Organization and evolution of variable region genes of the human immunoglobulin heavy chain

We have isolated 23 different cosmid clones of the heavy-chain variable region genes (VH) of human immunoglobulin. These clones encompass about 1000 X 10(3) base-pairs of DNA containing 61 VH genes. Characterization of the 23 clones by Southern blot hybridization showed that VH genes belonging to different families were physically linked in many regions. Cluster 71, which was analyzed in detail, comprised seven VH segments arranged in the same orientation with different intervals. This clone contained internal homology regions, each carrying two VH segments of different families. Comparison of the nucleotide sequences of VH segments within each family showed that profiles of accumulation of mutations in framework (FR) and complementarity-determining (CDR) regions were different. CDR had more mutations at amino-acid-substituting positions than at silent positions, whereas FR had the reverse distribution of mutations. Five out of seven VH segments of this cluster were pseudogenes containing various mutations. VH pseudogenes were classified into two distinct groups; one with a few replacement mutations (conserved pseudogenes), and the other with rather extensive mutations (diverged pseudogenes). The possibility that conserved pseudogenes serve as a reservoir of VH segments is discussed.     

Concerted evolution of the immunoglobulin VH gene family

With the aim of understanding the concerted evolution of the immunoglobulin VH multigene family, a phylogenetic tree for the DNA sequences of 16 mouse and five human germ line genes was constructed. This tree indicates that all genes in this family have undergone substantial evolutionary divergence. The most closely related genes so far identified in the mouse genome seem to have diverged about 6 million years (MY) ago, whereas the most distantly related genes diverged about 300 MY ago. This suggests that gene duplication caused by unequal crossing-over or gene conversion occurs very slowly in this gene family. The rate of occurrence of gene duplication in the VH gene family has been estimated to be 5 x 10(-7) per gene per year, which seems to be at least about 100 times lower than that for the rRNA gene family. This low rate of concerted evolution in the VH gene family helps retain intergenic genetic variability that in turn contributes to antibody diversity. Because of accumulation of destructive mutations, however, about one-third of the mouse and human VH genes seem to have become nonfunctional. Many of these pseudogenes have apparently originated recently, but some of them seem to have existed in the genome for more than 10 MY. The rate of nucleotide substitution for the complementarity-determining regions (CDRs) is as high as that of pseudogenes. This suggests that there is virtually no purifying selection operating in the CDRs and that germ line mutations are effectively used for generating antibody diversity.      

Immunoglobulin heavy chain constant and heavy chain variable region genes in phylogenetically diverse species of bony fish

Summary Genomic DNA from 18 phylogenetically diverse species of bony fish was hybridized with probes specific for the channel catfish immunoglobulin heavy chain constant (CH) gene, as well as with immunoglobulin heavy chain variable (VH) probes specific for five channel catfish VH gene families. The results showed that CH probes strongly hybridized only to genomic fragments from other catfish species. In contrast, restricted DNA from most other species hybridized with at least two channel catfish VH probes. In those species whose DNA hybridized with multiple VH probes, the restriction pattern of hybridizing fragments was probe-dependent. These studies suggest that (1) the CH gene defined in channel catfish appears to share similarity only with CH genes in other catfish species, (2) families of VH genes appear to have diverged in early phylogenetic lineages of teleosts, and (3) VH genes similar to those defined in catfish appear to be widely represented in phylogenetically diverse species of teleosts.     

Organization of variable region segments of the human immunoglobulin heavy chain: duplication of the D5 cluster within the locus and interchromosomal translocation of variable region segments.

We have studied the organization of variable region (V) genes of the human immunoglobulin heavy chain (H) by cosmid cloning. We isolated two independent immunoglobulin D5 clusters (D5-a and D5-b) from cosmid libraries of the human genome. Restriction maps of these two regions showed that downstream 15 kb portions of the 55 kb overlap were different although upstream 40 kb portions were almost identical. Four more D segments, (DM, DXP, DA and DK) were found around the D5 segment in the conserved region of each cluster. Nucleotide sequences of the corresponding D segments from each cluster were almost identical and they encoded potentially functional D regions. Analysis using human-rodent somatic cell hybrids demonstrated that both clusters were located in the immunoglobulin heavy chain (H) locus on chromosome 14, suggesting that the D5-a and D5-b regions evolved by internal duplication within this locus. We also isolated a 60 kb DNA region carrying four VH segments, designated as VH-F region, which was located on chromosome 16. Nucleotide sequences of the four VH segments were determined. Two of them encoded potentially functional VH segments, and the other two were pseudogenes. Some more VH segments were found to be located outside chromosome 14, by Southern blot hybridization of human-rodent hybrid cell DNAs. These results provide further evidence that the human VH locus has undergone recent reorganization.     

Examination of four HLA class I pseudogenes. Common events in the evolution of HLA genes and pseudogenes.

