This work acknowledges a Faculty Early Career Grant from the University of Newcastle (CM).

注意:实验涉及使用放射性同位素32 p和没有工作应该进行，直到所有适当的安全条件已经具备。一般人员都必须参加安全课程，并接受之前，使用放射性试剂的实验监督的做法。请佩戴个人防护装备进行反应时（热释光剂量计，防护眼镜，手套，放射室实验室外套，全长裤，封闭趾鞋）。 1.试验材料的制备<ol><li>获取所需的蛋白质转录分析。 <ol><li>对于这里所描述的实验中，过量产生和纯化大肠杆菌大肠杆菌 RNA聚合酶在实验室中描述11，或获得自商业来源（材料清单）。 注意:两个提供类似的结果。对于天然或亲和标记的RNA聚合酶的纯化的其他方法也可以使用12,13。适马/努沙登加​​因素可为d获得旁切在以前的工作14,15。 </li></ol></li><li>焦碳酸二乙酯的制备（DEPC）处理的水 注意:可替换地，可以从商业来源（材料清单）获得无RNase水。 <ol><li>加入1ml焦碳酸二乙酯（DEPC），以1升纯化水（0.1％体积/体积）并混合过夜。 </li><li>高压灭菌DEPC水和提示两次。 </li></ol></li><li> DNA模板制备<ol><li>使用质粒纯化试剂盒根据制造商的说明（材料清单）纯化质粒pSG1154 16和/或pIA226 17。 </li><li>以放大决胜转录测定的模板（360 bp的片段包含在P XYL启动子区域），装配在冰上的PCR反应包括25微升2×PCR混合物（材料清单），每个引物2微升（5μM） :5&#39;-CAAAGCCTGTCGGAATTGG-3&#39;（正向）和5&#39;-CCCATTAACATCACCATC-3&#39;（反向），1微升pSG1154质粒公关eparation（250μM）和20μl纯化水）。 <ol><li>或者可以选择，对于单轮转录实验，扩增447 bp的片段从pIA226 17涵盖了λP R启动区。设置用25微升2×PCR混合物，2微升的各引物（5μM）在冰上的PCR反应:5&#39;-GTGCTCATACGTTAAATCT-3&#39;（正向）和5&#39;-CAGTTCCCTACTCTCGCATG-3&#39;（反向），1微升pIA226 17质粒制备（250μM）和20μl纯化水）。 注意:此模板缺乏胞嘧啶，直到+26 NT位置，这是从转录起始位点的26 日核苷酸。因此，在使用该模板将允许启动，启动子间隙和在+26核苷酸失速，以形成一个稳定的转录延伸复合物（EC），当只有ATP，UTP和GTP存在于反应转录。 </li></ol></li><li>设置了满足以下条件的PCR反应:95℃;℃，30秒，50℃，30秒，72℃30秒，重复30个循环。 </li><li>纯化使用根据一个PCR清理试剂盒（材料清单）制造商的指导产品并在用于转录分析100μl的转录缓冲液洗脱。 </li><li>确定使用fluorospectrometer（材料清单）模板DNA的浓度。 </li></ol></li><li> NTP准备 注意:的NTPs可从不同的商业来源（材料列表）来获得。 <ol><li>溶解于DEPC处理过的水的NTPs（≥99％纯），以使1M的库存，等分试样1ml溶液进入1.5毫升管（50微升在每个管），并存储在-20℃。 </li><li>为了α-32 p UTP（3000居里/毫摩尔）在实验前32 P具有14.29天的半衰期短。 注意:使用阿尔法 - 标记的UTP避免比较不同长度带的强度。 </li></ol></li><li> RNA梯子准备<ol><li>组装后续在室温下ING反应:0.5微克的RNA模板混合（材料清单），80毫米的HEPES缓冲液pH7.5的12毫氯化镁 ，10毫摩尔NaCl，10毫摩尔DTT，2毫摩尔ATP / CTP / GTP，0.5mM的UTP，2微升α-32 p UTP，0.5微升T7 RNA聚合酶（材料清单），DEPC处理过的水到20微升。 </li><li>允许反应在37℃进行1小时。立即使用或在-80℃保存。 </li></ol></li></ol> 2. RNA测序凝胶的制备<ol><li>凝胶制备<ol><li>清洗从测序凝胶装置（材料清单）用70％的乙醇与纯净水两次板，并用梳子和两个间隔件组装的单元格。 注意:一个盘可以与硅化试剂（材料列表）进行包衣，以促进在步骤4.2凝胶去除。 </li><li>溶解33.6克脲在22ml水和16.4 5倍的Tris /硼酸盐/ EDTA（TBE）缓冲液（54克亚磷酸三（羟甲基）氨基甲烷的溶液27.5克硼酸10毫升1M的乙二胺四乙酸（EDTA），pH为7.4在1L水）用磁力搅拌器。 </li><li>将16毫升40％丙烯酰胺/双丙烯酰胺溶液（19:1）的尿素溶液，并通过0.45μm的过滤器进行过滤。 </li><li>添加514微升10％（重量/体积）新鲜制备的过硫酸铵在水中，随后51.4微升N，N，N&#39;，N&#39;四甲基乙二胺（TEMED），以过滤后的溶液。 </li><li>轻轻颠倒凝胶溶液以避免过度通气和立即顺利注入从底部喷射孔的水平定位预包装测序细胞。注意避免在注射过程中制作气泡。 </li><li>允许1-1.5小时，直到凝胶在转录实验使用前完全设置。当与在测序细胞的顶部水的湿纸巾包装的凝胶可以在室温下贮存两年或三天。 </li></ol></li></ol> 3.转录反应<ol><li>转录延长暂停试验<ol><li>一个转录延伸复合物的形成<ol><li>加入1μlRNAP核心酶（转录缓冲1μM股票）以1.5 ml管。 </li><li>加入1μl纯化的Σ-70因子（σ70;在转录缓冲液为3μM股票）的RNA聚合酶，并在冰上孵育10分钟，以形成RNA聚合酶全酶。 </li><li>添加在转录缓冲液3微升500nM的纯化的模板DNA（来自pIA226 PCR产物λP R启动）到RNAP全酶，并在37ºC孵育5分钟，以形成开放的复杂。 </li><li>制备反应成分的混合物中，用5微升10×转录缓冲液（材料清单另外150毫升管:400毫的Tris-HCl，1.6M的氯化钾，100mM的MgCl 2的，10毫摩尔DTT，50％甘油，pH值7.9中DEPC处理水），2微升250μM每个GTP和UTP，1微升1毫米ATP和1微升α-32 p UTP（3000居里/毫摩尔）的。 DEPC处理水加至41微升。 没有德:RNase抑制剂（材料清单）可以被用于最小化RNase污染。 </li><li>该混合物溶液转移到含有开放的复杂的管，并在37ºC孵育15分钟，以形成转录延伸复杂在+26停止核苷酸。 </li><li>移动至反应至室温（〜21℃）并加入1微升的2.5毫克/毫升利福平至50微克/毫升的最终浓度。 </li><li>加入1微升0.1mM的纯化的NusA到反应混合物中的孵育在室温下5分钟。 </li><li>添加2微升250微米的CTP到反应混合物中，以使50微升的最终体积，并使用定时器测量的反应时间。 </li><li>转移5μl反应溶液加入到2微升的RNA凝胶上样缓冲液（95％甲酰胺，0.05％溴酚蓝和0.05％二甲苯青），在30，60，120，240，480，和1200秒以猝灭反应。 </li><li>处置的放射性废物的续32磷UTP提示和管ainer。 </li></ol></li></ol></li><li>转录起始含量<ol><li>在开放的复杂的形成<ol><li> σ70稀释纯化至0.5微米。 </li><li>重复步骤3.1.1.1至3.1.1.3使用0.5微米（而不是3μM）σ70精制后，DNA模板（从pSG1154 PCR产物在P XYL启动子区）。 </li></ol></li><li>转录起始试验的抑制<ol><li>添加旨在防止RNA聚合酶全酶形成到RNAP核心酶之前加入σ70（步骤3.1.1.1之后）抑制剂，接着步骤3.1.1.2和3.1.1.3检查抑制剂是否能够破坏全酶形成。 </li><li>另外，步骤3.1.1.2后，添加抑制剂来检查它是否能够竞争性抑制σ70结合RNA聚合酶。 </li><li>使用二甲基亚砜，抑制剂的溶剂相同量，而不是抑制剂小号滴答控制的反应。 </li></ol></li><li>在反应完成<ol><li>溶于水肝素到在-20℃的储备浓度为1mg / ml和库存。 注意:肝素是DNA的竞争者能够螯合尚未形成一个开放的复杂RNA聚合酶。一旦开放的复杂形成，肝素不能进入RNA聚合酶的DNA结合位点，并从预形成的开放的复杂抑制转录。因此，肝素只需要单轮试验;它并不需要用于多轮测定。 </li><li>制备使用5微升的10×转录缓冲液（材料清单），1微升的1毫克/毫升肝素溶液，1微升10毫每个ATP，GTP，和CTP，2.5mM的UTP的，和1μl的α-32的反应混合物P UTP（3000居里/毫摩尔）。 DEPC处理水加至45微升。 </li><li>混合与转录开放的复杂，脉冲自旋反应混合物中并在37℃孵育20分钟。 </li><li>转移反应的10微升这样lution入5微升的RNA凝胶上样缓冲液（95％甲酰胺，0.05％溴酚蓝和0.05％二甲苯青），以淬灭反应。 </li></ol></li></ol></li></ol> 4. RNA凝胶的运行与发展<ol><li>运行变性聚丙烯酰胺凝胶<ol><li>运行在50ºC在1×TBE缓冲液中的凝胶和50瓦为约1.5小时，直至凝胶的温度和缓冲液是稳定的，在50ºC。 </li><li>在90ºC30秒热样品并且将它们移动到冰之前装载到凝胶上。 </li><li>负载样品插入凝胶的顶部旁边含有2微升的RNA梯一个车道的孔（1:100稀释）。 </li><li>在50瓦和50ºC在1×TBE缓冲液中运行该凝胶直到溴酚蓝染料到达〜2/3的向下的凝胶。 </li></ol></li><li>凝胶处理<ol><li>打开缓慢而仔细地上部板从测序细胞中分离出来。采取非常谨慎，以避免破坏凝胶。 </li><li>切chromatograpHY纸（材料清单）从顶部至溴酚蓝染料匹配凝胶尺寸。用一个盖革计数器确认放射性标记的RNA转录物的大致位置。 </li><li>小心盖上滤纸上的凝胶，然后用力按压，直到凝胶粘附在滤纸。切断并丢弃凝胶底部放射性废物的未掺入的NTPs运行到凝胶底部，它可以是非常热。 </li><li>放置在真空干燥机的干燥床的类似大小的一片滤纸，并小心地放置凝胶涂布纸到它与凝胶的一面朝上。 </li><li>覆盖保鲜膜和干燥在60℃下1.5小时的凝胶。 </li><li>用保鲜膜包住凝胶涂覆的滤纸的另一侧，并将其放置于在成像情况下的光激励荧光体板，用适当的曝光时间，这可以从1小时变化到过夜根据反应的效率。 </li><li>图像使用荧光板根据制造商的说明的成像器扫描仪。决定根据软件说明书使用ImageJ软件相对带强度。 </li><li>用一个盖革计数器来检查放射性污染在工作区，擦拭可疑物品和面积与组织和清洗剂和处置的组织中的处置容器。 </li></ol></li></ol>

