这项工作由加拿大自然科学和工程研究委员会资助-发现补助金 (RGPIN-2017-05463), 加拿大创新基金会-约翰 r. 埃文斯领袖基金 (35017), 校园艾伯塔省创新计划和艾伯塔省部经济发展和贸易。

注意: RNA 极易被 ribonucleases (RNases) 降解。采取标准的预防措施, 保持 RNA 完好无损。经常更换手套。在协议的所有步骤中使用过滤过的吸管提示、无核酸酶 plasticware 和无核酸酶化学品。使用无核酸酶或二乙基焦碳酸 (DEPC) 处理水的所有解决方案。
1. 解决方案的准备工作
<ol><li>准备 Toeprinting 缓冲器:20 毫米三盐酸 (pH 7.6), 100 毫米 KOAc, 2.5 毫米毫克 (华侨)2, 5% (瓦特/v) 蔗糖, 2 毫米 dithiothreitol (德勤), 0.5 毫米胺。<ol>
<li>在-20 摄氏度的单用途整除数中存储数码和胺, 以避免重复的冻融循环。 注: 在使用前应立即将胺和 toeprinting 缓冲器添加到该缓冲区中。缺乏胺的解决方案可以存储在-20 摄氏度。</li>
	</ol>
</li>
	<li>制备整除数85毫米 5 '-guanylyl imidodiphosphate (GMP-PNP) 和91毫米三磷酸腺苷 (ATP)。把整除数-20 摄氏度。</li>
	<li>准备450毫升 6% polyacrylamide-7M 尿素凝胶混合: 67.5 毫升40% 丙烯酰胺: 双丙烯酰胺 (19:1), 189 克尿素, 112.5 毫升 5x (三/硼酸/thylenediaminetetraacetic 酸 (EDTA)) 和120毫升水。在37摄氏度的水浴或热板上搅拌, 溶解尿素。筛选解决方案 (e. g, 0.2 微米硝化棉真空过滤器)。 注: 解决方案可存储在4摄氏度, 至少一个月。 注意: 单体丙烯酰胺是一种可以通过皮肤吸收的神经毒素。小心避免皮肤接触 (i. e), 戴上手套, 实验室大衣, 眼睛保护)。聚丙烯酰胺也应小心处理, 因为聚合可能不会进行100% 完成。</li>
	<li>制备甲酰胺负载染料: 95% 甲酰胺, 18 毫米 EDTA, 0.025% (w/v) 月桂酸钠 (SDS), 0.025% (w/v) 溴酚蓝, 0.025% (瓦特/v) 二甲苯蓝。存储在-20 摄氏度。</li>
	<li>溶解 1 nmol 底漆 (5 ' CTCGATATGTGCATCTGTA; 5 ' 末端标记以 IRDye 800) 入 100 ul 水为 10 pmol/ul 的工作集中。存储在-20 °c 在单一使用整除数 (大约10µL), 保护免受光。</li>
</ol>2. mRNA 的制备
<ol><li>从适当的模板 (i. e、基因组 dna 或质粒 dna) 放大 dna 模板, 以聚合酶链反应 (PCR) 合成 mRNA。使用高保真 DNA 聚合酶根据制造商的指示, 以以下反应条件 (35 个周期): 熔化, 98 °c, 十年代;退火, 53 °c, 二十年代;延伸, 72 °c, 三十年代。 注: 前底漆 (5 ' AAGCTTAATACGACTCACTATAG) 包含 T7 启动子序列, 允许 RNA 转录;反向底漆 (5 ' T51GAATTCGGATCCGACCGTGG) 包括51嘧啶残留物, 为转录 RNA 提供一个聚尾。请注意, RNA 也可以从质粒 DNA 中转录为体外。</li>
	<li>使用适当的转录试剂盒体外转录含有 IRES 的 rna 或 DNA 模板中的标记 rna。按照制造商的说明进行操作。在标准的 20 ul 反应量中准备 RNA 样品。治疗新合成的 RNA 与2单位的 RNase 无 DNase 30 分钟37摄氏度。</li>
	<li>将 DNase 处理的 RNA 稀释至110µL, 核酸酶无水加110µL 酸苯酚, 涡 5 s 和离心机在室温下 2万 x g 的3分钟。去除100µL 的水相到一个新的1.5 毫升离心管, 添加10µL 的3米醋酸钠, 和涡旋 2 s. 添加3容量100% 乙醇, 涡流 5 s, 并在-20 °c 一夜之间沉淀 RNA。</li>
