Binding of nuclear factors to the proximal and distal CACCC motifs of the beta-globin gene promoter: implications for the -101 (C-->T) 'silent' beta-thalassemia mutation.
Binding of nuclear factors to the proximal and distal CACCC motifs of the beta-globin gene promoter: implications for the -101 (C-->T) 'silent' beta-thalassemia mutation.
复制标题
核因子与β-珠蛋白基因启动子的近端和远端CACCC基序的结合：对-101（C-->T）“沉默”β-地中海贫血突变的影响。
DOI：
10.1159/000204237
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发表时间：
1994
影响因子：
2.4
通讯作者：
Huisman,TH
中科院分区：
文献类型：
--
作者：
Baysal,E;Ribeiro,ML;Huisman,TH
我们用凝胶滞留实验研究了核蛋白与β-珠蛋白基因启动子区域中复制的CACCC盒的结合。使用适当的突变寡核苷酸研究了β-地中海贫血突变对这两个共同序列的影响(-88C→T突变和-101C→T突变)。当与人红白血病细胞的核蛋白孵育时，含有单一和/或复制CACCC盒的人工合成的寡核苷酸产生5条延缓条带。竞争实验表明，B1和B2是高度特异的复合体，而B3、B4和B5是非特异性的。特异性反式作用因子的结合是稳定的，只有相对较高浓度的竞争对手DNA才能竞争。近端的CACCC盒(如-88N探针)在核蛋白的结合中似乎是必不可少的，而NT-88(C→T)的突变完全取消了结合。相反，远端的CACCC盒无论是正常的(-101N)还是突变的(-101M)都没有结合活性。与Sp1寡核苷酸的完全竞争，即使在低浓度下，也表明与Sp1共同序列的高亲和力结合。标记的-88N、-88N/-101N与标记的Sp1探针的结合模式非常相似，进一步证明了条带B1和B2可能是由于SPL蛋白，一种普遍表达的反式激活剂。
We have used the gel retardation assay to investigate the binding of nuclear proteins to the duplicated CACCC boxes in the β-globin gene promoter region. The effect of β-thalassemia mutations affecting both of these consensus sequences (the -88 C→T and -101 C→T mutations) were studied by using appropriate mutant oligonucleotides. Upon incubation with nuclear proteins from human erythroleukemia cells, the synthetic oligonucleotides containing single and/or duplicated CACCC box(es) generated five retarded bands. Bands B1 and B2 appeared to be highly specific complexes as determined by competition experiments whereas bands B3, B4 and B5 were nonspecific. The binding of the specific trans-acting factors was stable and could be competed out only by relatively high concentrations of competitor DNA. The proximal CACCC box (as in the -88N probe) appeared to be essential in the binding of nuclear proteins and a mutation at nt -88 (C→T) abolished binding completely. In contrast, the distal CACCC box showed no binding activity either as normal (-101N) or as mutant (-101M). A full competition with the Sp1 oligonucleotide, even at low concentrations, suggested a high affinity binding to the Sp1 consensus sequence. The close similarities in the binding patterns of the labelled -88N, -88N/-101N and that of the labelled Sp1 probe add further credence to the possibility that bands B1 and B2 may be due to the Spl protein, a ubiquitously expressed trans-activator.