MOLECULAR ANALYSIS OF POINT MUTATIONS IN CHINESE HAMSTER OVARY CELLS

我们使用转gpt基因的中国仓鼠卵巢(CHO)细胞系AS52， 用于突变的分子研究。扩增突变的gpt序列 利用聚合酶链式反应(PCR)和DNA测序 分析以生成诱导突变的序列谱。我们 研究了两类化合物引起的突变， 一)直接作用诱变化疗药物阿多斯莱辛(ADZ)和 5-氮胞苷(5AC)，以及II)已知或可疑的人类致癌物 需要代谢激活，如烟草特有的亚硝胺， NNK。ADZ是一种有效的抗肿瘤药物和有效的DNA烷基化 探员。ADZ主要在N3位形成大体积DNA加合物 并且显示TTA*或AAA*的序列特异性，其中 (a*)是任一3碱基靶标的3‘位置上的修饰腺嘌呤。 虽然Adz在AS52细胞中诱导了大部分缺失突变，但Point 突变也会被诱发。至少有一个突变热点 生成AT&>TA转换以及几个插入、小删除 并且观察到似乎是针对 包含3个重叠的共识ADZ结合位点的序列。5AC 是AS52细胞中GC&GT；CG颠倒突变的有效诱导者。这 罕见的突变类型涉及5AC：C错配对 复制。由于C：C错对不会被原核生物或 真核聚合酶，这些数据表明结构 5AC：C错对的干扰可能允许聚合酶在 活着。此外，5AC似乎诱导了一类6TG抗性 不携带gpt结构基因突变的菌落。许多. 这些克隆回复产生频率高得多的功能性gpt基因。 高于预期(l0e-4至l0e-2)。一般来说，这样的“变种人”会这样做。 细胞在6TG培养液中生长时不表达gpt mRNA。 越来越多地表明这些突变的产生是由于 染色质结构修饰。NNK主要诱导GC&GT；AT 携带功能性人细胞色素P450-A6基因的AS52细胞的转变。 这些数据表明，NNK的CYP2A6羟基化主要产生 一种导致DNA甲基化的中间体，导致观察到的 GC&>AT转换。

We use the gpt transgenic Chinese hamster ovary (CHO) cell line, AS52, for molecular studies of mutation. Mutant gpt sequences are amplified using the polymerase chain reaction (PCR) followed by DNA sequence analysis to generate a sequence spectrum of induced mutations. We have studied mutations induced by two general classes of compounds, i) direct acting mutagenic chemotherapeutic agents, Adozelesin (ADZ) and 5-azacytidine (5AC), and ii) known or suspect human carcinogens that require metabolic activation, such as the tobacco specific nitrosamine, NNK. ADZ is a potent antitumor agent and an efficient DNA alkylating agent. ADZ forms bulky DNA adducts predominantly at the N3 position of adenine and shows a sequence specificity of TTA* or AAA*, where (A*) is the modified adenine in the 3' position of either 3-base target. While ADZ induces mostly deletion mutations in AS52 cells, point mutations are induced as well. At least one mutational hotspot which yields AT>TA transversions as well as several insertions, small deletions and rearrangements are observed that appear to be targeted to a sequence containing 3-overlapping consensus ADZ binding sites. 5AC is a potent inducer GC>CG transversion mutations in AS52 cells. This rarely observed type of mutation implicates 5AC:C mispairs during replication. Since C:C mispairs are not extended by prokaryotic or eukaryotic polymerases, these data suggest that structural perturbations of the 5AC:C mispair may allow polymerase extension in vivo. In addition, 5AC appears to induce a class of 6TG resistant colonies that do not carry gpt structural gene mutations. Many of these clones revert to yield a functional gpt gene at frequencies much higher than expected (l0e-4 to l0e-2). In general, such "mutants" do not express gpt mRNA when the cells are grown in 6TG medium and data are accumulating to suggest that these mutants arise as a result of chromatin structural modifications. NNK induces predominantly GC>AT transitions in AS52 cells carrying a functional human CYP2A6 gene. These data suggest that CYP2A6 hydroxylation of NNK mostly generates an intermediate that results in DNA methylation leading to the observed GC>AT transitions.