Evidence for multiple distinct major histocompatibility complex class I lineages in teleostean fish

In the context of studies on the expression of MhcCyca-Z sequences of the common carp, PCR amplifications of exon 4 were performed on cDNA obtained from pooled thymi of 20 carp F1 individuals. Five recombinant clones (Cyca-TC3, -TC13, -TC15, -TC17 and -TC18) were found to be 96% similar to the exon 4 region of Cyca-ZA1. Each of the five sequences was unique, and differed in a few positions in both the nucleotide and the derived amino acid sequences from any of the Cyca-Z sequences known to date. These data suggest that multiple Z genes per locus are present in the carp, which are transcribed in the thymus. In the course of analysing the amplified Cyca-Z sequences, serendipity yielded a clone, Cyca-TC16, containing a class I-like sequence substantially different from any other carp class I sequence. The predicted amino acid sequence of Cyca-TC16 was most similar to the class I genes (Lach-U) from the coelacanth (42-46% amino acid identity). Cyca-TC16 contains three conserved beta 2-microglobulin contact residues, and the secondary structure was predicted by computer algorithms to be similar to that of the alpha 3 domain of HLA-A2. Phylogenetic analysis shows that carp class I sequences reside in four distinct clusters: (i) Cyca-Z, Cyca-TC3, -TC13, -TC15, -TC17 and -TC18 together with Caau-Z from ginbuna crucian carp, (ii) Cyca-U with Bree-U (zebrafish) and Sasa-p30 (Atlantic salmon), (iii) Cyca-TC16 with Lach-U (coelacanth), and (iv) Cyca-C4.