The genetics of UV tolerance in Chlamydomonas zygospores

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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.