<ol>
	<li>Mooney, R. A., Artsimovitch, I., Landick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+processing+by+RNA+polymerase:+recognition+of+regulatory+signals+during+RNA+chain+elongation.">Information processing by RNA polymerase: recognition of regulatory signals during RNA chain elongation.</a> J. Bacteriol. 180 (13), 3265-3275 (1998).</li><li>Burgess, R. R., Anthony, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+sigma+docks+to+RNA+polymerase+and+what+sigma+does.">How sigma docks to RNA polymerase and what sigma does.</a> Curr. Opin. Microbiol. 4 (2), 126-131 (2001).</li><li>Gusarov, I., Nudler, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+intrinsic+transcription+termination+by+N+and+NusA:+the+basic+mechanisms.">Control of intrinsic transcription termination by N and NusA: the basic mechanisms.</a> Cell. 107 (4), 437-449 (2001).</li><li>Ha, K. S., Toulokhonov, I., Vassylyev, D. G., Landick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NusA+N-terminal+domain+is+necessary+and+sufficient+for+enhancement+of+transcriptional+pausing+via+interaction+with+the+RNA+exit+channel+of+RNA+polymerase.">The NusA N-terminal domain is necessary and sufficient for enhancement of transcriptional pausing via interaction with the RNA exit channel of RNA polymerase.</a> J. Mol. Biol. 401 (5), 708-725 (2010).</li><li>Yang, X., Lewis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+interaction+between+RNA+polymerase+and+the+elongation+factor+NusA.">The interaction between RNA polymerase and the elongation factor NusA.</a> RNA Biol. 7 (3), 272-275 (2010).</li><li>Artsimovitch, I., Henkin, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+approaches+to+analysis+of+transcription+termination.">In vitro approaches to analysis of transcription termination.</a> Methods. 47 (1), 37-43 (2009).</li><li>Decker, K. B., Hinton, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+regulation+at+the+core:+Similarities+among+bacterial,+archaeal,+and+eukaryotic+RNA+polymerases.">Transcription regulation at the core: Similarities among bacterial, archaeal, and eukaryotic RNA polymerases.</a> Annu. Rev. Microbiol. 67, 113-139 (2013).</li><li>Artsimovitch, I., Vassylyev, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+easy+to+stop+RNA+polymerase?.">Is it easy to stop RNA polymerase?.</a> Cell Cycle. 5 (4), 399-404 (2006).</li><li>Murakami, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+biology+of+bacterial+RNA+polymerase.">Structural biology of bacterial RNA polymerase.</a> Biomolecules. 5 (2), 848-864 (2015).</li><li>Ma, C., Yang, X., Lewis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+transcription+as+a+target+for+antibacterial+drug+development.">Bacterial transcription as a target for antibacterial drug development.</a> Microbiol. Mol. Biol. Rev. 80 (1), 139-160 (2016).</li><li>Yang, X., Ma, C., Lewis, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vector+system+that+allows+simple+generation+of+mutant+Escherichia+coli+RNA+polymerase.">A vector system that allows simple generation of mutant Escherichia coli RNA polymerase.</a> Plasmid. 75, 37-41 (2014).</li><li>Burgess, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+method+for+the+large+scale+purification+of+Escherichia+coli+deoxyribonucleic+acid-dependent+ribonucleic+acid+polymerase.">A new method for the large scale purification of Escherichia coli deoxyribonucleic acid-dependent ribonucleic acid polymerase.</a> J. Biol. Chem. 244, 6160-6167 (1969).</li><li>Artsimovitch, I., Svetlov, V., Murakami, K. S., Landick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-overexpression+of+Escherichia+coli+RNA+polymerase+subunits+allows+isolation+and+analysis+of+mutant+enzymes+lacking+lineage-specific+sequence+insertions.">Co-overexpression of Escherichia coli RNA polymerase subunits allows isolation and analysis of mutant enzymes lacking lineage-specific sequence insertions.</a> J. Biol. Chem. 278 (14), 12344-12355 (2003).</li><li>Ma, C., Yang, X., Lewis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+transcription+inhibitor+of+RNA+polymerase+holoenzyme+formation+by+structure-based+drug+design:+From+in+silico+screening+to+validation.">Bacterial transcription inhibitor of RNA polymerase holoenzyme formation by structure-based drug design: From in silico screening to validation.</a> ACS Infect. Dis. 2, 39-46 (2016).</li><li>Ma, C., Mobli, M., Yang, X., Keller, A. N., King, G. F., Lewis, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+polymerase-induced+remodelling+of+NusA+produces+a+pause+enhancement+complex.">RNA polymerase-induced remodelling of NusA produces a pause enhancement complex.</a> Nucleic Acids Res. 43 (5), 2829-2840 (2015).</li><li>Lewis, P. J., Marston, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GFP+vectors+for+controlled+expression+and+dual+labelling+of+protein+fusions+in+Bacillus+subtilis.">GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis.</a> Gene. 227 (1), 101-110 (1999).</li><li>Artsimovitch, I., Landick, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pausing+by+bacterial+RNA+polymerase+is+mediated+by+mechanistically+distinct+classes+of+signals.">Pausing by bacterial RNA polymerase is mediated by mechanistically distinct classes of signals.</a> Proc. Natl. Acad. Sci. 97 (13), 7090-7095 (2000).</li><li>Vassylyev, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+bacterial+RNA+polymerase+holoenzyme+at+2.6+&#197;+resolution.">Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 &#197; resolution.</a> Nature. 417, 712-719 (2002).</li><li>Artsimovitch, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+transcription+regulation+by+alarmone+ppGpp.">Structural basis for transcription regulation by alarmone ppGpp.</a> Cell. 117 (3), 299-310 (2004).</li><li>Bordier, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+rifampicin-resistant+RNA+synthesis+by+rifampicin-RNA+polymerase+complexes.">Inhibition of rifampicin-resistant RNA synthesis by rifampicin-RNA polymerase complexes.</a> FEBS lett. 45 (1-2), 259-262 (1974).</li><li>Tang, G. Q., Anand, V. S., Patel, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+assay+to+measure+the+real-time+kinetics+of+nucleotide+incorporation+during+transcription+elongation.">Fluorescence-based assay to measure the real-time kinetics of nucleotide incorporation during transcription elongation.</a> J. Mol. Biol. 405 (3), 666-678 (2011).</li></ol>

