title: Construction and comparison of yeast whole-cell biosensors regulated by two RAD54 promoters capable of detecting genotoxic compounds.
abstract: Two yeast enhanced green fluorescence protein (yEGFP) yeast reporter vectors, pR1558-yEGFP and pR406-yEGFP, which are regulated by two RAD54 promoters containing 406-bp and 1558-bp DNA sequences, respectively, were constructed using molecular biological techniques and transformed into yeast for the screening of genotoxins. The constructed biosensors were named W303-1A/R1558-yEGFP and W303-1A/R406-yEGFP. To quantify biosensor performance, both transformed yeast cells were exposed to multiple doses of genotoxins including methylmethane sulfonate (MMS; a DNA alkylating agent), 4-nitroquinoline-N-oxide (4-NQO; a DNA cleavage agent), 5-fluorouracil (5-Fu; an inhibitor of polymerases and topoisomerases) and colchicine and canavanine (affecting other biochemical activities). The yeast bioassay performance was analyzed using fluorescence-activated cell sorting (FACS) and Multi-Mode Reader in a 96-well black microplate. The observed W303-1A/R1558-yEGFP dose-effect relationship was more obvious and the maximum inductions were 5.96-fold (MMS), 2.19-fold (4-NQO) and 2.71-fold (5-Fu); the corresponding values for W303-1A/R406-yEGFP were 2.53-, 1.50- and 1.91-fold, respectively. It is suggested that it is best to select the entire RAD54 promoter when constructing recombinant yeast cells for screening mutagens.
authors.affiliation: a Preventive Medicine Department , Yangzhou Medical College, Yangzhou University , Yangzhou , Jiangsu , China.
authors.forename: Yongjie
authors.initials: Y
authors.lastname: Tian
authors.affiliation: a Preventive Medicine Department , Yangzhou Medical College, Yangzhou University , Yangzhou , Jiangsu , China.
authors.forename: Yixin
authors.initials: Y
authors.lastname: Lu
authors.affiliation: a Preventive Medicine Department , Yangzhou Medical College, Yangzhou University , Yangzhou , Jiangsu , China.
authors.forename: Xiuju
authors.initials: X
authors.lastname: Xu
authors.affiliation: a Preventive Medicine Department , Yangzhou Medical College, Yangzhou University , Yangzhou , Jiangsu , China.
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.affiliation: a Preventive Medicine Department , Yangzhou Medical College, Yangzhou University , Yangzhou , Jiangsu , China.
authors.forename: Tianqi
authors.initials: T
authors.lastname: Zhou
authors.affiliation: a Preventive Medicine Department , Yangzhou Medical College, Yangzhou University , Yangzhou , Jiangsu , China.
authors.forename: Xiangming
authors.initials: X
authors.lastname: Li
mesh_terms: D015374:Biosensing Techniques / Q000379:methods*; D004249:DNA Damage; D004265:DNA Helicases / Q000235:genetics*; D045643:DNA Repair Enzymes / Q000235:genetics*; D005434:Flow Cytometry; D005470:Fluorometry; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D009153:Mutagens / Q000633:toxicity*; D010957:Plasmids; D011401:Promoter Regions, Genetic; D012441:Saccharomyces cerevisiae / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics*; D020798:Two-Hybrid System Techniques
chemical_list: D009153:Mutagens; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; C075468:RAD54 protein, S cerevisiae; D004265:DNA Helicases; D045643:DNA Repair Enzymes
keywords: RAD54 promoter; chemical mutagen; dose-effect relationship; yEGFP; yeast cell