Giant Panda Genomic Data Provide Insight into the Birth-and-Death Process of Mammalian Major Histocompatibility Complex Class II Genes

To gain an understanding of the genomic structure and evolutionary history of the giant panda major histocompatibility complex (MHC) genes, we determined a 636,503-bp nucleotide sequence spanning the MHC class II region. Analysis revealed that the MHC class II region from this rare species contained 26 loci (17 predicted to be expressed), of which 10 are classical class II genes (1 DRA, 2 DRB, 2 DQA, 3 DQB, 1 DYB, 1 DPA, and 2 DPB) and 4 are non-classical class II genes (1 DOA, 1 DOB, 1 DMA, and 1 DMB). The presence of DYB, a gene specific to ruminants, prompted a comparison of the giant panda class II sequence with those of humans, cats, dogs, cattle, pigs, and mice. The results indicated that birth and death events within the DQ and DRB-DY regions led to major lineage differences, with absence of these regions in the cat and in humans and mice respectively. The phylogenetic trees constructed using all expressed alpha and beta genes from marsupials and placental mammals showed that: (1) because marsupials carry loci corresponding to DR, DP, DO and DM genes, those subregions most likely developed before the divergence of marsupials and placental mammals, approximately 150 million years ago (MYA); (2) conversely, the DQ and DY regions must have evolved later, but before the radiation of placental mammals (100 MYA). As a result, the typical genomic structure of MHC class II genes for the giant panda is similar to that of the other placental mammals and corresponds to BTNL2∼DR1∼DQ∼DR2∼DY∼DO_box∼DP∼COL11A2. Over the past 100 million years, there has been birth and death of mammalian DR, DQ, DY, and DP genes, an evolutionary process that has brought about the current species-specific genomic structure of the MHC class II region. Furthermore, facing certain similar pathogens, mammals have adopted intra-subregion (DR and DQ) and inter-subregion (between DQ and DP) convergent evolutionary strategies for their alpha and beta genes, respectively.     

Evolutionary relationships of class II major-histocompatibility-complex genes in mammals

The major histocompatibility complex (MHC) class II molecule consists of noncovalently associated alpha and beta chains. In mammals studied so far, the class II MHC can be divided into a number of regions, each containing one or more alpha-chain genes (A genes) and beta-chain genes (B genes), and it has been known for some time that orthologous relationships exist between genes in corresponding regions from different mammalian species. A phylogenetic analysis of DNA sequences of class II A and B genes confirmed these relationships; but no such orthologous relationship was observed between the B genes of mammals and those of birds. Thus, the class II regions have diverged since the separation of birds and mammals (approximately 300 Mya) but before the radiation of the placental mammalian orders (60-80 Mya). Comparison of the phylogenetic trees for A and B genes revealed an unexpected characteristic of DP-region genes: DPB genes are most closely related to DQB genes, whereas DPA chain genes are most closely related to DRA-chain genes. Thus, the DP region seems to have originated through a recombinational event which brought together a DQB gene and a DRA gene (perhaps approximately 120 Mya). The 5' untranslated region of all class II genes includes sequences which are believed to be important in regulating class II gene expression but which are not conserved in known pseudogenes. These sequences are conserved to an extraordinary degree in the human DQB1 gene and its mouse homologue A beta 1, suggesting that regulation of expression of this locus may play a key role in expression of the entire class II MHC.     

Marsupial MHC class II beta genes are not orthologous to the eutherian beta gene families

The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the beta genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II beta sequences with eutherian beta gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II beta molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial beta-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.     

Exon-intron structure of the <Emphasis Type="Italic">Xist</Emphasis> gene in elephant,armadillo, and the ancestor of placental mammals

The Xist gene belongs to the class of long noncoding regulatory RNA genes which play a key role in the process of inactivation of one of the X chromosomes in females of placental mammals. Based on inter-specific comparative sequence analysis performed using a set of bioinformatic programs and approaches, the exon-intron gene structure was first described in two species, elephant and armadillo, belonging to the most primitive placental mammal groups, Afrotheria and Xenarthra. Using multiple sequence alignment of the species representing all main groups of placental mammals (12 species), consensus sequence of the ancestral gene was reconstructed. In the gene structure four evolutionary conserved regions with the identity level of 90% and the sizes of more than 100 bp were identified. Substantial contribution of transposable elements to the gene origin, as well as mosaic evolution of certain elements of the Xist locus was demonstrated. It is likely that the ancestral gene consisted of ten exons and was formed before the radiation of placental mammals, in the period from 140 to 105 Myr ago.     

