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The V-J recombination of T cell receptor-gamma genes is blocked in interleukin-7 receptor-deficient mice.

Maki K, Sunaga S, Ikuta K - J. Exp. Med. (1996)

Bottom Line: PCR analysis indicated that the V-J recombination of all the V gamma genes is severely hampered in the mutant mice.The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected.These data demonstrated that the V-J recombination of the TCR gamma genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma gene rearrangement.

View Article: PubMed Central - PubMed

Affiliation: Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, University of Tokyo, Japan.

ABSTRACT
IL-7R-deficient mice have severely impaired expansion of early lymphocytes and lack gamma delta T cells. To elucidate the role of IL-7R on gamma delta T cell development, we analyzed the rearrangements of TCR-alpha, beta, gamma, and delta genes in the thymus of the IL-7R-deficient mice. Southern blot analysis with a J gamma 1 probe revealed that more than 70% of J gamma 1 and J gamma 2 alleles are recombined to form distinct V gamma 1.2-J gamma 2 and V gamma 2-J gamma 1 fragments in control mice. On the contrary, no such recombination was detected in the mutant mice. The rearrangements in the TCR-alpha, beta, and delta loci were comparably observed in control and mutant mice. PCR analysis indicated that the V-J recombination of all the V gamma genes is severely hampered in the mutant mice. The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected. These data demonstrated that the V-J recombination of the TCR gamma genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma gene rearrangement.

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TCR gene rearrangements in the thymus of  IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/− mice;  lane 2, thymocytes from IL-7R  −/− mice; lane 3, E14.1 ES  cells. The position of HindIIIdigested phage λ DNA fragments was shown on the right.  (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jγ1 (left), the Jβ2  (middle), and the RAG-2 (right)  probes. (B) Thymocyte DNA  was digested with EcoRI. A  Southern blot was sequentially  hybridized with the Jδ1 (left), the  Jα1 (middle), and the RAG-2  (right) probes.
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Figure 1: TCR gene rearrangements in the thymus of IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/− mice; lane 2, thymocytes from IL-7R −/− mice; lane 3, E14.1 ES cells. The position of HindIIIdigested phage λ DNA fragments was shown on the right. (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jγ1 (left), the Jβ2 (middle), and the RAG-2 (right) probes. (B) Thymocyte DNA was digested with EcoRI. A Southern blot was sequentially hybridized with the Jδ1 (left), the Jα1 (middle), and the RAG-2 (right) probes.

Mentions: To examine whether the signal from IL-7R affects the V–J recombination, we compared the rearrangement of the TCR γ genes between the thymocytes of IL-7R +/− and −/− mice. The thymocyte DNA from 4-wk-old mice was digested with HindIII or EcoRI, and a Southern blot was hybridized with the Jγ1 probe (Fig. 1 A, left). The Jγ1 probe allows the analysis of DNA rearrangements involving not only Jγ1 but also Jγ2 and Jγ3 gene segments (15). The ES cell DNA showed a 6.6-kb Jγ1, a 9.0-kb Jγ3, and a 11.7-kb Jγ2 germline fragment. The thymocyte DNA from IL-7R +/− mice showed decreased intensity of Jγ1 and Jγ2 germline fragments compared with embryonic stem (ES) cell DNA. Furthermore, a 3.6-kb Vγ1.2–Jγ2 and a 1.4-kb Vγ2–Jγ1 fragment was clearly detected in IL-7R +/− mice. Quantification of the radioactivity revealed that 71% and 74% of Jγ1 and Jγ2 alleles, respectively, were rearranged in thymocytes. Because γδ T cells are only 0.3% of total thymocytes (12), the majority of the Vγ1.2–Jγ2 and Vγ2–Jγ1 recombined fragments are derived from αβ T cells or precursor cells. On the other hand, no fragment derived from Vγ1.2–Jγ2 or Vγ2–Jγ1 recombination was detected in IL-7R −/− mice (Fig. 1 A). This result demonstrates that Vγ1.2–Jγ2 and Vγ2–Jγ1 rearrangements are almost completely blocked in αβ T cells in IL-7R-deficient mice.


The V-J recombination of T cell receptor-gamma genes is blocked in interleukin-7 receptor-deficient mice.

