Name: enhanced yeast one-hybrid screens to identify transcription factor binding to human dna sequences
Uploaded: 2019-02-11T15:00:00
Description: Watch this Scientific Journal Video about Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences at JoVE.com

这项工作得到了国家卫生研究院 [r35-gm128625 至 j. i. f. b.] 的支持。

1. 准备工作
<ol><li>
sc-u-h 盘 (150 毫米 petri 菜) 请注意:这些板材将用于生长 dna 诱饵酵母菌株。<ol>
<li>在920毫升的水中溶解辍学混合物、酵母氮碱 (ynb)、腺嘌呤半硫酸盐和硫酸铵, 用 5mm naoh 将 ph 值降至 5.9 (约为每升介质1毫升; 成分见表 2 )。倒进一个2升的烧瓶, 并添加一个搅拌酒吧。</li>
		<li>在第二个2升烧瓶中, 将琼脂添加到950毫升的水中 (不要添加搅拌棒, ...

<ol>
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S., 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=Yeast+one-hybrid+assays+for+gene-centered+human+gene+regulatory+network+mapping.">Yeast one-hybrid assays for gene-centered human gene regulatory network mapping.</a> Nature Methods. 8 (12), 1050-1052 (2011).</li><li>Sahni, N., 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=Widespread+macromolecular+interaction+perturbations+in+human+genetic+disorders.">Widespread macromolecular interaction perturbations in human genetic disorders.</a> Cell. 161 (3), 647-660 (2015).</li><li>Fuxman Bass, J. I., Reece-Hoyes, J. S., Walhout, A. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymolyase-Treatment+and+Polymerase+Chain+Reaction+Amplification+from+Genomic+and+Plasmid+Templates+from+Yeast.">Zymolyase-Treatment and Polymerase Chain Reaction Amplification from Genomic and Plasmid Templates from Yeast.</a> Cold Spring Harbor Protocols. (12), (2016).</li><li>Deplancke, B., 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=A+gene-centered+C.+elegans+protein-DNA+interaction+network.">A gene-centered C. elegans protein-DNA interaction network.</a> Cell. 125 (6), 1193-1205 (2006).</li><li>Reece-Hoyes, J. S., 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=Extensive+rewiring+and+complex+evolutionary+dynamics+in+a+C.+elegans+multiparameter+transcription+factor+network.">Extensive rewiring and complex evolutionary dynamics in a C. elegans multiparameter transcription factor network.</a> Molecular Cell. 51 (1), 116-127 (2013).</li><li>Carrasco Pro, S., 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=Global+landscape+of+mouse+and+human+cytokine+transcriptional+regulation.">Global landscape of mouse and human cytokine transcriptional regulation.</a> Nucleic Acids Research. 46 (18), 9321-9337 (2018).</li><li>Whitfield, T. W., 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=Functional+analysis+of+transcription+factor+binding+sites+in+human+promoters.">Functional analysis of transcription factor binding sites in human promoters.</a> Genome Biology. 13 (9), R50 (2012).</li><li>Fuxman Bass, J. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+factor+binding+to+Caenorhabditis+elegans+first+introns+reveals+lack+of+redundancy+with+gene+promoters.">Transcription factor binding to Caenorhabditis elegans first introns reveals lack of redundancy with gene promoters.</a> Nucleic Acids Research. 42 (1), 153-162 (2014).</li><li>Hens, K., 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=Automated+protein-DNA+interaction+screening+of+Drosophila+regulatory+elements.">Automated protein-DNA interaction screening of Drosophila regulatory elements.</a> Nature Methods. 8 (12), 1065-1070 (2011).</li><li>Gubelmann, C., 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=A+yeast+one-hybrid+and+microfluidics-based+pipeline+to+map+mammalian+gene+regulatory+networks.">A yeast one-hybrid and microfluidics-based pipeline to map mammalian gene regulatory networks.</a> Molecular Systems Biology. 9, 682 (2013).</li><li>Brady, S. M., 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=A+stele-enriched+gene+regulatory+network+in+the+Arabidopsis+root.">A stele-enriched gene regulatory network in the Arabidopsis root.</a> Molecular Systems Biology. 7, 459 (2011).</li><li>Pruneda-Paz, J. 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与以前的库筛选或基于转换的排列筛选方法相比, 这里描述的机器人 ay1h 交配筛选方法大大提高了识别绑定到感兴趣的 dna 区域的 tf 集的吞吐量。此外, 由 ey1h 检测检测到的 tf-dna 相互作用是高度可重复的, 因为检测到的90% 的相互作用对每个 tf 测试的所有四个菌落都呈阳性, 90% 的相互作用在同一酵母 dna-诱饵菌株的独立屏幕上重新检测,12,<sup class=...

