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

Abstract

Homology-based Ig gene conversion is a major mechanism for Ab diversification in chickens and the Rad54 DNA repair protein plays an important role in this process. In mice, although gene conversion appears to be rare among endogenous Ig genes, Ab H chain transgenes undergo isotype switching and gene conversion-like sequence transfer processes that also appear to involve homologous recombination or gene conversion. Furthermore, homology-based DNA repair has been suggested to be important for somatic mutation of endogenous mouse Ig genes. To assess the role of Rad54 in these mouse B cell processes, we have analyzed H chain transgene isotype switching, sequence transfer, and somatic hypermutation in mice that lack RAD54. We find that Rad54 is not required for either transgene switching or transgene hypermutation. Furthermore, even transgene sequence transfers that are known to require homology-based recombinations are Rad54 independent. These results indicate that mouse B cells must use factors for promoting homologous recombination that are distinct from the Rad54 proteins important in homology-based chicken Ab gene recombinations. Our findings also suggest that mouse H chain transgene sequence transfers might be more closely related to an error-prone homology-based somatic hypermutational mechanism than to the hyperconversion mechanism that operates in chicken B cells.

Sequence Transfers between Variable Regions in a Mouse Antibody Transgene Can Occur by Gene Conversion

“…3 shows the 591A6 transgene-derived ␥-chain mRNA VDJ sequence compared with the R16.7 and 2B4 VDJ sequences present in the VVC transgene. The block of nucleotide changes in the expressed 591A6 VDJ that corresponds to the sequence of the 2B4 VDJ indicates a transgene sequence transfer similar to those described previously (25,28,29).…”

“…However, extensive efforts to obtain IgG-producing anti-Ars hybridomas after immunization of these offspring were unsuccessful, indicating that the single VVC transgene copy in the ES cell clone was not capable of interchromosomal switch recombination. Because all sequence transfers in multicopy VVC transgenic mice have been found in IgG-producing B cells (25,28,29), and because there is no information indicating whether such transfers can be found in IgMproducing B cells, these mice were not studied further. A third ES clone carrying about two copies of the VVC construct ( Fig.…”

“…The AD8 reagent reacts with H chains that carry the R16.7 VDJ CDR2 sequence but not with H chains having the 2B4 VDJ CDR2 sequence (25,28). Transgene-expressing B cells from VVC transgenic mice generally express the AD8-reactive idiotope whereas those VVC B cells that exhibit transgene sequence transfers often do not express the AD8-reactive idiotope (25,28,29). Candidate hybridomas expressing anti-Ars IgG Abs that were not reactive with AD8 were further screened by sequence analyses of the expressed H chain VDJ from RT-PCR products.…”

“…Transgene sequence transfers in immunized VV29 blood lymphocytes were assessed using an RT-PCR/Southern blot assay that has been described previously (28,29). In this assay, PCR products that hybridize with the 2B4 probe are indicative of sequence transfers in the VVC transgene.…”

“…Four days after the fourth immunization, splenocytes were obtained for hybridoma production. Sequence transfers in splenocytes and blood lymphocytes from immunized transgenic mice were detected using RT-PCR/Southern blot assays that have been described previously (28,29). Immunized transgenic mice exhibiting sequence transfers as indicated by the RT-PCR/Southern blot assay were then used to produce hybridomas.…”