| 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. ...
[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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| Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing
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Author:
Date:
2017-06-05
[Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence using specific antibodies. Unlike biochemical and NGS-based methods, DNA combing provides unique information on cell-to-cell variations in DNA replication profiles, including initiation and elongation. Finally, this assay can be used to monitor the effect of DNA lesions on fork progression, ...
[摘要] DNA梳理方法允许在沿着硅烷涂覆的玻璃盖玻片拉伸的单个DNA分子的水平上分析DNA复制。在DNA提取前，进行的DNA合成用胸苷的卤化类似物标记。使用特异性抗体通过免疫荧光可视化复制轨迹。与生物化学和基于NGS的方法不同，DNA梳理提供了DNA复制谱中细胞间细胞变化的独特信息，包括引发和延长。最后，该测定可用于监测DNA损伤对叉进展，停止和重新启动的影响。
背景 在称为复制起点的真核染色体上的数千个位点处启动DNA合成。原始激活遵循由检查点激酶和染色质的表观遗传修饰（Prioleau和MacAlpine，2016）控制的定义良好的复制计时程序。复制叉在正常S阶段经常停顿。叉停止是由多个事件引起的，例如DNA损伤，紧密结合的蛋白质复合物和高表达基因的转录（Tourriere和Pasero 2007; Zeman and Cimprich，2013）。真核生物已经制定了不同的策略来应对这种复制压力，包括修复机制来重新启动捕获的叉子和激活休眠复制起源以抢救终末抓捕的叉。
＆NBSP; ...
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| Large-scale Phenotypic Profiling of Gene Deletion Mutants in Candida glabrata
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Author:
Date:
2015-07-20
[Abstract] Here, we describe a method enabling the phenotypic profiling of genome-scale deletion collections of fungal mutants to detect phenotypes for various stress conditions. These stress conditions include among many others antifungal drug susceptibility, temperature-induced and osmotic as well as heavy metal or oxidative stress. The protocol was extensively used to phenotype a collection of gene deletion mutants in the human fungal pathogen Candida glabrata (C. glabrata) (Schwarzmüller et al., 2014).
[摘要] 在这里，我们描述了一种使表型分析的基因组规模删除集合的真菌突变体检测表型的各种应力条件的方法。 这些胁迫条件包括许多其它抗真菌药物敏感性，温度诱导和渗透以及重金属或氧化应激。 该方案广泛用于表达人真菌病原体光滑假丝酵母（ glabrata ）中基因缺失突变体的集合（Schwarzmüller等人 >，2014）。
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