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The resolution of the gar genome affords an opportunity to examine the diversification and functional specialization of immune effector molecules at a distant and potentially informative point in phylogenetic development. Although innate immunity is effected by a particularly large number of different families of molecules, the focus here is to provide detailed characterization of several families of innate receptors that are encoded in large multigene families, for which orthologous forms can be identified in other species of bony fish but not in other vertebrate groups as well as those for which orthologs are present in other vertebrate species. The results indicate that although teleost fish and the gar, as a holostean reference species, share gene families thought previously to be restricted to the teleost fish, the manner in which the members of the multigene families of innate immune receptors have undergone diversification is different in these two major phylogenetic radiations. It appears that both the total genome duplication and different patterns of genetic selection have influenced the derivation and stabilization of innate immune genes in a substantial manner during the course of vertebrate evolution.

We have analyzed the available genome and transcriptome resources from the coelacanth in order to characterize genes involved in adaptive immunity. Two highly distinctive IgW-encoding loci have been identified that exhibit a unique genomic organization, including a multiplicity of tandemly repeated constant region exons. The overall organization of the IgW loci precludes typical heavy chain class switching. A locus encoding IgM could not be identified either computationally or by using several different experimental strategies. Four distinct sets of genes encoding Ig light chains were identified. This includes a variant sigma-type Ig light chain previously identified only in cartilaginous fishes and which is now provisionally denoted sigma-2. Genes encoding α/β and γ/δ T-cell receptors, and CD3, CD4, and CD8 co-receptors also were characterized. Ig heavy chain variable region genes and TCR components are interspersed within the TCR α/δ locus; this organization previously was reported only in tetrapods and raises questions regarding evolution and functional cooption of genes encoding variable regions. The composition, organization and syntenic conservation of the major histocompatibility complex locus have been characterized. We also identified large numbers of genes encoding cytokines and their receptors, and other genes associated with adaptive immunity. In terms of sequence identity and organization, the adaptive immune genes of the coelacanth more closely resemble orthologous genes in tetrapods than those in teleost fishes, consistent with current phylogenomic interpretations. Overall, the work reported described herein highlights the complexity inherent in the coelacanth genome and provides a rich catalog of immune genes for future investigations.

Humoral immunity is effected through the rearrangement of immunoglobulin (Ig) genes in individual somatic cells committed to the B lymphocyte lineage. Haplotype or allelic exclusion restricts B lymphocytes to the expression of a single Ig receptor that can sustain further somatic modification. In most species, a specific Ig chain is encoded at a single genetic locus. However, in cartilaginous fish, hundreds of independent Ig heavy- (IgH) and Ig light-chain (IgL) gene loci are present, many of which are joined in the germ line. Ig gene transcripts have been amplified from single peripheral blood lymphocytes isolated from the clearnose skate (Raja eglanteria) using reverse-transcription PCR, and a single productive IgH transcript was detected in the majority of cells analyzed. Similarly, only a single IgL transcript was detected in over half of the individual cells. Taken together, these findings suggest that a mechanism for haplotype exclusion arose early in the evolution of antibody diversity and is independent of a single genetic locus.

The adaptive immune system arose in ancestors of the jawed vertebrates approximately 500 million years ago. Homologs of immunoglobulins (Igs), T-cell antigen receptors (TCRs), major histocompatibility complex I (MHC I) and MHC II, and the recombination-activating genes (RAGs) have been identified in all extant classes of jawed vertebrates; however, no definitive homolog of any of these genes has been identified in jawless vertebrates or invertebrates. RAG-mediated recombination and associated junctional diversification of both Ig and TCR genes occurs in all jawed vertebrates. In the case of Igs, somatic variation is expanded further through class switching, gene conversion, and somatic hypermutation. Although the identity of the 'primordial' receptor that was interrupted by the recombination mechanism in jawed vertebrates may never be established, many different families of genes that exhibit predicted characteristics of such a receptor have been described both within and outside the jawed vertebrates. Recent data from various model systems point toward a continuum of immune receptor diversity, encompassing many different families of recognition molecules whose functions are integrated in an organism's response to pathogenic invasion. Various approaches, including both genomic and protein-functional analyses, currently are being applied in jawless vertebrates, protochordates, and other invertebrate deuterostome systems and may yield definitive evidence regarding the presence or absence of adaptive immune homologs in species lacking adaptive immune systems. Such studies have the potential for uncovering previously unknown mechanisms of generating receptor diversity.

Novel immune-type receptor (NITR) genes, which initially were identified in the Southern pufferfish (Spheroides nephelus), encode products which consist of an extracellular variable (V) and V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail, which typically possesses an immunoreceptor tyrosine-based inhibition motif (ITIM). Multiple NITR genes have been identified in close, contiguous chromosomal linkage. The V regions of NITRs resemble prototypic forms defined for immunoglobulin (Ig) and T-cell antigen receptor (TCR), are present in multiple families and exhibit regionalized variation in sequence, which also occurs in Ig and TCR. Comparisons of exons encoding transmembrane and cytoplasmic regions of multiple NITRs suggest that exon shuffling has factored in the diversification of the NITR gene complex. Zebrafish (Danio rerio) NITRs exhibit many of these characteristics. NITRs that have been identified in additional species of bony fish demonstrate additional variation in the number of extracellular domains as well as in the presence of intramembranous charged residues, cytoplasmic tails and ITIMs. The presence in NITRs of V regions that are related closely to those found in Ig and TCR, as well as regulatory motifs and other structural features that are characteristic of immune inhibitory receptors encoded at the leukocyte receptor cluster, suggests that the NITRs are representative of an integral stage in the evolution of innate and adaptive immune function.

