Research in Ansari lab was supported by the research grant (No. MCB 1020911) from National Science Foundation.

注:在研究文章中Medler，S和安萨里，A 2日报道在使用此方法。 1.培养和收集细胞<ol><li>从新鲜划线酿酒酵母板开始在YPD培养基（10克/升酵母提取物，20g / L的蛋白胨，2％葡萄糖）的细胞的5毫升培养。 </li><li>生长细胞过夜在轨道摇床在30℃和250rpm。 </li><li>稀释过夜生长的培养的100倍，以YPD培养基100mL的最终体积的250毫升带挡板的烧瓶中。 注:共100毫升的文化，每TRO反应需要;如果需要多个反应，必须使用培养物的体积较大。 </li><li>生长细胞为0.8的光密度- 1.0在600nm（A 600〜0.8 - 1.0）。 </li><li>离心在820 xg离心在4℃下5分钟收获细胞在无菌50mL的锥形管中。 </li><li>悬浮细胞在10毫升冰冷的TMN的卜FFER（10毫米的Tris-HCl pH为7.5，5mM的MgCl 2的，100毫摩尔NaCl），并在冰上孵育5分钟。 </li><li>离心机在820×g离心5分钟，在4℃以沉淀细胞。轻轻通过抽吸去除上清液。 </li></ol> 2.透化和准备的细胞运行的分析<ol><li>通过上下吹打轻轻用截短P 1000尖悬浮在1毫升0.6％十二烷基肌氨酸钠溶液的细胞。细胞悬液转移到一个冷却的1.5毫升离心管中。 </li><li>轻轻摇动细胞在4℃下于一个平台摇床25分钟。 注:微量离心管应该被打包到50mL锥形管中在冰冷保持冷温度，从而避免在此阶段的启动子产生的任何新的起始事件的可能性。 </li><li>用台式冷冻离心机离心细胞以1200 xg离心6分钟，4℃。 </li><li>轻轻吸出上清液从每除去任何过量的液体meabilized细胞沉淀。 </li></ol> 3.转录运行的反应<ol><li>添加转录150μL的运行上缓冲液（50mM的Tris-HCl pH值7.5，100mM的氯化钾，10mM的MgCl 2的，2毫摩尔DTT（二硫苏糖醇），0.75毫每个ATP，CTP，GTP，和BrUTP，200单位核糖核酸酶抑制剂）的透化细胞沉淀。 </li><li>通过上下吹打使用截断的第200页尖轻轻拌匀。 注:如果处理多个样本，把一切都在冰上，直至所有样本都准备好，然后进行下一个步骤。 </li><li>在水浴孵育5分钟，在30℃。 注:调整为1〜5分钟之间温育的时间，以确保BrUTP的最佳掺入新生RNA。 </li><li>停止加入冷准备使用的RNA分离试剂500μL的反应。孵育在室温下5分钟。 </li></ol> 4. RNA提取<ol><li>加的酸洗的玻璃珠200微升至细胞suspensi上（步骤3.4），并在旋涡混合器剧烈摇晃20分钟，4℃。 </li><li>使用热压22克针穿刺微量离心管的底部，将其放置在一个15毫升的无菌锥形管的顶部。 </li><li>离心机在4℃下300×g离心1分钟，以收集细胞裂解物。转移细胞裂解物到1.5mL离心管中。 </li><li>添加冷的RNA分离试剂500μL，和200微升氯仿至细胞裂解物。在4℃下混合在涡旋混合器上并离心，内容物在16168×g离心20分钟。 </li><li>上层水相转移到新的离心管中。 </li><li>添加冷的酚/氯仿的等体积/异戊醇（125:24:1）的pH 4-5。剧烈摇动在涡旋混合器上并离心16168 xg离心在4℃下15分钟。 </li><li>传送上层水含有相的RNA到一个新的离心管中。重复步骤4.6两次。 </li><li>到最后含有RNA的水相，加5 M氯化钠STOCk以得到0.3 M的最终浓度添加3倍的冷乙醇的体积以沉淀RNA。 <ol><li>孵育1小时，或在-20℃过夜。 注: - 当抗BrdU珠粒孵育阻断（参见步骤5.3）4.11执行步骤4.9。 </li></ol></li><li>离心机在16168 xg离心在4℃下20分钟。去除上清，重悬在100微升DEPC（焦碳酸二乙酯）水的RNA沉淀。 </li><li>使用RNA提取试剂盒迷你的RNA从非法人BrUTP核苷酸分开。洗脱从用100微升的无RNase水列中的RNA。 </li><li>孵育在65℃水浴中的RNA的5分钟，然后转移到冰上至少2分钟。 </li></ol> 5. BrUTP标记的RNA的亲和纯化<ol><li>以25μL抗BrdU落户珠，加500 0.25X SSPE（盐水磷酸钠磷酸EDTA）结合缓冲液（20X SSPE的微升:3M氯化钠，0.2M的NaH 2 PO 4 </sub&gt;，0.02摩尔EDTA，pH值7.4，1mM EDTA中，0.05％吐温，37.5毫摩尔NaCl）。离心机在4℃下200×g离心30秒，并去除上清。 </li><li>重复步骤5.1三次。 </li><li>加入500微升封闭缓冲液（1×结合缓冲液，0.1％聚乙烯吡咯烷酮，1毫克超纯牛血清白蛋白（BSA））将珠并轻轻1摇动 - 在4℃在一个平台上摇动2小时。 </li><li>两次用结合缓冲液500微升洗珠，如在步骤5.1中描述。 </li><li>加入400微升的结合缓冲液至珠。 </li><li>转移100μLRNA（从步骤4.11）直接向珠。在4℃下2小时在一个平台摇床 - 温和振荡1孵育。 </li><li>用500微升结合缓冲液，低盐缓冲液500微升（0.2×SSPE，1毫摩尔EDTA，0.05％吐温），高盐缓冲液的500微升（0.25×SSPE，1毫摩尔EDTA，0.05％吐温洗涤珠粒顺序，的100mM NaCl）中，并两次用TET缓冲液500微升（1×TE，0.05％吐温）中，在纺200在每次洗涤之间于4℃xg离心30秒。 </li><li>用150微升洗脱从珠BrUTP标记的RNA顺序，两次和一次用200μl洗脱缓冲液（20mM DTT，150 mM氯化钠，的50mM的Tris-HCl pH为7.5，1mM EDTA中，0.1％SDS）中的，通过温育对于在42℃的水浴中，随后通过短的自旋来从上清液中分离珠4分钟。 </li></ol> 6.沉淀BrUTP标记的RNA <ol><li>加入500微升冷的酚/氯仿/异戊醇（125:24:1）pH为4 - 5到纯化BrUTP亲和性标记的RNA。在4℃下混合在涡旋混合器上并离心16168×g离心15分钟。 </li><li>上层水相转移到新的离心管中。 </li><li>加入5 M氯化钠库存得到冷乙醇0.3 M和3倍体积的最终浓度，以沉淀RNA。 </li><li>孵育在-20℃过夜。 </li><li>收集通过离心分离沉淀的RNA在16168×g离心30分钟，在4℃下&lt;/ LI&gt; <li>除去上清液，并允许沉淀风干10分钟。 </li><li>重悬DEPC水26μLRNA沉淀。 </li><li>通过使用分光光度计测定260nm处的吸光度确定最终的RNA浓度。此外，确定的260/280比例。 A比值〜2指示纯RNA样品。 </li><li>直到需要用于cDNA合成在-80存储在等分试样的RNA样品℃。 </li></ol> 7. BrUTP标记的RNA的反转录<ol><li>调整RNA浓度为100纳克/μLDEPC使用水。使用RNA的0.5微克每个cDNA合成反应。 注:RNA模板的浓度可以变化为0.5至1.0微克，以达到最佳的cDNA合成。 </li><li>扭转使用如图3A所示设计的多聚（A）位点（终止位点）下游的多个反向引转录的RNA。 </li><li>对于每个逆转录反应，加入5微升RNA templatE（0.5微克），1微升dNTP混合物（各10毫米），反向引物C，D，E，F或G的2微升（50μM各）（ 见图3A）和无核酸酶的水。在每个管中的最终的反应体积为13微升。 注意:反转录反应的数量将取决于用于cDNA合成反向引数。例如，对于ASC1被使用的五种反向引，因此5逆转录反应成立。标签用于cDNA合成的反向引物的基础上，在管为C，D，E，F和G。 </li><li>孵育含RNA模板，dNTP混合物和引物在热循环仪的管在65℃下5分钟，以除去任何二级结构构象，然后冷却至4℃至少2分钟。 </li><li>添加1μL的逆转录酶（200U /μL），4微升缓冲液和2微升的0.1M DTT的含有RNA模板和引物各管（步骤7.4）。轻轻吹打向上和向下混合。 </li><li>通过在42℃保温60分钟在热循环反应混合物中进行逆转录。 </li><li>灭活在65℃下的逆转录酶20分钟。 </li><li>存储的cDNA在-20℃或进行下一步骤。 </li></ol> 8.扩增的cDNA <ol><li>从步骤7.8进行，每一cDNA的制备（标为C，D，E，F和G为ASC1）的扩增的PCR反应如下:从步骤7.8，1.0微升正向引物B（10μM），1.0微升3.0微升cDNA模板反向引物C或D或E或F或G（10μM），2.5微升10×聚合酶PCR缓冲液，0.5微升dNTP混合物（各10毫摩尔），1.0微升MgCl 2的（2.5毫摩尔），15.5微升的PCR级水，和0.5微升热稳定聚合酶。 注:5至1::cDNA模板从1的不同稀释液100进行测试，以优化的PCR产物的产率。单启动子近端的正向引物将被用于扩增cDNA的使用不同的反向引获得。例如，位于该启动子附近的单个正向引物B组合物用于与从C到基伍反向引为ASC1。 BC，BD，BE，BF和BG中分别标为C，D，E，F和G管被用作如图3A所示。 </li><li>使用以下热循环参数运行的PCR:1个循环95℃2分钟（热启动聚合酶的活化），30 - 40个循环95℃30秒（变性），54℃15秒（退火） ，68℃1分钟（延伸），和1个循环68℃5分钟（最终延伸）。 注:退火温度将取决于具体的引物被用来变化和延长时间将取决于预期的产物大小而有所不同。 </li><li>通过电泳在0.8％，1.5％，或取决于产品的预期大小的2％琼脂糖凝胶上分离PCR产物。 </li><li>如在El Kaderi 等人所述量化PCR产物。 ，2012 11。 注:另外，采用实时定量RT-qPCR的策略，以量化新生成绩单的数量。 </li></ol>

