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Derived amino acid sequences of NS4 genes. The amino acid sequences of most of the cloned NS4 genes shown in Fig. 2 are compared with R4. Dots indicate identity, dashes indicate gaps. R19 and S32 contain stop codons (asterisks), and one nucleotide was left out of CDR3 in R19 in order to obtain a reading frame that is comparable to the other sequences (indicated by a hyphen). R6 carried too many disruptions and is not shown.
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The variable (V), (diversity [D]), and joining (J) region recombinases (recombination activating genes [RAGs]) can perform like transposases and are thought to have initiated development of the adaptive immune system in early vertebrates by splitting archaic V genes with transposable elements. In cartilaginous fishes, the immunoglobulin (Ig) light...
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... Over the past several decades, evidence for germline-joined Ig elements have also been reported outside of Chondrichthyans in lineages as evolutionarily divergent as chickens (Gallus gallus) (Reynaud et al. 1989) and channel catfishes (Ictalurus punctatus) (Ghaffari and Lobb 1999). These germline joined IgH and IgL loci may, therefore, reflect the structure of a shared ancestral Ig gene that has escaped the influence of the RAG1/2 machinery, or joined loci derived from unjoined genes reflecting the result of RAG-mediated genome editing within the germline (Yoder and Litman 2000;Lee et al. 2000;van den Berg et al. 2004). Given the rapid proliferation of high quality reference genomes in the last several years, testing these competing hypotheses now represents an achievable and promising avenue of future research. ...
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.
... The first six HHC1 cDNAs (Fig. 6, top) belong to a subfamily that we shall refer to as "HHC1 Group 1." The Group 1 GL cluster contains a single D that is a 17 bp "GL-joining" of D1 and D2, like a recombined D1D2, shown as the reference sequence in Fig. 6, bottom (accession number MN958905). GL-joining is a nonsomatic phe-nomenon that has been observed frequently within cartilaginous fish Ig clusters [37,38] and suggested to occur through RAG1/2 activity in the germ cells because some joint sequences are consistent with P-nucleotide insertions [39]. P nucleotides are a signature of RAG1/2 activity, being generated through the hairpin DNA intermediate [40]. ...
In addition to conventional immunoglobulin, camelids and cartilaginous fish express a special class of antibody that consists only of heavy (H) chain (HCAbs). In the holocephalan elephantfish there are two HCAb classes, one of which has evolved surprising features. The H chain genes in cartilaginous fish are organized as 20–200 minigenes, or clusters, each consisting of VH, 1–3 DH, JH gene segments with one set of constant region exons. We report that HHC2 (holocephalan H chain antibody 2) evolved from IgM H chain clusters, but its DH gene segments have diverged considerably. The three DH in HHC2 clusters are A-rich, so that 1–3 potential reading frames for each DH encode lysine and arginine. All three are incorporated into the rearranged VDJ, ensuring the ligand-binding site carries multiple basic residues, as cDNA sequences demonstrate. The electropositive character in HHC2 CDR3 is accompanied by a paucity of aromatic amino acids, the latter feature at variance to the established, interactive role of tyrosine not only in ligand-binding but generally at interfaces of protein complexes. The selection for these divergent HHC2 features challenges currently accepted ideas on what determines antibody reactivity and molecular recognition.
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... The entire locus is made up of many repeats of such clusters or miniloci (15,66,67) where V(D)J recombination occurs within a cluster and not between clusters, resulting in a limited combinatorial diversity (68). Some of the Ig clusters in cartilaginous fish contain fully prejoined (germline-joined) VJ or VDJ gene segments or partially pre-joined VD-J combinations indicating the possible activity of RAG machinery in the germline (69,70). The pre-joined Ig genes are expressed during early developmental stages, and thus may provide protection to the progeny of this animal group (71). ...
The appearance of adaptive immunity in jawed vertebrates is termed the immunological ‘Big Bang’ because of the short evolutionary time over which it developed. Underlying it is the recombination activating gene (RAG)-based V(D)J recombination system, which initiates the sequence diversification of the immunoglobulins and lymphocyte antigen receptors. It was convincingly argued that the RAG1 and RAG2 genes originated from a single transposon. The current dogma postulates that the V(D)J recombination system was established by the split of a primordial vertebrate immune receptor gene into V and J segments by a RAG1/2 transposon, in parallel with the domestication of the same transposable element in a separate genomic locus as the RAG recombinase. Here, based on a new interpretation of previously published data, we propose an alternative evolutionary hypothesis suggesting that two different elements, a RAG1/2 transposase and a Transib transposon invader with RSS-like terminal inverted repeats, co-evolved to work together, resulting in a functional recombination process. This hypothesis offers an alternative understanding of the acquisition of recombinase function by RAGs and the origin of the V(D)J system.
