这项工作得到了FAPESP赠款（15/06477-9）的支持。

1. 酵母总提取物制备
<ol><li>用适当的氨基酸或核基，将酵母细胞与TAP标签9 融合成1 L YNB-胶质（酵母氮基补充2%m/v葡萄糖）的拼接因子之一， 在这种情况下，腺苷（20 μg/mL）、柳辛（30微克/mL）、色氨酸（30微克/mL），最高可达OD600 = 1.0。</li>
	<li>在 4 °C 下以 17，000 x g 的 10 分钟在 3 个 500 mL 离心机瓶中离心，从 1 L 培养物中收集细胞，然后用 10 mL 冷无菌水洗两次。</li>
	<li>将收集的酵母细胞重新用于冷缓冲器 A 的 1/10 细胞体积 （10 mmol L-1 HEPES pH = 7.9， 1.5 mmol L-1 MgCl2， 50 mmol L-1 KCl， 5% v/v 甘油， 0.5 mmol L-1 DTT， EDTA 无蛋白酶抑制剂鸡尾酒）.。</li>
	<li>将小滴细胞悬浮液氮冷冻。小滴可以通过管道重用电池溶液和直接将 50 μL 滴入液氮中获得。 警告:液氮可能会在与皮肤或眼睛接触时造成伤害。使用拨配的安全设备进行处理。 注:协议可以在此处暂停。电池悬浮液的冻结滴可以存储在 -80 °C 下。</li>
	<li>准备酵母细胞解解，在球磨机设备中研磨，以20 Hz/s为6个周期，持续3分钟。 注:每个周期后，将容器浸入液氮中，以避免熔化。协议可以在此处暂停。冷冻提取物可储存在 -80 °C 下。 酵母拼接提取物也可以使用同质化物解解球体10，或使用迫击炮和害虫11，12。</li>
	<li>将含有这些提取物的管子在室温下放入水中，偶尔摇晃，从而融化提取物。</li>
	<li>离心机提取物在 45，000 x g 1 h 在 4 °C.</li>
	<li>通过BCA方法13量化清除超高纳特的蛋白质含量。</li>
	<li>准备提取物的无名小品，快速将其冷冻在液氮中，并储存在 -80 °C 下。 注:协议可以在此处暂停。</li>
</ol>2. 甘油梯度制备
<ol><li>在缓冲区 A 中准备两个甘油溶液，一个包含 10% v/v，另一个包含 30% v/v 甘油。</li>
	<li>在 30% v/v 甘油溶液中将交叉链接剂谷胱甘肽添加到 0.1% v/v，并混合以实现均质化。 注意事项:使用适当的安全设备处理烟罩内的谷胱甘肽。</li>
	<li>在 12 mL 离心机管（14 x 89 mm）底部加入 6 mL 的冷 10% v/v 甘油溶液。</li>
	<li>加入6 mL的冷30%v/v甘油溶液，辅以谷胱甘肽，将注射器连接到长针上，在管底提供梯度主设备，就在10%v/v甘油溶液下面。 注意:另外，10% v/v 甘油溶液可以在 30% v/v 甘油/谷胱甘肽溶液的顶部小心地管道。</li>
	<li>使用梯度主设备生成连续密度梯度。 注:在梯度主设备中，管子被放置在一个适当的机架中，并根据制造商关于确定参数（时间/角度/速度）的建议进行短暂旋转。在这项工作中，我们使用: 2:25 分钟/81.5°/11 rpm。</li>
	<li>在添加细胞提取物之前，小心地在顶部添加 200 μL 的 7% v/v 甘油/缓冲垫。 注:垫子的甘油浓度必须低于用于创建线性梯度的浓度较低的甘油溶液。</li>
</ol>3. 提取离心机
<ol><li>将大约 2 毫克的总蛋白质加载到每个 12 mL 线性甘油梯度 10%至 30% 谷胱甘肽的顶部，并配有垫子。</li>
	<li>将管子放在预冷却的回转桶转子中。 注意:轻轻处理管子，以避免在超中心处理前混合。</li>
	<li>离心机在 194，000 x g 为 16 h 在 4 °C.</li>
	<li>Aliquot 每个 12 mL 管在 24 500 μL 分数使用适应的 Econosystem 或小心管道。 注:经过改造的 EconoSystem 由渗透泵、紫外线探测器和连接到管穿孔装置的分数收集器组成。沉降剖面可以通过测量280纳米的吸收度来监测。</li>
	<li>使用 40% v/v 甘油溶液通过渗透泵将甘油 10%+30% 梯度从管子推至分数收集器。 注:协议可以在此处暂停。将分数存储在 -80 °C 下，直到使用。</li>
	<li>将每个分数的 50 μL 直接加载到点斑或槽斑点上的硝基纤维素膜上。使用抗体对CBP（1:6，000，检测TAP标签的CBP部分）和与马萝卜过氧化酶（1:15，000）结合的抗兔IgG作为二级抗体，通过免疫膨胀检测蛋白质。</li>
</ol>

