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. 1996 Feb;16(2):609–618. doi: 10.1128/mcb.16.2.609

T-cell receptor alpha locus V(D)J recombination by-products are abundant in thymocytes and mature T cells.

F Livak 1, D G Schatz 1
PMCID: PMC231040  PMID: 8552089

Abstract

In addition to the assembled coding regions of immunoglobulin and T-cell receptor (TCR) genes, the V(D)J recombination reaction can in principle generate three types of by-products in normal developing lymphocytes: broken DNA molecules that terminate in a recombination signal sequence or a coding region (termed signal or coding end molecules, respectively) and DNA molecules containing fused recombination signal sequences (termed reciprocal products). Using a quantitative Southern blot analysis of the murine TCR alpha locus, we demonstrate that substantial amounts of signal end molecules and reciprocal products, but not coding end molecules, exist in thymocytes, while peripheral T cells contain substantial amounts of reciprocal products. At the 5' end of the J alpha locus, 20% of thymus DNA exists as signal end molecules. An additional 30 to 40% of the TCR alpha/delta locus exists as remarkably stable reciprocal products throughout T-cell development, with the consequence that the TCR C delta region is substantially retained in alpha beta committed T cells. The disappearance of the broken DNA molecules occurs in the same developmental transition as termination of expression of the recombination activating genes, RAG-1 and RAG-2. These findings raise important questions concerning the mechanism of V(D)J recombination and the maintenance of genome integrity during lymphoid development.

