Sequences at the somatic recombination sites of immunoglobulin light-chain genes.

The entire nucleotide sequence of a 1.7-kilobase embryonic DNA fragment containing five joining (J) DNA segments for mouse immunoglobulin kappa chain gene has been determined. Each J DNA segment can encode amino acid residues 96--108. Comparison of one of the five J DNA sequences with those of an embryonic variable (V) gene and a complete kappa chain gene permitted localisation of a precise recombination site. The 5'-flanking regions of J DNA segments could form an inverted stem structure with the 3'-non-coding region of embryonic V genes. This hypothetical structure and gel-blotting analysis of total embryo and myeloma DNA suggest that the somatic recombination may be accompanied by excision of an entire DNA segment between a V gene and a J DNA segment. Antibody diversity may in part be generated by modulation of the precise recombination sites.     

A novel type of aberrant recombination in immunoglobulin genes and its implications for V-J joining mechanism

Functional kappa light chain genes are formed during B-lymphocyte differentiation by the joining of initially separate V and J gene segments. It has been suggested that the intervening DNA is deleted, however the recent reports of what appear to be the reciprocal products of V and J recombination (back-to-back conserved V and J flanking sequences, called f-fragments) in DNA from mature lymphocytes make a simple deletion model unlikely. An alternative scheme involving unequal sister chromatid exchange has been proposed, supported by the evidence that the f-fragments seem to have segregated from the chromosome carrying the reciprocal complete kappa light chain gene (this and other schemes are briefly reviewed in ref. 8). We report here the analysis of a mouse myeloma (MOPC 41), in which a productive (kappa+) and a non-productive (kappa-) rearrangement has occured, which may help to clarify the mechanism of V-J joining. The aberrant rearrangement has led to the joining of a J1 gene segment to a sequence unrelated to any V gene (L10), and which in the germ line is flanked by a sequence resembling a V region recombination signal sequence. In this case no segregation of the reciprocal recombination products (kappa-41 and f41), which is a required step in sister chromatid exchange models, has taken place. An inversion model provides the simplest explanation of this J rearrangement.     

Somatic variants of murine immunoglobulin lambda light chains

Studies of the murine lambda light chains produced by myeloma cells provided the first evidence for somatic point mutation of germ-line variable (V) region genes. An examination of the variable regions of 19 lambda 1 chains revealed seven which differed from a common sequence by one to three amino acid substitutions. Subsequently, one of these presumed somatic variants of the single lambda 1 V gene was characterized by DNA sequence analysis of the rearranged functional gene. The predicted DNA sequence alteration was observed and no silent mutation was evident. These studies of lambda chain variants suggested that the hypervariable, complementarity-determining regions (CDRs) ht be a preferred site of somatic mutation because all seven characterized variants contained substitutions only in these regions. By contrast, comparisons of closely related kappa chain variable region amino acid sequences, and more recently VK and VH genes, have suggested that somatic mutation probably occurs in codons for both framework and CDR residues. To examine this apparent discrepancy between the sites of somatic mutations in lambda and kappa genes, we have determined the nucleotide sequence of two lambda 1 gene from hybridomas and a lambda 2 gene from a myeloma. These sequences demonstrate that somatic mutation in lambda genes can occur in both the framework and CDR residues.     

An unusual translocation of immunoglobulin gene segments in variants of the mouse myeloma MPC11

Immunoglobulin light chain genes of the mouse are composed in germ-line DNA of four separate segments, the leader, V (variable), J (joining) and C (constant) segments. In immunocompetent cells a V and J gene segment are joined by a site-specific recombination event. In variants of the mouse myeloma MPC11 a so-called kappa (k) light chain fragment is expressed which consists of the MOPC321 leader peptide, joined to the kappa constant region peptide. Using the Southern blotting technique we found that the gene coding for the light chain fragment has apkparently been generated by an aberrant translocation of a V gene segment identical or very similar to the MOPC321 V gene segment into the large intervening sequence between the J and the C gene segments. The resulting deletion of the splice signals of the J segments could be the reason for the observed splicing between leader and C region sequences, a phenomenon which may be of general interest for the understanding of the splicing mechanism.     

