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首页> 外文期刊>Molecular and Cellular Biology >Molecular cloning and characterization of NF-IL3A, a transcriptional activator of the human interleukin-3 promoter.
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Molecular cloning and characterization of NF-IL3A, a transcriptional activator of the human interleukin-3 promoter.

机译：NF-IL3A的分子克隆和表征，NF-IL3A是人白介素3启动子的转录激活因子。

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To isolate transcription factors important in the regulation of the human interleukin-3 (IL-3) gene, we screened a lambda gt11 cDNA library, constructed from phytohemagglutinin-stimulated human T-cell RNA, with a probe containing regulatory sequences in the upstream region of the IL-3 gene (located from bp -165 to -128 and referred to as the DNase I footprint A region). We isolated a 0.96-kb cDNA that encoded a basic amino acid domain and a leucine zipper domain and used the "rapid amplification and cloning of 3' ends" technique to isolate the 3' half of the cDNA clone, generating a 1.9-kb full-length cDNA clone. Using in vitro-translated protein, which we call NF-IL3A, we defined the IL-3 promoter sequences bound by NF-IL3A in DNase I footprinting assays as TAATTACGTCTG and, using gel shift assays, defined ATTACG as the minimal sequence required for binding of NF-IL3A in vitro. Proteins that bind to the NF-IL3A binding site are found in both unstimulated and stimulated T-cell lines in similar amounts, although the level of NF-IL3A mRNA increases after T-cell activation in several mature T-cell lines. The NF-IL3A protein is nearly identical to a recently identified transcriptional repressor protein, E4BP4, and NF-IL3A binds specifically to regulatory sequences in both the adenovirus E4 promoter and the human gamma interferon promoter. Cotransfection experiments demonstrate that introduction of an expression vector containing the NF-IL3A cDNA into resting T cells transactivates IL-3 promoter-chloramphenicol acetyltransferase gene plasmids that contain the A region; this effect requires the presence of an intact NF-IL3A binding site. One or more copies of the A region also confer NF-IL3A responsiveness on a heterologous promoter in T cells. NF-IL3A appears to play an important role in the expression of IL-3 by T cells.
机译：为了分离对人类白介素3（IL-3）基因调控重要的转录因子，我们筛选了由植物血凝素刺激的人类T细胞RNA构建的λgt11cDNA文库，并在上游区域包含了调控序列的探针IL-3基因（位于bp -165至-128之间，称为DNase I足迹A区）的片段。我们分离出编码基本氨基酸结构域和亮氨酸拉链结构域的0.96 kb cDNA，并使用“ 3'末端的快速扩增和克隆”技术来分离cDNA克隆的3'一半，从而产生1.9 kb的全长长cDNA克隆。使用我们称为NF-IL3A的体外翻译蛋白，在DNase I足迹分析中将被NF-IL3A结合的IL-3启动子序列定义为TAATTACGTCTG，并使用凝胶位移分析将ATTACG定义为结合所需的最小序列NF-IL3A的体外培养。在未刺激和刺激的T细胞系中都发现了与NF-IL3A结合位点结合的蛋白质，其含量相似，尽管在一些成熟的T细胞系中，T细胞活化后NF-IL3A mRNA的水平增加了。 NF-IL3A蛋白与最近鉴定的转录阻遏蛋白E4BP4几乎相同，并且NF-IL3A特异性结合腺病毒E4启动子和人γ干扰素启动子中的调控序列。共转染实验表明，将含有NF-IL3A cDNA的表达载体导入静止的T细胞可激活包含A区的IL-3启动子-氯霉素乙酰转移酶基因质粒。该作用需要完整的NF-IL3A结合位点的存在。 A区的一个或多个副本还赋予T细胞中异源启动子NF-IL3A响应性。 NF-IL3A似乎在T细胞表达IL-3中起重要作用。

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