这项研究的部分得到了德意志 Forschungsgemeinschaft (SFB 1027, A6) 的资助。

注意: 一般实验工作流的概述如图 1所示。
警告: 对人类来说, 贸易协定是剧毒的。安全实验室许可 S2 (生物安全等级2当量) 是必要的。在整个实验中请戴上手套。
1. 在大肠杆菌中他标记的异种的异源表达
<ol>
	<li>用表达质粒 pET24-RTA(其) 6或空向量 pET24a(+)使用标准的电穿孔协议9,10转换大肠杆菌单元格。含有空质粒的细胞充当阴性对照。</li>
	<li>在含有卡那霉素的阳性克隆 (100 µg/毫升) lb 板后, 在5毫升kan培养基 (lb 培养基与100µg/毫升卡那霉素) 中接种含有区域内表达质粒或空向量的细胞, 并在37° c 和 220 rpm 下孵育 24 h。</li>
	<li>补充 1 L LB坎培养基与5毫升培养和孵育细胞在37° c 和 220 rpm 直到细胞达到了 OD600 0.8-1.0 (大约 3-4 h)。此后, 将培养温度降低到28° c, 并在 H2O 中准备1米的 isopropyl-β-D-1-thiogalactopyranoside (IPTG) 库存解决方案。</li>
	<li>通过在1毫米的最终浓度中加入 IPTG, 诱导大肠杆菌的表达。</li>
	<li>在 3.5 h 在28° c 和220转每分钟, 收获细胞由离心在 1万 x g 和4° c 为 10 min, 洗涤药丸二次以5毫升的捆绑缓冲 (500 毫米氯化钠, 10 毫米咪唑, 并且20毫米在2PO4, pH = 7.4) 在 1万 x g 和4° c 为 10 min, 和重颗粒在5毫升结合缓冲器。 注意: 协议在这个阶段可以暂停, 并且细胞可以被存放在80° c 几天。</li>
</ol>
2. 通过亲和层析纯化其标记的贸易量
<ol>
	<li>几种细胞在冰使用以下协议: 十五年代脉冲 (20 微米), 三十年代暂停。重复此步骤五次。</li>
	<li>离心分离器在 2.1万 x 和4° c 为15分钟和过滤上清液使用无菌注射器过滤系统 (0.2 µm 孔大小)。 注: 成功声样品的细胞颗粒显示透明边框。</li>
	<li>使用配备了5毫升 Ni2 +的亲和柱的自动净化系统, 从未消毒的过滤后的大肠杆菌上清纯化其标记的含量。一般而言, 使用1毫升/分钟的洗脱速度, 冷却整个净化系统, 以防止效率毒素的结合和毒素活性的丧失。 注意: 在表 1中列出了有效的贸易协定净化参数。参见贝克尔et al。有关详细信息,9。<ol>
		<li>简单地, 用 20 mL 的绑定缓冲区平衡关联列来删除存储缓冲区。用注射器将无菌过滤的上清液涂抹在亲和柱上。</li>
		<li>用25-35 毫升的结合缓冲器清洗柱, 从柱上移除未绑定的蛋白质。执行洗涤步骤, 直到 280 nm 的紫外线吸收接近初始 uv 值。</li>
		<li>洗在20-35 毫升的洗脱缓冲液 (500 毫米咪唑, 500 毫米氯化钠, 20 毫米,2PO4, pH = 7.2), 并保持样品在冰上 (图 2A和图 2B)。 注: 在紫外线吸收的情况下, 该组分的洗脱率明显增加。请注意, 专门收集这个分数, 以防止污染与非特异性结合蛋白。</li>
		<li>使用20µL 洗样品执行马斯亮蓝色染色11或西方印迹分析12,13 (可选)。使用初级抗他的抗体 (1:1, 000) 和第二 anti-mouse-IgG-HRP (1:10,000) 来检测和验证样品纯度 (图 3)。</li>
		<li>淡化洗在5毫升脱盐柱和平衡样品中的0.8 米山梨醇中的含量。<ol>
			<li>用脱盐柱取代纯化系统的亲和柱, 先平衡20毫升0.8 米山梨醇的柱。</li>
			<li>通过注射器将洗的比例分数应用到柱子上。当紫外线吸收开始增加 (图 2C) 时, 用100毫升0.8 米山梨醇和洗的盐水在15毫升管中清洗该柱。</li>
