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2.
Philos Trans R Soc Lond B Biol Sci ; 356(1405): 91-7, 2001 Jan 29.
Article in English | MEDLINE | ID: mdl-11205336

ABSTRACT

This paper discusses two aspects of immunoglobulin (Ig) gene hypermutation. In the first approach, a transcription termination signal is introduced in an Ig light chain transgene acting as a mutation substrate, and transgenic lines are generated with control and mutant transgenes integrated in tandem. Analysis of transcription levels and mutation frequencies between mutant and control transgenes clearly dissociates transcription elongation and mutation, and therefore argues against models whereby specific pausing of the RNA polymerase during V gene transcription would trigger an error-prone repair process. The second part reports the identification of two novel beta-like DNA polymerases named Pol lambda and Pol mu, one of which (Pol mu) represents a good candidate for the Ig mutase due to its higher lymphoid expression and its similarity with the lymphoid enzyme terminal deoxynucleotidyl transferase. Peculiar features of the expression of this gene, including an unusual splicing variability and a splicing inhibition in response to DNA-damaging agents, are discussed.


Subject(s)
DNA-Directed DNA Polymerase/physiology , Intramolecular Transferases/physiology , Mutation , Transcription, Genetic , Animals , DNA Nucleotidylexotransferase/physiology , DNA Polymerase beta/physiology , Humans , Immunoglobulins/genetics
3.
Proc Natl Acad Sci U S A ; 98(3): 1166-70, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11158612

ABSTRACT

Somatically mutated IgM(+)-only and IgM(+)IgD(+)CD27(+) B lymphocytes comprise approximately 25% of the human peripheral B cell pool. These cells phenotypically resemble class-switched B cells and have therefore been classified as postgerminal center memory B cells. X-linked hyper IgM patients have a genetic defect characterized by a mutation of the CD40L gene. These patients, who do not express a functional CD40 ligand, cannot switch Ig isotypes and do not form germinal centers and memory B cells. We report here that an IgM(+)IgD(+)CD27(+) B cell subset with somatically mutated Ig receptors is generated in these patients, implying that these cells expand and diversify their Ig receptors in the absence of classical cognate T-B collaboration. The presence of this sole subset in the absence of IgM(+)-only and switched CD27(+) memory B cells suggests that it belongs to a separate diversification pathway.


Subject(s)
B-Lymphocytes/immunology , CD40 Antigens/genetics , CD40 Ligand/genetics , Genes, Immunoglobulin , Immunoglobulin M/genetics , Immunologic Deficiency Syndromes/genetics , Mutation , Adolescent , Adult , Alternative Splicing , B-Lymphocyte Subsets/immunology , CD40 Antigens/immunology , CD40 Ligand/immunology , Child , Child, Preschool , Codon, Terminator , Fetal Blood/immunology , Gene Rearrangement , Humans , Immunoglobulin A/blood , Immunoglobulin D/genetics , Immunoglobulin G/blood , Immunoglobulin M/blood , Immunologic Deficiency Syndromes/blood , Immunologic Deficiency Syndromes/immunology , Infant, Newborn , Reference Values , Sequence Deletion
4.
Nucleic Acids Res ; 28(18): 3684-93, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10982892

ABSTRACT

We describe here two novel mouse and human DNA polymerases: one (pol lambda) has homology with DNA polymerase beta while the other one (pol mu) is closer to terminal deoxynucleotidyltransferase. However both have DNA polymerase activity in vitro and share similar structural organization, including a BRCT domain, helix-loop-helix DNA-binding motifs and polymerase X domain. mRNA expression of pol lambda is highest in testis and fetal liver, while expression of pol mu is more lymphoid, with highest expression both in thymus and tonsillar B cells. An unusually large number of splice variants is observed for the pol mu gene, most of which affect the polymerase domain. Expression of mRNA of both polymerases is down-regulated upon treatment by DNA damaging agents (UV light, gamma-rays or H(2)O(2)). This suggests that their biological function may differ from DNA translesion synthesis, for which several DNA polymerase activities have been recently described. Possible functions are discussed.


Subject(s)
DNA-Directed DNA Polymerase/chemistry , Alternative Splicing , Amino Acid Sequence , Animals , Cell Line , Cloning, Molecular , DNA Damage , DNA Polymerase beta/chemistry , DNA Polymerase beta/classification , DNA, Complementary/isolation & purification , DNA-Directed DNA Polymerase/classification , DNA-Directed DNA Polymerase/isolation & purification , Escherichia coli , Gene Expression Regulation, Enzymologic , Humans , Mice , Molecular Sequence Data , Phylogeny , Protein Conformation , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Tissue Distribution , Tumor Cells, Cultured
6.
J Exp Med ; 189(9): 1443-50, 1999 May 03.
Article in English | MEDLINE | ID: mdl-10224284

ABSTRACT

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.