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The authors have nothing to disclose.

这里使用的链特异性TRO协议是改编自哺乳动物细胞12用于GRO-SEQ（全球运行在测序）分析的协议。我们成功修改了协议，来研究转录新生在芽殖酵母。要具体分析转录终止缺陷，我们通过摆脱RNA水解步骤的进一步调整的协议。这使我们能够特异性检测来自子区靠近转录起始位点开始的全长新生转录物。 有迹象表明，应以最大的谨慎进行以获得有意义的结果在协议的几个关键步骤。首先，该协议必须以指数生长的酵母细胞中进行。在对数期的细胞（A 600〜0.8 - 1.0）得到为现阶段的多数细胞的最好的结果是活跃生长并表现出强大的转录。其次，RNA分离圣4℃ - 应在2之间的温度尽可能快地进行EP。第三，前溴-UMP的亲和纯化标记的RNA，未掺入BrUTP应当从RNA制备除去。我们使用了RNA试剂盒获得从纯化RNA制备摆脱BrUTP的。四，利用强大的逆转录酶是为协议的顺利完成的关键。 这里使用的特定链-TRO协议有过Northern杂交技术优势，在检测结束的缺陷，因为它是更为敏感，速度更快，具有更高的分辨率。该协议还比传统的TRO协议更好，因为它可以区分反义转录物有义转录物，并且如果观察到的结果是由于启动子启动的转录或异常转录从启动子下游区域开始。的链特异性TRO协议有一定的局限性。它是更费时费力比稳定ST吃了基因检测技术。它需要大量的细胞。因此，低转印基因的转录分析可能是一个问题。 我们已经成功地使用这种技术来研究在RNA聚合酶II转录周期终止缺陷芽殖酵母2。该方法可以扩展由RNA聚合酶I和RNA聚合酶III在酵母研究终止转录为好。适当修改，该方法也可以适用于研究转录终止缺陷在高等真核生物。总体而言，该技术是相对便宜的，高度可再现，并且需要在一个分子生物学实验室中使用的标准设备。

