Jawed Vertebrates Research Articles

A Comprehensive Analysis of Fibrillar Collagens in Lamprey Suggests a Conserved Role in Vertebrate Musculoskeletal Evolution.

Vertebrates have distinct tissues which are not present in invertebrate chordates nor other metazoans. The rise of these tissues also coincided with at least one round of whole-genome duplication as well as a suite of lineage-specific segmental duplications. Understanding whether novel genes lead to the origin and diversification of novel cell types, therefore, is of great importance in vertebrate evolution. Here we were particularly interested in the evolution of the vertebrate musculoskeletal system, the muscles and connective tissues that support a diversity of body plans. A major component of the musculoskeletal extracellular matrix (ECM) is fibrillar collagens, a gene family which has been greatly expanded upon in vertebrates. We thus asked whether the repertoire of fibrillar collagens in vertebrates reflects differences in the musculoskeletal system. To test this, we explored the diversity of fibrillar collagens in lamprey, a jawless vertebrate which diverged from jawed vertebrates (gnathostomes) more than five hundred million years ago and has undergone its own gene duplications. Some of the principal components of vertebrate hyaline cartilage are the fibrillar collagens type II and XI, but their presence in cartilage development across all vertebrate taxa has been disputed. We particularly emphasized the characterization of genes in the lamprey hyaline cartilage, testing if its collagen repertoire was similar to that in gnathostomes. Overall, we discovered thirteen fibrillar collagens from all known gene subfamilies in lamprey and were able to identify several lineage-specific duplications. We found that, while the collagen loci have undergone rearrangement, the Clade A genes have remained linked with the hox clusters, a phenomenon also seen in gnathostomes. While the lamprey muscular tissue was largely similar to that seen in gnathostomes, we saw considerable differences in the larval lamprey skeletal tissue, with distinct collagen combinations pertaining to different cartilage types. Our gene expression analyses were unable to identify type II collagen in the sea lamprey hyaline cartilage nor any other fibrillar collagen during chondrogenesis at the stages observed, meaning that sea lamprey likely no longer require these genes during early cartilage development. Our findings suggest that fibrillar collagens were multifunctional across the musculoskeletal system in the last common ancestor of vertebrates and have been largely conserved, but these genes alone cannot explain the origin of novel cell types.

Evolutionary History of TOPIIA Topoisomerases in Animals.

TOPIIA topoisomerases are required for the regulation of DNA topology by DNA cleavage and re-ligation and are important targets of antibiotic and anticancer agents. Humans possess two TOPIIA paralogue genes (TOP2A and TOP2B) with high sequence and structural similarity but distinct cellular functions. Despite their functional and clinical relevance, the evolutionary history of TOPIIA is still poorly understood. Here we show that TOPIIA is highly conserved in Metazoa. We also found that TOPIIA paralogues from jawed and jawless vertebrates had different origins related with tetraploidization events. After duplication, TOP2B evolved under a stronger purifying selection than TOP2A, perhaps promoted by the more specialized role of TOP2B in postmitotic cells. We also detected genetic signatures of positive selection in the highly variable C-terminal domain (CTD), possibly associated with adaptation to cellular interactions. By comparing TOPIIA from modern and archaic humans, we found two amino acid substitutions in the TOP2A CTD, suggesting that TOP2A may have contributed to the evolution of present-day humans, as proposed for other cell cycle-related genes. Finally, we identified six residues conferring resistance to chemotherapy differing between TOP2A and TOP2B. These six residues could be targets for the development of TOP2A-specific inhibitors that would avoid the side effects caused by inhibiting TOP2B. Altogether, our findings clarify the origin, diversification and selection pressures governing the evolution of animal TOPIIA.

