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1
Aster, J. C., and J. Sklar. "Interallelic V(D)J trans-rearrangement within the beta T cell receptor gene is infrequent and occurs preferentially during attempted D beta to J beta joining." Journal of Experimental Medicine 175, no. 6 (June 1, 1992): 1773–82. http://dx.doi.org/10.1084/jem.175.6.1773.
Анотація:
Previous work has demonstrated that intergenic V(D)J rearrangement, a process referred to as trans-rearrangement, occurs at an unexpectedly high frequency. These rearrangements generate novel V(D)J combinations which could conceivably have some role in the normal immune system, and since they probably arise through chromosomal rearrangements akin to those associated with lymphoid neoplasia, they may also serve as a model for investigating recombinational events which underlie oncogenesis. In view of the existence of a mechanism that permits relatively frequent intergenic trans-rearrangements, it seems reasonable that interallelic trans-rearrangements involving segments belonging to each of the two alleles of a single antigen receptor gene might also occur. To determine the frequency of such rearrangements, we examined thymocytes of F1 progeny of a cross between SWR mice, which have a deletion spanning 10 of the known V beta segments, and NZW mice, which have a deletion involving all J beta 2 segments. Rearranged TCR-beta genes containing V beta segments from the NZW chromosome and J beta segments from the SWR chromosome were amplified from the DNA of F1 thymocytes with the polymerase chain reaction. Using this approach, we found that such rearrangements are relatively uncommon, being present in about 1 in 10(5) thymocytes, a frequency lower than that of V gamma/J beta intergenic trans-rearrangements. The ratio of conventional cis-rearrangement to interallelic trans-rearrangement for any particular V beta segment appears to be about 10(4):1. The structure of the junctions in all trans-rearrangements analyzed closely resembles conventional cis-rearrangements, indicating involvement of V(D)J recombinase in the ultimate joining event. However, in contrast to cis-rearrangements, a strong bias for inclusion of D beta 1 segments over D beta 2 segments was noted, suggesting that interallelic trans-rearrangement may occur preferentially during attempted D-J joining. J beta 2 segment usage in trans-rearrangements also appeared to differ from that expected from previously studied cis-rearrangements. The results have implications with respect to the events and timing of conventional cis-rearrangement during thymocyte differentiation, and the prevalence of various types of trans-rearrangements.
2
Hendrickson, E. A., V. F. Liu, and D. T. Weaver. "Strand breaks without DNA rearrangement in V (D)J recombination." Molecular and Cellular Biology 11, no. 6 (June 1991): 3155–62. http://dx.doi.org/10.1128/mcb.11.6.3155.
Анотація:
Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.
3
Hendrickson, E. A., V. F. Liu, and D. T. Weaver. "Strand breaks without DNA rearrangement in V (D)J recombination." Molecular and Cellular Biology 11, no. 6 (June 1991): 3155–62. http://dx.doi.org/10.1128/mcb.11.6.3155-3162.1991.
Анотація:
Somatic gene rearrangement of immunoglobulin and T-cell receptor genes [V(D)J recombination] is mediated by pairs of specific DNA sequence motifs termed signal sequences. In experiments described here, retroviral vectors containing V(D)J rearrangement cassettes in which the signal sequences had been altered were introduced into wild-type and scid (severe combined immune deficiency) pre-B cells and used to define intermediates in the V(D)J recombination pathway. The scid mutation has previously been shown to deleteriously affect the V(D)J recombination process. Cassettes containing a point mutation in one of the two signal sequences inhibited rearrangement in wild-type cells. In contrast, scid cells continued to rearrange these cassettes with the characteristic scid deletional phenotype. Using these mutated templates, we identified junctional modifications at the wild-type signal sequences that had arisen from strand breaks which were not associated with overall V(D)J rearrangements. Neither cell type was able to rearrange constructs which contained only a single, nonmutated, signal sequence. In addition, scid and wild-type cell lines harboring cassettes with mutations in both signal sequences did not undergo rearrangement, suggesting that at least one functional signal sequence was required for all types of V(D)J recombination events. Analysis of these signal sequence mutations has provided insights into intermediates in the V(D)J rearrangement pathway in wild-type and scid pre-B cells.
4
Lauzurica, P., and M. S. Krangel. "Temporal and lineage-specific control of T cell receptor alpha/delta gene rearrangement by T cell receptor alpha and delta enhancers." Journal of Experimental Medicine 179, no. 6 (June 1, 1994): 1913–21. http://dx.doi.org/10.1084/jem.179.6.1913.
Анотація:
To analyze the regulation of gene rearrangement at the T cell receptor (TCR) alpha/delta locus during T cell development, we generated transgenic mice carrying a human TCR delta gene minilocus. We previously showed that the presence of the TCR delta enhancer (E delta) within the J delta 3-C delta intron was required to activate a specific step (V-D to J) of transgene rearrangement, and that rearrangement was activated equivalently in the precursors of alpha beta and gamma delta T cells. To further explore the role of transcriptional enhancers in establishing the developmental pattern of gene rearrangement at the TCR alpha/delta locus, we substituted the TCR alpha enhancer (E alpha) in place of E delta within the transgenic minilocus. We found that V-D-J rearrangement of the E alpha+ minilocus was restricted to the alpha beta T cell subset. Further, we found that although V-D-J rearrangement of the E delta+ minilocus was initiated in the fetal thymus by day 14.5, V-D-J rearrangement of the E alpha+ minilocus did not occur until fetal day 16.5. Finally, whereas V-D-J rearrangement of the E delta+ minilocus is essentially completed within the triple negative population of postnatal thymocytes, V-D-J rearrangement of the E alpha+ minilocus is only initiated late within this population. Since the properties of minilocus rearrangement under the control of E delta and E alpha parallel the properties of V delta-D delta-J delta and V alpha-J alpha rearrangement at the endogenous TCR alpha/delta locus, we conclude that these enhancers play an important role in orchestrating the developmental program of rearrangements at this locus.
5
Bain, Gretchen, William J. Romanow, Karen Albers, Wendy L. Havran, and Cornelis Murre. "Positive and Negative Regulation of V(D)J Recombination by the E2A Proteins." Journal of Experimental Medicine 189, no. 2 (January 18, 1999): 289–300. http://dx.doi.org/10.1084/jem.189.2.289.
Анотація:
A key feature of B and T lymphocyte development is the generation of antigen receptors through the rearrangement and assembly of the germline variable (V), diversity (D), and joining (J) gene segments. However, the mechanisms responsible for regulating developmentally ordered gene rearrangements are largely unknown. Here we show that the E2A gene products are essential for the proper coordinated temporal regulation of V(D)J rearrangements within the T cell receptor (TCR) γ and δ loci. Specifically, we show that E2A is required during adult thymocyte development to inhibit rearrangements to the γ and δ V regions that normally recombine almost exclusively during fetal thymocyte development. The continued rearrangement of the fetal Vγ3 gene segment in E2A-deficient adult thymocytes correlates with increased levels of Vγ3 germline transcripts and increased levels of double-stranded DNA breaks at the recombination signal sequence bordering Vγ3. Additionally, rearrangements to a number of Vγ and Vδ gene segments used predominately during adult development are significantly reduced in E2A-deficient thymocytes. Interestingly, at distinct stages of T lineage development, both the increased and decreased rearrangement of particular Vδ gene segments is highly sensitive to the dosage of the E2A gene products, suggesting that the concentration of the E2A proteins is rate limiting for the recombination reaction involving these Vδ regions.
6
Dombret, H., P. Loiseau, JC Bories, and F. Sigaux. "Unexpected consistent involvement of V beta gene segments in inappropriate T-cell receptor beta gene rearrangements occurring in B- lineage acute lymphoblastic leukemias." Blood 80, no. 10 (November 15, 1992): 2614–21. http://dx.doi.org/10.1182/blood.v80.10.2614.2614.
Анотація:
Abstract T-cell receptor beta (TCR beta) gene rearrangements occur in a third of early B-cell acute lymphoblastic leukemias (ALLs). V, D, and J segments involved in these inappropriate rearrangements remain unknown and are of interest, both because partial D beta J beta and complete V beta D beta J beta recombinations occur at distinct stages of thymic maturation and because these rearrangements are regulated differently. We have therefore studied in detail seven cases of B-lineage ALL that show inappropriate clonal TCR beta gene rearrangements. Analysis of genomic DNA by Southern hybridization with C beta, J beta 1, V beta 8, and V beta 11 probes suggested the involvement of V beta segment in tumor cell rearrangements. A complete genomic library constructed from one case was screened with a C beta probe, and the TCR beta gene rearrangement was cloned and fully sequenced to show an out of frame V beta 2.2-J beta 2.6 recombination. TCR beta gene rearrangements occurring in other cases were further analyzed by polymerase chain reaction (PCR) using J beta and V beta primers and the resulting specific PCR products were sequenced. Evidence of clonal V beta rearrangements was obtained in all cases. These unexpected findings represent the first definitive demonstration that complete V beta(D beta)J beta rearrangements can occur in B-lineage cells and contrast with the previously reported lack of V beta(D beta)J beta rearrangement in B cells from V beta-J beta-C beta-E mu transgenic mice. In the context of increasing evidence that rearrangements are linked to transcription of unrearranged gene segments, these data prompt a search in B-lineage ALL cells for the presence of germline V beta transcripts whose deregulated expression may be linked to early transforming events.
