化学遗传相互作用的快速鉴定<em>酿酒酵母</em
Summary
Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.
Abstract
确定生物活性的化学物质的作用方式是感兴趣的范围广泛的学术，医药，和工业的科学家。 酿酒酵母，或出芽酵母，是一个模型真核生物的量的〜6000基因缺失突变体和亚效等位基因必需基因的完整集合突变体是市售的。突变体的这些集合可以被用来系统地检测化学基因的相互作用，必要即基因容忍的化学物质。这个信息，反过来，报告了化合物的作用可能模式。在这里，我们描述了一个协议，用于快速识别在芽殖酵母化学基因的相互作用。我们证明使用化疗剂5-氟尿嘧啶（5-FU），其具有定义良好的作用机制的方法。我们的研究结果表明，都需要在5-FU的存在下，这是与以往的米一致增殖外来体和DNA修复酶的核TRAMP核糖核酸icroarray基于条形码化学遗传方法和5-FU的RNA和DNA代谢产生不利影响的认识。这些高通量筛选所需的验证方案也有所说明。
Introduction
的遗传工具，并在模式生物酿酒酵母可利用的资源已启用大规模功能基因组学的研究，它们共同提供了新的见解的基因如何充当网络来履行生物系统的要求。这些工具的基础是在酵母1,2-一套完整的所有开放读框的非必需基因缺失的协作创作。一个显着的观察是，只有约20％的酵母基因时，作为标准的实验室条件下培养单倍体所需的可行性。这凸显了一个细胞通过替代生物途径的利用率，减缓基因组扰动的能力。遗传突变体是可行的单独的，但致命的组合，信号连接或收敛平行生物途径和形式描述的生物学功能的基因相互作用网络。带有条件TE的发展必需基因的mperature敏感和亚效等位基因的等位基因的技术还没有被限制在非必需基因3,4的研究。这个概念已经在基因组规模已经应用产生偏见的遗传相互作用地图说明如何参与类似的细胞过程的基因聚集在一起5。
基因网络的化学扰动模拟基因缺失（ 图1）6。查询生长抑制化合物对缺失株的高密度阵列，用于超敏反应标识化学遗传相互作用信息， 即所需要容忍化学应力的基因的列表。像遗传相互作用，化学文库的大规模的屏幕显示，与动作一起簇7的类似模式的化合物。因此，通过建立一个化合物的化学遗传相互作用轮廓的作用方式，可以通过将其与LAR比较推断GE规模合成的遗传和化学遗传相互作用数据集8,9。
大型化学遗传筛选，其中化合物的分数审问，已经由条码竞争测定进行。在这种方法中，缺失菌株的汇总收集于小体积的含有化学介质中生长集体几代。由于每个缺失突变窝藏一个独特的遗传条形码，个别突变体缺失菌株的池内的生存能力/生长被微阵列或高通量测序10跟踪。
通过监测生长在含有生物活性化合物的固体琼脂物理阵列突变体菌落大小推断健身也识别化学遗传相互作用11,12的有效方法。这种方法提供了一种具有成本效益的替代竞争为基础的筛选，非常适合用于测定化学品的小库。这里所概述是用于制造在酿酒化学遗传相互作用的列表的简单方法酵母不依赖于分子生物学操作或基础设施，它仅需要一个酵母删除集合，机器人或手动钉扎装置中，并自由可用的图像分析软件。
Protocol
Representative Results
Discussion
这里列出的是产生化学遗传相互作用的配置文件的方法。该方法是简单的：通过比较在对化学品的存在和不存在完整基因缺失集合各菌株的菌落的大小，要容忍化学损伤所需的所有基因被识别。所得敏感菌株的列表的注释富集分析然后可以用于提供洞察行动的化学物质的模式。虽然该协议已被优化的芽殖酵母，它也可以适用于与其他阵列微生物缺失的集合，如大肠杆菌使用大肠杆菌?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
研究在CJN实验室由NSERC，加拿大癌症协会研究所（CCSRI），以及加拿大乳腺癌基金会（BC-育空分公司）工作的资助。
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Yeast Extract | BioBasic | G0961 | For YEPD liquid/solid media add to 1% final concentration (w/v) |
| Tyrptone Powder | BD Biosciences | 211820 | For YEPD liquid/solid media add to 2% final concentration (w/v) |
| Dextrose | Anachemia | 31096-380 | For YEPD liquid/solid media add to 2% final concentration (w/v) – Do not autoclave. Prepare 20% stock solution, filter sterilize, and add to media after autoclaving. |
| Agar A | Bio Basic | FB0010 | For YEPD solid media add to 2% final concentration (w/v) |
| G418 | A.G. Scientific Inc. | G-1033 | Prepare 1000x stock at 200mg/ml in dH2O and filter sterilize. |
| 12 well plate | Greiner Bio One | 655180 | |
| 5ml culture tubes | Evergreen Scientific | 222-2376-080 | |
| 10cm Petri Dish | VWR | 25384-302 | |
| ROTOR HDA | Singer Instruments | ROT-001 | high-throughput microbial array pinning robot |
| PLUSPLATE© Petri Dish | Singer Instruments | PLU-001 | Box of 200 dishes |
| 384 Short-Pin RePad | Singer Instruments | RP-MP-384 | Box of 1000 pads |
| 1536 Short-Pin RePad | Singer Instruments | RP-MP-1536 | Box of 1000 pads |
| Alternative Pinning Tools: | |||
| Fully Automated Robtic Systems | S&P robotics | http://www.sprobotics.com | Several automated colony handling robitic and imagining systems available. |
| Manual Pinning Tools | V&P Scientific | http://www.vp-scientific.com | Handheld replication tools and accessories. |
References
- Giaever, G., et al. Functional profiling of the Saccharomyces cerevisiae genome. Nature. 418, 387-391 (2002).
