Sequence variability at three MHC loci of finless porpoises (<Emphasis Type="Italic">Neophocaena phocaenoides</Emphasis>)

Major histocompatibility complex (MHC) class II DQB and DRA genes and class I gene of finless porpoises (Neophocaena phocaenoides) were investigated by single-strand conformation polymorphism and sequence analysis. The DRA, DQB, and MHC-I loci each contained 5, 14, and 34 unique sequences, respectively, and considerable sequence variation was found at the MHC-I and DQB loci. Gene duplication was manifested as three to five distinct sequences at each of the DQB and MHC-I loci from some individuals, and these sequences at each of the two loci separately clustered into four groups (cluster A, B, C, and D) based on the phylogenetic trees. Phylogenetic reconstruction revealed a trans-species pattern of evolution. Relatively high rates of non-synonymous (dN) vs synonymous (dS) substitution in the peptide-binding region (PBR) suggested balancing selection for maintaining polymorphisms at the MHC-I and DQB loci. In contrast, one single locus with little sequence variation was detected in the DRA gene, and no non-synonymous substitutions in the PBR indicated no balancing selection on this gene.     

Sequence Polymorphism and Evolution of Three Cetacean MHC Genes

Sequence variability at three major histocompatibility complex (MHC) genes (DQB, DRA, and MHC-I) of cetaceans was investigated in order to get an overall understanding of cetacean MHC evolution. Little sequence variation was detected at the DRA locus, while extensive and considerable variability were found at the MHC-I and DQB loci. Phylogenetic reconstruction and sequence comparison revealed extensive sharing of identical MHC alleles among different species at the three MHC loci examined. Comparisons of phylogenetic trees for these MHC loci with the trees reconstructed only based on non-PBR sites revealed that allelic similarity/identity possibly reflected common ancestry and were not due to adaptive convergence. At the same time, trans-species evolution was also evidenced that the allelic diversity of the three MHC loci clearly pre-dated species divergence events according to the relaxed molecular clock. It may be the forces of balancing selection acting to maintain the high sequence variability and identical alleles in trans-specific manner at the MHC-I and DQB loci.     

中华白海豚3 个MHC 基因座位遗传变异的初步分析

主要组织相容性复合体(Major histocompatibility complex,MHC) 基因是由一组紧密连锁的基因组成,是哺乳动物免疫系统中最重要的组成部分。本文选择3 个MHC 基因座位的第二外元,即:MHC-I 类基因和II 类基因的DRA 和DQB 座位,初步调查濒危物种中华白海豚的遗传变异。共鉴定了2 个DRA、2 个DQB 和7 MHC-I等位基因。DRA 座位遗传变异非常低,而DQB 和MHC-I 座位具有相对较高水平的遗传变异。并且,在DQB 和MHC-I 基因座位的假定的抗原结合位点(Antigen binding sites,ABS),非同义替代明显大于同义替代,提示平衡选择(Balancing selection)维持这两个座位的多态性,而在DRA 座位上,并没有检测到平衡选择。系统发生分析表明中华白海豚的MHC 等位基因没有聚在一起,而是和其他的物种聚在一起,符合MHC 跨种进化(Transspecies evolution)的模式。     

两种鹤科动物MHC-Ⅰ基因高变区的对比分析

主要组织相容性复合体（MHC）是脊椎动物基因组中高度多态的基因家族,其编码产物在脊椎动物免疫系统中起着重要作用。受湿地生境污染和破坏等因素的影响,全球现存鹤科（Gruidae）动物大都已处于受胁状态。为了解鹤科动物MHC-I基因序列信息,本研究设计通用引物对灰鹤（Grus grus）和肉垂鹤（Bugeranus carunculatus）MHC-I基因进行分离。结果从1只灰鹤和1只肉垂鹤血液基因组中分别分离到2条和3条长约1 500 bp的序列片段,这暗示鹤科动物至少存在两个MHC-I基因座位。分离到的核苷酸序列均可翻译成正常的氨基酸,表明它们具有一定的生物学功能。MHC-I抗原结合区的核苷酸和氨基酸变异率,灰鹤分别为5.0%和9.6%,肉垂鹤为9.1%和14.6%。两种鹤抗原肽结合位点的非同义替代率与同义替代率的比值分别为7.348 8和2.145 2,表明其受到强烈的正选择作用。贝叶斯系统发生树显示,鹤科动物MHC-I基因并未按物种聚类,暗示MHC-I基因跨物种多态性的存在。本研究获得的MHC-I基因通用引物及序列信息,可为今后濒危鹤科动物的保护遗传学研究奠定基础。     

High similarity at three MHC loci between the baiji and finless porpoise: trans-species or convergent evolution?

