Characterization of arrangement and expression of the T cell receptor gamma locus in the sandbar shark

Abstract

Ig and T cell receptor (TCR) genes consist of separate genomic elements, which must undergo rearrangement and joining before a functional protein can be expressed. Considerable plasticity in the genomic arrangement of these elements has occurred during the evolution of the immune system. In tetrapods, all Ig and TCR chain elements are arranged as translocons. In teleosts, the Ig heavy and TCR chains are translocons, but light chain genes may occur as clusters. However, in chondrichthyes, all of the Ig light and heavy chain genes are arranged as clusters. These clusters vary in number from <10 to several hundred, depending on isotype and species. Here, we report that the germ-line gene for the TCR gamma chain in a chondrichthyan, the sandbar shark (Carcharhinus plumbeus), is present as a single locus arranged in a classic translocon pattern. Thus, the shark utilizes 2 types of genomic arrangements, the unique cluster organization for Ig genes and the "conventional" translocon organization for TCR genes. The TCR gamma translocon contains at least 5 V region genes, 3 J segment genes, and 1 C segment. As expected, the third hypervariable segment (CDR3), formed by the rearrangement of the Vgamma and Jgamma segments, contributed the major variability in the intact V region structure. Our data also suggest that diversity may be generated by mutation in the V regions.

Fig. 1.

Sequence comparisons of the TCR γ C, J, and V regions identified in cDNA clones. (A) Derived amino acid sequence of the C region and comparison with skate (Raja eglanteria) and human sequences. Only one C region was found in the shark TCR γ cDNA. Identities with the shark sequence are shaded. Domains are indicated by arrows above the diagram, and were identified by comparison with the human sequence. Conserved residues in the TM domain are underlined. Percentage identities are listed on the right. (B) Three J families were identified by cDNA sequence analysis. (C) Five V families, designated GV1, GV2, GV3, GV4, and GV5, were identified by cDNA sequence analysis. Derived amino acid sequences are shown. Putative leader segments are shown in lowercase and were identified by using the SignalP 3.0 program (49). Identical residues are indicated by using both shaded and open boxes. The percentage identities are shown in the matrix on the right.

Fig. 2.

Southern blottings of the shark TCR γ locus. Sandbar shark spleen DNA was digested by BamHI (lane 1), EcoRI (lane 2), and HindIII (lane 3) independently. The probe was full-length C region isolated by PCR of cDNA.

Fig. 3.

Comparison of shark and human TCR γ loci. (A) Genomic map of the shark TCR γ locus. V, J, and C segments are arranged in a translocon. RSS sequences at the 3′ end of V segments and the 5′ end of the J regions are highlighted by white (RSS 23) and black (RSS 12) triangles. The RSS sequence at the 5′ end of J1 region is shown in gray, because it has not been possible to derive an accurate sequence for an ≈15-bp segment in this region because multiple PCR products show variation in length and sequence. We conclude that this variation represents PCR error. The genomic structure showing the intron present in all of the V segments is illustrated by using the GV2 segment. (B) Diagram comparing the layout of the shark and human TCR γ chain loci. Dark boxes are functional V regions, gray boxes are pseudogenes, vertical lines are J segments, and open boxes are constant regions. The depiction of the human gene is adapted from the Gene View page at the ImMunoGeneTics website (http://imgt.cines.fr).

Fig. 4.

Junctional diversity at the CDR3 segments of GV1-containing V regions. Sequences of shark TCR γ cDNA at the V/J junction of GV1 joined to all 3 J segments are shown. The parent genomic sequences are underlined above each group. Deletions (by endonuclease) are shown by asterisks, and nucleotides in lowercase indicate additions (by terminal deoxynucleotidyltransferase).

Fig. 5.

Phylogenetic tree, derived from C region comparisons, showing the relationship between TCR γ, Ig λ light chains, and TCR β chains. Sequences were aligned by using MAFFT (44, 45), and the tree was constructed with MEGA4 (48) using the neighbor-joining method (50). Percentage bootstrap values (1,000 replicates) are shown at the major interior branch points (for all of the bootstrap values, see Fig. S4). The horizontal length is proportional to the distance score generated by the computer program. The IgC1 domain amino acid sequences, corresponding to residues 1 to ≈125 using the IMGT numbering scheme (51), of λ light chains and TCR γ and β chains from different species were used to build this tree. The shark TCR γ sequence is from this work; all other sequences were obtained from GenBank.