Tao L, Nielsen T, Friedlander P, Zannis-Hadjopoulos M, Price G
McGill Cancer Centre, McGill University, Montreal, Quebec, H3G 1Y6, Canada.
J Mol Biol. 1997 Oct 31;273(3):509-18. doi: 10.1006/jmbi.1997.1352.
A modification of the extrusion method for the isolation of nascent DNA from mammalian cells and a PCR-based assay has been used in order to compare the in vivo activities of DNA replication origins in different cell lines. Conventional PCR was firstly applied to detect the chromosomal activities of several known (origins associated with c-myc, hsp70, beta-globin, immunoglobulin mu-chain enhancer) and putative DNA replication origins (autonomously replicating sequences obtained from enriched libraries of human origins of DNA replication from normal and transformed cells) in four human cell lines (HeLa, NSF, WI-38 and SK-MG-1). Then, in nascent DNA samples from normal skin fibroblast (NSF) and HeLa cells, abundance of DNA sequences in the regions of five of these origins was determined by competitive PCR. Our results suggest that autonomously replicating sequences NOA3, S14, S3 and F15 are associated with functional chromosomal origins of replication. Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 are approximately twice as active in HeLa cells as in NSF cells. The described approach can facilitate the identification of origins which may be differentially active in normal cells and transformed cells or in different cell types.
为了比较不同细胞系中DNA复制起点的体内活性，人们采用了一种从哺乳动物细胞中分离新生DNA的挤压方法的改进方法以及一种基于PCR的检测方法。首先应用常规PCR检测四种人类细胞系（HeLa、NSF、WI-38和SK-MG-1）中几个已知的（与c-myc、hsp70、β-珠蛋白、免疫球蛋白μ链增强子相关的起点）和假定的DNA复制起点（从正常和转化细胞的人类DNA复制起点富集文库中获得的自主复制序列）的染色体活性。然后，在来自正常皮肤成纤维细胞（NSF）和HeLa细胞的新生DNA样本中，通过竞争性PCR确定这些起点中五个区域的DNA序列丰度。我们的结果表明，自主复制序列NOA3、S14、S3和F15与功能性染色体复制起点相关。起点活性的定量比较表明，与c-myc和NOA3相关的起点在HeLa细胞中的活性约为NSF细胞中的两倍。所描述的方法有助于鉴定在正常细胞和转化细胞或不同细胞类型中可能具有不同活性的起点。
