CHARACTERIZATION OF AN EPISOME PRODUCED IN HAMSTER-CELLS THAT AMPLIFY A TRANSFECTED CAD GENE AT HIGH-FREQUENCY - FUNCTIONAL EVIDENCE FOR A MAMMALIAN REPLICATION ORIGIN
CHARACTERIZATION OF AN EPISOME PRODUCED IN HAMSTER-CELLS THAT AMPLIFY A TRANSFECTED CAD GENE AT HIGH-FREQUENCY - FUNCTIONAL EVIDENCE FOR A MAMMALIAN REPLICATION ORIGIN
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DOI：
10.1128/mcb.7.5.1740
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发表时间：
1987-05-01
影响因子：
5.3
通讯作者：
WAHL, GM
中科院分区：
文献类型：
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作者：
CARROLL, SM;GAUDRAY, P;WAHL, GM
在先前的研究中（G. M. Wahl、B. Robert de Saint Vincent 和 M. L. De Rose, Nature (London) 307:516-520, 1984），我们使用 CAD 粘粒的基因转移来证明基因位置深刻影响扩增频率。一种转化体 T5 扩增捐赠的 CAD 基因的频率比其他分析的转化体高至少 100 倍。 T5和两个耐药衍生物中的CAD基因被定位在染色体上。在本报告中，我们表明 T5 的亚克隆产生含有捐赠的 CAD 基因的染色体外分子（CAD 附加体）。凝胶电泳表明CAD附加体大约为250至300 kilobase对，多种方法表明它是一个共价闭合环。我们发现 CAD 附加体半保守复制，大约每个细胞周期复制一次。由于包含大部分 CAD 附加体的 CAD 粘粒在转染到细胞中时不会自主复制，因此我们的结果表明，要么 (i) 生成附加体的过程导致 DNA 复制的细胞起点与 CAD 序列连接，要么 (ii) 附加体内的特定重排产生了功能起点。讨论了这些结果对基因扩增机制和微小染色体起源的影响。
In a previous study (G. M. Wahl, B. Robert de Saint Vincent, and M. L. De Rose, Nature (London) 307:516-520, 1984), we used gene transfer of a CAD cosmid to demonstrate that gene position profoundly affects amplification frequency. One transformant, T5, amplified the donated CAD genes at a frequency at least 100-fold higher than did the other transformants analyzed. The CAD genes in T5 and two drug-resistant derivatives were chromosomally located. In this report, we show that a subclone of T5 gives rise to an extrachromosomal molecule (CAD episome) containing the donated CAD genes. Gel electrophoresis indicated that the CAD episome is approximately 250 to 300 kilobase pairs, and a variety of methods showed that it is a covalently closed circle. We show that the CAD episome replicates semiconservatively and approximately once per cell cycle. Since the CAD cosmid, which comprises most of the CAD episome, does not replicate autonomously when transfected into cells, our results indicate that either (i) the process which generated the episome resulted in a cellular origin of DNA replication being linked to the CAD sequences or (ii) specific rearrangements within the episome generated a functional origin. The implications of these results for mechanisms of gene amplification and the genesis of minute chromosomes are discussed.