裂殖酵母中不稳定遗传耐药菌株的筛选及其运用

doi:10.16288/j.yczz.24-266

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Hereditas(Beijing) ›› 2025, Vol. 47 ›› Issue (5): 589-599.doi: 10.16288/j.yczz.24-266

• Research Article • Previous Articles Next Articles

Screening and application of unstable genetically resistant strains in fission yeast

Lufeng Dan¹^,²(), Yiwen Chu¹^,², Xinrong Wang¹^,², Xiangwei He³()

1. School of Pharmacy, Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu 610106, China
2. Antibiotic Research and Re-Evaluation Key Laboratory of Sichuan Province, Chengdu University, Chengdu 610106, China
3. Institute of Life Sciences, Zhejiang University, Hangzhou 310000, China

Received:2024-10-14 Revised:2024-12-18 Online:2025-04-25 Published:2025-01-07
Contact: Xiangwei He E-mail:danlufeng@zju.edu.cn;1187853534@qq.com
Supported by:
National Natural Science Foundation of China(32400477);Natural Science Foundation of Sichuan Province(2023NSFSC1231)

Abstract

Abstract:

Reversible alterations at DNA sequence or epigenetic levels can result in phenotypes that are unstably inherited. The reversibility of these inheritable changes might be uniquely beneficial for adaption to possible fluctuations in environment. However, unstable changes are always ignored for the genetic instability in traditional studies, especially in the cause of drug resistance. In this study, we conduct a specific genetic screen in fission yeast using rapamycin (+caffeine) and obtain 173 resistant isolates. In contrast to the common strategy of isolating stable genetic mutants, we passage the cell culture with rapamycin resistance on drug free condition and test the resistance of offspring every five days, and obtain 14 strains that exhibit unstable resistance to rapamycin (the drug resistance is lost randomly among the cell progenies without drug selection pressure). Further studies show that the unstable genetic resistance of some strains is regulated by reversible DNA sequence alterationat the ssp1 gene locus. This study provides new insights and relevant scientific basis for the regulatory mechanism of unstable drug resistance in the process of rapamycin as a clinical anti-tumor drug, and a new possible target for solving the problem of drug resistance.

Key words: drug resistance, genetic instability, reversible mutation, rapamycin

Lufeng Dan, Yiwen Chu, Xinrong Wang, Xiangwei He. Screening and application of unstable genetically resistant strains in fission yeast[J]. Hereditas(Beijing), 2025, 47(5): 589-599.

Figures/Tables 9

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References 31

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菌株名称	基因型	来源
446 (野生型)	h^- ade6-210, leu1-32, ura4D	何向伟实验室
447 (野生型)	h⁺, ade6-216, leu1-32, ura4D	何向伟实验室
gaf1∆	h^?, gaf1∆::kanMX6, ade6∆::hphMX6, cnt2::ade6-natMX6, leu1-32, ura4D	基因敲除库
Epm101	h^-, ssp1^MTD-AGCCA, ade-210, leu1-32, ura4D	本研究筛选
Epm102	h^-, ssp1^MTD-GCTTT, ade-210, leu1-32, ura4D	本研究筛选
Epm201-206	h⁺, ssp1^MTD-GTCGTCCG, ade-210, leu1-32, ura4D	本研究筛选
Epm103-108	h^-, ade-210, leu1-32, ura4, 未知改变	本研究筛选
Epm102-R	h^-, ssp1^MTD-GCTTT, ade-210, leu1-32, ura4D	本研究分离
Epm102-S	h^-, ade6-210, leu1-32, ura4D	本研究分离
ssp1∆	h^-, ssp1∆::kanMX6, leu1-32, ura4D	本研究构建
Epm101-wt	h^-, ssp1^wt::kanMX6, leu1-32, ura4D	本研究构建
Epm102-wt	h^-, ssp1^wt::kanMX6, leu1-32, ura4D	本研究构建

咖啡因浓度(mmol/L)	涂布10³个细胞时的菌落数	涂布10⁴个细胞时的菌落数	涂布10⁵个细胞时的菌落数
0 mmol/L(雷帕霉素：0 ng/mL)	10³	10⁴	10⁵
6 mmol/L(雷帕霉素：100 ng/mL)	2~20	10⁴	10⁵
8 mmol/L(雷帕霉素：100 ng/mL)	0	0~2	2~20
10 mmol/L(雷帕霉素：100 ng/mL)	0	0	0~10

菌株	无药处理第1天	无药处理第5天	无药处理第10天	无药处理第15天	无药处理第20天
WT	0%	0%	0%	0%	0%
gaf1△	100%	100%	100%	100%	100%
Epm101	100%	100%	93.5%	9.7%	0%
Epm102	100%	100%	97.5%	35%	0%
Epm201	100%	97%	90.8%	7.8%	0%
Epm202	100%	100%	100%	67%	1%
Epm203	100%	100%	80%	5%	0%
Epm204	100%	100%	99%	74%	1%
Epm205	99.4%	95.4%	55.8%	0.6%	0%

回交	回交比(R∶S )	杂交	杂交比(R∶S )
Epm201×WT	2∶2 (37/42)	Epm101×Epm201	4∶0 (19/19)
Epm203×WT	2∶2 (38/39)	Epm102×Epm201	4∶0 (25/26)
Epm205×WT	2∶2 (46/46)	Epm101×Epm205	4∶0 (20/20)
Epm206×WT	2∶2 (36/36)	Epm102×Epm205	4∶0 (24/24)
Epm103×WT	2∶2 (23/24)	Epm201×Epm205	4∶0 (36/37)
Epm104×WT	2∶2 (37/37)	Epm203×Epm205	4∶0 (26/26)
Epm105×WT	2∶2 (16/17)	Epm106×Epm103	4∶0 (16/16)
Epm106×WT	2∶2 (30/30)	Epm106×Epm104	4∶0 (26/26)
Epm107×WT	2∶2 (26/27)	Epm106×Epm105	4∶0 (17/17)
Epm108×WT	2∶2 (20/20)	Epm106×Epm107	4∶0 (20/20)
Epm103×WT	2∶2 (23/24)	Epm106×Epm108	4∶0 (15/15)
		Epm104×Epm105	4∶0 (11/11)
		Epm206×Epm106	3∶1 (11/17)，2∶2 (6/17)

Screening and application of unstable genetically resistant strains in fission yeast

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