Sponge homologs of vertebrate protein tyrosine kinases and frequent domain shufflings in the early evolution of animals before the parazoan–eumetazoan split

The protein tyrosine kinases (PTKs) diverged specifically in animal lineages by gene duplications and domain shufflings to form a large protein family comprising diverse subfamilies with distinct domain organizations and functions. On the basis of a phylogenetic tree inferred from a comparison of the shared kinase domains, we previously showed that gene duplications that gave rise to diverse subfamilies predate the divergence of parazoans and eumetazoans. There is, however, still a possibility that, although the kinase domain duplications are ancient events, the domain shufflings that gave rise to different subfamilies with distinct domain organization are more recent event than the kinase domain duplications. To clarify this problem, we have determined the complete sequences of 15 sponge PTKs and have compared the domain organizations of these sponge PTKs and those of eumetazoans. For each of ten sponge PTKs out of 15 analyzed here, a possible eumetazoan (human and Drosophila) ortholog has been identified. The sponge and eumetazoan orthologs are virtually identical in domain organization and belong to the same subfamily in the PTK family tree for each of ten orthologous pairs, except for one subfamily in which a considerable deletions and/or insertions of domains are observed. This result suggests that most, if not all, of the domain shufflings, together with gene duplications, are very old, going back to dates before the parazoan–eumetazoan split, the earliest divergence among extant animal phyla.     

Kinesin-related genes from diplomonad,sponge, amphioxus,and cyclostomes: divergence pattern of kinesin family and evolution of giardial membrane-bounded organella

To understand the question of whether divergence of eukaryotic genes by gene duplications and domain shufflings proceeded gradually or intermittently during evolution, we have cloned and sequenced Giardia lamblia cDNAs encoding kinesins and kinesin-related proteins and have obtained 13 kinesin-related cDNAs, some of which are likely homologs of vertebrate kinesins involved in vesicle transfer to ER, Golgi, and plasma membrane. A phylogenetic tree of the kinesin family revealed that most gene duplications that gave rise to different kinesin subfamilies with distinct functions have been completed before the earliest divergence of extant eukaryotes. This suggests that the complex endomembrane system has arisen very early in eukaryotic evolution, and the diminutive ER and Golgi apparatus recognized in the giardial cells, together with the absence of mitochondria, might be characters acquired secondarily during the evolution of parasitism. To understand the divergence pattern of the kinesin family in the lineage leading to vertebrates, seven more Unc104-related cDNAs have been cloned from sponge, amphioxus, hagfish, and lamprey. The divergence pattern of the animal Unc104/KIF1 subfamily is characterized by two active periods in gene duplication interrupted by a considerably long period of silence, instead of proceeding gradually: animals underwent extensive gene duplications before the parazoan-eumetazoan split. In the early evolution of vertebrates around the cyclostome-gnathostome split, further gene duplications occurred, by which a variety of genes with similar structures over the entire regions were generated. This pattern of divergence is similar to those of animal genes involved in cell-cell communication and developmental control.     

Protein Tyrosine Kinase cDNAs from Amphioxus, Hagfish, and Lamprey: Isoform Duplications Around the Divergence of Cyclostomes and Gnathostomes

Animals evolved a variety of gene families involved in cell–cell communication and developmental control by gene duplication and domain shuffling. Each family is made up of several subtypes or subfamilies with distinct structures and functions, which diverged by gene duplications and domain shufflings before the divergence of parazoans and eumetazoans. Since the separation from protostomes, vertebrates expanded the multiplicity of members (isoforms) in the same subfamily by further gene duplications in their early evolution before the fish–tetrapod split. To know the dates of isoform duplications more closely, we have conducted isolation and sequencing cDNAs encoding the fibroblast growth factor receptor, Eph, src, and platelet-derived growth factor receptor subtypes belonging to the protein tyrosine kinase family from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Lampetra reissneri, a lamprey. From a phylogenetic tree of each subfamily inferred from a maximum likelihood (ML) method, together with a bootstrap analysis based on the ML method, we have shown that the isoform duplications frequently occurred in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes by gene duplications and possibly chromosomal duplications. Received: 28 April 1998 / Accepted: 30 June 1999     

