Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.
Efficient recovery and sequencing of mutant genes from mammalian chromosomal DNA.
复制标题
从哺乳动物染色体 DNA 中有效回收突变基因并对其进行测序。
DOI：
10.1073/pnas.83.10.3356
复制
发表时间：
1986
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;Jagadeeswaran,P;Davidson,RL
陈晓明，陈晓明，陈晓明，等。将大肠杆菌黄嘌呤（鸟嘌呤）磷酸核糖基转移酶基因（gpt）导入到pZip-NeoSV(X)1载体中构建逆转录病毒穿梭载体[j]。该载体被包装成感染性病毒，然后用于感染次黄嘌呤（鸟嘌呤）磷酸核糖基转移酶缺陷小鼠细胞系。分离出表达gpt基因的细胞系，发现这些细胞含有原病毒形式的单一整合载体拷贝。用甲磺酸乙酯或BrdUrd处理这些细胞系，产生6-硫鸟嘌呤抗性（Sgur）突变体的频率增加了10倍以上。在COS细胞融合后，完整的gpt基因已从许多Sgur细胞系中恢复，并作为质粒的一部分引入大肠杆菌。三个突变基因的完整DNA序列已经确定。其中两个突变基因有一个碱基替换，而第三个突变基因有34个碱基对的缺失。该系统对分析哺乳动物细胞诱变的特异性和化学诱变的分子机制具有重要意义。与其他穿梭载体系统相比，该系统的一个潜在重要特征是，突变是在整合到哺乳动物染色体中的基因中诱导的，而不是在作为自主复制质粒一部分存在的基因中诱导的。
A retroviral shuttle vector was constructed by introducing the Escherichia coli xanthine (guanine) phosphoribosyltransferase gene (gpt) into the pZip-NeoSV(X)1 vector [Cepko, C. L., Roberts, B. E. & Mulligan, R. C. (1984) Cell 37, 1053-1062]. This vector was packaged into infectious virus which then was used to infect a hypoxanthine (guanine) phosphoribosyltransferase-deficient mouse cell line. Cell lines that expressed the gpt gene were isolated, and it was found that these cells contained a single integrated copy of the vector in a proviral form. Treatment of these cell lines with either ethyl methanesulfonate or BrdUrd produced a greater than 10-fold increase in the frequency of 6-thioguanine-resistant (Sgur) mutants. Intact gpt genes have been recovered from a number of Sgur cell lines after COS cell fusion and introduced into E. coli as part of a plasmid. The complete DNA sequences of three mutant genes have been determined. Two of the mutant genes have a single base substitution, whereas the third has a 34-base-pair deletion. This system should be valuable for analyzing mutagenic specificity and the molecular mechanisms of chemical mutagenesis in mammalian cells. A potentially important feature of the system relative to other shuttle-vector systems is that the mutations are induced in genes integrated into mammalian chromosomes rather than in genes existing as part of autonomously replicating plasmids.