Expression and hypermutation of human immunoglobulin kappa genes.
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Alleles↗
Biomedical subjects
H G Zachau
Publications and source records attributed to H G Zachau.
In memoriam Alexander A. Bayev, 1904-1994.
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History, 20th Century↗
Searching for non-V kappa transcripts from the human immunoglobulin kappa locus.
The 76 V kappa (variable) gene segments of the human immunoglobulin kappa locus (Ig kappa) were cloned in two contigs. Within each contig the distances between the genes range from a few hundred bp to 31 kb. We have now studied the question of whether transcripts are produced from intergenic regions, from regions near the kappa locus or from V kappa orphon regions. RNAs from several cell lines were converted to cDNAs which were then hybridized to the cosmid and phage lambda clones of the locus and of the orphon regions; the conditions were chosen such that the hybridization of transcripts with repetitive elements was minimized. The expression of a rearranged V kappa gene in a lymphoid cell line was readily detected. Also, a region 46 kb downstream from C kappa (constant) and a cDNA clone from a non-lymphoid cell line hybridizing to this region were studied in some detail. No transcripts were found, however, to be derived from the intergenic regions of the germ-line kappa locus which is in keeping with current ideas on the evolution of the V kappa multigene family.
Amino Acid Sequence↗
Comparative mapping of DNA probes derived from the V kappa immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization.
Fluorescence in situ hybridization (FISH) of cosmid clones of human V kappa gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the kappa locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V kappa I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V kappa region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution.
Animals↗
The immunoglobulin kappa locus of primates.
The immunoglobulin kappa genes of nonhuman primates were studied by using sequence information and hybridization probes derived from the human kappa gene regions. The following results were obtained: (1) V kappa gene probes of the three major human kappa subgroups hybridized to restriction nuclease digests of DNA from the chimpanzees Pan troglodytes (PTR) and Pan paniscus (PPA), the gorilla Gorilla gorilla (GGO), the orangutan Pongo pygmaeus (PPY), the macaque Macaca mulatta (MMU), the marmoset Callithrix geoffrei (CGE), and the bushbaby Galago demidovii (GDE), yielding patterns of decreasing similarity to the patterns of the human V kappa multigene family. (2) The C kappa gene segments of PTR, GGO, and PPY were 99.6, 97, and 93%, respectively, identical in sequence to the human C kappa gene. A V kappa gene in PTR, GGO, PPY, and MMU was 98, 96, 96, and 95%, respectively, identical to the most C kappa proximal V kappa gene, called B3. The other two J kappa-C kappa proximal V kappa genes in human, B1 and B2, hybridize to restriction fragments of sizes identical to that of DNA from humans and great apes. (3) The long-range restriction maps of the human (HSA), PTR, and GGO kappa loci as established by pulsed-field gel electrophoresis (PFGE) are quite homologous. According to the maps, however, and to hybridization studies with 11 duplication-differentiating probes, there is only one copy of the locus in PTR and GGO. This means that the duplication of large parts of the kappa locus as found in humans occurred after the branch-point of human and great ape evolution.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
A potentially functional V kappa gene at a distance of 1.5 Mb from the immunoglobulin kappa locus.
An amplicon that is highly homologous to a part of the human kappa locus was found at the 3' or telomeric side of C kappa at a distance of about 1.5 Mb. From analysis of sequence divergence, it is concluded that the amplicon was formed after the duplication of the kappa locus, which may have taken place about 1 million years ago. A V kappa gene of subgroup III within the amplicon turned out to have no defects in its sequence and to have a 5'-3' orientation opposite to the one of J kappa C kappa. Theoretically, a functional V kappa-J kappa-C kappa gene could be formed by an inversion mechanism, but no rearrangement products were observed so far.
Base Sequence↗
The human immunoglobulin kappa locus. Characterization of the duplicated A regions.
The central regions of the kappa locus, the so-called A regions, have been fully characterized on cosmid and phage lambda clones. The regions, which are parts of the C kappa-proximal and -distal copies of the locus and are, therefore, called Ap and Ad regions, comprise about 140 kb each and contain together 30 V kappa genes and pseudogenes. The A regions have been linked on their 5' sides to the O regions and on their 3' sides to the L regions. Chromosomal walking has eliminated a previous gap in the Ap region. Detailed restriction maps of the Ap and Ad regions and the sequences of 9 V kappa genes are reported. Four events, which have occurred in evolution probably after the duplication of the A region, were identified: the insertion of an Alu element in Ad; the insertion of part of a LINE element in Ap; the deletion of a 17.5-kb fragment including one V kappa gene from Ap; the sequence divergence of duplicated V kappa gene regions which ranges among the five pairs studied here from 0 to 14 bp per kb and converted two genes to pseudogenes while their duplicates stayed functional. An analysis of the A regions of the lymphoid cell lines RPMI 6140 and GM607 confirmed the previous finding that the V kappa-J kappa rearrangement in these cell lines had occurred by deletion and inversion mechanisms, respectively. Thus, the structural data contribute to the understanding of the evolution and the functioning of the A regions of the kappa locus.
