An Approach to monitor environmental pollutants: Survey of it's target in cells by using transformed yeast

为了改进环境污染物毒性筛选的方法，利用96孔板进行了基因组DNA文库转导酵母和筛选对环境污染物具有抗性或敏感性基因的酵母细胞。该基因缺失菌株可用于镉毒性的筛选。利用这些材料和方法，研究了细胞内监测环境污染物所涉及的生物紊乱指数的指标。据报道，GFA1为酵母细胞提供了对甲基汞的抗性，而MSN2的过度表达则起到了过敏性作用。GFA1编码L-谷氨酰胺-D-果糖-6-磷酸氨基转移酶，是甲基汞的靶分子，是一种必需的酶。MSN_2是参与酿酒酵母多应激反应的转录激活子。为了研究msn2依赖的对甲基汞的敏化方式，减少甲基…的基因寻找了更多因MSN_2过表达而增强的汞毒性。在本研究中，酵母过表达MS2抑制了GEA1的转录水平。因此，甲基汞的毒性增强可能是由于GEA1转录本水平降低所致。通过对MSN_2中各结构域的分析，推测细胞质中的某些功能可能参与了酵母对甲基汞的过敏反应。通过筛选和分析与MSN2相关的基因缺失的酵母菌，结果表明，MSN2的过表达可能抑制了转录因子SOK2和SOK1的功能，并与MSN2相互作用，导致GFA1转录水平下降。对260株在350μM氯化镉溶液中生长受到抑制的基因缺失酵母菌，在0-20μM氯化镉溶液中进行了剂量依赖性生长试验。其中，与液泡蛋白运输相关的VPS63、VPS68或VAM3的缺失与镉的抗性有关，也与甲基汞有关。较少

To improve procedures for screening toxicity of environmental pollutants, transfection of yeast with a genomic DNA library and screening of yeast cells transformed with a resistant or a sensitive gene to environmental pollutants were performed by using 96well plate for culture. The gene deletion strains were useful for screening of cadmium toxicity. By using these materials and methods, intracellular targets were investigated for monitoring indices of biological disorder involved in environmental pollutants. It was reported that GFA1 provides methylmercury resistance to yeast cells, while Msn2 overexpression works for hypersensitivity. GFA1 encodes L-glutamineD-fructose-6-phosphate amidotransferase (GFAT), which is an essential enzyme, and is the target molecule of methylmercury. Msn2 is the transcriptional activator for gene involvement in the multi stress response in Saccharomyces cerevisiae. To examine the way of Msn2 dependent sensitization to methylmercury, genes that reduce methy … More lmercury toxicity enhanced by Msn2-overexpressing were searched. In the present study, yeast overexpression of Msn2 suppressed the levels of GEA1 transcript. Thus, it is suggested that the enhanced toxicity of methylmercury might be due to reduction of levels of GEA1 transcript. By analysis of each domain in Msn2, it was suggested that some functions in cytoplasm might be involved in hypersensitivity of yeast to methymercury. The results by screening and analyzing yeast lacking genes related to Msn2 indicated a possibility that overexpression of Msn2 suppresses the function of Sok2 and Sok1, transcription factors and interacting with Msn2, and it causes reduction in the levels of GFA1 transcript. Two hundred sixty strains of gene deletion yeasts, which were growth-inhibited in 350μM cadmium chloride, were checked for dose-dependent growth under 0-20μM CdCl_2. Yeast gained cadmium resistance in some gene deletion strains. Among them, the deletion of VPS63, VPS68 or VAM3 related to vacuolar protein trafficking gives cadmium resistance as well as methylmercury. Less