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TES

TES

Company: Sigma-Aldrich
Catalog#: T1375
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ChIP-seq Experiment and Data Analysis in the Cyanobacterium Synechocystis sp. PCC 6803
Author:
Date:
2018-06-20
[Abstract]  Nitrogen is an essential nutrient for all living organisms. In cyanobacteria, a group of oxygenic photosynthetic bacteria, nitrogen homeostasis is maintained by an intricate regulatory network around the transcription factor NtcA. Although mechanisms controlling NtcA activity appear to be well understood, the sets of genes under its control (i.e., its regulon) remain poorly defined. In this protocol, we describe the procedure for chromatin immunoprecipitation using NtcA antibodies, followed by DNA sequencing analysis (ChIP-seq) during early acclimation to nitrogen starvation in the cyanobacterium Synechocystis sp. PCC 6803 (hereafter Synechocystis). This protocol can be extended to analyze any DNA-binding protein in cyanobacteria for which suitable antibodies ... [摘要]  氮是所有生物体的必需营养素。 在蓝细菌中,一组含氧光合细菌通过围绕转录因子NtcA的错综复杂的调节网络维持氮稳态。 尽管控制NtcA活性的机制似乎已被很好地理解,但其控制下的基因集(即它的调节子)仍然没有很好的定义。 在该协议中,我们描述了使用NtcA抗体进行染色质免疫沉淀的过程,随后在蓝藻Synechocystis sp。早期适应氮饥饿期间进行DNA测序分析(ChIP-seq)。 PCC 6803(以下简称<集气囊)。 该协议可以扩展到分析蓝细菌中存在合适抗体的任何DNA结合蛋白。

【背景】为了维持体内平衡,细菌经常需要响应环境变化来调整基因表达。许多这些调整是由转录因子(TF)控制的,这些转录因子可以感知代谢信号并激活或抑制目标基因。然而,反映传统上费力的任务来表征TFs在体内的活性和范围,我们对它们在细菌中的结合位点的了解仍然有限。直到最近,染色质免疫沉淀与高通量测序分析的结合为快速确定基因组水平调节子打开了大门。特别是,ChIP-seq使用下一代测序(NGS)的能力来并行识别大量DNA序列。与微阵列相比,ChIP-seq的一个有吸引力的特征是对某些区域如启动子序列没有限制,并且可以研究整个基因组的TF结合位点。

在蓝细菌中,氮同化和代谢的全球调节剂是NtcA,属于CRP(cAMP受体蛋白)家族的TF(Herrero等人,2001)。在集胞蓝细菌中,NtcA通过将二聚体结合至包含共有序列GTAN ...

Isolation of Tomato Fruit Chromoplasts and Determination of ATP Levels
Author:
Date:
2014-08-05
[Abstract]  It has recently been reported that tomato fruit chromoplasts can synthesize ATP de novo using an ATP synthase complex harboring an atypical γ-subunit which is also present in a variety of plant species. However many aspects related with the biochemical processes underlying this process remain largely unknown. Here we describe detailed protocols for the isolation of tomato fruit chromoplasts and the determination of ATP levels (end-point measurements) and ATP synthesis rates (kinetic measurements) in these organelles using bioluminescent luciferin/luciferase based assays. [摘要]  最近报道了番茄果实色素可以使用含有非典型γ亚基的ATP合酶复合物来合成ATP,所述亚基也存在于多种植物物种中。 然而,与这个过程基础的生化过程相关的许多方面仍然很大程度上未知。 在这里我们描述了隔离番茄果实色质体和ATP水平(终点测量)和ATP合成率(动力学测量)在这些细胞器中使用基于生物发光荧光素/荧光素酶的测定的确定详细协议。

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