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The Journal of Immunology, 1999, 162: 903-910.
Copyright © 1999 by The American Association of Immunologists

The Mechanism of Chromosome 7 Inversion in Human Lymphocytes Expressing Chimeric {gamma}ß TCR1

Christelle Retière*, Franck Halary*, Marie-Alix Peyrat*, Françoise Le Deist{dagger}, Marc Bonneville* and Marie-Martine Hallet2,*

* Institut National de la Santé et de la Recherche Médicale U463, Institut de Biologie, Nantes, France; and {dagger} Institut National de la Santé et de la Recherche Médicale U132, Hopital Necker, Enfants Malades, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Functional chimeric TCR chains, encoded by V{gamma}J{gamma}Cß or V{gamma}JßCß hybrid gene TCR, are expressed at the surface of a small fraction of {alpha}ß T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR {gamma} and ß loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between Dß and J{gamma} genic segments generates C{gamma}Vß and CßV{gamma} hybrid loci. Within the C{gamma}Vß locus, secondary rearrangements between V{gamma} and J{gamma} or V{gamma} and Jß elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the CßV{gamma} locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same Dß-3' recombination signal sequence can be used in successive rearrangements with J{gamma} and Jß genic segments, thus showing that a signal joint has been involved in a secondary recombination event.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Acommon recombination system mediates Ig and TCR gene rearrangements in lymphoid cells (for a review see 1 . In each locus, the Rag1/Rag2 protein complex recognizes and cleave recombination signal sequence (RSS)3 consisting of highly conserved heptamer and nonamer motifs and a spacer region of 12 or 23 nucleotides (2, 3, 4, 5). These sequences are found at the 3' end of V segments, the 5' end of J segments, and at both ends of D segments of Ig and TCR loci.

Ig and TCR gene rearrangements occur in a cell lineage-dependent fashion (i.e., in B and T cells, respectively) at specific developmental stages. The mechanisms controlling activation of recombination involve both regulation of expression of members of the recombination complex (e.g., Rag1/Rag2 proteins are detected almost exclusively in immature lymphoid cells) and control of Ig and TCR locus accessibility (2, 6, 7, 8, 9, 10, 11). Although such a process classically involves RSS from the same locus, strong conservation of the Ig and TCR recombination machinery predisposes immature lymphoid cells to recombinations between different loci through translocation or inversion events (12, 13, 14, 15, 16, 17, 18, 19, 20). In this respect, "trans" rearrangements involving elements belonging to Ig and TCR or to distinct TCR loci have been described in both normal or malignant lymphoid cells. In particular, hybrid Ag-receptor genes formed by interlocus recombination between TCR {gamma} and ß elements are detected in PBL from normal individuals, and their occurrence is greatly increased (10- to 100-fold) in patients with the inherited disease ataxia-telangiectasia (A-T) (21, 22, 23, 24, 25).

We recently demonstrated that hybrid genes formed by interlocus recombination between V{gamma} and (D)Jß elements can generate chimeric functional chains at the surface of normal {alpha}ß T lymphocytes (26). Nevertheless, it could not be established whether diverse hybrid TCR {gamma}ß productive rearrangements resulted from common inversion 7 (inv(7)) (p15;q35) breakpoints involving or not classical heptamer/nonamer RSS flanking TCR elements or directly from diverse recombination events between V{gamma} and (D)Jß elements. In the present study, we have located the inv(7) (p15;q35) breakpoints of T cell clones expressing chimeric V{gamma}Cß-chains by restriction map analysis. By amplifying and sequencing ß{gamma} junctional flanking segments, we demonstrated that productive hybrid V{gamma}Cß rearrangements are preceded by translocation events involving classical RSS flanking 3'-Dß and diverse J{gamma} elements. Our results suggested that secondary rearrangements occurred in the VßC{gamma} locus of A-T lymphocytes expressing hybrid V{gamma}Cß TCR. Finally, we provide evidence for the occurrence of productive rearrangements involving signal joints in one T cell clone.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells

Lines 36, 37, 70, 71, 73, 76, and 83 are V{gamma}2, 3, or 4+ {alpha}ß+. They were previously sorted from 2 to 5 x 106 healthy donor PBL with 23D12 (anti-V{gamma}2–3-4) and BMA031 (pan ß) mAb as previously described (26, 27, 28, 29). The estimated efficiency of this commonly used sorting method was 30–70%. T cell clones expressing chimeric {gamma}ß-chains were generated from these lines by limiting dilution (26, 30), and the sequences of their productive V{gamma}(DJ)Cß and V{alpha}J{alpha}C{alpha} transcripts have been described elsewhere (26). The probability of monoclonality for growing colonies was >95%. With this method, no bias was observed during the short bulk culture preceding the cloning, and the plating efficiency was around 70%. Lines 101, 102, 103, 104, 105, 106, and 107 are also V{gamma}2, 3, or 4+ {alpha}ß+. These lines were sorted from A-T donor PBL and cultured in the same way. Cellular clones were generated from lines 104 and 105 by limiting dilution. Each T cell clone was referred to by a PBL sorted line number and a clone-specific number.

Southern blot analysis

DNA extracted from clone cells was digested by EcoRI or KpnI and Southern blots were conducted as previously described (31) with Hybond N+ transfer membrane (Amersham, Les Ulis, France). Hybridizations were performed with the J{gamma} probe pH60 containing 700 bp HindIII-EcoRI from the J{gamma}1 genomic region (32, 33, 34). This probe cross-hybridizes with the J{gamma}2 but not with the additional J{gamma} segments. Southern blots were also performed on PCR-amplified DNA fragments corresponding to various Dß-J{gamma} breakpoint regions of normal and A-T-sorted lines. In this case, hybridizations were performed either with a Dß1 or with a Dß2 probe internal to the amplified target.

PCR on genomic DNA and sequencing of junctional regions

PCR (1 cycle of denaturation for 9 mn at 94°C, annealing for 1 mn at 58°C, and extension for 1 mn at 72°C; and 30 cycles of denaturation for 1 mn at 94°C, annealing for 1 mn at 58°C, and extension for 1 mn at 72°C) were conducted on 0.5 µg of DNA extracted from clones or from V{gamma}2, 3, or 4+-sorted PBL lines by using various Dß, Jß, and J{gamma} primers, which are reported in Table IGo. DNA fragments amplified from clones were directly sequenced according to the USB kit procedure (USB, Cleveland, OH). Those obtained from cell lines were cloned in pBlueScript KS+ (Stratagene, La Jolla, CA), and sequences were conducted on recombinant plasmids purified from bacterial clones.


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  		Table I. Primers for PCR and sequencing

 
Amplification and sequencing of TCR transcripts

Preparation of A-T T cell clone RNA, reverse transcription, and PCR amplification were performed as previously described (29) using V{gamma}I (27) and Cß (26) primers. cDNA-amplified fragments were directly sequenced according to the USB kit procedure.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Localization and PCR amplification of inv(7) breakpoint regions

To characterize the primary recombination event(s) leading to the formation of hybrid TCR, Southern blots were conducted on four T cell clones derived from three healthy donors’ PBL and expressing V{gamma}Cß productive rearrangements. All clones carried common unassignable DNA fragments hybridizing with the J{gamma} probe pH60 in EcoRI (3 kb) and in KpnI (2.4 kb) digests (Fig. 1GoA and Table IIGo). Because a consequence of a V{gamma}Jß rearrangement is the exclusion of all J{gamma} segments from the V{gamma}Jß hybrid locus, these unassignable fragments presumably corresponded to the reciprocal VßC{gamma} inversion products, and their presence indicated that classical or cryptic recombination sequences involved in the recombination process were located either in the {gamma} genomic region hybridizing with the pH60 probe or in an upstream proximal flanking segment. Then, RSS flanking J{gamma}1 or J{gamma}2 elements were the likely targets for primary interlocus recombination events (Table IIGo).



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  		FIGURE 1. Characterization of inv(7) Vß-C{gamma} breakpoint region(s) in four trans-rearranged T-cell clones. A, Southern blot analysis of the four clones digested by EcoRI or KpnI and hybridized with the pH60 probe; #, points out the unattributable bands. B, Deduced restriction map and localization of the Vß-C{gamma} breakpoint region(s). C, Schematic representation of the two hypothetical chromosome 7 inversion events.

