Taira T, Iguchi-Ariga S M, Ariga H
Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Mol Cell Biol. 1994 Sep;14(9):6386-97. doi: 10.1128/mcb.14.9.6386-6397.1994.
A general and sensitive method for the mapping of initiation sites of DNA replication in vivo, developed by Vassilev and Johnson, has revealed replication origins in the region of simian virus 40 ori, in the regions upstream from the human c-myc gene and downstream from the Chinese hamster dihydrofolate reductase gene, and in the enhancer region of the mouse immunoglobulin heavy-chain gene. Here we report that the region containing the promoter of the human heat shock protein 70 (hsp70) gene was identified as a DNA replication origin in HeLa cells by this method. Several segments of the region were cloned into pUC19 and examined for autonomously replicating sequence (ARS) activity. The plasmids carrying the segments replicated episomally and semiconservatively when transfected into HeLa cells. The segments of ARS activity contained the sequences previously identified as binding sequences for a c-myc protein complex (T. Taira, Y. Negishi, F. Kihara, S. M. M. Iguchi-Ariga, and H. Ariga, Biochem. Biophys. Acta 1130:166-174, 1992). Mutations introduced within the c-myc protein complex binding sequences abolished the ARS activity. Moreover, the ARS plasmids stably replicated at episomal state for a long time in established cell lines. The results suggest that the promoter region of the human hsp70 gene plays a role in DNA replication as well as in transcription.
瓦西列夫和约翰逊开发的一种用于体内DNA复制起始位点定位的通用且灵敏的方法，揭示了猿猴病毒40 ori区域、人c-myc基因上游区域、中国仓鼠二氢叶酸还原酶基因下游区域以及小鼠免疫球蛋白重链基因增强子区域中的复制起点。在此我们报告，通过该方法，包含人热休克蛋白70（hsp70）基因启动子的区域在HeLa细胞中被鉴定为DNA复制起点。该区域的几个片段被克隆到pUC19中，并检测其自主复制序列（ARS）活性。携带这些片段的质粒转染到HeLa细胞后能以附加体形式进行半保留复制。具有ARS活性的片段包含先前被鉴定为c-myc蛋白复合物结合序列的序列（T. Taira、Y. Negishi、F. Kihara、S. M. M. Iguchi-Ariga和H. Ariga，《生物化学与生物物理学报》1130:166 - 174，1992）。在c-myc蛋白复合物结合序列内引入的突变消除了ARS活性。此外，ARS质粒在已建立的细胞系中能以附加体状态长期稳定复制。结果表明，人hsp70基因的启动子区域在DNA复制以及转录中都发挥作用。
