INDUCIBLE REPAIR OF NEAR-UV RADIATION DAMAGE

这项研究计划的总体目标是确定基因，使用 操纵子融合，涉及由 近紫外-B（NUV-B; 290-320 nm）和近紫外-A（NUV-1; 320-400纳米）辐射损伤，并确定那些条件， 调节这些基因的表达。 NUV的生物反应 辐射包括明显不同于 远紫外光（FUV; 200-290 nm）。 E.杆菌 科普NUV压力可能与其他全球氧化应激有关 管理系统; NUV应激蛋白可能对生物体至关重要 生存和完整的正常细胞功能，在类似的问题， 热休克蛋白 有证据表明这些压力反应 在原核细胞和真核细胞中的活性可以相同。 额外的NUV辐射，特别是NUV-B（290-320 nm）可能会增加 由于臭氧消耗，这将增加 皮肤黑素瘤 日光浴灯会增加人体对NUV-A的暴露 辐射 这项研究将首先集中在识别修复 低能量密度宽谱NUV辐射诱导的操纵子 利用Tn 5-lac，一种将lac Z表达融合到外源基因的转座子， 发起人。 这项研究的工作假设是，katF和/或毛皮 是一个可诱导的调控网络的成员， 参与NUV的特定基因子集的协调表达 保护和修复。 其他可能的候选者是apaH、oxyR、soxR 和oxoR。 具体目的：（1）分离Tn 5lac操纵子融合体， 在低注量率（225 W/m2）下， 鉴定影响转录调节的条件 这些操纵子。 研究设计和方法：修复操纵子将 通过利用P1：Tn 5 lac构建随机操纵子融合体鉴定。 Tn 5是将lacZ表达融合到外源启动子的转座子， 仅与Tn 5以外的启动子进行转录融合。 将换能器复制平板接种在X-gal上，并进行以下循环： 15分钟NUV，然后30分钟无NUV，持续72小时。 Tn 5紫胶 显示NUV诱导的操纵子融合体将被转导至菌株 含有已知影响对NUV敏感性的基因突变 (katE，katG，dam，polA，sodA，sodB，xthA），以及在含有 已知是氧化应激调节因子的基因突变（oxyR， soxR、oxoR、apaH、fur、katF），然后筛选对 转录调控 辐射率、通量和比辐射率 还将测试波长（NUV-A与NUV-B）。 特别强调 将集中在铁吸收调节子（毛皮）的影响， katF基因对乳糖的转录调控。 其他可能 还将使用Tn 10构建调节突变。 所有Tn 5 lac操纵子融合体的转录调控和有前景的 调节突变体将详细研究通过测量水解 在液体培养物中通过lac Z基因产物β-半乳糖苷酶的ONPG 同时在人工NUV下或在自然条件下连续搅拌， 阳光 他们将在各种不同的注量率下进行筛查 和不同的波长范围（NUV-B和NUV-A），以确定那些 诱导或抑制参与调节的基因的条件， 修复和保护细胞免受NUV胁迫。

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.