 

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  		Table II. Analysis of Southern blots performed from the DNA of trans-rearranged T cell clones

 
Using the above results and known restriction maps of the TCR ß and TCR {gamma} loci (34, 35), we then located the hypothetical inv(7) breakpoint in the ß locus in the vicinity of the Dß1 segment (Fig. 1GoB). The repercussions of such an inversion involving either the J{gamma}1 and Dß1 or the J{gamma}2 and Dß1 RSS on chromosome 7 structure are summarized in Fig. 1GoC. To test our hypothesis, PCR were conducted on DNA extracted from all available T cell clones carrying TCR ß/{gamma} trans rearrangements. Fragments of the expected size (0.4 kb) were amplified from all but three clones with DB1 and JG1/2 primers (Table IGo), which are located upstream of Dß1 and downstream of J{gamma}1 or 2 gene segments, respectively. Hence, occurrence of inversion events involving J{gamma}1/2 and Dß1 RSS in these cells was confirmed.

Three negative T cell clones (36.1, 36.6, and 36.17) did not exhibit the previously described 3-kb EcoRI and 2.4-kb KpnI unassignable bands when their blotted DNA was hybridized to the pH60 probe (data not shown). Instead, they all displayed common redundant bands of germline size, which were necessarily assigned to the VßC{gamma} hybrid locus of the inverted chromosome 7 (Table IIGo). By hypothesizing that Dß and J{gamma} classical RSS were also involved in the inversion mechanisms of these three clones, three possible restriction maps of the inversion region could be designed (Fig. 2Go). The only map consistent with the blotting results was obtained with a recombination event involving the RSS flanking the J{gamma}P1 segment (Fig. 2GoA). This hypothetical structure of the VßC{gamma} hybrid locus was confirmed by amplifying a 0.18-kb fragment in the three clones using DB1 and JP1 primers (Table IGo), the latter being localized downstream of the J{gamma}P1 genic element.



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  		FIGURE 2. Restriction maps of breakpoint regions generated from interlocus recombination between (A) Dß1 and J{gamma}P1, (B) Dß1 and J{gamma}P, and (C) Dß1 and J{gamma}P2 genic segments.

 
Sequence characteristics of the inv(7) ß{gamma} breakpoint regions

As expected, the sequences obtained from the PCR-amplified fragments confirmed that recombinations occurred either between a Dß1 and a J{gamma}2 segment (seven clones from five donors) or between a Dß1 and a J{gamma}P1 segment (three clones from one donor). They corresponded to a classical Dß1–3' and J{gamma} RSS assembly with junctional N diversity, which indicated that the inversion was mediated by "classical" Rag1/Rag2 recombination complexes (Table IIIGo). These results led us to conclude that the inversion recombination process had generated a signal joint formed by these RSS in the reciprocal hybrid V{gamma}Cß locus before productive rearrangements between V{gamma} and Jß. They excluded any use of the Dß1 gene segment, localized in the VßC{gamma} locus, in secondary recombination processes into the V{gamma}Cß locus.


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  		Table III. Sequences of inversion Vß-C{gamma} breakpoint regions in trans-rearranged T cell clones

 
Evidence of a signal joint involvement in a productive rearrangement

Among the 10 T cell clones studied, one of them (clone 71.13) exhibited several interesting features. Although its functional chimeric transcripts carried rearranged V{gamma}4J{gamma}P2 elements, their differential splicing involved Cß2 instead of Cß1 exon (Fig. 3GoB). This Cß1 exclusion from the 71.13 mature transcript suggested occurrence of a third recombination event leading to Cß1 deletion from the genomic DNA and consequently involving the 5' RSS of a genic element localized between Cß1 and Cß2 segments. Moreover, Southern blot analysis of 73.13-digested DNA revealed unassignable weak bands hybridizing to the J{gamma} probe pH60 in EcoRI (6.5 kb) and KpnI (5 kb) digests (Fig. 1Go and Table IIGo). This suggested the existence of genomic restriction fragments carrying short pH60 complementary sequences. Sequences presenting these characteristics were generated in the V{gamma}Cß locus of all cells presenting a J{gamma}1 or 2-Dß trans rearrangement and corresponded to the genomic regions localized upstream of signal joints formed with J{gamma}1 or two RSS. They were deleted in V{gamma}Jß secondary recombination process but remained at the 5' end of the signal joint generated from J{gamma}2-Dß inversion when a V{gamma}4J{gamma}P2 recombination occurred. Using the restrictions maps of the TCR ß and {gamma} loci, we could determine which rearrangement(s) could generate the above unassignable fragments. The only consistent hypothesis suggested the occurrence of a recombination event between the Dß1–3' RSS of the signal joint generated by the earlier inversion event and the Jß2.1 element. To address this, a PCR was conducted on the 71.13 clone DNA with J{gamma} and Jß2 primers (Table IGo) located upstream of J{gamma}2 and downstream of Jß2.1 RSS. Sequence analysis of the amplified fragment revealed a junctional region containing the nonamer motif and part of the spacer of the J{gamma}2 RSS associated to the Jß2–1 segment, with exonuclease trimming of the two DNA extremities and N nucleotide insertion (Fig. 3GoB). Therefore, the only explanation consistent with these results was that, after a previous interlocus recombination between the Dß1–3' and the J{gamma}2 RSS, two secondary rearrangements occurred in the C{gamma}Vß hybrid locus (Fig. 3GoA). One of them involved the 3' RSS of the signal joint formed by the Dß1J{gamma}2 inversion and the Jß2.1 RSS (deletion 1), and the other one involved V{gamma}4 and J{gamma}P2 elements (deletion 2 yielding a coding joint). Therefore, those data show the involvement of a signal joint in a secondary recombination event.



