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.      

Evolution of the rat immunoglobulin gamma heavy-chain gene family

The sequences of the four immunoglobulin gamma heavy chains of the rat (gamma 1, gamma 2a, gamma 2b, gamma 2c) have been determined. These sequences reveal that the rat genes have evolved differently from the closely related mouse gamma genes (gamma 1, gamma 2a, gamma 2b, gamma 3): in rat two of the four genes (gamma 2a and gamma 1) are 94% homologous to each other and best resemble the single mouse gamma 1 gene. Rat gamma 2b is equivalent to the mouse gamma 2a/gamma 2b pair as regards both nucleotide sequence and antibody effector functions whilst rat gamma 2c resembles mouse gamma 3. In evolutionary terms this suggests the existence of a set of three common C gamma genes before separation of rat and mouse as individual species. In addition, two independent duplication events must have occurred after species separation affecting different constant regions; this yielded rat gamma 2a and gamma 1 as a recently evolved pair and mouse gamma 2a and gamma 2b as a different pair. Furthermore, the sequence comparisons reveal several other features of interest; rat IgG2b lacks two amino acids in CH1 which are conserved in all other sequenced gamma chains. Residues believed to be essential for monocyte interaction (FcRI) are retained only in rat gamma 2b and not in the other rat gamma genes whilst a particular motif involved in C1q interaction shows a variation in both rat IgG1 and rat IgG2a which has not been observed previously.     

H-gene (histocompatibility) mutations induced by triethylenemelamine in the mouse

Based on a simple dermal-graft procedure, the H-test for histo-compatibility mutations screens the mouse “H-system” of not less than 29 and perhaps considerably more than 100 loci that are scattered throughout the genome. Graft-tests with normally heterozygous or hemizygous loci permit scoring mutations as gains, losses, and gains+losses. Tests with normally homozygous loci screen only for gains, but further analysis can detect accompanying losses. Assayed by the H-test, triethylenemelamine (TEM) increased the spontaneous mutation rate per generation by approximately four- to five-fold in the case of BALB/c spermatogonia and F₁ hybrid oocytes (BALB/c females × C57BL/6 males), but had a much smaller effect, if any, on the rate of C57BL/6 spermatogonia. Single intraperitoneal doses of 2.4–4.0 mg/kg were given.     

Germline repertoire of the immunoglobulin V H 3 family in rhesus monkeys

 To facilitate molecular studies of antibody responses in rhesus monkeys (Macaca mulatta), we cloned and sequenced germline segments from its largest and most diverse immunoglobulin heavy-chain gene family, V _H 3. Using a PCR-based approach, we characterized 29 sequences, 20 with open reading frames (ORFs) and 9 pseudogenes. The leader sequences, introns, exons, and recombination signal sequences of M. mulatta V _H 3 gene segments are not strictly identical to those of humans, but the mature coding regions demonstrate, on average, greater than 90% sequence similarity. Although the framework regions are more highly conserved, the complementarity-determining regions (CDRs) also show strong similarities, and their predicted three-dimensional structures resemble those of their human homologues. In one instance, homologous macaque and human CDR1 sequences were 100% identical at the nucleotide level, and some CDR2s shared nucleotide identity as high as 96.5%. However, some rhesus V _H 3 ORFs have unusual structural features, including atypical CDR lengths and uncommon amino acids at structurally crucial positions. The similarity of rhesus and human V _H 3 homologues reinforces the notion that humoral immunity in this nonhuman primate species is an appropriate system for modeling human antibody responses. Received: 10 August 1999 / Revised: 30 December 1999     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

Conservation and divergence of immunoglobulin VH pseudogenes.

The 12 immunoglobulin VH pseudogenes, that have been characterized to date, differ from most pseudogenes of other multigene families in two aspects: (i) they carry only one (11 cases) or at the most two (1 case) deleterious mutations and (ii) they show no evidence of increased divergence from intact VH genes. We describe here the first immunoglobulin VH pseudogene that does not have these characteristics. This pseudogene accumulated numerous deleterious mutations and diverged considerably from other genes of the VH gene family to which it belongs. In possible contrast to the other VH pseudogenes, this pseudogene seems to be selectively neutral. We discuss the implications of the characterization of this diverged VH pseudogene in relation to our understanding of the genetic mechanisms that generate diversity among germline immunoglobulin VH genes.     