Protein Tyrosine Phosphatases from Amphioxus, Hagfish, and Ray: Divergence of Tissue-Specific Isoform Genes in the Early Evolution of Vertebrates

Since separation from fungi and plants, multicellular animals evolved a variety of gene families involved in cell-cell communication from a limited number of ancestral precursors by gene duplications in two separate periods of animal evolution. In the very early evolution of animals before the separation of parazoans and eumetazoans, animals underwent extensive gene duplications by which different subtypes (subfamilies) with distinct functions diverged. The multiplicity of members (isoforms) in the same subtype increased by further gene duplications (isoform duplications) in the first half of chordate evolution before the fish-tetrapod split; different isoforms are virtually identical in structure and function but differ in tissue distribution. From cloning and phylogenetic analyses of four subfamilies of the protein tyrosine kinase (PTK) family, we recently showed extensive isoform duplications in a limited period around or just before the cyclostome-gnathostome split. To obtain a reliable estimate for the divergence time of vertebrate isoforms, we have conducted isolation of cDNAs encoding the protein tyrosine phosphatases (PTPs) from Branchiostoma belcheri, an amphioxus, Eptatretus burgeri, a hagfish, and Potamotrygon motoro, a ray. We obtained 33 different cDNAs in total, most of which belong to known PTP subfamilies. The phylogenetic analyses of five subfamilies based on the maximum likelihood method revealed frequent isoform duplications in a period around or just before the gnathostome-cyclostome split. An evolutionary implication was discussed in relation to the Cambrian explosion.     

Multiple receptor-like kinase cDNAs from liverwort Marchantia polymorpha and two charophycean green algae, Closterium ehrenbergii and Nitella axillaris: Extensive gene duplications and gene shufflings in the early evolution of streptophytes

Plant receptor-like kinases (RLKs) comprise a large family with more than several hundred members in vascular plants. The RLK family is thought to have diverged specifically in the plant kingdom, and no family member has been identified in other lineages except for animals and Plasmodium, both of which have RLK related families of small size. To know the time of divergence of RLK family members by gene duplications and domain shufflings, comprehensive isolations of RLK cDNAs were performed from a nonvascular plant, liverwort Marchantia polymorpha and two charophycean green algae, Closterium ehrenbergii, and Nitella axillaris, thought to be the closest relatives to land plants. We obtained twenty-nine, fourteen, and thirteen RLK related cDNAs from M. polymorpha, C. ehrenbergii, and N. axillaris, respectively. The amino acid sequences of these RLKs were compared with those of vascular plants, and phylogenetic trees were inferred by GAMT, a genetic algorithm-based maximum likelihood (ML) method that outputs multiple trees, together with best one. The inferred ML trees revealed ancient gene duplications generating subfamilies with different domain organizations, which occurred extensively at least before the divergence of vascular and nonvascular plants. Rather it remains possible that the extensive gene duplications occurred during the early evolution of streptophytes. Multicellular-specific somatic embryogenesis receptor kinase (SERK) involved in somatic embryogenesis was found in a unicellular alga C. ehrenbergii, suggesting the evolution of SERK by gene recruitment of a unicellular gene.     

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.     

