MOLECULAR ANALYSIS OF DELETION MUTATIONS IN CHINESE HAMSTER OVARY CELLS

基因组重排是最严重的突变之一 哺乳动物细胞，参与了癌症的发生和发展过程 多种人类遗传性疾病的病因学。虽然许多诱变剂和 致癌物诱导基因组重排，大多数哺乳动物的突变 检测大规模基因组的能力是有限的。 突变。然而，基因组重排往往对细胞是致命的， 这种重排导致活细胞的机制值得 进一步研究。研究基因组重排的机制 我们使用了转基因中国仓鼠卵巢细胞系AS52，该细胞系 稳定携带细菌gpt基因的单一功能拷贝 整合到CHO基因组中。AS52细胞被很好地定义为 能够检测到由各种碎裂原引起的突变和 在其他哺乳动物中未检测到诱变剂的模拟放射性物质 突变检测系统，显然是由于基因组的位置 细菌gpt转基因的整合。我们正在刻画 Gpt整合的基因组位置，并定义了一个非功能性的 与功能GPT紧密连锁的GPT/质粒相关序列 吉恩。为了快速区分删除和重排 推测的点突变，我们已经开发出了引物和反应条件 用于AS52基因组DNA的多重聚合酶链式反应。我们的多重聚合酶链式反应产生 来自dhfr基因(与gpt无关)以及来自 功能性和非功能性gpt序列。不起作用的 GPT序列可作为快速区分小分子的标记 当DHFR序列用作内部聚合酶链式反应时的缺失/重排 控制力。如果聚合酶链式反应的条件合适，那么我们应该 观察dhfr的聚合酶链式反应片段，同时功能和 不起作用的gpt相关的聚合酶链式反应片段允许区分较大的 刻度删除/重新排列事件。因此，我们现在可以快速区分 完全删除(即大规模基因组重排)部分 删除(即较小规模的基因组重排) 只需使用聚合酶链式反应即可获得突变。我们目前正在对分子进行表征 碱基类似物5-氮杂胞苷诱导的缺失的精细结构，以及 化疗药物U73,975。

Genomic rearrangements are among the most serious mutations arising in mammalian cells, implicated in the process of carcinogenesis and in the etiology of a variety of human genetic disorders. While many mutagens and carcinogens induce genomic rearrangements, most mammalian mutagenesis assays are limited in their ability to detect large scale genomic mutations. Often genomic rearrangements are lethal to the cell, however, the mechanisms by which such rearrangements result in viable cells deserve further study. To study the mechanisms by which genomic rearrangements arise, we use the transgenic Chinese hamster ovary cell line, AS52, which carries a single functional copy of the bacterial gpt gene stably integrated into the CHO genome. AS52 cells are well defined as being capable of detecting mutations induced by a variety of clastogens and radio-mimetic agents that are not detected as mutagens in other mammalian mutational assay systems, apparently due to the location of the genomic integration of the bacterial gpt transgene. We are characterizing the genomic site of gpt integration and have defined a nonfunctional gpt/plasmid related sequence that is closely linked to the functional gpt gene. In order to rapidly distinguish deletions and rearrangements from putative point mutations, we have developed primers and reaction conditions useful for multiplex PCR of AS52 genomic DNA. Our multiplex PCR yields sequences from the dhfr gene (which is not linked to gpt) as well as from both the functional and the nonfunctional gpt sequences. The nonfunctional gpt sequence can serve as a marker for rapidly distinguishing smaller deletions/rearrangements while the dhfr sequences serve as an internal PCR control. If the PCR conditions are appropriate then we should always observe the dhfr PCR fragment, while loss of both the functional and the nonfunctional gpt related PCR fragments allows the distinction of larger scale deletion/rearrangement events. Thus, we can now rapidly distinguish complete deletions (i.e., large scale genomic rearrangements) from partial deletions (i.e., smaller scale genomic rearrangements), from point mutations simply using PCR. We are presently characterizing the molecular fine structure of deletions induced by the base analog, 5-azacytidine, and the chemotherapeutic agent, U73,975.