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.     

Mechanism of selection of class II recombinant murine leukemia viruses in the highly leukemic strain CWD

The development of spontaneous lymphomas in CWD mice is associated with the expression of endogenous ecotropic murine leukemia viruses (MuLV) and the formation of recombinant viruses. However, the pattern of substitution of nonecotropic sequences within the envelope genes of the CWD class II recombinant viruses differs from that seen in class I recombinant MuLVs of AKR, C58, and HRS mice. To determine how CWD host genes might influence the envelope gene structure of the recombinant viruses, we characterized the responses of these mice to two different types of exogenous MuLVs. Neonatal mice injected the HRS class I recombinant PTV-1 became infected and developed T-cell lymphomas more rapidly than controls did. The inoculation of CWD mice with the leukemogenic AKR ecotropic virus SL3-3 led to the formation of recombinant MuLVs with a novel genetic structure and class II-like envelope genes, although SL3-3 generates class I recombinants in other strains. These results suggest that the absence of class I recombinant MuLVs in CWD mice is not related to the restriction of the replication or oncogenicity of class I viruses or to the absence of an appropriate ecotropic virus that can generate class I recombinants. More likely, the genes of CWD mice that direct the formation or selection of class II recombinant viruses affect the process of recombination between the ecotropic and nonecotropic envelope gene sequences.     

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Identification of new HLA class I region genes by sample sequencing

 Although many human major histocompatibility genes have been identified, relatively few have been localized to the class I region. We searched for new class I region genes by sample sequencing, a process in which short stretches of random genomic sequence are generated from cosmids and then compared with sequences deposited in nucleotide databases. Four class I region cosmids were isolated for sample sequencing by screening a chromosome 6 specific cosmid library with probes derived from specific class I region genes or with overlapping class I region yeast artificial chromosomes. Cosmids were sonnicated to produce fragments of 0.5 – 1 kilobases, subcloned, and sequenced using an automated sequencer. Sequences were then compared with nucleotide sequences deposited in the GenBank databases using the BLASTN algorithm. A number of potential new class I region genes were identified, including a cDNA with similarity to the tre oncogene, the trans-activating factor SC1 (TCF19), and a member of the interferon inducible 1 – 8 gene family. These observations suggest that sample sequencing is an efficient method for identifying new class I region genes, which can be applied to other regions of the genome and to other species, and support previous observations that the class I region contains a variety of genes other than those encoding HLA antigens. Received: 10 December 1996 / Revised: 7 January 1997     

MHC class I genes in a New World primate, the cotton-top tamarin (Saguinus oedipus), have evolved by an active process of loci turnover

 Lymphocytes of a New World primate, the cotton-top tamarin (Saguinus oedipus), express classical G–related major histocompatibility complex (MHC) class I molecules with unusually limited polymorphism and variability. Three G-related loci, an F locus, an E locus, and two pseudogenes (So-N1 and So-N3) have been identified by cDNA library screening and extensive PCR analysis of both cDNA and genomic DNA from the cotton-top tamarin. Furthermore, each genus of the subfamily Callitrichinae (tamarins and marmosets) appears to express its own unique set of MHC class I genes, likely due to a rapid turnover of loci. The rapid emergence of unique MHC class I genes in the Callitrichinae genera, resulting from an active process of duplication and inactivation of loci, may account for the limited diversity of the MHC class I genes in the cotton-top tamarin. To determine the nature of the entire complement of MHC class I genes in the cotton-top tamarin, we synthesized a genomic DNA library and screened it with MHC class I-specific probes. We isolated nine new MHC class I pseudogenes from this library. These newly isolated tamarin G–related MHC class I pseudogenes are not closely related to any of their functional counterparts in the tamarin, suggesting that they do not share a recent common ancestral gene with the tamarin's currently expressed MHC class I loci. In addition, these tamarin sequences display a high rate of nonsynonymous substitutions in their putative peptide binding region. This indicates that the genes from which they have derived were likely subject to positive selection and, therefore, were once functional. Our data support the notion that an extremely high rate of loci turnover is largely responsible for the limited diversity of the MHC class I genes in the cotton-top tamarin. Received: 15 September 1997 / Revised: 2 July 1998     

