Simpson K, Huxley C
Department of Biochemistry and Molecular Genetics, Imperial College School of Medicine at St. Mary's, London, UK.
Nucleic Acids Res. 1996 Dec 1;24(23):4693-9. doi: 10.1093/nar/24.23.4693.
The development of a system for shuttling DNA cloned as yeast artificial chromosomes (YACs) between yeast and mammalian cells requires that the DNA is maintained as extrachromosomal elements in both cell types. We have recently shown that circular YACs carrying the Epstein-Barr virus origin of plasmid replication (oriP) are maintained as stable, episomal elements in a human kidney cell line constitutively expressing the viral transactivator protein EBNA-1. Here, we demonstrate that a 90-kb episomal YAC can be isolated intact from human cells by a simple alkaline lysis procedure and shuttled back into Saccharomyces cerevisiae by spheroplast transformation. In addition, we demonstrate that the 90-kb YAC can be isolated intact from yeast cells. The ability to shuttle large, intact fragments of DNA between yeast and human cells should provide a powerful tool in the manipulation and analysis of functional regions of mammalian DNA.
开发一种用于在酵母和哺乳动物细胞之间穿梭以酵母人工染色体（YAC）形式克隆的DNA的系统，要求DNA在这两种细胞类型中都作为染色体外元件维持。我们最近表明，携带爱泼斯坦-巴尔病毒质粒复制起点（oriP）的环状YAC在持续表达病毒反式激活蛋白EBNA-1的人肾细胞系中作为稳定的附加型元件维持。在此，我们证明通过简单的碱裂解程序可以从人细胞中完整分离出一个90 kb的附加型YAC，并通过原生质球转化将其穿梭回酿酒酵母中。此外，我们证明可以从酵母细胞中完整分离出90 kb的YAC。在酵母和人细胞之间穿梭大的完整DNA片段的能力应该为哺乳动物DNA功能区域的操作和分析提供一个强大的工具。
