Y. Tanaka 对 Y2H 进行了技术改进。这项工作得到了 JSPS KAKENHI Grant Number JP22K06336 和大阪发酵研究所的支持。

1. 突变体库的建设
<ol><li>设置聚合酶链反应 （PCR）（示例如下所示）。通常制备 50 μL 的反应物，将其分成 10 份 5 μL 的等分试样，并在 PCR 仪4 中扩增目标基因片段。 注意:用户应选择适当的聚合酶以有效地引入突变。如果要扩增的DNA片段较短，则需要错误率较高的聚合酶，例如缺乏校对活性的Taq聚合酶。如果要扩增的DNA片段很长，则需要更高保真度的聚合酶来减少突变的数量。使用常规 Taq 聚合酶4 扩增 30 个周期后，每几百个碱基对发生一次突变。在代表性结果中，使用了不同的Taq聚合酶（参见 材料表），其保真度高于常规Taq聚合酶，突变率为600个碱基对中的1。PCR混合物的示例、引物详细信息和反应条件在 补充文件1中提供。</li>
	<li>当PCR完成后，合并反应的所有等分试样，确认目标DNA片段已使用琼脂糖凝胶电泳等法扩增，并纯化DNA5。</li>
	<li>使用"无缝"克隆策略将步骤 1.2 中生成的 DNA 片段与适当的 Y2H 载体组装（图 1）。 注:无缝克隆系统，如Gibson组装6，最大限度地减少 了通过 自连接产生的空载体对构建的突变体文库的污染。 表 1 中提供了 Y2H 载体的列表。它们可以在组装前通过 BamHI 消化来制备。所有质粒均可从国家生物资源项目 - 酵母中获得（见 材料表）。</li>
	<li>将步骤1.3中产生的反应混合物引入根据高效大肠杆菌转化方案制备的大肠杆菌感受态细胞中（例如，Inoue等人7）。将所有感受态细胞铺展在含有适当抗生素的几个LB平板（1%胰蛋白胨，0.5%酵母提取物，1%NaCl和2%琼脂）上（参见材料表）。此时，将少量（总反应的~1/100）涂抹在同一选择板上以计算文库滴度。将板在37°C孵育过夜。</li>
	<li>一旦出现大量 大肠杆 菌菌落，使用一次性塑料环从板表面刮取所有细胞。使用常用方法（如碱性裂解8）从这些细胞中回收质粒 DNA。同时，计算在板上散布转化混合物的小等分试样后出现的菌落数。使用此数字估计库中独立克隆的总数。 注意:此时，选取一些出现在铺有转化反应小等分试样的板上的几个独立菌落（4-6），将它们分开培养，并从中分离质粒，以确认几乎所有克隆都携带所需的DNA片段5。许多商业试剂盒可用于从 大肠杆菌中分离质粒。撒开小等分试样后出现在板上的菌落数量可以手动计算。</li>
	<li>测量文库DNA库的浓度。通常，几百纳克的文库DNA就足以在下一步中获得数千个转化子。 注意:建议使用与 DNA 结合的荧光染料测量 DNA 浓度，因为 A260 的测量经常不准确。</li>
</ol>2. 用突变体文库和复制铺板转化酵母