Exon-intron structure of the Xist gene in elephant, armadillo, and the ancestor of placental mammals

The Xist gene belongs to the class of long noncoding regulatory RNA genes which play a key role in the process of inactivation of one of the X chromosomes in females of placental mammals. Based on interspecific comparative sequence analysis performed using a set ofbioinformatic programs and approaches, the exon-intron gene structure was first described in two species, elephant and armadillo, belonging to the most primitive placental mammal groups, Afrotheria and Xenarthra. Using multiple sequence alignment of the species representing all main groups of placental mammals (12 species), consensus sequence of the ancestral gene was reconstructed. In the gene structure four evolutionary conserved regions with the identity level of 90% and the sizes of more than 100 bp were identified. Substantial contribution of transposable elements to the gene origin, as well as mosaic evolution of certain elements of the Xist locus was demonstrated. It is likely that the ancestral gene consisted often exons and was formed before the radiation of placental mammals, in the period from 140 to 105 Myr ago.     

The class II genes of the rat MHC

Genes that encode class II Ag from the MHC of the rat, the RT1 region, have been isolated as a series of cosmid clones. The cosmids define two clusters, each of which contains three identifiable sequences; one homologous to alpha-chain and two to beta-chain genes. Both the serologically identified rat class II Ag have been expressed in mouse L cell fibroblasts after the introduction of each alpha-chain gene along with a beta-chain gene from the same cluster. There are substantial homologies to the I region of the mouse H-2 complex in the presence, location, orientation, and expression of the six identified sequences from the rat RT1, supporting the view that the overall organization of the two gene complexes has remained conserved since the species separated.     

Comparative organization and the origin of noncoding regulatory RNA genes from X-chromosome inactivation center of human and mouse

The Xist gene belongs to the class of long noncoding regulatory RNA genes which play a key role in the process of inactivation of one of the X chromosomes in females of placental mammals. Based on inter-specific comparative sequence analysis performed using a set of bioinformatic programs and approaches, the exon-intron gene structure was first described in two species, elephant and armadillo, belonging to the most primitive placental mammal groups, Afrotheria and Xenarthra. Using multiple sequence alignment of the species representing all main groups of placental mammals (12 species), consensus sequence of the ancestral gene was reconstructed. In the gene structure four evolutionary conserved regions with the identity level of 90% and the sizes of more than 100 bp were identified. Substantial contribution of transposable elements to the gene origin, as well as mosaic evolution of certain elements of the Xist locus was demonstrated. It is likely that the ancestral gene consisted of ten exons and was formed before the radiation of placental mammals, in the period from 140 to 105 Myr ago.     

Concerted evolution of the immunoglobulin VH gene family

With the aim of understanding the concerted evolution of the immunoglobulin VH multigene family, a phylogenetic tree for the DNA sequences of 16 mouse and five human germ line genes was constructed. This tree indicates that all genes in this family have undergone substantial evolutionary divergence. The most closely related genes so far identified in the mouse genome seem to have diverged about 6 million years (MY) ago, whereas the most distantly related genes diverged about 300 MY ago. This suggests that gene duplication caused by unequal crossing-over or gene conversion occurs very slowly in this gene family. The rate of occurrence of gene duplication in the VH gene family has been estimated to be 5 x 10(-7) per gene per year, which seems to be at least about 100 times lower than that for the rRNA gene family. This low rate of concerted evolution in the VH gene family helps retain intergenic genetic variability that in turn contributes to antibody diversity. Because of accumulation of destructive mutations, however, about one-third of the mouse and human VH genes seem to have become nonfunctional. Many of these pseudogenes have apparently originated recently, but some of them seem to have existed in the genome for more than 10 MY. The rate of nucleotide substitution for the complementarity-determining regions (CDRs) is as high as that of pseudogenes. This suggests that there is virtually no purifying selection operating in the CDRs and that germ line mutations are effectively used for generating antibody diversity.      

