我们想感谢卡罗琳价格博士科学讨论和有见地的建议。这项工作是由美国国立卫生研究院授予AI066095（PI李:碧波）的支持。

1。检测T。布氏端粒G -悬使用母语-凝胶杂交3 原理（图1）:在自然条件下，结束标记（CCCTAA）4（TELC）寡核苷酸探针只能杂交的单链端粒G -悬地区。杂交强度悬的长度成正比。变性，中和后，相同的探测器将能够杂交整个端粒区域。杂交强度代表总端粒DNA量和可用于装载控制。这个实验的两个标准控制杂交使用末端标记（TTAGGG）4（TELG）寡核苷酸探针，它应该不会产生任何信号为T 。 布氏细胞不会有端粒的C型悬结构，以及对待具体3&#39; -单链外切酶，如埃克索我或埃克索T的基因组DNA，以杂交之前，应消除本地TELC杂交信号。 第1步。准备DNA的脉冲场凝胶电泳（PFGE）插头将脉冲电泳分离完整的染色体。因此，基因组DNA作为DNA的插头准备。 <ol><li>开始用100毫升的血液形成细胞（1.5 -2万细胞/ mL）或20毫升的procyclic细胞（10万细胞/ mL）。 </li><li>溶解1.6％低熔点（LMP）的琼脂糖在L缓冲液（0.01 mol三•CL pH值7.6，0.02 M氯化钠，0.1 M EDTA pH值8.0），并保持它温暖，在50 ° C. </li><li>离心收集细胞在1.5 krpm，4℃10分钟。删除与L缓冲到终浓度为5万细胞/ mL上清和悬浮细胞沉淀。 </li><li>细胞悬液，在42-50 ° C孵育10分钟。 </li><li>加入1体积LMP琼脂糖1细胞的体积。充分混匀，但很快。 </li><li>分装85μL细胞悬液，以每一个一次性的插件模具（BioRad公司）以及。 </li><li>经过5-10分钟，或直至插头是凝固的，转让插入15 m​​L锥形管，用5 mL大号缓冲/ 1％Sarkosyl。加入100μL250 mg / mL的蛋白酶K或蛋白酶K粉捏，拌匀，孵育50 ° 48个小时的彗星。 </li><li>丢弃的缓冲区，用5毫升的L缓冲插头两次重复蛋白酶K消化48小时。新鲜的大号缓冲区清洗插头了。商店插头在L缓冲液在4 ° C。这些插头应使用2个星期内。 </li></ol>第2步。不同的完整的T 。 布氏染色体使用脉冲场凝胶电泳<ul><li>准备琼脂糖凝胶<ol><li>准备2-3 0.5 × TBE缓冲液和凝胶运行室的一名厨师DRII（BioRad公司）单位转移到大号。设置运行温度，启动水泵，然后开始冷却装置。 </li><li>准备在0.5 × TBE缓冲液100毫升1.2％琼脂糖凝胶。酷琼脂糖而设立的凝胶托盘。 </li><li>决定看样订货。请记住，包括DNA标准。干梳和它在一个平面上奠定。每个DNA插件放置到适当的齿。过度缓冲吸删除这样的插件会粘到牙齿周围的DNA插头。 </li><li>倒入琼脂糖凝胶不插入梳子。让降温到40-50℃（几分钟），慢慢地进入凝胶的附加插头插入梳子。确保插头不滑出梳子。让凝胶巩固。从凝胶中取出梳子缓慢。 </li></ol></li><li>脉冲场凝胶电泳 运行在一个厨师DRII单位凝胶:2.5伏/厘米，700 S - 1500小号脉冲，在12 ° C为120小时。 </li></ul>第3步。染色和destain琼脂糖凝胶 1μg/ mL的0.5 × TBE在室温溴乙锭在1小时凝胶染色，然后在0.5 × TBE没有乙锭与温和的摇摆为1小时的甲基溴。以凝胶的图片。 第4步。干琼脂糖凝胶琼脂糖凝胶放在两层3毫米的Whatman纸，并用保鲜膜覆盖的凝胶。干凝胶在室温（凝胶不应该加热超过50 ° C，以避免任何的DNA样本变性）。分别为2和4小时的干燥后更换用干的湿的Whatman论文。保持干燥，直到凝胶完全干燥。这可能需要通宵取决于干燥器。 第5步。标记寡核苷酸探针 1μL50纳克/微升TELC或TELG寡核苷酸1μL10 ×多聚核苷酸激酶（PNK）缓冲液，5μL的混合γ[32P] ATP 2μL，DDH 2和1μLT4 PNK。在37 ° C为1小时。反应混合物中加入90μLTNES缓冲区（10毫米三氯，pH值8.0，氯化钠100毫米，10毫米EDTA，pH值8.0，1％SDS）。 虽然标签oligoes，准备一个G - 25柱:投入3毫升注射器一些玻璃棉和它紧紧与枪头的东西。经Sephadex G - 25罚款（autocl填充注射器aved在TE）3毫升大关。让解决重量。为了净化探针:负载探头列的顶部。洗700 TNES缓冲液，洗脱600μLTNES缓冲区。另外，净化使用一个小型的快速旋转探针™列（罗氏应用科学部）。 第6步:杂交<ol><li>简言之湿干凝胶在蒸馏水洗去任何的Whatman纸卡凝胶</li><li>杂交袋的凝胶，并添加20毫升hybridiztion缓冲区（0.25米娜2 HPO 4 PH7.2; 1毫米EDTA; 7％SDS，1％BSA，通过0.22微米过滤器过滤）。水浴中孵育50℃，温和的摇摆至少1小时。 </li><li>取出预杂交缓冲。煮沸5分钟，探针，并把它添加到25毫升新鲜的杂交缓冲液，过滤消毒用0.22μm的注射器过滤器的组合，直接添加的杂交组合杂交袋。密封袋浅容器用温水和地点。将整个容器的入水洗澡。在50℃过夜孵育，用温和的摇摆。 </li><li>切在杂交袋的小开。倒出尽可能多的杂交组合，并保存在50 mL锥形管。保持在-20 ° C冷冻探头从杂交袋中取出凝胶，并把它在一个容器中。 在此步骤中的一切都将热，需要彻底清洗（长凳上，屏幕，剪刀，其他手续。请一定要戴双层手套！！ </li><li>在30分钟的4 × SSC洗涤凝胶在50 ° C。更换新鲜的缓冲液洗，重复两次。 </li><li>洗4 x SSC/0.1％的SDS凝胶在50 ° C。 </li><li>凝胶放在了一块3毫米的Whatman纸，轻轻擦干凝胶。 </li><li>用保鲜膜包裹的凝胶和揭露〜3天，以phosphorimager </li><li>扫描的phosphorimager </li><li>在变性缓冲液在室温下（1.5 M氯化钠，0.5 M的氢氧化钠）中孵育30分钟凝胶</li><li>凝胶和缓冲液中清洗（3 M氯化钠，0.5 M的三•/ Cl的pH值7.0）在室温下30分钟</li><li>在蒸馏水冲洗3分钟</li><li>前在55℃杂交1小时</li><li>重复使用相同的探针杂交组合，含有55 ° C过夜杂交。 </li><li>洗净上文所述，除了在55 ° C。 </li><li>包装凝胶和揭露phosphorimager〜2小时。 </li><li>扫描phosphorimager。正常化之前变性，变性后获得的，获得的杂交信号。 </li></ol> 2。确定端粒的终端适配器结扎 6核苷酸结扎原则（图2A及2B）:5&#39;末端标记独特的寡核苷酸的3&#39;结束的G悬。这是通过退火具体的互补指南寡核苷酸。只有独特/指导寡适配器轴承兼容的端粒G -悬端序列的序列，将结扎。 为 T 布氏 ，端粒可能终止在6个不同的核苷酸TTAGGG重复之一。因此，使用6个不同的指导oligoes（TG1 TG6，图2A）。结扎后，DNA限制性内切酶酶切和琼脂糖凝胶上解决。 <ol><li>独特的寡核苷酸激酶治疗。 混合3μL10倍PNK缓冲，6μL10 pmole /μL独特的寡核苷酸，5μL的γ[32 P] ATP，DDH 2 O 14μL，2μLT4 PNK（10 U） 。在37 ° C为1小时孵育的混合物。 </li><li>删除非法人热核苷酸。 使用Qiaquick核苷酸去除试剂盒，以消除非法人热（ATP）和结束标记的寡核苷酸洗脱，洗脱缓冲液60μL（终浓度会〜1 pmole /μL）。 </li><li>退火独特的寡核苷酸指导寡核苷酸（创建适配器）。 添加10μL纯化独特的寡核苷酸2μL每个导寡核苷酸1μL，退火缓冲液（200 mM氯化钠，100毫米的Tris - HCl pH值8.0）。放入85℃加热块管和关闭热块。让冷却到RT管和热块。这将需要几个小时。如果有急事，传输管后5分钟至65 ° C，然后37 ° C，然后让它冷却到RT。 </li><li>结扎适配器总DNA。 2.5μL完整的基因组DNA，加4μL5 ×连接酶缓冲液，10μL退火寡核苷酸，2.5μLDDH 2，1μLT4 DNA连接酶。孵育16℃过夜。 </li><li>酶切 20μL连接产物加入3μL10 × NEB缓冲液4，1.5μLALUI和姆博伊每个，4μLDDH 2 O在37℃过夜孵育的混合物。 </li><li>电泳。 每个样品中加入3μL10 ×橙G染料（50％的甘油，0.5％，橙G）。 0.2微克/ ml溴化乙锭，装入0.5 × TBE一个20X20厘米0.7％琼​​脂糖凝胶上的DNA样本。在30V运行30分钟，然后是Continue具有更高的电压，但比120V共有1000 V HR。以用尺子图片旁边的DNA标记的凝胶。 </li><li>干凝胶和揭露。 DE81滤纸和两层3毫米的Whatman纸层琼脂糖凝胶，并用保鲜膜覆盖的凝胶。干凝胶在室温下。公开phosphorimager通宵干凝胶。 </li></ol> 3。结扎介导的引物延伸6 原则（图2A和2C）:一个独特的寡核苷酸是结扎3&#39;与特定的终端序列的G -悬，取决于其在适配器（指导）寡核苷酸的互补序列。经纯化后，标记的指导寡核苷酸，退火G-overhang/unique寡是使用了T4 DNA聚合酶，缺乏链置换和5&#39; - 3&#39;核酸外切酶的活动扩展到SS - DS交界的引物。因此，引物延伸产品的G -悬的长度。 <ol><li>引导和独特的oligoes激酶治疗<ol class="ualpha"><li>混合使用10μL10 × PNK缓冲液，20μL，5 ×连接酶缓冲液（其中包含10毫米ATP），3μLT4 PNK（30 U），47μLDDH 2 20μL10 pmole /μL独特的寡核苷酸。在37 ° C 60分钟的孵育的混合物。 </li><li>混合1μL10 pmole /μL的指导寡核苷酸1μL，2μL的10 × PNK缓冲γ[32 P] ATP，1μLT4 PNK（10 U），和5μLDDH 2 O在37 ° C 60分钟的孵育的混合物。 </li></ol></li><li>退火寡核苷酸指导和独特的寡核苷酸 加入10μL磷酸化的独特的寡核苷酸和结束标记引导寡核苷酸在1.5 mL Eppendorf管（独特的2:1的比例，引导寡确保所有指导寡核苷酸退火10μL（10 pmole）（20 pmole） ）。按照相同的步骤中的第二号议定书退火oligoes。 </li><li>端粒末端结扎的退火指南/独特的寡核苷酸适配器 按照同样的步骤第二号议定书的DNA结扎。 </li><li> 1 / 10体积的醋酸钠沉淀DNA和2.5体积的冰冷乙醇在-80 ° C为30分钟。 12 krpm，4℃离心15分钟，DNA沉淀。 70％的乙醇清洗DNA溶于10μLDDH 2 O托盘</li><li>引物延伸 每10μL的DNA样本，加2μL10 × T4 DNA聚合酶缓冲，1μL1 mg / mL的BSA，1μL25毫米的dATP，dCTP，dTTP的每1μLT4 DNA聚合酶（3 U），和3μLDDH 2 O 请与聚合酶，缓冲等主组合，每个DNA样本等份适量的主结构。在30℃孵育30分钟。然后添加1.2μL0.5M EDTA立即反应。 </li><li>电泳 运行在10％丙烯酰胺/ 7米urea/1xTBE凝胶的延伸产品。装入4μL每个样品。煮沸10分钟在装货前的样品。在800V 1X TBE运行，直到蓝色染料到达凝胶底部。干凝胶在65℃30分钟30分钟，然后在室温下暴露phosphorimager 2小时。 要准备100毫升10％的聚丙烯酰胺凝胶，尿素41克和21毫升40％的丙烯酰胺溶液（丙烯酰胺/双丙烯酰胺29:1）溶于蒸馏水30毫升，加入10毫升10倍TBE。