HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bentolila, L. A. Articles by Wu, G. E. Search for Related Content PubMed PubMed Citation Articles by Bentolila, L. A. Articles by Wu, G. E.

Extensive Junctional Diversity in Ig Light Chain Genes from Early B Cell Progenitors of µMT Mice¹

Laurent A. Bentolila^*, Stacy Olson, Aaron Marshall, François Rougeon^*, Christopher J. Paige, Noëlle Doyen^* and Gillian E. Wu^2,

^* Unité de Génétique et Biochimie du Développement, Unité de Recherche Associée, Centre National de la Recherche Scientifique 1960, Département d’Immunologie, Institut Pasteur, Paris, France; and Department of Immunology, University of Toronto, and Ontario Cancer Institute, Toronto, Canada

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nontemplated (N) nucleotide additions contribute significantly to the junctional diversity of all Ag receptor chains in adult mice except Ig light (L) chains, primarily because terminal deoxynucleotidyl transferase (TdT) expression is turned off at the time of their rearrangement in pre-B cells. However, because some Ig L chain gene rearrangements are detectable earlier during B cell ontogeny when TdT expression is thought to be maximal, we have examined the junctional processing of - and -chain genes of CD45(B220)⁺CD43⁺ pro-B cells from µMT mice. We found that both and coding junctions formed in these B cell precursors were extensively diversified with N-region additions. Together, these findings demonstrate that Ig L chain genes are equally accessible to TdT in pro-B cells as Ig heavy chain genes. Surprisingly, however, the two L chain isotypes differed in the pattern of N addition, which was more prevalent at the -chain locus. We observed the same diversity pattern in pre-B cells from TdT-transgenic mice. These results suggest that some aspects of TdT processing could be influenced by factors intrinsic to the sequence of Ig genes and/or the process of V(D)J recombination itself.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Agreat deal of the diversity of the Ig V region gene is created during the joining of V, D, and J gene segments at the heavy (H)³ chain locus and of V and J gene segments for the and light (L) chain loci in the developing B lymphocyte 1, 2 . The combined assortment of a large number of these gene segments is further enhanced by the deletion or the addition of nucleotides at their junctions 3 . The removal of an apparently random number of nucleotides is commonly attributed to exonuclease activity 4 . The addition of nucleotides fall into two categories, template-dependent (palindromic (P) nucleotides) 5 or template-independent (nontemplated (N) nucleotides) 6, 7, 8 , which are in the latter case added almost exclusively by the short alternatively spliced isoform of the enzyme terminal deoxynucleotidyl transferase (TdT) 9, 10, 11, 12, 13 .

In mice, TdT is differentially expressed during development, with TdT transcripts not being found until the first week after birth 14 . In adults, TdT expression is tightly controlled during the ordered Ig V(D)J recombination events. TdT gene expression is found during the early B lineage pathway at stages where Ig H chain rearrangements predominate, but is absent from the next stage where most L chain rearrangements occur 15 . As a result, Ig L chain genes display much more restricted junctional heterogeneity than Ig H chain genes and significantly lack N regions 1, 16, 17, 18 . However, it has been demonstrated that a small fraction of B cells appear to vary from this ordered process 19, 20 as 3–10% of rearrangements can be detected during the early B lineage pathway (pro-B cells characterized by the coexpression of CD45R(B220) and CD43), in which TdT expression is thought to be maximal. Contrary to expectations, extensive analysis of the junctional diversity of these initial L chain gene rearrangements did not display much more junctional heterogeneity 21 . This observation was rather puzzling given the evidence that constitutive expression of TdT in a transgenic mice model demonstrated that both H and L chain genes were equally accessible to TdT throughout B cell differentiation when TdT is present during the recombination process 22 .

Because N addition is such an important component of the CDR3 region and hence of Ig diversity, we set out to determine the regulation of N region addition in B cell precursors undergoing early Ig L chain rearrangements. We chose to examine the µMT mouse model that carries a targeted disruption of one of the membrane exons of the µ chain (µMT) 23 . In the µMT mouse, B lineage differentiation is arrested at the late pro-B stage 23, 24 . Thus, only CD43⁺ pro-B cells—including Hardy’s fractions A, B, and C—are found in the bone marrow. However, these pro-B cells have almost the same frequency of early L chain gene rearrangements as normal mice 19 . We analyzed the junctional diversity of the Ig L chain gene rearrangements from whole µMT bone marrow and compared them to those obtained from normal and TdT-transgenic bone marrow. We found that the B cell precursors that rearrange L chain genes at the early CD45R(B220)⁺CD43⁺ stage of development in µMT mice exhibit extensive N region diversity, comparable to that found in TdT-transgenic mice. This analysis reveals much heterogeneity in early Ig L chain junctions and demonstrates that endogenous TdT can process Ig L chain gene segments as well as H chain. Interestingly, we found isotypic differences in TdT processing of coding ends between and gene segments in both µMT and TdT-transgenic-generated sequences. We discuss how this observation has implications for the accessibility of TdT during the recombination process.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mice