			<li>在4° c 下贮存洗的含量。 注: 当电导增加时, 直接停止含盐量的分数取样, 以避免盐分污染。脱盐过程的参数列于表1。</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>将洗 1万 x g 和4° c 的30-180 分钟集中度用 10 kDa 截止自旋柱到1-2 毫升的最终体积, 并在4° c 下储存样品3-4 周。 警告: 不要冻结样品, 因为冷冻会导致整个贸易协定活动的完全丧失。</li>
	<li>使用常规蛋白质测定试剂盒确定蛋白质浓度。蛋白质浓度应在 1-1. 5 毫克/毫升的范围内。 注: 如果蛋白质浓度太高 (#62; 5 毫克/毫升), 就会沉淀。</li>
</ol>
3. 酵母转化和细胞壁去除
<ol>
	<li>利用 gfp 报告质粒 pRS315-K28SP-gfp6使用标准醋酸锂转化方法14转换野生型或选定的酵母缺失突变体。在0.087% ° c 下孵育亮氨酸辍学 (d-/o) 葡萄糖板 (2% 葡萄糖, 1.5% 琼脂, 0.5% 硫酸铵, 0.17% 酵母氮基 (YNB) 和30的无亮氨酸混合) 的细胞, 用于阳性克隆选择的2-3 天。</li>
	<li>从每个板块挑选3不同的酵母克隆, 在100毫升的亮氨酸糖培养基中接种克隆 (2% 糖, 0.5% 硫酸铵, 0.17% YNB 和0.087% 的无亮氨酸的混合, 在 220 rpm 和30° c 到 OD600 = 1.0-2.0 (2-4 x 107细胞/毫升)。 注意: 不同酵母缺失菌株的细胞生长是不同的。通过分光光度计监视 OD600 。</li>
	<li>要计算 od600值, 请将样本稀释到 od600 = 0.1-0.3 (1:5 到1:10 稀释), 并在分光光度计中测量外径600 。作为参考, 使用 H2o 辅以相应数量的亮氨酸 raffionose 培养基。</li></ol>最后, 重 OD600 = 125 在5毫升的无菌水 (5 x 108细胞/毫升)。
	<li>离心细胞在25° c 为 5 min 在 1万 x g, 洗涤细胞两次与5毫升无菌水, 和重细胞在50毫升 spheroplasting 缓冲 (0.8 米山梨醇, 10 毫米三盐酸 (pH 7.5), 10 毫米 CaCl2, 2 毫米糖 (德勤), 和200µg/毫升溶解酶)。</li>
	<li>孵育50毫升培养在 100 rpm 和30° c 为 90 min. 可选: 每15分钟监测 spheroplast 形成的相衬显微镜。在显微镜下, 细胞应该有一个深半透明的灰色颜色。明亮 (折光) 细胞没有充分地被消化, 而鬼魂 (苍白灰色细胞与少许, 如果任何, 内部结构) 是 over-digested 的。</li>
	<li>检查每个样品的 spheroplast 制备效果。
	<ol>
		<li>完成步骤3.5 后, 混合4µL 的 spheroplasted 50 mL 文化与496µL spheroplasting 缓冲区 (约 2 x 106原生质) 和离心机为10分钟 400 x g。</li>
		<li>重颗粒在10毫升 H2O 蒸馏, 漩涡样品三十年代, 和板材出10µL 的样品上亮氨酸 d-/O 葡萄糖板 (2% 葡萄糖, 1.5% 琼脂, 0.5% 硫酸铵, 0.17% YNB, 和0.087% 的 d/o 混合没有亮氨酸)。</li>
		<li>孵育细胞3天在30° c。计算板上生长细胞菌落总数。对于数据评估, 只使用效率高于98% 的样本 (总细胞菌落数和 #60; 40 殖民地/板块)。</li>
	</ol>
	</li>
	<li>离心机细胞从步骤3.5 在 400 x g 和4° c 为10分钟和洗涤细胞两次与5毫升稳定亮氨酸 d/o 糖培养基 (0.8 M 山梨醇, 2% 糖, 0.5% 硫酸铵, 0.17% YNB, 和0.087% 辍学混合没有亮氨酸)。重细胞在5毫升稳定的亮氨酸 d/o 糖培养基 (1 x 108细胞/毫升), 并直接使用细胞的 GFP 记者化验测量 (4 节)。</li>

