Name: high-throughput screening for protein-based inheritance in s. cerevisiae
Uploaded: 2017-08-08T14:00:00
Description: Watch this Scientific Journal Video about High-throughput Screening for Protein-based Inheritance in S. cerevisiae at JoVE.com

我々 は、彼らの思慮深いコメントの校閲者と同様に、本稿で使用される試金の開発に彼らの支援のため Sohini Chakrabortee、サンドラ ・ ジョーンズ、ダビド ガルシア、Bhupinder Bhullar、アメリア チャン、彼女はリチャードとスーザン ・ リンドキストをありがちましょう。

1. Initial Overexpression
<ol>
	<li>Transform the yeast cells (in this case, BY4741 MAT a haploids) previously grown in YPD liquid (10 g of yeast extract, 20 g of peptone, and 20 g of glucose per 1 L) with the desired candidate constructs from the FLEXGene ORFeome library (yeast ORFs under the control of a galactose-inducible promoter in the URA3-marked centromericplasmid backbone, pBY01129).</li>
	<li>Use autoclaved toothpicks to pick four separate colonies from these...

<ol>
	<li>Halfmann, R., Alberti, S., 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=Prions,+protein+homeostasis,+and+phenotypic+diversity.">Prions, protein homeostasis, and phenotypic diversity.</a> Trends Cell Biol. 20 (3), 125-133 (2010).</li><li>Byers, J. S., Jarosz, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pernicious+pathogens+or+expedient+elements+of+inheritance:+the+significance+of+yeast+prions.">Pernicious pathogens or expedient elements of inheritance: the significance of yeast prions.</a> PLoS Pathog. 10 (4), 1003992 (2014).</li><li>Jarosz, D. F., 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=Cross-kingdom+chemical+communication+drives+a+heritable,+mutually+beneficial+prion-based+transformation+of+metabolism.">Cross-kingdom chemical communication drives a heritable, mutually beneficial prion-based transformation of metabolism.</a> Cell. 158 (5), 1083-1093 (2014).</li><li>True, H. L., 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+yeast+prion+provides+a+mechanism+for+genetic+variation+and+phenotypic+diversity.">A yeast prion provides a mechanism for genetic variation and phenotypic diversity.</a> Nature. 407 (6803), 477-483 (2000).</li><li>Suzuki, G., Shimazu, N., Tanaka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+prion,+Mod5,+promotes+acquired+drug+resistance+and+cell+survival+under+environmental+stress.">A yeast prion, Mod5, promotes acquired drug resistance and cell survival under environmental stress.</a> Science. 336 (6079), 355-359 (2012).</li><li>Hou, F., 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=MAVS+forms+functional+prion-like+aggregates+to+activate+and+propagate+antiviral+innate+immune+response.">MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response.</a> Cell. 146 (3), 448-461 (2011).</li><li>Cai, X., 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=Prion-like+polymerization+underlies+signal+transduction+in+antiviral+immune+defense+and+inflammasome+activation.">Prion-like polymerization underlies signal transduction in antiviral immune defense and inflammasome activation.</a> Cell. 156 (6), 1207-1222 (2014).</li><li>Majumdar, 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=Critical+role+of+amyloid-like+oligomers+of+Drosophila+Orb2+in+the+persistence+of+memory.">Critical role of amyloid-like oligomers of Drosophila Orb2 in the persistence of memory.</a> Cell. 148 (3), 515-529 (2012).</li><li>Khan, M. R., 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=Amyloidogenic+Oligomerization+Transforms+Drosophila+Orb2+from+a+Translation+Repressor+to+an+Activator.">Amyloidogenic Oligomerization Transforms Drosophila Orb2 from a Translation Repressor to an Activator.</a> Cell. 163 (6), 1468-1483 (2015).</li><li>Fioriti, L., 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=The+Persistence+of+Hippocampal-Based+Memory+Requires+Protein+Synthesis+Mediated+by+the+Prion-like+Protein+CPEB3.">The Persistence of Hippocampal-Based Memory Requires Protein Synthesis Mediated by the Prion-like Protein CPEB3.</a> Neuron. 86 (6), 1433-1448 (2015).</li><li>Derkatch, I. L., Bradley, M. E., Hong, J. Y., Liebman, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prions+affect+the+appearance+of+other+prions:+the+story+of+[PIN(+)].">Prions affect the appearance of other prions: the story of [PIN(+)].</a> Cell. 106 (2), 171-182 (2001).</li><li>Alberti, S., Halfmann, R., King, O., Kapila, A., 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+systematic+survey+identifies+prions+and+illuminates+sequence+features+of+prionogenic+proteins.">A systematic survey identifies prions and illuminates sequence features of prionogenic proteins.