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Selected References

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  1. Alt F. W., Oltz E. M., Young F., Gorman J., Taccioli G., Chen J. VDJ recombination. Immunol Today. 1992 Aug;13(8):306–314. doi: 10.1016/0167-5699(92)90043-7. [DOI] [PubMed] [Google Scholar]
  2. Blunt T., Finnie N. J., Taccioli G. E., Smith G. C., Demengeot J., Gottlieb T. M., Mizuta R., Varghese A. J., Alt F. W., Jeggo P. A. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell. 1995 Mar 10;80(5):813–823. doi: 10.1016/0092-8674(95)90360-7. [DOI] [PubMed] [Google Scholar]
  3. Borgulya P., Kishi H., Uematsu Y., von Boehmer H. Exclusion and inclusion of alpha and beta T cell receptor alleles. Cell. 1992 May 1;69(3):529–537. doi: 10.1016/0092-8674(92)90453-j. [DOI] [PubMed] [Google Scholar]
  4. Chien Y. H., Iwashima M., Kaplan K. B., Elliott J. F., Davis M. M. A new T-cell receptor gene located within the alpha locus and expressed early in T-cell differentiation. 1987 Jun 25-Jul 1Nature. 327(6124):677–682. doi: 10.1038/327677a0. [DOI] [PubMed] [Google Scholar]
  5. Danska J. S., Pflumio F., Williams C. J., Huner O., Dick J. E., Guidos C. J. Rescue of T cell-specific V(D)J recombination in SCID mice by DNA-damaging agents. Science. 1994 Oct 21;266(5184):450–455. doi: 10.1126/science.7524150. [DOI] [PubMed] [Google Scholar]
  6. Dudley E. C., Petrie H. T., Shah L. M., Owen M. J., Hayday A. C. T cell receptor beta chain gene rearrangement and selection during thymocyte development in adult mice. Immunity. 1994 May;1(2):83–93. doi: 10.1016/1074-7613(94)90102-3. [DOI] [PubMed] [Google Scholar]
  7. Egerton M., Scollay R., Shortman K. Kinetics of mature T-cell development in the thymus. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2579–2582. doi: 10.1073/pnas.87.7.2579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elliott J. F., Rock E. P., Patten P. A., Davis M. M., Chien Y. H. The adult T-cell receptor delta-chain is diverse and distinct from that of fetal thymocytes. Nature. 1988 Feb 18;331(6157):627–631. doi: 10.1038/331627a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Fujimoto S., Yamagishi H. Isolation of an excision product of T-cell receptor alpha-chain gene rearrangements. Nature. 1987 May 21;327(6119):242–243. doi: 10.1038/327242a0. [DOI] [PubMed] [Google Scholar]
  11. Hesse J. E., Lieber M. R., Mizuuchi K., Gellert M. V(D)J recombination: a functional definition of the joining signals. Genes Dev. 1989 Jul;3(7):1053–1061. doi: 10.1101/gad.3.7.1053. [DOI] [PubMed] [Google Scholar]
  12. Holman P. O., Roth M. E., Huang M., Kranz D. M. Characterization of transcripts from unrearranged V alpha 8 genes in the thymus. J Immunol. 1993 Aug 15;151(4):1959–1967. [PubMed] [Google Scholar]
  13. Hong S. C., Chelouche A., Lin R. H., Shaywitz D., Braunstein N. S., Glimcher L., Janeway C. A., Jr An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain. Cell. 1992 Jun 12;69(6):999–1009. doi: 10.1016/0092-8674(92)90618-m. [DOI] [PubMed] [Google Scholar]
  14. Jameson S. C., Nakajima P. B., Brooks J. L., Heath W., Kanagawa O., Gascoigne N. R. The T cell receptor V alpha 11 gene family. Analysis of allelic sequence polymorphism and demonstration of J alpha region-dependent recognition by allele-specific antibodies. J Immunol. 1991 Nov 1;147(9):3185–3193. [PubMed] [Google Scholar]
  15. Jouvin-Marche E., Hue I., Marche P. N., Liebe-Gris C., Marolleau J. P., Malissen B., Cazenave P. A., Malissen M. Genomic organization of the mouse T cell receptor V alpha family. EMBO J. 1990 Jul;9(7):2141–2150. doi: 10.1002/j.1460-2075.1990.tb07383.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kirchgessner C. U., Patil C. K., Evans J. W., Cuomo C. A., Fried L. M., Carter T., Oettinger M. A., Brown J. M. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science. 1995 Feb 24;267(5201):1178–1183. doi: 10.1126/science.7855601. [DOI] [PubMed] [Google Scholar]
  17. Kitamura D., Roes J., Kühn R., Rajewsky K. A B cell-deficient mouse by targeted disruption of the membrane exon of the immunoglobulin mu chain gene. Nature. 1991 Apr 4;350(6317):423–426. doi: 10.1038/350423a0. [DOI] [PubMed] [Google Scholar]
  18. Koop B. F., Wilson R. K., Wang K., Vernooij B., Zallwer D., Kuo C. L., Seto D., Toda M., Hood L. Organization, structure, and function of 95 kb of DNA spanning the murine T-cell receptor C alpha/C delta region. Genomics. 1992 Aug;13(4):1209–1230. doi: 10.1016/0888-7543(92)90039-u. [DOI] [PubMed] [Google Scholar]