Maki K, Sunaga S, Ikuta K - J. Exp. Med. (1996)

TCR gene rearrangements in the thymus of  IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/− mice;  lane 2, thymocytes from IL-7R  −/− mice; lane 3, E14.1 ES  cells. The position of HindIIIdigested phage λ DNA fragments was shown on the right.  (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jγ1 (left), the Jβ2  (middle), and the RAG-2 (right)  probes. (B) Thymocyte DNA  was digested with EcoRI. A  Southern blot was sequentially  hybridized with the Jδ1 (left), the  Jα1 (middle), and the RAG-2  (right) probes.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196379&req=5

Figure 1: TCR gene rearrangements in the thymus of IL-7R-deficient mice. Lane 1, thymocytes from IL-7R +/− mice; lane 2, thymocytes from IL-7R −/− mice; lane 3, E14.1 ES cells. The position of HindIIIdigested phage λ DNA fragments was shown on the right. (A) Thymocyte DNA was digested with HindIII. A Southern blot was sequentially hybridized with the Jγ1 (left), the Jβ2 (middle), and the RAG-2 (right) probes. (B) Thymocyte DNA was digested with EcoRI. A Southern blot was sequentially hybridized with the Jδ1 (left), the Jα1 (middle), and the RAG-2 (right) probes.
Mentions: To examine whether the signal from IL-7R affects the V–J recombination, we compared the rearrangement of the TCR γ genes between the thymocytes of IL-7R +/− and −/− mice. The thymocyte DNA from 4-wk-old mice was digested with HindIII or EcoRI, and a Southern blot was hybridized with the Jγ1 probe (Fig. 1 A, left). The Jγ1 probe allows the analysis of DNA rearrangements involving not only Jγ1 but also Jγ2 and Jγ3 gene segments (15). The ES cell DNA showed a 6.6-kb Jγ1, a 9.0-kb Jγ3, and a 11.7-kb Jγ2 germline fragment. The thymocyte DNA from IL-7R +/− mice showed decreased intensity of Jγ1 and Jγ2 germline fragments compared with embryonic stem (ES) cell DNA. Furthermore, a 3.6-kb Vγ1.2–Jγ2 and a 1.4-kb Vγ2–Jγ1 fragment was clearly detected in IL-7R +/− mice. Quantification of the radioactivity revealed that 71% and 74% of Jγ1 and Jγ2 alleles, respectively, were rearranged in thymocytes. Because γδ T cells are only 0.3% of total thymocytes (12), the majority of the Vγ1.2–Jγ2 and Vγ2–Jγ1 recombined fragments are derived from αβ T cells or precursor cells. On the other hand, no fragment derived from Vγ1.2–Jγ2 or Vγ2–Jγ1 recombination was detected in IL-7R −/− mice (Fig. 1 A). This result demonstrates that Vγ1.2–Jγ2 and Vγ2–Jγ1 rearrangements are almost completely blocked in αβ T cells in IL-7R-deficient mice.

Bottom Line: PCR analysis indicated that the V-J recombination of all the V gamma genes is severely hampered in the mutant mice.The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected.These data demonstrated that the V-J recombination of the TCR gamma genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma gene rearrangement.

View Article: PubMed Central - PubMed

Affiliation: Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, University of Tokyo, Japan.

ABSTRACT
IL-7R-deficient mice have severely impaired expansion of early lymphocytes and lack gamma delta T cells. To elucidate the role of IL-7R on gamma delta T cell development, we analyzed the rearrangements of TCR-alpha, beta, gamma, and delta genes in the thymus of the IL-7R-deficient mice. Southern blot analysis with a J gamma 1 probe revealed that more than 70% of J gamma 1 and J gamma 2 alleles are recombined to form distinct V gamma 1.2-J gamma 2 and V gamma 2-J gamma 1 fragments in control mice. On the contrary, no such recombination was detected in the mutant mice. The rearrangements in the TCR-alpha, beta, and delta loci were comparably observed in control and mutant mice. PCR analysis indicated that the V-J recombination of all the V gamma genes is severely hampered in the mutant mice. The mRNA of RAG-1, RAG-2, Ku-80, and terminal deoxynucleotidyl transferase (TdT) genes was equally detected between control and mutant thymi, suggesting that the expression of common recombination machinery is not affected. These data demonstrated that the V-J recombination of the TCR gamma genes is specifically blocked in the IL-7R-deficient mice and suggested the presence of highly specific regulation for TCR gamma gene rearrangement.

Show MeSH
Related in: MedlinePlus