Subject(s)
B-Lymphocytes , Gene Rearrangement, B-Lymphocyte , Immunoglobulin Joining Region/genetics , Immunoglobulin kappa-Chains/genetics , Sequence Deletion , Alleles , Animals , B-Lymphocytes/cytology , Cell Line , Gene Targeting , Germ Cells , Mice , Mutagenesis , Transcription, Genetic
7.
Proc Natl Acad Sci U S A ; 95(22): 13135-40, 1998 Oct 27.
Article in English | MEDLINE | ID: mdl-9789054

ABSTRACT

Common Variable Immuno-Deficiency (CVID) is the most common symptomatic primary antibody-deficiency syndrome, but the basic immunologic defects underlying this syndrome are not well defined. We report here that among eight patients studied (six CVID and two hypogammaglobulinemic patients with recurrent infections), there is in two CVID patients a dramatic reduction in Ig V gene somatic hypermutation with 40-75% of IgG transcripts totally devoid of mutations in the circulating memory B cell compartment. Functional assays of the T cell compartment point to an intrinsic B cell defect in the process of antibody affinity maturation in these two cases.


Subject(s)
B-Lymphocytes/immunology , Common Variable Immunodeficiency/genetics , Genes, Immunoglobulin , Immunoglobulin Variable Region/genetics , Point Mutation , Transcription, Genetic , Adult , Agammaglobulinemia/genetics , Agammaglobulinemia/immunology , Age of Onset , Antigens, CD/analysis , Antigens, CD19/analysis , Base Sequence , Burkitt Lymphoma/genetics , Burkitt Lymphoma/immunology , Child , Cloning, Molecular , Common Variable Immunodeficiency/immunology , Flow Cytometry , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Introns , Middle Aged , Molecular Sequence Data , Mutagenesis , Reference Values , Sequence Alignment , Sequence Homology, Nucleic Acid , Tumor Cells, Cultured
8.
Eur J Immunol ; 28(9): 2809-16, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9754568

ABSTRACT

We previously showed that the V-J intervening sequence of the chicken lambda immunoglobulin locus contains a strong silencer that acts both on transcription and rearrangement. We show here that the transcriptional silencer activity can be ascribed to a minimal 150-bp fragment. The rearrangement silencing activity was previously shown by the replacement of the V-J intervening sequence with a neutral DNA fragment that dramatically increased the rate of rearrangement of the transgene. Insertion of the minimal silencer in this neutral fragment is shown here to result in a marked decrease in rearrangement of the transgenic construct. Strikingly, deletion of 28 bp from the 150-bp fragment abolished most of the transcriptional silencing activity and had a similar effect on rearrangement. These results conclusively correlate the silencing activity on both rearrangement and transcription.


Subject(s)
Gene Rearrangement , Genes, Immunoglobulin , Immunoglobulin lambda-Chains/genetics , Transcription, Genetic/immunology , Animals , Chickens , Molecular Sequence Data
9.
Immunity ; 9(2): 257-65, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9729046

ABSTRACT

As the rate of Ig gene hypermutation approximates the level of nucleotide discrimination of DNA polymerases (10(-3) to 10(-4)), a local inhibition of proofreading and mismatch repair during semiconservative replication could generate the mutations introduced by the process. To address this question, we have constructed transgenic mice that carry a hypermutation substrate containing a "polymerase slippage trap": an Ig gene with a mono or dinucleotide tract inserted in its V region. The low amount of slippage events as compared to the number of mutations, the absence of transient misalignment mutations at the border of the repeats, and the dissociation between the amount of frameshifts and mutations when the transgene is put on mismatch repair-deficient genetic backgrounds, suggest that Ig gene hypermutation occurs by an error-prone short patch DNA synthesis taking place outside global DNA replication.


