Skip to main content
NCBI home page
The Journal of Experimental Medicine logoLink to The Journal of Experimental Medicine
. 1988 Mar 1;167(3):954–973. doi: 10.1084/jem.167.3.954

Point mutations cause the somatic diversification of IgM and IgG2a antiphosphorylcholine antibodies [published erratum appears in J Exp Med 1990 Aug 1;172(2):669]

PMCID: PMC2188873  PMID: 3127529

Abstract

The genetic mechanism responsible for the somatic diversification of two mAbs was determined. The two PC-binding hybridomas were representative of events early and late in the immune response. The P28 cell line that produces an IgM antibody and thus represents events early in the immune response, was found to have 3 bp changes in its heavy chain variable (VH) region, with some changes in antibody affinity or specificity. The RP93 cell line that produces an IgG2a antibody and thus represents later events in the immune response, was found to have 9 bp changes in its VH region resulting in decreased affinity for PC and altered specificity. Oligonucleotides specific for linked base changes in the second hypervariable regions of both of these antibodies were used to look for previously undescribed V regions or other donor sequences that could have been responsible for these base changes. Since no donor sequences were found, we have concluded that somatic point mutation rather than gene conversion, V region replacement or the expression of an unidentified germline VH region gene is truly responsible for at least some of the somatic diversification of these antibodies.

