Construction of mammalian artificial chromosomes using YAC

着丝粒是真核生物染色体分离所必需的染色体结构域。虽然DNA被认为是哺乳动物细胞功能着丝粒结构的主要决定因素，但迄今为止还没有关于基本DNA结构的结论性信息。本研究的目的是利用酵母人工染色体（YAC）技术克隆具有功能的人类着丝粒DNA。我们已经在人类21号染色体上发现了两个超级碱基大小的阿尔法DNA结构域，阿尔法21- i和阿尔法21- ii。alpha21-I含有大量的CENP-B盒子，即CENP-B的结合位点，间隔高且有规律，而alpha21-II含有很少，如果有的话。我们利用重组负酵母菌克隆了取自alpha21- i_1和alpha21-II结构域的100 kb阿尔法DNA。我们与Dr.Cooke合作，构建了包含人类端粒序列和哺乳动物细胞可选择标记的YAC臂载体，并在体内用这些臂重组替代了alphoid YAC的两条臂。我们用这些含有YAC的人端粒和蛋白体转化培养的人细胞系，获得了含有这两种YAC的转化子。对转化细胞系的FISH分析表明，α 21- i型YAC的转化子含有一条稳定维持的哺乳动物人工染色体，而α 21- ii型YAC的转化子则整合在端粒或着丝粒上。这些结果表明，重要的着丝粒DNA序列位于α 21- i区。

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 intormation on the essential DNA structure is available until now. The purpose of this work is to clone functional human centromere DNA using technology of the yeast artificial chromosome (YAC). We have found previously two megabase-sized domains of alphoid DNA,alpha21-I and alpha21-II,in the human chromosome 21. The alpha21-I contained many CENP-B boxes, binding sites of CENP-B,at high and regular intervals, while the alpha21-II contained very few, if any. We have cloned into YAC 100 kb alphoid DNA derived from alpha21-Iand alpha21-II domain using a recombination minus yeast strain. In collaboration with Dr.Cooke, we constructed YAC arm vectors containing human telomere sequences and selectable markers for mammalian cells and both arms of alphoid YAC were replaced with these arms by recombination in vivo. We transformed a cultured human cell line with these human telomere and alphoid containing YAC and obtained transformants with both YACs. FISH analyzes of the transformed cell lines showed that transformants of alpha21-I YAC contained a stably maintained mammalian artificial chromosome but those of alpha21-II YAC was integrated at telomere or centromere. These results indicated that the essential centromere DNA sequence resides in alpha21-I region.