Marine Iguana Class I Figure 1. Map of the Amcr-UA class I gene based on genomic and cDNA data. All exons are scaled relative to each other. Introns are also scaled relative to each other, but are more condensed than exons. Asterisk in the a-3 amino acid sequence represents the termination signal. Numbers above arrows show the position of primers used in this study and correspond to the numbering scheme in Table 1 – except for primer 11, which has been published elsewhere [18]. doi:10.1371/journal.pone.0002859.g001 dNTP, 0.5 mM gene-specific primer, 16 Phusion HF Buffer with MgCl2. Because the melting temperature of the gene-specific primer was .72uC, a two-step PCR was carried out with an initial denaturation (denat) of 98uC for 30 s followed by 30 cycles of 98uC for 10 s and 72uC for 60 s, and a final extension (ext) of 72uC for 10 min. Nested 39 RACE was carried out under the same conditions as the initial PCR using the gene-specific primer Ig- NC-F2, the nested oligo dT adaptor primer provided, and 1 ml of PCR product from the initial 39 RACE. Sequence data derived from 39 RACE products were then used to design two gene-specific reverse primers (MHC17H3-5R-R1 and MHC17H3-5R-R2) in the 39 UTR for use in 59 RACE. In the first round of PCR, an adaptor-specific primer provided by the kit was used in combination with the gene-specific primer MHC17H3-5R-R1. PCR was performed in a 50 ml reaction with the following reagents and concentrations: 2.5 U Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 200 mM of each dNTP, 0.2 mM gene-specific primer, 2 mM MgSO4, 16 High Fidelity PCR Buffer. Cycling conditions were as follows: initial denat at 94uC for 2 min followed by 5 cycles of 94uC for 30 s and 72uC for 2 min; 5 cycles of 94uC for 30 s and 70uC for 2 min; 23 cycles of 94uC for 30 s, 59uC for 30 s, and 68uC for 2 min; and a final ext for 10 min at 68uC. The nested 59 RACE step was carried out with the nested 59 adaptor primer and the primer MHC17H3- 5R-R2 under the following conditions: initial denat at 94uC for 2 min; 25 cycles with 30 s of denat at 94uC, 30 s of annealing at 61uC, 2 min ext at 68uC; and a final ext of 68uC for 20 min. The nested RACE products from both the 59 and 39 procedures were ligated into the pCR4-TOPO vector, transformed, and grown overnight on LB plates following the manufacturer’s protocol for the TOPO TA Cloning Kit for Sequencing (Invitrogen). Approximately ten clones each from 59 and 39 RACE were added to 25 ml of water and heated at 94uC for 10 min to lyse cells. 1 ml of this solution was added to a 25 ml PCR reaction containing 1.5 U AmpliTaq Gold DNA Polymerase (Applied Biosystems, Foster City, CA, USA), 200 mM of each dNTP, 1 mM standard T3 and T7 primers, 1.5 mM MgCl2, and 16 PCR Buffer without MgCl2. Cycling conditions were as follows: initial denat at 94uC for 10 min, followed by 30 cycles with 30 s of denat at 94uC, 30 s of annealing at 55uC, and 90 s ext at 72uC; and a final ext of 72uC for 10 min. PCR products were purified by filtration using either the QIAquick PCR purification kit (Qiagen; Valencia, CA, USA) or Millipore (Billerica, MA, USA) filter plate and sequenced using a 3730 DNA Analyzer (Applied Biosystems). PCR amplification of Amcr-UA from genomic DNA RACE products yielded a complete coding class I sequence designated Amcr-UA. (see Results). In order to amplify this gene from genomic DNA, total DNA was extracted from the whole blood of a single marine iguana using standard phenol-chloroform extraction [19]. Primers designed in the 59 and 39 UTR of the marine iguana cDNA sequence were used to amplify the corresponding genomic fragment from the total DNA (Table 1). This individual was not the same as the one used in RNA extraction and RACE but did come from the same population (Santa Cruz) and is likely to be closely related. PCR conditions were similar to those used in 39 RACE with the Phusion enzyme, except that the two-step ext time was increased to 4 min. Due to the large size of the resultant fragment (,8 kb), numerous internal primers (available upon request) had to be designed in addition to the original PCR primers in order to obtain the complete sequence. Population-level data of Amcr-UA PCR targeting a portion of exon 3, which corresponds to the a-2 domain, and intron 3 was performed on genomic DNA from 65 marine iguanas representing three different islands in the Gala´pagos archipelago: Santa Cruz, N = 31; Isabela, N = 18; Fernandina, N = 16. Exon 3 was targeted because it is known to be highly variable in class Ia genes and contains sites that are involved in antigen recognition [20]. Therefore, we felt that the presence or absence of nucleotide variation here would be satisfactory for determining whether this gene is polymorphic or not. The forward primer described in Radtkey et al. [18] was used in combination with the primer MHCI-Int-R1 in a 25 ml PCR reaction with the following reagents and conditions: 2.5 U AmpliTaq DNA Poly- merase (Applied Biosystems), 200 mM of each dNTP, 1 mM of each primer, 1.5 mM MgCl2, and 16 PCR Buffer without MgCl2. Cycling conditions were as follows: initial denat at 94uC for 5 min, followed by 33 cycles with 30 s of denat at 94uC, 30 s of annealing at 59uC, and 60 s ext at 72uC; and a final ext of 72uC for 10 min. PCR products were purified and sequenced as described above. Iguaninae species data collection A genomic fragment homologous to the Amcr-UA sequence was targeted in six other species in the same subfamily as Amblyrhynchus (Iguaninae): Conolophus subcristatus, Cyclura carinata, Cyclura cornuta, Cyclura rileyi, Ctenosaura defensor, and Ctenosaura clarki (for all table and figures, taxa are labeled with the following abbreviations: Amcr = Amblyrhynchus cristatus; Cosu = Conolophus subcristatus; Cyco = - Cyclura cornuta; Cyca = Cyclura carinata; Cyri = Cyclura rileyi; Ctde = Cte- PLoS ONE | www.plosone.org 3 August 2008 | Volume 3 | Issue 8 | e2859