Orthologous nature of mammaliam insulin genes

Summary Investigation of 7 insulin sequences from a bony fish, a bird, and 5 mammalian species showed that guinea pig and coypu insulin, that have a strongly divergent primary and quarternary structure, are not the result of gene duplication in an ancient vertebrate or invertebrate ancestor but that they diverged from the other mammals after divergence of the mammals from the other vertebrates. After this divergence both insulins underwent evolution at a highly increased rate.     

Phylogenetic analysis of vertebrate and invertebrate Delta/Serrate/LAG-2 (DSL) proteins

Delta/Serrate/LAG-2 (DSL) proteins are putative transmembrane signaling molecules that regulate cell differentiation in metazoans. DSL proteins are characterized by the presence of a motif unique to these proteins, the DSL motif, and a variable number of tandemly repeated copies of an epidermal growth factor-like (EGF) motif. We have completed a phylogenetic analysis of 15 DSL proteins from eight species. Our findings reveal that at least one gene duplication occurred prior to the divergence of the Drosophila melanogaster and vertebrate lineages, with subsequent duplications in vertebrates. The three known Caenorhabditis elegans proteins likely arose by two independent duplications in the nematode lineage. Analysis of EGF repeats suggests that EGF 2 has been conserved among DSL proteins in vertebrates and D. melanogaster. The sequences of two EGF repeats have been perfectly conserved in vertebrate orthologs: EGF 2 in Delta and EGF 15 in Jagged/Serrate. Finally, the linear order of EGF repeats has been conserved in the vertebrate Jagged/Serrate orthologs and vertebrate Delta orthologs.     

Evolution of the proteasome components

 A phylogenetic analysis of proteasome subunits revealed two major families (α and β) which originated by an ancient gene duplication prior to the divergence of archaebacteria and eukaryotes. Numerous gene duplications have subsequently occurred in eukaryotes; at least nine of these duplications were shown to have occurred prior to the divergence of animals and fungi. In mammals, two genes encoding proteasome subunits (LMP2 and LMP7) are located in the major histocompatibility complex (MHC) region and play a specific role in generation of peptides for presentation by class I MHC molecules. Phylogenetic analysis of LMP7 and related sequences from mammals and lower vertebrates indicated that this locus arose by gene duplication prior to the divergence of jawed and jawless vertebrates; the time of this duplication was estimated to have been about 600 million years ago. The evolutionary history of the proteasome subunits provides support for a model of the evolution of new gene function postulating that, after gene duplication, the proteins encoded by daughter loci can adapt to specialized functions previously performed by the product of a single generalized ancestral locus. Received: 19 August 1996 / Revised: 24 December 1996     

Gene duplication events producing muscle (M) and brain (B) isoforms of cytoplasmic creatine kinase: cDNA and deduced amino acid sequences from two lower chordates.

Creatine kinase (CK) is coded for by at least four loci in higher vertebrates--two cytoplasmic isoforms, muscle (M) and brain (B), and two mitochondrial isoforms, sarcomeric and ubiquitous. M is expressed primarily in skeletal muscle, while B is expressed in a variety of cells, including cardiac and smooth muscle fibers, neurons, transport epithelia, and photoreceptors. M and B subunits form very stable homodimers (MM [M-CK], BB [B-CK]) and heterodimers (MB). M-CK is capable of binding to the M line of the myofibril, thereby creating an energy transfer microcompartment; BB and MB CKs are not. M- and B-like CKs are present in all vertebrates yet examined, including fish. Cytoplasmic, dimeric CKs are widely distributed in the invertebrates. The only available amino acid sequence for an invertebrate dimeric CK, that of the protostome polychaete Chaetopterus variopedatus, is just as similar to the vertebrate M isoform as to the B isoform. Echinoderms lack dimeric, cytoplasmic CKs, which appear to be replaced by a dimeric arginine kinase which evolved secondarily from CK. Thus, it is likely that the gene duplication event producing the M and B isoforms occurred after the divergence of the chordates from echinoderms. To narrow down the timing of this duplication event, we obtained the cDNA and deduced amino acid sequences of dimeric CKs from the tunicate Ciona intestinalis (subphylum Urochordata) and the lancelet Branchiostoma floridae (subphylum Cephalochordata). Our results show that these CKs are strikingly similar to both invertebrate and vertebrate CKs. However, phylogenetic analyses by neighbor-joining and parsimony show that these two enzymes appeared to have diverged before the point of divergence of the M and B isoforms. Thus, the gene duplication event for formation of the muscle and brain isoforms of CK most likely occurred during the radiation of the fish, a time noted for gene duplication events at a variety of other loci.     

