Analysis of genomic and expressed major histocompatibility class Ia and class II genes in a hexaploid Lake Tana African ‘large’ barb individual (<Emphasis Type="Italic">Barbus intermedius</Emphasis>)

Expression of too many co-dominant major histocompatibility complex (MHC) alleles is thought to be detrimental to proper functioning of the immune system. Polyploidy of the genome will increase the number of expressed MHC genes unless they are prone to a silencing mechanism. In polyploid Xenopus species, the number of MHC class I and II genes has been physically reduced, as it does not increase with higher ploidy genomes. In the zebrafish some class IIB loci have been silenced, as only two genomically bona fide loci, DAA/DAB and DEA/DEB, have been described. Earlier studies indicated a reduction in the number of genomic and expressed class II MHC genes in a hexaploid African large barb. This prompted us to study the number of MHC genes present in the genome of an African large barb individual (Barbus intermedius) in relation to those expressed, adopting the following strategy. Full-length cDNA sequences were generated from mRNA and compared with partial genomic class Ia and II sequences generated by PCR using the same primer set. In addition, we performed Southern hybridizations to obtain a verification of the number of class I and IIB genes. Our study revealed three ₂-microglobulin, five class Ia, four class IIA, and four class IIB genes at the genomic level, which were shown to be expressed in the hexaploid barb individual. The class Ia and class II data indicate that the ploidy status does not correlate with the presence and expression of these MHC genes.     

Role of thyroid hormone in the ontogeny and morphological diversification of <Emphasis Type="Italic">Barbus intermedius sensu</Emphasis> Banister, 1973 of Lake Tana in Ethiopia

The role of thyroid hormone in the ontogeny of the Large African barb Barbus intermedius sensu Banister, 1973 (Teleostei; Cyprinidae)—the presumed ancestral species for the species flock of the large African Barbs of Lake Tana (Ethiopia)—has been studied. It has been shown that the thyroid hormone influences the timing and rate of many morphogenetic processes and a change in its level causes heterochronies, affecting the definitive morphology of fish. Thus, it can be assumed that even a slight change in activity of the thyroid axis during ontogeny can be the cause of wide morphological variability in B. intermedius and may underlie explosive diversification of the species flock of Large African Barbs of Lake Tana.     

Evolution of MHC class II &; chain-encoding genes in the Lake Tana barbel species flock (Barbus intermedius complex)

Major histocompatibility complex (MHC) class II protein polymorphism is maintained in allelic lineages which evolve in a trans-specific manner, passing from one species to descendant species. Selection pressure on peptide binding residues should be greatest during speciation, when organisms move into new environments and their MHC molecules encounter new pathogens. The isolation ofMHC genes from teleost fishes, the most diverse group of vertebrates, has created possibilities for testing this hypothesis. The large barbels of Lake Tana have undergone an adaptive radiation within the last 5 million years, producing 14 morphotypes which inhabit different ecological niches within the lake. We studied the variability in class II beta chain-encoding genes of four of these morphotypes using polymerase chain reaction amplification and DNA sequencing. The sequences obtained were orthologous to four of the known class II genes from the common carp, from which barbels diverged approximately 32 million years ago. When subjected to phylogenetic analysis, the 48 sequences clustered into groups which represent allelic lineages. A comparison of nonsynonymous and synonymous substitutions between the peptide binding region codons and non-peptide binding region codons of these sequences revealed that they are under strong selective pressure. The nucleotide sequence data reported in this paper have been submitted to the EMBL and GenBank databases and have been assigned the accession numbers Z49869, Z49870, X93654-X93694, and X93894-X93898     

Gene duplication and gene conversion in class II MHC genes of New Zealand robins (Petroicidae)