Amino Acid Sequence↗
Ongoing V kappa-J kappa recombination after formation of a productive V kappa-J kappa coding joint.
V kappa genes of man can recombine with the J kappa gene segments either by an inversion or by a deletion mechanism. Back-to-back fusion products of the respective recombination signal sequences (signal joints) are retained on the chromosome after the formation of a V kappa-J kappa coding joint by an inversion. Our knowledge of the structure of the human kappa locus and the application of the polymerase chain reaction allowed us now to establish a direct relationship between different kappa recombination products in the lymphoid cell line JI. Two consecutive inversions fully explain the existence of two coding joints and two signal joints on the same chromosome of this cell line. Although the initially formed coding joint is productively rearranged and expressed, a second V kappa-J kappa rearrangement took place which leads to an aberrant joint. In this process a J kappa gene segment of the signal joint that had been created in the first V kappa-J kappa joining was used as the recombination target. The sequence of the two rearrangements is unequivocal since a product of the first (productive) reaction is a partner in the second (aberrant) one.
Amino Acid Sequence↗
Of orphons and UHOs. Delimitation of the germline repertoire of human immunoglobulin kappa genes.
Two problems in defining the germline repertoire of immunoglobulin kappa genes were investigated. One concerns putative transposed V kappa genes (orphons), the other one weak hybridization signals which may or may not turn out to be V kappa genes (UHOs). It was shown by sequencing that the three V kappa genes Z2, Z3 and Z4 are very closely related to the Z1 and V118 genes and to two other genes which had been localized on chromosomes 1 and 22, i.e. outside the kappa locus on chromosome 2. It is therefore likely that also the Z2-Z4 genes are orphons and not part of the kappa locus. Two UHOs turned out not to contain V kappa-like structures. This together with previous results makes it likely that we have detected all germline V kappa genes with the available hybridization probes.
Amino Acid Sequence↗
Human immunoglobulin genes of the kappa type. The long-range map of an orphon V kappa gene region.
As was previously shown by Zimmer et al. (EMBO J. 9, 1535-1542, 1990 and Biol. Chem. Hoppe-Seyler 371, 939-951, 1990), the so-called W regions comprising 11 V kappa pseudogenes are located on the long arm of chromosome 2, very closely to the centromere. They are probably derived by a pericentric inversion and amplification events from gene regions of the kappa locus, which is located on the short arm of chromosome 2 also very closely to the centromere. The restriction map of the W regions was now extended from the previous 1.2 Mb to 4.3 Mb and, at the same time, revised with respect to certain features. This was made possible by a new hybridization probe specific for the Wc region and by the improved resolution and extended range of pulsed field gel electrophoresis. On the basis of the long-range maps of the W regions and the kappa locus the V kappa genes of the kappa locus have to be at least 2.5 Mb apart. This distance can be taken also as a minimal estimate for the size of the centromere DNA of chromosome 2; it is quite possible that the size is much larger.
Chromosome Mapping↗
The human antibody V region repertoire to the type B capsular polysaccharide of Haemophilus influenzae.
The V region repertoire of the human antibody response to the type b capsular polysaccharide of Haemophilus influenzae (Hib-PS) is being defined at the molecular level using antibodies purified from serum of immunized adults. The VH of this response is restricted to the VHIII subgroup while the VL can be divided into two categories. The most common VL, expressed in > 90% of adults and usually constituting the majority of a subjects anti-Hib-PS antibody response, is restricted to the product of a single V kappa II gene known as A2 that probably lacks somatic mutations. The product of the A2 gene is invariably joined to one of several J kappa products by an inserted arginine at the V kappa-J kappa junction. In contrast to the restricted nature of the dominant VL clonotype, the second category of VL constitutes a heterogeneous group of at least seven different VL gene products that often contain somatic mutations and generally exhibit crossreactivity with a related polysaccharide from E. coli. Elucidation of anti-Hib-PS V regions at the molecular level will permit examination of structure-function relationships among these clinically important antibodies and should make the V region repertoire to Hib-PS a useful model for studying human V gene responses.