Summary: The rearranging antigen binding receptors, immunoglobulin heavy (IgH) and light (IgL) chains and the four classes of T-cell antigen receptors (TCR) are found in all contemporary species of jawed vertebrates examined thus far. Ig genes have undergone marked changes in organization and mechanisms ot diversification during vertebrate phylogeny: whereas TCR genes, which are found in species as phylogenetically removed as man and cartilaginous fishes (e.g. skate), are generally similar in terms of structure, diversification and, presumably, function. The patterns of Ig divergence in cartilaginous fish are informative as to both the potential for genetic variation and the mechanisms that bring about such change. No evidence has been found for homologs of cither Ig, TCR, recombination activating gene (RAG) I or RAG2 in jawless vertebrates or invertebrates. Thus, a phylogenetic demarcation exists in terms of the presence and absence of the rearranging antigen binding receptor genes. It is presumed that the rearranging antigen binding receptors arose from a non-rearranging predecessor. The recent discovery of non-rearranging homologs of antigen binding receptor genes in several species offers insight into alternative forms of recognition, relationships between adaptive and innate mechanisms of immunity, and the origins of antigen recognition.

The transcription pattern of the heavy chain immunoglobulin gene locus was analyzed in a 6-month-old female with agammaglobulinemia characterized by the absence of mature B cells in peripheral blood, arrested B cell development in the bone marrow and lack of germinal center development. DNA sequencing provided no evidence of mutations within the coding region of the Bruton's tyrosine kinase gene. Polymerase chain reaction-generated cDNA libraries from blood and bone marrow were screened initially using JH and CH oligodeoxynucleotide probes and VH family-specific probes. Only 10% of the transcripts constituted mature VDJC mu recombinations. Ninety percent of the cDNA were sterile immunoglobulin transcripts comprised of: DJC mu (DH-JHC mu), JC mu (JH-C mu), EC mu (enhancer spliced to C mu), SC mu and IC mu [corresponding to switch (S) and intron (I) regions spliced to C mu]. In the mature immunoglobulin transcripts, VH use indicated germline expression with little evidence of somatic mutation. All cDNA were of the C mu type. Different D segments, D-D joining events and unknown D-like elements were noted in the DJC mu and VDJC mu transcripts. This pattern of immunoglobulin rearrangements, along with the phenotypic cell surface antigen characteristics (CD19-), suggest that an earlier arrest in B cell development than is characteristic of Bruton's X-linked agammaglobulinemia has occurred in this patient.

The immunoglobulin (Ig) heavy chain variable (VH) gene family of Heterodontus francisci (horned shark), a phylogenetically distant vertebrate, is unique in that VH, diversity (DH), joining (JH) and constant region (CH) gene segments are linked closely, in multiple individual clusters. The V regions of 12 genomic (liver and gonad) DNA clones have been sequenced completely and three organization patterns are evident: (i) VH-D1-D2-JH-CH with unique 12/22 and 12/12 spacers in the respective D recombination signal sequences (RSSs); VH and JH segments have 23 nucleotide (nt) spacers, (ii) VHDH-JH-CH, an unusual germline configuration with joined VH and DH segments and (iii) VHDHJH-CH, with all segmental elements being joined. The latter two configurations do not appear to be pseudogenes. Another VH-D1-D2-JH-CH gene possesses a D1 segment that is flanked by RSSs with 12 nt spacers and a D2 segment with 22/12 spacers. Based on the comparison of spleen, VH+ cDNA sequences to a germline consensus, it is evident that both DH segments as well as junctional and N-type diversity account for Ig variability. In this early vertebrate, the Ig genes share unique properties with higher vertebrate T-cell receptor as well as with Ig and may reflect the structure of a common ancestral antigen binding receptor gene.

A two-step procedure employing gel filtration and anion exchange chromatography has been utilized to isolate LMW immunoglobulin from the horned shark, Heterodontus francisci. Light chains obtained by complete reduction and alkylation of the parent protein have been compared by several analytical techniques. Amino acid composition data implies a limited degree of variation in the light chains isolated from individual animals. Polyacrylamide gel electrophoresis of the CNBr digests of the light chains reveal indistinguishable banding profiles of the major peptides. Isoelectric focusing indicates limited heterogeneity in the light chain spectrotype and identity in the pI of the majority of bands detectable by staining. The suggested degree of structural similarity in the light chains of this phylogenetically primitive shark is discussed in terms of the evolutionary position of the species and current theories concerning the origins of structural diversity in immunoglobulins.