MBE Advance Access originally published online on January 24, 2006
Molecular Biology and Evolution 2006 23(5):949-956; doi:10.1093/molbev/msj099
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Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005 |
MHC Class I Genes in the Tuatara (Sphenodon spp.): Evolution of the MHC in an Ancient Reptilian Order
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* Allan Wilson Centre for Molecular Ecology and Evolution, School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand; and Evolutionary Biology Unit, Australian Museum, Sydney, Australia
E-mail: hilary.miller{at}vuw.ac.nz.
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Abstract |
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The major histocompatibility complex (MHC) is an extremely dynamic region of the genome, characterized by high polymorphism and frequent gene duplications and rearrangements. This has resulted in considerable differences in MHC organization and evolution among vertebrate lineages, particularly between birds and mammals. As nonavian reptiles are ancestral to both mammals and birds, they occupy an important phylogenetic position for understanding these differences. However, little is known about reptile MHC genes. To address this, we have characterized MHC class I sequences from the tuatara (Sphenodon spp.), the last survivor of an ancient order of reptiles, Sphenodontia. We isolated two different class I cDNA sequences, which share 93% sequence similarity with each other but are highly divergent from other vertebrate MHC genes. Southern blotting and polymerase chain reaction amplification of class I sequences from seven adult tuatara plus a family group indicate that these sequences represent at least two to three loci. Preliminary analysis of variation among individuals from an island population of tuatara indicates that these loci are highly polymorphic. Maximum likelihood analysis of reptile MHC class I sequences indicates that gene duplication has occurred within reptilian orders. However, the evolutionary relationships among sequences from different reptilian orders cannot be resolved, reflecting the antiquity of the major reptile lineages.
Key Words: MHC class I • reptile • Sphenodon • gene duplication • polymorphism
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Introduction |
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TOPAbstractIntroductionMethodsResultsDiscussionAcknowledgementsReferences |
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The major histocompatibility complex (MHC) is a highly variable multigene family found in all jawed vertebrates (Hedrick 1994
). The classical class I and II MHC genes play a central role in disease resistance as they encode cell-surface glycoproteins that present short peptides, usually of bacterial or viral origin, to T cells (Klein 1986). The high polymorphism observed at classical MHC loci is thought to result from balancing selection driven by past and present exposure to disease (Meyer and Thomson 2001). Class I molecules comprise a single polypeptide chain, encoded by a single MHC gene, which is associated with a ß2-microglobulin molecule, whereas class II molecules form a heterodimer with each polypeptide chain encoded by a separate gene.
The MHC region is thought to evolve under a birth-and-death model, characterized by repeated gene duplication and loss (Nei, Gu, and Sitnikova 1997). This results in considerable variation in organization and copy number of MHC genes among vertebrate lineages (reviewed in Kelley, Walter, and Trowsdale 2005), particularly for class I genes, which appear to undergo more rapid turnover than class II genes (Takahashi, Rooney, and Nei 2000; Piontkivska and Nei 2003). There are major differences in MHC structure and complexity between nonmammalian and mammalian vertebrates (Kulski et al. 2002). In eutherian mammals, the MHC region is large (spanning 4 Mb in humans, MHC Sequencing Consortium 1999) and contains many duplicated genes and pseudogenes. In contrast, the chicken has a simple arrangement of MHC genes with few gene duplications (Kaufman et al. 1999), similar to that seen in ancient vertebrate lineages, such as the frog Xenopus laevis and the shark Ginglyostoma cirratum (Kulski et al. 2002; Kelley, Walter, and Trowsdale 2005). However, multiple duplications of both class I and II genes are evident in the MHC of other birds such as passerines (Westerdahl, Wittzell, and von Schantz 1999; Miller and Lambert 2004b) and quail (Shiina et al. 2004).
MHC genes have been extensively characterized in mammals, birds, amphibians, and fish, but few studies to date have reported MHC sequences from nonavian reptiles, and the genomic arrangement of MHC genes in this group is unknown. Nonavian reptiles are represented by four orders: Squamata (lizards and snakes), Sphenodontia (tuatara), Crocodylia (crocodilians), and Chelonia (turtles). Birds are within the class Reptilia as they form a monophyletic group with crocodilians. As sister taxa to both mammals and birds, nonavian reptiles provide the link between ancient ectothermic lineages (fish and amphibians) and the modern endotherms (mammals and birds). Thus, analysis of reptile MHC genes will enable us to gain insights into the ancestral arrangement of MHC genes from which all mammalian and bird sequences evolved and aid in determining when the simple MHC organization seen in ancient lineages changed to the more complex arrangement seen in eutherian mammals.
