这项工作得到国家卫生研究院的支持: NIH R21 EB021870-01A1 和 NSF 研究项目赠款: 1517110。

1. 映射 Fastq 文件
注: DEEPN 软件以及许多生物信息学程序使用 dna 序列数据, 其中每个序列读取已被映射为其在参考 DNA 的位置。可以使用多种映射程序, 包括此处使用 HISTAT2 程序生成. sam 文件在后续步骤中使用的 MAPster 接口。
<ol><li>将序列数据映射到基因组的正确版本。对于 Y2H 的小鼠源库, 使用 UCSC mm10 基因组;对于那些使用人类基因的人, 使用 UCSC hg38 参考基因组, 为酿酒酵母基因, 使用 UCSC SacCer3 参考基因组。</li>
	<li>
安装 MAPster。
	<ol>
<li>下载 MAPster 软件并安装。可以使用 web 浏览器在下面找到该软件: https://github.com/emptyewer/MAPster/releases。HISAT2 在基于 Unix 的系统 (如苹果 Macintosh) 上运行。因此, MAPster 程序只会在兼容的系统 (如 Apple Macintosh 和 linux) 上运行。 注: 苹果 Mac 的系统要求有: OSX 10.10 +, > 4 gb RAM, > 500 Gb 磁盘空间, 以及用于下载参考基因组的 internet 访问。如果用户的企业具有限制管理员权限和权限的安全协议, 则可能需要与机构 IT 人员协商。</li>
	</ol>
</li>
	<li>
通过 "主" 选项卡输入所需的文件和参数 (图 1)。选择适当的 "配对" 按钮, 以对形式或以 FASTQ 作为默认文件格式输入文件。
	<ol>
<li>对于 DEEPN 分析, 将 "配对" 选项改为 "Off" 以单读格式运行。</li>
		<li>只需将文件拖放到相应的窗口中, 即可将其加载到 MAPster 中。</li>
		<li>选择与 Y2H 猎物库插入源对应的参考 DNA/基因组源。来自多个模型生物体的索引基因组在 "基因组" 框中列出, 可以从约翰霍普金斯大学计算生物学中心自动下载。参考基因组将存储在本地以供以后使用。</li>
		<li>指示在 "线程" 框下用于映射程序的计算机进程数, 因为 HISAT2 支持多线程处理。MAPster 将搜索计算机并建议默认情况下可用的最大处理器数。</li>
		<li>指定输出文件名。此文件名将在整个 DEEPN 过程中使用, 因此建议不带空格或特殊字符的简短但描述性的名称。指定使用 "打开输出目录" 按钮输出映射文件的文件夹。</li>
		<li>选择适当的文件和参数后, 使用 "添加到队列" 按钮将映射作业添加到作业队列中。可以删除主窗口中的文件名并替换为与新示例相对应的文件, 并在提供相应的输出文件名后将它们添加到队列中。</li>
		<li>一旦所有作业都输入到作业队列中, 请单击 "运行队列" 按钮。 注意: 一旦将映射作业放置在队列中, 选择该作业会导致参数设置显示在 "作业参数" 窗口中, 并且命令行语句中的所有参数都将显示在 "作业命令" 窗口中。输出选项包括指示是否保留无法对齐的读取, 并指定每个读取所允许的主对齐次数。来自 MAPster 的默认输出文件是 SAM 格式 (如". SAM" 文件)。它将包含为该示例指定的 fastq 文件中的所有序列读取, 其中包括 (映射的) 和未 (未映射) 成功映射到指定 geome 的程序。</li>
	</ol>
</li>
</ol>2. 使用 DEEPN 软件进行生物信息学处理
注意: DEEPN 软件目前正在编译, 用于包含小鼠 cdna 序列、人类 cdna 序列或S. 酵母基因组 DNA 序列的猎物库。DEEPN 接受标准的. sam 文件格式, 并且可以接受包含映射和未映射的读取或单独文件的 sam (. sam) 文件, 用于每个未映射和映射的读取。
<ol><li>下载 DEEPN 软件并安装。可以使用 web 浏览器在下面找到该软件: https://github.com/emptyewer/DEEPN/releases。选择与计算平台匹配的版本并下载。要安装, 请打开下载的安装包。 注意: DEEPN 的版本可用于 PC、Mac 和 Linux sysrems。Mac 和 PC 系统应该有 > 500 gb 硬盘空间和 > 4 gb RAM。</li>
