Increased transcription levels induce higher mutation rates in a hypermutating cell line
Increased transcription levels induce higher mutation rates in a hypermutating cell line
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DOI：
10.4049/jimmunol.166.8.5051
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发表时间：
2001-04-15
影响因子：
4.4
通讯作者：
Olsson, C
中科院分区：
文献类型：
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作者：
Bachl, J;Carlson, C;Olsson, C
除了V（D）J重组之外，体细胞超突变是产生Ab库的巨大多样性的另一主要机制。点突变被引入IG基因的可变区，其速率比基因组的其余部分高出百万倍。我们已经使用了一个绿色荧光蛋白（GFP）为基础的逆转试验，以确定转录的作用，在突变机制的超突变细胞系18-81。含有提前终止密码子的GFP转基因从诱导型tet-on操纵子转录。使用诱导型启动子使我们能够研究GFP转基因在不同转录水平的突变性。通过用流式细胞术分析超突变细胞系的稳定转染子，可以通过GFP阳性回复突变细胞的出现来测量提前终止密码子处的突变率。在这里，我们表明GFP转基因的突变率与其转录水平相关。GFP转基因转录水平的增加导致提前终止密码子的点突变率增加。用组蛋白去乙酰化酶的特异性抑制剂阿司他丁A处理超突变转染克隆，导致突变率额外增加2倍。最后，使用北方印迹分析，我们表明，激活诱导的胞苷脱氨酶，在体内超突变机制的一个必不可少的trans-factor，转录在超突变细胞系18-81。免疫学杂志，2001年。
Somatic hypermutation, in addition to V(D)J recombination, is the other major mechanism that generates the vast diversity of the Ab repertoire. Point mutations are introduced in the variable region of the Ig genes at a million-fold higher rate than in the rest of the genome. We have used a green fluorescent protein (GFP)-based reversion assay to determine the role of transcription in the mutation mechanism of the hypermutating cell line 18-81. A GFP transgene containing a premature stop codon is transcribed from the inducible tet-on operon. Using the inducible promoter enables us to study the mutability of the GFP transgene at different transcription levels. By analyzing stable transfectants of a hypermutating cell line with flow cytometry, the mutation rate at the premature stop codon can be measured by the appearance of GFP-positive revertant cells. Here we show that the mutation rate of the GFP transgene correlates with its transcription level. Increased transcription levels of the GFP transgene caused an increased point mutation rate at the premature stop codon. Treating a hypermutating transfection clone with trichostatin A, a specific inhibitor of histone deacetylase, caused an additional 2-fold increase in the mutation rate. Finally, using Northern blot analysis we show that the activation-induced cytidine deaminase, an essential trans-factor for the in vivo hypermutation mechanism, is transcribed in the hypermutating cell line 18-81. The Journal of Immunology, 2001.