Search

Immune gene evolution can be critical to species survival in the face of infectious disease. In particular, polymorphism in the genes of the major histocompatibility complex (MHC) helps vertebrates combat novel and diverse pathogens by increasing the number of pathogen-derived proteins that can initiate the host's acquired immune response. In this study, we used a combination of presumably adaptive and neutral markers to investigate MHC evolution in populations of five salamander species within the Ambystoma velasci complex, a group consisting of 15 recently diverged species, several of which are endangered. We isolated 31 unique MHC class II β alleles from 75 total individuals from five species in this complex. MHC heterozygosity was significantly lower than expected for all five species, and we found no clear relationship between number of MHC alleles and species range, life history, or level of heterozygosity. We inferred a phylogeny representing the evolutionary history of Ambystoma MHC, with which we found signatures of positive selection on the overall gene, putative peptide-binding residues, and allelic lineages. We identified several instances of trans-species polymorphism, a hallmark of balancing selection observed in other groups of closely related species. In contrast, we did not detect comparable allelic diversity or signatures of selection on neutral loci. Additionally, we identified 17 supertypes among the 44 unique Ambystoma alleles, indicating that these sequences may encode functionally distinct MHC variants. We therefore have strong evidence that positive selection is a major evolutionary force driving patterns of MHC polymorphism in this recently radiated species complex.

In mammals, the T-cell receptor (TCR) complex expressed on mature T-cells consists of α/β or γ/δ clonotypic heterodimers non-covalently associated with four invariant chains forming the CD3 complex (CD3γ, CD3δ, CD3ε and CD3ζ). The TCR is the unit implicated in the antigenic peptide recognition whereas the CD3 subunits present as three different dimers (δ-ε, γ-ε and ζ-ζ) in the receptor complex participate to the signal transduction and are indispensable for the expression of the TCR at the cell surface. We report the cloning, characterization and expression analysis of CD3γ/δ and CD3ε genes in an amphibian urodele, the Mexican axolotl. Amino acid comparisons show that important motifs and residues were preserved between the axolotl CD3 chains and various vertebrate CD3ε, CD3γ, CD3δ and CD3γ/δ chains. During ontogeny, CD3ε transcripts are first detected in the dorsal region of tail-bud embryos before thymus organogenesis. CD3γ/δ transcripts are first detected in the head of 4-week-old larvae. A cross-reactive polyclonal anti-CD3ε antibody was used for the co-immunoprecipitation of the two CD3 proteins of 25 and 29 kDa, respectively, associated with the 90-kDa αβ TCR heterodimer.

Nontemplate (N)-nucleotide addition by the terminal dideoxynucleotidyl transferase (TdT) at the junctions of rearranging V( D) J gene segments greatly contribute to antigen-receptor diversity. TdT has been identified in several vertebrate species, where it is highly conserved. We report here the isolation of two forms of TdT mRNA in an amphibian, the Mexican axolotl. The isoform TdT1 shares all of the conserved structural motifs required for TdT activity and displays an average of 50-58% similarity at the amino acid level with TdT of other species. The second axolotl TdT variant ( TdT2) differs from TdT1 by a 57-amino acid deletion located between amino acids 165-222 of TdT1, including the first helix-hairpin-helix DNA-binding motif. During ontogeny, TdT products are first detected in the head of 6-week-old larvae and further in the head and trunk of 8-month-old larvae. These developmental stages correspond to the first detection of RAG1 and antigen-receptor (TCRbeta and IgHmicro) products in axolotl larvae. Our results suggest that in contrast to mammalian development, N diversity occurs early in axolotl development to diversify the primary repertoire. Phylogenetic analyses reveal that TdT and DNA polymerase mu(Pol mu) genes are closely related, and that both enzymes were already present in the common ancestor of jawed vertebrates.

We report here the structure of cDNA clones encoding axolotl light chains of the lambda type. A single IGLC gene and eight different potential IGLV genes belonging to four different families were detected. The axolotl Cgamma domain has several residues or stretches of residues that are typically conserved in mammalian, avian, and Xenopus Cgamma, but the KATLVCL stretch, which is well conserved in the Cgamma and T-cell receptor Cbeta domains of many vertebrate species, is not well conserved. All axolotl Vgamma sequences closely match several human and Xenopus Vgamma-like sequences and, although the axolotl Cgamma and Vgamma sequences are very like their tetrapod homologues, they are not closely related to nontetrapod L chains. Southern blot experiments suggested the presence of a single IGLC gene and of a limited number of IGLV genes, and analysis of IGLV-J junctions clearly indicated that at least three of the IGLJ segments can associate with IGLV1, IGLV2, or IGLV3 subgroup genes. The overall diversity of the axolotl Vgamma CDR3 junctions seems to be of the same order as that of mammalian Vgamma chains. However, a single IGLV4 segment was found among the 45 cDNAs analyzed. This suggests that the axolotl IGL locus may have a canonical tandem structure, like the mammalian IGK or IGH loci. Immunofluorescence, immunoblotting, and microsequencing experiments strongly suggested that most, if not all L chains are of the gamma type. This may explain in part the poor humoral response of the axolotl.

The developmental expression of the RAG1 gene in the Mexican axolotl hematopoietic organs was studied. RAG1 mRNAs were first detected in trunk extracts from 6-week-old larvae, and in head and trunk extracts of 8- and 9-week-old larvae. RAG1 is expressed in the thymus at all stages of development, until its natural involution after 12 months of age. In contrast, although RAG1 transcripts were present in the spleen and liver of the young larvae, they were not detected in the liver after 4.5 months and in the spleen after 8 months. No RAG1 mRNA expression was observed in the spleens or livers of 24-month-old hyperimmunized axolotls. The developmental expression of the RAG2 protein was also analyzed in axolotl thymus, spleen, and liver extracts using specific anti-RAG2 antibodies. RAG2 was readily detected at 7 months, but not in hematopoietic organs of 12- and 24-month-old axolotls. The presence of RAG1 transcripts was limited to the sub-capsular area of the thymus lobes, as detected by in situ hybridization. Discrete clusters of labeled cells were observed in the spleen sections, and a relatively large number of labeled cells were located in the hepatic peripheral hematopoietic layer of 3-month-old axolotls. The first appearance of RAG1 gene products in the axolotl hematopoietic organs is thus well correlated with the first production of rearranged T-cell and B-cell receptor mRNAs, 40-60 days after fertilization.

Class I major histocompatibility complex (Mhc) cDNA clones were isolated from axolotl mRNA by polymerase chain reaction (PCR) and by screening a cDNA phage library. The nucleotide and predicted amino acid sequences show definite similarities to the Mhc class Ialpha molecules of higher vertebrates. Most of the amino acids in the peptide binding region that dock peptides at their N and C termini in mammals are conserved. Several amino acids considered to be important for the interaction of beta2-microglobulin with the Mhc alpha chain are also conserved in the axolotl sequence. The fact that axolotl class I A cDNAs are ubiquitously expressed and highly polymorphic in the alpha1 and alpha2 domains suggests the classical nature of axolotl class I A genes.