生物医学领域的一个核心问题是确定调节每一个人基因的机制。转录是控制基因表达水平的第一步, 它是由每个基因特有的转录因子 (tf) 集调节的。考虑到人类编码 & gt;1,500 tf1,2, 确定一整套控制每个基因表达的 tf 仍然是一个开放的挑战。
两种类型的方法可用于映射 tf-dna 相互作用: tf 为中心的方法3和以 dna 为中心的方法 3 (图 1 a)。在以 tf 为中心的方法中, 研究了与基因组 dna 区域结合或确定其 dna 结合特异性的感兴趣的 tf。这些方法包括染色质免疫沉淀 (chip), 然后是高通量测序、蛋白质结合微阵列和 selex4,5,6。在以 dna 为中心的方法中, 研究了感兴趣的 dna 序列, 以确定与 dna 序列结合的 tf 集。这种方法应用最广泛的是酵母单杂交 (y1h) 检测, 其中 tf 和 dna 区域之间的相互作用在酵母细胞核的环境中使用报告基因7,8,9进行测试。
y1h 检测涉及两个组成部分: "dna 诱饵" (例如, 促进剂、增强剂、消音器等) 和 "tf-猎物", 可用于对报告基因激活9、10 (图 1 b) 进行筛选。dna 诱饵是克隆的上游两个报告基因 (lacz和his3) 和两个 dna 诱饵:: 记者结构被集成到酵母基因组中, 产生染色质的 ' dna 诱饵菌株 '。tf-猎物编码在表达 tf 融合到酵母 gal4 tf 的激活域 (ad) 的质粒中, 被引入 dna 诱饵菌株中, 用于 tf-dna 的相互作用。如果 tf-猎物与 dna 诱饵序列结合, 那么 tf-猎物中存在的 ad 将导致两个记者基因的激活。因此, 可以选择具有正相互作用的细胞在缺乏组氨酸的板材上生长, 并克服竞争性抑制剂, 3-氨基-1, 2, 4-三唑 (3-at), 并可视化为蓝色菌落在 x-ger 的存在。由于使用了强效酵母 gal4 ad, y1h 检测可以检测涉及转录激活剂和抑制剂的相互作用。此外, 考虑到 tf-preys 是由强酵母启动子 (adh1) 表达的, 即使是内源性表达水平较低的 tf 也可以检测到相互作用, 而 chip 11,12很难检测到这些 tf.
大多数已发布的执行 y1h 检测的协议都是基于将 tf 前置入酵母 dna 诱饵菌株, 方法是转换集合 tf-猎物库, 然后进行选择、群体选择和排序, 以识别相互作用的 tf, 或者通过改变单个克隆8,9。这些都是耗时的协议, 限制了每个研究人员可以测试的 dna 序列的数量。最近对 y1h 检测的改进被称为增强 y1h (ey1h), 通过使用高密度阵列 (hda) 机器人平台将酵母 dna-诱饵菌株与酵母 dna-诱饵菌株组合在一起, 每个菌株都表达了不同的表达, 从而显著提高了筛选吞吐量tf-猎物 10,13 (图 1c)。这些屏幕采用 1, 536个菌落格式, 只需三个盘子即可测试大多数人体的 tf, 这些 tf 为四元。此外, 考虑到 tf-dna 相互作用是以对等的方式进行测试的, 这种方法允许比较 dna 诱饵 (如两个非编码单核苷酸变种) 之间和不同 tf 或 tf 变种之间的相互作用11,12 ,14。
利用 ey1h 检测, 我们已经描述了迄今为止最大的人类和以 dna为中心的 tf-dna 相互作用网络。特别是, 我们确定了246个人类发展促进者和 283个 tf12之间的 2 2230 种互动。此外, 我们还使用 ey1h 检测发现了改变后的 tf 结合到109种与遗传性疾病 (如发育畸形、癌症和神经紊乱) 相关的单核苷酸非编码变异。最近, 我们使用 ey1h 来描述一个网络, 该网络由 2, 576 ·c. elegans基因启动子和 366个 tf11之间的 21, 714个相互作用组成。这个网络有助于揭示数十种线虫的功能作用。
产生 dna 诱饵污渍和评估背景记者活动水平的协议已在其他地方报道过 15、16、17。在这里, 我们描述了一个 ey1h 管道, 它可以用来筛选任何人类基因组 dna 区域, 以对抗 1, 086个人类 tf 阵列。一旦产生酵母 dna-诱饵菌株, 并在相应的板块上发现 tf-猎物阵列, 整个协议可以在两周内执行 (表 1)。更重要的是, 该协议可以并行化, 以便单个研究人员可以同时筛选 60个 dna 诱饵序列。为了证明该协议, 我们筛选了两个细胞因子基因 ccl15 和 il17f 的启动子。此外, 我们还显示了失败屏幕的结果, 以说明在执行 ay1h 检测时可能出现的问题类型以及如何对其进行故障排除。

