MOLECULAR ANALYSIS OF DELETION MUTATIONS IN CHINESE HAMSTER OVARY CELLS

基因组重排是最严重的突变， 哺乳动物细胞，涉及的过程中的致癌作用，并在 各种人类遗传疾病的病因学。 虽然许多诱变剂和 致癌物诱导基因组重排，大多数哺乳动物诱变 测定在检测大规模基因组DNA的能力方面是有限的， 突变。 然而，基因组重排通常对细胞是致命的， 这种重排导致活细胞的机制值得研究， 进一步研究 研究基因组重排的机制 因此，我们使用转基因中国仓鼠卵巢细胞系AS 52， 稳定地携带细菌GPT基因的单一功能拷贝 整合到CHO基因组中。 AS 52细胞被明确定义为 能够检测由多种断裂素诱导的突变， 在其他哺乳动物中未检出诱变剂的放射性模拟剂 突变分析系统，显然是由于基因组的位置， 细菌GPT转基因的整合。 我们正在描述 gpt整合的基因组位点，并已确定了一个非功能性 与功能性GPT紧密相连的GPT/质粒相关序列 基因 为了快速区分缺失和重排， 为了检测推定的点突变，我们开发了引物和反应条件 用于AS 52基因组DNA多重PCR。 我们的多重PCR 来自dhfr基因（其不与gpt连接）的序列以及来自 功能性和非功能性GPT序列。 该非功能性 gpt序列可以作为快速区分较小 缺失/重排，而dhfr序列用作内部PCR 控制 如果PCR条件合适，那么我们应该始终 观察dhfr PCR片段，而功能和 非功能性gpt相关PCR片段允许区分较大的 缩放删除/重排事件。 因此，我们现在可以快速区分 完全缺失（即，大规模基因组重排） 缺失（即，小规模的基因组重排），从点 基因突变的方法。 我们目前正在鉴定 由碱基类似物5-氮杂胞苷诱导的缺失的精细结构， 化疗剂U 73975

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.