Whole-genome duplications spurred the functional diversification of the globin gene superfamily in vertebrates

It has been hypothesized that two successive rounds of whole-genome duplication (WGD) in the stem lineage of vertebrates provided genetic raw materials for the evolutionary innovation of many vertebrate-specific features. However, it has seldom been possible to trace such innovations to specific functional differences between paralogous gene products that derive from a WGD event. Here, we report genomic evidence for a direct link between WGD and key physiological innovations in the vertebrate oxygen transport system. Specifically, we demonstrate that key globin proteins that evolved specialized functions in different aspects of oxidative metabolism (hemoglobin, myoglobin, and cytoglobin) represent paralogous products of two WGD events in the vertebrate common ancestor. Analysis of conserved macrosynteny between the genomes of vertebrates and amphioxus (subphylum Cephalochordata) revealed that homologous chromosomal segments defined by myoglobin + globin-E, cytoglobin, and the α-globin gene cluster each descend from the same linkage group in the reconstructed proto-karyotype of the chordate common ancestor. The physiological division of labor between the oxygen transport function of hemoglobin and the oxygen storage function of myoglobin played a pivotal role in the evolution of aerobic energy metabolism, supporting the hypothesis that WGDs helped fuel key innovations in vertebrate evolution.     

Evolution of the globin gene family in deuterostomes: lineage-specific patterns of diversification and attrition

In the Metazoa, globin proteins display an underlying unity in tertiary structure that belies an extraordinary diversity in primary structures, biochemical properties, and physiological functions. Phylogenetic reconstructions can reveal which of these functions represent novel, lineage-specific innovations, and which represent ancestral functions that are shared with homologous globin proteins in other eukaryotes and even prokaryotes. To date, our understanding of globin diversity in deuterostomes has been hindered by a dearth of genomic sequence data from the Ambulacraria (echinoderms + hemichordates), the sister group of chordates, and the phylum Xenacoelomorpha, which includes xenoturbellids, acoelomorphs, and nemertodermatids. Here, we report the results of a phylogenetic and comparative genomic analysis of the globin gene repertoire of deuterostomes. We first characterized the globin genes of the acorn worm, Saccoglossus kowalevskii, a representative of the phylum Hemichordata. We then integrated genomic sequence data from the acorn worm into a comprehensive analysis of conserved synteny and phylogenetic relationships among globin genes from representatives of the eight lineages that comprise the superphylum Deuterostomia. The primary aims were 1) to unravel the evolutionary history of the globin gene superfamily in deuterostomes and 2) to use the estimated phylogeny to gain insights into the functional evolution of deuterostome globins. Results of our analyses indicate that the deuterostome common ancestor possessed a repertoire of at least four distinct globin paralogs and that different subsets of these ancestral genes have been retained in each of the descendant organismal lineages. In each major deuterostome group, a different subset of ancestral precursor genes underwent lineage-specific expansions of functional diversity through repeated rounds of gene duplication and divergence. By integrating results of the phylogenetic analysis with available functional data, we discovered that circulating oxygen-transport hemoglobins evolved independently in several deuterostome lineages and that intracellular nerve globins evolved independently in chordates and acoelomorph worms.     

Phylogenetic analysis of the cadherin superfamily.

Cadherins are a multigene family of proteins which mediate homophilic calcium-dependent cell adhesion and are thought to play an important role in morphogenesis by mediating specific intercellular adhesion. Different lines of experimental evidence have recently indicated that the site responsible for mediating adhesive interactions is localized to the first extracellular domain of cadherin. Based upon an analysis of the sequence of this domain, I show that cadherins can be classified into three groups with distinct structural features. Furthermore, using this sequence information a phylogenetic tree relating the known cadherins was assembled. This is the first such tree to be published for the cadherins. One cadherin subtype, neural cadherin (N-cadherin), shows very little sequence divergence between species, whereas all other cadherin subtypes show more substantial divergence, suggesting that selective pressure upon this domain may be greater for N-cadherin than for other cadherins. Phylogenetic analysis also suggests that the gene duplications which established the main branches leading to the different cadherin subtypes occurred very early in their history. These duplications set the stage for the diversified superfamily we now observe.     

