Structural evolution of Otx genes in craniates

Using a degenerate PCR approach, we performed an exhaustive search of Otx genes in the reedfish Erpetoichthys calabaricus, the dogfish Scyliorhinus canicula, and the hagfish Myxine glutinosa. Three novel Otx genes were identified in each of these species, and their deduced protein sequences were determined over a large C-terminal fragment located immediately downstream of the homeodomain. Like their lamprey and osteichthyan counterparts, these nine genes display a tandem duplication of a 20--25-residue C-terminal domain, which appears to be a hallmark of all craniate Otx genes identified thus far, including the highly divergent Crx gene. Phylogenetic analyses show that, together with their osteichthyan counterparts, the dogfish and reedfish genes can be classified into three gnathostome orthology classes. Two of the three genes identified in each of these species belong to the Otx1 and Otx2 orthology classes previously characterized in osteichthyans. The third one unambiguously clusters with the Otx5/Otx5b genes recently characterized in Xenopus laevis, thus defining a novel orthology class. Our results also strongly suggest that the highly divergent Crx genes identified in humans, rodents, and oxen are the mammalian representatives of this third class. The hagfish genes display no clear relationships to the three gnathostome orthology classes, but one of them appears to be closely related to the LjOtxA gene, previously identified in Lampetra japonica. Taken together, these data support the hypothesis that the Otx multigene families characterized in craniates all derive from duplications of a single ancestral gene which occurred after the splitting of cephalochordates but prior to the gnathostome radiation. Using site-by-site sequence comparisons of the gnathostome Otx proteins, we also identified structural constraints selectively acting on each of the three gnathostome orthology classes. This suggests that specialized functions for each of these orthology classes were fixed in the gnathostome lineage prior to the splitting between osteichthyans and chondrichthyans.     

Evolution of MHC class I genes in the European badger (Meles meles)

The major histocompatibility complex (MHC) plays a central role in the adaptive immune system and provides a good model with which to understand the evolutionary processes underlying functional genes. Trans-species polymorphism and orthology are both commonly found in MHC genes; however, mammalian MHC class I genes tend to cluster by species. Concerted evolution has the potential to homogenize different loci, whereas birth-and-death evolution can lead to the loss of orthologs; both processes result in monophyletic groups within species. Studies investigating the evolution of MHC class I genes have been biased toward a few particular taxa and model species. We present the first study of MHC class I genes in a species from the superfamily Musteloidea. The European badger (Meles meles) exhibits moderate variation in MHC class I sequences when compared to other carnivores. We identified seven putatively functional sequences and nine pseudogenes from genomic (gDNA) and complementary (cDNA) DNA, signifying at least two functional class I loci. We found evidence for separate evolutionary histories of the α1 and α2/α3 domains. In the α1 domain, several sequences from different species were more closely related to each other than to sequences from the same species, resembling orthology or trans-species polymorphism. Balancing selection and probable recombination maintain genetic diversity in the α1 domain, evidenced by the detection of positive selection and a recombination event. By comparison, two recombination breakpoints indicate that the α2/α3 domains have most likely undergone concerted evolution, where recombination has homogenized the α2/α3 domains between genes, leading to species-specific clusters of sequences. Our findings highlight the importance of analyzing MHC domains separately.     

Phylogenetic classification of prokaryotic and eukaryotic Sir2-like proteins

Sirtuins (Sir2-like proteins) are present in prokaryotes and eukaryotes. Here, two new human sirtuins (SIRT6 and SIRT7) are found to be similar to a particular subset of insect, nematode, plant, and protozoan sirtuins. Molecular phylogenetic analysis of 60 sirtuin conserved core domain sequences from a diverse array of organisms (including archaeans, bacteria, yeasts, plants, protozoans, and metazoans) shows that eukaryotic Sir2-like proteins group into four main branches designated here as classes I-IV. Prokaryotic sirtuins include members of classes II and III. A fifth class of sirtuin is present in gram positive bacteria and Thermotoga maritima. Saccharomyces cerevisiae has five class I sirtuins. Caenorhabditis elegans and Drosophila melanogaster have sirtuin genes from classes I, II, and IV. The seven human sirtuin genes include all four classes: SIRT1, SIRT2, and SIRT3 are class I, SIRT4 is class II, SIRT5 is class III, and SIRT6 and SIRT7 are class IV.     