TdT-transgenic mice were maintained by back crossing heterozygous transgenic males to normal C57BL/6 females (Institute Pasteur, Paris, France). Integration of the TdT transgene was determined by Southern blot and PCR analysis as previously described 22 . Control mice used in this study represent TdT-negative litter mates of TdT-transgenic mice. Originally coming from K. Rajewsky 23 , µMT mice were a kind gift of L. Schultz (Bar Harbor, ME). µMT mice were maintained as a homozygous strain (Ontario Cancer Institute, Toronto, Canada) and verified by immunofluorescent staining of peripheral blood and flow cytometry.

Cell surface staining and flow cytometry

Before mAb-fluorescent staining, bone marrow cells were depleted of red cells in ACK solution (155 mM NH₄Cl, 0.1 mM EDTA, 10 mM KHCO₃, pH 7.3). Single-cell suspension was then stained simultaneously with phycoerythrin-labeled anti-CD43 (mAb S7; PharMingen, San Diego, CA) and biotin-labeled anti-CD45R(B220) (mAb RA3–6B2; PharMingen), followed by a second step of quantum red-streptavidin (Sigma, St. Louis, MO). This combination permits the discrimination of the pro-B cell subsets from the most mature ones as previously described 25 . Dead cells were eliminated from the analysis by propidium iodide exclusion. Fluorescence was measured with a FACScan flow cytometer (Becton Dickinson, Sunnyvale, CA) using the CellQuest software.

DNA analysis

Genomic DNA was prepared from tail tips for genotyping of the TdT-transgenic line as previously described 26 . Genomic DNA was prepared from bones (femur and tibia) of control, TdT-transgenic, or µMT mice for PCR analysis of L chain gene rearrangements using the same procedure.

L chain gene PCR assay

PCR reactions contained a total of 2.4 µg of DNA except for µMT samples where up to 4.8 µg of bone marrow DNA was used. A same DNA sample was subjected to both and L chain gene PCR assay. The PCR assay used a conserved V primer located in the leader intron (V5', 5'-GCCTTTCTACACTGCAGTGGGTATGCAACAAT-3') and a degenerate J consensus primer (J3'cons, 5'-AGCCACT(C/T)ACCTAGGACAGT(C/G)A(C/G)(C/T)TTGGTTCC-3') designed to amplify most of the rearrangements. Reactions were performed essentially as described previously 22 except for µMT bone marrow DNA samples where the amplification was raised up to 35 cycles. We amplified VJ rearrangements using the V41 segment and the J1 segment. The PCR assay used a V41 primer located in the CDR1 region (V41, 5'-GCAAGTCAGGACATTGGTAGTAGC-3') and a J1 primer (J1, 5'-GAAGCCACAGACATAGACAACGG-3') located 3' of the J1 segment 27 . PCR reactions were performed in a final volume of 100 µl of 1x PCR buffer with a 100-µM concentration of each deoxynucleoside triphosphate, a 1-µM concentration of each primer, and a 2.5-mM concentration of MgCl₂. PCR amplification was initiated after the reaction reached a temperature of 85°C by adding 5 U of Taq DNA polymerase. The amplification consisted of 35 cycles of 1) 1 min denaturation at 94°C; 2) 1 min annealing at 65°C; and 3) 1 min primer extension at 72°C. A 10 min 72°C primer extension step was appended. An aliquot (2 µl) of the PCR reaction was cloned by using the Invitrogen TA Cloning System (Invitrogen, San Diego, CA). DNA preparations from white colonies were sequenced using the Pharmacia T7 polymerase sequencing kit (Pharmacia LKB, Piscataway, NJ)) and an internal V41 primer located in the FR3 region (V41int., 5'-GTGGCAGTAGGTCTGGGTCAG-3') according to the manufacturers’ instructions. All junctions represent independent recombination events, i.e., where there are identical sequences, they come from different PCR amplifications.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
TdT expression in pro-B cells of µMT mice increases the junctional diversity of L chain gene rearrangements