In this study, we aimed to investigate the evolutionary relationships between MHC class I sequences of tuatara (Sphenodon spp.) and those of other vertebrates. The tuatara is the last survivor of the order Sphenodontia, which are usually regarded as sister to squamates (Rest et al. 2003). Sphenodontids diverged from other reptiles about 230 MYA and were globally widespread until the late Cretaceous (65–80 MYA). They are now found only on the offshore islands of New Zealand and are represented by two species: Sphenodon guntheri, found only on North Brother Island, Cook Strait and Sphenodon punctatus which is present on islands in Cook Strait and off the northeastern coast of New Zealand's North Island (Daugherty et al. 1990). Stimulation of tuatara peripheral blood mononuclear cells (PBMCs) with a T-cell mitogen elicits a typical T-cell–dependent immune response (Burnham et al. 2005), indicating the presence of functional MHC genes, and previous work in our laboratory has found evidence for at least four expressed class II genes (Miller, Belov, and Daugherty 2005). As reptilian MHC class I sequences have previously only been reported for squamates (Grossberger and Parham 1992; Radtkey et al. 1996), our study helps fill an important gap in the evolutionary history of the MHC.
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Methods |
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Isolation of MHC Class I cDNA Sequences
MHC class I cDNA sequences were isolated from tuatara PBMC cDNA library, constructed from a single adult female tuatara (S. punctatus) as described in Miller, Belov, and Daugherty (2005)
. To construct a probe for screening the cDNA library, a fragment spanning 210 bp of MHC class I exon 3 was amplified from genomic DNA from a single adult male tuatara using degenerate primers (RepMHC1F1 5'-GTTSCAGYDGATGTDYGGCTGYGA-3' and RepMHC1R1 5'-CTCGABRCASIYBYSCTCCAGRTA-3') designed from aligned reptile (including birds), amphibian, fish, and mammalian MHC class I sequences. Polymerase chain reaction (PCR) amplification was performed in a 25-µl reaction containing 10 mM Tris-HCl, 50 mM KCl pH 8.3, 1.5 mM MgCl2, 200 µM each deoxyribonucleotide triphosphate (dNTP), 0.8 µM each primer, 0.5 units of Taq polymerase (Roche, Auckland, New Zealand), and 1 µl DNA, for 40 cycles of 94°C, 30 s; 60°C, 20 s; and 72°C, 30 s. PCR products were cloned into a pGEM-T Easy vector (Promega, Madison, Wisc.), and six positive clones were sequenced using the BigDye Terminator Cycle Sequencing kit (version 3.1) and analyzed on an ABI3730 Genetic Analyzer. All six clones of the exon 3 fragment had the same sequence, and similarity to MHC class I sequences was confirmed using Blast (National Center for Biotechnology Information). One positive clone (named tutMHC1ex3) was randomly selected for screening the cDNA library. Filters containing approximately 5 x 105 plaque-forming units were hybridized overnight with the tutMHC1ex3 probe and then washed as described in Miller, Belov, and Daugherty (2005). Positive clones were sequenced as described above using internal primers where necessary.
Southern Blot Hybridization
High molecular weight genomic DNA was isolated from whole blood sampled from wild S. punctatus from Stephens Island (Takapourewa), Cook Strait, and from animals housed at Victoria University of Wellington, which were derived from eggs taken from natural nests on Stephens Island. Samples from two S. guntheri individuals from North Brother Island, Cook Strait were also used. DNA was extracted using standard phenol-chloroform methods and digested overnight with PvuII. Southern hybridizations were performed as described previously (Miller, Belov, and Daugherty 2005), using the tutMHC1ex3 probe. Membranes were washed at 65°C in 2x standard saline citrate (SSC), 0.1% sodium dodecyl sulfate (SDS); followed by 1x SSC, 0.1% SDS, 30 min and 0.1x SSC, 0.1% SDS, 30 min; and then exposed to Kodak Biomax MR film for up to 2 weeks.
Analysis of MHC Class I Variation
MHC class I exon 2 sequences were amplified from genomic DNA of seven adult tuatara from Stephens Island plus a family group comprising mother, father, and four offspring. PCR products were amplified using the primers MHC1ex2F1 (5'-GCTATTTCTACACGGGGGTGTC-3') and MHC1ex2R1 (5'-CGGTCTGGCTCTGGTTGTAGC-3'). PCR amplification was performed in a 25-µl reaction using the Expand HiFi PCR system (Roche) with 1.5 mM MgCl2, 1 M betaine, 200 µM each dNTP, 0.4 µM each primer, and 1 µl genomic DNA, for 30 cycles of 95°C, 30 s; 55°C, 20 s; and 72°C 30 s. PCR products were cloned into a pGEM-T Easy vector (Promega), and 15–20 clones per individual were analyzed for MHC variation using single-strand conformation polymorphism (SSCP) analysis (Orita et al. 1989) prior to sequencing. SSCP analysis was performed as described in Miller and Lambert (2004a) using an 8% polyacrylamide gel (37.5:1 acrylamide:bisacrylamide) with 5% glycerol. Electrophoresis was carried out in 0.5x tris-borate-EDTA at 7 W for 5 h using a Protean II xi Vertical Electrophoresis Cell (BioRad, Auckland, New Zealand), and gels were stained with SYBR Gold (Molecular Probes, Eugene, Ore.). From each gel, one representative clone of each SSCP-banding pattern was chosen for sequencing. Plasmids were prepared using a HighPure plasmid purification kit and sequenced as described above. Sequences that differed by only 1–2 bp and could not be verified in an independent PCR were discarded as these are likely to have resulted from PCR errors.
Data Analysis
Sequences were edited and assembled using Sequencher 4.2 (GeneCodes Corporation, Ann Arbor, Mich.) and BioEdit 7.0.1 (Hall 1999). Sequences were aligned using ClustalX version 1.83 (Thompson et al. 1997). Amino acid alignments were performed using the BLOSUM matrix, with gap penalties (gap opening 10, gap extension 2) optimized using TuneClustalX (Hall 2004). Maximum likelihood trees were constructed using PHYML (Guindon and Gascuel 2003) using the general-time-reversible model with gamma-distributed rates. GenBank accession numbers of sequences used for comparisons are as follows: Ameiva lizard (Ameiva ameiva) M81094–M81097; water snake (Nerodia sipedon) SC1 M81099
[GenBank]
; Chinese soft-shelled turtle (Pelodiscus sinensis) AB185243; chicken (Gallus gallus) B-F10 X12780
[GenBank]
; great reed warbler (Acrocephalus arundinaceus) cN3 AJ005503; axolotl (Ambystoma mexicanum) Amme-3 U83137
[GenBank]
; African clawed frog (X. laevis) UAA-1f L20733
[GenBank]
; zebrafish (Danio rerio) Brre-UBA NM131471; rainbow trout (Oncorhynchus mykiss) Omny-UBA AF287487; nurse shark (Ginglymostoma cirratum) Gici-UAA AF220063; human HLA-B7 (Homo sapiens) U29057
[GenBank]
, HLA-Cw D50852
[GenBank]
, HLA-A U07161
[GenBank]
; mouse (Mus musculus) H2-D1 NM010380, H2-K XM207061, H2-Q1 NM010390; possum (Trichosurus vulpecula) Truv-UB AF359509; wallaby (Macropus rufogriseus) Maru-UB*01 L04952
[GenBank]
; and platypus (Ornithorhynchus anatinus) Oran2-1 AY112715. Tuatara sequences reported in this paper are available in the GenBank database under accession numbers DQ145777–DQ145789.
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Results |
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TOPAbstractIntroductionMethodsResultsDiscussionAcknowledgementsReferences |
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Isolation and Characterization of MHC Class I Sequences from Tuatara
Six positive clones containing full-length MHC class I sequences were isolated from the tuatara cDNA library after screening with the tutMHC1ex3 probe. Two different sequences were present among the six clones and were named Sppu-U*01 (1860 bp; clones T1.3, T1.7b, and T1.17) and Sppu-U*02 (1849 bp; clones T1.10, T1.12, and T1.16). Homology of the sequences to class I MHC genes was confirmed by protein-protein Blast searching. These clones spanned the entire coding region of 1065 bp plus 37 bp of the 5'-untranslated region (UTR) and the entire 3' UTR, including the polyadenylation signal.