The authors have nothing to disclose.

在所有生物中，转录是一严格调节的过程。 在体外已经开发转录测定，以用于测试的转录因子，小分子和转录抑制剂的影响提供了平台。在该方法中纸张，被描述为一般细菌转录的测定法。因为它们允许沿时间线中的所有转录产物可视化转录实验与成绩单的测序凝胶电泳结合是机理研究非常重要。其结果是，这种方法是能够识别的在反应条件下小的变化的影响，并露出动作的一个可行的机...

转录是在其中RNA是从一个特定的DNA模板合成的过程。在真核细胞中有三个不同的RNAPs:RNA聚合酶我转录rRNA的前体，RNA聚合酶II负责mRNA和某些小核RNA的合成，和5S rRNA基因和tRNA的合成是通过RNA聚合酶III进行的。在细菌中，有负责RNA的所有类的转录只有一个RNAP。有转录三个阶段:起始，延伸和终止。转录是在细胞中最高度调节过程中的一个。 在转录周期各阶段表示基因表达1的调节一个检查点。为起始，RNA聚合酶具有与σ因子相关联，以形成全酶，这是需要以引导酶特定网站的所谓启动子2以形成一个开放的启动子复合物。随后，大套房的转录因子负责监管Ô在延伸和终止阶段˚FRNA聚合酶活性。这里研究的转录因子是高度保守的和必需的蛋白质，的NusA。它参与rRNA的合成3-5中调节转录暂停和终止，以及抗终止。 体外转录测定法已被开发作为有力工具转录6中学习复杂的调节步骤。在一般情况下，DNA的包括一个启动子区的线性片段是必需的作为模板进行转录。 DNA模板通常是通过PCR或通过线性化的质粒产生的。纯化的蛋白质和的NTPs（包括一个放射性标记的NTP为检测的目的），然后加入和产品分析培养所需要的时间以下。使用相应的模板和反应条件，一直在使用这种方法，它使得transcr详细的分子特征研究转录的各个阶段iption在过去的半个世纪7。在与RNA聚合酶的三维结构信息的组合，它也是可能探测由抗生素和抗生素引线转录抑制的分子机制，以及在新的，改进的药物8-10开发使用此信息。 在这项工作中，我们提供的转录分析如何可以被用来确定由转录延伸/终止因子的NusA，以及如何能够确定的一个新的转录起始抑制剂引线的作用机制的调节机制的例子。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Obtain the proteins required for transcription assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli RNAP</td>
			<td>Epicentre</td>
			<td>S90250</td>
			<td></td>
		</tr>
		<tr>
			<td>Preparation of DEPC-treated water</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl pyrocarbonate (DEPC)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>D5758</td>
			<td></td>
		</tr>
		<tr>
			<td>RNase-free water&#160;</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Ambion Nuclease-Free Water</td>
			<td>ThermoFisher</td>
			<td>AM9937</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA template preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard&#160;Plus&#160;SV Minipreps DNA Purification System</td>
			<td>Promega</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>ACCUZYME Mix</td>
			<td>Bioline</td>
			<td>BIO-25028</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Clean-Up System&#160;</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 3300 fluorospectrometer</td>
			<td>Thermo Scientific</td>
			<td>ND-3300</td>
			<td></td>
		</tr>
		<tr>
			<td>NTP Preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A6559</td>
			<td></td>
		</tr>
		<tr>
			<td>UTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>U1006</td>
			<td></td>
		</tr>
		<tr>
			<td>GTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G3776</td>
			<td></td>
		</tr>
		<tr>
			<td>CTP</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>C9274</td>
			<td></td>
		</tr>
		<tr>
			<td>High Purity rNTPs</td>
			<td>GE Healthcare</td>
			<td>27-2025-01</td>
			<td></td>
		</tr>
		<tr>
			<td>&#945;-32P UTP&#160;</td>
			<td>PerkinElmer&#160;&#160;</td>
			<td>BLU007C001MC</td>
			<td>Radioactive compound</td>
		</tr>
		<tr>
			<td>RNA ladder preparation&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Novagen Perfect RNA Marker Template Mix 0.1&#8211;1 kb</td>
			<td>Millipore</td>
			<td>69003</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>H7006</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>T7 RNAP</td>
			<td>Promega</td>
			<td>P2075</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel preparation</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sequi-Gen GT nucleic acid sequencing cell&#160;</td>
			<td>Bio-Rad&#160;&#160;</td>
			<td>165-3804</td>
			<td></td>
		</tr>
		<tr>
			<td>Sigmacote&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>SL2</td>
			<td></td>
		</tr>
		<tr>
			<td>urea&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>U6504</td>
			<td></td>
		</tr>
		<tr>
			<td>tris(hydroxymethyl)aminomethane&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>154563</td>
			<td></td>
		</tr>
		<tr>
			<td>boric acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>B7901</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>ED</td>
			<td></td>
		</tr>
		<tr>
			<td>40% Acrylamide/bis-acrylamide&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A9926</td>
			<td></td>
		</tr>
		<tr>
			<td>ammonium persulfate&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#697;&#8242;,N&#697;&#8242;-Tetramethylethylenediamine (TEMED)&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N",N"-Tetramethylethylenediamine (TEMED) </td>
			<td>Sigma-Aldrich </td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription Assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>rifampicin&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>R3501</td>
			<td></td>
		</tr>
		<tr>
			<td>formamide&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F9037</td>
			<td></td>
		</tr>
		<tr>
			<td>bromophenol blue&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>B0126</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene cyanol&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>X4126</td>
			<td></td>
		</tr>
		<tr>
			<td>heparin&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>84020</td>
			<td></td>
		</tr>
		<tr>
			<td>RNasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td></td>
		</tr>
		<tr>
			<td>Transcription buffer&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>P9541</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>M2393</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>DTT-RO</td>
			<td></td>
		</tr>
		<tr>
			<td>glycerol&#160;&#160;</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>G5516</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Whatman&#160;3MM Chr Chromatography Paper</td>
			<td>Fisher Scientific</td>
			<td>05-714-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Radioactive decontaminant</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Decon 90</td>
			<td>decon</td>
			<td>decon90</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel Treatment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon Trio+ imager</td>
			<td>GE Healthcare Life Sciences</td>
			<td>63-0055-89</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro transcription assays and their application in drug discovery

