Marine Iguana Class I Figure 2. 39 sequence map of the Amcr-UA fragment generated from cDNA. Possible polyadenylation sites are labeled with a solid underline. The asterisk marks the termination signal at the end of exon 4 (a-3 domain). The eleven amino acid residues that mark the unusual extension of the a- 3 domain are labeled above their respective coding nucleotide sequences. The sequence of primer MHC17H3-5R-R1 is labeled with a broken underline; this was the reverse primer used to generate the genomic DNA sequence and shows how far the genomic sequence stretches downstream relative to the cDNA. doi:10.1371/journal.pone.0002859.g002 which are highly conserved across vertebrates and are known to be involved in the binding of N- and C-termini of antigenic peptides [24,35,36]. Seven of these residues are shared between the Amcr- UA sequence (positions in Amcr-UA: Y9, Y62, Y124, T144, W148, Y160, and Y175) and the common reference type found in the classical Human HLA-A, -B, and -C, molecules. Moreover, one of the two remaining positions (R87) matches the predominant character state in non-mammalian vertebrates, including lizard, fish, birds, tuatara, and amphibians. Several studies have used the presence of these conserved peptide-binding residues in different species to distinguish classical antigen-presenting class I genes from nonclassical class I genes with alternative functions [36–38]. Other features that are shared between the marine iguana sequence and nearly all class I genes of other vertebrates include four cysteine residues at positions 103, 165, 204, and 260, which are involved in intradomain disulfide bridge formation, two salt bridges at positions H5-D31 and H95-D120, and the highly conserved FYP motif at positions 209–211 in the a-3 domain. Class I genes of most species contain a NQS or NQT glycosylation site near the end of the a1 domain; however, the Amcr-UA sequence contains a histidine (H) rather than a glutamine (Q) at position 90, which is not the case in other vertebrates, including the classical sequences from the three iguanine species as well as the Ameiva lizard. But this residue is not known to be important for N-linked glycan formation. The amino acid residues which correspond to the CD8 binding site of the HLA-A locus are at positions 224–230 in the Amcr-UA fragment, but three of the seven residues are non-polar, which would be unexpected at these sites for a classical class I gene; however this region is known to be highly variable among species, and the coevolution of the CD8 ligand and its class I binding site is not well understood [24]. Genomic sequence of Amcr-UA Amplification of the Amcr-UA gene was carried out on marine iguana genomic DNA using specific primers designed in the 59 and 39 UTR of the cDNA sequence. The resulting PCR product was approximately 8 kb and was identical to the cDNA in all areas of overlap, except for a single nucleotide position in the expected signal peptide. Some degree of mismatch between the cDNA and genomic sequences was anticipated since these fragments were derived from different individuals. A small portion (,100 bp) of intron 1 in the amplified genomic fragment could not be sequenced from either direction after several attempts. Secondary structure analysis revealed that the ,80 bp before and after the unsequenced region are highly complementary (,80%) and could form a secondary loop. However, even after addition of DMSO, which is known to relax secondary structure during cycle sequencing, there was still a consistent and steep drop-off of chromatogram peaks in this region. The structure of the genomic fragment is shown in Figure 1. This sequence revealed that the 11 amino acid residues (plus stop codon) that extend from the expected end of the a-3 domain are encoded in the same exon as the a-3 domain itself (Figure 1). In other vertebrates, separate exons usually code for the Tm/Cyt and a-3 domains. Yet the ‘‘EEP’’ amino acid motif, which marks the beginning of the Tm/Cyt domain in the classical class I sequences from the three iguanine species and the Ameiva lizard, is part of the a-3 encoding exon in Amcr-UA, and might be the result of some type of genomic rearrangement. Besides the missing Tm/Cyt domains, the position of exons and introns are the same as in the majority of class I genes, where the signal peptide and each of the three extracellular domains (a-1, a- 2, and a-3) are encoded by separate exons with intervening introns. The missing fragment from intron 1 was estimated to be ,100 bp by comparing the band size to a DNA ladder on an agarose gel. Relationship of Amcr-UA to Class I sequences of other vertebrates Over the three extracellular domains, the amino acid identity between the Amcr-UA sequence and other published vertebrate class I genes ranges from 33% in the Rainbow trout (Oncorhynchus mykiss) to between 56.2% and 60.0% for the iguanine UB sequences (Table 2). Considered separately, the a-2 and a-3 domains are most similar to the classical iguanine sequences, but the a-1 domain is approximately as close to the tuatara (45.5%– 50.0%) and Ameiva lizard (46.7%) as it is to the iguanine UB sequences (43.5%–50%). Phylogenetic reconstruction was carried out on exon 4 nucleotide sequences from a similar set of taxa as in Miller et al. [22]. In this previous study, bootstrap values supporting many of the class I gene clusters among vertebrates were low, as is the case in our study when the Amcr-UA and iguanine UB fragments are included (Figure 4). However, there is strong nodal support for the monophyly of all squamate class I sequences (A. cristatus, C. subcristatus, I. iguana, A. ameiva, and N. sipedon) included in the PLoS ONE | www.plosone.org 5 August 2008 | Volume 3 | Issue 8 | e2859