ANTIBODY CLASS SWITCH RECOMBINASE ACTIVITY IS B-CELL STAGE-SPECIFIC AND FUNCTIONS STOCHASTICALLY IN THE ABSENCE OF TARGETED ACCESSIBILITY CONTROL

Chromosomally integrated retroviral switch (S) substrates have been de veloped to reveal switch recombinase-like activities (SRLA) in pre-B a nd mature B cell lines. Switch substrate retrovectors (SSR) contain a long-terminal repeat-driven neomycin (Neo) gene for proviral chromosom al maintenance (pre- and post-S recombination) and a CMV promoter-driv en, chimeric hygromycin-thymidine kinase (Hytk) gene (flanked by S-mu and S(gamma)2b recombination targets) to select for (ganciclovir) and against (hygromycin B)S region recombination, The retro-substrates' st rong, constitutive promoters ensure that variations in cellular switch recombinase activities are independent of S region accessibility cont rol, By initially selecting for proviral integrants in hygromycin foll owed by shifting into neomycin + ganciclovir to select for S sequence- mediated deletions, switch recombinations can be specifically forestal led in B cell lines whilst most switch-incompetent cells do not surviv e secondary selection. A qualitative, direct PCR assay reveals that SS R recombinations are stochastic in B cell lines generating a product a rray akin to natural CH class switching. A semi-quantitative DC-PCR as say detects a significant recombinase activity only in a restricted se t of late stage pre-B and mature B cell lines, BCL1B1 mature B cells h ave the highest level of recombinase activity with 25% or more of prov iral integrants accumulating S-mu/S<(gamma>)S2b substrate recombinatio ns within 10-14 cell generations, The SSR recombinase assay can be per formed in a transient fashion wherein extensive, B cell-specific recom bination can be visualized within only a few cell divisions post provi ral integration, We propose that switch recombinase activity becomes a ctivated during B cell ontogeny independent of or prior to the acquisi tion of C-H locus accessibility and that endogenous S segment targetin g to pre-existing recombinase requires a level of accessibility beyond transcriptional activation.