酵母模型揭示胁迫因子驱动基因组变异的研究进展

doi:10.13560/j.cnki.biotech.bull.1985.2023-0723

摘要/Abstract

摘要：

基因组变异是遗传疾病发生和物种演化的分子基础，这个过程受到细胞内外源理化因子的共同作用。模式生物酿酒酵母（Saccharomyces cerevisiae）基因组小且易于开展分子遗传操作，在探究基因组变异进化调控机制的相关研究中应用广泛。本文总结了酵母模型中典型的DNA变异检测遗传体系，包括利用报告基因检测DNA突变率和红白扇形菌落筛选染色体重组子等；讨论了高通量测序技术在检测自发性和胁迫因子诱导基因组变异中的应用；综述了运用酵母模型揭示温度波动、氧化压力、抗肿瘤药物、金属离子和辐射等胁迫因子对基因组稳定性的影响及遗传机制的研究进展。酵母在多种胁迫条件下均会发生适应性进化现象，特定的染色体结构变异是适应性背后的重要遗传机制之一。在酵母中结合遗传筛选体系和高通量分析手段阐释细胞胁迫因子与基因组变异的关联机制，可为全面理解生物基因组不稳定机理和物种进化规律提供新的视角。

关键词: 环境胁迫, 酿酒酵母, DNA突变, 基因组不稳定, 适应性进化

Abstract:

Genomic alteration is the molecular basis for the occurrence of genetic diseases and species evolution. This process is affected by the joint action of endogenous and exogenous physical and chemical factors of the cell. The genome in model organism Saccharomyces cerevisiae is small, thus it is easy for carrying out molecular genetic manipulation, and is widely used in research related to exploring the evolutionary regulation mechanism of genome variation. This article sums the typical DNA mutation detection genetic system in yeast models, including the use of reporter genes to detect DNA mutation rates and red and white/red sectoring colonies to screen chromosomal recombinants. Further the article also discussed the application of high-throughput sequencing technology in detecting spontaneous and stress factor-induced genomic alterations. Moreover, this review summarizes the research progresses in using S. cerevisiae to investigate the impacts of temperature fluctuations, oxidative stress, anti-tumor drug, heavy metal ions, radiation, and other stressors on genome stability and genetic mechanisms. Yeast cells are prone to undergo adaptive evolution under multiple stressful conditions, and specific chromosomal structural variations is an important genetic mechanism behind the adaptation. It may provide new insights for a comprehensive understanding of the impact of stressors on genome stability and the evolutionary patterns of species under different environments by combining genetic screening systems and high-throughput analysis methods in yeast to elucidate the relationship between cellular stress factors and genome variations.

Key words: environmental stress, Saccharomyces cerevisiae, DNA mutation, genomic instability, adaptive evolution

祝瑛萱, 李克景, 何敏, 郑道琼. 酵母模型揭示胁迫因子驱动基因组变异的研究进展[J]. 生物技术通报, 2023, 39(11): 191-204.

ZHU Ying-xuan, LI Ke-jing, HE Min, ZHENG Dao-qiong. Research Progress in the Exploring Genomic Variations Driven by Stress Factors Using the Yeast Model[J]. Biotechnology Bulletin, 2023, 39(11): 191-204.

图/表 6

图1 酵母红白扇形菌落系统筛选染色体交换重组子该二倍体酵母的ade2-1等位基因是赭石突变体，一份拷贝的SUP4-o基因可以部分抑制赭石突变，亲株菌落呈粉色。染色体交换重组事件可使一个子细胞具有两份拷贝的SUP4-o，在平板上形成白色菌落；另一个子细胞没有SUP4-o基因，形成的菌落显红色

Fig. 1 Yeast white/red sectoring colony system to screen chromosomal crossover recombinants The homologous allele ade2-1 in the diploid yeast strain is ocher mutant. One copy of the SUP4-o gene can partially suppress this ochre mutation of ade2-1, making the colonies appear pink. Chromosomal crossover events can lead to one daughter cell having two copies of the SUP4-o gene, causing the colonies to appear white; another daughter cell without the SUP4-o gene would result in red colonies

