The Genetics of UV Tolerance in Chlamydomonas Zygospores

描述（由申请人提供）：紫外线（UV）辐射会直接损伤DNA，并通过与许多其他细胞成分的相互作用促进自由基的形成。因此，太阳辐射有助于致癌、免疫抑制、冠心病和衰老。细胞对UV诱导的应激的反应包括回避和修复。当暴露在太阳辐射下时，植物会合成具有保护作用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。该绿色的披衣菌，Chlamyelocyanica产生一个厚壁接合孢子，是显着抵抗环境压力。与接合孢子壁相关的酚类成分可能是接合孢子耐紫外线能力增强的原因。我们将使用各种遗传，组织化学，生物化学和超微结构的方法来表征几个抗紫外线和紫外线敏感的接合孢子突变株。我们的目标是确定单个接合孢子壁成分的性质或数量的变化是否与突变株的UV抗性增加或降低相关。遗传方法将包括测试突变等位基因之间的上位相互作用，并使用紫外线抗性降低的突变株作为选择抑制突变的遗传背景，通过增强保护性化合物的合成来重建紫外线耐受性。突变体（和野生型）菌株的表征将包括使用荧光显微镜来监测接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识别主要类别的壁聚合物，以及旨在评估过氧化物酶在紫外线保护化合物形成中的潜在作用的抑制剂研究。本研究的另一个目标是进一步完善将外源DNA导入C. monoica细胞--这是随后克隆接合孢子特异性基因的第一步，该基因负责增强紫外线耐受性。我们将克隆C. monoica用异源C. reinhardtii并将使用克隆的基因转化硝酸盐还原酶缺陷型C. monoica。克隆的硝酸还原酶基因然后可用于插入诱变，以及用于转化方案的进一步优化。我们的研究将深入了解陆地真核生物为适应与陆地生命有关的紫外线辐射增加以及平流层臭氧层持续消耗而使用的机制。

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.