	<li>离心机在 > 2万 x g 30 分钟4摄氏度, 并丢弃上清。用500µL 的冰冷 70% (v/v) 乙醇清洗颗粒, 重复离心。吸入尽可能多的上清, 空气干燥的颗粒为 5-10 分钟. 小心不要把球团取出来。</li>
	<li>并用重悬 RNA 在适当的容量核酸酶水中产生0.5 微克/ul 的工作浓度。这可以存储在-80 摄氏度或立即使用。</li>
</ol>3. Toeprinting 反应
<ol><li>混合 15 ul 的兔子网织裂解 (RRL, 不核酸酶处理), 1 ul (40 单位) 的 RNase 抑制剂, 1 µL 91 毫米 ATP (1.82 毫米最后) 和 1 ul GMP-PNP (85 毫米决赛) 在1.7 毫升离心管。孵育30°c 5 分钟。 注: RRL 应 aliquoted 用于单一用途, 以避免重复冻融。临界控制是缺乏 RRL 的反应, 为了阐明反向转录的自然停顿由于 RNA 的次要结构。添加 toeprinting 缓冲区以替换此控件的 RRL。</li>
	<li>添加0.5 µg (1 µL) RNA 和孵育30°c 5 分钟。</li>
	<li>添加22µL 的 Toeprinting 缓冲和孵化在30摄氏度3分钟。</li>
	<li>添加0.5 µL (5 pmol) 的 IRDye 标记底漆和孵化在冰上10分钟。</li>
	<li>添加2µL 25 毫米 dNTPs (最终浓度: 1 毫米), 2 µL 100 毫米毫克 (华侨)2, 1 µL 的禽 Myeloblastosis 病毒逆转录酶 (AMV RT) 和3.5 µL Toeprinting 缓冲。最后的音量是 50 ul。</li>
	<li>孵育反应在30°c 45 分钟。</li>
	<li>添加200µL 的核酸酶水, 并立即提取与250µL 25:24:1 phenol:chloroform:isoamyl 酒精 (pH 约 8.0)。涡流 5 s 和离心机在 2万 x g 在室温下3分钟。去除水相到一个新的1.5 毫升离心管, 增加3容量100% 乙醇, 漩涡 5 s, 并且沉淀在-20 °c 隔夜。</li>
	<li>离心机在 > 2万 x g 30 分钟4摄氏度, 并丢弃上清。用500µL 的冰冷 70% (v/v) 乙醇清洗 DNA 颗粒。离心机在 > 2万 x g 15 分钟4摄氏度。吸入尽可能多的上清, 空气干燥颗粒为 5-10 分钟. 小心不要把球团取出来。</li>
	<li>在 6 ul 的无核酸酶水中溶解颗粒, 加入3的甲酰胺负载染料 ul。</li>
</ol>4. 测序反应
<ol><li>使用 DNA 模板从2.1 和 IRDye 标记底漆从步骤3.4 的标准排序反应, 使用 dideoxynucleotides (ddNTPs) 作为链终结器。使用适当的 DNA 测序套件, 并按照制造商的指示。</li>
	<li>混合 6 ul 的每一个排序反应与 3 ul 的甲酰胺负载染料。</li>
</ol>5. 序凝胶和电泳的制备
注: 本协议使用荧光凝胶为基础的成像仪和21厘米 x 23 厘米 x 0.2 毫米凝胶, 但可以适应其他音序器或凝胶大小, 如果需要的话。
<ol><li>彻底清洁0.2 毫米垫片以及短 (23 x 25 厘米) 和长 (30 x 25 厘米) 玻璃板与100% 乙醇。空气干燥。</li>
	<li>混合30毫升 6% polyacrylamide-7M 尿素凝胶混合 200 ul 10% (瓦特/v) 过硫酸铵 (APS) 和 20 ul 的 tetramethylethylenediamine (TEMED)。倒入凝胶, 小心避免气泡, 并插入 ' 鲨鱼牙齿 ' 凝胶梳子。允许凝胶在室温下聚合1小时。</li>
	<li>装配测序仪, 用1x 填充储层。</li>
	<li>在1500伏前运行15分钟, 直到达到55摄氏度的最佳温度。</li>
	<li>将样品/负载染料混合 (从步骤3.9 或 4.2) 加热到85摄氏度, 5 分钟. 将 1 ul 加载到排序凝胶上。</li>
	<li>用1500伏的凝胶8小时。这台机器将实时读取波段。</li>
	<li>拆卸设备, 处理丙烯酰胺凝胶和运行缓冲器。</li>
</ol>