大豆KNOX基因家族的结构和表达分析

KNOX基因家族编码同源异型盒蛋白, 在植物生长发育过程中起重要调控作用。利用生物信息学手段在全基因组水平上对大豆(Glycine max)KNOX家族基因进行鉴定和分类, 并分析其基因结构、蛋白同源结构域特征以及基因表达方式。研究结果表明: 大豆中的27个GmKNOX基因可以分为GmKNOX I和GmKNOX II两个亚类, 其中GmKNOX I类可分为3个主要的进化支, GmKNOX II类分为2个主要的进化支; 26个GmKNOX基因不均匀地分布在16条染色体上, GmKNOX27尚无法定位。不同组织表达谱的分析表明: GmKNOX I类基因表达部位比较集中, 以茎顶端分生组织中表达量最高; 而GmKNOX II类基因的表达特异性较GmKNOX I类低, 表达部位更广泛。     

KNOX homeobox genes potentially have similar function in both diploid unicellular and multicellular meristems, but not in haploid meristems

Members of the class 1 knotted-like homeobox (KNOX) gene family are important regulators of shoot apical meristem development in angiosperms. To determine whether they function similarly in seedless plants, three KNOX genes (two class 1 genes and one class 2 gene) from the fern Ceratopteris richardii were characterized. Expression of both class 1 genes was detected in the shoot apical cell, leaf primordia, marginal part of the leaves, and vascular bundles by in situ hybridization, a pattern that closely resembles that of class 1 KNOX genes in angiosperms with compound leaves. The fern class 2 gene was expressed in all sporophyte tissues examined, which is characteristic of class 2 gene expression in angiosperms. All three CRKNOX genes were not detected in gametophyte tissues by RNA gel blot analysis. Arabidopsis plants overexpressing the fern class 1 genes resembled plants that overexpress seed plant class 1 KNOX genes in leaf morphology. Ectopic expression of the class 2 gene in Arabidopsis did not result in any unusual phenotypes. Taken together with phylogenetic analysis, our results suggest that (a) the class 1 and 2 KNOX genes diverged prior to the divergence of fern and seed plant lineages, (b) the class 1 KNOX genes function similarly in seed plant and fern sporophyte meristem development despite their differences in structure, (c) KNOX gene expression is not required for the development of the fern gametophyte, and (d) the sporophyte and gametophyte meristems of ferns are not regulated by the same developmental mechanisms at the molecular level.     

MetaPhOrs: orthology and paralogy predictions from multiple phylogenetic evidence using a consistency-based confidence score

Reliable prediction of orthology is central to comparative genomics. Approaches based on phylogenetic analyses closely resemble the original definition of orthology and paralogy and are known to be highly accurate. However, the large computational cost associated to these analyses is a limiting factor that often prevents its use at genomic scales. Recently, several projects have addressed the reconstruction of large collections of high-quality phylogenetic trees from which orthology and paralogy relationships can be inferred. This provides us with the opportunity to infer the evolutionary relationships of genes from multiple, independent, phylogenetic trees. Using such strategy, we combine phylogenetic information derived from different databases, to predict orthology and paralogy relationships for 4.1 million proteins in 829 fully sequenced genomes. We show that the number of independent sources from which a prediction is made, as well as the level of consistency across predictions, can be used as reliable confidence scores. A webserver has been developed to easily access these data (http://orthology.phylomedb.org), which provides users with a global repository of phylogeny-based orthology and paralogy predictions.     

Genomic and Phylogenetic Analysis of the Human CD1 and HLA Class I Multicopy Genes

The human CD1 proteins belong to a lipid-glycolipid antigen-presenting gene family and are related in structure and function to the MHC class I molecules. Previous mapping and DNA hybridization studies have shown that five linked genes located within a cluster on human chromosome 1q22-23 encode the CD1 protein family. We have analyzed the complete genomic sequence of the human CD1 gene cluster and found that the five active genes are distributed over 175,600 nucleotides and separated by four expanded intervening genomic regions (IGRs) ranging in length between 20 and 68 kb. The IGRs are composed mostly of retroelements including five full-length L1 PA sequences and various pseudogenes. Some L1 sequences have acted as receptors for other subtypes or families of retroelements. Alu molecular clocks that have evolved during primate history are found distributed within the HLA class I duplicated segments (duplicons) but not within the duplicons of CD1. Phylogeny of the alpha3 domain of the class I-like superfamily of proteins shows that the CD1 cluster is well separated from HLA class I by a number of superfamily members including MIC (PERB11), HFE, Zn-alpha2-GP, FcRn, and MR1. Phylogenetically, the human CD1 sequences are interspersed by CD1 sequences from other mammalian species, whereas the human HLA class I sequences cluster together and are separated from the other mammalian sequences. Genomic and phylogenetic analyses support the view that the human CD1 gene copies were duplicated prior to the evolution of primates and the bulk of the HLA class I genes found in humans. In contrast to the HLA class I genomic structure, the human CD1 duplicons are smaller in size, they lack Alu clocks, and they are interrupted by IGRs at least 4 to 14 times longer than the CD1 genes themselves. The IGRs seem to have been created as "buffer zones" to protect the CD1 genes from disruption by transposable elements.     

