Lebecque S G, Gearhart P J
Department of Biochemistry, Johns Hopkins University School of Hygiene and Public Health, Baltimore, Maryland 21205.
J Exp Med. 1990 Dec 1;172(6):1717-27. doi: 10.1084/jem.172.6.1717.
To investigate why somatic mutations are spatially restricted to a region around the rearranged V(D)J immunoglobulin gene, we compared the distribution of mutations flanking murine V gene segments that had rearranged next to either proximal or distal J gene segments. 124 nucleotide substitutions, nine deletions, and two insertions were identified in 32,481 bp of DNA flanking the coding regions from 17 heavy and kappa light chain genes. Most of the mutations occurred within a 2-kb region centered around the V(D)J gene, regardless of which J gene segment was used, suggesting that the structural information for mutation is located in sequences around and within the V(D)J gene, and not in sequences downstream of the J gene segments. The majority of mutations were found within 300 bp of DNA flanking the 5' side of the V(D)J gene and 850 bp flanking the 3' side at a frequency of 0.8%, which was similar to the frequency in the coding region. The frequency of flanking mutations decreased as a function of distance from the gene. There was no evidence for hot spots in that every mutation was unique and occurred at a different position. No mutations were found upstream of the promoter region, suggesting that the promoter delimits a 5' boundary, which provides strong evidence that transcription is necessary to generate mutation. The 3' boundary was approximately 1 kb from the V(D)J gene and was not associated with a DNA sequence motif. Occasional mutations were located in the nuclear matrix association and enhancer regions. The pattern of substitutions suggests that there is discrimination between the two DNA strands during mutation, in that the four bases were mutated with different frequencies on each strand. The high frequency of mutations in the 3' flanking region and the uniqueness of each mutation argues against templated gene conversion as a mechanism for generating somatic diversity in murine V(D)J genes. Rather, the data support a model for random point mutations where the mechanism is linked to the transcriptional state of the gene.
为了研究体细胞突变为何在空间上局限于重排的V(D)J免疫球蛋白基因周围的区域，我们比较了在与近端或远端J基因片段相邻重排的小鼠V基因片段侧翼的突变分布。在17个重链和κ轻链基因编码区侧翼的32,481 bp DNA中，鉴定出124个核苷酸替换、9个缺失和2个插入。无论使用哪个J基因片段，大多数突变都发生在以V(D)J基因为中心的2 kb区域内，这表明突变的结构信息位于V(D)J基因周围和内部的序列中，而不是在J基因片段下游的序列中。大多数突变出现在V(D)J基因5'侧翼300 bp的DNA范围内以及3'侧翼850 bp的范围内，频率为0.8%，这与编码区的频率相似。侧翼突变的频率随着与基因距离的增加而降低。没有证据表明存在热点，因为每个突变都是独特的，且发生在不同的位置。在启动子区域上游未发现突变，这表明启动子划定了一个5'边界，有力地证明了转录对于产生突变是必要的。3'边界距离V(D)J基因约1 kb，且与DNA序列基序无关。偶尔有突变位于核基质结合区和增强子区域。替换模式表明在突变过程中两条DNA链之间存在差异，因为每条链上四个碱基的突变频率不同。3'侧翼区域的高突变频率以及每个突变的独特性表明，模板化基因转换不是小鼠V(D)J基因产生体细胞多样性的机制。相反，这些数据支持一种随机点突变模型，其机制与基因的转录状态相关。