Phylogenetic evidence for pollinator-driven diversification of angiosperms

Since Darwin, the diversity of flowers has been attributed to selection by pollinators. Although pollinators commonly act as selective agents on floral traits, determining the extent to which they have influenced angiosperm diversification requires a historical perspective. Here we review recent studies that combine species-level phylogenies with pollinator data and show that pollinator shifts are common, being associated with at least a quarter of documented divergence events. However, shift frequency and directionality vary extensively, owing to variation in intrinsic factors such as floral features and phylogenetic history, as well as extrinsic factors such as interactions with local pollinator assemblages. Despite technical advances, phylogenies remain limited in their power to distinguish among various pollinator-driven evolutionary processes.     

Phylogenetic and functional characterization of the hAT transposon superfamily

Transposons are found in virtually all organisms and play fundamental roles in genome evolution. They can also acquire new functions in the host organism and some have been developed as incisive genetic tools for transformation and mutagenesis. The hAT transposon superfamily contains members from the plant and animal kingdoms, some of which are active when introduced into new host organisms. We have identified two new active hAT transposons, AeBuster1, from the mosquito Aedes aegypti and TcBuster from the red flour beetle Tribolium castaneum. Activity of both transposons is illustrated by excision and transposition assays performed in Drosophila melanogaster and Ae. aegypti and by in vitro strand transfer assays. These two active insect transposons are more closely related to the Buster sequences identified in humans than they are to the previously identified active hAT transposons, Ac, Tam3, Tol2, hobo, and Hermes. We therefore reexamined the structural and functional relationships of hAT and hAT-like transposase sequences extracted from genome databases and found that the hAT superfamily is divided into at least two families. This division is supported by a difference in target-site selections generated by active transposons of each family. We name these families the Ac and Buster families after the first identified transposon or transposon-like sequence in each. We find that the recently discovered SPIN transposons of mammals are located within the family of Buster elements.     

Phylogenetic analysis reveals wide distribution of globin X

The vertebrate globin gene repertoire consists of seven members that differ in terms of structure, function and phyletic distribution. While hemoglobin, myoglobin, cytoglobin, and neuroglobin are present in almost all gnathostomes examined so far, other globin genes, like globin X, are much more restricted in their phyletic distribution. Till today, globin X has only been found in teleost fish and Xenopus. Here, we report that globin X is also present in the genomes of the sea lamprey, ghost shark and reptiles. Moreover, the identification of orthologs of globin X in crustacean, insects, platyhelminthes, and hemichordates confirms its ancient origin.     

Genomic organization and gene expression of the multiple globins in Atlantic cod: conservation of globin-flanking genes in chordates infers the origin of the vertebrate globin clusters

Background  

The vertebrate globin genes encoding the α- and β-subunits of the tetrameric hemoglobins are clustered at two unlinked loci. The highly conserved linear order of the genes flanking the hemoglobins provides a strong anchor for inferring common ancestry of the globin clusters. In fish, the number of α-β-linked globin genes varies considerably between different sublineages and seems to be related to prevailing physico-chemical conditions. Draft sequences of the Atlantic cod genome enabled us to determine the genomic organization of the globin repertoire in this marine species that copes with fluctuating environments of the temperate and Arctic regions.     

Dating cryptodiran nodes: origin and diversification of the turtle superfamily Testudinoidea

The superfamily Testudinoidea is the most diverse and widely distributed clade of extant turtles. Surprisingly, despite an extensive fossil record, and increasing amount of molecular data available, the temporal origin of this group is still largely unknown. To address this issue, we used a comprehensive molecular dataset to perform phylogenetic and molecular dating analyses, as well as seven fossil constraints to calibrate the ages of the nodes in the phylogeny. The molecular dataset includes the complete mitochondrial genomes of 37 turtle species, including newly sequenced mitochondrial genomes of Phrynops hilarii, Emys orbicularis, Rhinoclemmys punctularia, and Chelonoidis nigra, and four nuclear markers. Our results revealed that the earliest divergences within crown testudinoids occurred around 95.0 Mya, in the early Late Cretaceous, earlier than previously reported, raising new questions about the historical biogeography of this group.     

