| Real-time Base Excision Repair Assay to Measure the Activity of the 8-oxoguanine DNA Glycosylase 1 in Isolated Mitochondria of Human Skin Fibroblasts
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Author:
Date:
2021-03-20
[Abstract] 7,8-dihydro-8-oxoguanine (8-oxoG) is one of the most common and mutagenic oxidative DNA damages induced by reactive oxygen species (ROS). Since ROS is mainly produced in the inner membranes of the mitochondria, these organelles and especially the mitochondrial DNA (mtDNA) contained therein are particularly affected by this damage. Insufficient elimination of 8-oxoG can lead to mutations and thus to severe mitochondrial dysfunctions. To eliminate 8-oxoG, the human body uses the enzyme 8-oxoguanine DNA glycosylase 1 (OGG1), which is the main antagonist to oxidative damage to DNA. However, previous work suggests that the activity of the human OGG1 (hOGG1) decreases with age, leading to an age-related accumulation of 8-oxoG. A better understanding of the exact mechanisms of hOGG1 could lead ...
[摘要] [摘要] 7,8-二氢-8-氧鸟嘌呤（8-oxoG）是由活性氧（ROS）引起的最常见且诱变的氧化DN A损伤之一。由于ROS主要在线粒体的内膜中产生，因此这些细胞器，特别是其中所含的线粒体DNA（mtDNA）受到这种损害的特别影响。消除8-oxoG可能会导致突变，从而导致严重的线粒体功能障碍。为了消除8-oxoG，人体使用了8-氧代鸟嘌呤DNA糖基化酶1（OGG1），它是DNA氧化损伤的主要拮抗剂。但是，先前的研究表明，人类OGG1的活性（h OGG1）随着年龄的增长而减少，导致与年龄相关的8-oxoG积累。更好地了解hOGG1的确切机制可能会导致发现新的靶标，因此对于开发预防性疗法具有重要意义。因此，我们开发了一种实时碱基切除修复测定法，该测定法采用了专门设计的双链报告寡核苷酸来测量分离的线粒体裂解物中hOGG1的活性。这里介绍的该系统与经典测定法不同，在经典测定法中，可以通过实时测量hOGG1活性通过变性丙烯酰胺凝胶进行终点测定。另外，为了确定该双功能酶的每个酶促步骤的活性（N-糖基化酶和AP-裂解酶活性），还可以进行解链曲线分析。使用各种离心步骤从人成纤维细胞中分离线粒体后，将其裂解，然后与专门设计的报告寡核苷酸一起孵育。hOGG1活性的后续测量是在常规实时PCR系统中进行的。
[背景]人体是永久的损害案例。每天约10 ...
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| Tethered Chromosome Conformation Capture Sequencing in Triticeae: A Valuable Tool for Genome Assembly
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Author:
Date:
2018-08-05
[Abstract] Chromosome conformation capture sequencing (Hi-C) is a powerful method to comprehensively interrogate the three-dimensional positioning of chromatin in the nucleus. The development of Hi-C can be traced back to successive increases in the resolution and throughput of chromosome conformation capture (3C) (Dekker et al., 2002). The basic workflow of 3C consists of (i) fixation of intact chromatin, usually by formaldehyde, (ii) cutting the fixed chromatin with a restriction enzyme, (iii) religation of sticky ends under diluted conditions to favor ligations between cross-linked fragments or those between random fragments and (iv) quantifying the number of ligations events between pairs of genomic loci (de Wit and de Laat, 2012). In the original 3C protocol, ligation frequency was ...
[摘要] 染色体构象捕获测序（Hi-C）是一种全面询问细胞核中染色质三维定位的有效方法。 Hi-C的发展可以追溯到染色体构象捕获的分辨率和通量的连续增加（3C）（Dekker et al。，2002）。 3C的基本工作流程包括（i）通常用甲醛固定完整的染色质，（ii）用限制酶切割固定的染色质，（iii）在稀释条件下重新连接粘性末端，以促进交联片段之间的连接或随机片段之间的那些和（iv）量化基因组基因座对之间的连接事件的数量（de Wit和de Laat，2012）。在最初的3C方案中，通过半定量PCR扩增对应于少量基因组位点（“一对一”）的选定连接接头来测量连接频率（Dekker et al。，2002 ）。然后，染色体构象捕获芯片（4C）和染色体构象捕获碳复制（5C）技术扩展3C以分别以“一对多”或“多对多”方式计算结扎事件。 Hi-C（Lieberman-Aiden et al。，2009）最终将3C与下一代测序相结合（Metzker，2010）。此处，在再连接之前，用生物素标记的核苷酸类似物填充粘性末端以在后续步骤中富集具有连接连接的片段。然后对Hi-C文库进行高通量测序，并将得到的读数映射到参考基因组，允许以“多对多”方式确定接触概率，其分辨率仅受限制性位点的分布限制和阅读深度。 Hi-C的首次应用是阐明人类基因组中的全球染色质折叠原理（Lieberman-Aiden et ...
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| Investigating the Assembly Status of the Plastid Encoded Polymerase Using BN-PAGE and Sucrose Gradient Centrifugation
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Author:
Date:
2016-07-20
[Abstract] The plastid encoded polymerase (PEP) represents a major transcription machinery in mature chloroplasts (Liere et al., 2011; Zhelyazkova et al., 2012). The proper assembly of this multi-subunit complex is important for plant growth and development (Pfalz and Pfannschmidt, 2013). The PEP polymerase can be purified from soluble and from membrane-bound (also named transcriptionally active chromosome, TAC) fractions. Blue Native polyacrylamide gel electrophoresis (BN-PAGE) and sucrose gradient sedimentation followed by immunoblot analyses is used to detect the status of the PEP complex assembly.
[摘要] 质体编码聚合酶（PEP）代表成熟叶绿体中的主要转录机制（Liere等人，2011; Zhelyazkova等人，2012）。 这种多亚基复合物的正确装配对于植物生长和发育是重要的（Pfalz和Pfannschmidt，2013）。 PEP聚合酶可以从可溶性和膜结合（也称为转录活性染色体，TAC）级分中纯化。 蓝色使用天然聚丙烯酰胺凝胶电泳（BN-PAGE）和蔗糖梯度沉淀，随后进行免疫印迹分析来检测PEP复合物组装体的状态。
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