这项工作是由人类前沿科学计划授予RGP0024。

1。 DNA模板制备及体外转录<ol><li>感兴趣的基因被克隆在T7和/或如SP6的转录子。 </li><li> 在体外转录模板DNA线性适当的限制酶切割的ORF终止密码子和/或mRNA的3&#39;端下游。一个需要验证完整的线性质粒DNA的琼脂糖凝胶电泳上运行酶切产品。 </li><li>线性质粒用于体外转录反应。不同浓度的DNA模板可以进行测试，以确定最佳的DNA浓度在体外转录。一般来说，Ambion公司的MEGAscript高产转录试剂盒（Ambion公司/生命科技，大岛，NY），1微克的线性的DNA产生mRNA的40-60微克。 在体外转录完成后，制造商的指令（Ambion公司/生命科技，大IslanD，NY）。 </li><li>继在体外转录的mRNA氯化锂沉淀纯化，根据制造商的指示（Ambion公司的MEGAscript高产转录试剂盒）。 </li><li>丙烯酰胺或琼脂糖凝胶电泳进一步证实mRNA的完整性。 </li></ol> 2， 在体外无细胞翻译 在体外翻译使用ŕabbitŕeticulocyte裂解物，RRL（Promega公司，麦迪逊，威斯康星州）按照以下步骤： <ol><li>准备在体外翻译反应以下制造商的指示100微升。简单地说，加入2μL氨基酸混合物减蛋氨酸（1毫米），10个单位，20微居里放射性核糖核酸酶抑制剂（Invitrogen公司/生命科技，大岛，NY）[35 S的 -蛋氨酸（MP Biomedicals核酸自由水，梭伦，俄亥俄州），70μL兔网织红细胞裂解液（Promega公司，麦迪逊，威斯康星州）。 </li><li>孵育日ê反应在30°C 5分钟预先热烈的翻译反应。新增2微克的mRNA的翻译反应，并在30°C孵育5分钟。 </li><li>停止反应，放置在冰的翻译反应。 </li></ol> 3。分离新生多肽在体外翻译反应<ol><li>要隔离新生多肽势必核糖体（多聚核糖体），翻译反应分层4.5毫升30％甘油的顶部在10毫米的Tris-HCl缓冲液pH值7.6，含有氯化钾，100毫米10毫米MGCI 2，100,000 XĞ离心1 TLA-110转子为1小时（贝克曼公司，布雷亚，CA）在4°C </li><li>进一步仔细去除上清。 </li><li>新生多肽polysom​​al颗粒悬浮在一个小体积（10-15微升）1毫米的Tris-HCl缓冲液pH值7.6，含0.5毫克/毫升核糖核酸酶A（Invitrogen公司/生命科技，大岛，NY），孵育30分钟，在37°C。为了提高的肽酰-tRNA酯键的水解，氢氧化钠加入到终浓度为10毫米，和额外的30分钟继续孵化。 </li></ol> 体外翻译反应mRNA和组装不相同的条件下新生肽隔离已完成，将作为一个重要的控制。翻译反应嘌呤（S）前的密度梯度离心提取物治疗，可以作为一个额外的控制。 4。解决新生多肽的Tris-甘氨酸SDS-PAGE <ol><li>解决孤立的新生多肽的Tris-甘氨酸SDS-PAGE电泳分析。请注意材料的数量，加载在凝胶上蛋白标记的效率，对蛋白质的翻译和许多其他参数的效率依赖。装材料的数量应调整的经验，以便在BES牛逼的可视化和个别新生肽链的分离。 </li><li>干凝胶电泳后是固定的，采用真空凝胶代尔受到显影。新生多肽的分布观察使用phosphoimaging。 </li></ol> 5。代表结果牛γ-B晶体在兔网织红细胞裂解以及在大肠杆菌中被翻译大肠杆菌 S30的提取系统（Promega公司，麦迪逊，威斯康星州），其次是新生肽链的隔离。 图1描述了参与隔离核糖体结合的新兴链的步骤。在本研究的目的是测试，如果改变tRNA基因（称为是本质的不同，在哺乳动物（RRL）和原核系统（ 大肠杆菌 ）因密码子的生物体之间的偏差的差异），即翻译剧目的环境改变沿mRNA的核糖体运动和对翻译暂停站点分布。改变核糖体的运动导致的翻译，以及其相对强度过程中积累的新生多肽的大小分配的变化。