Immunoglobulin variable region hypermutation in hybrids derived from a pre-B- and a myeloma cell line.
Immunoglobulin variable region hypermutation in hybrids derived from a pre-B- and a myeloma cell line.
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来自前 B 细胞系和骨髓瘤细胞系的杂交体中的免疫球蛋白可变区超突变。
DOI：
10.1073/pnas.92.14.6304
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发表时间：
1995
影响因子：
11.1
通讯作者：
Scharff,MD
中科院分区：
文献类型：
--
作者：
Green,NS;Rabinowitz,JL;Zhu,M;Kobrin,BJ;Scharff,MD
免疫球蛋白基因可变 (V) 区的体细胞突变在体内发生的速率估计为每代每 bp 10(-3) 到 10(-4) 之间。为了在体外研究这一过程，用在 V 区 (Vn) 或恒定区 (Cn) 中含有无义突变的 mu 重链构建体转染 18.81 前 B 细胞系和通过将 18.81 与 NSO 骨髓瘤融合伴侣融合而衍生的杂交体。使用 ELISA 斑点测定法检测分泌 IgM 的单细胞，通过回复分析对突变进行定量。波动分析显示，V区突变在18.81细胞中以每代每bp 5.8 x 10(-6)的平均速率自发发生，而在选定的18.81-NSO杂交体中，以每代每bp 1.6 x 10(-3)至5.8 x 10(-4)的速率大幅增加。 Vn 构建体在转基因小鼠中也频繁恢复，表明它包含足够的信息，可以在体内和体外以高速率突变。对恢复基因的序列分析表明，恢复是由于点突变造成的。由于这些转染基因中发生的突变的速率和性质与体内报道的相似，因此应该可以使用该系统来鉴定导致 V 区体细胞超突变的顺式作用序列和反式作用因子。
Somatic mutation of the variable (V) regions of immunoglobulin genes occurs in vivo at rates that have been estimated to be between 10(-3) and 10(-4) per bp per generation. To study this process in vitro, the 18.81 pre-B-cell line and hybrids derived by fusing 18.81 to the NSO myeloma fusion partner were transfected with a mu heavy-chain construct containing a nonsense mutation in the V region (Vn) or the constant region (Cn). Mutation was quantitated by reversion analysis using the ELISA spot assay to detect single cells secreting IgM. Fluctuation analysis revealed that V-region mutations spontaneously occurred in 18.81 cells at an average rate of 5.8 x 10(-6) per bp per cell generation and in selected 18.81-NSO hybrids at greatly increased rates of 1.6 x 10(-3) to 5.8 x 10(-4) per bp per generation. The Vn construct also reverted frequently in transgenic mice, indicating that it contained sufficient information to mutate at high rates both in vivo and in vitro. Sequence analysis of reverted genes revealed that reversion was due to point mutations. Since the rates and nature of the mutations that are occurring in these transfected genes are similar to those reported in vivo, it should be possible to use this system to identify the cis-acting sequences and trans-acting factors that are responsible for V-region somatic hypermutation.