The HLA class I gene family in lymphoblastoid cell line 721 has been studied in detail and a number of sequences in addition to the classical genes have been identified. The cloning, characterization, and nucleotide sequences of four sequences, all full length HLA class I pseudogenes, are described in this report. These pseudogenes, contained within 5.4-, 5.9-, 7.0-, and 9.2-kb HindIII fragments, each have the class I exon-intron structure as well as class I homology in their 5' and 3' flanking regions. However, all four sequences have one or more substitutions that perturb the coding region, leaving little doubt that they are in fact pseudogenes. Comparisons among these sequences and the HLA class I genes revealed that their homology with the class I genes is patchwork. Thus, although some regions have diverged, other contiguous intron-exon sequences are highly conserved. Comparisons in the 5' regions indicate that the pseudogene promoters more closely resemble the classical HLA promoters than the nonclassical promoters as none of the unique structural features found in the HLA-E, -F, or -G regulatory regions are present in any of the pseudogene promoters. Further comparisons revealed that at least two putative gene conversion events, similar to those hypothesized to have occurred in the evolution of some HLA genes, may have occurred in the evolution of some of the pseudogenes. These and other hypothetical events in the evolution of the class I gene family are discussed.     

Evolution of immunoglobulin VH pseudogenes in chickens

In chickens, there is a single functional gene (VH1) coding for the heavy chain variable region of immunoglobulins, and immunoglobulin diversity is generated by gene conversion of the VH1 gene by many variable region pseudogenes (psi VH's) that exist on the 5' side of the VH1 gene. To understand the evolution of this unique genetic system, we conducted statistical analyses of VH1 and psi VH genes together with functional VH genes from other higher vertebrate species. The results indicate, first, that chicken VH genes are all closely related to one another and were derived relatively recently from an ancestral gene belonging to one of the three major groups of VH genes in higher vertebrates. Second, the rate of nonsynonymous substitution is slightly higher than that of synonymous substitution in the complementarity- determining regions (CDRs), which suggests that diversity-enhancing selection has operated in the CDRs even for pseudogenes. However, both the rates of synonymous and nonsynonymous substitution are higher in the CDRs than in the framework regions (FRs), apparently because of an interaction between positive selection and meiotic gene conversion in the CDRs. Third, a dot matrix analysis of the psi VH genes and genomic diversity (D) genes has indicated that the 3' end of psi VH genes is attached by D-gene-like sequences, and this region of psi VH genes has high similarity with D gene sequences. This suggests that V and D genes were fused at some point of evolutionary time and this fused element multiplied by gene duplication. Finally, two alternative hypotheses of explaining the evolution of the chicken VH gene system are presented.      

Is Esterase-P Encoded by a Cryptic Pseudogene in Drosophila Melanogaster?

We have amplified and sequenced the gene encoding Esterase-P (Est-P) in 10 strains of Drosophila melanogaster. Three premature termination codons occur in the coding region of the gene in two strains. This observation, together with other indirect evidence, leads us to propose that Est-P may be a pseudogene in D. melanogaster. Est-P would be a ``cryptic' pseudogene, in the sense that it retains intact the coding sequence (without stop codons and other alterations usually observed in pseudogenes) in most D. melanogaster strains. We conjecture that the β-esterase cluster may consist in other Drosophila species of functional and nonfunctional genes. We also conjecture that the rarity of detected pseudogenes in Drosophila may be due to the difficulty of discovering them, because most of them are cryptic.     

Germ line maintenance of the pseudogene donor pool for somatic immunoglobulin gene conversion in chickens.

Somatic immunoglobulin diversity is generated in avian species by sequential gene conversion of variable (V) gene segments of the immunoglobulin heavy- and light-chain loci during B-cell development. The germ line pools of donor sequence information for somatic V-region gene conversion are found in families of V pseudogenes, located 5' of the single functional V gene of each locus. The sequence relationships among the pseudogenes (psi VL) and functional VL1 gene of the chicken light-chain alleles in three inbred strains were compared to determine the extent of diversity within the germ line pseudogene cluster. Numerous differences were observed. For example, compared with the previously reported CB allele and the G4 allele, the S3 allele contains two intact pseudogenes between psi VL16 and psi VL18. These two adjacent psi VL gene segments (psi VL17a and psi VL17b) could have given rise to the psi VL17 segment of the G4 and CB alleles by homologous recombination. The majority of other sequence polymorphisms among the psi VL alleles appear to be the result of meiotic gene conversion. The incidence of untemplated mutations within psi VL segments is significantly lower than the incidence of mutation within the pseudogene flanking regions. Together with the observations that most psi VL segments have open reading frames and lack stop codons, these data support the hypothesis that the psi VL cluster resembles a functional multigene family maintained by evolutionary selection for its functional role in generating somatic antibody diversity. Meiotic gene conversion events within the psi VL cluster serve both to introduce diversity by the exchange of short segments between family members and to prevent the accumulation of random mutations.     