In contrast to mammals, the evolution of MHC genes in birds appears to be characterized by high rates of gene duplication and concerted evolution. To further our understanding of the evolution of passerine MHC genes, we have isolated class II B sequences from two species of New Zealand robins, the South Island robin (Petroica australis australis), and the endangered Chatham Island black robin (Petroica traversi). Using an RT-PCR based approach we isolated four transcribed class II B MHC sequences from the black robin, and eight sequences from the South Island robin. RFLP analysis indicated that all class II B loci were contained within a single linkage group. Analysis of 3-untranslated region sequences enabled putative orthologous loci to be identified in the two species, and indicated that multiple rounds of gene duplication have occurred within the MHC of New Zealand robins. The orthologous relationships are not retained within the coding region of the gene, instead the sequences group within species. A number of putative gene conversion events were identified across the length of our sequences that may account for this. Exon 2 sequences are highly diverse and appear to have diverged under balancing selection. It is also possible that gene conversion involving short stretches of sequence within exon 2 adds to this diversity. Our study is the first report of putative orthologous MHC loci in passerines, and provides further evidence for the importance of gene duplication and gene conversion in the evolution of the passerine MHC.Nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers AY258333–AY258335, AY428561–AY428570, and AY530534–AY530535     

Structure,diversity and evolutionary patterns of expressed MHC class II<Emphasis Type="Italic">B</Emphasis> genes in chub (<Emphasis Type="Italic">Squalius cephalus</Emphasis>), a cyprinid fish species from Europe

The polymorphism of exon 2 of the DAB genes (major histocompatibility complex [MHC] class IIB) was investigated for the first time in the freshwater cyprinid fish species, Squalius cephalus, in the wide range of its distribution in Europe. We identified 111 different MHC class IIB variants in 15 chub populations distributed from Finland to Spain. The sequence analysis showed that many structurally important amino acid sites that were conserved among tetrapods were also conserved in chub. The analysis of recombination indicated that it does not play an important role in producing and maintaining the variation of DAB genes analyzed in the present study. The exon 2 was shown to be subjected to intense positive selection. Phylogenetic analysis and sequence identities suggest the presence of two class IIB loci (DAB1-like and DAB3-like) in chub. Nevertheless, the presence of three DAB3-like sequence variants in several individuals indicates the duplication of the DAB3 gene. A contrasting selection pattern was found in DAB1-like and DAB3-like genes, which suggests the potential functional differences between these genes. Some DAB sequence variants were shared among the populations of different mtDNA lineages. The phylogenetic analyses did not confirm any biogeographical pattern of the genetic structure of MHC IIB in chub, which is in line with balancing selection and trans-species polymorphism in MHC genes. Nevertheless, cluster analysis based on the presence/absence of DAB sequence variants in the populations showed the phylogeophraphical pattern corresponding to the mtDNA lineages, which indicates that neutral selection can partially explain the MHC IIB evolution in chub.     

2004 Nomenclature for the chicken major histocompatibility (<Emphasis Type="Italic">B</Emphasis> and <Emphasis Type="Italic">Y</Emphasis> ) complex

The first standard nomenclature for the chicken (Gallus gallus) major histocompatibility (B) complex published in 1982 describing chicken major histocompatibility complex (MHC) variability is being revised to include subsequent findings. Considerable progress has been made in identifying the genes that define this polymorphic region. Allelic sequences for MHC genes are accumulating at an increasing rate without a standard system of nomenclature in place. The recommendations presented here were derived in workshops held during International Society of Animal Genetics and Avian Immunology Research Group meetings. A nomenclature for B and Y (Rfp-Y) loci and alleles has been developed that can be applied to existing and newly defined haplotypes including recombinants. A list of the current standard B haplotypes is provided with reference stock, allele designations, and GenBank numbers for corresponding MHC class I and class II sequences. An updated list of proposed names for B recombinant haplotypes is included, as well as a list of over 17 Y haplotypes designated to date.     

Isolation, characterization and evolution of ovine major histocompatibility complex class II DRA and DQA genes

Four full-length ovine major histocompatibility complex (MHC) class II A cDNA clones coding for new alleles of DRA, DQA1 and DQA2 genes were isolated from two ovine Λgt10 cDNA libraries. The derived amino acid sequences of these clones resemble class II A molecules from other species in both size and structure. Restriction fragment length polymorphism analysis, using an Ovar-DRA probe on DNA from Merino and Romney sheep revealed only limited polymorphism in contrast to the high levels of polymorphism revealed by Ovar-DQA probes. Comparison of the predicted amino acid sequences for the three ovine A genes with class II A genes from five other species revealed that the most variable region of the molecule is the signal peptide. Although virtually every amino acid site shows variation, within or between species, there are some blocks of highly conserved residues. Within gene comparisons of nucleotide differences reveal that the greatest number of changes is found between the alleles of Ovar-DQA1 and -DQA2 genes and the least between Ovar-DRA1 alleles. Phylogenetic analysis of class IIA sequences from several species place DRA and DQA genes on two distinct branches, with Ovar-DRA1 and BOLA-DRA, and Ovar-DQA1 and BOLA-DQA being most similar on their respective branches.     