Evolutionary Dynamics of the Interferon-Induced Transmembrane Gene Family in Vertebrates

Vertebrate interferon-induced transmembrane (IFITM) genes have been demonstrated to have extensive and diverse functions, playing important roles in the evolution of vertebrates. Despite observance of their functionality, the evolutionary dynamics of this gene family are complex and currently unknown. Here, we performed detailed evolutionary analyses to unravel the evolutionary history of the vertebrate IFITM family. A total of 174 IFITM orthologous genes and 112 pseudogenes were identified from 27 vertebrate genome sequences. The vertebrate IFITM family can be divided into immunity-related IFITM (IR-IFITM), IFITM5 and IFITM10 sub-families in phylogeny, implying origins from three different progenitors. In general, vertebrate IFITM genes are located in two loci, one containing the IFITM10 gene, and the other locus containing IFITM5 and various numbers of IR-IFITM genes. Conservation of evolutionary synteny was observed in these IFITM genes. Significant functional divergence was detected among the three IFITM sub-families. No gene duplication or positive selection was found in IFITM5 sub-family, implying the functional conservation of IFITM5 in vertebrate evolution, which is involved in bone formation. No IFITM5 locus was identified in the marmoset genome, suggesting a potential association with the tiny size of this monkey. The IFITM10 sub-family was divided into two groups: aquatic and terrestrial types. Functional divergence was detected between the two groups, and five IFITM10-like genes from frog were dispersed into the two groups. Both gene duplication and positive selection were observed in aquatic vertebrate IFITM10-like genes, indicating that IFITM10 might be associated with the adaptation to aquatic environments. A large number of lineage- and species-specific gene duplications were observed in IR-IFITM sub-family and positive selection was detected in IR-IFITM of primates and rodents. Because primates have experienced a long history of viral infection, such rapid expansion and positive selection suggests that the evolution of primate IR-IFITM genes is associated with broad-spectrum antiviral activity.     

Evolutionary history of the enolase gene family

The enzyme enolase [EC 4.2.1.11] is found in all organisms, with vertebrates exhibiting tissue-specific isozymes encoded by three genes: alpha (alpha), beta (beta), and gamma (gamma) enolase. Limited taxonomic sampling of enolase has obscured the timing of gene duplication events. To help clarify the evolutionary history of the gene family, cDNAs were sequenced from six taxa representing major lineages of vertebrates: Chiloscyllium punctatum (shark), Amia calva (bowfin), Salmo trutta (trout), Latimeria chalumnae (coelacanth), Lepidosiren paradoxa (South American lungfish), and Neoceratodus forsteri (Australian lungfish). Phylogenetic analysis of all enolase and related gene sequences revealed an early gene duplication event prior to the last common ancestor of living organisms. Several distantly related archaebacterial sequences were designated as 'enolase-2', whereas all other enolase sequences were designated 'enolase-1'. Two of the three isozymes of enolase-1, alpha- and beta-enolase, were discovered in actinopterygian, sarcopterygian, and chondrichthian fishes. Phylogenetic analysis of vertebrate enolases revealed that the two gene duplications leading to the three isozymes of enolase-1 occurred subsequent to the divergence of living agnathans, near the Proterozoic/Phanerozoic boundary (approximately 550Mya). Two copies of enolase, designated alpha(1) and alpha(2), were found in the trout and are presumed to be the result of a genome duplication event.     