</a> Cell. 137 (1), 146-158 (2009).</li><li>Chernoff, Y. O., Lindquist, S. L., Ono, B., Inge-Vechtomov, S. G., Liebman, S. 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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=[URE3]+as+an+altered+URE2+protein:+evidence+for+a+prion+analog+in+Saccharomyces+cerevisiae.">[URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae.</a> Science. 264 (5158), 566-569 (1994).</li><li>Sondheimer, N., 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=Rnq1:+an+epigenetic+modifier+of+protein+function+in+yeast.">Rnq1: an epigenetic modifier of protein function in yeast.</a> Mol Cell. 5 (1), 163-172 (2000).</li><li>Balbirnie, M., Grothe, R., Eisenberg, D. 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Y., 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=Prion-inducing+domain+2-114+of+yeast+Sup35+protein+transforms+in+vitro+into+amyloid-like+filaments.">Prion-inducing domain 2-114 of yeast Sup35 protein transforms in vitro into amyloid-like filaments.</a> Proc Natl Acad Sci U S A. 94 (13), 6618-6622 (1997).</li><li>Shorter, 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=Hsp104+catalyzes+formation+and+elimination+of+self-replicating+Sup35+prion+conformers.">Hsp104 catalyzes formation and elimination of self-replicating Sup35 prion conformers.</a> Science. 304 (5678), 1793-1797 (2004).</li><li>Ferreira, P. C., Ness, F., Edwards, S. R., Cox, B. S., Tuite, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+elimination+of+the+yeast+[PSI+]+prion+by+guanidine+hydrochloride+is+the+result+of+Hsp104+inactivation.">The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation.</a> Mol Microbiol. 40 (6), 1357-1369 (2001).</li><li>Caudron, F., Barral, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+super-assembly+of+Whi3+encodes+memory+of+deceptive+encounters+by+single+cells+during+yeast+courtship.">A super-assembly of Whi3 encodes memory of deceptive encounters by single cells during yeast courtship.</a> Cell. 155 (6), 1244-1257 (2013).</li><li>Wickner, R. B., Edskes, H. K., Shewmaker, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+find+a+prion:+[URE3],+[PSI+]+and+[beta].">How to find a prion: [URE3], [PSI+] and [beta].</a> Methods. 39 (1), 3-8 (2006).</li><li>Chernoff, Y. O., Derkach, I. L., Inge-Vechtomov, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicopy+SUP35+gene+induces+de-novo+appearance+of+psi-like+factors+in+the+yeast+Saccharomyces+cerevisiae.">Multicopy SUP35 gene induces de-novo appearance of psi-like factors in the yeast Saccharomyces cerevisiae.</a> Curr Genet. 24 (3), 268-270 (1993).</li><li>Brown, J. C., 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+heritable+switch+in+carbon+source+utilization+driven+by+an+unusual+yeast+prion.">A heritable switch in carbon source utilization driven by an unusual yeast prion.</a> Genes Dev. 23 (19), 2320-2332 (2009).</li><li>Coustou, V., Deleu, C., Saupe, S., Begueret, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+protein+product+of+the+het-s+heterokaryon+incompatibility+gene+of+the+fungus+Podospora+anserina+behaves+as+a+prion+analog.">The protein product of the het-s heterokaryon incompatibility gene of the fungus Podospora anserina behaves as a prion analog.</a> Proc Natl Acad Sci U S A. 94 (18), 9773-9778 (1997).</li><li>Sopko, R., 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=Mapping+pathways+and+phenotypes+by+systematic+gene+overexpression.">Mapping pathways and phenotypes by systematic gene overexpression.</a> Mol Cell. 21 (3), 319-330 (2006).</li><li>Chakrabortee, S., 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=Intrinsically+Disordered+Proteins+Drive+Emergence+and+Inheritance+of+Biological+Traits.">Intrinsically Disordered Proteins Drive Emergence and Inheritance of Biological Traits.</a> Cell. 167 (2), 369-381 (2016).</li><li>Hu, Y., 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=Approaching+a+complete+repository+of+sequence-verified+protein-encoding+clones+for+Saccharomyces+cerevisiae.">Approaching a complete repository of sequence-verified protein-encoding clones for Saccharomyces cerevisiae.</a> Genome Res. 17 (4), 536-543 (2007).</li><li>Swain, P. 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最初の酵母プリオンは、彼らの異常な表現型と継承の複雑なパターンによって識別されました。これらのプリオンの特性は、アルゴリズムと計算ツール追加プリオン蛋白質のため画面を構築するそれから使用されました。ここで説明する方法は対照的は実験的一時的な過剰発現タンパク質の立体構造の状態でエンコードされた永続的な変更は安定を作成するに依存しています。ただし、任意?...

