Tracing the origin of fish immunoglobulins.

We have studied the origin of immunoglobulin genes in fish. There are two evolutionary lines of bony fish, Actinopterygii and Sarcopterygii. The former gave rise to most of the current fish and the latter to the animals that went to land. Non-teleost actinopterygians are significant evolutionary, sharing a common ancestor with sarcopterygians. There are three different immunoglob- ulin isotypes in ray-finned fish: IgM, IgD and IgT. We deduce that translocon formation in im- munoglobulins genes occurred already in non-teleost Actinopterygii. We establish a relationship between no teleosts and teleostean fish at the domain level of different immunoglobulins. We found two evolutionary lines of immunoglobulin. A line that starts from Immunoglobulin M and another from an ancestral Immunoglobulin W. The M line is stable, and the W line gives rise to the IgD of the fish. Immunoglobulin T emerges by recombination between both lines.

Immunoglobulins genes in Neoceratodus forsteri and Protopterus annectens explain the origin of the immunoglobulins of the animals that passed ashore.

Sarcopterygian fishes are a taxon of bony fishes. They include lungfish and coelacanths (six species of lungfish and two species of coelacanths). Evolutionary adaptations arose with these fish, such as the appearance of lungs and paired lobed fins that help them move over the bottom of the sea. In the Devonian period, they came ashore, and tetrapods (amphibians, reptiles, and mammals) arose from them. Within immunology, they can teach us about the emergence of immunoglobulins D, A/X, and Y already present in amphibians. We have studied the genes of the immunoglobulins in the fish Sarcopterygii Neoceratodus forsteri and Protopterus annectens. In the first fish, we find that several loci for the constant chains of immunoglobulins are distributed across 4 chromosomes. We have found four genes for IgM, a gene for IgW and a gene for IgN. In the second, we find one locus with genes for IgN and IgM and another with one gene for IgW. With these sequences, together with those obtained in other publications, we have been able to study the possible evolution and emergence of immunoglobulin classes. We conclude that there are two evolutionary lineages, one focused on IgM and very conservative, and the other focused on IgW, which allows high variability. In the case of the animals that went to land, their IgD is formed only by domains whose origin is in the W lineage. IgA/X and IgY are unique since they arose from the recombination between the two evolutionary lineagess (M and W). In both IgA/X and IgY, the CH1 and CH2 domains come from domains whose origin is the W lineage, while their CH3 and CH4 derive from the M lineage.

Gouania willdenowi is a teleost fish without immunoglobulin genes.

Immunoglobulin (Ig) genes encode antibodies in jawed vertebrates. They are essential elements of the adaptive immune response. Ig exists in soluble form or as part of the B cell membrane antigen receptor (BCR). Studies of Ig genes in fish genomes reveal the absence of Ig genes in Gouania willdenowi by deletion of the entire Ig locus from the canonical chromosomal region. The genes coding for integral BCR proteins, CD79a and CD79b, are also absent. Genes exist for T α/ß lymphocyte receptors but not for the T γ/δ receptors. The results of the genomic analysis are independently corroborated with RNA-Seq transcriptomes from other Gobiesocidae species. From the transcriptome studies, Ig is also absent from these other Gobiesocidae species, Acyrtus sp. and Tomicodon sp. Present evidence suggests that Ig is missing from all species of the Gobiesocidae family.

Immunoglobulin T genes in Actinopterygii.

In teleost fishes, there are three immunoglobulin isotypes named immunoglobulin M (IgM), D (IgD), and T (IgT). IgT was the last to be described in teleost fishes, and it is specific to them. From recent fish genomes, we identified and studied the immunoglobulin heavy chain genes in Actinopterygii. For this analysis, a custom bioinformatics and machine learning pipeline, we call CHfinder, was developed that identifies the exons coding for the CH domains of fish immunoglobulins. Some IgT in teleost and holostean fish found from this systematic study have not been previously described. Phylogenetic analysis of the deduced amino acid sequences of the IgT CH1 exons reveals they are similar to the CH1 of IgM. This analysis also shows that the other three domains (CH2, CH3, and CH4) were not the result of recent IgM duplication processes in Actinopterygii, demonstrating that it is an immunoglobulin of earlier origin. The bioinformatics program, CHfinder, is publicly available at https://github.com/compimmuno/CHfinder.

