Name: determination of s-phase duration using 5-ethynyl-2'-deoxyuridine incorporation in saccharomyces cerevisiae
Uploaded: 2022-10-21T13:30:00
Description: Watch this Scientific Journal Video about Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae at JoVE.com

作者希望感谢国家研究局（ANR）和癌症研究协会（ARC）向JDD提供博士奖学金，并感谢国家研究机构（ANR）的财政支持（赠款ANR-18-CE12-0018-01）。细胞术和显微镜检查在蒙彼利埃MRI BioCampus成像设施中进行。

1. 酿酒酵母 细胞培养
注意：使用的酵母菌株列于表1中
注意：可以通过不同的方式监控 S 阶段持续时间。根据要解决的问题，细胞可以在G1 停滞后异步或同步生长。
<ol><li>来自异步生长的 酿酒酵母 细胞 注意：该方法允许确定异步生长的细胞群中S期细胞的百分比。通过确定倍增时间，可以推断S期（和其...

<ol>
	<li>Bell, S. P., 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=Chromosome+duplication+in+Saccharomyces+cerevisiae.">Chromosome duplication in Saccharomyces cerevisiae.</a> Genetics. 203 (3), 1027-1067 (2016).</li><li>Kitamura, E., Blow, J. J., Tanaka, T. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+reveals+replication+of+individual+replicons+in+eukaryotic+replication+factories.">Live-cell imaging reveals replication of individual replicons in eukaryotic replication factories.</a> Cell. 125 (7), 1297-1308 (2006).</li><li>Marston, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome+segregation+in+budding+yeast:+Sister+chromatid+cohesion+and+related+mechanisms.">Chromosome segregation in budding yeast: Sister chromatid cohesion and related mechanisms.</a> Genetics. 196 (1), 31-63 (2014).</li><li>Lengronne, A., Schwob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+CDK+inhibitor+Sic1+prevents+genomic+instability+by+promoting+replication+origin+licensing+in+late+G1.">The yeast CDK inhibitor Sic1 prevents genomic instability by promoting replication origin licensing in late G1.</a> Molecular Cell. 9 (5), 1067-1078 (2002).</li><li>Tanaka, S., Diffley, J. F. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deregulated+G+1-cyclin+expression+induces+genomic+instability+by+preventing+efficient+pre-RC+formation.">Deregulated G 1-cyclin expression induces genomic instability by preventing efficient pre-RC formation.</a> Genes &amp; Development. 16 (20), 2639-2649 (2002).</li><li>Teixeira, L. 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=Cyclin+E+deregulation+promotes+loss+of+specific+genomic+regions.">Cyclin E deregulation promotes loss of specific genomic regions.</a> Current Biology. 25 (10), 1327-1333 (2015).</li><li>Slater, M. L., Sharrow, S. O., Gart, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+cycle+of+Saccharomyces+cerevisiae+in+populations+growing+at+different+rates.">Cell cycle of Saccharomyces cerevisiae in populations growing at different rates.</a> Proceedings of the National Academy of Sciences of the United States of America. 74 (9), 3850-3854 (1977).</li><li>McNeil, J. B., Friesen, 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=Expression+of+the+herpes+simplex+virus+thymidine+kinase+gene+in+Saccharomyces+cerevisiae.">Expression of the herpes simplex virus thymidine kinase gene in Saccharomyces cerevisiae.</a> Molecular and General Genetics. 184 (3), 386-393 (1981).</li><li>Vernis, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+an+efficient+thymidine+salvage+pathway+in+Saccharomyces+cerevisiae.">Reconstitution of an efficient thymidine salvage pathway in Saccharomyces cerevisiae.</a> Nucleic Acids Research. 31 (19), 120 (2003).</li><li>Salic, A., Mitchison, T. 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+chemical+method+for+fast+and+sensitive+detection+of+DNA+synthesis+in+vivo.">A chemical method for fast and sensitive detection of DNA synthesis in vivo.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2415-2420 (2008).</li><li>Bruschi, C. V., McMillan, J. N., Coglievina, M., Esposito, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genomic+instability+of+yeast+cdc6-1/cdc6-1+mutants+involves+chromosome+structure+and+recombination.">The genomic instability of yeast cdc6-1/cdc6-1 mutants involves chromosome structure and recombination.</a> Molecular and General Genetics. 249 (1), 8-18 (1995).</li><li>Feng, L., Wang, B., Wu, L., Jong, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+control+of+Mcm5+interaction+with+chromatin+in+cdc6-1+mutated+in+CDC-NTP+motif.">Loss control of Mcm5 interaction with chromatin in cdc6-1 mutated in CDC-NTP motif.</a> DNA and Cell Biology. 19 (7), 447-457 (2000).</li><li>Saner, 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=Stochastic+association+of+neighboring+replicons+creates+replication+factories+in+budding+yeast.">Stochastic association of neighboring replicons creates replication factories in budding yeast.</a> Journal of Cell Biology. 202 (7), 1001-1012 (2013).</li><li>Pasero, P., Braguglia, D., Gasser, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ORC-dependent+and+origin-specific+initiation+of+DNA+replication+at+defined+foci+in+isolated+yeast+nuclei.">ORC-dependent and origin-specific initiation of DNA replication at defined foci in isolated yeast nuclei.</a> Genes &amp; Development. 11 (12), 1504-1518 (1997).</li><li>Lengronne, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+S+phase+progression+globally+and+locally+using+BrdU+incorporation+in+TK++yeast+strains.">Monitoring S phase progression globally and locally using BrdU incorporation in TK+ yeast strains.</a> Nucleic Acids Research. 29 (7), 1433-1442 (2001).</li><li>Magiera, M. M., Gueydon, E., Schwob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+replication+and+spindle+checkpoints+cooperate+during+S+phase+to+delay+mitosis+and+preserve+genome+integrity.">DNA replication and spindle checkpoints cooperate during S phase to delay mitosis and preserve genome integrity.</a> The Journal of Cell Biology. 204 (2), 165-175 (2014).</li><li>Talarek, N., Petit, J., Gueydon, E., Schwob, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EdU+incorporation+for+FACS+and+microscopy+analysis+of+DNA+replication+in+budding+yeast.">EdU incorporation for FACS and microscopy analysis of DNA replication in budding yeast.</a> Methods in Molecular Biology. 1300, 105-112 (2015).</li><li>Ivanova, T., 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=Budding+yeast+complete+DNA+synthesis+after+chromosome+segregation+begins.">Budding yeast complete DNA synthesis after chromosome segregation begins.</a> Nature Communications. 11 (1), 2267 (2020).</li></ol>