Using quaternary structures to assess the evolutionary history of proteins: the case of the aspartate carbamoyltransferase

Many evolutionary scenarios describing the history of proteins are based solely on phylogenetic studies. We have designed a new approach that allows ascertainment of such questionable scenarios by taking into account quaternary structures: we used aspartate carbamoyltransferase (ATCase) as a case study. Prokaryotic ATCases correspond to different classes of quaternary structures according to the mode of association of the catalytic PyrB subunit with other polypeptides, either the PyrI regulatory subunit (class B) or a dihydroorotase (class A), which may be active (PyrC, subclass A1) or inactive (PyrC', subclass A2). Class C is uniquely made up of trimers of PyrB. The PyrB phylogenetic tree is not congruent with the tree of life, but it became coherent when we recognized the existence of two families of ATCases, ATC I and ATC II. Remarkably, a very strong correlation was found between the pattern of PyrB phylogenetic clustering and the different classes of quaternary structures of ATCases. All class B ATCases form a clade in family ATC II, which also contains all eukaryotic sequences. In contrast, family ATC I is made up of classes A and C. These results suggest unexpected common ancestry for prokaryotic B and eukaryotic ATCases on the one hand, and for A and C on the other. Thus, the emergence of specific quaternary structures appears to have been a more recent event than the separation into the ATC I and ATC II families. We propose that different evolutionary constraints, depending on the identity of the partners interacting in the different kinds of holoenzymes, operated in a concerted way on the ancestral pyrB genes and the respective associated genes pyrI or pyrC, so as to maintain appropriate inter-polypeptides interactions at the level of quaternary structure. The process of coevolution of genes encoding proteins interacting in various holoenzymes has been assessed by calculating the correlation coefficient between their respective phylogenetic trees. Our approach integrating data obtained from the separate fields of structural biology and molecular evolution could be useful in other cases where pure statistical data need to receive independent confirmation.     

Phylogenomics of Reichenowia parasitica, an alphaproteobacterial endosymbiont of the freshwater leech Placobdella parasitica

Although several commensal alphaproteobacteria form close relationships with plant hosts where they aid in (e.g.,) nitrogen fixation and nodulation, only a few inhabit animal hosts. Among these, Reichenowia picta, R. ornata and R. parasitica, are currently the only known mutualistic, alphaproteobacterial endosymbionts to inhabit leeches. These bacteria are harbored in the epithelial cells of the mycetomal structures of their freshwater leech hosts, Placobdella spp., and these structures have no other obvious function than housing bacterial symbionts. However, the function of the bacterial symbionts has remained unclear. Here, we focused both on exploring the genomic makeup of R. parasitica and on performing a robust phylogenetic analysis, based on more data than previous hypotheses, to test its position among related bacteria. We sequenced a combined pool of host and symbiont DNA from 36 pairs of mycetomes and performed an in silico separation of the different DNA pools through subtractive scaffolding. The bacterial contigs were compared to 50 annotated bacterial genomes and the genome of the freshwater leech Helobdella robusta using a BLASTn protocol. Further, amino acid sequences inferred from the contigs were used as queries against the 50 bacterial genomes to establish orthology. A total of 358 orthologous genes were used for the phylogenetic analyses. In part, results suggest that R. parasitica possesses genes coding for proteins related to nitrogen fixation, iron/vitamin B translocation and plasmid survival. Our results also indicate that R. parasitica interacts with its host in part by transmembrane signaling and that several of its genes show orthology across Rhizobiaceae. The phylogenetic analyses support the nesting of R. parasitica within the Rhizobiaceae, as sister to a group containing Agrobacterium and Rhizobium species.     

Phylogenetic analysis of the Wnt gene family. Insights from lophotrochozoan members

The Wnt gene family encodes secreted signaling molecules that control cell fate specification, proliferation, polarity, and movements during animal development. We investigate here the evolutionary history of this large multigenic family. Wnt genes have been almost exclusively isolated from two of the three main subdivisions of bilaterian animals, the deuterostomes (which include chordates and echinoderms) and the ecdysozoans (e.g., arthropods and nematodes). However, orthology relationships between deuterostome and ecdysozoan Wnt genes, and, more generally, the phylogeny of the Wnt family, are not yet clear. We report here the isolation of several Wnt genes from two species, the annelid Platynereis dumerilii and the mollusc Patella vulgata, which both belong to the third large bilaterian clade, the lophotrochozoans (which constitute, together with ecdysozoans, the protostomes). Multiple phylogenetic analyses of these sequences with a large set of other Wnt gene sequences, in particular, the complete set of Wnt genes of human, nematode, and fly, allow us to subdivide the Wnt family into 12 subfamilies. At least nine of them were already present in the last common ancestor of all bilaterian animals, and this further highlights the genetic complexity of this ancestor. The orthology relationships we present here open new perspectives for future developmental comparisons.     

