Evolutionary dynamics of the human immunoglobulin kappa locus and the germline repertoire of the Vkappa genes.

We have determined the entire nucleotide sequence of the human immunoglobulin kappa locus, comprising a total of 1,010,706 nucleotides. The 76 Vkappa genes found by a hybridization-based approach and their classification in 7 families were confirmed. A Vkappa orphon located near the locus was also sequenced. In addition, we identified 55 novel Vkappa relics and truncated pseudogenes, which establish 5 new families. Among these 132 Vkappa genes, 46 have open reading frames. According to the databases and the literature, 32 unique Vkappa genes and 5 identical gene pairs form VJ-joints, 27 unique genes and 4 gene pairs are transcribed, and 25 unique genes and 4 gene pairs produce functional proteins. The Vkappa gene locus contains a 360-kb inverted duplication, which harbors 118 Vkappa genes. A comparison of the duplicated Vkappa genes suggests positive selection on the complementarity-determining regions of the duplicated genes by point mutations. The entire duplication unit was divided into 13 blocks, each of which has its distinct nucleotide sequence identity to its duplication counterpart (98.1 - 99.9 %). An inversion-mediated mechanism is suggested to generate the high-homology blocks. Based on the homology blocks and the mutation rates, the inverted duplication is assumed to have taken place approximately 5 million years ago. An orphon Vkappa gene near the kappa locus and a cluster of five Vkappa orphons on chromosome 22 have no counterparts within the kappa locus. This suggests possible mechanisms of the transposition of orphon Vkappa genes.

The immunoglobulin lambda light chain enhancer consists of three modules which synergize in activation of transcription.

V(D)J rearrangement, high level expression and somatic hypermutation of assembled Ig genes is tightly controlled by a number of regulatory sequence elements located in the vicinity of the J-, (D)-, and C-gene segments. During B cell maturation these elements become accessible to binding of trans-acting factors, reflecting the opening of the chromatin structure through an as yet unidentified mechanism. The mapping of regions of an altered chromatin structure (DNase I hypersensitivity) therefore is a powerful approach in identifying regulatory sequence elements. We here show that the human Ig lambda enhancer consists of three modules previously identified by us as DNase I-hypersensitive sites HSS-1, -2, and -3. The three sequence elements synergize in transcriptional activation of a reporter gene and together constitute a powerful tissue-specific enhancer which is a much stronger transcriptional activator than the kappa enhancers alone or in combination. We further show that the accessibility of the kappa and lambda enhancer elements for DNase I in the chromatin of a pre-B cell line (207) correlates with the transcriptional enhancer activities of kappa and lambda enhancer constructs. This finding is in support of an ordered model for Ig light chain activation (kappa before lambda).

V lambda and J lambda-C lambda gene segments of the human immunoglobulin lambda light chain locus are separated by 14 kb and rearrange by a deletion mechanism.

We have cloned a region of 124 kb of the human immunoglobulin lambda light chain locus on chromosome 22 encompassing seven V lambda and seven J-C lambda gene segments. No further C lambda gene segment was found in a region of 35 kb downstream of C lambda 7, which encodes the Ke+Oz- isotype. The C lambda proximal V lambda gene segment V lambda III. 1 is located 14.5 kb upstream of C lambda 1. The five sequenced V lambda genes have the same transcriptional orientation as the J-C lambda gene segments which is likely to be true for the majority of the V lambda gene segments in the human lambda locus and which suggests a deletion mechanism for DNA rearrangement. This is supported by hybridization of V lambda gene probes to germ-line and rearranged DNA from lambda light chain-producing cell lines. Sequences of 23 cDNA clones allow to establish a V lambda subgroup classification based on nucleic acid sequence data and an estimate of the J-C lambda usage.

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.

The V-J-intergenic region of the human kappa locus.

The 23-kb region between the V kappa and J kappa gene clusters was investigated in some detail. The region was found to be free of V kappa genes or V kappa gene-like structures, confirming the previous supposition that the V kappa gene B3 is the J kappa proximal V kappa gene. The B3-J kappa distance of 23 kb was found to be the same in the DNAs of several individuals. A HindIII restriction fragment length polymorphism was detected within this region. A sequence of 533 bp located approximately in the middle of the region has a highly homologous counterpart (called homox) on another chromosome. The two sequences are 96% identical. Possible mechanisms for the generation of such a duplicate are discussed.

Linking of the human immunoglobulin VK and JKCK regions by chromosomal walking.

The linking of the human VK and JKCK gene regions (abbreviations in ref. 1) by chromosomal walking is reported. Hybridization experiments with the DNA of a somatic cell hybrid containing the region between JKCK and the telomer show that none of the major VK gene clusters is located downstream of CK. The distance between the VK and JK genes was found to be 23 kb. The JK proximal VK gene is the B3 gene which is the only representative of subgroup IV in the genome. This gene and the neighbouring B2 gene (accompanying paper) are arranged in opposite orientation to JKCK and can therefore rearrange only by an inversion mechanism. This finding is used, together with previous data, to delineate the rearrangement processes in the Burkitt lymphoma derived cell line BL21 as comprising an inversion in the first and a deletion in the second step.

N segment insertion and region-directed somatic hypermutation in a kappa gene of a t(2;8) chromosomal translocation.

