On-line version ISSN 1678-4685
Genet. Mol. Biol. vol. 21 n. 2 São Paulo June 1998
Physical map and one-megabase sequencing of the human immunoglobulin lambda locus
Geraldo A.S. Passos Jr.
Grupo de Imunogenética Molecular, Departamento de Genética, Faculdade de Medicina de Ribeirão Preto, USP, 14040-900 Ribeirão Preto, SP, Brasil. Fax: 016 633 0069. E-mail: email@example.com and Faculdade de Odontologia de Ribeirão Preto, USP, Departamento de Morfologia, Disciplina de Genética, 14040-904 Ribeirão Preto, SP, Brasil. http://www.rgm.fmrp.usp.br/imunogenetica
The human immunoglobulin lambda (IGL) locus is located on chromosome 22q11.1-q11.2 and contains the genes responsible for the immunoglobulin lambda light chains. This locus was recently mapped (physical map) and its 1-Mb DNA totally sequenced. In this review we focus on the characterization of the v-lambda genes, its chromosomal location, genomics and sequencing of the IGL locus.
The interface between molecular genetics and immunology has recently led to important advances in human genome research. In 1996 the human T-cell receptor (TCR) b locus (685 kb) was totally sequenced (Rowen et al., 1996).
Less than one year after this achievement, the record in genome sequencing was beat. Today the largest contiguous human DNA sequence published is the one-megabase sequence from chromosome 22q11.1-q11.2, including the immunoglobulin lambda variable locus (IGL) which contains the genes responsible for the immunoglobulin lambda light chains (Kawasaki et al., 1997). In fact, the study of the IGL locus is an illustrative example of genomics applied to immunology.
Immunoglobulins (Ig) are among the most extensively studied protein classes. Thousands of amino acid and nucleotide sequences are available for different Ig subclasses and animal species (Kabat et al., 1991; Pladlan, 1994). All the parts of antibody molecules have been visualized, including the hinge (Harris et al., 1992). Very recently, data banks specialized in immunoglobulin and TCR sequences have been organized and available on the Internet (http://imgt.cnusc.fr:8104 and www.ebi.ac.uk/imgt).
An immunoglobulin molecule is characterized by two polypeptide heavy chains and two light chains, held together by disulfide bonds. The functional molecule assumes a Y shape (Padlan, 1994).
Each heavy and light chain contains a region of intense variability in the amino acid sequence at the amino end (variable region or V) and a constant region at the carboxy end (C). The V regions interact with the antigens by non-covalent binding.
The variable regions contain three intervals of hypervariability, the complementarity-determining regions (CDR 1, 2 and 3) and four less variable intervals in the sequence, the frameworks (FR 1, 2 and 3). There are five classes of immunoglobulins, IgG, IgM, IgA, IgD and IgE, defined by the primary structure of the constant region of the heavy chains.
The light chains are of two types: the kappa (k) and lambda (l) chains bind exclusively to one of the five heavy chain classes to form a complete antibody molecule, for example IgGk or IgGl.
Serum antibody light chains are 95% kappa and 5% lambda in mice and 60% kappa and 40% lambda in man (Kabat et al., 1991; Padlan 1994).
Though lambda chains are important components of human serum antibodies, the IGL locus only recently has been totally mapped and sequenced on chromosome 22q11.1-q11.2 (immunoglobulin lambda locus) (Frippiat et al., 1995; Kawasaki et al., 1995, 1997).
Locating the human IGL locus
The human IGL locus was located on chromosome 22 by analyzing a group of human/rodent somatic cell hybrids and cloned Cl gene probes by the Southern method (McBride et al., 1982). A cytogenetic visualization was obtained by in situ hybridization on the 22q11 band (de la Chapelle et al., 1983; Mattei et al., 1991). Probes of Vl genes were cloned from human genomic libraries (Anderson et al., 1984) and their copy number was estimated to be around 70 genes by Southern blots (Lai et al., 1989).
Sequencing Vl genes and nomenclature
Several germline Vl genes were isolated and lambda chains from antibodies sequenced during the past 10 years.
Functional Vl genes, ORF sequences and pseudogenes have been identified from these sequences (Dariavach et al., 1987; Alexandre et al., 1989; Brockly et al., 1989; Siminovitch et al., 1989; Bernard et al., 1990; Frippiat et al ., 1990; Vasicek and Leder, 1990; Bauer Jr. and Blomberg, 1991; Combriato and Klobeck, 1991; Daley et al., 1992a,b; Winkler et al., 1992; Williams and Winter, 1993; Stiernholm et al., 1994).
