Position of the rearranged V kappa and its 5' flanking sequences determines the location of somatic mutations in the J kappa locus

Somatic hypermutation is known to occur in the VJ kappa exon and its flanking sequences, yet little is known about the hypermutation mechanism or its exact target within the rearranged locus. Mutations may occur at the same frequency, spanning a region from the leader intron to 3' of J kappa 5, regardless of which J is chosen for VJ rearrangement. Another possibility is that mutations may be limited to the rearranged VJ kappa and its immediate flanking sequences. To distinguish between these possibilities, the JC introns of 21 alleles with V kappa rearranged to J kappa 1 were sequenced, and mutations were located. The frequency of mutations was determined for different sections of the intron and compared with the frequencies of mutations found in the JC intron of a set of VJ kappa 5 alleles. The results showed that mutations were concentrated in and around the rearranged VJ, regardless of whether J kappa 1 or J kappa 5 was used. These data imply that the hypermutational mechanism focuses on rearranged V genes.     

Somatic point mutations in unrearranged immunoglobulin gene segments encoding the variable region of lambda light chains.

Somatic point mutations are usually found in the coding and flanking regions of functionally and aberrantly rearranged immunoglobulin variable region gene segments. Mutations in the unrearranged V gene segments of myelomas or hybridomas have not been described so far. We have cloned and sequenced unrearranged V lambda gene segments from several cell lines. There were no nucleotide changes in four unrearranged V lambda segments: one V lambda 1 from a lambda 3-producing hybridoma and one V lambda 2 from a lambda 1-producing myeloma (J558) and two V lambda 2 from a kappa-producing myeloma (P3X63). However, we found somatic mutations in the unrearranged V lambda segments from the lambda 2-producing myeloma MOPC315. The unrearranged V lambda 1 gene segment had two mutations in the coding region and the unrearranged V lambda 2 had one mutation in the 3' flanking region. We also cloned and sequenced the unrearranged J lambda and C lambda gene segments of MOPC315 and found no sequence alterations. This is consistent with the notion that the overall mutation rate is not higher in this cell line. Therefore, we suggest that the somatic hypermutation system can use unrearranged V gene segments as substrates. The extensive sequencing required for this work revealed a number of errors in the reported nucleotide sequences of the Ig lambda locus in BALB/c mice.     

V gene rearrangement is required to fully activate the hypermutation mechanism in B cells

Nonproductively rearranged H and L chain loci of B cell hybridoma lines expressing heavily mutated antibodies were cloned and partially sequenced. The results confirm earlier data showing that somatic point mutations are as frequent in nonproductively rearranged loci containing a rearranged V gene as in productively rearranged loci. They establish in addition that in nonproductive H chain loci which bear a DJH rearrangement the frequency of somatic mutations is more than 10 times lower (0.2%) than in VDJH loci expressed by the same cells (2.5%). Thus, the hypermutation mechanism operating in B cell differentiation is targeted at V genes rearranged to the J locus and may require nucleotide sequences associated with both V and J elements in order to be fully activated. An inversion of the JH2 segment was detected in one DJH locus. This inversion appears to be the result of a secondary joining event occurring occasionally in the course of B cell development.     

Mouse immunoglobulin gene rearrangements. The sequence of a nonexpressed lambda 3-chain gene

A functionally defective lambda 3-immunoglobulin chain gene has been cloned from plasmacytoma HOPC-1 (gamma 2b, lambda 1). The lambda 3 gene resulted from the juxtaposition of the germline V lambda 1 sequence with a J lambda 3 C lambda 3 gene segment. DNA sequencing of the rearranged V lambda 1 J lambda 3 exon showed the presence of a single base pair deletion at the site of V-J joining. The alteration in the reading frame caused by this deletion generated a stop codon at the 3' end of J lambda 3, thus rendering this gene nonfunctional for light chain production. In addition, a one-point mutation in the J lambda 3-C lambda 3 intron distinguishes the rearranged gene from the unrearranged counterpart. The implications that this rearrangement has in terms of the mechanism of somatic mutations and of selective proliferation of B cells mediated by antigen stimulation are discussed.     

