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. 1988 Feb 1;167(2):372–388. doi: 10.1084/jem.167.2.372

Rearrangement of exogenous immunoglobulin VH and DJH gene segments after retroviral transduction into immature lymphoid cell lines

PMCID: PMC2188853  PMID: 2831291

Abstract

A model substrate for the joining of Ig VH and DJH elements has been constructed in a retroviral vector carrying a selectable marker whose expression is independent of the arrangement of the resident Ig gene segments. The substrate was introduced into lymphoid and nonlymphoid cells, and site-specific recombination between the VH and DJH elements was monitored by a direct hybridization assay. Joining of the exogenous gene segments was observed in cell lines representative of three distinct stages in early B cell differentiation. Rearrangement was not observed in three cell lines derived from mature B cells, or in a fibroblastoid cell line. The VH and DJH elements were initially arranged so that the VH-DJH junction and the recombined flanking sequences could be recovered after rearrangement. By molecular cloning and nucleotide sequence determination, VH-DJH junctions formed upon rearrangement of the substrate were found to resemble closely similar junctions in functional H chain genes. The joining of VH and DJH elements was observed to be asymmetric; loss of nucleotides occurred at the coding joints, but not at the junctions between flanking sequences. Our results suggest that Ig H and L chain gene segments are joined by a common mechanism that is more active in B cell precursors than in mature B cells. These observations provide further evidence that the rearrangement of Ig gene segments occurs by a nonreciprocal recombinational mechanism. The model substrate described here is likely to be of use in defining the nucleotide sequences that mediate rearrangement and in examining the developmental specificity of this process.

