MECHANISM OF FORMATION OF TERMINAL DELETIONS

末端缺失的形成机制

• 批准号: 6342571
• 负责人: John P. Murnane
• 金额: $ 29.14万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 2002-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6342571
• 关键词: DNA repair; apoptosis; chromosome deletion; embryonic stem cell; genetic markers; genetically modified animals; laboratory mouse; nucleic acid sequence; p53 gene /protein; plasmids; telomerase; telomere

Terminal deletions are responsible for approximately 10 percent of human genetic diseases resulting from chromosome aberrations. All terminal deletions resulting in genetic diseases that have been analyzed thus far have resulted from new telomeres being added directly onto the ends of broken chromosomes. The restoration of lost telomeres, termed chromosome healing, has been studied extensively in single cell organisms, however, very little is known about this process in mammalian cells. This project involves the use of an assay system developed in mouse embryonic stem (ES) cells to study the mechanism of formation of terminal deletions and the genes involved in chromosome healing. This system utilizes plasmid sequences containing selectable marker genes and an I-SceI endonuclease recognition sequence integrated immediately adjacent to a telomere. The I-SceI recognition sequence is used to specifically introduce double-strand breaks and the selectable marker genes are used to identify cells that have terminal deletions on the end of the marked chromosome. Electroporation of an expression vector containing the gene for the I-SceI endonuclease produced terminal deletions resulting from chromosome healing. The new telomeres are added on with the loss of little or no DNA from the end of the chromosome at sites with short homologies to the telomeric repeat sequences. The new telomeres are much shorter than in the parental cell line, and increase in length with time in culture, consistent with de novo synthesis by telomerase. In the current grant proposal we will utilize this assay system to investigate the mechanism of chromosome healing, the factors that influence this process, and the importance of chromosome healing in the cellular response to DNA double-strand breaks. The formation of terminal deletions by chromosome healing will be studied in mouse cell lines with deficiencies in telomerase, DNA double-strand-break repair and cell cycle regulation to determine the role of these pathways in this process. The ES cell clones will also be used to establish transgenic mice, which will provide a method for studying chromosome healing in a variety of different cell types and for investigating chromosome healing in different genetic backgrounds. Using these approaches we will be able to test the hypothesis that chromosome healing is a cell-type specific mechanism that can influence the cellular response to DNA double-strand breaks. The ability of this system to monitor telomere length and the rate of telomere loss will also provide a means of investigating the role of different genes in maintaining telomere stability.

终末缺失是染色体畸变导致的大约10％的人类遗传疾病。到目前为止，所有导致遗传疾病的终末缺失都是由于新的端粒直接添加到破裂的染色体末端而导致的。 在单细胞生物中已经广泛研究了被称为染色体愈合的丢失的端粒的恢复，但是，在哺乳动物细胞中，这一过程知之甚少。 该项目涉及在小鼠胚胎茎（ES）细胞中开发的测定系统来研究末端缺失形成的机理以及涉及染色体愈合的基因。该系统利用含有可选标记基因的质粒序列和紧邻端粒附近的I-SCEI核酸内切酶识别序列。 I-SCEI识别序列用于特异性引入双链断裂，可选标记基因用于识别在标记染色体末端具有末端缺失的细胞。 含有I-SCEI核酸内切酶基因的表达载体的电穿孔产生了由染色体愈合引起的末端缺失。 添加了新的端粒，在染色体末端几乎没有DNA的损失，而在端粒重复序列的同源性短的位置。 新的端粒比父母细胞系中的短得多，并且随着培养时间的时间而增加，与端粒酶从头合成一致。 在当前的赠款建议中，我们将利用该测定系统来研究染色体愈合的机制，影响该过程的因素以及染色体愈合在细胞对DNA双链断裂的反应中的重要性。 将研究染色体愈合的末端缺失的形成，将在小鼠细胞系中研究，端粒酶缺陷，DNA双链破裂修复和细胞周期调节，以确定这些途径在此过程中的作用。 ES细胞克隆还将用于建立转基因小鼠，该小鼠将提供一种研究各种不同细胞类型中染色体愈合以及在不同遗传背景下染色体愈合的方法。 使用这些方法，我们将能够检验以下假设：染色体愈合是一种细胞类型的特定机制，可以影响细胞对DNA双链断裂的反应。 该系统监测端粒长度和端粒损失率的能力也将提供一种研究不同基因在维持端粒稳定性中的作用的方法。