Evolution of human immunoglobulin kappa J region genes

Immunoglobulin kappa chain variable region genes are assembled from two discontinuous DNA segments, a V and a J gene. The J region genes, in addition to encoding amino acid positions 96-108 of the kappa polypeptide chain, also provide sequences required for both DNA and RNA splicing reactions. For purposes of evolutionary comparison and to establish the complexity of the kappa J region locus in man, we have determined an approximately 3000 basepair nucleotide sequence in a cloned human DNA fragment that encodes the germline distinct J region segments. Significant blocks of homology have been tightly maintained between this region and an analogous segment of the mouse genome. In particular, the short sequences, GGTTTTTGT and CACTGTG, thought to be involved in V-J recombination, are the most highly conserved regions (97% homology). In addition, from heteroduplex data and computer analysis of the nucleotide sequences, it is clear that the mouse J3 sequence, a pseudogene, is not present in the human cluster. This can be explained by a duplication event in the mouse J region gene cluster that may have been the result of unequal crossing over between homologous chromosomes.     

DNA sequence of the constant gene region of the mouse immunoglobulin kappa chain.

The constant (C; ref. 3) gene segment of the immunoglobulin kappa light chain and about 1 kb upstream as well as downstream of the segment have been sequenced. The sequences of the C gene segment itself and parts of the upstream region were determined both in liver and in myeloma T DNA clones derived from the same mouse inbred strain. The sequences were identical, i.e. no somatic mutations were detected. Two sites in the region not coding for protein are discussed as possible targets of aberrant variable (V) gene translocations. Doublet frequencies were calculated in the approx. 2500 bp of the C region sequence reported in this paper and in the approx. 3400 bp of two rearranged V gene regions.     

The human kappa deleting element and the mouse recombining segment share DNA sequence homology.

We have used cloned mouse and human DNA probes to identify regions of conserved homology between the human and murine DNA segments, (termed kappa deleting element (kde) and recombining segment (RS) respectively) which are frequently recombined in lambda-producing B cells. Heteroduplex analysis indicated extensive homology in the region immediately downstream of the recombination site of both segments. This was confirmed by Southern and direct nucleotide sequence analyses. Fifty percent homology was detected within the 500 nucleotides that neighbour the recombination points in the kde and RS segments. These results indicate that the kde and RS sequences are evolutionarily conserved and may be functionally relevant to normal B cell development.     

DNA sequence comparisons of the human, mouse, and rabbit immunoglobulin kappa gene

A comparative analysis between human, mouse, and rabbit immunoglobulin (Ig) kappa-gene DNA sequences is presented. New formulas for determining the expected length and variance of the longest block identity (a succession of matching nucleotides) between multiple random sequences are given and are used to establish statistical criteria for ascertaining the significance of block identities shared in r out of s sequences. The statistically significant block identities within and between the Ig-kappa-gene sequences are ascertained, and alignment maps based on these similarities are constructed. The human and rabbit sequences (especially in the noncoding regions) and the human and mouse sequences (on the coding regions) show a similarity much stronger than that between the mouse and rabbit sequences. The existence of several highly significant shared oligonucleotides occurring in alignment with each other or with respect to the J- and C-gene segments suggests a configuration of multiple control sites. Discussion and interpretations of the form and distribution of the block identities are given.      

Evolutionary relationship between human and mouse immunoglobulin kappa light chain variable region genes

Similar to the Igh-V multigene family, the human or mouse Igk-V repertoirer is a distorted continuum of homologous genes that may be grouped into families displaying >80% nucleic acid sequence similarity among their members. systematic interspecies sequence comparisons reveal that most human Igk-V gene families exhibit clear homology to mouse Ogk-V families (sequence similarity >74%). A hypothetical phylogenetic tree of Igk-V genes predicts that a minimum of seven Igk-V genes/families predate mammalian radiation. In two cases, several interrelated mouse Igk-V families exhibit phylogenetic equidistance with just one human Igk-V family, implying a more pronounced divergence for the elevated number of Igk-V gene families in the mouse. Mouse-human Igk-V comaprisons, moreover, illustrate how expansion, contraction, and perhaps deletion of Igk-V gene families shape the Igk-V repertoire during mammalian evolution.     

Purification and characterization of a protein that binds to the recombination signal sequence of the immunoglobulin J kappa segment.

A protein that binds to the recombination signal sequence (RS) of the immunoglobulin J kappa segment was purified almost to homogeneity from the nuclear extract of a murine pre-B cell line 38B9. A similar binding protein was found in lymphoid cell lines but not in non-lymphoid cell lines. The binding activity was associated with a polypeptide with a molecular weight of 60,000. DNase I footprinting analysis demonstrated that this binding protein interacted with the heptamer and several 3' bases close to the heptamer. The Kd value of the J kappa RS binding protein to the J kappa RS was 1 nM. One base substitution in the heptamer of the J kappa RS greatly reduced the affinity of the J kappa RS binding protein. The high specificity of the binding site of the J kappa RS binding protein suggests that this protein may be involved in V-J recombination.     

