Dijkwel Pieter A, Wang Shuntai, Hamlin Joyce L
Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Mol Cell Biol. 2002 May;22(9):3053-65. doi: 10.1128/MCB.22.9.3053-3065.2002.
Previous radiolabeling and two-dimensional (2-D) gel studies of the dihydrofolate reductase (DHFR) domain of Chinese hamster cells have suggested that replication can initiate at any one of a very large number of inefficient sites scattered throughout the 55-kb intergenic spacer region, with two broad subregions (ori-beta and ori-gamma) preferred. However, high-resolution analysis by a PCR-based nascent strand abundance assay of the 12-kb subregion encompassing ori-beta has suggested the presence of a relatively small number of fixed, highly efficient initiation sites distributed at infrequent intervals that correspond to genetic replicators. To attempt to reconcile these observations, two different approaches were taken in the present study. In the first, neutral-neutral 2-D gel analysis was used to examine replication intermediates in 31 adjacent and overlapping restriction fragments in the spacer, ranging in size from 1.0 to 18 kb. Thirty of 31 fragments displayed the complete bubble arcs characteristic of centered origins. Taking into account overlapping fragments, these data suggest a minimum of 14 individual start sites in the spacer. In the second approach, a quantitative early labeled fragment hybridization assay was performed in which radioactive origin-containing DNA 300 to 1,000 nucleotides in length was synthesized in the first few minutes of the S period and used to probe 15 clones distributed throughout the intergenic spacer but separated on average by more than 1,000 bp. This small nascent DNA fraction hybridized to 14 of the 15 clones, ranging from just above background to a maximum at the ori-beta locus. The only silent region detected was a small fragment lying just upstream from a centered matrix attachment region--the same region that was also negative for initiation by 2-D gel analysis. Results of both approaches suggest a minimum of approximately 20 initiation sites in the spacer (two of them being ori-beta and ori-gamma), with ori-beta accounting for a maximum of approximately 20% of initiations occurring in the spacer. We believe that the results of all experimental approaches applied to this locus so far can be fitted to a model in which the DHFR origin consists of a 55-kb intergenic zone of potential sites that are used with very different efficiencies and which are separated in many cases by a few kilobases or less.
先前对中国仓鼠细胞二氢叶酸还原酶（DHFR）结构域的放射性标记和二维（2-D）凝胶研究表明，复制可以在散布于整个55 kb基因间隔区的大量低效位点中的任何一个起始，其中两个较宽的子区域（ori-β和ori-γ）更受青睐。然而，通过基于PCR的新生链丰度测定法对包含ori-β的12 kb子区域进行的高分辨率分析表明，存在相对少量的固定、高效起始位点，这些位点以不频繁的间隔分布，对应于基因复制子。为了试图调和这些观察结果，本研究采用了两种不同的方法。第一种方法是使用中性-中性二维凝胶分析来检查间隔区中31个相邻且重叠的限制片段中的复制中间体，片段大小从1.0 kb到18 kb不等。31个片段中的30个显示出以中心为原点的完整泡状弧特征。考虑到重叠片段，这些数据表明间隔区中至少有14个独立的起始位点。第二种方法是进行定量早期标记片段杂交测定，其中在S期的最初几分钟内合成了长度为300至1000个核苷酸的含放射性原点的DNA，并用于探测分布在整个基因间隔区但平均间隔超过1000 bp的15个克隆。这个少量的新生DNA片段与15个克隆中的14个杂交，从略高于背景水平到在ori-β位点达到最大值。唯一检测到的沉默区域是位于一个中心基质附着区域上游的一个小片段——该区域通过二维凝胶分析也显示起始为阴性。两种方法的结果都表明间隔区中至少约有20个起始位点（其中两个是ori-β和ori-γ），ori-β在间隔区发生的起始中最多占约20%。我们认为，到目前为止应用于该位点的所有实验方法的结果都可以拟合到一个模型中，其中DHFR原点由一个55 kb的潜在位点基因间隔区组成，这些位点的使用效率差异很大，并且在许多情况下相隔几千碱基或更少。