The nucleotide sequence and evolution of ovine MHC class II B genes: DQB and DRB

The nucleotide sequences of one Ovar-DQB gene, excluding exon 1 and parts of the introns, and one Ovar-DRB pseudogene are presented. The structure of the Ovar-DQB gene is typical of a major histocompatibility complex (MHC) class II B gene and demonstrats considerable sequence similarity with that of humans including such characteristics as the less common polyadenylation signal, ATTAAA. The ovine sequence has a typical 5' acceptor splice signal for exon 5, thus potentially encoding a full length cytoplasmic tail. The Ovar-DRB gene identified in this study was found to be a pseudogene, lacking a defined exon 2 and containing premature termination codons in both exons 3 and 4. The 3' donor splice site of exon 3 is also atypical. A purine-pyrimidine microsatellite repeat, (dCdA)₁₅, in the 3' region of the pseudogene may be a hotspot for recombination within the ovine DR subregion.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M33306 and M33307. Address correspondence and offprint requests to: M. R. Brandon.     

Mitochondrial DNA phylogeography of the Labeobarbus intermedius complex (Pisces,Cyprinidae) from Ethiopia

Mitochondrial DNA phylogeography of populations of the Labeobarbus intermedius complex (hexaploid barb) was investigated using 88 complete and 71 partial cytochrome b (cytb) sequences originating from 21 localities in five major drainages in Ethiopia and two localities in northern Kenya. The samples included 14 of the 15 Labeobarbus species described from Lake Tana. Discrete phylogeographic analyses of 159 cytb sequences employing Bayesian Markov Chain Monte Carlo (MCMC) simulations using Bayesian evolutionary analysis by sampling trees (BEAST) supported the monophyly of the L. intermedius complex, including the Lake Tana species. This analysis, in combination with statistical parsimony analysis, identified two mitochondrial DNA lineages within the complex. Divergence dating employing coalescent simulations suggested that the geographic split in the L. intermedius complex that led to the formation of these lineages occurred during the Pleistocene (c. 0·5 M b.p. ), consistent with the timing of volcano‐tectonic events postulated to have shaped the current landscape of East Africa.     

Evolutionary origins of retroposon lineages of Mhc class II Ab alleles

Major histocompatibility complex (Mhc) class II Ab genes have evolved into three distinct lineages. While lineage 2 alleles differ from lineage 1 alleles by the insertion of a retroposon in intron 2, the basis for the extremely large intron 2 in lineage 3 alleles has heretofore been undetermined. In this report, we demonstrate by nucleotide sequencing that the genomic sequences of prototypic alleles from all three lineages diverge significantly and that lineage 3 is derived from lineage 2 by two insertional events in intron 2. One insert, composed of a member of B1 short interspersed repetitive elements (SINEs), occurs 508 base pairs (bp) 3 of exon 2, and the other, 1141 bp 3 of exon 2 within the retroposon that distinguishes lineage 2 from lineage 1. To assess the evolutionary stability of these lineages and the extent of ancestral polymorphisms of Ab within Mus species, we extended our restriction site polymorphism analysis to include 86 alleles from 120 independently derived H2 haplotypes from 12 separate species and subspecies of Mus. A phylogenetic tree revealing the relationships of these Ab alleles with respect to restriction site polymorphisms, but excluding the retroposon insertions, demonstrated that these lineages have distinctive genomic structures beyond the retroposon polymorphisms. In summary, these mouse Ab genes were produced from successive retroposon insertion events. Lineage 1 and 2 were detected in a variety of Mus species, including Mus caroli, indicating that these lineages diverged more than 2 million years ago. Lineage 3 alleles were found only in the Mus musculus subspecies, suggesting that it diverged from lineage 2 more recently. These results indicate that all three lineages of Ab have persisted through several speciation events in the genus Mus.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M60562     