Somatic DNA rearrangement generates functional rat immunoglobulin kappa chain genes: the J kappa gene cluster is longer in rat than in mouse

The kappa immunoglobulin (Ig) genes from rat kidney and from rat myeloma cells were cloned and analyzed. In kidney DNA one C kappa species is observed by Southern blotting and cloning in phage vectors; this gene most likely represents the embryonic configuration. In the IR52 myeloma DNA two C kappa species are observed: one in the same configuration seen in kidney and one which has undergone a rearrangement. This somatic rearrangement has brought the expressed V region to within 2.7 kb 5' of the C kappa coding region; the rearrangement site is within the J kappa cluster which we have mapped. The rat somatic Ig rearrangement, therefore, closely resembles that seen in mouse Ig genes. In the rat embryonic fragment two J kappa segments were mapped at 2 and 4.3 kb 5' from the C kappa coding region. Therefore, the rat J kappa cluster extends over about 2.3 kb, a region much longer than the 1.4 kb of the mouse and human J kappa clusters. In the region between C kappa and the expressed J kappa of IR52 myeloma DNA, and XbaI site present in the embryonic kappa gene has been lost. A somatic mutation has therefore occurred in the intervening sequence DNA approx. 0.7 kb 3' from the V/J recombination site. Southern blots of rat kidney DNA hybridized with different rat V kappa probes showed non-overlapping sets of bands which correspond to different subgroups, each composed of 8-10 closely related V kappa genes.     

Complete nucleotide sequence of mouse immunoglobulin mu gene and comparison with other immunoglobulin heavy chain genes

We have determined the complete nucleotides sequence (2168 bases) of the immunoglobulin mu gene cloned from newborn mouse DNA. The cloned 13kb fragment contained the entire constant region gene sequence that is interrupted by three intervening sequences at the junction of domains as previously shown in the gamma 1, gamma 2 b and alpha genes. The amino acid sequence predicted by the nucleotide sequence agrees with that of the mu chain secreted by a myeloma MOPC104E except for 8 residues out of 448 residues. The homologous domains of the mu, gamma 1 and gamma 2b genes are more similar to each other than the different domains of the mu genes are. The result implicates that the class of the immunoglobulin heavy chain genes diverged after the heavy chain genes established the multi-domain structure. The short intervening sequences of the mu and gamma genes are more conserved than the coding sequences except for the COOH-terminal domains. The results implicate that the nucleotide sequence of the intervening sequence is under selective pressure, possibly to maintain a secondary structure of the nuclear RNA to be spliced.     

The nucleotide sequence of a cDNA clone containing the entire coding region for mouse X-chromosome-linked phosphoglycerate kinase

A clone containing cDNA for X chromosome-linked phosphoglycerate kinase (PGK-1) was isolated from a mouse myeloma cDNA library. The nucleotide (nt) sequence of the cDNA has been determined, and the amino acid (aa) sequence of the enzyme thereby deduced. At the nt level, the coding region of mouse PGK cDNA has 93% homology with human X-linked cDNA and 60% homology with the yeast gene. Mouse PGK-1 protein contains 416 aa and is 98%, 96% and 64% homologous with human, horse, and yeast enzyme sequences, respectively.     

The nucleotide sequence of a human immunoglobulin C gamma1 gene

We report the nucleotide sequence of a gene encoding the constant region of a human immunoglobulin gamma 1 heavy chain (C gamma 1). A comparison of this sequence with those of the C gamma 2 and C gamma 4 genes reveals that these three human C gamma genes share considerable homology in both coding and noncoding regions. The nucleotide sequence differences indicate that these genes diverged from one another approximately 608 million years ago. An examination of hinge exons shows that these coding regions have evolved more rapidly than any other areas of the C gamma genes in terms of both base substitution and deletion/insertion events. Coding sequence diversity also is observed in areas of CH domains which border the hinge.     

Characterization of a mouse DNA clone containing an immunoglobulin variable region gene.

A 4.8 kilobase mouse embryo DNA fragment was inserted into a phage lambda genome and was subsequently characterized by electron microscopy, restriction enzyme mapping and partial DNA sequencing. This fragment contains a 400 base sequence which is homologous to that of an immunoglobulin light lambda chain mRNA which spans 3.3 to 3.7 kilobases from one end of the fragment. Restriction enzyme mapping as well as partial nucleotide sequencing of the 3' terminal of the homology region confirm the previous conclusion [Tonegawa, Brack, Hozumi and Schuller, Proc. Natl. Acad. Sci. USA. 74, 3518-3522 (1977)] that the cloned DNA fragment contains a Vlambda gene sequence which is separate from any Clambda sequence.     

The rabbit kappa 1 b5 immunoglobulin gene: another J region gene cluster with only one functional J gene segment?

