Predominant VH genes expressed in innate antibodies are associated with distinctive antigen-binding sites. (1/283)

Antibodies to phosphatidylcholine (PtC), a common constituent of mammalian and bacterial cell membranes, represent a large proportion of the natural antibody repertoire in mice. Previous studies of several mouse strains (e.g., C57BL/6) have shown that anti-PtC antibodies are mainly encoded by the VH11 and VH12 immunoglobulin heavy chain variable region gene families. We show here, however, that VH11 and VH12 encode only a small proportion of the anti-PtC antibodies in BALB/c mice. Instead, VHQ52-encoded antibodies predominate in this strain. In addition, two-thirds of the cells expressing VHQ52 family genes use a single gene (which, interestingly, has been previously shown to predominate in the anti-oxazolone response). We also show here that in anti-PtC antibodies from all strains, the distinctive antigen-binding sites associated with VHQ52 differ substantially from those associated with VH11 and VH12. That is, VHQ52-containing transcripts preferentially use the joining region JH4 rather than JH1 and exhibit more diverse complementarity-determining region 3 (CDR3) junctions with more N-region nucleotide additions at the gene segment junctions. Thus, the VH gene family that predominates in the anti-PtC repertoire differs among mouse strains, whereas the distinctive VHDJH rearrangements (CDR3, JH) associated with each VH gene family are similar in all strains. We discuss these findings in the context of a recent hypothesis suggesting that CDR3 structure, independent of VH framework, is sufficient to define the specificity of an antibody.  (+info)

Evidence that a single replication fork proceeds from early to late replicating domains in the IgH locus in a non-B cell line. (2/283)

In non-B cell lines, like the murine erythroleukemia cell line (MEL), the most distal IgH constant region gene, C alpha, replicates early in S; other heavy chain constant region genes, joining and diversity segments, and the most proximal Vh gene replicate successively later in S in a 3' to 5' direction proportional to their distance from C alpha. In MEL, replication forks detected in the IgH locus also proceed in the same 3' to 5' direction for approximately 400 kb, beginning downstream of the IgH 3' regulatory region and continuing to the D region, as well as within the Vh81X gene. Downstream of the initiation region is an early replicating domain, and upstream of Vh81X is a late replicating domain. Hence, the gradual transition between early and late replicated domains can be achieved by a single replication fork.  (+info)

Intermolecular V(D)J recombination is prohibited specifically at the joining step. (3/283)

V(D)J recombination, normally an intramolecular process, assembles immunoglobulin and T cell receptor genes from V, D, and J coding segments. Oncogenic chromosome translocations can result from aberrant rearrangements, such as occur in intermolecular V(D)J recombination. How this is normally prevented remains unclear; DNA cleavage, joining, or both could be impaired when the recombination signal sequences (RSS) are located in trans, on separate DNA molecules. Here, we show that both trans cleavage and joining of signal ends occur efficiently in vivo. Unexpectedly, trans joining of coding ends is severely impaired (100-to 1000-fold), indicating that protection against intermolecular V(D)J recombination is established at the joining step. These findings suggest a novel surveillance mechanism for eliminating cells containing aberrant V(D)J rearrangements.  (+info)

A targeted deletion of a region upstream from the Jkappa cluster impairs kappa chain rearrangement in cis in mice and in the 103/bcl2 cell line. (4/283)

We have shown previously that a mutation of the KI-KII site immediately 5' to J(kappa)1 on the mouse immunoglobulin light chain kappa locus reduces the rearrangement level in cis, although it does not affect transcription. Here we deleted by homologous recombination in mouse embryonic stem cells a 4-kb DNA fragment, located immediately upstream of the KI-KII element, which contains the promoter of the long germline transcript. Analysis of gene-targeted heterozygous mouse splenic B cells showed a strong decrease in rearrangement for the allele bearing the deletion. When both the KI-KII mutation and the 4-kb deletion were present on the same allele, the overall reduction in rearrangement was stronger than with the 4-kb deletion alone underlying the role of these two elements in the regulation of rearrangement. The same deletion was performed by homologous recombination on one allele of the rearrangement-inducible mouse 103/bcl2-hygro(R) pre-B cell line, and resulted in a similar reduction in the induction of rearrangement of the mutated allele. This result validates this cell line as an in vitro model for studying the incidence of gene-targeted modifications of the kappa locus on the regulation of rearrangement.  (+info)