Organization of the constant-region gene family of the mouse immunoglobulin heavy chain

We cloned overlapping DNA segments that encompass the region from the immunoglobulin JH segments to the C gamma 3 gene of BALB/c mouse. We have now cloned the entire region (about 200 kilobases) of the constant-region gene family of the immunoglobulin heavy chain, the organization of which is 5'-JH-6.5 kb-C mu-4.5 kb-C delta-55 kb-C gamma 3-34 kb-C gamma 1-21 kb-C gamma 2b-15 kb-C gamma 2a-14 kb-C epsilon-12 kb-C alpha-3'. Using these cloned DNAs, we have characterized several structural features of the constant-region gene loci. There are no other J region segments except for those at the 5' side of the C mu gene. The S region is 5' to each CH gene except for the C delta gene, and the nucleotide sequences of the S region share some homology. There is no reasonably conserved pseudogene. There are at least two species of reiterated sequences scattered in these loci. Cloning and Southern blot hybridization analyses indicate that the general organizations of the heavy-chain gene loci of BALB/c and C57BL/6 mice, which have many different serological markers, are fundamentally similar but different in the lengths of S regions. Restriction enzyme cleavage maps of the whole constant-region gene loci were constructed with respect to eight restriction endonucleases.     

Analysis of immunoglobulin heavy chain V-region genes belonging to the V NP-gene family.

A method was devised to clone immunoglobulin VH-region genes located on selected restriction fragments from genomic DNA directly into M13 vectors for subsequent nucleotide sequence analysis. Ten recombinant M13 clones representing four so far unknown VH-region genes of the VNP-gene family have been analysed. Sequence comparison shows that these genes are closely related to other VH-genes of the VNP gene family. One of the VH-genes exhibits a so far unobserved unusual length of 100 2/3 codons and appears to be functional. Analysis of the variation of the isolated VH-genes suggests that framework and complementarity determining regions are exposed to separate types of selective pressures.     

Comparative analysis of processed pseudogenes in the mouse and human genomes

Pseudogenes are important resources in evolutionary and comparative genomics because they provide molecular records of the ancient genes that existed in the genome millions of years ago. We have systematically identified approximately 5000 processed pseudogenes in the mouse genome, and estimated that approximately 60% are lineage specific, created after the mouse and human diverged. In both mouse and human genomes, similar types of genes give rise to many processed pseudogenes. These tend to be housekeeping genes, which are highly expressed in the germ line. Ribosomal-protein genes, in particular, form the largest sub-group. The processed pseudogenes in the mouse occur with a distinctly different chromosomal distribution than LINEs or SINEs - preferentially in GC-poor regions. Finally, the age distribution of mouse-processed pseudogenes closely resembles that of LINEs, in contrast to human, where the age distribution closely follows Alus (SINEs).     

Evolution of trnF(GAA) pseudogenes in cruciferous plants

In several studies we used the 5′-trnL(UAA)–trnF(GAA) region of the chloroplast DNA for phylogeographic reconstructions, gene diversity calculations and phylogenetic analyses among the genera Arabidopsis and Boechera. Despite the fact that extensive gene duplications are rare within the chloroplast genome of higher plants, within several genera of the Brassicaceae the anticodon domain of the trnF(GAA) gene exhibit extensive gene duplications with 1–12 tandemly repeated copies in close 5′-proximity of the functional gene. A recent re-examination and additional analysis of trnL(UAA)–trnF(GAA) regions from numerous cruciferous taxa not only reveal extensive trnF gene duplications, but also favour the hypothesis that in cruciferous taxa at least four independent phylogenetic lineages are characterized by these pseudogenes. Among these lineages there is one major clade of taxa carrying pseudogenes indicating an ancient split in crucifer evolution. In two case studies, Boechera and Arabidopsis, intra- and inter-molecular recombinations have been shown to be the reason for the reciprocal exchange of several similar motifs. However, functional constraints might favour two to three or five to six copies as shown for Arabidopsis and Boechera. Herein, we compare the occurrence and distribution of pseudogene copy number in the framework of a comprehensive survey of cpDNA haplotype variation in Boechera, the former genus Cardaminopsis and Arabidopsis thaliana and comment on the value of such kind of mutations in phylogenetic and evolutionary reconstructions.     

