A functional screen for copper homeostasis genes identifies a pharmacologically tractable cellular system. Saccharomyces cerevisiae

Copper is an essential component of cytochrome C oxidase (i.e. complex IV of the electron transport chain), and is thus critical for the survival of aerobic organisms. If copper is not properly regulated in the body however, it can be extremely cytotoxic and genetic mutations that compromise copper homeostasis result in severe clinical phenotypes. Understanding how cells maintain optimal copper levels is therefore highly relevant to human health. We found that addition of copper to culture medium leads to increased respiratory growth of yeast, a phenotype which we then systematically and quantitatively measured in 5050 homozygous diploid deletion strains using microarrays. Overall design: In the yeast homozygous gene deletion collection, both copies of every non-essential gene are deleted in a diploid strain. Each strain contains unique 20-bp “barcode” sequences flanked by common priming sites, allowing the relative abundance of individual strains to be quantitatively monitored within a pool of competitively-grown strains. To identify genes that are important for Cu-dependent respiratory growth, we measured the fitness of 5050 homozygous deletion strains in parallel in rich media (i.e. Yeast Extract, Peptone) using either dextrose, ethanol, glycerol, or lactate as a carbon source, in the presence or absence of 500 µM CuSO4 for 9 generations. Each condition was tested in triplicate. Please note that there is a large percentage of null-values (i.e. Not Available) in the respiration deficient-pool samples because the respiration deficient pool consists of only a small subset of the complete pool (represented by 331 barcodes instead of 9937). For our analysis we discarded the remaining barcodes by assigning the null-value to them.

铜是细胞色素C氧化酶（即电子传递链复合物IV）的必需组成成分，因此对需氧生物的存活至关重要。但若体内铜稳态调控失衡，铜会产生极强的细胞毒性；而破坏铜稳态的基因突变会引发严重的临床表型。因此，阐明细胞维持最佳铜水平的机制对人类健康研究具有重要意义。

我们发现，向培养基中添加铜可增强酵母的呼吸生长表型，随后我们利用微阵列（microarrays）技术，对5050株纯合二倍体基因缺失菌株的该表型进行了系统性定量检测。

实验总体设计：在酵母纯合基因缺失文库中，二倍体菌株的每个非必需基因的两个拷贝均被敲除。每株菌株均带有由通用引物序列侧翼连接的独特20 bp"条形码（barcode）"序列，从而可在竞争性培养的菌株混合池中，对单株菌株的相对丰度进行定量监测。

为筛选出铜依赖型呼吸生长相关的关键基因，我们分别以葡萄糖、乙醇、甘油或乳酸作为碳源，在酵母提取物与蛋白胨的富营养培养基中，并行检测5050株纯合二倍体基因缺失菌株的适合度：设置添加或不添加500 μM硫酸铜（CuSO4）的两组，连续传代培养9代。每个实验条件均设置三次生物学重复。

请注意，呼吸缺陷型混合样本中存在大量空值（即Not Available，不可用数据），这是因为呼吸缺陷型混合池仅包含完整混合池的极小一部分（仅对应331个条形码序列，而非总文库的9937个）。在后续分析中，我们将其余条形码对应的样本赋值为空值并予以剔除。