Evidence that the Igkappa gene MAR regulates the probability of premature V-J joining and somatic hypermutation. (5/283)

The Igkappa gene contains an evolutionarily conserved nuclear matrix association region (MAR) adjacent to the intronic enhancer. To test for the function of this MAR, we created mouse lines with a targeted MAR deletion. In MAR knockout animals, the immune system was normal in nearly all respects, including the distributions of various B cell populations and Ab levels. However, in pro-B cells, enhanced rearrangement was noted on the MAR- allele in heterozygotes. In addition, the efficiencies for targeting and generating somatic mutations were reduced on MAR-deleted alleles. These results provide evidence for the MAR negatively regulating the probability of premature rearrangement and positively regulating the probability of somatic hypermutation.  (+info)

IgM heavy chain complementarity-determining region 3 diversity is constrained by genetic and somatic mechanisms until two months after birth. (6/283)

Due to the greater range of lengths available to the third complementarity determining region of the heavy chain (HCDR3), the Ab repertoire of normal adults includes larger Ag binding site structures than those seen in first and second trimester fetal tissues. Transition to a steady state range of HCDR3 lengths is not complete until the infant reaches 2 mo of age. Fetal constraints on length begin with a genetic predilection for use of short DH (D7-27 or DQ52) gene segments and against use of long DH (e.g., D3 or DXP) and JH (JH6) gene segments in both fetal liver and fetal bone marrow. Further control of length is achieved through DH-specific limitations in N addition, with D7-27 DJ joins including extensive N addition and D3-containing DJ joins showing a paucity of N addition. DH-specific constraints on N addition are no longer apparent in adult bone marrow. Superimposed upon these genetic mechanisms to control length is a process of somatic selection that appears to ensure expression of a restricted range of HCDR3 lengths in both fetus and adult. B cells that express Abs of an "inappropriate" length appear to be eliminated when they first display IgM on their cell surface. Control of N addition appears aberrant in X-linked agammaglobulinemia, which may exacerbate the block in B cell development seen in this disease. Restriction of the fetal repertoire appears to be an active process, forcing limits on the diversity, and hence range of Ab specificities, available to the young.  (+info)

Concanavalin A stimulation enhanced secondary VlambdaJlambda rearrangement in some human plasma B cells without up-regulation of recombination-activating gene expression and Vlambda germline transcription. (7/283)

Light chain shifting is a phenomenon that occurs in certain human antibody-producing plasma B cells which, when stimulated with concanavalin A (Con A), shift production of the original light chain to new light chains. Here we investigated the effect of Con A stimulation on these light chain shift-inducible cells. Analysis of transcripts and VJ-coding joints for new light chains revealed that a leaky amount of secondary VlambdaJlambda rearrangement occurs spontaneously, without Con A stimulation, and that Con A stimulation markedly increases VJ-coding joints and transcripts for new light chains. It was also shown that new light chain producers, which have carried out secondary rearrangement, do not further rearrange their light chain genes, even when stimulated with Con A. Recombination-activating gene (RAG) products and Vlambda germline transcription were constitutively expressed in these cell lines and their expression levels were not affected by Con A stimulation. These results suggest that Con A stimulation enhanced secondary VlambdaJlambda rearrangement, but this was not a result of the up-regulation of RAG expression and Vlambda germline transcription, which are believed to be sufficient for the process of VlambdaJlambda rearrangement.  (+info)

Four of five RAG-expressing JCkappa-/- small pre-BII cells have no L chain gene rearrangements: detection by high-efficiency single cell PCR. (8/283)

Single cell PCR assays have been further developed that detect over 80% of all VkappaJkappa, VkappaRS, and VlambdaJlambda rearrangements at efficiencies between 70% and 90%. These IgL chain gene rearrangement assays were used with small pre-BII cells that develop in comparably high numbers in the bone marrow of wild-type, Ckappa-deficient, and JCkappa-deficient homozygous and heterozygous mice. In all of these mice, only 15%-25% of all small pre-BII cells carry VlambdaJlambda rearrangements. These results confirm that lambdaL chain gene rearrangements occur independently of kappaL chain gene rearrangement and expression. They also show that a large part of the small pre-BII cells that express the rearrangement machinery can develop without IgL chain gene rearrangements.  (+info)