In µMT mice, one of the membrane exons of the gene encoding the µ-chain constant region was disrupted by gene targeting 23 . B cells are absent in homozygous animals; their development being arrested at the late pro-B cell stage due to the lack of surface µ expression. Fig. 1 shows flow cytometric profiles of lymphoid cells from µMT and C57BL/6 bone marrow. In µMT mice, there is a large population of CD45R(B220)⁺CD43⁺ pro-B cells, but no pre-B cell maturation as shown by the absence of CD45R(B220)⁺CD43^- cells. Rare VJ rearrangements have been detected in this population 19, 21 . Because these cells strongly express TdT 15 , we investigated the junctional diversity of these early -chain rearrangements.

View larger version (25K): [in this window] [in a new window]   FIGURE 1. Flow cytometry of total bone marrow showing separation of B lineage subsets in normal C57BL/6 and µMT mice. The CD45R(B220)+CD43+ fraction includes the pro-B cell subsets, while the CD45(B220)+CD43- fraction contains the more mature cells. In µMT mice, B lineage differentiation is blocked at the pro-B cell stage.

View larger version (31K): [in this window] [in a new window]   FIGURE 2. Sequences of V41J1 junctional regions of whole bone marrow DNA from adult mice. Junctional sequences were derived from µMT (A) and wild-type control (B) mice. Sequences are aligned with the germline sequence given on top. The numbering of codons is given according to Kabat et al. (18). Repeated junctions are shown only if they were obtained in at least two independent amplifications. Nucleotides that could have been encoded by either the V41 or the J1 germline segments are written in parentheses and have been arbitrarily placed on the J side. P region nucleotides are underlined. Nongermline-encoded nucleotides (N) are located at the V41J1 junction. In-frame rearrangements are indicated by (+), and out-of-frame rearrangements are indicated by (-).

Extensive N region diversity in L chain gene rearrangements in pro-B cells of µMT mice

Given the evidence that rearrangements in the Ig locus was also detectable in fractions B and C of pro-B cells in normal mice 19 , we investigated the junctional diversity of early L chain gene rearrangements in µMT mice. We used a PCR assay capable of amplifying most of the rearrangements (see Materials and Methods) because of the infrequent usage of as compared with L chains in murine B cells. We have shown previously that N regions are almost totally absent in junctions of normal mice 22 . Fig. 3 shows nucleotide sequences of VJ rearrangements obtained from whole bone marrow of µMT animals. Some 55% of joints have N insertions with an average of 4.4 nucleotides added per junction (Fig. 3 and Table I). Some exceptionally long insertions up to 14 nucleotides are also observed. As observed with VJ rearrangements, both productive and nonproductive VJ rearrangements occur at roughly equal frequencies (Fig. 3, 45 and 55%, respectively). These data further demonstrate that the presence of TdT is sufficient for N diversity to occur at any rearranging L chain gene in CD45R(B220)⁺CD43⁺ early B cell progenitors of µMT mice. Surprisingly however, N nucleotide additions were more frequent and almost four times longer in than in -chain genes.

View larger version (34K): [in this window] [in a new window]   FIGURE 3. Sequences of VJ junctional regions of whole bone marrow DNA from adult µMT mice. Sequences are obtained from the same DNA preparations that yielded the -chain joints and are displayed as described in Fig. 2.

We have shown previously that enforced TdT expression beyond the pre-B/B cells transition in transgenic mice results in extensive N region diversity in L chain rearrangements both in bone marrow and peripheral B cells 22 . However, junctional diversity of L chain rearrangements were not examined in this study. Fig. 4 shows nucleotide sequences of VJ rearrangements obtained from whole bone marrow of adult TdT-transgenic mice. Some 44% of the junctions have N additions, while 73% of joints do so (Table I). The average is 1.3 nucleotides added per junction as opposed to 2.6 nucleotides added per junctions (Table I). These results are similar to those found for pro-B cells of µMT mice. Altogether, these data demonstrate that the specific pattern of N region additions associated with each L chain isotype is not restricted to a particular developmental stage of B cell differentiation. Rather, and L chain genes undergo similar TdT-mediated N diversification both in µMT pro-B cells and in TdT-transgenic pre-B cells with N additions being always more prevalent at the -chain locus. We concluded that -chain rearrangements are differently processed by TdT as compared with .