By comparing all the Vl protein sequences and sequences deduced from codons, Chuchana et al. (1990) defined seven Vl subgroups designated IGLV1 to IGLV7 following the recommendations of the Human Gene Mapping Committees, where IG is immunoglobulin, L is lambda light chain, V is variable and 1 is subgroup. The different genes in the same subgroup are designated S1, S2, etc.
However, at this time there is still limited genomic information about the IGL locus. All we know is the organization of the Jl-Cl region, located 14 kb downstream of the Vl genes, spanning 40-55 kb of DNA, depending on the haplotype (Combriato and Klobeck, 1991).
We know the positions of 69 germline Vl sequences including functional genes and pseudogenes distributed among ten IGLV subgroups (Kawasaki et al., 1997). The genomic methodology applied to analyze the IGL locus permitted Frippiat et al. (1995) and Kawasaki et al. (1995) to construct physical maps by means of contigs of YACs, BACs and cosmids. Frippiat's map located 52 Vl functional genes from 10 Vl families and seven Jl and Cl genes covering 1140 kb, with the Vl genes being arranged within 800 kb.
The IGL locus was divided into three clusters: cluster A (proximal to the Cl1 gene) comprises genes from the IGLV2 and IGLV3 families, cluster B comprises genes from the IGLVl, IGLV5, IGLV7 and IGLV9 families and cluster C (distal to Cl1) comprises genes from the IGLV4, IGLV6, IGLV8 and IGLV10 families (Figure 1). The clustered feature of the IGL locus differs from that of the human immunoglobulin heavy (Cook et al., 1994) and kappa light chain (Zachau, 1993), for which genes from different families are interspersed. The 52 mapped Vl genes were identified as EcoR1 fragments, hybridizing strongly to the Vl family-specific probes.
Figure 1 - Schematic representation of the human IGL locus on chromosome 22q11.1-q11.2. This figure was taken from the Immunogenetics Data Base on the Internet http://imgt.cnusc.fr:8104/textes/maps/human/IGL.html.
A unique Vl8 gene (IGL8S1, acc Z73650) was mapped in a 3.7-kb EcoR1 fragment on cluster C. However, Southern-EcoR1-RFLP studies on a Brazilian population have revealed two additional fragments bearing Vl8 sequences (Passos et al., 1977). In a study of human/rodent somatic cell hybrid panel DNA, Frippiat et al. (1997) and Queiroz et al. (1997) showed that IGLV8 is a gene family with members that are duplicated and translocated from chromosome 22 to 8, providing the first evidence for the dispersion of Vl genes outside the major locus (orphan genes).
Non-Vl genes within the IGL locus
Other sequences unrelated to the Vl genes, such as the VpreB gene, which encodes part of the surrogate light chain, the g-glutamyl transpeptidase gene (GGT) and the BCRL4 pseudogene, have also been mapped within the IGL locus and the t(8;22)(q24;q11) chromosomal translocation breakpoint of the BL-2 Burkitt's lymphoma.
Divergence between the two maps
The Kawasaki map spanned about 1020 kb on 22q11, revealing that the IGL locus is 911 kb in length. This map located 69 unique EcoRI-HindIII gene segments that hybridize to Vl gene probes. This author divided the IGL locus into five clusters (regions I-V). These clusters are separated by a 40-kb interval.
Some regions in the Frippiat map are not well resolved, which may be the reason the distribution of the Vl gene segments does not corroborate the data from Kawasaki.
The main priority in the Frippiat map was the functional genes, those that continued to be hybridized after high stringency washes of the Southern membranes, while in the Kawasaki map all the possible Vl sequences, hybridizing weakly or strongly to the probes, were mapped. Moreover, these authors do not use the same nomenclature. The Vl sequences in the Kawasaki map were numbered 1 to 69, beginning proximal to the Cl1 gene. The basic organization of the locus is the same in the two physical maps, with the Vl genes towards the centromeric region and the Jl-Cl segments towards the telomeric region.
The IGL locus is totally sequenced
The contig methodology applied by Kawasaki's group, overlapping 189 cosmid clones to construct the physical map of the IGL locus, opened the possibility for its total sequencing. Kawasaki et al. (1997) then demonstrated the longest contiguous human DNA sequence ever published. A total of 1,025,415 bases of this nucleotide sequence, including the entire human IGL locus, were determined.
