Name: labelling and visualization of mitochondrial genome expression products in baker's yeast saccharomyces cerevisiae
Uploaded: 2021-04-11T14:00:00
Description: Watch this Scientific Journal Video about Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae at JoVE.com

这项研究由俄罗斯基础研究基金会资助，赠款编号为18-29-07002。P.K.得到俄罗斯联邦科学和高等教育部国家分配的支持，赠款编号为AAAA-A16-116021660073-5。M.V.P.得到俄罗斯联邦科学和高等教育部的支持，赠款编号为075-15-2019-1659（库尔恰托夫基因组研究中心方案）。这项工作部分是在莫斯科国立大学发展方案框架下购买的设备上完成的。I.C、S.L.和M.V..B还得到了莫斯科国立大学授予的"诺亚方舟领先科学学校"的资助。

1. 酵母文化准备
<ol><li>用适当的介质在新鲜盘子上冷冻库存培养物上的条纹酵母。将盘子放入30°C的文化孵化器中，24-48小时。 注意：让对温度敏感的突变体在允许的温度下生长。</li>
	<li>接种酵母培养物在2 mL的YPGal介质（2%的肽，1%酵母提取物，2%的辣椒素）从新鲜条纹在15 mL管，并孵育他们过夜搅拌在200 rpm在30°C。</li>
	<li>以 600 nm （OD 600） 的波长测量培养的光学密度。</li>
	<li>以无?...

<ol>
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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioactive+labeling+of+mitochondrial+translation+products+in+cultured+cells.">Radioactive labeling of mitochondrial translation products in cultured cells.</a> Methods in Molecular Biology. 837, 207-217 (2012).</li><li>Kuzmenko, A., 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=Aim-less+translation:+loss+of+Saccharomyces+cerevisiae+mitochondrial+translation+initiation+factor+mIF3/Aim23+leads+to+unbalanced+protein+synthesis.">Aim-less translation: loss of Saccharomyces cerevisiae mitochondrial translation initiation factor mIF3/Aim23 leads to unbalanced protein synthesis.</a> Science Reports. 6, 18749 (2016).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature Methods. 9 (7), 671-675 (2012).</li><li>Keil, 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=Oxa1-ribosome+complexes+coordinate+the+assembly+of+cytochrome+c+oxidase+in+mitochondria.">Oxa1-ribosome complexes coordinate the assembly of cytochrome c oxidase in mitochondria.</a> Journal of Biological Chemistry. 287 (41), 34484-34493 (2012).</li><li>Singhal, R. 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U., 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=Coa3+and+Cox14+are+essential+for+negative+feedback+regulation+of+COX1+translation+in+mitochondria.">Coa3 and Cox14 are essential for negative feedback regulation of COX1 translation in mitochondria.</a> The Journal of Cell Biology. 191 (1), 141-154 (2010).</li><li>Bietenhader, 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=Experimental+relocation+of+the+mitochondrial+ATP9+gene+to+the+nucleus+reveals+forces+underlying+mitochondrial+genome+evolution.">Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution.</a> PLoS Genetics. 8 (8), e1002876 (2012).</li><li>Couvillion, M. T., Churchman, L. 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基因表达研究在现代生命科学中占有核心地位。已开发出许多方法，为这一复杂的过程提供见解。在这里，我们描述了允许在面包师酵母S.脑线粒体中获取蛋白质生物合成的方法。它通常用于比较突变酵母菌株线粒体中的mRNA的翻译效率与野生类型，以获得研究突变的后果。这是研究人员在研究带有突变的酵母细胞线粒体功能时进行的基本实验之一，该细胞被建议影响线粒体8、...