装配玻璃板。浇筑前的凝胶，加1毫升10％的APS和100μLTEMED。倒入胶，并尽量避免任何气泡。 </li></ol> 4。代表性的成果: 检测T。布氏端粒G -悬结构，使用母语-凝胶杂交完整的T。布氏由脉冲场凝胶电泳分离的染色体显示在图3A和3B（左侧面板）T 。 布氏细胞通常含有minichromosomes〜100份，所有具有类似规模（50-150 KB），并迁移到相同的位置（MC）凝胶。由于这一事实后，与TELC寡核苷酸探针的杂交，端粒的G -悬信号是最突出minichromosomes（图3A，中间面板）的。与TELG寡核苷酸探针杂交，一般不会产生任何杂交信号（图3B，中间面板），因为 T。 布氏细胞没有端粒的C悬结构。变性和中，与TELC或TELG寡核苷酸探针杂交后，应揭露对所有的染色体末端端粒信号（图3A及3B，右面板）。 确定端粒的终端适配器结扎核苷酸载入等量的DNA在每个泳道是这个实验是必不可少的，这是用EB染色的凝胶甲基溴。如图4A所示，左边的面板就是一个例子。 在这个协议，结束标记独特的寡核苷酸和引导寡适配器只结扎染色体末端端粒的G -出挑指导寡兼容的序列结束。四<html> NCE独特的寡核苷酸末端标记，将放射性结扎产品，并给予强烈的信号。如图4A所示，右侧面板，巷1给出了一个强烈的信号，表明大量的unique/TG1适配器已结扎端粒结束。 TG1 5&#39;CCCTAA3"的结束序列。因此，端粒的G -出挑这个适配器的一端结扎5&#39;TTAGGG3"。没有观察结扎产品的大量使用其他引导oligoes，表明端粒在TTAGGG 结尾 T中占主导地位布氏细胞。 治疗埃克索我或埃克索T，它是特定的核酸3&#39;悬的基因组DNA，造成损失的结扎产品（图右侧面板中，4A，泳道2和3），表明结扎确实从端粒摹悬结构结扎介导的引物延伸不结扎的末端标记的指导寡核苷酸是长22新台币。载入结束标记引导寡核苷酸作为一个阴性对照和大小标记。 （图4B，11巷，星号表示结束标记指导寡）。通常，我们也观察到这种消极的控制（图4B，11巷，开放的三角形），这是最有可能的残留物非变性适配器〜44 NT带。结扎后的染色体末端，延长产品的大小反映的G -悬结构的长度。大多数T。布氏端粒在5&#39;TTAGGG3年底的G -出挑"（图4A）。然而，这些G -出挑似乎很短（仅10 NT长），产品从结扎TG1指导寡延长是远远长于指导寡（图4B，10巷），但他们允许结扎TG1适配器（图4A，右面板中，泳道1）。虽然只有少量TG4适配器连接到端粒末端（图4A，右面板中，6车道），结扎TG4延伸产品更长（图4B，7巷，箭头头部），最长的产品被~~ 55新台币。因此，我们观察到端粒两个类型的G -悬在 T 布氏细胞。主要端粒的G -出挑5&#39;TTAGGG3"的结束，而是只〜10 NT长。很少端粒G -出挑5&#39;GGGTTA3结束"，但可高达40 NT长。两种类型的G -悬敏感埃克索T（或埃克索我）治疗（图4A，右面板中，2巷，3，和7;图4B，1巷，箭）。 <img src="/files/ftp_upload/1959/1959fig1.jpg" alt="图1" /> 图1。本地凝胶杂交原理，左，自然条件下，结束标记TELC寡核苷酸探针只能与单链的G -丰富的端粒悬杂交。杂交的强度反映了端粒的G -悬的长度。右，变性后，东芝电梯有限公司寡核苷酸探针将与所有的端粒DNA杂交。代表总额的端粒DNA杂交的强度，并用于装载控制，以及最后的G -悬水平计算除以本地杂交信号，通过后变性获得的。 <img src="/files/ftp_upload/1959/1959fig2.jpg" alt="图2" /> 图2。适配器结扎，结扎介导的引物延伸的原则 （A）序列的独特性和六个指导oligoes。 （二）确定端粒的终端适配器结扎核苷酸。独特的寡核苷酸末端标记（标有红色星号），退火，以指导oligoes和结扎端粒。只有当独特/指导适配器熊3&#39;悬，是与终端的端粒序列将予以结扎适配器兼容。 （三）在结扎介导的引物延伸，引导寡末端标记（标有红色星号）和退火处理，以独特的寡核苷酸之前结扎的染色体末端。只有轴承与G -悬兼容的3&#39;悬独特/指导适配器将予以结扎。 ^表示结扎phophordiester债券。去除unligated寡核苷酸对后，将进行引物延伸，用T4 DNA聚合酶，缺乏链置换和5&#39; - 3&#39;核酸外切酶的活动。扩展引导寡核苷酸的最终长度，因此反映的G -悬的长度。 <img src="/files/ftp_upload/1959/1959fig3.jpg" alt="图3" /> 图3 T。布氏端粒G -悬结构分析本地凝胶杂交 （一）在凝胶与TELC寡核苷酸探针杂交。 （二） - 凝胶与TELG寡核苷酸探针杂交。在这两种（A）和（b）留，Ethedium溴化染色PFG。中东，原生的杂交结果。右，变性后的杂交结果。野生类型 T 布氏细胞。完好或埃克索我治疗的基因组DNA的脉冲电泳分离。开放的三角形代表的megabase染色体;集成电路，中等大小的染色体;管委会，minichromosomes。 "/&gt; 图4 T。布氏端粒结构的G -悬结扎介导的引物延伸分析 （一）大多数T.布氏端粒G -出挑终止5&#39;TTAGGG3"。左，Ethedium溴化染色的DNA凝胶。装在每个泳道DNA的量大致相等。权利，接触相同的凝胶干燥后的结果。完整，出我的治疗，或埃克索处理的T - DNA连接到6个不同的结束标记的唯一/指导寡适配器表示。 （二）T布氏端粒短的G -出挑。完整或EXO T型治疗的基因组DNA克隆到六个不同的独特/指导的寡核苷酸引物使用T4 DNA聚合酶的延伸适配器。末端标记TG4寡核苷酸作为阴性对照组（11巷）加载。寡核苷酸本身运行在NT（*）22日也给了一个微弱的信号在44〜NT（Δ）。只有unique/TG4适配器取得了明显长于指导寡核苷酸（箭头，泳道7）延伸产品。