	<li>
打开 DEEPN 软件。从主窗口 (图 2) 从顶部选择框中选择相应的猎物库信息。通过单击 "工作文件夹" 按钮并导航到文件夹/目录, 可以选择已处理文件的文件夹。如果需要, 可以创建一个新的文件夹/目录。一旦选择了 "工作文件夹", DEEPN 将创建三个子文件夹, 标题为 unmapped_sam_files、mapped_sam_files 和 sam_files。
	<ol>
<li>如果使用包含映射和未映射读取的. sam 文件 (如 MAPster 程序的默认设置所生成的), 请将它们放在 "sam_files" 文件夹中。否则将. sam 文件放入 unmapped_sam_files 和 mapped_sam_files 相应。</li>
	</ol>
</li>
	<li>通过点击 "基因计数 + 连接制造" 按钮启动处理。 注意: 处理将开始与基因计数模块, 将使用映射位置来计算多少读取对应于每个基因。然后, 接合器将提取连接序列 (从 Gal4-activation 域直接熔化的序列), 并使用爆破算法对其进行识别。这将创建一组完整的文件夹, 如图 3所示。处理时间取决于序列数据文件的大小和数量以及使用的计算机的处理速度。典型的时间范围从 12–30 h 为实验数据集2.5亿读。基因计数程序和 Junction_Make 程序可以单独启动, 点击 "基因计数" 按钮或 "连接做" 按钮。</li>
	<li>
下载并安装 Stat_Maker (https://github.com/emptyewer/DEEPN/releases)。这是一个为 DEEPN 数据集设计的统计分析包, 当前只在 Unix Mac 系统上工作。
	<ol>
<li>打开 Stat_Maker 并单击按钮 "验证安装" (图 4)。如果第一次运行, Stat_Maker 将通过从 internet 上拉出这些资源自动安装 R、锯齿和 Bioconductor。一旦检测到 R、锯齿和 Bioconductor, Stat_Maker 将变为活动状态并允许用户进一步输入。</li>
		<li>单击 "选择文件夹" 按钮以导航到 DEEPN 处理的工作文件夹。Stat_Maker 将自动查找并列出在窗口中进行统计分析的文件。</li>
		<li>将相应的文件从上面的 "文件列表" 窗口拖放到下面的文件窗口中, 每个向量和诱饵数据集以及每个增长条件: 非选定 (他的 + 媒体) 和选定的 (他的媒体)。重要的是, Stat_Maker 需要单独的空矢量的重复数据集、两个非选定种群样本和选定的两个样本。这给出了实验中的可变性的估计。</li>
		<li>单击 "运行" 按钮。根据计算机的速度, 计算将在5–15分钟之间进行。</li>
	</ol>
</li>
	<li>查看 Stat_Maker 输出中的结果, 这些数据被放在标有 "Stat_Maker 结果" 的主工作文件夹中的新文件夹中。 注意: 结果在一个 CSV (逗号分隔值) 文件中找到, 可以在公共电子表格程序中打开。Stat_Maker 将排名的基因命中, 可能是差异丰富后, 选择与诱饵的兴趣在空 pTEF-GBD (图 5)。表中还列出了每个数据集的读取百分比, 其中在上游、下游或开放阅读框架内发现基因插入, 以及是否在正确的平移阅读框架内找到该基因。通常, DEEPN 将捕获一个诱饵的强健的 Y2H 相互作用的一部分, 给定的 cdna, 是从适当的阅读框架的相应的蛋白质或是在其相应的开放阅读框架的一部分的 cdna。扫描 Stat_Maker 的组合输出将简化这些无关命中的检测和消除。</li>
	<li>
要查看每个潜在候选者的数据, 请打开 DEEPN 软件, 选择相应的猎物库信息, 然后使用 "工作文件夹" 正确的工作文件夹。
	<ol>
<li>单击 "爆炸查询" 按钮。这将加载一个新窗口 (图 6)。在顶部文本框中, 键入基因名称或基因库 NM 数以选择感兴趣的候选基因。这些基因名称对应于 StatMaker 输出文件中列出的名称。输入或返回, 启动感兴趣的基因检索。</li>
		<li>使用 "选择数据集" 菜单选择要用于分析的数据集。通常, 这些都包括向量和在非选择性条件下生长的诱饵样本, 以及在选择条件下生长的诱饵样本。最初, 数据集需要几分钟的时间来加载, 但是, 对相同数据集的后续查询将会迅速进行。Blast_Query 将沿感兴趣的序列显示融合点, 以及每个融合点的丰富程度。这可以使用 "结果" 选项卡或使用 "绘图" 选项卡的图形格式显示在表格格式中。通过单击右上方的 "保存. csv" 按钮, 可以将这些结果导出到. csv 文件。</li>