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作者没有利益冲突的披露。

Toeprinting 是一种强有力的技术, 直接测量一个 RNA 的能力, 以支持在高度控制条件下的翻译起始配合物的形成。该协议描述了一种 toeprinting 哺乳动物 rna 的简化技术。兔网织红细胞裂解剂 (RRL) 是一种方便的核糖体、eIFs、引发 tRNA 和 IRES反式作用因子 (ITAFs) 的来源。实验者提供他们的选择 RNA, 也可以补充 toeprinting 反应与他们选择的特定辅助因子。例如, 48S vs 80S 平移起始配合物可以根据 ...

由于翻译消耗了大部分的细胞能量, 所以翻译被严格管理的1是有意义的。相反, 失调的翻译, 以及随之而来的蛋白质输出的改变, 经常被观察到应激反应和疾病状态, 如癌症1,2。平移控制的一个主要优点是细胞能够改变蛋白质输出的速度, 以响应各种刺激3。因此, 翻译规则是一个重要的机制, 可以影响细胞生存和死亡1,2,3。在翻译步骤中, 启动是最受高度管制和复杂的3。简单地说, 大多数真核基因包含5是7G cap, 几乎总是对他们的翻译至关重要。cap 依赖启动需要真核启动因子 eIF4E, eIF4A 和 eIF4G (cap 识别复合体), 以互动的 5 ' 末端的 mRNA。43S preinitiation 核糖体复合体, 包含 eIF2-bound 引发器 tRNA 和 eIF3, 被招募到 5 ' 末端的 mRNA通过eIF4G 与 eIF3 的相互作用。preinitiation 复合体被认为扫描 mRNA, 由 eIF4A (RNA 旋) 辅助, 直到开始密码子 (AUG) 的位置。48S 起爆复合体随后形成, tRNA 被交付到核糖体的 P 位点。最后, 60S 和40S 核糖体亚基联合形成80S 起始复合体, 后跟翻译伸长1,3,4。相比之下, 内部核糖体进入站点 (IRESes) 通过将40S 核糖体亚基直接招募到起始密码子3, 绕过 5 ' cap 的要求。由于主要的真核致动因子 (eIFs) 的修饰, 生理应激条件减弱了全球 mRNA 的转化。然而, 非规范的翻译启动机制允许选择性地翻译某些基因经常编码的应力反应蛋白, 和失调的非规范化的翻译启动是牵连到疾病的状态, 如癌症1,2. 谨慎的 RNA 管理元素, 如蜂窝 IRESes, 允许翻译这样的基因2,3。
翻译控制的一个特别有趣的方面是了解给定 mRNA 的规范化与非规范化翻译的机制。Toeprinting 是一种技术, 允许详细的机械研究的翻译启动特定 rna在体外。toeprinting 的总体目标是评估一个有兴趣的 RNA 的能力, 以核化在各种条件下与核糖体形成的翻译起始复合体, 以确定哪些序列、结构元素或附件有效的翻译启动需要考虑因素。例如, 核糖体的招募可能会受到阻碍, 因为没有 5 ' 盖帽, 但刺激的存在 IRES。
该技术的原理是体外转录一个感兴趣的 RNA, 在含有翻译成分 (或纯化成分) 的细胞萃取物的存在下孵化它, 以允许起始络合物形成, 并反转转录具有特定底漆的 RNA。稳定的 RNA 核糖体复合物将导致反向转录在核糖体的 3 ' 边缘-所谓的 ' toeprint '5,6,7。
在本协议中, eIFs 的核糖体亚基、tRNAs 和 IRES反式作用因子 (ITAFs) 由兔网织红细胞裂解 (RRL) 方便地贡献。该协议的另一个优点是使用荧光标记的底漆和荧光凝胶为基础的成像仪, 而不是核素底漆。这消除了额外的步骤, 包括 radiolabeling 底漆, 以及干燥的凝胶和暴露在一个强化的屏幕。荧光带是实时记录, 因为凝胶运行, 允许更大的分辨率。名额 X 链细胞凋亡蛋白 (XIAP) IRES RNA 作为一个例子在这里, 虽然上限基因也可以分析此技术8。
与西方分析不同的是, 它测量了细胞裂解物中翻译过程的最终输出, toeprinting 是一种体外方法, 用于测量 RNA 上的翻译起始复杂形成。这种简化方法允许对调节翻译起始的基板或因素进行高度详细的研究 (e. g., 有上限或未覆盖的 mRNA, IRES 结构, 存在或没有聚尾, 提供特定的蛋白质因素,等)。因此, toeprinting 可以用来研究不同的翻译模式8或 mRNA 结构的影响, 如 IRESes, 对蛋白质合成9,10。