Subject(s)
DNA Probes/genetics , DNA Replication/genetics , DNA/biosynthesis , Genes, Immunoglobulin/genetics , Microsatellite Repeats/genetics , Mutation/genetics , Animals , B-Lymphocytes/physiology , DNA Repair/genetics , DNA-Directed DNA Polymerase , Frameshift Mutation/genetics , Immunoglobulin Variable Region/genetics , Immunoglobulin kappa-Chains/genetics , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Mice, Transgenic , Peyer's Patches/cytology , Time Factors , Transgenes/genetics
10.
Immunity ; 9(1): 127-34, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9697842

ABSTRACT

Primary responses to the hapten phenyloxazolone and chronic responses to environmental antigens occurring in Peyer's patches were analyzed in two different mismatch repair-deficient backgrounds. Paradoxically, whereas primary responses were found normal in MSH2- and only slightly diminished in PMS2-deficient mice, mutations in Peyer's patch B cells from both k.o. animals were reduced three times, the subset of Peyer's patch B cells with highly mutated sequences being specifically missing in the mismatch repair-deficient context. Strikingly, germinal center B cells from Peyer's patches of k.o. animals showed microsatellite instability at an unprecedented level. We thus propose that the amount of DNA damages generated prevents these cells from recycling in germinal centers and that mismatch repair deficiency is only the indirect cause of the lower mutation incidence observed.


Subject(s)
Adenosine Triphosphatases , B-Lymphocytes , DNA Repair Enzymes , DNA Repair , DNA-Binding Proteins , Genes, Immunoglobulin , Mutation , Proteins/physiology , Proto-Oncogene Proteins/physiology , Animals , Base Sequence , Cell Line , Germinal Center , Haptens/administration & dosage , Mice , Mice, Knockout , Microsatellite Repeats , Mismatch Repair Endonuclease PMS2 , Molecular Sequence Data , MutS Homolog 2 Protein , Oxazolone/administration & dosage , Oxazolone/analogs & derivatives , Peyer's Patches , Proteins/genetics , Proto-Oncogene Proteins/genetics
12.
J Immunol ; 159(7): 3093-5, 1997 Oct 01.
Article in English | MEDLINE | ID: mdl-9317102

ABSTRACT

The sheep genome contains 60 to 90 V lambda genes distributed in least 6 different families, whereas 37 V lambda genes and 10 families exist in humans. Comparison with human V lamda sequences indicates that sheep V lambda genes display less overall sequence divergence, but are closer to the genes most frequently used in the human peripheral repertoire. In both species, 2 to 3 genes contribute half of the expressed sequences. Therefore, similar large combinatorial potential and restriction of the expressed repertoire can exist in two species whose strategy of diversification differs widely (ongoing rearrangement throughout life in human bone marrow vs postrearrangement diversification during early development in sheep ileal Peyer's patches).


Subject(s)
Antibody Diversity , Digestive System/immunology , Gene Rearrangement, B-Lymphocyte , Genes, Immunoglobulin , Immunoglobulin Variable Region/genetics , Lymphoid Tissue/immunology , Multigene Family/immunology , Animals , Humans
13.
Semin Immunol ; 8(3): 125-9, 1996 Jun.
Article in English | MEDLINE | ID: mdl-8738911

Subject(s)
Mutation , Animals , Humans
14.
Science ; 271(5254): 1416-20, 1996 Mar 08.
Article in English | MEDLINE | ID: mdl-8596914

ABSTRACT

Transcriptional regulatory elements have been shown to be necessary but not sufficient for the developmental regulation of immunoglobulin gene rearrangement in mouse precursor B cells. In the chicken lambda light chain locus, additional elements in the V-J intervening sequence are involved in negative and positive regulation of rearrangement. Here, mutation of the mouse homolog of a chicken element, located in the V(K)-J(K) intervening sequence upstream of the J(K) cluster, was shown to significantly decrease rearrangement. This cis-acting recombination-enhancing element affects the rearrangement process without being involved in regulating transcription.


Subject(s)
Gene Rearrangement, B-Lymphocyte , Genes, Immunoglobulin , Immunoglobulin J-Chains/genetics , Immunoglobulin kappa-Chains/genetics , Regulatory Sequences, Nucleic Acid , Alleles , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Base Sequence , Chimera , Enhancer Elements, Genetic , Gene Rearrangement, T-Lymphocyte , Gene Targeting , Immunoglobulin Variable Region/genetics , Introns , Mice , Mice, Inbred C57BL , Mice, SCID , Molecular Sequence Data , Mutation , Recombination, Genetic , Stem Cells
15.
Immunol Today ; 17(2): 92-7, 1996 Feb.
Article in English | MEDLINE | ID: mdl-8808057

ABSTRACT

Diversification strategies for immunoglobulins vary widely in different species. Here, Jean-Claude Weill and Claude-Agnès Reynaud argue that V(D)J recombination arose as a means for achieving allelic exclusion rather than diversity, and postulate that the choice of a diversification strategy is selected along with a specific site of B-cell differentiation. They propose that somatic mutation and gene conversion represent analogous molecular strategies occurring in specific chromatin accessibility contexts.


