Fitzsimmons S P, Rotz B T, Shapiro M A
Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, MD 20892, USA.
J Immunol. 1998 Sep 1;161(5):2290-300.
The mouse has approximately 140 germline V kappa genes, and functional V kappa exons are expressed at roughly equivalent levels in the preimmune repertoire. We have examined the expression of individual members of the V kappa 10 family. V kappa 10A and V kappa 10B genes have been utilized in numerous hybridomas and myelomas, while V kappa 10C has not. In this study, we have cloned the V kappa 10C gene and shown that it is structurally functional, has the expected promoter elements and recombination signal sequences, and that it is capable of recombination. V kappa 10C mRNA, however, is present at levels at least 1000-fold lower than V kappa 10A and V kappa 10B in adult spleens. While there are no sequence differences in the octamer or TATA box between V kappa 10C and V kappa 10A, there are three nucleotide changes in the promoter region. These promoters equally drive the expression of a reporter gene in B cells or plasma cells, but the V kappa 10A promoter is able to drive expression in pre-B cell lines significantly better than the V kappa 10C promoter (p < 0.05). V kappa 10C rearrangements can be detected in bone marrow and splenic DNA. Therefore, the lack of V kappa 10C expression may reflect the inability of V kappa 10C-rearranged cells to undergo positive or negative selection. Our results suggest that the available Ab repertoire is shaped not only by the number of structurally functional genes, but also by the ability of assembled genes to be expressed at critical points during B cell maturation.
小鼠大约有140个种系Vκ基因，功能性Vκ外显子在免疫前库中以大致相当的水平表达。我们研究了Vκ10家族单个成员的表达情况。Vκ10A和Vκ10B基因已在众多杂交瘤和骨髓瘤中被利用，而Vκ10C基因则未被利用。在本研究中，我们克隆了Vκ10C基因，并表明它在结构上具有功能，具有预期的启动子元件和重组信号序列，并且能够发生重组。然而，在成年脾脏中，Vκ10C mRNA的水平比Vκ10A和Vκ10B至少低1000倍。虽然Vκ10C和Vκ10A之间的八聚体或TATA框没有序列差异，但启动子区域有三个核苷酸变化。这些启动子在B细胞或浆细胞中同样驱动报告基因的表达，但Vκ10A启动子在pre - B细胞系中驱动表达的能力明显优于Vκ10C启动子（p < 0.05）。在骨髓和脾脏DNA中可以检测到Vκ10C重排。因此，Vκ10C表达的缺乏可能反映了Vκ10C重排细胞无法进行阳性或阴性选择。我们的结果表明，可用的抗体库不仅由结构上有功能的基因数量决定，还由组装基因在B细胞成熟关键阶段的表达能力决定。