Complex evolutionary history and diverse domain organization of SET proteins suggest divergent regulatory interactions

? Plants and animals possess very different developmental processes, yet share conserved epigenetic regulatory mechanisms, such as histone modifications. One of the most important forms of histone modification is methylation on lysine residues of the tails, carried out by members of the SET protein family, which are widespread in eukaryotes. ? We analyzed molecular evolution by comparative genomics and phylogenetics of the SET genes from plant and animal genomes, grouping SET genes into several subfamilies and uncovering numerous gene duplications, particularly in the Suv, Ash, Trx and E(z) subfamilies. ? Domain organizations differ between different subfamilies and between plant and animal SET proteins in some subfamilies, and support the grouping of SET genes into seven main subfamilies, suggesting that SET proteins have acquired distinctive regulatory interactions during evolution. We detected evidence for independent evolution of domain organization in different lineages, including recruitment of new domains following some duplications. ? More recent duplications in both vertebrates and land plants are probably the result of whole-genome or segmental duplications. The evolution of the SET gene family shows that gene duplications caused by segmental duplications and other mechanisms have probably contributed to the complexity of epigenetic regulation, providing insights into the evolution of the regulation of chromatin structure.     

Evolution of gene families and relationship with organismal evolution: rapid divergence of tissue-specific genes in the early evolution of chordates

To determine a possible relationship between organismal and molecular evolution, the divergence patterns of gene families were examined by taking special notice of functional difference, tissue distribution, and intracellular localization of the members. A phylogenetic analysis of 25 different gene families revealed interesting patterns of divergence of these families: Most gene duplications giving rise to different functions antedate the vertebrates-arthropods separation. On the other hand, in a group of members carrying virtually identical function to one another but differing in tissue distribution (tissue- specific isoform), most gene duplications have occurred independently in each of vertebrates and arthropods after the separation of the two animal groups. In family members encoding molecules localizing in cell compartments (compartmentalized isoforms), the gene duplications antedate the animals-fungi separation. In the cases of the Ca2+ pump and rab subfamilies, the compartmentalized isoforms were shown to have diverged during the early evolution of eukaryotes. A phylogenetic analysis of the tissue-specific isoforms from 26 different subfamilies revealed extensive gene duplications and rapid rates of amino acid substitutions in the early evolution of chordates before the separation of fishes and tetrapods. On the contrary, the genetic variations are relatively low in the later period. This pattern of evolution observed at the molecular level is correlated well with that of tissue evolution based on fossil evidence and morphological data, and thus evolution at the two levels may be related.      

Pattern of the divergence of olfactory receptor genes during tetrapod evolution

The olfactory receptor (OR) multigene family is responsible for the sense of smell in vertebrate species. OR genes are scattered widely in our chromosomes and constitute one of the largest gene families in eutherian genomes. Some previous studies revealed that eutherian OR genes diverged mainly during early mammalian evolution. However, the exact period when, and the ecological reason why eutherian ORs strongly diverged has remained unclear. In this study, I performed a strict data mining effort for marsupial opossum OR sequences and bootstrap analyses to estimate the periods of chromosomal migrations and gene duplications of OR genes during tetrapod evolution. The results indicate that chromosomal migrations occurred mainly during early vertebrate evolution before the monotreme-placental split, and that gene duplications occurred mainly during early mammalian evolution between the bird-mammal split and marsupial-placental split, coinciding with the reduction of opsin genes in primitive mammals. It could be thought that the previous chromosomal dispersal allowed the OR genes to subsequently expand easily, and the nocturnal adaptation of early mammals might have triggered the OR gene expansion.     

Evolution of keratin genes: Different protein domains evolve by different pathways

Summary Intermediate filaments are composed of a family of proteins that evolved from a common ancestor. The proteins consist of three domains: a central, alpha-helical domain similar in all intermediate filaments, bracketed by two domains that are variable in length and structure. Within the intermediate-filament family, several subfamilies have been recognized by immunologic and nucleic acid hybridization techniques. In this paper we present the sequence of the genomic DNA coding for a 65-kilodalton human keratin and compare it with the sequences of other intermediate-filament proteins. While the central, alpha-helical domains of these proteins show homologies that indicate a common ancestor, the sequences of the variable terminal domains indicate that the variable domains evolved through a series of tandem duplications and possibly by gene-conversion mechanisms.     