<ol>
	<li>Munoz-Jordan, J. L., Cross, G. A., de Lange, T., Griffith, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=t-loops+at+trypanosome+telomeres.">t-loops at trypanosome telomeres.</a> EMBO J. 20, 579-588 (2001).</li><li>Li, B., Espinal, A., Cross, G. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trypanosome+telomeres+are+protected+by+a+homologue+of+mammalian+TRF2.">Trypanosome telomeres are protected by a homologue of mammalian TRF2.</a> Mol Cell Biol. 25, 5011-5021 (2005).</li><li>Wellinger, R. J., Wolf, A. J., Zakian, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+telomeres+acquire+single-strand+TG1-3+tails+late+in+S+phase.">Saccharomyces telomeres acquire single-strand TG1-3 tails late in S phase.</a> Cell. 72, 51-60 (1993).</li><li>McElligott, R., Wellinger, R. 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+terminal+DNA+structure+of+mammalian+chromosomes.">The terminal DNA structure of mammalian chromosomes.</a> EMBO J. 16, 3705-3714 (1997).</li><li>Harrington, L. A., Greider, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomerase+primer+specificity+and+chromosome+healing.">Telomerase primer specificity and chromosome healing.</a> Nature. 353, 451-454 (1991).</li><li>Jacob, N. K., Skopp, R., Price, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=G-overhang+dynamics+at+Tetrahymena+telomeres.">G-overhang dynamics at Tetrahymena telomeres.</a> Embo J. 20, 4299-4308 (2001).</li><li>Barry, J. D., McCulloch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigenic+variation+in+trypanosomes:+enhanced+phenotypic+variation+in+a+eukaryotic+parasite.">Antigenic variation in trypanosomes: enhanced phenotypic variation in a eukaryotic parasite.</a> Adv Parasitol. 49, 1-70 (2001).</li><li>Yang, X., Figueiredo, L. M., Espinal, A., Okubo, E., Li, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RAP1+is+essential+for+silencing+telomeric+variant+surface+glycoprotein+genes+in+Trypanosoma+brucei.">RAP1 is essential for silencing telomeric variant surface glycoprotein genes in Trypanosoma brucei.</a> Cell. 137, 99-109 (2009).</li></ol>

