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. [unreadable] [unreadable] [unreadable]