MOLECULAR ANALYSIS OF DELETION MUTATIONS IN CHINESE HAMSTER OVARY CELLS

我们研究了gpt基因座上的大规模基因组重排。 中国仓鼠卵巢(CH0)AS52细胞株。AS52细胞中gpt基因的定位 对碎裂基因引起的基因组重排反应更快 或仿放射剂，允许恢复和表征 在其他可选择的基因座上没有恢复的突变体，如hprt。我们检测到 由于改变了聚合酶链式反应图谱或 一个改变过的南方污点。这些突变株产生带有 重新排列的序列可以直接测序。抗肿瘤药物， 阿多司匹林(ADZ)，可导致约90%的大规模缺失或 用聚合酶链式反应检测重排。然而，相当数量的 也可以观察到较小的删除、插入或重新排列。 这些重组包括一个带有3个碱基侧翼的202个碱基对的插入物 重复，几个小的缺失(9-13个碱基对)都带有侧翼序列 重复序列、重复的相邻3碱基直接重复序列和一些 短缺失(1-5个碱基)，没有侧翼的同源序列。合而为一 突变体，一种包括约100个碱基对的复制的重排 似乎是在序列同源性的5个碱基区介导的 其中最后3个碱基包括ADZ共识结合序列。 这样的突变可能是由于Adz-DNA加合物阻止了 聚合酶导致呼吸和异常的复温 合成链，在同一模板链上或在 复制姐妹染色单体。这些数据表明，在大多数情况下 删除需要较短的序列同源性， 插入和复制。此外，我们正在克隆 GPT在AS52细胞中的整合，以更准确地定义 观察到大规模的基因组重排。目前，我们不能 区分染色体内缺失，有丝分裂重组， 基因/染色体转换或非整倍体和重复。通过 描述gpt整合的位置并定义多态 基因组序列的侧翼，我们将能够评估类型和 环境引起的特定重排的频率 诱变剂和致癌物。我们目前正在描述几个 来源于AS52基因组文库的Lambda克隆。

We study large scale genomic rearrangements at the gpt locus in the Chinese hamster ovary (CH0) AS52 cell line. The gpt locus in AS52 cells is more responsive to genomic rearrangements induced by clastogenic or radiomimetic agents allowing for the recovery and characterization of mutants not recovered at other selectable loci, like hprt. We detect such rearrangements as the result of either an altered PCR profile or an altered Southern blot. Those mutants that yield a PCR product with rearranged sequences can be sequenced directly. The antitumor agent, Adozelesin (ADZ), induces approximately 90% large scale deletions or rearrangements as detected by PCR. However, a substantial number of smaller deletions, insertions or rearrangements are observed as well. These rearrangements include a 202-bp insert with a 3-base flanking repeat, several small deletions (9-13 bp) all with flanking sequence repeats, deletions of repeated adjacent 3-base direct repeats and a few short deletions (1-5 bp) without flanking homologous sequences. In one mutant, a rearrangement that includes a duplication of about 100 bp appears to be mediated at a 5-base region of sequence homology in which the last 3 bases include an ADZ consensus binding sequence. Such a mutant may arise as a result of ADZ-DNA adduct blocking the polymerase resulting in the breathing and aberrant reannealing of the synthesized strand, either on the same template strand or on a replicating sister chromatid. These data suggest that in most cases short stretches of sequence homology are required for deletions, insertions and duplications. In addition, we are cloning the site of the gpt integration in AS52 cells to more accurately define the types of large scale genomic rearrangements observed. Presently, we cannot distinguish between intrachromosomal deletions, mitotic recombination, gene/chromosome conversion or aneuploidy and reduplication. By characterizing the site of gpt integration and defining polymorphic flanking genomic sequences, we will be able to assess the type and frequency of specific rearrangements induced by environmental mutagens and carcinogens. We are presently characterizing several lambda clones derived from an AS52 genomic library.