乐队的相对强度反映的暂停时间。新生的多肽的分离和16.5％T的6％C处的Tris-甘氨酸SDS-PAGE（图2）解决。 γ-B晶体是一个20 kDa蛋白。 RRL和E.非相同的长度和强度的新生多肽的观察大肠杆菌系统表明，在这两个系统中沿mRNA的核糖体的运动遵循不同的翻译动力学。例如，我们观察到在大肠杆菌中的17.7 kDa的突出带大肠杆菌裂解，而不是在RRL系统，这意味着有一个额外的mRNA的3&#39;-端区域（编码C-端区域γ-B晶体），平移暂停网站，当它在大肠杆菌中翻译大肠杆菌系统。我们注意到，γ-B晶体港口串联精氨酸密码子（AGG的153和AGA 154）频繁在哺乳动物系统，但被称为是极其罕见的，并在大肠杆菌中慢慢翻译一种新型的新生肽在大肠杆菌中积累大肠杆菌。大肠杆菌系统（〜17.7 kDa的图2.2），可能造成在这些串联的稀有密码子的核糖体暂停。请注意没有带可以观察到的mRNA（ 图2.3）的情况下，和，嘌呤治疗消除大部分新生链从核糖体（ 图2.3）。长期治疗与嘌呤（15分钟），删除所有新生链（数据未显示）。这证实了观测到的多肽产品，而不是与核糖体cosedimenting mRNPs核糖体相关的新兴连锁。 如上所述，密码子偏好以及组织特定的有机体。这里描述的有代表性的例子，清楚地表明，改变环境/密码子的tRNA的翻译，即剧目偏见可能导致改变的核糖体沿mRNA的运动。显然，这个简单的技术，不仅可以使沿mRNA的翻译暂停网站的识别，而且还允许快速比较不同系统之间的翻译暂停网站。 <img alt="图1" src="/files/ftp_upload/4026/4026fig1.jpg" /> 图1。原理解释隔离核糖体结合新生多肽所涉及的步骤。牛伽玛-B的晶状体（528个碱基对）的ORF序列克隆在pBSKS T7启动子下游+。 在体外转录线性模板DNA，用EcoRI治疗。线性模板，用于在体外转录。 T7 RNA聚合酶的转录下游的伽马 - B晶体蛋白基因的T7启动子在DNA模板是公认的。使用氯化锂沉淀法纯化表达。用于纯化的mRNA 在体外翻译。以下孵化，翻译反应1小时30％的甘油溶液的顶部和分层离心4°100,000 XĞC至隔离核糖体结合新生多肽。 <img alt="图2" src="/files/ftp_upload/4026/4026fig2.jpg" /> 图2。牛伽马在兔网织红细胞裂解液（RRL） 和 E-B的翻译晶状体新生多肽的分离大肠杆菌裂解液 100μL反应RRL 或 E。2微克的mRNA混合大肠杆菌 S30的提取系统（Promega公司，麦迪逊，威斯康星州）根据制造商的指令为5分钟与20微居里[35]-蛋氨酸和培养在30°C间。 4.5毫升30％甘油的顶部在10毫米的Tris-HCl缓冲液pH值7.6以下孵化翻译反应呈层状，含100毫米氯化钾，10毫米MGCI 2，在4℃和100,000 XĞ离心1小时1 TLA-110转子（Beckman Coulter公司，股份有限公司，布雷亚，CA）的。孤立polyribosome颗粒是resuspended在20μL1毫米的Tris-HCl pH值7.6，含0.5毫克/毫升核糖核酸（Invitrogen公司/生命科技，大岛，NY）的治疗与NaOH（如在协议部分所述）。新生多肽ţ16.5％，6％C处的Tris-甘氨酸SDS-PAGE凝胶上解决。凝胶干燥和为显影暴露。暴露后凝胶使用GE Healthcare的台风成像扫描仪扫描。 图2.1显示了隔离和解决翻译后的反应做了两个不同的系统（RRL和大肠杆菌 ）的新生多肽的凝胶。 图2.2凝胶图像定量TL分析V2005软件，这里作为一个例子，说明在两个系统中积累的新生多肽的分子量和强度，可以很容易地分析和比较图2.3显示了两套控制：SDS-PAGE分离和解决翻译后的新生多肽反应组装没有mRNA和/或之后的翻译反应治疗嘌呤（5分钟，终浓度。1毫米），反应前离心。

