Diversity and evolution of MHII β genes in a non-model percid species—The Eurasian perch (Perca fluviatilis L.)
Introduction
The major histocompatibility complex (MHC) multigene family is functionally involved in the innate and adaptive immune response (Klein, 1986a). In all jawed vertebrates studied to date, specific genes of the MHC encode glycoprotein receptors that constitute a central component of the vertebrate immune system (Flajnik and Du Pasquier, 2004). These highly polymorphic genes consist of two major subclasses – class I and class II genes (Klein, 1986a).
The class II receptors are heterodimers, consisting of two transmembrane proteins (α and β chain), which are encoded by separate genes. They predominantly present antigens from extra-cellular pathogens, such as bacteria or parasites (Trowsdale, 1993). For antigen presentation the antigenic peptides are anchored by specific amino acid (aa) residues (‘Peptide Binding Residues’, PBR) located in the extra-cellular antigen recognition site (ARS), which is encoded in exon II of the respective gene (Engelhard, 1994). The antigen/receptor complexes are then transported to the cell surface, thereby the antigen is presented to CD4+ bearing T-cells that finally trigger the adaptive immune response. MHC genes that differ at their PBR are able to bind a diverse array of antigens, and, hence, the variability of the PBR critically influences the individual immune response (Hedrick and Kim, 2000).
Homologous genes of both MHC gene classes can be found in representatives of all gnathostomata, and, hence, the proto-MHC likely originated at least 500 million years ago (Flajnik and Kasahara, 2001). The class I and class II genes are tightly linked in humans (The MHC sequencing consortium, 1999), and most other vertebrate classes (Kelley et al., 2005). The only known exception are the teleost fish, where class I and II genes are not linked (Bingulac-Popovic et al., 1997, Malaga-Trillo et al., 1998, Hansen et al., 1999, Sato et al., 2000, Sambrook et al., 2005), and, hence, in teleosts these genes are mostly referred to as major histocompatibility genes (MH genes), rather than MHComplex genes (Stet et al., 2003). Furthermore, when more than one class II locus occurs in a given teleost, they can be dispersed throughout the genome and even on separate chromosomes (Sambrook et al., 2002, Sambrook et al., 2005, Phillips et al., 2003). In general, the genomes of Euteleostei are characterized by an expansion of gene families (Robinson-Rechavi et al., 2001). This trend can also be observed in regard to the MH class II loci, since their number varies substantially among different teleost species, ranging from a single MHII β locus in salmonids (Shum et al., 2001, Stet et al., 2002), to presumably four loci in sticklebacks (Reusch and Langefors, 2005) to up to13 loci in cichlids (Malaga-Trillo et al., 1998).
Doherty and Zinkernagel (1975) first proposed a central role of pathogen mediated positive selection for the maintenance of the MHC diversity. Later, Hughes and Nei (1988) proposed that if the MHC diversity is maintained by positive selection related to antigen recognition such selection should act specifically on the amino acids involved in antigen recognition (i.e. PBR), thereby increasing the PBR variability. Accordingly, a higher number of non-synonymous than synonymous substitutions should be found specifically in those codons encoding the PBR. They confirmed their hypothesis in a study of human and mouse MHC II genes (Hughes and Nei, 1989), and since then their findings were supported by numerous studies of species from all vertebrate classes (Klein et al., 1993, Hughes et al., 1994, Hughes and Hughes, 1995, Apanius et al., 1997, Edwards et al., 1998, Hughes and Yeager, 1998, Figueroa et al., 2000, Bernatchez and Landry, 2003).
