Table 2. Percent amino acid identities between Amcr-UA and class I sequences from other vertebrates. Species Percent Identity with Amcr-UA a3 52.2 56.5 53.3 48.4 36.3 36.3 40.7 44.0 34.8 37.4 38.0 34.8 31.5 38.0 32.6 34.8 32.2 24.7 24.7 a1, a2, a3 56.2–57.8 57.8–60.0 57.8–59.5 53.3 44.1–45.6 43.1 47.4 44.4 44.4 41.9 45.2 45.7 42.4 44.3 43.2 45.8 39.2 33.0 34.8 a1 43.5–46.7 45.7–50.0 46.7–48.9 a2 74.7–75.8 68.1–75.8 73.6–76.7 45.5–50.0 50.5 46.7 40.9 45.5 43.2 43.2 44.3 43.7 44.8 43.7 40.0 40.9 46.7 41.4 27.6 64.8 52.2 56.0 46.2 54.9 44.0 53.8 57.8 52.2 54.9 56.0 56.0 44.0 40.7 51.6 Marine iguana (Amcr-UB) Land iguana (Cosu-UB) Green iguana (Igig-UB) Ameiva Lizard (LC1) Tuatara (Sppu-U) Chicken (Gaga-B-F10) Mallard (Anpl-Du2) Warbler (Acar-cN3) Axolotl (Amme-3) Xenopus (Xela-UAA-1f) Human (Hosa-HLA-A) Mouse (Mumu-Q10) Mouse (Mumu-H2K) Wallaby (Maru-UB*01) Platypus (Oran-2-1) Possum (Trvu-UB) Nurse Shark (Gici-UAA) Rainbow Trout (Onmy-UBA) 33.3 Zebrafish (Dare-UBA) Values were calculated for each extracellular domain separately and for all three domains combined. doi:10.1371/journal.pone.0002859.t002 Polymorphism of Amcr-UA in Amblyrhynchus cristatus populations The majority of exon 3 (248 bp) and the first 235 bp of intron 3 were amplified from 65 marine iguanas from three separate islands representing distinct parts of the Gala´pagos archipelago. Direct sequencing of PCR products for 62 of these individuals generated a single sequence with no double peaks, which is identical to the full-length genomic fragment. Due to the limited polymorphism exhibited at the population level, these data are not displayed graphically. But it is worth noting that two individuals possess a double peak at a single nucleotide position in exon 3, where one base matches the main Amcr-UA type and the other results in a nonsynonymous amino acid change (from threonine to serine at amino acid position 133). Another individual possesses double peaks at two nucleotide positions, one site in exon 3 and another in intron 3; here also, the change in the exon is nonsynonymous (from isoleucine to valine at amino acid position 168) Patterns of variation between Iguana species Primers designed on the Amcr-UA sequence successfully amplified the majority of exon 3 (248 bp) and the entire intron 3 (,1,030 bp) of six other species from the Iguaninae subfamily, including a representative of Conolophus (Gala´pagos land iguana), which is thought to be the sister genus of the monospecific Amblyhrynchus [39,40]. No stop codons were found in the amino acid sequence of any of the species examined. Uncorrected pairwise genetic distance (p-distance) between species was calculated separately for the exon and intron (Table 3). With the exception of Conolophus and Ctenosaura clarki, the p-distance was lower in exon 3 than in intron 3 for all pairwise comparisons, even Marine Iguana Class I though the a-2 domain is known to be polymorphic and under strong diversifying selection in classical class I genes [20]. There were 19 polymorphic nucleotide positions in the alignment of iguanine UA exon 3 sequences, but only five of these variable sites contributed to nonsynonymous differences between one or more species (Figure 5). As a result, only three of the 82 amino acid positions were polymorphic. While the low divergence of exon 3 compared to intron 3 suggests that the structure of this gene may be conserved, there was insufficient exon variation to carry out dN/dS-based analyses as a means of detecting purifying selection acting on this locus. Discussion The basic structure of class I loci is highly conserved across vertebrates. Typically, a single gene encodes a large a chain that contains separate exons for the two membrane-distal peptide-binding domains (a-1 and a-2), a third exon for the membrane-proximal Ig- like domain (a-3), and a number of additional, smaller exons that are responsible for the transmembrane (Tm) and cytoplasmic (Cyt) domains [1]. The Tm/Cyt domains are essential for the standard antigen presentation function of class I molecules because they anchor the receptor structure into the cell membrane of altered self- cells where they can be recognized by CD8+ TC cells [1,2]. However, in the Amcr-UA cDNA sequence, the Tm/Cyt domains are largely missing, except for an 11 amino acid extension of the a-3 domain that is followed by a stop codon. The first three residues of this extension are identical to the start of the Tm domain identified in iguanine UB and Ameiva lizard class I sequences, but the other eight residues show no apparent match between these sequences. Interestingly, the genomic data did not reveal the presence of a Tm/Cyt coding region in between the unexpected early stop codon and the downstream 39 UTR sequence, and there is no evidence of a cryptic, hidden, or vestigial splice site in the 39 end of the gene. Therefore, the lack of these domains in the cDNA is not likely to be a result of incomplete transcription or a splicing event. Rather, it seems apparent that the Tm/Cyt region is simply absent at this locus. One possible explanation for the match between the Amcr-UA a-3 extension and the start of the iguanine UB and Ameiva lizard Tm sequences (the ‘‘EEP’’ motif) is that the ancestor of Amcr-UA underwent a genomic rearrangement in which a small portion of the Tm-containing exon was transferred to the end of the a-3 encoding exon. Characterization and comparison of similar genes across other squamates may reveal the pattern and timing of such a modification. In particular, it would be interesting to obtain full- length sequences of Amcr-UA orthologues from the other closely related iguana species used in this study to determine whether they possess the same truncation at the 39 end of the CDS. However, it is also conceivable that intact exons coding for Tm/ Cyt domains do exist downstream of the available genomic sequence and simply are not transcribed, at least not at easily detectable levels in the blood. Therefore, future work should involve the use of large insert vectors to explore adjacent stretches of genomic DNA in order to rule out this possibility. In addition, it is equally important to measure expression levels of Amcr-UA in different tissues, and perform a more exhaustive search for a full-length version of this molecule containing a membrane-spanning domain. Despite the apparent lack of Tm/Cyt domains, which are necessary for classical MHC antigen presentation, the results presented here suggest that the Amcr-UA sequence represents a functional class I gene and not a pseudogene. The presence of the gene in the cDNA pool shows that it is expressed at some level in the blood. In addition, both the genomic and cDNA sequences do not contain any additional stop codons in the CDS, as might be PLoS ONE | www.plosone.org 7 August 2008 | Volume 3 | Issue 8 | e2859