体外转录测定法已被开发并广泛使用了许多年，以研究参与转录的分子机制。这个过程需要多亚基DNA依赖性RNA聚合酶（RNAP）等一系列的，其作用的基因表达过程中调节RNA聚合酶的活性转录因子。放射性成绩单的测序凝胶电泳用于提供关于如何转录所得，哪些参数可以影响它的详细机理信息。在本文中，我们描述了协议来研究必不可少延伸因子的NusA如何调控转录暂停，以及一个以确定抗菌剂通过抑制RNA聚合酶全酶形成指定转录起始方法。这些方法可用于一个作为平台的额外方法的发展探索转录因子的作用机制仍然不清楚，以及新的抗菌阿根TS靶向转录这是在抗生素研发的未充分利用的药物靶标。

转录效率可以通过测量在不同时间点的频段辐射水平来确定。的暂停检测来测试启用暂停，终止的可视化的NusA因子的功能，和径流制品（ 图1A）。在的NusA的N-末端结构域的存在（的NusA NTD;氨基酸残基1-137）时，RNA产物的外观相比缺乏的NusA对照实验显著延迟。的的NusA片段，或改变的一系列氨基酸的丙氨酸的氨基酸残基104-137（螺旋3）缺失（残基K36，K37，R104，Q10...

Watch this Scientific Journal Video about In Vitro Transcription Assays and Their Application in Drug Discovery at JoVE.com

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 ...

in-vitro-transcription-assays-and-their-application-in-drug-discovery

Research

JoVE Journal

Genetics

In Vitro Transcription Assays and Their Application in Drug Discovery

15.0K 次观看.  University of Newcastle. In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

视频: In Vitro Transcription Assays and Their Application in Drug Discovery

siRNA Transfection and EMSA Analyses on Freshly Isolated Human Villous Cytotrophoblasts

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend diseases related to pregnancy. In this protocol, human primary villous cytotrophoblasts freshly isolated from placentas through a standard DNase/trypsin protocol are microporated with small interfering RNA (siRNA). This approach provided greater efficiency for siRNA transfection when compared to a lipofection-based method. Transfected cells can subsequently be analyzed by standard Western blot within a time frame of 3-4 days post-transfection. In addition, using cultured primary villous cytotrophoblasts, Electrophoretic Mobility Shift Assay (EMSA) analysis was optimized and performed on extracts from days 1 to 4. The use of these cultured primary cells and the protocol described allow for an evaluation of the implication of specific genes and transcription factors in the process of villous cytotrophoblast differentiation into a syncytiotrophoblast-like cell layer. However, the limited time span allowable in culture precludes the use of methods requiring more time, such as generation of a stable cell population. Therefore testing of this cell population requires highly optimized gene transfer protocols.

This protocol describes a method for efficiently transfecting siRNA in freshly isolated human villous cytotrophoblasts using microporation and identifying DNA-protein complexes in these cells. Transfected cells can be monitored by Western blot and EMSA analyses during the 4-day culture time.

在新鲜分离的人代绒毛滋养细胞转染和EMSA分析

Human primary villous cytotrophoblasts are a very useful source of primary cells to study placental functions and regulatory mechanisms, and to comprehend ...

科研

遗传学

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most mutagenesis-based screens, researchers have relied on the use of toxic chemicals, carcinogens, or irradiation, which requires designated equipment, safety setup, and/or disposal of hazardous materials. We have developed a simple approach to induce heritable mutations in C. elegans using germline-expressed histone-miniSOG, a light-inducible potent generator of reactive oxygen species. This mutagenesis method is free of toxic chemicals and requires minimal laboratory safety and waste management. The induced DNA modifications include single-nucleotide changes and small deletions, and complement those caused by classical chemical mutagenesis. This methodology can also be used to induce integration of extrachromosomal transgenes. Here, we provide the details of the LED setup and protocols for standard mutagenesis and transgene integration.

