Pelletier R, Price G B, Zannis-Hadjopoulos M
McGill Cancer Centre, McGill University, Montréal, Québec H3G 1YG, Canada.
J Cell Biochem. 1999 Sep 15;74(4):562-75.
Ors12, a mammalian autonomously replicating sequence (812 bp), was previously isolated by extrusion of African green monkey (CV-1 cells) nascent DNA from active replication bubbles. It contains a region of alpha-satellite extending 168-bp from the 5'-end, and a nonrepetitive portion extending from nucleotide position 169 to nucleotide 812 that is present in less than nine copies per haploid genome. Ors12 is capable of transient autonomous DNA replication in vivo and in vitro, associates with the nuclear matrix in a cell cycle-dependent manner, and hybridizes at the centromeric region of six CV-1 cell chromosomes as well as a marker chromosome. To demonstrate that DNA replication initiates at ors12 at a native chromosomal locus, a 14.2 kb African green monkey genomic clone was isolated and sequence information was obtained that allowed us to generate eight sets of PCR primers spanning a region of 8 kb containing ors12. One set of primers occurred inside ors12. These primers were used to amplify nascent DNA strands from asynchronously growing CV-1 and African green monkey kidney (AGMK) cells, using noncompetitive and competitive PCR-based mapping methodologies. Both assays showed that DNA replication in vivo initiates preferentially in a 2.3 kb region containing ors12, as well as at a second site located 1.7 kb upstream of ors12. This study provides the first demonstration of genomic function for a centromeric mammalian origin of DNA replication, originally isolated by nascent strand extrusion.
Ors12是一段哺乳动物自主复制序列（812碱基对），先前通过从活跃复制泡中挤出非洲绿猴（CV-1细胞）新生DNA而分离得到。它包含一个从5'端延伸168碱基对的α卫星区域，以及一个从核苷酸位置169延伸至核苷酸812的非重复部分，该部分在每个单倍体基因组中的拷贝数少于9个。Ors12能够在体内和体外进行瞬时自主DNA复制，以细胞周期依赖的方式与核基质结合，并与6条CV-1细胞染色体的着丝粒区域以及一条标记染色体杂交。为了证明DNA复制在天然染色体位点的ors12处起始，分离了一个14.2 kb的非洲绿猴基因组克隆，并获得了序列信息，这使我们能够生成8组跨越包含ors12的8 kb区域的PCR引物。其中一组引物位于ors12内部。这些引物用于使用基于非竞争性和竞争性PCR的定位方法，从异步生长的CV-1细胞和非洲绿猴肾（AGMK）细胞中扩增新生DNA链。两种检测方法均表明，体内DNA复制优先在包含ors12的2.3 kb区域以及ors12上游1.7 kb处的第二个位点起始。本研究首次证明了最初通过新生链挤出分离得到的着丝粒哺乳动物DNA复制起点的基因组功能。
