nosaura defensor; Ctcl = Ctenosaura clarki). This fragment corresponds to the majority of exon 3, the entire intron 3, and a small portion of exon 4 in the Amcr-UA sequence, and was amplified in two separate but overlapping fragments. The first fragment, which spans exon 3 and the first part of intron 3, is identical to the one amplified in the population sample of Amblyrhynchus, and was generated using the same PCR protocol described above. The second fragment covers the second part of intron 3 and the very beginning of exon 4. The forward primer used to amplify this fragment was the same for all taxa (MHC1-Int-F2), but a different reverse primer (MHC1-R4) was used to amplify Amblyrhynchus and Conolophus specimens than for the other species (MHC1-R6). The PCR reagent concentrations for both primer pairs were the same as for the exon 3/intron 3 fragment, but the cycling conditions for the MHC1-Int-F2/MHC1-R4 primer combination were as follows: initial denat at 94uC for 5 min, followed by 35 cycles with 40 s of denat at 94uC, 40 s of annealing at 60uC, and 2 min 30 s ext at 72uC; and a final ext of 72uC for 15 min. The PCR cycling conditions for the MHC1-Int-F2/MHC1-R6 primer combination were 94uC for 5 min for initial denat, followed by 35 cycles with 45 s of denat at 94uC, 45 s of annealing at 55uC, and 60 s ext at 72uC; and a final ext of 72uC for 10 min. Data analysis Sequences from the Amcr-UA genomic fragment were aligned in the program SEQUENCHER 4.2.2 (Gene Codes Corporation, Ann Arbor, MI, USA). Comparison of the genomic and cDNA sequences was used to identify the exon/intron structure of Amcr- UA. The program MUSCLE v3.6 [21] was used to produce full- length amino acid and exon 4 (a-3 domain) nucleotide alignments for Amcr-UA and the following vertebrate class I sequences from GenBank: Gala´pagos marine iguana (A. cristatus), Amcr-UB*01 EU604308, Amcr-UB*02 EU604309, Amcr-UB*03 EU604310, Amcr-UB*0401 EU604311, Amcr-UB*0402 EU604312; Gala´pagos land iguana (C. subcristatus), Cosu-UB*0101 EU604313, Cosu- UB*0102 EU604314, Cosu-UB*02 EU604315, Cosu-UB*03 EU604316; Green iguana (Iguana iguana), Igig-UB*0101 EU604317, Igig-UB*0102 EU604318, Igig-UB*02 EU604319; Ameiva lizard, LC5 M81095, LC25 M91097; Northern water snake (Nerodia sipedon), SC1 M81099; Chinese soft-shelled turtle (Pelodiscus sinensis), AB185243; Chicken (Gallus gallus), B-F10 X12780; Mallard (Anas platyrhynchos), Du2 AB115242; Great reed warbler (Acrocephalus arundinaceus), cN3 AJ005503; Axolotl (Ambys- toma mexicanum), Amme-3 U83137; African clawed frog (Xenopus laevis), UAA-1f L20733; Mouse (Mus musculus), H2K L36312, H2-D1 NM_010380, H2-Q1 NM_010390, H2-Q10 NM_010391; Walla- by (Macropus rufogriseus), Maru-UB*01 L04952; Platypus (Ornithor- hynchus anatinus), Oran2-1 AY112715; Possum (Trichosurus vulpecula), Trvu-UB AF359509; Rainbow trout (Oncorhynchus mykiss), Onmy- UBA AF287487; Zebrafish (Danio rerio), Dare-UBA NM131471; Human, HLA-B7 U29057, HLA-Cw D50852. This set of sequences is similar to the one used in a study of tuatara class I genes by Miller et al. [22] as well as the recent study of iguanine class Ia loci [16], and was chosen for consistency. For the protein data, percent identity between Amcr-UA and other vertebrate class I genes was derived from p-distance values calculated separately for each structural domain in the program MEGA 4.0 [23]. Conserved vertebrate class I amino acid positions were identified following Kaufman et al. [24]. The exon 4 alignment provided the basis for Bayesian, maximum likelihood (ML), and neighbor-joining (NJ) phylogenetic reconstruction. The program MRMODELTEST v2 [25], which is based on code from the MODELTEST software [26], was used to Marine Iguana Class I compare the fit of different nucleotide substitution models to the vertebrate dataset. The general time reversible model (GTR) with additional parameters for gamma distribution and fraction of invariable sites provided the best fit to the data according to both the hierarchical likelihood ratio test and the Akaike information criterion. This model was implemented in a Bayesian framework using the program MRBAYES [27] as well as in ML reconstruction using the TREEFINDER software [28]. For Bayesian analysis, the default software settings were used, and the search was run for 2,000,000 generations with the first 10% of parameter samples discarded as burn-in. In the ML analysis, 1,000 bootstrap replicates were run to assess support for specific nodes. NJ search and bootstrap analysis were carried out in PAUP using the GTR substitution model with 500 replicates. Sequence fragments spanning the majority of exon 3 and all of intron 3 in the seven iguanine species were aligned in MEGA, and p-distance values were calculated separately for the exon and intron. Sequences generated in this study were deposited in GenBank under the following accession numbers.: EU839663 (full-length Amcr-UA cDNA); EU839664 (Amcr-UA genomic fragment); EU839665-EU839670 (Iguaninae exon 3/intron 3). Results Characterization of cDNA sequences from Amblyrhynchus cristatus Multiple clones were sequenced from PCR products generated by both 59and 39 RACE of marine iguana cDNA. For 39 RACE, resulting clones carried two unique class I-like sequences. One of these became the subjected of another study [16], while the other was the focus of this paper, and was used to design specific 59 RACE primers. The sequence of this 39 RACE clone was identical in the area of overlap with the single class I fragment obtained from 59 RACE. When aligned, these sequences comprised a 1,294 bp fragment which spanned the complete coding sequence (CDS) as well as the 59 and 39 UTRs of a single class I sequence type (Figure 1). This gene was labeled Amcr-UA based on established nomenclature rules [29]. Although the 39 UTR sequence reaches the site of polyadenylation, neither of the canonical polyadenylation signals (AATAAA or ATTAAA) were found. However, there are several alternative motifs at a range of positions in the 39 UTR that are known to be involved in polyadenylation, including AATACA, AATATA, GATAAA or ATAAA, and AAGAAA, which are located 64, 101, 131, and 150 bp upstream of the poly(A) region respectively (Figure 2; see [30–34] for characterization of alternative polyadenylation sites). One of the most striking features of the Amcr-UA sequence is the absence of the transmembrane (Tm) and cytoplasmic (Cyt) domains that are characteristic of most classical and nonclassical class I MHC genes. The CDS extends only 11 amino acid residues downstream from the expected end of the a-3 domain. The first three of these residues (EEP) are identical to the first three Tm domain residues of the classical UB genes from the three iguanine species as well as the Ameiva lizard (LC1) (Figure 3); but the other eight residues have no clear homology to any of the other loci. There was no evidence of a longer 39 RACE fragment that might contain a full-length version of the truncated Amcr-UA sequence. While the structure and composition of class I genes are quite variable within and between vertebrate groups, the Amcr-UA sequence possesses many of the conserved amino acid residues that are characteristic of class I genes, and even some residues that are common in classical class I genes (indicated by asterisks in Figure 3). For example, there are nine class I a chain residues PLoS ONE | www.plosone.org 4 August 2008 | Volume 3 | Issue 8 | e2859