A detailed molecular analysis of both reciprocal recombination products of the variant t(2;8) chromosomal translocation of the Burkitt lymphoma derived cell line JI and their germline counterparts was carried out. The breakpoint on chromosome 8 is localized 28 kb to the 3' side of the c-myc protooncogene, the breakpoint on chromosome 2 was found to be within an aberrantly rearranged VK gene (abbreviations ref. 1). Novel features of the immunoglobulin moiety involved in this process include insertion of extra nucleotides in the V-J junction which have the characteristics of a N segment as it has been found up to now only in heavy chain and T cell receptor genes; the occurrence of somatic mutations in 8q+ and not in 2p-. These data allow a reconstruction of the course of events in the cell line JI; remarkable sequence regularities at the chromosomal breakpoints consisting of symmetrically placed dinucleotides and elements related to the hepta- and nonanucleotide recombinase recognition sequences are discussed in the context of the translocation mechanism.

Reciprocal recombination products of VK-JK joining reactions in human lymphoid cell lines.

The recombination process that joins a VK to a JK segment of an immunoglobulin gene generates a second, reciprocal recombination product called f fragment. In this second product the regions flanking the VK and JK segments in the germline are joined in a head to head fashion. We now analysed f fragments in the human lymphoid cell lines Daudi, JI and IARC/BL41. All three f fragments contain JK1 flanks; the VK derived moiety of f Daudi and f41 could be traced back to known germline VK genes. There is a precise head to head joining of the heptanucleotide signal sequences in f Daudi and fJI while in f41 six nucleotides are present between the signal sequences. In contrast to the VK-JK recombination products, the f fragments were found to lack somatic mutations. The structures of the f fragments are discussed in the context of the VK-JK rearrangement mechanism.

Human immunoglobulin kappa light chain genes of subgroups II and III.

The first complete sequences of functionally rearranged VK genes (abbreviations ref. 1) of subgroups II and III are reported. The genes have been cloned from lymphoid cell lines synthesizing KII or KIII light chains as evidenced from immunochemical analyses with anti-VK subgroup-specific antisera. These data, together with the sequence of a KIV gene (described in the accompanying paper) and those of previously published KI genes make possible a comparison of genes representative of the four known V region subgroups of human K light chains. The VKII gene is distinguished from the VKI, VKIII, and VKIV genes by a much longer intron within the leader sequence: 426 bp vs ca. 120-220 bp. Blot hybridization experiments with human DNA digests using probes from the KII and KIII genes and from the respective upstream regions help to define subgroup specific probes and hybridization conditions.

Subgroup IV of human immunoglobulin K light chains is encoded by a single germline gene.

The series of studies on the human K light chain genes of the various subgroups is concluded by this report on the isolation and nucleotide sequence determination of a functional VKIV gene (abbreviations ref. 1) and its germline counterpart. The rearranged gene which stems from a lymphoid cell line and the germline gene differ in four nucleotides which can be attributed to somatic mutations; three of the mutations are clustered in CDR3. The germline gene regions of two unrelated individuals were identical over a stretch of 1267 bp. By hybridization experiments it is shown that the human K locus contains only one VKIV gene. In 16 lymphoid cell lines studied here, the VKIV gene is frequently deleted or aberrantly rearranged which may be a consequence of peculiarities of its function and/or its structural organization.

Immunoglobulin genes of the kappa light chain type from two human lymphoid cell lines are closely related.

As a first step in our studies of functionally rearranged K genes of man we cloned the germline JK-CK region from placenta DNA employing a mouse JK clone as hybridization probe. Subclones of the human JK-CK region were then used to characterize and clone the rearranged K genes of the lymphoid cell lines Walker and Daudi. The Walker cell line contains one rearranged and one germline K allele (K+,KO; ref. 1). Only one K gene was found in Daudi cells (K+). Restriction mapping and DNA sequencing showed, that the rearranged K genes from both cell lines are closely related. These features make the two cell lines particularly suitable for studies on the chromatin structure of K light chain genes. The 5' flanks of the two genes (388 bp) are identical while there is a 12% divergence between the VK gene segments themselves. This situation may reflect somatic mutation processes and/or gene conversion like events.

Three different missense suppressor mutations affecting the tRNA GGG Gly species of Escherichia coli.

The Escherichia coli suppressor mutation, supT, has been shown to cause a C --> U substitution in the middle position of the tRNA(GGG) (Gly) anticodon. This is the same tRNA species that is altered by the glyUsu(AGA) mutation studied previously. This finding indicates that the supT mutant tRNA reads the glutamic acid codon, GAG. The supT suppressor has also been converted to a new suppressor, called glyUsu(GAA), which will suppress the GAA mutation, trpA46. The in vivo suppression efficiencies of each of these three missense suppressors has been measured and are as follows: glyUsu(AGA), 3.6%; supT, 1.6%; and glyUsu(GAA), 0.4%. Mistranslation by these mutant glycine tRNA species has no adverse affects on cell growth since cultures possessing the suppressors grow as fast as cells without. The supT tRNA species can be observed as a peak in the profile of glycyl-tRNA fractionated on a RPC-5 chromatographic column, indicating that the mutant tRNA can be aminoacylated with reasonable efficiency. This finding contrasts with previous findings concerning the glyUsu(AGA) mutant tRNA which is not significantly aminoacylated under the same conditions.