衰老研究（远方），遗传性疾病基金会（高密度纤维板）和威廉·伍德基金会从美国联邦赠款支持在Duennwald实验室工作。

1。 polyQ蛋白膨胀有毒蛋白在酵母中的表达系统分析建立了一个polyQ蛋白膨胀蛋白所必需的生产在酵母中的毒性7精确的氨基酸序列。这种有毒的polyQ蛋白膨胀蛋白包含一个氨基末端的标志，标签从原始序列的亨廷顿蛋白质，polyQ蛋白地区和羧基端融合荧光蛋白（GFP或CFP资格由17个氨基酸，见图1 A）。表达的蛋白质与polyQ蛋白46 glutamines或更多扩展（例如72和103 glutamines的）生产酵母中的毒性控制下的诱导和相对强GAL1启动7，9时表示。 如前所述，polyQ蛋白在酵母中的毒性是唯一明显的细胞携带的朊蛋白构象的蛋白质Rnq1p [RNQ +]，如酵母菌株W303 9。有毒polyQ蛋白膨胀蛋白ŕecapitulates中央polyQ蛋白在酵母细胞生物学方面的，如polyQ蛋白长度依赖毒性（见下文）和聚集（图1）。值得注意的是，有毒polyQ蛋白膨胀蛋白不杀死酵母细胞立即而不是削弱或逮捕的细胞周期和细胞及试产，从而减缓或抑制酵母片或液体培养（未发表资料）殖民地的增长。 2。对有毒polyQ蛋白膨胀蛋白与酵母细胞的潜在问题 polyQ蛋白扩张，否则有毒蛋白质的酵母细胞中表达，可能不会显示任何增长的缺陷9。这些抑制器polyQ蛋白毒性的遗传性质，不会出现简单的孟德尔遗传突变的基础上，从而可以发生的频率比较高（未公布结果）。我们推测，这些自发的抑制是造成类似[RNQ]在确定polyQ蛋白toxicity.Thes功能不明的朊病毒固化polyQ蛋白毒性的é自发抑制器可危及任何实验，旨在刻画polyQ蛋白毒性或识别和描述polyQ蛋白的毒性修饰符取得圆满成功。 为了避免这些自发抑制频繁发生，请按照下述的预防措施，这已被证明是非常有效的: <ol><li>使用鲜酵母细胞毒性实验。不要长时间储存​​的酵母。经常从冷冻股票检索鲜酵母殖民地。 </li><li>经常监测荧光显微镜有毒polyQ蛋白膨胀蛋白的表达和聚集。 </li><li>保持在镇压开始前任何毒性测量polyQ蛋白的毒性在所有时代的毒性（即含葡萄糖为唯一碳源的选择性培养基）的表达媒体的酵母细胞。 </li><li>每个polyQ蛋白毒性实验中使用的至少有三个独立的转化。&lt;/ LI&gt; </ol> 3。生长实验<ol><li>对于每个实验条件下，接种一个殖民地polyQ蛋白膨胀在3毫升选择性酵母媒体有毒蛋白细胞窝藏葡萄糖作为唯一碳源的酵母从三个独立的转化。 </li><li>这些文化过夜孵育30°C。在这些条件下，细胞不表达polyQ蛋白膨胀蛋白，因为葡萄糖在中期抑制其表达。不要让这些文化长满;保持外径600（600 nm波长的光吸收）低于1隔夜文化。 </li><li>我们经常使用三种不同的化验，以监察表达有毒polyQ蛋白膨胀蛋白的酵母细胞的生长缺陷: </li></ol> 3.1平板上的生长<ol><li>选择性稀释隔夜酵母培养（220转震动成长）600 0.0005 OD（即一个OD600值= 0.5文化1:1000稀释）直径7含葡萄糖。 </li><li>均匀地涂抹上含有葡萄糖为唯一碳源的选择性介质和含半乳糖为唯一碳源的选择性培养基板一板（直径10厘米）50μl各摊薄文化的（CA 700菌落每盘）。 </li><li>在30°C孵育三到四天的板孵化后，每块板的照片和葡萄糖和半乳糖板菌落数菌落计数的数量。在理想的条件下，应该没有或只有很少的殖民地上的半乳糖板时，利用酵母细胞表达一种剧毒polyQ蛋白膨胀蛋白（103Q，图2a）。 </li></ol> 3.2合模分析此法比电镀法上面介绍更多的定量，从而可以揭示polyQ蛋白毒性哪怕是细微的差异与同一板块上的相同的实验。 <ol><li>稀释的overn生长在培养基葡萄糖0.1的外径600的飞行文化。 </li><li>吸取200μL无菌96孔板和这些摊薄文化的准备五个五倍串行无菌水稀释，使用多通道移液器。 </li><li>用青蛙过河（也称为去污剂与8×6针细胞悬液转移）转移到选择性培养基含葡萄糖作为唯一碳源和含半乳糖为唯一碳源的选择性培养基平板板的细胞悬液。 </li><li>允许板晾干后方可孵化30°Ç三到四天。 </li><li>孵化后，每块板的照片（图2b）。 </li></ol> 3.3。监测polyQ蛋白在酵母中的毒性液体培养的生长此协议是最定量这里描述的三个实验（OD600值的数字），甚至可以检测polyQ蛋白的毒性非常细微的差别。上述业主立案法团然而，自发抑制currence，可能产生误导的结果。因此，我建议至少有一个以上所述的两个电镀检测相结合的检测。我们建议使用这些实验BioscreenC仪器。 BioscreenC是仪器自动测量光密度在100孔板酵母培养，而培养与定义搅拌定义的温度。日益增长的酵母细胞，并测量其光密度等方法也可申请。 <ol><li>从最小的媒体含有葡萄糖作为唯一碳源，在3毫升无菌水三次洗的酵母细胞。 </li><li>稀释与半乳糖的外径600 0.1在培养基上生长的过夜培养。 </li><li>填补300μl酵母菌文化的每个井，100井BioscreenC板。 </li><li>打开简易BIOSCREEN实验方案。确定要监视（包括空白和中唯一的样本数量控制），设定温度至30°C，设置实验的长度为3天，设置了测量时间间隔为15分钟，设置过滤器以600海里/布朗，并设置每次测量前15秒震动模式中等强度。 </li><li>的BioscreenC文书和所附的软件会产生在实验过程中所采取的每个数据点的Excel电子表格。 </li><li>准备与Excel的生长曲线，每个样品和比较不同样本（图2c）的增长。 10日前已提供BioscreenC实验产生的数据进行分析的详细描述。 </li></ol> 4。代表结果<img alt="图1" src="/files/ftp_upload/3461/3461fig1.jpg" /> 图1。 polyQ蛋白酵母模型。荧光显微镜一）有毒polyQ蛋白膨胀的蛋白质，B的示意图），显示酵母细胞中表达了一个简短的，无毒的polyQ蛋白扩张蛋白（25Q，左侧面板）和酵母细胞长，有毒polyQ蛋白膨胀蛋白（103Q，右图）。 <img alt="图2" src="/files/ftp_upload/3461/3461fig2.jpg" /> 图2。代表表达polyQ蛋白膨胀蛋白质的酵母细胞的生长实验结果。一）电镀法。约700个酵母细胞伸长平板上，三天在30℃孵育上游面板显示板含葡萄糖培养基，即有毒polyQ蛋白膨胀蛋白（103Q）的表达， 不诱导。较低的面板显示含有半乳糖培养基酵母片，即有毒polyQ蛋白膨胀蛋白表达诱导 。请注意，这里显示实验中，没有发生自发抑制B）合模试验。五串行5倍稀释的酵母细胞，窝藏一个无毒polyQ蛋白蛋白（25Q）或有毒polyQ蛋白膨胀proteiN（103Q）被发现葡萄糖板压制表达蛋白（左侧面板）或半乳糖诱导其表达的板块。板，然后孵育3天30°C。C）BioscreenC实验。酵母细胞中表达一个无毒polyQ蛋白蛋白（25Q）或有毒polyQ蛋白膨胀蛋白（103Q）文化的增长，由监察BioscreenC仪器。在主文本描述的实验条件和分析的BioscreenC实验。