Insights into the evolution of IG genes in Amphibians and reptiles.

Immunoglobulins are essential proteins of the immune system to neutralize pathogens. Gene encoding B cell receptors and antibodies (Ig genes) first appeared with the emergence of early vertebrates having a jaw, and are now present in all extant jawed vertebrates, or Gnathostomata. The genes have undergone evolutionary changes. In particular, genomic structural changes corresponding to genes of the adaptive immune system were coincident or in parallel with the adaptation of vertebrates from the sea to land. In cartilaginous fish exist IgM, IgD/W, and IgNAR and in bony fish IgM, IgT, IgD. Amphibians and reptiles witnessed significant modifications both in the structure and orientation of IG genes. In particular, for these amphibians and Amniota that adapted to land, IgM and IgD genes were retained, but other isotypes arose, including genes for IgA(X)1, IgA(X)2, and IgY. Recent progress in high throughput genome sequencing is helping to uncover the IG gene structure of all jawed vertebrates. In this work, we review the work and present knowledge of immunoglobulin genes in genomes of amphibians and reptiles.

Iterative Variable Gene Discovery from Whole Genome Sequencing with a Bootstrapped Multiresolution Algorithm.

In jawed vertebrates, variable (V) genes code for antigen-binding regions of B and T lymphocyte receptors, which generate a specific response to foreign pathogens. Obtaining the detailed repertoire of these genes across the jawed vertebrate kingdom would help to understand their evolution and function. However, annotations of V-genes are known for only a few model species since their extraction is not amenable to standard gene finding algorithms. Also, the more distant evolution of a taxon is from such model species, and there is less homology between their V-gene sequences. Here, we present an iterative supervised machine learning algorithm that begins by training a small set of known and verified V-gene sequences. The algorithm successively discovers homologous unaligned V-exons from a larger set of whole genome shotgun (WGS) datasets from many taxa. Upon each iteration, newly uncovered V-genes are added to the training set for the next predictions. This iterative learning/discovery process terminates when the number of new sequences discovered is negligible. This process is akin to "online" or reinforcement learning and is proven to be useful for discovering homologous V-genes from successively more distant taxa from the original set. Results are demonstrated for 14 primate WGS datasets and validated against Ensembl annotations. This algorithm is implemented in the Python programming language and is freely available at http://vgenerepertoire.org.

Immunoglobulin genes in Primates.

Five classes of immunoglobulins are known to exist in mammals. The number of isotypes of classes G, E and A varies among species for unknown reasons. Here, a study of the presence of immunoglobulin genes in Primates was carried out from the genomes and transcriptomes deposited in the NCBI repository. For this, a machine learning application based upon neural networks was implemented that scans the genomes and identifies the exon sequences that encode the immunoglobulin CH domains. From these exons, the immunoglobulins that each species possess can be inferred. Also, the presence of sequences outside the IGHC locus was found which were produced by retrotranscription of RNA that are probably not viable. From this study, the distribution of immunoglobulin genes across primate orders is described in detail. In Prosimians, IgD genes are not found; in Platyrrhines, a gene is identified for each of the immunoglobulin classes but the IgD gene does not have the CH2 exon; in the Cercopithecidae family, a gene is detected for each class in the Colobinae family, while in Cercopithecidae the genes for IgG have been duplicated several times. In hominids, a greater number of duplications that include the genes that code for IgA and IgE are observed. These results indicate that from the appearance of the Cercopithecidae, there is an evolutionary instability in the Ig locus.

Immunoglobulin and T cell receptor genes in Chinese crocodile lizard Shinisaurus crocodilurus.

