The quasi-monoclonal (QM) mouse has a functionally rearranged H chain gene inserted into its natural position in the IgH locus. In this position, the H chain gene is subject to many of the same activities as normally arranged H chain genes, including somatic hypermutation, VH gene replacement, and class switch recombination. Here, we have used this mouse strain to determine some of the rules that govern the V(D)J recombination activity of the IgH locus in thymus. We focused on the requirements for VH gene replacement. In normal mice, thymic DJH rearrangements are common, but VDJH rearrangements are not. We found intermediate products of VH replacement in double-positive CD4+CD8+ cells of the QM thymus, demonstrating that the inserted VH gene was accessible and ruling out the possibility that a VH gene per se cannot be rearranged in the thymus. We found transcripts from the knocked-in H chain gene of QM, but no μ H chain protein was detectable in thymocytes. Cloning and sequencing of these transcripts revealed that some had been generated by VH gene replacement. Corresponding signal joints could also be identified. These results suggest that neither a B cell-specific signal nor an Ig protein are necessary to activate VH-to-VDJH joining in thymocytes. Possible mechanisms remaining to account for overcoming the barrier to VH joining in thymocytes include the insertion of a transcriptionally active gene segment and/or the inactivation of a silencer.

In the mouse, the primary repertoire of B and T cell Ag receptor (BCR3 and TCR) genes is generated in the bone marrow and thymus. This is an ordered, regulated process that ensures that the appropriate gene segments are arranged at the appropriate stage; functional TCR gene rearrangements do not occur in B cells, nor functional BCR gene rearrangements in T cells (1). Ag per se is thought not to play a role in this process. Although further diversification in the periphery is commonly thought to be rare for the TCR (2, 3), somatic hypermutation and isotype switching are common for the BCR (4, 5, 6).

Understanding the generation of TCR and BCR diversity has been aided by mutant mouse strain studies. To name a few, the μMT, the recombination-activating genes (RAG)1/2 −/−, SCID, PMS2−/−, Eh−/−, and the L chain and H chain transgenics (7, 8, 9, 10, 11, 12, 13, 14). The strains with knocked-in prerearranged H chain genes have been particularly informative for studies questioning which diversification mechanisms are used by mice with near-monoclonal repertoires (15, 16). One aspect of diversification exposed by these mice is the extent to which V gene replacement can be used. V gene replacement at the IgH locus is the activity wherein a new upstream VH replaces the one in the original VHDJH rearrangement, with a loss of the intervening DNA including the original VH (17, 18, 19, 20, 21). VH replacement is mediated by the embedded heptamer, a sequence near the 3′ end of most VH, possibly in conjunction with a nonstandard nonamer (22, 23, 24, 25). It is the only known mechanism of receptor editing at the IgH locus (18). Because VH replacement was not detected in RAG-deficient mice (26), it is likely to be RAG mediated and hence take place only in cells that have RAG activity. Most VH, Vκ, Vα, and Vβ gene segments contain a sequence that qualifies as an embedded heptamer (27), and any cell with a sufficient level of RAG activity might undergo V gene replacement.

Because the new incoming VH segment replaces all but a few bases of the original VH, it has been difficult to assess how extensive a role VH replacement plays in generating diversity. However, an intermediate in the replacement reaction—the signal-end intermediate of the original VH—can be assessed (28, 29, 30). The signal end, which is blunt and 5′-phosphorylated, can be detected by a ligation-mediated PCR (LM-PCR) assay (28). We have reported the existence of such intermediates in the spleen and bone marrow of quasi-monoclonal (QM) mice (31), one of the knock-in strains that bear a VHDJH gene segment inserted into the natural location in the IgH locus (16, 32, 33, 34).

We have used thymocytes from a QM mouse that bears a knocked-in IgH to help answer questions about the requirements to diversify the IgH gene. Based on a number of studies including another knocked-in mouse model (22), it has been proposed that receptor editing, and hence V gene replacement, is regulated by Ag-driven stimuli through the BCR. We decided to examine the IgH locus in developing thymocytes, where, in normal mice, DJH rearrangements are frequent and rearrangements extending to the VH region are not (35, 36, 37, 38, 39). Indeed, although DJH alleles are sometimes transcribed, Dμ protein has not been detected. The QM thymus, where by definition B lineage-specific signals cannot be, presented an ideal model system to determine whether Ag-driven stimuli are absolutely required for VH replacement.