Full Text

The Full Text of this article is available as a PDF (1.7 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alt F. W. Antibody diversity. New mechanism revealed. 1986 Aug 28-Sep 3Nature. 322(6082):772–773. doi: 10.1038/322772a0. [DOI] [PubMed] [Google Scholar]
  2. Alt F. W., Yancopoulos G. D., Blackwell T. K., Wood C., Thomas E., Boss M., Coffman R., Rosenberg N., Tonegawa S., Baltimore D. Ordered rearrangement of immunoglobulin heavy chain variable region segments. EMBO J. 1984 Jun;3(6):1209–1219. doi: 10.1002/j.1460-2075.1984.tb01955.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Amit A. G., Mariuzza R. A., Phillips S. E., Poljak R. J. Three-dimensional structure of an antigen-antibody complex at 2.8 A resolution. Science. 1986 Aug 15;233(4765):747–753. doi: 10.1126/science.2426778. [DOI] [PubMed] [Google Scholar]
  4. Ayares D., Chekuri L., Song K. Y., Kucherlapati R. Sequence homology requirements for intermolecular recombination in mammalian cells. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5199–5203. doi: 10.1073/pnas.83.14.5199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berek C., Milstein C. Mutation drift and repertoire shift in the maturation of the immune response. Immunol Rev. 1987 Apr;96:23–41. doi: 10.1111/j.1600-065x.1987.tb00507.x. [DOI] [PubMed] [Google Scholar]
  6. Berek C., Schreier M. H., Sidman C. L., Jaton J. C., Kocher H. P., Cosenza H. Phosphorylcholine-binding hybridoma proteins of normal and idiotypically suppressed BALB/c mice I. Characterization and idiotypic analysis. Eur J Immunol. 1980 Apr;10(4):258–263. doi: 10.1002/eji.1830100407. [DOI] [PubMed] [Google Scholar]
  7. Bothwell M. A., Howlett G. J., Schachman H. K. A sedimentation equilibrium method for determining molecular weights of proteins with a tabletop high speed air turbine centrifuge. J Biol Chem. 1978 Apr 10;253(7):2073–2077. [PubMed] [Google Scholar]
  8. Chang S. P., Perlmutter R. M., Brown M., Heusser C. H., Hood L., Rittenberg M. B. Immunologic memory to phosphocholine. IV. Hybridomas representative of Group I (T15-like) and Group II (non-T15-like) antibodies utilize distinct VH genes. J Immunol. 1984 Mar;132(3):1550–1555. [PubMed] [Google Scholar]
  9. Chesebro B., Metzger H. Affinity labeling of a phosphorylcholine binding mouse myeloma protein. Biochemistry. 1972 Feb 29;11(5):766–771. doi: 10.1021/bi00755a014. [DOI] [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Claflin J. L., Davie J. M. Clonal nature of the immune response to phosphorylcholine. IV. Idiotypic uniformity of binding site-associated antigenic determinants among mouse antiphosphorylcholine antibodies. J Exp Med. 1974 Sep 1;140(3):673–686. doi: 10.1084/jem.140.3.673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clarke S. H., Huppi K., Ruezinsky D., Staudt L., Gerhard W., Weigert M. Inter- and intraclonal diversity in the antibody response to influenza hemagglutinin. J Exp Med. 1985 Apr 1;161(4):687–704. doi: 10.1084/jem.161.4.687. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Clarke S. H., Kenny J. J., Sieckmann D. G., Rudikoff S. Amino acid sequence of a phosphocholine-binding antibody from an immune defective CBA/N mouse employing the T15 VH region associated with unusual DH, JH, and V kappa segments. J Immunol. 1984 Mar;132(3):1544–1549. [PubMed] [Google Scholar]
  14. Crews S., Griffin J., Huang H., Calame K., Hood L. A single VH gene segment encodes the immune response to phosphorylcholine: somatic mutation is correlated with the class of the antibody. Cell. 1981 Jul;25(1):59–66. doi: 10.1016/0092-8674(81)90231-2. [DOI] [PubMed] [Google Scholar]
  15. Desaymard C., Giusti A. M., Scharff M. D. Rat anti-T15 monoclonal antibodies with specificity for VH- and VH-VL epitopes. Mol Immunol. 1984 Oct;21(10):961–967. doi: 10.1016/0161-5890(84)90154-8. [DOI] [PubMed] [Google Scholar]
  16. Diamond B., Scharff M. D. Somatic mutation of the T15 heavy chain gives rise to an antibody with autoantibody specificity. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5841–5844. doi: 10.1073/pnas.81.18.5841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gearhart P. J., Bogenhagen D. F. Clusters of point mutations are found exclusively around rearranged antibody variable genes. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3439–3443. doi: 10.1073/pnas.80.11.3439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Geliebter J., Zeff R. A., Melvold R. W., Nathenson S. G. Mitotic recombination in germ cells generated two major histocompatibility complex mutant genes shown to be identical by RNA sequence analysis: Kbm9 and Kbm6. Proc Natl Acad Sci U S A. 1986 May;83(10):3371–3375. doi: 10.1073/pnas.83.10.3371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Griffiths G. M., Berek C., Kaartinen M., Milstein C. Somatic mutation and the maturation of immune response to 2-phenyl oxazolone. Nature. 1984 Nov 15;312(5991):271–275. doi: 10.1038/312271a0. [DOI] [PubMed] [Google Scholar]
  20. Hartman A. B., Rudikoff S. VH genes encoding the immune response to beta-(1,6)-galactan: somatic mutation in IgM molecules. EMBO J. 1984 Dec 1;3(12):3023–3030. doi: 10.1002/j.1460-2075.1984.tb02249.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Hieter P. A., Max E. E., Seidman J. G., Maizel J. V., Jr, Leder P. Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments. Cell. 1980 Nov;22(1 Pt 1):197–207. doi: 10.1016/0092-8674(80)90168-3. [DOI] [PubMed] [Google Scholar]
  22. Howlett G. J., Markov G. Ligand-affinity sedimentation equilibrium using an air-driven ultracentrifuge. Arch Biochem Biophys. 1980 Jul;202(2):507–514. doi: 10.1016/0003-9861(80)90456-7. [DOI] [PubMed] [Google Scholar]
  23. Jackson D. C., Howlett G. J., Nestorowicz A., Webster R. G. The equilibrium constant for the interaction between a monoclonal Fab fragment and an influenza virus neuraminidase. J Immunol. 1983 Mar;130(3):1313–1316. [PubMed] [Google Scholar]
  24. Kawakami T., Takahashi N., Honjo T. Complete nucleotide sequence of mouse immunoglobulin mu gene and comparison with other immunoglobulin heavy chain genes. Nucleic Acids Res. 1980 Sep 11;8(17):3933–3945. doi: 10.1093/nar/8.17.3933. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kim S., Davis M., Sinn E., Patten P., Hood L. Antibody diversity: somatic hypermutation of rearranged VH genes. Cell. 1981 Dec;27(3 Pt 2):573–581. doi: 10.1016/0092-8674(81)90399-8. [DOI] [PubMed] [Google Scholar]
  26. Kleinfield R., Hardy R. R., Tarlinton D., Dangl J., Herzenberg L. A., Weigert M. Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma. 1986 Aug 28-Sep 3Nature. 322(6082):843–846. doi: 10.1038/322843a0. [DOI] [PubMed] [Google Scholar]
  27. Kocher H. P., Berek C., Jaton J. C. The immune response of BALB/c mice to phosphorylcholine is restricted to a limited number of VH- and VL-isotypes. Mol Immunol. 1981 Dec;18(12):1027–1033. doi: 10.1016/0161-5890(81)90018-3. [DOI] [PubMed] [Google Scholar]
  28. Kocher H. P., Berek C., Schreier M. H., Cosenza H., Jaton J. C. Phosphorylcholine-binding hybridoma proteins of normal and idiotypically suppressed BALB/c mice II. Variable region N-terminal amino acid sequences. Eur J Immunol. 1980 Apr;10(4):264–267. doi: 10.1002/eji.1830100408. [DOI] [PubMed] [Google Scholar]
  29. Krawinkel U., Zoebelein G., Brüggemann M., Radbruch A., Rajewsky K. Recombination between antibody heavy chain variable-region genes: evidence for gene conversion. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4997–5001. doi: 10.1073/pnas.80.16.4997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kwan S. P., Max E. E., Seidman J. G., Leder P., Scharff M. D. Two kappa immunoglobulin genes are expressed in the myeloma S107. Cell. 1981 Oct;26(1 Pt 1):57–66. doi: 10.1016/0092-8674(81)90033-7. [DOI] [PubMed] [Google Scholar]
  31. Livant D., Blatt C., Hood L. One heavy chain variable region gene segment subfamily in the BALB/c mouse contains 500-1000 or more members. Cell. 1986 Nov 7;47(3):461–470. doi: 10.1016/0092-8674(86)90603-3. [DOI] [PubMed] [Google Scholar]
  32. Malipiero U. V., Levy N. S., Gearhart P. J. Somatic mutation in anti-phosphorylcholine antibodies. Immunol Rev. 1987 Apr;96:59–74. doi: 10.1111/j.1600-065x.1987.tb00509.x. [DOI] [PubMed] [Google Scholar]
  33. Manser T., Wysocki L. J., Margolies M. N., Gefter M. L. Evolution of antibody variable region structure during the immune response. Immunol Rev. 1987 Apr;96:141–162. doi: 10.1111/j.1600-065x.1987.tb00513.x. [DOI] [PubMed] [Google Scholar]
  34. Matthyssens G., Rabbitts T. H. The sequence at the 3' terminus of mouse immunoglobulin secreted mu chain messenger RNA determined from cloned cDNA. Nucleic Acids Res. 1980 Feb 25;8(4):703–713. [PMC free article] [PubMed] [Google Scholar]
  35. Max E. E., Maizel J. V., Jr, Leder P. The nucleotide sequence of a 5.5-kilobase DNA segment containing the mouse kappa immunoglobulin J and C region genes. J Biol Chem. 1981 May 25;256(10):5116–5120. [PubMed] [Google Scholar]
  36. Newell N., Richards J. E., Tucker P. W., Blattner F. R. J genes for heavy chain immunoglobulins of mouse. Science. 1980 Sep 5;209(4461):1128–1132. doi: 10.1126/science.6250219. [DOI] [PubMed] [Google Scholar]
  37. Perlmutter R. M., Crews S. T., Douglas R., Sorensen G., Johnson N., Nivera N., Gearhart P. J., Hood L. The generation of diversity in phosphorylcholine-binding antibodies. Adv Immunol. 1984;35:1–37. doi: 10.1016/s0065-2776(08)60572-6. [DOI] [PubMed] [Google Scholar]
