REPAIR AND RECOMBINATION OF BROKEN CHROMOSOMES

染色体异常，如易位、缺失和 在肿瘤细胞中经常观察到基因扩增。 在许多 在某些情况下，这些异常导致癌基因的激活， 肿瘤抑制基因的丢失，以及癌基因的过度表达， 耐药基因 最近的证据表明， DNA损伤，染色体断裂，可能与这些的形成有关。 异常 尽管哺乳动物细胞已经被证明可以修复或 修复断裂的染色体，这些染色体的确切性质或机制 过程尚未阐明。 在确定的困难，在 在DNA序列水平上，发生断裂的地方或预测断裂的地方， 断裂的发生一直是研究修复的主要障碍 流程. 鉴定一个断裂的染色体， 在CHO细胞中扩增DHFR基因，其断裂位点已被 在DNA水平上绘制，提供了一个研究分子生物学的系统， 修复或愈合断裂染色体的机制。 总站 将克隆和测序序列，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，一种在确定的DNA中产生双链断裂的方法， 序列中的染色体提出。 引入一个FLP 识别和切割位点进入CHO染色体中的确定位置 而FLP蛋白的诱导表达应该会产生这样的一种 定义break。 该系统将用于研究CHO的能力 细胞愈合或修复断裂。 荧光原位 杂交将用于跟踪发生在 染色体水平。 愈合或修复的末端将被克隆和测序 以确定这些修复事件的确切性质。 这 位点特异性切割系统也将提供一种研究 游离末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断裂也将被 直接用这个系统测试。

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.