Author:
Date:
2018-10-05
[Abstract] Ribonucleoprotein particles (mRNPs) are complexes consisting of mRNAs and RNA-binding proteins (RBPs) which control mRNA transcription localization, turnover, and translation. Some mRNAs within the mRNPs have been shown to undergo degradation or storage. Those transcripts can lack general mRNA elements, like the poly(A) tail or 5’ cap structure, which prevent their identification through the application of widely-used approaches like oligo(dT) purification. Here, we describe a modified cross-linking affinity purification protocol (cCLAP) based on existing cross-linking and immunoprecipitation (CLIP) methods to isolate mRNAs which could be deadenylated, decapped and/or partially degraded in mRNPs, opening the possibility to detect different types of non-coding RNAs (ncRNAs). Once isolated, ...
[摘要] 核糖核蛋白颗粒(mRNP)是由mRNA和RNA结合蛋白(RBP)组成的复合物,其控制mRNA转录定位,转换和翻译。已显示mRNP内的一些mRNA经历降解或储存。那些转录物可能缺乏一般的mRNA元件,如poly(A)尾或5'帽结构,这通过应用广泛使用的方法如oligo(dT)纯化来阻止它们的鉴定。在这里,我们描述了基于现有的交联和免疫沉淀(CLIP)方法的修饰的交联亲和纯化方案(cCLAP),以分离mRNP中可被去腺苷酸化,去除和/或部分降解的mRNA,从而开启了检测不同的可能性。非编码RNA(ncRNA)的类型。分离后,将RNA进行衔接子连接,然后进行下一代测序(NGS)。由于快速有效的交联和淬灭步骤,该方案也适用于瞬时诱导的mRNP颗粒。实例包括由外在应激物触发的处理体(PB)或应力颗粒(SG)。其重现性和广泛应用使该方案成为研究特定RNP的RNA组成的有用且有力的工具。 【背景】mRNP内转录物的表征对于理解细胞转录和转录后过程至关重要。通过交联和免疫沉淀,然后通过RNA-Seq从mRNP颗粒中分离RNA已经成为鉴定mRNA靶标的常用方法(Tagwerker et al。,2006; Hafner et al。,2010; Kishore et al。,2011)。 ...
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Author:
Date:
2017-07-20
[Abstract] Cyanobacteria synthesize a variety of chemically-different, high-value biopolymers such as glycogen (polyglucose), poly-β-hydroxybutyrate (PHB), cyanophycin (polyamide of arginine and aspartic acid) and volutin (polyphosphate) under excess conditions. Especially under unbalanced C to N ratios, glycogen and in some cyanobacterial genera also PHB are massively accumulated in the progression of the general nitrogen stress response. Several different technologies have been established for in situ and in vitro PHB analysis from different microbial sources. In this protocol, a rapid and reliable spectrophotometric method is described for PHB quantification in the cyanobacterium Synechocystis sp. PCC 6803 upon nitrogen deprivation as described in (Damrow et ...
[摘要] 蓝细菌合成各种化学上不同的高价值生物聚合物,如糖原(polyglucose),poly-β-β-羟基丁酸酯(PHB),蓝藻素(精氨酸和天冬氨酸的聚酰胺)和挥发物(多磷酸盐) 在超额条件下。 特别是在不平衡的C至N比下,糖原和一些蓝藻属中,PHB在一般氮应激反应进程中大量积累。 已经针对不同微生物来源的原位实验和/或从体外分析,建立了几种不同的技术。 在该协议中,描述了用于蓝藻(Stechocystis)的PHB定量的快速可靠的分光光度法。 如(Damrow等人,2016)中描述的氮缺乏的PCC 6803。 【背景】非重氮营养蓝细菌,例如集胞藻(Synechocystis) PCC 6803通过漂白来解决缺乏组合的氮源,这是一种被称为褪绿的过程(Allen和Smith,1969)。这种驯化反应的特征在于四个主要的结构和形态变化:(i)类囊体层之间的电子致密糖原夹杂物(直径约40nm)的大量积累伴随着(ii)藻糖酵母天线复合物的降解, (iii)类囊体膜层的拆卸,包括数量减少和包装密度,和(iv)形成不同的电子透明PHB颗粒(直径约400-500nm)(Damrow等人,2016)。由于不存在分解代谢酶和PHB缺陷型突变体的明显表型,所以在几种物种中合成的蓝细菌PHB代谢的生理功能是相当不透明的(Beck等人,2012; van ,2010; Damrow等人,2016; ...
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