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.

Keywords:

• RAD54 promoter
• yeast cell
• chemical mutagen
• yEGFP
• dose-effect relationship

Acknowledgements

The authors would like to thank Mr. Yang Kun from the Gene Company Limited in China for valuable technical assistance.

Disclosure statement

Part of this work was supported by Yi Xin Lu, who is studying for a Master degree in our college.

Additional information

Funding

This study was supported by The National Natural Science Foundation of China (No. 81241099).