FINE MAPPING OF A REPLICATION ORIGIN OF HUMAN DNA
FINE MAPPING OF A REPLICATION ORIGIN OF HUMAN DNA
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DOI：
10.1073/pnas.91.15.7119
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发表时间：
1994-07-19
影响因子：
11.1
通讯作者：
FALASCHI, A
中科院分区：
文献类型：
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作者：
GIACCA, M;ZENTILIN, L;FALASCHI, A
开发了一种高灵敏度的方法，用于鉴定哺乳动物细胞单拷贝复制子中双向DNA合成的起源。该方法不需要细胞同步化或透化，需要在新合成的DNA样品中通过竞争性PCR程序对沿给定基因组区域沿着的相邻DNA片段的丰度进行绝对定量。该程序被用于定位DNA复制的起始位点，该起始位点位于编码核纤层蛋白B2的人19号染色体的13.7-kb区域中，核纤层蛋白B2在HL-60细胞中S期开始后立即复制。在此区域内，DNA复制起始于一个474 bp的区域，该区域对应于核纤层蛋白B2基因的3'非编码端和该基因与另一个高度转录的核纤层蛋白B2基因的5'端之间的非转录间隔区。
A highly sensitive procedure was developed for the identification of the origin of bidirectional DNA synthesis in single-copy replicons of mammalian cells. The method, which does not require cell synchronization or permeabilization, entails the absolute quantification, by a competitive PCR procedure in newly synthesized DNA samples, of the abundance of neighboring DNA fragments distributed along a given genomic region. This procedure was utilized for mapping the start site of DNA replication in a 13.7-kb region of human chromosome 19 coding for lamin B2, which is replicated immediately after the onset of S phase in HL-60 cells. Within this region, DNA replication initiates in a 474-bp area corresponding to the 3' noncoding end of the lamin B2 gene and the nontranscribed spacer between this gene and the 5' end of another highly transcribed one, This localization was obtained both in aphidicolin-synchronized and in exponentially growing HL-60 cells.