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Selected References

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  1. Alessandrini A., Pierce J. H., Baltimore D., Desiderio S. V. Continuing rearrangement of immunoglobulin and T-cell receptor genes in a Ha-ras-transformed lymphoid progenitor cell line. Proc Natl Acad Sci U S A. 1987 Apr;84(7):1799–1803. doi: 10.1073/pnas.84.7.1799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alt F. W., Enea V., Bothwell A. L., Baltimore D. Activity of multiple light chain genes in murine myeloma cells producing a single, functional light chain. Cell. 1980 Aug;21(1):1–12. doi: 10.1016/0092-8674(80)90109-9. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Alt F., Rosenberg N., Lewis S., Thomas E., Baltimore D. Organization and reorganization of immunoglobulin genes in A-MULV-transformed cells: rearrangement of heavy but not light chain genes. Cell. 1981 Dec;27(2 Pt 1):381–390. doi: 10.1016/0092-8674(81)90421-9. [DOI] [PubMed] [Google Scholar]
  5. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  6. Bernard O., Gough N. M. Nucleotide sequence of immunoglobulin heavy chain joining segments between translocated VH and mu constant regions genes. Proc Natl Acad Sci U S A. 1980 Jun;77(6):3630–3634. doi: 10.1073/pnas.77.6.3630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Blackwell T. K., Alt F. W. Site-specific recombination between immunoglobulin D and JH segments that were introduced into the genome of a murine pre-B cell line. Cell. 1984 May;37(1):105–112. doi: 10.1016/0092-8674(84)90305-2. [DOI] [PubMed] [Google Scholar]
  8. Blackwell T. K., Moore M. W., Yancopoulos G. D., Suh H., Lutzker S., Selsing E., Alt F. W. Recombination between immunoglobulin variable region gene segments is enhanced by transcription. Nature. 1986 Dec 11;324(6097):585–589. doi: 10.1038/324585a0. [DOI] [PubMed] [Google Scholar]
  9. Bothwell A. L., Paskind M., Reth M., Imanishi-Kari T., Rajewsky K., Baltimore D. Heavy chain variable region contribution to the NPb family of antibodies: somatic mutation evident in a gamma 2a variable region. Cell. 1981 Jun;24(3):625–637. doi: 10.1016/0092-8674(81)90089-1. [DOI] [PubMed] [Google Scholar]
  10. Cepko C. L., Roberts B. E., Mulligan R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell. 1984 Jul;37(3):1053–1062. doi: 10.1016/0092-8674(84)90440-9. [DOI] [PubMed] [Google Scholar]
  11. Chien Y. H., Gascoigne N. R., Kavaler J., Lee N. E., Davis M. M. Somatic recombination in a murine T-cell receptor gene. Nature. 1984 May 24;309(5966):322–326. doi: 10.1038/309322a0. [DOI] [PubMed] [Google Scholar]
  12. Colbère-Garapin F., Horodniceanu F., Kourilsky P., Garapin A. C. A new dominant hybrid selective marker for higher eukaryotic cells. J Mol Biol. 1981 Jul 25;150(1):1–14. doi: 10.1016/0022-2836(81)90321-1. [DOI] [PubMed] [Google Scholar]
  13. Davies J., Jimenez A. A new selective agent for eukaryotic cloning vectors. Am J Trop Med Hyg. 1980 Sep;29(5 Suppl):1089–1092. doi: 10.4269/ajtmh.1980.29.1089. [DOI] [PubMed] [Google Scholar]
  14. Deev S. M., Combriato G., Klobeck H. G., Zachau H. G. Reciprocal recombination products of VK-JK joining reactions in human lymphoid cell lines. Nucleic Acids Res. 1987 Jan 12;15(1):1–14. doi: 10.1093/nar/15.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Desiderio S. V., Yancopoulos G. D., Paskind M., Thomas E., Boss M. A., Landau N., Alt F. W., Baltimore D. Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature. 1984 Oct 25;311(5988):752–755. doi: 10.1038/311752a0. [DOI] [PubMed] [Google Scholar]
  16. Early P., Huang H., Davis M., Calame K., Hood L. An immunoglobulin heavy chain variable region gene is generated from three segments of DNA: VH, D and JH. Cell. 1980 Apr;19(4):981–992. doi: 10.1016/0092-8674(80)90089-6. [DOI] [PubMed] [Google Scholar]
  17. Feddersen R. M., Van Ness B. G. Double recombination of a single immunoglobulin kappa-chain allele: implications for the mechanism of rearrangement. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4793–4797. doi: 10.1073/pnas.82.14.4793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  19. Gough N. M., Bernard O. Sequences of the joining region genes for immunoglobulin heavy chains and their role in generation of antibody diversity. Proc Natl Acad Sci U S A. 1981 Jan;78(1):509–513. doi: 10.1073/pnas.78.1.509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Graham F. L., van der Eb A. J. A new technique for the assay of infectivity of human adenovirus 5 DNA. Virology. 1973 Apr;52(2):456–467. doi: 10.1016/0042-6822(73)90341-3. [DOI] [PubMed] [Google Scholar]
  21. Hamano T., Kim K. J., Leiserson W. M., Asofsky R. Establishment of B cell hybridomas with B cell surface antigens. J Immunol. 1982 Oct;129(4):1403–1406. [PubMed] [Google Scholar]
  22. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Hoffmann J. W., Steffen D., Gusella J., Tabin C., Bird S., Cowing D., Weinberg R. A. DNA methylation affecting the expression of murine leukemia proviruses. J Virol. 1982 Oct;44(1):144–157. doi: 10.1128/jvi.44.1.144-157.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Holmes K. L., Pierce J. H., Davidson W. F., Morse H. C., 3rd Murine hematopoietic cells with pre-B or pre-B/myeloid characteristics are generated by in vitro transformation with retroviruses containing fes, ras, abl, and src oncogenes. J Exp Med. 1986 Aug 1;164(2):443–457. doi: 10.1084/jem.164.2.443. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Honjo T., Habu S. Origin of immune diversity: genetic variation and selection. Annu Rev Biochem. 1985;54:803–830. doi: 10.1146/annurev.bi.54.070185.004103. [DOI] [PubMed] [Google Scholar]
  27. Höchtl J., Müller C. R., Zachau H. G. Recombined flanks of the variable and joining segments of immunoglobulin genes. Proc Natl Acad Sci U S A. 1982 Mar;79(5):1383–1387. doi: 10.1073/pnas.79.5.1383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Karn J., Matthes H. W., Gait M. J., Brenner S. A new selective phage cloning vector, lambda 2001, with sites for XbaI, BamHI, HindIII, EcoRI, SstI and XhoI. Gene. 1984 Dec;32(1-2):217–224. doi: 10.1016/0378-1119(84)90049-0. [DOI] [PubMed] [Google Scholar]
  29. Kavaler J., Davis M. M., Chien Y. Localization of a T-cell receptor diversity-region element. Nature. 1984 Aug 2;310(5976):421–423. doi: 10.1038/310421a0. [DOI] [PubMed] [Google Scholar]
  30. Kim K. J., Kanellopoulos-Langevin C., Merwin R. M., Sachs D. H., Asofsky R. Establishment and characterization of BALB/c lymphoma lines with B cell properties. J Immunol. 1979 Feb;122(2):549–554. [PubMed] [Google Scholar]
  31. Korman A. J., Frantz J. D., Strominger J. L., Mulligan R. C. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2150–2154. doi: 10.1073/pnas.84.8.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kurosawa Y., Tonegawa S. Organization, structure, and assembly of immunoglobulin heavy chain diversity DNA segments. J Exp Med. 1982 Jan 1;155(1):201–218. doi: 10.1084/jem.155.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kurosawa Y., von Boehmer H., Haas W., Sakano H., Trauneker A., Tonegawa S. Identification of D segments of immunoglobulin heavy-chain genes and their rearrangement in T lymphocytes. Nature. 1981 Apr 16;290(5807):565–570. doi: 10.1038/290565a0. [DOI] [PubMed] [Google Scholar]
  34. Lewis S., Gifford A., Baltimore D. DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes. Science. 1985 May 10;228(4700):677–685. doi: 10.1126/science.3158075. [DOI] [PubMed] [Google Scholar]
  35. Lewis S., Gifford A., Baltimore D. Joining of V kappa to J kappa gene segments in a retroviral vector introduced into lymphoid cells. 1984 Mar 29-Apr 4Nature. 308(5958):425–428. doi: 10.1038/308425a0. [DOI] [PubMed] [Google Scholar]
  36. Lewis S., Rosenberg N., Alt F., Baltimore D. Continuing kappa-gene rearrangement in a cell line transformed by Abelson murine leukemia virus. Cell. 1982 Oct;30(3):807–816. doi: 10.1016/0092-8674(82)90285-9. [DOI] [PubMed] [Google Scholar]
  37. Litman G. W., Berger L., Murphy K., Litman R., Hinds K., Erickson B. W. Immunoglobulin VH gene structure and diversity in Heterodontus, a phylogenetically primitive shark. Proc Natl Acad Sci U S A. 1985 Apr;82(7):2082–2086. doi: 10.1073/pnas.82.7.2082. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Maki R., Kearney J., Paige C., Tonegawa S. Immunoglobulin gene rearrangement in immature B cells. Science. 1980 Sep 19;209(4463):1366–1369. doi: 10.1126/science.6774416. [DOI] [PubMed] [Google Scholar]
