Tandem Repetitive D Domains of the Sperm Ligand Zonadhesin Evolve Faster in the Paralogue Than in the Orthologue Comparison

Gene duplication is regarded as an important evolutionary mechanism creating genetic and phenotypic novelty. At the same time, the evolutionary mechanisms following gene duplication have been a subject of much debate. Here we analyze the sequence evolution of zonadhesin, a mammalian sperm ligand that binds to the oocyte zona pellucida in a species-specific manner. In pig, rabbit, and primates, precursor zonadhesin comprises, among others, one partial and four complete tandem repetitive D domains. The mouse precursor is distinguished by 20 additional partial D3 domains consisting of 120 amino acids each. This gene structure allows sequence comparison in both paralogues and orthologues. Detailed sequence analysis reveals that D domains evolve faster across paralogues than orthologues. Moreover, at the codon level, partial D3 paralogues of mouse show evidence of positive selection, whereas the corresponding orthologues do not. Individual posttranslational motif patterns and positive selection point to neofunctionalization of partial D3 paralogues of mouse, rather than subfunctionalization. However, as we found additional evidence for homogenization by partial gene conversion, sequence evolution of partial D3 paralogues of mouse might be better described as a combination of divergent and convergent evolution. So far, the divergence at the codon level has outbalanced the convergence at the level of smaller fragments. The probable driving force behind the evolutionary patterns observed is sexual selection. We finally discuss whether the functional determination influences the evolutionary regime acting on sperm ligands and egg receptors, respectively. [Reviewing Editor: Dr. Yves Van de Peer]     

Pattern of divergence of amino acid sequences encoded by paralogous genes in human and pufferfish

We used phylogenetic analyses of protein families containing two or more pairs of orthologues in the genomes of human and pufferfish (Takifugu rubripes) to test the hypothesis that these sequences show a strong signal of polyploidization events hypothesized to have occurred early in vertebrate history. In order to test for evidence of two distinct rounds of polyploidization (the 2R hypothesis), we compared the pattern of amino acid sequence divergence of proteins encoded by genes duplicated just prior to the most recent common ancestor of human and pufferfish with that of proteins encoded genes duplicated earlier. These sequence divergences were statistically indistinguishable, contrary to the prediction of the 2R hypothesis. The variance of amino acid sequence divergences between paralogues was significantly greater than expected from that of orthologues in the same families. Estimation of gene duplication times assuming a molecular clock provided earlier estimates than expected, suggesting that it may not be appropriate to time the duplication of paralogues using rate estimates derived from orthologous comparisons. Overall, the results indicate that amino acid sequences do not provide a strong signal supporting the hypothesis that gene duplications early in vertebrate history occurred by polyploidization. On the other hand, the data are easily explained under an alternative model that gene duplications occurred at different times in different vertebrate gene families.     

Identifying concerted evolution and gene conversion in mammalian gene pairs lasting over 100 million years

Background  

Concerted evolution occurs in multigene families and is characterized by stretches of homogeneity and higher sequence similarity between paralogues than between orthologues. Here we identify human gene pairs that have undergone concerted evolution, caused by ongoing gene conversion, since at least the human-mouse divergence. Our strategy involved the identification of duplicated genes with greater similarity within a species than between species. These genes were required to be present in multiple mammalian genomes, suggesting duplication early in mammalian divergence. To eliminate genes that have been conserved due to strong purifying selection, our analysis also required at least one intron to have retained high sequence similarity between paralogues.     

Evolution of thesrc-related protein tyrosine kinases

A phylogenetic analysis ofsrc-related protein tyrosine kinases (PTKs) showed that one group of these genes is quite ancient in the animals, its divergence predating the divergence of the diploblast and triploblast phyla. Three other major groupings of genes were found to predate the divergence of protostome and deuterostome phyla. Most knownsrc-related PTKs of mammals were found to belong to five well-differentiated families: srcA, srcB, abl, csk, and tec. One srcA gene (fyn) has an alternatively spliced seventh exon which shows a different pattern of relationship from the remainder of the gene; this suggests that this exon may have been derived by a recombinational event with another gene, perhaps one related tofgr. The recently published claim that mammalian members of this family expressed in the nervous system evolve more slowly at nonsynonymous nucleotide sites than do those expressed in the immune system was not supported by an analysis of 13 pairs of human and mouse orthologues. Rather, T-cell-specificsrc-related PTKs were found to have higher rates of nonsynonymous substitution than were those having broader expression. This effect was particularly marked in the peptide binding site of the SH2 domain. While the SH2 binding site was highly conserved among paralogous mammalian members of the srcA and srcB subfamilies, no such effect was seen in the comparison of paralogous members of the csk and tec subfamilies. This suggests that, while the peptide binding function of SH2 is conserved within both srcA and srcB subfamilies, paralogous members of the csk and tec subfamilies have diverged functionally with respect to peptide recognition by SH2.     

