Ronda T. Litman

α, β, γ, and δ T Cell Antigen Receptor Genes Arose Early in Vertebrate Phylogeny

Abstract A series of products were amplified using a PCR strategy based on short minimally degenerate primers and R. eglanteria (clearnose skate) spleen cDNA as template. These products were used as probes to select corresponding cDNAs from a spleen cDNA library. The cDNA sequences exhibit significant identity with prototypic α, β, γ, and δ T cell antigen receptor (TCR) genes. Characterization of cDNAs reveals extensive variable region diversity, putative diversity segments, and varying degrees of junctional diversification. This demonstrates expression of both α/β and γ/δ TCR genes at an early level of vertebrate phylogeny and indicates that the three major known classes of rearranging antigen receptors were present in the common ancestor of the present-day jawed vertebrates.

The spotted gar genome illuminates vertebrate evolution and facilitates human-teleost comparisons

To connect human biology to fish biomedical models, we sequenced the genome of spotted gar (Lepisosteus oculatus), whose lineage diverged from teleosts before teleost genome duplication (TGD). The slowly evolving gar genome has conserved in content and size many entire chromosomes from bony vertebrate ancestors. Gar bridges teleosts to tetrapods by illuminating the evolution of immunity, mineralization and development (mediated, for example, by Hox, ParaHox and microRNA genes). Numerous conserved noncoding elements (CNEs; often cis regulatory) undetectable in direct human-teleost comparisons become apparent using gar: functional studies uncovered conserved roles for such cryptic CNEs, facilitating annotation of sequences identified in human genome-wide association studies. Transcriptomic analyses showed that the sums of expression domains and expression levels for duplicated teleost genes often approximate the patterns and levels of expression for gar genes, consistent with subfunctionalization. The gar genome provides a resource for understanding evolution after genome duplication, the origin of vertebrate genomes and the function of human regulatory sequences.

Immune-type receptor genes in zebrafish share genetic and functional properties with genes encoded by the mammalian leukocyte receptor cluster.

An extensive, highly diversified multigene family of novel immune-type receptor (nitr) genes has been defined in Danio rerio (zebrafish). The genes are predicted to encode type I transmembrane glycoproteins consisting of extracellular variable (V) and V-like C2 (V/C2) domains, a transmembrane region and a cytoplasmic tail. All of the genes examined encode immunoreceptor tyrosine-based inhibition motifs in the cytoplasmic tail. Radiation hybrid panel mapping and analysis of a deletion mutant line (b240) indicate that a minimum of ≈40 nitr genes are contiguous in the genome and span ≈0.6 Mb near the top of zebrafish linkage group 7. One flanking region of the nitr gene complex shares conserved synteny with a region of mouse chromosome 7, which shares conserved synteny with human 19q13.3-q13.4 that encodes the leukocyte receptor cluster. Antibody-induced crosslinking of Nitrs that have been introduced into a human natural killer cell line inhibits the phosphorylation of mitogen-activated protein kinase that is triggered by natural killer-sensitive tumor target cells. Nitrs likely represent intermediates in the evolution of the leukocyte receptor cluster.

Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor α in zebrafish.

Abstract. The zebrafish (Danio rerio) has become a significant model for understanding the developmental regulation of gene expression and holds considerable potential for characterizing the development of the immune system. Using a number of different approaches, including heterologous hybridization and short-primer PCR, cDNAs for three different classes of light-chain genes were identified and characterized. The zebrafish light chains are similar to trout type 1, trout type 2, and catfish type F, respectively. T-cell antigen receptor α (TCRα) was also identified and characterized. A high proportion of unusual transcripts including sterile transcripts, germline VJC transcripts, aberrant splice forms, and V-V transcripts were encountered in the immunoglobulin and TCR cDNAs examined. The light-chain and TCRα loci each consist of multiple families of V gene segments, apparent even from the small numbers of cDNAs of each isotype sequenced. The gene sequences reported provide an essential set of markers of both B- and T-cell lineages that will facilitate investigations of immune system development.

Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny

The prototypic chondrichthyan immunoglobulin (Ig) light chain type (type I) isolated from Heterodontus francisci (horned shark) has a clustered organization in which variable (V), joining (J), and constant (C) elements are in relatively close linkage (V-J-C). Using a polymerase chain reaction-based approach on a light chain peptide sequence from the holocephalan, Hydrolagus colliei (spotted ratfish), it was possible to isolate members of a second light chain gene family. A probe to this light chain (type II) detects homologs in two orders of elasmobranchs, Heterodontus, a galeomorph and Raja erinacea (little skate), a batoid, suggesting that this light chain type may be present throughout the cartilaginous fishes. In all cases, V, J, and C regions of the type II gene are arranged in closely linked clusters typical of all known Ig genes in cartilaginous fishes. All representatives of this type II gene family are joined in the germline. A third (kappa-like) light chain type from Heterodontus is described. These findings establish that a degree of light chain class complexity comparable to that of the mammals is present in the most phylogenetically distant extant jawed vertebrates and that the phenomenon of germline-joined (pre-rearranged) genes, described originally in the heavy chain genes of cartilaginous fishes, extends to light chain genes.

Extraordinary variation in a diversified family of immune-type receptor genes

Immune inhibitory receptor genes that encode a variable (V) region, a unique V-like C2 (V/C2) domain, a transmembrane region, and a cytoplasmic tail containing immunoreceptor tyrosine-based inhibition motifs (ITIMs) have been described previously in two lineages of bony fish. In the present study, eleven related genes encoding distinct structural forms have been identified in Ictalurus punctatus (channel catfish), a well characterized immunological model system that represents a third independent bony fish lineage. Each of the different genes encodes an N-terminal V region but differs in the number of extracellular Ig domains, number and location of joining (J) region-like motifs, presence of transmembrane regions, presence of charged residues in transmembrane regions, presence of cytoplasmic tails, and/or distribution of ITIM(s) within the cytoplasmic tails. Variation in the numbers of genomic copies of the different gene types, their patterns of expression, and relative levels of expression in mixed leukocyte cultures (MLC) is reported. V region-containing immune-type genes constitute a far more complex family than recognized originally and include individual members that might function in inhibitory or, potentially activatory manners.

Identification and characterization of T-cell antigen receptor-related genes in phylogenetically diverse vertebrate species.

Characterization of the structure, multiplicity, organization, and cell lineage-specific expression of T-cell receptor (TCR) genes of nonmammalian vertebrate species is central to the understanding of the evolutionary origins of rearranging genes of the vertebrate immune system. We recently described a polymerase chain reaction (PCR) strategy that relies on short sequence similarities shared by nearly all vertebrate TCR and immunoglobulin (Ig) variable (V) regions and have used this approach to isolate a TCR beta (TCRB) homolog from a cartilaginous fish. Using these short PCR products as probes in spleen cDNA and genomic libraries, we were able to isolate a variety of unique TCR and TCR-like genes. Here we report the identification and characterization of a chicken TCR gamma (TCRG) homolog, apparent Xenopus and pufferfish TCR alpha (TCRA) homologs, and two horned shark TCR delta (TCRD)-like genes. In addition, we have identified what could be a novel representative of the Ig gene super-family in the pufferfish. This method of using short, minimally degenerate PCR primers should speed progress in the phylogenetic investigations of the TCR and related genes and lend important insights into both the origins and functions of these unique gene systems.