A genetic model for the evolution of the glycosphingolipids

The glycosphingolipids have been found in many animal tissues, but the complexity of their molecular structure varies considerably among the different phyla. Relatively simple structures have been found in invertebrate species, while the most complex have been demonstrated in brain tissue of modern fishes and amphibians. The data on the phylogenetic distribution of the glycosphingolipids has been interpreted to indicate that a significant number of gene duplications, involving many different structural genes, may have occurred during a few specific periods of vertebrate evolution. The transition from invertebrate to jawless vertebrate, the divergence of rays and skates from true sharks, the advent of modern bony fishes and the transition from aquatic to terrestrial vertebrates, each could have veen accompained by duplications of genes involved in the synthesis and degradation of glycosphingolipids. The evolutionary study of such a multi-enzyme system may be one means to detect alterations in the genome as a whole. The apparent correspondence in time of these gene duplications involved in glycosphingolipid metabolism and periods of rapid vertebrate evolution which may have been accompanied by significant increases in the amount of cellular DNA suggests that such changes may have occurred via the mechanism of tetraploidization.     

Some aspects of isozymes of lactate dehydrogenase, malate dehydrogenase and glucosephosphate isomerase in fish

1. The present paper reports some aspects of the isozymes of LDH, MDH and GPI in fish. 2. In Petromyzontiformes LDH is encoded by a single Ldh-A gene locus. In Myxiniformes and in most vertebrates LDH is encoded by two gene loci, A and B. A third Ldh-C locus is characteristic of the bony fishes Actinopterygii. 3. In fish the MDH isozymes are generally encoded by three gene loci Mdh-M, Mdh-A and Mdh-B. 4. In most diploid bony fish the GPI is controlled by two independent gene loci Gpi-A and Gpi-B. 5. The relationships of isozymes with evolution of vertebrates, tissual specificity, ontogenetic changes, with physiological and metabolic roles are discussed.     

Hox cluster organization in the jawless vertebrate Petromyzon marinus

Large-scale gene amplifications may have facilitated the evolution of morphological innovations that accompanied the origin of vertebrates. This hypothesis predicts that the genomes of extant jawless fish, scions of deeply branching vertebrate lineages, should bear a record of these events. Previous work suggests that nonvertebrate chordates have a single Hox cluster, but that gnathostome vertebrates have four or more Hox clusters. Did the duplication events that produced multiple vertebrate Hox clusters occur before or after the divergence of agnathan and gnathostome lineages? Can investigation of lamprey Hox clusters illuminate the origins of the four gnathostome Hox clusters? To approach these questions, we cloned and sequenced 13 Hox cluster genes from cDNA and genomic libraries in the lamprey, Petromyzon marinus. The results suggest that the lamprey has at least four Hox clusters and support the model that gnathostome Hox clusters arose by a two-round-no-cluster-loss mechanism, with tree topology [(AB)(CD)]. A three-round model, however, is not rigorously excluded by the data and, for this model, the tree topologies [(D(C(AB))] and [(C(D(AB))] are most parsimonious. Gene phylogenies suggest that at least one Hox cluster duplication occurred in the lamprey lineage after it diverged from the gnathostome lineage. The results argue against two or more rounds of duplication before the divergence of agnathan and gnathostome vertebrates. If Hox clusters were duplicated in whole-genome duplication events, then these data suggest that, at most, one whole genome duplication occurred before the evolution of vertebrate developmental innovations.     