Both Sppu-U*01 and Sppu-U*02 encode proteins of 355 amino acids, both of which contain the conserved cysteines, salt-bridge–forming residues, glycosylation site, and ß2-microglobulin contact residues expected of functional, classical MHC class I genes (fig. 1). The conserved residues that bind the antigenic peptide in classical class I MHC molecules are also present. As in other cold-blooded vertebrates (fish, amphibians, and lizards) examined to date, the putative CD8 contact site between amino acids 219–229 is not conserved (Sammut, Laurens, and Tournefier 1997).
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Sppu-U*01 and Sppu-U*02 have 93% nucleotide (87.6% amino acid) identity overall. The 3' UTR,
-3 (immunoglobulin like), and cytoplasmic/transmembrane domains of the two sequences are almost identical (95.1%, 98.8%, and 98.9% nucleotide identity, respectively). The -2 domain also had high similarity between clones (92.6% nucleotide identity), however, similarity in this domain with the tutMHC1ex3 probe, which was constructed from a different individual, was lower (80%–82% nucleotide identity). The -1 domain has lowest similarity with 79.7% nucleotide (68.1% amino acid) identity. Sppu-U*01 has an insertion of two amino acids between residues 13 and 14 in the -1 domain, however, its leader peptide is two amino acids shorter than that of Sppu-U*02, so the proteins are of the same length overall.
Relationship of Tuatara MHC Class I to Other Vertebrates
Sequence identities between Sppu-U*01 and other vertebrate MHC class I amino acid sequences are given in table 1. Percent amino acid identity ranges from 30.8% to 42.7% (43%–53.5% nucleotide identity). Highest sequence identity overall is with the Gici-UAA (Nurse shark) sequence. The only full-length nonavian reptile sequence available for comparison was the Ameiva lizard sequence LC1. This sequence had the highest identity to Sppu-U*01 in the -1 and -2 domains but did not have the highest overall identity to the tuatara sequences. A phylogenetic tree of exon 4 sequences reflects the high sequence divergence between the tuatara sequences and other vertebrates (fig. 2). Exon 4 was used for phylogenetic analysis as it codes for the immunoglobulin-like -3 domain, which is generally highly conserved, and also enabled the inclusion of sequences from the Chinese soft-shelled turtle P. sinensis and the water snake N. sipedon, for which only partial sequences were available. On this tree, all the reptile (including birds) and mammalian sequences form a monophyletic group, and where there are multiple sequences from within a reptilian order (e.g., for squamates and crocodilians/birds), these sequences cluster together with strong bootstrap support. However, bootstrap values for nodes separating each reptilian order are extremely low, so the relationships among major reptile lineages cannot be discerned.
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Southern Blot Analysis
Hybridization of the tutMHC1ex3 probe with PvuII-digested genomic DNA produced two to four strongly hybridizing bands plus up to three weakly hybridizing bands per individual (fig. 3). There was no evidence of fixed differences between S. punctatus and S. guntheri. As the probe tutMHC1ex3 has 80%–82% nucleotide identity with the cDNA sequences, the strongly hybridizing bands are likely to represent these loci and the weakly hybridizing bands may represent nonclassical class I genes.
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MHC Class I Variation in Tuatara
The primers MHC1ex2F1 and MHC1ex2R1 were designed from conserved sites within the cDNA sequences and amplify a product of either 252 or 258 bp. This product spans most of the
-1 domain (exon 2), including the 6-bp indel identified in the cDNA sequences. We used these primers to investigate whether these loci are polymorphic in seven adult tuatara from the Stephens Island population plus a family group comprising mother, father, and four offspring. The Stephens Island population was chosen for this analysis as it is large (30,000–50,000 individuals, Newman 1982) and has moderate levels of microsatellite DNA variation (Aitken et al. 2001). PCR products were cloned and then screened using SSCP, and up to six clones per individual were sequenced. A total of 11 different sequences, including sequences identical to those isolated from the cDNA library, were found among the seven Stephens Island tuatara (fig. 4), and each individual had a different genotype overall (table 2). Each individual had two to five different sequences, indicating that two to three class I loci are present. Most individuals had one to two "short" alleles of 252 bp plus one to two "long" alleles containing the 6-bp insertion. The exceptions to this were one individual that had two short alleles and three long alleles and another individual that had only two short alleles.
