Structural and Functional Analyzes of Human Artificial Chromosomes

人类人工染色体的结构和功能分析

• 批准号: 09480185
• 负责人: MASUMOTO Hiroshi
• 金额: $ 8.51万
• 依托单位: Nagoya University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 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-09480185/
• 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 alphoid 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 alphoid DNA can induce de novo assembly of active centromere/kinetochore structures on artificial chromosomes.

为了定义功能性的人工丝粒序列，人工染色体被构造为可重复的DNA分子。将哺乳动物的端粒重复序列和可选标记物引入酵母人工染色体（YAC）中，这些染色体（YACS）含有来自人类染色体的21 Centromere区域中的载体DNA。当将这些修饰的YAC引入人类培养的细胞中时，带有α21-I基因座的字母DNA的YAC，含有高频和常规重复阵列的CENP-B框，有效地形成了人工染色体，在缺乏选择和束缚的Centromere/Kintromere/Kinetochor Proteep-cenp-cenp-cenp-a，Cenp-a，Cenp-a，Cenp-a，Cenp-a，Cenp-a，Cenp-a，Cenp-a，Cenp-a，Cenp-a CENP -E。人造染色体1-5 MB，由引入的YAC DNA的多聚体组成，在中期板上对齐，并在后期正确隔离到相对的杆子。广泛的细胞学分析强烈表明，它们尚未获得宿主序列，并且在所有情况下都是通过从头机制形成的。相比之下，从未使用含有α21-II基因座的α21-II基因座的Alphoid DNA产生人造染色体，该基因座不含CENP-B盒，并且具有较少的正常序列排列。我们得出的结论是，α21-I字母DNA可以在人工染色体上诱导活性centromere/Kinetochore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)." ENBO 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。”

H.Masumoto: "Assay of centromere function using a human artificial chromosome." Chromosoma. 107. 406-416 (1998)

H.Masumoto：“使用人类人工染色体测定着丝粒功能。”

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

M.Ikeno 等人：“基于 YAC 的哺乳动物人工染色体的构建。”

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

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