Genome-wide identification and characterization of NLR genes in lamprey (Lethenteron reissneri) and their responses to lipopolysaccharide/poly(I:C) challenge

The nucleotide oligomerization domain (NOD)-like receptor (NLR) is a relatively conserved receptor family involved in natural immunity that plays a key role in the resistance to pathogen invasion and regulation of the innate immune response. Lethenteron reissneri (lamprey) is a representative species of existing ancient jawless vertebrates. Studies of the evolutionary relationship of immune system-related molecules in lampreys can provide an important reference for the origin and evolution of innate immunity. However, the characterization and evolutionary patterns of the NLR family remain unclear in the lamprey genome. Based on the genome database of L. reissneri, we identified nine NLR genes, characterized their functional domains and chromosomal positions, and constructed a network comprising the results of gene structure and gene-collinearity analyses. Comparative genomics studies suggest that Lr-NODa and Lr-NODb most likely share the common ancestor of NOD1 and NOD2 in jawed vertebrates, and that Lr-NODb may have been generated by lamprey-specific tandem duplication of Lr-NODa. Additionally, phylogenetic analysis of the NLRC subfamily suggests that Lr-NLRC3a has ancestral traits and may be derived from the common ancestor of another vertebrate NLRC subfamily. Further analysis of the formation of the NLRC subfamily has shown that exon shuffling, domain recombination, and chromosome rearrangement play important roles in its structural evolution. Furthermore, real-time quantitative polymerase chain reaction shows that most NLR genes in lamprey are highly expressed in the immune tissues of the heart, gill, and supraneural body, with these genes also showing significant responses to polyinosinic-polycytidylic acid infection. These results indicate that NLR genes are involved in the immune protection of L. reissneri and provide an important theoretical foundation for studies of the functional evolution of vertebrate NLRs involved in the innate immune system.

The Evolution of Oxytocin and Vasotocin Receptor Genes in Jawed Vertebrates: A Clear Case for Gene Duplications Through Ancestral Whole-Genome Duplications.

The neuronal and neuroendocrine peptides oxytocin (OT) and vasotocin (VT), including vasopressins, have six cognate receptors encoded by six receptor subtype genes in jawed vertebrates. The peptides elicit a broad range of responses that are specifically mediated by the receptor subtypes including neuronal functions regulating behavior and hormonal actions on reproduction and water/electrolyte balance. Previously, we have demonstrated that these six receptor subtype genes, which we designated VTR1A, VTR1B, OTR, VTR2A, VTR2B and VTR2C, arose from a syntenic ancestral gene pair, one VTR1/OTR ancestor and one VTR2 ancestor, through the early vertebrate whole-genome duplications (WGD) called 1R and 2R. This was supported by both phylogenetic and chromosomal conserved synteny data. More recently, other studies have focused on confounding factors, such as the OTR/VTR orthologs in cyclostomes, to question this scenario for the origin of the OTR/VTR gene family; proposing instead less parsimonious interpretations involving only one WGD followed by complex series of chromosomal or segmental duplications. Here, we have updated the phylogeny of the OTR/VTR gene family, including a larger number of vertebrate species, and revisited seven representative neighboring gene families from our previous conserved synteny analyses, adding chromosomal information from newer high-coverage genome assemblies from species that occupy key phylogenetic positions: the polypteriform fish reedfish (Erpetoichthys calabaricus), the cartilaginous fish thorny skate (Amblyraja radiata) and a more recent high-quality assembly of the Western clawed frog (Xenopus tropicalis) genome. Our analyses once again add strong support for four-fold symmetry, i.e., chromosome quadruplication in the same time window as the WGD events early in vertebrate evolution, prior to the jawed vertebrate radiation. Thus, the evolution of the OTR/VTR gene family can be most parsimoniously explained by two WGD events giving rise to the six ancestral genes, followed by differential gene losses of VTR2 genes in different lineages. We also argue for more coherence and clarity in the nomenclature of OT/VT receptors, based on the most parsimonious scenario.

Evolutionary diversification of the canonical Wnt signaling effector TCF/LEF in chordates.