7
Dombret, H., P. Loiseau, JC Bories, and F. Sigaux. "Unexpected consistent involvement of V beta gene segments in inappropriate T-cell receptor beta gene rearrangements occurring in B- lineage acute lymphoblastic leukemias." Blood 80, no. 10 (November 15, 1992): 2614–21. http://dx.doi.org/10.1182/blood.v80.10.2614.bloodjournal80102614.
Анотація:
T-cell receptor beta (TCR beta) gene rearrangements occur in a third of early B-cell acute lymphoblastic leukemias (ALLs). V, D, and J segments involved in these inappropriate rearrangements remain unknown and are of interest, both because partial D beta J beta and complete V beta D beta J beta recombinations occur at distinct stages of thymic maturation and because these rearrangements are regulated differently. We have therefore studied in detail seven cases of B-lineage ALL that show inappropriate clonal TCR beta gene rearrangements. Analysis of genomic DNA by Southern hybridization with C beta, J beta 1, V beta 8, and V beta 11 probes suggested the involvement of V beta segment in tumor cell rearrangements. A complete genomic library constructed from one case was screened with a C beta probe, and the TCR beta gene rearrangement was cloned and fully sequenced to show an out of frame V beta 2.2-J beta 2.6 recombination. TCR beta gene rearrangements occurring in other cases were further analyzed by polymerase chain reaction (PCR) using J beta and V beta primers and the resulting specific PCR products were sequenced. Evidence of clonal V beta rearrangements was obtained in all cases. These unexpected findings represent the first definitive demonstration that complete V beta(D beta)J beta rearrangements can occur in B-lineage cells and contrast with the previously reported lack of V beta(D beta)J beta rearrangement in B cells from V beta-J beta-C beta-E mu transgenic mice. In the context of increasing evidence that rearrangements are linked to transcription of unrearranged gene segments, these data prompt a search in B-lineage ALL cells for the presence of germline V beta transcripts whose deregulated expression may be linked to early transforming events.
8
Hendrickson, E. A., M. S. Schlissel, and D. T. Weaver. "Wild-type V(D)J recombination in scid pre-B cells." Molecular and Cellular Biology 10, no. 10 (October 1990): 5397–407. http://dx.doi.org/10.1128/mcb.10.10.5397.
Анотація:
Homozygous mutation at the scid locus in the mouse results in the aberrant rearrangement of immunoglobulin and T-cell receptor gene segments. We introduced a retroviral vector containing an inversional immunoglobulin rearrangement cassette into scid pre-B cells. Most rearrangements were accompanied by large deletions, consistent with previously characterized effects of the scid mutation. However, two cell clones were identified which contained perfect reciprocal fragments and wild-type coding joints, documenting, on a molecular level, the ability of scid pre-B cells to generate functional protein-coding domains. Subsequent rearrangement of the DGR cassette in one of these clones was accompanied by a deletion, suggesting that this cell clone had not reverted the scid mutation. Indeed, induced rearrangement of the endogenous kappa loci in these two cell clones resulted in a mixture of scid and wild-type V-J kappa joints, as assayed by a polymerase chain reaction and DNA sequencing. In addition, three immunoglobulin mu- scid pre-B cell lines showed both scid and wild-type V-J kappa joins. These experiments strongly suggest that the V(D)J recombinase activity in scid lymphoid cells is diminished but not absent, consistent with the known leakiness of the scid mutation.
9
Hendrickson, E. A., M. S. Schlissel, and D. T. Weaver. "Wild-type V(D)J recombination in scid pre-B cells." Molecular and Cellular Biology 10, no. 10 (October 1990): 5397–407. http://dx.doi.org/10.1128/mcb.10.10.5397-5407.1990.
Анотація:
Homozygous mutation at the scid locus in the mouse results in the aberrant rearrangement of immunoglobulin and T-cell receptor gene segments. We introduced a retroviral vector containing an inversional immunoglobulin rearrangement cassette into scid pre-B cells. Most rearrangements were accompanied by large deletions, consistent with previously characterized effects of the scid mutation. However, two cell clones were identified which contained perfect reciprocal fragments and wild-type coding joints, documenting, on a molecular level, the ability of scid pre-B cells to generate functional protein-coding domains. Subsequent rearrangement of the DGR cassette in one of these clones was accompanied by a deletion, suggesting that this cell clone had not reverted the scid mutation. Indeed, induced rearrangement of the endogenous kappa loci in these two cell clones resulted in a mixture of scid and wild-type V-J kappa joints, as assayed by a polymerase chain reaction and DNA sequencing. In addition, three immunoglobulin mu- scid pre-B cell lines showed both scid and wild-type V-J kappa joins. These experiments strongly suggest that the V(D)J recombinase activity in scid lymphoid cells is diminished but not absent, consistent with the known leakiness of the scid mutation.
10
Agard, Emily A., and Susanna M. Lewis. "Postcleavage Sequence Specificity in V(D)J Recombination." Molecular and Cellular Biology 20, no. 14 (July 15, 2000): 5032–40. http://dx.doi.org/10.1128/mcb.20.14.5032-5040.2000.
Анотація:
ABSTRACT Unintended DNA rearrangements in a differentiating lymphocyte can have severe, oncogenic consequences, but the mechanisms for avoiding pathogenic outcomes in V(D)J recombination are not well understood. The first level at which fidelity is instituted is in discrimination by the recombination proteins between authentic and inauthentic recombination signal sequences. Nevertheless, this discrimination is not absolute and cannot fully eliminate targeting errors. To learn more about the basis of specificity during V(D)J recombination, we have investigated whether it is possible for the recombination machinery to detect an inaccurately targeted sequence subsequent to cleavage. These studies indicate that even postcleavage steps in V(D)J recombination are sequence specific and that noncanonical sequences will not efficiently support the resolution of recombination intermediates in vivo. Accordingly, interventions after a mistargeting event conceivably occur at a late stage in the joining process and the likelihood may well be crucial to enforcing fidelity during antigen receptor gene rearrangement.
11
Livák, F., S. C. Welsh, C. J. Guidos, I. N. Crispe, J. S. Danska, and D. G. Schatz. "Transient restoration of gene rearrangement at multiple T cell receptor loci in gamma-irradiated scid mice." Journal of Experimental Medicine 184, no. 2 (August 1, 1996): 419–28. http://dx.doi.org/10.1084/jem.184.2.419.
Анотація:
The developmental arrest of thymocytes from scid mice, deficient in variable, (diversity), and joining, or V(D)J recombination, can be overcome by sublethal gamma-irradiation. Since previous studies focused on restoration of rearrangement of the T cell receptor (TCR) beta locus, productive rearrangement of which is selected for, we sought to examine to what extent locus specificity and cellular selection contributed to the observed effects. We report here that irradiation of newborn scid mice induces normal V-D-J rearrangements of the TCR delta locus, which like TCR beta, is also actively rearranged in CD(4-)CD(8-) (double negative) thymocytes. In contrast, no complete V-J alpha rearrangements were detected. Instead, we detected substantial levels of hairpin-terminated coding ends at the 5' end of the J alpha locus, demonstrating that TCR alpha rearrangements manifest the effects of the scid mutation. Irradiation, therefore, transiently compensates for the effects of the scid mutation in a locus-nonspecific manner in thymocytes, resulting in a burst of normal TCR beta and delta rearrangements. Irradiation also allows the development of cells that can initiate but fail to complete V(D)J recombination events at the TCR alpha locus, which is normally inaccessible in scid thymocytes.
12
Lauzurica, P., and M. S. Krangel. "Enhancer-dependent and -independent steps in the rearrangement of a human T cell receptor delta transgene." Journal of Experimental Medicine 179, no. 1 (January 1, 1994): 43–55. http://dx.doi.org/10.1084/jem.179.1.43.