真核转录周期包括三个主要步骤;起始，延伸和终止。转录由RNA聚合酶II终止包括两个不同的，相互依存的步骤3。第一步包括裂解，多腺苷酸化和mRNA的释放从模板，并紧接着第二个步骤，通过从所述模板的聚合酶脱开标记。适当的终止是聚合酶的转录起始/再引发在回收至关重要的，用于防止干扰的下游基因的转录，并为通过限制普遍非编码RNA 4,5转录保持异常转录检查。尽管最近的进展，终端是迄今为止RNA聚合酶II转录周期的最不被理解的一步。一个可靠的终止试验是研究机制underlyin至关重要通过体内 RNA聚合酶II转录克终止。 许多实验方法是用来检测在生理条件下可主动转录基因终止缺陷。这些措施包括Northern杂交，终止因子芯片（染色质免疫沉淀）和传统TRO检测。每种技术具有一定的优点和缺点。所使用的传统的TRO测定是在体内 1检测的转录终止缺陷的最可靠和灵敏的方法。该测定本地化于细胞内的基因的不同区域的转录活性聚合酶分子。在正常情况下，该测定显示了转录接合聚合酶分子严格分布的基因（ 图1A）的启动子和终止子区域之间。在有缺陷的终止，然而，聚合酶是无法读取终止信号并在该基因（ 图1B）的3&#39;端下游的区域进行检测。 转录终止缺陷主要表现在从启动子近侧区发起感-转录聚合酶存在，并且是无法读取终止信号，继续转录的基因的3&#39;末端的下游区， 如图2A。与真核基因组中表现出感压倒性普遍存在的转录以及反义方向和周围的基因6，7，8，9，10的实现，它很快变得清晰，传统的TRO测定无法检测，如果聚合酶信号一个基因的下游代表的启动子启动的转录物（ 图2A）或发起索姆的异常的RNAewhere在基因或终止子元件（ 图2B），或从基因（ 图2C）的3&#39;末端的反义方向的聚合酶转录的下游的中间位置。如果观测到的通读聚合酶信号表示启动子启动的扩展感的mRNA或发起检查中的基因的下游的反义转录物的链特异性TRO测定此处描述的使用BrUTP可以区分。我们已经成功地使用这种方法来证明在芽殖酵母2中转录终止Kin28激酶的作用。这里采用的办法，我们表明，Rna14需要在芽殖酵母ASC1的转录终止。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Phenol -chloroform-isoamyl alcohol mixture</td>
			<td>Sigma-Aldrich</td>
			<td>77619</td>
			<td>pH 4-5</td>
		</tr>
		<tr>
			<td>TRIzol reagent</td>
			<td>Life technologies</td>
			<td>15596-026</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase Inhibitor, human placenta</td>
			<td>New England Biolabs</td>
			<td>M0307S</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-BrdU beads</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-32323AC</td>
			<td></td>
		</tr>
		<tr>
			<td>Protoscript II</td>
			<td>New England Biolabs</td>
			<td>M03368L</td>
			<td>or an equivalent enzyme</td>
		</tr>
		<tr>
			<td>Advantage 2 polymerase Mix</td>
			<td>Clontech</td>
			<td>639201</td>
			<td>or an equivalent enzyme</td>
		</tr>
		<tr>
			<td>5-Bromouridine 5&#39; triphosphate sodium salt</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-214314A</td>
			<td></td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>New England Biolabs</td>
			<td>N0451AA</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td></td>
			<td>N0454AA</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td></td>
			<td>N0452AA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-pure bovine serum albumin (BSA)</td>
			<td>MC Labs</td>
			<td>UBSA-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Asc1 B</td>
			<td></td>
			<td></td>
			<td>AGATTCGTCGGTCACAAGTCC</td>
		</tr>
		<tr>
			<td>Asc1 C</td>
			<td></td>
			<td></td>
			<td>GAACTTTATACATATTCTTAGTT 
			AGCAGTC</td>
		</tr>
		<tr>
			<td>Asc1 D</td>
			<td></td>
			<td></td>
			<td>TGTACATATGTATTTTCGCAGCA</td>
		</tr>
		<tr>
			<td>Asc1 E</td>
			<td></td>
			<td></td>
			<td>GCCAAGGAGACTGAATTTAATG</td>
		</tr>
		<tr>
			<td>Asc1 F</td>
			<td></td>
			<td></td>
			<td>CTATGGAATGGGGGTTTTAAG</td>
		</tr>
		<tr>
			<td>Asc1 G</td>
			<td></td>
			<td></td>
			<td>GGTTATGGCAGACATGCCAC</td>
		</tr>
		<tr>
			<td>5s cDNA</td>
			<td></td>
			<td></td>
			<td>AGATTGCAGCACCTGAGT</td>
		</tr>
		<tr>
			<td>5&#8217; 5s reverse</td>
			<td></td>
			<td></td>
			<td>GGTTGCGGCCATATCTAC</td>
		</tr>
		<tr>
			<td>3&#8217; 5s reverse</td>
			<td></td>
			<td></td>
			<td>TGAGTTTCGCGTATGGTC</td>
		</tr>
	</tbody>
</table>