The animal sialyltransferases and sialyltransferase-related genes: a phylogenetic approach

The animal sialyltransferases are Golgi type II transmembrane glycosyltransferases. Twenty distinct sialyltransferases have been identified in both human and murine genomes. These enzymes catalyze transfer of sialic acid from CMP-Neu5Ac to the glycan moiety of glycoconjugates. Despite low overall identities, they share four conserved peptide motifs [L (large), S (small), motif III, and motif VS (very small)] that are hallmarks for sialyltransferase identification. We have identified 155 new putative genes in 25 animal species, and we have exploited two lines of evidence: (1) sequence comparisons and (2) exon-intron organization of the genes. An ortholog to the ancestor present before the split of ST6Gal I and II subfamilies was detected in arthropods. An ortholog to the ancestor present before the split of ST6GalNAc III, IV, V, and VI subfamilies was detected in sea urchin. An ortholog to the ancestor present before the split of ST3Gal I and II subfamilies was detected in ciona, and an ortholog to the ancestor of all the ST8Sia was detected in amphioxus. Therefore, single examples of the four families (ST3Gal, ST6Gal, ST6GalNAc, and ST8Sia) have appeared in invertebrates, earlier than previously thought, whereas the four families were all detected in bony fishes, amphibians, birds, and mammals. As previously hypothesized, sequence similarities among sialyltransferases suggest a common genetic origin, by successive duplications of an ancestral gene, followed by divergent evolution. Finally, we propose predictions on these invertebrates sialyltransferase-related activities that have not previously been demonstrated and that will ultimately need to be substantiated by protein expression and enzymatic activity assays.     

Computational identification and systematic analysis of the ACR gene family in Oryza sativa

Based on sequence similarity search and domain detection, nine ACT domain repeat protein-coding genes (the "ACR" genes) in rice were identified, which were mainly distributed on the chromosomes 2, 3, 4, and 8. An InterPro database search indicated that four copies of the ACT domain linearly occupied the entire polypeptide. The first three ACT domains were linked by two different sequences. However, the fourth ACT domain was extremely close to ACT3. Gene structure comparisons showed large differences in exon numbers, from three to eight, among members of the rice ACR gene family. In addition, it appeared that gene duplication might be operative when the compositions of exons and introns were analyzed. Phylogenetic analysis divided the ACR gene family into five distinct groups, and this division was generally according to the expression patterns of the ACR genes. The Arabidopsis and rice ACR proteins were clustered across together, suggesting that these ACR genes might originate from an ancient common ancestor. Notably, the identification of orthologues and paralogues would be useful for rice gene functional annotation.     

Phylogenetic analysis of the Wnt gene family and discovery of an arthropod wnt-10 orthologue

Wnt genes encode a conserved family of secreted signaling proteins that play many roles in arthropod and vertebrate development. We have investigated both the phylogenetic history and molecular evolution of this gene family. We have identified a novel Wnt gene in a diversity of arthropods that it is likely an orthologue of the vertebrate Wnt-10 group. Wnt-10 is one of only two cases in which orthology between protostome and deuterostome genes could be consistently assigned based on our analyses. Despite difficulties in assessing orthologies, all of our trees suggest that the most recent common ancestor of protostomes and deuterostomes possessed more than the five Wnt genes known from either arthropods or nematodes. This suggests that Wnt gene loss has occurred during protostome evolution. In addition, we examined the rate of amino acid evolution in the two arthropod/deuterostome orthology groups we identified. We found little rate variation across taxa, with the exception that Drosophila Wnt-1 is evolving more rapidly than all vertebrate and most arthropod orthologues.     

DEVELOPMENTAL DECOUPLING OF ALTERNATIVE PHENOTYPES: INSIGHTS FROM THE TRANSCRIPTOMES OF HORN‐POLYPHENIC BEETLES

Developmental mechanisms play an important role in determining the costs, limits, and evolutionary consequences of phenotypic plasticity. One issue central to these claims is the hypothesis of developmental decoupling, where alternate morphs result from evolutionarily independent developmental pathways. We address this assumption through a microarray study that tests whether differences in gene expression between alternate morphs are as divergent as those between sexes, a classic example of developmental decoupling. We then examine whether genes with morph‐biased expression are less conserved than genes with shared expression between morphs, as predicted if developmental decoupling relaxes pleiotropic constraints on divergence. We focus on the developing horns and brains of two species of horned beetles with impressive sexual‐ and morph‐dimorphism in the expression of horns and fighting behavior. We find that patterns of gene expression were as divergent between morphs as they were between sexes. However, overall patterns of gene expression were also highly correlated across morphs and sexes. Morph‐biased genes were more evolutionarily divergent, suggesting a role of relaxed pleiotropic constraints or relaxed selection. Together these results suggest that alternate morphs are to some extent developmentally decoupled, and that this decoupling has significant evolutionary consequences. However, alternative morphs may not be as developmentally decoupled as sometimes assumed and such hypotheses of development should be revisited and refined.     

