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首页> 外文期刊>Journal of Virology >Requirements for Excision and Amplification of Integrated Viral DNA Molecules in Polyoma Virus-Transformed Cells
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Requirements for Excision and Amplification of Integrated Viral DNA Molecules in Polyoma Virus-Transformed Cells

机译：多环病毒转化细胞中综合病毒DNA分子的切除和扩增的要求

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The integration of polyoma virus DNA into the genome of transformed rat cells generally takes place in a tandem head-to-tail arrangement. A functional viral large tumor antigen (T-Ag) renders this structure unstable, as manifested by free DNA production and excision or amplification of the integrated viral DNA. All of these phenomena involve the mobilization of precise genomic “units,” suggesting that they result from intramolecular homologous recombination events occurring in the repeated viral DNA sequences within the integrated structures. We studied polyoma ts-a-transformed rat cell lines, which produced large T-Ag but contained less than a single copy of integrated viral DNA. In all of these lines, reversion to a normal phenotype (indicative of excision) was extremely low and independent of the presence of a functional large T-Ag. The revertants were either phenotypic or had undergone variable rearrangements of the integrated sequences that seemed to involve flanking host DNA. In two of these cell lines (ts-a 4A and ts-a 3B), we could not detect any evidence of amplification even after 2 months of propagation under conditions permissive for large T-Ag. An amplification event was detected in a small subpopulation of the ts-a R5-1 line after 2 months of growth at 33°C. This involved a DNA fragment of 5.1 kilobases, consisting of the left portion of the viral insertion and about 2.5 kilobases of adjacent host DNA sequences. None of these lines spontaneously produced free viral DNA, but after fusion with 3T3 mouse fibroblasts, R5-1 and 4A produced a low level of heterogeneous free DNA molecules, which contained both viral and flanking host DNA. In contrast, the ts-a 9 cell line, whose viral insertion consists of a partial tandem of ～1.2 viral genomes, underwent a high rate of excision or amplification when propagated at temperatures permissive for large T-Ag function. These results indicate that the high rate of excision and amplification of integrated viral genomes observed in polyoma-transformed rat cells requires the presence of regions of homology (i.e., repeats) in the integrated viral sequences. Therefore, these events occur via homologous intramolecular recombination, which is promoted directly or indirectly by the large viral T-Ag.
机译：多瘤病毒DNA进入转化大鼠细胞基因组的整合通常在串联头尾布置中进行。功能性病毒大肿瘤抗原（T-Ag）使这种结构不稳定，如通过自由DNA生产和综合病毒DNA的切除或扩增表现出的这种结构。所有这些现象涉及动员精确的基因组“单位”，表明它们是由在综合结构中的重复的病毒DNA序列中发生的分子内同源重组事件导致。我们研究了PolyoMa Ts - A -Transformed大鼠细胞系，其产生大的T-AG，但含有少于单一的集成病毒DNA拷贝。在所有这些线中，逆转到正常表型（指示切除）的逆转非常低，并且与功能性大T-AG的存在无关。还原剂是表型或经过综合序列的可变重排，似乎涉及侧翼宿主DNA。在这些细胞系中的两个（ TS - A 4A和 TS - A 3B），我们无法检测到即使在大型T-AG允许的条件下繁殖2个月后扩增的任何证据。在33℃的2个月的生长后2个月后，在小亚群中检测到扩增事件在 Ts - R5-1系中。这涉及5.1千碱基的DNA片段，由病毒插入的左侧部分和约2.5千碱基的相邻宿主DNA序列组成。这些系列没有任何自发性产生的游离病毒DNA，但在用3T3小鼠成纤维细胞融合后，R5-1和4A产生低水平的非均相游离DNA分子，其含有病毒和侧翼宿主DNA。相反， ts - a 9个细胞系，其病毒插入由〜1.2病毒基因组的部分串联组成，在传播时经历了高的切除或放大速率温度允许大型T-AG功能。这些结果表明，在多瘤转化大鼠大鼠大鼠细胞中观察到的综合病毒基因组的高速率和扩增需要在综合病毒序列中存在同源性（即，重复）的区域。因此，这些事件通过同源分子内重组发生，其通过大病毒T-Ag直接或间接促进。

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