A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds

HE Ying; XIA Xingya; WEI Jiali; ZHENG Feng

doi:10.11665/j.issn.1000-5048.20210213

Journal of China Pharmaceutical University > 2021 > 52(2): 236-244. > DOI: 10.11665/j.issn.1000-5048.20210213

HE Ying, XIA Xingya, WEI Jiali, ZHENG Feng. A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds[J]. Journal of China Pharmaceutical University, 2021, 52(2): 236-244. DOI: 10.11665/j.issn.1000-5048.20210213

Citation:

PDF (2269 KB)

A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds

1.
Department of Pharmaceutical Analysis, China Pharmaceutical University, Nanjing 210009, China
2.
Key Laboratory of Drug Quality Control and Pharmacovigilance, Ministry of Education, China Pharmaceutical University, Nanjing 210009, China

Funds: This study was supported by the National Natural Science Foundation of China (No.82073815)

More Information

Received Date: November 15, 2020
Revised Date: January 08, 2021

Graphical Abstract

Abstract

Abstract

pdr5 and snq2 gene knockout was constructed by overlap PCR, and the effects of pdr5 and snq2 mutations on the accuracy and sensitivity of RNR2 promoter-regulated yeast cell sensors in detecting genotoxic compounds were studied. The yeast cell sensors of wild-type, single-gene mutation of pdr5, single-gene mutation of snq2, and double-gene mutation of pdr5 and snq2 were studied. The cell growth inhibition and the fluorescence induction factor of the yeast cell sensors exposed to a series of concentrations of methyl methanesulfonate(MMS), ethyl methanesulfonate(EMS), cisplatin, 4-nitroquinoline-N-oxide(4NOQ), 5-fluorouracil(5-FU), hydroxyurea, salicylic acid and glucose solution were investigated. The results showed that overlap PCR method could efficiently construct the mutant yeast cell sensor. The accuracy of cell sensors of single-gene mutation of snq2 and double-gene mutation of pdr5 and snq2 were both 100%, higher than that of cell sensors of wild-type and single-gene mutation of pdr5 (87.5%). The yeast cell sensor of double-gene mutation of pdr5 and snq2 showed the highest sensitivity in detecting genotoxicity. This study provides guidance for the construction of high accuracy and sensitivity yeast cell sensor, and foundation for further functional research of yeast cell membrane transporter gene pdr5 and snq2.
- overlap PCR,
- yeast cell sensor,
- RNR2 promoter,
- genotoxicity

FullText(HTML)

References (21)

References

[1]	. J Pharm Biomed Anal， 2018， 156： 307-312.
[2]	Sun YQ， Zhang XY， Yan YM， et al. Identification and genotoxicity evaluation of two carbamate impurities in rasagiline［J］. RSC Adv， 2016， 6（108）： 106268-106274.
[3]	Xian ZR， Sun CM， Luo XF， et al. Profiling of genotoxic impurities in a lidocaine hydrochloride injection［J］. J China Pharm Univ（中国药科大学学报）， 2020， 51（4）： 466-471.
[4]	Friedberg EC. DNA damage and repair［J］. Nature， 2003， 421（6921）： 436-440.
[5]	Jarque S， Bittner M， Blaha L， et al. Yeast biosensors for detection of environmental pollutants： current state and limitations［J］. Trends Biotechnol， 2016， 34（5）： 408-419.
[6]	Lan J， Rahman SM， Gou N， et al. Genotoxicity assessment of drinking water disinfection byproducts by DNA damage and repair pathway profiling analysis［J］. Environ Sci Technol， 2018， 52（11）： 6565-6575.
[7]	Lan J， Hu M， Gao C， et al. Toxicity assessment of 4-methyl-1-cyclohexanemethanol and its metabolites in response to a recent chemical spill in west Virginia， USA［J］. Environ Sci Technol， 2015， 49（10）： 6284-6293.
[8]	Walmsley RM， Billinton N. How accurate is in vitro prediction of carcinogenicity？［J］. Br J Pharmacol， 2011， 162（6）： 1250-1258.
[9]	Chen YL， Feng JJ， Yang HX， et al. Determination of genotoxic impurities of alkyl methanesulfonates in methanesulfonic acid by gas chromatography-mass spectrometry［J］. J China Pharm Univ（中国药科大学学报）， 2020， 51（4）： 472-478.
[10]	Eki T. Yeast-based genotoxicity tests for assessing DNA alterations and DNA stress responses： a 40-year overview［J］. Appl Microbiol Biotechnol， 2018， 102（6）： 2493-2507.
[11]	Lan JQ， Gou N， Rahman SM， et al. A quantitative toxicogenomics assay for high-throughput and mechanistic genotoxicity assessment and screening of environmental pollutants［J］. Environ Sci Technol， 2016， 50（6）： 3202-3214.
[12]	Bui VN， Nguyen TTH， Mai CT， et al. Procarcinogens - determination and evaluation by yeast-based biosensor transformed with plasmids incorporating RAD54 reporter construct and cytochrome P450 genes［J］. PLoS One， 2016， 11（12）： e0168721.
[13]	Mewes HW， Albermann K， B?hr M， et al. Overview of the yeast genome［J］. Nature， 1997， 387（6632 suppl）： 7-65.
[14]	Sancar A， Lindsey-Boltz LA， Unsal-Ka?maz K， et al. Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints［J］. Annu Rev Biochem， 2004， 73： 39-85.
[15]	Bui VN， Nguyen TTH， Bettarel Y， et al. Genotoxicity of chemical compounds identification and assessment by yeast cells transformed with GFP reporter constructs regulated by the PLM2 or DIN₇ promoter［J］. Int J Toxicol， 2015， 34（1）： 31-43.
[16]	Nordlund P， Reichard P. Ribonucleotide reductases［J］. Annu Rev Biochem， 2006， 75（1）： 681-706.
[17]	Lu Y， Tian Y， Wang R， et al. Dual fluorescent protein-based bioassay system for the detection of genotoxic chemical substances in Saccharomyces cerevisiae［J］. Toxicol Mech Methods， 2015， 25（9）： 698-707.
[18]	Dimitrov M， Venkov P， Pesheva M. The positive response of Ty1 retrotransposition test to carcinogens is due to increased levels of reactive oxygen species generated by the genotoxins［J］. Arch Toxicol， 2011， 85（1）： 67-74.
[19]	Wei T， Zhang C， Xu X， et al. Construction and evaluation of two biosensors based on yeast transcriptional response to genotoxic chemicals［J］. Biosens Bioelectron， 2013， 44： 138-145.
[20]	Li C， Lou H. From gene editing to genome reconstitution： evolving techniques in yeast［J］. Hereditas（遗传）， 2015， 37（10）： 1021-1028.
[21]	Blagus T， Zager V， Cemazar M， et al. A cell-based biosensor system HepG2CDKN1A-DsRed for rapid and simple detection of genotoxic agents［J］. Biosens Bioelectron， 2014， 61： 102-111.

Cited By

Cited by

Periodical cited type(0)

Other cited types(1)

Get Citation

PDF

XML

Article views (135) PDF downloads (545) Cited by(1)

Turn off MathJax

Article Contents

Abstract

References

A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds

Abstract

References

Cited by

Periodical cited type(0)

Other cited types(1)

Catalog

Export File

Citation

Format

Content