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 developed to reveal switch recombinase-like activities (SRLA) in pre-B and mature B cell lines. Switch substrate retrovectors (SSR) contain a long-terminal repeat-driven neomycin (Neo) gene for proviral chromosomal maintenance (pre- and post-S recombination) and a CMV promoter-driven, 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' strong, constitutive promoters ensure that variations in cellular switch recombinase activities are independent of S region accessibility control. By initially selecting for proviral integrants in hygromycin followed by shifting into neomycin + ganciclovir to select for S sequence-mediated deletions, switch recombinations can be specifically forestalled in B cell lines whilst most switch-incompetent cells do not survive secondary selection. A qualitative, direct PCR assay reveals that SSR recombinations are stochastic in B cell lines generating a product array akin to natural GH class switching. A semi-quantitative DC-PCR assay detects a significant recombinase activity only in a restricted set of late stage pre-B and mature B cell lines. BCL1B1 mature B cells have the highest level of recombinase activity with 25% or more of proviral integrants accumulating S mu/S gamma 2b substrate recombinations within 10-14 cell generations. The SSR recombinase assay can be performed in a transient fashion wherein extensive, B cell-specific recombination can be visualized within only a few cell divisions post proviral integration. We propose that switch recombinase activity becomes activated during B cell ontogeny independent of or prior to the acquisition of CH locus accessibility and that endogenous S segment targeting to pre-existing recombinase requires a level of accessibility beyond transcriptional activation.
Ballantyne
J
J
Graduate Program in Genetics, Institute for Cell and Developmental Biology, State University of New York, Stony Brook 11794-5215, USA.
Henry
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D016475:3T3 Cells; D000818:Animals; D016175:B-Lymphocyte Subsets / Q000166:cytology / Q000201:enzymology* / Q000821:virology; D002454:Cell Differentiation / Q000276:immunology; D004254:DNA Nucleotidyltransferases / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000276:immunology; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006921:Hygromycin B / Q000494:pharmacology; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007127:Immunoglobulin Constant Regions / Q000235:genetics; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D051379:Mice; D009355:Neomycin / Q000494:pharmacology; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012190:Retroviridae / Q000235:genetics; D012680:Sensitivity and Specificity; D013269:Stochastic Processes; D013379:Substrate Specificity / Q000235:genetics / Q000276:immunology; D013937:Thymidine Kinase / Q000378:metabolism; D045567:VDJ Recombinases
D007127:Immunoglobulin Constant Regions; D007143:Immunoglobulin Heavy Chains; D006921:Hygromycin B; D013937:Thymidine Kinase; D004254:DNA Nucleotidyltransferases; D045567:VDJ Recombinases; D009355:Neomycin