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PMID: 9224606 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Analysis of immunoglobulin Sgamma3 recombination breakpoints by PCR: implications for the mechanism of isotype switching.

Nucleic acids research ·Vol. 25 ·No. 15 ·1997-08-01 ·Pages 3066-73

Du J, Zhu Y, Shanmugam A, Kenter AL

Abstract

The molecular mechanism of immunoglobulin switch recombination is poorly understood. Switch recombination occurs between pairs of switch regions located upstream of the constant heavy chain genes. Previously we showed that switch recombination breakpoints cluster to a defined subregion in the Sgamma3, Sgamma1 and Sgamma2b tandem repeats. We have developed a strategy for direct amplification of Smu/Sgamma3 composite fragments as well as Smu and Sgamma3 regions by PCR. This assay has been used to analyze the organization of Smu, Sgamma3 and a series of Smu/Sgamma3 recombination breakpoints from hybridomas and normal mitogen-activated splenic B cells. DNA sequence analysis of the switch fragments showed direct joining of Smu and Sgamma3 without deletions or duplications. Mutations were found in two switch junctions on both sides of the crossover point, suggesting that template switching is the most likely model for the mechanism of switch recombination. Statistical analysis of the positions of the recombination breakpoints in the Sgamma3 tandem repeat indicates the presence of two sub-clusters, suggesting non-random usage of DNA substrate in the recombination reaction.

MeSH Terms
Animals B-Lymphocytes/cytology Base Sequence Blotting, Southern Cell Line Cells, Cultured Female Hybridomas Immunoglobulin G/biosynthesis,genetics Immunoglobulin Heavy Chains/genetics Immunoglobulin Switch Region/genetics Immunoglobulin mu-Chains/genetics Lipopolysaccharides/pharmacology Mice Mice, Inbred BALB C Mitogens/pharmacology Molecular Sequence Data Polymerase Chain Reaction Recombination, Genetic Sequence Analysis, DNA Spleen/cytology
Chemicals
Immunoglobulin G Immunoglobulin Heavy Chains Immunoglobulin mu-Chains Lipopolysaccharides Mitogens
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Du J
Department of Microbiology and Immunology (M/C 790), University of Illinois College of Medicine, 835 South Wolcott Avenue, Chicago, IL 60612-7344, USA.
Zhu Y
Shanmugam A
Kenter A L
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Article Info
Journal
Nucleic acids research
Abbr.
Nucleic Acids Res
ISSN
0305-1048
DOI
10.1093/nar/25.15.3066
Published
1997-08-01
Pages
3066-73
Language
English
Region
England
NLM ID
0411011
PMCID
PMC146845
Subset
IM
Grants
NIAID NIH HHS · R21 AI106328 · United States
NIGMS NIH HHS · GM39231 · United States
External Links
PubMed source DOI: 10.1093/nar/25.15.3066 PMC Full Text View journal details
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