Here, we demonstrate VH replacement in thymocytes. Thus, neither activation of the BCR nor of any other B cell-specific stimulus is absolutely required for VH gene replacement. The question remains as to why in normal thymocytes the VH locus is not open to rearrangement, whereas in the knocked-in thymocytes it is.

QM mice are heterozygous at the IgH locus. One homologue has a knocked-in, rearranged VDJH containing VH17.2.25; on the other, all JH segments have been knocked out (34). QM mice are also homozygous for an inactive allele at the κ L chain locus (33).

Thymocytes were isolated from 4- to 8-wk-old QM mice by standard methods and stained with a combination of three Abs (PharMingen, San Diego, CA): PE-conjugated anti-B220, FITC-conjugated anti-CD4, and biotin-conjugated anti-CD8 developed with streptavidin-Quantum Red (Sigma, St. Louis, MO). Populations of stained cells were sorted with a FACStarPlus equipped with Turbo Sort using Lysis2 software (Becton Dickinson, Mountain View, CA). Sorted samples were reanalyzed with the same machine and the same Abs to check purity. Cells were then counted and prepared for DNA extraction.

For the FACS data shown in Fig. 5, cells of QM and C57BL/6 mice were triple-stained with the following Abs: FITC-conjugated anti-CD4 (PharMingen), PE-conjugated anti-CD8 (PharMingen), and biotin-conjugated anti-μ H chain (mAb 33-60). Staining with biotinylated Abs was revealed using Quantum Red-conjugated streptavidin (Sigma). To detect surface and cytoplasmic μ protein, cells were stained with PE-conjugated anti-CD3 (PharMingen), μ H chain-specific FITC-conjugated anti-IgM (Southern Biotechnology Associates, Birmingham, AL), and biotin-conjugated anti-μ H chain (mAb 33-60). For cytoplasmic μ protein, surface proteins were stained using standard methods followed by a 1-h incubation with ice-cold 70% ethanol and intermittent vortexing. Cells were washed twice and then incubated with FITC-conjugated anti-IgM for 45 min on ice to detect intracellular μ. The data were analyzed with the CellQuest program (Becton Dickinson).

FIGURE 5.

FACS analysis of thymocytes and splenocytes from QM and C57BL/6 mice: Ig μ H chain protein is not expressed in thymocytes. A, Thymocytes stained for CD4, CD8, and μ, and for CD3ε and cytoplasmic μ. Staining profiles in QM and control C57BL/6 mice are comparable. No significant surface or cytoplasmic μ H chain protein was detected in thymocytes of either strain. B, Total splenocytes from QM and C57BL/6 mice stained for cytoplasmic and surface μ. There are normal numbers of cells expressing surface and cytoplasmic μ H chain protein in both strains. Specificity and sensitivity of the anti-μ reagent and the streptavidin-Quantum Red reagents are shown.

FIGURE 5.

FACS analysis of thymocytes and splenocytes from QM and C57BL/6 mice: Ig μ H chain protein is not expressed in thymocytes. A, Thymocytes stained for CD4, CD8, and μ, and for CD3ε and cytoplasmic μ. Staining profiles in QM and control C57BL/6 mice are comparable. No significant surface or cytoplasmic μ H chain protein was detected in thymocytes of either strain. B, Total splenocytes from QM and C57BL/6 mice stained for cytoplasmic and surface μ. There are normal numbers of cells expressing surface and cytoplasmic μ H chain protein in both strains. Specificity and sensitivity of the anti-μ reagent and the streptavidin-Quantum Red reagents are shown.

Close modal

All PCR primers and probes used in this study are listed in Fig. 1.

FIGURE 1.

PCR primers and oligomer probes used in this study.

FIGURE 1.

PCR primers and oligomer probes used in this study.

Close modal

DNA from cell lysates (1.5 μg) was ligated to the BW linker (40) (8 ng) with 2 U T4 ligase (Life Technologies, Rockville, MD) for 16 h at 16°C and heated to 95°C for 15 min. The first round of PCR was performed with 300–400 ng ligated DNA, 15 ng each of primers BW-1HR (28) and VHA, 10 mM Tris (pH 8.3), 50 mM KCl, 0.5% Triton X-100, 2 mM MgCl2, and 2.5 U Taq polymerase (Boehringer Mannheim, Indianapolis, IN). A “touch down and hot start” PCR program was used: 15 cycles of 30 s denaturation at 94°C, 1 min annealing at 56°C, and 1 min elongation at 72°C, then another 15 cycles in which the annealing temperature is decreased to 53°C. The second round of PCR was done under the same conditions, with 1 μl of a 1/50 dilution of the first PCR and 8 ng each of BW and nested primer VHB.