Wnt signaling is essential during animal development and regeneration, but also plays an important role in diseases such as cancer and diabetes. The canonical Wnt signaling pathway is one of the most conserved signaling cascades in the animal kingdom, with the T‐cell factor/lymphoid enhancer factor (TCF/LEF) proteins being the major mediators of Wnt/β‐catenin‐regulated gene expression. In comparison with invertebrates, vertebrates possess a high diversity of TCF/LEF family genes, implicating this as a possible key change to Wnt signaling at the evolutionary origin of vertebrates. However, the precise nature of this diversification is only poorly understood. The aim of this study is to clarify orthology, paralogy, and isoform relationships within the TCF/LEF gene family within chordates via in silico comparative study of TCF/LEF gene structure, molecular phylogeny, and gene synteny. Our results support the notion that the four TCF/LEF paralog subfamilies in jawed vertebrates (gnathostomes) evolved via the two rounds of whole‐genome duplications that occurred during early vertebrate evolution. Importantly, gene structure comparisons and synteny analysis of jawless vertebrate (cyclostome) TCFs suggest that a TCF7L2‐like form of gene structure is a close proxy for the ancestral vertebrate structure. In conclusion, we propose a detailed evolutionary path based on a new pre‐whole‐genome duplication vertebrate TCF gene model. This ancestor gene model highlights the chordate and vertebrate innovations of TCF/LEF gene structure, providing the foundation for understanding the role of Wnt/β‐catenin signaling in vertebrate evolution.

Molecular evolution of hatching enzymes and their paralogous genes in vertebrates

BackgroundHatching is identified as one of the most important events in the reproduction of oviparous vertebrates. The genes for hatching enzymes, which are vital in the hatching process, are conserved among vertebrates. However, especially in teleost, it is difficult to trace their molecular evolution in detail due to the presence of other C6astacins, which are the subfamily to which the genes for hatching enzymes belong and are highly diverged. In particular, the hatching enzyme genes are diversified with frequent genome translocations due to retrocopy.ResultsIn this study, we took advantage of the rapid expansion of whole-genome data in recent years to examine the molecular evolutionary process of these genes in vertebrates. The phylogenetic analysis and the genomic synteny analysis revealed C6astacin genes other than the hatching enzyme genes, which was previously considered to be retained only in teleosts, was also retained in the genomes of basal ray-finned fishes, coelacanths, and cartilaginous fishes. These results suggest that the common ancestor of these genes can be traced back to at least the common ancestor of the Gnathostomata. Moreover, we also found that many of the C6astacin genes underwent multiple gene duplications during vertebrate evolution, and the results of gene expression analysis in frogs implied that genes derived from hatching enzyme genes underwent neo-functionalization.ConclusionsIn this study, we describe in detail the molecular evolution of the C6astacin gene in vertebrates, which has not been summarized previously. The results revealed the presence of the previously unknown C6astacin gene in the basal-lineage of jawed vertebrates and large-scale gene duplication of hatching enzyme genes in amphibians. The comprehensive investigation reported in this study will be an important basis for studying the molecular evolution of the vertebrate C6astacin genes, hatching enzyme, and its paralogous genes and for identifying these genes without the need for gene expression and functional analysis.

Hagfish to Illuminate the Developmental and Evolutionary Origins of the Vertebrate Retina.

The vertebrate eye is a vital sensory organ that has long fascinated scientists, but the details of how this organ evolved are still unclear. The vertebrate eye is distinct from the simple photoreceptive organs of other non-vertebrate chordates and there are no clear transitional forms of the eye in the fossil record. To investigate the evolution of the eye we can examine the eyes of the most ancient extant vertebrates, the hagfish and lamprey. These jawless vertebrates are in an ideal phylogenetic position to study the origin of the vertebrate eye but data on eye/retina development in these organisms is limited. New genomic and gene expression data from hagfish and lamprey suggest they have many of the same genes for eye development and retinal neurogenesis as jawed vertebrates, but functional work to determine if these genes operate in retinogenesis similarly to other vertebrates is missing. In addition, hagfish express a marker of proliferative retinal cells (Pax6) near the margin of the retina, and adult retinal growth is apparent in some species. This finding of eye growth late into hagfish ontogeny is unexpected given the degenerate eye phenotype. Further studies dissecting retinal neurogenesis in jawless vertebrates would allow for comparison of the mechanisms of retinal development between cyclostome and gnathostome eyes and provide insight into the evolutionary origins of the vertebrate eye.

