Research Project
2153814
Solar near-ultraviolet radiation (near-UV; 290-4OOnm), because of its abundance, is perhaps one of the most mutagenic agents to which organisms are exposed. In addition to genetic effects, near-UV influences biological systems in unique and often subtle ways. Many animal, microbial, and plant biochemical reactions may be induced, cued, and modulated by near-UV in the organism's developmental, growth and behavioral activities. Near-UV generates excess active oxygen species that have been implicated in a wide variety of environmental and health effects including premature aging, circulatory diseases, rheumatoid arthritis, and induction of cancers, cataracts and repression of the immune system. In addition to the "ozone effect," many humans receive excessive near-UV from natural sunlight and from sun lamps. The broad Objective of this research is to identify specific biological components involved in damage, protection and recovery from excess near-UV radiation. The experimental approach is to analyze mutants of Escherichia coli cells that are defective in dealing with protection and recovery from near-UV assault. Because characterization of genes under control of sigma factor rpoS (katF) may be key to understanding the damage and recovery processes, research will focus on the biological role(s) of the rpoS gene products. Specifically, we plan: (1) To determine unique DNA base changes that result from near-UV radiation especially in the absence or overproduction of cellular antioxidants and repair proteins (e.g., catalases, superoxide dismutases, and exonuclease III). (2). To characterize the roles of rpoS-regulated genes and their products in sensitization, protection and recovery from near-UV stress and in mutagenesis; to identify new rpoS regulated genes. (3). To determine regulatory overlaps of rpoS with other known regulons (e.g. oxidative stress, superoxide stress, anaerobic stress, iron regulation). (4). To determine the transcriptional/post-transcriptional roles of rpoS, including the identification of rpoS sequence segments that are involved in the interactions with some, but not all, promoters of genes under rpoS control, thus explaining variable phenotypes. (5) To identify immunochemically the functional domain(s) of rpoS protein using DNA binding assay, in vitro transcription assay, and monoclonal antibody against synthetic peptide epitopes of rpoS protein. (6) To determine the genetic relationship between "near-UV death" and "stationary-phase death" in rpoS, and other mutants. (7) To assess the near-UV effects at fluences that are anticipated with ca. 15% depletion of stratospheric ozone. The experimental design and methods will utilize (1) genetic engineering techniques, including polymerase chain reactions (PCR), DNA footprinting and DNA sequencing; (2) biochemical assays, primarily for hydroperoxidases, superoxide dismutase, ribonucleotide reductase, glutathione reductase, and alkyl peroxidase, and (3) immunological assays.
