Ma C, Leu T H, Hamlin J L
Department of Biochemistry, University of Virginia School of Medicine, Charlottesville 22908.
Mol Cell Biol. 1990 Apr;10(4):1338-46. doi: 10.1128/mcb.10.4.1338-1346.1990.
We recently showed that replication initiates in the early S period at two closely spaced zones in the 240-kilobase (kb) dihydrofolate reductase (DHFR) amplicon of the methotrexate-resistant Chinese hamster ovary cell line CHOC 400. Both of these initiation loci (ori-beta and ori-gamma) have previously been cloned in a recombinant cosmid. In this study, we identified a third early-firing initiation locus (ori-alpha) in the much larger DHFR amplicon of the independently isolated methotrexate-resistant Chinese hamster cell line DC3F-A3/4K (A3/4K). We describe the molecular cloning of this newly identified locus and demonstrate by chromosomal walking that ori-alpha lies approximately 240 kb upstream from ori-beta. Using overlapping cosmid clones for more than 450 kb of DNA sequence from this region of the DHFR domain, we have monitored the replication pattern of the amplicons in synchronized A3/4K cells. These studies suggest that ori-alpha, ori-beta, and ori-gamma are the only early-firing initiation sites in this 450-kb sequence. In addition, we have been able to roughly localize the termini between ori-alpha and ori-beta and between ori-alpha and the next origin in the 5' direction. Thus, we have now isolated the equivalent of three early-firing replicons (including their origins) from a well-characterized chromosomal domain. With these tools, it should be possible to determine those properties that are shared by the origins and termini of different replicons and which are therefore likely to be functionally significant.
我们最近发现，在耐甲氨蝶呤的中国仓鼠卵巢细胞系CHOC 400的240千碱基（kb）二氢叶酸还原酶（DHFR）扩增子中，复制在S期早期的两个紧密间隔区域起始。这两个起始位点（ori-β和ori-γ）此前已被克隆到一个重组黏粒中。在本研究中，我们在独立分离的耐甲氨蝶呤的中国仓鼠细胞系DC3F-A3/4K（A3/4K）更大的DHFR扩增子中鉴定出第三个早期起始位点（ori-α）。我们描述了这个新鉴定位点的分子克隆，并通过染色体步移证明ori-α位于ori-β上游约240 kb处。利用来自DHFR结构域该区域超过450 kb DNA序列的重叠黏粒克隆，我们监测了同步化的A3/4K细胞中扩增子的复制模式。这些研究表明，ori-α、ori-β和ori-γ是这个450 kb序列中仅有的早期起始位点。此外，我们已经能够大致定位ori-α与ori-β之间以及ori-α与5'方向的下一个起始点之间的末端。因此，我们现在从一个特征明确的染色体结构域中分离出了相当于三个早期起始复制子（包括它们的起始点）。有了这些工具，应该能够确定不同复制子的起始点和末端所共有的那些特性，因此这些特性可能具有功能上的重要意义。
