Molecular mecbanism for determination ofdynamics and positioning in nucleus of vertebrates

确定脊椎动物细胞核动力学和定位的分子机制

• 批准号: 12440212
• 负责人: IKEMURA Toshimichi
• 金额: $ 7.36万
• 依托单位: Natiomal Institute of Genetics
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12440212/
• 关键词: Triplex; Polypurine; CENP; FISH; Nuclear organization; chromosome band boundary; Replication timing; DNA複製タイミング; ポリピリミジン

The single-strand formation and transmolecular triplex formation are thought to enable sequences spaced distantly along the genome to associate with each other and organize nuclear DNA into ordered configurations. Triplex-forming DNAs in the human interphase nucleus were analyzed by combining immunodetection by antitriplex antibodies and fluorescence in situ "nondenaturing " hybridization employing polypurine/polypyrimidine (PuPy) -tract probes. The "nondenaturing" hybridization technique, which has been used to detect RNA, could detect single-stranded DNAs in nondenatured nuclei. Triplexes visualized differentially with distinct PuPy-tract probes were associated spatially with centromere sequences in the interphase nucleus in a sequence-specific manner. Therefore, we were interested in centromore formation and function. To determine the precise functions of centromere proteins, we examined their roles by generating a conditional loss-of-function mutant in the chicken DT40 cell line. We concluded that both CENP-I and CENP-H are necessary for localization of CENP-C but not CENP-A to the centromere. We also performed living cell analysis of cells expressing GFP-or YFP- tagged centromere proteins to observe the dynamics of chromosomes.

单链形成和跨分子三链体形成被认为使得沿基因组间隔较远的序列能够彼此关联并将核DNA组织成有序的结构。通过将抗三链体抗体的免疫检测与使用聚嘌呤/聚嘧啶(PuPy)-束探针的荧光原位“非变性”杂交相结合，分析了人间期细胞核中形成三链体的DNA。用于检测RNA的“非变性”杂交技术可以检测非变性细胞核中的单链DNA。使用不同的 PuPy-tract 探针进行差异可视化的三链体以序列特异性方式与间期核中的着丝粒序列在空间上相关。因此，我们对中心粒的形成和功能感兴趣。为了确定着丝粒蛋白的精确功能，我们通过在鸡 DT40 细胞系中产生条件性功能丧失突变体来检查它们的作用。我们的结论是，CENP-I 和 CENP-H 都是 CENP-C 定位到着丝粒所必需的，而不是 CENP-A 到着丝粒的定位。我们还对表达 GFP 或 YFP 标记的着丝粒蛋白的细胞进行了活细胞分析，以观察染色体的动态。

项目成果

Y. Watanabe: "Chromosome-wide assessment of replication timing for human chromosomes 11q and 21q : disease-related genes in timing-switch regions"Human Molecular Genetics. 11. 13-21 (2002)

Y. Watanabe：“人类染色体 11q 和 21q 复制时序的全染色体评估：时序转换区域中的疾病相关基因”人类分子遗传学。

野上正弘: "Relative locations of the centromere and imprinted SNRPN gene within chromosome 15 territories during the cell cycle in HL60 cells"Journal of Cell Science. 113. 2157-2165 (2000)

Masahiro Nogami：“HL60 细胞细胞周期中 15 号染色体区域内着丝粒和印记 SNRPN 基因的相对位置”《细胞科学杂志》113. 2157-2165 (2000)。

T. Shina: "Genomic anatomy of a premier major histocompatibility complex paralogous region on chromosome 1q21-q22"Genome Research. 11. 789-802 (2001)

T. Shina：“染色体 1q21-q22 上首要主要组织相容性复合体旁系同源区域的基因组解剖”基因组研究。

Shina, T.: "Genomic anatomy of a premier major histocompatibility complex paralogous region on chromosome 1q21-q22"Genome Research. 11. 789-802 (2001)

Shina, T.：“染色体 1q21-q22 上首要主要组织相容性复合体旁系同源区域的基因组解剖”基因组研究。

Kanaya, S., Yamada, Y, Kinouchi M., Kudo, Y, and Ikemura, T: "Codon usage and tRNA genes in eukaryotes : Correlation of coaorf usage diversity with translation efficiency and with CG-0inudfcotidゥ usage as assessed by multivariate analysis."J. Mol. Evol. 5

Kanaya, S.、Yamada, Y、Kinouchi M.、Kudo, Y 和 Ikemura, T：“真核生物中的密码子使用和 tRNA 基因：通过多变量分析评估 coaorf 使用多样性与翻译效率以及 CG-0inudfcotidu 使用的相关性.”J. Mol. Evol. 5