布氏锥虫引起睡在人类的疾病。这种疾病，如果不及时治疗，必然是致命的 T 。 布氏在哺乳动物宿主细胞进行定期抗原变异，以逃避宿主的免疫攻击7。因此，它是非常难以消除T。布氏一旦感染细胞的建立。最近，我们已经证明， 端粒 T的表达调控发挥了重要作用布氏表面抗原基因8。因此，重要的是进一步了解在 T端粒维持端粒的?...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>SeaKem LE Agarose</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>50004</td>
<td>&nbsp;</td>
<tr>
<td>Agarose Type VII (low melting agarose)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A4018</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K, recombinant, PCR grade</td>
<td>&nbsp;</td>
<td>Roche Diagnostic</td>
<td>03 115801001</td>
<td>&nbsp;</td>
<tr>
<td>Exo I</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0293</td>
<td>&nbsp;</td>
<tr>
<td>Exo T</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0265</td>
<td>&nbsp;</td>
<tr>
<td>T7 exonuclease</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0263</td>
<td>&nbsp;</td>
<tr>
<td>T4 DNA polymerase</td>
<td>&nbsp;</td>
<td>NEB</td>
<td>M0203</td>
<td>&nbsp;</td>
<tr>
<td>QIAquick nucleotide removal kit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>28304</td>
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		</div>