A uniform deleting element mediates the loss of kappa genes in human B cells

Human immunoglobulin light-chain genes become rearranged in an ordered fashion during pre-B-cell development such that rearrangement generally occurs in kappa genes before lambda genes (refs 1,2). This ordered process includes an unanticipated deletion of the constant kappa (C kappa) gene and kappa enhancer sequence which precedes lambda rearrangement, and the site of this deletional recombination was located 3' to the joining (J kappa) segments in 75% of cases studied. We have now characterized the recombinational element responsible for this event on three separate alleles and found them to be identical. This kappa-deleting element recombined site-specifically with a palindromic signal (CACAGTG) located in the J kappa-C kappa intron. All losses of C kappa genes in other human B cells were mediated by this determinant, including the 25% of instances when this element recombined with sequences 5' to J kappa. In contrast, the kappa-deleting element remained in its germline form on all successful kappa-producing alleles. Moreover, kappa loss is an evolutionarily conserved event, as the kappa-deleting element appears to be the human homologue of the murine RS sequence. Our results suggest that this element may help ensure isotypic and allelic exclusion of light chains and may be involved in the ordered use of human light-chain genes.     

Recombination between an expressed immunoglobulin heavy-chain gene and a germline variable gene segment in a Ly 1+ B-cell lymphoma

The early stages of murine B-cell differentiation are characterized by a series of immunoglobulin gene rearrangements which are required for the assembly of heavy(H) and light(L)-chain variable regions from germline gene segments. Rearrangement at the heavy-chain locus is initiated first and consists of the joining of a diversity (DH) gene segment to a joining (JH) gene segment. This forms a DJH intermediate to which a variable (VH) gene segment is subsequently added. Light-chain gene rearrangement follows and consists of the joining of a VL gene segment to a JL gene segment: once a productive light-chain gene has been formed the cell initiates synthesis of surface immunoglobulin M (sIgM) receptors (reviewed in ref. 1). These receptors are clonally distributed and may undergo further diversification either by somatic mutation or possibly by continued recombinational events. Such recombinational events have been detected in the Ly 1+ B-cell lymphoma NFS-5, which has been shown to rearrange both lambda and H-chain genes subsequent to the formation of sIgM (mu kappa) molecules. Here we have analysed a rearrangement of the productive allele of NFS-5 and found that it is due to a novel recombination event between VH genes which results in the replacement of most or all of the coding sequence of the initial VHQ52 rearrangement by a germline VH7183 gene. Embedded in the VH coding sequence close to the site of the cross-over is the sequence 5' TACTGTG 3', which is identical to the signal heptamer found 5' of many DH gene segments. This embedded heptamer is conserved in over 70% of known VH genes. We suggest that this heptamer mediates VH gene replacement and may play an important part in the development of the antibody repertoire.     

Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.     

Germline transmission of genetically modified primordial germ cells

Primordial germ cells (PGCs) are the precursors of sperm and eggs. In most animals, segregation of the germ line from the somatic lineages is one of the earliest events in development; in avian embryos, PGCs are first identified in an extra-embryonic region, the germinal crescent, after approximately 18 h of incubation. After 50-55 h of development, PGCs migrate to the gonad and subsequently produce functional sperm and oocytes. So far, cultures of PGCs that remain restricted to the germ line have not been reported in any species. Here we show that chicken PGCs can be isolated, cultured and genetically modified while maintaining their commitment to the germ line. Furthermore, we show that chicken PGCs can be induced in vitro to differentiate into embryonic germ cells that contribute to somatic tissues. Retention of the commitment of PGCs to the germ line after extended periods in culture and after genetic modification combined with their capacity to acquire somatic competence in vitro provides a new model for developmental biology. The utility of the model is enhanced by the accessibility of the avian embryo, which facilitates access to the earliest stages of development and supplies a facile route for the reintroduction of PGCs into the embryonic vasculature. In addition, these attributes create new opportunities to manipulate the genome of chickens for agricultural and pharmaceutical applications.     