Evolutionary implications of lactate dehydrogenases (LDHs) of hagfishes compared to lampreys: LDH cDNA sequences from Eptatretus burgeri, Paramyxine atami and Eptatretus okinoseanus

Heart muscles of hagfishes Paramyxine atami and Eptatretus okinoseanus express the B4 isozyme of lactate dehydrogenase [L-LDH: NAD oxidoreductase, EC 1.1.1.27] (LDH-B4) whereas their skeletal muscles express LDH-A4. To examine the relationship of hagfish LDHs to lamprey and other vertebrate LDHs, we determined the cDNA sequences of LDH-A from three hagfishes and compared them with previously published sequences. A phylogenic tree shows that hagfishes diverged just after lampreys. The deduced amino acid sequences showed ten regions common to all vertebrate LDHs examined, i.e., the active site, the pocket recognizing the substrate-coenzyme complex, part of a loop at the surface, and the substrate binding site. The cyclostomate-specific regions (S1, S2) were located in the neighborhood of the active site loop. Three regions, IGS1, IGS2 and IGS3, seem to have altered their structures during the differentiation of LDH isozymes, and the regions remain in LDH-B of vertebrates hitherto examined. IGS2 and IGS3, which are in the neighborhood of the active site, may regulate catalytic activity. There were differences in six amino acid residues (6, 10, 20, 156, 269, and 341) in LDHs of hagfishes. These differences might reflect the tolerance to high pressure and low temperature of LDHs from hagfishes at different habitat depths.     

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.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. MHC Class I genes in the Tuatara (Sphenodon spp.): evolution of the MHC in an ancient reptilian order

The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.     

Selection on coding regions determined Hox7 genes evolution

The important role of Hox genes in determining the regionalization of the body plan of the vertebrates makes them invaluable candidates for evolutionary analyses regarding functional and morphological innovation. Gene duplication and gene loss led to a variable number of Hox genes in different vertebrate lineages. The evolutionary forces determining the conservation or loss of Hox genes are poorly understood. In this study, we show that variable selective pressures acted on Hox7 genes in different evolutionary lineages, with episodes of positive selection occurring after gene duplications. Tests for functional divergence in paralogs detected significant differentiation in a region known to modulate HOX7 protein activity. Our results show that both positive and negative selection on coding regions are influencing Hox7 genes evolution.     

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.     

Timing of genome duplications relative to the origin of the vertebrates: did cyclostomes diverge before or after?

Two rounds of whole-genome duplications are thought to haveplayed an important role in the establishment of gene repertoiresin vertebrates. These events occurred during chordate evolutionafter the split of the urochordate and cephalochordate lineagesbut before the radiation of extant gnathostomes (jawed vertebrates).During this interval, diverse agnathans (jawless fishes), includingcyclostomes (hagfishes and lampreys), diverged. However, thereis no solid evidence for the timing of these genome duplicationsin relation to the divergence of cyclostomes from the gnathostomelineage. We conducted cDNA sequencing in diverse early vertebratesfor members of homeobox-containing (Dlx and ParaHox) and othergene families that would serve as landmarks for genome duplications.Including these new sequences, we performed a molecular phylogeneticcensus using the maximum likelihood method for 55 gene families.In most of these gene families, we detected many more gene duplicationsbefore the cyclostome–gnathostome split, than after. Manyof these gene families (e.g., visual opsins, RAR, Notch) havemultiple paralogs in conserved, syntenic genomic regions thatmust have been generated by large-scale duplication events.Taken together, this indicates that the genome duplicationsoccurred before the cyclostome–gnathostome split. We proposethat the redundancy in gene repertoires possessed by all vertebrates,including hagfishes and lampreys, was introduced primarily bygenome duplications. Apart from subsequent lineage-specificmodifications, these ancient genome duplication events mightserve generally to distinguish vertebrates from invertebratesat the genomic level.     