	</ol>
</li>
</ol>3. 核查 DEEPN 确定的候选人
注意: DEEPN 和 Stat_Maker 的目的是确定候选基因, 从而给予积极的 Y2H 互动。验证这种 Y2H 交互可以使用传统的二进制 Y2H 格式, 利用与空 Gal4-activation 域 "猎物" 质粒配对的诱饵质粒, 并与携带该基因/cDNA 片段的捕食质粒进行配对。在 Y2H 选择的酵母种群 DNA 混合物中分离出感兴趣的实际质粒是不可行的。然而, 一个可以计算重建什么基因/cDNA 片段是产生 Y2H 相互作用, 设计引物的 5 ' 和 3 ' 的两端的片段, 并扩大该片段从 DNA 分离的酵母种群。本节介绍如何查找候选猎物片段的 5 ' 和 3 ' 结尾。
<ol><li>打开 DEEPN 软件, 然后选择对应于项目的 "选择参数" 和 "选择工作文件夹" 的参数。通过单击 "爆炸查询" 按钮启动 Blast_Query 模块。</li>
	<li>
在顶部文本框中键入感兴趣基因的名称或其基因库 "NM" 号。从下拉菜单中选择与所选酵母填充相对应的数据集, 以便在 "结果" 选项卡中检索接合位置表。默认情况下, Blast_Query 将根据数据集中的丰度顺序排列不同的位置, 并通过数据库中找到的连接总数的 ppm 进行量化。
	<ol>
<li>找到最丰富的位置, 是 "在 ORF"和 "框架"。位置值对应于该基因的核苷酸位置与 NCBI 参考序列 ("NM" 号) 在顶部文本框中找到。此序列可从基因库 (https://www.ncbi.nlm.nih.gov/nuccore/) 中检索, 也可以从 Blast_Query 窗口中的下文本框中复制。 注: 一个例子可以在图 6, 中间面板中找到。在中心数据集中, "结果" 显示为最丰富的连接点: "位置": 867;' #Junctions ': 20033.821;"查询开始", 1;CDS: 在 ORF;和 ' 框架 ': 在框架。核苷酸867的基因库 NCBI 参考序列 NM_019648 是猎物片段的开始。</li>
	</ol>
</li>
	<li>如果查询起始为 1, 则设计该底漆的 5 ' 末端, 其中包括与位置编号相对应的核苷酸, 并将25核苷酸从该位置向下延伸 (图 7)。如果查询起始值大于 1, 则表明 Gal4 激活域和猎物序列之间存在额外的核苷酸, 并且该底漆应根据查询起始数值进一步下游。</li>
	<li>从 DEEPN 窗口单击 "分析数据" 下的 "读取深度" 按钮。打开 "读取深度" 窗口后, 在顶部文本框中键入 NCBI 参考序列 (NM) 编号或基因名称。使用下拉菜单选择包含感兴趣的丰富基因的相关数据集。使用左侧的表格和右侧的图形显示, 以确定在与感兴趣的基因对应的数据中发现了多少读取 (图 7B)。</li>
	<li>设计一个 3 ' 端底漆, 将捕获的序列的基因片段的读取深度计算。如果大量的读取超出了 ORF 和停止密码子, 设计的底漆, 使它包括停止密码子和区域只是在上游的停止密码。如果基因序列不延伸到过去的停止密码子, 使用结果表找到最遥远的 3 ' 区域, 可以检测和使用这个位置作为最远的 3 ' 位置放置底漆。 注意: 读深度程序扫描的间隔时间, 以找到匹配的特定基因/cDNA 感兴趣的序列。这有助于预测在样本中最丰富的猎物片段的 5 ' 和 3 ' 末端是什么。沿序列长度的读取深度的波动是正常的, 如图 7所示。如果读取深度清楚地超过了停止密码子, 它表明, 猎物片段延伸到停止密码子, 因此 3 ' 底漆可以简单地对应于停止密码子周围的区域。</li>
	<li>
每一个基因执行50µL PCR 反应。每个反应包含 25 pmol 的每向前和反向底漆匹配的猎物-图书馆质粒 (见材料表)。反应还包括25µL 高保真 2x PCR 主混合, 5 µg 的 DNA 样本, 水多达50µL。
	<ol>
<li>放大反应为25个周期以3分钟的延长的时间在72°c, 退火温度55°c 三十年代, 并且变性在98°c 十年代. 在98摄氏度的三十年代变性之前骑自行车, 在5摄氏度时跟随72分钟孵化。</li>
	</ol>
</li>
</ol>