线粒体是深陷真核细胞代谢的细胞器。他们的电子传输链为细胞提供ATP，ATP是用于多种生化途径的主要能量货币。此外，他们参与凋亡，脂肪酸和血红素合成，和其他过程。线粒体功能障碍是人类疾病的著名来源。它可以是由于核基因或线粒体基因的突变，编码细胞器2的结构或调节成分。贝克的酵母 糖精 是研究线粒体基因表达的一个很好的模型有机体，原因有几个。首先，他们的基因组被完全测序3，有很好的注释，由于与这种生物体进行的长期调查历史，大量的数据已经在文献中可用。其次，由于其快速的生长速度和高效的同源重组系统，其核基因组的处理相对快速和容易。第三，面包师的酵母 S.脑膜 是少数利用线粒体基因组进行操作的生物之一。最后，面包师的酵母是一种气样-厌食性有机体，它允许分离和研究呼吸缺陷突变体，因为它们可以在含有可发酵碳源的介质中生长。
我们描述了研究面包师酵母 S.cerevisiae 的线粒体基因表达的方法在转化水平4。它的主要原则来自几个观察。首先，酵母线粒体基因组只编码八种蛋白质：细胞色素c氧化酶的三个亚单位（Cox1p， 考克斯2p和Cox3p），ATP合成酶（Atp6p、Atp8p和Atp9p）的三个子单位，ubiquinol-细胞色素c氧化酶、细胞色素b（细胞）和线粒体核糖体蛋白Var1p5的子单位。这个数字很小，在适当条件下，所有凝胶上的电光可以分离。其次，线粒体核糖体属于原核糖体类，而不是真核生物6，因此，酵母细胞质和线粒体核糖体对抗生素的敏感性不同。它允许用环氧化物抑制细胞质转化，为标记的氨基酸（35S-甲基氨酸）仅在线粒体转化产品中结合提供条件。因此，该实验提供了有关线粒体蛋白中氨基酸的吸收率的信息，反映了八种产品中线粒体转化的整体效率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>M3148</td>
			<td></td>
		</tr>
		<tr>
			<td>Acrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>A9099</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacteriological agar</td>
			<td>Sigma-Aldrich</td>
			<td>A5306&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Biowave Cell Density Meter CO8000</td>
			<td>BIOCHROM US BE</td>
			<td>80-3000-45</td>
			<td></td>
		</tr>
		<tr>
			<td>BRAND standard disposable cuvettes</td>
			<td>Sigma-Aldrich</td>
			<td>Z330361</td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Sigma-Aldrich</td>
			<td>288306&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>cycloheximide</td>
			<td>Sigma-Aldrich</td>
			<td>C1988&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Galactose</td>
			<td>Sigma-Aldrich</td>
			<td>G5388&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G7021&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>digital block heater</td>
			<td>Thermo Scientific</td>
			<td>88870001</td>
			<td></td>
		</tr>
		<tr>
			<td>EasyTag L-[35S]-Methionine, 500&#181;Ci (18.5MBq), Stabilized Aqueous Solution</td>
			<td>Perkin Elmer</td>
			<td>NEG709A500UC</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Centrifuge 5425</td>
			<td>Thermo Scientific</td>
			<td>13-864-457</td>
			<td></td>
		</tr>
		<tr>
			<td>GE Storage Phosphor Screens</td>
			<td>Sigma-Aldrich</td>
			<td>GE29-0171-33</td>
			<td></td>
		</tr>
		<tr>
			<td>L-methionine</td>
			<td>Sigma-Aldrich</td>
			<td>M9625&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethylethylenediamine</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N&#8242;-Methylenebisacrylamide</td>
			<td>Sigma-Aldrich</td>
			<td>M7279</td>
			<td></td>
		</tr>
		<tr>
			<td>New Brunswick Innova 44/44R Shaker Incubator</td>
			<td>New Brunswick Scientific</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peptone from meat, bacteriological</td>
			<td>Millipore</td>
			<td>91249&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenylmethanesulfonyl fluoride</td>
			<td>Sigma-Aldrich</td>
			<td>P7626&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660nm Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>22662</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Protean II xi cell</td>
			<td>Bio-Rad</td>
			<td>1651802</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride from Streptomyces alboniger</td>
			<td>Sigma-Aldrich</td>
			<td>P8833</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td>221465</td>
			<td></td>
		</tr>
		<tr>
			<td>Storm 865 phosphor imager</td>
			<td>GE Healthcare</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma-Aldrich</td>
			<td>93352&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum Heated Gel Dryer</td>
			<td>Cleaver Scientific</td>
			<td>CSL-GDVH</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Sigma-Aldrich</td>
			<td>Y1625&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

labelling and visualization of mitochondrial genome expression products in baker's yeast saccharomyces cerevisiae

线粒体是能够有氧呼吸的真核细胞的基本细胞。它们含有圆形基因组和基因表达装置。面包师酵母的线粒体基因组编码八种蛋白质：细胞色素c氧化酶的三个亚单位（Cox1p， 考克斯2p和考克斯3p），ATP合成酶（Atp6p、Atp8p和Atp9p）的三个子单位，是紫基酚-细胞色素c氧化酶、细胞色素b（细胞色素b）和线粒体核糖体蛋白Var1p的子单位。这里描述的方法的目的是用 35S甲氨酸专门标记这些蛋白质，通过电磷分离它们，并将信号可视化为屏幕上的离散带。该过程涉及几个步骤。首先，酵母细胞在含角质的介质中培养，直到它们到达晚期对数生长阶段。其次，环素酰胺治疗阻断细胞质转化，仅允许 35S甲基氨酸在线粒体翻译产品中结合。然后，所有蛋白质都从酵母细胞中提取，并通过聚丙烯酰胺凝胶电泳分离。最后，凝胶干燥并与存储磷屏幕一起孵育。屏幕扫描在荧光仪上，显示带子。该方法可用于比较突变酵母菌株线粒体中单个多肽的生物合成率与野生菌型，这有助于研究线粒体基因表达缺陷。此协议提供了有关所有酵母线粒体 mRNA 翻译速率的宝贵信息。然而，它需要几个控制和额外的实验，以作出适当的结论。

