REPAIR AND RECOMBINATION OF BROKEN CHROMOSOMES

染色体异常，如易位、缺失和 在肿瘤细胞中经常可以观察到基因扩增。在许多 这些异常导致癌基因的激活， 肿瘤抑制基因的丢失，癌基因和 耐药基因。最近的证据表明，一种单一形式的 DNA损伤，染色体断裂，可能与这些疾病的形成有关 异常现象。尽管哺乳动物细胞已被证明能够修复或 修复断裂的染色体，这些染色体的确切性质或机制 这些过程还没有被阐明。很难确定，在 DNA序列水平，在哪里发生断裂或在预测哪里发生断裂 断裂是否会发生一直是研究修复的主要障碍 流程。 一例染色体断裂的鉴定 DHFR基因在CHO细胞中的扩增，其断裂位点已被 在DNA水平上的定位，为分子研究提供了一个系统 修复或修复断裂染色体的机制。终点站 序列将被克隆和测序，以确定是否有任何序列 例如端粒重复序列，已经被添加到断裂的自由端。 为了研究修复和愈合的各种机制 当它们发生时，一种在已定义的DNA中产生双链断裂的方法 提出了染色体中的序列。引入One FLP 识别和切割到CHO染色体上的特定位置 而FLP蛋白的诱导表达应该产生这样一种 定义的中断。该系统将被用于研究CHO的能力 细胞在断裂发生时进行修复或修复。原位荧光 杂交将被用来跟踪发生在 染色体水平。修复或修复的末端将被克隆和测序 以确定这些维修事件的确切性质。这 特定部位的切割系统也将提供一种研究手段 游离端的同源重组及其参与 双链断裂修复。最后，提出了双链的作用 介导缺失和基因扩增事件的中断也将是 直接使用该系统进行了测试。

Chromosome abnormalities such as translocations, deletions, and gene amplifications are frequently observed in tumor cells. In many cases, these abnormalities result in the activation of oncogenes, the loss of tumor suppressor genes, and the overexpression of oncogenes and drug resistance genes. Recent evidence indicates that a single form of DNA damage, chromosome breaks, may be involved in the formation of these abnormalities. Although mammalian cells have been shown to repair or heal broken chromosomes, the precise nature or mechanism of these processes has not been elucidated. The difficulty in determining, at the DNA sequence level, where a break has occurred or in predicting where a break will occur has been the major obstacle in studying the repair processes. The identification of a broken chromosome resulting from amplification of the DHFR gene in CHO cells, whose break site has been mapped at the DNA level, provides a system for the study of the molecular mechanisms of repair or healing of broken chromosomes. The terminus sequence will be cloned and sequenced to determine whether any sequences such as telomeric repeats, have been added to the free end of the break. In order to study the various mechanisms of repair and healing as they occur, a means of creating a double-strand break in a defined DNA sequence in a chromosome is proposed. The introduction of one FLP recognition and cleavage site into a defined location in a CHO chromosome and the induced expression of the FLP protein should create such a defined break. This system will be used to study the ability of CHO cells to heal or repair the break as it occurs. Fluorescent in situ hybridization will be used to track the events that occur at the chromosomal level. Healed or repaired ends will be cloned and sequenced to determine the precise nature of these repair events. This site-specific cleavage system will also provide a means of studying homologous recombination of free ends and its involvement in double-strand break repair. Finally the proposed role of double-strand breaks mediating deletion and gene amplifications events will also be tested directly with this system.