| DNA Fiber Assay upon Treatment with Ultraviolet Radiations
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Author:
Date:
2017-06-05
[Abstract] Genome stability is continuously challenged by a wide range of DNA damaging factors. To promote a correct DNA repair and cell survival, cells orchestrate a coordinated and finely tuned cascade of events collectively known as the DNA Damage Response (DDR). Ultra Violet (UV) rays are among the main environmental sources of DNA damage and a well recognized cancer risk factor. UV rays induce the formation of toxic cyclobutane-type pyrimidine dimers (CPD) and [6-4]pyrimidine-pyrimidone (6-4PP) photoproducts which trigger the activation of the intra-S phase cell cycle checkpoint (Kaufmann, 2010) aimed at preventing replication fork collapse, late origin firing, and stabilizing fragile sites (Branzei and Foiani, 2009). To monitor the activation of the intra-S phase checkpoint in response to UV ...
[摘要] 基因组稳定性不断受到DNA损伤因素的广泛影响。为了促进正确的DNA修复和细胞存活，细胞协调统一称为DNA损伤反应（DDR）的协调和精细调整的事件级联。超紫外线（UV）是DNA损伤的主要环境来源之一，也是公认的癌症危险因素。紫外线诱导形成毒性环丁烷型嘧啶二聚体（CPD）和[6-4]嘧啶嘧啶酮（6-4PP）光产物，其触发S期细胞周期检查点的活化（Kaufmann，2010），目的在于防止复制叉崩溃，晚期起火和稳定脆弱场所（Branzei和Foiani，2009）。为了监测响应于UV型C（UVC）暴露的S-S相检查点的激活，DNA纤维测定可用于分析新的起始点和DNA合成速率（Jackson等， ，1998; Merrick等人，2004; Alfano等人，2016）。 DNA纤维测定技术在90年代被设想，然后通过使用并入新生DNA链中的胸苷类似物（如CldU和IdU）进一步开发。通过用这些类似物以连续模式处理细胞，可以通过携带不同荧光团的特异性抗体来观察细胞，可以监测复制叉活性并评估其如何受到紫外辐射或其他试剂的影响。
背景 ...
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| MNase Digestion for Nucleosome Mapping in Neurospora
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Author:
Date:
2016-06-05
[Abstract] Digestion of chromatin by micrococcal nuclease MNase followed by high throughput sequencing allows us to determine the location and occupancy of nucleosomes on the genome. Here in this protocol we have described optimized conditions of MNase digestion of filamentous fungus Neurospora crassa chromatin without a requirement of a nuclear fractionation step.
[摘要] 通过微球菌核酸酶MNase消化染色质，然后高通量测序允许我们确定核小体在基因组上的位置和占据。 在这个协议中，我们描述了MNase消化丝状真菌粗糙链孢霉染色体的优化条件，而不需要核分馏步骤。
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| RNA Chromatin Immunoprecipitation (RNA-ChIP) in Caenorhabditis elegans
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Author:
Date:
2014-12-20
[Abstract] The RNA chromatin immunoprecipitation assay (RNA-ChIP) allows detection and quantification of RNA–protein interactions using in vivo cross-linking with formaldehyde followed by immunoprecipitation of the RNA–protein complexes. Here we describe the RNA–ChIP protocol that we have adapted for Caenorhabditis elegans (C. elegans) to detect interaction between the nuclear Argonaute CSR-1 (chromosome segregation and RNAi deficient) protein and its target nascent RNAs. We have used a transgenic strain expressing a recombinant long isoform of CSR-1 protein fused with N-terminal 3x FLAG epitope.
[摘要] RNA染色质免疫沉淀测定（RNA-ChIP）允许使用体内与甲醛交联，随后RNA-蛋白复合物的免疫沉淀来检测和定量RNA-蛋白质相互作用。 在这里我们描述我们已经适应于秀丽隐杆线虫（ C. elegans ）的RNA-ChIP协议以检测核Argonaute CSR-1（染色体分离和RNAi缺陷 ）蛋白及其目标新生RNA。 我们使用表达与N-末端3x FLAG表位融合的CSR-1蛋白的重组长同种型的转基因菌株。
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