IDENTIFICATION OF THE DNA-BINDING SITE FOR NGFI-B BY GENETIC SELECTION IN YEAST
IDENTIFICATION OF THE DNA-BINDING SITE FOR NGFI-B BY GENETIC SELECTION IN YEAST
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DOI：
10.1126/science.1925541
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发表时间：
1991-05-31
期刊：
影响因子：
56.9
通讯作者：
MILBRANDT, J
中科院分区：
文献类型：
--
作者：
WILSON, TE;FAHRNER, TJ;MILBRANDT, J
建立了酵母DNA结合蛋白的体内选择系统，用于鉴定类固醇甲状腺激素受体超家族成员NGFI-B蛋白的DNA结合位点。通过选择含有GCN4结合位点的DNA片段来验证该技术的可行性，GCN4是一种特性良好的酵母转录激活剂。NGFI-B的DNA结合域作为LexA-NGFI-B-GAL4嵌合激活剂的一部分表达，然后用于分离含有NGFI-B结合位点的大鼠基因组DNA片段。NGFI-B应答元件（NBRE）与雌激素和甲状腺激素受体以及激素受体样蛋白COUP-TF、CF1和H-2RIIBP识别的元件相似，但在功能上有所不同。在哺乳动物细胞中的共转染实验表明，NGFI-B可以激活NBRE的转录，无论是否具有其假定的配体结合结构域。
An in vivo selection system for isolating targets of DNA binding proteins in yeast was developed and used to identify the DNA binding site for the NGFI-B protein, a member of the steroid-thyroid hormone receptor superfamily. The feasibility of the technique was verified by selecting DNA fragments that contained binding sites for GCN4, a well-characterized yeast transcriptional activator. The DNA binding domain of NGFI-B, expressed as part of a LexA-NGFI-B-GAL4 chimeric activator, was then used to isolate a rat genomic DNA fragment that contained an NGFI-B binding site. The NGFI-B response element (NBRE) is similar to but functionally distinct from elements recognized by the estrogen and thyroid hormone receptors and the hormone receptor-like proteins COUP-TF, CF1, and H-2RIIBP. Cotransfection experiments in mammalian cells demonstrated that NGFI-B can activate transcription from the NBRE with or without its putative ligand binding domain.