  19. Landau N. R., Schatz D. G., Rosa M., Baltimore D. Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol Cell Biol. 1987 Sep;7(9):3237–3243. doi: 10.1128/mcb.7.9.3237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Leu T. M., Schatz D. G. rag-1 and rag-2 are components of a high-molecular-weight complex, and association of rag-2 with this complex is rag-1 dependent. Mol Cell Biol. 1995 Oct;15(10):5657–5670. doi: 10.1128/mcb.15.10.5657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lewis S. M. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparative analyses. Adv Immunol. 1994;56:27–150. doi: 10.1016/s0065-2776(08)60450-2. [DOI] [PubMed] [Google Scholar]
  22. Lin W. C., Desiderio S. Cell cycle regulation of V(D)J recombination-activating protein RAG-2. Proc Natl Acad Sci U S A. 1994 Mar 29;91(7):2733–2737. doi: 10.1073/pnas.91.7.2733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lindsten T., Fowlkes B. J., Samelson L. E., Davis M. M., Chien Y. H. Transient rearrangements of the T cell antigen receptor alpha locus in early thymocytes. J Exp Med. 1987 Sep 1;166(3):761–775. doi: 10.1084/jem.166.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Livak F., Petrie H. T., Crispe I. N., Schatz D. G. In-frame TCR delta gene rearrangements play a critical role in the alpha beta/gamma delta T cell lineage decision. Immunity. 1995 Jun;2(6):617–627. doi: 10.1016/1074-7613(95)90006-3. [DOI] [PubMed] [Google Scholar]
  25. Malissen M., Trucy J., Jouvin-Marche E., Cazenave P. A., Scollay R., Malissen B. Regulation of TCR alpha and beta gene allelic exclusion during T-cell development. Immunol Today. 1992 Aug;13(8):315–322. doi: 10.1016/0167-5699(92)90044-8. [DOI] [PubMed] [Google Scholar]
  26. Mombaerts P., Anderson S. J., Perlmutter R. M., Mak T. W., Tonegawa S. An activated lck transgene promotes thymocyte development in RAG-1 mutant mice. Immunity. 1994 Jul;1(4):261–267. doi: 10.1016/1074-7613(94)90077-9. [DOI] [PubMed] [Google Scholar]
  27. Mombaerts P., Clarke A. R., Rudnicki M. A., Iacomini J., Itohara S., Lafaille J. J., Wang L., Ichikawa Y., Jaenisch R., Hooper M. L. Mutations in T-cell antigen receptor genes alpha and beta block thymocyte development at different stages. Nature. 1992 Nov 19;360(6401):225–231. doi: 10.1038/360225a0. [DOI] [PubMed] [Google Scholar]
  28. Mombaerts P., Iacomini J., Johnson R. S., Herrup K., Tonegawa S., Papaioannou V. E. RAG-1-deficient mice have no mature B and T lymphocytes. Cell. 1992 Mar 6;68(5):869–877. doi: 10.1016/0092-8674(92)90030-g. [DOI] [PubMed] [Google Scholar]
  29. Oettinger M. A., Schatz D. G., Gorka C., Baltimore D. RAG-1 and RAG-2, adjacent genes that synergistically activate V(D)J recombination. Science. 1990 Jun 22;248(4962):1517–1523. doi: 10.1126/science.2360047. [DOI] [PubMed] [Google Scholar]
  30. Okazaki K., Davis D. D., Sakano H. T cell receptor beta gene sequences in the circular DNA of thymocyte nuclei: direct evidence for intramolecular DNA deletion in V-D-J joining. Cell. 1987 May 22;49(4):477–485. doi: 10.1016/0092-8674(87)90450-8. [DOI] [PubMed] [Google Scholar]
  31. Okazaki K., Sakano H. Thymocyte circular DNA excised from T cell receptor alpha-delta gene complex. EMBO J. 1988 Jun;7(6):1669–1674. doi: 10.1002/j.1460-2075.1988.tb02994.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Petrie H. T., Livak F., Burtrum D., Mazel S. T cell receptor gene recombination patterns and mechanisms: cell death, rescue, and T cell production. J Exp Med. 1995 Jul 1;182(1):121–127. doi: 10.1084/jem.182.1.121. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Petrie H. T., Livak F., Schatz D. G., Strasser A., Crispe I. N., Shortman K. Multiple rearrangements in T cell receptor alpha chain genes maximize the production of useful thymocytes. J Exp Med. 1993 Aug 1;178(2):615–622. doi: 10.1084/jem.178.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rabbitts T. H. Chromosomal translocations in human cancer. Nature. 1994 Nov 10;372(6502):143–149. doi: 10.1038/372143a0. [DOI] [PubMed] [Google Scholar]
  35. Raulet D. H., Spencer D. M., Hsiang Y. H., Goldman J. P., Bix M., Liao N. S., Zijstra M., Jaenisch R., Correa I. Control of gamma delta T-cell development. Immunol Rev. 1991 Apr;120:185–204. doi: 10.1111/j.1600-065x.1991.tb00592.x. [DOI] [PubMed] [Google Scholar]
  36. Roth D. B., Menetski J. P., Nakajima P. B., Bosma M. J., Gellert M. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell. 1992 Sep 18;70(6):983–991. doi: 10.1016/0092-8674(92)90248-b. [DOI] [PubMed] [Google Scholar]
  37. Roth D. B., Nakajima P. B., Menetski J. P., Bosma M. J., Gellert M. V(D)J recombination in mouse thymocytes: double-strand breaks near T cell receptor delta rearrangement signals. Cell. 1992 Apr 3;69(1):41–53. doi: 10.1016/0092-8674(92)90117-u. [DOI] [PubMed] [Google Scholar]
