The genetics of UV tolerance in Chlamydomonas zygospores

描述（由申请人提供）：暴露于紫外线（UV）辐射会导致DNA的直接损伤，并通过与各种细胞成分的相互作用增强有害自由基的形成。因此，太阳辐射有助于致癌、免疫抑制、冠心病和衰老。我们对细胞如何应对紫外线诱导的应激感兴趣，包括修复和避免机制。在对太阳辐射的反应中，植物合成了保护性的吸收紫外线的酚类化合物（防晒霜），其中许多是有效的抗氧化剂。这项工作的长期目标是鉴定单细胞绿藻单衣单胞菌合子孢子耐紫外线特性增加的基因和基因产物。在营养胁迫下，单根草营养细胞分化为有性活性配子。配子融合形成的合子发育成一个厚壁合子。我们的主要方法是分离和表征合子特异性突变体，这些突变体显示出远高于或远低于我们的野生型菌株的抗紫外线水平。紫外线敏感菌株也将用于分离恢复（或增强）野生型耐受性水平的遗传抑制因子。其他具体目标包括：(a)比较合子孢子和营养细胞对紫外线辐射和活性氧的最大抗性水平；(b)利用荧光法、分光光度法和高效液相色谱法测定合子孢子和合子壁提取物中吸收紫外线的酚类化合物的存在；(c)根据吸收紫外线壁相关成分的类型和数量，从遗传学、超微结构（通过电子显微镜）和生化角度表征相关突变菌株。提出的工作的一个重要的最终目标是开发利用外源可选择标记基因转化单孢菌营养细胞的方案，这是通过补充敏感菌株克隆抗紫外线基因的第一步。我们的研究将深入了解陆生真核生物如何适应陆地上生命所特有的紫外线辐射的增强暴露——这种暴露与平流层臭氧层的消耗同步继续增加。

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.