<ol><li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+genetic+system+to+detect+protein-protein+interactions%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li>
<li>Rajagopala, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mapping+the+Protein-Protein+Interactome+Networks+Using+Yeast+Two-Hybrid+Screens%5BTitle%5D)+AND+%22Advances+in+Experimental+Medicine+and+Biology%22%5BJournal%5D)">Mapping the Protein-Protein Interactome Networks Using Yeast Two-Hybrid Screens.</a> Advances in Experimental Medicine and Biology. 883, 187-214 (2015).</li>
<li>Weimann, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+Y2H-seq+approach+defines+the+human+protein+methyltransferase+interactome%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">A Y2H-seq approach defines the human protein methyltransferase interactome.</a> Nature Methods. 10 (4), 339-342 (2013).</li>
<li>Yachie, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Pooled-matrix+protein+interaction+screens+using+Barcode+Fusion+Genetics%5BTitle%5D)+AND+%22Molecular+Systems+Biology%22%5BJournal%5D)">Pooled-matrix protein interaction screens using Barcode Fusion Genetics.</a> Molecular Systems Biology. 12 (4), 863(2016).</li>
<li>Trigg, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((CrY2H-seq%3A+a+massively+multiplexed+assay+for+deep-coverage+interactome+mapping%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">CrY2H-seq: a massively multiplexed assay for deep-coverage interactome mapping.</a> Nature Methods. , (2017).</li>
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<li>Kim, D., Langmead, B., Salzberg, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((HISAT%3A+a+fast+spliced+aligner+with+low+memory+requirements%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">HISAT: a fast spliced aligner with low memory requirements.</a> Nature Methods. 12 (4), 357-360 (2015).</li>
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</ol>