... making it structurally convergent with mammalian IgG) predominates in the plasma of neonatal animals and pups (Rumfelt et al., 2001). This IgM form, IgM1 gj, forms both monomers and dimers, has an entirely germline-joined (VDJ) V domain (Rumfelt et al., 2001) and preferentially associates with a germline-joined L chain ( (Lee et al., 2000) Flajnik & Hsu, unpublished data), giving this Ig a completely predetermined binding site. As pups age the levels of pIgM and mIgM gradually increase to become the dominant Ig forms. ...
... Like the H chains, shark Ig L chain genes are organized in a cluster configuration (Greenberg et al., 1993;Shamblott and Litman, 1989). Of these loci in nurse shark, κ and σ-cart contain both germline-joined and unjoined or split clusters, σ contains no germline-joined clusters, and all λ clusters are germline-joined (Fig. 5) (Criscitiello and Flajnik, 2007;Fleurant et al., 2004;Lee et al., 2000;Rast et al., 1994). The number of clusters of each isotype, like Ig H chains, varies between shark species, as does the proportion of split versus germline-joined clusters (Fleurant et al., 2004;Hohman et al., 1992;Rast et al., 1994). ...
... It has been proposed that germline-joined Igs arose as a consequence of RAG activity in the germline (Lee et al., 2000), however, it should be noted that other scenarios have not been entirely ruled out; for example, retrotransposition of a rearranged VDJ transcript back into the germline is a possibility, but the presence of a split leader (Anderson et al., 1995) makes this explanation far less likely. While it might be thought that germline-joined L chains would restrict the antibody repertoire, λ L chain loci hypermutate at high rates in an antigen-driven fashion that results in tandem mutations, leading to diverse sequences despite a lack of junctional diversity (Lee et al., 2002). ...
Cartilaginous fishes, comprising the chimeras, sharks, skates, and rays, split from the common ancestor with other jawed vertebrates approx. 450 million years ago. Being the oldest extant taxonomic group to possess an immunoglobulin (Ig)-based adaptive immune system, examination of this group has taught us much about the evolution of adaptive immunity, as well as the conserved and taxon-specific characteristics of Igs. Significant progress has been made analyzing sequences from numerous genomic and transcriptomic data sets. These findings have been supported by additional functional studies characterizing the Igs and humoral response of sharks and their relatives. This review will summarize what we have learned about the genomic organization, protein structure, and in vivo function of these Ig isotypes in cartilaginous fishes and highlight the areas where our knowledge is still lacking.
... In many of these clusters the segments are partially recombined in the germline to D-J, V-D or even to fully recombined V-D-J elements. This tells us that in the ancestors of these fish, RAG recombinase must have been expressed in the germline, and raises the possibility that RAG recombination arose as a strategy to generate new, germline encoded, innate immune system receptors [48]. Perhaps RAG-based adaptive immunity in gnathostomes arose as a result of a molecular misunderstanding, when RAG, instead of being expressed in the germline, was suddenly expressed instead in lymphocyte precursors. ...
The receptors of innate immune systems evolve slowly over time. Those that confer some fitness benefit will be naturally selected, and so become the common property of the succeeding generations. In contrast, the receptors of the so-called adaptive immune systems are generated somatically within each individual, by moving evolution from the level of the germline to that of somatic cells. As a result, each individual ends up with a repertoire of adaptive immune receptors that is as distinctive as are their fingerprints. Unlike the fingerprint, however, the repertoire of adaptive immunity in an individual is constantly changing. Adaptive immune systems come in two fundamentally different forms that differ both in the nature and in the source of the pathogen-sensing element. On the one hand are those immune systems that use nucleic acids as the pathogen sensors. In these cases the sensor is only formed after infection, and the key information needed to build it is derived from the pathogen. On the other hand are those “anticipatory” systems that use proteins as pathogen sensors. Here the sensors have been formed prior to infection with the pathogen, and the information used to form the sensors is entirely host derived.