Optogenetic Random Mutagenesis Using Histone-miniSOG in C. elegans

Genetically-encoded histone-miniSOG induces genome-wide heritable mutations in a blue&#160;light-dependent manner. This mutagenesis method is simple, fast, free of toxic chemicals, and well-suited for forward genetic screening and transgene integration.

光遗传学随机突变用组蛋白miniSOG在<em&gt;℃。线虫</em

Forward genetic screening in model organisms is the workhorse to discover functionally important genes and pathways in many biological processes. In most ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

饲养并在隐藏甲壳虫双链RNA介导的基因敲除，<em&gt;白腹皮蠹</em

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to sleep behavior. In vertebrates, circadian rhythm is controlled by a molecular oscillator that functions in both the suprachiasmatic nucleus (SCN; central pacemaker) and individual cells comprising most peripheral tissues. More importantly, disruption of circadian rhythm by exposure to light-at-night, environmental stressors and/or toxicants is associated with increased risk of chronic diseases and aging. The ability to identify agents that can disrupt central and/or peripheral biological clocks, and agents that can prevent or mitigate the effects of circadian disruption, has significant implications for prevention of chronic diseases. Although rodent models can be used to identify exposures and agents that induce or prevent/mitigate circadian disruption, these experiments require large numbers of animals. In vivo studies also require significant resources and infrastructure, and require researchers to work all night. Thus, there is an urgent need for a cell-type appropriate in vitro system to screen for environmental circadian disruptors and enhancers in cell types from different organs and disease states. We constructed a vector that drives transcription of the destabilized luciferase in eukaryotic cells under the control of the human PERIOD 2 gene promoter. This circadian reporter construct was stably transfected into human mammary epithelial cells, and circadian responsive reporter cells were selected to develop the in vitro bioluminescence assay. Here, we present a detailed protocol to establish and validate the assay. We further provide details for proof of concept experiments demonstrating the ability of our in vitro assay to recapitulate the in vivo effects of various chemicals on the cellular biological clock. The results indicate that the assay can be adapted to a variety of cell types to screen for both environmental disruptors and chemopreventive enhancers of circadian clocks.

In Vitro Bioluminescence Assay to Characterize Circadian Rhythm in Mammary Epithelial Cells

An in vitro bioluminescence assay to determine cellular circadian rhythm in mammary epithelial cells is presented. This method utilizes mammalian cell reporter plasmids expressing destabilized luciferase under the control of the PERIOD 2 gene promoter. It can be adapted to other cell types to evaluate organ-specific effects on circadian rhythm.

体外生物荧光法对乳腺上皮细胞昼夜节律的表征

The circadian rhythm is a fundamental physiological process present in all organisms that regulates biological processes ranging from gene expression to ...

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in humans, but more tools and research efforts are needed to better elucidate the mechanisms involved. For over a century, the genetically highly tractable model of Drosophila has been instrumental in the discovery of key genes and molecular pathways that proved to be highly conserved across species. Many biological processes and disease mechanisms are functionally conserved in the fly, such as development (e.g., body plan, heart), cancer, and neurodegenerative disease. Recently, the study of obesity and secondary pathologies, such as heart disease in model organisms, has played a highly critical role in the identification of key regulators involved in metabolic syndrome in humans.
Here, we propose to use this model organism as an efficient tool to induce obesity, i.e., excessive fat accumulation, and develop an efficient protocol to monitor fat content in the form of TAGs accumulation. In addition to the highly conserved, but less complex genome, the fly also has a short lifespan for rapid experimentation, combined with cost-effectiveness. This paper provides a detailed protocol for High Fat Diet (HFD) feeding in Drosophila to induce obesity and a high throughput triacylglyceride (TAG) assay for measuring the associated increase in fat content, with the aim to be highly reproducible and efficient for large-scale genetic or chemical screening. These protocols offer new opportunities to efficiently investigate regulatory mechanisms involved in obesity, as well as provide a standardized platform for drug discovery research for rapid testing of the effect of drug candidates on the development or prevention of obesity, diabetes and related metabolic diseases.

High Fat Diet Feeding and High Throughput Triacylglyceride Assay in Drosophila Melanogaster

This is a high fat diet feeding protocol to induce obesity in Drosophila, a model for understanding fundamental molecular mechanisms implicated in lipotoxicity. It also provides a high throughput triacylglyceride assay for measuring fat accumulation in Drosophila and potentially other (insect) models under various dietary, environmental, genetic or physiological conditions.

高脂膳食喂养和高通量三测定在果蝇中的含量

Heart disease is the number one cause of human death worldwide. Numerous studies have shown strong connections between obesity and cardiac malfunction in ...

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 ...

