Construction of mammalian artificial chromosomes using YAC

着丝粒是真核细胞染色体分离所必需的染色体区域。虽然DNA被认为是哺乳动物细胞中功能着丝粒结构的主要决定因素，但到目前为止还没有关于基本DNA结构的确凿信息。本工作的目的是利用酵母人工染色体(YAC)技术克隆人着丝粒DNA。我们以前已经在人类21号染色体上发现了两个百万碱基大小的阿尔法DNA结构域，α21-I和α21-II。Alpha21-I以较高和规则的间隔包含许多CENP-B盒，即CENP-B的结合部位，而Alpha21-II包含很少的CENP-B盒。我们利用重组酵母菌株克隆了来自α21-I和α21-II结构域的100kb的alphid DNA。我们与库克博士合作，构建了包含人端粒序列和哺乳动物细胞可选择标记的YAC臂载体，并通过体内重组将这些臂替换为Alphid YAC的双臂。我们用这些含有YAC的人端粒和α蛋白转化了培养的人细胞系，并获得了含有这两种YAC的转化子。对转化细胞系的FISH分析表明，alpha21-Iyac的转化子含有一个稳定维持的哺乳动物人工染色体，而alpha21-iyac的转化子则整合在端粒或着丝粒。这些结果表明，着丝粒DNA的基本序列位于α21-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.