A dynamic history of gene duplications and losses characterizes the evolution of the SPARC family in eumetazoans

The vertebrates share the ability to produce a skeleton made of mineralized extracellular matrix. However, our understanding of the molecular changes that accompanied their emergence remains scarce. Here, we describe the evolutionary history of the SPARC (secreted protein acidic and rich in cysteine) family, because its vertebrate orthologues are expressed in cartilage, bones and teeth where they have been proposed to bind calcium and act as extracellular collagen chaperones, and because further duplications of specific SPARC members produced the small calcium-binding phosphoproteins (SCPP) family that is crucial for skeletal mineralization to occur. Both phylogeny and synteny conservation analyses reveal that, in the eumetazoan ancestor, a unique ancestral gene duplicated to give rise to SPARC and SPARCB described here for the first time. Independent losses have eliminated one of the two paralogues in cnidarians, protostomes and tetrapods. Hence, only non-tetrapod deuterostomes have conserved both genes. Remarkably, SPARC and SPARCB paralogues are still linked in the amphioxus genome. To shed light on the evolution of the SPARC family members in chordates, we performed a comprehensive analysis of their embryonic expression patterns in amphioxus, tunicates, teleosts, amphibians and mammals. Our results show that in the chordate lineage SPARC and SPARCB family members were recurrently recruited in a variety of unrelated tissues expressing collagen genes. We propose that one of the earliest steps of skeletal evolution involved the co-expression of SPARC paralogues with collagenous proteins.     

Structure and expression of conserved Wnt pathway components in the demosponge Amphimedon queenslandica

Wnt-signalling plays a critical role in animal development, and its misregulation results in serious human diseases, including cancer. While the Wnt pathway is well studied in eumetazoan models, little is known about the evolutionary origin of its components and their functions. Here, we have identified key machinery of the Wnt-β-catenin (canonical)-signalling pathway that is encoded in the Amphimedon queenslandica (Demospongiae; Porifera) genome, namely Wnt, Fzd, SFRP, Lrp5/6, Dvl, Axin, APC, GSK3, β-catenin, Tcf, and Groucho. Most of these genes are not detected in the choanoflagellate and other nonmetazoan eukaryotic genomes. In contrast, orthologues of some of key components of bilaterian Wnt-planar cell polarity and Wnt/Ca(2+) are absent from the Amphimedon genome, suggesting these pathways evolved after demosponge and eumetazoan lineages diverged. Sequence analysis of the identified proteins of the Wnt-β-catenin pathway has revealed the presence of most of the conserved motifs and domains responsible for protein-protein and protein-DNA interactions in vertebrates and insects. However, several protein-protein interaction domains appear to be absent from the Amphimedon Axin and APC proteins. These are also missing from their orthologues in the cnidarian Nematostella vectensis, suggesting that they are bilaterian novelties. All of the analyzed Wnt pathway genes are expressed in specific patterns during Amphimedon embryogenesis. Most are expressed in especially striking and highly dynamic patterns during formation of a simple organ-like larval structure, the pigment ring. Overall, our results indicate that the Wnt-β-catenin pathway was used in embryonic patterning in the last common ancestor of living metazoans. Subsequently, gene duplications and a possible increase in complexity of protein interactions have resulted in the precisely regulated Wnt pathway observed in extant bilaterian animals.     

Mouse homologues of Shisa antagonistic to Wnt and Fgf signalings

In an effort to identify Otx2 targets in mouse anterior neuroectoderm we identified a gene, mShisa, which is homologous to xShisa1 that we previously reported as a head inducer in Xenopus. mShisa encodes an antagonist against both Wnt and Fgf signalings; it inhibits these signalings cell-autonomously as xShisa1 does. The mShisa expression is lost or greatly reduced in Otx2 mutant visceral endoderm, anterior mesendoderm and anterior neuroectoderm. However, mShisa mutants exhibited no defects in head development. Shisa is composed of five subfamilies, but normal head development in mShisa mutants is unlikely to be explained in terms of the compensation of mShisa deficiency by its paralogues or by known Wnt antagonists in anterior visceral endoderm and/or anterior mesendoderm.     

