Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.
Multiple point mutations in a shuttle vector propagated in human cells: evidence for an error-prone DNA polymerase activity.
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在人类细胞中传播的穿梭载体中的多个点突变：容易出错的 DNA 聚合酶活性的证据。
DOI：
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发表时间：
1987
影响因子：
11.1
通讯作者：
K. Kraemer
中科院分区：
文献类型：
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作者：
M. Seidman;A. Bredberg;S. Seetharam;K. Kraemer
通过使用含有抑制性 tRNA 标记基因的穿梭载体质粒 pZ189，在人成纤维细胞和淋巴样细胞的 DNA 序列水平上研究了诱变。在一系列实验中，回收了 62 个质粒，这些质粒在 160 个碱基对的标记基因中具有 2 至 6 个碱基取代。将经过紫外线处理的 pZ189 通过修复能力强的人成纤维细胞系后回收的突变质粒中，大约有 20-30% 含有这些多重突变。相比之下，紫外线处理的 pZ189 通过切除修复缺陷（着色性干皮病）系的传代仅产生 2% 的多碱基取代突变体。在标记附近未修饰的 pZ189 中引入单链切口，然后通过色素性干皮病细胞，产生约 66% 的多碱基取代突变体。在包含标记基因的 160 个碱基对区域中发现了多个突变，但在相邻的 170 个碱基对区域中很少发现。将经过紫外线处理或带切口的 pZ189 通过修复能力强的人类 B 细胞系，也会在 20-33% 的突变质粒中产生多个碱基取代突变。对这些多重突变的解释是，它们是由容易出错的聚合酶在填补空白时产生的。这些突变具有免疫球蛋白高变区突变所表现出的许多特性。
Mutagenesis was studied at the DNA-sequence level in human fibroblast and lymphoid cells by use of a shuttle vector plasmid, pZ189, containing a suppressor tRNA marker gene. In a series of experiments, 62 plasmids were recovered that had two to six base substitutions in the 160-base-pair marker gene. Approximately 20-30% of the mutant plasmids that were recovered after passing ultraviolet-treated pZ189 through a repair-proficient human fibroblast line contained these multiple mutations. In contrast, passage of ultraviolet-treated pZ189 through an excision-repair-deficient (xeroderma pigmentosum) line yielded only 2% multiple base substitution mutants. Introducing a single-strand nick in otherwise unmodified pZ189 adjacent to the marker, followed by passage through the xeroderma pigmentosum cells, resulted in about 66% multiple base substitution mutants. The multiple mutations were found in a 160-base-pair region containing the marker gene but were rarely found in an adjacent 170-base-pair region. Passing ultraviolet-treated or nicked pZ189 through a repair-proficient human B-cell line also yielded multiple base substitution mutations in 20-33% of the mutant plasmids. An explanation for these multiple mutations is that they were generated by an error-prone polymerase while filling gaps. These mutations share many of the properties displayed by mutations in the immunoglobulin hypervariable regions.