在全基因组范围内筛选酵母中砷抗性相关基因

Genome-wide Identification of Genes Whose Disruption Confer Resistance to Arsenic in Saccharomyces cerevisiae

砷化物是广泛应用的抗癌药物,特别是对白血病有显著疗效.然而,治疗过程中病人会因对砷化物具有耐药性而影响治疗效果,而目前对于砷抗性机制尚缺全面深入的研究.利用酵母作为模式生物,使用不同浓度的砷对由4757个酵母缺失型突变体组成的菌株库进行筛查.共鉴定出104个基因/开放阅读框(ORF),其缺失导致酵母对砷的抗性增加.生物信息学分析结果提示,这些基因与mRNA分解代谢、应激反应、组蛋白乙酰化和蛋白质合成及分解代谢等功能有关.同时这些基因中多于半数具有哺乳动物同源类似物.所以,进一步研究这些基因有望为人类砷化物的耐药性及毒性机制研究提供富有价值的新线索.

Arsenic is highly effective in treating acute promyelocytic leukemia （APL）, especially for relapsed patients. However, the treatment is highly affected by the resistance of the drug by patients, while the arsenic-resistance mechanism has not been well studied. A genome-wide screen was performed against a pool of 4 757 Saccharomyces cerevisiae mutants, each with one different gene individually deleted, to isolate genes that may mediate cellular resistance to arsenic. A one-step selection method was used. An aliquot of the pooled yeast library was plated on YPD agar plates supplemented with 3 mmol/L sodium arsenite. The genomic DNAs of the arsenic resistant strains were separately extracted, and amplified by PCR to get DNA fragments with UPTAG. The corresponding deleted genes were identified by comparing the PCR-amplified sequences with the UPTAG sequences from the Saccharomyces Genome Deletion Project. Mutations were identified in 104 genes/ORFs showing resistance to arsenic as compared to the wild type strain. To rule out the possibility that the resistant phenotype of these mutants is a result of arsenic-induced mutation during the screening process, the individual deletion strains from the mutant collection were picked up and tested individually for arsenic resistance using the spot assay. Of the 104 mutants identified in the screen, all exhibited significantly more resistance than the wild-type cells. Among the verified strains, 32 mutants turned out to have stronger phenotype that is resistant to 5 mmol/L arsenite. Five of the 32 mutants （FPS1, TMA20, UPF3, YAL066W, YOR309C） showed resistance to 7 mmol/L arsenite. The phenotype data were mapped onto the regulatory network. Bioinformatic studies of the genes revealed four neighborhoods, including mRNA catabolism, response to stress, histone acetylation, and protein synthesis and catabolism.