Molecular requirements for immunoglobulin heavy chain constant region gene switch recombination revealed with switch substrate retroviruses

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Abstract
We have employed a retroviral vector, ZN(Sμ,/Sγ2b)tk1, as a means of introducing immunoglobulin heavy chain (IgH) swltch (S) region sequences into B cell lines to directly measure their switch recombinase activities. in an earlier study, we demonstrated that retrovector Sμ,−Sγ2b, recombination events occurred in two thymidine kinase (tk)-negative murine pre-B cell lines (18-8 and 38B9) upon selection in bromodeoxyuridine (BUdR) media for the loss of an Htk gene inserted in between the vector's Sμ and Sγ2b sequences. Here we have used this assay system to show that the 300-18 murine pre-B cell line possesses a very high level of switch-recombinase activity (>1 event in 2500 cells/generation) while a terminally differentiated, antibody-secreting hybridoma line (A39R 1.1) has no detectable recombinase activity. Both Sμ and Sγ2b segments are required for switch region-mediated deletions. Retrovectors harboring only an Sμ2b segment or an Sμ segment and a portion of the murine c-myc gene in place of Sγ2b sequences were both non-recombinagenic in this assay system. Nucleotide sequence analysis of six retrovector S segment recombinants, recovered from ZN(Sμ/Sγ2b)tk1-infected 18-8 and 39B9 pre-B lines, did not reveal homology at their sites of recomblnatlon. We conclude that: (1) S segment repetitive sequences play an essential but indirect role in IgCH gene switch-recombination, which occurs by an illegitimate, non-homologous mechanism; (2) the c-myc gene is not a significant target for switch -recombination; and (3) since endogenous Sμ and Sγ2b rearrangements were not observed in populations and clones of pre-B cells expressing a high level of swttch-recombinase activity, multiple factors (presumably contributed in part by the degree of S segment accessibllity) in addition to S recomblnase activity are required for CH class swltchlng.

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