<ol>
	<li>Soto, C., Estrada, L. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+misfolding+and+neurodegeneration.">Protein misfolding and neurodegeneration.</a> Arch. Neurol. 65, 184-189 (2008).</li><li>Ross, C. A., Tabrizi, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington's+disease:+from+molecular+pathogenesis+to+clinical+treatment.">Huntington's disease: from molecular pathogenesis to clinical treatment.</a> Lancet Neurol. 10, 83-98 (2011).</li><li>Zoghbi, H. Y., Orr, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamine+repeats+and+neurodegeneration.">Glutamine repeats and neurodegeneration.</a> Annu. Rev. Neurosci. 23, 217-247 (2000).</li><li>Meriin, A. 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没有利益冲突的声明。

这手稿概述了三种互补的实验方法来衡量polyQ蛋白表达有毒polyQ蛋白膨胀蛋白的酵母细胞减少增长的基础上在模式生物酵母毒性。在酵母中的工作提供了深刻的见解成蛋白质错误折叠的基本细胞和分子机制及其随后的毒性，包括错误折叠和毒性polyQ蛋白膨胀蛋白9,11,12。在这里提出的协议为基础的实验已经帮助识别和描述polyQ蛋白毒性增强或抑制基因，polyQ蛋白毒性的小分子调节剂和细胞机制...

<table border="1"><tbody><tr><td> 仪器名称 </td><td> 公司 </td><td> 目录编号 </td></tr><tr><td>青蛙过河（6X8针） </td><td> V＆P科学，圣地亚哥</td><td>副总裁407的AH </td></tr><tr><td> BioscreenC </td><td> growthcurves美国</td><td> 5101370 </td></tr><tr><td> 100蜂窝板</td><td> growthcurves美国</td><td> 9602550 </td></tr></tbody></table>

growth assays to assess polyglutamine toxicity in yeast

蛋白质错误折叠与人类的许多疾病，特别是神经退行性疾病，如阿尔茨海默氏症，帕金森氏病，亨廷顿氏病1。亨廷顿氏病（HD），是由异常扩张的多聚谷氨酰胺（polyQ蛋白）区域内的蛋白质亨廷顿。亨廷顿蛋白质的polyQ蛋白膨胀达到异常的构象（即它错误折叠），并导致细胞毒性2。至少有八个进一步的神经退行性疾病造成polyQ蛋白的扩展，包括脊髓小脑性共济失调和肯尼迪的疾病3。 模式生物酵母促进polyQ蛋白毒性的细胞和分子基础的重要见解，包括polyQ蛋白毒性内和分子间的因素的影响，并在细胞受损的细胞通路的识别表达polyQ蛋白膨胀蛋白质3-8。重要polyQ蛋白毒性在酵母中发现的许多方面，LY，复制在其他的实验系统，并一定程度上在血液透析病人的样本，从而表明polyQ蛋白毒性支撑的基本机制的发现酵母模型的意义。 直接和相对简单的方法来确定polyQ蛋白在酵母毒性是衡量表达polyQ蛋白膨胀蛋白质的酵母细胞的生长缺陷。这手稿描述了三种互补的实验方法，确定酵母测量表达polyQ蛋白膨胀蛋白质的酵母细胞的生长，polyQ蛋白毒性。前两个实验方法监测酵母的生长，对板的第三种方法监测液体酵母文化使用BioscreenC文书的增长。 此外，这个手稿描述实验处理酵母polyQ蛋白模型时可能出现的困难，并概述了战略，这将有助于避免或最大限度地减少这些困难。这里所描述的协议，可以用来识别和特征的遗传途径和调节polyQ蛋白毒性的小分子。此外，所述的检测可以作为模板在酵母模型由其他疾病相关的错误折叠的蛋白质引起的毒性的准确分析。