analysis of termination of transcription using brutp-strand-specific transcription run-on (tro) approach

这份手稿描述了在体内检测转录终止缺陷的协议。这里描述使用BrUTP的具体链-TRO协议是在生理条件下分析转录终止缺陷强大的实验方法。像传统的TRO测定法，它依赖于超出基因作为转录终止缺陷1的指示器的3&#39;端的转录活性的聚合酶的存在。它克服了与传统的TRO测定遇到的两个主要问题。首先，它可以检测是否聚合酶通过终止信号读出的是，从启动子近侧区启动转录的一个，或者如果它被简单地表示该发起非特异性从身体某处一个普遍地转录聚合酶或3&#39;该基因的末端。其次，它可以区分，如果转录活性聚合酶信号超出终止区是真正的通读义mRNA转录聚合酶或终止子启动的非编码反义RNA的信号。简单地说，该协议涉及透的指数生长的酵母细胞中，从而允许在体内启动在BrUTP核苷酸的存在下伸长，由亲和方法纯化BrUTP标记的RNA的转录，逆转录纯化的新生RNA和扩增的cDNA用链特异性的引物侧翼启动子和基因2的终止子区域。

为了证明在检测转录终止缺陷BrUTP链特异性TRO过程的适用性，我们使用RNA14的终止缺陷，温度敏感突变体称为rna14-1。 Rna14在由RNA聚合酶II转录终止的作用被使用在选择的基因1的终结者近端区域检测RNA传统TRO检测证明。超出在非允许温度下rna14-1突变体的终止子区域的RNA信号的检测被解释为终止缺陷。所观察到的信号，然而，可以归因于从附近的某处基因的3&#39;端开始转录， 如图2B中的普遍地转录聚合酶。要最终证实Rna14在终端的作用，我们在rna14-1进行链特异性BrUTP-TRO突变体，并在37℃下的同基因野生型菌株。我们预计，在野生型菌株，该TRO信号将所述启动子和终止子区域之间的制约， 如图1A所示 。在rna14-1突变体，但是，聚合酶将通过终止信号读和TRO信号将超出该基因的3&#39;末端， 如图1B和2A来检测。 简要地说，我们长大的rna14-1突变体及其等基因野生型细胞在25℃至对数中期，然后转移细胞到37℃三小时。转录运行上测定进行掺入BrUTP到如上所述通过一个主动转录RNA聚合酶合成的新生RNA。所述BrUTP标记的RNA纯化和使用引物C，D，E，F和G在图3A所示逆转录。相应的cDNA进行PCR扩增USI纳克引物对BC，BD，BE，BF，和BG和分级在琼脂糖凝胶上（ 图3B）。凝胶的量化示于图3C。的PCR扩增产物从引物对存在BE，BF，和BG在rna14-1突变反射通过表型读出终止（ 图3B，泳道11 - 13;以及图3C中 ，区域E，F和G）。在野生型细胞中，然而，TRO信号被限制，直到终止元件（ 图3B，泳道2和3;和图3C，区域C和D）。没有可检测的TRO信号超出终止区（ 图3B，泳道4 - 6;和图3C，区域E，F和G）。因此，我们可以检测到通过在突变，但不是在野生型细胞中的终止信号读出的启动子启动的新生转录物，从而确认Rna14是一个终止因子。 图1:转录运行的（TRO）分析检测转录活性RNA聚合酶存在于基因的不同区域。 （A）当终止是正常的，不存在聚合酶信号超出终止区。 （b）在有缺陷的终止中，聚合酶是无法读取终止信号和可以超越该基因的3&#39;端来检测。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55446/55446fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/55446/55446fig2.jpg" /> 图2:钢绞线特定TRO分析。的链特异性TRO测定可以检测一个转录活性聚合酶存在超出T的3&#39;末端他的基因是一个真正的终端缺陷，由于发起人发起聚合酶通过终止信号（A）读书，或只是在感觉方向上普遍地转录聚合酶转录（B），或者是反义非编码RNA转录聚合酶启动从3&#39;端（C）。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55446/55446fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55446/55446fig3.jpg" /> 图3:Rna14是一个终止因子聚合酶通过读取在rna14-1突变的终止信号。 （A）中示出的正向引物B和五个反向引，C，D，E，F和G的位置ASC1基因的示意性描绘用于纯化BrUTP标记的RNA的cDNA合成。 （B）中从反向引C，D，E制备的cDNA，F和G分别使用引物对BC，BD，BE，BF和BG PCR扩增。上述（C）在野生型和rna14-1细胞（B）中所示的TRO数据的在37℃的定量分析。 的5S RNA作为归一化控制。误差条表示基于最小的三次试验的标准偏差的一个完整单元。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55446/55446fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach at JoVE.com