Evolution of the regulators of G-protein signaling multigene family in mouse and human

The regulators of G-protein signaling (RGS) proteins are important regulatory and structural components of G-protein coupled receptor complexes. RGS proteins are GTPase activating proteins (GAPs) of Gi-and Gq-class Galpha proteins, and thereby accelerate signaling kinetics and termination. Here, we mapped the chromosomal positions of all 21 Rgs genes in mouse, and determined human RGS gene structures using genomic sequence from partially assembled bacterial artificial chromosomes (BACs) and Celera fragments. In mice and humans, 18 of 21 RGS genes are either tandemly duplicated or tightly linked to genes encoding other components of G-protein signaling pathways, including Galpha, Ggamma, receptors (GPCR), and receptor kinases (GPRK). A phylogenetic tree revealed seven RGS gene subfamilies in the yeast and metazoan genomes that have been sequenced. We propose that similar systematic analyses of all multigene families from human and other mammalian genomes will help complete the assembly and annotation of the human genome sequence.     

Mapping of the mouse Tdgf1 gene and Tdgf pseudogenes

Teratocarcinoma-derived growth factor-1 (Tdgf1), a member of the ``EGF family' of growth factors, is expressed during mouse gastrulation in the forming mesoderm and later in the truncus arteriosus of the developing heart. In humans, TDGF1 is highly expressed in germ cell tumors and in colon and mammary carcinomas. In mouse, one gene (Tdgf1) and two pseudogenes (Tdgf1-ps1 and Tdgf1-ps2) have been isolated and characterized. Tdgf1 corresponds to the gene expressed in F9 teratocarcinoma cells. Tdgf1-ps1 and Tdgf1-ps2 are two intronless sequences with all the characteristics of retroposons. In the present paper, we assign the chromosomal location for Tdgf1, Tdgf1-ps1, and Tdgf1-ps2 sequences to Chromosomes (Chrs) 9, 16, and 17, respectively. Two previously described mouse mutants, scant hair (sch) and fur deficient (fd), map near the Tdgf1 gene. Analysis of their DNA coding region provided no evidence that Tdgf1 could be the responsible gene for these phenotypes. Finally, analysis of the DNA from several Mus musculus strains and from Mus spretus mice revealed a highly variable restriction pattern and the absence of the Tdgf1-ps1 genomic sequence from the Mus spretus genome. Received: 23 November 1996 / Accepted: 17 February 1997     

Genomic organization of the mouse T cell receptor V alpha family.

Based on the analysis of V alpha gene segment deletions in a panel of T lymphomas, we have constructed a map of the mouse T cell receptor alpha/delta region and assigned the relative position of 72 distinct V gene segments. Three major observations have emerged from such studies. First, members of a given V alpha subfamily are not organized in discrete units along the chromosome but largely interspersed with members of other V alpha subfamilies. Second, analysis of the deletion map suggests the existence of repetitive patterns (V alpha clusters) in the chromosomal distribution of the V alpha gene segments. Third, the present-day organization of the V alpha/delta region may be readily explained by a series of sequential duplications involving three ancestral V alpha clusters. Direct evidence for the existence of these unique structural features has been gained by cloning approximately 370 kb of DNA and positioning 26 distinct V alpha gene segments belonging to six different subfamilies. Finally, the relationships existing between the V alpha/delta gene segment organization and usage are discussed in terms of position-dependent models.     

The Z family, a group of transposed human immunoglobulin V kappa genes

A group of highly homologous transposed human V kappa I genes, which we call the Z family, was characterized. To date four members, ZI-ZIV, comprising about 230 kb, have been analyzed on cosmid clones. The largest region (ZI) has a length of 85 kb. The Z regions show extensive homology to each other according to restriction maps and hybridization data. In each Z region a solitary V kappa I gene was found. No V kappa genes of other subgroups were detected by hybridization. The nucleotide sequence of the ZI gene revealed a non-processed V kappa I pseudogene. Hybridization experiments with DNAs from rodent/human cell hybrids and other experimental data indicate that some and possibly all members of the Z family lie outside of the kappa locus which is located on chromosome 2; they have been transposed to other chromosomes. Because of their separation from the J kappa C kappa gene segment, the Z genes can be classified as pseudogenes independent of their sequences. We postulate that the Z family arose by amplification event(s). The Z regions can also be regarded as a small family of very long repetitive sequences.