Анотація:
The rearrangement and expression of T cell receptor (TCR) gene segments occurs in a highly ordered fashion during thymic ontogeny of T lymphocytes. To study the regulation of gene rearrangement within the TCR alpha/delta locus, we generated transgenic mice that carry a germline human TCR delta minilocus that includes V delta 1, V delta 2, D delta 3, J delta 1, J delta 3, and C delta segments, and either contains or lacks the TCR delta enhancer. We found that the enhancer-positive construct rearranges stepwise, first V to D, and then V-D to J. Construct V-D rearrangement mimics a unique property of the endogenous TCR delta locus. V-D-J rearrangement is T cell specific, but is equivalent in alpha/beta and gamma/delta T lymphocytes. Thus, either there is no commitment to the alpha/beta and gamma/delta T cell lineages before TCR delta gene rearrangement, or if precommitment occurs, it does not operate directly on TCR delta gene cis-acting regulatory elements to control TCR delta gene rearrangement. Enhancer-negative mice display normal V to D rearrangement, but not V-D to J rearrangement. Thus, the V-D to J step is controlled by the enhancer, but the V to D step is controlled by separate elements. The enhancer apparently controls access to J delta 1 but not D delta 3, suggesting that a boundary between two independently regulated domains of the minilocus lies between these elements. Within the endogenous TCR alpha/delta locus, this boundary may represent the 5' end of a chromatin regulatory domain that is opened by the TCR delta enhancer during T cell development. The position of this boundary may explain the unique propensity of the TCR delta locus to undergo early V to D rearrangement. Our results indicate that the TCR delta enhancer performs a crucial targeting function to regulate TCR delta gene rearrangement during T cell development.
13
Yeo, Tiong Chia, Dong Xia, Samar Hassouneh, Xuexian O. Yang, Daniel E. Sabath, Karl Sperling, Richard A. Gatti, Patrick Concannon, and Dennis M. Willerford. "V(D)J rearrangement in Nijmegen breakage syndrome." Molecular Immunology 37, no. 18 (December 2000): 1131–39. http://dx.doi.org/10.1016/s0161-5890(01)00026-8.
14
Wang, Yui-Hsi, Zhixin Zhang, Peter D. Burrows, Hiromi Kubagawa, S. Louis Bridges, Harry W. Findley, and Max D. Cooper. "V(D)J recombinatorial repertoire diversification during intraclonal pro-B to B-cell differentiation." Blood 101, no. 3 (February 1, 2003): 1030–37. http://dx.doi.org/10.1182/blood-2002-06-1828.
Анотація:
Abstract The initial B-cell repertoire is generated by combinatorial immunoglobulin V(D)J gene segment rearrangements that occur in a preferential sequence. Because cellular proliferation occurs during the course of these rearrangement events, it has been proposed that intraclonal diversification occurs during this phase of B-cell development. An opportunity to examine this hypothesis directly was provided by the identification of a human acute lymphoblastic leukemic cell line that undergoes spontaneous differentiation from pro-B cell to the pre-B and B-cell stages with concomitant changes in the gene expression profile that normally occur during B-cell differentiation. After confirming the clonality of the progressively differentiating cells, an analysis of immunoglobulin genes and transcripts indicated that pro-B cell members marked by the same DJ rearrangement generated daughter B cells with multiple VH and VL gene segment rearrangements. These findings validate the principle of intraclonal V(D)J diversification during B-cell generation and define a manipulable model of human B-cell differentiation.
15
Sikes, Michael L., Cristina C. Suarez, and Eugene M. Oltz. "Regulation of V(D)J Recombination by Transcriptional Promoters." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 2773–81. http://dx.doi.org/10.1128/mcb.19.4.2773.
Анотація:
ABSTRACT Enhancer elements potentiate the rearrangement of antigen receptor loci via changes in the accessibility of gene segment clusters to V(D)J recombinase. Here, we show that enhancer activity per se is insufficient to target T-cell receptor β miniloci for DβJβ recombination. Instead, a promoter situated 5′ to Dβ1 (PDβ) was required for efficient rearrangement of chromosomal substrates. A critical function for promoters in regulating gene segment accessibility was further supported by the ability of heterologous promoters to direct rearrangement of enhancer-containing substrates. Importantly, activation of a synthetic tetracycline-inducible promoter (Ptet) positioned upstream from the Dβ gene segment was sufficient to target recombination of miniloci lacking a distal enhancer element. The latter result suggests that DNA loops, generated by interactions between flanking promoter and enhancer elements, are not required for efficient recognition of chromosomal gene segments by V(D)J recombinase. Unexpectedly, the Ptet substrate exhibited normal levels of rearrangement despite its retention of a hypermethylated DNA status within the DβJβ cluster. Together, our findings support a model in which promoter activation, rather than intrinsic properties of enhancers, is the primary determinant for regulating recombinational accessibility within antigen receptor loci.
16
Pasqual, Nicolas, Maighréad Gallagher, Catherine Aude-Garcia, Mélanie Loiodice, Florence Thuderoz, Jacques Demongeot, Rod Ceredig, Patrice Noël Marche та Evelyne Jouvin-Marche. "Quantitative and Qualitative Changes in V-J α Rearrangements During Mouse Thymocytes Differentiation". Journal of Experimental Medicine 196, № 9 (28 жовтня 2002): 1163–74. http://dx.doi.org/10.1084/jem.20021074.
Анотація:
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 α 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 α 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 α repertoire size and suggest a scenario for V usage during differentiation.
17
Dudley, Darryll D., JoAnn Sekiguchi, Chengming Zhu, Moshe J. Sadofsky, Scott Whitlow, Jeffrey DeVido, Robert J. Monroe, Craig H. Bassing, and Frederick W. Alt. "Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice." Journal of Experimental Medicine 198, no. 9 (October 27, 2003): 1439–50. http://dx.doi.org/10.1084/jem.20030627.
Анотація:
RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.
18
Inlay, Matthew A., Tongxiang Lin, Heather H. Gao, and Yang Xu. "Critical roles of the immunoglobulin intronic enhancers in maintaining the sequential rearrangement of IgH and Igk loci." Journal of Experimental Medicine 203, no. 7 (June 19, 2006): 1721–32. http://dx.doi.org/10.1084/jem.20052310.
Анотація:
V(D)J recombination of immunoglobulin (Ig) heavy (IgH) and light chain genes occurs sequentially in the pro– and pre–B cells. To identify cis-elements that dictate this order of rearrangement, we replaced the endogenous matrix attachment region/Igk intronic enhancer (MiEκ) with its heavy chain counterpart (Eμ) in mice. This replacement, denoted EμR, substantially increases the accessibility of both Vκ and Jκ loci to V(D)J recombinase in pro–B cells and induces Igk rearrangement in these cells. However, EμR does not support Igk rearrangement in pre–B cells. Similar to that in MiEκ−/− pre–B cells, the accessibility of Vκ segments to V(D)J recombinase is considerably reduced in EμR pre–B cells when compared with wild-type pre–B cells. Therefore, Eμ and MiEκ play developmental stage-specific roles in maintaining the sequential rearrangement of IgH and Igk loci by promoting the accessibility of V, D, and J loci to the V(D)J recombinase.
19
Oltz, E. M., F. W. Alt, W. C. Lin, J. Chen, G. Taccioli, S. Desiderio, and G. Rathbun. "A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination." Molecular and Cellular Biology 13, no. 10 (October 1993): 6223–30. http://dx.doi.org/10.1128/mcb.13.10.6223.
Анотація:
Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.
20
Oltz, E. M., F. W. Alt, W. C. Lin, J. Chen, G. Taccioli, S. Desiderio, and G. Rathbun. "A V(D)J recombinase-inducible B-cell line: role of transcriptional enhancer elements in directing V(D)J recombination." Molecular and Cellular Biology 13, no. 10 (October 1993): 6223–30. http://dx.doi.org/10.1128/mcb.13.10.6223-6230.1993.
Анотація:
Rapid analysis of mechanisms that regulate V(D)J recombination has been hampered by the lack of appropriate cell systems that reproduce aspects of normal prelymphocyte physiology in which the recombinase is activated, accessible antigen receptor loci are rearranged, and rearrangement status is fixed by termination of recombinase expression. To generate such a system, we introduced heat shock-inducible V(D)J recombination-activating genes (RAG) 1 and 2 into a recombinationally inert B-cell line. Heat shock treatment of these cells rapidly induced high levels of RAG transcripts and RAG proteins that were accompanied by a parallel induction of V(D)J recombinase activity, strongly suggesting that RAG proteins have a primary role in V(D)J recombination. Within hours after induction, these cells began to rearrange chromosomally integrated V(D)J recombination substrates but only if the substrates contained an active transcriptional enhancer; substrates lacking an enhancer were not efficiently rearranged. Activities necessary to target integrated substrates for rearrangement were provided by two separate lymphoid-specific transcriptional enhancers, as well as an active nonlymphoid enhancer, unequivocally demonstrating that such elements enhance both transcription and V(D)J recombinational accessibility.
