Characterization and analysis of Chinese hamster ovary cell ERCC1 mutant alleles
Characterization and analysis of Chinese hamster ovary cell ERCC1 mutant alleles
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DOI：
10.1093/mutage/13.4.357
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发表时间：
1998-07-01
期刊：
影响因子：
2.7
通讯作者：
Nairn, RS
中科院分区：
文献类型：
--
作者：
Rolig, RL;Lowery, MP;Nairn, RS
从3个中国仓鼠卵巢（CHO）亲本（CHO- aa8、CHO- at3 -2和CHO-9）和10个ERCC1突变细胞系中分离出ERCC1编码序列并进行了鉴定。两种突变细胞系表现出核苷酸添加或缺失，从而产生移码突变，7种突变细胞系表现出点突变，导致过渡或翻转，包括无义突变和产生内含子/外显子剪接错误的突变。其中一个突变体（UV201）被暂时分配到ERCC1互补组1 (CG1)，其编码序列未检测到突变。在9个ERCC1突变等位基因中，有2个突变位于ERCC1蛋白的XpA结合区；Ercc1蛋白n端未发现突变。Northern杂交分析结果显示，ERCC1 mRNA的相对表达水平在亲本细胞系之间以及从亲本细胞系衍生的突变细胞系之间均存在显著差异。CHO Ercc1特异性抗体的Western分析在每个亲本细胞系和UV201中检测到Ercc1蛋白。在所有其他突变体中观察到的Ercc1蛋白水平的显著降低支持了Ercc1突变可能破坏这种多肽稳定的假设。
We isolated and characterized the ERCC1 coding sequence from three Chinese hamster ovary (CHO) parental (CHO-AA8, CHO-AT3-2 and CHO-9) and 10 ERCC1 mutant cell lines, Two general classes of mutations were observed: two mutant cell lines exhibited nucleotide additions or deletions to produce frameshift mutations and seven mutant cell lines exhibited point mutations that resulted in transitions or transversions, including nonsense mutations and mutations that generated intron/exon splicing errors. One mutant (UV201) which had been provisionally assigned to ERCC1 complementation group 1 (CG1) had no detectable mutation in its coding sequence. Of the nine ERCC1 mutant alleles characterized two mutations were identified in the XpA binding region of the Ercc1 protein; no mutations were found in the N-terminal portion of the Ercc1 protein. Results of Northern hybridization analysis showed that the relative levels of ERCC1 mRNA differed significantly both among the parental cell lines rind among the mutant cell lines derived from each parental cell line. Western analysis with a CHO Ercc1-specific antibody detected Ercc1 protein in each of the parental cell lines and also in UV201. The marked reduction in Ercc1 protein levels observed in all the other mutants examined supports the hypothesis that ERCC1 mutations may destabilize this polypeptide.