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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure,+function+and+regulation+of+spliceosomal+RNA+helicases.">Structure, function and regulation of spliceosomal RNA helicases.</a> Current Opinion in Cell Biology. 24, 431-438 (2012).</li><li>Kastner, 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=GraFix:+sample+preparation+for+single-particle+electron+cryomicroscopy.">GraFix: sample preparation for single-particle electron cryomicroscopy.</a> Nature Methods. 5 (1), 53-55 (2008).</li><li>Stark, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Grafix:+stabilization+of+fragile+macromolecular+complexes+for+single+particle+cryo-EM.">Grafix: stabilization of fragile macromolecular complexes for single particle cryo-EM.</a> Methods in Enzymology. 481, 109-126 (2010).</li><li>Yan, C., Wan, R., Bai, R., Huang, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+yeast+activated+spliceosome+at+3.5+&#197;+resolution.">Structure of a yeast activated spliceosome at 3.5 &#197; resolution.</a> Science. 353 (6302), 895-904 (2016).</li><li>Ohi, M. D., 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=Proteomics+analysis+revelas+stable+multiprotein+complexes+in+both+fission+and+budding+yeasts+containing+Myb-related+Cdc5p/Cef1p,+novel+pre-mRNA+splicing+factors,+and+snRNAs.">Proteomics analysis revelas stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.</a> Molecular and Cellular Biology. 22 (7), 2011-2024 (2002).</li><li>Lourenco, R. F., Leme, A. F. P., Oliveira, C. 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作者没有利益冲突。

蛋白质蛋白和核糖核酸-蛋白质相互作用可以通过交联剂稳定下来。由此产生的复合物必须稳定，能够承受甘油梯度的超中心化。此外，缓冲条件应允许交互，但必须足够严格，以避免非特定的绑定。在下面显示的实验中，我们使用了已经为体外拼接反应15建立的缓冲液。
应针对分析的复合物优化离心的速度和时间。我们测试了不同的沉积条件，在 94，000 x <e...

拼接是一个高度动态的过程，需要以协调的方式约束和释放多种因素。这些拼接因子包括RNA结合蛋白、ATPases、螺旋酶、蛋白激酶和磷酸盐、无用性韧带等1、2、3:为了让分子重新排列发生，其中一些因素与拼接体子复杂性非常短暂地结合，使得这些RNP中间复合物的隔离和识别极具挑战性。
在这里， 我们使用Grafix方法4，5来稳定酵母拼接因子Cwc24与B行为复合物6的相互作用，以便识别与该子复合物相关的其他因素，并确定是否 泛素韧带Prp19在Cwc24的结合或释放到19（NTC）复合物和激活前内电的5'端和第一次转录反应中起任何作用地方。使大分子沿着甘油梯度使大分子接触到交叉链接器日益集中的优点是它避免了复合物交叉链路4、5，从而避免了聚合物的形成。
这种方法被用作蛋白质同位素沉淀和拉下测定的补充，尽管允许对大型复合物进行隔离，但对于在大型动态复合物7、8中维持瞬态相互作用可能并不可靠。在甘油梯度中使用固定试剂可以稳定这些因素的结合，从而确认特定蛋白质与拼接亚复合物的相互作用。由于所选的交联器在化学上不可逆转，因此在梯度离心后，通过点斑对恢复分数中的蛋白质进行分析。

