Yang T P, Caskey C T
Institute for Molecular Genetics, Baylor College of Medicine, Houston, Texas 77030.
Mol Cell Biol. 1987 Aug;7(8):2994-8. doi: 10.1128/mcb.7.8.2994-2998.1987.
We investigated the conformation of the X-linked mouse hypoxanthine-guanine phosphoribosyltransferase gene (HPRT) promoter region both in chromatin from the active and inactive X chromosomes with DNase I and in naked supercoiled DNA with S1 nuclease. A direct comparison of the chromatin structures of the active and inactive mouse HPRT promoter regions was performed by simultaneous DNase I treatment of the active and inactive X chromosomes in the nucleus of interspecies hybrid cells from Mus musculus and Mus caroli. Using a restriction fragment length polymorphism to distinguish between the active and inactive HPRT promoters, we found a small but very distinct difference in the DNase I sensitivity of active versus inactive chromatin. We also observed a single DNase I-hypersensitive site in the immediate area of the promoter which was present only on the active X chromosome. Analysis of the promoter region by S1 nuclease digestion of supercoiled plasmid DNA showed an S1-sensitive site which maps adjacent to or within the DNase I-hypersensitive site found in chromatin but upstream of the region minimally required for normal HPRT gene expression.
我们用脱氧核糖核酸酶I研究了活性和非活性X染色体染色质中X连锁小鼠次黄嘌呤-鸟嘌呤磷酸核糖基转移酶基因（HPRT）启动子区域的构象，并用S1核酸酶研究了裸露超螺旋DNA中的该区域构象。通过对小家鼠和野鼠种间杂交细胞核中的活性和非活性X染色体同时进行脱氧核糖核酸酶I处理，对活性和非活性小鼠HPRT启动子区域的染色质结构进行了直接比较。利用限制性片段长度多态性来区分活性和非活性HPRT启动子，我们发现活性染色质与非活性染色质在脱氧核糖核酸酶I敏感性上存在微小但非常明显的差异。我们还在启动子的紧邻区域观察到一个单一的脱氧核糖核酸酶I超敏感位点，该位点仅存在于活性X染色体上。通过对超螺旋质粒DNA进行S1核酸酶消化来分析启动子区域，结果显示一个S1敏感位点，该位点位于染色质中发现的脱氧核糖核酸酶I超敏感位点附近或其内部，但在正常HPRT基因表达所需的最小区域的上游。