Family relationships of murine major histocompatibility complex class I genes. Sequence of the T2Aa pseudogene, a member of gene family 3

The major histocompatibility complex of the mouse contains numerous class I genes, most of which are encoded in the Qa and Tla regions. By hybridizations, the murine class I genes have been classified into three major families (Rogers, J. H. (1985a) Immunogenetics 21, 343-353). As yet, complete sequences are available only for members of family 1 (several H-2 and Qa genes) or family 2 (the pseudoallelic Tla genes T3b and T13c). We here present the complete nucleotide sequence of a gene from the Tla region that belongs to family 3. This gene, T2Aa, is a pseudogene by several criteria. The general structure of the gene is nonetheless well preserved. A comparison of the T2Aa sequence to those of other murine class I genes confirms the classification into three gene families. Members of gene families 2 and 3, located in the Tla region, are no more similar to each other than to family 1 (the H-2 and Qa2,3 genes). This suggests that families 2 and 3 were both created by ancient duplications of the functionally important family 1 genes. The fact that families 2 and 3 have diverged extensively both from family 1 and from each other may suggest that they are devoid of function.     

Systematic Sequencing of the Human HLA-A/HLA-F Region: Establishment of a Cosmid Contig and Identification of a New Gene Cluster within 37 kb of Sequence

The class I region of the human histocompatibility complex is characterized by a high density of genes and pseudogenes and a complex structural organization. To elucidate the complete structure of the HLA-A/HLA-F region with a view to defining its contents in genes and pseudogenes, we developed a strategy of systematic sequencing. This report describes the establishment of a cosmid contig spanning most of the region and the analysis of a 37-kb sequence from one of the cosmids. Four new genes, organized with the HCG-V gene in a clustered structure, have been identified. Two of these contain a zinc finger motif characteristic of DNA-binding proteins. The former, a member of the C3HC4 protein family, is highly expressed in prostate and contains a B30-2-like sequence identified in several genes mapped within the class I region. The latter, which is ubiquitously expressed, is the human equivalent of the yeast polymerase I A12.2 subunit and of the murine tctex6 gene. Of the two other genes, one remains an anonymous gene with no particular feature, while the fourth, specifically expressed in testis, is the human equivalent of the murine tctex4 gene. This cluster, located in a region corresponding to a syntenic unit between mouse and human, appears to be highly conserved.     

The dominant MHC class I gene is adjacent to the polymorphic<Emphasis Type="Italic"> TAP2</Emphasis> gene in the duck,<Emphasis Type="Italic"> Anas platyrhynchos</Emphasis>

We are investigating the expression and linkage of major histocompatibility complex (MHC) class I genes in the duck (Anas platyrhynchos) with a view toward understanding the susceptibility of ducks to two medically important viruses: influenza A and hepatitis B. In mammals, there are multiple MHC class I loci, and alleles at a locus are polymorphic and co-dominantly expressed. In contrast, in lower vertebrates the expression of one locus predominates. Southern-blot analysis and amplification of genomic sequences suggested that ducks have at least four loci encoding MHC class I. To identify expressed MHC genes, we constructed an unamplified cDNA library from the spleen of a single duck and screened for MHC class I. We sequenced 44 positive clones and identified four MHC class I sequences, each sharing approximately 85% nucleotide identity. Allele-specific oligonucleotide hybridization to a Northern blot indicated that only two of these sequences were abundantly expressed. In chickens, the dominantly expressed MHC class I gene lies adjacent to the transporter of antigen processing (TAP2) gene. To investigate whether this organization is also found in ducks, we cloned the gene encoding TAP2 from the cDNA library. PCR amplification from genomic DNA allowed us to determine that the dominantly expressed MHC class I gene was adjacent to TAP2. Furthermore, we amplified two alleles of the TAP2 gene from this duck that have significant and clustered amino acid differences that may influence the peptides transported. This organization has implications for the ability of ducks to eliminate viral pathogens.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers AY294416–22     