21
Bailey, S. N., and N. Rosenberg. "Assessing the pathogenic potential of the V(D)J recombinase by interlocus immunoglobulin light-chain gene rearrangement." Molecular and Cellular Biology 17, no. 2 (February 1997): 887–94. http://dx.doi.org/10.1128/mcb.17.2.887.
Анотація:
Chromosomal translocations involving antigen receptor genes and oncogenes have been observed in several forms of lymphoid malignancy. Observations of their lymphocyte-restricted occurrence and a molecular analysis of some translocation breakpoints have suggested that some of these rearrangements are generated by V(D)J recombinase activity. However, a direct correlation between this activity and the generation of such rearrangements has never been established. In addition, because these aberrant rearrangements are usually detected only after a tumor has been formed, the frequency with which the recombinase machinery generates translocations has never been assessed directly. To approach these issues, immunoglobulin light-chain gene rearrangements were induced in pre-B cells transformed by temperature-sensitive mutants of Abelson murine leukemia virus and PCR was used to identify interlocus recombinants. Vlambda Jkappa and Vkappa Jlambda rearrangements as well as signal joints resulting from the recombination of Vlambda and Jkappa coding elements were recovered and were found to be similar in structure to conventional intrachromosomal joints. Because these products were detected only when the cells were undergoing active intralocus rearrangement, they provide direct evidence that translocations can be generated by the V(D)J recombinase machinery. Dilution analyses revealed that interlocus rearrangements occur about 1,000 times less frequently than conventional intralocus rearrangements. Considering the large numbers of lymphocytes generated throughout life, aberrant rearrangements generated by the V(D)J recombinase may be relatively common.
22
Wucherpfennig, K. W., Y. J. Liao, M. Prendergast, J. Prendergast, D. A. Hafler, and J. L. Strominger. "Human fetal liver gamma/delta T cells predominantly use unusual rearrangements of the T cell receptor delta and gamma loci expressed on both CD4+CD8- and CD4-CD8- gamma/delta T cells." Journal of Experimental Medicine 177, no. 2 (February 1, 1993): 425–32. http://dx.doi.org/10.1084/jem.177.2.425.
Анотація:
Substantial numbers of both alpha/beta and gamma/delta T cells are present in human fetal liver, which suggests a role of the fetal liver in T cell development. The diversity of fetal liver T cell receptor (TCR) gamma and delta chain rearrangements was examined among both CD4+CD8- and CD4-CD8- gamma/delta T cell clones. In addition, TCR delta chain transcripts from three fetal livers were sequenced after polymerase chain reaction amplification of TCR delta chains with V delta 1 or V delta 2 rearrangements. Five of six fetal liver gamma/delta T cell clones had a V delta 2-D delta 3-J delta 3 gene rearrangement with limited junctional diversity; three of these clones had an unusual CD4+CD8- phenotype. V delta 2-D delta 3-J delta 3 gene rearrangements were also common among both in-frame and out-of-frame transcripts from three fetal livers, indicating that they are the result of an ordered rearrangement process. TCR gamma chain sequences of the fetal liver gamma/delta T cell clones revealed V gamma 1-J gamma 2.3, V gamma 2-J gamma 1.2, and V gamma 3-J gamma 1.1 rearrangements with minimal incorporation of template-independent N region nucleotides. TCR gamma chain rearrangements found in these fetal liver T cell clones were different from those that have been observed among early thymic gamma/delta T cell populations, while similar TCR delta chain rearrangements are found among gamma/delta T cells from both sites. These data demonstrate that the fetal liver harbors gamma/delta T cell populations distinct from those found in the fetal thymus, suggesting that the fetal liver is a site of gamma/delta T cell development in humans. These unusual T cell populations may serve a specific function in the fetal immune system.
23
Staunton, J. E., and D. T. Weaver. "scid cells efficiently integrate hairpin and linear DNA substrates." Molecular and Cellular Biology 14, no. 6 (June 1994): 3876–83. http://dx.doi.org/10.1128/mcb.14.6.3876.
Анотація:
The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.
24
Staunton, J. E., and D. T. Weaver. "scid cells efficiently integrate hairpin and linear DNA substrates." Molecular and Cellular Biology 14, no. 6 (June 1994): 3876–83. http://dx.doi.org/10.1128/mcb.14.6.3876-3883.1994.
Анотація:
The scid mouse mutation affects V(D)J rearrangement and double-strand break repair. scid V(D)J rearrangement is characterized by defective coding joint formation which prevents the development of mature B and T cells. Hairpin DNA has been implicated in the formation of V(D)J coding joints. We found scid cells to be proficient in hairpin processing in the context of DNA integration. In addition, we found that the scid defect did not impair integration of linear DNA via nonhomologous recombination. Therefore, hairpin processing and integration of DNA into the genome are distinct from hypersensitivity to ionizing radiation and the defect in V(D)J recombination.
25
Retter, Marc W., and David Nemazee. "Receptor Editing Occurs Frequently during Normal B Cell Development." Journal of Experimental Medicine 188, no. 7 (October 5, 1998): 1231–38. http://dx.doi.org/10.1084/jem.188.7.1231.
Анотація:
Allelic exclusion is established in development through a feedback mechanism in which the assembled immunoglobulin (Ig) suppresses further V(D)J rearrangement. But Ig expression sometimes fails to prevent further rearrangement. In autoantibody transgenic mice, reactivity of immature B cells with autoantigen can induce receptor editing, in which allelic exclusion is transiently prevented or reversed through nested light chain gene rearrangement, often resulting in altered B cell receptor specificity. To determine the extent of receptor editing in a normal, non-Ig transgenic immune system, we took advantage of the fact that λ light chain genes usually rearrange after κ genes. This allowed us to analyze κ loci in IgMλ+ cells to determine how frequently in-frame κ genes fail to suppress λ gene rearrangements. To do this, we analyzed recombined VκJκ genes inactivated by subsequent recombining sequence (RS) rearrangement. RS rearrangements delete portions of the κ locus by a V(D)J recombinase-dependent mechanism, suggesting that they play a role in receptor editing. We show that RS recombination is frequently induced by, and inactivates, functionally rearranged κ loci, as nearly half (47%) of the RS-inactivated VκJκ joins were in-frame. These findings suggest that receptor editing occurs at a surprisingly high frequency in normal B cells.
26
Macintyre, E., L. d'Auriol, F. Amesland, P. Loiseau, Z. Chen, L. Boumsell, F. Galibert, and F. Sigaux. "Analysis of junctional diversity in the preferential V delta 1-J delta 1 rearrangement of fresh T-acute lymphoblastic leukemia cells by in vitro gene amplification and direct sequencing." Blood 74, no. 6 (November 1, 1989): 2053–61. http://dx.doi.org/10.1182/blood.v74.6.2053.2053.
Анотація:
Abstract To define the junctional diversity of T-cell antigen receptor delta gene rearrangements in fresh T-acute lymphoblastic cells and to correlate cell phenotype with the coding potential of rearrangements, we determined the junctional nucleotide sequences of 13 T-cell antigen receptor delta gene rearrangements involving the preferentially rearranged V (V delta 1) and J (J delta 1) segments using in vitro gene amplification and direct sequencing. We showed that, as in gamma delta+ cell lines, extensive junctional diversity exists in these clones and that this diversity is due both to random nucleotide deletions/additions and to the use of at least two D delta segments. We also showed that a high percentage of these rearrangements are potentially translatable (7:13) and that such functional rearrangements occur in both surface CD3+ and CD3- cells. Comparison of alpha beta versus gamma delta surface expression demonstrates that all CD3+ T acute lymphoblastic leukemias with a functional V delta 1-J delta 1 rearrangement express a surface gamma delta receptor and are recognized by the anti-delta monoclonal antibody delta TCS1, whereas a control CD3+ gamma delta+ leukemic case that had not undergone V delta 1 rearrangement was delta TCS1-. In addition, expression of this monoclonal antibody is not restricted by V gamma or C gamma usage or by the covalent or noncovalent link between gamma and delta chains.
27
Macintyre, E., L. d'Auriol, F. Amesland, P. Loiseau, Z. Chen, L. Boumsell, F. Galibert, and F. Sigaux. "Analysis of junctional diversity in the preferential V delta 1-J delta 1 rearrangement of fresh T-acute lymphoblastic leukemia cells by in vitro gene amplification and direct sequencing." Blood 74, no. 6 (November 1, 1989): 2053–61. http://dx.doi.org/10.1182/blood.v74.6.2053.bloodjournal7462053.