Novel Sequence Discovery by Subtractive Genomics

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a larger genomic context. Subtractive genomics enables a researcher to isolate a target sequence of interest (T) by comprehensive sequencing and subtracting out known genetic elements (reference, R). The method can be used to identify novel sequences such as mitochondria, chloroplasts, viruses, or germline restricted chromosomes, and is particularly useful when T cannot be easily isolated from R. Beginning with the comprehensive genomic data (R + T), the method uses Basic Local Alignment Search Tool (BLAST) against a reference sequence, or sequences, to remove the matching known sequences (R), leaving behind the target (T). For subtraction to work best, R should be a relatively complete draft that is missing T. Since sequences remaining after subtraction are tested through quantitative Polymerase Chain Reaction (qPCR), R does not need to be complete for the method to work. Here we link computational steps with experimental steps into a cycle that can be iterated as needed, sequentially removing multiple reference sequences and refining the search for T. The advantage of subtractive genomics is that a completely novel target sequence can be identified even in cases in which physical purification is difficult, impossible, or expensive. A drawback of the method is finding a suitable reference for subtraction and obtaining T-positive and negative samples for qPCR testing. We describe our implementation of the method in the identification of the first gene from the germline-restricted chromosome of zebra finch. In that case computational filtering involved three references (R), sequentially removed over three cycles: an incomplete genomic assembly, raw genomic data, and transcriptomic data.

The purpose of this protocol is to use a combination of computational and bench research to find novel sequences that cannot be easily separated from a co-purifying sequence, which may be only partially known.

减法基因组学的新序列发现

Subtractive genomics can be used in any research where the goal is to identify the sequence of a gene, protein, or general region that is embedded in a ...

Characterizing Histone Post-translational Modification Alterations in Yeast Neurodegenerative Proteinopathy Models

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives each year. Effective treatment options able to halt disease progression are lacking. Despite the extensive sequencing efforts in large patient populations, the majority of ALS and PD cases remain unexplained by genetic mutations alone. Epigenetics mechanisms, such as the post-translational modification of histone proteins, may be involved in neurodegenerative disease etiology and progression and lead to new targets for pharmaceutical intervention. Mammalian in vivo and in vitro models of ALS and PD are costly and often require prolonged and laborious experimental protocols. Here, we outline a practical, fast, and cost-effective approach to determining genome-wide alterations in histone modification levels using Saccharomyces cerevisiae as a model system. This protocol allows for comprehensive investigations into epigenetic changes connected to neurodegenerative proteinopathies that corroborate previous findings in different model systems while significantly expanding our knowledge of the neurodegenerative disease epigenome.

This protocol outlines experimental procedures to characterize genome-wide changes in the levels of histone post-translational modifications (PTM) occurring in connection with the overexpression of proteins associated with ALS and Parkinson&#39;s disease in Saccharomyces cerevisiae models. After SDS-PAGE separation, individual histone PTM levels are detected with modification-specific antibodies via Western blotting.

酵母神经退行性蛋白病模型中组蛋白后转化修饰改变的表征

Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and Parkinson&#39;s disease (PD), cause the loss of hundreds of thousands of lives ...

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant viruses they vector. Despite numerous studies of the biology of these species in different environments, a key life history parameter, offspring sex ratios, has received little attention, yet is important for predicting population dynamics. The primary sex ratio (sex ratio at oviposition) of Bemisia tabaci has never been reported but can be found by determining the egg fertilization rate of this haplodiploid insect. The technique involves the dechorionation of eggs with bleach, a series of fixation steps, and the application of the general DNA fluorescent stain, DAPI (4&#8242;,6-diamidino-2-phenylindole, a DNA-binding fluorescent dye), to bind to female and male pronuclei. Here, we present the technique, and an example of its application, to test whether an endosymbiotic bacterium, Rickettsia sp. nr. bellii, influenced the primary sex ratio of B. tabaci. This method may assist in population studies of whiteflies, or in determining if sex allocation exists with certain environmental stimuli.

Determining the Egg Fertilization Rate of Bemisia tabaci Using a Cytogenetic Technique

We present a simple cytogenetic technique using 4&#8242;,6-diamidino-2-phenylindole (DAPI) to determine the fertilization rate and primary sex ratio of the haplodiploid invasive pest Bemisia tabaci.

用细胞遗传学技术测定芽孢杆菌的受精率

A few species of sap-sucking whiteflies are some of the most damaging terrestrial pests worldwide because of the crop damage they inflict and plant ...

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.&#160;

Presented here are protocols for in vitro biochemical assays using biotin labels that may be widely&#160;applicable for studying protein-nucleic acid interactions.

使用生物素标签研究蛋白质-核酸相互作用的体外生化分析

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental ...