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. 1992 Aug 1;89(15):6978–6982. doi: 10.1073/pnas.89.15.6978

Quantitation of immunoglobulin mu-gamma 1 heavy chain switch region recombination by a digestion-circularization polymerase chain reaction method.

C C Chu 1, W E Paul 1, E E Max 1
PMCID: PMC49628  PMID: 1495989

Abstract

B lymphocytes expressing surface IgM with or without IgD may switch to the expression of other isotypes (IgG, IgA, or IgE) in the course of immune responses. Analyses of genomic DNA from cloned myelomas and hybridomas have shown that the isotype switch is accompanied by a rearrangement characterized by deletion of DNA between the switch (S) region of the mu gene and that associated with the new isotype, resulting in the formation of a composite S region. Measurement of this deletional rearrangement has been difficult in populations of normal B cells but would be useful for investigating the mechanism of the rearrangement and determining whether deletional rearrangement is responsible for all instances of class switching. We have developed a sensitive assay for deletional rearrangement that we designate the digestion-circularization polymerase chain reaction (PCR). In this assay, genomic DNA is digested with a restriction enzyme that recognizes sites that flank the recombined composite S region. The digested DNA is then ligated at low concentrations to favor the formation of circles. The ligation joins the 5' and 3' ends of each restriction fragment, making it possible to amplify by PCR across the ligated restriction site by using appropriate primers. From DNA that has undergone deletional rearrangement, a single-sized PCR product is produced and can be quantitated. We demonstrate here that the digestion-circularization PCR assay can detect S mu-S gamma 1 rearrangements in B cells cultured with lipopolysaccharide and interleukin 4. The assay is sensitive enough to quantitate switched cells constituting only 1-2% of the population.

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Selected References

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  1. Buonanno A., Mudd J., Merlie J. P. Isolation and characterization of the beta and epsilon subunit genes of mouse muscle acetylcholine receptor. J Biol Chem. 1989 May 5;264(13):7611–7616. [PubMed] [Google Scholar]
  2. Chan M. A., Benedict S. H., Dosch H. M., Hui M. F., Stein L. D. Expression of IgE from a nonrearranged epsilon locus in cloned B-lymphoblastoid cells that also express IgM. J Immunol. 1990 May 1;144(9):3563–3568. [PubMed] [Google Scholar]
  3. Chen Y. W., Word C., Dev V., Uhr J. W., Vitetta E. S., Tucker P. W. Double isotype production by a neoplastic B cell line. II. Allelically excluded production of mu and gamma 1 heavy chains without CH gene rearrangement. J Exp Med. 1986 Aug 1;164(2):562–579. doi: 10.1084/jem.164.2.562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collins F. S., Weissman S. M. Directional cloning of DNA fragments at a large distance from an initial probe: a circularization method. Proc Natl Acad Sci U S A. 1984 Nov;81(21):6812–6816. doi: 10.1073/pnas.81.21.6812. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gillies S. D., Morrison S. L., Oi V. T., Tonegawa S. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell. 1983 Jul;33(3):717–728. doi: 10.1016/0092-8674(83)90014-4. [DOI] [PubMed] [Google Scholar]
  6. Gilliland G., Perrin S., Blanchard K., Bunn H. F. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2725–2729. doi: 10.1073/pnas.87.7.2725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gritzmacher C. A. Molecular aspects of heavy-chain class switching. Crit Rev Immunol. 1989;9(3):173–200. [PubMed] [Google Scholar]
  8. Köhler G., Milstein C. Derivation of specific antibody-producing tissue culture and tumor lines by cell fusion. Eur J Immunol. 1976 Jul;6(7):511–519. doi: 10.1002/eji.1830060713. [DOI] [PubMed] [Google Scholar]
  9. MacKenzie T., Dosch H. M. Clonal and molecular characteristics of the human IgE-committed B cell subset. J Exp Med. 1989 Feb 1;169(2):407–430. doi: 10.1084/jem.169.2.407. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Mowatt M. R., Dunnick W. A. DNA sequence of the murine gamma 1 switch segment reveals novel structural elements. J Immunol. 1986 Apr 1;136(7):2674–2683. [PubMed] [Google Scholar]
  11. Murphy L. D., Herzog C. E., Rudick J. B., Fojo A. T., Bates S. E. Use of the polymerase chain reaction in the quantitation of mdr-1 gene expression. Biochemistry. 1990 Nov 13;29(45):10351–10356. doi: 10.1021/bi00497a009. [DOI] [PubMed] [Google Scholar]
  12. Nolan-Willard M., Berton M. T., Tucker P. Coexpression of mu and gamma 1 heavy chains can occur by a discontinuous transcription mechanism from the same unrearranged chromosome. Proc Natl Acad Sci U S A. 1992 Feb 15;89(4):1234–1238. doi: 10.1073/pnas.89.4.1234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Perlmutter A. P., Gilbert W. Antibodies of the secondary response can be expressed without switch recombination in normal mouse B cells. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7189–7193. doi: 10.1073/pnas.81.22.7189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Shapira S. K., Jabara H. H., Thienes C. P., Ahern D. J., Vercelli D., Gould H. J., Geha R. S. Deletional switch recombination occurs in interleukin-4-induced isotype switching to IgE expression by human B cells. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7528–7532. doi: 10.1073/pnas.88.17.7528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Shimizu A., Nussenzweig M. C., Han H., Sanchez M., Honjo T. Trans-splicing as a possible molecular mechanism for the multiple isotype expression of the immunoglobulin gene. J Exp Med. 1991 Jun 1;173(6):1385–1393. doi: 10.1084/jem.173.6.1385. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Snapper C. M., Finkelman F. D., Stefany D., Conrad D. H., Paul W. E. IL-4 induces co-expression of intrinsic membrane IgG1 and IgE by murine B cells stimulated with lipopolysaccharide. J Immunol. 1988 Jul 15;141(2):489–498. [PubMed] [Google Scholar]
  17. Tanaka T., Chu C. C., Paul W. E. An antisense oligonucleotide complementary to a sequence in I gamma 2b increases gamma 2b germline transcripts, stimulates B cell DNA synthesis, and inhibits immunoglobulin secretion. J Exp Med. 1992 Feb 1;175(2):597–607. doi: 10.1084/jem.175.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Triglia T., Peterson M. G., Kemp D. J. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucleic Acids Res. 1988 Aug 25;16(16):8186–8186. doi: 10.1093/nar/16.16.8186. [DOI] [PMC free article] [PubMed] [Google Scholar]

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