<ol><li>将突变文库引入宿主酵母菌株（TAT-7（L40-ura3）9,10 MATa，leu2-3,112，trp1-901，his3-Δ200，ade2-101，gal80Δ，LYS2:（lexAop）4-HIS3，ura3:（lexAop）8-lacZ）使用约100ng DNA进行Y2H测定。用"诱饵"质粒预转化宿主细胞。<ol><li>转化程序后，将酵母细胞悬浮在无菌水中，并将不同量的等分试样散布在适当的平板上（通常是缺乏亮氨酸和色氨酸的SC培养基:SC-LW [0.67%酵母氮基，2%葡萄糖，1.546g / L SC双滴混合物-Leu -Trp和2%琼脂，参见 材料表]）。将这些板在30°C下孵育过夜。 注:标准方案，例如醋酸锂-PEG 方法11 ，具有 ~5 × 106 个对数生长的细胞，足以用于酵母的转化。也可以使用另一种转化效率高的方法，例如电穿孔。</li>
	</ol></li>
	<li>第二天，当出现微小的菌落时，选择具有适当细胞数量（500-2000）的板，并按以下步骤从中进行复制。</li>
	<li>将两张滤纸放在复制块上，制成多个复制品（培养基为含有卡那霉素的SC-LW和SC-LW）（见 材料表）。在30°C孵育1-2天。 注:卡那霉素 （G418） 的浓度为 600 μg/mL。不要使用天鹅绒进行复制电镀，因为菌落的分辨率会丢失。</li>
	<li>一旦菌落生长，比较板并选择候选板。如果使用 lacZ 作为报告基因，则执行Y2H颜色测定（参见步骤3）。 注意:对于 Y2H 报告基因， lacZ 通常比 HIS3 更擅长检测弱相互作用。</li>
</ol>3. Y2H显色法
<ol><li>将一张预先切成适当尺寸的滤纸放在使用步骤 2 制备的复制板上。注意不要让气泡在滤纸和板之间形成。 注意: 使用 4A 号滤纸或 50 级滤纸（见 材料表）。任何含有卡那霉素的SC-LW或SC-LW板，都是通过复制电镀制成的，可用于颜色测定。</li>
	<li>当板上的滤纸完全湿润时（当滤纸的颜色完全改变时），在上面放几张纸巾，使其与滤纸接触，以去除多余的水分。当纸巾吸收水分并变色时，请取下纸巾。重复此过程两到三次。</li>
	<li>用镊子捡起滤纸的边缘，迅速将其从板上撕下，浸入液氮中，然后冷冻。如果没有液氮，将滤纸放在塑料托盘上，菌落面朝上，然后立即将其放入冰箱（-20°C至-80°C）中完全冷冻。</li>
	<li>取出冷冻的滤纸，将其放在干燥的纸巾上，菌落面朝上解冻。解冻后，立即将滤纸（菌落面朝上）放在另一张滤纸上，该滤纸已放置在塑料托盘或可密封容器上，并用含有X-gal（5-溴-5-氯-3-吲哚基-β-D-半乳糖苷）和2-巯基乙醇9（参见材料表）的Z-缓冲液（60mM Na2HPO 4,40mM NaH2PO4,10mM KCl和1mM MgSO 4，pH 7.0）浸泡).注意不要让气泡在顶部和底部滤纸之间形成。</li>
	<li>盖上装有滤纸的塑料托盘，将其放入密闭容器或塑料袋中。关闭密闭容器或塑料袋，在37°C下孵育，直到出现蓝色。</li>
	<li>当出现蓝色时，将滤纸（菌落面朝上）放在放置在干净塑料托盘上的约 500 μL 终止溶液 （1 M Na2CO3） 上。停止溶液快速浸泡;因此，请立即取下过滤器，将其放在新鲜的纸巾上，菌落面朝上，然后晾干。</li>
	<li>更换纸巾并且过滤器足够干燥后，使用扫描仪或其他设备捕获数据。</li>
</ol>4. 候选克隆的恢复和确认