Comparative genomics reveals evolutionary loss of epiplakin in cetaceans

The adaptation of vertebrates to different environments was associated with changes in the molecular composition and regulation of epithelia. Whales and dolphins, together forming the clade cetaceans, have lost multiple epithelial keratins during or after their evolutionary transition from life on land to life in water. It is unknown whether the changes in keratins were accompanied by gain or loss of cytoskeletal adapter proteins of the plakin family. Here we investigated whether plakin proteins are conserved in cetaceans and other vertebrates. Comparative analysis of genome sequences showed conservation of dystonin, microtubule actin crosslinking factor 1 (MACF1), plectin, desmoplakin, periplakin and envoplakin in cetaceans. By contrast, EPPK1 (epiplakin) was disrupted by inactivating mutations in all cetaceans investigated. Orthologs of EPPK1 are present in bony and cartilaginous fishes and tetrapods, indicating an evolutionary origin of EPPK1 in a common ancestor of jawed vertebrates (Gnathostomes). In many vertebrates, EPPK1 is flanked by an as-yet uncharacterized gene that encodes protein domains homologous to the carboxy-terminal segment of MACF1. We conclude that epiplakin, unlike other plakins, was lost in cetaceans.

Evolution of Brain-Expressed Biogenic Amine Receptors into Olfactory Trace Amine-Associated Receptors.

The family of trace amine-associated receptors (TAARs) is distantly related to G protein-coupled biogenic aminergic receptors. TAARs are found in the brain as well as in the olfactory epithelium where they detect biogenic amines. However, the functional relationship of receptors from distinct TAAR subfamilies and in different species is still uncertain. Here, we perform a thorough phylogenetic analysis of 702 TAAR-like (TARL) and TAAR sequences from 48 species. We show that a clade of Tarl genes has greatly expanded in lampreys, whereas the other Tarl clade consists of only one or two orthologs in jawed vertebrates and is lost in amniotes. We also identify two small clades of Taar genes in sharks related to the remaining Taar genes in bony vertebrates, which are divided into four major clades. We further identify ligands for 61 orphan TARLs and TAARs from sea lamprey, shark, ray-finned fishes, and mammals, as well as novel ligands for two 5-hydroxytryptamine receptor 4 orthologs, a serotonin receptor subtype closely related to TAARs. Our results reveal a pattern of functional convergence and segregation: TARLs from sea lamprey and bony vertebrate olfactory TAARs underwent independent expansions to function as chemosensory receptors, whereas TARLs from jawed vertebrates retain ancestral response profiles and may have similar functions to TAAR1 in the brain. Overall, our data provide a comprehensive understanding of the evolution and ligand recognition profiles of TAARs and TARLs.

On the relationship between extant innate immune receptors and the evolutionary origins of jawed vertebrate adaptive immunity.

For over half a century, deciphering the origins of the genomic loci that form the jawed vertebrate adaptive immune response has been a major topic in comparative immunogenetics. Vertebrate adaptive immunity relies on an extensive and highly diverse repertoire of tandem arrays of variable (V), diversity (D), and joining (J) gene segments that recombine to produce different immunoglobulin (Ig) and T cell receptor (TCR) genes. The current consensus is that a recombination-activating gene (RAG)-like transposon invaded an exon of an ancient innate immune VJ-bearing receptor, giving rise to the extant diversity of Ig and TCR loci across jawed vertebrates. However, a model for the evolutionary relationships between extant non-recombining innate immune receptors and the V(D)J receptors of the jawed vertebrate adaptive immune system has only recently begun to come into focus. In this review, we provide an overview of non-recombining VJ genes, including CD8β, CD79b, natural cytotoxicity receptor 3 (NCR3/NKp30), putative remnants of an antigen receptor precursor (PRARPs), and the multigene family of signal-regulatory proteins (SIRPs), that play a wide range of roles in immune function. We then focus in detail on the VJ-containing novel immune-type receptors (NITRs) from ray-finned fishes, as recent work has indicated that these genes are at least 50 million years older than originally thought. We conclude by providing a conceptual model of the evolutionary origins and phylogenetic distribution of known VJ-containing innate immune receptors, highlighting opportunities for future comparative research that are empowered by this emerging evolutionary perspective.