Thus, the emerging view is that the high nucleotide diversity observed in the PBR is generated by positive selection, mediated by a host–pathogen arms race (Hedrick and Kim, 2000, Hedrick, 2002, Wegner et al., 2003, Dionne et al., 2007, Dionne et al., 2009) Most researchers agree about the central role of positive (diversifying) selection for the maintenance of the MHC diversity (Klein et al., 1993, Hughes et al., 1994, Hughes and Hughes, 1995, Apanius et al., 1997, Bernatchez and Landry, 2003). However, the molecular mechanisms that generate new allelic variants in the MHC genes are still controversial (Klein, 1986b, Hughes et al., 1993, Ohta, 1995, Shum et al., 2001, Richman et al., 2003, Bos and Waldman, 2006, Schaschl et al., 2006). It was originally proposed that the high MHC variation originates solely from point mutations (Klein, 1986a). However, if point mutations are the sole mechanism generating the high allelic diversity observed in the MHC, then the mutation rate should be significantly higher than in other gene loci, which has not been supported by sequence analyses (Satta et al., 1993). Alternatively, repeated non-reciprocal recombination (gene-conversion) could contribute to the observed high allelic diversity (Martinsohn et al., 1999, Ohta, 1999). Evidence for gene-conversion among MHC alleles has been found in several vertebrate species, including teleosts (Högstrand and Böhme, 1994, Zangenberg et al., 1995, Reusch et al., 2004, Reusch and Langefors, 2005, Schaschl et al., 2005). These studies also indicate that both intra- and inter-locus gene-conversion contributes to the allelic diversity observed in the MHC genes (Ohta, 1995, Reusch et al., 2004, Reusch and Langefors, 2005). For example, Reusch and Langefors (2005) suggests that in stickleback Gasterosteus aculeatus, a teleost species with multiple MHC class II β loci, inter-locus gene-conversion contributes significantly to the allelic diversity of the MHII β1 domain. It has also been suggested that gene-conversion might be an important mechanism for generating new MHII β alleles, particularly in species where a high number of MHC loci provide a ‘broad reservoir’ of genetic diversity (Andersson and Mikko, 1995) – such as in teleost fish.
The evolution of the MHII β genes may differ between different vertebrate classes (Edwards et al., 1995) and possibly also among different teleost lineages (Aguilar and Garza, 2007). The current knowledge in teleosts is mostly restricted to a small number of species with only limited information available about non-model teleost species. Hence, a broader taxonomic coverage is warranted to expand our understanding of the diversification of this gene family among different teleost lineages. To this end, we investigated the diversity, evolution, and molecular structure of the MHII β genes in a non-model percid species – the Eurasian perch (Perca fluviatilis L.). The specific aims of this study were, firstly, to approximate the putative number of MHII β loci present in perch, and, secondly, to characterize the role of positive selection and gene-conversion for the generation of the allelic diversity. Thirdly, we compared the tertiary structure and amino-acid composition of the β1 domain in perch with other teleost species and tetrapods to investigate possible differences among vertebrate lineages.
Section snippets
Sample collection
To characterize the MHII β1 domain in perch, 58 specimens were sampled from 12 geographic locations of Lake Constance (Germany) with about five individuals per location. Fin clips were taken, and preserved in approximately 10 volumes of p.a. grade ethanol absolute (Riedel de Haen GmbH, Germany). For molecular analyses whole genomic DNA was isolated from a small piece of preserved fin tissue (approx. 1 mm2) with a modified high-salt DNA extraction protocol following Aljanabi and Martinez (1997).
Molecular methods
Processing of sequence files
Intron I and most of the β1 domain of the MHII β genes of 58 specimens were amplified by combining one specific forward primer with one of three specific reverse primers (Table 1). Except for PfluMH4R, the other two reverse primers amplified multiple bands ranging in size from 300 bp to 700 bp. Our cloning and sequencing procedure detected sequences in the size range of all the bands (data not shown). A mean ± SD of 8.12 ± 7.79 clones (min = 4, max = 47) were sequenced per individual, and a total of 471
Genetic diversity of the β1 domain
This study provides evidence for the occurrence of multiple MHII β loci in perch. This was namely reflected in a three- to eight-fold higher pm in our dataset (0.19 ± 0.01) as compared to the variation found in population samples of the β1 domain in salmonids, which posses a single MHII β locus (Miller and Withler, 1996: pm = 0.06; Miller et al., 1997: pm = 0.03; Kim et al., 1999: pm = 0.02). Moreover, the phylogenetic analysis of the exon II sequences identified eight distinct clusters (Fig. 2A,
Conclusions
This study provides evidence that the non-model percid species Eurasian perch (Perca fluviatilis L.) has at least eight MHII β gene loci that exhibit a high allelic variability (28 alleles in 58 individuals analysed). The tertiary structure of the β1 domain in perch is similar to other vertebrates. However, our results indicate some teleost specific differences in the amino acid composition. Finally, both positive selection and gene-conversion events (i.e. intra- and intergenic sequence
Acknowledgements
The authors would like to thank Reiner Eckmann, Elisabeth Gross, Melanie Hempel, Arne Nolte, and Jonatan Blais for help and support. Moreover, Dave Gerrard and Kai Müller are acknowledged for help with the data analysis. The corrections and comments of Andrew Clarke substantially improved earlier versions of this manuscript. Additionally we want to thank two anonymous reviewers for their comments on the manuscript. This project was financially supported by the Deutsche Forschungsgemeinschaft
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