<table><tbody><tr><td>Anti-Calmodulin Binding Protein Epitope</td><td>Millipore</td><td>07-482</td><td></td></tr><tr><td>ECL anti-Rabbit IgG</td><td>GE Healthcare</td><td>NA934</td><td></td></tr><tr><td>EconoSystem</td><td>Bio-Rad</td><td>1-800-424-6723</td><td>Parts of the EconoSystem used: peristaltic pump, the UV detector and the fraction collector</td></tr><tr><td>EDTA-free Protease Inhibitor Cocktail</td><td>Roche</td><td>11873580001</td><td></td></tr><tr><td>Fraction Recovery System</td><td>Beckman Coulter</td><td>270-331580</td><td>Tube-perforating device that was connected to the parts of the EconoSystem</td></tr><tr><td>Gradient Master Model 107ip</td><td>Biocomp</td><td>107-201M</td><td></td></tr><tr><td>Mixer Mill MM 200</td><td>Retsch</td><td>207460001</td><td>Ball Mill device</td></tr><tr><td>Rotor F12-6x500Lex</td><td>Thermo Scientific</td><td>096-062375</td><td></td></tr><tr><td>Sorvall RC 6 Plus Centrifuge</td><td>Thermo Scientific</td><td>36-101-0816</td><td></td></tr><tr><td>Swinging Bucket Rotor P40ST</td><td>Hitachi</td><td></td><td></td></tr><tr><td>Ultracentrifuge CP 80 NX</td><td>Hitachi</td><td>901069</td><td></td></tr><tr><td>Ultra-Clear Centrifuge Tubes (14 x 89 mm)</td><td>Beckman Coulter</td><td>344059</td><td></td></tr></tbody></table>

utilization of grafix for the detection of transient interactors of saccharomyces cerevisiae spliceosome subcomplexes

mRNA 前拼接是一个非常动态的过程，在组装、RNA 处理和复杂组件的释放过程中涉及许多拼接体子复合物的分子重新排列。甘油梯度离心已用于分离蛋白质或RNP（核蛋白）复合物，用于功能和结构研究。在这里，我们描述了Grafix（梯度固定）的利用，这是首次开发用于净化和稳定单粒子低温电子显微镜的大分子复合物，以识别暂时与拼接体复合物结合的拼接因子之间的相互作用。此方法基于将样品离心到固定试剂的浓度增加，以稳定复合物。在甘油梯度上装载酵母总提取物离心后，通过点斑分析回收的分数，以识别拼接体子复合物并确定单个拼接因子的存在。

为了分析 Cwc24-TAP 的沉积剖面，并确定 Grafix 方法是否有效稳定其与拼接亚复杂物的结合，我们通过甘油梯度的离心分离了表达 Cwc24-TAP 的细胞的总酵母提取物，无论是否存在谷胱甘肽作为交联剂。然后，通过带抗体对 TAP 标签的 CBP 部分进行带抗体的插槽斑点分析 24 个 500 μL 分数的样本。结果显示，在没有交联器的情况下，Cwc24集中在分数5和13之间（图1B），相对应于约80至20...

Watch this Scientific Journal Video about Utilization of Grafix for the Detection of Transient Interactors of Saccharomyces cerevisiae Spliceosome Subcomplexes at JoVE.com

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

在这里，我们描述了Grafix（梯度固定）的利用，这是一种在交行器面前的甘油梯度离心，用于识别暂时与拼接体复合物结合的拼接因子之间的相互作用。

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, ...

utilization-grafix-for-detection-transient-interactors-saccharomyces

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

3.5K 次观看.  University of Sao Paulo. 在这里，我们描述了Grafix（梯度固定）的利用，这是一种在交行器面前的甘油梯度离心，用于识别暂时与拼接体复合物结合的拼接因子之间的相互作用。

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

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

科研

遗传学

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 ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

生物学

ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. Whether disease-related or functionally selected, many of these mutations have been studied using growth assays in the model organism Saccharomyces cerevisiae (yeast). The splicing-specific copper growth assay, or ACT1-CUP1 assay, provides a comprehensive analysis of mutation at the phenotypic level. The ACT1-CUP1 assay utilizes reporters that confer copper tolerance when correctly spliced. Thus, in the presence of copper, changes in yeast viability correlate to changes in mRNA production through splicing. In a typical experiment, the yeast spliceosome is challenged with different non-consensus splicing reporters and the splicing factor mutation of interest to detect any synergetic or antithetical impact on splicing. Here a full description of copper plate preparation, plating of yeast cells, and data evaluation are given. A selection of complimentary experiments is described, highlighting the versatility of the ACT1-CUP1 reporters. The ACT1-CUP1 assay is a handy tool in the splicing toolbox thanks to the direct read-out of mutational effect(s) and the comparative possibilities from the continuing use in the field.