examination of the telomere g-overhang structure in trypanosoma brucei

端粒的G -悬结构已被确定在许多真核生物，包括酵母，脊椎动物和布氏锥虫。它作为基板从头端粒DNA合成的端粒酶，因此，重要的端粒维持 T 。 布氏是一种原生动物寄生虫引起的睡眠疾病在人类和牛的那加那病。一旦被感染的哺乳动物主机， T.定期交换机布氏细胞的表面抗原，以逃避宿主的免疫攻击。我们最近表明的T.布氏端粒的结构起着至关重要的作用，在调控的表面抗原基因的表达，这是 T的关键布氏的发病机制。然而，T.布氏端粒的结构还没有被广泛研究，由于这个专门的结构分析方法的局限性。我们现在已经成功地通过在凝胶杂交和连接介导的引物延长端粒G -悬结构， 在 T核苷酸测定端粒终端适配器结扎方法检查方法的原生布氏细胞。在这里，我们将详细描述了协议，并比较其不同的优势和局限。

Watch this Scientific Journal Video about Examination of the Telomere G-overhang Structure in Trypanosoma brucei at JoVE.com

布氏锥虫端粒 G 突出结构的检测免疫学和感染|视频

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

端粒是染色体的稳定至关重要，并介导的端粒酶的端粒维持端粒G -悬结构是必不可少的。最近，我们通过两种方法检测端粒G -悬结构<em&gt;锥虫布氏</em&gt;，这是原生 - 凝胶杂交和连接介导的引物延伸，这将是描述。

审核端粒G -悬结构锥虫布氏</em

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

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Examination of the Telomere G-overhang Structure in Trypanosoma brucei

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14.2K 次观看.  Cleveland State University. 端粒是染色体的稳定至关重要，并介导的端粒酶的端粒维持端粒G -悬结构是必不可少的。最近，我们通过两种方法检测端粒G -悬结构锥虫布氏，这是原生 - 凝胶杂交和连接介导的引物延伸，这将是描述。

视频: Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

科研

免疫与感染

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Examination of Thymic Positive and Negative Selection by Flow Cytometry

A healthy immune system requires that T cells respond to foreign antigens while remaining tolerant to self-antigens. Random rearrangement of the T cell receptor (TCR) &alpha; and &beta; loci generates a T cell repertoire with vast diversity in antigen specificity, both to self and foreign. Selection of the repertoire during development in the thymus is critical for generating safe and useful T cells. Defects in thymic selection contribute to the development of autoimmune and immunodeficiency disorders1-4. 
T cell progenitors enter the thymus as double negative (DN) thymocytes that do not express CD4 or CD8 co-receptors. Expression of the &alpha;&beta;TCR and both co-receptors occurs at the double positive (DP) stage. Interaction of the &alpha;&beta;TCR with self-peptide-MHC (pMHC) presented by thymic cells determines the fate of the DP thymocyte. High affinity interactions lead to negative selection and elimination of self-reactive thymocytes. Low affinity interactions result in positive selection and development of CD4 or CD8 single positive (SP) T cells capable of recognizing foreign antigens presented by self-MHC5.
Positive selection can be studied in mice with a polyclonal (wildtype) TCR repertoire by observing the generation of mature T cells. However, they are not ideal for the study of negative selection, which involves deletion of small antigen-specific populations. Many model systems have been used to study negative selection but vary in their ability to recapitulate physiological events6. For example, in vitro stimulation of thymocytes lacks the thymic environment that is intimately involved in selection, while administration of exogenous antigen can lead to non-specific deletion of thymocytes7-9. Currently, the best tools for studying in vivo negative selection are mice that express a transgenic TCR specific for endogenous self-antigen. However, many classical TCR transgenic models are characterized by premature expression of the transgenic TCR&alpha; chain at the DN stage, resulting in premature negative selection. Our lab has developed the HYcd4 model, in which the transgenic HY TCR&alpha; is conditionally expressed at the DP stage, allowing negative selection to occur during the DP to SP transition as occurs in wildtype mice10.
Here, we describe a flow cytometry-based protocol to examine thymic positive and negative selection in the HYcd4 mouse model. While negative selection in HYcd4 mice is highly physiological, these methods can also be applied to other TCR transgenic models. We will also present general strategies for analyzing positive selection in a polyclonal repertoire applicable to any genetically manipulated mice.

We present a flow cytometry-based method to examine T cell development in vivo using genetically manipulated mice on a wildtype or T cell receptor transgenic background.

