High resolution analysis of interchromosomal nuclear compartmentalization in situ and in vivo

原位和体内染色体间核区室化的高分辨率分析

• 批准号: 5107531
• 负责人: Professor Dr. Peter Lichter
• 金额: --
• 依托单位: Abteilung Molekulare Genetik
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 1998
• 资助国家: 德国
• 起止时间: 1997-12-31 至 2006-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/5107531?language=en
• 关键词: resolution; analysis; interchromosomal; nuclear; compartmentalization

In order to investigate the topological organziation of chromosomes in the cell nucleus at the level of electron microscopy (EM), protocols were elaborated within the fist funding period to allow delineation of chromosomes and subchromosomal regions. Since the method to visualize segregated chromosomes after BrdU incorporation via immunostaining revealed the best results, this method will be utilized in experiments co-delineating chromosomes with other markers, such as i) endogenous RNAs, nuclear bodies etc. as well as ii) ectopically expressed filament proteins as marker for interchromosomal space and iii) intra-chromosomal regions generated by integrated lac-operator sequences and visualized via lac-repressor-GFP fusion protein detected by immuno-EM. Since the integrity of the intra- and interchromosomal fine structure in the nucleus is dependent on the hydration state, we will also focus on the adaption of EM-cryotechnology to gain maximum structural information with minimum pertubation of specimen. The anticipated results will have important implications for the understanding i) of chromosome organization within interphase nuclei and ii) of the higher order nuclear architecture and subcompartmentilization. Even more importantly, these analyses will allow to assess the relationship between genome function, such as gene expression, and nuclear structure.

为了在电子显微镜（EM）水平上研究细胞核中染色体的拓扑组织，在第一个资助期内制定了方案，以划定染色体和亚染色体区域。由于在BrdU掺入后通过免疫染色使分离的染色体可视化的方法显示了最佳结果，因此该方法将用于与其他标志物共描绘染色体的实验中，所述其他标志物例如i）内源RNA，核体等，以及ii）异位表达的丝蛋白作为染色体间空间的标记物，以及iii）由整合的lac-operator序列产生并通过lac-operator可视化的染色体内区域免疫电镜检测阻遏物-GFP融合蛋白。由于细胞核中染色体内和染色体间精细结构的完整性取决于水合状态，因此我们还将重点关注EM冷冻技术的适应性，以最小的样本扰动获得最大的结构信息。预期的结果将有重要意义的理解i）间期核内的染色体组织和ii）的高级核结构和亚区室化。更重要的是，这些分析将允许评估基因组功能（如基因表达）与核结构之间的关系。