图2 DNA同源重组诱导杂合二倍体基因组杂合性丢失 A：同源重组修复姐妹染色体上双链断裂形成内部LOH，也称基因转换；B：同源重组修复姐妹染色体上双链断裂形成末端LOH，也称染色体交换。在断点附近形成3∶1的基因转换区域（黑框）；C：G1期染色体断裂会使两条姐妹染色体上均存在DNA断裂。这种情况经修复后在断点处形成4∶0的基因转换区域

Fig. 2 LOH events induced by DNA homologous recombination in heterozygous diploid A: The interstitial LOH or gene conversion is resulted from repairing the DSB（double-strand DNA breaks）on a sister chromatid via homologous recombination. B: A 3∶1 pattern of the crossover associated with gene conversion tract（black rectangle）indicates repairing the DSB on one sister chromatid via homologous recombination. C: Chromosome breakage during the G1 phase results in DNA breaks on both sister chromosomes. A 4∶0 gene conversion district is formed at the breakpoint after this situation is repaired

图3 利用SNP位点的相对测序深度分析基因组变异事件 A：染色体三体畸变事件；B：染色体单体畸变事件；C：染色体内部片段缺失；D：染色体末端缺失；E：基因转换引起内部杂合性丢失；F：染色体交换引起末端杂合性丢失。蓝色和红色的线条或点代表同源染色体或同源染色体上的SNP位点。SNP位点的相对测序深度0、0.5和1分别代表0、1和2份DNA拷贝

Fig. 3 Mapping genomic alterations by analyzing the relative sequencing coverages of SNP sites A: Chromosomal trisomy events. B: Chromosomal monosomy events. C: An internal deletion event in chromosome. D: A terminal deletion event in chromosome. E: Internal loss of heterozygosity（LOH）caused by gene conversion events. F: Crossover induced terminal LOH. The blue and red lines or points represent homologous chromosomes or SNP sites. The relative coverage value 0, 0.5, and 1 indicate zero, one, and two copies of DNA, respectively

图4 不同化合物处理酵母细胞诱导的特征性变异及遗传机制 A：溴酸钾诱导细胞产生ROS将碱基G氧化成8-oxo-G，其偏好与A配对并引起G > T的特征性突变；B：MMS和博来霉素处理细胞诱导产生无碱基AP位点。DNA聚合酶δ和Rev1具有识别AP位点的能力并分别偏好掺入A和C，造成G > T和T > G两类特征性变异。Rev1插入的C在DNA聚合酶ζ作用与AP位点邻近的G配对，这种模板滑步现象会引起碱基T的缺失；C：顺铂处理诱导C > T或C缺失的特征性突变

Fig. 4 Characteristic mutations and genetic mechanisms of yeast cells induced by different compounds A: Potassium bromate induces cells to produce ROS, which oxidizes base G to 8-oxo-G that prefers to pair with A, causing G > T. B: MMS and bleomycin treatment induces the generation of AP sites without base. DNA polymerase δ and Rev1 can recognize AP sites and prefer to insert A and C, respectively, causing G > T and T > G. The C inserted by Rev1 pairs with the G adjacent to the AP site when DNA polymerase ζ acts, this template slippage will cause one base T deletion. C: Cisplatin treatment induces C > T substitution or C deletion

表1 酵母基因组变异驱动适应性进化的研究

Table 1 Examples of evolutionary adaptation induced by genomic alternations in yeast

