Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells.
Effects of flanking base sequences on 5-bromodeoxyuridine mutagenesis in mammalian cells.
复制标题
侧翼碱基序列对哺乳动物细胞中 5-溴脱氧尿苷诱变的影响。
DOI：
10.1007/bf01233065
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发表时间：
1991
期刊：
影响因子：
--
通讯作者：
Davidson,RL
中科院分区：
文献类型：
--
作者：
Kresnak,MT;Davidson,RL
在携带大肠杆菌gpt基因单拷贝作为穿梭载体整合到染色体DNA中的小鼠A9细胞中，分析了结合依赖的5-溴脱氧尿苷（BrdU）诱导突变的分子机制。在brdu诱导的逆转分析中，利用4个独立衍生的GPT -突变体，在整合gptgene中存在单碱基变化，以测试四种不同设置下鸟嘌呤残基的相对易变性：GG双偶的5 ‘和3 ’鸟嘌呤残基，GGGG四联体的3 '鸟嘌呤残基，以及GGG三联体的中间鸟嘌呤残基。两个突变系具有GG双链序列，其中双链的任一鸟嘌呤残基上的GC→AT转换导致GPT酶活性恢复，而不恢复野生型DNA序列。结果表明，这两个品系都能通过BrdU结合依赖性突变有效地恢复，对这两个品系的许多独立衍生的回复性基因进行测序表明，90%以上的回复性是由双联体3 '鸟嘌呤残基的GC→AT转变引起的。BrdU诱导的另外两个GPT -突变系的逆转表明，GGGG四重奏序列的3 ‘鸟嘌呤残基是有效突变的，而GGG三重奏序列的中间鸟嘌呤残基通过BrdU结合依赖的突变比GG双偶或GGGG四重奏序列的3 ’鸟嘌呤残基的变异性至少低10倍。用烷基化剂甲烷磺酸乙酯处理后，所有4个突变系均具有相同的可逆性。本研究的结果定义了brdu诱导的合并依赖性突变的序列特异性机制，并证明了在基因精确位点上使用反转分析来确定序列特异性效应。
The molecular mechanisms of incorporation-dependent, 5-bromodeoxyuridine (BrdU)-induced mutagenesis were analyzed in murine A9 cells that possess a single copy of theEscherchia coli gptgene integrated into the chromosomal DNA as part of a shuttle vector. Four independently derived GPT−mutants with single base changes within the integratedgptgene were utilized in BrdU-induced reversion analyses to test the relative mutability of guanine residues in four different settings: the 5′ and 3′ guanine residues of a GG doublet, the 3′ guanine residue of a GGGG quartet, and the middle guanine residue of a GGG triplet. Two of the mutant lines possessed GG doublet sequences in which a GC→AT transition at either guanine residue of the doublet leads to restoration of GPT enzyme activity without restoring wild-type DNA sequence. Both lines were shown to be effectively reverted by BrdU incorporation-dependent mutagenesis, and sequencing of thegptgenes from numerous independently derived revertants of both lines demonstrated that greater than 90% of the revertants arose due to GC→AT transitions at the 3′ guanine residue of the doublet. BrdU-induced reversion of two additional GPT−mutant lines demonstrated that the 3′ guanine residue of a GGGG quartet is efficiently mutated, while the middle guanine residue of a GGG triplet sequence is at least 10-fold less mutable by BrdU incorporation-dependent mutagenesis than the 3′ guanine residue of a GG doublet or GGGG quartet. All four mutant lines tested were equally revertible by treatment with the alkylating agent ethyl methane sulfonate. The results from this study define a sequence-specific mechanism for BrdU-induced, incorporation-dependent mutagenesis and demonstrate the use of reversion analysis for the determination of sequence specific effects at precise sites within a gene.