Mhc class II B gene evolution in East African cichlid fishes

 A distinctive feature of essential major histocompatibility complex (Mhc) loci is their polymorphism characterized by large genetic distances between alleles and long persistence times of allelic lineages. Since the lineages often span several successive speciations, we investigated the behavior of the Mhc alleles during or close to the speciation phase. We sequenced exon 2 of the class II B locus 4 from 232 East African cichlid fishes representing 32 related species. The divergence times of the (sub)species ranged from 6000 to 8.4 million years. Two types of evolutionary analysis were used to elucidate the pattern of exon 2 sequence divergence. First, phylogenetic methods were applied to reconstruct the most likely evolutionary pathways leading from the last common ancestor of the set to the extant sequences, and to assess the probable mechanisms involved in allelic diversification. Second, pairwise comparisons of sequences were carried out to detect differences seemingly incompatible with origin by nonparallel point mutations. The analysis revealed point mutations to be the most important mechanism behind allelic divergences, with recombination playing only an auxiliary part. Comparison of sequences from related species revealed evidence of random allelic (lineage) losses apparently associated with speciation. Sharing of identical alleles could be demonstrated between species that diverged 2 million years ago. The phylogeny of the exon was incongruent with that of the flanking introns, indicating either a high degree of convergent evolution at the peptide-binding region-encoding sites, or intron homogenization. Received: 6 December 1999 / Revised: 15 February 2000     

Speciation of endemic Lake Tana barbs (Cyprinidae, Ethiopia) driven by trophic resource partitioning; a molecular and ecomorphological approach

A unique species flock of large barbs (Barbus spp.) from Lake Tana is presented, from the level of fish stocks to molecules. Evidence is given for the species status of 14 morphotypes of large barbs. They distinctly differ in: (1) head and body morphometrics, (2) food preferences, (3) distribution patterns, (4) maximal body size, (5) spawning area and period, and (6) molecular genetic characters. Most types show early morphological divergence at small size. Major Histocompatibility Complex (MHC) genes, encoding cell membrane proteins involved in defence against pathogens, were found to be diagnostic for the species' genetic identity. A strong selective pressure on particular amino acid positions in the MHC protein sequence most probably arose in response to different pathogen loads from the newly invaded ecological niches after formation of Lake Tana, ca. two million years ago.Arguments for a sympatric origin of this species flock are discussed. An evolutionary scenario suggests a riverine ancestral Barbus intermedius invading Lake Tana after its formation by volcanic blocking of the Blue Nile river and its isolation by waterfalls. Specialisation for particular food types and disruptive selection on many feeding structures are hypothesized as the evolutionary drive in speciation. The causal relationship between the diversity in feeding structures and food types is explained from experiments and models. As an example, the potential food niche of three barb species is predicted from parameter values measured from a large set of feeding structures and tested against the actual gut contents. The co-occurrence of eight piscivorous barb species is unique for cyprinid fish, which lack oral jaw teeth and a stomach. The significance of this aquatic ecosystem as a multidisciplinary evolutionary laboratory and the need for a wise balance between exploitation and conservation is stressed.     

Survey of major histocompatibility complex class II diversity in pig-tailed macaques

Pig-tailed macaques (Macaca nemestrina) serve as important models for human infectious disease research. Major histocompatibility complex (MHC) class II molecules are important to this research since they present peptides to CD4+ T cells. Despite the importance of characterizing the MHC-II alleles expressed in model species like pig-tailed macaques, to date, less than 150 MHC-II alleles have been named for the six most common classical class II loci (DRA, DRB, DQA, DQB, DPA, and DPB) in this population. Additionally, only a small percentage of these alleles are full-length, making it impossible to use the known sequence for reagent development. To address this, we developed a fast, high-throughput method to discover full-length MHC-II alleles and used it to characterize alleles in 32 pig-tailed macaques. By this method, we identified 128 total alleles across all six loci. We also performed an exon 2-based genotyping assay to validate the full-length sequencing results; this genotyping assay could be optimized for use in determining MHC-II allele frequencies in large cohorts of pig-tailed macaques.     