Анотація:
To define the junctional diversity of T-cell antigen receptor delta gene rearrangements in fresh T-acute lymphoblastic cells and to correlate cell phenotype with the coding potential of rearrangements, we determined the junctional nucleotide sequences of 13 T-cell antigen receptor delta gene rearrangements involving the preferentially rearranged V (V delta 1) and J (J delta 1) segments using in vitro gene amplification and direct sequencing. We showed that, as in gamma delta+ cell lines, extensive junctional diversity exists in these clones and that this diversity is due both to random nucleotide deletions/additions and to the use of at least two D delta segments. We also showed that a high percentage of these rearrangements are potentially translatable (7:13) and that such functional rearrangements occur in both surface CD3+ and CD3- cells. Comparison of alpha beta versus gamma delta surface expression demonstrates that all CD3+ T acute lymphoblastic leukemias with a functional V delta 1-J delta 1 rearrangement express a surface gamma delta receptor and are recognized by the anti-delta monoclonal antibody delta TCS1, whereas a control CD3+ gamma delta+ leukemic case that had not undergone V delta 1 rearrangement was delta TCS1-. In addition, expression of this monoclonal antibody is not restricted by V gamma or C gamma usage or by the covalent or noncovalent link between gamma and delta chains.
28
Zhao, Lijuan, Richard L. Frock, Zhou Du, Jiazhi Hu, Liang Chen, Michael S. Krangel, and Frederick W. Alt. "Orientation-specific RAG activity in chromosomal loop domains contributes to Tcrd V(D)J recombination during T cell development." Journal of Experimental Medicine 213, no. 9 (August 15, 2016): 1921–36. http://dx.doi.org/10.1084/jem.20160670.
Анотація:
T cell antigen receptor δ (Tcrd) variable region exons are assembled by RAG-initiated V(D)J recombination events in developing γδ thymocytes. Here, we use linear amplification–mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) to map hundreds of thousands of RAG-initiated Tcrd D segment (Trdd1 and Trdd2) rearrangements in CD4−CD8− double-negative thymocyte progenitors differentiated in vitro from bone marrow–derived hematopoietic stem cells. We find that Trdd2 joins directly to Trdv, Trdd1, and Trdj segments, whereas Trdd1 joining is ordered with joining to Trdd2, a prerequisite for further rearrangement. We also find frequent, previously unappreciated, Trdd1 and Trdd2 rearrangements that inactivate Tcrd, including sequential rearrangements from V(D)J recombination signal sequence fusions. Moreover, we find dozens of RAG off-target sequences that are generated via RAG tracking both upstream and downstream from the Trdd2 recombination center across the Tcrd loop domain that is bounded by the upstream INT1-2 and downstream TEA elements. Disruption of the upstream INT1-2 boundary of this loop domain allows spreading of RAG on- and off-target activity to the proximal Trdv domain and, correspondingly, shifts the Tcrd V(D)J recombination landscape by leading to predominant V(D)J joining to a proximal Trdv3 pseudogene that lies just upstream of the normal boundary.
29
Lindsten, T., B. J. Fowlkes, L. E. Samelson, M. M. Davis, and Y. H. Chien. "Transient rearrangements of the T cell antigen receptor alpha locus in early thymocytes." Journal of Experimental Medicine 166, no. 3 (September 1, 1987): 761–75. http://dx.doi.org/10.1084/jem.166.3.761.
Анотація:
The dull Ly-1 double-negative (Ly-1dull, Lyt-2-, L3T4-) subpopulation appears to be the major precursor group of T lymphocytes in the thymus. In examining the status of the alpha, beta, and gamma chain genes for T cell receptors (TCR) in this population of cells and hybridomas made from them, we find that all of these loci appear to begin DNA rearrangements in a nearly simultaneous fashion. In the case of the gamma genes, these involve V gamma----J gamma C gamma gene rearrangements; with the beta chain genes, both D beta----J beta C beta rearrangement and V beta----D beta J beta C beta rearrangements are evident; and in the case of the alpha locus, assayed in part by pulsed-field gel electrophoresis, they take the form of a novel series of rearrangements occurring 80 kb or more 5' to the C alpha gene. These alpha locus rearrangements are well away from any of the J alpha gene segments found in cDNA clones to date and are deleted in most mature thymocytes and functional T cell lines. Therefore they appear to represent a distinct class of rearrangement that occurs before V alpha----J alpha joining. These distinctions between the character of the TCR gene rearrangements in these cells represent useful markers in further distinguishing different stages of T cell differentiation within this compartment of early T cells. In addition, the unexpected discovery of clonal rearrangements so far away from any of the expressed J alpha gene segments, and at a stage where there is little or no stable C alpha RNA present, has interesting implications for the hierarchy of TCR gene expression.
30
Krangel, M. S., H. Yssel, C. Brocklehurst, and H. Spits. "A distinct wave of human T cell receptor gamma/delta lymphocytes in the early fetal thymus: evidence for controlled gene rearrangement and cytokine production." Journal of Experimental Medicine 172, no. 3 (September 1, 1990): 847–59. http://dx.doi.org/10.1084/jem.172.3.847.
Анотація:
The rearrangement and expression of human T cell receptor (TCR)-gamma and -delta gene segments in clonal and polyclonal populations of early fetal and postnatal human TCR-gamma/delta thymocytes were examined. The data suggest that the TCR-gamma and -delta loci rearrange in an ordered and coordinated fashion. Initial rearrangements at the TCR-delta locus join V delta 2 to D delta 3, and initial rearrangements at the TCR-gamma locus join downstream V gamma gene segments (V gamma 1.8 and V gamma 2) to upstream J gamma gene segments associated with C gamma 1. These rearrangements are characterized by minimal junctional diversity. At later times there is a switch at the TCR-delta locus such that V delta 1 is joined to upstream D delta gene segments, and a switch at the TCR-gamma locus such that upstream V gamma gene segments are joined to downstream J gamma gene segments associated with C gamma 2. These rearrangements are characterized by extensive junctional diversity. Programmed rearrangement explains in part the origin of discrete subpopulations of peripheral blood TCR-gamma/delta lymphocytes that have been defined in previous studies. In addition, cytokine production by early fetal and postnatal TCR-gamma/delta thymocyte clones was examined. Fetal thymocyte clones produced significant levels of IL-4 and IL-5 following stimulation, whereas postnatal thymocyte clones did not produce these cytokines. Thus, these cell populations may represent functionally distinct subsets as well.
31
Sun, Amy, Tatiana I. Novobrantseva, Maryaline Coffre, Susannah L. Hewitt, Kari Jensen, Jane A. Skok, Klaus Rajewsky, and Sergei B. Koralov. "VH replacement in primary immunoglobulin repertoire diversification." Proceedings of the National Academy of Sciences 112, no. 5 (January 21, 2015): E458—E466. http://dx.doi.org/10.1073/pnas.1418001112.
Анотація:
The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.
32
Engler, P., E. Klotz, and U. Storb. "N region diversity of a transgenic substrate in fetal and adult lymphoid cells." Journal of Experimental Medicine 176, no. 5 (November 1, 1992): 1399–404. http://dx.doi.org/10.1084/jem.176.5.1399.
Анотація:
The rearrangement of immunoglobulin (Ig) and T cell receptor (TCR) genes requires the activity of an as yet undefined V(D)J recombinase. One component of the recombinase appears to be a terminal transferase which may be involved in the addition of untemplated nucleotides (N regions) to the V(D)J joints. It has been observed that rearranged Ig and TCR genes isolated from fetal liver have few if any N regions, whereas in the adult mouse, these genes have a large number of untemplated nucleotides. The presence of N regions greatly alters the composition of the third hypervariable, complementarity determining region of the respective proteins, thus playing a major role in the conformation of the binding site. It was possible that, for functional reasons, N region-containing Ig and TCR genes were not permissible at the fetal stage of development. We have produced transgenic mice with a rearrangement test gene which, after V-J recombination, does not result in the production of functional Ig or TCR proteins. We report here that the rearrangement products have no N regions in fetal liver, but that the majority of joints in adult lymphoid tissues have N additions. The study is also an interesting demonstration of the randomness of rearrangements and the enormous variability that can be created from a single pair of V and J sequences.
33
Okada, A., M. Mendelsohn, and F. Alt. "Differential activation of transcription versus recombination of transgenic T cell receptor beta variable region gene segments in B and T lineage cells." Journal of Experimental Medicine 180, no. 1 (July 1, 1994): 261–72. http://dx.doi.org/10.1084/jem.180.1.261.
