Co-existence of somatic hypermutation and gene conversion in hypervariable regions of single Igkappa clones.

Abstract

In the rabbit, recent investigations have provided evidence that gene conversion leads to the generation of diversity of heavy chain rearranged VH-DH-JH genes. No data have been published on a similar mechanism for rabbit light chains. In our laboratory, we initially infected rabbits with Trypanosoma brucei, which stimulates B-cell hyperplasia and hypergammaglobulinaemia. The heterozygous rabbits exhibited the Ckappa1 b4 and b9 kappa light chain allotypes. After reverse transcription of mRNA, and cloning and sequencing of cDNA, the Vkappa-Jkappa-Ckappa genes provided evidence for both somatic hypermutation and gene conversion. We saw that in each of the b4 and b9 kappa light chain cDNA, CDR1 and CDR3 carried both point mutation and provisional gene conversion traits. In the CDR2 region, point mutation and gene conversion inserts were observed in the b4 genes, with only gene conversion in two b9 genes. In the CDR regions, although some genes exhibited only somatic hypermutation or gene conversion, others showed linkage of both somatic hypermutation and gene conversion in the same sequence. This also marks the first time that somatic hypermutation and gene conversion in the same cloned CDR region has been observed in Vkappa1 genes; however, it has been seen earlier in rabbit heavy chain VH sequences. Furthermore, the addition of several codons to the CDR3 segment by gene conversion may have provided a mechanism for length variation. In addition, we demonstrated that Jkappa and framework region segments contained examples of somatic hypermutation. Confirmation of gene conversion necessitates that donor sequences be identified as providing the templated inserts. Thus after cloning two pseudogenes we found putative CDR3 donor segments for two CDR3 rearranged genes. The results offer additional mechanisms for the generation of diversity among rearranged rabbit kappa light chain genes. Whether there is a relationship or influence of gene conversion upon somatic hypermutation or vice versa is not discernable at present.