Two DRA alleles and six MHC-I alleles were identified from a group of 15 baiji (Lipotes vexillifer), the most threatened cetacean in the world. Little sequence variation was detected at the DRA locus but extensive variation at the MHC-I locus. In combination with data at the DQB locus previously reported, three MHC loci exon 2 of the baiji all revealed striking similarity with those of the finless porpoise. Especially, some identical alleles shared by both species at the MHC-I and DQB loci suggested the convergent evolution as a consequence of common adaptive solutions to similar environmental pressures in the Yangtze River. As for DRA locus, the identity alleles were shared not only by baiji and finless porpoise but by some other cetacean species of the families Phocoenidae and Delphinidae, suggesting trans-species evolution on this gene.     

Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.     

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     

Design of a predicted MHC restricted short peptide immunodiagnostic and vaccine candidate for Fowl adenovirus C in chicken infection

Fowl adenoviruses (FAdVs) are the ethiologic agents of multiple pathologies in chicken. There are five different species of FAdVs grouped as FAdV-A, FAdV-B, FAdV-C, FAdV-D, and FAdV-E. It is of interest to develop immunodiagnostics and vaccine candidate for Peruvian FAdV-C in chicken infection using MHC restricted short peptide candidates. We sequenced the complete genome of one FAdV strain isolated from a chicken of a local farm. A total of 44 protein coding genes were identified in each genome. We sequenced twelve Cobb chicken MHC alleles from animals of different farms in the central coast of Peru, and subsequently determined three optimal human MHC-I and four optimal human MHC-II substitute alleles for MHC-peptide prediction. The potential MHC restricted short peptide epitope-like candidates were predicted using human specific (with determined suitable chicken substitutes) NetMHC MHC-peptide prediction model with web server features from all the FAdV genomes available. FAdV specific peptides with calculated binding values to known substituted chicken MHC-I and MHC-II were further filtered for diagnostics and potential vaccine epitopes. Promiscuity to the 3/4 optimal human MHC-I/II alleles and conservation among the available FAdV genomes was considered in this analysis. The localization on the surface of the protein was considered for class II predicted peptides. Thus, a set of class I and class II specific peptides from FAdV were reported in this study. Hence, a multiepitopic protein was built with these peptides, and subsequently tested to confirm the production of specific antibodies in chicken.     

Cetacean mitochondrial DNA control region: sequences of all extant baleen whales and two sperm whale species

The sequence of the mitochondrial control region was determined in all 10 extant species commonly assigned to the suborder Mysticeti (baleen or whalebone whales) and to two odontocete (toothed whale) species (the sperm and the pygmy sperm whale). In the mysticetes, both the length and the sequence of the control region were very similar, with differences occurring primarily in the first approximately 160 bp of the 5' end of the L-strand of the region. There were marked differences between the mysticete and sperm whale sequences and also between the two sperm whales. The control region, less its variable portion, was used in a comparison including the 10 mysticete sequences plus the same region of an Antarctic minke whale specimen and the two sperm whales. The difference between the minke whales from the North Atlantic and the Antarctic was greater than that between any acknowledged species belonging to the same genus (Balaenoptera). The difference was similar to that between the families Balaenopteridae (rorquals) and Eschrichtiidae (gray whales). The findings suggest that the Antarctic minke whale should have a full species status, B. bonaerensis. Parsimony analysis separated the bowhead and the right whale (family Balaenidae) from all remaining mysticetes, including the pygmy right whale. The pygmy right whale is usually included in family Balaenidae. The analysis revealed a close relationship between the gray whale (family Eschrichtiidae) sequence and those of the rorquals (family Balaenopteridae). The gray whale was included in a clade together with the sei, Bryde's, fin, blue, and humpback whales. This clade was separated from the two minke whale types, which branched together.      