Seminested PCR was performed with 100 ng DNA to amplify the signal-end deletion product from a recombination signal sequence (RSS) fusion of the embedded heptamer of VH17.2.25 to the RSS of a member of the VHJ558 family. The first 30 cycles of amplification used the 5′ primer VHB and a 3′ J558 RSS consensus primer. The primer hybridization temperature starts at 56°C and decreases to 53°C. The second round of PCR uses 1 μl of a 1/50 dilution of the first PCR as template DNA, with the same conditions except that the 5′ nested primer is VHC.

RNA was purified with Tri-Reagent (Molecular Research Center, Cincinnati, OH). First-strand cDNA was synthesized using standard methods with avian myeloblastosis virus-reverse transcriptase (Boehringer Mannheim). PCR conditions using the primers described were: 30 μl volume, 30 cycles of 30 s at 95°C, 30 s at 55°C, 1 min at 72°C.

PCR products were run on 1% agarose gels. DNA was transferred to Hybond-N (Amersham, Arlington Heights, IL) by capillary blotting and cross-linked with standard protocols. Southern blots were probed with VHall (see Figs. 2 and 3) or Cμ1-5′ (see Fig. 6), exposed to PhosphoImager plates for 4 h, and analyzed with a Storm PhosphoImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). The TA-cloning method (Invitrogen, San Diego, CA) was used according to the manufacturer’s protocol to clone PCR products. A T7 sequencing kit (Pharmacia, Piscataway, NJ) was used to sequence clones in both directions by the dideoxy method.

FIGURE 2.

A, Strategy of the LM-PCR. The signal-end intermediates are amplified with 5′ primers VHA and VHB specific for VH17.2.25 of the QM mouse and a 3′ primer specific for the BW linker. B, Analysis of VH replacement intermediates in bone marrow (BM), spleen (Spl), and thymus (Thy) of QM mice by the LM-PCR assay. Ethidium bromide-stained agarose gel of amplification products of 511 bp. Below, The Southern blot probed with VHall, a primer specific for VH segments.

FIGURE 2.

A, Strategy of the LM-PCR. The signal-end intermediates are amplified with 5′ primers VHA and VHB specific for VH17.2.25 of the QM mouse and a 3′ primer specific for the BW linker. B, Analysis of VH replacement intermediates in bone marrow (BM), spleen (Spl), and thymus (Thy) of QM mice by the LM-PCR assay. Ethidium bromide-stained agarose gel of amplification products of 511 bp. Below, The Southern blot probed with VHall, a primer specific for VH segments.

Close modal
FIGURE 3.

Analysis of VH replacement intermediates in thymocytes of QM mice at four stages of T cell development by the LM-PCR assay. The amplification strategy for VH replacements is the same as in Fig. 2. Actin DNA was amplified as a quantification and DNA quality control. A, Ethidium bromide-stained VH amplification products. B, Southern blot of A probed with VHall. C, Ethidium bromide-stained actin amplification products. D, Amplification of transcripts by RT-PCR from sorted QM DP thymocytes, T cell marker CD3ε, neo, and the B cell marker CD19. The fourth lane is the control without reverse transcriptase. The PCR lane is amplification of DP thymocyte cell lysis DNA with the CD19 primers.

FIGURE 3.

Analysis of VH replacement intermediates in thymocytes of QM mice at four stages of T cell development by the LM-PCR assay. The amplification strategy for VH replacements is the same as in Fig. 2. Actin DNA was amplified as a quantification and DNA quality control. A, Ethidium bromide-stained VH amplification products. B, Southern blot of A probed with VHall. C, Ethidium bromide-stained actin amplification products. D, Amplification of transcripts by RT-PCR from sorted QM DP thymocytes, T cell marker CD3ε, neo, and the B cell marker CD19. The fourth lane is the control without reverse transcriptase. The PCR lane is amplification of DP thymocyte cell lysis DNA with the CD19 primers.

Close modal
FIGURE 6.

Detection of transcripts from unsorted thymocytes. A, RAG1 and control without reverse transcriptase; B, V-Cμ and CD3ε; C, Southern blot of B probed with Cμ1-5′.

FIGURE 6.

Detection of transcripts from unsorted thymocytes. A, RAG1 and control without reverse transcriptase; B, V-Cμ and CD3ε; C, Southern blot of B probed with Cμ1-5′.

