| Biochemical Analysis of Dimethyl Suberimidate-crosslinked Yeast Nucleosomes
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Author:
Date:
2018-03-20
[Abstract] Nucleosomes are the fundamental unit of eukaryotic chromosome packaging, comprised of 147 bp of DNA wrapped around two molecules of each of the core histone proteins H2A, H2B, H3, and H4. Nucleosomes are symmetrical, with one axis of symmetry centered on the homodimeric interaction between the C-termini of the H3 molecules. To explore the functional consequences of nucleosome symmetry, we designed an obligate pair of H3 heterodimers, termed H3X and H3Y, allowing us to compare cells with single or double H3 alterations. Our biochemical validation of the heterodimeric X-Y interaction included intra-nucleosomal H3 crosslinking using dimethyl suberimidate (DMS). Here, we provide a detailed protocol for the use of DMS to analyze yeast nucleosomes.
[摘要] 核小体是真核染色体包装的基本单元，由围绕核心组蛋白H2A，H2B，H3和H4中的每一个的两个分子包裹的147bp DNA组成。 核小体是对称的，一个对称轴以H3分子的C-末端之间的同源二聚体相互作用为中心。 为了探索核小体对称性的功能性后果，我们设计了一对特异性H3异二聚体，称为H3X和H3Y，使我们能够比较具有单一或双重H3改变的细胞。 我们对异二聚体X-Y相互作用的生物化学验证包括使用二甲基琥珀三酸酯（DMS）进行的核内H3交联。 在这里，我们提供了使用DMS来分析酵母核小体的详细方案。
【背景】组蛋白的翻译后修饰影响染色体生物学的各个方面，包括转录，复制，修复和重组。因为核小体包含每个核心组蛋白的两个拷贝，所以修饰可以是对称的（在两个H3尾部上的相同修饰，例如，在核小体内的两个H3尾部上的K27me（Voigt等人
对于单个核小体内H3X-H3Y相互作用的生化验证，我们生成了表达细菌生物素连接酶BirA，N-末端V5-标记的H3X和N-末端生物素接受表位标记的H3Y的酵母菌株（Beckett等人， 1999）。 ...
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| Ribosomal RNA N-glycosylase Activity Assay of Ribosome-inactivating Proteins
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Author:
Date:
2017-03-20
[Abstract] Ribosome-inactivating proteins (RIPs) are enzymes that irreversibly inactivate ribosomes as a consequence of their N-glycosylase (EC 3.2.2.22) activity. The enzyme cleaves the N-glycosidic bond between the adenine No. 4324 from the 28S rRNA and its ribose in rat ribosomes (or the equivalent adenine in sensitive ribosomes from other organisms). This adenine is located in the α-sarcin-ricin loop (SRL) that is crucial for anchoring the elongation factor (EF) G and EF2 on the ribosome during mRNA-tRNA translocation in prokaryotes and eukaryotes, respectively. RIPs have been isolated mainly from plants and examples of these proteins are ricin or Pokeweed Antiviral Protein (PAP). These proteins, either alone or as a part of immunotoxins, are useful tools for cancer therapy. The following ...
[摘要] 核糖体失活蛋白（RIP）是由于其N-糖基化酶（EC 3.2.2.22）活性而不可逆地灭活核糖体的酶。酶从28S rRNA的腺嘌呤编号4324和大鼠核糖体中的核糖（或来自其他生物体的敏感核糖体中的等同腺嘌呤）切割N-糖苷键。该腺嘌呤位于α-原丝素 - 蓖麻毒素环（SRL）中，这对于在原核生物和真核生物中的mRNA-tRNA易位期间分别在核糖体上锚定延伸因子（EF）G和EF2至关重要。 RIP主要从植物中分离出来，这些蛋白质的实例是蓖麻毒蛋白或者口服抗病毒蛋白（PAP）。这些蛋白质，单独或作为免疫毒素的一部分，是癌症治疗的有用工具。以下方案描述了当通过在聚丙烯酰胺凝胶上电泳在来自兔网织红细胞裂解物的RIP处理的脱嘌呤RNA在酸性苯胺存在下孵育时检测释放的RNA片段的方法。发布的片段（Endo的片段）是RIP的动作的诊断。
Endo和Tsurugi首先在大鼠核糖体中描述了RIP对真核28S rRNA的N-糖基化酶活性，蓖麻毒素（Endo and ...
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| ACE-score-based Analysis of Temporal miRNA Targetomes During Human Cytomegalovirus Infection Using AGO-CLIP-seq
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Author:
Date:
2016-04-20
[Abstract] Although temporal regulation of gene expression during the course of infection is known to be critical for determining the outcome of host-virus interactions, systematic temporal analysis of the miRNA targetomes during productive viral infection has been technically challenging due to the large range of miRNA-mRNA cross-talks at the host-virus interface. High-confidence quantifying models of the suppression efficacy in targeting sites by integrating bioinformatics with Argonaute-crosslinking and immunoprecipitation followed by high-throughput sequencing (AGO-CLIP-seq) (Chi et al., 2009) data have been poorly developed. To accurately identify miRNA target sites and calculate the targeting efficacy of miRNA-target interactions, we developed a new bioinformatic quantitation method, ...
[摘要] 尽管已知在感染过程中基因表达的时间调节对于确定宿主 - 病毒相互作用的结果是至关重要的，但是在生产性病毒感染期间对miRNA targetomes的系统时间分析在技术上是具有挑战性的，因为大范围的miRNA- mRNA在主机 - 病毒接口交叉对话。数据通过将生物信息学与Argonaute-交联和免疫沉淀接着高通量测序（AGO-CLIP-seq）数据（Chi等人，2009）数据结合，已经不发达。为了准确地鉴定miRNA靶位点并计算miRNA-靶相互作用的靶向效果，我们开发了新的生物信息学定量方法，即AGO-CLIP-seq富集（ACE） - 评分算法（Kim等， 2015）。在我们的AGO-CLIP-seq分析中包括未感染的对照可以显着提高病毒或人miRNA的真实靶位点识别的准确性，并且在我们的ACE评分方法中提取生产性人巨细胞病毒（HCMV）感染期间的生理学显着变化。因此，我们建议我们新的基于ACE评分的方法可以应用于各种miRNA targetome研究，这将在其他类型的时间背景下进行，如发展阶段，细胞因子或病原体的免疫刺激和其他病毒。
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