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</table>

toeprinting analysis of translation initiation complex formation on mammalian mrnas

翻译起始是蛋白质合成的速率限制步骤, 是细胞调节蛋白质输出的关键点。调控蛋白质合成是细胞应激反应的关键, 失调是许多疾病状态的中心, 如癌症。例如, 虽然细胞应力导致了全球翻译的抑制, 通过衰减上限依赖性的启动, 某些应力反应蛋白有选择地翻译成与盖帽无关的方式。谨慎的 RNA 调控元素, 如细胞内核糖体进入站点 (IRESes), 允许翻译这些特定的基因。这种基因的识别, 以及它们的调控机制的特性, 一直是分子生物学中的一个关键领域。Toeprinting 是一种研究 RNA 结构和功能的方法, 它与翻译起始有关。toeprinting 的目的是评估体外转录 RNA 在各种条件下与核糖体形成稳定的配合物的能力, 以确定核糖体中涉及的序列、结构元素或辅助因素绑定-一个前游标有效的翻译启动。除了其他技术, 如西方分析和 polysome 分析, toeprinting 允许一个强大的特点的机制, 调整翻译的启动。

我们以前描述了 XIAP IRES 支持与 cap 无关的翻译启动的体外8,10的能力。Toeprinting 是讯问 XIAP IRES 起爆复合体机械细节的关键技术。对包含 XIAP IRES (图 1A) 的 mRNA 进行编码的 DNA 构造是体外转录并受到 toeprinting 分析。此处使用的 XIAP IRES mRNA 的变种变体在图 1<stron...

Watch this Scientific Journal Video about Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs at JoVE.com

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

哺乳动物基因翻译起始复合体形成的 Toeprinting 分析

Toeprinting 的目的是测量体外转录 RNA 的能力, 在各种条件下形成与核糖体的翻译起始配合物。该协议描述了一种 toeprinting 哺乳动物 RNA 的方法, 可用于研究 cap 依赖和 IRES 驱动的翻译。

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

toeprinting-analysis-translation-initiation-complex-formation-on

Research

JoVE Journal

Biochemistry

12.4K 次观看.  University of Lethbridge. Toeprinting 的目的是测量体外转录 RNA 的能力, 在各种条件下形成与核糖体的翻译起始配合物。该协议描述了一种 toeprinting 哺乳动物 RNA 的方法, 可用于研究 cap 依赖和 IRES 驱动的翻译。

视频: Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

从Cryomilled哺乳动物细胞中的蛋白质复合物亲和捕获

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

科研

生物化学

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

巨囊泡封装微球通过一个水包油乳液的离心制备

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Quantitative Immunofluorescence to Measure Global Localized Translation

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and organogenesis, as well as in multiple diseases. Numerous publications have convincingly shown that specific mechanisms tightly regulate mRNA translation. Increased interest in the translation-induced regulation of protein expression has led to the development of novel methods to study and follow de novo protein synthesis in cellulo. However, most of these methods are complex, making them costly and often limiting the number of mRNA targets that can be studied. This manuscript proposes a method that requires only basic reagents and a confocal fluorescence imaging system to measure and visualize the changes in mRNA translation that occur in any cell line under various conditions. This method was recently used to show localized translation in the subcellular structures of adherent cells over a short period of time, thus offering the possibility of visualizing de novo translation for a short period during a variety of biological processes or of validating changes in translational activity in response to specific stimuli.

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

定量荧光测量全球本地化翻译

The mechanisms regulating mRNA translation are involved in various biological processes, such as germ line development, cell differentiation, and ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

碳水化合物转运基质结合蛋白 SP0092 的生化和结构表征

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

一种简单的高通量化学筛选方法在秀丽线虫中的应用

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

从茶中提取的新建细胞悬浮培养基中六种系统杀虫剂的代谢研究(山茶西宁西斯L.叶

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

用于分析依赖帽的翻译动力学的 体外 单分子成像测定

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.