<table border="0" cellpadding="0" cellspacing="0" style="width:1195px;" width="1195">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">3-Amino-1,2,4-triazole (3AT) ~95 % TLC</td>
			<td style="width:223px;">Sigma</td>
			<td style="width:103px;">A8056-100G</td>
			<td style="width:315px;">Competitive inhibitor for products of HIS3 gene</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Adenine sulfate (hemisulfate), dihydrate</td>
			<td>US Biologicals</td>
			<td>A0865</td>
			<td>Required for proper yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agar High Gel Strength - Bacteriological grade</td>
			<td>American International Chemical</td>
			<td>AGHGUP</td>
			<td>Nutritive media for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate</td>
			<td>US Biologicals</td>
			<td>A1450</td>
			<td>Nitrogen source in synthetic yeast media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D+ Glucose Anhydrous</td>
			<td>US Biologicals</td>
			<td>G3050</td>
			<td>Required for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Drop-Out Mix minus His, Leu, Tryp and Uracil, adenine rich w/o yeast nitrogen base</td>
			<td>US Biologicals</td>
			<td>D9540-02</td>
			<td>Synthetic complete media required for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">edge Multiparameter pH Meter</td>
			<td style="width:223px;">Hanna Instruments</td>
			<td>HI2020-01</td>
			<td>To measure pH of selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flat Toothpicks 750ct</td>
			<td style="width:223px;">Diamond</td>
			<td style="width:103px;"></td>
			<td>To streak yeasts on petridishes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">Glass Beads</td>
			<td style="width:223px;">Walter Stern</td>
			<td style="width:103px;">100C</td>
			<td>To spreak yeast when making lawns</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol &#8805;99%</td>
			<td>Millipore Sigma</td>
			<td>G9012-1L</td>
			<td>Required to make frozen yeast stocks</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Histidine</td>
			<td>US Biologicals</td>
			<td>H5100</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Leucine</td>
			<td>US Biologicals</td>
			<td>L2020-05</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Tryptophan</td>
			<td>Sigma</td>
			<td>T-0254</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N-Dimethylformamide</td>
			<td>Sigma</td>
			<td>319937-1L</td>
			<td>To make X-gal solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Omnipense Elite</td>
			<td style="width:223px;">Wheaton</td>
			<td>W375030-A</td>
			<td>For dispensing accurate volumes of media into Singer plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Peptone, Bacteriological</td>
			<td>American International Chemical</td>
			<td>PEBAUP</td>
			<td>Protein source required for yeast growth</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish, 150x15 mm</td>
			<td>VWR</td>
			<td>10753-950</td>
			<td>For growing yeast baits for screening</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">PlusPlates</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">PLU-003</td>
			<td>To make rectangular agar plates to use with Singer Robot</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">Precision Low Temperature BOD Refrigerated Incubator</td>
			<td style="width:223px;">ThermoFisher Scientific</td>
			<td>PR205745R</td>
			<td>To incubate yeast plates at constant temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 1,536 short</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-005</td>
			<td>To transfer the TF-prey array, mate yeast, and transfer yeast to diploid selection and readout plates</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 384 short</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-004</td>
			<td>To transfer TF-prey array from 384 to 1,536 colony format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 96 long</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-001</td>
			<td>To transfer TF-prey array from glycerol stock to agar plate</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">RePads 96 short</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;">REP-002</td>
			<td>To transfer TF-prey array from 96 to 384 colony format</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:555px;">Singer HDA RoToR robot</td>
			<td style="width:223px;">Singer Instruments</td>
			<td style="width:103px;"></td>
			<td>For transfering yeast in high-throughput manner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide (Pellets/Certified ACS)</td>
			<td>Fisher</td>
			<td>S318-1</td>
			<td>For adjusting pH of selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate dibasic heptahydrate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-203402C</td>
			<td>Required for LacZ reporter activity on X-gal&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate monobasic monohydrate</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-202342B</td>
			<td>Required for LacZ reporter activity on X-gal&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Uracil</td>
			<td>Sigma</td>
			<td>U0750-100G</td>
			<td>For yeast growth selection in selective media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal (5-Bromo-4-chloro-3-indoxyl-beta-Dgalactopyranoside)</td>
			<td>Gold Biotechnology</td>
			<td>X4281C100</td>
			<td>&#946;-galactosidase turns colorless X-gal blue to detect protein-DNA interaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Extract</td>
			<td>US Biologicals</td>
			<td>Y2010</td>
			<td>Nutritious medium for growth and propagation of yeast</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Yeast Nitrogen Base (powder) w/o AA, carbohydrate and w/o AS&#160;</td>
			<td>US Biologicals</td>
			<td>Y2030</td>
			<td>Required for vigorous yeast growth</td>
		</tr>
	</tbody>
</table>