Анотація:
We have tested the ability of the T cell receptor beta (TCR-beta) transcriptional enhancer (E beta) to confer transcriptional activation and tissue-specific V(D)J recombination of TCR-beta V, D, and J segments in a transgenic minilocus recombination substrate. We find that the minimal E beta element, as previously shown for a DNA segment that contained the E mu element, promotes a high level of substrate D to J beta rearrangement in both B and T cells, but only promotes V beta to DJ beta rearrangement in T cells. Thus, both the E mu and E beta elements similarly direct V(D)J recombination of this substrate in vivo, supporting a general role for transcriptional enhancers in the normal regulation of this rearrangement process. Surprisingly, however, we found that both the V beta and DJ beta portion of the constructs were transcribed in an enhancer-dependent fashion (conferred by either E mu or E beta) in both B and T lineage cells, including normal precursor B cells propagated in culture. These findings indicate that, at least in some contexts, transcriptional activation, per se, is not sufficient to confer V(D)J recombinational accessibility to a substrate V gene segment.
34
Fish, S. M., and M. J. Bosma. "Abnormal deletions in the T-cell receptor delta locus of mouse thymocytes." Molecular and Cellular Biology 14, no. 7 (July 1994): 4455–64. http://dx.doi.org/10.1128/mcb.14.7.4455.
Анотація:
Separate genetic elements (V, D, and J) encode the variable regions of lymphocyte antigen receptors. During early lymphocyte differentiation, these elements rearrange to form contiguous coding segments (VJ and VDJ) for a diverse array of variable regions. Rearrangement is mediated by a recombinase that recognizes short DNA sequences (signals) flanking V, D, and J elements. Signals flank both the 5' and 3' sides of each D element, thereby allowing assembly of a functional VDJ gene. However, in rearrangements involving the D delta 2 and J delta 1 elements of the mouse T-cell receptor delta (TCR delta) locus, we unexpectedly found that the D delta 2 element and a portion of its 5' signal are often deleted. Approximately 50% of recovered D delta 2 to J delta 1 rearrangements from thymocytes of adult wild-type mice showed such deletions. An additional 20% of the rearrangements contained standard D delta 2-J delta 1 coding junctions but showed some loss of nucleotides from the 5' D delta 2 signal. This loss was clearly associated with another event involving a site-specific cleavage at the 5' signal/coding border of D delta 2 and rejoining of the modified signal and coding ends. The abnormal loss of D delta 2 and a portion of the 5' D delta 2 signal was infrequently observed in D delta 2-to-J delta 1 rearrangements recovered from neonatal mice. The possible basis and significance of this age-dependent phenomenon are discussed.
35
Fish, S. M., and M. J. Bosma. "Abnormal deletions in the T-cell receptor delta locus of mouse thymocytes." Molecular and Cellular Biology 14, no. 7 (July 1994): 4455–64. http://dx.doi.org/10.1128/mcb.14.7.4455-4464.1994.
Анотація:
Separate genetic elements (V, D, and J) encode the variable regions of lymphocyte antigen receptors. During early lymphocyte differentiation, these elements rearrange to form contiguous coding segments (VJ and VDJ) for a diverse array of variable regions. Rearrangement is mediated by a recombinase that recognizes short DNA sequences (signals) flanking V, D, and J elements. Signals flank both the 5' and 3' sides of each D element, thereby allowing assembly of a functional VDJ gene. However, in rearrangements involving the D delta 2 and J delta 1 elements of the mouse T-cell receptor delta (TCR delta) locus, we unexpectedly found that the D delta 2 element and a portion of its 5' signal are often deleted. Approximately 50% of recovered D delta 2 to J delta 1 rearrangements from thymocytes of adult wild-type mice showed such deletions. An additional 20% of the rearrangements contained standard D delta 2-J delta 1 coding junctions but showed some loss of nucleotides from the 5' D delta 2 signal. This loss was clearly associated with another event involving a site-specific cleavage at the 5' signal/coding border of D delta 2 and rejoining of the modified signal and coding ends. The abnormal loss of D delta 2 and a portion of the 5' D delta 2 signal was infrequently observed in D delta 2-to-J delta 1 rearrangements recovered from neonatal mice. The possible basis and significance of this age-dependent phenomenon are discussed.
36
Hikida, Masaki, та Hitoshi Ohmori. "Rearrangement of λ Light Chain Genes in Mature B Cells In Vitro and In Vivo. Function of Reexpressed Recombination-activating Gene (RAG) Products". Journal of Experimental Medicine 187, № 5 (2 березня 1998): 795–99. http://dx.doi.org/10.1084/jem.187.5.795.
Анотація:
V(D)J (V, variable; D, diversity; J, joining) combination of immunoglobulin (Ig) genes established in premature B cells has been thought to be conserved throughout differentiation at mature stages. However, germinal center (GC) B cells have been shown to reexpress recombination-activating gene (RAG)-1 and RAG-2 proteins in immunized mice. Here, we present several lines of evidence indicating that RAG proteins thus induced are functional as the V(D)J recombinase. DNA excision product reflecting Vλ1 to Jλ1 rearrangement was generated in parallel with the expression of RAG genes in mature mouse B cells that were activated in vitro with LPS and IL-4. Similar λ chain gene rearrangement was observed in the draining lymph node of immunized mice. Further, B cells that underwent λ gene rearrangement were shown by in situ PCR to be localized within GCs. Thus, secondary rearrangement of Ig genes (receptor editing) can occur in mature B cells.
37
Hsu, Lih-Yun, Hong-Erh Liang, Kristen Johnson, Chulho Kang, and Mark S. Schlissel. "Pax5 Activates Immunoglobulin Heavy Chain V to DJ Rearrangement in Transgenic Thymocytes." Journal of Experimental Medicine 199, no. 6 (March 8, 2004): 825–30. http://dx.doi.org/10.1084/jem.20032249.
Анотація:
Mice deficient for the B cell–restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5−/− pro–B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in β-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.
38
Nakajima, P. B., and M. J. Bosma. "Characterization of excised DNA intermediates associated with V(D)J recombination at the T-cell receptor delta locus." Molecular and Cellular Biology 17, no. 5 (May 1997): 2631–41. http://dx.doi.org/10.1128/mcb.17.5.2631.
Анотація:
Lymphocyte development requires the assembly of antigen receptor genes through the specialized process of V(D)J recombination. This process is initiated by cleavage at the junction between coding segments (V, D, and J) and the recombination signal sequences that border these segments, resulting in generation of double-strand break intermediates. We have used a two-dimensional gel system to characterize broken molecules arising from V(D)J recombination at the T-cell receptor (TCR) delta locus and have identified linear species excised by Ddelta1-Ddelta2 and V-Ddelta2 rearrangement in thymus DNA. Relatively few (approximately 10) V-Ddelta2-excised linear species were detected in DNA from fetal thymocytes. The sizes of these species corresponded to the estimated distances between Ddelta2 and the V gene segments utilized by gammadelta T cells and indicated that both Ddelta2-proximal and -distal V gene segments are targeted for V-Ddelta2 rearrangement. Similar-sized species were observed in DNA from thymocytes of scid mice in which T-cell development is arrested prior to TCR expression. Since previous studies suggest that the TCR alpha/delta locus encodes more than 100 V gene segments, our results indicate that a few select V gene segments are predominantly targeted for rearrangement to Ddelta2, and this primarily accounts for the restricted Vdelta gene repertoire of gammadelta T cells.
39
Breit, TM, IL Wolvers-Tettero, A. Beishuizen, MA Verhoeven, ER van Wering, and JJ van Dongen. "Southern blot patterns, frequencies, and junctional diversity of T-cell receptor-delta gene rearrangements in acute lymphoblastic leukemia." Blood 82, no. 10 (November 15, 1993): 3063–74. http://dx.doi.org/10.1182/blood.v82.10.3063.3063.
Анотація:
Abstract Southern blot analysis of T-cell receptor (TCR)-delta gene rearrangements is useful for diagnostic studies on the clonality of lymphoproliferative diseases. We have developed 18 new TCR-delta gene probes by use of the polymerase chain reaction (PCR) techniques. Application of these probes for detailed analysis of the TCR-delta genes in normal control samples, 138 T-cell acute lymphoblastic leukemias (T-ALL), and 91 precursor B-ALL allowed us to determine the TCR-delta gene restriction map for five restriction enzymes, as well as the Southern blot restriction enzyme patterns of all theoretically possible TCR-delta gene rearrangements. Based on this information, it appeared that 97% of all 213 detected TCR-delta gene rearrangements in our series of ALL could be detected by use of the TCRDJ1 probe and that the majority (76%) of the 213 rearrangements could be identified precisely. In T-ALL, we found a strong preference for the complete rearrangements V delta 1-J delta 1 (33%), V delta 2-J delta 1 (10%), and V delta 3-J delta 1 (7%) and the incomplete rearrangement D delta 2- J delta 1 (11%). In precursor B-ALL, the majority of rearrangements consisted of V delta 2-D delta 3 (72%) and D delta 2-D delta 3 (10%). The junctional diversity of these 6 preferential TCR-delta rearrangements was analyzed and showed an extensive junctional insertion (approximately 30 nucleotides) for complete V delta-J delta rearrangements, whereas incomplete rearrangements had correspondingly smaller junctional regions. The detailed TCR-delta gene restriction map and probes presented here, in combination with the Southern blot patterns of TCR-delta gene rearrangements, are important for TCR-delta gene studies in ALL; all TCR-delta gene rearrangements can be detected and the majority can be identified precisely. Identification of rearrangements is a prerequisite for subsequent PCR analysis of TCR- delta gene junctional regions, eg, for detection of minimal residual disease during follow-up of ALL patients.
