Construction of mammalian artificial chromosomes using YAC

为了定义一个有功能的人类着丝粒序列，我们构建了一个人工染色体作为一个可复制的DNA分子。将哺乳动物端粒重复序列和一个可选择的标记引入酵母人工染色体(YAC)中，YAC中含有来自人类21号染色体着丝粒区域的Aphid DNA。当这些修饰的YAC被导入人培养细胞时，YAC具有来自Alpha21-I基因座的Alphid DNA，含有高频的CENP-B盒和规则的重复阵列，有效地形成人工染色体，在没有选择和结合着丝粒/着丝粒/着丝粒蛋白CENP-A、CENP-B、CENP-C和CENP-E的情况下保持稳定。人工染色体1-5Mb由导入的YAC DNA的多聚体组成，在中期平板上排列，并在后期正确地分离到相反的两极。广泛的细胞学分析强烈表明，它们没有获得宿主序列，在所有情况下都是通过从头机制形成的。相比之下，人工染色体从未用含有来自alpha21-II基因座的Alphid DNA的改良YAC产生，该基因座不包含CENP-B盒，并且序列排列不那么规则。我们的结论是，α21-I配基DNA可以诱导人工染色体上活性着丝粒/着丝粒结构的从头组装。

In order to define a functional human centromere sequence, an artificial chromosome was constructed as a reproducible DNA molecule. Mammalian telomere repeats and a selectable marker were introduced into yeast artificial chromosomes (YACs) containing aiphoid DNA from the human chromosome 21 centromere region in a recombination-deficient yeast host. When these modified YACs were introduced into human cultured cells, a YAC with the alphoid DNA from alpha21-I locus, containing CENP-B boxes at a high frequency and a regular repeat array, efficiently formed artificial chromosomes which were maintained stably in the absence of selection and bound centromere/kinetochore proteins, CENP-A, CENP-B, CENP-C and CENP-E.The artificial chromosomes 1 - 5 Mb in size and composed of multimers of the introduced YAC DNA, aligned at metaphase plates and segregated to opposite poles correctly in anaphase. Extensive cytological analyses strongly suggested that they had not acquired host sequences and were formed in all cases by a de novo mechanism. In contrast, artificial chromosomes were never produced with a modified YAC containing alphoid DNA from the alpha21-II locus which contains no CENP-B boxes and has a less regular sequence arrangement. We conclude that alpha21-I aiphoid DNA can induce de novo assembly of active centromere/kinetochore structures on artificial chromosomes.