The genetics of UV tolerance in Chlamydomonas zygospores

描述(由申请人提供)：暴露在紫外线(UV)辐射下会直接损伤DNA，并通过与各种细胞成分的相互作用加强破坏性自由基的形成。因此，太阳辐射会导致癌症、免疫抑制、冠心病和衰老。我们感兴趣的是细胞如何对紫外线诱导的应激做出反应，包括修复和避免机制。作为对太阳辐射的反应，植物会合成保护性的紫外线吸收酚类化合物(防晒霜)，其中许多是有效的抗氧化剂。这项拟议工作的长期目标是鉴定导致单细胞绿藻衣藻合子孢子紫外线耐受性增强的基因和基因产品。在营养胁迫下，单胞藻营养细胞分化为有性行为的配子。配子融合形成的合子发育成厚壁合子孢子。我们的主要方法是分离和鉴定那些表现出远高于或远低于我们野生型菌株的紫外线抗性水平的合孢子特异性突变体。对紫外线敏感的菌株也将用于分离恢复(或增强)野生型耐受性的遗传抑制基因。其他具体目标包括：(A)比较合孢子与营养细胞对紫外线辐射和活性氧的最大抗性水平；(B)利用荧光、分光光度和高效液相色谱检测合孢子和合孢子壁提取物中是否存在紫外线吸收酚类化合物；(C)从基因、超微结构(通过电子显微镜)和生化方面鉴定相关突变菌株的紫外线吸收壁相关成分的类型和数量。这项拟议工作的一个重要的最终目标是开发使用外来可选择标记基因转化单胞菌营养细胞的方案--这是随后通过补充敏感菌株来克隆抗紫外线基因的第一步。我们的研究将深入了解陆地真核生物如何适应陆地生命受到紫外线辐射的增加--这种暴露与平流层臭氧层的耗尽平行地继续增加。

DESCRIPTION (provided by applicant): Exposure to ultraviolet (UV) radiation causes direct lesions in DNA and enhances the formation of damaging free radicals through its interaction with a variety of cellular components. As a consequence, solar radiation contributes to carcinogenesis, immunosuppression, coronary heart disease, and aging. We are interested in how cells respond to UV-induced stress, including both repair and avoidance mechanisms. In response to solar radiation, plants synthesize protective UV-absorbing phenolic compounds (sunscreens), many of which are potent antioxidants. The long-term goal of the proposed work is the identification of genes and gene products responsible for the increased UV tolerance characteristic of zygospores of the unicellular green alga, Chlamydomonas monoica. Under nutrient stress, C. monoica vegetative cells differentiate into sexually active gametes. The zygote formed from gamete fusion develops into a heavily walled zygospore. Our primary approach is to isolate and characterize zygospore-specific mutants that show UV-resistance levels well above, or well below that of our wildtype strains. UV-sensitive strains will also be used for the isolation of genetic suppressors that restore (or enhance) wildtype levels of tolerance. Additional specific aims include (a) comparing maximal resistance levels for zygospores vs. vegetative cells to both UV radiation and reactive oxygen species; (b) assaying zygospores, and zygospore wall extracts for the presence of UV-absorbing phenolic compounds using fluorometry, spectrophotometry and HPLC, (c) characterizing relevant mutant strains genetically, ultrastructurally (by electron microscopy), and biochemically in terms of the types and quantities of UV-absorbing wall-associated components. An important final goal of the proposed work is the development of protocols for transforming C. monoica vegetative cells using foreign selectable marker genes -- a first step toward the subsequent cloning of UV-resistance genes by complementation of sensitive strains. Our studies will provide insight into the mechanisms whereby terrestrial eukaryotic organisms have adapted to the enhanced exposure to ultraviolet radiation characteristic of life on land -- an exposure that continues to increase in parallel with depletion of the stratospheric ozone layer.