enhanced yeast one-hybrid screens to identify transcription factor binding to human dna sequences

识别调节每个人类基因的转录因子 (tf) 集是一项艰巨的任务, 需要整合许多实验和计算方法。其中一种方法是酵母单杂交 (y1h) 检测, 在该方法中, 利用报告基因在酵母细胞核环境中测试 tf 和 dna 区域之间的相互作用。y1h 检测涉及两个组成部分: "dna 诱饵" (例如, 促进剂、增强剂、消音器等) 和 "tf-猎物", 可用于记者基因激活的筛选。大多数用于执行 y1h 屏幕的已发布协议都是基于将 tf-猎物库或阵列转换为 dna 诱饵酵母菌株。在这里, 我们描述了一个被称为增强 y1h (ey1h) 检测的管道, 在这种情况下, tf-dna 相互作用通过将 dna 诱饵菌株与使用高密度阵列 (hda) 机器人平台排列的 tf-猎物菌株进行交配来进行测试, 该平台允许在 1 536个测试中进行筛选菌落格式。这使得吞吐量大幅增加 (60个 dna 诱饵序列对 & gt;1,000 tf 每个研究人员需要两周时间) 和可重复性。我们通过针对 1, 086个人 tf 的阵列测试人类启动子序列来说明不同类型的预期结果, 以及屏幕过程中可能出现的问题以及如何进行故障排除的示例。

在分析 ey1h 检测结果时, 应考虑三个主要因素: dna-诱饵株的背景报告活性、与 tf-dna 相互作用相对应的报告活性强度以及阳性菌落的数量。dna 诱饵菌株的背景报告活动 (即自体活动) 是指读出板中酵母菌落的整体生长和颜色, 即使在没有 tf-猎物的情况下也是如此。理想情况下, 非自动反应菌株显示背景白色或浅棕色, 与菌落的积极互动是较大和蓝色的。自体活性 dna 诱饵菌株显示酵母生长在缺乏组氨?...

Watch this Scientific Journal Video about Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences at JoVE.com

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

在这里, 我们提出了一个增强的酵母单杂交筛选方案, 以确定转录因子 (tf), 可以绑定到人类 dna 区域感兴趣。该方法采用高通量筛选管道, 可以在一次实验中询问 tf 和 gt;1,000 的结合。

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 ...

