Differences between germ-line and rearranged immunoglobulin Vκ coding sequences suggest a localized mutation mechanism

To study the distribution of somatic mutation, we determined nucleotide sequences of rearranged Alchain genomic DNA from four hybridomas obtained from C57BL/6 mice that had been immunized with (4-hydroxy-3-nitrophenyl)acetyl-conjugated chicken gamma globulin. In total, 114 nucleotide substitutions were observed, with neither insertion nor deletion. Sixty-one mutations occurred in the variable-joining region genes (VAI-JA1) and 49 in joiningconstant (JAI-CAl) introns. Although frequency decreased with distance from the VAI-JAl coding region, somatic mutations occurred in the entire JAI-CAI intron and even in the CAI region. We found four nucleotide substitutions in CA, genes, all of which were replacement mutations. Therefore, the mechanism responsible for somatic mutation is operative into the CA, exons. Nucleotide sequences of rearranged but inactive A2-chain genes from two hybridomas were also examined and compared with those of Al-chain genes. The clustering of replacement mutations in complementarity-determining regions in the inactive A2-chain genes similar to the active Al-chain genes suggested a mechanism that induces somatic mutation preferentially in this region even in the absence of antigenic selection.Sequence diversity in the variable (V) domains of immunoglobulins is generated by random recombination of V, diversity (D), and joining (J) gene segments of heavy (H) chains or V and J segments of light (L) chains (1). Insertion of the N region and flexibility in the recombination sites also result in the variation in the V domain sequences (2). Somatic mutation introduces additional variation to VH and VL domains (1, 3-6). It has been shown that somatic mutation is triggered by stimulation with thymus-dependent antigens (3, 4, 6, 7) and is found in rearranged immunoglobulin genes both active and inactive (8)(9)(10)(11)(12). The somatic mutation mechanism is as yet unclear, although several models have been proposed (13-15). To evaluate possible mechanisms, information on the distribution of somatic mutations is undoubtedly required (5,(16)(17)(18)(19). Both murine and human immunoglobulin gene sequences show a lack of somatic mutation in the constant (C) region exons of H and K chains (5,17,20,21). However, Cleary et al. (22) reported the occurrence of somatic mutation in the human CA exon. Because of the complex genetics and gene structure of human immunoglobulins, it is rather difficult to distinguish between nucleotide variations arising from somatic mutation and those from other mechanisms.Murine A chains are particularly useful for examining the distribution of somatic mutations because of their simple gene structures (23-25). The A-chain genes possess only two VL genes (VA1 and VA2), which recombine with JA1, JA2, orJA3.Selective recombination of VA, to JA1CA1 or JA3-CA3 has been commonly observed (26,27). Therefore, a variation in sequence as a result of somatic mutation could be easily distinguished from that generated by other mechanisms.Furthermore, the J-C introns of A-chain genes di...

Section: Introductionmentioning

confidence: 99%

Somatic mutation in constant regions of mouse lambda 1 light chains.

Motoyama

Okada

Azuma

1991

“…Somatic diversification includes junctional diversity, the flexible joining of gene segments at different sites (10,11), and N-region diversification, the addition of random nucleotides at rearranged gene segment junctions (10,12). Somatic hypermutation, an increased frequency of nucleotide substitution occurring in and around immunoglobulin V genes (13,14), occurs very infrequently, if at all, in T-cell receptor genes (15)(16)(17)(18).…”

mentioning

confidence: 99%

“…Second, the published mouse Vp gene segments fall into 16 V. subfamilies, as defined by nucleic acid hybridization with germ-line DNA on Southern blots, of which 13 contain just a single member (15)(16)(17)22). In contrast, the immunoglobulin VH and VK subfamilies, generally defined as sets of V gene segments >75% homologous (23), range in size from 4 to 1000 (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21).…”

mentioning

confidence: 99%

Diversity and structure of human T-cell receptor beta-chain variable region genes.