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes.

使用BrUTP链特异性转录运行的（TRO）方法终止转录分析

This manuscript describes a protocol for detecting transcription termination defect in vivo. The strand-specific TRO protocol using BrUTP described here ...

analysis-termination-transcription-using-brutp-strand-specific

Nanyang Technological University

Research

JoVE Journal

Genetics

Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on (TRO) Approach

9.8K 次观看.  Wayne State University. We describe a basic experimental approach for analysis of termination of transcription by RNA polymerase II in vivo using BrUTP by the strand-specific transcription run-on (TRO) approach in budding yeast. This protocol can be extended to study transcription termination by other RNA polymerases both in yeast and higher eukaryotes. 

视频: Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

体外转录测定及其在药物研究中的应用

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

科研

遗传学

Metagenomic Analysis of Silage

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of the microbial communities present within them. Two main metagenomic approaches exist; sequencing the 16S rRNA gene coding region, which exhibits sufficient variation between taxa for identification, and shotgun sequencing, in which genomes of the organisms that are present in the sample are analyzed and ascribed to "operational taxonomic units"; species, genera or families depending on the extent of sequencing coverage. 
In this study, shotgun sequencing was used to analyze the microbial community present in cattle silage and, coupled with a range of bioinformatics tools to quality check and filter the DNA sequence reads, perform taxonomic classification of the microbial populations present within the sampled silage, and achieve functional annotation of the sequences. These methods were employed to identify potentially harmful bacteria that existed within the silage, an indication of silage spoilage. If spoiled silage is not remediated, then upon ingestion it could be potentially fatal to the livestock.

Metagenomics was used to investigate the microbiome of silage cattle feed. Analysis was performed by shotgun sequencing. This approach was used to characterize the composition of the microbial community within the cattle feed.

青贮的宏基因组学分析

Metagenomics is defined as the direct analysis of deoxyribonucleic acid (DNA) purified from environmental samples and enables taxonomic identification of ...

