REGULATION OF HUMAN B GLOBIN GENE IN ERYTHROID CELLS

地中海贫血综合征和镰状细胞性贫血的基因治疗需要 引入的珠蛋白基因模仿了原位正常的功能 珠蛋白基因既有组织特异性，又有转录效率。 DNA介导的基因转移实验表明，有效的 人β-珠蛋白转基因的转录需要存在 一个远在上游的增强剂。在基因组中，这种红系特有的 独立于发育阶段的增强子位于DNase1超级 胚胎表型珠蛋白基因-11Kb 5‘端的过敏性位点(HS2) 因此，成人β-珠蛋白基因的-54kb 5‘。HS2增强子 指导红系特异性长增强子转录本的合成 它们似乎在增强子功能和 下游顺式连锁基因的转录激活。 这项拟议工作的长期目标是研究分子 这个远上游的增强子在54 kb DNA上激活的机制 红系细胞中的胚胎、胎儿和成体珠蛋白基因 各自的发育阶段。具体目的是检验这一假设。 通过跟踪和合成长增强子转录本 转录机制是必需的，并构成以下条件的前提 类β珠蛋白基因在转录水平上的激活 红血球生成。以下建议的实验旨在测试 这一假设的精选方面。 (1)跟踪与环路相对重要性的评估 远上游HS2增强子的作用机制。《长河》 增强子转录，从而跟踪机制，将被阻止 转录终止子。这种阻断对表达的影响 将对顺式连锁CAT基因进行研究。(2)增强剂分析 含HS2的CAT载体导入红系后的转录 处于不同发育阶段的细胞。(3)内源性Long的检测 未转染的K562细胞和原代人BFU-1细胞中的增强子转录 E‘s和CFU-E’s。(4)转录增强子作用的检测 26个碱基的HS2增强子核心和结合到 核心。(5)研究两个相邻区域的协同效应 HS2驱动的增强子上的DNA酶1超敏感位点HS3和HS4 抄写。通过结合寡核苷酸合成，聚合酶链式反应， 重组CAT载体的构建、转染实验、CAT检测 和RNA保护试验，在生物意义上的增强子 启动上游长记录，因此跟踪机制将 被评估。

Gene therapy for the thalassemia syndromes and sickle cell anemia requires that the introduced globin gene mimic the function of the in situ normal globin gene both in tissue specificity and efficiency of transcription. DNA-mediated gene transfer experiments have shown that efficient transcription of the human beta-globin transgene requires the presence of a far upstream enhancer. In the genome, this erythroid specific and development-stage independent enhancer is located in a DNase 1 super hypersensitive site (HS2) at -11Kb 5' of the embryonic epsilon globin gene and thus at -54 Kb 5' of the adult beta-globin gene. The HS2 enhancer directs the synthesis of erythroid specific, long enhancer transcripts which appear to play a pivotal role in enhancer function and transcriptional activation of the downstream cis-linked gene. The long-term objective of the proposed work is to study the molecular mechanism by which this far upstream enhancer activates across 54 Kb of DNA the embryonic, fetal and adult globin genes in erythroid cells at the respective developmental stage. The specific aim is to test the hypothesis that the synthesis of long enhancer transcripts by a tracking and transcription mechanism is required, and constitutes a precondition, for the transcriptional activation of the beta-like globin genes during erythropoiesis. The following proposed experiments are designed to test selected aspects of this hypothesis. (1) Assessment of the relative importance of the tracking v.s. the looping mechanisms in the function of the far upstream HS2 enhancer. The long enhancer transcripts, and thus the tracking mechanism, will be blocked by transcriptional terminators. The effect of this blockage on the expression of the cis-linked CAT gene will be studied. (2) Analysis of enhancer transcription in HS2-containing CAT plasmids transfected into erythroid cells at different developmental stages. (3) Detection of endogenous long enhancer transcripts in non-transfected K562 cells and primary human BFU- E's and CFU-E's. (4) Examination of the role in enhancer transcription of a 26 bp HS2 enhancer core and of the transcription factors that bind to the core. (5) Investigation of the synergistic effects of two neighboring DNase 1 hypersensitive sites HS3 and HS4 on the HS2-driven enhancer transcription. By a combination of oligonucleotide synthesis, PCR, recombinant CAT plasmids construction, transfection experiments, CAT assays and RNA protection assays, in the biological significance of the enhancer initiated upstream long transcript and thus of the tracking mechanism will be evaluated.