IDENTIFICATION OF NUCLEOTIDE PREFERENCES IN DNA-SEQUENCES RECOGNIZED SPECIFICALLY BY C-ETS-1 PROTEIN
IDENTIFICATION OF NUCLEOTIDE PREFERENCES IN DNA-SEQUENCES RECOGNIZED SPECIFICALLY BY C-ETS-1 PROTEIN
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DOI：
10.1093/nar/20.4.699
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发表时间：
1992-02-25
影响因子：
14.9
通讯作者：
OWEN, MJ
中科院分区：
文献类型：
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作者：
WOODS, DB;GHYSDAEL, J;OWEN, MJ
原癌基因 Ets-1 是 c-Ets 基因家族的成员，最初通过其与禽成红细胞增多症病毒 E26 的 v-ets 基因的序列同源性来鉴定。 Ets 样因子的特征是保守的 85 个氨基酸结构域，该结构域似乎对于结合富含嘌呤的 DNA 序列至关重要。通过免疫沉淀从随机寡核苷酸库中选择与 Ets-1 结合的序列，并使用聚合酶链式反应进行扩增。通过该程序富集的寡核苷酸被克隆到质粒中并测序。 DNA 序列比对显示 GGAA 和 GGAT 核心的比例约为 1.4:1。优选的序列在 GGAW 核心的 5' 和 3' 处被鉴定，将结合位点延伸至 ACMGGAWRTT。对与 GGAA 和 GGAT 核心相关的侧翼序列的分析揭示了差异，这些差异可能补偿了含有 GGAT 核心的结合位点通常较低的亲和力。最后，对一个特定 Ets-1 结合位点的突变分析用于确定核心内某些核苷酸结合的相对重要性，并表明 Ets-1 和密切相关的 Ets-2 蛋白与相似序列结合。
The protooncogene Ets-1 is a member of the c-Ets family of genes originally identified through their sequence homology to the v-ets gene of the avian erythroblastosis virus E26. Ets-like factors are characterised by a conserved 85 amino acid domain which appears to be essential for binding to purine rich DNA sequences. Sequences binding to Ets-1 were selected from a random oligonucleotide pool by immunoprecipitation and amplified using the Polymerase Chain Reaction. Oligonucleotides enriched by this procedure were cloned in plasmids and sequenced. Alignment of DNA sequences revealed GGAA and GGAT cores at about a 1.4:1 ratio. Preferred sequences were identified both 5' and 3' of the GGAW core, extending the binding site to ACMGGAWRTT. Analysis of the flanking sequences associated with GGAA and GGAT cores revealed differences which may have compensated for the generally lower affinity of binding sites containing a GGAT core. Lastly mutational analysis of one particular Ets-1 binding site was used to establish the relative importance for binding of some nucleotides within the core and to show that Ets-1 and the closely related Ets-2 proteins bind to similar sequences.