Immunoglobulin kappa light chain gene promoter and enhancer are not responsible for B-cell restricted gene rearrangement.

We have produced transgenic mice which synthesize chimeric mouse-rabbit immunoglobulin (Ig) kappa light chains following in vivo recombination of an injected unrearranged kappa gene. The exogenous gene construct contained a mouse germ-line kappa variable (V kappa) gene segment, the mouse germ-line joining (J kappa) locus including the enhancer, and the rabbit b9 constant (C kappa) region. A high level of V-J recombination of the kappa transgene was observed in spleen of the transgenic mice. Surprisingly, a particularly high degree of variability in the exact site of recombination and the presence of non germ-line encoded nucleotides (N-regions) were found at the V-J junction of the rearranged kappa transgene. Furthermore, unlike endogenous kappa genes, rearrangement of the exogenous gene occurred in T-cells of the transgenic mice. These results show that additional sequences, other than the heptamer-nonamer signal sequences and the promoter and enhancer elements, are required to obtain stage- and lineage- specific regulation of Ig kappa light chain gene rearrangement in vivo.     

Somatic diversification of the chicken immunoglobulin light chain gene is limited to the rearranged variable gene segment

Previous studies have shown that the chicken lambda immunoglobulin light chain gene undergoes a single rearrangement that results in functional VJ joining of the unique variable (V lambda 1) and joining (J lambda) coding regions. The immunologic repertoire of lambda genes is created through extensive sequence diversification within the rearranged locus during B cell development in the bursa of Fabricius. This sequence diversification was detected only at the rearranged V lambda 1 segment and not within the 5' leader sequence, the J lambda segment, or the unrearranged V lambda 1 segment. The selective diversification of the rearranged V lambda 1 segment was associated with unique DNAase I-hypersensitive sites on the rearranged allele. While probes for V lambda 1 sequences detect multiple homologous V lambda segments, probes for both the 5' leader and J lambda segments fail to detect homologous sequences. Taken together, these results suggest that a highly selective process, possibly gene conversion, operates during B cell ontogeny to generate diversity within the lambda gene.     

Two human gamma-crystallin genes are linked and riddled with Alu-repeats

A human genomic cosmid clone, pHcos gamma-1, has been isolated containing two closely linked gamma-crystallin genes, oriented in the same direction. The sequence of these genes and their 5' and 3' flanking regions has been determined. The coding regions of both genes are interrupted by two introns. The first introns (94 and 100 bp, respectively) are located in the 5' region of the genes. The second introns (2.82 and 0.95 kb, respectively) divide the genes into two halves, each encoding a structural domain of the gamma-crystallin protein. The coding regions of the two genes show 80% homology. Due to a mutation in the splice acceptor site of the second intron of the first gene, the coding region of its third exon is 3 bp longer than that of the second gene. In the flanking regions several conserved sequence elements were found, including those elements that are known to be necessary for the correct expression of eukaryotic genes. The flanking and intronic regions of the genes contain 'simple sequence' DNA and Alu repeats. The Alu repeats are usually clustered, contain truncated elements, and are often located near simple sequence DNA.     

Burkitt's lymphoma is a malignancy of mature B cells expressing somatically mutated V region genes.

BACKGROUND: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents. MATERIALS AND METHODS: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined. RESULTS: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines). CONCLUSIONS: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.     

V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer.

