Switch recombination in a transfected plasmid occurs preferentially in a B cell line that undergoes switch recombination of its chromosomal Ig heavy chain genes.
Ab class switching is induced upon B cell activation in vivo by immunization or infection or in vitro by treatment with mitogens, e. g. LPS, and results in the expression of different heavy chain constant region (CH) genes without a change in the Ab variable region. This DNA recombination event allows Abs to alter their biological activity while maintaining their antigenic specificity. Little is known about the molecular mechanism of switch recombination. To attempt to develop an assay for enzymes, DNA binding proteins, and DNA sequences that mediate switch recombination, we have constructed a plasmid DNA substrate that will undergo switch recombination upon stable transfection into the surface IgM+ B cell line (I.29 mu), a cell line capable of undergoing switch recombination of its endogenous genes. We demonstrate that recombination occurs between the two switch regions of the plasmid, as assayed by PCRs across the integrated plasmid switch regions, followed by Southern blot hybridization. Nucleotide sequence analysis of the PCR products confirmed the occurrence of S mu-S alpha recombination in the plasmid. Recombination of the plasmid in I.29 mu cells does not require treatment with inducers of switch recombination, suggesting that recombinase activity is constitutive in I.29 mu cells. Recombination does not require high levels of transcription across the switch regions of the plasmid. Fewer recombination events are detected in four different B and T cell lines that do not undergo switch recombination of their endogenous genes.
Stavnezer
J
J
Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester 01655, USA. janet.stavnezer@banyan.ummed.edu
Bradley
S P
SP
Rousseau
N
N
Pearson
T
T
Shanmugam
A
A
Waite
D J
DJ
Rogers
P R
PR
Kenter
A L
AL
D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D002875:Chromosomes / Q000276:immunology*; D003001:Cloning, Molecular; D015326:Gene Rearrangement, B-Lymphocyte, Heavy Chain / Q000235:genetics*; D017578:Immunoglobulin Class Switching / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007134:Immunoglobulin Switch Region / Q000235:genetics*; D007137:Immunoglobulin alpha-Chains / Q000235:genetics / Q000302:isolation & purification; D007148:Immunoglobulin mu-Chains / Q000235:genetics / Q000302:isolation & purification; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D009154:Mutation / Q000276:immunology; D010954:Plasmacytoma; D010957:Plasmids / Q000138:chemical synthesis / Q000235:genetics / Q000276:immunology*; D016133:Polymerase Chain Reaction; D013601:T-Lymphocytes / Q000276:immunology / Q000378:metabolism; D014158:Transcription, Genetic / Q000276:immunology; D014162:Transfection / Q000235:genetics / Q000276:immunology*; D014407:Tumor Cells, Cultured
D007143:Immunoglobulin Heavy Chains; D007137:Immunoglobulin alpha-Chains; D007148:Immunoglobulin mu-Chains