The overall goal of this research program is to identify genes, using operon fusions, that are involved in a unique repair system induced by near-ultraviolet-B (NUV-B; 290-320 nm) and near-ultraviolet-A (NUV-1; 320-400 nm) radiation damage, and to identify those conditions which regulate the expression of these genes. The biological response of NUV irradiation include effects that are distinctly different from those of far-ultraviolet light (FUV; 200-290 nm). Mechanisms utilized by E. coli to cope with NUV stress may link up with other global oxidative stress management systems; NUV-stress proteins may be vital for an organisms survival and integral to normal cell functioning in a similar matter to heat shock proteins. Evidence indicates that these stress response activities may be the same in both prokaryotic to eukaryotic cells. Additional NUV radiation, particularly NUV-B (290-320 nm) may increase as a result of ozone depletion; this will elevate the incidence of cutaneous melanoma. Tanning lamps increase human exposure to NUV-A radiation. This study will initially concentrate on identifying repair operons induced by low fluence levels of broad-spectrum NUV irradiation utilizing Tn5-lac, a transposon that fuses lac Z expression to exogenous promoters. The working hypothesis for this study is that katF and/or fur are members of an inducible, regulatory network that controls the coordinated expression of a specific subset of genes involved in NUV protection and repair. Other possible candidates are apaH, oxyR, soxR and oxoR.
Specific aims : (1) Isolation of Tn5 lac operon fusions that are induced by NUV at low fluence rates (225 W/m2) and (2) the identification of conditions which affect the transcriptional regulation of these operons. Research Design and Methods: Repair operons will be identified by constructing random operon fusions utilizing P1:Tn5 lac. Tn5 is transposon that fuses lacZ expression to exogenous promoters and makes only transcriptional fusions to promoters outside Tn5. Transductants will be replica plated on X-gal and subjected to cycles of 15 minutes NUV followed by 30 minutes without NUV for 72 hours. Tn5 lac operon fusions showing induction by NUV will be transduced to strains containing mutations in genes known to influence sensitivity to NUV (katE, katG, dam, polA, sodA, sodB, xthA), and in strains containing mutations in genes known to be regulators for oxidative stress (oxyR, soxR, oxoR, apaH, fur, katF), then screened for effects on transcriptional regulation. Radiation rate, fluence and specific wavelengths (NUV-A vs. NUV-B) will also be tested. Particular emphasis will be focused on the influence of the ferric uptake regulon (fur) and the katF genes on transcriptional regulation of lac. Other possible regulatory mutations will also be constructed using Tn10. Transcriptional regulation of all Tn5 lac operon fusions and promising regulatory mutants will be studied in detail by measuring hydrolysis of ONPG by the lac Z gene product, beta-galactosidase in liquid cultures while continuously stirred under artificial NUV or under natural sunlight. They will be screened under a wide variety of fluence rates and different wavelength ranges (NUV-B and NUV-A) to determine those conditions that induce or suppress genes involved in regulating, repairing and protecting cells from NUV stress.
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