<ol><li>从复制品的原始板（或未用于颜色测定的复制板）中选择白色和 KanR 候选克隆。 示例如图 1C 所示。通过在含有卡那霉素的SC-LW培养基上将它们重新划线为小斑块，并在30°C下孵育1-2天，确认候选克隆是白色和KanR 。至少制作两组或在第二天进行复制。 注意:应选择白色菌落，因为淡蓝色菌落可能会保留相互作用。</li>
	<li>一旦候选克隆重新生长良好，如步骤3中所述，使用步骤4.1中生成的至少一个板重复颜色测定，并确认它们保持白色并且不会变成蓝色（图2A）。 注意:重新划线候选细胞时，不要携带大量细胞。细胞过多导致无法区分 KanR 和 KanS 克隆。</li>
	<li>从板残渣中取出仍然是白色和步骤4.2中产生的KanR 的克隆，并在1mL选择培养基（SC-L或SC-W，取决于用于文库构建的载体）中独立生长过夜。</li>
	<li>将培养物转移到微管中并通过离心（~15,000 × g，在室温下 10-30 秒）收集细胞。按如下方式从细胞沉淀中分离全 DNA。</li>
	<li>将细胞沉淀悬浮在含有1μL2-巯基乙醇和20mg / mL酶解酶100T的400μL裂解缓冲液（2%Triton X-100,1%SDS，100mM NaCl，10mM Tris-Cl（8.0）和1mM EDTA中（参见 材料表），并在37°C孵育30分钟以消化细胞壁。此时，每 5-10 分钟倒置试管轻轻混合。</li>
	<li>当细胞悬液变得不透明或半透明时，加入等体积的苯酚 - 氯仿 - 异戊醇（25:24:1）混合物，并通过中等速度涡旋10-20秒充分混合。</li>
	<li>在微量离心机中以全速（~15,000× g，室温）离心5分钟，并在新的微管中回收含有DNA的水相。加入 0.8 体积的异丙醇，倒置试管充分混合。</li>
	<li>将试管在室温下放置 2-3 分钟，然后在微量离心机中以全速（~15,000 × g，室温）离心 4-5 分钟以沉淀 DNA。</li>
	<li>除去上清液，用约500μL的70%乙醇洗涤沉淀物。完全去除乙醇并短暂干燥沉淀物。</li>
	<li>向沉淀物中加入 20 μL TE 缓冲液（10 mM Tris-Cl （8.0） 和 1 mM EDTA），短暂混合，并在室温下离开试管过夜以溶解沉淀物。 注意:如果用户赶时间，请将试管保持在 37-50 °C。 DNA 沉淀物将在几个小时内溶解。</li>
	<li>一旦沉淀完全溶解，用少量的这种DNA溶液转化 大肠杆菌 。 注:如果使用转化效率高的大 肠杆菌 ，则约 0.5 μL DNA 溶液就足够了。</li>
	<li>当 出现大肠杆菌 菌落时，选择几个独立的菌落，培养它们，并通过标准方法（例如碱性裂解）回收质粒。</li>
	<li>将步骤4.12中获得的质粒DNA引入含有诱饵质粒的Y2H宿主细胞中。当转化体出现时，通过颜色测定法（它们应显示为白色）和蛋白质印迹4 （它们应表达所需大小的蛋白质）进行检查。 注:后碱性方法12 是从酵母制备全细胞提取物的一种简单快速的方法。</li>
	<li>如果满足上述两个标准，则通过核苷酸测序4确定克隆的突变位点。</li>
</ol>

