Vassilev L, Johnson E M
Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029.
Mol Cell Biol. 1990 Sep;10(9):4899-904. doi: 10.1128/mcb.10.9.4899-4904.1990.
Studies on origins of DNA replication in mammalian cells have long been hampered by a lack of methods sensitive enough for the localization of such origins in chromosomal DNA. We have employed a new method for mapping origins, based on polymerase chain reaction amplification of nascent strand segments, to examine replication initiated in vivo near the c-myc gene in human cells. Nascent DNA, pulse-labeled in unsynchronized HeLa cells, was size fractionated and purified by immunoprecipitation with anti-bromodeoxyuridine antibodies. Lengths of the nascent strands that allow polymerase chain reaction amplification were determined by hybridization to probes homologous to amplified segments and used to calculate the position of the origin. We found that DNA replication through the c-myc gene initiates in a zone centered approximately 1.5 kilobases upstream of exon I. Replication proceeds bidirectionally from the origin, as indicated by comparison of hybridization patterns for three amplified segments. The initiation zone includes segments of the c-myc locus previously reported to drive autonomous replication of plasmids in human cells.
长期以来，由于缺乏足够灵敏的方法来定位哺乳动物细胞中DNA复制的起始位点，对其起源的研究一直受到阻碍。我们采用了一种基于新生链片段聚合酶链反应扩增的新方法来绘制起始位点图谱，以检测人类细胞中c-myc基因附近的体内复制起始情况。在未同步化的HeLa细胞中进行脉冲标记的新生DNA，通过用抗溴脱氧尿苷抗体进行免疫沉淀进行大小分级和纯化。通过与与扩增片段同源的探针杂交来确定允许聚合酶链反应扩增的新生链长度，并用于计算起始位点的位置。我们发现，通过c-myc基因的DNA复制起始于外显子I上游约1.5千碱基处的一个区域。如对三个扩增片段的杂交模式进行比较所示，复制从起始位点双向进行。起始区域包括先前报道可驱动质粒在人类细胞中自主复制的c-myc基因座片段。