- Winzeler, E. A., et al. Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis. Science. 285, 901-906 (1999).
- Breslow, D. K., et al. A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Methods. 5, 711-718 (2008).
- Li, Z., et al. Systematic exploration of essential yeast gene function with temperature-sensitive mutants. Nat Biotechnol. 29, 361-367 (2011).
- Costanzo, M., et al. The genetic landscape of a cell. Science. 327, 425-431 (2010).
- Parsons, A. B., et al. Integration of chemical-genetic and genetic interaction data links bioactive compounds to cellular target pathways. Nat Biotechnol. 22, 62-69 (2004).
- Parsons, A. B., et al. Exploring the mode-of-action of bioactive compounds by chemical-genetic profiling in yeast. Cell. 126, 611-625 (2006).
- Hillenmeyer, M. E., et al. The chemical genomic portrait of yeast: uncovering a phenotype for all genes. Science. 320, 362-365 (2008).
- Hillenmeyer, M. E., et al. Systematic analysis of genome-wide fitness data in yeast reveals novel gene function and drug action. Genome Biol. 11, R30 (2010).
- Smith, A. M., et al. Competitive genomic screens of barcoded yeast libraries. J Vis Exp. (54), (2011).
- Bowie, D., Parvizi, P., Duncan, D., Nelson, C. J., Fyles, T. M. Chemical-genetic identification of the biochemical targets of polyalkyl guanidinium biocides. Organic & Biomolecular Chemistry. 11, 4359-4366 (2013).
- Alamgir, M., Erukova, V., Jessulat, M., Azizi, A., Golshani, A. Chemical-genetic profile analysis of five inhibitory compounds in yeast. BMC Chem Biol. 10 (6), (2010).
- Amberg, D. C., Burke, D., Strathern, J. N. . Cold Spring Harbor Laboratory. Methods In Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual. , (2005).
- Baryshnikova, A., et al. Synthetic genetic array (SGA) analysis in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Methods Enzymol. 470, 145-179 (2010).
- Young, B. P., Loewen, C. J. Balony: a software package for analysis of data generated by synthetic genetic array experiments. BMC Bioinformatics. 14, 354 (2013).
- Wagih, O., et al. SGAtools: one-stop analysis and visualization of array-based genetic interaction screens. Nucleic Acids Res. 41, W591-W596 (2013).
- Dittmar, J. C., Reid, R. J., Rothstein, R. ScreenMill: a freely available software suite for growth measurement, analysis and visualization of high-throughput screen data. BMC Bioinformatics. 11, 353 (2010).
- Longley, D. B., Harkin, D. P., Johnston, P. G. 5-fluorouracil: mechanisms of action and clinical strategies. Nat Rev Cancer. 3, 330-338 (2003).
- Lum, P. Y., et al. Discovering modes of action for therapeutic compounds using a genome-wide screen of yeast heterozygotes. Cell. 116, 121-137 (2004).
- Toussaint, M., Conconi, A. High-throughput and sensitive assay to measure yeast cell growth: a bench protocol for testing genotoxic agents. Nat Protoc. 1, 1922-1928 (2006).
- Robinson, M. D., Grigull, J., Mohammad, N., Hughes, T. R. FunSpec: a web-based cluster interpreter for yeast. BMC Bioinformatics. 3, 35 (2002).
- Huang da, W., Sherman, B. T., Lempicki, R. A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat Protoc. 4, 44-57 (2009).
- Huang da, W., Sherman, B. T., Lempicki, R. A. Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists. Nucleic Acids Res. 37, 1-13 (2009).
- Hong, E. L., et al. Gene Ontology annotations at SGD: new data sources and annotation methods. Nucleic Acids Res. 36, D577-D581 (2008).
- Fang, F., Hoskins, J., Butler, J. S. 5-fluorouracil enhances exosome-dependent accumulation of polyadenylated rRNAs. Mol Cell Biol. 24, 10766-10776 (2004).
- Baba, T., et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2, 0008 (2006).
- Nichols, R. J., et al. Phenotypic landscape of a bacterial cell. Cell. 144, 143-156 (2011).
- Dai, J., et al. Probing nucleosome function: a highly versatile library of synthetic histone H3 and H4 mutants. Cell. 134, 1066-1078 (2008).