Close modal

Using the LM-PCR assay (Fig. 2,A), we searched for VH signal-end intermediates at the IgH locus in DNA from the thymus of QM mice. For comparison, the same strategy was applied in parallel to spleen and bone marrow DNA (Fig. 2,B). Southern blots were probed with VHall, a primer specific for VH segments, to confirm the specificity of the 511-bp amplification product obtained from spleen, bone marrow, and thymus (Fig. 2,B). Then, from each tissue, the amplification product was cloned. From the thymus samples, nine plasmid clones were sequenced and then compared with three clones from the bone marrow and seven from the spleen. Most sequences (15/19) were signal-end intermediates of VH17.2.25, the VH knocked into QM mice. As in the bone marrow (31), every VH17.2.25 cleavage product in the thymus was a true VH replacement intermediate; the product ended at the 3′-embedded heptamer at the last G of the TACTGTG motif (Table I). We conclude that VH replacement intermediates can be generated at the IgH locus in the thymus of QM mice.

Table I.

Overview of the sequences obtained by LM-PCR in the three distinct organs

Bone MarrowSpleenThymus
Total number of sequences 
Number of sequences cut at the embedded heptamer 
Number of sequences that are not VQM 
Bone MarrowSpleenThymus
Total number of sequences 
Number of sequences cut at the embedded heptamer 
Number of sequences that are not VQM 

To identify the cells in which VH replacement takes place, we sorted thymus cells into subsets based on CD4 and CD8 expression. B220+ cells, which comprised <0.2% of the original population, were first eliminated by sorting. From the B220 population, double-negative CD4CD8, DP CD4+CD8+, and the two single positives CD4+CD8 and CD4CD8+ were isolated. For the double-negative fraction, two sorts were pooled to obtain enough cells. Upon re-analysis, the purity of the sorted cell fractions was at least 99%.

The four fractions were analyzed by LM-PCR for the presence of linker-ligated VH17.2.25 segments (Fig. 3,A). After seminested PCR, an amplification product was evident only in the DP fraction both by ethidium bromide staining (Fig. 3,A) and by Southern analysis with VHall (Fig. 3,B). The absence of signal in the other fractions was confirmed by prolonged exposure of the blots. In parallel, the actin gene was amplified by PCR (Fig. 3,C); this confirmed the quantity and quality of DNA used from the four fractions. Using a sensitive RT-PCR assay for CD19 RNA, a well-expressed marker of the B lineage, we could not detect CD19 cDNA in DP cells; as a control, CD19 DNA was easily detected in the same cells (Fig. 3 D). This result ruled out the possibility that the replacement intermediates were in contaminating B cells and leads to the conclusion that VH replacement intermediates can be generated at the IgH locus in DP thymocytes of QM mice.

To confirm that the free signal-end intermediates detected were generated in VH replacement events, we searched for signal joints, specifically for the embedded heptamer of VH17.2.25 fused to an RSS from VHJ558, the largest mouse VH gene family. Using 5′ primers VHB and VHC, specific for VH17.2.25, and a 3′ J558 RSS primer, a band of the expected size, 473 bp, was found in the QM DP thymocyte and splenic cell lysates (Fig. 4). This band was absent in a C57BL/6 splenic lysate; that is, the primers used are specific for the product and do not nonspecifically amplify germline VHJ558 genes present in both QM and C57BL/6. We conclude that fused signal-end products generated by VH replacement at the IgH locus are present in DP thymocytes of QM mice.

FIGURE 4.

Analysis of VH signal joint products by PCR in QM DP thymus cells. VH deletion products are present in QM thymus and spleen, but not in normal C57BL/6 spleen.

FIGURE 4.

Analysis of VH signal joint products by PCR in QM DP thymus cells. VH deletion products are present in QM thymus and spleen, but not in normal C57BL/6 spleen.

Close modal

To determine whether an external signal through the BCR might promote VH replacement in thymocytes from QM mice, we looked for evidence of μ protein in these cells. Thymocytes from QM and C57BL/6 control mice were stained with anti-CD4, anti-CD8, anti-CD3, and anti-μ (Fig. 5,A). There were no detectable surface μ+/CD4+ or μ+/CD8+ thymocytes in either strain. When stained for CD3ε and cytoplasmic μ, there were no double-stained thymocytes. Because T cells do not express μ-associated proteins required for surface expression, including Igα, the absence of stable μ protein in thymocytes is not surprising. Nonetheless, the absence of μ protein suggests that H chain protein is not required for VH replacement. The sensitivity and specificity of the FACS was assessed using splenocytes from the same animals. As would be expected, the splenocytes of the two strains have cytoplasmic and surface μ+ cells in comparable numbers (Fig. 5 B).

