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
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