International Immunology, Vol 9, 963-974, Copyright © 1997 by Oxford University Press
J Ballantyne, DL Henry and KB Marcu
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.
ARTICLES
Antibody class switch recombinase activity is B cell stage specific and functions stochastically in the absence of 'targeted accessibility' control [published erratum appears in Int Immunol 1997 Nov;9(11):1773]
Graduate Program in Genetics, Institute for Cell and Developmental Biology, State University of New York, Stony Brook 11794-5215, USA.
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