Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event
Establishment of an oriP replicon is dependent upon an infrequent, epigenetic event
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DOI：
10.1128/mcb.21.13.4149-4161.2001
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发表时间：
2001-07-01
影响因子：
5.3
通讯作者：
Sugden, B
中科院分区：
文献类型：
--
作者：
Leight, ER;Sugden, B
当提供病毒蛋白 EBNA-1 时，含有 oriP（Epstein-Barr 病毒的潜在复制起点）的质粒支持在选定细胞克隆中的有效复制，在去除选择后每代细胞以 2% 至 4% 的比率丢失（A. L. Kirchmaier 和 B. Sugden, J. Virol. 69:1280-1283, 1995 ；B. Sugden 和 N. Warren, Mel. 69:1280-1283, 1995）。生物医学。 5:85-94, 1988)。我们将这些质粒称为已建立的复制子，因为它们支持有效的 DNA 合成和划分每个细胞周期。出乎意料的是，我们发现，将 oriP 质粒引入 EBNA-1 阳性细胞群后，oriP 质粒会复制，但在转染后 2 周内从细胞中急剧丢失（每代细胞丢失率 >25%）。通过对这些不同观察结果的研究，我们发现只有 1% 至 10% 的用表达 EBNA-1 和潮霉素磷酸转移酶的 oriP 质粒转染的细胞产生耐药克隆，其中建立了 oriP 复制子。这些耐药细胞克隆中表现在遗传或表观遗传水平上的可遗传改变并不是 oriP 建立的基础，因为新引入的 oriP 质粒会复制，但也会从这些细胞中迅速丢失。此外，oriP 质粒中的遗传改变与建立无关，因为从已建立的细胞克隆中分离的 oriP 质粒，在大肠杆菌中繁殖，并重新引入 EBNA-1 阳性细胞中，同样是低效建立的。我们的研究结果表明 oriP 复制子在 EBNA-1 阳性细胞系中本质上不稳定。相反，oriP 复制子的建立是由随机的表观遗传事件赋予复制子的，该事件很少发生，因此仅在少数细胞中检测到。
Plasmids containing oriP, the latent origin of replication for Epstein-Barr virus, support efficient replication in selected cell clones when the viral protein EBNA-1 is provided, being lost at a rate of 2 to 4% per cell generation after removal of selection (A. L. Kirchmaier and B. Sugden, J. Virol. 69:1280-1283, 1995; B. Sugden and N. Warren, Mel. Biol. Med. 5:85-94, 1988). We refer to these plasmids as established replicons in that they support efficient DNA synthesis and partitioning each cell cycle. Unexpectedly, we have found that upon introduction of oriP plasmids into a population of EBNA-1-positive cells, oriP plasmids replicate but are lost precipitously from cells during 2 weeks posttransfection (>25% rate of loss per cell generation). Upon investigation of these disparate observations, we have found that only 1 to 10% of cells transfected with an oriP plasmid expressing EBNA-1 and hygromycin phosphotransferase give rise to drug-resistant clones in which the oriP replicon is established. A hereditable alteration in these drug-resistant cell clones, manifested at the genetic or epigenetic level, does not underlie the establishment of oriP, as newly introduced oriP plasmids replicate but are also lost rapidly from these cells. In addition, agenetic alteration in the oriP plasmid is not responsible for establishment, as oriP plasmids isolated from an established cell clone, propagated in Escherichia coli, and reintroduced into EBNA-1-positive cells are likewise established inefficiently. Our findings demonstrate that oriP replicons are not intrinsically stable in EBNA-1-positive cell lines. Rather, the establishment of an oriP replicon is conferred upon the replicon by a stochastic, epigenetic event that occurs infrequently and, therefore, is detected in only a minority of cells.