Analysis of sequence specificity of 5-bromodeoxyuridine-induced reversion in cells containing multiple copies of a mutant gpt gene.
Analysis of sequence specificity of 5-bromodeoxyuridine-induced reversion in cells containing multiple copies of a mutant gpt gene.
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分析含有多个突变 gpt 基因拷贝的细胞中 5-溴脱氧尿苷诱导的逆转的序列特异性。
DOI：
10.1007/bf01233163
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发表时间：
1992
期刊：
影响因子：
--
通讯作者：
Davidson,RL
中科院分区：
文献类型：
--
作者：
Rotstein,JB;Kresnak,MT;Samadashwily,GM;Davidson,RL
为了研究突变的分子机制，将具有特定变化的突变基因导入哺乳动物细胞，并将转化的细胞用于逆转分析将是有利的。在本文中，我们描述了一种有效的方法来分析在拥有突变靶基因的多个副本的细胞中发生的逆转事件。这种方法包括用聚合酶链式反应(PCR)扩增染色体整合的目标基因，并对扩增产物进行限制性内切酶消化。可以在保留突变序列的10-20个拷贝的基因的背景中识别产生或破坏包含原始突变位置的限制性内切酶识别序列的单个逆转事件。用这种方法，我们分析了5-溴脱氧尿苷(BrdU)在中国仓鼠卵巢细胞系中诱导的回复突变，该细胞系拥有含有特定改变的突变细菌gpt基因的多个副本。这项研究的结果不仅证明了该方法在分析具有多个突变目的基因拷贝的转染体中单基因拷贝逆转的有效性，而且还证明了BrdU诱导的突变在中国仓鼠细胞中的序列特异性与以前在小鼠细胞中观察到的相同。
For studies on molecular mechanisms of mutagenesis, it would be advantageous to transfer mutant genes with specific alterations into mammalian cells and use the transformed cells in reversion analyses. In the present paper, we describe an efficient method for analyzing reversion events occurring in cells that possess multiple copies of a mutational target gene. This method involves amplification of the chromosomally integrated target genes with the polymerase chain reaction (PCR) and restriction endonuclease digestion of the amplified product. Single reversion events that either create or destroy restriction endonuclease recognition sequences that encompass the site of the original mutation can be identified in a background of 10–20 copies of the gene that retain the mutant sequence. Using this method, we have analyzed revertants induced by 5-bromodeoxyuridine (BrdU) in a Chinese hamster ovary cell line that possesses multiple copies of a mutant bacterial gpt gene containing a specific alteration. The results of this study not only demonstrate the effectiveness of this method for analyzing reversion of a single gene copy in transfectants possessing multiple copies of a mutant target gene, but also demonstrate that the sequence specificity for BrdU-induced mutations is the same in Chinese hamster cells as previously observed with mouse cells.