Evolutionary dynamics of leucine-rich repeat receptor-like kinases and related genes in plants：A phylogenomic approach

Leucine-rich repeat （LRR） receptor-like kinases （RLKs）, evolutionarily related LRR receptor-like proteins （RLPs） and receptor-like cytoplasmic kinases （RLCKs） have important roles in plant signaling, and their gene subfamilies are large with a complicated history of gene duplication and loss. In three pairs of closely related lineages, including Arabidopsis thaliana and A. lyrata （Arabidopsis）, Lotus japonicus, and Medicago truncatula （Legumes）, Oryza sativa ssp. japonica, and O. sativa ssp. indica （Rice）, we find that LRR RLKs comprise the largest group of these LRR-related subfamilies, while the related RLCKs represent the smal est group. In addition, comparison of orthologs indicates a high frequency of reciprocal gene loss of the LRR RLK/LRR RLP/RLCK subfamilies. Furthermore, pairwise comparisons show that reciprocal gene loss is often associated with lineage-specific duplication（s） in the alternative lineage. Last, analysis of genes in A. thaliana involved in development revealed that most are highly conserved orthologs without species-specific duplication in the two Arabidopsis species and originated from older Arabidopsis-specific or rosid-specific duplications. We discuss＆amp;nbsp;potential pitfal s related to functional prediction for genes that have undergone frequent turnover （duplications, losses, and domain architecture changes）, and conclude that prediction based on phylogenetic relationships wil likely outperform that based on sequence similarity alone.     

Promoter analysis of MADS-box genes in eudicots through phylogenetic footprinting

The MIKC MADS-box gene family has been shaped by extensive gene duplications giving rise to subfamilies of genes with distinct functions and expression patterns. However, within these subfamilies the functional assignment is not that clear-cut, and considerable functional redundancy exists. One way to investigate the diversity in regulation present in these subfamilies is promoter sequence analysis. With the advent of genome sequencing projects, we are now able to exert a comparative analysis of Arabidopsis and poplar promoters of MADS-box genes belonging to the same subfamily. Based on the principle of phylogenetic footprinting, sequences conserved between the promoters of homologous genes are thought to be functional. Here, we have investigated the evolution of MADS-box genes at the promoter level and show that many genes have diverged in their regulatory sequences after duplication and/or speciation. Furthermore, using phylogenetic footprinting, a distinction can be made between redundancy, neo/nonfunctionalization, and subfunctionalization.     

Slicing a protease: structural features of the ATP-dependent Lon proteases gleaned from investigations of isolated domains

ATP-dependent Lon proteases are multi-domain enzymes found in all living organisms. All Lon proteases contain an ATPase domain belonging to the AAA(+) superfamily of molecular machines and a proteolytic domain with a serine-lysine catalytic dyad. Lon proteases can be divided into two subfamilies, LonA and LonB, exemplified by the Escherichia coli and Archaeoglobus fulgidus paralogs, respectively. The LonA subfamily is defined by the presence of a large N-terminal domain, whereas the LonB subfamily has no such domain, but has a membrane-spanning domain that anchors the protein to the cytoplasmic side of the membrane. The two subfamilies also differ in their consensus sequences. Recent crystal structures for several individual domains and sub-fragments of Lon proteases have begun to illuminate similarities and differences in structure-function relationships between the two subfamilies. Differences in orientation of the active site residues in several isolated Lon protease domains point to possible roles for the AAA(+) domains and/or substrates in positioning the catalytic residues within the active site. Structures of the proteolytic domains have also indicated a possible hexameric arrangement of subunits in the native state of bacterial Lon proteases. The structure of a large segment of the N-terminal domain has revealed a folding motif present in other protein families of unknown function and should lead to new insights regarding ways in which Lon interacts with substrates or other cellular factors. These first glimpses of the structure of Lon are heralding an exciting new era of research on this ancient family of proteases.     