4. GFP 报告仪测定96井板
<ol>
	<li>将步骤3.7 中获得的酵母细胞原生质成 96-井板 (200 µL/井)。</li>
	<li>添加70µL 稳定的亮氨酸 d/o 糖培养基含有负控制 (洗的镍2 +亲和纯化细胞裂解 IPTG 诱导的大肠杆菌细胞表达空矢量) 或纯化的区域贸易协定在最后的贸易量集中5µM ( 对应于每井160克/升)。</li>
	<li>立即添加30µL 稳定的半乳糖溶液 (30% 半乳糖, 0.8 米山梨醇), 以诱导 GFP 的表达, 并随后开始测量。 注: 每次试验至少进行3技术复制, 每株酵母菌3次生物复制。</li>
	<li>完成样品制备后 (步骤 4.1-4.3), 把 96-井板在一个荧光阅读器, 并开始测量。使用 GFP 荧光检测所需的 475/509 nm 过滤器集。执行测量在30° c, 120 转每分钟, 并且以震动直径1毫米在时间窗口 20 h (测量的区间 10 min)。 注意: GFP 过滤器集通常在所有读卡器系统中都可用。温度、时间窗、测量间隔和浓度可根据自身需要进行调整。有代表性的结果如图 4所示。</li>
	<li>可选: 在质量控制的测量中使用额外的内部控制。通过添加30µL 稳定亮氨酸 d/o 糖培养基而不是30% 半乳糖 (无 GFP 诱导) 来准备负控制。此外, 添加70µL 0.8 米山梨醇稳定 G418 溶液 (300 µg/毫升)。蛋白质翻译抑制剂 G418 作为积极控制蛋白质抑制, 因为它防止 GFP 在酵母表达。</li>
	<li>根据以下公式计算 20 h 时间点的相对 GFP 荧光百分比 (请参见图 4A): <img alt="Equation" src="/files/ftp_upload/56588/56588eq1.jpg"> 其中 NC 为负控件
	<ol>
		<li>另外, 确定相对 GFP 荧光为每个测量点 (在这种情况下 10 min, 也参见步骤 4.4) 使用上述等式。如图 4B所示, 通过在时间 (x 轴) 上印迹 GFP 荧光强度 (y-axis) 来创建图形。</li>
	</ol>
	</li>
</ol>

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作者没有什么可透露的。

在执行上述协议时, 我们建议以下建议, 以取得成功的实验结果。
对于异源蛋白的表达, 重要的是不超过 IPTG 浓度的1毫米。IPTG 的浓度和 #62; 1 mM 抑制启动子诱导的贸易量表达, 导致毒素产量降低。此外, 细胞不应在高于28° c 的温度下培养, 以防止包涵体形成、低效折叠和毒素失活。在较低的温度 (例如, 20-28 ° c) 是可能的, 也导致生物活性毒素的生产。然而, 额外的温度降...