考试流式细胞仪检测胸腺阳性和阴性选择

A healthy immune system requires that T cells respond to foreign  antigens while remaining tolerant to self-antigens. Random rearrangement of the  T cell ...

Culturing Microglia from the Neonatal and Adult Central Nervous System

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and pathological processes such as CNS maturation in development, multiple sclerosis, and spinal cord injury. Microglia can be activated and recruited to action by neuronal injury or stimulation, such as axonal damage seen in MS or ischemic brain trauma resulting from stroke. These immunocompetent members of the CNS are also thought to have roles in synaptic plasticity under non-pathological conditions. We employ protocols for culturing microglia from the neonatal and adult tissues that are aimed to maximize the viable cell numbers while minimizing confounding variables, such as the presence of other CNS cell types and cell culture debris. We utilize large and easily discernable CNS components (e.g. cortex, spinal cord segments), which makes the entire process feasible and reproducible. The use of adult cells is a suitable alternative to the use of neonatal brain microglia, as many pathologies studied mainly affect the postnatal spinal cord. These culture systems are also useful for directly testing the effect of compounds that may either inhibit or promote microglial activation. Since microglial activation can shape the outcomes of disease in the adult CNS, there is a need for in vitro systems in which neonatal and adult microglia can be cultured and studied.

We outline methods for the efficient and quick isolation/culture of viable microglia from the neonatal cerebral cortex and adult spinal cord. The dissection and plating of cortical microglia can be accomplished within 90 minutes, with the subsequent microglial harvest taking place ~ 10 days following the initial dissection.

培养小胶质细胞的新生和成年的中枢神经系统

Microglia are the resident macrophage-like cells of the central nervous system (CNS) and, as such, have critically important roles in physiological and ...

Isolation, Characterization and Functional Examination of the Gingival Immune Cell Network

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident immune cell populations in the oral mucosa and gingiva. We have established a technique for the isolation and study of immune cells from murine gingival tissues, an area of constant microbial exposure and a vulnerable site to a common inflammatory disease, periodontitis. Our protocol allows for a detailed phenotypic characterization of the immune cell populations resident in the gingiva, even at steady state. Our procedure also yields sufficient cells with high viability for use in functional studies, such as the assessment of cytokine secretion ex vivo. This combination of phenotypic and functional characterization of the gingival immune cell network should aid towards investigating the mechanisms involved in oral immunity and periodontal homeostasis, but will also advance our understanding of the mechanisms involved in local immunopathology.

We have established a technique for the isolation, phenotypic characterization and functional analysis of immune cells from murine gingiva.

分离，鉴定和牙龈免疫细胞网络的功能性考试

Immune cell networks in tissues play a vital role in mediating local immunity and maintaining tissue homeostasis, yet little is known of the resident ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) cycles between a tsetse vector and a mammalian host. It evades the mammalian host immune system by periodically switching the dense, variant surface glycoprotein (VSG) that covers its surface. The detection of antigenic variation in Trypanosoma brucei can be both cumbersome and labor intensive. Here, we present a method for quantifying the number of parasites that have 'switched' to express a new VSG in a given population. The parasites are first stained with an antibody against the starting VSG, and then stained with a secondary antibody attached to a magnetic bead. Parasites expressing the starting VSG are then separated from the rest of the population by running the parasites over a column attached to a magnet. Parasites expressing the dominant, starting VSG are retained on the column, while the flow-through contains parasites that express a new VSG as well as some contaminants expressing the starting VSG. This flow-through population is stained again with a fluorescently labeled antibody against the starting VSG to label contaminants, and propidium iodide (PI), which labels dead cells. A known number of absolute counting beads that are visible by flow cytometry are added to the flow-through population. The ratio of beads to number of cells collected can then be used to extrapolate the number of cells in the entire sample. Flow cytometry is used to quantify the population of switchers by counting the number of PI negative cells that do not stain positively for the starting, dominant VSG. The proportion of switchers in the population can then be calculated using the flow cytometry data.

Detection of Trypanosoma brucei Variant Surface Glycoprotein Switching by Magnetic Activated Cell Sorting and Flow Cytometry

African trypanosomes grown in vitro undergo antigenic variation at a low rate, such that populations are made up of parasites expressing a dominant variant surface glycoprotein (VSG) type and a small population of "switched" variants. This protocol describes a fast method for detecting and quantifying these populations.

检测<em&gt;布氏锥虫</em&gt;变体表面糖蛋白通过磁性细胞分选和流式细胞仪开关

Trypanosoma brucei, a protozoan parasite that causes both Human and Animal African Trypanosomiasis (known as sleeping sickness and nagana, respectively) ...