使用酵母双杂交技术的生物分子相互作用研究 

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Leanna, C. A., Hannink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reverse+two-hybrid+system:+A+genetic+scheme+for+selection+against+specific+protein/protein+interactions.">The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions.</a> Nucleic Acids Res. 24 (17), 3341-3347 (1996).</li><li>Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., Boeke, 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=Reverse+two-hybrid+and+one-hybrid+systems+to+detect+dissociation+of+protein-protein+and+DNA-protein+interactions.">Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.</a> Proc Natl Acad Sci U S A. 93 (19), 10315-10320 (1996).</li><li>Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+isolation+of+interaction-null/impaired+mutants+using+the+yeast+two-hybrid+technique.">An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.</a> Genes Cells. 24 (12), 781-788 (2019).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. 4th edn. , (2012).</li><li>Gibson, 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=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Birnboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>Bartel, P. L., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein-protein+interactions+using+two-hybrid+system.">Analyzing protein-protein interactions using two-hybrid system.</a> Meth Enzymol. 254, 241-263 (1995).</li><li>Hollenberg, S. M., Sternglanz, R., Cheng, P. F., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol Cell Biol. 15 (7), 3813-3822 (1995).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Meth Enzymol. 350, 87-96 (2002).</li><li>Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+protein+extraction+from+yeast.">Rapid and reliable protein extraction from yeast.</a> Yeast. 16 (9), 857-860 (2000).</li><li>Van Deursen, F., Sengupta, S., De Piccoli, G., Sanchez-Diaz, A., Labib, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+associates+with+the+loaded+DNA+helicase+at+replication+origins+and+defines+a+novel+step+in+its+activation.">Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.</a> EMBO J. 31 (9), 2195-2206 (2012).</li><li>Watase, G., Takisawa, H., Kanemaki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+plays+a+role+in+functioning+of+the+eukaryotic+replicative+DNA+helicase,+cdc45-mcm-gins.">Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, cdc45-mcm-gins.</a> Current Biology: CB. 22 (4), 343-349 (2012).</li><li>Miyazawa-Onami, M., Araki, H., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-initiation+complex+assembly+functions+as+a+molecular+switch+that+splits+the+mcm2-7+double+hexamer.">Pre-initiation complex assembly functions as a molecular switch that splits the mcm2-7 double hexamer.</a> EMBO Rep. 18 (10), 1752-1761 (2017).</li></ol>

提交人声明他们没有利益冲突。

本文介绍如何使用 Y2H 检测分离相互作用无效/受损的突变体。这种突变体是分析目标蛋白质功能的有力工具。为了分离此类突变体，以前通过修饰 Y2H 宿主菌株 2,3 开发了 rY2H 测定法。然而，他们并没有大大减少劳动量。相比之下，可以使用这种方法分离突变体，而无需大量的劳动力。在这种方法中，理论上，对Y2H载体的修饰可确保所有候选突变体都是?...