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

这手稿描述了三个评估毒性的多聚谷氨酰胺（polyQ蛋白）扩张蛋白在酵母的补充协议<em&gt;酿酒酵母</em&gt;。这些协议可以很容易地进行修改，以监察在酵母中错误折叠的蛋白质的毒性。

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

growth-assays-to-assess-polyglutamine-toxicity-in-yeast

Research

JoVE Journal

Biology

13.5K 次观看.  Boston Biomedical Research Institute. 这手稿描述了三个评估毒性的多聚谷氨酰胺（polyQ蛋白）扩张蛋白在酵母的补充协议酿酒酵母。这些协议可以很容易地进行修改，以监察在酵母中错误折叠的蛋白质的毒性。

视频: Growth Assays to Assess Polyglutamine Toxicity in Yeast

Mating-based Overexpression Library Screening in Yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

基于交配的酵母超表达文库筛选

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

科研

遗传学

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

癌症研究

Identification of Host Pathways Targeted by Bacterial Effector Proteins using Yeast Toxicity and Suppressor Screens

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated biological pathways to the benefit of the bacterium. Identification of putative bacterial effector proteins has become more manageable due to advances in bacterial genome sequencing and the advent of algorithms that allow in silico identification of genes encoding secretion candidates and/or eukaryotic-like domains. However, identification of these important virulence factors is only an initial step. Naturally, the goal is to determine the molecular function of effector proteins and elucidate how they interact with the host. In recent years, techniques like the yeast two-hybrid screen and large-scale immunoprecipitations coupled with mass spectrometry have aided in the identification of protein-protein interactions. Although identification of a host binding partner is the crucial first step toward elucidating the molecular function of a bacterial effector protein, sometimes the host protein is found to have multiple biological functions (e.g., actin, clathrin, tubulin), or the bacterial protein may not physically bind host proteins, depriving the researcher of crucial information about the precise host pathway being manipulated. A modified yeast toxicity screen coupled with a suppressor screen has been adapted to identify host pathways impacted by bacterial effector proteins. The toxicity screen relies on a toxic effect in yeast caused by the effector protein interfering with the host biological pathways, which often manifests as a growth defect. Expression of a yeast genomic library is used to identify host factors that suppress the toxicity of the bacterial effector protein and thus identify proteins in the pathway that the effector protein targets. This protocol contains detailed instructions for both the toxicity and suppressor screens. These techniques can be performed in any lab capable of molecular cloning and cultivation of yeast and Escherichia coli.

Bacterial pathogens secrete proteins into the host that target crucial biological processes. Identifying the host pathways targeted by bacterial effector proteins is key to addressing molecular pathogenesis. Here, a method using a modified yeast suppressor and toxicity screen to elucidate host pathways targeted by toxic bacterial effector proteins is described.

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

Intracellular bacteria secrete virulence factors called effector proteins into the host cytosol that act to subvert host proteins and/or their associated ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

高通量酵母质粒过表达屏幕

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

生物学

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s disease (HD) is principally driven by a CAG nucleotide repeat that encodes an expanded glutamine tract in huntingtin protein. Thus, the inhibition of polyglutamine (polyQ) aggregation and, in particular, aggregation-associated neurotoxicity is a useful strategy for the prevention of HD and other polyQ-associated conditions. This paper introduces generalized experimental protocols to assess the neuroprotective capacity of test compounds against HD using established polyQ transgenic Caenorhabditis elegans models. The AM141 strain is chosen for the polyQ aggregation assay as an age-associated phenotype of discrete fluorescent aggregates can be easily observed in its body wall at the adult stage due to muscle-specific expression of polyQ::YFP fusion proteins. In contrast, the HA759 model with strong expression of polyQ-expanded tracts in ASH neurons is used to examine neuronal death and chemoavoidance behavior. To comprehensively evaluate the neuroprotective capacity of target compounds, the above test results are ultimately presented as a radar chart with profiling of multiple phenotypes in a manner of direct comparison and direct viewing.

Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity

Here, we present a protocol to assess the neuroprotective activities of test compounds in Caenorhabditis elegans, including polyglutamine aggregation, neuronal death, and chemoavoidance behavior, as well as an exemplary integration of multiple phenotypes.