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

使用了大气颗粒物引起的染色体畸变分析<em&gt;体外</em&gt;系统

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

使用染色质免疫沉淀分析c-KIT配体启动子

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In bacteria, the impact of the genetic context on the function of a genetic element can be difficult to assess. Several mechanisms, including topological effects, transcriptional interference from neighboring genes, and/or replication-associated gene dosage, may affect the function of a given genetic element. Here, we describe a method that permits the random integration of a DNA element into the chromosome of Escherichia coli and select the most favorable locations using a simple growth competition experiment. The method takes advantage of a well-described transposon-based system of random insertion, coupled with a selection of the fittest clone(s) by growth advantage, a procedure that is easily adjustable to experimental needs. The nature of the fittest clone(s) can be determined by whole-genome sequencing on a complex multi-clonal population or by easy gene walking for the rapid identification of selected clones. Here, the non-coding DNA region DARS2, which controls the initiation of chromosome replication in E. coli, was used as an example. The function of DARS2 is known to be affected by replication-associated gene dosage; the closer DARS2 gets to the origin of DNA replication, the more active it becomes. DARS2 was randomly inserted into the chromosome of a DARS2-deleted strain. The resultant clones containing individual insertions were pooled and competed against one another for hundreds of generations. Finally, the fittest clones were characterized and found to contain DARS2 inserted in close proximity to the original DARS2 location.

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal position.

用新的转介导法测定大肠杆菌中 DNA 元素的最佳染色体位置 (s)

The optimal chromosomal position(s) of a given DNA element was/were determined by transposon-mediated random insertion followed by fitness selection. In ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for example, during cellular differentiation, morphogenesis, and functional stimulation (such as activation of immune cells), or after exposure to drugs and other factors from the local environment. Depending on the stimulus and cell type, these changes occur rapidly and at any possible level of gene regulation. Displaying all molecular processes of a responding cell to a certain type of stimulus/drug is one of the hardest tasks in molecular biology. Here, we describe a protocol that enables the simultaneous analysis of multiple layers of gene regulation. We compare, in particular, transcription factor binding (Chromatin-immunoprecipitation-sequencing (ChIP-seq)), de novo transcription (4-thiouridine-sequencing (4sU-seq)), mRNA processing, and turnover as well as translation (ribosome profiling). By combining these methods, it is possible to display a detailed and genome-wide course of action.
Sequencing newly transcribed RNA is especially recommended when analyzing rapidly adapting or changing systems, since this depicts the transcriptional activity of all genes during the time of 4sU exposure (irrespective of whether they are up- or downregulated). The combinatorial use of total RNA-seq and ribosome profiling additionally allows the calculation of RNA turnover and translation rates. Bioinformatic analysis of high-throughput sequencing results allows for many means for analysis and interpretation of the data. The generated data also enables tracking co-transcriptional and alternative splicing, just to mention a few possible outcomes.
The combined approach described here can be applied for different model organisms or cell types, including primary cells. Furthermore, we provide detailed protocols for each method used, including quality controls, and discuss potential problems and pitfalls.

This protocol describes the combinatorial use of ChIP-seq, 4sU-seq, total RNA-seq, and ribosome profiling for cell lines and primary cells. It enables tracking changes in transcription-factor binding, de novo transcription, RNA processing, turnover and translation over time, and displaying the overall course of events in activated and/or rapidly changing cells.

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

全基因组对真核生物转录误差的监测

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

Reverse Transcription-Loop-mediated Isothermal Amplification (RT-LAMP) Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth defects. The goal of this protocol is to quickly detect ZIKV in both human and mosquito samples. The current gold standard for ZIKV detection is quantitative reverse transcription PCR (qRT-PCR); reverse transcription loop-mediated isothermal amplification (RT-LAMP) may allow for a more efficient and low-cost testing without the need for expensive equipment. In this study, RT-LAMP is used for ZIKV detection in various biological samples within 30 min, without first isolating the RNA from the sample. This technique is demonstrated using ZIKV infected patient urine and serum, and infected mosquito samples. 18S ribosomal ribonucleic acid and actin are used as controls in human and mosquito samples, respectively.

Reverse Transcription-Loop-mediated Isothermal Amplification RT-LAMP Assay for Zika Virus and Housekeeping Genes in Urine, Serum, and Mosquito Samples

This protocol provides an efficient and low-cost method to detect Zika virus or control targets in human urine and serum samples or in mosquitoes by reverse transcription loop-mediated isothermal amplification (RT-LAMP). This method does not require RNA isolation and can be done within 30 min.

反向转录-环介导的等温放大 (RT 灯) 测定尿液、血清和蚊子标本中的 Zika 病毒和管家基因

Infection with Zika virus (ZIKV) can be asymptomatic in adults, however, infection during pregnancy can lead to miscarriage and severe neurological birth ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...