Inactivation of human keratin genes: the spectrum of mutations in the sequence of an acidic keratin pseudogene

Keratins are cytoskeletal proteins encoded by a multigene family. We have identified the first human keratin pseudogene and determined its complete nucleotide sequence. Sequence comparisons indicate that the pseudogene arose from a very recent duplication of the 50-kd keratin (K14) gene. The coding and the intron sequences of the two genes are 95% and 93% identical, respectively. Although the sequence of the regulatory region in the pseudogene is virtually identical to that in the 50-kd functional gene, several deleterious mutations have been identified in the pseudogene. There are three frameshifts in the coding regions, one of which is a perfect 8-bp duplication. A single-base-pair deletion in the first exon and a single-base-pair insertion in the penultimate exon also result in frameshifts. The three remaining deleterious mutations interfere with the mRNA processing signals: two alter the intron/exon boundaries, and the third disrupts the polyadenylation signal. These mutations clearly identify the sequence as a human keratin pseudogene.     

Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes

Two rabbit germ-line VH gene segments have been isolated from a recombinant phage DNA library. Nucleotide sequence analysis indicates that both of the genes share structural and regulatory features common to mouse and human VH genes, although one appears to be a pseudogene. Comparison of the protein sequences encoded by these genes to the protein sequences of rabbit immunoglobulin V regions indicates that both genes encode VH a-negative-like molecules. Quantitative genomic blot analysis with a VH probe capable of recognizing most, if not all, germ-line VH genes indicates that there are approximately 100 VH genes in the haploid genome of rabbits. The average spacing between the germ-line VH genes was determined to be approximately 6 kb. The molecular basis for the allelic inheritance of rabbit VH allotypes is discussed in view of the structural organization of germ-line VH genes.     

Molecular evolution of cdc2 pseudogenes in spruce (Picea)

The p34^cdc2 protein and other cyclin-dependent protein kinases (CDK) are important regulators of eukaryotic cell cycle progression. We have previously cloned a functional cdc2 gene from Picea abies and found it to be part of a family of related sequences, largely consisting of pseudogenes. We now report on the isolation of partial cdc2 pseudogenes from Picea engelmannii and Picea sitchensis, as well as partial functional cdc2 sequences from P. engelmannii, P. sitchensis and Pinus contorta. A high level of conservation between species was detected for these sequences. Phylogenetic analyses of pseudogene and functional cdc2 sequences, as well as the presence of shared insertions or deletions, support the division of most of the cdc2 pseudogenes into two subfamilies. New cdc2 pseudogenes appear to have been formed in Picea at a much higher rate than they have been obliterated by neutral mutations. The pattern of nucleotide changes in the cdc2 pseudogenes, as compared to a presumed ancestral functional cdc2 gene, was similar to that previously found in mammalian pseudogenes, with a strong bias for the transitions C to T and G to A, and the transversions C to A and G to T.     

Identification of two human ferritin H genes on the short arm of chromosome 6

We have found by analyses of human-hamster hybrid cells that two human ferritin H genes lie near the locus of the iron storage disease idiopathic hemochromatosis on chromosome 6p. One of these genes was isolated and shown to be a processed pseudogene. Comparison of its sequence with those of other ferritin H pseudogenes indicates that they may be derived from a functional H gene other than that on chromosome 11.     

Simple DNA sequences in homologous flanking regions near immunoglobulin VH genes: a role in gene interaction?

Five closely related immunoglobulin VH genes (subgroup II) were compared by sequencing of several kb of DNA. In three of the genes homology greater than 75% was found along an area of 4 kb that includes the coding region. The homology in flanking regions is only slightly lower than that in the coding sequences. Two other genes, which are located on the same EcoRI fragment, show high homology to the first three genes in the coding and immediately flanking regions. In more distant flanking regions no homology is found with the first three genes. This indicates that their evolutionary history differs from that of the other three genes. A region of simple DNA sequence composed of repetitive TCC and TCA elements was found at a distance of approximately 380 bp upstream from the initiator ATG of these VH genes. This region is the site where the two sets of genes abruptly start to diverge. The structure of the simple DNA sequence in the various VH genes suggests that it may be involved in gene interaction. We propose that both simple DNA sequences and homology in flanking regions serve a function in the correction of VH genes, which seem to be rather free to diverge and drift into pseudogenes. A correction mechanism may help this gene family to maintain its two major features, multiplicity and diversity.     

The snRNP E protein multigene family contains five pseudogenes with common mutations.

Sequence data from three previously-uncharacterized members of the snRNP E protein multigene family suggest that each is a non-transcribed processed pseudogene, even though one clone has the potential to code for a full-length protein with greater than 90% similarity to previously-characterized E protein cDNAs. Each of the newly-analyzed family members is without introns, contains a tract of polyadenylic acid residues, and is flanked by short direct repeats. In addition, the three sequences all contain point mutations that distinguish them from the E protein coding sequence. Seven point mutations are common to the three sequences described here and to two previously-described E protein pseudogenes. Although all of these mutations are transitions, only 5 of 7 could have been generated by deamination of methylated cytosines in inactive genes. Thus, the common mutations in the pseudogenes suggest an origin other than the expressed gene that we have described. Allelic variants for two of the pseudogenes were detected and repetitive elements are located near four of the five E protein pseudogenes that have been characterized.