Lamprey possess both V1R and V2R olfactory receptors, but only V1Rs are expressed in olfactory sensory neurons

The sense of smell employs some of the largest gene families in the genome to detect and distinguish a multitude of different odors. Within vertebrates, 4 major olfactory receptor families have been described; of which, only 3 (OR, TAAR-like, and V1R) were found already in lamprey, a jawless vertebrate. The forth family (V2R) was believed to have originated later, in jawed vertebrates. Here we have delineated the entire vomeronasal receptor repertoire in 3 lamprey species. We report the presence of 6 v1r and 2 v2r genes in Lethenteron camtschaticum, arctic lamprey, and Lampetra fluviatilis, river lamprey (6 and 1, respectively, in sea lamprey, Petromyzon marinus). Three v1r genes but no v2r genes were found to be expressed in olfactory sensory neurons in the characteristic sparse expression pattern. Our results show the olfactory function of some V1Rs already in lamprey and, unexpectedly, an early origin of the V2R family in the shared ancestor of jawed and jawless vertebrates. However, lamprey v2r genes appear not to have acquired an olfactory function yet, thus dissociating the evolutionary origin of the family from the onset of a function as olfactory receptor.

Big insight from the little skate: Leucoraja erinacea as a developmental model system.

The vast majority of extant vertebrate diversity lies within the bony and cartilaginous fish lineages of jawed vertebrates. There is a long history of elegant experimental investigation of development in bony vertebrate model systems (e.g., mouse, chick, frog and zebrafish). However, studies on the development of cartilaginous fishes (sharks, skates and rays) have, until recently, been largely descriptive, owing to the challenges of embryonic manipulation and culture in this group. This, in turn, has hindered understanding of the evolution of developmental mechanisms within cartilaginous fishes and, more broadly, within jawed vertebrates. The little skate (Leucoraja erinacea) is an oviparous cartilaginous fish and has emerged as a powerful and experimentally tractable developmental model system. Here, we discuss the collection, husbandry and management of little skate brood stock and eggs, and we present an overview of key stages of skate embryonic development. We also discuss methods for the manipulation and culture of skate embryos and illustrate the range of tools and approaches available for studying this system. Finally, we summarize a selection of recent studies on skate development that highlight the utility of this system for inferring ancestral anatomical and developmental conditions for jawed vertebrates, as well as unique aspects of cartilaginous fish biology.

IMGT/3Dstructure-DB: T-Cell Receptor TR Paratope and Peptide/Major Histocompatibility pMH Contact Sites and Epitope.

T-cell receptors (TR), the antigen receptors of T cells, specifically recognize peptides presented by the major histocompatibility (MH) proteins, as peptide/MH (pMH), on the cell surface. The structure characterization of the trimolecular TR/pMH complexes is crucial to the fields of immunology, vaccination, and immunotherapy. IMGT/3Dstructure-DB is the three-dimensional (3-D) structure database of IMGT®, the international ImMunoGenetics information system®. By its creation, IMGT® marks the advent of immunoinformatics, which emerged at the interface between immunogenetics and bioinformatics. The IMGT® immunoglobulin (IG) and TR gene and allele nomenclature (CLASSIFICATION axiom) and the IMGT unique numbering and IMGT/Collier-de-Perles (NUMEROTATION axiom) are the two founding breakthroughs of immunoinformatics. IMGT-ONTOLOGY concepts and IMGT Scientific chart rules generated from these axioms allowed IMGT® bridging genes, structures, and functions. IMGT/3Dstructure-DB contains 3-D structures of IG or antibodies, TR and MH proteins of the adaptive immune responses of jawed vertebrates (gnathostomata), IG or TR complexes with antigens (IG/Ag, TR/pMH), related proteins of the immune system of any species belonging to the IG and MH superfamilies, and fusion proteins for immune applications. The focus of this chapter is on the TR V domains and MH G domains and the contact analysis comparison in TR/pMH interactions. Standardized molecular characterization includes "IMGT pMH contact sites" for peptide and MH groove interactions and "IMGT paratopes and epitopes" for TR/pMH complexes. Data are available in the IMGT/3Dstructure database, at the IMGT Home page http://www.imgt.org .