RNA splicing generates a variant light chain from an aberrantly rearranged kappa gene

Both C kappa regions in MPC 11 cells are rearranged into active transcripion units, one producing a normal kappa chain and the other an internally deleted kappa fragment lacking a V region. The gene coding for the kappa fragment mRNA is aberrantly rearranged and lacks a site for V leads to C kappa splicing. An alternative splicing event which deletes the V region from the nuclear RNA precursor generates the kappa fragment mRNA.     

p63 protects the female germ line during meiotic arrest

Meiosis in the female germ line of mammals is distinguished by a prolonged arrest in prophase of meiosis I between homologous chromosome recombination and ovulation. How DNA damage is detected in these arrested oocytes is poorly understood, but it is variably thought to involve p53, a central tumour suppressor in mammals. While the function of p53 in monitoring the genome of somatic cells is clear, a consensus for the importance of p53 for germ line integrity has yet to emerge. Here we show that the p53 homologue p63 (refs 5, 6), and specifically the TAp63 isoform, is constitutively expressed in female germ cells during meiotic arrest and is essential in a process of DNA damage-induced oocyte death not involving p53. We also show that DNA damage induces both the phosphorylation of p63 and its binding to p53 cognate DNA sites and that these events are linked to oocyte death. Our data support a model whereby p63 is the primordial member of the p53 family and acts in a conserved process of monitoring the integrity of the female germ line, whereas the functions of p53 are restricted to vertebrate somatic cells for tumour suppression. These findings have implications for understanding female germ line fidelity, the regulation of fertility and the evolution of tumour suppressor mechanisms.     

Major reorganization of immunoglobulin VH segmental elements during vertebrate evolution

In mammals, the immunoglobulin heavy-chain variable region (VH) locus is organized in a linear fashion; individual VH, diversity (DH), joining (JH) and constant (CH) region segments are linked in separate regions. During somatic development, coding segments flanked by characteristic short recombination signal sequences, separated by intervening sequence regions that may exceed 2,000 kilobases (kb), are recombined. Combinatorial joining of different segments as well as imprecision in this process contribute to the diversity of the primary antibody response; subsequent mutation further alters functionally rearranged genes. This basic somatic reorganization mechanism is shared by six major families of genes encoding antigen receptors. Previously, we have shown that multiple germline genes and mammalian-like recombination signal sequences are associated with the VH gene family of Heterodontus francisci (horned shark), a primitive elasmobranch. Studies presented here demonstrate that segmental reorganization involving mammalian-like DH and JH segments occurs in the lymphoid tissues of this species. In marked contrast to the mammalian system, we find multiple instances of close linkage (approximately 10 kb) between individual VH, DH, JH, and CH segments. This unique organization may limit combinatorial joining and be a factor in the restricted antibody response of this lower vertebrate.     

Gene conversion between duplicated genetic elements in yeast

The mitotic recombination behaviour of a duplication of the his4 region on chromosome III in the yeast Saccharomyces cerevisiae was studied. The major recombination event between the duplicated segments is gene conversion unassociated with reciprocal recombination. The rad52-1 mutation preferentially decreases mitotic gene conversion. These results suggest that mitotic gene conversion may occur by a different pathway from that occurring in meiosis. This mitotic gene conversion may be important in yeast mating type interconversion and the maintenance of sequence homogeneity in families of repeated eukaryotic genes.     

