Construction of mammalian artificial chromosomes using YAC

为了确定一个功能性的人类着丝粒序列，人工染色体被构建为一个可重复的DNA分子。将哺乳动物端粒重复序列和选择性标记引入重组缺陷型酵母宿主中含有来自人21号染色体着丝粒区域的类α DNA的酵母人工染色体（YAC）中。当将这些修饰的YAC引入人培养细胞中时，具有来自α 21-I基因座的α DNA、含有高频率的CENP-B盒和规则重复序列的YAC有效地形成人工染色体，其在不存在选择和结合的着丝粒/动粒蛋白质CENP-A、CENP-B、CENP-C和CENP-E.人工染色体大小为1 - 5 Mb，由导入的YAC DNA的多聚体组成，在中期平板上对齐，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，并且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α 21-II基因座的α DNA的修饰的YAC产生，所述α 21-II基因座不含CENP-B盒并且具有较不规则的序列排列。我们的结论是，α 21-I类DNA可以诱导活性着丝粒/动粒结构在人工染色体上从头组装。

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.