Nielsen T O, Cossons N H, Zannis-Hadjopoulos M, Price G B
Department of Pathology, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
J Cell Biochem. 2000 Jan;76(4):674-85. doi: 10.1002/(sici)1097-4644(20000315)76:4<674::aid-jcb15>3.0.co;2-2.
pYACneo, a 15.8-kb plasmid, contains a bacterial origin, G418-resistance gene, and yeast ARS, CEN, and TEL elements. Three mammalian origins have been cloned into this circular vector: 343, a 448-bp chromosomal origin from a transcribed region of human chromosome 6q; X24, a 4.3-kb element containing the hamster DHFR origin of bidirectional replication (oribeta), and S3, a 1.1-kb human anti-cruciform purified autonomously replicating sequence. The resulting constructs have been transfected into HeLa cells, and G418-resistant subcultures were isolated. The frequency of G418-resistant transformation was 1.7-8.7 times higher with origin-containing YACneo than with vector alone. After >45 generations under G418 selection, the presence of episomal versus integrated constructs was assessed by fluctuation assay and by PCR of supercoiled, circular, and linear genomic cellular DNAs separated on ethidium bromide-cesium chloride gradients. In stable G418-resistant subcultures transfected with vector alone or with linearized constructs, as well as in some subcultures transfected with circular origin-containing constructs, resistance was conferred by integration into the host genome. However, several examples were found of G418-resistant transfectants maintaining the Y.343 and the YAC.S3 circular constructs in a strictly episomal state after long-term culture in selective medium, with 80-90% stability per cell division. The episomes were found to replicate semiconservatively in a bromodeoxyuridine pulse-labeling assay for </=130 cell generations after transfection. Furthermore, after </=172 cell generations rescued episomal DNA could be isolated intact and unrearranged, and could be used to retransform bacteria. These versatile constructs, containing mammalian origins, have the capacity for further modification with human telomere or large putative centromere elements, in an effort to move towards construction of a human artificial chromosome.
pYACneo是一种15.8千碱基对的质粒，包含一个细菌复制起点、G418抗性基因以及酵母自主复制序列（ARS）、着丝粒（CEN）和端粒（TEL）元件。三个哺乳动物复制起点已被克隆到这个环状载体中：343，一个来自人类6号染色体q转录区域的448碱基对的染色体复制起点；X24，一个包含仓鼠二氢叶酸还原酶双向复制起点（oribeta）的4.3千碱基对元件，以及S3，一个1.1千碱基对的人抗十字形纯化自主复制序列。将得到的构建体转染到HeLa细胞中，并分离出G418抗性亚培养物。含复制起点的YACneo的G418抗性转化频率比单独使用载体时高1.7至8.7倍。在G418选择下经过>45代培养后，通过波动试验以及对在溴化乙锭-氯化铯梯度上分离的超螺旋、环状和线性基因组细胞DNA进行PCR，评估游离型与整合型构建体的存在情况。在单独用载体或线性化构建体转染的稳定G418抗性亚培养物中，以及在用含环状复制起点的构建体转染的一些亚培养物中，抗性是通过整合到宿主基因组中来赋予的。然而，发现了几个例子，即在选择性培养基中长时间培养后，G418抗性转染子以严格的游离型状态维持Y.343和YAC.S3环状构建体，每个细胞分裂的稳定性为80 - 90%。在转染后进行的溴脱氧尿苷脉冲标记试验中，发现游离型在≤130个细胞代中以半保留方式复制。此外，在≤172个细胞代后，可以完整且未重排地分离出拯救的游离型DNA，并可用于再次转化细菌。这些含有哺乳动物复制起点的通用构建体，有能力用人端粒或大型假定着丝粒元件进行进一步修饰，以努力朝着构建人类人工染色体的方向发展。