Squamata are reptiles that diverged from mammals 300 million years ago. During this period, the immunoglobulin (IG) and T lymphocyte receptor (TCR) genes evolved parallel to mammals. However, unlike mammals whose IG/TCR locus has retained a constant structure throughout evolution, Squamata have witnessed duplications, losses, and/or gains in the domains of their immunoglobulin genes. The recent genome sequencing of Shinisaurus crocodilurus, a representative species of the oldest reptiles, provides an opportunity to contrast the structure of IG and TCR genes from previously studied Squamata. This study revealed ten immunoglobulin genes: five genes for immunoglobulin M (IgM), two for immunoglobulin D (IgD), one for immunoglobulin D2 (IgD2), and two for immunoglobulin Y (IgY). As in other Squamata, there are genes for the λ light chain (IGLV) but not for the κ chain (IGKV). Here, the data shows that in some IgM genes, the cysteine needed to bind the λ chains does not exist, but we present evidence for possible non-covalent binding to the light chain. With respect to TCR, one gene is detected for the α constant chain (TRAC) and two genes for the ß constant chain (TRBC); one of which is located in the locus of the variable regions of the heavy chain. As in the rest of the Squamata, genes for the γ/δ T cell receptor were not found. The V gene repertoire is found to be consistent with all other Squamata with few V genes for beta chain of TCR.

Genomic structure and expression of immunoglobulins in Squamata.

The Squamata order represents a major evolutionary reptile lineage, yet the structure and expression of immunoglobulins in this order has been scarcely studied in detail. From the genome sequences of four Squamata species (Gekko japonicus, Ophisaurus gracilis, Pogona vitticeps and Ophiophagus hannah) and RNA-seq datasets from 18 other Squamata species, we identified the immunoglobulins present in these animals as well as the tissues in which they are found. All Squamata have at least three immunoglobulin classes; namely, the immunoglobulins M, D, and Y. Unlike mammals, however, we provide evidence that some Squamata lineages possess more than one Cµ gene which is located downstream from the Cδ gene. The existence of two evolutionary lineages of immunoglobulin Y is shown. Additionally, it is demonstrated that while all Squamata species possess the λ light chain, only Iguanidae species possess the κ light chain.

Amphibians have immunoglobulins similar to ancestral IgD and IgA from Amniotes.

We studied the immunoglobulin genes from either the genomes or RNAs of amphibians. In particular, we obtained data from one frog genome (Nanorana parkeri) and three transcriptomes of the Caudata order (Andrias davidianus, Notophthalmus viridescens and Cynops pyrrhogaster). Apart from the immunoglobulins IgM and IgY previously described, we identified several IgD related immunoglobulins. The species N. parkeri, N. viridescens and C. pyrrhogaster have two IgD genes, while Andrias davidianus has three such genes. The three Caudata species have long IgD immunoglobulins similar to IgD of reptiles, and could be an ancient relic from the common ancestor of IgD of all mammals and reptiles. We also found two IgA isotypes. The results suggest that one of the IgA may be the ancestor of IgA in crocodiles and birds, while the other could be the ancestor IgA found in mammals. These results provide information that could help understand the evolution of immunoglobulins in terrestrial vertebrates.

Evolution of V genes from the TRV loci of mammals.

Information concerning the evolution of T lymphocyte receptors (TCR) can be deciphered from that part of the molecule that recognizes antigen presented by major histocompatibility complex (MHC), namely the variable (V) regions. The genes that code for these variable regions are found within the TCR loci. Here, we describe a study of the evolutionary origin of V genes that code for the α and ß chains of the TCR loci of mammals. In particular, we demonstrate that most of the 35 TRAV and 25 TRBV conserved genes found in Primates are also found in other Eutheria, while in Marsupials, Monotremes, and Reptiles, these genes diversified in a different manner. We also show that in mammals, all TRAV genes are derived from five ancestral genes, while all TRBV genes originate from four such genes. In Reptiles, the five TRAV and three out of the four TRBV ancestral genes exist, as well as other V genes not found in mammals. We also studied the TRGV and TRDV loci from all mammals, and we show a relationship of the TRDV to the TRAV locus throughout evolutionary time.