Divergence in spatial expression patterns and in response to stimuli of tandem-repeat paralogues encoding a novel class of proline-rich proteins in Oryza sativa

Gene duplication has been recognized as a major route to supply raw sequences for genome novelty in evolution, but the mechanism underlying the retention of duplicate genes is not fully understood yet. Divergence in spatial expression patterns was investigated here and in response to stimuli of the four members of a rice proline-rich protein gene family (OsPRP1), which encode a class of proline-rich proteins. The four paralogues are tandemly organized within a 20 kbp range of chromosome 10 without any interval of other open reading frames and with a median K(S) value of 0.474. These paralogues showed little similarity in their regulatory regions but high conservation in coding regions. Search of an intergenomic cis-element database predicted their promoter regions with divergent cis-element fingerprints. Further expression analyses involving different tissues/organs and nine types of stimuli by a promoter::GUS-fusion strategy revealed that the four paralogues were expressed mainly in vascular cylinders of different organs and showed diversity in tissue/organ specificity and in response to these stimuli, with some overlapping expression. Furthermore, these data show that OsPRP1.2 appeared to inherit most of the functions from their multifunctional progenitor, whereas the other three genes diverged after duplication events. Thus, the retention of paralogues in a multigene family seems to require a more complicated diversification process than originally thought. In addition, the promoter::GUS strategy is a powerful way to explore function divergence of a tandem-repeat gene family.     

Comparison of the gap segmentation gene hunchback between Drosophila melanogaster and Drosophila virilis reveals novel modes of evolutionary change.

We have cloned and sequenced a large portion of the hunchback (hb) locus from Drosophila virilis. Comparison with the Drosophila melanogaster hb sequence shows multiple strong homologies in the upstream and downstream regions of the gene, including most of the known functional parts. The coding sequence is highly conserved within the presumptive DNA-binding finger regions, but more diverged outside of them. The regions of high divergence are correlated with regions which are rich in short direct repeats (regions of high 'cryptic simplicity'), suggesting a significant influence of slippage-like mechanisms in the evolutionary divergence of the two genes. Staining of early D.virilis embryos with an hb antibody reveals conserved and divergent features of the spatial expression pattern at blastoderm stage. It appears that the basic expression pattern, which serves as the gap gene function of hb, is conserved, while certain secondary expression patterns, which have separate functions for the segmentation process, are partly diverged. Thus, both slippage driven mutations in the coding region, which are likely to occur at higher rates than point mutations and the evolutionary divergence of secondary expression patterns may contribute to the evolution of regulatory genes.     

Analyses of synteny between Arabidopsis thaliana and species in the Asteraceae reveal a complex network of small syntenic segments and major chromosomal rearrangements

Comparative genomic studies among highly divergent species have been problematic because reduced gene similarities make orthologous gene pairs difficult to identify and because colinearity is expected to be low with greater time since divergence from the last common ancestor. Nevertheless, synteny between divergent taxa in several lineages has been detected over short chromosomal segments. We have examined the level of synteny between the model species Arabidopsis thaliana and species in the Compositae, one of the largest and most diverse plant families. While macrosyntenic patterns covering large segments of the chromosomes are not evident, significant levels of local synteny are detected at a fine scale covering segments of 1-Mb regions of A. thaliana and regions of <5 cM in lettuce and sunflower. These syntenic patches are often not colinear, however, and form a network of regions that have likely evolved by duplications followed by differential gene loss.     

Genomic organisation, embryonic expression and biochemical interactions of the zebrafish junctional adhesion molecule family of receptors

The mammalian JAM family is composed of three cell surface receptors. Interactions between the proteins have well-characterised roles in inflammation and tight junction formation, but little is known about their function in early development. Recently, we identified a role for jamb and jamc in zebrafish myocyte fusion. Genome duplication in the teleost lineage raised the possibility that additional JAM family paralogues may also function in muscle development. To address this, we searched the zebrafish genome to identify potential paralogues and confirmed their homology, bringing the total number of zebrafish jam family members to six. We then compared the physical binding properties of each paralogue by surface plasmon resonance and determined the gene expression patterns of all zebrafish jam genes at different stages of development. Our results suggest a significant sub-functionalisation of JAM-B and JAM-C orthologues with respect to binding strength (but not specificity) and gene expression. The paralogous genes, jamb2 and jamc2, were not detected in the somites or myotome of wild-type embryos. We conclude that it is unlikely that the paralogues have a function in primary myogenesis.