Zhang K, Cheah H K, Saxon A
Hart and Louise Lyon Laboratory, Division of Clinical Immunology and Allergy, University of California Los Angeles School of Medicine, 90024-1680.
J Immunol. 1995 Mar 1;154(5):2237-47.
Analysis of the Ig switch (S) region structure from many stable isotype-switched B cells reveals that the majority of the ultimate switch recombination sites are located at the 5' end, or even upstream of the 5' end of the Smu region. These findings strongly contrast with results from switch circle analysis which reveal that the primary switch recombination sites are preferentially distributed in the middle or at the 3' end of the Smu region. Secondary deletion within recombined chimeric S regions has been proposed as a mechanism to account for the different results. We directly tested whether secondary deletion of rearranged chimeric S regions indeed occurs in human cells. Circular DNAs representing secondary deletion events in gamma switched cells were isolated and characterized by using a specially designed PCR-based approach. Nucleotide sequence analysis revealed that all clones had the S gamma 1-Smu-S gamma 1 structure. Thus, these cloned fragments resulted from secondary deletion/recombination events within a chimeric Smu-S gamma 1 switch region, i.e., rearrangement between its 5' Smu and 3' S gamma 1. Analysis of the Smu region from stimulated B cells also revealed that some Smu regions undergo an internal deletion/rearrangement between the 5' and 3' ends. These results definitively demonstrate that secondary deletion/recombination of chimeric S regions of isotype-switched B cells occurs. Such secondary deletion/recombination events potentially can be responsible for isotype stabilization of switched B cells, as the active Ig gene may have an insufficient amount of retained chimeric switch sequence to serve as a substrate for further S-S recombination.
对许多稳定的同种型转换B细胞的Ig转换（S）区结构分析表明，大多数最终的转换重组位点位于Smu区5'端，甚至在其5'端上游。这些发现与转换环分析结果形成强烈对比，后者显示初级转换重组位点优先分布在Smu区的中间或3'端。已提出重组嵌合S区中的二级缺失作为解释不同结果的一种机制。我们直接测试了重排的嵌合S区的二级缺失是否确实发生在人类细胞中。通过使用一种专门设计的基于PCR的方法，分离并鉴定了代表γ转换细胞中二级缺失事件的环状DNA。核苷酸序列分析表明，所有克隆均具有Sγ1-Smu-Sγ1结构。因此，这些克隆片段是由嵌合Smu-Sγ1转换区内的二级缺失/重组事件产生的，即其5'Smu与3'Sγ1之间的重排。对受刺激B细胞的Smu区分析还表明，一些Smu区在5'和3'端之间发生内部缺失/重排。这些结果明确证明了同种型转换B细胞的嵌合S区发生二级缺失/重组。这种二级缺失/重组事件可能负责转换B细胞的同种型稳定，因为活性Ig基因可能保留的嵌合转换序列量不足，无法作为进一步S-S重组的底物。
