REGULATIN OF GLOBIN GENE EXPRESSION BY 5' DNA SEQUENCES

为了更好地理解人类珠蛋白合成的转变 从胚胎到胎儿再到成体珠蛋白，我们正在研究一种人类细胞系， K562，受阻于从胎儿到成人的珠蛋白合成过程。K562 细胞不能合成β-珠蛋白基因。之前的研究来自 该实验室已经证明K562细胞中的分子缺陷似乎是 在反式作用因素中。最有可能的可能性是 抑制子是连续合成的，它与DNA结合在 β-珠蛋白启动子，阻止转录，或者是一种激活因子，它 是不合成β-珠蛋白转录所必需的，或者是K562 细胞既合成抑制物，又缺乏激活剂。 为了检验这些假设，我们使用了一种名为p-beta-GLCAT的质粒，它 包含人β-珠蛋白启动子和额外的5‘DNA融合到一个 易于检测的基因，CAT(氯霉素乙酰转移酶)。这个 融合基因，β-CAT在K562细胞中是不活跃的，所以我们试图 使用几种方法激活它。我们激活β-CAT的实验 无论是顺式的，带有SV40增强子的，还是反式的，带有E1a蛋白的， 在K562细胞上不成功，但在中国仓鼠身上成功 细胞，这与K562细胞中存在抑制子是一致的。进一步 对这一模型的支持来自β-CAT的5‘端缺失突变体，它 表明某些5‘序列缺失确实允许β-CAT 在K562细胞中表达。因此，似乎存在一种负面的监管 β-珠蛋白启动子附近的区域。实验正在进行中，试图 鉴定推测的抑制子的DNA结合部位，并鉴定 抑制物分子本身。

In order to better understand the switching of human globin synthesis from embryonic to fetal to adult globin, we are studying a human cell line, K562, blocked in the transition from fetal to adult globin synthesis. K562 cells are unable to synthesize beta-globin mRNA. Previous studies from this laboratory have shown the molecular defect in K562 cells appears to be in a trans-acting factor. The most likely possibilities are that a repressor is continuously synthesized which binds to DNA near the beta-globin promoter, preventing transcription, or that an activator which is necessary for beta-globin transcription is not synthesized, or that K562 cells both synthesize a repressor and lack an activator. To test these hypotheses we are using a plasmid, p-beta-GLCAT, which contains the human beta-globin promoter and additional 5' DNA fused to an easily assayable gene, CAT (chloramphenicol acetyl transferase). The fusion gene, beta-CAT, is inactive in K562 cells so we are attempting to activate it using several approaches. Our experiments to activate beta-CAT either in cis, with the SV40 enhancer, or in trans, with the E1A protein, have been unsuccessful in K562 cells but successful in Chinese hamster cells, consistent with the presence of a repressor in K562 cells. Further support for this model comes from 5' deletion mutants of beta-CAT, which indicate that deletion of certain 5' sequences does allow beta-CAT expression in K562 cells. Thus, there seems to be a negative regulatory region near the beta-globin promoter. Experiments are underway to try to identify the DNA binding site of the putative repressor and to identify the repressor molecule itself.