Frequent segmental sequence exchanges and rapid gene duplication characterize the<Emphasis Type="Italic"> MHC</Emphasis> class I genes in lemurs

Major histocompatibility complex (MHC) class I genes have complicated and profound evolutionary histories. To reconstruct and better understand their histories, partial class I genes (exon 2–intron 2–exon 3) were sequenced in a sampling of prosimians (Strepsirhini, Primates). In total, we detected 117 different sequences from 36 Malagasy prosimians (lemurs) and 1 non-Malagasy prosimian (galago) representing 4 families, 7 genera, and 13 species. Unlike the MHC class II genes (MHC-DRB), MHC class I genes show a generally genus-specific mode of evolution in lemurs. Additionally, no prosimian class I loci were found to be orthologous to HLA genes, even at highly conserved loci (such as HLA-E, HLA-F). Phylogenetic analysis indicates that nucleotide diversity among loci was very small and the persistence time of the polymorphisms was short, suggesting that the origin of the lemur MHC class I genes detected in this study was relatively recent. The evolutionary mode of these genes is similar to that of classic HLA genes, HLA-A, HLA-B, and HLA-C, in terms of their recent origin and rarity of pseudogenes, and differs from them with respect to the degree of gene duplications. From the viewpoint of MHC genes evolution, some interlocus sequence exchanges were apparently observed in the lemur lineage upon phylogenetic and amino acid motif analyses. This is also in contrast to the evolutionary mode of HLA genes, where intralocus exchanges have certainly occurred but few interlocus exchanges have taken place. Consequently, the gene conversion model for explaining the generation of the MHC diversity among different loci can be thought to play more important roles in the evolution of lemur MHC class I genes than in that of HLA genes.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Construction of novel class I histocompatibility antigens by interspecies exon shuffling

Human and mouse class I histocompatibility antigens share considerable structural homology at both the protein and DNA sequence level. This homology has allowed the production of hybrid class I molecules by the reciprocal exchange of DNA sequences corresponding to equivalent domains of HLA-B7 and either H-2Ld or H-2Dd. It is shown that these genes give rise to protein products that are stably expressed on the surface of murine L cells after DNA-mediated gene transfer. These proteins express only those monoclonal antibody-defined H-2 determinants that are expected based on their genetic construction. The molecules have allowed the localization of a number of polymorphic and monomorphic HLA-specific epitopes. In all but one case, expression of an epitope on a domain does not appear to be influenced by the replacement of adjacent human domains with their murine equivalents, suggesting a considerable degree of structural independence of the domains. Cells expressing the hybrid molecules have also been tested as targets for a panel of HLA-B7-specific cytotoxic T cell clones. The results show that the polymorphic determinants recognized by these clones map to the alpha 1 and alpha 2 domains of the HLA-B7 molecule. No evidence for an influence of species-related amino acid sequence differences in the third extracellular domain on T cell recognition was seen. The results are discussed in light of the proposed domain structure of the class I proteins and the potential use of such molecules for further functional studies.     

Gene organization of the quail major histocompatibility complex (MhcCoja) class I gene region

 Class I genomic clones of the quail (Coturnix japonica) major histocompatibility complex (MhcCoja) were isolated and characterized. Two clusters spanning the 90.8 kilobase (kb) and 78.2 kb class I gene regions were defined by overlapping cosmid clones and found to contain at least twelve class I loci. However, unlike in the chicken Mhc, no evidence for the existence of any Coja class II gene was obtained in these two clusters. Based on comparative analysis of the genomic sequences with those of the cDNA clones, Coja-A, Coja-B, Coja-C, and Coja-D (Shiina et al. 1999), these twelve loci were assigned to represent one Coja-A gene, two Coja-B genes (Coja-B1 and -B2), four Coja-C genes (Coja-C1-C4), four Coja-D genes (Coja-D1-D4), and one new Coja-E gene. A class I gene-rich segment of 24.6 kb in which five of these genes (Coja-B1, -B2, -D1, -D2 and -E) are densely packed were sequenced by the shotgun strategy. All of these five class I genes are very compact in size [2089 base pairs (bp)–2732 bp] and contain no apparent genetic defect for functional expression. A transporter associated with the antigen processing (TAP) gene was identified in this class I gene-rich segment. These results suggest that the quail class I region is physically separated from the class II region and characterized by a large number of the expressible class I loci (at least seven) in contrast to the chicken Mhc, where the class I and class II regions are not clearly differentiated and only at most three expressed class I loci so far have been recognized. Received: 9 March 1998 / Revised: 12 October 1998     