Microscopic Characterization of the Mucous Cells and Their Mucin Secretions in the Alimentary Canal of the Blackmouth Catshark Galeus melastomus (Chondrichthyes: Elasmobranchii)

Sharks belong to the most primitive group of jawed vertebrates and have some special structural and functional features such as a cartilaginous skeleton, a spiral intestinal valve, and a rectal gland for osmoregulation. In January 2020, ten specimens of Galeus melastomus, the Blackmouth catshark, were collected from the Gulf of Asinara (North Sardinia, Italy) and the entire alimentary canal was studied using histochemical reactions to characterize the mucous cell types. In the alimentary canal of G. melastomus, mucous cells mainly secrete a mixture of acidic and neutral mucins. Of the acidic mucins, only the carboxylated type was present in mucous cells of the stomach, while the sulfated type predominated in the esophagus and the intestines. The use of lectins revealed a distribution of sugar residues in mucins related to cellular activities of the different regions of the catshark alimentary canal. The current study is the first report to characterize the intestinal mucous cells of G. melastomus and to provide quantitative data on their different populations in the alimentary canal.

Ankfn1-mutant vestibular defects require loss of both ancestral and derived paralogs for penetrance in zebrafish.

How and to what degree gene duplication events create regulatory innovation, redundancy, or neofunctionalization remain important questions in animal evolution and comparative genetics. Ankfn1 genes are single copy in most invertebrates, partially duplicated in jawed vertebrates, and only the derived copy retained in most mammals. Null mutations in the single mouse homolog have vestibular and neurological abnormalities. Null mutation of the single Drosophila homolog is typically lethal with severe sensorimotor deficits in rare survivors. The functions and potential redundancy of paralogs in species with two copies are not known. Here, we define a vestibular role for Ankfn1 homologs in zebrafish based on the simultaneous disruption of each locus. Zebrafish with both paralogs disrupted showed vestibular defects and early lethality from swim bladder inflation failure. One intact copy at either locus was sufficient to prevent major phenotypes. Our results show that vertebrate Ankfn1 genes are required for vestibular-related functions, with at least partial redundancy between ancestral and derived paralogs.

Discovery of an ancient MHC category with both class I and class II features

Two classes of major histocompatibility complex (MHC) molecules, MHC class I and class II, play important roles in our immune system, presenting antigens to functionally distinct T lymphocyte populations. However, the origin of this essential MHC class divergence is poorly understood. Here, we discovered a category of MHC molecules (W-category) in the most primitive jawed vertebrates, cartilaginous fish, and also in bony fish and tetrapods. W-category, surprisingly, possesses class II-type α- and β-chain organization together with class I-specific sequence motifs for interdomain binding, and the W-category α2 domain shows unprecedented, phylogenetic similarity with β2-microglobulin of class I. Based on the results, we propose a model in which the ancestral MHC class I molecule evolved from class II-type W-category. The discovery of the ancient MHC group, W-category, sheds a light on the long-standing critical question of the MHC class divergence and suggests that class II type came first.

Identification of Glycan-Specific Variable Lymphocyte Receptors Using Yeast Surface Display and Glycan Microarrays.

