Cloning of functional human centromere DNA by YAC technology

本工作的目的是利用酵母人工染色体（YAC）技术克隆具有功能的人着丝粒DNA。虽然DNA被认为是哺乳动物细胞中功能性着丝粒结构的主要决定因素，但迄今为止，关于DNA的基本结构还没有结论性的信息。为了解决这个问题，我们之前分析了人类21号染色体着丝粒卫星DNA的远程组织，特别强调了包含CENP-B识别序列（CENP-B盒）的阿尔法DNA的分布。我们已经在21号染色体中发现了两个兆碱基大小的α DNA结构域（α 21-I和α 21-II）：α 21-I以规则的间隔包含许多CENP-B盒，而α 21-II包含很少的CENP-B盒。在这项与P.Hieter博士的国际合作中，我们将来自21号染色体的α 21-I和α 21-II区域的100 kb大小的DNA克隆到YAC中。我们发现这些α-YAC克隆在重组减酵母菌株中稳定保持。然后，我们将人端粒序列引入到α YAC克隆的两个载体臂中，以便这些YAC克隆可以作为哺乳动物人工染色体（MAC）在哺乳动物细胞中维持，如果它们含有功能性着丝粒DNA的话。我们已经获得了一些同时具有alpha 21-I YAC和alpha 21-II YAC的转化体。我们目前正在分析掺入的alphoid YAC的核内状态。

The purpose of this work is to clone functional human centromeric DNA using the technology of the yeast artificial chromosome (YAC).Centromere is the chromosomal domain essential to the segregation of eukaryotic chromosomes. Although DNA is believed to be the primary determinant of the functional centromere structure in mammalian cells, no conclusive information on the essential DNA structure is available until now. To solve this problem, we previously analyzed long range organization of the centromeric satellite DNA of the human chromosome 21, in specific emphasis on the distribution of alphoid DNA containing the recognition sequence of CENP-B,the CENP-B box. We have found two megabase-sized domains of alphoid DNA (alpha21-I and alpha21-II) in the chromosome 21 : alpha21-I,contained many CENP-B boxes at regular intervals and alpha21-II containing very few, if any, CENP-B boxes. In this international collaboration with Dr.P.Hieter, we performed the cloning into YAC of 100 kb size DNA from the alpha21-I and alpha21-II region of the chromosome 21. We found that these alphoid YAC clones were stably maintained in the recombination minus yeast strains. We then introduced the human telomere sequences into the two vector arms of the alphoid YAC clones so that these YAC clones could be maintained in the mammalian cells as mammalian artificial chromosomes (MAC), if they contained functional centromere DNA.We introduced resulting clones into a human cell line by microinjection and lipofection. We have obtained a few transformants with both alpha21-I YAC and alpha21-II YAC.We are currently analyzing intranuclear state of incorporated alphoid YACs.