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没有利益冲突的声明。

对于重复性的结果， 体外转录和翻译反应中使用的组件的质量和浓度是关键。在目前的研究中，我们已经使用市售的包和提取物，提供高度可重复性的数据，如果小心处理。然而，翻译能力的提取物可准备从细胞的选择，如果需要的话。质量可以影响mRNA的翻译，所以它是最重要的测试之前使用它在体外翻译的mRNA的完整性。 此外， 在体外翻译反应的时间?...

<table border="1"><tbody><tr><td> 试剂名称/盒 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td> MEGAscript T7高产转录试剂盒</td><td> Ambion公司</td><td> AM1333 </td></tr><tr><td>核糖核酸酶抑制剂</td><td> Invitrogen公司</td><td> 15518012 </td></tr><tr><td>反式[35]标签</td><td> MP的Biomedicals </td><td> 0151006 </td></tr><tr><td>核糖核酸酶- </td><td> Invitrogen公司</td><td> 12091 </td></tr><tr><td>兔网织红细胞裂解系统，核酸治疗</td><td> Promega公司</td><td> L4960 </td></tr><tr><td> 大肠杆菌 S30提取物系统的线性模板</td><td> Promega公司</td><td> L1030 </td></tr><tr><td>离心</td><td>贝克曼</td><td>舰TLX超速离心机</td></tr><tr><td>存储磷放射自显影Ÿ </td><td> GE医疗集团</td><td>台风9410可变模式成像</td></tr><tr><td>新生多肽分析软件</td><td> GE医疗集团</td><td>图像定量TL，V2005 </td></tr></tbody></table>

isolation of ribosome bound nascent polypeptides in vitro to identify translational pause sites along mrna

翻译伸长率非均匀。 mRNA的二级结构，密码子的使用和mRNA的相关蛋白可能会改变核糖体的运动上的信息进行审查，见1。然而，它现在被广泛接受的同义密码子使用的非均匀平移伸长率的主要原因。同义密码子使用相同的频率。比其他2更频繁使用一些密码子的同义密码子的使用存在的偏见。密码子偏性是有机体以及组织特异性2,3。此外，密码子的使用频率是4同源tRNA的浓度成正比。因此，经常使用的密码子，将有相应的tRNA高出许多，这进一步意味着，频繁的密码子将被翻译速度比不经常。因此，将减缓地区稀有密码子（潜在暂停网站）丰富的mRNA作为一项规则上的信息核糖体运动GE和事业积累的新生肽5-8各自的大小。这些暂停的网站可以有功能的蛋白表达的影响， 审查的 mRNA稳定性和蛋白质折叠9。事实上，它表明，扶贫等暂停网站，可以改变的mRNA上的核糖体运动和随后可能会影响效率的翻译共同体内蛋白质折叠1,7,10,11。为了了解在体内的蛋白质折叠的过程中，在细胞中，蛋白质的合成过程，它必须获得全面的见解，在翻译延伸到沿mRNA的核糖体运动的影响密码子使用/ tRNA的内容最终加上。 在这里，我们描述一个简单的方法可以用来定位主要翻译为一个给定的mRNA翻译各种无细胞系统6-8暂停网站。这个程序是基于隔离accumulati新生多肽在体外的靶mRNA的翻译过程中核糖体纳克。理由是，在低频率的密码子，增加停留时间的增加相应大小的新生肽的核糖体结果。 在体外转录的mRNA存在放射性标记的氨基酸在体外翻译反应让新生链的检测。以孤立核糖体结合的新生多肽复合物的翻译反应顶端30％的甘油溶液离心分层。 polysom​​al颗粒新生多肽进一步用核糖核酸酶A和SDS-PAGE解决。这种技术可能可以被用于任何蛋白质，并允许沿mRNA的核糖体运动和检测的主要暂停网站分析。此外，该协议可以适应学习的因素和条件，可以改变核糖体的运动，从而可能还可以改变日é功能/蛋白质的构象。

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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

描述的一种技术，以确定mRNA的翻译暂停网站。本程序是基于对新生多肽在体外培养的靶mRNA的翻译，其次是使用变性凝胶电泳新生链的大小分析核糖体积累的隔离。

核糖体束缚新生多肽的分离<em&gt;在体外</em&gt;确定沿mRNA的翻译暂停网站

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

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Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

16.2K 次观看.  Cleveland State University. 描述的一种技术，以确定mRNA的翻译暂停网站。本程序是基于对新生多肽在体外培养的靶mRNA的翻译，其次是使用变性凝胶电泳新生链的大小分析核糖体积累的隔离。

视频: Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR剪辑 - 一个方法来确定全转录组学，RNA结合蛋白的结合位点