Subject(s)
B-Lymphocytes/immunology , Gene Rearrangement, B-Lymphocyte , Immunoglobulin Variable Region/genetics , Animals , Genes, Immunoglobulin , Humans , Mutation
17.
Cell ; 80(1): 115-25, 1995 Jan 13.
Article in English | MEDLINE | ID: mdl-7813007

ABSTRACT

Somatic hypermutation of light chain V genes during development of B cells in sheep ileal Peyer's patches was studied in three experimental conditions: in sterile fragments of the ileum surgically isolated from the gut during fetal life, in germ-free sheep, and in animals thymectomized during early fetal life. The somatic mutation pattern was found identical to control tissues in all three experiments. The same age-dependent amount of mutations, a higher than theoretical R/S ratio in complementarity-determining regions (CDRs), and a similar clustering of mutations in CDRs were observed. The mechanism, as estimated from the silent mutation pattern, appears to target mutations to CDRs; moreover, the major V lambda genes have a specific codon usage with a high purine content at the first two bases of the codons and a low content at the third position, which, together with a specific targeting of mutations to purines, favors replacement mutations in CDRs.


Subject(s)
Gene Rearrangement , Genes, Immunoglobulin , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Mutation , Peyer's Patches/immunology , Animals , Antigens/immunology , B-Lymphocytes/immunology , Base Composition , Base Sequence , Codon , Germ-Free Life , Immunoglobulin Joining Region/genetics , Immunoglobulin lambda-Chains/genetics , Molecular Sequence Data , Peyer's Patches/embryology , Phenotype , Sheep/embryology , Sheep/genetics , Sheep/immunology , Thymectomy
18.
Semin Immunol ; 6(3): 165-73, 1994 Jun.
Article in English | MEDLINE | ID: mdl-7948956

ABSTRACT

Chicken immunoglobulin lambda light chain rearrangement is regulated by different controlling elements. One negative control element, acting as a strong transcriptional silencer, is located in the V-J intervening sequence which is excised during the rearrangement process. Positive control elements include the V lambda promoter, the enhancer of transcription (3' of C lambda), and one (or two) putative antisilencer element(s) located on each side of the silencer. It is proposed that these antisilencer elements counteract transiently the silencer thus allowing rearrangement of one allele in chicken B cell progenitors. The implications of this regulation for mouse B cell development are discussed.


Subject(s)
Gene Expression Regulation/genetics , Gene Rearrangement, B-Lymphocyte, Light Chain/genetics , Immunoglobulin lambda-Chains/genetics , Regulatory Sequences, Nucleic Acid/genetics , Animals , Chickens , Down-Regulation/genetics , Transcription, Genetic , Up-Regulation/genetics
20.
EMBO J ; 12(12): 4615-23, 1993 Dec.
Article in English | MEDLINE | ID: mdl-8223471

ABSTRACT

The chicken Ig lambda light chain locus is composed of a single V gene closely linked (1.8 kb) to a single J-C unit in its natural configuration. In mice transgenic for this locus, the transgene becomes rearranged in B cells and to a much lesser extent in T cells. Modifications were introduced in the transgene in order to characterize elements which target the recombinase to the Ig loci. In the absence of either the promoter or the enhancer located 3' of C lambda, rearrangement of the transgene is reduced 20- to 100-fold. Moreover, rearrangement is increased 5-fold when the DNA segment between V lambda and J lambda ('Uo segment'), which is deleted during the joining process, is replaced by a neutral DNA segment of equal length. The Uo segment behaved as a strong transcriptional silencer when tested in a CAT assay in vitro. Control transgenic mice harbouring only the two 3 bp mutations that introduced restriction sites at both ends of the Uo segment to allow for its replacement were also analysed. Rearrangement was reduced 10- to 100-fold in B cells from such transgenic lines. A model is proposed whereby the sites of these two mutations would function by counteracting transiently the repressing effect of the silencer, thus giving access of the chicken light chain locus to the recombinase.


Subject(s)
Enhancer Elements, Genetic , Gene Rearrangement, B-Lymphocyte, Light Chain , Immunoglobulin G/genetics , Promoter Regions, Genetic , Regulatory Sequences, Nucleic Acid , Animals , B-Lymphocytes , Base Sequence , Cell Line , Chickens , Cloning, Molecular , DNA , Mice , Mice, Transgenic , Molecular Sequence Data , Mutagenesis , Polymerase Chain Reaction , Transcription, Genetic
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