作者没有什么可透露的

这里描述的软件套件允许一个从 DEEPN 实验中完全处理和分析高通量 DNA 测序数据。第一个程序使用的是 MAPster, 它采取的 DNA 序列读取标准的 fastq 文件, 并将其位置映射到参考 DNA 的下游处理由一个完整的信息学程序, 包括 DEEPN 软件。MAPster 接口的实用程序以及它对多个作业进行排队、组合输入文件、coveniently 名称输出文件的能力, 加上底层 HISAT2 程序7 it 控件的速度, 提供了一种易?...

一种发现蛋白质相互作用的方法是酵母 2-杂交 (Y2H) 试验, 它利用工程酵母细胞生长, 只有当一个兴趣蛋白质绑定到一个相互作用的伙伴1的片段。在大规模并行高通量测序的帮助下, 现在可以进行多 Y2H 交互的检测。有几种格式被描述2,3,4,5 , 包括一个我们开发的人口是分批种植的条件下, 选择酵母含有质粒, 产生积极的 Y2H 互动6。我们开发的工作流称为 DEEPN (动态富集, 用于评估蛋白质网络), 它识别出相同的猎物库中的差异 interactomes, 以识别与一种蛋白质 (或域) 的交互作用的蛋白质。另一种蛋白质或构象不同的突变域。该工作流的主要步骤之一是正确处理和分析 DNA 测序数据。有些信息可以通过计算每种基因在选择 Y2H 交互前后的次数, 比如 RNA 序列实验的方式来收集。但是, 可以从这些数据集中提取更多的深入信息, 包括有关特定蛋白质的子域的信息, 该子域能够产生 Y2H 的交互作用。此外, 虽然 DEEPN 方法是有价值的, 但分析许多样本复制可能是繁琐和昂贵的。使用一个专门为 DEEPN 数据集开发的统计模型来缓解这个问题, 因为复制的数量有限6。为了处理和分析 DNA 测序数据集可靠、完整、健壮、可供没有生物信息学专业知识的调查人员使用, 我们开发了一套涵盖所有分析步骤的软件程序。
这套单机版的软件程序在台式计算机上运行, 包括 MAPster、DEEPN 和 Stat_Maker。MAPster 是一个图形用户界面, 允许每个 fastq 文件排队使用 HISAT2 程序7映射到基因组, 生成一个标准. sam 文件, 以便在下游应用程序中使用。DEEPN 有几个模块。它分配和计数的读数对应的特定基因类似的 RNA 序列类型量化使用模块 ' 基因计数 '。它还提取与 Gal4 转录域和猎物序列相对应的序列, 并整理这些结点的位置, 以便通过比较表和图 (使用模块 "Junction_Make") 进行检查。模块 ' Blast_Query ' 允许简单的检查, 定量, 并比较连接 Gal4 连接序列。Stat_Maker 以统计学的方式评估每种基因浓缩数据的读数, 以此来确定可能 Y2H 命中的优先级。在这里, 我们描述如何使用这些软件程序, 并充分分析 DNA 序列数据从 DEEPN Y2H 实验。DEEPN 版本可在 PC、Mac 和 Linux 系统上运行。其他程序, 如映射程序 MAPster 和 DEEPN 统计模块 Stat_Maker 依赖于在 Unix 下运行的子例程, 仅在 Mac 和 linux 系统上可用。

<table><tbody><tr><td>Mapster</td><td></td><td></td><td>https://github.com/emptyewer/MAPster/releases</td></tr><tr><td>DEEPN software</td><td></td><td></td><td>https://github.com/emptyewer/DEEPN/releases</td></tr><tr><td>Statmaker</td><td></td><td></td><td>https://github.com/emptyewer/DEEPN/releases</td></tr><tr><td>Minimum computer system</td><td>Apple</td><td></td><td>Mac Intel Core i5 or better</td></tr><tr><td></td><td>-</td><td></td><td>4 Gb RAM or better</td></tr><tr><td></td><td>-</td><td></td><td>500 Gb Disk spce or better</td></tr><tr><td></td><td>-</td><td></td><td>OS 10.10 or higher</td></tr><tr><td></td><td>Dell</td><td></td><td>Intel i5-7400 or better</td></tr><tr><td></td><td>-</td><td></td><td>4 Gb RAM or better</td></tr><tr><td></td><td>-</td><td></td><td>500 Gb Disk spce or better</td></tr><tr><td></td><td>-</td><td></td><td>Windows 7 or higher</td></tr></tbody></table>

