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     

Molecular cloning of cDNA that encode MHC class I molecules from a New World primate (Saguinus oedipus). Natural selection acts at positions that may affect peptide presentation to T cells

To investigate the evolutionary pressures that drive the generation of polymorphism in primate MHC class I molecules, three cDNA that encode MHC class I alleles from a New World monkey, the cotton-top tamarin (Saguinus oedipus), were cloned and sequenced. These tamarin MHC class I alleles contained amino acid substitutions not found in any of the previously sequenced human MHC class I alleles. Moreover, the majority of these unique amino acid substitutions was located in the Ag recognition site at positions that have been shown to be critical in the presentation of viral peptides to T cells in mice and humans. These data suggest that selective pressures on MHC class I molecules preferentially act on the Ag recognition site and that the peptide binding or presenting functions of these molecules may drive the generation of MHC class I polymorphism. The novel Ag recognition sites of the tamarin MHC class I molecules, in addition to their restricted polymorphism, might account for the unusual susceptibility of the cotton-top tamarin to human pathogens.     

Two different primate species express an identical functional MHC class I allele

 The products of the highly polymorphic and variable major histocompatibility complex (MHC) class I loci play a crucial role in host defenses against infectious disease. While similar alleles have been found in closely related species, sharing of a functional MHC class I allele between two species has never been reported. Here we show that an identical functional MHC class I molecule is present in two different primate species with an approximate divergence time of 0.7 million years. Lymphocytes from the red-crested tamarin (Saguinus geoffroyi) expressed an MHC class I allele (Sage-G ^* 01) that was identical in coding sequence to an MHC class I allele (Saoe-G ^* 08) found in the cotton-top tamarin (Saguinus oedipus). Furthermore, influenza virus-specific cytotoxic T lymphocytes (CTLs) generated in the cotton-top tamarin killed lymphocytes expressing the influenza virus nucleoprotein (NP) from the red-crested tamarin. Since the influenza virus NP epitope is bound by Saoe-G^*08 in the cotton-top tamarin, it is likely that this molecule is functional in both species. These data provide the first evidence that functional MHC class I molecules can be maintained entirely intact in two separate species. Received: 6 June 1997 / Revised: 21 July 1997     

Three different MHC class I molecules bind the same CTL epitope of the influenza virus in a primate species with limited MHC class I diversity

One of the most remarkable features of the MHC class I loci of most outbred mammalian populations is their exceptional diversity, yet the functional importance of this diversity remains to be fully understood. The cotton-top tamarin (Saguinus oedipus) is unusual in having MHC class I loci that exhibit both limited polymorphism and sequence variation. To investigate the functional implications of limited MHC class I diversity in this outbred primate species, we infected five tamarins with influenza virus and defined the CTL epitopes recognized by each individual. In addition to an immunodominant epitope of the viral nucleoprotein (NP) that was recognized by all individuals, two tamarins also made a response to the same epitope of the matrix (M1) protein. Surprisingly, these two tamarins used different MHC class I molecules, Saoe-G*02 and -G*04, to present the M1 epitope. In addition, CTLs from one of the tamarins recognized target cells that expressed neither Saoe-G*02 nor -G*04, but, rather, a third MHC class I molecule, Saoe-G*12. Sequence analysis revealed that Saoe-G*12 differs from both Saoe-G*02 and -G*04 by only two nucleotides and was probably generated by recombination between these two alleles. These results demonstrate that at least three of the tamarin's MHC class I molecules can present the same epitope to virus-specific CTLs. Thus, four of the tamarin's 12 MHC class I molecules bound only two influenza virus CTL epitopes. Therefore, the functional diversity of cotton-top tamarin's MHC class I loci may be even more limited than their genetic diversity suggests.     

The T-cell receptor β chain-encoding gene repertoire of a New World primate species,the cotton-top tamarin

 The New World primate, the cotton-top tamarin (Saguinus oedipus), expresses major histocompatibility complex (MHC) class I molecules with limited diversity. The uniqueness of the cotton-top tamarin MHC class I loci may contribute to this species’ unusual susceptibility to viral infections and high incidence of ulcerative colitis. As a prelude to examining the effect of this limited MHC class I diversity on the tamarin CD8⁺ T-cell receptor (TCR) repertoire, we identified expressed tamarin TCR β chain (TCRB) cDNAs by anchored and inverse polymerase chain reaction. Sequence alignments and phylogenetic comparisons with human and rhesus macaque sequences identified homologues of 21 human variable (V) gene families. Only single variable region genes were identified in each of these tamarin VB families, with the exception of the VB 5, 9, and 13 families which were comprised of two or three distinct members. The multiple genes within these three VB families do not appear to have separate human homologues, but rather aligned equally well to a single human gene from their respective VB families. These genes appear to have arisen, therefore, by duplication of certain VB genes in the tamarin ancestors following their divergence from the lineage leading to Old World primates and hominoids. Homologues of 12 of the 13 human joining (J) region genes were also identified in the tamarin. Comparison of the proportion of nonsynonymous (p_N) and synonymous (p_S) substitutions occurring per site within tamarin variable region genes demonstrated a reduction in p_N in the framework regions compared with p_N in the presumed MHC contact regions (CDR1 and CDR2). Taken together, these findings illustrate that the TCR β chain-encoding genes of the cotton-top tamarin are similar in structure and degree of complexity compared with their Old World primate and human counterparts. Received: 19 July 1996 / Revised: 12 August 1996     

