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Abstract
Introduction
Protocol
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Biology

基于蛋白质的继承在酿酒酵母中的高通量筛选

Published: August 08, 2017
doi:
10.3791/56069
,
1Department of Developmental Biology,Stanford University School of Medicine, 2Department of Chemical and Systems Biology,Stanford University School of Medicine

Summary

此协议说明一种高通量方法来功能屏幕在酿酒酵母中的蛋白质基础继承。

Abstract

对后代是容易接近的生物信息的编码通常被通过 DNA 序列的变化。长寿遗传编码的蛋白质构象 （而不是序列） 长期以来被视为范式转移，但很少。这种表观遗传的元素的最佳的特征的示例包括朊病毒，拥有可以推动新的表型的遗传表现的自组装行为。许多原型朊病毒显示醒目的富含 Q 的 N 序列偏见和组装成淀粉样蛋白的折叠。这些不寻常的特征已通知筛选确定新的朊病毒蛋白的大多数努力。然而，至少三个已知的朊病毒 （包括创始的朊病毒，PrPSc） 不要怀有这些生化特性。因此，我们开发了一种替代的方法，探讨基于蛋白质的继承，基于属性的大规模行动的范围： 瞬态的朊病毒蛋白过度表达增加，他们获得自我模板构象的频率。本文介绍了一种分析酵母 ORFeome 能够引出基于蛋白质的继承方法。使用此策略，我们以前发现 > 1%的酵母蛋白质能燃料出现的生物性状，寿命长，性能稳定，并比遗传突变更频繁地出现。这种方法可以从事高吞吐量，横跨整个 ORFeomes 或作为特定遗传网络或环境刺激有针对性的筛选模式。正如前的遗传屏幕定义许多发展和信号转导途径，这些技术提供一种方法来探讨基于蛋白质的继承在生物过程中的影响。

Introduction

生物系统经常经历的瞬态波动蛋白质丰富。这些是否有持久的影响，在塑造的有机体或未来几代人的表型仍不清楚。这个生物学的著名实例涉及一类罕见的蛋白质，朊病毒，开车遗传性基因组原封不动的出现。相反，这些临的 teinaceous 和在发感染粒子传输通过自我延续的变化对蛋白质构象1，2的表型。这种类型的继承被发现不寻常的继承模式的破坏性的神经退行性疾病的原因。然而，在生物体从真菌到哺乳动物3，4，，56，7，8，，910研究以来发现朊病毒样元素可以赋予自适应值。尽管如此，朊病毒已被视为美丽的向往但罕见生物古怪。

这个普遍的智慧是部分举行因为表征蛋白质基础继承长期以来一直受一小套的示例。最近系统筛查努力扩大这张照片明显确定几个新的善意取得制度朊病毒11和几乎两打蛋白域12与燃料朊病毒样构象转化的能力。然而，因为这些方法一般集中在强氨基酸序列偏见，朊病毒被发现分享创始酵母朊病毒 [PSI+]13，14，[URE3]15和 [RNQ+]11，16的生化性质。这些措施包括： 1） 模块化域，富含长高分子绵延的天冬酰胺 (N) 和谷氨酰胺 (Q) 2） 组装成淀粉样 [朊病毒+] 构象17，，1819，和 3） 完成 disaggregase 忠实传播从母亲到女儿13，，2021Hsp104 函数依赖。事实上，许多善意朊病毒，包括 [噶尔+]，[Het-s]，和甚至原始朊蛋白 (PrPSc)，将错过下这样严格的标准。也许更重要的是，他们将无法捕获任何新的机制的蛋白质基础继承22。因此，真正的生物学宽度，这种现象可能是在自然比以前想象中更为常见。

为了调查这一问题，采用高通量蛋白质组全战略。所有的朊病毒，包括 PrPSc，[噶尔+]，标志和 [Het-s]，是瞬变的因果蛋白过度表达强烈增加的朊病毒采集15，23，，2425，26率。我们利用此功能，以便系统地问，跨整个酵母 ORFeome，如果稳定的蛋白质基础、 表观遗传状态可以由瞬态诱发个别蛋白质的表达。众所周知，蛋白过度表达可以改变表型27。然而朊病毒蛋白是不寻常的因为他们临时生产过剩产生许多数百代后的初始的过度表达是可遗传的表型变化。我们以前采取了利用此功能，以及基于蛋白质的遗传元件，以查明的蛋白质能够重新 heritably 接线表型景观而不改变基因组28几十件不寻常的继承模式。虽然有一些蛋白质被以前称为朊病毒，大多数没有，彰显了这种方法来发现新的基于蛋白质的继承形式。