酵母是细胞周期研究的主要模式生物，但其S期的表征长期以来一直受到阻碍，因为它无法结合外源性核苷，如BrdU，其用作DNA复制的示踪剂。为酵母配备高表达的单纯疱疹胸苷激酶（TK）并添加人核苷转运蛋白（hENT）在很大程度上解决了这个问题15，16。与抗BrdU抗体不同，EdU比BrdU更通用，因为它使用小荧光叠氮化物和Click化学进行检测，适用于透化的酵?...

通过有丝分裂的基因组稳定性是通过将一组完整且相等的染色体传递给两个产生的细胞后代来确保的。这依赖于在细胞周期的每个阶段的给定时间内发生的一系列事件的准确完成。在 G 1 中，复制来源是在招募几个许可因素（包括 Cdc61）后获得许可的。在S阶段，全基因组复制从多个活跃的复制起源开始，并由复制机器执行，这些机器聚集在显微镜下可见的灶中，称为复制工厂2。在M期，重复的姐妹染色单体附着并双向定向在有丝分裂纺锤体上，以允许它们分离到有丝分裂细胞3的相对极点。每个阶段的调控、正确完成和持续时间是确保基因组稳定性的关键。事实上，过早退出这些阶段中的任何一个都会导致基因组不稳定。例如，由出芽酵母CDK抑制剂Sic1缺失或G 1细胞周期蛋白过表达诱导的较短的G1将改变随后的S期4，5，6。因此，这些失调，无论是否与复制应激相关，都会导致染色体断裂、重排和错误分离4，5，6。因此，监测S期的持续时间以及更广泛地说，监测细胞周期其他阶段的持续时间对于识别不同突变体和不同应激条件下发生的缺陷可能至关重要。
测量细胞周期阶段持续时间的传统方法包括简单的DNA含量流式细胞术（图1A），并依赖于拟合算法（大多数细胞术软件中可用），用于将群体从1C和2C峰分离为G1，S和G2 + M相级分。然后将分数乘以总体倍增时间7。然而，该方法仅给出估计值，需要在给定级分内均匀的细胞大小分布，并且不适用于同步培养。为了研究哺乳动物细胞中的S期持续时间，已经开发并广泛使用了几种胸苷类似物，包括EdU。它们从细胞外培养基中摄取并被胸苷激酶（以下简称TK）磷酸化，使它们可用于DNA聚合酶，以将它们掺入DNA合成位点（复制，重组，修复）。为了绕过 酿酒 酵母细胞中TK基因的缺失，酵母菌株已被设计为允许单纯疱疹病毒TK8 和人平衡核苷转运蛋白（hENT1）9的稳定和组成性表达。一旦掺入 DNA 中，EdU 就会通过 选择性 Click 反应进行检测，该反应将其炔烃部分化学偶联到叠氮化物修饰的荧光染料10。
本文提供了两种优化的综合协议，用于使用 EdU 对异步和同步 TK-hENT1 工程细胞进行脉冲标记，以便通过显微镜和流式细胞术精确可视化和测量 DNA 复制持续时间和动态，以及细胞周期其他阶段的持续时间，在单细胞和群体水平上具有高空间和时间分辨率。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>&#945;-factor&#160;</td>
			<td>Genescript</td>
			<td>RP01002</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Euromedex</td>
			<td>04-100--812-E</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper sulfate</td>
			<td>Sigma</td>
			<td>C1297</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-sulfo-Cyanine5 azide (Cy5 azide)</td>
			<td>Interchim</td>
			<td>FP-JV6320</td>
			<td>Alternative to Alexa647-Azide</td>
		</tr>
		<tr>
			<td>Dy-530 azide</td>
			<td>Dyomics</td>
			<td>&#160;530-10</td>
			<td></td>
		</tr>
		<tr>
			<td>EdU (5-ethynyl-2&#8217;-deoxyuridine)</td>
			<td>Carbosynth</td>
			<td>NE08701</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>Carlo Erba reagents</td>
			<td>P013A10D16</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>L- ascorbic acid</td>
			<td>Sigma</td>
			<td>A4544</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide</td>
			<td>Sigma</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Euromedex</td>
			<td>EU0090</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase</td>
			<td>SIGMA</td>
			<td>R5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sytox Green</td>
			<td>Invitrogen</td>
			<td>S-7020</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td>OLS</td>
			<td>CASY</td>
			<td></td>
		</tr>
		<tr>
			<td>Flow cytometer</td>
			<td>Agilent</td>
			<td>NovoSampler Pro</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaking incubator</td>
			<td>Infors</td>
			<td>444-4230</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Shaking water bath</td>
			<td>Julabo</td>
			<td>SW22</td>
			<td>or equivalent</td>
		</tr>
		<tr>
			<td>Sonicator</td>
			<td>Sonics</td>
			<td>Vibra cell</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-field microscopy</td>
			<td>Leica</td>
			<td>THUNDER Imager</td>
			<td>or equivalent</td>
		</tr>
	</tbody>
</table>

determination of s-phase duration using 5-ethynyl-2'-deoxyuridine incorporation in saccharomyces cerevisiae

真核DNA复制是一个高度调控的过程，可确保在染色体分离之前正确复制细胞的遗传蓝图。由于DNA合成缺陷是染色体重排的基础，因此监测DNA复制已成为了解基因组不稳定基础的关键。 酿酒酵母 是研究细胞周期调控的经典模型，但关键的DNA复制参数，如S期或S期持续时间的细胞比例，仍然难以确定。该协议在工程TK-hENT1酵母细胞中使用5-乙炔基-2'-脱氧尿苷（EdU）（胸苷类似物）的短且无毒脉冲，然后通过Click反应进行检测，以允许通过显微镜和流式细胞术在单细胞和群体水平上以高空间和时间分辨率可视化和定量DNA复制。该方法可以识别酵母突变体的S期和细胞周期进程中以前被忽视的缺陷，从而允许表征对确保基因组稳定性至关重要的新参与者。

