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Alternative TitleResearch on A Novel Genotoxic Chemical Detection System Based on the Transcriptional Response to DNA Damage in Yeast
Thesis Advisor戴和平 ; 肖伟
Degree Grantor中国科学院水生生物研究所
Place of Conferral水生生物研究所
KeywordRnr3-lacz 酵母 遗传毒性试验 灵敏度 细胞壁 甘露糖蛋白 通透性 Pdr途径 外排泵蛋白 转录因子 前致癌物 Cyp450 代谢活化 全细胞生物催化剂 Dna氧化性损伤剂 Yap1 转录激活因子 氧化应急反应 Dna损伤反应
Abstract遗传毒性化学物的检测对于环境保护及癌症预防意义重大。基于酵母RNR3的DNA损伤转录响应所建立的RNR3-lacZ遗传毒性检测系统具有如下优势:该检测系统即具有真核生物的特点,又具有原核生物细菌易于培养和基因操作的特点,能够检测不同类型的遗传毒性化学物,其灵敏度高于细菌SOS测试方法,也能互补Ames实验。但由于酵母细胞的通透性问题,以及过于简单的代谢活化系统,阻碍了该检测系统的实际应用。为了使酵母RNR3-lacZ检测系统更加适用于环境检测,需要进一步提高其检测灵敏度,扩大其检测范围,我们在以下几方面尝试对该系统进行了遗传改造: 1、提高酵母细胞的通透性: 细胞壁、细胞膜的通透性以及PDR途径(pleiotropic drug resistance)会限制外源化学物在酵母细胞内的积累,我们通过基因敲除的方法改变酵母细胞壁、细胞膜结构或抑制细胞膜外排泵蛋白的作用来提高RNR3-lacZ系统灵敏度。 结果显示失活编码细胞壁甘露糖蛋白的CWP1和CWP2基因可以明显提高酵母细胞壁的通透性,提高RNR3-lacZ检测系统对大分子量化学物的灵敏度;失活编码主要外排泵蛋白的SNQ2和PDR5基因则可提高RNR3-lacZ检测系统对不同分子量大小化学物的灵敏度。我们希望组合两种改造途径进一步提高RNR3-lacZ系统灵敏度。 2、提高酵母的代谢活力: 许多遗传毒性化学物必须经代谢活化,形成中间代谢产物才具遗传毒性。而酵母细胞缺少代谢关键酶细胞色素P450(CYP),不能活化这类化学物。我们通过遗传改造,使酵母表达人源CYP1A1或1A2。所得到的有代谢能力的酵母可以代谢活化芳香胺化学物2-aminofluorene (2-AF)和2-acetyl aminofluorene (2-AAF),继而诱导RNR3-lacZ表达,但是不能够活化苯并芘(benzo[a]pyrene,BaP)。 然而将表达人源CYP1A1的细胞壁突变酵母作为全细胞生物催化剂可以活化苯并芘(benzo[a]pyrene,BaP),并通过Ames实验检测到其致突变性。为了能延长酵母培养时间,积累大量的CYP4501A1用于代谢活化BaP,继而诱导RNR3表达,我们更换了报告基因,构建了酵母RNR3-EGFP系统。 3、提高酵母氧化性损伤的压力: 外源化学物常常通过释放氧自由基损伤DNA,并可能致癌或导致衰老发生。然而,酵母RNR3-lacZ检测系统对DNA氧化性损伤剂的检测灵敏度很差。我们通过失活氧化应急反应中的一种关键转录激活因子YAP1明显提高了酵母RNR3-lacZ系统检测DNA氧化性损伤剂的能力。此外,我们也观察到YAP1或另一种重要的转录激活因子SKN7的失活显著提高了酵母RNR3-lacZ系统对MMS的灵敏度。MMS是一种烷化剂,以往并没有报道它可以通过释放氧自由基的方式损伤DNA。 总之,我们通过不同生物学途径的改造显著提高了酵母RNR3-lacZ遗传毒性检测系统的灵敏度。而且在系统改造过程中观察到了一些独特的生物学现象,为该检测系统及其它类似检测系统的实际应用提供了更多的实验基础。我们的研究也为研究真核生物细胞对DNA损伤的反应机制提供了一些启示。
Other AbstractIt is important to detect genotoxic chemicals for environment protection and cancer prevention. A genotoxic testing system of RNR3-lacZ based on yeast RNR3 transcriptional response to DNA damage has shown some advantages: this testing system has not only traits of eukaryotes, but also traits of prokaryotic bacteria for easy culture and gene operation, which can detect various kinds of genotoxic chemicals and appears to be more sensitive than other similar tests in microorganisms, such as bacterial SOS chromotest assay, and is complementary to the Ames Test. Due to the permeability of yeast cells and its simpler metabolic activation system, this system has been compromised for practical applications. In order to apply this testing system for environmental detection, further enhancement of detection sensitivity and spectrum is desired. In this desertation, we have attempted genetic manipulation in the following areas: 1. Enhance permeability of yeast cells: Cell wall, cell membrane and the PDR pathway (pleiotropic drug resistance) limit the accumulation of genotoxic chemicals in the cell. We try to alter the cell wall and membrane structure, or inhibit the membrane efflux function through gene deletion to enhance the RNR3-lacZ system sensitivity. The results show that inactivation of both CWP1 and CWP2 gene encoding cell wall mannoproteins has profound effects on the cell wall structure and permeability, and markedly enhances the system sensitivity to high molecular weight compounds. Menawhile, inactivation of both SNQ2 and PDR5 genes encoding membrane efflux proteins enhances the system sensitivity to compounds with any molecular weights. We hope to incorporate two pathways to further enhance the systeme sensitivity. 2. Enhance metabolic activity of yeast cells: Many chemical carcinogens require metabolic activation before they show genotoxicity. Because of lack of the key metabolic enzyme cytochrome P450 (CYP), the yeast is unable to activate these chemicals. We genetically engineered the yeast to produce human CYP1A1 or 1A2. The gained metabolic competent yeast can activate aromatic amine 2-aminofluorene (2-AF) or 2-acetyl aminofluorene (2-AAF) to induce RNR3-lacZ, but failed to activate benzo[a]pyrene (BaP). However, BaP can be metabolically activated by yeast expressiong human CYPlAl as whole cell biocatalyst and show mutagenesis in Ames test. In order to achieve a log-term activation, we created a RNR3-EGFP system in a hope to accumulate sufficient amount of CYP4501A1 for metabolic activation of BaP that leads to induction of RNR3. 3. Enhance oxidative stress of yeast cells: Many chemicals damage DNA through the release of reactive oxygen species (ROS), which maybe cause cancer or aging. However, the yeast RNR3-lacZ show poor sensitivity to DNA oxidative agents. Inactivation of YAP1, a key transcriptional activator in the oxidative stress response, markedly enhanced RNR3-lacZ sensitivity to oxidative agents. Furthermore, inactivation of YAP1 or SKN7, another transcriptional activator in the oxidative stress response, also enhanced RNR3-lacZ sensitivity to MMS. MMS as been regarded as an alkylating agent and not previously reported to cause DNA damage through ROS. In summary, we had enhanced sensitivity of the yeast RNR3-lacZ genotoxic testing system through altering different biological pathways and observed some new phenomena, which provide more research groundwork for this as well as other similar testing systems into practical applications. This study also provides insights into the mechanisms of cellular response to DNA damage in eukaryotes
Document Type学位论文
Recommended Citation
GB/T 7714
张敏. 一种基于酵母DNA损伤转录响应的新型遗传毒性化学物检测系统的研究[D]. 水生生物研究所. 中国科学院水生生物研究所,2008.
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