... In many of these clusters the segments are partially recombined in the germline to D-J, V-D or even to fully recombined V-D-J elements. This tells us that in the ancestors of these fish, RAG recombinase must have been expressed in the germline, and raises the possibility that RAG recombination arose as a strategy to generate new, germline encoded, innate immune system receptors [48]. Perhaps RAG-based adaptive immunity in gnathostomes arose as a result of a molecular misunderstanding, when RAG, instead of being expressed in the germline, was suddenly expressed instead in lymphocyte precursors. ...
All defence systems have much in common. No matter whether we are thinking in terms of defending a mediaeval castle from greedy neighbours or a multicellular eukaryote from attack by pathogens, the basic story is always the same. The defence system employed will consist of three parts. The first part provides information about whether a dangerous situation is developing. In the case of the castle, sentries will provide this necessary information. In the case of an animal’s innate immune system, soluble extracellular receptor molecules, and cell-associated sensors expressed by macrophages and other innate sentinel cells will detect incipient infections or other deviations from the homeostatic norm.
... Both PRARP1 and PRARP2 in chickens are single-copy genes that exhibit low polymorphism As IgSF members usually show high variations in either gene number or sequence diversity (polymorphism), Southern blotting using probes corresponding to the V exon was conducted to determine whether PRARP1 and PRARP2 are present in the genome as single or multiple copies and, more importantly, whether there are highly homologous genes present in split forms as observed in nurse shark NS4 Ig L chain loci (42). The results revealed a single restriction fragment in each lane, demonstrating that both PRARP1 and PRARP2 were present as single-copy genes and that only the preassembled configuration of these genes can be found in the whole genome (Fig. 1D). ...
... Marsupials contain a TCRm that uses a prejoined Vmj whose origin might be due to retrotransposition (56,57), which differs from the finding that the LP portion is encoded by an independent exon for each one of the PRARP genes. A V H 3.1 fused to D H in the germline of the IgH locus was also observed in the opossum and was hypothesized to be caused by ectopic expression of the V(D)J recombination enzymatic machinery in germ cells as confirmed by the generation of germline-joined Ig V L in sharks (42,58). Obviously, these germline VJ-joined genes should have originated from the rearrangement of BCR-or TCR-encoding genes. ...
In this study, we identified a pair of nonrearranging VJ-joined Ig superfamily genes, termed putative remnants of an Ag receptor precursor (PRARP) genes, in chicken. Both genes encode a single V-set Ig domain consisting of a canonical J-like segment and a potential immunoreceptor tyrosine-based inhibitory or switch motif in the cytoplasmic region. In vitro experiments showed that both genes were expressed at the cell surface as membrane proteins, and their recombinant products formed a monomer and a disulfide-linked homodimer or a heterodimer. These two genes were mainly expressed in B and T cells and were upregulated in response to stimulation with poly(I:C) in vitro and vaccination in vivo. Orthologs of PRARP have been identified in bony fish, amphibians, reptiles, and other birds, and a V-C1 structure similar to that of Ig or TCR chains was found in all these genes, with the exception of those in avian species, which appear to contain degenerated C1 domains or divergent Ig domains. Phylogenetic analyses suggested that the newly discovered genes do not belong to any known immune receptor family and appear to be a novel gene family. Further elucidation of the functions of PRARP and their origin might provide significant insights into the evolution of the immune system of jawed vertebrates.
... In many of these clusters the segments are partially recombined in the germline to D-J, V-D or even to fully recombined V-D-J elements. This tells us that in the ancestors of these fish, RAG recombinase must have been expressed in the germline, and raises the possibility that RAG recombination arose as a strategy to generate new, germline encoded, innate immune system receptors [48]. Perhaps RAG-based adaptive immunity in gnathostomes arose as a result of a molecular misunderstanding, when RAG, instead of being expressed in the germline, was suddenly expressed instead in lymphocyte precursors. ...
Immunology is a nodal subject that links many areas of biology. It permeates the biosciences, and also plays crucial roles in diagnosis and therapy in areas of clinical medicine ranging from the control of infectious and autoimmune diseases to tumour therapy. Monoclonal antibodies and small molecule modulators of immunity are major factors in the pharmaceutical industry and now constitute a multi billion dollar business. Students in these diverse areas are frequently daunted by the complexity of immunology and the astonishing array of unusual mechanisms that go to make it up.
