Antibody class switch recombinase activity is B cell stage specific and functions stochastically in the absence of 'targeted accessibility' control
| Autor: | J, Ballantyne, D L, Henry, K B, Marcu |
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| Rok vydání: | 1997 |
| Předmět: |
Recombination
Genetic Stochastic Processes Genetic Vectors B-Lymphocyte Subsets Gene Rearrangement B-Lymphocyte Heavy Chain Cell Differentiation Neomycin 3T3 Cells Immunoglobulin Class Switching Polymerase Chain Reaction Sensitivity and Specificity Thymidine Kinase Gene Expression Regulation Enzymologic Substrate Specificity Mice Retroviridae DNA Nucleotidyltransferases Animals Humans Hygromycin B Immunoglobulin Constant Regions Immunoglobulin Heavy Chains VDJ Recombinases HeLa Cells |
| Zdroj: | International immunology. 9(7) |
| ISSN: | 0953-8178 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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