From unsorted QM thymocytes, we monitored μ H chain mRNA by RT-PCR using primers VHall and Cμ-ex1, with RAG1 and CD3ε assayed in parallel as controls (Fig. 6). IgH transcripts that included the knocked-in VHDJH were present.

To determine whether some of these transcripts resulted from VH replacement in DP thymocytes, we amplified and sequenced μ transcripts from the DP fraction. The 5′ primer VHall can amplify any VH, and the 3′ primer is complementary to JH4. Each cloned VDJCμ rearrangement was digested with the restriction enzymes StuI, which is unique to VH17.2.25, and EcoRV, which is unique to the vector. Clones that were not digested by this combination of enzymes were potential new VHDJH rearrangements.

Of 19 VDJCμ clones from two independent experiments of two mice each, six had lost the restriction sites and were sequenced. All six were new rearrangements (Fig. 7). Five clones resulted from VH replacement, with the new VH gene segment being from four VH families, including VHJ558. The V segment of clone 5 differed from VH17.2.25 at only 2 nt, but had been processed (3 N nt added, 6 nt deleted from D) at the V-D junction. Clone 5 could be either an open-and-shut rearrangement of the knocked-in QM VDJH4, which is generally a rare event (41), or a VH gene replacement using the endogenous VH17.2.25. The same 6 nt were deleted from the D segment of clone 3. Clones 7Q and 1Q (and another clone with identical sequence to 1Q) have coding-end processing typical of that previously observed in VH replacements. In the two 1Q sequences, there is an additional D segment, DSP2.2; in B lineage cells from the QM mouse, additional DH (V-D-DJH4) are frequently found (16, 32). From the sequences of these transcripts, we conclude that VH replacement occurs in DP thymocytes of the QM mouse.

FIGURE 7.

Alignment of VHDJH rearrangements amplified by RT-PCR from DP thymocytes. Based on a GenBank search, each incoming VH gene segment was attributed to a VH family. QM denotes the knocked-in H chain of the QM mouse. In clones 1, 3, 1Q (found twice), and 7Q, the QM VH has been replaced. Clone 5 is either a VH replacement by the endogenous VH17.2.25 gene or an open-and-shut rearrangement. Clone 1Q possesses a new incoming DH segment, which is double-underscored.

FIGURE 7.

Alignment of VHDJH rearrangements amplified by RT-PCR from DP thymocytes. Based on a GenBank search, each incoming VH gene segment was attributed to a VH family. QM denotes the knocked-in H chain of the QM mouse. In clones 1, 3, 1Q (found twice), and 7Q, the QM VH has been replaced. Clone 5 is either a VH replacement by the endogenous VH17.2.25 gene or an open-and-shut rearrangement. Clone 1Q possesses a new incoming DH segment, which is double-underscored.

Close modal

Much is already known about the generation of diversity in the TCR and BCR. When all species that have an adaptive immune system are surveyed, one has the sense that any available mechanism is used, one of the charms of the specific immune system. The chicken has few functional V gene segments and it makes extensive use of gene conversion to diversify its repertoire, a mechanism that takes advantage of the 20-fold or so excess of pseudogenes in the chicken genome (42). The horned shark relies on multiple copies of complete IgH genes, some of which are split (and thus require V(D)J recombination for their generation) and some of which are fused and a ready component of diversity (43). The constructed VDJH knock-in strains have revealed available mechanisms in the mouse. In the periphery of these strains, VH gene replacement is about as common as somatic hypermutation, perhaps because a single VH replacement changes Ag specificity (32). The extent of its usage in unmanipulated strains is not known because VH replacement leaves little of the first VH to mark the event (15, 32, 44). By analyzing VH gene replacement in the thymus, a tissue that does not usually undergo VH joining, we were able to question the requirements for evoking this mechanism without the overshadowing processes of selection.

Some of the elements regulating V-to-D joining are different from those regulating D-to-J joining (45, 46, 47). We can think of two possibilities as to why VH replacement occurs during thymic development in the QM strain but is not detectable in unmanipulated strains. First, it may well be that the VH promoter in the proximity of its J-C intron enhancer leads to a complex that opens up the VH region, making it accessible to recombination (48, 49). Second, silencers and barriers to recombination have been detected in a few species and loci (45, 50, 51, 52). It may well be that the insertion of knocked-in prerearranged H chain genes (16) disrupted a barrier or silencer that functioned to prevent VH-to-DH joining in the T lineage, thus allowing VH-to-DH joining in DP thymocytes.

