The Genetics of UV Tolerance in Chlamydomonas Zygospores

描述(申请人提供)：紫外线(UV)辐射对DNA造成直接损害，并通过其与许多其他细胞成分的相互作用促进自由基的形成。因此，太阳辐射会导致癌症、免疫抑制、冠心病和衰老。细胞对紫外线胁迫的反应包括回避和修复。当暴露在太阳辐射下时，植物会合成保护性的吸收紫外线的酚类化合物(天然防晒剂)，其中许多是有效的抗氧化剂。绿色藻类，衣藻，产生一种厚壁的合子孢子，对环境压力具有显著的抵抗力。与合孢子壁相关的酚类成分可能与合孢子对紫外线的耐受性增加有关。我们将使用各种遗传、组织化学、生化和超微结构方法对几个抗紫外线和紫外线敏感的合孢子突变株进行鉴定。我们的目标是确定单个合孢子壁成分的性质或数量的变化是否与突变菌株的紫外线抗性增强或降低相关。遗传方法将包括测试突变等位基因之间的上位性相互作用，并使用紫外线抗性降低的突变菌株作为选择抑制突变的遗传背景，这些突变通过促进保护性化合物的合成来重建对紫外线的耐受性。突变(和野生型)菌株的特征将包括使用荧光显微镜监测合孢子发育过程中紫外线吸收化合物的积累，使用特定的组织化学染色来鉴定主要类型的壁状聚合物，以及旨在评估过氧化物酶在形成防紫外线化合物中的潜在作用的抑制剂研究。这项拟议工作的另一个目标是进一步完善将外源DNA导入莫尼卡细胞的转化方案--这是随后克隆负责增强紫外线耐受性的合孢子特异基因的第一步。我们将利用莱茵哈迪尔锥虫的一个异源探针克隆莫尼卡锥虫的硝酸还原酶基因，并将所克隆的基因用于转化一株硝酸还原酶缺陷的莫尼卡锥虫。克隆的硝酸还原酶基因可用于插入突变，也可用于进一步优化转化方案。我们的研究将为陆地真核生物用来适应与陆地生命相关的紫外线辐射暴露增加以及平流层臭氧层持续耗尽所使用的机制提供洞察。

DESCRIPTION (provided by applicant): Ultraviolet (UV) radiation causes direct damage to DNA and promotes the formation of free radicals through its interaction with many other cellular components. As a consequence, solar radiation contributes to carcinogenesis, immunosuppression, coronary heart disease, and aging. Cellular responses to UV-induced stress include both avoidance and repair. When exposed to solar radiation, plants synthesize protective UV- absorbing phenolic compounds (natural sunscreens), many of which are potent antioxidants. The green alga, Chlamydomonas manoica produces a heavily walled zygospore that is remarkably resistant to environmental stress. Phenolic components associated with the zygospore wall may be responsible for the increased UV tolerance of zygospores. We will characterize several UV-resistant and UV-sensitive zygospore mutant strains using a variety of genetic, histochemical, biochemical and ultrastructural approaches. Our goal is to determine if changes in the nature or quantity of individual zygospore wall components correlate with increased or decreased UV resistance in the mutant strains. Genetic approaches will include testing for epistatic interactions between mutant alleles, and using mutant strains with reduced UV resistance as the genetic background for selection of suppressor mutations that re-establish UV tolerance by enhancing synthesis of protective compounds. Characterization of mutant (and wildtype) strains will include the use of fluorescence microscopy to monitor the accumulation of UV-absorbing compounds during zygospore development, the use of specific histochemical stains to identify major classes of wall polymers, and inhibitor studies aimed at evaluating the potential role of peroxidases in the formation of UV-protective compounds. An additional goal of the proposed work is to further refine transformation protocols for introducing exogenous DNA into C. monoica cells - a first step toward the subsequent cloning of the zygospore-specific genes responsible for enhanced UV tolerance. We will clone the nitrate-reductase gene of C. monoica using a heterologous probe from C. reinhardtii and will use the cloned gene to transform a nitrate-reductase-defective strain of C. monoica. The cloned nitrate reductase gene can then be used for insertional mutagenesis, as well as for further optimization of transformation protocols. Our studies will provide insight into mechanisms used by terrestrial eukaryotic organisms to adapt to the increased exposure to UV radiation associated with life on land, and with the continuing depletion of the stratospheric ozone layer.