CHARACTERIZATION OF THE GENE PRODUCTS OF THE ETS LOCUS

从CEM细胞中纯化的ETS1蛋白用于筛选最适ETS1蛋白 DNA结合序列(PU)G/C C/a G G A A G T/c(Py)。该序列 CCGGAAGT(ETS1-3)优于CAGGAAGT(PEA3)，比例为5：1。量化 电泳迁移率改变分析(EMSA)表明，纯化的 ETS1蛋白与ETS1-3或PEA3寡核苷酸探针结合 高亲和力，纯化的ETS1具有不同的结合能力 用于ETS1-3和PEA3寡核苷酸探针。ETS1蛋白与2-5结合 ETS1-3是PEA3探头的三倍。竞争性绑定 实验表明，ETS1-3和PEA3探针有效地竞争了 ETS1-3的结合。然而，核心DNA结合序列的变化 从GGAA到AGAA消除了竞争。人ETS1蛋白的筛选 来自随机寡核苷酸混合物的相同DNA序列，正如 果蝇E74A蛋白(Ets中最分化的成员之一 家族)，强烈表明所有含有ETS 85的蛋白质 氨基酸结构域(定义ETS家族的序列)将与 相同的序列。

The ETS1 protein purified from CEM cells was used to select its optimum DNA binding sequence (pu) G/C C/a G G A A G T/c (py). The sequence CCGGAAGT (ETS1-3) was preferred 5:1 over CAGGAAGT (PEA3). Quantitative electrophoretic mobility shift assays (EMSA) indicate that the purified ETS1 protein binds to either ETS1-3 or PEA3 oligonucleotide probes with high affinity, and that the purified ETS1 has different binding capacities for ETS1-3 and PEA3 oligonucleotide probes. The ETS1 protein binds 2-5 times more to the ETS1-3 than the PEA3 probe. Competitive binding experiments show that the ETS1-3 and PEA3 probes effectively compete for the binding of ETS1-3. However, change of the core DNA-binding sequence from GGAA to AGAA eliminates competition. The human ETS1 protein selected the same DNA sequence from a mixture of random oligonucleotides, as did the Drosophila E74A protein (one of the most divergent members of the ETS family), strongly suggesting that all proteins containing the ETS 85 amino acid domain (sequences which define the ETS family) will bind to the same sequence.