Amino Acid Sequence↗
Clonal characterization of the human IgG antibody repertoire to Haemophilus influenzae type b polysaccharide. IV. The less frequently expressed VL are heterogeneous.
We previously demonstrated that the human anti-Haemophilus influenzae type b polysaccharide (Hib-PS) VL repertoire is dominated by a product of the V kappa II gene, A2, and that V kappa II-A2 anti-Hib-PS antibodies have little or no somatic mutation in VL. To further study this VL repertoire, we studied non-A2 anti-Hib-PS antibodies that were identified either serologically or by amino-terminal amino acid sequence analysis. Of 15 non-A2 anti-Hib-PS antibodies from 12 vaccinated adults, we found four V lambda, five V kappa I, one non-A2 V kappa II, four V kappa III, and one V kappa IV antibodies. As expected, all but two of these subjects also produced V kappa II-A2 antibodies. Interestingly, one of these subjects lacks the A2 gene in the germ line. However, both subjects who did not produce detectable V kappa II antibody did produce normal amounts of total anti-Hib-PS antibody after vaccination. Candidate V kappa genes for the non-A2 antibodies were identified by comparison of up to 60 VL amino acid residues, including CDR1 and CDR2, with all sequenced V kappa genes. V kappa I antibodies appear to be products of three newly sequenced V kappa I genes, O8, O18, and L11, that are reported here. The O8 and O18 genes encode identical amino acid sequences. The non-A2 V kappa II antibody is a likely product of the A1 or A17 genes, the V kappa III antibodies are likely products of the A27 gene, and the V kappa IV antibody is a product of the single V kappa IV gene, B3. Unlike V kappa II-A2 antibodies, the V kappa I, V kappa III, and V kappa IV antibodies differed by one to five CDR residues from the germ line product of the candidate genes, suggesting the presence of somatic mutations. Thus, anti-Hib-PS antibodies can be divided into two types, the most frequently observed A2 antibodies with little or no somatic mutation and non-A2 antibodies that likely contain somatic mutations.
Amino Acid Sequence↗
The human immunoglobulin kappa locus. Characterization of the duplicated O regions.
Two large regions of the human immunoglobulin kappa locus, the so-called O regions, have been characterized on cosmid and phage lambda clones. The two regions are very similar but not identical duplicates belonging to the C kappa proximal (p) and the distal (d) copies of the kappa locus. The Op and Od regions comprise contigs of 90 and 120 kb, respectively, and contain 20 V kappa genes and pseudogenes which have been sequenced. Three pairs of V kappa genes were found to be practically identical in the duplicates while allotypic differences, at least for two of the genes, are considerable. The similarities between the duplicate genes may be related to the fact that the two copies of the kappa locus are arranged in a palindrome-like fashion with the 5' sides of the O regions pointing towards each other (C kappa J kappa B Lp Ap Op-Od Ad Ld). This may have contributed to equalizing the sequences. Beyond Op and Od no further V kappa genes were found within about 80 kb. Instead, repetitive DNA sequences have been localized there, the structures of which suggest that they may have been involved in the evolution of the V kappa gene-containing regions. The V kappa pseudogene containing W regions, that had been transposed in evolution from the short to the long arm of chromosome 2 by a pericentric inversion, may have been derived from the O regions according to structural homologies between defined sections of the O and W regions.
Base Sequence↗
Polymorphisms and haplotypes in the human immunoglobulin kappa locus.
By comparing the restriction patterns of the DNA from 23 unrelated individuals 16 polymorphisms were defined which allowed us to differentiate between the duplicated copies Op, Ap, Lp and Od, Ad, Ld of the kappa locus (p for the C kappa proximal, d for the distal copy). Some of these duplication-differentiating polymorphisms or DDP revealed also allelic differences between individuals; they are therefore restriction fragment length polymorphism (RFLP) markers at the same time. Three RFLP in the single copy B-J kappa-C kappa region were included into the study. Three basic haplotypes were derived from the combined genotype data, haplotypes N, G and 11. The latter haplotype in which the whole distal copy of the kappa locus is missing was found three times among the 46 haploid genomes studied. The genotypes of the family members of an individual who is homozygous for haplotype 11 are consistent with Mendelian inheritance. Haplotypes N and G are distinguished from each other by eight RFLP markers. Six additional haplotypes, which were found in one or several individuals each, can be derived from the basic haplotypes N and G by hypothetical recombination and/or mutation events.