The trouble with flippers: a report on the prevalence of digital anomalies in Cetacea

The forelimbs of cetaceans (whales, dolphins, and porpoises) are unique among mammals as the digits exhibit hyperphalangy, and the entire limb is encased in a soft tissue flipper that functions to generate lift. The typical morphology of cetacean digits has been well documented by detailed anatomical studies. This study however furthers our understanding of cetacean forelimb anatomy by conducting a taxonomically broad survey of cetacean digital anomalies. Forelimb radiographs from museum collections provided the basis upon which we calculated the prevalence and documented the morphology of cetacean digital abnormalities. Results indicated that 11% (n = 255) of toothed whales displayed some type of aberrant ossification: the majority of these cases displayed a fusion of elements within a single digital ray, whereas cases exhibiting branched digits were rare. A small sample of baleen whale radiographs (n = 6) contained the only documented case of baleen whale polydactyly in a specimen of the gray whale (Eschrichtius). Furthermore, some Balaenoptera specimens displayed ossified elements within the interdigital spaces that lacked attachment to the adjacent digits and carpus. In addition, we speculated on the role that several genes may have played in creating cetacean digital anomalies. © 2009 The Linnean Society of London, Zoological Journal of the Linnean Society, 2009, 155 , 722–735.     

Molecular identification of novel alpha- and gammaherpesviruses from cetaceans stranded on Japanese coasts

Herpesviral infections have been documented in some cetaceans; however, they have not yet been identified in species in the western North Pacific. In the present study, 178 tissue samples from 76 stranded cetacean individuals were tested for the presence of herpesviruses. Herpesvirus genomic DNA fragments surrounding the DNA polymerase gene were amplified in samples from four individuals. TA cloning and direct sequencing of these DNA fragments revealed the presence of two novel alphaherpesviruses, and two novel gammaherpesviruses in the four cetacean individuals. The alphaherpesviruses were associated with the lung tissue of a false killer whale (Pseudorca crassidens), and with the mucus of a melon-headed whale (Peponocephala electra). The gammaherpesviruses were found in the lymph tissues of a Stejneger's beaked whale (Mesoplodon stejnegeri) and a sperm whale (Physeter macrocephalus). The phylogenetic tree using amino acid sequences of the DNA polymerase gene supported the inclusion of the novel viruses identified here in a single monophyletic group containing alphaherpesviruses from other Atlantic cetacean species. Conversely, the novel gammaherpesviruses formed an independent clade distant from other known cetacean gammaherpesviruses.     

Bacterial proteins can be processed by macrophages in a transporter associated with antigen processing-independent, cysteine protease-dependent manner for presentation by MHC class I molecules

MHC class I molecules present peptides derived primarily from endogenously synthesized proteins on the cell surface as ligands for CD8+ T cells. However, CD8+ T cell responses to extracellular bacteria, virus-infected, or tumor cells can also be elicited because certain professional APC can generate peptide/MHC class I (MHC-I) complexes from exogenous sources. Whether the peptide/MHC-I complexes are generated because the exogenous proteins enter the classical cytosolic, TAP-dependent MHC-I processing pathway or an alternate pathway is controversial. Here we analyze the generation of peptide/MHC-I complexes from recombinant Escherichia coli as an exogenous Ag source that could be delivered to the phagosomes or directly into the cytosol. We show that peritoneal and bone marrow macrophages generate peptide/MHC-I complexes by the classical as well as an alternate, but relatively less efficient, TAP-independent pathway. Using a novel method to detect proteolytic intermediates we show that the generation of the optimal MHC-I binding peptide in the alternate pathway requires cysteine as well as other protease(s). This alternate TAP-independent pathway also operates in vivo and provides a potential mechanism for eliciting CD8+ T cell responses to exogenous Ags.     