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Sequences of V41J1 junctional regions of whole bone marrow DNA from adult TdT-transgenic mice. Sequences are obtained from the same DNA preparations that previously yielded the -chain joints (see Fig. 4B in Ref. 22). Sequences are displayed as described in Figs. 2 and 3. Nucleotides in bold can represent mutational events or PCR artifacts.

We asked whether the coding-end sequence may influence the N region addition process as was shown previously for the nucleotide deletions 29, 30, 31 . The composition of the sequences in terms of the frequency of A/T vs G/C nucleotides in the first 10 nucleotides flanking the coding ends were considered. V41 and J1 coding ends lack A/T stretches as do the majority of V and J coding ends (Table II and 18 . In contrast, coding ends fall into two major prototypes, which are either A/T rich (J2, J3, V1, and V2) or either G/C rich (J1) (Table II). As shown in Table III, the presence of at least one A/T stretch in one of the coding-end partners correlates with a high level of N diversity (V1J1, V1J3, and V2J2), while stretches of G/C at both coding flanks tend to lower the level of N diversity in the coding joint (V41J1). These results suggest that the coding-end sequence could influence the N region addition process and could account for at least some of the / processing differences.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In both systems, we found N region additions at the V-J junction in L chain genes. Some exceptionally long insertions were found in Ig gene rearrangements of µMT bone marrow cells. Because these cells do not express a µ H chain on their surface, the pool of junctional sequences likely represents a truly unselected sequence library reflecting the potential diversity. The paucity of such long N region insertions both in wild-type 32 or in TdT-transgenic animals (this work and 22 suggest that negative selection of cells with such CDR3 regions in normal lymphoid development is likely occurring at the stage when there is membrane expression of the functional Ag receptor 33, 34 .

The µMT mutation also prevents complete B cell differentiation from occurring in bone marrow and results in a single population of pro-B cells defined by the expression of CD45R(B220) and CD43. The L chain rearrangements found at this stage are susceptible to N region additions. In a previous report, no significant increase of N nucleotide additions was observed at VJ junctions from BALB/c-sorted CD45(B220)⁺CD43⁺ B cell progenitors 21 . This is likely to be due to subtle differences in the cell populations examined. Indeed, a recent RT-PCR analysis of TdT expression in bone marrow B cell fractions from normal mice revealed that the previously described sharp down-regulation of TdT expression at the pre-B stage (fraction D) actually occurs in the CD43⁺ transitional early pre-B subset (fraction C') 35 , where most of the initial rearrangements are detected 19 . Sorted CD45R(B220)⁺CD43⁺ cells from normal mice would also include this pre-B fraction C' with the consequence of diluting the output of joints containing N regions. No such effect should be operating in µMT mice because they contain only fraction A to the late pro-B cell fraction C but lack fraction C' 19 .

A second surprising feature of this study was isotypic differences in the processing of the coding ends. We show that additions were on the average more prevalent at than joints and that the number of nucleotides was at least twice as long in as in . Indeed, the low frequencies and length of N region additions in -chain genes were remarkably constant in both pro-B cells of µMT and pre-B cells of TdT-transgenic mice when compared with -chain genes. Therefore, this dichotomy is independent of the stage of B cell differentiation. Rather, we would like to suggest that the differential processing patterns of the two L chain isotypes might be due to factors intrinsic to the sequence of these genes and/or could be a general mechanistic feature of the V(D)J recombination itself. We considered two main hypotheses to explain this result.

It is first possible that particular motifs in the coding-end sequence influence the N region addition process. Indeed, we observed that high occurrence of N diversity tends to take place when stretches of A/T are present in at least one coding-end partner ( isotype), while the presence of G/C stretches in both coding-end partners ( isotype) tends to limit the TdT processing. In vitro, it has been shown that the nucleotide sequence adjacent to the exposed end considerably influences the incorporation of nucleotides at the 3'-end of duplex DNA 36, 37, 38 . The labeling efficiency of different duplex DNA fragments can correlate inversely with the number of G/C base pairs. The authors suggested that this could be determined by the degree of "terminal breathing" at the exposed ends. Interestingly, the length of nucleotide deletion also correlates with the presence of internal stretches of A/T nucleotides both in vitro and in vivo 29, 30, 31 . Base pairing interaction may also influence the processing by acting on the outcome of the hairpin opening, which may ultimately constrain TdT’s ability to modify DNA ends. Perhaps the tight paired structures formed by G/C stretches dictate where the hairpin termini is nicked (so that very G/C-rich sequences are nicked at the tip) generating blunt ends that are poor TdT substrates. In contrast, A/T-rich coding termini, because of "terminal breathing" can be nicked off center, providing a better substrate for TdT additions.