Phylogenetic analyses alone are insufficient to determine whether genome duplication(s) occurred during early vertebrate evolution

The widely accepted notion that two whole-genome duplications occurred during early vertebrate evolution (the 2R hypothesis) stems from the fact that vertebrates often possess several genes corresponding to a single invertebrate homolog. However the number of genes predicted by the Human Genome Project is less than twice as many as in the Drosophila melanogaster or Caenorhabditis elegans genomes. This ratio could be explained by two rounds of genome duplication followed by extensive gene loss, by a single genome duplication, by sequential local duplications, or by a combination of any of the above. The traditional method used to distinguish between these possibilities is to reconstruct the phylogenetic relationships of vertebrate genes to their invertebrate orthologs; ratios of invertebrate-to-vertebrate counterparts are then used to infer the number of gene duplication events. The lancelet, amphioxus, is the closest living invertebrate relative of the vertebrates, and unlike protostomes such as flies or nematodes, is therefore the most appropriate outgroup for understanding the genomic composition of the last common ancestor of all vertebrates. We analyzed the relationships of all available amphioxus genes to their vertebrate homologs. In most cases, one to three vertebrate genes are orthologous to each amphioxus gene (median number=2). Clearly this result, and those of previous studies using this approach, cannot distinguish between alternative scenarios of chordate genome expansion. We conclude that phylogenetic analyses alone will never be sufficient to determine whether genome duplication(s) occurred during early chordate evolution, and argue that a "phylogenomic" approach, which compares paralogous clusters of linked genes from complete amphioxus and human genome sequences, will be required if the pattern and process of early chordate genome evolution is ever to be reconstructed.     

Birth-and-death evolution in primate MHC class I genes: divergence time estimates

The major histocompatibility complex (MHC) is a multigene family that mediates the host immune response by helping T lymphocytes to recognize and respond to foreign antigens. The high degree of polymorphism and a quick turnover of the genetic loci make the evolution of MHC genes an intriguing subject of study. To understand the evolutionary pattern of this multigene family, we studied the phylogeny and divergence times of six functional MHC class I loci from primate species. On the phylogenetic trees, locus F occupies the most basal position among these loci. Our results suggest that the F locus diverged from the other MHC class I loci about 46-66 MYA. The major diversification of the other class I loci was estimated to have occurred at about 35-49 MYA, which is before the time of separation of Old World-New World monkeys. The gene duplication leading to the classical C locus in great apes appears to have occurred about 21-28 MYA. At approximately the same time the duplication of the B locus occurred in macaques. The oldest allelic lineages of A, B, and C loci in humans seem to have appeared at least 14-19, 10-15, and 13-17 MYA, respectively. Our phylogenetic analysis supports the hypothesis that the nonclassical locus F has diverged from the rest of class I loci very early in primate evolution. The overall phylogenetic pattern observed among class I genes is consistent with the model of birth-and-death evolution.     

Recent duplication of the common carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci

Genome duplications may have played a role in the early stages of vertebrate evolution, near the time of divergence of the lamprey lineage. Additional genome duplication, specifically in ray-finned fish, may have occurred before the divergence of the teleosts. The common carp (Cyprinus carpio) has been considered tetraploid because of its chromosome number (2n = 100) and its high DNA content. We studied variation using 59 microsatellite primer pairs to better understand the ploidy level of the common carp. Based on the number of PCR amplicons per individual, about 60% of these primer pairs are estimated to amplify duplicates. Segregation patterns in families suggested a partially duplicated genome structure and disomic inheritance. This could suggest that the common carp is tetraploid and that polyploidy occurred by hybridization (allotetraploidy). From sequences of microsatellite flanking regions, we estimated the difference per base between pairs of alleles and between pairs of paralogs. The distribution of differences between paralogs had two distinct modes suggesting one whole-genome duplication and a more recent wave of segmental duplications. The genome duplication was estimated to have occurred about 12 MYA, with the segmental duplications occurring between 2.3 and 6.8 MYA. At 12 MYA, this would be one of the most recent genome duplications among vertebrates. Phylogenetic analysis of several cyprinid species suggests an evolutionary model for this tetraploidization, with a role for polyploidization in speciation and diversification.