An automated algorithm for extracting functional immunologic V-genes from genomes in jawed vertebrates.

Variable (V) domains of immunoglobulins (Ig) and T cell receptors (TCR) are generated from genomic V gene segments (V-genes). At present, such V-genes have been annotated only within the genome of a few species. We have developed a bioinformatics tool that accelerates the task of identifying functional V-genes from genome datasets. Automated recognition is accomplished by recognizing key V-gene signatures, such as recombination signal sequences, size of the exon region, and position of amino acid motifs within the translated exon. This algorithm also classifies extracted V-genes into either TCR or Ig loci. We describe the implementation of the algorithm and validate its accuracy by comparing V-genes identified from the human and mouse genomes with known V-gene annotations documented and available in public repositories. The advantages and utility of the algorithm are illustrated by using it to identify functional V-genes in the rat genome, where V-gene annotation is still incomplete. This allowed us to perform a comparative human-rodent phylogenetic analysis based on V-genes that supports the hypothesis that distinct evolutionary pressures shape the TCRs and Igs V-gene repertoires. Our program, together with a user graphical interface, is available as open-source software, downloadable at http://code.google.com/p/vgenextract/.

IgH loci of American alligator and saltwater crocodile shed light on IgA evolution.

Immunoglobulin loci of two representatives of the order Crocodylia were studied from full genome sequences. Both Alligator mississippiensis and Crocodylus porosus have 13 genes for the heavy chain constant regions of immunoglobulins. The IGHC locus contains genes encoding four immunoglobulins M (IgM), one immunoglobulin D (IgD), three immunoglobulins A (IgA), three immunoglobulins Y (IgY), and two immunoglobulins D2 (IgD2). IgA and IgD2 genes were found in reverse transcriptional orientation compared to the other Ig genes. The IGHD gene contains 11 exons, four of which containing stop codons or sequence alterations. As described in other reptiles, the IgD2 is a chimeric Ig with IgA- and IgD-related domains. This work clarifies the origin of bird IgA and its evolutionary relationship with amphibian immunoglobulin X (IgX) as well as their links with mammalian IgA.

Immunoglobulin light chains in medaka (Oryzias latipes).

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.

Immunoglobulin genes of the turtles.

The availability of reptile genomes for the use of the scientific community is an exceptional opportunity to study the evolution of immunoglobulin genes. The genome of Chrysemys picta bellii and Pelodiscus sinensis is the first one that has been reported for turtles. The scanning for immunoglobulin genes resulted in the presence of a complex locus for the immunoglobulin heavy chain (IGH). This IGH locus in both turtles contains genes for 13 isotypes in C. picta bellii and 17 in P. sinensis. These correspond with one immunoglobulin M, one immunoglobulin D, several immunoglobulins Y (six in C. picta bellii and eight in P. sinensis), and several immunoglobulins that are similar to immunoglobulin D2 (five in C. picta belli and seven in P. sinensis) that was previously described in Eublepharis macularius. It is worthy to note that IGHD2 are placed in an inverted transcriptional orientation and present sequences for two immunoglobulin domains that are similar to bird IgA domains. Furthermore, its phylogenetic analysis allows us to consider about the presence of IGHA gene in a primitive reptile, so we would be dealing with the memory of the gene that originated from the bird IGHA. In summary, we provide a clear picture of the immunoglobulins present in a turtle, whose analysis supports the idea that turtles emerged from the evolutionary line from the differentiation of birds and the presence of the IGHA gene present in a common ancestor.

Snakes antibodies.