  38. Potter M., Pumphrey J. G., Walters J. L. Growth of primary plasmacytomas in the mineral oil-conditioned peritoneal environment. J Natl Cancer Inst. 1972 Jul;49(1):305–308. [PubMed] [Google Scholar]
  39. Primi D., Barbier E., Drapier A. M., Cazenave P. A. Paucity of phosphorylcholine-specific clones in B cells expressing the VHT15 gene product. J Exp Med. 1986 Dec 1;164(6):2107–2112. doi: 10.1084/jem.164.6.2107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Primi D., Cazenave P. A. Lack of expression of the VHS107 gene family in the lipopolysaccharide-sensitive B cell subset of X-linked immunodeficiency-defective mice. J Exp Med. 1986 Jul 1;164(1):357–362. doi: 10.1084/jem.164.1.357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Primi D., Drapier A. M., Cazenave P. A. Highly preferential VH-VL pairing in normal B cells results in antigen-independent selection of the available repertoire. J Immunol. 1987 Mar 1;138(5):1607–1612. [PubMed] [Google Scholar]
  42. Reth M., Gehrmann P., Petrac E., Wiese P. A novel VH to VHDJH joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy-chain production. 1986 Aug 28-Sep 3Nature. 322(6082):840–842. doi: 10.1038/322840a0. [DOI] [PubMed] [Google Scholar]
  43. Reynaud C. A., Anquez V., Grimal H., Weill J. C. A hyperconversion mechanism generates the chicken light chain preimmune repertoire. Cell. 1987 Feb 13;48(3):379–388. doi: 10.1016/0092-8674(87)90189-9. [DOI] [PubMed] [Google Scholar]
  44. Rodwell J. D., Gearhart P. J., Karush F. Restriction in IgM expression. IV. Affinity analysis of monoclonal anti-phosphorylcholine antibodies. J Immunol. 1983 Jan;130(1):313–316. [PubMed] [Google Scholar]
  45. Rudikoff S., Giusti A. M., Cook W. D., Scharff M. D. Single amino acid substitution altering antigen-binding specificity. Proc Natl Acad Sci U S A. 1982 Mar;79(6):1979–1983. doi: 10.1073/pnas.79.6.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sakano H., Maki R., Kurosawa Y., Roeder W., Tonegawa S. Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature. 1980 Aug 14;286(5774):676–683. doi: 10.1038/286676a0. [DOI] [PubMed] [Google Scholar]
  47. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Selsing E., Storb U. Somatic mutation of immunoglobulin light-chain variable-region genes. Cell. 1981 Jul;25(1):47–58. doi: 10.1016/0092-8674(81)90230-0. [DOI] [PubMed] [Google Scholar]
  49. Spira G., Bargellesi A., Teillaud J. L., Scharff M. D. The identification of monoclonal class switch variants by sib selection and an ELISA assay. J Immunol Methods. 1984 Nov 30;74(2):307–315. doi: 10.1016/0022-1759(84)90298-9. [DOI] [PubMed] [Google Scholar]
  50. Thammana P., Scharff M. D. Immunoglobulin heavy chain class switch from IgM to IgG in a hybridoma. Eur J Immunol. 1983 Aug;13(8):614–619. doi: 10.1002/eji.1830130803. [DOI] [PubMed] [Google Scholar]
  51. Todd I., Chang S. P., Perlmutter R. M., Aebersold R., Heusser C. H., Hood L., Rittenberg M. B. Immunologic memory to phosphocholine. V. Hybridomas representative of group II antibodies utilize V kappa 1-3 gene(s). J Immunol. 1984 Mar;132(3):1556–1560. [PubMed] [Google Scholar]
  52. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  53. Weigert M. G., Cesari I. M., Yonkovich S. J., Cohn M. Variability in the lambda light chain sequences of mouse antibody. Nature. 1970 Dec 12;228(5276):1045–1047. doi: 10.1038/2281045a0. [DOI] [PubMed] [Google Scholar]
  54. Wittner M. K., Bach M. A., Köhler H. Immune response to phosphorylcholine. IX. Characterization of hybridoma anti-TEPC15 antibodies. J Immunol. 1982 Feb;128(2):595–599. [PubMed] [Google Scholar]
  55. Yamawaki-Kataoka Y., Miyata T., Honjo T. The complete nucleotide sequence of mouse immunoglobin gamma 2a gene and evolution of heavy chain genes: further evidence for intervening sequence-mediated domain transfer. Nucleic Acids Res. 1981 Mar 25;9(6):1365–1381. doi: 10.1093/nar/9.6.1365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Zack D. J., Morrison S. L., Cook W. D., Dackowski W., Scharff M. D. Somatically generated mouse myeloma variants synthesizing IgA half-molecules. J Exp Med. 1981 Nov 1;154(5):1554–1569. doi: 10.1084/jem.154.5.1554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zeff R. A., Gopas J., Steinhauer E., Rajan T. V., Nathenson S. G. Analysis of somatic cell H-2 variants to define the structural requirements for class I antigen expression. J Immunol. 1986 Aug 1;137(3):897–903. [PubMed] [Google Scholar]

Articles from The Journal of Experimental Medicine are provided here courtesy of The Rockefeller University Press

RESOURCES