Codon bias and gene expression of mitochondrial ND2 gene in chordates

Background: Mitochondrial ND gene, which encodes NADH dehydrogenase, is the first enzyme of the mitochondrial electron transport chain. Leigh syndrome, a neurodegenerative disease caused by mutation in the ND2 gene (T4681C), is associated with bilateral symmetric lesions in basal ganglia and subcortical brain regions. Therefore, it is of interest to analyze mitochondrial DNA to glean information for evolutionary relationship. This study highlights on the analysis of compositional dynamics and selection pressure in shaping the codon usage patterns in the coding sequence of MT-ND2 gene across pisces, aves and mammals by using bioinformatics tools like effective number of codons (ENC), codon adaptation index (CAI), relative synonymous codon usage (RSCU) etc. Results: We observed a low codon usage bias as reflected by high ENC values in MT-ND2 gene among pisces, aves and mammals. The most frequently used codons were ending with A/C at the 3^rd position of codon and the gene was AT rich in all the three classes. The codons TCA, CTA, CGA and TGA were over represented in all three classes. The F1 correspondence showed significant positive correlation with G, T3 and CAI while the F2 axis showed significant negative correlation with A and T but significant positive correlation with G, C, G3, C3, ENC, GC, GC1, GC2 and GC3. Conclusions: The codon usage bias in MTND2 gene is not associated with expression level. Mutation pressure and natural selection affect the codon usage pattern in MT-ND 2 gene.     

Early diversification of the TNF superfamily in teleosts: genomic characterization and expression analysis

The TNF superfamily (TNFSF) of proteins are cytokines involved in diverse immunological and developmental pathways. Little is known about their evolution or expression in lower vertebrate species. Bioinformatic searches of Zebrafish, Tetraodon, and Fugu genome and other teleost expressed sequence tag databases identified 44 novel gene sequences containing a TNF homology domain. This work reveals the following: 1) teleosts possess orthologs of BAFF, APRIL, EDA, TWEAK, 4-1BBL, Fas ligand, LIGHT, CD40L, RANKL, and possibly TL1A; 2) the BAFF-APRIL subfamily is enriched by a third member, BALM, unique to fish; 3) orthologs of lymphotoxins alpha and beta were not clearly identified in teleosts and are substituted by a related ligand, TNF-New; 4) as many as four TRAIL-like genes are present in teleosts, as compared with only one in mammals; and 5) T cell activation ligands OX40L, CD27L, CD30L, and GITRL were not identified in any fish species. Finally, we characterize mRNA expression of TNFSF members CD40L, LIGHT, BALM, APRIL, Fas ligand, RANKL, TRAIL-like, and TNF-New in rainbow trout, Oncorhynchus mykiss, immune and nonimmune tissues. In conclusion, we identified a total of 14 distinct TNFSF members in fishes, indicating expansion of this superfamily before the divergence of bony fish and tetrapods, approximately 360-450 million years ago. Based on these findings, we extend a model of TNFSF evolution and the co-emergence of the vertebrate adaptive immune system.     