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  		FIGURE 3. Analysis of the recombination events leading to the structure of the TCRß and {gamma} loci in the 71–13 trans-rearranged T-cell clone. A, Schematic representation of the three rearrangements events that have occurred during differentiation of 71–13 T cells. B, Nucleotide sequences of junctional regions corresponding to the deletions 1 and 2. The nonamer sequence of the 5'-J{gamma}2 RSS is underlined.

 
Possible involvement of all the Dß and J{gamma} genic segments in the inv(7) process

To detect interlocus recombination events involving other Dß and/or J{gamma} RSS, PCR were conducted on genomic DNA extracted from V{gamma}2–3-4+-sorted lines from seven healthy and seven A-T donors. For these screening experiments, primers located upstream of Dß1 or 2 segments and downstream of RSS flanking the five J{gamma} elements were designed (Table IGo). To ensure that only authentic DßJ{gamma} fragments were scored, the PCR products were blotted and hybridized with Dß1 or Dß2 internal probes (Fig. 4Go, A and B). Our results, summarized in Table IVGo, show that all RSS flanking 3'-Dß and 5'-J{gamma} could be involved in such rearrangement processes, although preferential involvement of Dß1 and J{gamma}1 or 2 segments was noted, mainly in lines sorted from healthy donors. Nevertheless, not all possible inversion events were detected in a given sorted line, thus presumably attesting their marked oligoclonality.



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  		FIGURE 4. Southern blot detection of the various Dß-J{gamma} inversion events in V{gamma}2, 3, or 4+ PBL lines from healthy and A-T donors. PCR amplified fragments with the two Dß and the five J{gamma} primers are revealed by hybridization with an internal (A) Dß1 or (B) Dß2 probe.

 

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  		Table IV. PCR detection of Dß-J{gamma} inversion events in V{gamma}2, 3, or 4+ PBL lines from healthy and A-T donors

 
To test the occurrence of multiple inversion events involving the same Dß and J{gamma} elements, Dß2J{gamma}1 or 2 (normal line 73), Dß1J{gamma}1 or 2 (A-T line 103), and Dß1J{gamma}P (A-T line 105) junctional sequences were determined on bacterial clones obtained from suitable amplified fragments (Table VGo). Only one or two different clones were isolated from each analyzed DNA fragment, suggesting either that they were amplified from mono- or oligoclonal T lymphocyte subpopulations.


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  		Table V. Sequences of amplified and cloned Dß-J{gamma} fragments

 
Such results were consistent with previous data showing that cells expressing productively trans-rearranged TCR genes were infrequent in healthy donors (26). However, because V{gamma}Cß hybrid genes have been found 10- to 100-fold more frequently in A-T patients (22, 23, 24), we expected that the PCR products from V{gamma}Cß+ cell lines would have been highly diverse. In fact, only one or two DßJ{gamma} junctions were found in such PCR products (Table VGo). Therefore, we undertook further analysis of T cell clones generated from two A-T lines.