Isolation of major histocompatibility complex Class I genes from the tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) plays an essential role in the adaptive immune system of vertebrates through antigen recognition. Although MHC genes are found in all vertebrates, the MHC region is dynamic and has changed throughout vertebrate evolution, making it an important tool for comparative genomics. Marsupials occupy an important position in mammalian phylogeny, yet the MHC of few marsupials has been studied in detail. We report the isolation and analysis of expressed MHC Class I genes from the tammar wallaby, a model marsupial used extensively for the study of mammalian reproduction, genetics, and immunology. We determined that there are at least 11 Class I loci in the tammar genome and isolated six expressed Class I sequences from spleen and testes cDNA libraries, representing at least four loci. Two of the Class I sequences contain substitutions at sites known to be important for antigen binding, perhaps impacting their ability to bind peptides, or the types of peptide to which they bind. Phylogenetic analysis of tammar wallaby Class I sequences and other mammalian Class I sequences suggests that some tammar wallaby and red-necked wallaby loci evolved from common ancestral genes.     

Rod Monochromacy and the Coevolution of Cetacean Retinal Opsins

Cetaceans have a long history of commitment to a fully aquatic lifestyle that extends back to the Eocene. Extant species have evolved a spectacular array of adaptations in conjunction with their deployment into a diverse array of aquatic habitats. Sensory systems are among those that have experienced radical transformations in the evolutionary history of this clade. In the case of vision, previous studies have demonstrated important changes in the genes encoding rod opsin (RH1), short-wavelength sensitive opsin 1 (SWS1), and long-wavelength sensitive opsin (LWS) in selected cetaceans, but have not examined the full complement of opsin genes across the complete range of cetacean families. Here, we report protein-coding sequences for RH1 and both color opsin genes (SWS1, LWS) from representatives of all extant cetacean families. We examine competing hypotheses pertaining to the timing of blue shifts in RH1 relative to SWS1 inactivation in the early history of Cetacea, and we test the hypothesis that some cetaceans are rod monochomats. Molecular evolutionary analyses contradict the “coastal” hypothesis, wherein SWS1 was pseudogenized in the common ancestor of Cetacea, and instead suggest that RH1 was blue-shifted in the common ancestor of Cetacea before SWS1 was independently knocked out in baleen whales (Mysticeti) and in toothed whales (Odontoceti). Further, molecular evidence implies that LWS was inactivated convergently on at least five occasions in Cetacea: (1) Balaenidae (bowhead and right whales), (2) Balaenopteroidea (rorquals plus gray whale), (3) Mesoplodon bidens (Sowerby''s beaked whale), (4) Physeter macrocephalus (giant sperm whale), and (5) Kogia breviceps (pygmy sperm whale). All of these cetaceans are known to dive to depths of at least 100 m where the underwater light field is dim and dominated by blue light. The knockout of both SWS1 and LWS in multiple cetacean lineages renders these taxa rod monochromats, a condition previously unknown among mammalian species.     

Porcine major histocompatibility complex (MHC) class I molecules and analysis of their peptide-binding specificities

In all vertebrate animals, CD8⁺ cytotoxic T lymphocytes (CTLs) are controlled by major histocompatibility complex class I (MHC-I) molecules. These are highly polymorphic peptide receptors selecting and presenting endogenously derived epitopes to circulating CTLs. The polymorphism of the MHC effectively individualizes the immune response of each member of the species. We have recently developed efficient methods to generate recombinant human MHC-I (also known as human leukocyte antigen class I, HLA-I) molecules, accompanying peptide-binding assays and predictors, and HLA tetramers for specific CTL staining and manipulation. This has enabled a complete mapping of all HLA-I specificities (“the Human MHC Project”). Here, we demonstrate that these approaches can be applied to other species. We systematically transferred domains of the frequently expressed swine MHC-I molecule, SLA-1*0401, onto a HLA-I molecule (HLA-A*11:01), thereby generating recombinant human/swine chimeric MHC-I molecules as well as the intact SLA-1*0401 molecule. Biochemical peptide-binding assays and positional scanning combinatorial peptide libraries were used to analyze the peptide-binding motifs of these molecules. A pan-specific predictor of peptide–MHC-I binding, NetMHCpan, which was originally developed to cover the binding specificities of all known HLA-I molecules, was successfully used to predict the specificities of the SLA-1*0401 molecule as well as the porcine/human chimeric MHC-I molecules. These data indicate that it is possible to extend the biochemical and bioinformatics tools of the Human MHC Project to other vertebrate species.     

