| Gene conversion of rabbit IgH genes has thus far only been described as a means of generating a diverse primary antibody repertoire, and has only been reported to occur in young rabbit gut associated lymphoid tissue (GALT) follicles. Although rabbit GALT requires exogenous factors to develop, the agent(s) and mechanism by which somatic gene conversion is induced in GALT remain elusive.;I have asked whether rabbit GALT is the only specialized microenvironment for the induction of somatic gene conversion of rabbit IgH genes, or whether there is evidence for somatic gene conversion in other peripheral locations in response to immunization, as is the case for somatic hypermutation of mouse and human Ig genes.;I first analyzed clonally-related VDJ gene sequences from FITC-specific hybridomas derived at d. 3 of a secondary immune response. Out of 40 total antigen-specific hybridoma VDJ gene sequences analyzed, I obtained seven small sets of clonally-related VDJ gene sequences. Within these clonally-related sequences, there was ongoing somatic point mutation, as evidenced by differences between related clones in the D-regions. In addition, there were four potential ongoing gene conversion-like events between the related clones, one of which was quite convincing. This putative gene conversion spanned 45 nucleotides, and resulted in 19 mutations from germline, including a codon insertion. Moreover, this gene conversion-like event was found in one of two clonally-related antigen-specific VDJ gene sequences, indicating that somatic gene conversion was induced during the course of this immune response.;From the germinal center VDJ genes we were able to establish several lineages of clonally-related sequences. Three small lineages were derived from germinal centers scraped at d. 5 of a tertiary immune response, and two more extensive lineages were built from germinal centers obtained at d. 14 of a primary immune response.;Within these lineages of germinal center VDJ genes, we found evidence for both ongoing somatic point mutation in the D-regions, and several examples of ongoing somatic gene conversion-like events that matched within the V-regions. In order to be certain that the gene conversion events are due to in vivo generated mutation processes, rather than PCR-chimeric artifacts, the sequences need to be confirmed in independent PCR amplification reactions. We have not yet been able to confirm any of the clonally-related sequences containing putative gene conversion events in this fashion, so these data are not yet conclusive. However, these data are consistent with the observations found within the clonally-related, antigen-specific hybridoma VDJ genes, of both antigen-induced somatic point mutation and somatic gene conversion. (Abstract shortened by UMI.). |