A molecular programme for the specification of germ cell fate in mice

Germ cell fate in mice is induced in proximal epiblast cells by the extra-embryonic ectoderm, and is not acquired through the inheritance of any preformed germ plasm. To determine precisely how germ cells are specified, we performed a genetic screen between single nascent germ cells and their somatic neighbours that share common ancestry. Here we show that fragilis, an interferon-inducible transmembrane protein, marks the onset of germ cell competence, and we propose that through homotypic association, it demarcates germ cells from somatic neighbours. Using single-cell gene expression profiles, we also show that only those cells with the highest expression of fragilis subsequently express stella, a gene that we detected exclusively in lineage-restricted germ cells. The stella positive nascent germ cells exhibit repression of homeobox genes, which may explain their escape from a somatic cell fate and the retention of pluripotency.     

DNase I hypersensitive sites in the chromatin of human mu immunoglobulin heavy-chain genes

An immunoglobulin polypeptide chain is encoded by multiple gene segments that lie far apart in germ-line DNA and must be brought together to allow expression of an immunoglobulin gene active in B lymphocytes. For the immunoglobulin heavy chain genes, one of many variable (V) region genes becomes joined to one of several diversity (D) segments which are fused to one of several joining (J) segments lying 5' of the constant region (C) genes. Here we show that the rearranged mu genes of an IgM-producing human B-lymphocyte cell line exhibit pancreatic deoxyribonuclease (DNase I) hypersensitive sites in the JH-C mu intron that are absent in naked DNA or the chromatin of other differentiated cell types. DNA sequence analysis reveals that the major hypersensitive site maps to a conserved region of the JH-C mu intron recently shown to function as a tissue-specific enhancer of heavy-chain gene expression. A similar association of an enhancer-like element with a DNase I hypersensitive site has been reported for the mouse immunoglobulin light-chain J kappa-C kappa intron. These results implicate disruption of local chromatin structure in the mechanism of immunoglobulin enhancer function.     

The joining of V and J gene segments creates antibody diversity

The variable regions of mouse kappa (kappa) chains are coded for by multiple variable (V) gene segments and multiple joining (J) gene segments. The V kappa gene segments code for residues 1 to 95; the J kappa gene segments code for residues 96 to 108 (refs 1-3). This gene organisation is similar to that encoding the V lambda regions. Diversity in V kappa regions arises from several sources: (1) there are multiple germ-line V kappa gene segments and J kappa gene segments; (2) combinatorial joining of V kappa gene segments with different germline J kappa gene segments; and possibly, (3) somatic point mutation, as postulated for V lambda gene segments. Also, from a comparison of the number of germ-line J kappa gene segments and amino acid sequences, it has been suggested that J kappa region sequences may be determined by the way V kappa and J kappa gene segments are joined. This report supports this model by directly associating various J kappa sequences with given J kappa gene segments.     

Ablation of XRCC2/3 transforms immunoglobulin V gene conversion into somatic hypermutation

After gene rearrangement, immunoglobulin V genes are further diversified by either somatic hypermutation or gene conversion. Hypermutation (in man and mouse) occurs by the fixation of individual, non-templated nucleotide substitutions. Gene conversion (in chicken) is templated by a set of upstream V pseudogenes. Here we show that if the RAD51 paralogues XRCC2, XRCC3 or RAD51B are ablated the pattern of diversification of the immunoglobulin V gene in the chicken DT40 B-cell lymphoma line exhibits a marked shift from one of gene conversion to one of somatic hypermutation. Non-templated, single-nucleotide substitutions are incorporated at high frequency specifically into the V domain, largely at G/C and with a marked hotspot preference. These mutant DT40 cell lines provide a tractable model for the genetic dissection of immunoglobulin hypermutation and the results support the idea that gene conversion and somatic hypermutation constitute distinct pathways for processing a common lesion in the immunoglobulin V gene. The marked induction of somatic hypermutation that is achieved by ablating the RAD51 paralogues is probably a consequence of modifying the recombination-mediated repair of such initiating lesions.