Gene conversion–like sequence transfers in a mouse antibody transgene: antigen selection allows sensitive detection of V region interactions based on homology

Abstract

Gene conversion is important for antibody diversification in chickens, rabbits and cows. In mice, however, conversion events appear to be infrequent among endogenous antibody genes. DNA sequence transfer events that resemble gene conversions have been reported for a mouse H chain transgene (VVC(mu)) that contains two closely spaced homologous VDJ segments. Surprisingly, these reported VVC(mu) sequence transfers were found frequently among mouse B cells responding to immunization. Transgene sequence transfers could be occurring at high frequency in responding VVC(mu) B cells or could be occurring at lower frequency with subsequent amplification by preferential antigen selection. To distinguish these possibilities, we have analyzed a second transgene (InVVC(mu)) that is identical to VVC(mu) except that the two VDJ regions have been exchanged in position. We find that transgene sequence transfers are much less frequent among responding B cells in InVVC(mu) mice, demonstrating the importance of selection in the frequent transgene conversions observed in VVC(mu) mice. These results suggest that mice, like other species, can use gene conversion to diversify antibodies. Such diversification events are apparently infrequent, however, and might only be detected among endogenous Ig genes with a favorable arrangement of V genes and an antigenic stimulation that selects cells with conversions. For both VVC(mu) and InVVC(mu) mice, conversion-like sequence transfers are strongly correlated with somatic hypermutation. Based on these results, we hypothesize that, in mice, gene conversions represent infrequent alternative reactions of a homology-based DNA repair process that is central in the somatic hypermutational mechanism.

Gene Conversion-Like Sequence Transfers Between Transgenic Antibody V Genes Are Independent of RAD54

“…Fourteen of these clones contained inserts derived from the transgene VDJ segments and five of the fourteen clones displayed sequence transfers (Fig. 5) like those that have been previously reported for other immunized VVC mice (12,13). All five of the RT-PCR products that had transgene conversion also had somatic mutations.…”

“…Studies have also indicated that homology-based sequence transfers might play a significant role in Ig gene somatic hypermutation. Transgene V gene sequence transfers have been found to be frequently associated with high levels of somatic hypermutation (12,13), suggesting that homology-based recombination and somatic hypermutation might share some mechanistic steps (13,14). Furthermore, DNA cleavages associated with somatic hypermutation of endogenous Ig genes are predominantly found at the G 2 cell cycle stage; this observation is consistent with the hypothesis that homologybased repair might play a role in the hypermutational mechanism (15).…”

“…The role of Rad54 in the gene conversion-like sequence transfers that occur in VVC transgenic mice was analyzed using an RT-PCR/Southern blot assay (13). In this assay, splenocyte RNAs were amplified by RT-PCR using primers that amplify transgenederived VDJ sequences from IgG-producing B cells.…”

“…The RT-PCR/Southern blot assay has been used to assess gene conversion-like sequence transfers in immunized VVC transgenic mice (13). Fig.…”

“…Clones were sequenced at the Tufts University core facility using the leader specific primer mentioned above. Dot blot analyses of cloned PCR products were performed as described previously (13).…”