The ACT1-CUP1 assay, a copper growth assay, provides a quick readout of precursor messenger RNA (pre-mRNA) splicing and the impact mutant splicing factors have on spliceosomal function. This study provides a protocol and highlights the customization possible to address the splicing question of interest.

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

Mutations introduced in the spliceosome or its substrate have significantly contributed to our understanding of the intricacies of spliceosomal function. ...

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by homologous recombination (HR). In addition, HR represents an important mechanism of replication fork rescue following stalling or collapse. The regulation of the many reversible and irreversible steps of this complex pathway promotes its fidelity. The physical analysis of the recombination intermediates formed during HR enables the characterization of these controls by various nucleoprotein factors and their interactors. Though there are well-established methods to assay specific events and intermediates in the recombination pathway, the detection of D-loop formation and extension, two critical steps in this pathway, has proved challenging until recently. Here, efficient methods for detecting key events in the HR pathway, namely DNA double-stranded break formation, D-loop formation, D-loop extension, and the formation of products via break-induced replication (BIR) in Saccharomyces cerevisiae are described. These assays detect their relevant recombination intermediates and products with high sensitivity and are independent of cellular viability. The detection of D-loops, D-loop extension, and the BIR product is based on proximity ligation. Together, these assays allow for the study of the kinetics of HR at the population level to finely address the functions of HR proteins and regulators at significant steps in the pathway.

Detection of Homologous Recombination Intermediates via Proximity Ligation and Quantitative PCR in Saccharomyces cerevisiae

The D-loop capture (DLC) and D-loop extension (DLE) assays utilize the principle of proximity ligation together with quantitative PCR to quantify D-loop formation, D-loop extension, and product formation at the site of an inducible double-stranded break in Saccharomyces cerevisiae.

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

DNA damage, including DNA double-stranded breaks and inter-strand cross-links, incurred during the S and G2 phases of the cell cycle can be repaired by ...

Complementation of Splicing Activity by a Galectin-3 - U1 snRNP Complex on Beads

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of galectin-3 into the splicing pathway is addressed in this paper. Sedimentation of HeLa cell nuclear extracts on 12%-32% glycerol gradients yields fractions enriched in an endogenous ~10S particle that contains galectin-3 and U1 snRNP. We now describe a protocol to deplete nuclear extracts of U1 snRNP with concomitant loss of splicing activity. Splicing activity in the U1-depleted extract can be reconstituted by the galectin-3 - U1 snRNP particle trapped on agarose beads covalently coupled with anti-galectin-3 antibodies. The results indicate that the galectin-3 - U1 snRNP - pre-mRNA ternary complex is a functional E complex leading to intermediates and products of the splicing reaction and that galectin-3 enters the splicing pathway through its association with U1 snRNP. The scheme of using complexes affinity- or immuno-selected on beads to reconstitute splicing activity in extracts depleted of a specific splicing factor may be generally applicable to other systems.

This article describes the experimental procedures for (a) depletion of U1 snRNP from nuclear extracts, with concomitant loss of splicing activity; and (b) reconstitution of splicing activity in the U1-depleted extract by galectin-3 - U1 snRNP particles bound to beads covalently coupled with anti-galectin-3 antibodies.

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

Classic depletion-reconstitution experiments indicate that galectin-3 is a required splicing factor in nuclear extracts. The mechanism of incorporation of ...

生物化学

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

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

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

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

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 ...

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. ...