Structural and Evolutionary Relationships Among Chitinases of Flowering Plants

The analysis of nuclear-encoded chitinase sequences from various angiosperms has allowed the categorization of the chitinases into discrete classes. Nucleotide sequences of their catalytic domains were compared in this study to investigate the evolutionary relationships between chitinase classes. The functionally distinct class III chitinases appear to be more closely related to fungal enzymes involved in morphogenesis than to other plant chitinases. The ordering of other plant chitinases into additional classes mainly relied on the presence of auxiliary domains—namely, a chitin-binding domain and a carboxy-terminal extension—flanking the main catalytic domain. The results of our phylogenetic analyses showed that classes I and IV form discrete and well-supported monophyletic groups derived from a common ancestral sequence that predates the divergence of dicots and monocots. In contrast, other sequences included in classes I* and II, lacking one or both types of auxiliary domains, were nested within class I sequences, indicating that they have a polyphyletic origin. According to phylogenetic analyses and the calculation of evolutionary rates, these chitinases probably arose from different class I lineages by relatively recent deletion events. The occurrence of such evolutionary trends in cultivated plants and their potential involvement in host–pathogen interactions are discussed. Received: 5 July 1996 / Accepted: 9 January 1997     

In silico peptide-binding predictions of passerine MHC class I reveal similarities across distantly related species, suggesting convergence on the level of protein function

The major histocompatibility complex (MHC) genes are the most polymorphic genes found in the vertebrate genome, and they encode proteins that play an essential role in the adaptive immune response. Many songbirds (passerines) have been shown to have a large number of transcribed MHC class I genes compared to most mammals. To elucidate the reason for this large number of genes, we compared 14 MHC class I alleles (α1–α3 domains), from great reed warbler, house sparrow and tree sparrow, via phylogenetic analysis, homology modelling and in silico peptide-binding predictions to investigate their functional and genetic relationships. We found more pronounced clustering of the MHC class I allomorphs (allele specific proteins) in regards to their function (peptide-binding specificities) compared to their genetic relationships (amino acid sequences), indicating that the high number of alleles is of functional significance. The MHC class I allomorphs from house sparrow and tree sparrow, species that diverged 10 million years ago (MYA), had overlapping peptide-binding specificities, and these similarities across species were also confirmed in phylogenetic analyses based on amino acid sequences. Notably, there were also overlapping peptide-binding specificities in the allomorphs from house sparrow and great reed warbler, although these species diverged 30 MYA. This overlap was not found in a tree based on amino acid sequences. Our interpretation is that convergent evolution on the level of the protein function, possibly driven by selection from shared pathogens, has resulted in allomorphs with similar peptide-binding repertoires, although trans-species evolution in combination with gene conversion cannot be ruled out.     

Arabidopsis JAGGED LATERAL ORGANS Acts with ASYMMETRIC LEAVES2 to Coordinate KNOX and PIN Expression in Shoot and Root Meristems

Organ initiation requires the specification of a group of founder cells at the flanks of the shoot apical meristem and the creation of a functional boundary that separates the incipient primordia from the remainder of the meristem. Organ development is closely linked to the downregulation of class I KNOTTED1 LIKE HOMEOBOX (KNOX) genes and accumulation of auxin at sites of primordia initiation. Here, we show that Arabidopsis thaliana JAGGED LATERAL ORGANS (JLO), a member of the LATERAL ORGAN BOUNDARY DOMAIN (LBD) gene family, is required for coordinated organ development in shoot and floral meristems. Loss of JLO function results in ectopic expression of the KNOX genes SHOOT MERISTEMLESS and BREVIPEDICELLUS (BP), indicating that JLO acts to restrict KNOX expression. JLO acts in a trimeric protein complex with ASYMMETRIC LEAVES2 (AS2), another LBD protein, and AS1 to suppress BP expression in lateral organs. In addition to its role in KNOX regulation, we identified a role for AS2 in regulating PINFORMED (PIN) expression and auxin transport from embryogenesis onwards together with JLO. We propose that different JLO and AS2 protein complexes, possibly also comprising other LBD proteins, coordinate auxin distribution and meristem function through the regulation of KNOX and PIN expression during Arabidopsis development.