为了确定S期持续时间，更广泛地说，G 1和G2 + M的持续时间（方案步骤1.1），酿酒酵母W303野生型细胞（WT，表1）在SC培养基中异步生长7小时。每小时监测一次细胞浓度以确定倍增时间（图2B）。在这些生长条件下，计算的倍增时间为25°C下的120分钟±13分钟（表2）。当细胞处于指数期（2× 106-5×106细胞/ mL）时，?...

Watch this Scientific Journal Video about Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae at JoVE.com

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

我们描述了两种互补的方案，以使用EdU（一种胸苷类似物）准确确定 酿酒酵母 的S期持续时间，该EdU在 体内 掺入并通过显微镜和流式细胞术使用Click化学进行检测。它可以轻松表征突变体中DNA复制的持续时间和被忽视的复制缺陷。

使用酿酒酵母中的5-乙炔基-2'-脱氧尿苷掺入测定S相持续时间

Eukaryotic DNA replication is a highly regulated process that ensures that the genetic blueprint of a cell is correctly duplicated prior to chromosome ...

该协议允许精确确定同步出芽酵母细胞中的S相持续时间。这是一种快速、简单且可重复的方法。此外，它足够灵敏，可以检测经典方案无法检测到的轻度合成缺陷。这种方法对于许多研究出芽酵母细胞周期调节的研究人员很有用。展示该协议的将是高级研究员Nicolas Talarek和实验室的博士生Juan De Dios Barba Tena。在10毫升SC培养基中以低细胞浓度接种酿酒酵母细胞，以30摄氏度的速度进行过夜培养，以每分钟130转的速度进行轨道搅拌。第二天，将细胞稀释在20毫升新鲜SC培养基中，终浓度为每毫升10至6个细胞的两到三倍。在40微升每毫升1毫克α因子稀释在水中。然后在30摄氏度下培养细胞，以每分钟130转的速度进行轨道搅拌，持续一小时。重复此步骤一次。在光学显微镜下可视化细胞以监测G1停滞。如果超过 90% 的单元格显示 shmoo，而其他单元格是圆形的未对接单元格，则继续。将样品在1， 500G下离心三分钟，然后用真空移液管弃去上清液并将细胞重悬于20毫升SC培养基中。重复此步骤一次。每五分钟收集两次一毫升细胞，然后进行EDU标记。释放后 30 分钟加入 400 微升 α 因子。将一毫升细胞培养物转移到含有一微升10毫摩尔EDU的两毫升微量离心管中，通过倒置充分混合。为了在Pi EDU双变量事实上区分EDU阳性细胞和EDU阴性细胞，将另外一毫升细胞培养物转移到含有一微升DMSO的两毫升微量离心管中。在 30 摄氏度下在摇动的水浴中搅拌孵育三到五分钟。通过加入100微升100%乙醇来停止反应。在微量离心机中以10， 000G沉淀细胞两分钟。使用真空移液器除去上清液。将细胞沉淀重悬于500微升70%乙醇中，并通过涡旋充分混合。