40
Breit, TM, IL Wolvers-Tettero, A. Beishuizen, MA Verhoeven, ER van Wering, and JJ van Dongen. "Southern blot patterns, frequencies, and junctional diversity of T-cell receptor-delta gene rearrangements in acute lymphoblastic leukemia." Blood 82, no. 10 (November 15, 1993): 3063–74. http://dx.doi.org/10.1182/blood.v82.10.3063.bloodjournal82103063.
Анотація:
Southern blot analysis of T-cell receptor (TCR)-delta gene rearrangements is useful for diagnostic studies on the clonality of lymphoproliferative diseases. We have developed 18 new TCR-delta gene probes by use of the polymerase chain reaction (PCR) techniques. Application of these probes for detailed analysis of the TCR-delta genes in normal control samples, 138 T-cell acute lymphoblastic leukemias (T-ALL), and 91 precursor B-ALL allowed us to determine the TCR-delta gene restriction map for five restriction enzymes, as well as the Southern blot restriction enzyme patterns of all theoretically possible TCR-delta gene rearrangements. Based on this information, it appeared that 97% of all 213 detected TCR-delta gene rearrangements in our series of ALL could be detected by use of the TCRDJ1 probe and that the majority (76%) of the 213 rearrangements could be identified precisely. In T-ALL, we found a strong preference for the complete rearrangements V delta 1-J delta 1 (33%), V delta 2-J delta 1 (10%), and V delta 3-J delta 1 (7%) and the incomplete rearrangement D delta 2- J delta 1 (11%). In precursor B-ALL, the majority of rearrangements consisted of V delta 2-D delta 3 (72%) and D delta 2-D delta 3 (10%). The junctional diversity of these 6 preferential TCR-delta rearrangements was analyzed and showed an extensive junctional insertion (approximately 30 nucleotides) for complete V delta-J delta rearrangements, whereas incomplete rearrangements had correspondingly smaller junctional regions. The detailed TCR-delta gene restriction map and probes presented here, in combination with the Southern blot patterns of TCR-delta gene rearrangements, are important for TCR-delta gene studies in ALL; all TCR-delta gene rearrangements can be detected and the majority can be identified precisely. Identification of rearrangements is a prerequisite for subsequent PCR analysis of TCR- delta gene junctional regions, eg, for detection of minimal residual disease during follow-up of ALL patients.
41
Lewis, S. M., E. Agard, S. Suh, and L. Czyzyk. "Cryptic signals and the fidelity of V(D)J joining." Molecular and Cellular Biology 17, no. 6 (June 1997): 3125–36. http://dx.doi.org/10.1128/mcb.17.6.3125.
Анотація:
V(D)J recombination is responsible for the de novo creation of antigen receptor genes in T- and B-cell precursors. To the extent that lymphopoiesis takes place throughout an animal's lifetime, recombination errors present an ongoing problem. One type of aberrant rearrangement ensues when DNA sequences resembling a V(D)J joining signal are targeted by mistake. This study investigates the type of sequence likely to be subject to mistargeting, the level of joining-signal function associated with these sequences, and the number of such cryptic joining signals in the genome.
42
Cibotti, R., J. P. Cabaniols, C. Pannetier, C. Delarbre, I. Vergnon, J. M. Kanellopoulos, and P. Kourilsky. "Public and private V beta T cell receptor repertoires against hen egg white lysozyme (HEL) in nontransgenic versus HEL transgenic mice." Journal of Experimental Medicine 180, no. 3 (September 1, 1994): 861–72. http://dx.doi.org/10.1084/jem.180.3.861.
Анотація:
We have previously produced a transgenic mouse line for hen egg lysozyme (HEL), an experimental model for analyzing tolerance to self-antigens at the peptide level. We have now characterized transgenic mice with HEL blood levels below 2 ng/ml, where significant T cell proliferative responses to HEL and its immunodominant peptide were observed. This HEL-low transgenic model was chosen because it mimics physiological conditions in which autoreactive T lymphocytes, recognizing self-components expressed at very low levels, persist without inducing a break in tolerance. Furthermore, in H-2d mice, HEL-specific T lymphocytes are triggered by a single immunodominant region, allowing us to compare the HEL-specific T cell V beta repertoires of transgenic and nontransgenic animals against a single peptide presented as self or foreign, respectively. We found that a V beta 8.2-D beta 1-J beta 1.5 rearrangement is found in response to HEL in all nontransgenic mice, whereas this V beta-restricted response is absent in HEL-low transgenic animals. At the nucleotide level, this rearrangement results from the trimming of the genomic segments during VDJ or DJ joining, without N additions, suggesting that the dominant rearrangement is selected early during fetal or neonatal life, before the expression of terminal deoxynucleotidyl transferase. In HEL-low transgenic mice, no dominant rearrangements are found as alternatives to the one observed in normal mice. Instead, each transgenic animal uses a different set of V beta-J beta combinations in its response to the immunodominant HEL peptide. In nontransgenic mice, besides the dominant V beta 8.2-D beta 1-J beta 1.5 combination, minor V beta repertoires were found which differed in each animal and were distinct from the rearrangements used by individual transgenic mice. These findings suggest that the T cell response to an immunodominant peptide involves a "public" V beta repertoire found in all animals and a "private" one which is specific to each individual.
43
Ghosh, Jimut Kanti, William J. Romanow та Cornelis Murre. "Induction of a Diverse T Cell Receptor γ/δ Repertoire by the Helix-Loop-Helix Proteins E2a and Heb in Nonlymphoid Cells". Journal of Experimental Medicine 193, № 6 (19 березня 2001): 769–76. http://dx.doi.org/10.1084/jem.193.6.769.
Анотація:
During specific stages of thymocyte development, the T cell receptor (TCR) locus is assembled from variable (V), diversity (D), and joining (J) gene segments. Proper TCR γ and δ V(D)J rearrangement during thymocyte development requires the presence of the E2A proteins. Here we show that E2A and a closely related protein, HEB, in the presence of recombination activating gene (RAG)1 and RAG2, each have the ability to activate TCR γ and δ rearrangement in human kidney cells. The coding joints are diverse, contain nucleotide deletions, and occasionally show the presence of P nucleotides. Interestingly, only a subset of V, D, and J segments are targeted by the E2A and HEB proteins. Thus, E2A and HEB permit localized accessibility of the TCR γ and δ loci to the recombination machinery. These data indicate that a distinct but diverse TCR repertoire can be induced in nonlymphoid cells by the mere presence of the V(D)J recombinase and the transcriptional regulators, E2A and HEB.
44
Fondell, J. D., and K. B. Marcu. "Transcription of germ line V alpha segments correlates with ongoing T-cell receptor alpha-chain rearrangement." Molecular and Cellular Biology 12, no. 4 (April 1992): 1480–89. http://dx.doi.org/10.1128/mcb.12.4.1480.
Анотація:
M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombination-activating genes RAG-1 and RAG-2 are differentially expressed, with RAG-2 detectable only by polymerase chain reaction, implying that very low levels of one of these gene products are sufficient to complement the other to facilitate VJ rearrangements. These findings provide the first direct evidence for an accessibility model of antigen receptor rearrangement in T cells.
45
Fondell, J. D., and K. B. Marcu. "Transcription of germ line V alpha segments correlates with ongoing T-cell receptor alpha-chain rearrangement." Molecular and Cellular Biology 12, no. 4 (April 1992): 1480–89. http://dx.doi.org/10.1128/mcb.12.4.1480-1489.1992.