按照上述协议，我们从两个 S. Cerevisiae 菌株中分配线粒体翻译产品：野生类型（WT）和 AIM23 基因的突变轴承删除（AIM23Δ），编码线粒体翻译启动因子 3 （表 1）8.线粒体翻译产品在SDS-PAAG9中被放射性标记和分离。样本在饱和前每2.5分钟收集一次，以建立一个时间过程（图1A）。凝胶在为期5天的展览（图...

Watch this Scientific Journal Video about Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae at JoVE.com

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

贝克的酵母线粒体基因组编码了八种多肽。当前协议的目标是将所有这些都标记为单独的频段，然后将其可视化为单独的频段。

贝克酵母糖精中线粒体基因组表达产品的标记和可视化

Mitochondria are essential organelles of eukaryotic cells capable of aerobic respiration. They contain circular genome and gene expression apparatus. A ...

线粒体是能够有氧呼吸的真核细胞的基本细胞。他们的功能障碍是众所周知的人类疾病来源。贝克的酵母线粒体含有循环基因组编码八种蛋白质。在这段视频中，我们描述了通过对转化产品进行放射性标记和随后通过凝胶电泳分离来检测酵母线粒体中蛋白质生物合成的协议。该程序包括几个主要步骤。新鲜盘子上冷冻库存培养物中的斯特雷克酵母， 具有适当的介质。将盘子放在文化孵化器中30度，24至48小时。在 15 毫升管中的新鲜条纹中以两毫升的介质接种这些培养物，并在 30 度的 200 RPM 下一夜之间孵育它们。测量600纳米波长的培养物的光学密度。在室温下，在 9，000 G 的无菌管调色板酵母细胞中，以相当于 0.2 吸附装置的体积为例，时间为 30 秒。丢弃此超自然。通过涡流5秒钟，用0.5毫升无菌水清洗细胞。在室温下，9000 G的皮莱特酵母细胞可容纳30秒。丢弃超自然人。在两毫升的新鲜擦拭中稀释细胞。孵化边缘在 200 RPM 和 30 度，直到光学密度达到 1.5 到 1.9 从 1.5 到 1.9 值。传输相当于微型离心机管中一个光学单元的培养体积。在 3000 G 下旋转管子一分钟。丢弃超自然人。通过涡流5秒钟，用0.5毫升无菌水清洗细胞。在室温下，在9000G的帕莱特酵母细胞30秒内丢弃超母细胞。在 0.5 毫升无菌转化缓冲中重新悬浮酵母细胞。将悬架置于 15 毫升管中。在细胞悬浮中加入环氧酰胺，最终浓度为每毫升0.2毫克孵化，在200 RPM和30度下进行5分钟边缘，以抑制细胞溶解。从 25 到 30 添加 S35 甲氨酸 S35 甲氨酸的 25 到 30 微库到细胞悬浮和孵育 30 分钟边缘在 200 RPM 和 30 度。添加无标签的冷甲基氨酸和博里霉素，停止标签孵化10分钟边缘采取在200 RPM和30度。通过离心收集酵母细胞，在9000G下30秒。通过涡流5秒钟，用0.5毫升无菌水清洗细胞。在室温下，9000 G的皮莱特酵母细胞可容纳30秒。丢弃超自然人。在调色板和漩涡中加入 75 微升裂解缓冲器，时间为 5 到 10 秒。添加 500 微升 0.5 摩尔三轮缓冲区 0.5 摩尔三轮缓冲与 pH 6.8。pH 6.8.漩涡短暂。在样品中加入600微升甲醇。漩涡五秒钟。在样品中加入150微升氯仿。漩涡五秒钟。在 12，000 G.小心地用取样器丢弃 opaface 时，将样品离心两分钟。在样品中加入600微升甲醇。通过多次倒置管子小心混合。离心机样品两分钟，在12，000 G.丢弃超自然。空气在80度下将调色板干燥两分钟。溶解60微升莱姆利样品缓冲器中的沉淀蛋白。在 14 度下加热样品 10 分钟。导致17.5%莱姆利SDS多丙烯酰胺凝胶。将每个样品的 15 微升装入口袋。在冷室中运行凝胶，直到蓝色染料达到凝胶淋巴的约 65%。用 Kumasi 亮蓝色染色凝胶，并进行扫描或拍照，这需要作为负载控制。干燥凝胶干燥机中的凝胶。将其与存储磷屏幕一起保存在盒式磁带中三到五天。在磷成像仪上扫描屏幕。按照上述协议，我们从两个糖精脑菌株中分配线粒体翻译产品。Aim23基因编码线粒体翻译启动因子三的野生类型和突变变种删除。线粒体翻译产品已经在变性聚丙烯酰胺凝胶中积极标记和分离。样本在饱和前每两分半钟收集一次，以建立时间过程。凝胶在为期五天的展览后被染色、干燥和筛选。在成功的实验中，图片演示了根据标准模式分配的八个波段。然而，根据应变和实验条件，单个频段的强变程度可能变化很大。每个波段对应于一个翻译产品。数据表明，突变菌株能够线粒体蛋白合成，因为所有出现在野生类型的产品都可见于这种突变体中。然而，波段的特性不同于野生类型，这意味着Aim23的删除会影响线粒体基因表达。库马西酿造的蓝色污渍，并作为负载控制。它们的数据结果可以量化，使用图像 G 或图像泉软件识别菌株或实验条件之间的差异。对于这些，与每个产品对应的信号比率与总信号进行计算。平均值和标准偏差至少在三个独立实验中计算。在去年的实验中，对合成蛋白的动能进行了研究。样品在标签反应被冷甲氨酸和博里霉素停止后，在指示的时间点采集。这种控制对于估计产品的稳定性是必要的。免疫染色与抗波林一个抗体是一个加载控制。我们描述了这种方法，它经常用于研究酵母线粒体中的蛋白质生物合成。此协议相对简单且操作速度较快。同时，它允许获得关于四个酵母线粒体mRNA的转化率的宝贵信息，这是非常有利的。然而，它有几个限制，需要额外的实验，并在于这种州际水平的蛋白质和mRNA。它可以在线粒体翻译系统中提供有关这些功能的信息，但应使用更先进的技术来发现该机制。