We have assessed the importance of the immunoglobulin heavy chain (IgH) intron enhancer for recombination of variable gene segments (V, D and J) during B cell development. We generated chimeric mice with embryonic stem cells lacking the intron enhancer from one of their IgH loci. The IgH intron enhancer was substituted by a short oligonucleotide through homologous recombination using the 'Hit and Run' procedure. V(D)J recombination occurred less frequently on mutant alleles, but was not blocked completely. Quantitative polymerase chain reaction analyses demonstrated that 15-30% of the mutated loci in mature B cells were unrearranged, in striking contrast to the wild-type alleles. The remainder of the mutated loci underwent D-J (65-80%) as well as V-DJ rearrangements, although the latter were less frequent (3-6%). These results are in line with previous data which showed that the V(D)J recombination machinery is modulated through cis-regulatory elements within the intron enhancer. However, our data predict the existence of additional cis-regulatory element(s) which, together with the intron enhancer, are required to activate the V(D)J recombination machinery fully. Such cis-regulatory element(s) might function as an enhancer of recombination or as a locus control region regulating the accessibility of the IgH locus.     

Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci

The complete nucleotide sequence has been determined for the intron, its junctions and the flanking exon regions of the 21S rRNA gene in three genetically characterized strains differing by their omega alleles (omega+, omega- and omega n) and by their chloramphenicol-resistant mutations at the rib-1 locus. Comparison of these DNA sequences shows that: --omega+ differs from omega- and omega n by the presence of the intron (1143 bp), as well as by a second and unexpected mini-insert (66 bp) located 156 bp upstream within the exon, whose nature and functions are still unknown but whose striking palindromic structure may suggest a mitochondrial transposable element. --The two mutations C321R and C323R correspond to two different monosubstitutions, 56 bp apart in the omega- and omega n strains but separated by the intron in the omega+ strains. In relation to previous genetic results, a model is discussed assuming that the interactions of two different regions or genetic loci determine the chloramphenicol resistance, one of which contains the omega n mutations. --A long uninterrupted coding sequence able to specify a 235 amino acid polypeptide exists within the intron. This remarkable observation gives new insight into the origin of the mitochondrial introns and raises the question of the possible functions of intron-encoded polypeptides. Finally, sequence comparisons with evolutionarily distant organisms, showing that different rRNA introns are inserted at different positions of an otherwise highly conserved region of the gene, suggest a recent insertion of these introns and a mechanism for splicing after the assembly of the large ribosomal subunit.     

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.

     

Identification and characterization of new long conserved noncoding sequences in vertebrates

Comparative sequence analyses have identified highly conserved genomic DNA sequences, including noncoding sequences, between humans and other species. By performing whole-genome comparisons of human and mouse, we have identified 611 conserved noncoding sequences longer than 500 bp, with more than 95% identity between the species. These long conserved noncoding sequences (LCNS) include 473 new sequences that do not overlap with previously reported ultraconserved elements (UCE), which are defined as aligned sequences longer than 200 bp with 100% identity in human, mouse, and rat. The LCNS were distributed throughout the genome except for the Y chromosome and often occurred in clusters within regions with a low density of coding genes. Many of the LCNS were also highly conserved in other mammals, chickens, frogs, and fish; however, we were unable to find orthologous sequences in the genomes of invertebrate species. In order to examine whether these conserved sequences are functionally important or merely mutational cold spots, we directly measured the frequencies of ENU-induced germline mutations in the LCNS of the mouse. By screening about 40.7 Mb, we found 35 mutations, including mutations at nucleotides that were conserved between human and fish. The mutation frequencies were equivalent to those found in other genomic regions, including coding sequences and introns, suggesting that the LCNS are not mutational cold spots at all. Taken together, these results suggest that mutations occur with equal frequency in LCNS but are eliminated by natural selection during the course of evolution.     

Intron-size constraint as a mutational mechanism in Rothmund-Thomson syndrome

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder caused by deleterious mutations in the RECQL4 gene on chromosome 8. The RECQL4 gene structure is unusual because it contains many small introns <100 bp. We describe a proband with RTS who has a novel 11-bp intronic deletion, and we show that this mutation results in a 66-bp intron too small for proper splicing. Constraint on intron size may represent a general mutational mechanism, since human-genome analysis reveals that approximately 15% of genes have introns <100 bp and are therefore susceptible to size constraint. Thus, monitoring of intron size may allow detection of mutations missed by exon-by-exon approaches.