Construction of mammalian artificial chromosomes using YAC

为了确定一个功能性的人类着丝粒序列，构建了一个人工染色体作为一个可复制的DNA分子。在重组缺陷酵母宿主中，将哺乳动物端粒重复序列和一个可选择标记引入含有人类21号染色体着丝粒区非类人体DNA的酵母人工染色体（YACs）中。当这些修饰的YAC被引入人培养细胞时，一个含有来自alpha21-I位点的阿尔法体DNA的YAC，包含高频的CENP-B盒子和规则的重复阵列，有效地形成人工染色体，这些人工染色体在没有选择的情况下稳定地维持，并结合着丝粒/着丝粒蛋白CENP-A， CENP-B， CENP-C和CENP-E。人工染色体大小为1 - 5mb，由引入的YAC DNA的多聚体组成，在中期板上排列，在后期正确地分离到对极。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新生机制形成的。相比之下，人工染色体从未由含有alpha21-II基因座的阿尔法DNA的修饰YAC产生，该基因座不含CENP-B盒，序列排列不太规则。我们得出结论，α 21- i非类人体DNA可以诱导人工染色体上活性着丝粒/着丝粒结构的重新组装。

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.