V{gamma}Cß and VßC{gamma} rearrangements in clones generated from two A-T PBL lines

Twenty-six cellular clones were generated from line 104 and 14 were generated from line 105. In all clones, cells expressed at their surface hybrid V{gamma}Cß TCR. As shown in Table VIGo, all clones obtained from a given line presented the same V{gamma}Cß productive trans rearrangement with poor nibbling and without N nucleotide addition. This result suggested clonal amplifications in populations from which these T cells were derived. Although DNA extracted from all clones were amplified with control primers, PCR performed with the oligonucleotides previously used to detect the various DßJ{gamma} inversion fragments in PBL lines were negative for all clones of line 104 and for six clones of line 105. However, Dß1J{gamma}P PCR fragments were obtained from the other eight clones generated from line 105. They presented the same junctional sequence with poor nibbling and without N nucleotide addition (Table VIGo). Other PCR trying were performed without success on the DNA purified from the negative clones to test the occurrence of direct generation of V{gamma}Cß productive genes by inversion (data not shown).


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  		Table VI. Analysis of V{gamma}-Cß and Vß-C{gamma} rearrangement status of A-T cellular clones generated from V{gamma}2, 3, or 4+ PBL Lines

 
These results suggested a frequent occurrence of secondary V(D)J rearrangements, resulting in a deletion of primary inversion events in the nonexpressed VßC{gamma} locus of A-T patients, and could explain the oligoclonal characteristics of the Dß-J{gamma} PCR targets in A-T-sorted lines.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The experiments presented in this report demonstrate that the trans rearrangements yielding functional V{gamma}Cß genes are preceded by a primary inv(7) event occurring between Dß-3' and J{gamma} recombination signal sequences. Although a bias for inclusion of Dß1 and J{gamma}1/2 genic elements is found in several cell lines, the two Dß1 and five J{gamma} are potential targets for this first step mechanism, which, as attested by exonuclease nibbling and nontemplated nucleotide insertion, is mediated by a classical activity of the Rag1/Rag2 complex.

During lymphocyte development, this recombinase activity, present in all lymphoid cells, is primarily controlled by the accessibility of RSS-flanking genic elements of Ig and TCR loci. This accessibility is regulated via cis-acting elements such as promoters, enhancers, and silencers and has been shown to correlate with DNA hypomethylation, changes in chromatin structure, and transcriptional activation (6, 7, 8, 9, 10, 11). Some years ago, it has been postulated that during T cell differentiation, both V{gamma}J{gamma} and DßJß rearrangements occur simultaneously (36, 37). This synchronism in accessibility of {gamma} and ß loci allows occurrence of the observed inversion events at presumably low frequencies, part of which only yielding functional products. Data obtained from a mouse model have shown a preferential Dß to Jß joining in interallelic trans rearrangements (38). The DßJ{gamma} structure of the inv(7) junctions is consistent with these results and with the timing of conventional cis rearrangements during thymocyte differentiation.

In our study, the lack of secondary recombination events within the CßV{gamma} locus allowed us to characterize DßJ{gamma} inversion breakpoints in the ten analyzed clones generated from healthy donors. This result suggests that, at least in these cells, rearrangements were blocked in all the nonexpressed CßV{gamma} hybrid genes. In the TCR ß locus, complete V to (D)J rearrangements appear to be controlled by specific cis-regulating elements (9, 39, 40). The lack of ß enhancer in the 3' region flanking the Cß-V{gamma} locus could prevent occurrence of complete rearrangements and explain our data. As the TCR ß V domain is not necessary to induce a signal for allelic exclusion (41), another possible explanation is that the expressed V{gamma}Cß-chain could mediate inhibition of further rearrangements in both the reciprocal VßC{gamma} locus and the ß locus on the other allele.

The frequency of V{gamma}Cß TCR genes formed by interlocus recombination was shown to be 10- to 100-fold higher in A-T patients (22, 23, 24) and 5- to 10-fold higher in agriculture workers exposed to pesticides than in healthy donors (42). This marked increase of hybrid gene frequency was shown to correlate with predisposition to lymphoid malignancies, and was thus considered as a nonpathogenic epiphenomenon linked to dysregulation of DNA repair processes (42, 43, 44). An assay based on measurement of the frequency of such hybrid Ag-receptor genes in PBL has been proposed to identify populations at risk for lymphocyte-specific genomic instability (42).