由毒素产生菌感染的病人是每个社会保健系统的严重的医疗和经济负担, 特别是因为有效的治疗治疗仍然很大程度上缺失。为了发展新的治疗策略, 在医学上相关的 a/b 毒素, 如霍乱毒素, 志贺毒素, 或蓖麻毒的复杂中毒机制, 需要在分子水平上充分了解, 基于新的强有力的化验, 必须实施。
近年来, 一些研究试图通过使用耗时和成本密集型的方法, 如放射性毒素标记1,2以及 siRNA-based 筛选来分析酵母和哺乳动物细胞中的 a/b 毒素转运接近3。在某些情况下, 毒素贩运已被可视化的在体内荧光显微镜后, 与荧光, 量子点, 或荧光蛋白的单个毒素亚基的化学和/或遗传耦合4,5。不幸的是, 这种修改往往导致不活泼和/或改变的自然性质的毒素。另一种可以间接回答各种科学问题的优雅方法是使用基于酶的记者系统, 如lacZ、荧光或荧光蛋白 (如GFP 或Discosoma sp. 红色荧光蛋白 (dsRed))。
在这篇手稿中, 一个简单的协议被描述为 extracellularly 在S. 酿酒酵母中的细胞内传输所需的细胞成分进行识别。因此, 荧光-报告质粒含有一个 N 终端 ER 输入信号, 其次是 gfp 作为蛋白质生物合成传感器, 这间接地测量了在体内的 gfp 荧光发射后的蛋白质的翻译抑制. 翻译6。如果与野生型相比, 在特定的酵母缺失突变体中, 吞和/或胞内贩运是消极的 (或积极的), 这可以通过 GFP 荧光发射的增加 (或减少) 检测6。
目前, 所有分析酵母细胞中的区域内转运的方法都被限制在 ER-to-cytosol 的转运过程中。在这样的人工系统中, 含有 ER 输入信号的含氟贸易协定是由一个可诱导的启动子表达的, 导致自杀型表现为1,7。虽然在本手稿中所描述的实验装置中, 蓖麻蛋白的细胞结合 B 亚基同样缺失, 因此, 不完全代表蓖麻毒素 holotoxin 中毒的自然情况8, 从等离子体中运毒膜通过高尔基体对 ER 的仪器可以与这种新的检测严密模仿。有趣的是, 在试点研究中取得的初步结果表明, 贸易协定所使用的贩运途径显示出与蓖麻毒 holotoxin 的中毒路线有惊人的相似之处。
总之, 所描述的方法可以用来确定特定的作用, 选择细胞蛋白在吞和贩运酵母。此外, 这种实验装置可以很容易地适应其他的核糖体灭活毒素产生和分泌的各种酵母和细菌物种, 如 zymocin 或志贺毒素。

