Preparation of Yeast tRNA Sample for NMR Spectroscopy
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Author:
Date:
2020-06-20
[Abstract] Transfer RNAs (tRNAs) are heavily decorated with post-transcriptional modifications during their biosynthesis. To fulfil their functions within cells, tRNAs undergo a tightly controlled biogenesis process leading to the formation of mature tRNAs. In addition, functions of tRNAs are often modulated by their modifications. Although the biological importance of post-transcriptional RNA modifications is widely appreciated, methods to directly detect their introduction during RNA biosynthesis are rare and do not easily provide information on the temporal nature of events. To obtain information on the tRNA maturation process, we have developed a methodology, using NMR as a tool to monitor tRNA maturation in a non-disruptive and continuous fashion in cellular extracts. By following the ...
[摘要] [摘要 ] 转移RNA(tRNA )在其生物合成过程中大量修饰有转录后修饰。为了在细胞内履行其功能,tRNA 经历了严格控制的生物生成过程,导致了成熟的tRNA 的形成。此外,tRNA的功能通常是虽然转录后修饰RNA的生物学重要性被广泛理解,方法直接检测它们的RNA生物合成过程中引入是罕见的,并且不容易提供上events.To的时间特性信息获取的信息的tRNA 成熟 在此过程中,我们开发了一种方法,使用NMR作为监测细胞提取物中tRNA 成熟的无中断和连续方式。通过模型酵母tRNA 的时间分辨NMR 成熟,我们发现修饰是该方法的实施需要对具有不同修饰状态的tRNA 样品进行NMR光谱学分析,以鉴定各个修饰的NMR特征。此处将介绍用于NMR光谱分析修饰途径的tRNA 样品的生产,并在酵母tRNA Phe 上进行例证,但可以通过更改构建体的序列扩展到其他tRNA 。该方案描述了未修饰的生产通过体外转录获得tRNA 样品,并通过在大肠杆菌中重组表达tRNA 产生修饰的tRNA 样品。大肠杆菌。
[背景 ] 在生活的各个领域,合成和RNA的成熟包括在特定地点的核苷酸的转录后的化学修饰。在不同的RNA家族,tRNA基因不仅显示最高多种化学修饰,而且密度最高每转录修饰(〜中经修饰的核苷酸8-25%的tRNA 各种生物体的)(Boccaletto ...
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Selective Isolation of Retroviruses from Extracellular Vesicles by Intact Virion Immunoprecipitation
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Author:
Date:
2018-09-05
[Abstract] There exists a wide variety of techniques to isolate and purify viral particles from cell culture supernatants. However, these techniques vary greatly in ease of use, purity, yield and impact on viral structural integrity. Most importantly, it is becoming evident that secreted extracellular vesicles (EVs) co-purify with retroviruses using nearly all purification methods due to nearly indistinguishable biophysical characteristics such as size, buoyant density and nucleic acid content. Recently, our group has illustrated a means of isolating intact and highly enriched retroviral virions from EV-containing cell supernatants using an immunoprecipitation approach targeting the viral envelope glycoprotein of the Moloney Murine Leukemia Virus (Renner et al., 2018). This technique, that ...
[摘要] 存在多种从细胞培养上清液中分离和纯化病毒颗粒的技术。然而,这些技术在易用性,纯度,产量和对病毒结构完整性的影响方面差异很大。最重要的是,由于几乎无法区分的生物物理特征,例如大小,浮力密度和核酸含量,使用几乎所有纯化方法分泌的细胞外囊泡(EV)与逆转录病毒共同纯化变得明显。最近,我们小组已经阐明了一种利用针对Moloney鼠白血病病毒的病毒包膜糖蛋白的免疫沉淀方法从含有EV的细胞上清液中分离完整和高度富集的逆转录病毒病毒颗粒的方法(Renner et al。, 2018)。这种技术,我们称之为完整的病毒粒子免疫沉淀(IVIP),使我们能够表征这些逆转录病毒表面上表位的可及性,并评估病毒包膜中病毒编码的整合膜蛋白Glycogag(gPr80)的方向。该方案的正确实施使得能够快速,简单且可重复地制备完整且高度纯化的逆转录病毒颗粒,其没有可检测的EV污染物。
【背景】广泛使用的分离逆转录病毒的方法,如人类免疫缺陷病毒(HIV)和小鼠白血病病毒(MLV),包括沉淀,色谱,超滤,超速离心,以及各种其他粒子分离方法(评论在Nestola et al。,2015)。虽然每种技术都有其特定的优点,缺点和局限性,但所有方法的共同关注点是细胞分泌的细胞外囊泡(EV)的共同纯化。
EV构成由所有细胞类型分泌的膜衍生囊泡的异质群体(Yanez-Mo ...
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