Constraints on haplotype structure and variable gene frequencies suggest a functional hierarchy within cattle MHC class I

Six major histocompatibility complex (MHC) classical class I genes have been identified in cattle, and up to three of these are expressed in variable combinations on different haplotypes. The origin and functional significance of this genetic complexity is unknown. However, an improved assembly of the cattle genome, an expanded database of full-length cDNA sequences and high-resolution frequency data concerning expressed class I genes in an economically important cattle breed combine to provide a new opportunity to study the significance of cattle MHC class I diversity. Analysis of these new data supports assignment of alleles to six discrete genes and further shows that all these classical genes share a common ancestor with a single non-classical gene, NC1. While haplotype structure is variable, with thirteen gene configurations identified, there are nevertheless clear constraints relating to both the number and combination of genes. Haplotypes expressing two classical genes are most frequently observed, and the classical class I gene 2 is almost invariably present. The frequency data support the dominance of gene 2, showing that close to 100?% of individuals carry at least one copy. This indicates a hierarchy in the functional importance of particular genes and haplotype structures. Haplotype frequency in cattle populations is therefore likely to impact on differential disease susceptibilities. This knowledge will be important for development of informed breeding strategies aimed at increasing the ability of cattle to survive in the face of future unpredictable pathogen exposure.     

Olfactory receptors in aquatic and terrestrial vertebrates

In species representing different levels of vertebrate evolution, olfactory receptor genes have been identified by molecular cloning techniques. Comparing the deduced amino-acid sequences revealed that the olfactory receptor gene family of Rana esculenta resembles that of Xenopus laevis, indicating that amphibians in general may comprise two classes of olfactory receptors. Whereas teleost fish, including the goldfish Carassius auratus, possess only class I receptors, the `living fossil' Latimeria chalumnae is endowed with both receptor classes; interestingly, most of the class II genes turned out to be pseudogenes. Exploring receptor genes in aquatic mammals led to the discovery of a large array of only class II receptor genes in the dolphin Stenella Coeruleoalba; however, all of these genes were found to be non-functional pseudogenes. These results support the notion that class I receptors may be specialized for detecting water-soluble odorants and class II receptors for recognizing volatile odorants. Comparing the structural features of both receptor classes from various species revealed that they differ mainly in their extracellular loop 3, which may contribute to ligand specificity. Comparing the number and diversity of olfactory receptor genes in different species provides insight into the origin and the evolution of this unique gene family. Accepted: 29 July 1998     

PHYLOGENY OF ZYGNEMOPHYCEAE BASED ON COXIII GENE SEQUENCE DATA

Molecular phylogenetic analysis of the conjugating green algae (Class Zygnemophyceae) using nuclear (SSU rDNA) and chloroplast (rbcL) gene sequences has resolved hypotheses of relationship at the class, order, and family levels, but several key questions will require data from additional genes. Based on SSU and rbcL sequences, the Zygnemophyceae and Desmidiales are monophyletic, and families of placoderm desmids are distinct clades (Desmidiaceae, Peniaceae, Closteriaceae, and Gonatozygaceae). In contrast, the Zygnemataceae and Mesotaeniaceae are paraphyletic, although whether these two traditional families constitute a clade is uncertain. In addition, relationships of genera within families have proven resistant to resolution with these two oft‐used genes. We have sequenced the coxIII gene from the mitochondrial genome to address some of these ambiguous portions of the phylogeny of conjugating green algae. The coxIII gene is more variable than rbcL or SSU rDNA and offers greater resolving power for relationships of genera. We present preliminary analyses of coxIII sequences from each of the traditional families of Zygnemophyceae and contrast the resulting topologies with those derived from nuclear and chloroplast genes.     