Characterization of the rhesus macaque (Macaca mulatta) equivalent of HLA-F

Nucleotide sequence analysis of rhesus macaque major histocompatibility complex class I cDNAs allowed the identification of the orthologue of HLA-F, designated Mamu-F. Comparison of Mamu-F with earlier published human and chimpanzee orthologues demonstrated that these sequences share a high degree of similarity, both at the nucleotide and amino acid level, whereas a New World monkey (cotton-top tamarin) equivalent is more distantly related. Exon 7, encoding one of the cytoplasmatic domains, is absent for all primate Mhc-F cDNA sequences analyzed so far. In contrast to the human, chimpanzee, and rhesus macaque equivalents, the cotton-top tamarin Saoe-F gene seems to have accumulated far more nonsynomynous than synonymous differences.The nucleotide sequence data reported in this paper have been submitted to the Genbank nucleotide sequence database and have been assigned the accession number Z 21819.     

Genetically distinct cell populations in naturally occurring bone marrow-chimeric primates express similar MHC class I gene products

The cotton-top tamarin (Saguinus oedipus) is a naturally occurring "A" + "B"----"A" bone marrow-chimeric species. These primates usually are born as dizygotic twins and, due to placental vascular anastomoses, develop sharing each others' bone marrow elements. Strikingly, almost 50% of the PBL of a member of a twin pair are derived from the hematopoietic stem cells of its cotwin. To clarify the mechanisms underlying the maintenance of tolerance in these stable chimeras, MHC gene products have been biochemically characterized in cloned, genetically distinct, male and female lymphocytes from two male/female cotton-top tamarin twin pairs. Extensive MHC class II sharing between the genetically distinct cell populations was not seen in the two twin pairs. This was consistent with the MHC class II polymorphism seen in the species. However, the MHC class I gene products expressed by one member of a twin pair were almost identical to those expressed by its cotwin. A human minisatellite probe demonstrated restriction fragment length polymorphism in DNA from these animals, indicating extensive polymorphism. Thus, MHC class I sharing did not occur due to inbreeding in these animals. Additionally, another bone marrow-chimeric primate species, the common marmoset (Callithrix jacchus), expresses MHC class I molecules with low levels of variation. These studies suggest that the stable chimerism of bone marrow-chimeric primates may be facilitated by MHC class I similarity between the genetically distinct bone marrow derived-cell populations in their circulation.     

Sequence and evolution of cattle MHC class I cDNAs: concerted evolution has not taken place in cattle

To explore genetic mechanisms responsible for major histocompatibility complex (MHC) class I evolution in the artiodactyls, we cloned and sequenced MHC class I cDNAs from a Bos taurus bull heterozygous for cattle MHC (BoLA) class I serological specificities w2 and w30. Four unique cDNAs were found, indicating the presence of at least two MHC class I loci. Analysis of these four cDNAs and all previously published BoLA cDNA sequences suggested that there may be three cattle MHC class I loci. Additionally, comparison of all of the BoLA class I cDNAs to MHC class I cDNAs of other artiodactyls showed that some of the BoLA class I cDNAs were more similar to certain sheep cDNAs than they were to other cattle cDNAs. These data indicate that each BoLA class I locus has evolved independently after an ancestral gene duplication event and that inter-locus segmental exchange o or concerted evolution has not occurred rapidly enough to cause extensive divergence between the orthologous MHC class I loci of sheep and 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 L02832–L02835. Correspondence to: T. L. Garber at the present address.     

src-specific immunity in inbred chickens bearing v-src DNA- and RSV-induced tumors

Serological data identify a single major histocompatibility complex (MHC) class I locus in cattle. Molecular data, however, demonstrate the presence of at least two cattle MHC (BoLA) class I loci. To investigate the number of transcribed BoLA class I genes, we amplified cattle cDNA by using a single MHC class I-specific primer that hybridized to a conserved region of exon 4 and a non-specific 3 primer. Six BoLA class I cDNAs have been cloned and sequenced from a Bos taurus bull heterozygous for BoLA class I serological antigens, demonstrating the presence of a minimum of three loci. Sequence comparisons suggested that one of these cDNAs may be an unexpressed allele or the product of a nonclassical locus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers U01186 and U01187.     

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded. Correspondence to: N. Yuhki     

Evolutionary origin and diversification of the mammalian CD1 antigen genes.