informatic analysis of sequence data from batch yeast 2-hybrid screens

我们已经适应了酵母 2-杂交试验, 同时揭示了在一个单一的屏幕上大量的瞬时和静态蛋白质相互作用, 利用高通量的短读 DNA 测序。所产生的序列数据集不仅可以跟踪在选择的阳性酵母 2-混合相互作用的人群中丰富的基因, 而且还提供了足够的相互作用的蛋白质相关子域的详细信息。在这里, 我们描述了一整套独立的软件程序, 允许非专家执行所有的生物信息学和统计步骤, 以处理和分析 DNA 序列 fastq 文件从一批酵母 2-杂交试验。这些软件包含的处理步骤包括: 1) 映射和计数序列读数对应于酵母 2-混合猎物库中编码的每个候选蛋白;2) 评估浓缩剖面的统计分析程序;和 3) 工具, 以检查在编码区域内的平移帧和位置的每个丰富的质粒, 编码的相互作用的蛋白质的兴趣。

映射 fastq 数据: 第一步 在几乎所有的包括 DEEPN 的应用中, 初始输出是一个短序列读取的文件, 必须通过对基因组、transcriptomic 或其他参考 DNA8进行映射。最近, 开发了 HISAT2 对齐程序, 使用最先进的索引算法来显著提高映射速度7,9。HISAT2 在台式计算机上高效运行, 可以在几分钟内映射一个典型大小?...

Watch this Scientific Journal Video about Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens at JoVE.com

Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

酵母菌群的深度测序为阳性酵母 2-混合相互作用可能产生大量的信息关于相互作用的伙伴蛋白。在这里, 我们描述了具体的生物信息学工具的操作和定制的更新软件来分析这些屏幕的序列数据。

间歇酵母 2-混合筛序列数据的信息化分析

We have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing ...

informatic-analysis-of-sequence-data-from-batch-yeast-2-hybrid-screens

Research

JoVE Journal

Genetics

7.1K 次观看.  University of Iowa. 酵母菌群的深度测序为阳性酵母 2-混合相互作用可能产生大量的信息关于相互作用的伙伴蛋白。在这里, 我们描述了具体的生物信息学工具的操作和定制的更新软件来分析这些屏幕的序列数据。

视频: Informatic Analysis of Sequence Data from Batch Yeast 2-Hybrid Screens

A Modified Yeast-one Hybrid System for Heteromeric Protein Complex-DNA Interaction Studies

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between proteins such as transcription factors (TFs) and their DNA target. The method presented here utilizes the underlying concept of the Y1H but is modified further to study and validate protein complexes binding to their target DNA. Hence, it is referred to as the modified yeast one-hybrid (Y1.5H) assay. This assay is cost effective and can be easily performed in a regular laboratory setting. Albeit using a heterologous system, the described method could be a valuable tool to test and validate the heteromeric protein complex binding to their DNA target(s) for functional genomics in any system of study, especially plant genomics.

The modified yeast one-hybrid assay described here is an extension of the classical yeast one-hybrid (Y1H) assay to study and validate the heteromeric protein complex-DNA interaction in a heterologous system for any functional genomics study.

用于异构蛋白复合物 -  DNA相互作用研究的修饰的酵母一杂交系统

Over the years, the yeast one-hybrid assay has proven to be an important technique for the identification and validation of physical interactions between ...