  38. Roth D. B., Zhu C., Gellert M. Characterization of broken DNA molecules associated with V(D)J recombination. Proc Natl Acad Sci U S A. 1993 Nov 15;90(22):10788–10792. doi: 10.1073/pnas.90.22.10788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schatz D. G., Oettinger M. A., Baltimore D. The V(D)J recombination activating gene, RAG-1. Cell. 1989 Dec 22;59(6):1035–1048. doi: 10.1016/0092-8674(89)90760-5. [DOI] [PubMed] [Google Scholar]
  40. Schlissel M., Constantinescu A., Morrow T., Baxter M., Peng A. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5'-phosphorylated, RAG-dependent, and cell cycle regulated. Genes Dev. 1993 Dec;7(12B):2520–2532. doi: 10.1101/gad.7.12b.2520. [DOI] [PubMed] [Google Scholar]
  41. Shinkai Y., Rathbun G., Lam K. P., Oltz E. M., Stewart V., Mendelsohn M., Charron J., Datta M., Young F., Stall A. M. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement. Cell. 1992 Mar 6;68(5):855–867. doi: 10.1016/0092-8674(92)90029-c. [DOI] [PubMed] [Google Scholar]
  42. Taccioli G. E., Gottlieb T. M., Blunt T., Priestley A., Demengeot J., Mizuta R., Lehmann A. R., Alt F. W., Jackson S. P., Jeggo P. A. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science. 1994 Sep 2;265(5177):1442–1445. doi: 10.1126/science.8073286. [DOI] [PubMed] [Google Scholar]
  43. Takeshita S., Toda M., Yamagishi H. Excision products of the T cell receptor gene support a progressive rearrangement model of the alpha/delta locus. EMBO J. 1989 Nov;8(11):3261–3270. doi: 10.1002/j.1460-2075.1989.tb08486.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Thompson S. D., Manzo A. R., Pelkonen J., Larche M., Hurwitz J. L. Developmental T cell receptor gene rearrangements: relatedness of the alpha/beta and gamma/delta T cell precursor. Eur J Immunol. 1991 Aug;21(8):1939–1950. doi: 10.1002/eji.1830210824. [DOI] [PubMed] [Google Scholar]
  45. Thompson S. D., Pelkonen J., Hurwitz J. L. First T cell receptor alpha gene rearrangements during T cell ontogeny skew to the 5' region of the J alpha locus. J Immunol. 1990 Oct 1;145(7):2347–2352. [PubMed] [Google Scholar]
  46. Tonegawa S. Somatic generation of antibody diversity. Nature. 1983 Apr 14;302(5909):575–581. doi: 10.1038/302575a0. [DOI] [PubMed] [Google Scholar]
  47. Tough D. F., Sprent J. Turnover of naive- and memory-phenotype T cells. J Exp Med. 1994 Apr 1;179(4):1127–1135. doi: 10.1084/jem.179.4.1127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wang K., Klotz J. L., Kiser G., Bristol G., Hays E., Lai E., Gese E., Kronenberg M., Hood L. Organization of the V gene segments in mouse T-cell antigen receptor alpha/delta locus. Genomics. 1994 Apr;20(3):419–428. doi: 10.1006/geno.1994.1196. [DOI] [PubMed] [Google Scholar]
  49. Wilson R. K., Koop B. F., Chen C., Halloran N., Sciammis R., Hood L. Nucleotide sequence analysis of 95 kb near the 3' end of the murine T-cell receptor alpha/delta chain locus: strategy and methodology. Genomics. 1992 Aug;13(4):1198–1208. doi: 10.1016/0888-7543(92)90038-t. [DOI] [PubMed] [Google Scholar]
  50. Winoto A., Baltimore D. Separate lineages of T cells expressing the alpha beta and gamma delta receptors. Nature. 1989 Mar 30;338(6214):430–432. doi: 10.1038/338430a0. [DOI] [PubMed] [Google Scholar]
  51. Woffendin C., Yang Z. Y., Udaykumar, Xu L., Yang N. S., Sheehy M. J., Nabel G. J. Nonviral and viral delivery of a human immunodeficiency virus protective gene into primary human T cells. Proc Natl Acad Sci U S A. 1994 Nov 22;91(24):11581–11585. doi: 10.1073/pnas.91.24.11581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wysocki L. J., Sato V. L. "Panning" for lymphocytes: a method for cell selection. Proc Natl Acad Sci U S A. 1978 Jun;75(6):2844–2848. doi: 10.1073/pnas.75.6.2844. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhu C., Roth D. B. Characterization of coding ends in thymocytes of scid mice: implications for the mechanism of V(D)J recombination. Immunity. 1995 Jan;2(1):101–112. doi: 10.1016/1074-7613(95)90082-9. [DOI] [PubMed] [Google Scholar]
  54. de Villartay J. P., Hockett R. D., Coran D., Korsmeyer S. J., Cohen D. I. Deletion of the human T-cell receptor delta-gene by a site-specific recombination. Nature. 1988 Sep 8;335(6186):170–174. doi: 10.1038/335170a0. [DOI] [PubMed] [Google Scholar]
  55. van Gent D. C., McBlane J. F., Ramsden D. A., Sadofsky M. J., Hesse J. E., Gellert M. Initiation of V(D)J recombination in a cell-free system. Cell. 1995 Jun 16;81(6):925–934. doi: 10.1016/0092-8674(95)90012-8. [DOI] [PubMed] [Google Scholar]

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