CD1 antigens are cell-surface glycoproteins which have a molecular structure which is similar (consisting of extracellular domains alpha 1, alpha 2, and alpha 3, a transmembrane portion, and a cytoplasmic tail) to that of class I MHC molecules. Phylogenetic analysis of mammalian CD1 DNA sequences revealed that these genes are more closely related to the class I major histocompatibility complex (MHC) than to the class II MHC and that mammalian genes are more closely related to avian class I MHC genes than they are to mammalian class I MHC genes. The CD1 genes form a multigene family with different numbers of genes in different species (five in human, eight in rabbit, and two in mouse). Known CD1 genes are grouped into the following three families, on the basis of evolutionary relationship: (1) the human HCD1B gene and a partial sequence from the domestic rabbit, (2) the human HCD1A and HCD1C genes, and (3) the human HCD1D and HCD1E genes plus the two mouse genes and a sequence from the cottontail rabbit. The alpha 1 and alpha 2 domains of CD1 are much less conserved at the amino acid level than are the corresponding domains of class I MHC molecules, but the alpha 3 domain of CD1 seems to be still more conserved than the well-conserved alpha 3 domain of class I MHC molecules. Furthermore, in the human CD1 gene family, interlocus exon exchange has homogenized alpha 3 domains of all CD1 genes except HCD1C.     

Segmental exchange between MHC class I genes in a higher primate: recombination in the gorilla between the ancestor of a human non-functional gene and an A locus gene

Classical human major histocompatibility complex (MHC) class I molecules are the products of highly diverse gene loci. It has been suggested that segmental exchange may play a role in the generation of diversity at the antigen recognition site of MHC class I molecules. Here we present the cloning, sequencing and expression of two gorilla A locus cDNAs. One of these cDNAs shows remarkable similarity to the non-functional HLA-AR locus gene (5.4-LBF) only in exon 2. The remainder of the cDNA, however, is most closely related to other classical higher primate A locus genes. This suggests that a segmental exchange may have occurred between the ancestor of the non-functional HLA-AR gene and a classical gorilla A locus gene. Furthermore, the recombination event resulting in Gogo-A3 has affected its antigen recognition site. These data, therefore, demonstrate that segmental exchange can generate diversity at the antigen recognition sites of primate MHC class I molecules and suggest that non-functional genes can contribute to the generation of diversity of classical MHC class I genes.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data base and have been assigned the accession numbers X54375 (Gogo-A3) and X54376 (Gogo-A4). Address correspondence and offprint requests to: D. I. Watkins.     

Syrian hamsters express diverse MHC class I gene products

MHC class I glycoproteins are highly diverse in most species. The Syrian hamster has long been thought to express monomorphic MHC class I molecules and thus be an exception to this rule. Here we show that Syrian hamsters express diverse MHC class I gene products. The nucleotide sequences of the alpha 1 and alpha-2 domains of classical Syrian hamster MHC class I molecules are highly variable and show evidence of having been under selective pressures at their Ag recognition sites. Interestingly, none of the Syrian hamster class I genes was closely related to their counterparts in the mouse. These observations suggest that Syrian hamsters in the wild may express diverse MHC class I molecules.     

A novel instance of class I modification (cim) affecting two of three rat class I RT1-A molecules within one MHC haplotype

MHC class I expression by rats of the RT1(o), RT1(d), and RT1(m) MHC haplotypes was investigated. Identical, functional cDNAs were obtained from RT1(o) and BDIX (RT1(dv1)) rats for three MHC class I molecules. RT1-A1(o/d) and -A2(o/d) are closely related in sequence to other cloned rat class Ia genes that have been shown to map to the RT1-A region, while RT1-A3 degrees is highly homologous to a class I gene identified by sequencing an RT1-A(n) genomic contig and is named A3(n). Detailed analysis of the three molecules was undertaken using serology with mAbs, two-dimensional gel analysis of immunoprecipitates, and killing assays using cytotoxic T cells. Arguments are presented suggesting that A1 degrees is the principal MHC class Ia (classical) restricting element of this haplotype. A2 degrees, which is highly cross-reactive with A1 degrees, and A3 degrees probably play more minor or distinct roles in Ag presentation. Unexpectedly, cDNAs encoding exactly the same three molecules were cloned from rats of the RT1(m) haplotype, an MHC that until now was thought to possess unique class Ia genes. RT1(m) contains the TAP-B allele of the TAP transporter, and we present evidence that functional polymorphism in rat TAP has an even greater impact on the expression of RT1-A1 degrees and -A2 degrees than it does on RT1-A(a) in the established case of class I modification (cim). Historically, this led to the misclassification of RT1(m) class Ia molecules as separate and distinct.     

Molecular analyses of five new chimpanzee MHC class I alleles: implications for differences between evolutional mechanisms of HLA-A, -B, and -C loci.

In order to study the origin of the polymorphism of MHC class I molecules, we have cloned and sequenced five new Patr-A, -B, and -C loci alleles from two chimpanzees. Previous studies of sequence comparison between Patr and HLA class I alleles revealed that many of the sequence motifs were shared and the origin of class I molecules predated the divergence of chimpanzees and humans. These findings are confirmed by our current study. Additionally, our data suggest significant differences between mechanisms of evolution of the A, B, and C loci: (1) The B locus is characterized by frequent nucleotide substitutions, whereas the A and C loci are relatively more conserved; (2) However, unlike the A locus, the alpha2 domains of the C locus sequenced appear to produce MHC polymorphism between these species. These differences might imply the distinctive contributions of each locus during the evolutionary history.     

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