科研

生物化学

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

酵母 2-混合筛在批次中比较蛋白质相互作用

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

一种在裂变酵母中的深度固序辅助自发抑制屏

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

遗传学

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

强化酵母单杂交筛网识别与人 dna 序列结合的转录因子

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the strength of linkage and association mapping methods, which trace the relationship between DNA sequence variants and phenotype across recombinant progeny from matings between individuals of a species. These approaches, although powerful, are not well suited to trait differences between reproductively isolated species. Here we describe a new method for genome-wide dissection of natural trait variation that can be readily applied to incompatible species. Our strategy, RH-seq, is a genome-wide implementation of the reciprocal hemizygote test. We harnessed it to identify the genes responsible for the striking high temperature growth of the yeast Saccharomyces cerevisiae relative to its sister species S. paradoxus. RH-seq utilizes transposon mutagenesis to create a pool of reciprocal hemizygotes, which are then tracked through a high-temperature competition via high-throughput sequencing. Our RH-seq workflow as laid out here provides a rigorous, unbiased way to dissect ancient, complex traits in the budding yeast clade, with the caveat that resource-intensive deep sequencing is needed to ensure genomic coverage for genetic mapping. As sequencing costs drop, this approach holds great promise for future use across eukaryotes.

Genetic Mapping of Thermotolerance Differences Between Species of Saccharomyces Yeast via Genome-Wide Reciprocal Hemizygosity Analysis

Reciprocal hemizygosity via sequencing (RH-seq) is a powerful new method to map the genetic basis of a trait difference between species. Pools of hemizygotes are generated by transposon mutagenesis and their fitness is tracked through competitive growth using high-throughout sequencing. Analysis of the resulting data pinpoints genes underlying the trait.

通过全基因组互惠性分析,对糖母菌菌种间热耐性差异的遗传图谱

A central goal of modern genetics is to understand how and why organisms in the wild differ in phenotype. To date, the field has advanced largely on the ...

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 ...

生物学

Unravelling the Function of a Bacterial Effector from a Non-cultivable Plant Pathogen Using a Yeast Two-hybrid Screen

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria have evolved different strategies to manipulate their host metabolism for their own benefit. This bacterial manipulation is often coupled with severe symptom development or the death of the affected plants. Determining the specific bacterial molecules responsible for the host manipulation has become an important field in microbiological research. After the identification of these bacterial molecules, called &#34;effectors,&#34; it is important to elucidate their function. A straightforward approach to determine the function of an effector is to identify its proteinaceous binding partner in its natural host via a yeast two-hybrid (Y2H) screen. Normally the host harbors numerous potential binding partners that cannot be predicted sufficiently by any in silico algorithm. It is thus the best choice to perform a screen with the hypothetical effector against a whole library of expressed host proteins. It is especially challenging if the causative agent is uncultivable like phytoplasma. This protocol provides step-by-step instructions for DNA purification from a phytoplasma-infected woody host plant, the amplification of the potential effector, and the subsequent identification of the plant&#39;s molecular interaction partner with a Y2H screen. Even though Y2H screens are commonly used, there is a trend to outsource this technique to biotech companies that offer the Y2H service at a cost. This protocol provides instructions on how to perform a Y2H in any decently equipped molecular biology laboratory using standard lab techniques.

Bacterial effector proteins are important for establishing successful infections. This protocol describes the experimental identification of proteinaceous binding partners of a bacterial effector protein in its natural plant host. Identifying these effector interactions via yeast two-hybrid screens has become an important tool in unravelling molecular pathogenicity strategies.

从非耕种植物病原菌利用酵母双杂交筛选Unravelling一个细菌效应中的作用

Unravelling the molecular mechanisms of disease manifestations is important to understand pathologies and symptom development in plant science. Bacteria ...

免疫与感染

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.

化学遗传相互作用的快速鉴定<em&gt;酿酒酵母</em

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

使用酵母双杂交技术的生物分子相互作用研究 

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

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

作者聚焦:增强酵母中相互作用零突变体的分离

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

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 ...