Chromosome Mapping↗
Megabase inversions in the human genome as physiological events.
The genes of the immunoglobulin kappa light chains are assembled during B-cell differentiation by somatic recombination of one of the V kappa (variable) gene segments and the J kappa-C kappa (joining-constant) gene region. This seems to occur by deletion of the DNa between V kappa and J kappa-C kappa if they are arranged in germ-line DNA in the same transcriptional polarity or by inversion of a fragment containing the V kappa gene if the polarities are opposite. We have cloned 75 V kappa genes and pseudogenes of the human kappa locus and linked them in large contigs. There seem to be no more than 85 such genes, less than 50 of these being potentially functional. Thirty-eight of the cloned genes have the same transcriptional polarity as J kappa-C kappa and are part of the so-called J kappa proximal cluster; 35 genes in a distal cluster (the result of a duplication event in evolution) have a polarity that was suggested to be opposite to the one of J kappa-C kappa. We now show that the V kappa genes of the proximal cluster rearrange by a deletion mechanism whereas the others join J kappa-C kappa by inversion of megabase-sized DNA fragments.
Base Sequence↗
A human immunoglobulin kappa orphon without sequence defects may be the product of a pericentric inversion.
The VK gene segments that have been transposed from the kappa locus on the short arm of chromosome 2 at 2p11-12 to other chromosomal sites are called orphons. The 18 VK orphons sequenced up to now carry defects and are to be considered pseudogenes. We now describe the VKI gene segment V108 whose sequence is without any defects and which was localized to the long arm of chromosome 2 at 2q12-14 by in situ hybridization. The V108 region may have been transposed from the short to the long arm of chromosome 2 by a pericentric inversion. Possible reasons for the conservation of its sequence are discussed. In spite of its bona fide sequence V108 is considered to be an unlikely candidate for a VK-JK rearrangement and subsequent functional expression.
Amino Acid Sequence↗
Structural features of transposed human VK genes and implications for the mechanism of their transpositions.
The genes encoding the variable, joining and constant regions of human immunoglobulin light chains have been localized to the short arm of chromosome 2. However, several VK genes lie outside of the locus: a single copy cluster of five VK genes is located on chromosome 22; an isolated but amplified VkI gene is found on chromosome 1; and several isolated VkI genes are on as-yet-unidentified chromosomes other than chromosome 2. Vk genes not contained within the kappa locus are termed orphons. We have attempted to gain insight into the mechanism of transposition of both the chromosome 22 cluster and the several amplified VkI genes by searching in the kappa locus for a parent copy of the former, and by analyzing the junctions between transposed VKI-containing segments and adjacent non-amplified regions. The chromosome 22 orphon cluster must have been non-duplicatively transposed. Sequence features at the junctions of this and other orphon regions are direct and inverted repeats, and, in one case, an Alu repeat. These unusual features may have predisposed the orphon regions to transposition by serving as target sites for enzymes involved in recombination.
Base Sequence↗
The V kappa gene repertoire in the human germ line.
The question of how many V kappa gene segments exist in the human germ line was addressed. Seventy-five V kappa genes of the kappa locus and twenty-five V kappa genes localized outside of the locus ("orphons") had been cloned previously; 67 of the genes and 19 of the orphons had already been sequenced yielding 36 and 1 potentially functional V kappa genes, respectively, the remaining ones being pseudogenes. We now (a) determined the relative hybridization intensities of the cloned V kappa genes and orphons, (b) identified the bands in blot hybridizations of genomic DNA digests with the cloned genes and orphons, (c) determined the band intensities in the genomic DNA digests from two individuals and one cell line, (d) normalized the results with the help of the C kappa gene segment which is present in the haploid genome in one copy, (e) compared the genomic blot hybridization patterns with patterns of equimolar mixtures of the cloned V kappa genes and orphons, and (f) defined the bands and fractional intensities in bands that could not be assigned to cloned genes or orphons. From the resulting data we conclude that there are 5-7 still uncloned V kappa genes in germ-line DNA in addition to the 75 known V kappa genes and in addition to the 25 orphons 12-15 orphon candidates. It appears that the rheumatoid factor light chains of the Wa and 6B6.6 idiotypes are coded for by one V kappa III gene each. It is concluded that the kappa locus comprises no more than 50 potentially functional genes and no more than 85 V kappa genes altogether.
Blotting, Southern↗