Evolution of a new nonclassical MHC class I locus in two Old World primate species

 HLA-G is a nonclassical major histocompatibility complex (MHC) class I molecule that is expressed only in the human placenta, suggesting that it plays an important role at the fetal-maternal interface. In rhesus monkeys, which have similar placentation to humans, the HLA-G orthologue is a pseudogene. However, rhesus monkeys express a novel placental MHC class I molecule, Mamu-AG, which has HLA-G-like characteristics. Phylogenetic analysis of AG alleles in two Old World primate species, the baboon and the rhesus macaque, revealed limited diversity characteristic of a nonclassical MHC class I locus. Gene trees constructed using classical and nonclassical primate MHC class I alleles demonstrated that the AG locus was most closely related to the classical A locus. Interestingly, gene tree analyses suggested that the AG alleles were most closely related to a subset of A alleles which are the products of an ancestral interlocus recombination event between the A and B loci. Calculation of the rates of synonymous and nonsynonymous substitution at the AG locus revealed that positive selection was not acting on the codons encoding the peptide binding region. In exon 4, however, the rate of nonsynonymous substitution was significantly lower than the rate of synonymous substitution, suggesting that negative selection was acting on these codons. Received: 22 April 1998 / Revised: 15 July 1998     

Molecular characterization of three Mhc class II B haplotypes in the ring-necked pheasant

We investigated the class II B genes in free-ranging population of the ring-necked pheasant Phasianus colchicus by a combination of restriction fragment length polymorphism (RFLP), polymerase chain reaction (PCR), and DNA sequencing. Special attention was paid to the variation in the second exon, which encodes the peptide-binding ₁-domain. The population was introduced, but it still exhibited major histocompatibility complex polymorphism with at least three segregating class II B haplotypes and consequently six genotypes. We found two class II B genes associated with each haplotype. The class II B genes of birds had until then only been molecularly characterized in the domestic chicken. the pheasant genes were highly variable, although one of the amplified sequences was found in two different haplotypes. Taken together, the most polymorphic positions (residues 37 and 38) were not identical in any of the predicted protein sequences, but all except one of the motifs had already been foud in the domestic chicken. Structurally important features in mammalian class II B genes were generally conserved also in the pheasant sequences, but the loss of a potential salt bridge constituent (Arg₇₂) in several sequences may suggest a slightly different structure of the adjacent parts of the peptide-binding groove. The pheasant genes are most closely related to the so called B-LBII family in the chicken, indicating that this represents a major line of development among avian class II B genes.The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession numbers X75403-X75407. Correspondence to: H. Wittzell, Department of Theoretical Ecology, Ecology Building, Lund University, S-223 62 Lund, Sweden.     

Support for the minimal essential MHC hypothesis: a parrot with a single,highly polymorphic MHC class II <Emphasis Type="Italic">B</Emphasis> gene

We characterized the MHC class II B gene in the green-rumped parrotlet, Forpus passerinus. Three approaches were used: polymerase chain reaction amplification using primers complementary to conserved regions of exon 2, sequencing clones from a genomic library, and amplification of exon 2 using species-specific primers. All three methods indicate that there is only a single class II B locus in this species and no pseudogenes. We suggest that this is the ancestral state for birds. The gene is highly polymorphic; 33 alleles were found in a sample of 25 individuals. Variation in exon 2 is concentrated in the peptide binding residues which show a significant excess of non-synonymous substitutions consistent with the operation of selection in maintaining this extraordinary polymorphism. Genomic clones show that major histocompatibility complex (MHC) gene organization is different from that of chickens; the class II A locus is close to II B. These data provide support for the hypothesis that the bird MHC constitutes a “minimal essential MHC” for responding to infectious disease.     

Molecular characterization and phylogenetic analysis of Wx genes from three Taeniatherum diploid species

In wheat seeds, starch synthase I or the Waxy protein is an enzyme involved in amylose synthesis. The gene encoding this enzyme is Wx and in this study, eight novel Wx alleles were identified in three diploid Taeniatherum species. The variability of these alleles was evaluated, and their nucleotide sequences were compared with those of homologous alleles from wheat. Two types of Taeniatherum Wx alleles were detected in three diploid species Ta. caput-medusae, Ta. asperum, and Ta. crinitum. A phylogenetic analysis indicates that the Taeniatherum Wx alleles were more closely related to Wx alleles from Aegilops species with C, D, M, and U genomes than to Wx alleles of other species. These alleles represent a potential genetic resource that may be useful in wheat breeding programs.