Distinct patterns of IgH structure and organization in a divergent lineage of chrondrichthyan fishes

Abstract Immunoglobulin heavy chain (IgH) genes in representative chondrichthyan fishes (sharks and skates) consist of independently functioning clusters, containing separate variable (VH), diversity (DH), and joining (JH) region elements and constant (CH) region exons. IgH loci have been characterized in Hydrolagus colliei (spotted ratfish), a modern representative of a major independent chondrichthyan lineage. Three distinct families of IgH gene clusters were identified. The most numerous genes consist of unjoined VH-D1-D2-JH segments that correspond to the most abundant Hydrolagus spleen (cDNA) transcripts which apparently arise from a diversified gene family. In the second cluster type, VH, DH, and JH segments are germline-joined, whereas the CH exons exhibit typical organization. This gene type is found in only a few copies per haploid genome and both transmembrane and secretory transcripts have been identified. A third cluster type has been identified that consists of unjoined VH elements but lacks a typical CH1 exon, which is substituted with a second CH2-like exon. Transcripts from this third cluster type also appear to derive from a diversified gene family. Genomic D regions of the two unjoined clone types exhibit structural differences that are consistent with incorporation of recombination machinery-mediated events. Genomic library screening indicates that 90% of VH+ clones are truncated, nearly identical pseudogenes (lacking JH and CH). These studies demonstrate an early phylogenetic origin for the cluster type of gene organization and document extensive organizational diversification within an apparent single class of IgH genes.

A long form of the skate IgX gene exhibits a striking resemblance to the new shark IgW and IgNARC genes

Abstract Differential screening has been used to identify cDNAs encoding a long form of IgX in Raja eglanteria (clearnose skate). Comparisons of the IgX long form with the previously described short-form IgX cDNAs and the genomic IgX locus indicate that the V and two 5′C regions of the short and long forms of IgX are >90% identical at the nucleotide level. Differences between the V sequences of the long- and short-form IgX genes are concentrated in complementarity determining regions, suggesting that these forms are derived through alternative splicing of the same genomic loci or transcription of highly related loci. The extreme conservation of nucleotide sequence, including third position codons, among different cDNAs as well as the near identity of nucleotide sequence in the intervening sequences of germline IgX, IgX short-form sterile transcripts and IgX long-form sterile transcripts indicate that the multiple IgX loci are recently diverged from one another and/or are under intense gene correction. Phylogenetic analyses of the known cartilaginous fish immunoglobulin loci demonstrate that the long form of IgX is orthologous to IgW/IgNARC (NARC) and is most consistent with: 1) the divergence of the IgX/IgW/NARC and IgM-like loci from a common ancestral locus prior to the divergence of the cartilaginous/bony fish lineages and 2) the divergence of the NAR locus from the IgX/IgW/NARC gene(s) after the cartilaginous/bony fish split but prior to the shark/skate split, approximately 220 million years ago.

Genomic complexity of the variable region-containing chitin-binding proteins in amphioxus

BackgroundThe variable region-containing chitin-binding proteins (VCBPs) are found in protochordates and consist of two tandem immunoglobulin variable (V)-type domains and a chitin-binding domain. We previously have shown that these polymorphic genes, which primarily are expressed in the gut, exhibit characteristics of immune genes. In this report, we describe VCBP genomic organization and characterize adjacent and intervening genetic features which may influence both their polymorphism and complex transcriptional repertoire.ResultsVCBP genes 1, 2, 4, and 5 are encoded in a single contiguous gene-rich chromosomal region and VCBP3 is encoded in a separate locus. The VCBPs exhibit extensive haplotype variation, including copy number variation (CNV), indel polymorphism and a markedly elevated variation in repeat type and density. In at least one haplotype, inverted repeats occur more frequently than elsewhere in the genome. Multi-animal cDNA screening, as well as transcriptional profilingusing a novel transfection system, suggests that haplotype-specific transcriptional variants may contribute to VCBP genetic diversity.ConclusionThe availability of the Branchiostoma floridae genome (Joint Genome Institute, Brafl1), along with BAC and PAC screening and sequencing described here, reveal that the relatively limited number of VCBP genes present in the amphioxus genome exhibit exceptionally high haplotype variation. These VCBP haplotypes contribute a diverse pool of allelic variants, which includes gene copy number variation, pseudogenes, and other polymorphisms, while contributing secondary effects on gene transcription as well.