Development of Human Artificial Chromosome

我们一直对哺乳动物细胞中维持线性染色体所需的基本元素的结构和功能感兴趣。着丝粒和端粒结构是这些染色体功能所必需的元素。我们一直在研究人类（alphoid DNA和CENP-B）和小鼠染色体着丝粒区域的DNA序列和DNA-蛋白相互作用。通过与howard Cooke博士的国际合作，我们希望在酵母中构建哺乳动物人工染色体（MAC）并将其转移到哺乳动物细胞中，其优点是MAC可以被拯救回酵母宿主。近年来，我们开发了将数百kb的阿尔法体DNA和小鼠次要卫星DNA片段不经重排克隆到酵母人工染色体（YACs）中的方法，并成功获得了几个含有阿尔法体DNA或小鼠次要卫星DNA的稳定的YAC克隆。Cooke的团队开发了在酵母细胞中用人类端粒序列修饰的YAC载体改造YAC克隆的更多端粒序列的方法。在改造过程中，YAC的插入DNA也有很高的重排率。因此，在改造的同时，我们开始应用体外消化和结扎方法来交换YACs的端粒序列。他们还开发了YAC DNA扩增和分离方法，并通过显微注射或脂肪转染将几种端粒修饰的YAC引入哺乳动物细胞。结果表明，这两种方法都是有效的，可以用于分析系统。选择端粒酶活性高的受体细胞是我们研究进展的必要条件，因为修饰后的YACs的稳定性很大程度上取决于细胞中端粒酶的活性。作为MAC发展的下一步，我们现在正在改造脂蛋白YACs和小鼠小卫星YACs，并将这些修饰的YACs引入哺乳动物细胞。少

We have been interested in the structures and the functions of the essential elements required for the maintenance of linear chromosomes in mammalian cells. The centromere and the telomere structures are the elements necessary for such chromosome functions. We have been studying the DNA sequences and the DNA-protein interaction at the centromere regions of human (alphoid DNA and CENP-B) and mouse chromosomes. Through this international collaboration with Dr.Howard Cooke, we hope to construct mammalian artificial chromosome (MAC) in yeast and transfer them to mammalian cells with the advantage that MAC can be rescued back into the yeast host.Recently we have developed cloning methods for several hundred kb fragments of alphoid DNA and mouse minor satellite DNA into Yeast Artificial Chromosomes (YACs) without the rearrangement and succeeded in obtaining several stable YAC clones containing alphoid DNA or mouse minor satellite DNA.Dr.Cooke's group developed methods for retrofitting the te … More lomere sequences of YAC clones with YAC vectors modified with human telomere sequences in the yeast cells. During the retrofitting process the insert DNA of YAC also rearranged with very high rate. Therefore, in parallel with the retrofitting we began to apply in vitro digestion and ligation methods to exchange the telomere sequence of YACs. They also developed the YAC DNA amplification and isolation methods and introduced several telomere modified YACs into mammalian cells using microinjection or lipofection. Both the methods for YAC introduction turned out to be efficient and be able to use for assay systems. It was necessary for the progress of our study to choose a recipient cell with high telomerase activity because the stability of the modified YACs greatly depended on the telomerase activity in the cells. As the next step towards the development of MAC, we are now retrofitting alphoid YACs and mouse minor satellite YACs and going to introduce these modified YACs into mammalian cells. Less