表型效应 Phenotypic effect	变异类型 Variation type	遗传机理 Genetic mechanism	参考文献 References
H₂O₂耐受性提高	Ⅳ号染色体扩增	编码硫氧还蛋白过氧化物酶的TSP2基因拷贝数上升	[70]
H₂O₂耐受性提高	VII号染色体上572-734 kb区域扩增	编码过氧化氢酶T的 CTT1基因拷贝数上升	[24]
热胁迫耐受性提高	III号染色体扩增	该染色体上至少14个与耐热相关基因表达量上调	[75]
博来霉素耐受性提高	SKY1突变， XIII号染色体右臂缺失	SKY1基因编码蛋白激酶，其缺失可抑制膜转运蛋白活性，减少药物进入细胞	[16]
乙醇耐受性提高	III号染色体扩增	未知	[76]
对亚硫酸盐环境耐受性提高	VIII和XVI号染色体易位，XV和XVI号染色体易位	染色体易位引起SSU1基因表达量提高，SSU1基因编码可泵出亚硫酸盐的膜蛋白	[77]
氮限制环境耐受性提高	含有GAP1基因区域形成eccDNA	GAP1基因编码氨基酸透性酶	[73]
尿囊素利用能力提高	DAL4基因所在区域扩增	DAL4基因编码尿囊素通透酶	[78]
尿素利用能力提高	DUR3基因所在区域扩增	DUR3基因编码尿素通透酶	[78]
冻融耐受性增强	AQY2基因所在区域扩增	AQY2基因介导水分子跨膜运输通道	[79]
氟康唑耐受性增强	ERG11基因所在区域扩增	ERG11基因编码羊毛甾醇14α-去甲基化酶，可与氟康唑结合发挥耐药性作用	[80]
雷帕霉素耐受性提高	XII号染色体扩增	维持核糖体DNA拷贝数	[71]
NaCl耐受性提高	Gcn20基因所在区域缺失	未知	[74]
5-氟胞嘧啶耐受性提高	FCY2基因所在区域扩增缺失	FCY2基因编码胞嘧啶通透酶	[74]
葡萄糖限制环境耐受性提高	IV号染色体扩增	己糖转运蛋白编码基因HXT6和HXT7扩增	[81]

表1 酵母基因组变异驱动适应性进化的研究

Table 1 Examples of evolutionary adaptation induced by genomic alternations in yeast

表型效应 Phenotypic effect	变异类型 Variation type	遗传机理 Genetic mechanism	参考文献 References
H₂O₂耐受性提高	Ⅳ号染色体扩增	编码硫氧还蛋白过氧化物酶的TSP2基因拷贝数上升	[70]
H₂O₂耐受性提高	VII号染色体上572-734 kb区域扩增	编码过氧化氢酶T的 CTT1基因拷贝数上升	[24]
热胁迫耐受性提高	III号染色体扩增	该染色体上至少14个与耐热相关基因表达量上调	[75]
博来霉素耐受性提高	SKY1突变， XIII号染色体右臂缺失	SKY1基因编码蛋白激酶，其缺失可抑制膜转运蛋白活性，减少药物进入细胞	[16]
乙醇耐受性提高	III号染色体扩增	未知	[76]
对亚硫酸盐环境耐受性提高	VIII和XVI号染色体易位，XV和XVI号染色体易位	染色体易位引起SSU1基因表达量提高，SSU1基因编码可泵出亚硫酸盐的膜蛋白	[77]
氮限制环境耐受性提高	含有GAP1基因区域形成eccDNA	GAP1基因编码氨基酸透性酶	[73]
尿囊素利用能力提高	DAL4基因所在区域扩增	DAL4基因编码尿囊素通透酶	[78]
尿素利用能力提高	DUR3基因所在区域扩增	DUR3基因编码尿素通透酶	[78]
冻融耐受性增强	AQY2基因所在区域扩增	AQY2基因介导水分子跨膜运输通道	[79]
氟康唑耐受性增强	ERG11基因所在区域扩增	ERG11基因编码羊毛甾醇14α-去甲基化酶，可与氟康唑结合发挥耐药性作用	[80]
雷帕霉素耐受性提高	XII号染色体扩增	维持核糖体DNA拷贝数	[71]
NaCl耐受性提高	Gcn20基因所在区域缺失	未知	[74]
5-氟胞嘧啶耐受性提高	FCY2基因所在区域扩增缺失	FCY2基因编码胞嘧啶通透酶	[74]
葡萄糖限制环境耐受性提高	IV号染色体扩增	己糖转运蛋白编码基因HXT6和HXT7扩增	[81]

图5 不同环境胁迫因子诱导基因组产生差异化的特征性变异模式多种胞外胁迫因子均会引起DNA损伤，包括DNA断裂、碱基损伤和交联等，进而诱导不同类型的变异形式。遗传筛选体系和全基因组测序是运用酵母模型检测环境因子诱导基因组变异的重要技术方法

Fig. 5 Different environmental stressors result in differentiated and characteristic variations in genomes Various extracellular stressors can cause DNA damage, including DNA breaks, base damage, and cross-linking, thereby inducing different types of variations. Genetic screening systems and whole-genome sequencing are crucial technical methods for detecting environmental factors induced genomic alterations in yeast

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