生物学的システムはよくタンパク質豊富で過渡変動を経験します。これらは有機体のまたは将来の世代の表現型を形作る上で永続的な影響をあるかどうかは不明します。この生物の最も有名なインスタンスを含むタンパク質、プリオンは、ゲノム修正なしの遺伝性の特性の出現をドライブの珍しいクラス。代わりに、これらのプロの teinaceous とのfectious 粒子タンパク質構造1,2の自己永続的変更による表現型を送信します。この型を継承は、壊滅的な神経変性疾患の異常な継承のパターンの原因として発見されました。しかし、菌類から哺乳類3,4,5,6,7,8,9,10まで及ぶ有機体の研究は以来プリオンのような要素が適応値を与えることができること明らかに。それにもかかわらず、プリオンは、魅惑的で珍しい生物風変わりなしかしとして表示されています。
この相場の知恵は、継承の蛋白質ベースの特性は長い例の小さなセットによって制限されているので一部で開催されます。最近の組織的スクリーニングの努力は大幅燃料プリオンのような構造変換する能力を持ついくつかの新しいbona fideプリオン11およびほぼ 2 つのダース蛋白質ドメイン12を識別することによってこの画像を拡大しています。ただし、これらのアプローチは、一般的に強力なアミノ酸シーケンス バイアスを当てている、ので、発見されているプリオンは創立酵母プリオン [PSI+]13,14、[URE3]15[RNQ+]11,16の生化学的性質を共有します。これらが含まれます: 1) モジュラー ドメイン アスパラギン (N) とグルタミン (Q)、2)、アミロイド [プリオン+] 構造17,18,に19アセンブリの長い高分子の伸張に富んでいる、3) 完了 disaggregase 母から娘13,20,21に忠実な伝播のための Hsp104 関数に依存します。確かに、多くbona fideプリオンを含む [ガー+] [ヘット s]、元プリオン (PrPSc) にも、このような厳しい条件下では見逃されること。おそらくもっと重要なは、彼らことはできません蛋白質ベースの継承22の任意の新規メカニズムをキャプチャします。したがって、このような現象の真の生物学的幅は、はるか以前の仮定より自然で一般的な可能性があります。
この問題を調べるためには、高スループット、プロテオーム的戦略が採用されました。PrPSc[ガー+] を含む全てのプリオンの特徴 [ヘット-s] は、原因タンパク質の一過性発現が強くプリオン買収15,23,24,25,26の率を増加することとします。一過性個々 のタンパク質の発現を誘導することによって安定した蛋白質ベース、エピジェネティック状態を開始できる場合、体系的に全体にわたって酵母の ORFeome、お願い、この機能の利用をしました。その蛋白の過剰発現は、表現型27を変更できますをよく知られています。しかし、彼らの一時的な過剰世代初期の過剰発現後の数百の遺伝性は表現型の変化を生成するため、プリオン蛋白質は通常ありません。以前、ホリールード ゲノム28を変えないで表現型の風景を再配線することができる蛋白質の多数を識別するために、タンパク質ベース遺伝的要素の異常な継承のパターンだけでなく、この機能を利用しました。いくつかの特定蛋白質以前として知られていたプリオン、ほとんどなかった、タンパク質ベースの継承の新たな形態を明らかにするこのアプローチの力を強調します。