Isolation of Mhc Class II DMA and DMB cDNA Sequences in a Marsupial: The Gray Short-Tailed Opossum (Monodelphis domestica)

We report the cDNA sequences for the DMA and DMB family of Mhc genes of the gray short-tailed opossum. Until now DM sequences were available only in eutherian mammals. The marsupial sequences indicate that both members of the family are old and probably diverged from other classical class II families about the time of the radiation of jawed vertebrates some 450 million years ago. We examine the evolutionary rates of equivalent sets of classical and nonclassical genes to check for rate heterogeneity. We find the α-1 domain of the DR genes to be untypically conservative in its evolutionary mode. The DM genes appear to evolve at rates typical of other class II genes, indicating that their placement at the root of class II gene evolutionary trees may be justified. Received: 2 March 1998 / Accepted: 2 June 1998     

A survey of type I interferons from a marsupial and monotreme: implications for the evolution of the type I interferon gene family in mammals

Sequence data for type I interferons (IFNs) have previously only been available for birds and eutherian ('placental') mammals, but not for the other two groups of extant mammals, the marsupials and monotremes. This has left a large gap in our knowledge of the evolutionary and functional relationships of what is a complex gene family in eutherians. In this study, a PCR-based survey of type I IFN genes from a marsupial, the tammar wallaby (Macropus eugenii), and a monotreme, the short-beaked echidna (Tachyglossus aculeatus), was conducted. Along with Southern blot and phylogenetic analysis, this revealed a large number of type I IFN genes for the wallaby, rivalling that of eutherians, but relatively few type I IFN genes in the echidna. The wallaby genes include both IFNA and IFNB orthologues, indicating that the gene duplication leading to these subtypes occurred prior to the divergence of marsupials and eutherians some 130 million years ago. Results from this study support the idea that the expansion of type I IFN gene complexity in mammals coincides with a concomitant expansion in the functionality of these molecules. For example, this expansion in complexity may have, at least partially, facilitated the evolution of viviparity in marsupials and eutherians. Other evolutionary aspects of these sequences are also discussed.     

Assignment of orthologous relationships among mammalian alpha-globin genes by examining flanking regions reveals a rapid rate of evolution

In order to study the relationships among mammalian alpha-globin genes, we have determined the sequence of the 3' flanking region of the human alpha 1 globin gene and have made pairwise comparisons between sequenced alpha-globin genes. The flanking regions were examined in detail because sequence matches in these regions could be interpreted with the least complication from the gene duplications and conversions that have occurred frequently in mammalian alpha-like globin gene clusters. We found good matches between the flanking regions of human alpha 1 and rabbit alpha 1, human psi alpha 1 and goat I alpha, human alpha 2 and goat II alpha, and horse alpha 1 and goat II alpha. These matches were used to align the alpha-globin genes in gene clusters from different mammals. This alignment shows that genes at equivalent positions in the gene clusters of different mammals can be functional or nonfunctional, depending on whether they corrected against a functional alpha-globin gene in recent evolutionary history. The number of alpha-globin genes (including pseudogenes) appears to differ among species, although highly divergent pseudogenes may not have been detected in all species examined. Although matching sequences could be found in interspecies comparisons of the flanking regions of alpha- globin genes, these matches are not as extensive as those found in the flanking regions of mammalian beta-like globin genes. This observation suggests that the noncoding sequences in the mammalian alpha-globin gene clusters are evolving at a faster rate than those in the beta-like globin gene clusters. The proposed faster rate of evolution fits with the poor conservation of the genetic linkage map around alpha-globin gene clusters when compared to that of the beta-like globin gene clusters. Analysis of the 3' flanking regions of alpha-globin genes has revealed a conserved sequence approximately 100-150 bp 3' to the polyadenylation site; this sequence may be involved in the expression or regulation of alpha-globin genes.      

