Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.
Promoter of the mouse gene encoding calcium-independent nitric oxide synthase confers inducibility by interferon gamma and bacterial lipopolysaccharide.
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DOI：
10.1084/jem.177.6.1779
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发表时间：
1993-06-01
期刊：
影响因子：
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通讯作者：
Nathan C
中科院分区：
文献类型：
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作者：
Xie QW;Whisnant R;Nathan C
诱导型一氧化氮合酶（iNOS）可以通过多种类型的哺乳动物细胞对多种协同作用的信号作出反应而表达，包括细胞因子和微生物产物。我们之前的研究表明，干扰素γ (ifn - γ)和脂多糖（LPS）在转录水平上诱导小鼠巨噬细胞中的iNOS。从小鼠基因组文库中，我们从iNOS基因的5'侧区克隆了一个1749 bp的片段，并使用S1核酸酶定位和引物扩展来确定其mRNA转录起始位点。mRNA起始位点之前有一个TATA盒和至少22个寡核苷酸元件，这些元件与细胞因子或细菌产物诱导其他基因的转录因子结合的一致序列同源。其中包括10份ifn - γ反应元件；γ活化位点3份；核因子κ B、干扰素刺激反应元件、激活蛋白1、肿瘤坏死因子反应元件各2份；和一个X盒子。将全部或下游1 / 2或1 / 3的iNOS区域连接到编码氯霉素乙酰转移酶的报告基因的质粒转染到RAW264.7巨噬细胞样细胞系细胞中。所有这些构建体都具有LPS诱导iNOS启动子的能力，但只有包含所有1,749 bp的构建体具有ifn - γ + LPS的协同诱导能力。
Inducible nitric oxide synthase (iNOS) can be expressed by many types of mammalian cells in response to diverse signals acting synergistically, including cytokines and microbial products. We previously showed that induction of iNOS in mouse macrophages by interferon gamma (IFN-gamma) and lipopolysaccharide (LPS) was at the transcriptional level. From a mouse genomic library, we now cloned a 1,749-bp fragment from the 5'-flanking region of the iNOS gene, and used S1 nuclease mapping and primer extension to identify the mRNA transcription start site within it. The mRNA initiation site is preceded by a TATA box and at least 22 oligonucleotide elements homologous to consensus sequences for the binding of transcription factors involved in the inducibility of other genes by cytokines or bacterial products. These include 10 copies of IFN-gamma response element; 3 copies of gamma-activated site; 2 copies each of nuclear factor-kappa B, IFN-alpha-stimulated response element, activating protein 1, and tumor necrosis factor response element; and one X box. Plasmids in which all or the downstream one half or one third of this region of iNOS were linked to a reporter gene encoding chloramphenicol acetyltransferase were transfected into cells of the RAW264.7 macrophage-like line. All these constructs conferred inducibility of the iNOS promoter by LPS, but only the construct containing all 1,749 bp conferred synergistic inducibility by IFN-gamma plus LPS.