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。
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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此问题会将 2 微米质粒蛋白表达在将来用于实验。最后,每个诱导的朊病毒有其自己独特的增长型集,将不会在每个条件明显测定。因此,不同的条件和剂量测试数目限制点击次数。
重要的是,并非所有类型的基于蛋白质的继承将同样都恢复使用此方法。不能有效地或没有毒性中高表达的蛋白质显然将不断地错过了。毛干不稳定的元素,如"mnemons,"将永远不会传播到女儿后初始过度表达22。与此相反的是,理论上可以通过瞬时表达42,43诱导其他类型的长寿命双稳态开关。然而,这些国家通常是不依赖于蛋白质稳态机械或传染性通过"种子"的蛋白质。此外,依赖于其他伴侣 (外热休克蛋白 70 和 Hsp104) 或蛋白质代谢网络的传播更多的军备的朊病毒会失败这里描述的伴侣依赖项化验。最后,也形成淀粉样蛋白的低丰度蛋白质可能有传染性利率低于检测限在蛋白质转换设置。
此协议说明技术诱导蛋白表达,以及进一步下游步骤来验证是否每个导致表观遗传状态稳定基于蛋白质的表观遗传态是善意取得制度朊病毒。这个例子说明了本文 Psp1,是一种蛋白质,显示"朊病毒样"氨基酸偏差,并从理论上讲可以恢复以前使用示例开发生物信息学算法。然而,Psp1 无法形成淀粉样蛋白和其不寻常的伴侣依赖 (Hsp104) 便会有迅速取消它从进一步的分析,从而消除它从朊病毒审议。然而,本文介绍的筛选技术是不可知论者对这些假设,集中精力继承的基本模式和充分性的蛋白质独自来传输相应的表型。事实上,绝大多数的恢复,这种方法基于蛋白质的继承没有富含 Q 的 N 序列偏见。
此方法用于探测整个酵母 ORFeome 为其引出不带偏见的方式使用压力 (25 毫米氯化镉,1 毫米氯化钴,2 毫米硫酸铜、 1 毫米酰胺、 氟康唑 0.2 毫米、 50 毫米羟基脲、 20 毫米氯化锰、 0.75 毫米百草枯、 50 毫米 radicicol,80 J/m2 UV 照射数目基于蛋白质的继承的能力和硫酸锌 10 毫米)。然而,这种方法可以容易地修改屏幕遗传网络或特定的细胞反应以更有针对性的方式。为例子,功能相关的蛋白质或所有蛋白质调节在离散信号网络可以通过瞬时表达诱导和筛选与应激源与他们的生理功能有关。与此相反的是,一套更全面的应激源调查是否特定细胞的反应自然进化,港湾朊病毒开关可以用来筛选蛋白质一大套。最后,虽然我们已进行这些研究酵母,许多方面的实验结果 (例如,瞬态蛋白表达、 伴侣"固化,"等) 可以推广到其他模型系统在将来。例如,哺乳动物的组织文化是服从表达质粒为基础的系统,通过和荧光灶可能也用于作为读出遗传自组装,如前面所述28。此外,从其他生物的蛋白质编码序列可以在酵母中表达和测试他们的能力争取朊病毒样继承使用此处所述的方法。
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Disclosures
作者没有透露。
Acknowledgments
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
Name | Company | Catalog Number | Comments |
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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