We used three independent approaches that established the existence of VH replacement in the thymocytes of QM mice: 1) signal-end intermediates in VH replacement were identified with the LM-PCR assay; 2) fused signal-end joints generated by VH replacement were amplified by normal PCR; and 3) μ transcripts of genes with VH replacement were amplified by RT-PCR and sequenced. These results mean that VH replacement can diversify an IgH receptor while not necessarily requiring a BCR-mediated external signal. Because no μ protein could be demonstrated in thymocytes, it would seem unlikely that a μ-mediated internal signal would be needed. Moreover, the absence of CD19 transcripts in QM thymocytes reinforces the FACS data and ensures that no contaminating B cells were present. These data do not rule out the possibility that VH replacement can be induced through signals via the BCR. In addition, VH replacement might occur during the normal course of early B cell development, implicating a pathway other than signaling through the mature surface BCR. In this regard, we have found evidence in QM bone marrow of intermediates of VH replacement in the pro-B cell compartment (data not shown). Thus, we favor, as do others, the hypothesis that VH replacement is a mechanism that can be used to diversify the B cell repertoire (19).

It would follow that receptor editing is a selective rather than an instructional event. That is, in the B lineage, VH replacement may occur in any cell in which RAG is still expressed. The cell is not instructed to replace its VH segment after the first VDJ-encoded protein has been tested and found wanting; instead, after that protein is found wanting, some of the cells that have undergone VH replacement are selected.

We thank Charley Steinberg for discussions and early help with the preparation of this manuscript and Queenie Lam and Stacy Hirano for excellent technical assistance.

1

R.G., D.M., and G.E.W. were supported by the Medical Research Council of Canada, the Terry Fox Marathon of Hope, the Leukemia Society of Canada, and the Cancer Research Society of Canada. F.E.B. is a Special Fellow of the Leukemia Society of America. M.C. and M.W. were supported by National Institutes of Health Grant R01 AI41570, a Howard Hughes Transgenic Mouse Grant, and the Junta Nacional de Investigação Científica e Tecnológica of Portugal.

3

Abbreviations used in this paper: BCR, B cell receptor; QM, quasi-monoclonal; RAG, recombination activating genes; LM-PCR, ligation-mediated PCR; DP, double- positive; RSS, recombination signal sequences.