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

Age-related misfolding and aggregation of pathogenic proteins are responsible for several neurodegenerative diseases. For example, Huntington&#39;s ...

神经科学

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

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

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

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

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 ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

基于蛋白质的继承在酿酒酵母中的高通量筛选

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

A Method to Study &#945;-Synuclein Toxicity and Aggregation Using a Humanized Yeast Model

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy bodies containing misfolded and aggregated &#945;-synuclein. &#945;-synuclein is an abundant presynaptic protein that regulates synaptic vesicle trafficking, but the accumulation of its proteinaceous inclusions results in neurotoxicity. Recent studies have revealed that various genetic factors, including bacterial chaperones, could reduce the formation of &#945;-synuclein aggregates in vitro. However, it is also important to monitor the anti-aggregation effect in the cell to apply this as a potential treatment for the patients. It would be ideal to use neuronal cells, but these cells are difficult to handle and take a long time to exhibit the anti-aggregation phenotype. Therefore, a quick and effective in vivo tool is required for the further evaluation of in vivo anti-aggregation activity. The method described here was used to monitor and analyze the anti-aggregation phenotype in the humanized yeast Saccharomyces cerevisiae, which expressed human &#945;-synuclein. This protocol demonstrates in vivo tools that could be used for monitoring &#945;-synuclein-induced cellular toxicity, as well as the formation of &#945;-synuclein aggregates in cells.

An in vivo physiological model of &#945;-synuclein is required to study and understand the pathogenesis of Parkinson's disease. We describe a method to monitor the cytotoxicity and aggregate formation of &#945;-synuclein using a humanized yeast model.

一种基于人源酵母模型研究α-突触核蛋白毒性和聚集的方法

Parkinson's disease is the second most common neurodegenerative disorder and is characterized by progressive cell death caused by the formation of Lewy ...

PolyQ Aggregation Assay to Identify Neuroprotective Effect of a Compound in Worms

Source: Wang, Q. et al. <a href="https://app.jove.com/v/63081">Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity.</a> J. Vis. Exp. (2021)
This video describes an in vivo method to screen the protective potential of neuroprotective test compounds in reducing polyQ aggregation in body wall muscle cells of nematodes and ameliorating aggregation-associated proteotoxicity.

PolyQ 聚集检测试剂盒鉴定化合物在蠕虫中的神经保护作用

Source: Wang, Q. et al. Caenorhabditis elegans as a Model System for Discovering Bioactive Compounds Against Polyglutamine-Mediated Neurotoxicity. J. Vis. ...

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

摘要

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此视频中的章节

基于交配的酵母超表达文库筛选

酵母作为开发功能分析研究人类P53的机箱

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

高通量酵母质粒过表达屏幕

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

成长型测定和遗传需求生化确认的蛋白质降解

基于蛋白质的继承在酿酒酵母中的高通量筛选

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

一种基于人源酵母模型研究α-突触核蛋白毒性和聚集的方法

PolyQ 聚集检测试剂盒鉴定化合物在蠕虫中的神经保护作用

生长实验，以评估在酵母中的多聚谷氨酰胺毒性

摘要

探索更多视频

此视频中的章节

基于交配的酵母超表达文库筛选

酵母作为开发功能分析研究人类P53的机箱

使用酵母毒性和抑制器屏幕识别细菌效应蛋白的宿主通路

高通量酵母质粒过表达屏幕

秀丽隐杆线虫 作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统

成长型测定和遗传需求生化确认的蛋白质降解

基于蛋白质的继承在酿酒酵母中的高通量筛选

荧光蛋白对酵母中多谷氨酰胺毒性测定的融合伙伴的影响

一种基于人源酵母模型研究α-突触核蛋白毒性和聚集的方法

PolyQ 聚集检测试剂盒鉴定化合物在蠕虫中的神经保护作用

秀丽隐杆线虫作为发现生物活性化合物对抗聚谷氨酰胺介导的神经毒性的模型系统