Our previous analysis of an immunoglobulin gene encoding the rabbit kappa chain of b4 allotype revealed that of the five J-like sequences in the J kappa cluster of this gene, only one, J2, appeared to be functional. This unusual ratio of J pseudogenes to functional J genes is unique among all J clusters of light and heavy chain genes of all species examined to date, including the cluster from the rabbit kappa 2 isotype, and must have consequences for diversity generation of b4 immunoglobulins. The fact that the only two known b5 J kappa sequences are different from the functional J2 of the b4 allotype prompted investigation of the b5 J kappa cluster to determine whether it resembled the b4 cluster, or the more typical mouse or human J kappa clusters. Our analysis of the b5 gene reveals a J kappa cluster strikingly similar to that of b4; apparent defects occur in all J sequences except J2. Although J2 is apparently functional, it differs from the J2 of the b4 locus by four nucleotide and three amino acid substitutions. The unusually high degree of sequence similarity previously observed between the b4 and b5 loci in the noncoding (vs the coding) regions extends through the newly sequenced DNA segment and remains an enigma.     

The Drosophila RBP-J kappa gene encodes the binding protein for the immunoglobulin J kappa recombination signal sequence.

We previously isolated a cDNA encoding the 60-kDa murine protein (RBP-J kappa protein) that specifically binds to the immunoglobulin J kappa recombination signal sequence. The RBP-J kappa gene is highly conserved in a wide variety of organisms including man, Xenopus, Drosophila, and yeast. We have isolated and characterized the Drosophila homologue of the RBP-J kappa gene. The Drosophila RBP-J kappa gene was mapped to the polytene region 35BC of chromosome 2. The nucleotide sequence of this gene indicates that it is not one of the known genes located in the 35 BC region. The nucleotide and amino acid sequences of the Drosophila and mouse RBP-J kappa genes are 60 and 75% homologous, respectively. The central 248-residue regions of RBP-J kappa proteins of the two species are 93% homologous and include the 40-residue integrase motif. The Drosophila RBP-J kappa protein expressed in COS cells bound to the J kappa recognition sequence with the same specificity as the murine counterpart. These results suggest that Drosophila may have a site-specific recombination system which utilizes the immunoglobulin recombination signal sequence. Implications for evolution of immunoglobulin gene rearrangement were also discussed.     

The organization of immunoglobulin variable kappa chain genes on mouse chromosome 6

One mouse with a known recombination (NAK) at the Igk locus on chromosome 6 and two new recombinants [B6.PL (7 NS) and B6.PL (85NS)] were examined using a series of probes, each of which is specific for a set of immunoglobulin (Ig) Vk genes. Under high stringency conditions, each probe detects from 1 to 19 Bam HI restriction endonuclease fragments (REFs) in genomic DNA by Southern transfer hybridization techniques. Analysis of the REF patterns indicate that the NAK recombination event occurred within the variable region of Igk. The REF patterns of the two B6.PL congenic mice provided two additional recombination events which could be examined. Although some of the REFs had shared mobility among the parental strains, at least 1 and up to 13 polymorphic REFs were present for a given probe among the NZB and AKR parental strains. The results from the NAK mouse indicate that at least some members of Vk4, Vk8, Vk10, and Vk21 were on one side of the recombination event linked to the Lyt-2 ^a and Igk-Efl ^a alleles of AKR, while the Vk9, Vk11, and Vk24 REF patterns came from the NZB parental strain linked to the Igk-Ef2 ^b (Vk1) allele. The two B6.PL congenics produced a refined map on the Lyt-2, Lyt-3 side of the Vk region. The B6.PL (85NS) mice retained the Vk21 REF pattern of the Lyt-2 ^a, Lyt-3 ^a donor strain PL/J, while displaying the C57BL/6 REF pattern for the other Vk gene groups tested. The B6.PL (75NS) mice retained the REF patterns of PL/J for Vk21 and Ef-1, indicating a third recombination. This indicates the Vk gene order is (Lyt-2; Vk21); Ef-1; (Vk4; Vk8; Vk10); and (Vk9; Vk11; Vk24; Ef-2).     

Evolution of immunoglobulin kappa chain variable region genes in vertebrates

The major source of immunoglobulin diversity is variation in DNA sequence among multiple copies of variable region (V) genes of the heavy- and light-chain multigene families. In order to clarify the evolutionary pattern of the multigene family of immunoglobulin light kappa chain V region (V kappa) genes, phylogenetic analyses of V kappa genes from humans and other vertebrate species were conducted. The results obtained indicate that the V kappa genes so far sequenced can be grouped into three major monophyletic clusters, the cartilaginous fish, bony fish and amphibian, and mammalian clusters, and that the cartilaginous fish cluster first separated from the rest of the V kappa genes and then the remaining two clusters diverged. The mammalian V kappa genes can further be divided into 10 V kappa groups, 7 of which are present in the human genome. Human and mouse V kappa genes from different V kappa groups are intermingled rather than clustered on the chromosome, and there are a large number of pseudogenes scattered on the chromosome. This indicates that the chromosomal locations of V kappa genes have been shuffled many times by gene duplication, deletion, and transposition in the evolutionary process and that many genes have become nonfunctional during this process. This mode of evolution is consistent with the model of birth-and-death evolution rather than with the model of concerted evolution. An analysis of duplicate V kappa functional genes and pseudogenes in the human genome has indicated that pseudogenes evolve faster than functional genes but that the rate of nonsynonymous nucleotide substitution in the complementarity-determining regions of V kappa genes has been enhanced by positive Darwinian selection.