Pellegrini S, Dailey L, Basilico C
Cell. 1984 Apr;36(4):943-9. doi: 10.1016/0092-8674(84)90044-8.
Cells transformed by Polyoma virus (Py) can undergo a high rate of excision or amplification of integrated viral DNA sequences, and these phenomena require the presence of homology (i.e., repeats) within the viral insertion as well as a functional viral large T antigen (T-Ag). To determine whether the main role of large T-Ag in excision and amplification was replicative or recombination-promoting, we studied transformed rat cell lines containing tandem insertions of a ts-a Py molecule (encoding a thermolabile large T-Ag) with a deletion of the origin of viral DNA replication. Culturing of these cells at the temperature permissive for large T-Ag function did not result in any detectable excision or amplification of integrated Py sequences. We then introduced into origin-defective lines a recombinant plasmid containing the viral origin of replication and the gene coding for resistance to the antibiotic G418. All G418-resistant clones analyzed readily amplified the integrated plasmid molecules when grown under conditions permissive for large T-Ag function, showing that these cells produced viral large T-Ag capable of promoting amplification in trans of DNA sequences containing the Py origin. These observations strongly suggest that Polyoma large T antigen promotes excision or amplification of viral DNA by initiating replication at the integrated origin, providing a favorable substrate for subsequent recombination.
由多瘤病毒（Py）转化的细胞可对整合的病毒DNA序列进行高频率的切除或扩增，而这些现象需要病毒插入片段内存在同源性（即重复序列）以及功能性的病毒大T抗原（T-Ag）。为了确定大T抗原在切除和扩增中的主要作用是复制性的还是促进重组的，我们研究了含有ts-a Py分子（编码一种温度敏感型大T抗原）串联插入且缺失病毒DNA复制起点的转化大鼠细胞系。在允许大T抗原发挥功能的温度下培养这些细胞，并未导致整合的Py序列出现任何可检测到的切除或扩增。然后，我们将一个含有病毒复制起点和编码对抗生素G418抗性基因的重组质粒导入缺乏复制起点的细胞系中。当在允许大T抗原发挥功能的条件下生长时，所有分析的G418抗性克隆都能轻易地扩增整合的质粒分子，这表明这些细胞产生的病毒大T抗原能够促进含有Py起点的DNA序列进行反式扩增。这些观察结果强烈表明，多瘤病毒大T抗原通过在整合起点处启动复制来促进病毒DNA的切除或扩增，为后续重组提供了有利的底物。