增强酵母单杂交测定的目标是识别与感兴趣的DNA序列结合的转录因子集。该技术的优点是增强酵母单杂交测定可以在一次实验中评估1000多个转录因子。相反，染色质免疫沉淀一次只评估一个转录因子。此方法是功能基因组学的关键工具，用于识别与基因启动剂和增强剂结合的转录因子的序列，并识别与非编码变异结合的改变转录因子。演示这个程序的将是刘兴博士，一个博士后从我的实验室。用冰上的 TF 猎物阵列解冻酵母甘油库存板。使用 12 通道移液器重新发送酵母，并在 1 到 3 分钟内继续执行下一步。要发现酵母进入Sc减去色氨酸矩形板，使用高密度阵列机器人选择多井96板作为来源，96个阿加板作为目标，和96长针垫。针垫不能重复使用，应丢弃。选择"现货多"程序，每 96 个井板制作两个副本。不要使用回收或重新循环选项，以避免冷冻库存的回污染。此外，选择在源中上下旋转以混合酵母的选项。按照说明放置板的位置和时间，让机器人发现酵母到Sc减去色氨酸矩形板。袋斑点阵列，孵育它加糖侧在30摄氏度2至3天。要生成 384 个菌落阵列，在 Sc 减去色氨酸矩形板中，请选择 96 个阿加板作为源，选择 384 个加糖板作为目标，以及 96 个短销垫。然后选择 1：4 数组单源程序。这样，四个96个殖民地板块将合并为一个384个殖民地板块。请勿使用回收或重新循环选项，以避免不同板之间的污染。袋板和孵育斑点的384殖民地阵列加糖侧在30摄氏度2天。要在 Sc 减去色氨酸矩形板中生成 1536 个菌落阵列，请选择 384 个加糖板作为源，选择 1536 个加糖板作为目标，选择 384 个短销垫。然后选择1：4检测单源程序。目标是将每个殖民地复制到四个殖民地，以获得四倍。使用回收和重新访问选项，因为这需要复制每个殖民地 4 次。按照机器人的指示，让机器人在袋装和孵育斑点的1536殖民地阵列加糖侧在30摄氏度3天之前，发现酵母进入板。要放大 Sc 减去色氨酸矩形板中的 1536 菌落阵列，请选择 1536 个加糖板作为源，选择 1536 个加糖板作为目标，选择 1536 个短销垫。选择"复制多个"程序可复制 3 到 4 个副本。使用回收和重访选项，但在切换到阵列的不同板时扔掉垫，以避免交叉污染。允许机器人放大 Sc 减去色氨酸矩形板中的 1536 殖民地阵列。最后，将盘子包起来，在30摄氏度的温度下孵育斑点的1536殖民地阵列，用于交配步骤3天。要准备DNA诱饵菌株草坪交配，发现DNA酵母诱饵菌株在Sc减去乌拉西和血素板，并在30摄氏度生长3天。使用无菌牙签将酵母切成 15 厘米的 Sc 减 uracil 和组蛋白板，使每个板适合 12 到 16 种不同的菌株。在30摄氏度下孵育一天。第二天，使用无菌牙签将酵母条纹成15厘米的Sc减去uracil和组蛋白板，使每个盘子适合4种不同的菌株。在30摄氏度下孵育一天。孵育后，用无菌牙签刮酵母，确保不刮任何阿加。将酵母加入1.5毫升管中，加入500微升无菌水。现在添加 10 到 15 个无菌玻璃珠和酵母悬浮液到 YAPD 矩形板上。在四面八方彻底摇动一分钟，以确保酵母在盘子中传播。立即反转板，然后轻点使珠子进入盖子。取出并回收珠子。袋板，并在30摄氏度下孵育其加糖侧1至2天。然后，继续执行交配步骤。要配合酵母DNA诱饵和TF阵列菌株，请将TF阵列与机器人转移到雅普矩形板中。选择 1536 agar 板作为源和目标，以及 1536 短销垫。选择"复制多个"程序。选择 4 个源板和回收和重新访问选项。每个 TF 阵列板可用于传输 3 到 4 个 YAPD 板。用于交配的 TF 阵列板必须具有 2 到 3 天的旧时间，但不应更多，因为交配可能效率低下。要将 DNA 诱饵菌株的草坪转移到已经包含 TF 阵列的 YAPD 板，请选择 1536 agar 板作为源和目标以及 1536 短销垫。选择"复制多个"程序。在半径约为 0.6 毫米的源中使用随机偏移，以避免从同一位置服用酵母，并选择"目标混合"，以方便酵母菌株之间的接触。使用含有DNA诱饵菌株的草坪作为来源，使用含有斑点TF阵列的亚普德板作为目标，然后袋装板，在30摄氏度的温度下将它们孵育一天。要选择二倍体酵母，选择1536糖板作为来源和目标，并选择1536短针垫。选择"复制"程序。选择"在源上和目标上混合"。允许机器人将配合酵母从 YAPD 板转移到 Sc 减化板和色氨酸板，然后袋装板并在 30 摄氏度下将糖侧孵育 2 至 3 天。现在，选择 1536 阿加板作为源和目标以及 1536 短销垫。选择"复制"程序。使用机器人将二倍体酵母从 Sc 减去 uracil 和色氨酸板转移到读出矩形 3-AT 和 X-gal 的板中。袋板和孵育他们加糖侧在30摄氏度长达7天。对于具有高背景记者活动的DNA诱饵菌株，请于第2天、第3天和4天拍照。否则，在第 4 天和 7 天拍照。将 TF 阵列板保持室温，7 天后再次复制，进行新一轮筛查。为了说明使用增强酵母单杂交测定可以获得的结果类型，对CCL15和IL17F基因的启动区域进行了1086个人类TF阵列的筛选。CCL15启动子是非自动DNA诱饵的一个例子，其中相互作用，即使是弱的，可以很容易地检测。IL17F 启动子是具有不均匀背景报告器活动的自动活性 DNA 诱饵的一个例子，其中可以检测到某些交互，而对于多个 TF，不确定报告器活动是否高于背景。在进行增强酵母单杂交测定时，可能会出现几个问题。在这种情况下，殖民地太小，无法转移。通常，大约 95% 的 TF 猎物群显示正常生长。在此示例中，板的一部分没有酵母生长。此问题通常与在交配步骤中失败有关，如果 1536 销垫未能与 DNA 诱饵应变草坪、TF 阵列或交配板中的酵母接触。在这两个示例中，未检测到任何交互。此问题通常与记者基因（尤其是 LacZ）中意外的不激活突变有关，或者与掩盖相互作用的过高的自动性有关。在这种情况下，板呈现随机蓝点，这通常与细菌污染有关。最重要的事情要考虑的是适当的板准备。确保添加所有组件，并且加糖高度是均匀的，因为所有后续步骤都依赖于此。虽然大多数试剂并不危险，但必须时刻戴上手套，以避免样品和板材受到污染。与任何DNA结合检测一样，使用分析器检测、转录因子敲除或敲除等功能检测来验证所识别的相互作用非常重要，其次是测量目标基因的表达。这种方法允许识别用于调节特定生物过程中涉及的基因的转录因子，以及与非编码疾病相关变异交替结合的转录因子。

