Broad objectives are to: identify chemical and biological alterations (including specific DNA lesions) that result when photons from near-ultraviolet radiation (NUV) are absorbed by bacterial (Escherichia coli) cells; compare NUV alterations with those produced by H2O2 and/or superoxide anion (O2-), photoproducts of NUV; identify specific difference between cellular damage and repair from NUV stress from those of oxidative stress; compare the effects of NUV at environmentally normal sunlight fluences with the NUV fluences anticipated with ca. 15% depletion of stratospheric ozone.
The specific aims are to (1) identify specific proteins (using appropriate mutants and plasmid-containing strains) involved in H2O2, O2- and NUV stress and recovery; to identify differences in protein profile induced by the stresses; (2) identify the pathway of NUV mutagenesis; i.e., to identify the specific base pair changes produced by NUV; (3) identify cellular components with which HP1 and HP2 catalases, and superoxide dismutases (MnSOD and FeSOD) interact (e.g., outer membrane, inner membrane, periplasmic space, cytoplasm); (4) test the role of catalase in """"""""uptake delay"""""""" (cessation of amino acid transport) produced by NUV; (5) study mutants with membrane protein defects, and test for NUV and/or H2O2 sensitivity; (6) asses lethal and mutagenic effects of NUV fluences anticipated upon ca. 15% depletion of stratospheric ozone with solar and very high fluences. This research is important to human health, since activities of our technological society could reduce stratospheric ozone (a natural filter of solar UV), resulting in a projected 5-15% increase in 290-320 nm NUV exposure for the entire surface biome. Excess NUV could yield excess reactive oxygen species in cells. Reactive oxygen molecules have critical roles in both normal and abnormal cellular metabolism. In addition to solar NUV, humans are constantly exposed to toxic NUV via artificial illumination and reactive oxygen species via pollutants, disease and aging processes. The experimental procedures will be electrophoretic and amino acid sequence analysis (to identify unique proteins involved in recovery from NUV stress); DNA base sequencing (to determine mutagenic specificity of NUV); antibody assays and immuno-electron microscopy (to identify sub-cellular and molecular components involved in recovery from NUV; analysis of amino acid transport in cells damaged by NUV (to identify mechanism of """"""""transport delay"""""""" produced by NUV, and to identify defects in mutants that over-ride """"""""transport delay""""""""); analysis of results to assess importance of anticipated excess NUV on mutational and physiological activities of cells.
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