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Quantitative and qualitative changes in V-J alpha rearrangements during mouse thymocytes differentiation: implication for a limited T cell receptor alpha chain repertoire.

Pasqual N, Gallagher M, Aude-Garcia C, Loiodice M, Thuderoz F, Demongeot J, Ceredig R, Marche PN, Jouvin-Marche E - J. Exp. Med. (2002)

Bottom Line: Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development.Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways.These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunochimie, Commissariat à l'Energie Atomique, Institut National de la Santé et de la Recherche Médicale, Unité 548, Université Joseph Fourier, 38054 Grenoble Cedex 9, France.

ABSTRACT
Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR alpha repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3' side than 5' end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.

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Kinetics of V2, V6, V20, V12, and V19 rearrangements. (a) Rearrangements were analyzed by multiplex PCR on DNA extracted from thymi of 28-d-old, day 1 BALB/c mice, and FTOC from F16 after 6 d of culture. Specific V family primers are indicated on panels, these were used in conjunction with 9 J segments primers, as indicated on lanes. (b) Comparison of V2S2 versus V2S7 members at D28. PCR products were revealed with a pool of appropriate J-segments probes. Asterisks indicate nonspecific products, as determined by distance migration.
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fig4: Kinetics of V2, V6, V20, V12, and V19 rearrangements. (a) Rearrangements were analyzed by multiplex PCR on DNA extracted from thymi of 28-d-old, day 1 BALB/c mice, and FTOC from F16 after 6 d of culture. Specific V family primers are indicated on panels, these were used in conjunction with 9 J segments primers, as indicated on lanes. (b) Comparison of V2S2 versus V2S7 members at D28. PCR products were revealed with a pool of appropriate J-segments probes. Asterisks indicate nonspecific products, as determined by distance migration.

Mentions: We have previously demonstrated at the transcript level that, in addition to the opening of the J region of the TCRAD locus, there is a similar opening of the V region (17). We further investigated whether V localization is a factor that dictates the rearrangement to particular J segments. We availed of the multiplex technique to study the families identified in Fig. 1 that showed differential rearrangement to J56 or J27 genes. Thus, we compared in adult and newborn thymus two V families situated proximal to the J cluster, V6 (340 kb from Cα coding region) and V20 (380 kb) with two V families distal to the J cluster, V12 (1,420 kb) and V19 (1,580 kb). As expected from the results in Fig. 1, V6 and V20 rearrangements are mainly detected with proximal, whereas V12 and V19 rearrange with a more distal group of J segments (Fig. 4 a). Nevertheless, there is a notable difference in the range of utilization of proximal J segments between V6 and V20. In five different experiments, we detect V6 rearranged to J segments located between J61-J33 and V20 rearranged to J segments located between J6–J23. Taking into account that between the J61 gene and either J33 or J23, 24 or 32 J segments contain a classical RSS, we calculate that V6 and V20 can be recombined with 49 and 65% of the functional J segments, respectively.


Quantitative and qualitative changes in V-J alpha rearrangements during mouse thymocytes differentiation: implication for a limited T cell receptor alpha chain repertoire.

Pasqual N, Gallagher M, Aude-Garcia C, Loiodice M, Thuderoz F, Demongeot J, Ceredig R, Marche PN, Jouvin-Marche E - J. Exp. Med. (2002)

Kinetics of V2, V6, V20, V12, and V19 rearrangements. (a) Rearrangements were analyzed by multiplex PCR on DNA extracted from thymi of 28-d-old, day 1 BALB/c mice, and FTOC from F16 after 6 d of culture. Specific V family primers are indicated on panels, these were used in conjunction with 9 J segments primers, as indicated on lanes. (b) Comparison of V2S2 versus V2S7 members at D28. PCR products were revealed with a pool of appropriate J-segments probes. Asterisks indicate nonspecific products, as determined by distance migration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2194109&req=5

fig4: Kinetics of V2, V6, V20, V12, and V19 rearrangements. (a) Rearrangements were analyzed by multiplex PCR on DNA extracted from thymi of 28-d-old, day 1 BALB/c mice, and FTOC from F16 after 6 d of culture. Specific V family primers are indicated on panels, these were used in conjunction with 9 J segments primers, as indicated on lanes. (b) Comparison of V2S2 versus V2S7 members at D28. PCR products were revealed with a pool of appropriate J-segments probes. Asterisks indicate nonspecific products, as determined by distance migration.
Mentions: We have previously demonstrated at the transcript level that, in addition to the opening of the J region of the TCRAD locus, there is a similar opening of the V region (17). We further investigated whether V localization is a factor that dictates the rearrangement to particular J segments. We availed of the multiplex technique to study the families identified in Fig. 1 that showed differential rearrangement to J56 or J27 genes. Thus, we compared in adult and newborn thymus two V families situated proximal to the J cluster, V6 (340 kb from Cα coding region) and V20 (380 kb) with two V families distal to the J cluster, V12 (1,420 kb) and V19 (1,580 kb). As expected from the results in Fig. 1, V6 and V20 rearrangements are mainly detected with proximal, whereas V12 and V19 rearrange with a more distal group of J segments (Fig. 4 a). Nevertheless, there is a notable difference in the range of utilization of proximal J segments between V6 and V20. In five different experiments, we detect V6 rearranged to J segments located between J61-J33 and V20 rearranged to J segments located between J6–J23. Taking into account that between the J61 gene and either J33 or J23, 24 or 32 J segments contain a classical RSS, we calculate that V6 and V20 can be recombined with 49 and 65% of the functional J segments, respectively.

Bottom Line: Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development.Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways.These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunochimie, Commissariat à l'Energie Atomique, Institut National de la Santé et de la Recherche Médicale, Unité 548, Université Joseph Fourier, 38054 Grenoble Cedex 9, France.

ABSTRACT
Knowledge of the complete nucleotide sequence of the mouse TCRAD locus allows an accurate determination V-J rearrangement status. Using multiplex genomic PCR assays and real time PCR analysis, we report a comprehensive and systematic analysis of the V-J recombination of TCR alpha chain in normal mouse thymocytes during development. These respective qualitative and quantitative approaches give rise to four major points describing the control of gene rearrangements. (a) The V-J recombination pattern is not random during ontogeny and generates a limited TCR alpha repertoire; (b) V-J rearrangement control is intrinsic to the thymus; (c) each V gene rearranges to a set of contiguous J segments with a gaussian-like frequency; (d) there are more rearrangements involving V genes at the 3' side than 5' end of V region. Taken together, this reflects a preferential association of V and J gene segments according to their respective positions in the locus, indicating that accessibility of both V and J regions is coordinately regulated, but in different ways. These results provide a new insight into TCR alpha repertoire size and suggest a scenario for V usage during differentiation.

Show MeSH