Immunoglobulins are basic molecules of the immune system of vertebrates. In previous studies we described the immunoglobulins found in two squamata reptiles, Anolis carolinensis and Eublepharis macularius. Snakes are squamata reptiles too but they have undergone an extreme evolutionary process. We therefore wanted to know how these changes affected their immunoglobulin coding genes. To perform this analysis we studied five snake transcriptomes and two genome draft sequences. Sequences coding for immunoglobulin M (IgM), immunoglobulin D (IgD) and two classes of immunoglobulin Y (IgY - named IgYa and IgYb-) were found in all of them. Moreover, the Thamnophis elegans transcriptome and Python molurus genome draft sequences showed a third class of IgY, the IgYc, whose constant region only presents three domains and lacks the CH2. All data suggest that the IgYb is the evolutionary origin of this IgYc. An exhaustive search of the light chains were carried out, being lambda the only light chain found in snakes. The results provide a clear picture of the immunoglobulins present in the suborder Serpentes.

Immunoglobulin heavy chains in medaka (Oryzias latipes).

BACKGROUND: Bony fish present an immunological system, which evolved independently from those of animals that migrated to land 400 million years ago. The publication of whole genome sequences and the availability of several cDNA libraries for medaka (Oryzias latipes) permitted us to perform a thorough analysis of immunoglobulin heavy chains present in this teleost. RESULTS: We identified IgM and IgD coding ESTs, mainly in spleen, kidney and gills using published cDNA libraries but we did not find any sequence that coded for IgT or other heavy chain isotypes described in fish. The IgM - ESTs corresponded with the secreted and membrane forms and surprisingly, the latter form only presented two constant heavy chain domains. This is the first time that this short form of membrane IgM is described in a teleost. It is different from that identified in Notothenioid teleost because it does not present the typical splicing pattern of membrane IgM. The identified IgD-ESTs only present membrane transcripts, with Cµ1 and five Cδ exons. Furthermore, there are ESTs with sequences that do not have any VH which disrupt open reading frames. A scan of the medaka genome using transcripts and genomic short reads resulted in five zones within a region on chromosome 8 with Cµ and Cδ exons. Some of these exons do not form part of antibodies and were at times interspersed, suggesting a recombination process between zones. An analysis of the ESTs confirmed that no antibodies are expressed from zone 3. CONCLUSIONS: Our results suggest that the IGH locus duplication is very common among teleosts, wherein the existence of a recombination process explains the sequence homology between them.

Presence of an unique IgT on the IGH locus in three-spined stickleback fish (Gasterosteus aculeatus) and the very recent generation of a repertoire of VH genes.

This study describes the IGH locus in Gasterosteus aculeatus, with 10 genes encoding three immunoglobulin classes: IgT, IgM and IgD. These genes are organized into a structure with three repeats of IGHT-IGHM-IGHD separated by segments including the VH segments. There was also a fourth IGHT gene. IGHT encodes an antibody with three immunoglobulin domains. Comparative studies indicate it is related to IgT and IgZ and other antibodies located upstream of the IGHM in teleost fish. The IGHM and IGHD are similar to the ones described in teleost. The IGHM has four immunoglobulin domains while the IGHD seven and none is duplicated. The IGH locus of G. aculeatus has 49 VH segments located in four regions. They belonged to four families, whose members show a greater than 92% amino acid identity, indicating that VH families diversified recently. Phylogenetic reconstruction suggests they were originated from four VH segments that must have duplicated with the constant region genes, after that the four VH segments gave rise to the remaining segments. This suggests the presence of an active biological process that generates diversity in VH regions.

The immunoglobulin heavy chain locus in the reptile Anolis carolinensis.

We describe the entire immunoglobulin heavy chain (IgH) locus from the reptile Anolis carolinensis. The heavy chain constant (C(H)) region includes C mu, C delta and C upsilon genes. This is the first description of a C upsilon gene in the reptilian class. Variable (V(H)), diversity (D(H)) and joining (J(H)) genes are located 5' from the constant (C(H)) chain complex locus. The C mu and C upsilon genes encode antibodies with four immunoglobulin domains. The C delta gene encoded an 11 domain delta heavy chain as in Eublepharis macularius. Seventy V(H) genes, belonging to 28 families, were identified, and they can be sorted into five broader groups. The similarity of the organization of the reptilian genes with those of amphibians and mammals suggests the existence of a process of heavy chain genomic reorganization before the radiation of tetrapod vertebrates.