Construction of mammalian artificial chromosomes using YAC

使用 YAC 构建哺乳动物人工染色体

• 批准号: 09044213
• 负责人: MASUMOTO Hiroshi
• 金额: $ 1.22万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for international Scientific Research
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1998
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044213/
• 关键词: cetromere; teloreme; alphoid DNA; YAC; HAC; CENP-B

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.

为了定义功能性人类着丝粒序列，人工染色体被构建为可复制的 DNA 分子。将哺乳动物端粒重复序列和选择标记引入酵母人工染色体 (YAC) 中，该染色体含有来自重组缺陷酵母宿主中人类 21 号染色体着丝粒区域的 aphoid DNA。当这些修饰的YAC被引入人类培养细胞时，带有来自α21-I位点的αDNA、含有高频率的CENP-B盒和规则重复阵列的YAC，有效地形成人工染色体，并且在没有DNA的情况下保持稳定。选择并结合着丝粒/着丝粒蛋白、CENP-A、CENP-B、CENP-C 和 CENP-E。人工染色体大小为 1 - 5 Mb，由多聚体组成引入 YAC DNA，在中期平板上对齐并在后期正确分离到相反的两极。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 alpha21-II 基因座的 α21-II 基因座的阿尔法 DNA 的修饰 YAC 产生，该基因座不包含 CENP-B 框，并且序列排列不太规则。我们得出结论，α21-I aiphoid 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。

项目成果

Junji Iwahara: "A helix-turn-helix structure unit in human centromere protein B(CENP-B)." EMBO J.17. 827-837 (1998)

Junji Iwahara：“人类着丝粒蛋白 B (CENP-B) 中的螺旋-转角-螺旋结构单元。”

M.Ikeno: "Construction of YAC-based mammalian artificial chromosomes." Nature Biotech.16. 431-439 (1998)

M.Ikeno：“基于 YAC 的哺乳动物人工染色体的构建。”