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We analyzed segregation of alleles in a family group to determine whether the 6-bp indel was locus specific. A possible scheme of inheritance is shown in table 2, where one locus contains both long and short alleles and the other locus has only short alleles. However, this analysis was somewhat inconclusive because of the similarity in genotypes of the parents and lack of variation in the offspring. It is also possible that the PCR primers do not amplify all alleles. The long alleles have much lower nucleotide diversity (p-distance = 0.099 ± 0.014) than the shorter alleles (p-distance = 0.272 ± 0.020) and form a monophyletic group with high bootstrap support in maximum likelihood analysis (fig. 5). This tree topology is not simply due to the presence of the indel as the topology is the same if the indel is removed. These data suggest that the long alleles are of more recent origin than the short alleles.
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Tests for balancing selection which compare nonsynonymous and synonymous substitution rates among peptide-binding (PB) and non-PB sites were not significant (data not shown), probably due to the small number of highly divergent sequences. However, there was some indication of balancing selection on PB sites among the long alleles, as 61.5% of putative PB sites have amino acid changes, compared with 30.5% of non-PB codons. When all alleles are considered, PB sites have only slightly more amino acid changes than non-PB sites (77% vs. 74.5%) due to the high diversity among sequences overall.
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Discussion |
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TOPAbstractIntroductionMethodsResultsDiscussionAcknowledgementsReferences |
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Nonavian reptiles occupy an important phylogenetic position for understanding the evolutionary history of MHC genes. However, the study of MHC genes in nonavian reptiles has lagged behind that of other vertebrate lineages, possibly due to the absence of a model organism for molecular and cellular studies in this group. In this study, we have characterized class I cDNA sequences from the tuatara, the sole extant representative of the ancient reptilian order Sphenodontia, and found evidence for at least two polymorphic class I genes.
We isolated two different sequences from low-stringency hybridization of a tuatara cDNA library. These sequences are highly divergent from all other vertebrate MHC sequences but appear to represent classical class I loci as they contain amino acid motifs which are conserved in classical loci. The two cDNA sequences have a high level of similarity with each other, with the exception of the variable peptide-binding -1 domain (exon 2), where a 6-bp indel is present. These cDNA sequences may represent either two alleles of one gene or two separate genes, although pedigree analysis (see below, and table 2) suggests that they are alleles of the same gene. Preliminary data from the Stephens Island population indicate high levels of polymorphism in exon 2 at class I loci, and amino acid diversity was highest at PB sites suggesting balancing selection typical of classical MHC genes. The sequence divergence between the tutMHC1ex3 probe and cDNA sequences suggests that exon 3 is also polymorphic between individuals.
Analysis of exon 2 sequences in tuatara from the Stephens Island population revealed two sets of alleles in most individuals: alleles with the 6-bp insertion and those without. However, it is difficult to assign these alleles to loci. Analysis of segregation of alleles within a family group suggested that the long alleles represent one gene, but that this gene also has short alleles. The long alleles appear to have been recently derived from the short alleles as they have much lower nucleotide diversity than the shorter alleles and still form a monophyletic group. However, the finding of a single individual with three long alleles (five alleles in total) indicates that there may be more than one locus containing alleles with the 6-bp insertion. As each of these alleles was also found in another individual, they are unlikely to be the result of PCR artefacts. We were also unable to amplify any long alleles from another individual. It is possible that there is polymorphism in gene number among individuals, as has been found in some instances (e.g., Malaga-Trillo et al. 1998). However, we cannot rule out the possibility that our primers do not amplify all alleles with equal efficiency and that these results reflect the stochastic nature of the PCR.
Overall, our data indicate that tuatara have at least two to three classical class I MHC genes. Southern blot analysis revealed two to four strongly hybridizing bands, and all individuals genotyped had between two and five alleles. No major differences in MHC complexity between S. guntheri and S. punctatus were apparent in the Southern blot analysis. The few studies to date on lizard MHC genes indicate that tuatara have fewer expressed class I genes than their sister group, the squamates. A study of cDNA sequences from the Ameiva lizard found evidence for at least four expressed loci (Grossberger and Parham 1992) and Southern blot-restriction fragment length polymorphism studies on sand lizards (Olsson et al. 2003) and geckos (Radtkey et al. 1996) showed a higher number of hybridizing bands than we observed for tuatara (4 to 11 hybridizing bands for geckos and an average of 11 bands for sand lizards). This supports the hypothesis that ancient lineages have fewer genes because tuatara are remnants of a more ancient radiation of reptiles than the lizards. However, the tuatara MHC does not appear to have a simple organization overall, as we have previously found multiple duplicated copies of the classical class II genes (Miller, Belov, and Daugherty 2005).
