REGULATION OF GLOBIN GENE EXPRESSION BY UPSTREAM POSITIVE CONTROL DNA SEQUENCES

我们关注的是远端启动子的作用（高达~ 500 核苷酸）在人类β珠蛋白基因的调节中的作用。先前 实验（项目#ZOI DK 25045-05 LCB）基于瞬态 表达测定，其中重组质粒，含有一系列的 β珠蛋白的5'侧翼区的缺失突变体，其连接到 CAT报告基因和SV 40的终止信号， 转染到K562红系细胞中，随后进行CAT测定。这些 实验表明，从-233到-185（-200区域） 刺激β珠蛋白表达。值得注意的是， 含有结合核因子I（NFI）的共有序列， 这种蛋白质被发现是复制DNA所必需的， 腺病毒，并且似乎与CAAT盒无法区分 转录因子（CTF）。另一个，虽然部分，因此较弱 NFI站点位于-150区域。 结果表明：（1）存在一个 蛋白质-DNA结合，并映射到NF 1位点（通过两种凝胶位移 竞争测定和DNA酶I足迹测定）。2)纯化的NF 1 导致与K562相似的凝胶位移模式。3)抗NFI抗体 证实了NFI参与了这种结合。4)初始数据 表明NF 1与其他因子相互作用， 人类β-珠蛋白基因 在目前的项目中，我们专注于详细的结构分析 上述地区。我们正在寻找假定的顺式调节序列， 被潜在的调节蛋白（反式作用因子）识别， 并试图找出这些因素。

We are focusing on the role of the distal promoter (up to -500 nucleotides) in the regulation of the human beta globin gene. Previous experiments (project #ZOI DK 25045-05 LCB), were based on transient expression assays, in which recombinant plasmids, containing a series of deletion mutants of the 5' flanking region of the beta globin, linked to the CAT reporter gene and to the termination signal of the SV40, were transfected into K562 erythroid cells, followed by CAT assays. These experiments suggested that the region from -233 to -185 (the -200 region) stimulates the beta globin expression. Of note is that this region contains a consensus sequence for binding of nuclear factor I (NFI), a protein which was found to be necessary for the replication of the DNA of adenovirus, and appears to be indistinguishable from the CAAT box transcription factors (CTF). Another, although partial and hence weaker NFI site is located in the -150 region. The results, so far, can be summarized as follows: 1) There is a protein-DNA binding, and mapped to the NF1 site (by both gel shift competition assays and DNase I footprinting assay). 2) Purified NFl resulted in a similar gel shift pattern to K562. 3) Antibody anti-NFI confirmed the involvement of the NFI in this binding. 4) Initial data suggest that NF1 interacts with other factors in potential regulation of the human beta-globin gene. In the current project, we are focusing on a detailed structural analysis of the above region. We are looking for putative cis-regulatory sequences, recognized by potential regulatory proteins (trans-acting factors), as well as trying to identify these factors.