将样品在室温下以每分钟20次倾斜的速度在变速摇杆上放置至少一小时，以渗透细胞。在微量离心机中以10， 000g沉淀细胞两分钟，并用真空移液管弃去上清液。用500微升10%乙醇和PBS洗涤细胞两次。在微量离心机中以10， 000G沉淀细胞两分钟，并用真空移液管弃去上清液。将沉淀重悬于含有RNase A和蛋白酶K的200微升PBS中，在50摄氏度下孵育一至两个小时，偶尔摇动，或在37摄氏度下孵育过夜。在微量离心机中以10， 000G沉淀细胞两分钟，并用真空移液管弃去上清液。用500微升PBS洗涤细胞。然后沉淀细胞并丢弃上清液，如前所述。将细胞沉淀重悬于含有1%BSA的200微升PBS中。在室温下孵育30分钟。然后沉淀细胞，如前所述弃去上清液，并将沉淀重悬于含有1%BSA的300微升PBS中。将细胞分配到两个1.5毫升微量离心管中。第一个管应包含用于Click反应的200微升细胞培养物，第二个管应包含100微升用于Sytox绿色染色的细胞培养物。然后，如前所述沉淀细胞并丢弃上清液。对于系统毒素绿色染色，将细胞沉淀重悬于100微升PBS中。然后根据细胞浓度，将10至30微升转移到含有300微升Sytox绿色混合物的流式细胞仪管中。以40%至50%的振幅对样品进行两次超声处理两次，持续两秒钟在黑暗中放置，直到在流式细胞仪上处理样品。对于Click反应，用40微升新鲜制备的叠氮化物染料缓冲液重悬细胞沉淀。在室温下在黑暗中孵育60分钟。如前所述沉淀细胞并丢弃上清液。然后用300微升10%乙醇在PBS中洗涤细胞三次。接下来将细胞重悬于PBS中的100微升50微克/毫升碘化丙啶中，并在黑暗中放置10分钟。根据细胞浓度，将10至30微升细胞悬液转移到含有300微升50毫摩尔Tris HCl，pH 7.5的流式细胞仪管中。超声处理两次，每次两秒钟，振幅为 40 到 50。将样品放在黑暗中，直到在细胞仪上处理它们。使用488纳米的激发蓝色激光和530×30带通滤光片读取Sytox绿色样品。使用 488 纳米的激发蓝色激光和 Pi X 轴的 615 x 20 带通滤光片和 640 纳米的红色激发激光器、Y 轴的 660 x 20 带通滤光片在点图上读取巴伐利亚 Pi EDU 样品。在双变异EDU Pi细胞仪分析中观察到三个细胞群。在25摄氏度下，大约27%的细胞群处于G1期，29%处于S期，约44%处于G2加M期。在同步细胞中，S期的持续时间可能约为25分钟，具体取决于Sytox绿色流式细胞仪谱上的DNA含量从1C变为2C的时间。EDU脉冲标记准确确定了S相持续时间。释放15分钟后，检测到一小部分EDU阳性细胞，表明S期开始。通过S期的进展是通过巴伐利亚Pi EDU图中的细胞云向上和向右移动看到的。最后，在释放后35分钟，一小部分细胞为EDU阴性，但DNA量的两倍表明S期结束，细胞处于G2加M期。尽管观察到高度同步，但一些细胞在释放后60分钟完成S期，整个群体的S期在释放后约65分钟完成。保持完美的同步并防止细胞进入第二个S期至关重要。细胞可以用显微镜成像，其中可以监测和量化核DNA复制远见。该技术将有助于识别具有轻度S期缺陷以及细胞周期持续时间缺陷的忽视突变体。