1
Alt, F., G. Yancopoulos, T. K. Blackwell, C. Wood, E. Thomas, M. Boss, R. Coffman, N. Rosenberg, S. Tonegawa, D. Baltimore.
1984
. Ordered rearrangement of immunoglobulin heavy chain variable region segments.
EMBO J.
3
:
1209
2
Kisielow, P., H. von Boehmer.
1995
. Development and selection of T cells: facts and puzzles.
Adv. Immunol.
58
:
87
3
von Boehmer, H..
1990
. Developmental biology of T cells in T cell-receptor transgenic mice.
Annu. Rev. Immunol.
8
:
531
4
Tonegawa, S..
1983
. Somatic generation of antibody diversity.
Nature
302
:
575
5
Wabl, M., C. Steinberg.
1996
. Affinity maturation and class switching.
Curr. Opin. Immunol.
8
:
89
6
Weigert, M., I. M. Cesari, S. J. Yonkovich, M. Cohn.
1970
. Variability in the λ light chain sequences of mouse antibody.
Nature
228
:
1045
7
Kitamura, D., J. Roes, R. Kuhn, K. Rajewsky.
1991
. A B cell deficient mouse by targeted disruption of the membrane exon of the immunoglobulin μ chain class.
Nature
350
:
423
8
Mombaerts, P., J. Iacomini, R. S. Johnson, K. Herrup, S. Tonegawa, V. E. Papioannou.
1992
. RAG-1-deficient mice have no mature B and T lymphocytes.
Cell
68
:
869
9
Shinkai, Y., G. Rathbun, K. P. Lam, E. M. Oltz, V. Stewart, M. Mendelsohn, J. Charron, M. Datta, F. Young, A. M. Stall, F. Alt.
1992
. RAG-2-deficient mice lack mature lymphocytes owing to inability to initiate V(D)J rearrangement.
Cell
68
:
855
10
Bosma, M. J., A. M. Carroll.
1991
. The SCID mouse mutant: definition, characterization and potential uses.
Annu. Rev. Immunol.
9
:
323
11
Cascalho, M., J. Wong, C. Steinberg, M. Wabl.
1998
. Mismatch repair co-opted by hypermutation.
Science
279
:
1207
12
Phung, Q. H., D. B. Winter, R. Alrefai, P. J. Gearhart.
1999
. Hypermutation in Ig V genes from mice deficient in the MLH1 mismatch repair protein.
J. Immunol.
162
:
3121
13
Chen, J., F. Young, A. Bottaro, V. Stewart, R. K. Smith, F. W. Alt.
1993
. Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus.
EMBO J.
12
:
4635
14
Peters, A., U. Storb.
1996
. Somatic hypermutation of immunoglobulin genes is linked to transcription initiation.
Immunity
4
:
57
15
Lansford, R., J. P. Manis, E. Sonoda, K. Rajewsky, F. W. Alt.
1998
. Ig heavy chain class switching in Rag-deficient mice.
Int. Immun.
10
:
325
16
Cascalho, M., A. Ma, S. Lee, L. Masat, M. Wabl.
1996
. A quasi-monoclonal mouse.
Science
272
:
1649
17
Papavasiliou, F., R. Casellas, H. Suh, X. F. Qin, E. Besmer, R. Pelanda, D. Nemazee, K. Rajewsky, M. C. Nussenzweig.
1997
. V(D)J recombination in mature B cells: a mechanism for altering antibody responses.
Science
278
:
298
18
Chen, C., E. L. Prak, M. Weigert.
1997
. Editing disease-associated autoantibodies.
Immunity
6
:
97
19
Hertz, M., D. Nemazee.
1998
. Receptor editing and commitment in B lymphocytes.
Curr. Opin. Immunol.
10
:
208
20
Fanning, L., F. Bertrand, C. Steinberg, G. E. Wu.
1998
. Molecular mechanisms involved in receptor editing at the Ig heavy chain locus.
Int. Immun.
10
:
241
21
Qin, X. F., S. Schwers, W. Yu, F. Papavasiliou, H. Suh, A. Nussenzweig, K. Rajewsky, M. C. Nussenzweig.
1999
. Secondary V(D)J recombination in B-1 cells.
Nature
397
:
355
22
Chen, C., Z. Nagy, E. Prak, M. Weigert.
1995
. Immunoglobulin heavy chain gene replacement: a mechanism of receptor editing.
Immunity
3
:
747
23
Kleinfield, R., R. Hardy, D. Tarlinton, J. Dangl, L. A. Herzenberg, M. Weigert.
1986
. Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma.
Nature
322
:
843
24
Reth, M., P. Gehrmann, E. Petrac, P. Wiese.
1986
. A novel VH to VHDJH joining mechanism in heavy-chain-negative (null) pre-B cells results in heavy chain production.
Nature
322
:
840
25
Covey, L., P. Ferrier, F. A. Alt.
1990
. VH to VHDJH rearrangement is mediated by the internal VH heptamer.
Int. Immun.
2
:
579
26
Cascalho, M., D. Martin, J. Wong, Q. Lam, M. Wabl, G. E. Wu.
1999
. No VH gene replacement in a monoB RAG2−/− mouse.
Dev. Immun.
7
:
43
27
Kabat, E. A., T. T. Wu, M. Reid-Miller, H. M. Perry, and K. S. Gottesman. 1987. Sequences of Proteins of Immunological Interest, 4th Ed. National Institutes of Health Health Publication, U.S. Department of Health and Human Services, Bethesda, MD. p. 165.
28
Schlissel, M., A. Constantinescu, T. Morrow, M. Baxter, A. Peng.
1993
. Double-strand signal sequence breaks in V(D)J recombination are blunt, 5′-phosphorylated, RAG-dependent, and cell cycle regulated.