The jawless vertebrates (lamprey and hagfish) evolved a novel adaptive immune system with many similarities to that found in the jawed vertebrates, including the production of antigen-specific circulating antibodies in response to immunization. However, the jawless vertebrates use leucine-rich repeat (LRR)-based antigen receptors termed variable lymphocyte receptors (VLRs) for immune recognition, instead of immunoglobulin (Ig)-based receptors. VLR genes are assembled in developing lymphocytes through a gene conversion-like process, in which hundreds of LRR gene segments are randomly selected as template donors to generate a large repertoire of distinct antigen receptors, similar to that found within the mammalian adaptive immune system. Here we describe the development of a robust platform using immunized lampreys (Petromyzon marinus) for generating libraries of anti-carbohydrate (anti-glycan) variable lymphocyte receptor B, or VLRBs. The anti-carbohydrate VLRBs are isolated using a yeast surface display (YSD) expression platform and enriched by binding to glycan microarrays through the anti-glycan VLRB. This enables both the initial identification and enrichment of individual yeast clones against hundreds of glycans simultaneously. Through this enrichment strategy a broad array of glycan-specific VLRs can be isolated from the YSD library. Subsequently, the bound yeast cells are directly removed from the microarray, the VLR antibody clone is sequenced, and the end product is expressed as a VLR-IgG-Fc fusion protein that can be used for ELISA, Western blotting, flow cytometry, and immunomicroscopy. Thus, by combining yeast surface display with glycan microarray technology, we have developed a rapid, efficient, and novel method for generating chimeric VLR-IgG-Fc proteins that recognize a broad array of unique glycan structures with exquisite specificity.

Lamprey Variable Lymphocyte Receptor Monoclonal Antibodies for Whole-Cell Surface Antigens.

Lamprey antibodies, the variable lymphocyte receptor B proteins (VLRB), have unique properties that make them promising alternatives to jawed vertebrate immunoglobulin domain antibodies. These leucine-rich repeat proteins exhibit a diversity on par with that of jawed vertebrate antibodies but are structurally completely distinct. VLRB antibodies have been successfully raised to a variety of antigens. A procedure for high-throughput screening of full-length lamprey VLRB libraries using whole cells is described here. Lamprey antibodies against cell surface antigens can be generated and screened quickly using this method.

IMGT® databases, related tools and web resources through three main axes of research and development.

IMGT®, the international ImMunoGeneTics information system®, http://www.imgt.org/, is at the forefront of the immunogenetics and immunoinformatics fields with more than 30 years of experience. IMGT® makes available databases and tools to the scientific community pertaining to the adaptive immune response, based on the IMGT-ONTOLOGY. We focus on the recent features of the IMGT® databases, tools, reference directories and web resources, within the three main axes of IMGT® research and development. Axis I consists in understanding the adaptive immune response, by deciphering the identification and characterization of the immunoglobulin (IG) and T cell receptor (TR) genes in jawed vertebrates. It is the starting point of the two other axes, namely the analysis and exploration of the expressed IG and TR repertoires based on comparison with IMGT reference directories in normal and pathological situations (Axis II) and the analysis of amino acid changes and functions of 2D and 3D structures of antibody and TR engineering (Axis III).

Regulation by Progestins, Corticosteroids, and RU486 of Transcriptional Activation of Elephant Shark and Human Progesterone Receptors: An Evolutionary Perspective.

We investigated progestin and corticosteroid activation of the progesterone receptor (PR) from elephant shark, a cartilaginous fish belonging to the oldest group of jawed vertebrates. Comparison with the human PR provides insights into the evolution of steroid activation of the human PR. At 1 nM steroid, the elephant shark PR is activated by progesterone, 17-hydroxy-progesterone, 20β-hydroxy-progesterone, 11-deoxycorticosterone (21-hydroxyprogesterone), and 11-deoxycortisol. The human PR, in comparison, is activated at 1 nM steroid, only by progesterone and 11-deoxycorticosterone, indicating increased progestin and corticosteroid specificity during the evolution of the human PR. RU486, an important clinical antagonist of the human PR, did not inhibit progesterone activation of the elephant shark PR. Cys-528 in the elephant shark PR corresponds to Gly-722 in the human PR, which is essential for RU486 inhibition of the human PR. Confirming the importance of Cys-528 in the elephant shark PR, RU486 inhibited progesterone activation of the Cys528Gly mutant PR. To investigate the physiological relevance of Gly-722 in the human PR and Cys-528 in the elephant shark PR, we studied steroid activation of the Gly722Cys human PR and Cys528Gly elephant shark PR. Compared to the wild-type human PR, there was an increase in the activation of human Gly722Cys PR by11-deoxycortisol and a decrease in activation by corticosterone, which may have been important in selection for the mutation corresponding to the human glycine-722 PR that first evolved in the platypus PR, a basal mammal.