Phylogenetic dating and characterization of gene duplications in vertebrates: the cartilaginous fish reference

Vertebrates originated in the lower Cambrian. Their diversification and morphological innovations have been attributed to large-scale gene or genome duplications at the origin of the group. These duplications are predicted to have occurred in two rounds, the "2R" hypothesis, or they may have occurred in one genome duplication plus many segmental duplications, although these hypotheses are disputed. Under such models, most genes that are duplicated in all vertebrates should have originated during the same period. Previous work has shown that indeed duplications started after the speciation between vertebrates and the closest invertebrate, amphioxus, but have not set a clear ending. Consideration of chordate phylogeny immediately shows the key position of cartilaginous vertebrates (Chondrichthyes) to answer this question. Did gene duplications occur as frequently during the 45 Myr between the cartilaginous/bony vertebrate split and the fish/tetrapode split as in the previous approximately 100 Myr? Although the time interval is relatively short, it is crucial to understanding the events at the origin of vertebrates. By a systematic appraisal of gene phylogenies, we show that significantly more duplications occurred before than after the cartilaginous/bony vertebrate split. Our results support rounds of gene or genome duplications during a limited period of early vertebrate evolution and allow a better characterization of these events.     

The Evolution of the MAP Kinase Pathways: Coduplication of Interacting Proteins Leads to New Signaling Cascades

The MAP-kinase pathways are intracellular signaling modules that are likely to exist in all eukaryotes. We provide an evolutionary model for these signaling pathways by focusing on the gene duplications that have occurred since the divergence of animals from yeast. Construction of evolutionary trees with confidence assessed by bootstrap clearly shows that the mammalian JNK and p38 pathways arose from an ancestral hyperosmolarity pathway after the split from yeast and before the split from C. elegans. These coduplications of interacting proteins at the MAPK and MEK levels have since evolved toward substrate specificity, thus giving distinct pathways. Mammalian duplications since the split from C. elegans are often associated with divergent tissue distribution but do not appear to confer detectable substrate specificity. The yeast kinase cascades have undergone similar fundamental functional changes since the split from mammals, with duplications giving rise to central signaling components of the filamentous and hypoosmolarity pathways. Experimentally defined cross-talk between yeast pheromone and hyperosmolarity pathways is mirrored with corresponding cross-talk in mammalian pathways, suggesting the existence of ancient orthologous cross-talk; our analysis of gene duplications at all levels of the cascade is consistent with this model but does not always provide significant bootstrap support. Our data also provide insights at different levels of the cascade where conflicting experimental evidence exists. Received: 2 December 1998 / Accepted: 9 June 1999     

Expansion of the receptor-like kinase/Pelle gene family and receptor-like proteins in Arabidopsis

Receptor-like kinases (RLKs) are a family of transmembrane proteins with versatile N-terminal extracellular domains and C-terminal intracellular kinases. They control a wide range of physiological responses in plants and belong to one of the largest gene families in the Arabidopsis genome with more than 600 members. Interestingly, this gene family constitutes 60% of all kinases in Arabidopsis and accounts for nearly all transmembrane kinases in Arabidopsis. Analysis of four fungal, six metazoan, and two Plasmodium sp. genomes indicates that the family was represented in all but fungal genomes, indicating an ancient origin for the family with a more recent expansion only in the plant lineages. The RLK/Pelle family can be divided into several subfamilies based on three independent criteria: the phylogeny based on kinase domain sequences, the extracellular domain identities, and intron locations and phases. A large number of receptor-like proteins (RLPs) resembling the extracellular domains of RLKs are also found in the Arabidopsis genome. However, not all RLK subfamilies have corresponding RLPs. Several RLK/Pelle subfamilies have undergone differential expansions. More than 33% of the RLK/Pelle members are found in tandem clusters, substantially higher than the genome average. In addition, 470 of the RLK/Pelle family members are located within the segmentally duplicated regions in the Arabidopsis genome and 268 of them have a close relative in the corresponding regions. Therefore, tandem duplications and segmental/whole-genome duplications represent two of the major mechanisms for the expansion of the RLK/Pelle family in Arabidopsis.