Another cis-acting sequence that could be responsible for the differences in the length of N addition in and are the recombination signal sequences (RSS). When tested competitively in recombination vectors, RSS are used much less frequently than RSS. This behavior can be correlated to the deviation from the consensus sequence because RSS differ from the consensus by 3 base pairs while RSS have no more than 1 base pair substitution 28, 39 . A growing body of evidences suggest that RAG-1 and RAG-2 are involved in the final steps of the V(D)J recombination reaction 40, 41 , and thus would be present at the same time as TdT in the V(D)J recombination complex. The correlation between the recombinatorial weakness of the RSS and the length of N addition could then be viewed as a result of the "going and coming" of the TdT given its distributive nature 42, 43, 44 and the other components of the recombination complex formed around "weak" or "strong" RSS.

Experiments are in progress to test coding-end sequence and/or RSS effects on TdT activity by cotransfecting TdT-expressing vector and various / recombination competition substrates in fibroblasts.

	Acknowledgments

 
We thank Dr. Ana Cumano for many critical discussions, as well as for hosting G.E.W. as a sabbatical visitor in her laboratory at the Institut Pasteur. We also thank the people in her laboratory and Dr. Antonio Bandeira for help in completing this work. We also thank an anonymous referee for discussion suggestions.

	Footnotes

 
¹ This work was supported by grants from the Medical Research Council of Canada and the National Cancer Institute of Canada, Terry Fox Marathon of Hope. G.E.W. is an Medical Research Council of Canada Scientist. L.A.B is supported by a Roux Foundation Award from the Pasteur Institute. A.M. is supported by a Studentship Award from the Medical Research Council of Canada.

² Address correspondence and reprints requests to Dr. Gillian E. Wu, Ontario Cancer Institute, 610 University Ave, Room 8-113, Toronto, Ontario M5G 2 M9, Canada. E-mail address:

³ Abbreviations used in this paper: H, heavy; L, light; N, nontemplated; P, palindromic; TdT, terminal deoxynucleotidyl transferase; RSS, recombination signal sequence.

