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High-resolution yeast quiescence profiling in human-like media reveals interacting influences of auxotrophy and nutrient availability
bioRxiv - Genetics Pub Date : 2020-05-27 , DOI: 10.1101/2020.05.25.114801
Sean L Santos , Samantha Laflin , Audrie Broadway , Cosby Burnett , Joline Hartheimer , John W Rodgers , Daniel L Smith , John L Hartman

Yeast cells survive in stationary phase culture by entering quiescence, which is measured by colony forming capacity upon nutrient re-exposure. Yeast chronological lifespan (CLS) studies, employing the comprehensive collection of gene knockout strains, have correlated weakly between independent laboratories, which is hypothesized to reflect differential interaction between the deleted genes, auxotrophy, media composition and other assay conditions influencing quiescence. This hypothesis was investigated by high-throughput quiescence profiling of the parental prototrophic strain, from which the gene deletion strain libraries were constructed, and all possible auxotrophic allele combinations in that background. Defined media resembling human cell culture media promoted long-term quiescence, and was used to assess effects of glucose, ammonium sulfate, auxotrophic nutrient availability, Target of Rapamycin signaling, and replication stress. Frequent, high-replicate measurements of colony forming capacity from cultures aged past 60 days provided profiles of quiescence phenomena such as gasping and hormesis. Media acidification was assayed in parallel to assess correlation. Influences of leucine, methionine, glucose, and ammonium sulfate metabolism were clarified, and a role for lysine metabolism newly characterized, while histidine and uracil perturbations had less impact. Interactions occurred between glucose, ammonium sulfate, auxotrophy, auxotrophic nutrient limitation, aeration, TOR signaling, and/or replication stress. Weak correlation existed between media acidification and maintenance of quiescence. In summary, experimental factors, uncontrolled across previous genome-wide yeast CLS studies, influence quiescence and interact extensively, revealing quiescence as a complex metabolic and developmental process that should be studied in a prototrophic context, omitting ammonium sulfate from defined media, and employing highly replicable protocols.

中文翻译:

类似于人的培养基中的高分辨率酵母静态分析揭示营养缺陷和养分利用率的相互作用

酵母细胞通过进入静止状态而在固定相培养中存活,这通过营养物再暴露后的菌落形成能力来衡量。酵母时序寿命(CLS)研究采用了基因敲除菌株的全面收集,在独立实验室之间的相关性较弱,据推测,这反映了缺失基因,营养缺陷型,培养基成分和其他影响静止的测定条件之间的差异性相互作用。该假说是通过对亲本原养型菌株进行高通量静态分析来研究的,从中构建了基因缺失菌株文库,并建立了该背景下所有可能的营养型等位基因组合。类似于人类细胞培养基的特定培养基可促进长期静止,并用于评估葡萄糖,硫酸铵,营养缺陷型营养素的可用性,雷帕霉素信号转导的靶标和复制压力。对60天以上的培养物进行的菌落形成能力的频繁重复测量,提供了诸如喘气和兴奋等静止现象的概况。平行测定培养基酸化以评估相关性。阐明了亮氨酸,蛋氨酸,葡萄糖和硫酸铵代谢的影响,并新近表征了赖氨酸代谢的作用,而组氨酸和尿嘧啶摄动的影响较小。葡萄糖,硫酸铵,营养缺陷,营养缺陷型营养限制,通气,TOR信号传导和/或复制应激之间发生相互作用。介质酸化与维持静态之间存在弱相关性。总之,实验因素
更新日期:2020-05-27
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