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host microbiome community diversity and alter the microbial community composition. Many diseases such as antibiotic-associated enterocolitis, inflammatory bowel disease, and metabolic disorders have been linked to a disrupted microbiota. The complex interplay between host, microbiome, and antibiotics needs a tractable model for studying host-microbiome interactions. Our freshwater vertebrate fish serves as a useful model for investigating the universal aspects of mucosal microbiome structure and function as well as analyzing consequential host effects from altering the microbial community. Methods include host challenges such as infection by a known fish pathogen, exposure to fecal or soil microbes, osmotic stress, nitrate toxicity, growth analysis, and measurement of gut motility. These techniques demonstrate a flexible and useful model system for rapid determination of host phenotypes.

Examination of Host Phenotypes in Gambusia affinis Following Antibiotic Treatment

This study involves methods to reveal effects on a model fish host following alteration of the skin and gut microbiome communities&#160;composition by an antibiotic.

主机表型的考试<em&gt;食蚊鱼</em&gt;继抗生素治疗

The commonality of antibiotic usage in medicine means that understanding the resulting consequences to the host is vital. Antibiotics often decrease host ...

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous aspiration of cells and/or microorganisms from palpable masses, organs or effusions (fluid accumulation in a body cavity) using a thin needle similar to the regular needle used for the venous puncture. The material collected by FNA is in general highly cellular, and the retrieved aspirate is then smeared, air dried, wet-fixed, stained and observed under a microscope. In the clinical context, FNA is an important diagnostic tool that serves as a guide to the appropriate therapeutic management. Because it is simple, fast, minimally invasive and requires limited investment in the laboratory and human resources, it is extensively used by veterinary practitioners, mostly in domestic, but also in farm animals. In studies using animal models, FNA has the advantage that it can be performed repeatedly in the same animal, enabling longitudinal studies through the collection of cells from tumors and organs/tissues over the course of the disease. In addition to routine microscopy, retrieved material can also be used for immunocytochemistry, electron microscopy, biochemical analysis, flow cytometry, molecular biology or in vitro assays. FNA has been used to identify the protozoan parasite Trypanosoma brucei in the gonads of infected mice, opening the possibility for a future diagnosis in cattle.

Detection of Extravascular Trypanosoma Parasites by Fine Needle Aspiration

Fine Needle Aspiration is a technique, whereby cells are obtained from a lesion or organ using a thin needle. Aspirated material is smeared, stained and examined under a microscope for diagnosis or used for molecular biology, cytometry or in vitro analysis. It is cheap, simple, quick and causes minimal trauma.

细针吸入检测血管外胰腺瘤寄生虫

Fine Needle Aspiration (FNA) is a routine diagnostic procedure essential to both medical and veterinary practices. It consists of the percutaneous ...

Gene Knock-in by CRISPR/Cas9 and Cell Sorting in Macrophage and T Cell Lines

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to proteins of interest. Identification of gene functions in cell line models is important for gene discovery and exploration of cell-intrinsic mechanisms. However, genetic manipulations of immune cells such as T cells and macrophage cell lines using CRISPR/Cas9-mediated knock-in are difficult because of the low transfection efficiency of these cells, especially in a quiescent state. To modify genes in immune cells, drug-resistance selection and viral vectors are typically used to enrich for cells expressing the CRIPSR/Cas9 system, which inevitably results in undesirable intervention of the cells. In a previous study, we designed dual fluorescent reporters coupled to CRISPR/Cas9 that were transiently expressed after electroporation. This technical solution leads to rapid gene deletion in immune cells; however, gene knock-in in immune cells such as T cells and macrophages without the use of drug-resistance selection or viral vectors is even more challenging. In this article, we show that by using cell sorting to aid selection of cells transiently expressing CRISPR/Cas9 constructs targeting the Rosa26 locus in combination with a donor plasmid, gene knock-in can be achieved in both T cells and macrophages without drug-resistance enrichment. As an example, we show how to express human ACE2, a receptor of SARS-Cov-2, which is responsible for the current Covid-19 pandemic, in RAW264.7 macrophages by performing knock-in experiments. Such gene knock-in cells can be widely used for mechanistic studies.

This protocol uses fluorescent reporters and cell sorting to simplify knock-in experiments in macrophage and T cell lines. Two plasmids are used for these simplified knock-in experiments, namely a CRISPR/Cas9- and DsRed2-expressing plasmid and a homologous recombination donor plasmid expressing EBFP2, which is permanently integrated at the Rosa26 locus in immune cells.

基因敲击由CRISPR/Cas9和细胞排序在巨噬细胞和T细胞线

Functional genomics studies of the immune system require genetic manipulations that involve both deletion of target genes and addition of elements to ...