Tumor necrosis factor alpha and interleukin-1beta regulate the murine manganese superoxide dismutase gene through a complex intronic enhancer involving C/EBP-beta and NF-kappaB.

Manganese superoxide dismutase (MnSOD), a tumor necrosis factor (TNF)-inducible reactive oxygen-scavenging enzyme, protects cells from TNF-mediated apoptosis. To understand how MnSOD is regulated, transient transfections of promoter-reporter gene constructions, in vitro DNA binding assays, and in vivo genomic footprint (IVGF) analysis were carried out on the murine MnSOD gene. The results of this analysis identified a 238-bp region of intron 2 that was responsive to TNF and interleukin-1beta (IL-1). This TNF response element (TNFRE) had the properties of a traditional enhancer element that functioned in an orientation- and position-independent manner. IVGF of the TNFRE revealed TNF- and IL-1-induced factor occupancy of sites that could bind NF-kappaB and C/EBP. The 5' portion of the TNFRE bound C/EBP-beta in vitro and was both necessary and sufficient for TNF responsiveness with the MnSOD promoter or with a heterologous promoter when in an upstream position. The 3' end of the TNFRE bound both NF-kappaB and C/EBP but was not necessary for TNF responsiveness with the MnSOD promoter. However, this 3' portion of the TNFRE was required for the TNFRE to function as a downstream enhancer with a heterologous promoter. These data functionally separate the MnSOD TNFRE into a region responsible for TNF activation and one that mediates induction when it is downstream of a promoter.     

黑曲霉糖化酶高产和低产菌株糖化酶基因调控区的克隆及其分析比较

以PCR合成的糖化酶高产菌株黑曲霉(Asp. Niger)T21糖化酶基因5’近端非编码区588bp(EcoRI-BamHI)的序列为探针,从T21染色体DNA中克隆到近2.0kb的糖化酶基因5’端非编码区序列,并以此序列为探针从糖化酶低产菌株黑曲霉3.795(T21的诱变出发株)的染色体DNA中克隆到1.5kb的糖化酶基因5’端非编码区序列。该二序列的分析测定结果表明,其结构特征与文献报道的黑曲霉糖化酶基因5’端非编码区的基本一致,被称为“核心启动子”(Core promoter)的TATAAAT框及GCAAT框,分别在翻译起始点的-109bp及-178bp处。此外,在曲霉amdS,amyB基因中已发现有调控功能的CCAAT序列存在于-449bp和-799bp处。高产和低产菌株糖化酶基因5’端非编码区序列的分析比较结果表明,有9个部位的碱基发生了变化。此实验结果为进一步研究黑曲霉糖化酶基因在转录水平上的调控规律打下了基础。