生物分子之间的相互作用是生物现象中最基本的部分。蛋白质-蛋白质相互作用构成了这种相互作用的重要组成部分。因此，鉴定目标蛋白的相互作用伙伴对于进一步阐明目标蛋白质/基因的功能至关重要。酵母双杂交 （Y2H） 方法是一种用于鉴定体内蛋白质-蛋白质相互作用的常用技术 1。在该系统中，要测试其相互作用的两种蛋白质（X 和 Y）分别融合到 DNA 结合 （DB） 结构域和转录激活结构域 （AD）。DB-X 融合蛋白与 DB 结构域的识别序列结合;因此，当蛋白质 X 和 Y 相互作用时，AD-Y 融合蛋白就会接近识别序列。因此，识别序列下游的报告基因转录被激活。因此，报告基因活性的存在与否可用于确定蛋白质-蛋白质相互作用的存在与否1。
一旦确定了目标蛋白质的特定相互作用伙伴，就应进行进一步分析以阐明相互作用的生物学功能。为此，如果可以分离出损害或去除特定蛋白质-蛋白质相互作用的蛋白质突变体，它们将成为强大的工具。Y2H系统可以通过筛选"相互作用阴性"克隆直接用于分离此类突变体，从野生型"相互作用阳性"克隆开始。为了加速这一过程，开发了"反向"Y2H （rY2H） 系统 2,3。在 rY2H 系统中，宿主酵母菌株具有反选标记基因作为报告基因，这意味着酵母细胞仅在 AD-Y 和 DB-X 蛋白不相互作用时生长。
尽管 Y2H 和 rY2H 系统都允许分离相互作用阴性突变体，但分离突变体的过程非常费力，因为并非所有通过筛选获得的候选基因都携带所需类型的突变（通常是错义突变）。最严重的问题是，很大一部分候选基因存在移码或无义突变，因此有必要进行蛋白质印迹法以排除不需要的克隆。为了克服这个问题，已经开发了新的质粒载体4.在这些向量中，KanMX（一种耐药性标记物）位于 DB 域或 AD 下游的帧外。只有当目的基因入时，标记基因才会与DB结构域或AD一起进入框架。当在目的基因中引入随机突变时，不需要的突变体，例如具有移码或无义突变的突变体，可以通过进行耐药性选择轻松消除，并且携带理想错义突变的候选突变可以通过Y2H筛选轻松识别4。本文提出了一种方案，用于使用该策略分离目标蛋白质的相互作用无效/受损突变体。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA (8.0)</td>
			<td>Nacalai Tesque Inc.</td>
			<td>14347-21</td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS Solution</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>313-90275</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>135-07522</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>110-64785</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>021-07852</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Formedium</td>
			<td>AGR60</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>68-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HA-tag mAb-HRP-DirecT&#160;&#160;</td>
			<td>Medical &#38; Biological Laboratories Co. Ltd.</td>
			<td>M180-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon testes</td>
			<td>Merck KGaA.</td>
			<td>D1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck KGaA.</td>
			<td>9-0770-4-4L-J</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (Grade 50)</td>
			<td>Whatman, Cytiva</td>
			<td>1450-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (No.4A)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>01411090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for replicaplating (No.1)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>00011150</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418 Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>075-05962</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>230-37585</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>Nacalai Tesque Inc.</td>
			<td>20604-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>131-00405</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8226;12H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>10039-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31319-45</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31717-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td>AS ONE Corp.</td>
			<td>7-6200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>Nacalai Tesque Inc.</td>
			<td>25970-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid DNAs</td>
			<td>the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation Kit</td>
			<td>Nippon Genetics Co. Ltd.</td>
			<td>FG-90502</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol #4,000</td>
			<td>Nacalai Tesque Inc.</td>
			<td>11574-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SC double drop-out mix -Leu -Trp</td>
			<td>Formedium</td>
			<td>DSCK172</td>
			<td></td>
		</tr>
		<tr>
			<td>Seamless cloning kit (In-Fusion assembly )</td>
			<td>Takara Bio Inc.</td>
			<td>#639648</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>190-12865</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>3810-74-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase (GoTaq Green Master Mixes)</td>
			<td>Promega Corp.</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS (hydroxymethyl) aminomethane</td>
			<td>Formedium</td>
			<td>TRIS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque Inc.</td>
			<td>12967-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Nacalai Tesque Inc.</td>
			<td>35624-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (YNB)</td>
			<td>Formedium</td>
			<td>CYN0210</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>Nacalai Tesque Inc.</td>
			<td>07665-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating interaction-null&#47;impaired mutants using the yeast two-hybrid assay

蛋白质-蛋白质相互作用是构成生物现象的最基本过程之一。理解特定蛋白质-蛋白质相互作用的作用和功能的最简单和最好的方法之一是比较野生型的表型（具有相关的蛋白质-蛋白质相互作用）和缺乏相关相互作用的突变体的表型。因此，如果能够分离出这样的突变体，它们将有助于阐明相关的生物学过程。酵母双杂交 （Y2H） 程序是一种强大的方法，不仅可以检测蛋白质-蛋白质相互作用，还可以分离相互作用无效/受损的突变体。在本文中，提出了一种使用 Y2H 技术分离相互作用无效/受损突变体的协议。首先，将聚合酶链反应与高效的无缝克隆技术相结合，构建突变文库，有效地从文库中排除空载体;其次，通过Y2H测定筛选相互作用无效/受损的突变体。由于 Y2H 载体中的一个技巧，不需要的突变体，例如具有移码和无义突变的突变体，可以有效地从筛选过程中消除。这种策略很简单，因此可以应用于任何蛋白质组合，其相互作用可以被双杂交系统检测到。

最近，发现Pol2蛋白（Pol2-C）的C端半部分与Mcm10相互作用。这两种蛋白质对于启动 DNA 复制都是必需的，因此对于出芽酵母酿酒酵母 13,14,15 中的细胞生长都是必需的。为了帮助理解这种相互作用的生物学意义，使用此处描述的方法分离出与 Mcm10 没有/减少相互作用的 Pol2-C 突变体。
按照步骤1中描述的?...