Protocol

1. Initial Overexpression 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). Use autoclaved toothpicks to pick four separate colonies from these…

Representative Results

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 …

Discussion

他们不寻常的表型和令人费解的继承模式，确定了第一酵母朊病毒。这些朊病毒特征被用于生成算法和计算工具，屏幕为额外的朊病毒蛋白。在这里描述的方法相反，是实验和依赖瞬态的过度表达，创造一个持久变化一稳定状态编码的蛋白质构象。然而，如果”播种”朊病毒大会由为任何给定的蛋白质表达的效率是很低，这种蛋白质会不断出现作为过度屏幕这种类型的假阴性。一个此类修改要更正?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢他们协助制定检测用于本文，以及审阅者提出深思熟虑的意见 Sohini Chakrabortee、 桑德拉 · 琼斯、 大卫 · 加西亚、 Bhupinder Bhullar、 阿米莉亚昌、 理查德 · 她和苏珊林德奎斯特。

Materials

Guanidine hydrochloride Sigma Cat#G3272-25G Chemical
Manganese chloride Sigma Cat#M8054-100G Chemical
Ethidium bromide Sigma E1510 Chemical
5-Fluoroorotic Acid Sigma Cat#F5013-50MG Chemical
BY4741 MATa (his3Δ1 leu2Δ0 LYS2 met15Δ0 ura3Δ0) Winston et al., 1995; Brachmann et al., 1998 N/A Yeast strain
BY4741 MATα (his3Δ1 leu2Δ0 lys2Δ0 MET15 ura3Δ0) Winston et al., 1995; Brachmann et al., 1998 N/A Yeast strain
Hsp70 (K69M)  Jarosz et al., 2014b N/A Plasmid
FLEXGene library Hu et al., 2007 N/A Plasmid library
Dextrose (glucose) Fisher Scientific D16-3 Media component
Raffinose Sigma R0250-25G Media component
Galactose Fisher Scientific BP656-500 Media component
CSM Sunrise Science 1001-100 Media component
CSM-URA Sunrise Science 1004-100 Media component
CSM-LYS Sunrise Science 1032-100 Media component
CSM-MET Sunrise Science 1019-100 Media component
CSM-LYS-MET Sunrise Science 1035-100 Media component
yeast extract Fisher Scientific BP1422-2 Media component
peptone Research Products International P20240-5000 Media component
bacto-peptone BD 211677 Media component
glycerol EMD Millipore GX0185-2 Media component
yeast nitrogen base w/o amino acids BD 291920 Media component
agar IBI Scientific IB49172 Media component
Adenine sulfate Sigma A3159-25G Media component
Potassium acetate Sigma P1190-500G Media component
Uracil Sigma U0750-100G Media component
Histidine Sigma H8000-100G Media component
Leucine Sigma L8000-25G Media component
Lysine Sigma L5501-25G Media component
RNase I  Thermo Fisher Scientific EN0601 Enzyme
biotinylated DNase Thermo Fisher Scientific AM1906 Enzyme
zymolyase 100T (yeast lytic enzyme) Sunrise Science N0766555 Enzyme
Microplate reader BioTek Synergy H1 Equipment
Microplate stacker BioTek BioStack3 Equipment
Plate filler BiotTek EL406 Equipment
Liquid handling robot Beckman Coulter Biomek FX Equipment
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Tags

High-throughput ScreeningProtein-based InheritanceS. CerevisiaePrionProtein Over-expressionPhenotypic Memory96-well Plate384-well PlateLiquid Handling RobotOptical DensityManganese ChlorideSD-URASCal-URAST-URA
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Byers, J. S., Jarosz, D. F. High-throughput Screening for Protein-based Inheritance in S. cerevisiae. J. Vis. Exp. (126), e56069, doi:10.3791/56069 (2017).
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