Phylogenetic analysis of vertebrate MHC class I sequences shows that sequences within reptilian orders cluster together, but relationships among the different reptilian orders cannot be resolved. The tuatara sequences form a clearly separate clade from those of squamates. This result, along with the difference in gene number between lizards and tuatara described above, indicates that duplication of loci within orders has occurred after their split from a common ancestor approximately 230 MYA (Rest et al. 2003). Duplication of loci within phylogenetic lineages is a hallmark of birth-and-death evolution and has been documented in many other phylogenetic groups, for example, primates (Piontkivska and Nei 2003), teleost fish (Miller, Kaukinen, and Schulze 2002), and birds (Hess and Edwards 2002), where gene duplication within genera or species is common. Among the reptile lineages, more DNA sequence data from a range of species are required to determine the extent of gene duplications and whether they have occurred early or late in the evolution of each lineage.
Over longer evolutionary timescales, the relationships among class I sequences cannot be resolved. There is generally high sequence divergence between the tuatara sequences and those of other vertebrates. Surprisingly, the tuatara sequences had highest identity with the nurse shark Gici-UAA locus, with 42.7% amino acid identity overall (compared with 30%–40% identity with all other vertebrate sequences, including those from other reptiles). As the nurse shark represents the oldest class of jawed vertebrates, and hence the oldest MHC lineage (Ohta et al. 2000), this result suggests that the tuatara sequences have retained some characteristics of early ancestral MHC sequences. The lack of conservation among reptile MHC sequences is not surprising given the rapid evolution of class I genes and the age of the reptilian lineages. Under birth-and-death evolution, different genes may be duplicated and lost in different lineages, making it difficult to trace the evolutionary history of MHC genes throughout vertebrate evolution. The rate of gene duplication and loss is generally estimated to be higher for class I genes than class II genes (Nei, Gu, and Sitnikova 1997). For example, orthologous clusters of class II genes, estimated to have originated 170–200 MYA, have been identified in eutherian mammals (Takahashi, Rooney, and Nei 2000), whereas primate class I genes are estimated to have diverged 35–66 MYA (Piontkivska and Nei 2003). In general, class I orthologous loci cannot be identified among different mammalian orders, and differences in class I gene number and organization are common, sometimes even between genera (Kelley, Walter, and Trowsdale 2005). Reptiles split from their common ancestor with mammals approximately 300 MYA (Van Tuinen and Hadly 2004), and divergence of the major reptilian orders is thought to have occurred between 220 and 285 MYA (Rest et al. 2003). Therefore, it is unlikely that we will be able to identify orthologs of tuatara MHC sequences among other reptiles, because of the length of time the reptilian orders have been on separate evolutionary trajectories. If the rate of class I gene turnover is the same in reptiles as for mammals, identification of orthologous loci within reptile orders may also not be possible.
This study is a first step toward understanding the structure of the MHC in a nonavian reptile. Analysis of the genomic arrangement of MHC loci in tuatara plus characterization of additional MHC genes from other reptile lineages will be essential to understand the evolutionary history of reptile MHC genes and to understand how the differences between the mammalian and avian MHC arose.
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Acknowledgements |
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This research was approved by Victoria University of Wellington Animal Ethics Committee (2003R14 and 2003R16), the New Zealand Department of Conservation (WE/51/FAU and LIZ0410), and the Environmental Risk Management Authority (GMD03106). We thank Nicky Nelson, Sue Keall, Kim Burnham, and Anne Laflamme for assistance with sample preparation, Michael Green for laboratory assistance, Don Colgan and the staff of the Evolutionary Biology Unit, Australian Museum for providing laboratory facilities. We acknowledge the support of the Ngati Koata no Rangitoto ki te Tonga Trust and the Te Atiawa Trust. We also thank the organizers and participants of the 2005 Society for Molecular Biology and Evolution Young Investigators Workshop.
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Footnotes |
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1 Present address: Centre for Advanced Technologies in Animal Genetics and Reproduction, Faculty of Veterinary Science, University of Sydney, Sydney, Australia.
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References |
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