| Mating Based Split-ubiquitin Assay for Detection of Protein Interactions
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Author:
Date:
2017-05-05
[Abstract] The mating based split-ubiquitin (mbSUS) assay is an alternative method to the classical yeast two-hybrid system with a number of advantages. The mbSUS assay relies on the ubiquitin-degradation pathway as a sensor for protein-protein interactions, and it is suitable for the determination of interactions between full-length proteins that are cytosolic or membrane-bound. Here we describe the mbSUS assay protocol which has been used for detecting the interaction between K+ channel and SNARE proteins (Grefen et al., 2010 and 2015; Zhang et al., 2015 and 2016)
[摘要] 基于交配的分ubiquitin（mbSUS）测定是具有许多优点的经典酵母双杂交系统的替代方法。 mbSUS测定依赖于泛素降解途径作为蛋白质 - 蛋白质相互作用的传感器，并且它适用于测定细胞溶质或膜结合的全长蛋白质之间的相互作用。在这里，我们描述了已经用于检测K + 通道和SNARE蛋白之间的相互作用的mbSUS测定方案（Grefen等人，2010和2015; Zhang 等等，2015和2016）
背景 图1是mbSUS测定的概况。泛素部分被分成两半，N末端半突变（NubG）以避免重组。泛素部分（Cub）的C末端一半与转录报告基因复合物PLV（Protein A-LexA-VP16）连接。两种蛋白质（X和Y）分别与NubG和CubPLV融合产生蛋白质 - 蛋白质相互作用分析系统。转化后，酵母菌株THY.AP5含有NubG-X融合蛋白，而酵母菌株THY.AP4含有Y-CubPLV融合蛋白。在交配后，在二倍体酵母中，如果蛋白质X和Y彼此相互作用，则将重新组装功能性泛素，这导致PLV的切割。释放的转录蛋白复合物PLV可以开启报告基因（ADE2，HIS3），并允许酵母生长在选择性培养基上。
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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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