Evolution of major histocompatibility complex class I genes in Cetartiodactyls

Previous studies of cattle MHC have suggested the presence of at least four classical class I loci. Analysis of haplotypes showed that any combination of one, two or three genes may be expressed, although no gene is expressed consistently. The aim of this study was to examine the evolutionary relationships among these genes and to study their phylogenetic history in Cetartiodactyl species, including cattle and their close relatives. A secondary aim was to determine whether recombination had occurred between any of the genes. MHC class I data sets were generated from published sequences or by polymerase chain reaction from cDNA. Phylogenetic analysis revealed that MHC class I sequences from Cetartiodactyl species closely related to cattle were distributed among the main cattle gene "groups", while those from more distantly related species were either scattered (sheep, deer) or clustered in a species-specific manner (sitatunga, giraffe). A comparison between gene and species trees showed a poor match, indicating that divergence of the MHC sequences had occurred independently from that of the hosts from which they were obtained. We also found two clear instances of interlocus recombination among the cattle MHC sequences. Finally, positive natural selection was documented at positions throughout the alpha 1 and 2 domains, primarily on those amino acids directly involved in peptide binding, although two positions in the alpha 3 domain, a region generally conserved in other species, were also shown to be undergoing adaptive evolution.     

Analysis of MHC class I genes across horse MHC haplotypes

The genomic sequences of 15 horse major histocompatibility complex (MHC) class I genes and a collection of MHC class I homozygous horses of five different haplotypes were used to investigate the genomic structure and polymorphism of the equine MHC. A combination of conserved and locus-specific primers was used to amplify horse MHC class I genes with classical and nonclassical characteristics. Multiple clones from each haplotype identified three to five classical sequences per homozygous animal and two to three nonclassical sequences. Phylogenetic analysis was applied to these sequences, and groups were identified which appear to be allelic series, but some sequences were left ungrouped. Sequences determined from MHC class I heterozygous horses and previously described MHC class I sequences were then added, representing a total of ten horse MHC haplotypes. These results were consistent with those obtained from the MHC homozygous horses alone, and 30 classical sequences were assigned to four previously confirmed loci and three new provisional loci. The nonclassical genes had few alleles and the classical genes had higher levels of allelic polymorphism. Alleles for two classical loci with the expected pattern of polymorphism were found in the majority of haplotypes tested, but alleles at two other commonly detected loci had more variation outside of the hypervariable region than within. Our data indicate that the equine major histocompatibility complex is characterized by variation in the complement of class I genes expressed in different haplotypes in addition to the expected allelic polymorphism within loci.     

Genetic drift outweighs balancing selection in shaping post-bottleneck major histocompatibility complex variation in New Zealand robins (Petroicidae)

The Chatham Island black robin, Petroica traversi, is a highly inbred, endangered passerine with extremely low levels of variation at hypervariable neutral DNA markers. In this study we investigated variation in major histocompatibility complex (MHC) class II genes in both the black robin and its nonendangered relative, the South Island robin Petroica australis australis. Previous studies have shown that Petroica have at least four expressed class II B MHC genes. In this study, the sequences of introns flanking exon 2 of these loci were characterized to design primers for peptide-binding region (PBR) sequence analysis. Intron sequences were comprised of varying numbers of repeated units, with highly conserved regions immediately flanking exon 2. Polymerase chain reaction primers designed to this region amplified three or four sequences per black robin individual, and eight to 14 sequences per South Island robin individual. MHC genes are fitness-related genes thought to be under balancing selection, so they may be more likely to retain variation in bottlenecked populations. To test this, we compared MHC variation in the black robin with artificially bottlenecked populations of South Island robin, and with their respective source populations, using restriction fragment length polymorphism analyses and DNA sequencing of the PBR. Our results indicate that the black robin is monomorphic at class II B MHC loci, while both source and bottlenecked populations of South Island robin have retained moderate levels of variation. Comparison of MHC variation with minisatellite DNA variation indicates that genetic drift outweighs balancing selection in determining MHC diversity in the bottlenecked populations. However, balancing selection appears to influence MHC diversity over evolutionary timescales, and the effects of gene conversion are evident.