Research

JoVE Journal

Genetics

Methyl-binding DNA capture Sequencing for Patient Tissues

为患者组织甲基结合DNA序列捕获

Methylation is one of the essential epigenetic modifications to the DNA, which is responsible for the precise regulation of genes required for stable ...

科研

遗传学

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

Real-time Analysis of Transcription Factor Binding, Transcription, Translation, and Turnover to Display Global Events During Cellular Activation

转录因子结合、转录、翻译和营业额的实时分析在细胞激活过程中显示全球事件

Upon activation, cells rapidly change their functional programs and, thereby, their gene expression profile. Massive changes in gene expression occur, for ...

RNA Pull-down Procedure to Identify RNA Targets of a Long Non-coding RNA

rna 下拉法识别长非编码 rna 的 rna 靶点

Long non-coding RNA (lncRNA), which are sequences of more than 200 nucleotides without a defined reading frame, belong to the regulatory non-coding ...

Gene-targeted Random Mutagenesis to Select Heterochromatin-destabilizing Proteasome Mutants in Fission Yeast

基因靶向随机诱变在裂变酵母中选择染色质稳定蛋白酶突变体

Random mutagenesis of a target gene is commonly used to identify mutations that yield the desired phenotype. Of the methods that may be used to achieve ...

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

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 ...

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins

探索序列空间,识别调节RNA结合蛋白的结合位点

Gene regulation plays an important role in all cells. Transcriptional, post-transcriptional (or RNA processing), translational, and post-translational ...

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

In Vivo Forward Genetic Screen to Identify Novel Neuroprotective Genes in Drosophila melanogaster

在Vivo前向遗传屏幕识别新的神经保护基因在果蝇黑色素

There is much to understand about the onset and progression of neurodegenerative diseases, including the underlying genes responsible. Forward genetic ...

Pooled CRISPR-Based Genetic Screens in Mammalian Cells

哺乳动物细胞中基于CRISPR的聚集基因屏幕

Genome editing using the CRISPR-Cas system has vastly advanced the ability to precisely edit the genomes of various organisms. In the context of mammalian ...