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			<td height="21" style="height:21px;">Leucine</td>
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			<td>Biomek FX</td>
			<td>Equipment</td>
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high-throughput screening for protein-based inheritance in s. cerevisiae

将来の世代にアクセス可能な生物学的情報のエンコードは、DNA シーケンスの変更を介して達成される一般に。(シーケンスではなく) 蛋白質立体構造でエンコードされた長寿命の継承は、パラダイム シフトしますが、珍しいとして表示されている長い。などのエピジェネティックな要素の最高の特徴の例は、プリオンは、新しい表現型の遺伝的症状をドライブすることができます自己組織化挙動を所有しています。多くの典型的なプリオンは印象的な豊富な N/Q シーケンス バイアスを表示し、アミロイドの折りにアセンブルします。これらの珍しい特徴は、新しいプリオン蛋白質を識別するほとんどのスクリーニングの努力を知らせています。ただし、(創設のプリオン、PrPScを含む)、少なくとも 3 つの知られているプリオンは、これらの生化学的な特徴を港ないです。したがってプローブ質量作用のプロパティに基づいて蛋白質ベースの継承の範囲に代替法を開発した: プリオン蛋白質の一時的な過剰発現する彼らは自己テンプレート構造を取得頻度が増加します。本稿では、蛋白質ベースの継承を引き出す酵母 ORFeome の能力を分析するためのメソッドについて説明します。この戦略を使用して、我々 は以前ことを発見した &gt; イースト蛋白質の 1% は長寿命、安定した遺伝の突然変異よりも頻繁に起こったされ生物学的形質の出現を燃料でした。全体 ORFeomes 全体または特定の遺伝子ネットワークまたは環境刺激の標的スクリーニング パラダイムとして、高スループットでこの方法を採用できます。同様に前方遺伝的画面多数発達とシグナル伝達経路を定義する、これらの技術は生物学的過程での蛋白質ベースの継承の影響を調査するための方法論を提供します。

Protein overexpression is known to dramatically alter cellular phenotypes27. Indeed, with an initial screening approach, hundreds of new phenotypes were reproducibly recovered from the overexpression of clones from the yeast ORFeome using just ten stressors. However, the assays described above allow for the assessment of whether cells retain any long-term stable phenotypes following this overexpression. One protein capable of encoding such a state is Psp1. Psp1 is ...

Watch this Scientific Journal Video about High-throughput Screening for Protein-based Inheritance in S. cerevisiae at JoVE.com

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

このプロトコルでは、出芽酵母でタンパク質に基づく継承のため画面の機能的に高スループット方法論について説明します。

出芽酵母におけるタンパク質ベース継承の高速スクリーニング

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

Research

JoVE Journal

Biology

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

Assay for Adhesion and Agar Invasion in S. cerevisiae

S. cerevisiaeの接着と寒天の侵略のためのアッセイ

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

A high-throughput method to globally study the organelle morphology in S. cerevisiae

グローバルS. cerevisiaeの細胞小器官の形態を研究するためにハイスループット法

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

S. cerevisiaeの定量的プロテオミクスとタンパク質の複合体の同定

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

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

High-throughput Screening and Biosensing with Fluorescent C. elegans Strains

蛍光とハイスループットスクリーニングとバイオセンシング C.エレガンス菌株

High-throughput screening (HTS) is a powerful approach for identifying chemical modulators of biological processes.  However, many compounds identified in ...

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

High Throughput Screening of Fungal Endoglucanase Activity in Escherichia coli

における真菌エンドグルカナーゼ活性のハイスループットスクリーニング大腸菌</em

Cellulase enzymes (endoglucanases, cellobiohydrolases, and &beta;-glucosidases) hydrolyze cellulose into component sugars, which in turn can be converted ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Flow Cytometry-based Purification of S. cerevisiae Zygotes

のフローサイトメトリーベースの精製<em&gt; S.セレビ</em&gt;受精卵

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

内向き整流カリウムチャネルの小分子モジュレーターのためのハイスループットスクリーニング

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

で産生された組換えジスルフィド豊富な毒タンパク質に印加される高スループット定量的発現スクリーニングおよび精製<em&gt; E.大腸菌の</em

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...