Starting from Dobzhansky’s famous slogan, “Nothing in biology makes sense except in the light of evolution”, this book will serve to illuminate how evolutionary forces shaped immunity and thus provide an explanation for how many of its counter intuitive oddities arose. By doing so it will provide a conceptual framework on which students may organise the rapidly growing flood of immunological knowledge.
... In some species of sharks the IgL V and J genes, or IgH V, D and J genes, within a cluster are organized in the germline as separate gene segments, or in fused VJ, VD-J or VDJ configurations with the fused genes exhibiting sequence diversity at their junctions. The origin of the fused genes in the germline of cartilaginous fish is attributed to RAG activity in germ cells (20,21). The fused genes in sharks are only expressed early in development and were postulated to protect against infection by commonly encountered pathogens (21). ...
... Similarly, IgL V and J genes segments are arranged as both separate V and J gene segments, or in a fused VJ configuration. The rearrangement to a fused configuration affects only those genes within a cluster and their origin is attributed to RAG activity in germ cells (20,21). In bony fishes (teleosts), IgL genes are also organized in a cluster configuration with V, J, and C gene segments within a single cluster, but V and J genes are not found in a fused configuration (19). ...
... This differs from a cluster configuration in which only those genes in each cluster undergo rearrangement. Consequently, it is unlikely in channel catfish that these germline rearranged TCR genes are the result of RAG activity in germ cells, as proposed to occur in some sharks (20,21). Instead, we suggest that these germline integrated copies of rearranged TCR genes originated by conventional RAG mediated rearrangement of V(D)J genes during development of αβ T cells that subsequently underwent TE transposition to germline DNA (38). ...
Rearranged V(D)J genes coding for T cell receptor α and β chains are integrated into the germline genome of channel catfish. Previous analysis of expressed TCR Vβ2 repertoires demonstrated that channel catfish express multiple public clonotypes, which were shared among all the fish, following infection with a common protozoan parasite. In each case a single DNA sequence was predominately used to code for a public clonotype. We show here that the rearranged VDJ genes coding for these expressed public Vβ2 clonotypes can be amplified by PCR from germline DNA isolated from oocytes and erythrocytes. Sequencing of the Vβ2 PCR products confirmed that these expressed public Vβ2 clonotypes are integrated into the germline. Moreover, sequencing of PCR products confirmed that all five Vβ gene families and Vα1 have rearranged V(D)J genes with diverse CDR3 sequences integrated into the germline. Germline rearranged Vβ2 and Vβ4 genes retain the intron between the leader and Vβ sequence. This suggests that the germline rearranged TCR Vβ genes arose through VDJ rearrangement in T cells, and subsequently moved into the germline through DNA transposon mediated transposition. These results reveal a new dimension to the adaptive immune system of vertebrates, namely: the expression of evolutionarily conserved, rearranged V(D)J genes from the germline.
... Cluster organisation: Unexpectedly, a small percentage of Ig clusters were found to be partly (VD-J) or fully (VDJ or VJ) pre-joined in the germline, likely due to the expression of RAG in germ cells (Kokubu et al. 1988;Lee et al. 2000). While the antibodies produced by these "germline-joined" clusters have limited CDR3 diversity, they have been shown to have an expression advantage early in development (Rumfelt et al. 2001;Diaz et al. 2002) and so may play a protective role in neonates and pups. ...
The cartilaginous fishes (sharks, skates, rays, and chimaeras) hold a key evolutionary position, being the most distant group to mammals that possesses a “mammalian-like” adaptive immune system based on immunoglobulins (Ig) and T cell receptors (TCRs), which are somatically rearranged by recombination-activating gene (RAG) proteins, as well as polymorphic/polygenic major histocompatibility complex (MHC) molecules. Cartilaginous fishes are therefore an important research model to investigate the evolution of adaptive immunity and its interplay with the innate system. Despite this, cartilaginous fishes have historically been understudied; while early functional studies revealed sharks were able to produce a humoral response following immune stimulation, subsequent progress was hampered by bottlenecks in immune gene sequencing and a paucity of research tools (such as cell lines and monoclonal antibodies) for use in functional studies.