Research

JoVE Journal

Biology

Determination of S-Phase Duration Using 5-Ethynyl-2'-deoxyuridine Incorporation in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Dissection of Saccharomyces Cerevisiae Asci

剖析酿酒酵母 ASCI

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

科研

生物学

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

脂肪酸定量磷酸化刺激酿酒酵母</em

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

Microarray Analysis for Saccharomyces cerevisiae

微阵列分析FOR酿酒酵母</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

可视化和分析的mRNA分子，使用荧光<em&gt;在原位</em&gt;杂交<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

耦合检测监控蛋白复性<em&gt;酿酒酵母（Saccharomyces cerevisiae）</em

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

囊性纤维化跨膜电导调节蛋白表达纯化<em&gt;酿酒酵母</em

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

EPR Monitored Redox Titration of the Cofactors of Saccharomyces cerevisiae Nar1

EPR监控的辅助因子氧化还原滴定<em&gt;酿酒酵母</em&gt; Nar1

Electron Paramagnetic Resonance (EPR) monitored redox titrations are a powerful method to determine the midpoint potential of cofactors in proteins and to ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA的衰变率的测量<em&gt;酿酒酵母</em&gt;使用<em&gt; rpb1-1</em&gt;株

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

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. ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

成长型测定和遗传需求生化确认的蛋白质降解<em&gt;酿酒酵母</em

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...