Research

JoVE Journal

Biochemistry

Labelling and Visualization of Mitochondrial Genome Expression Products in Baker's Yeast Saccharomyces cerevisiae

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

核糖体剖面对芽殖酵母中翻译的全基因组定量

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

科研

生物化学

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液色谱结合串联质谱法对糖皮质酶细胞脂质的定量分析

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes

格拉菲克斯用于检测 糖精 的瞬态相互作用器

Pre-mRNA splicing is a very dynamic process that involves many molecular rearrangements of the spliceosome subcomplexes during assembly, RNA processing, ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

使用液体色谱加上串联质谱法的 糖核 酸菌的定量元细胞学

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

Analysis of Neutral Lipid Synthesis in Saccharomyces cerevisiae by Metabolic Labeling and Thin Layer Chromatography

通过代谢标记和薄层色谱分析 糖浆 中中性脂质合成

Neutral lipids (NLs) are a class of hydrophobic, chargeless biomolecules that play key roles in energy and lipid homeostasis. NLs are synthesized de ...

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

Preparation and Cryo-FIB micromachining of Saccharomyces cerevisiae for Cryo-Electron Tomography

冷冻电子断层扫描 的糖核 酸的准备和冷冻-FIB微加工

Today, cryo-electron tomography (cryo-ET) is the only technique that can provide near-atomic resolution structural data on macromolecular complexes in ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

萌芽酵母酿酒酵母中软骨蛋白定位的评估

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

下拉测定与细菌细胞共表达相结合，作为测试具有挑战性的蛋白质-蛋白质相互作用的省时工具

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

High-Throughput Quantification of the Synthesis and Distribution of mtDNA

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution

High-Throughput Image-Based Quantification of Mitochondrial DNA Synthesis and Distribution (Video) | JoVE 

基于图像的高通量线粒体DNA合成和分布定量

The vast majority of cellular processes require a continuous supply of energy, the most common carrier of which is the ATP molecule. Eukaryotic cells ...

Isolation of Mitochondrial Contact Sites

An Improved Method to Isolate Mitochondrial Contact Sites

Unveiling Mitochondrial Contact Sites and Architectural Insights (Video) | JoVE

一种分离线粒体接触位点的改进方法

Mitochondria are present in virtually all eukaryotic cells and perform essential functions that go far beyond energy production, for instance, the ...