Structure and evolution of a member of a new subfamily of GTP-binding proteins mapping to the human MHC class I region

A gene coding for a putative GTP-binding protein, MMR1, has been localized on band C of the murine Chr 17 within or close to the MHC (Denizot et al. 1992). Its human homolog, HSR1, localized to the human MHC class I region, is described in this paper. Its sequence, compared with MMR1, shows that the conceptual proteins encoded by these genes are highly homologous and have thus been subjected to high constraints during evolution. Furthermore, a detailed databank search with HSR1 leads to the characterization of a new subfamily of GTP-binding proteins, of which HSR1 and MMR1 are the only eukaryotic members. The precise localization of HSR1 within the human MHC class I region is also presented.     

Composite origin of major histocompatibility complex genes

Major histocompatibility complex WHO genes have now been cloned from representatives of all vertebrate classes except Agnatha. The recent accumulation of sequence data has given great insight into the course of evolution of these genes. Although the primary structure of the MHC genes varies greatly from class to class and also within the individual classes, the general features of the tertiary and quaternary structure have been conserved remarkably well during more than 400 million years.of evolution. The ancestral MHC genes may have been assembled from at least three structural elements derived from different gene families. Class II MHC genes appear to have been assembled first, and then to have given rise to class I genes.     

Short-range linkage relationships of the valyl-tRNA synthetase gene in Fugu rubripes

 The class III region of the human major histocompatibility complex (MHC) is gene-dense, averaging one gene every 10–20 kilobases (kb). Its gene order has been compared with other organisms. To extend this analysis further in another non-mammalian vertebrate, the compact genome of Fugu rubripes was investigated for the existence of orthologues of these class III genes and their relative arrangements. Orthologues of the M _r 70000 heat shock protein (HSP70) and valyl-tRNA synthetase genes have been isolated. They do not seem to be closely physically linked as compared with mammals (supported by longer-range analysis using pulsed field gel electrophoresis). Random shotgun sequencing of the two Fugu cosmids containing the gene encoding valyl-tRNA synthetase revealed sequences resembling genes encoding tenascin-X, the nuclear antigen A/Ro of Sjogren’s syndrome, and the Landsteiner-Wiener blood group glycoprotein. These linkage relationships recapitulate some mammalian data, albeit imperfectly. Tenascin-X has been located both in the human and mouse Mhc class III regions. Three copies of a sequence found in the gene encoding Sjogren’s syndrome nuclear antigen A/Ro have been identified in the human Mhc class I region; the mouse Mhc class I region contains one copy. It is postulated that a fragmented gene pattern had existed prior to convergence in the ancestral mammalian immune response-related Mhc region, and that some of these genes had belonged to the same linkage group. Received: 17 February 1997 / Revised: 25 March 1997     

The nucleotide sequence of the bovine MHC class II alpha genes:DRA,DQA, andDYA

The nucleotide sequence of the exons 2, 3, and 4, and parts of the intervening sequences of aBoLA-DRA and-DQA gene and one other class IIBoLA-A gene have been determined. The structure of theBoLA-DRA and-DQA gene was found to be very similar to that of the corresponding human HLA class II genes. An analysis of the structure of the other class IIBoLA-A gene showed that thisA gene was clearly very different from both the humanA genes and the bovineDRA andDQA genes. The results indicate that this other type of class IIA gene probably represents the class II gene that has already been identified in restriction fragment length polymorphism (RFLP) studies asBoLA-DYA. Since no clear homologue of this presumedBoLA-DYA gene was found among the human HLA class II genes, these results indicate that, at least as far as theA genes are concerned, a distinct class II gene is present in cattle.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M30117–M30120. Address correspondence and offprint requests to: J. van der Poel.     

Molecular characterization,balancing selection,and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074 bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8⁺ interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180 bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.