Phylogenetic analysis of the chromate ion transporter (CHR) superfamily

ChrA is a membrane protein that confers resistance to the toxic ion chromate through the energy-dependent chromate efflux from the cytoplasm. In the protein databases, ChrA is a member of the chromate ion transporter (CHR) superfamily, composed of at least several dozens of members, distributed in the three domains of life. The aim of this work was to perform a phylogenetic analysis of the CHR superfamily. An exhaustive search for ChrA homologous proteins was carried out at the National Center for Biotechnology Information database. One hundred and thirty-five sequences were identified as members of the CHR superfamily [77 long-chain sequences, or bidomains (LCHR), and 58 short-chain sequences, or monodomains (SCHR)], organized mainly as tandem pairs of genes whose resultant proteins probably possess oppositely oriented membrane topology. LCHR sequences were split into amino and carboxyl domains, and the resultant domains were aligned with the SCHR proteins. A phylogenetic tree was reconstructed using four different methods, obtaining similar results. The domains were grouped into three clusters: the SCHR proteins cluster, the amino domain cluster of LCHR proteins and the carboxyl domain cluster of LCHR proteins. These results, as well as differences in the genomic context of CHR proteins, enabled the proteins to be sorted into two families (SCHR and LCHR), and 10 subfamilies. Evidence was found suggesting an ancient origin of LCHR proteins from the fusion of two SCHR protein-encoding genes; however, some secondary events of fusion and fission may have occurred later. The separate distribution of the LCHR and SCHR proteins, differences in the genomic context in both groups and the fact that chromate transport has been demonstrated only in LCHR proteins suggest that the CHR proteins comprise two or more paralogous groups in the CHR superfamily.     

Evolution of a core gene network for skeletogenesis in chordates

The skeleton is one of the most important features for the reconstruction of vertebrate phylogeny but few data are available to understand its molecular origin. In mammals the Runt genes are central regulators of skeletogenesis. Runx2 was shown to be essential for osteoblast differentiation, tooth development, and bone formation. Both Runx2 and Runx3 are essential for chondrocyte maturation. Furthermore, Runx2 directly regulates Indian hedgehog expression, a master coordinator of skeletal development. To clarify the correlation of Runt gene evolution and the emergence of cartilage and bone in vertebrates, we cloned the Runt genes from hagfish as representative of jawless fish (MgRunxA, MgRunxB) and from dogfish as representative of jawed cartilaginous fish (ScRunx1–3). According to our phylogenetic reconstruction the stem species of chordates harboured a single Runt gene and thereafter Runt locus duplications occurred during early vertebrate evolution. All newly isolated Runt genes were expressed in cartilage according to quantitative PCR. In situ hybridisation confirmed high MgRunxA expression in hard cartilage of hagfish. In dogfish ScRunx2 and ScRunx3 were expressed in embryonal cartilage whereas all three Runt genes were detected in teeth and placoid scales. In cephalochordates (lancelets) Runt, Hedgehog and SoxE were strongly expressed in the gill bars and expression of Runt and Hedgehog was found in endo- as well as ectodermal cells. Furthermore we demonstrate that the lancelet Runt protein binds to Runt binding sites in the lancelet Hedgehog promoter and regulates its activity. Together, these results suggest that Runt and Hedgehog were part of a core gene network for cartilage formation, which was already active in the gill bars of the common ancestor of cephalochordates and vertebrates and diversified after Runt duplications had occurred during vertebrate evolution. The similarities in expression patterns of Runt genes support the view that teeth and placoid scales evolved from a homologous developmental module.     

High-performance liquid chromatography of globin chains in the identification of human globin gene abnormalities

This article summarizes experience and data obtained using a previously developed reverse-phase high-performance liquid chromatography method (J.B. Shelton, J.R. Shelton and W.A. Schroeder, J. Liq. Chromatogr. 7 (1984) 1969) in the study of a number of hemoglobinopathies in the Sardinian population. The occurrence and incidence of several abnormal hemoglobins are described, as well as aspects of the expression of abnormal gamma-globin gene arrangements and thalassemic genes.     