The basis for increased frequency of interlocus recombinations in the PBL of A-T patients is unknown, and explanations concerning abnormal V(D)J recombinase activity, perturbation of chromatine structure, or failure to eliminate lymphocytes carrying hybrid TCR genes have been proposed (42, 44). It is not clear at that stage whether the amplified T cell subsets harboring TCR trans rearrangements in A-T patients carry or not the same developmental and physiological characteristics than those of healthy individuals. Unexpectedly in the present study, the frequency of PCR-amplified DßJ{gamma} inversion events were very similar in lines sorted from healthy and A-T donors. An analysis of the status of both reciprocal hybrid loci in cellular clones generated from two A-T patients indicated dominance of clones expressing the same productive V{gamma}Cß rearrangement and strongly suggested that secondary V(D)J recombinations were not blocked in VßC{gamma} loci. Although we could not formally exclude that clonal amplifications resulted from in vitro selection, the introduction of a bias due to our sorting or culture protocols seemed quite unlikely and thus the amplified cells were probably already over-represented in vivo. Clonal amplifications of T cells subsets presenting cytogenetics defects have been described in A-T patients (for reviews see Refs. 41 and 42), and, as suggested by analysis of RT-PCR V{gamma}Cß products cloned in plasmid vectors (23), such amplifications could affect V{gamma}Cß sequence diversity within peripheral lymphocytes. Nevertheless, by demonstrating the existence of several V{gamma}Cß cDNA clones exhibiting an in-frame unique sequence, this previous study also suggested that the cell subset expressing V{gamma}Cß was heterogeneous (23). Such heterogeneity was not revealed from our analysis of A-T cellular clones. This apparent discrepancy is probably explained by the relatively limited number of clones studied here, which allowed analysis of dominant T cell population only. Irrespective of this issue, this clonal analysis allowed us to carefully control the status of both reciprocal hybrid loci and the timing of their subsequent rearrangements. Although all lymphocyte clones from the A-T line 105 expressed the same V{gamma}Cß transcript, our results strongly suggested that in about half of them the structure of the reciprocal VßC{gamma} locus was modified by secondary rearrangements. Consequently, these secondary recombinations had to occur after the productive one and the delay between the two events allowed at least one cell division. On the other hand, the same productive V{gamma}Cß was found in all clones from the A-T line 105 carrying a common inv(7) DßJ{gamma} sequence. These data are consistent with the hypothesis that productive rearrangements were almost synchronous with the first recombination events leading to inversion of {gamma} and ß loci. Nevertheless, because cells carrying out of frame but also productive V{gamma}Cß rearrangement might be excluded from the analyzed sample by in vivo and probably in vitro selection processes, conclusive information regarding the monoclonal or polyclonal nature of the productive trans rearrangements following a single inv(7) event could not be obtained from the present analysis.

Besides, our data demonstrate that a signal joint generated from an inversion mechanism mediated by the V(D)J recombinase can be involved in a secondary rearrangement. Such a multistep process has been already hypothesized but not formally proven in an human T cell leukemia from which the genomic junctional region has been amplified and sequenced (45). Indeed, the steps yielding to such a gene structure could not be clearly determined in this previous study because secondary V(D)J rearrangements occurred in both chromosome 7 hybrid loci.

Recent studies suggest close structural relationships between DNA-binding domains of the Rag-1 recombinase and a bacterial invertase (46) and fundamental similarities in the chemical mechanisms of V(D)J recombination and transposition (47). Nevertheless, in classical rearrangements the signal joint is lost with the excised DNA segment while in transposition, the mobile element is joined to a new target site and is able to move again. In intra- or interlocus rearrangement by inversion, coding, and signal joints are generated in the chromosomal DNA. Our findings, which demonstrate the involvement of a signal joint RSS generated from an inversion mechanism in a recombination process, strengthen the similarities of V(D)J recombination and transposition mechanisms.


   Acknowledgments
 
We thank the Centre Régional de Transfusion Sanguine de Nantes for blood samples.


   Footnotes
 
1 This work was supported by the Institut National de la Santé et de la Recherche Médicale and by the Association pour la Recherche sur le Cancer. Back

2 Address correspondence and reprint requests to Dr. M. M. Hallet, Institut National de la Santé et de la Recherche Médicale U463, Institut de Biologie, 9 quai Moncousu, 44093 Nantes cedex 1, France. E-mail address: Back

3 Abbreviations used in this paper: RSS, recombination signal sequence; A-T, ataxia-telangiectasia; inv(7), inversion 7. Back

Received for publication July 13, 1998. Accepted for publication October 2, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. Allam and D. Kabelitz
TCR trans-Rearrangements: Biological Significance in Antigen Recognition vs the Role as Lymphoma Biomarker
J. Immunol., May 15, 2006; 176(10): 5707 - 5712.
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