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

科研

生物学

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and hermaphrodites. In the earlier phase of gametogenesis, the germ cells of hermaphrodites differentiate into limited number of sperm - around 300 - and are stored in a small 'bag' called the spermatheca. Later on, hermaphrodites continually produce oocytes1. In contrast, males produce exclusively sperm throughout their adulthood. The males produce so much sperm that it accounts for &gt;50% of the total cells in a typical adult worm2. Therefore, isolating sperm from males is easier than from that of hermaphrodites.
Only a small proportion of males are naturally generated due to spontaneous non-disjunction of X chromosome3. Crossing hermaphrodites with males or more conveniently, the introduction of mutations to give rise to Him (High Incidence of Males) phenotype are some of strategies through which one can enrich the male population3.
Males can be easily distinguished from hermaphrodites by observing the tail morphology4. Hermaphrodite's tail is pointed, whereas male tail is rounded with mating structures.
Cutting the tail releases vast number of spermatids stored inside the male reproductive tract. Dissection is performed under a stereo microscope using 27 gauge needles. Since spermatids are not physically connected with any other cells, hydraulic pressure expels internal contents of male body, including spermatids2.
Males are directly dissected on a small drop of 'Sperm Medium'. Spermatids are sensitive to alteration in the pH. Hence, HEPES, a compound with good buffering capacity is used in sperm media. Glucose and other salts present in sperm media help maintain osmotic pressure to maintain the integrity of sperm.
Post-meiotic differentiation of spermatids into spermatozoa is termed spermiogenesis or sperm activation. Shakes5, and Nelson6 previously showed that round spermatids can be induced to differentiate into spermatozoa by adding various activating compounds including Pronase E. Here we demonstrate in vitro spermiogenesis of C. elegans spermatids using Pronase E.
Successful spermiogenesis is pre-requisite for fertility and hence the mutants defective in spermiogenesis are sterile. Hitherto several mutants have been shown to be defective specifically in spermiogenesis process7. Abnormality found during in vitro activation of novel Spe (Spermatogenesis defective) mutants would help us discover additional players participating in this event.

Isolation and In vitro Activation of Caenorhabditis elegans Sperm

A protocol for isolating and activating spermatids from male C. elegans is described here. Cutting the posterior end of male releases spermatids. The spermatids can be activated by addition of protease.

分离和<em在体外激活线虫精子

Males and hermaphrodites are the two naturally found sexual forms in the nematode C. elegans. The amoeboid sperm are produced by both males and ...

Using SCOPE to Identify Potential Regulatory Motifs in Coregulated Genes

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and motif position preference1. Each component algorithm is optimized to find a different kind of motif. By taking the best of these three approaches, SCOPE performs better than any single algorithm, even in the presence of noisy data1. In this article, we utilize a web version of SCOPE2 to examine genes that are involved in telomere maintenance. SCOPE has been incorporated into at least two other motif finding programs3,4 and has been used in other studies5-8. 
The three algorithms that comprise SCOPE are BEAM9, which finds non-degenerate motifs (ACCGGT), PRISM10, which finds degenerate motifs (ASCGWT), and SPACER11, which finds longer bipartite motifs (ACCnnnnnnnnGGT). These three algorithms have been optimized to find their corresponding type of motif. Together, they allow SCOPE to perform extremely well.
Once a gene set has been analyzed and candidate motifs identified, SCOPE can look for other genes that contain the motif which, when added to the original set, will improve the motif score. This can occur through over-representation or motif position preference. Working with partial gene sets that have biologically verified transcription factor binding sites, SCOPE was able to identify most of the rest of the genes also regulated by the given transcription factor.
Output from SCOPE shows candidate motifs, their significance, and other information both as a table and as a graphical motif map. FAQs and video tutorials are available at the SCOPE web site which also includes a "Sample Search" button that allows the user to perform a trial run. 
Scope has a very friendly user interface that enables novice users to access the algorithm's full power without having to become an expert in the bioinformatics of motif finding. As input, SCOPE can take a list of genes, or FASTA sequences. These can be entered in browser text fields, or read from a file. The output from SCOPE contains a list of all identified motifs with their scores, number of occurrences, fraction of genes containing the motif, and the algorithm used to identify the motif. For each motif, result details include a consensus representation of the motif, a sequence logo, a position weight matrix, and a list of instances for every motif occurrence (with exact positions and "strand" indicated). Results are returned in a browser window and also optionally by email. Previous papers describe the SCOPE algorithms in detail1,2,9-11.

A straight-forward and robust method to identify potential regulatory motifs in co-regulated genes is presented. SCOPE does not require any user parameters and returns motifs that represent excellent candidates for regulatory signals. The identification of such regulatory signals helps to understand the underlying biology.

使用范围，以确定潜在的监管图案协同调控基因

SCOPE is an ensemble motif finder that uses three component algorithms in parallel to identify potential regulatory motifs by over-representation and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

在体外转录和Gaussia荧光素酶基因的上限由HeLa细胞转染

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

作为一种工具来隔离核糖体的束缚多肽SECM逮捕序列

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

在体外合成在人体细胞修饰蛋白表达的mRNA的感应

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved.
Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.

Measuring Oxidative Stress Resistance of Caenorhabditis elegans in 96-well Microtiter Plates

C. elegans is an attractive model organism to study signal transduction pathways involved in oxidative stress resistance. Here we provide a protocol to measure oxidative stress resistance of C. elegans animals in liquid phase, using several oxidizing agents in 96 well plates.

测量氧化应激性<em&gt;秀丽隐杆线虫</em&gt;在96孔微量滴定板

Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

考试蛋白在哺乳动物细胞中绑定到新生的DNA使用的BrdU芯片插槽西方技术

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

体外sumo 法研究相扑 E3 连接活动

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily ...