Brewer A C, Enver T, Greaves D R, Allan J, Patient R K
Department of Biophysics, Cell and Molecular Biology, University of London--King's College, U.K.
J Mol Biol. 1988 Feb 20;199(4):575-85. doi: 10.1016/0022-2836(88)90302-6.
We have analysed the structure of the Xenopus beta globin gene 5' flanking region in erythroid and non-erythroid chromatin, in supercoiled plasmids and in minichromosomes assembled in HeLa cell transfections. We have identified two erythroid chromatin-specific, nuclease-hypersensitive sites (HSs), one centred on the cap site, the other located 1000 base-pairs further upstream. An (AT)n tract is located 200 base-pairs upstream from each of these sites. In supercoiled plasmids, the (AT)n tracts, and not the chromatin HSs, are preferentially cleaved by single strand and double strand-specific nucleases. Using restriction enzymes, we have looked at the structure of the cap site HS in minichromosomes assembled in HeLa cell transfections. We find that the structure is indistinguishable from that found in erythroid chromatin, thus reinforcing our previous suggestion, based only on DNase I studies, that the formation of this HS is not dependent on erythroid-specific factors. In view of this close structural mimicry of the situation in vivo, we have used the HeLa cell model system to study the sequences required for cap site HS formation. We find that deletion of the (AT)n tract immediately upstream influenced neither the formation of the HS nor transcription of the globin gene. Indeed, these features remained unaffected by further deletion of upstream sequences, including 50 base-pairs of the HS itself. In this construct, the dimensions of the HS remained the same as in the undeleted construct, with the plasmid sequences that replaced the deleted Xenopus sequences becoming hypersensitive. Thus, HS formation is directed by sequences downstream from --116 acting over a distance of at least 50 base-pairs.
我们分析了非洲爪蟾β珠蛋白基因5'侧翼区在红系和非红系染色质、超螺旋质粒以及在HeLa细胞转染中组装的微型染色体中的结构。我们鉴定出两个红系染色质特异性的核酸酶超敏位点（HSs），一个以帽位点为中心，另一个位于其上游1000个碱基对处。一个（AT）n序列位于这些位点中每一个的上游200个碱基对处。在超螺旋质粒中，（AT）n序列而非染色质HSs优先被单链和双链特异性核酸酶切割。利用限制性内切酶，我们研究了在HeLa细胞转染中组装的微型染色体中帽位点HS的结构。我们发现该结构与在红系染色质中发现的结构没有区别，从而强化了我们之前仅基于DNase I研究提出的观点，即这个HS的形成不依赖于红系特异性因子。鉴于这种与体内情况的紧密结构模拟，我们利用HeLa细胞模型系统研究帽位点HS形成所需的序列。我们发现紧邻上游的（AT）n序列的缺失既不影响HS的形成也不影响珠蛋白基因的转录。实际上，这些特征不受上游序列进一步缺失的影响，包括HS本身的50个碱基对。在这个构建体中，HS的尺寸与未缺失构建体中的相同，取代缺失的非洲爪蟾序列的质粒序列变得超敏。因此，HS的形成由-116下游的序列指导，作用距离至少为50个碱基对。
