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

Abstract

We have employed a retroviral vector, ZN(Smu/S gamma 2b)tk1, as a means of introducing immunoglobulin heavy chain (IgH) switch (S) region sequences into B cell lines to directly measure their switch-recombinase activities. In an earlier study, we demonstrated that retrovector Smu-S gamma 2b 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 Smu and S gamma 2b 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 (greater than 1 event in 2500 cells/generation) while a terminally differentiated, antibody-secreting hybridoma line (A39R 1.1) has no detectable recombinase activity. Both S mu and S gamma 2b segments are required for switch region-mediated deletions. Retrovectors harboring only an Smu segment or an Smu segment and a portion of the murine c-myc gene in place of S gamma 2b sequences were both non-recombinagenic in this assay system. Nucleotide sequence analysis of six retrovector S segment recombinants, recovered from ZN(Smu/S gamma 2b) tk1-infected 18-8 and 39B9 pre-B lines, did not reveal homology at their sites of recombination. 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 Smu and S gamma 2b rearrangements were not observed in populations and clones of pre-B cells expressing a high level of switch-recombinase activity, multiple factors (presumably contributed in part by the degree of S segment accessibility) in addition to S recombinase activity are required for CH class switching.