4 °C. Beads were collected and chromatins were eluted from the antibody/protein G magnetic beads by the elution buffer. Samples were de-crosslinked by incubation with NaCl and proteinase K for 2 h at 65 °C. DNA was purified using spin columns, followed by qPCR analysis (primers are listed in Table 2).
Neutrophil extracellular traps generation and degradation in patients with granulomatosis with polyangiitis and systemic lupus erythematosus
Published in Autoimmunity, 2019
Michal Przemyslaw Pruchniak, Magdalena Ostafin, Malgorzata Wachowska, Michal Jakubaszek, Brygida Kwiatkowska, Marzena Olesinska, Katarzyna Zycinska, Urszula Demkow
To determine the kinetics of extracellular-DNA degradation in vitro, a test which measures the activity of serum nucleases was used. Aliquots containing 620 ng of a human derived genomic DNA (11691112001, Roche) were incubated on black 96-well plates with DNase 1 and/or 10% human serum in DNase buffer (10 mM Tris-HCl pH 7.5, 10 mM MgCl2, 2 mM CaCl2, and 50 mM NaCl) for 3 h. In the next step, EDTA was added to a final concentration of 2 mM in order to stop DNase 1 activity. The DNA content in the solution was quantified using SYTOX green and a FLUOstar® Omega microplate reader and Omega Data Analysis software. DNA release values are presented as the RFUs, which reflect the free-DNA concentration. Micrococcal nuclease from Staphylococcus aureus (N3755-50UN, Sigma) was used as a positive control.
p300 Acetylates JHDM1A to inhibit osteosarcoma carcinogenesis
Published in Artificial Cells, Nanomedicine, and Biotechnology, 2019
Yongkun Wang, Baozhen Sun, Qiao Zhang, Hang Dong, Jingzhe Zhang
Mononucleosoma preparation was done in line with the previously described [19]. The nuclear pellet isolated from about 1 × 108 cells were mixed with lysis buffer to obtain oligonucleosomes. Then, the oligonucleosomes were digested with micrococcal nuclease (10 units/ml) for 10 min at 37 °C. After inactivation with 5 mM EDTA, the nuclease was centrifuged at 14000 rpm for 5 min. The supernatant was subjected to 10–40% glycerol gradient sedimentation. The mononucleosomes were derived from these supernatants through testing aliquots of fractions (digested with Proteinase K) on a 1% DNA agarose gel.