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Original Article

^a Department of Microbiology and Immunology, University of Illinois College of Medicine, Chicago, Illinois 60612
^b Department of Molecular Genetics and Microbiology and the Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Department of Microbiology/Immunology (M/C790), College of Medicine, University of Illinois, Chicago, IL 60612-7344.312-996-6415312-996-5293

amy.l.kenter{at}uic.edu

Immunoglobulin class switch recombination (SR) occurs by a B cell–specific, intrachromosomal deletional process between switch regions. We have developed a plasmid-based transient transfection assay for SR to test for the presence of transacting switch activities. The plasmids are novel in that they lack a eukaryotic origin of DNA replication. The recombination activity of these switch substrates is restricted to a subset of B cell lines that support isotype switching on their endogenous loci and to mitogen-activated normal splenic B cells. The factors required for extrachromosomal plasmid recombination are constitutively expressed in proliferating splenic B cells and in B cell lines capable of inducibly undergoing immunoglobulin SR on their chromosomal genes. These studies suggest that mitogens that induce switching on the chromosome induce accessibility rather than switch recombinase activity. Finally, we provide evidence for two distinct switching activities which independently mediate µ and µ3 SR.

Key Words: immunoglobulin • isotype switch • plasmid assay • transient transfection • PCR

Abbreviations used in this paper: dex, dextran-conjugated anti-IgD antibodies; β-gal, β-galactosidase; CAT, chloramphenicol acetyl transferase; DC, digestion-circularization; DNA-PK_{CS, DNA-dependent protein kinase catalytic subunit; DSB, double-stranded break; E, enhancer; gt, germline transcript; NF, nuclear factor; RAG, recombination activating gene; RRL, relative recombination level; S, switch; SR, switch recombination; TK, thymidine kinase gene.}

© 2000 The Rockefeller University Press

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