Name: growth assays to assess polyglutamine toxicity in yeast
Uploaded: 2012-03-05T12:00:00
Description: Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

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...

<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. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+machinery+is+involved+in+aggregation+of+proteins+with+expanded+polyglutamine+domains.">Endocytosis machinery is involved in aggregation of proteins with expanded polyglutamine domains.</a> FASEB J. 21, 1915-1925 (2007).</li><li>Giorgini, F., Guidetti, P., Nguyen, Q., Bennett, C. S., Muchowski, P. 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+genomic+screen+in+yeast+implicates+kynurenine+3-monooxygenase+as+a+therapeutic+target+for+Huntington+disease.">A genomic screen in yeast implicates kynurenine 3-monooxygenase as a therapeutic target for Huntington disease.</a> Nat. Genet. 37, 526-5231 (2005).</li><li>Duennwald, M. L., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impaired+ERAD+and+ER+stress+are+early+and+specific+events+in+polyglutamine+toxicity.">Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity.</a> Genes Dev. 22, 3308-3319 (2008).</li><li>Duennwald, M. L., Jagadish, S., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flanking+sequences+profoundly+alter+polyglutamine+toxicity+in+yeast.">Flanking sequences profoundly alter polyglutamine toxicity in yeast.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11045-1150 (2006).</li><li>Duennwald, M. L., Jagadish, S., Giorgini, F., Muchowski, P. J., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+network+of+protein+interactions+determines+polyglutamine+toxicity.">A network of protein interactions determines polyglutamine toxicity.</a> Proc. Natl. Acad. Sci. U.S.A. 103, 11051-116 (2006).</li><li>Meriin, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Huntington+toxicity+in+yeast+model+depends+on+polyglutamine+aggregation+mediated+by+a+prion-like+protein+Rnq1.">Huntington toxicity in yeast model depends on polyglutamine aggregation mediated by a prion-like protein Rnq1.</a> J. Cell. Biol. 157, 997-1004 (2002).</li><li>Murakami, C., Kaeberlein, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Yeast+Chronological+Life+Span+by+Outgrowth+of+Aged+Cells.">Quantifying Yeast Chronological Life Span by Outgrowth of Aged Cells.</a> J. Vis. Exp. (27), e1156-e1156 (2009).</li><li>Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beer+and+bread+to+brains+and+beyond:+can+yeast+cells+teach+us+about+neurodegenerative+disease.">Beer and bread to brains and beyond: can yeast cells teach us about neurodegenerative disease.</a> Neurosignals. 16, 52-62 (2008).</li><li>Giorgini, F., Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+genetic+modifiers+of+amyloid+toxicity+in+yeast.">Screening for genetic modifiers of amyloid toxicity in yeast.</a> Methods Enzymol. 412, 201-222 (2006).</li><li>Ehrnhoefer, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+tea+(-)-epigallocatechin-gallate+modulates+early+events+in+huntingtin+misfolding+and+reduces+toxicity+in+Huntington's+disease+models.">Green tea (-)-epigallocatechin-gallate modulates early events in huntingtin misfolding and reduces toxicity in Huntington's disease models.</a> Hum. Mol. Genet. 15, 2743-2751 (2006).</li><li>Cashikar, A. G., Duennwald, M., Lindquist, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chaperone+pathway+in+protein+disaggregation.+Hsp26+alters+the+nature+of+protein+aggregates+to+facilitate+reactivation+by+Hsp104.">A chaperone pathway in protein disaggregation. Hsp26 alters the nature of protein aggregates to facilitate reactivation by Hsp104.</a> J. Biol. Chem. 280, 23869-2375 (2005).</li></ol>

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 ...

Research

JoVE Journal

Biology

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

细菌III型效应，在酵母中的表达诱导的生长抑制的表型鉴定

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

科研

生物学

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

改良酵母双杂交系统，以确定蛋白与生长因子Progranulin的相互作用

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Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

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致命的甲壳类动物长期毒性试验卤虫franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

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方法来评估肌肉的亚细胞中<em&gt;温度。线虫</em

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Comparative Analysis of Human Growth Hormone in Serum Using SPRi, Nano-SPRi and ELISA Assays

人生长激素测定血清SPRI，纳米单层屋面和酶联免疫吸附测定比较分析

Sensitive and selective methods for the detection of human growth hormone (hGH) over a wide range of concentrations (high levels of 50-100 ng ml&#8722;1 ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

间隙连接通讯：天然产物的生物标志物的功能，以评估毒物和毒素的不利影响，以及健康的益处

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

ODELAY: A Large-scale Method for Multi-parameter Quantification of Yeast Growth

ODELAY：用于酵母生长多参数定量的大规模方法

Growth phenotypes of microorganisms are a strong indicator of their underlying genetic fitness and can be segregated into 3 growth regimes: lag-phase, ...

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

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 ...

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

Medium-throughput Screening Assays for Assessment of Effects on Ca2+-Signaling and Acrosome Reaction in Human Sperm

中通量筛选法评估对精子中 ca2 +信号和反应的影响

Ca2+-signaling is essential to normal sperm cell function and male fertility. Similarly, the acrosome reaction is vital for the ability of a human sperm ...

Examination of Mitotic and Meiotic Fission Yeast Nuclear Dynamics by Fluorescence Live-cell Microscopy

荧光活细胞显微镜对微孔裂变酵母核动力学的检测

Live-cell imaging is a microscopy technique used to examine cell and protein dynamics in living cells. This imaging method is not toxic, generally does ...