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of the cells. These adjustments are critical to eukaryotic cell survivability and &#39;damage control&#39; during fluctuating nutrient and salinity levels, temperature, and various chemical and radiation stresses. Due to the highly dynamic nature of the RNA-level responses, and the instability of many of the RNA:RNA and RNA:protein intermediates, obtaining a meaningful snapshot of the cytoplasmic RNA state is only possible with a limited number of methods. Transcriptome-wide, RNA-seq-based ribosome profiling-type experiments are among the most informative sources of data for the control of translation. However, absence of a uniform RNA and RNA:protein intermediate stabilization can lead to different biases, particularly in the fast-paced cellular response pathways. In this article, we provide a detailed protocol of rapid fixation applicable to eukaryotic cells of different permeability, to aid in RNA and RNA:protein intermediate stabilization. We further provide examples of isolation of the stabilized RNA:protein complexes based on their co-sedimentation with ribosomal and poly(ribo)somal fractions. The separated stabilized material can be subsequently used as part of ribosome profiling-type experiments, such as in Translation Complex Profile sequencing (TCP-seq) approach and its derivatives. Versatility of the TCP-seq-style methods has now been demonstrated by the applications in a variety of organisms and cell types. The stabilized complexes can also be additionally affinity-purified and imaged using electron microscopy, separated into different poly(ribo)somal fractions and subjected to RNA sequencing, owing to the ease of the crosslink reversal. Therefore, methods based on snap-chilling and formaldehyde fixation, followed by the sedimentation-based or other type of RNA:protein complex enrichment, can be of particular interest in investigating finer details of rapid RNA:protein complex dynamics in live cells.

Rapid In Vivo Fixation and Isolation of Translational Complexes from Eukaryotic Cells

We present a technique to rapidly stabilize translational (protein biosynthesis) complexes with formaldehyde crosslinking in live yeast and mammalian cells. The approach enables dissecting transient intermediates and dynamic RNA:protein interactions. The crosslinked complexes can be used in multiple downstream applications such as in deep sequencing-based profiling methods, microscopy, and mass-spectrometry.

从真核细胞 中快速体内 固定和分离转化复合物

Rapid responses involving fast redistribution of messenger(m)RNA and alterations of mRNA translation are pertinent to ongoing homeostatic adjustments of ...

Global Identification of Co-Translational Interaction Networks by Selective Ribosome Profiling

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded cellular environment. Ribosomes provide the platform for spatially and kinetically controlled binding of membrane-targeting factors, modifying enzymes, and folding chaperones. Even the assembly into high-order oligomeric complexes, as well as protein-protein network formation steps, were recently discovered to be coordinated with synthesis.
Here, we describe Selective Ribosome Profiling, a method developed to capture co-translational interactions in vivo. We will detail the various affinity purification steps required for capturing ribosome-nascent-chain complexes together with co-translational interactors, as well as the mRNA extraction, size exclusion, reverse transcription, deep-sequencing, and big-data analysis steps, required to decipher co-translational interactions in near-codon resolution.

Co-translational interactions play a crucial role in nascent-chain modifications, targeting, folding, and assembly pathways. Here, we describe Selective Ribosome Profiling, a method for in vivo, direct analysis of these interactions in the model eukaryote Saccharomyces cerevisiae.

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

In recent years, it has become evident that ribosomes not only decode our mRNA but also guide the emergence of the polypeptide chain into the crowded ...

格拉菲克斯用于检测糖精的瞬态相互作用器

摘要

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此视频中的章节

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

一种在裂变酵母中的深度固序辅助自发抑制屏

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

可视化和分析的mRNA分子，使用荧光

化学遗传相互作用的快速鉴定

从真核细胞中快速体内固定和分离转化复合物

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

格拉菲克斯用于检测 糖精 的瞬态相互作用器

摘要

探索更多视频

此视频中的章节

单分子荧光原位杂交 (smFISH) 在发芽酵母营养生长和减数分裂中的分析

一种在裂变酵母中的深度固序辅助自发抑制屏

鉴定与定量蛋白质组学在酿酒酵母中的蛋白质复合物

ACT1-CUP1测定确定出芽酵母中剪接体突变体的底物特异性敏感性

通过邻近连接和定量PCR检测酿酒酵母同源重组中间体

半乳糖凝集素-3 - U1 snRNP复合物在珠子上的剪接活性的补充

可视化和分析的mRNA分子，使用荧光

化学遗传相互作用的快速鉴定

从真核细胞 中快速体内 固定和分离转化复合物

通过选择性核糖体分析对共翻译相互作用网络进行全局鉴定

格拉菲克斯用于检测糖精的瞬态相互作用器

从真核细胞中快速体内固定和分离转化复合物