Concannon¹,

Pickering²,

Kung³

et al. 1986

139

The nucleotide sequences of 27 T-cell receptor P cDNA clones isolated from a human peripheral lymphocyte library were determined and compared to five additional published sequences. These cDNA clones represent 22 distinct Mouse 13-chain genes have at their disposal all of the diversification mechanisms that are used by immunoglobulin genes but one. These include germ-line diversity, the maintenance of a multiplicity of V, D, and J gene segments, combinational diversity, the joining of available gene segments in all possible combinations, and somatic diversification (8,9). Somatic diversification includes junctional diversity, the flexible joining of gene segments at different sites (10, 11), and N-region diversification, the addition of random nucleotides at rearranged gene segment junctions (10,12 (17), perhaps in keeping with the dual requirement of the T-cell receptor to recognize antigen in the context of a MHC molecule. In any case, much of the diversity in the j3 chain seems to be focused at the carboxyl end of the V region through the heavy use ofjunctional and N-region diversity, the unique ability to use Do segments in all three translational reading frames, and the use of a large family of JP segments that are, on average, considerably more diverse than JH or JK segments (15)(16)(17).We have analyzed the expression of 13-chain genes in human peripheral lymphocytes in order to ascertain whether the strategies for diversification observed in mouse ( chains are a standard feature ofthe T-cell immune response in higher vertebrates. We conclude that (i) the strategy of maintaining a limited repertoire of highly heterogeneous Va gene segments is shared between mice and humans; (it) 6598The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

“…Additional diversity is introduced into rearranged V-region genes by somatic mutations. One mechanism to generate diversity has been shown to be point mutations (2,(6)(7)(8)(9); a possible other one may be recombination between V-region genes (10,11). A case of somatic recombination between antibody H chain V-region genes has been described by Dildrop et at (12).…”

mentioning

confidence: 99%

Recombination between antibody heavy chain variable-region genes: evidence for gene conversion.

Krawinkel¹,

Zoebelein²,

Brüggemann³

et al. 1983

The murine hybridoma line B1-8.81 secretes monoclonal IgD Al antibodies specific for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP). The variable (V) region of these antibodies is defined by a characteristic pattern of idiotopes. A spontaneous V-region variant (BI-8.V1) with altered idiotope pattern was selected. The structural variation is confined to the V region of the heavy chain. It was shown previously that the variant V region is encoded by a gene that was generated by a crossover between the rearranged VDJ gene of the wild type (B1-8. 61) and a neighboring germ-line VH gene. In the present study the nucleotide sequence of coding and flanking regions of the VH gene expressed in variant B1-8.V1 was determined. Wild-type and variant VH genes differ at 15 positions in a region between leader sequence and codon 66. The sequence of the region carrying the substitutions is identical to the sequence of the corresponding region in a neighboring germ-line VH gene. This implies that the variant VH gene was generated by a mechanism of recombination more complicated than single crossover. Gene conversion as the mechanism of the recombination is discussed.Variable (V) regions of immunoglobulin heavy (H) and light (L) chains are encoded by polymorphic gene segments, which join as a result of DNA rearrangements in the precursors of antibody-producing cells (1-5). The combination of V, diversity (D), and joining (J) segments in active heavy chain V-region genes generates diversity particularly in the third hypervariable region (3, 4). Additional diversity is introduced into rearranged V-region genes by somatic mutations. One mechanism to generate diversity has been shown to be point mutations (2, 6-9); a possible other one may be recombination between V-region genes (10,11). A case of somatic recombination between antibody H chain V-region genes has been described by Dildrop et at (12). The present study extends this analysis.We use as a model system the hybridoma line B1-8.61 (13), which synthesizes an IgD Al antibody specific for the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP (19,20). Hybridization was visualized by autoradiography using prefogged x-ray films (21). DNA Probes and Recombinant Phage DNAs. The JH-region probe (J probe) is a 0.6-kilobase-pair (kb) Xba I/EcoRI restriction endonuclease fragment derived from the 3' end of the EcoRI fragment carrying the murine JH-region genes (22) (see Fig. 2). The VH-region probe (V probe) used in this study is derived from plasmid pAB'y2a-1 (8), which contains the cDNA encodingAbbreviations: H chain, immunoglobulin heavy chain; VH region, variable region of the immunoglobulin H chain; VH gene, nucleotides coding for the VH region through FR3; FR, framework segment of the V region; D, diversity; J, joining; CDR, complementarity-determining region; IVS, intervening sequence; NP, (4-hydroxy-3-nitrophenyl)acetyl;kb, kilobase pair(s); bp, base pair(s). 4997The publication costs of this article were defrayed in part by page charge payment. This article must therefor...