The sequence data revealed the organization of 36 potentially active genes, 33 pseudogenes and seven Jl-Cl segments. Among these 69 Vl sequences, 32 were newly discovered and confirmed the presence of the non-Vl genes within the locus.
The Vl genes were located within five gene-rich clusters and were divided into five clans, based on sequence identity. Clan I comprises IGLV1, IGLV2, IGLV6 and IGLV10 subgroup genes and pseudogenes IGLV(I) 20, 38, 42, 56, 63, 68 and 70; clan II, IGLV3 subgroup genes; clan III, IGLV7 and IGLV8 subgroup genes; clan IV, IGLV5 subgroup genes and pseudogenes IGL(IV) 53, 59, 64 and 65, and clan V, IGLV4 and IGLV9 subgroup genes, and pseudogenes IGLV(V) 58 and 66.
Internet data banks on immunogenetics have been organized
The increasing amount of data on DNA, cDNA and protein sequences, as well as physical maps of the immune response genes (Ig, TCR and MHC) of humans and mice led two important laboratories to organize informatized data banks on the Internet. These contain an exhaustive collection of sequences and maps. The "IMGT", international immunogenetics data base at Montpellier, France, was idealized and is coordinated by Professor Marie-Paule Lefranc (http://imgt.cnusc.fr:8104). This site contains a complete source of sequences of immunoglobulin DNA (kappa and lambda light chains) and T-cell receptors (TCR b, g and d) and a graphic representation of the physical maps. Special attention is given to the variable regions of Igs and TCRs at both the DNA and protein level. The sequences can be accessed by its accession numbers and compared by means of" DNAPLOT" interactive software.
The IMGT site represents a meticulous revision of the molecular immunogenetics of Ig and TCR, with the advantage of being constantly updated.
A graphic representation of the IGL locus (physical map) can be visualized at this site (Figure 1).
The "V BASE" (www.mrc-cpe.cam.ac.uk/imt-doc/vbase-home-page.html) is a comprehensive directory of all human germline variable regions. It contains thousands of sequences. This site was organized by Professor Ian Tomlinson and colleagues of the Centre for Protein Engineering at Cambridge, England. Among the various download options, the DNAPLOT software is available.
Expression of Vl genes in the B-cell repertoire
Diversity in the antibody repertoire in both heavy and light chains is generated by somatic events in DNA, such as V(D)J recombination and somatic hypermutation. Recombination at the IGL locus occurs by random deletions which approximate a given Vl gene to a Jll-Cl segment. The frequency of recombination between germline Vl, Jl and Cl genes contributes to the B-cell repertoire at the periphery. Ignatovich et al. (1997) characterized the l light chain gene expression using a library of l cDNA clones from peripheral blood lymphocytes. Most of the expressed l chains were derived from cluster A, closest to the Jl Cl segments. The use of the different Vl genes is highly variable, ranging from 0.02 to 27%. Of the nearly 30 functional segments, three (2a2, 1e and 2c) encode half the expressed Vl repertoire in normal individuals (Figure 2).
Figure 2 - Contribution of the immunoglobulin Vl gene families to the peripheral B cell repertoire. This figure was taken from Ignatovich et al. (1997), with permission.
Concluding remarks and perspectives
Over the last two years human genome research has been marked by historical achievements: the complete sequencing of the 685 kb of the TCR b locus by Rowen et al. (1996) and, more recently, of the entire IGL locus (1.0 Mb DNA) by Kawasaki et al. (1997). This knowledge opens new perspectives for further studies on the contribution of the germinative Vl genes to the B-cell repertoire under normal and pathological conditions, on V(D)J recombination, on DNA polymorphism among human populations, and on the evolution of the IGL locus.
G.A.S.P. Jr. received grants from FAPESP (No.95/9839-3) and CNPq-FAPESP (No. 96/5842-2). Publication supported by FAPESP. Figure 2 was reprinted from Ignatovich, O. et al. (J. Mol. Biol. 268: 69-77, 1997) with permission of the Academic Press Ltd., London.
O locus IGL humano está localizado no cromosomo 22q11.1-q11.2 e contém os genes responsáveis pelas cadeias leves de imunoglobulina tipo lambda. Este locus foi recentemente mapeado (mapa físico) e seu 1 Mb DNA totalmente sequenciado. Nesta revisão focamos os principais resultados de caracterização dos genes v-lambda, sua localização cromossômica, a genômica e seqüenciamento do locus IGL.
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(Received December 2, 1997)