<table>
	<tbody>
		<tr>
			<td>Bacterial and yeast strains</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>E coli BL21 DE3(Gold)</td>
			<td>Aligent Technologies</td>
			<td>230130</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742</td>
			<td>Euroscarf</td>
			<td>Y10000</td>
			<td></td>
		</tr>
		<tr>
			<td>S. cerevisiae BY4742 deletion mutants</td>
			<td>Dharmacon</td>
			<td>YSC1054</td>
			<td>whole collection</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids used in this protocol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pET24a(+) (Novagen)</td>
			<td>Millipore</td>
			<td>69772-3</td>
			<td></td>
		</tr>
		<tr>
			<td>pET-RTA(His6)</td>
			<td>Becker et al. (2016)3</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 20T</td>
			<td>USBio</td>
			<td>Z1000.250</td>
			<td>lytic enzyme for cell wall removal</td>
		</tr>
		<tr>
			<td>LB broth medium</td>
			<td>Thermo Scientific</td>
			<td>10855021</td>
			<td>15 g agar was added for plate production</td>
		</tr>
		<tr>
			<td>YNB</td>
			<td>Thermo Scientific</td>
			<td>DF0335-15-9</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A4418-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast drop-out mix supplemts without leucine</td>
			<td>Sigma-Aldrich</td>
			<td>Y1376-20G</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Sigma-Aldrich</td>
			<td>05040-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Sigma-Aldrich</td>
			<td>10197777001</td>
			<td></td>
		</tr>
		<tr>
			<td>D-raffinose</td>
			<td>Sigma-Aldrich</td>
			<td>83400-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>D-sorbitol</td>
			<td>Sigma-Aldrich</td>
			<td>S1876-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>D-galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G0750-10MG</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>Thermo Scientific</td>
			<td>11811031</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Sigma-Aldrich</td>
			<td>I6758-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Roth</td>
			<td>3899.1</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE ruler prestained</td>
			<td>Fermentas</td>
			<td>26616</td>
			<td>protein ladder used for Western analysis</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Material for RTA purification, desalting and reader measurements</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer Ultrospec 2100 pro</td>
			<td>Amersham</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Soniprep 150</td>
			<td>MSE</td>
			<td></td>
			<td>old model, other models available</td>
		</tr>
		<tr>
			<td>Fluoroskan Ascent</td>
			<td>Thermo Scientific</td>
			<td>5210470</td>
			<td>old model, not available anymore</td>
		</tr>
		<tr>
			<td>&#196;KTAPurifier</td>
			<td>Thermo Scientific</td>
			<td>28406266</td>
			<td>Product is discontinued and replaced</td>
		</tr>
		<tr>
			<td>HisTRAP HP column</td>
			<td>GE Healthcare</td>
			<td>17-5248-02</td>
			<td></td>
		</tr>
		<tr>
			<td>HiTRAP desalting column</td>
			<td>GE Healthcare</td>
			<td>11-0003-29</td>
			<td></td>
		</tr>
		<tr>
			<td>Midisart sterile filter</td>
			<td>Sartorius</td>
			<td>16534K</td>
			<td>0.2 &#181;m pore size</td>
		</tr>
		<tr>
			<td>BCA protein assay kit</td>
			<td>Pierce</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>660 nm assay kit</td>
			<td>Thermo Scientific</td>
			<td>22660</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plates</td>
			<td>Thermo Scientific</td>
			<td>260860</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies (optional)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Tetra-His</td>
			<td>Qiagen</td>
			<td>34670</td>
			<td>primary antibody; 1:1,000 dilution</td>
		</tr>
		<tr>
			<td>Anti-mouse-HRP</td>
			<td>Sigma-Aldrich</td>
			<td>A9044-2ML</td>
			<td>secondary antibody, 1:10,000 dilution</td>
		</tr>
	</tbody>
</table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

细菌和植物 A/B 毒素利用真核细胞的自然贩运途径达到其在胞的细胞内目标, 并最终杀死。这种/B 类毒素一般由酶活性 Asubunit (如、蓖麻毒素 A ()) 和一个或多个细胞结合 Bsubunit (s) 组成, 后者负责对特定细胞表面受体的毒素结合。我们目前对甲乙毒素如何能有效地令人陶醉的细胞的知识帮助科学家了解基本的细胞机制, 如吞和细胞内蛋白在更高的真核细胞中排序。从医学的角度来看, 同样重要的是要找出主要的毒素贩运路线, 为病人找到适当的治疗方案, 或最终发展治疗毒素的癌症治疗应用。
由于对哺乳动物细胞的 a/b 毒素贩运的全基因组分析是复杂、耗时和昂贵的, 因此在酵母模型生物体酿酒酵母中进行了关于 a/b 毒素转运的几项研究。尽管不那么复杂, 酵母和高真核细胞中的基本细胞过程是相似的, 而且在酵母中获得的结果往往可以转移到哺乳动物的情况。
在这里, 我们描述一个快速和易于使用的记者化验分析的细胞内贩运的酵母。这种新的检测方法的一个重要优点是, 不仅有机会从内质网 (er) 中研究胞, 而且从细胞膜到 ER 的吞和逆行毒素的转运。该分析利用了一个记者质粒, 允许间接测量在体内的绿色荧光蛋白 (GFP) 的荧光发射后,在活体翻译。由于贸易协定有效地防止了蛋白质生物合成的 28S rRNA depurination, 这一分析可以通过检测荧光发射的变化来识别细胞内的细胞间贸易中所涉及的宿主细胞膜蛋白。

本手稿中所描述的协议的一般工作流程在图 1中作了说明, 大致总结了成功的贸易协定纯化和随后的 GFP 报告实验的单一步骤。可以在协议中找到每个单独步骤的更详细的描述。图 2说明了通过亲和层析 (图 2A) 和空矢量控件 (图 2B) 成功进行的贸易协定净化的预期结果。...

Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

一个简单的荧光记者的检测方法, 用于识别蓖麻毒素所需的细胞成分 a 链 (贸易协定) 贩运酵母

在手稿中, 我们描述了使用模型荧光记者分析, 以确定涉及的细胞成分的贩运和杀伤过程中的毒素 a 亚基的植物毒蓖麻。

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

a-simple-fluorescence-based-reporter-assay-to-identify-cellular

Research

JoVE Journal

Biology

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

7.9K 次观看.  Saarland University. 在手稿中, 我们描述了使用模型荧光记者分析, 以确定涉及的细胞成分的贩运和杀伤过程中的毒素 a 亚基的植物毒蓖麻。

视频: A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

Pyrosequencing: A Simple Method for Accurate Genotyping

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a tremendous amount of data available comes an explosion of genotyping methods. Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. It also has the ability to identify tri-allelic, indels, short-repeat polymorphisms, along with determining allele percentages for methylation or pooled sample assessment. In addition, there is a standardized control sequence that provides internal quality control. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.


Pyrosequencing(R) is one of the most thorough yet simple methods to date used to analyze polymorphisms. This method has led to rapid and efficient single-nucleotide polymorphism evaluation including many clinically relevant polymorphisms. The technique and methodology of Pyrosequencing is explained.

焦磷酸测序：一个准确的基因分型的简单方法

Pharmacogenetic research benefits first-hand from the abundance of information provided by the completion of the Human Genome Project. With such a ...

科研

生物学

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

在酵母用于监测Mitophagy荧光显微镜检测酿酒酵母

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi筛选识别胚后的表型 C。线虫</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

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

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

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

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

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

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

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

为外植体含量的颅间充质细胞行为评定

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

A Quantitative Assay to Study Protein:DNA Interactions, Discover Transcriptional Regulators of Gene Expression, and Identify Novel Anti-tumor Agents

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, and multiwell-based assays are used to measure transcription factor activity. However, these assays are nonquantitative, lack specificity, may involve the use of radiolabeled oligonucleotides, and may not be adaptable for the screening of inhibitors of DNA binding. On the other hand, using a quantitative DNA-binding enzyme-linked immunosorbent assay (D-ELISA) assay, we demonstrate nuclear protein interactions with DNA using the RUNX2 transcription factor that depend on specific association with consensus DNA-binding sequences present on biotin-labeled oligonucleotides. Preparation of cells, extraction of nuclear protein, and design of double stranded oligonucleotides are described. Avidin-coated 96-well plates are fixed with alkaline buffer and incubated with nuclear proteins in nucleotide blocking buffer. Following extensive washing of the plates, specific primary antibody and secondary antibody incubations are followed by the addition of horseradish peroxidase substrate and development of the colorimetric reaction. Stop reaction mode or continuous kinetic monitoring were used to quantitatively measure protein interaction with DNA. We discuss appropriate specificity controls, including treatment with non-specific IgG or without protein or primary antibody. Applications of the assay are described including its utility in drug screening and representative positive and negative results are discussed.

We developed a quantitative DNA-binding, ELISA-based assay to measure transcription factor interactions with DNA. High specificity for the RUNX2 protein was achieved with a consensus DNA-recognition oligonucleotide and specific monoclonal antibody. Colorimetric detection with an enzyme-coupled antibody substrate reaction was monitored in real time.

定量检测，以研究蛋白质:DNA的相互作用，发现转录基因表达的调节，并确定新的抗肿瘤剂

Many DNA-binding assays such as electrophoretic mobility shift assays (EMSA), chemiluminescent assays, chromatin immunoprecipitation (ChIP)-based assays, ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

核酸的分离过滤:一种简单快速的DNA提取方法

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

使用微流体装置来衡量寿命和细胞表型单芽殖酵母细胞

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...