Development of Human Artificial Chromosome

我们一直感兴趣的结构和功能的基本要素所需的维持线性染色体在哺乳动物细胞。着丝粒和端粒的结构是这种染色体功能所必需的元件。我们一直在研究人类（α DNA和CENP-B）和小鼠染色体着丝粒区域的DNA序列和DNA-蛋白质相互作用。通过这次与霍华德库克博士的国际合作，我们希望在酵母中构建哺乳动物人工染色体（mammalian artificial chromosome，MAC）并将其转移到哺乳动物细胞中，利用MAC可以被拯救回酵母宿主的优点，最近我们发展了将几百kb的α DNA和小鼠小卫星DNA片段克隆到酵母人工染色体（Yeast artificial Chromosomes，YACs）中的方法。Cooke博士的研究小组开发了一种方法，可以对YAC基因进行改造， 关于我们 用酵母细胞中的人端粒序列修饰的YAC载体的YAC克隆的端粒序列。在改造过程中，YAC的插入DNA也发生了很高的重排率。因此，在改造的同时，我们开始应用体外消化和连接方法来交换YAC的端粒序列。他们还开发了YAC DNA扩增和分离方法，并使用显微注射或脂质转染将几种端粒修饰的YAC引入哺乳动物细胞。两种YAC导入方法都被证明是有效的，并且能够用于测定系统。选择端粒酶活性高的受体细胞是我们研究的必要条件，因为修饰后的YACs的稳定性在很大程度上取决于细胞中端粒酶活性的高低。作为MAC开发的下一步，我们现在正在改造α YAC和小鼠小卫星YAC，并将这些修饰的YAC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