IDENTIFICATION OF A SILENCER ELEMENT IN THE HUMAN EPSILON-GLOBIN GENE

负调控转录的DNA元件已经被发现， 在许多基因中发现。 其中一种DNA元素 抑制转录的位置和方向 独立的方式被称为消音器。 消音器已经 发现在大鼠胰岛素I基因，c-myc基因，小鼠β主要 珠蛋白基因、鸡溶菌酶基因和酵母中的，并出现发挥 在控制基因活动中起着重要作用。 我们研究了 人类ε-珠蛋白基因的上游控制区。 通过 缺失分析，我们发现一些上游区域有 监管职能。 特别是，两个负监管 在-177 bp和-392 bp之间，在-535 bp和-1400 bp之间 在-392~-535 bp之间有一个正调控区 已经被确认。 作为描述这些特征的第一步， 监管要素，我们集中在区域之间- 177和-392 bp。 该220 bp片段已在CAT中克隆 含有177 bp上游序列的表达质粒 在5 '和3'位置以及在两种意义上， 反义方向 一旦进入红白血病 K562和Hela细胞，含有220 bp DNA的质粒 片段的CAT活性显著低于 不含TNE片段的质粒。 这表明这块碎片 可以作为转录沉默器。 为了测试这是否 在异源系统中沉默器的功能，我们把这个 在tK启动子的5 '侧的片段。 当转染到 在HeLa细胞中，该沉默子也抑制tKCAT表达。 因此，在本发明中， 沉默子不仅对ε-珠蛋白基因起作用， 在其他推动者中发挥作用。

DNA elements which negatively regulate transcription have been identified in many genes. One kind of these DNA elements which inhibits transcription in an position - and orientation - independent manner has been termed silencer. Silencers have been found in the rat insulin I gene, c-myc gene, mouse beta major globin gene, chicken lysozyme gene and in yeast, and appear to play an important role in the control of gene activity. We have studied the upstream control region of the human epsilon-globin gene. By deletion analysis, we found that a number of upstream regions have regulatory functions. Particularly, two negative regulatory regions located between -177 bp and -392 bp, and -535 bp and -1400 bp, and one positive regulatory region between -392 and -535 bp have been identified. As a first step to characterize these regulatory elements, we have concentrated on the region between - 177 and -392 bp. This 220 bp fragment has been cloned in CAT expression plasmid containing 177 bp upstream sequences of the epsilon gene in both 5' and 3' positions and in both sense and antisense orientations. Upon introduction into the erythroleukemia K562 and Hela cells, the plasmids containing the 220 bp DNA fragment showed significantly lower CAT activity compared with the plasmid without tne fragment. This indicates that this fragment can act as a transcriptional silencer. To test whether this silencer functions in a heterologous system, we placed this fragment in the 5' side of the tK promoter. When transfected into HeLa cells, this silencer also inhibits tKCAT expression. Thus, the silencer not only functions on the epsilon-globin gene, it also functions on other promoters.