| EmPC-seq: Accurate RNA-sequencing and Bioinformatics Platform to Map RNA Polymerases and Remove Background Error
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Author:
Date:
2021-02-20
[Abstract] Transcription errors can substantially affect metabolic processes in organisms by altering the epigenome and causing misincorporations in mRNA, which is translated into aberrant mutant proteins. Moreover, within eukaryotic genomes there are specific Transcription Error-Enriched genomic Loci (TEELs) which are transcribed by RNA polymerases with significantly higher error rates and hypothesized to have implications in cancer, aging, and diseases such as Down syndrome and Alzheimer’s. Therefore, research into transcription errors is of growing importance within the field of genetics. Nevertheless, methodological barriers limit the progress in accurately identifying transcription errors. Pro-Seq and NET-Seq can purify nascent RNA and map RNA polymerases along the genome but cannot be ...
[摘要] [摘要]转录错误可通过改变表观基因组并引起mRNA的错误整合而严重影响生物体内的代谢过程,从而将其翻译为异常的突变蛋白。此外,真核基因组内有特定转录错误富集的基因组基因座(TEELs),它们由RNA聚合酶与显著更高的错误率转录并推测为具有影响在癌症,老化和疾病例如唐氏综合征和阿尔茨海默'秒。因此,在遗传学领域对转录错误的研究越来越重要。尽管如此,方法上的障碍限制了准确识别转录错误的进展。Pro-Seq和NET-Seq可以沿基因组纯化新生RNA并绘制RNA聚合酶,但不能用于鉴定转录突变。在这里,我们本背景误差模型耦合的精密核圆形测序上运行(EMPC -SEQ),一种方法COMBIN荷兰国际集团测定和圆形测序核上运行与背景误差模型精确地检测新生转录错误和有效地辨别TEELs基因组中。
[背景]核糖核苷酸错掺导致的转录错误在所有活生物体中无处不在(Carey,2015)。假设每个信使RNA(mRNA)可以翻译2-4千次(Schwanhausser et al。,2011),并且许多特殊RNA在给定时间每个细胞仅表达一次(Islam et al。,2011; Pelechano et al。,2011)。,2010),即使是关键残基的单个转录错误也会使特定蛋白质的表达产生很大差异。另外,转录错误可加速蛋白质聚集,导致人类中与年龄有关的疾病(van ...
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| K+ Release Assay and K+ Measurement in Oocyte Assay
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Author:
Date:
2020-11-05
[Abstract] The Xenopus oocyte is a powerful system for the exogenous expression and functional characterization of plant membrane transport proteins. Until now, a number of potassium transporters and channels have been identified in oocytes expression system by the two-electrode voltage clamp technology. It is difficult to characterize K+/H+ anti-transporters, especially, electroneutral transporter. The K+ efflux assay system enables easy, fast, large-scale measurement of the transporters activity without two-electrode voltage clamp technology. This protocol describes a technique to measure the efflux activity of potassium transporter in oocytes expressing system.
[摘要] [摘要]爪蟾卵母细胞是针对外源表达和植物膜转运蛋白的功能表征一个强大的系统。迄今为止,通过两电极电压钳技术已经在卵母细胞表达系统中发现了许多钾转运蛋白和钾通道。很难表征K + / H +反转运蛋白,尤其是电中性转运蛋白。K +外排测定系统无需两电极电压钳技术即可轻松,快速,大规模地测量转运蛋白的活性。该协议描述了一种测量卵母细胞表达系统中钾转运蛋白外排活性的技术。
[背景[非洲爪蟾卵母细胞中积累了酶,蛋白质和细胞器,这些都可以被利用来生产大量,正确翻译后修饰,正确定点的外来蛋白质(Miller and Zhou,2000)。因此,卵母细胞通过使用两电极电压钳技术提供了一种有效的表达系统,以功能上表征植物膜蛋白。在卵母细胞中表达的第一个植物钾转运蛋白是K +通道(Cao等,1992)。此后,大部分钾通道和转运已经在卵母细胞的研究(Schachtman等人,1994; V é RY等人,1994; Wang和吴,2013年)。但是一些K + / H +反转运蛋白是电中性的,两电极电压钳技术无法表征这些转运蛋白。为了克服这个问题,我们采用了一种新的方法来确定这些转运蛋白的活性,方法是测量孵育前后培养基中钾含量的变化。细胞内ķ +浓度为约70 - 150mM的(韦伯,1999年)。由表达的K +转运蛋白介导的K +外排可引起细胞内和细胞外K ...
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| Extracellular RNA Isolation from Biofilm Matrix of Pseudomonas aeruginosa
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Author:
Date:
2020-11-05
[Abstract] Most bacteria in natural ecosystems form biofilms-a bacterial community, surrounded by a polymer matrix that consists mostly of exopolysaccharides, proteins, and nucleic acids. Extracellular RNA as a matrix component is involved in biofilm formation-the fact that was confirmed by direct detection of extracellular RNA in the biofilm matrix, and by an interruption of the biofilm's structure with RNases. Number of protocols describing isolation of RNA from biofilm matrix are limited and usually involve uncommon equipment and reagents. Here we describe simple method for extracellular RNA isolation from biofilm matrix using basic laboratory reagent and equipment. Key steps of the protocol include separation of matrix and bacterial cells with high ionic solution of NaCl, RNA precipitation with ...
[摘要] [摘要]自然生态系统中的大多数细菌形成生物膜 –一个细菌群落,周围环绕着聚合物基质,该基质主要由胞外多糖,蛋白质和核酸组成。细胞外RNA作为基质成分参与生物膜的形成,这一事实已通过直接检测生物膜基质中的细胞外RNA以及通过RNase破坏生物膜结构而得到证实。。描述从生物膜基质中分离RNA的方案数量有限,通常涉及不常见的设备和试剂。在这里,我们描述了使用基本的实验室试剂和设备从生物膜基质分离细胞外RNA的简单方法。该方案的关键步骤包括用高离子浓度的NaCl溶液分离基质和细菌细胞,用LiCl沉淀RNA,并选择使用廉价的色谱柱进行质粒DNA分离,而不是使用专门的RNA试剂盒进行纯化。所描述的方案允许在不到一天的时间内(不包括生物膜生长的时间)分离适用于进一步的分子生物学程序(例如测序,RT-PCR和克隆)的细胞外RNA。
[背景]生物膜基质可抵抗不同的影响(抗菌药物,消毒剂,机械力),并为协调协调不同过程创造了环境(Svenningsen,2018年)。RNA存在于细胞外生物膜基质中,并形成RNA-DNA的主要交联弹性共聚物(Seviour等,2019)。用核糖核酸酶处理生物膜导致生物膜质量的重大损失,并强调了RNA对于维持生物膜完整性的重要性(Lee等人,2019)。同时,RNA在生物膜基质中的来源和作用仍未得到很好的研究。
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