Received for publication August 14, 1998. Accepted for publication November 4, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Tonegawa, S.. 1983. Somatic generation of antibody diversity. Nature 302:575.[Medline]
2. Lewis, S., M. Gellert. 1989. The mechanism of antigen receptor gene assembly. Cell 59:585.[Medline]
3. Lewis, S., A. Gifford, D. Baltimore. 1985. DNA elements are asymmetrically joined during the site-specific recombination of kappa immunoglobulin genes. Science 228:677.[Abstract/Free Full Text]
4. Lewis, S. M.. 1994. The mechanism of V(D)J joining: lessons from molecular, immunological, and comparatives analysis. Adv. Immunol. 56:27.[Medline]
5. Lafaille, J. J., A. DeCloux, M. Bonneville, Y. Takagaki, S. Tonegawa. 1989. Junctional sequences of T cell receptor genes: implications for T cell lineages and for a novel intermediate of V-(D)-J joining. Cell 59:859.[Medline]
6. Alt, F. W., D. Baltimore. 1982. Joining of immunoglobulin heavy chain gene segments: implications from a chromosome with evidence of three D-JH fusions. Proc. Natl. Acad. Sci. USA 79:4118.[Abstract/Free Full Text]
7. Desiderio, S. V., G. D. Yancopoulos, M. Paskind, E. Thomas, M. A. Boss, N. Landau, F. W. Alt, D. Baltimore. 1984. Insertion of N regions into heavy-chain genes is correlated with expression of terminal deoxytransferase in B cells. Nature 311:752.[Medline]
8. Landau, N. R., D. G. Schatz, M. Rosa, D. Baltimore. 1987. Increased frequency of N-region insertion in a murine pre-B-cell line infected with a terminal deoxynucleotidyl transferase retroviral expression vector. Mol. Cell. Biol. 7:3237.[Abstract/Free Full Text]
9. Kallenbach, S., N. Doyen, M. Fanton d’Andon, F. Rougeon. 1992. Three lymphoid-specific factors account for all junctional diversity characteristic of somatic assembly of T-cell receptor and immunoglobulin genes. Proc. Natl. Acad. Sci. USA 89:2799.[Abstract/Free Full Text]
10. Doyen, N., M. Fanton d’Andon, L. A. Bentolila, Q. T. Nguyen, F. Rougeon. 1993. Differential splicing in mouse thymus generates two forms of terminal deoxynucleotidyl transferase. Nucleic Acids Res. 21:1187.[Abstract/Free Full Text]
11. Gilfillan, S., A. Dierich, M. Lemeur, C. Benoist, D. Mathis. 1993. Mice lacking TdT: mature animals with an immature lymphocyte repertoire. Science 261:1175.[Abstract/Free Full Text]
12. Komori, T., A. Okada, V. Stewart, F. W. Alt. 1993. Lack of N regions in antigen receptor variable region genes of TdT-deficient lymphocytes. Science 261:1171.[Abstract/Free Full Text]
13. Bentolila, L. A., M. Fanton d’Andon, Q. T. Nguyen, O. Martinez, F. Rougeon, N. Doyen. 1995. The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization. EMBO J. 14:4221.[Medline]
14. Bogue, M., S. Gilfillan, C. Benoist, D. Mathis. 1992. Regulation of N-region diversity in antigen receptors through thymocyte differentiation and thymus ontogeny. Proc. Natl. Acad. Sci. USA 89:11011.[Abstract/Free Full Text]
15. Li, Y. S., K. Hayakawa, R. R. Hardy. 1993. The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver. J. Exp. Med. 178:951.[Abstract/Free Full Text]
16. Alt, F. W., K. Blackwell, G. D. Yancopoulos. 1987. Development of the primary repertoire. Science 238:1079.[Abstract/Free Full Text]
17. Heller, M., J. D. Owens, J. F. Mushinski, S. Rudikoff. 1987. Amino acids at the site of V-J recombination not encoded by germline sequences. J. Exp. Med. 166:637.[Abstract/Free Full Text]
18. Kabat, E. A., T. T. Wu, M. Reid-Miller, H. M. Perry, K. Gottesman. 1987. Sequences of Proteins of Immunological Interest 4th ed.165. National Institutes of Health Publication No. 165–462, Bethesda, Maryland.
19. Ehlich, A., S. Schaal, H. Gu, D. Kitamura, W. Müller, K. Rajewsky. 1993. Immunoglobulin heavy and light chain genes rearrange independently at early stages B cell development. Cell 72:695.[Medline]
20. Papavasiliou, F., M. Jankovic, M. C. Nussenzweig. 1996. Surrogate or conventional light chains are required for membrane immunoglobulin µ to activate the precursor B cell transition. J. Exp. Med. 184:2025.[Abstract/Free Full Text]
21. Victor, K. D., K. Vu, A. J. Feeney. 1994. Limited junctional diversity in light chains. J. Immunol. 152:3467.[Abstract]
22. Bentolila, L. A., G. E. Wu, F. Nourrit, M. Fanton d’Andon, F. Rougeon, N. Doyen. 1997. Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation. J. Immunol. 158:715.[Abstract]
23. 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 gene. Nature 350:423.[Medline]
