Donovan-Peluso M, Acuto S, Swanson M, Dobkin C, Bank A
Department of Medicine, Columbia University, College of Physicians and Surgeons, New York, New York 10032.
J Biol Chem. 1987 Dec 15;262(35):17051-7.
K562 cells express embryonic (epsilon) and fetal (gamma) globins and hemoglobins but not adult (beta) globin. To define the cis acting regulatory elements involved in the discrimination between gamma and beta genes, we have constructed chimeric genes composed of portions of gamma and beta and evaluated their expression in stable K562 transfectants. A gamma beta fusion gene containing gamma 5' sequences to the EcoRI site in exon 3 and beta sequences 3' is expressed at 10-40% that of the endogenous gamma level. In 50% of the lines, this fusion gene appropriately increases its expression in response to hemin, an inducer of endogenous globin gene expression in K562 cells. In contrast, a beta gamma fusion gene, containing beta sequences 5' to the EcoRI site in exon 3 and gamma sequences 3', is neither expressed nor correctly initiated. A beta gene containing gamma-intervening sequence (IVS) 2 accumulates an mRNA transcript when analyzed with a 3' beta probe. However, no correctly initiated beta mRNA is observed. A gamma gene with beta-IVS 2 is only inducible in one of six expressing clones. All the results are consistent with the presence of stage-specific trans acting factors in K562 cells that stimulate expression of gamma genes and suggest a significant role for gamma-IVS 2 in gamma gene expression.
K562细胞表达胚胎型（ε）和胎儿型（γ）珠蛋白及血红蛋白，但不表达成人型（β）珠蛋白。为了确定参与γ基因和β基因区分的顺式作用调控元件，我们构建了由γ基因和β基因部分组成的嵌合基因，并评估了它们在稳定的K562转染子中的表达。一个γβ融合基因，其包含从外显子3中的EcoRI位点到3'端的γ基因5'序列和β基因序列，其表达水平为内源性γ基因水平的10% - 40%。在50%的细胞系中，这种融合基因会因血红素（K562细胞内源性珠蛋白基因表达的诱导剂）而适当增加其表达。相比之下，一个βγ融合基因，其包含从外显子3中的EcoRI位点到3'端的β基因5'序列和γ基因序列，既不表达也不能正确起始。当用3'端β探针分析时，一个包含γ基因间隔序列（IVS）2的β基因会积累一个mRNA转录本。然而，未观察到正确起始的β mRNA。一个带有β-IVS 2的γ基因在六个表达克隆中只有一个是可诱导的。所有结果都与K562细胞中存在刺激γ基因表达的阶段特异性反式作用因子一致，并表明γ-IVS 2在γ基因表达中起重要作用。
