| Expression and Purification of Cyanobacterial Circadian Clock Protein KaiC and Determination of Its Auto-phosphatase Activity
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Author:
Date:
2017-02-20
[Abstract] Circadian rhythms are biological processes displaying an endogenous oscillation with a period of ~24 h. They allow organisms to anticipate and get prepared for the environmental changes caused mainly by the rotation of Earth. Circadian rhythms are driven by circadian clocks that consist of proteins, DNA, and/or RNA. Circadian clocks of cyanobacteria are the simplest and one of the best studied models. They contain the three clock proteins KaiA, KaiB, and KaiC which can be used for in vitro reconstitution experiments and determination of the auto-phosphatase activity of KaiC as described in this protocol.
[摘要] 昼夜节律是显示内源性振荡的生物过程，周期为〜24小时。它们允许生物体预期并准备好主要由地球旋转引起的环境变化。昼夜节律由由蛋白质，DNA和/或RNA组成的昼夜节律钟驱动。蓝藻的昼夜节律时钟是最简单的研究模型之一。它们含有三种时钟蛋白质KaiA，KaiB和KaiC，其可用于体外重组实验和本方案所述的KaiC的自磷酸酶活性的测定。
背景 行星地球的旋转导致〜24小时的昼夜振荡。为了适应并有效地利用环境的这种节奏变化，大多数（如果不是全部）生物体具有约24小时的内源性活动节律，这被称为昼夜节律。昼夜节律为这些生物提供进化优势。昼夜节律的长期破坏是非常有害的（Ma et al。，2013）。在人类中，包括癌症，高血压和睡眠障碍在内的许多疾病与昼夜节律紊乱密切相关（Shi等人，2013; Roenneberg和Merrow，2016）。
＆nbsp;昼夜节律由称为昼夜节律钟的内生节律发生器控制。功能性昼夜节律钟具有三个功能：接受环境信息，将环境提示转变为振荡信号，并将这些信号转发到下游调制器（Pattanayak和Rust，2014）。蓝细菌是具有良好研究的昼夜节律钟的最简单的生物体，其中振荡发生器由三种蛋白质控制：KaiA，KaiB和KaiC（Mackey等人，2011; Johnson＆amp; et al。 ...
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| Expression and Purification of the GRAS Domain of Os-SCL7 from Rice for Structural Studies
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Author:
Date:
2017-02-05
[Abstract] GRAS proteins, named after the first three members GAI, RGA and SRC, has been found in 294 embryophyta species and is represented by 1,035 sequences. They belong to a plant-specific protein family and play essential roles in plant growth and development. Proteins in this family are defined as minimally containing a conserved GRAS domain, which is about 350-450 resides and can be subdivided into five distinct motifs with their name derived from the most prominent amino acids: LRI (leucine-rich region I), VHIID, LRII (leucine-rich region II), PFYRE and SAW and mainly function in the interaction between GRAS proteins and their partners (Sun et al., 2012).By phylogenetic analysis, the GRAS family can be divided into more than ten subfamilies, of which SCL4/7 is one important subgroup ...
[摘要] 以前3个成员GAI，RGA和SRC命名的GRAS蛋白质已被发现在294个胚胎种，并由1,035个序列表示。它们属于植物特异性蛋白质家族，在植物生长和发育中起重要作用。该家族中的蛋白质被定义为最低限度地含有保守的GRAS结构域，其约为350-450个，并且可以细分为五个不同的基序，其名称源自最突出的氨基酸：LRI（富含亮氨酸的区域I），VHIID ，LRII（富含亮氨酸的区域II），PFYRE和SAW，并且主要在GRAS蛋白质与其配偶体之间的相互作用中起作用（Sun等人，2012）。通过系统发育分析，GRAS家族可以分为十多个亚科，其中SCL4 / 7是一个重要的亚组，对应对环境压力有作用。这里我们描述了Os-SCL7（水稻SCL4 / 7成员）的GRAS结构域的表达和纯化的详细方案，使我们能够使其结晶并确定其结构。
背景 GRAS蛋白是一个大家族，在植物发育和信号转导中起重要作用。结果表明，一些家庭成员如DELLAs起到GA响应植物生长的抑制作用，是GA信号通路（Murase等人，2008）中的关键调控目标，NSP1和NSP2起重要作用在调节结瘤发育和信号传导（Kaló等人，2005）中，蛋白质SCR和SHR一起在控制根和芽的径向图案中起重要作用（Helariutta et ...
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| Characterization of HBV Isolates from Patient Serum Samples and Cloning
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Author:
Date:
2015-12-20
[Abstract] Hepatitis B virus (HBV) mutants can lead to vaccine failure, diagnostic failure of HBV detection, increase viral replication and resistance to antiviral agents. To study the biological characteristics of these mutations may contribute to our knowledge on viral pathogenesis. Therefore, it is essential to isolate and characterize HBV strains from patients. Here we describe the experimental methods to isolate and clone HBV DNA from patient serum. The method will facilitate isolation and functional analysis of new HBV variants.
[摘要] 乙型肝炎病毒（HBV）突变体可导致疫苗失败，HBV检测的诊断失败，增加病毒复制和对抗病毒剂的抗性。 研究这些突变的生物学特性可能有助于我们对病毒发病机制的了解。 因此，必须从患者中分离和表征HBV病毒株。 在这里我们描述从患者血清中分离和克隆HBV DNA的实验方法。 该方法将促进新的HBV变体的分离和功能分析。
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