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换， 从胚胎到胎儿再到成人的珠蛋白，我们正在研究一种人类细胞系， K562，在从胎儿到成人珠蛋白合成的过渡中被阻断。 K562 细胞不能合成β-珠蛋白mRNA。 之前的研究 这个实验室已经表明K562细胞中的分子缺陷似乎是 在一个反式作用因子中。 最有可能的是， 阻遏物是连续合成的，它结合到DNA附近的 β-珠蛋白启动子，阻止转录，或激活剂， 是β-珠蛋白转录所必需的，或者K562 细胞既合成阻遏物又缺乏激活物。 为了验证这些假设，我们使用了一种质粒p-beta-GLCAT， 含有人β-珠蛋白启动子和另外的5 ′端DNA， 氯霉素乙酰转移酶基因CAT。 的 融合基因β-CAT在K562细胞中是无活性的，因此我们试图 用几种方法激活它。 我们激活β-CAT的实验 或者与SV 40增强子顺式连接，或者与E1 A蛋白反式连接， 在K562细胞中不成功，但在中国仓鼠中成功 细胞，与K562细胞中阻遏物的存在一致。 进一步 对该模型的支持来自β-CAT的5 ′缺失突变体， 表明某些5 ′序列缺失确实允许β-CAT 在K562细胞中表达。 因此，似乎有一个负面的监管 β-珠蛋白启动子附近的区域。 实验正在进行中， 鉴定推定阻遏物的DNA结合位点， 阻遏物分子本身。

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.