24. Kitamura, D., K. Rajewsky. 1992. Targeted disruption of µ chain membrane exon causes loss of heavy-chain allelic exclusion. Nature 356:154.[Medline]
25. Hardy, R. R., C. E. Carmack, S. A. Shinton, J. D. Kemp, K. Hayakawa. 1991. Resolution and characterization of Pro-B and Pre-Pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173:1213.[Abstract/Free Full Text]
26. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
27. Goodhardt, M., P. Cavelier, N. Doyen, S. Kallenbach, C. Babinet, F. Rougeon. 1993. Methylation status of immunoglobulin gene segments correlates with their recombination potential. Eur. J. Immunol. 23:1789.[Medline]
28. Ramsden, D. A., C. J. Paige, G. E. Wu. 1994. Light chain rearrangement in mouse fetal liver. J. Immunol. 153:1150.[Abstract]
29. Boubnov, N. V., Z. P. Wills, D. T. Weaver. 1993. V(D)J recombination coding junction formation without DNA homology: processing of coding termini. Mol. Cell. Biol. 13:6957.[Abstract/Free Full Text]
30. Nadel, B., S. Tehranchi, A. J. Feeney. 1995. Coding end processing is similar throughout ontogeny. J. Immunol. 154:6430.[Abstract]
31. Nadel, B., A. J. Feeney. 1997. Nucleotide deletion and P addition in V(D)J recombination: a determinant role of the coding-end sequence. Mol. Cell. Biol. 17:3768.[Abstract]
32. Roth, D. B., X. B. Chang, J. H. Wilson. 1989. Comparison of filler DNA at immune, nonimmune, and oncogenic rearrangements suggests multiple mechanisms of formation. Mol. Cell. Biol. 9:3049.[Abstract/Free Full Text]
33. ten Boekel, E., F. Melchers, A. G. Rolink. 1997. Changes in the V(H) gene repertoire of developing precursor B lymphocytes in mouse bone marrow mediated by the pre-B cell receptor. Immunity 7:357.[Medline]
34. ten Boekel, E., F. Melchers, A. G. Rolink. 1998. Precursor B cells showing H chain allelic inclusion display allelic exclusion at the level of pre-B cell receptor surface expression. Immunity 8:199.[Medline]
35. Wasserman, R., Y. S. Li, R. R. Hardy. 1997. Down-regulation of terminal deoxynucleotidyl transferase by Ig heavy chain in B lineage cells. J. Immunol. 158:1133.[Abstract]
36. Roychoudhury, R., C. P. Tu, R. Wu. 1979. Influence of nucleotide sequence adjacent to duplex DNA termini on 3' terminal labeling by terminal transferase. Nucleic Acids Res. 6:1323.[Abstract/Free Full Text]
37. Roychoudhury, R., R. Wu. 1980. Terminal transferase-catalyzed addition of nucleotides to the 3' termini of DNA. Methods Enzymol. 65:43.[Medline]
38. Deng, G., R. Wu. 1981. An improved procedure for utilizing terminal transferase to add homopolymers to the 3' termini of DNA. Nucleic Acids Res. 9:4173.[Abstract/Free Full Text]
39. Seidman, J. G., E. E. Max, P. Leder. 1979. A -immunoglobulin gene is formed by site-specific recombination without further somatic mutation. Nature 280:370.[Medline]
40. Agrawal, A., D. G. Schatz. 1997. RAG1 and RAG2 form a stable postcleavage synaptic complex with DNA containing signal ends in V(D)J recombination. Cell 89:43.[Medline]
41. Ramsden, D. A., T. T. Paull, M. Gellert. 1997. Cell-free V(D)J recombination. Nature 388:488.[Medline]
42. Bollum, F. J.. 1962. Oligodeoxyribonucleotide-primed reactions catalyzed by calf thymus polymerase. J. Biol. Chem. 237:1945.[Free Full Text]
43. Chang, L. M. S., F. J. Bollum. 1971. Enzymatic synthesis of oligodeoxynucleotides. Biochemistry 10:536.[Medline]
44. Michelson, A. M., S. H. Orkin. 1982. Characterization of the homopolymer tailing reaction catalyzed by terminal deoxynucleotidyl transferase. Implications for the cloning of cDNA. J. Biol. Chem. 257:14773.[Free Full Text]

This article has been cited by other articles:

				B. Gozalbo-Lopez, P. Andrade, G. Terrados, B. de Andres, N. Serrano, I. Cortegano, B. Palacios, A. Bernad, L. Blanco, M. A. R. Marcos, et al. A Role for DNA Polymerase {micro} in the Emerging DJH Rearrangements of the Postgastrulation Mouse Embryo Mol. Cell. Biol., March 1, 2009; 29(5): 1266 - 1275. [Abstract] [Full Text] [PDF]

				M. Fleurant, L. Changchien, C.-T. Chen, M. F. Flajnik, and E. Hsu Shark Ig Light Chain Junctions Are as Diverse as in Heavy Chains J. Immunol., November 1, 2004; 173(9): 5574 - 5582. [Abstract] [Full Text] [PDF]

				L. Delpy, C. Sirac, E. Magnoux, S. Duchez, and M. Cogne RNA surveillance down-regulates expression of nonfunctional {kappa} alleles and detects premature termination within the last {kappa} exon PNAS, May 11, 2004; 101(19): 7375 - 7380. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Bentolila, L. A. Articles by Wu, G. E. Search for Related Content PubMed PubMed Citation Articles by Bentolila, L. A. Articles by Wu, G. E.