Phylogenetic and biogeographic diversification of Rhus (Anacardiaceae) in the Northern Hemisphere

Sequences of internal transcribed spacers (ITS) of nuclear ribosomal DNA, the chloroplast ndhF gene, and chloroplast trnL-F regions (trnL intron, and trnL [UAA] 3' exon-trnF [GAA] intergenic spacer) were used for phylogenetic analyses of Rhus, a genus disjunctly distributed in Asia, Europe, Hawaii, North America, and Northern Central America. Both ITS and cpDNA data sets support the monophyly of Rhus. The monophyly of subgenus Rhus was suggested by the combined cpDNA and ITS data, and largely supported in the cpDNA data except that Rhus microphylla of subgenus Lobadium was nested within it. The monophyly of subgenus Lobadium was strongly supported in the ITS data, whereas the cpDNA data revealed two main clades within the subgenus, which formed a trichotomy with the clade of subgenus Rhus plus R. microphylla. The ITS and cpDNA trees differ in the positions of Rhus michauxii, R. microphylla, and Rhus rubifolia, and hybridization may have caused this discordance. Fossil evidence indicates that Rhus dates back to the early Eocene. The penalized likelihood method was used to estimate divergence times, with fossils of Rhus subgenus Lobadium, Pistacia and Toxicodendron used for age constraints. Rhus diverged from its closest relative at 49.1+/-2.1 million years ago (Ma), the split of subgenus Lobadium and subgenus Rhus was at 38.1+/-3.0 Ma. Rhus most likely migrated from North America into Asia via the Bering Land Bridge during the Late Eocene (33.8+/-3.1 Ma). Rhus coriaria from southern Europe and western Asia diverged from its relatives in eastern Asia at 24.4+/-3.2 Ma. The Hawaiian Rhus sandwicensis diverged from the Asian Rhus chinensis at 13.5+/-3.0 Ma. Subgenus Lobadium was inferred to be of North American origin. Taxa of subgenus Lobadium then migrated southward to Central America. Furthermore, we herein make the following three nomenclatural combinations: (1) Searsia leptodictya (Diels) T. S. Yi, A. J. Miller and J. Wen, comb. nov., (2) Searsia pyroides (A. Rich.) T. S. Yi, A. J. Miller and J. Wen, comb. nov., and (3) Searsia undulata (Jacq.) T. S. Yi, A. J. Miller and J. Wen, because our analyses support the segregation of Searsia from Rhus.     

Phylogenetic status and timescale for the diversification of Steno and Sotalia dolphins

Molecular data have provided many insights into cetacean evolution but some unsettled issues still remain. We estimated the topology and timing of cetacean evolutionary relationships using bayesian and maximum likelihood analyses of complete mitochondrial genomes. In order to clarify the phylogenetic placement of Sotalia and Steno within the Delphinidae, we sequenced three new delphinid mitogenomes. Our analyses support three delphinid clades: one joining Steno and Sotalia (supporting the revised subfamily Stenoninae); another placing Sousa within the Delphininae; and a third, the Globicephalinae, which includes Globicephala, Feresa, Pseudorca, Peponocephala and Grampus. We also conclude that Orcinus does not belong in the Globicephalinae, but Orcaella may be part of that subfamily. Divergence dates were estimated using the relaxed molecular clock calibrated with fossil data. We hypothesise that the timing of separation of the marine and Amazonian Sotalia species (2.3 Ma) coincided with the establishment of the modern Amazon River basin.     

Human globin gene expression after gene transfer

Human globin genes can be transferred into mouse and human erythroid cells in culture, and can be appropriately expressed at the mRNA level in these cells. A plasmid containing a human beta globin gene is expressed in mouse erythroleukemia cells (MELC), and another containing a human epsilon or gamma gene is expressed in human erythroleukemia (K562) cells. A neomycin resistance (neoR) gene on the plasmids has been used to select for those cells containing the transferred globin genes; this selection may favor the expression of the globin genes by providing chromosomal positions requiring neoR expression. Analyzing clones resistant to G418, a neomycin analogue, demonstrated globin mRNA expression and induction. Retroviral vectors have also been used to transfer and appropriately express human beta genes in MELC. In addition, a plasmid containing a dihydrofolate reductase (DHFR) gene as well as neoR and beta globin genes has been used to amplify and express beta globin mRNA in MELC. These experiments suggest that high level appropriate expression of human beta globin genes is feasible and provides potentially useful approaches to the long-range goal of gene therapy for sickle cell anemia and beta thalassemia.