related taxa, often possess exclusive sets of paralogous genes, it appears that class I lineages have undergone repeated, indepen- dent expansion and diversification events over the course of vertebrate evolution. This pattern has been shown to be congruent with a birth-and-death model of evolution, where loci are frequently duplicated and lost, even over short timescales [9]. However, concerted evolution is also thought to contribute to the close relationship of class I genes within species by the homogenization of even divergent lineages through inter-locus gene conversion [10,11]. Although the function of most nonclas- sical genes is not well understood, their presence within these independently expanded class I clades is a testament to their importance in vertebrate immunity. Since class I evolution is known to be highly erratic, large taxonomic gaps in MHC characterization prevent the examina- tion of the events and processes which have led to class I diversification. Therefore, a comparative phylogenetic approach may serve as an important tool for understanding class I history. This was recently demonstrated in the gray short-tailed opossum (Monodelphis domestica) where 11 class I loci were shown to have diverged after the split of marsupials from other mammals, yielding insight into the timing and breadth of class I differenti- ation within this lineage [12]. Among vertebrates, non-avian reptiles are the most poorly represented taxon in terms of MHC data. For example, the order Squamata, which includes lizards and snakes, contains over 7,000 species that have been independently evolving for over 200 million years [13–15]. Yet, only a single study characterizing full-length class I genes has been published in the last 15 years [16]. Squamates, together with the only two extant species in the order Sphenodontia, make up the superorder Lepidosauria, which is the other main lineage of amniotes in addition to archosaurs (birds and crocodilians) and mammals. Given the vast differences in the organization of mammalian and avian MHC regions [11,17], the description of squamate class I genes can therefore provide a firmer phylogenetic basis upon which to reconstruct the characteristics of the ancestral amniote MHC, an important launching point for understanding how the mode of MHC evolution differs among major vertebrate lineages. In this study, we use both cDNA and genomic sequence data to characterize a class I gene from the Gala´pagos marine iguana (Amblyrhynchus cristatus), a member of the squamate subfamily Iguaninae. We also present partial fragments of a similar gene Table 1. Gene-specific primers used in this study. Primer Name (1) MHCI-R3 (2) IgNC-F1 (3) IgNC-F2 (4) MHC17H3-5R-R1 (5) MHC17H3-5R-R2 (6) MHC1-Gr2-F1 (7) MHCI-Int-R1 (8) MHC1-Int2-F2 (9) MHC1-R4 (10) MHC1-R6 Sequence (59 to 39) ASGTAYYTCBBCAGCCACT TGYGAGCTGAGGAAAGATGGGAGCATAG TTACCARTGTGCTTATGATGGGAGGGAC TTCATGCAGTCCAAAGGCAGCAG TCTTCTGCCTTGCTTCTGTCAAATATGGAG ACCGAGAGGGTTGAGCTGGAGAG TGAGGCTGASAGAGTGTAATCCTCCC AGRTGTRTGTAATATATCATCCAGG CAGRCYGGYRTGMTCCACRCGSCAC TCTCCTTGGGGTAGAAGCCRTC Numbers before primer refer to location in the gene as indicated by arrows in Figure 1. Footnote: See Materials and methods for Iguaninae taxa abbreviations. doi:10.1371/journal.pone.0002859.t001 Marine Iguana Class I from other iguanine species. The marine iguana sequence possesses several hallmarks of a nonclassical locus and can serve as the basis for more detailed functional studies of class I genes in this group. We compare this sequence to the few others available from squamates, including those from a recent study of class Ia genes from marine iguanas and two other iguanines, the Gala´pagos land iguana (Conolophus subcristatus) and the common green iguana (Iguana iguana) [16]. The information provided here will hopefully aid in the collection of additional class I data from squamates and fill in the vast phylogenetic gap that is missing in our understanding of amniote MHC evolution. Materials and Methods RNA isolation and cDNA synthesis Total RNA was isolated from the blood of a single marine iguana from the island of Santa Cruz, Gala´pagos, using the Tri Reagent BD kit (Molecular Research Center, Cincinnati, OH, USA) and the protocol provided. Complementary DNA (cDNA) was synthesized using the SuperScript III RT enzyme and reagents provided in the GeneRacer kit (Invitrogen, Carlsbad, CA, USA). The complete kit protocol was followed for generating first-strand cDNA pools with intact 59 ends except that both random hexamers and the oligo dT primer provided in the kit were used in a single reverse transcription reaction to obtain full- length cDNA fragments. 59 and 39 RACE of marine iguana cDNA In order to design gene-specific primers for 39 RACE, a small fragment (201 base pairs [bp]) in the class I a-2 domain was amplified by PCR using a forward primer described in Radtkey et al. [18] and a degenerate primer (MHCI-R3) designed from an alignment of a range of vertebrate class I sequences (see Table 1 for primer list and sequences; see Figure 1 for primer locations). Sequences derived from the small, amplified fragment were used to design two forward-facing gene-specific primers for use in 39 RACE (IgNC-F1 and IgNC-F2). For the 39 RACE, nested PCR was performed on marine iguana first-strand cDNA. The first round of PCR utilized the oligo dT adaptor-specific primer provided in the GeneRacer kit and the primer Ig-NC-F1. The PCR was performed with the following reagents and concentrations: 1U Phusion High Fidelity DNA Polymerase (Finnzymes, Espoo, Finland), 200 mM of each Target Region Initial exon 3 genomic amplification 39 RACE 39 RACE (Nested) 59 RACE; Genomic fragment 59 RACE (Nested) Genomic fragment Iguaninae exon3/intron3 fragment Intron3/exon4 fragment (All Iguaninae) Intron3/exon4 fragment (Amcr; Cosu) Intron3/exon4 fragment (Cyco; Ctde; Cyca; Ctcl; Cyri) PLoS ONE | www.plosone.org 2 August 2008 | Volume 3 | Issue 8 | e2859