Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.
Sequence analysis of spontaneous mutations in a shuttle vector gene integrated into mammalian chromosomal DNA.
复制标题
整合到哺乳动物染色体 DNA 中的穿梭载体基因自发突变的序列分析。
DOI：
10.1073/pnas.84.10.3354
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发表时间：
1987
影响因子：
11.1
通讯作者：
Davidson,RL
中科院分区：
文献类型：
--
作者：
Ashman,CR;Davidson,RL
我们研究了携带可选择细菌基因的小鼠细胞自发突变的分子机制。小鼠细胞携带大肠杆菌黄嘌呤（鸟嘌呤）磷酸核糖基转移酶（gpt）基因，该基因位于逆转录病毒穿梭载体中，以前病毒形式整合到染色体DNA中。选择在gpt基因中具有自发突变的细胞作为对6-硫鸟嘌呤的抗性，然后与COS细胞融合以回收突变基因。在本研究中分离的总共77个独立的6-硫代鸟嘌呤抗性细胞系中，可以从43个突变系中拯救载体序列，并且确定所有43个这些系中的gpt基因的碱基序列。在测序的突变gpt基因中存在多种突变事件。最常见的突变事件是缺失（在43个突变基因中的29个中），其次是碱基替换突变（在43个突变基因中的11个中）。在缺失突变体中，绝大多数代表小于10个碱基对的缺失。实际上，29个缺失突变体中有19个缺失了3个碱基对，并且在3个碱基对缺失的突变体中，有16个独立突变体出现了非常强的缺失热点。所有19个3碱基对缺失导致天冬氨酸密码子的“框内”丢失。在碱基替换突变中，转换和颠换以大致相等的频率发生。我们的研究结果提出了一种可能性，即小的缺失代表了哺乳动物细胞中发生自发突变的主要机制。
We have studied the molecular mechanisms of spontaneous mutations in mouse cells carrying a selectable bacterial gene. The mouse cells carry the Escherichia coli xanthine (guanine) phosphoribosyltransferase (gpt) gene in a retroviral shuttle vector integrated into chromosomal DNA in a proviral form. Cells with spontaneous mutations in the gpt gene were selected as resistant to 6-thioguanine and then were fused with COS cells for recovery of the mutant genes. Out of a total of 77 independent 6-thioguanine-resistant cell lines isolated in this study, vector sequences could be rescued from 43 of the mutant lines, and the base sequences were determined for the gpt genes in all 43 of these lines. There was a variety of mutational events among the mutant gpt genes sequenced. The most frequent mutational event was a deletion (in 29 of the 43 mutant genes), and the next most frequent event was a base substitution mutation (in 11 of the 43 mutant genes). Among the deletion mutants, the great majority represent deletions of less than 10 base pairs. In fact, 19 of the 29 deletion mutants had deletions of 3 base pairs, and among the mutants with 3-base-pair deletions, there was a very strong deletion hot spot appearing in 16 independent mutants. All 19 of the 3-base-pair deletions resulted in the "in frame" loss of an aspartic acid codon. Among the base substitution mutations, transitions and transversions occurred with approximately equal frequency. Our results raise the possibility that small deletions represent the predominant mechanisms by which spontaneous mutations occur in mammalian cells.