DO beta: a new beta chain gene in HLA-D with a distinct regulation of expression

The HLA-D region of the human major histocompatibility complex encodes the genes for the alpha and beta chains of the DP, DQ and DR class II antigens. A cDNA clone encoding a new class II beta chain (designated DO) was isolated from a library constructed from mRNA of a mutant B-cell line having a single HLA haplotype. Complete cDNA clones encoding the four isotypic beta chains of the DR1, DQw1, DPw2 and putative DO antigens were sequenced. The DO beta gene was mapped in the D region by hybridization with DNA of HLA-deletion mutants. DO beta mRNA expression is low in B-cell lines but remains in mutant lines which have lost expression of other class II genes. Unlike other class II genes DO beta is not induced by gamma-interferon in fibroblast lines. The DO beta gene is distinct from the DP beta, DQ beta and DR beta genes in its pattern of nucleotide divergence. The independent evolution and expression of DO beta suggest that it may be part of a functionally distinct class II molecule.     

Evolution of Antennapedia-Class Homeobox Genes

Antennapedia (Antp)-class homeobox genes are involved in the determination of pattern formation along the anterior-posterior axis of the animal embryo. A phylogenetic analysis of Antp-class homeodomains of the nematode, Drosophila, amphioxus, mouse, and human indicates that the 13 cognate group genes of this gene family can be divided into two major groups, i.e., groups I and II. Group I genes can further be divided into subgroups A (cognate groups 1-2), B (cognate group 3), and C (cognate groups 4-8), and group II genes can be divided into subgroups D (cognate groups 9-10) and E (cognate groups 11-13), though this classification is somewhat ambiguous. Evolutionary distances among different amino acid sequences suggest that the divergence between group I and group II genes occurred ~1000 million years (MY) ago, and the five different subgroups were formed by ~600 MY ago, probably before the divergence of Pseudocoelomates (e.g., nematodes) and Coelomates (e.g., insects and chordates). Our results show that the genes that are phylogenetically close are also closely located in the chromosome, suggesting that the colinearity between the gene expression and gene arrangement was generated by successive tandem gene duplications and that the gene arrangement has been maintained by some sort of selection.     

Transposable elements as a source of genetic innovation: expression and evolution of a family of retrotransposon-derived neogenes in mammals

A family of functional neogenes called Mart, related to the gag gene of Sushi-like long terminal repeat retrotransposons from fish and amphibians, is present in the genome of human (11 genes) and other primates, as well as in mouse (11 genes), rat, dog (12 genes), cat, and cow. Mart genes have lost their capacity of retrotransposition through non-functionalizing rearrangements having principally affected long terminal repeats and pol open reading frame. Most Mart genes are located on the X chromosome in different mammals. Sequence database analysis suggested that Mart genes are present in opossum (marsupial), but absent from the genome of chicken. Hence, the Mart gene family might have been formed from Sushi-like retrotransposon(s) after the split of birds and mammals (310 myr ago), but before the divergence between placental mammals and marsupials (170 myr ago). RT-PCR analysis showed that at least six Mart genes are expressed during mouse embryonic development, with in situ hybridization analysis revealing rather ubiquitous expression patterns. Mart expression was also detected in adult mice, with some genes being expressed in all tissues tested, while others showed a much more restricted expression pattern. Although additional analysis will be required to establish the function of the retrotransposon-derived Mart neogenes, these observations support the evolutionary importance of retrotransposable elements as a source of genetic novelty.     

Increased rate of hair keratin gene loss in the cetacean lineage

Background

Hair represents an evolutionary innovation that appeared early on mammalian evolutionary history, and presumably contributed significantly to the rapid radiation of the group. An interesting event in hair evolution has been its secondary loss in some mammalian groups, such as cetaceans, whose hairless phenotype appears to be an adaptive response to better meet the environmental conditions. To determine whether different repertoire of keratin genes among mammals can potentially explain the phenotypic hair features of different lineages, we characterized the type I and II clusters of alpha keratins from eight mammalian species, including the hairless dolphin and minke whale representing the order Cetacea.

Results

We combined the available genomic information with phylogenetic analysis to conduct a comprehensive analysis of the evolutionary patterns of keratin gene clusters. We found that both type I and II gene clusters are fairly conserved among the terrestrial mammals included in this study, with lineage specific gene duplication and gene loss. Nevertheless, there is also evidence for an increased rate of pseudogenization in the cetacean lineage when compared to their terrestrial relatives, especially among the hair type keratins.

Conclusions

Here we present a comprehensive characterization of alpha-keratin genes among mammals and elucidate the mechanisms involved in the evolution of this gene family. We identified lineage-specific gene duplications and gene loss among the Laurasiatherian and Euarchontoglires species included in the study. Interestingly, cetaceans present an increased loss of hair-type keratin genes when compared to other terrestrial mammals. As suggested by the ‘less-is-more’ hypothesis, we do not rule out the possibility that the gene loss of hair-type keratin genes in these species might be associated to the hairless phenotype and could have been adaptive in response to new selective pressures imposed by the colonization of a new habitat. Our study provides support for the idea that pseudogenes are not simply ‘genomic fossils’ but instead have adaptive roles during the evolutionary process.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-869) contains supplementary material, which is available to authorized users.     

The Phylogenetic Informativeness of Nucleotide and Amino Acid Sequences for Reconstructing the Vertebrate Tree

To aid in future efforts to accurately reconstruct the vertebrate tree, a quantitative measure of phylogenetic informativeness was applied to nucleotide and amino acid sequences for a set of 11 genes. We identified orthologues and assembled published fossil-calibrated divergence times between taxa that had been sequenced for each gene. Rates of molecular evolution for each site were estimated to characterize the molecular evolutionary pattern of genes and to calculate the phylogenetic informativeness. The fast-evolving gene albumin yielded the highest informativeness over the period from 60 million years ago to 500 million years ago. In contrast, calmodulin yielded the lowest informativeness, presumably because functional constraint minimized substitutions in the amino acid sequence. The gene c-myc showed an intermediate level of informativeness. The nucleotide sequence of cytochrome b showed extremely high utility for recent epochs, but low utility for times before 100 million years ago. We ranked nine other genes for their utility during the epochs of the divergence of the muroid rodents, early placental mammals, early vertebrates, and early metazoa, yielding results consistent with, but more precise than, previous studies. Interestingly, DNA sequence always exceeded amino acid sequence in informativeness over all time scales, yet support values were at best moderately higher. For epochs not subject to strong phylogenetic conflict due to convergence, we advocate gleaning the additional power of the threefold increase in number of characters that is present for DNA sequences over resorting to the less noisy but less informative amino acid sequences.     

Phylogenetic analysis of vertebrate kininogen genes

Kininogens, the precursors of bradykinins, vary extremely in both structure and function among different taxa of animals, in particular between mammals and amphibians. This includes even the most conserved bradykinin domain in terms of biosynthesis mode and structure. To elucidate the evolutionary dynamics of kininogen genes, we have identified 19 novel amino acid sequences from EST and genomic databases (for mammals, birds, and fishes) and explored their phylogenetic relationships using combined amino acid sequence and gene structure as markers. Our results show that there were initially two paralogous kininogen genes in vertebrates. During their evolution, the original gene was saved with frequent multiplication in amphibians, but lost in fishes, birds, and mammals, while the novel gene was saved with multiple functions in fishes, birds, and mammals, but became a pseudogene in amphibians. We also propose that the defense mechanism against specific predators in amphibian skin secretions has been bradykinin receptor dependent. Our findings may provide a foundation for identification and structural, functional, and evolutionary analyses of more kininogen genes and other gene families.     

Allelic polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of Cynoglossus semilaevis (Cynoglossidae)

Major histocompatibility complex (MHC) genes play an important role in the immune response of vertebrates. Allelic polymorphism and evolutionary mechanism of MHC genes have been investigated in many mammals, but much less is known in teleosts. We examined the polymorphism, gene duplication and balancing selection of the MHC class II DAB gene of the half-smooth tongue sole (Cynoglossus semilaevis); 23 alleles were found in this species. Gene duplication manifested as three to six distinct sequences at each domain in the same individuals. Non-synonymous substitutions occurred at a significantly higher frequency than synonymous substitutions in the PBR domain, suggesting balancing selection for maintaining polymorphisms at the MHC II DAB locus. Many positive selection sites were found to act very intensely on antigen-binding sites of MHC class II DAB gene.