  39. Malissen M., Minard K., Mjolsness S., Kronenberg M., Goverman J., Hunkapiller T., Prystowsky M. B., Yoshikai Y., Fitch F., Mak T. W. Mouse T cell antigen receptor: structure and organization of constant and joining gene segments encoding the beta polypeptide. Cell. 1984 Jul;37(3):1101–1110. doi: 10.1016/0092-8674(84)90444-6. [DOI] [PubMed] [Google Scholar]
  40. Mann R., Baltimore D. Varying the position of a retrovirus packaging sequence results in the encapsidation of both unspliced and spliced RNAs. J Virol. 1985 May;54(2):401–407. doi: 10.1128/jvi.54.2.401-407.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  42. Max E. E., Seidman J. G., Leder P. Sequences of five potential recombination sites encoded close to an immunoglobulin kappa constant region gene. Proc Natl Acad Sci U S A. 1979 Jul;76(7):3450–3454. doi: 10.1073/pnas.76.7.3450. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mulligan R. C., Berg P. Expression of a bacterial gene in mammalian cells. Science. 1980 Sep 19;209(4463):1422–1427. doi: 10.1126/science.6251549. [DOI] [PubMed] [Google Scholar]
  44. Parker B. A., Stark G. R. Regulation of simian virus 40 transcription: sensitive analysis of the RNA species present early in infections by virus or viral DNA. J Virol. 1979 Aug;31(2):360–369. doi: 10.1128/jvi.31.2.360-369.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Perry R. P., Kelley D. E., Coleclough C., Kearney J. F. Organization and expression of immunoglobulin genes in fetal liver hybridomas. Proc Natl Acad Sci U S A. 1981 Jan;78(1):247–251. doi: 10.1073/pnas.78.1.247. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Pierce J. H., Aaronson S. A. BALB- and Harvey-murine sarcoma virus transformation of a novel lymphoid progenitor cell. J Exp Med. 1982 Sep 1;156(3):873–887. doi: 10.1084/jem.156.3.873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Pratt D., Subramani S. Nucleotide sequence of the Escherichia coli xanthine-guanine phosphoribosyl transferase gene. Nucleic Acids Res. 1983 Dec 20;11(24):8817–8823. doi: 10.1093/nar/11.24.8817. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Richardson K. K., Fostel J., Skopek T. R. Nucleotide sequence of the xanthine guanine phosphoribosyl transferase gene of E. coli. Nucleic Acids Res. 1983 Dec 20;11(24):8809–8816. doi: 10.1093/nar/11.24.8809. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Sakano H., Hüppi K., Heinrich G., Tonegawa S. Sequences at the somatic recombination sites of immunoglobulin light-chain genes. Nature. 1979 Jul 26;280(5720):288–294. doi: 10.1038/280288a0. [DOI] [PubMed] [Google Scholar]
  50. 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]
  51. Selsing E., Voss J., Storb U. Immunoglobulin gene 'remnant' DNA--implications for antibody gene recombination. Nucleic Acids Res. 1984 May 25;12(10):4229–4246. doi: 10.1093/nar/12.10.4229. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  53. Siu G., Kronenberg M., Strauss E., Haars R., Mak T. W., Hood L. The structure, rearrangement and expression of D beta gene segments of the murine T-cell antigen receptor. 1984 Sep 27-Oct 3Nature. 311(5984):344–350. doi: 10.1038/311344a0. [DOI] [PubMed] [Google Scholar]
  54. Steffen D., Weinberg R. A. The integrated genome of murine leukemia virus. Cell. 1978 Nov;15(3):1003–1010. doi: 10.1016/0092-8674(78)90284-2. [DOI] [PubMed] [Google Scholar]
  55. Steinmetz M., Altenburger W., Zachau H. G. A rearranged DNA sequence possibly related to the translocation of immunoglobulin gene segments. Nucleic Acids Res. 1980 Apr 25;8(8):1709–1720. doi: 10.1093/nar/8.8.1709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  57. Yancopoulos G. D., Alt F. W. Developmentally controlled and tissue-specific expression of unrearranged VH gene segments. Cell. 1985 Feb;40(2):271–281. doi: 10.1016/0092-8674(85)90141-2. [DOI] [PubMed] [Google Scholar]
  58. Yancopoulos G. D., Blackwell T. K., Suh H., Hood L., Alt F. W. Introduced T cell receptor variable region gene segments recombine in pre-B cells: evidence that B and T cells use a common recombinase. Cell. 1986 Jan 31;44(2):251–259. doi: 10.1016/0092-8674(86)90759-2. [DOI] [PubMed] [Google Scholar]
  59. Yancopoulos G. D., Desiderio S. V., Paskind M., Kearney J. F., Baltimore D., Alt F. W. Preferential utilization of the most JH-proximal VH gene segments in pre-B-cell lines. Nature. 1984 Oct 25;311(5988):727–733. doi: 10.1038/311727a0. [DOI] [PubMed] [Google Scholar]

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