Анотація:
M14T is a virally transformed immature T-cell line which continues to rearrange its T-cell antigen receptor (TCR) alpha-chain genes in vitro and thus represents a dynamic system for studying TCR assembly. In an effort to investigate whether the TCR alpha locus is accessible for V(D)J rearrangement events, we examined M14T cells for the presence of germ line TCR alpha transcripts. Several unrearranged V alpha segments were found to be transcriptionally active in M14T cells. By comparison, germ line V alpha transcripts are absent in nonlymphoid and pro-T-cell lines and barely detectable in mature T-cell lines, suggesting that this phenomenon is likely stage and tissue specific. We demonstrate a perfect correlation between transcriptionally active V alpha segments and their involvement in ongoing V alpha-to-J alpha rearrangements. In addition, data suggesting that the unrearranged J alpha locus is also transcriptionally active in the M14T line are presented. Furthermore, the recombination-activating genes RAG-1 and RAG-2 are differentially expressed, with RAG-2 detectable only by polymerase chain reaction, implying that very low levels of one of these gene products are sufficient to complement the other to facilitate VJ rearrangements. These findings provide the first direct evidence for an accessibility model of antigen receptor rearrangement in T cells.
46
Sollbach, A. E., and G. E. Wu. "Inversions produced during V(D)J rearrangement at IgH, the immunoglobulin heavy-chain locus." Molecular and Cellular Biology 15, no. 2 (February 1995): 671–81. http://dx.doi.org/10.1128/mcb.15.2.671.
Анотація:
Diversity in immunoglobulin antigen receptors is generated in part by V(D)J recombination. In this process, different combinations of gene elements are joined in various configurations. Products of V(D)J recombination are coding joints, signal joints, and hybrid junctions, which are generated by deletion or inversion. To determine their role in the generation of diversity, we have examined two sorts of recombination products, coding joints and hybrid junctions, that have formed by inversion at the mouse immunoglobulin heavy-chain locus. We developed a PCR assay for quantification and characterization of inverted rearrangements of DH and JH gene elements. In primary cells from adult mice, inverted DJH rearrangements are detectable but they are rare. There were approximately 1,100 to 2,200 inverted DJH coding joints and inverted DJH hybrid junctions in the marrow of one adult mouse femur. On day 16 of gestation, inverted DJH rearrangements are more abundant. There are approximately 20,000 inverted DJH coding joints and inverted DJH hybrid junctions per day 16 fetal liver. In fetal liver cells, the number of inverted DJH rearrangements remains relatively constant from day 14 to day 16 of gestation. Inverted DJH rearrangements to JH4, the most 3' JH element, are more frequently detected than inverted DJH rearrangements to other JH elements. We compare the frequencies of inverted DJH rearrangements to previously determined frequencies of uninverted DJH rearrangements (DJH rearrangements formed by deletion). We suggest that inverted DJH rearrangements are influenced by V(D)J recombination mechanistic constraints and cellular selection.
47
Liyanage, Marek, Zoë Weaver, Carrolee Barlow, Allen Coleman, Daniel G. Pankratz, Stacie Anderson, Anthony Wynshaw-Boris та Thomas Ried. "Abnormal rearrangement within the α/δ T-cell receptor locus in lymphomas from Atm-deficient mice". Blood 96, № 5 (1 вересня 2000): 1940–46. http://dx.doi.org/10.1182/blood.v96.5.1940.
Анотація:
Abstract Atm-deficient mice (Atm−/−) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm−/− mice. AllAtm−/− tumors are of thymic lymphoblastoid origin, display an immature CD3− and CD4+/CD8+ phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptorα/δ(Tcrα/δ) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% ofAtm−/− peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at theTcrα/δ locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcrα/δ locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks.
48
Liyanage, Marek, Zoë Weaver, Carrolee Barlow, Allen Coleman, Daniel G. Pankratz, Stacie Anderson, Anthony Wynshaw-Boris та Thomas Ried. "Abnormal rearrangement within the α/δ T-cell receptor locus in lymphomas from Atm-deficient mice". Blood 96, № 5 (1 вересня 2000): 1940–46. http://dx.doi.org/10.1182/blood.v96.5.1940.h8001940_1940_1946.
Анотація:
Atm-deficient mice (Atm−/−) recapitulate many aspects of the ataxia telangiectasia (AT) syndrome, including the susceptibility to tumors of lymphoid origin. To investigate the mechanism of tumorigenesis, we have examined a panel of 8 thymic lymphomas from Atm−/− mice. AllAtm−/− tumors are of thymic lymphoblastoid origin, display an immature CD3− and CD4+/CD8+ phenotype, and arise coincident with V(D)J recombination. Cytogenetically, all tumors are diploid or near diploid but exhibit multiple chromosome aberrations with an average of 4 abnormal chromosomes per tumor. All the tumors revealed chromosome 14 rearrangements precisely at the T-cell receptorα/δ(Tcrα/δ) locus, suggesting the involvement of V(D)J recombination in these translocations. In addition, 11.5% ofAtm−/− peripheral T cells showed chromosome 14 translocations, suggesting that rearrangements at theTcrα/δ locus occur early during tumor development in the absence of ATM. However, additional genetic aberrations are required for tumorigenesis. For example, translocations involving chromosome 12, often with chromosome 14 (more than 60%), and partial or complete trisomy of chromosome 15, with copy number increases of the c-myc oncogene were frequently observed. These observations suggest that ATM is required for normal rearrangement of the Tcrα/δ locus but not for V(D)J recombination at other loci. The mechanisms that lead to tumorigenesis may be due to the involvement of ATM in monitoring double-stranded DNA breaks.
49
McMurry, M. T., C. Hernandez-Munain, P. Lauzurica, and M. S. Krangel. "Enhancer control of local accessibility to V(D)J recombinase." Molecular and Cellular Biology 17, no. 8 (August 1997): 4553–61. http://dx.doi.org/10.1128/mcb.17.8.4553.
Анотація:
We have studied the role of transcriptional enhancers in providing recombination signal sequence (RSS) accessibility to V(D)J recombinase by examining mice carrying a transgenic human T-cell receptor (TCR) delta gene minilocus. This transgene is composed of unrearranged variable (Vdelta and Vdelta2), diversity (Ddelta3), joining (Jdelta1 and Jdelta3), and constant (Cdelta) gene segments. Previous data indicated that with the TCR delta enhancer (Edelta) present in the Jdelta3-Cdelta intron, V(D)J recombination proceeds stepwise, first V to D and then VD to J. With the enhancer deleted or mutated, V-to-D rearrangement is intact, but VD-to-J rearrangement is inhibited. We proposed that Edelta is necessary for J segment but not D segment accessibility and that J segment inaccessibility in the enhancerless minilocus resulted in the observed V(D)J recombination phenotype. In this study, we tested this notion by using ligation-mediated PCR to assess the formation of recombination-activating gene (RAG)-dependent double-strand breaks (DSBs) at RSSs 3' of Ddelta3 and 5' of Jdelta1. In five lines of mice carrying multicopy integrants of constructs that either lacked Edelta or carried an inactivated Edelta, the frequency of DSBs 5' of Jdelta1 was dramatically reduced relative to that in the wild type, whereas the frequency of DSBs 3' of Ddelta3 was unaffected. We interpret these results to indicate that Edelta is required for Jdelta1 but not Ddelta3 accessibility within the minilocus, and we conclude that enhancers regulate V(D)J recombination by providing local accessibility to the recombinase. cis-acting elements other than Edelta must maintain Ddelta3 in an accessible state in the absence of Edelta. The analysis of DSB formation in a single-copy minilocus integrant indicates that efficient DSB formation at the accessible RSS 3' of Ddelta3 requires an accessible partner RSS, arguing that RSS synapsis is required for DSB formation in chromosomal substrates in vivo.
50
Wang, Chiyu, Molly A. Bogue, Jonathan M. Levitt, and David B. Roth. "Irradiation-Mediated Rescue of T Cell–Specific V(D)j Recombination and Thymocyte Differentiation in Severe Combined Immunodeficient Mice by Bone Marrow Cells." Journal of Experimental Medicine 190, no. 9 (November 1, 1999): 1257–62. http://dx.doi.org/10.1084/jem.190.9.1257.
Анотація:
In SCID (severe combined immunodeficient) mice, proper assembly of immunoglobulin and T cell receptor (TCR) genes is blocked by defective V(D)J recombination so that B and T lymphocyte differentiation is arrested at an early precursor stage. Treating the mice with gamma irradiation rescues V(D)J rearrangement at multiple TCR loci, promotes limited thymocyte differentiation, and induces thymic lymphomas. These effects are not observed in the B cell lineage. Current models postulate that irradiation affects intrathymic T cell precursors. Surprisingly, we found that transfer of irradiated SCID bone marrow cells to unirradiated host animals rescues both TCR rearrangements and thymocyte differentiation. These data indicate that irradiation affects precursor cells at an earlier stage of differentiation than was previously thought and suggest new models for the mechanism of irradiation rescue.