Genes Dev.
7
:
2520
29
Schatz, D. G..
1997
. V(D)J recombination moves in vitro.
Semin. Immunol.
9
:
149
30
van Gent, D. C., J. F. McBlane, D. A. Ramsden, M. J. Sadofsky, J. E. Hesse, M. Gellert.
1996
. Initiation of V(D)J recombinations in a cell-free system by RAG1 and RAG2 proteins.
Curr. Top. Microbiol. Immunol.
217
:
1
31
Bertrand, F. E., R. Golub, G. E. Wu.
1998
. V(H) gene replacement occurs in the spleen and bone marrow of non-autoimmune quasi-monoclonal mice.
Eur. J. Immunol.
28
:
3362
32
Cascalho, M., J. Wong, M. Wabl.
1997
. VH gene replacement in hyperselected B cells of the quasimonoclonal mouse.
J. Immunol.
159
:
5795
33
Chen, J., M. Trounstine, C. Kurahara, F. Young, C. Kuo, Y. Xu, J. Loring, F. Alt, D. Huszar.
1993
. B cell development in mice that lack one or both immunoglobulin κ light chain genes.
EMBO J.
12
:
821
34
Chen, J., M. Trounstine, F. W. Alt, F. Young, C. Kurahara, J. F. Loring, D. Huszar.
1993
. Immunoglobulin gene rearrangement in B cell deficient mice generated by targeted deletion of the JH locus.
Int. Immunol.
5
:
647
35
Yu, W., Z. Misulovin, H. Suh, R. R. Hardy, M. Jankovic, N. Yannoutsos, M. C. Nussenzweig.
1999
. Coordinate regulation of RAG1 and RAG2 by cell type-specific DNA elements 5′ of RAG2.
Science
285
:
1080
36
Forster, A., M. Hobart, H. Hengartner, T. H. Rabbitts.
1980
. An immunoglobulin heavy-chain gene is altered in two T-cell clones.
Nature
286
:
897
37
Kemp, D. J., A. Wilson, A. W. Harris, K. Shortman.
1980
. The immunoglobulin μ constant region gene is expressed in mouse thymocytes.
Nature
286
:
168
38
Born, W., J. White, J. Kappler, P. Marrack.
1988
. Rearrangement of IgH genes in normal thymocyte development.
J. Immunol.
140
:
3228
39
Schlissel, M., A. Voronova, D. Baltimore.
1991
. Helix-loop-helix transcription factor E47 activates germ-line immunoglobulin heavy-chain gene transcription and rearrangement in a pre-T-cell line.
Genes Dev.
5
:
1367
40
Mueller, P. R., B. Wold.
1989
. In vivo footprinting of a muscle specific enhancer by ligation mediated PCR.
Science
246
:
780
41
Lewis, S. M., J. E. Hesse.
1991
. Cutting and closing without recombination in V(D)J joining.
EMBO J.
10
:
3631
42
Reynaud, C. A., V. Anquez, H. Grimal, J. C. Weill.
1987
. A hyperconversion mechanism generates the chicken light chain preimmune repertoire.
Cell
48
:
379
43
Litman, G. W., L. Berger, K. Murphy, R. Litman, K. Hinds, B. W. Erickson.
1985
. Immunoglobulin VH gene structure and diversity in Heterodontus, a phylogenetically primitive shark.
Proc. Natl. Acad. Sci. USA
82
:
2082
44
Taki, S., F. Schwenk, K. Rajewsky.
1995
. Rearrangement of upstream DH and VH genes to a rearranged Ig variable region gene inserted into the DQ52-JH region of the Ig heavy chain locus.
Eur. J. Immun.
25
:
1888
45
Ferrier, P., B. Krippl, T. K. Blackwell, A. J. W. Furley, H. Suh, A. Winoto, W. D. Cook, L. Hood, F. Constantini, F. W. Alt.
1990
. Separate elements control DJ and VDJ rearrangement in a transgenic recombination substrate.
EMBO J.
9
:
117
46
Kirch, S. A., G. A. Rathbun, M. A. Oettinger.
1998
. Dual role of RAG2 in V(D)J recombination: catalysis and regulation of ordered Ig gene assembly.
EMBO J.
17
:
4881
47
Larijani, M., C. C. Yu, R. Golub, Q. L. Lam, G. E. Wu.
1999
. The role of components of recombination signal sequences in immunoglobulin gene segment usage: a V81x model.
Nucleic Acids Res.
27
:
2304
48
O’Brien, D. P., E. M. Oltz, N. B. Van.
1997
. Coordinate transcription and V(D)J recombination of the κ immunoglobulin light-chain locus: NF-κB-dependent and -independent pathways of activation.
Mol. Cell Biol.
17
:
3477
49
McMurry, M. T., M. C. Hernandez, P. Lauzurica, M. S. Krangel.
1997
. Enhancer control of local accessibility to V(D)J recombinase.
Mol. Cell Biol.
17
:
4553
50
Cocea, L., A. Dahan, L. Ferradini, C. A. Reynaud, J. C. Weill.
1998
. Negative regulation of Ig gene rearrangement by a 150-bp transcriptional silencer.
Eur. J. Immunol.
28
:
2809
51
Ortiz, B. D., D. Cado, A. Winoto.
1999
. A new element within the T-cell receptor α locus required for tissue-specific locus control region activity.
Mol. Cell Biol.
19
:
1901
52
Diaz, P., D. Cado, A. Winoto.
1994
. A locus control region in the T cell receptor α/δ locus.
Immunity
1
:
207