Watch this Scientific Journal Video about Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast at JoVE.com

作者聚焦:增强酵母中相互作用零突变体的分离

生物分子的相互作用，如蛋白质-蛋白质相互作用，是生物学功能的分子基础。如果可以分离出特别缺乏相关相互作用的相互作用无效/受损突变体，它们将极大地有助于理解这种相互作用的功能。本文提出了一种有效的方法来分离相互作用无效/受损的突变体。

使用酵母双杂交试验分离相互作用无效/受损突变体

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

我们对了解真核生物细胞周期的调控机制感兴趣，特别是DNA复制、稳定胚芽维持和细胞增殖之间的关系。真核生物中细胞周期的进展受细胞周期蛋白依赖性激酶（CDK）的调节。DNA复制的启动就是这种情况。我们已经在CDK的底物上发现了一种启动DNA复制的方法。要了解您感兴趣的蛋白质的功能，比较野生型和突变体是一种直接而有效的方法。酵母双杂交试验，是简单的检测方法，将增强分离相互作用无效突变体的过程。这将有助于推动该领域的研究。

isolating-interaction-nullimpaired-mutants-using-yeast-two-hybrid

Research

JoVE Journal

Biology

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

341 次观看.  Kochi University of Technology. 我们对了解真核生物细胞周期的调控机制感兴趣，特别是DNA复制、稳定胚芽维持和细胞增殖之间的关系。真核生物中细胞周期的进展受细胞周期蛋白依赖性激酶（CDK）的调节。DNA复制的启动就是这种情况。我们已经在CDK的底物上发现了一种启动DNA复制的方法。要了解您感兴趣的蛋白质的功能，比较野生型和突变体是一种直接而有效的方法。酵母双杂交试验，是简单的检?...

视频: 作者聚焦:增强酵母中相互作用零突变体的分离

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

在芽殖酵母中的自我复制寿命测量

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

科研

生物学

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

一个定量评估的酵母使用电喷雾质谱法Lipidome

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

基于分体式泛素膜酵母双杂交（神话）系统：一个强大的工具用于识别蛋白质 - 蛋白质相互作用

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Yeast Colony Embedding Method

Patterning of different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms also display patterns of cell types. For example, in the yeast S. cerevisiae, sporulated cells and pseudohyphal cells are not uniformly distributed in colonies. The functional importance of patterning and the molecular mechanisms that underlie these patterns are still poorly understood.
One challenge with respect to investigating patterns of cell types in fungal colonies is that unlike metazoan tissue, cells in colonies are relatively weakly attached to one another. In particular, fungal colonies do not contain the same extensive level of extracellular matrix found in most tissues . Here we report on a method for embedding and sectioning yeast colonies that reveals the interior patterns of cell types in these colonies. The method can be used to prepare thick sections (0.5 &mu;) useful for light microscopy and thin sections (0.1 &mu;) suitable for transmission electron microscopy. Asci and pseudohyphal cells can easily be distinguished from ovoid yeast cells by light microscopy , while the interior structure of these cells can be visualized by EM.
The method is based on surrounding colonies with agar, infiltrating them with Spurr's medium, and then sectioning. Colonies with a diameter in the range of 1-2 mm are suitable for this protocol. In addition to visualizing the interior of colonies, the method allows visualization of the region of the colony that invades the underlying agar.

A method for embedding yeast colonies allowing sectioning for light and electron microscopy. This protocol allows determination of the distribution of sporulated cells and pseudohyphal cells within colonies providing a new tool toward understanding the organization of cell types within a fungal community.

酵母殖民地嵌入方法

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

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 ...

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 ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

从小鼠心脏中分离主黑素细胞样细胞

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

反向酵母双杂交系统，以确定哺乳动物核受体残留的互动与配体和/或拮抗剂

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

隔离增效变种的Hsp104利用酵母Proteinopathy模型

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...