REGULATION OF GLOBIN GENE EXPRESSION BY UPSTREAM POSITIVE CONTROL DNA SEQUENCES

人类β珠蛋白基因的表达调控仍然是 不是很清楚。我们实验室正在进行的研究(项目 #Z01 DK 25045-04 LCB)关注上游的643个碱基对 人类的β-珠蛋白基因。一系列的缺失分析， 然后是瞬时表达分析和氯霉素乙酰 转移酶(CAT)检测显示有3个DNA上游区域 监管的重要性。两个区域(介于-643和-490之间，以及 在-338到-233之间)被发现对基因有负面影响 表达，因为它们的缺失增加了CAT活性，以及 它们被定义为负控制区I(NCR1)和NCR2 分别进行了分析。删除第三个区域(介于-233和-185之间) 紧随其后的是CAT活性的急剧下降，这表明 这一部分的积极作用，因此被定义为积极控制 区域(PCR)。 当其他控制区目前正在调查时， 我们的重点是聚合酶链式反应。阳性对照效果为 在人红系细胞系(K562)中检测到，但在小鼠中未检测到 红白血病(Mel)或中国仓鼠(R1610)细胞系， 相反，这表明了人类红系组织的特异性活动 和NCR一起。这种特殊性，以及与 对NCR2的聚合酶链式反应增加了聚合酶链式反应可能含有增强子的可能性 序列，即能够增加启动子的DNA序列 活性，因此在一个位置的基因表达和 方向独立的方式。这一猜测是基于 将增强剂描述为组织特异性和受控的报告 抑制基因表达的封闭负控元件 在其他组织中，当抑制在特定的组织中被移除时 能够表达该基因的组织(前白蛋白肝细胞， β细胞中的胰岛素等)。 为了试图理解PCR的作用，我们克隆了它(100 BP)插入融合的-185缺失形式的两个BamH1位点 表达载体p-β-GLCAT。氯化高铁血红素对K562细胞的诱导作用 与新的DNA重组体一起可能回答了PCR的问题 作为增强剂。

The regulation of expression of the human beta globin gene is still not well understood. Ongoing studies in our laboratory (Project #Z01 DK 25045-04 LCB) focused on the 643 base pairs (bp) upstream from the human beta globin gene. A series of deletion analyses, followed by transient expression assays and chloramphenical acetyl transferase (CAT) assays revealed 3 upstream DNA regions of regulatory importance. Two regions (between -643 and -490, and between -338 and -233) were found to have a negative effect on gene expression, since their deletion increased the CAT activity, and they were defined as Negative Control Region I (NCR1) and NCR2 respectively. Deletion of a third region (between -233 and -185) was followed by a dramatic drop in CAT activity, suggesting a positive role of that segment, thus defined as Positive Control Region (PCR). While the other control regions are currently being investigated, we are focusing on the PCR. The positive control effect was detected in a human erythroid cell line (K562) but not in a mouse erythroleukemia (MEL) or a Chinese Hamster (R1610) cell lines, suggesting a human erythroid tissue specific activity, in contrast with the NCRs. This specificity, as well as the proximity of the PCR to NCR2 raise the possibility that PCR may contain an enhancer sequence, namely a DNA sequence capable of increasing the promoter activity and hence the gene expression in a position and orientation independent manner. This speculation is based on reports describing enhancers as tissue specific and controlled by close negative control elements which repress the gene expression in other tissues, while the repression is removed in the specific tissue enabling the gene to be expressed (prealbumin liver cells, insulin in beta cells, etc.). In an attempt to understand the PCR role, we are cloning it (100 bp) into two BamH1 sites of the -185 deleted form of the fusion plasmid p-beta-GLCAT. Transfection of hemin induced K562 cells with the new DNA recombinants may answer the question of the PCR as an enhancer.