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ANIMAL GENETICS |
* Animal and Food Sciences Division, Lincoln University, Canterbury, New Zealand and
and
Biochemistry Unit, Canterbury Health Laboratories, Christchurch, New Zealand
Abstract
Variation in the ovine DQA2 gene was investigated in approximately 2,000 sheep from six breeds. Fragments of DNA containing the ovine DQA2 exon 2 were amplified using PCR. Single-strand conformational polymorphism analysis and DNA sequence analysis were employed to detect genetic variation. Twenty-three nucleic acid sequences, encoding 22 DQA2 amino acid sequences, were identified. This increases the number of alleles identified from 10 to 23. In some cases, three or four unique sequences were isolated from individual sheep, suggesting that these DQA2 sequences may represent two loci. Phylogenetic tree analysis revealed that 5 of these 23 sequences were more closely related to cattle DQA3 or DQA4 sequences than to other sheep DQA2 sequences. These sequences clustered together and were called DQA2-like to differentiate them from other DQA2 sequences. There was no evidence of DQA5-like sequences in sheep. Information theory-based analysis indicated that some of the DQA2-like sequences had low information content at splice sites, suggesting that these alleles may have low functional activity. Allelic lineages were observed not only at the DQA2 locus, but also at the DQA2-like locus, supporting the trans-species mode of evolution of MHC genes. Comparison of the allelic sequences suggests that polymorphism seems to have arisen largely by point mutation and gene conversion, and a recent gene conversion event seems to have occurred between the DQA2 and DQA2-like loci. The high level of sequence polymorphism detected and varied number of loci demonstrate the extensive diversity of the ovine DQA2 gene.
Key Words: DQA2 • Major Histocompatibility Complex • Polymorphism • Sheep • Single-Strand Conformational Polymorphism
Introduction
The major histocompatibility complex (MHC) has a central role in the immune response of vertebrates, with the function of presenting antigenic peptides to T-cells. The class II gene region of the sheep MHC has an organization similar to that of humans (Scott et al., 1987
). Within this region, two subregions exhibit polymorphism—DR and DQ (Amills et al., 1998), but, in contrast to humans, high levels of polymorphism are observed in the DQ subregion (Escayg et al., 1996). This, together with the absence of a functional DP subregion (Scott et al., 1987; Deverson et al., 1991), suggests that DQ is important for antigen (Ag) presentation (Escayg et al., 1996). In cattle, it has been shown that DQ molecules present Ag to CD4+ T cells and the inter-haplotype pairing of DQ
and DQß molecules forms functional restriction elements (Glass et al., 2000).
Two DQA genes, called DQA1 and DQA2, have been identified in sheep (Scott et al., 1991), and they both are polymorphic (Escayg et al., 1996; Snibson et al., 1998). Seven alleles plus a null allele at the DQA1 locus and 16 alleles at the DQA2 locus have been identified by RFLP analyses (Wright and Ballingall, 1994; Escayg et al., 1996). Recently, sequence analysis has revealed 14 DQA1 sequences (Zhou and Hickford, 2004), but only 10 sequences of DQA2 have been characterized to date (Snibson et al., 1989).
In this study, variation in the ovine DQA2 gene was investigated in a large number of sheep from various breeds using single-strand conformational polymorphism (SSCP) analysis, cloning, and sequencing.
Materials and Methods
Sheep and DNA Sources
The sheep investigated in this study totaled approximately 2,000 from six breeds (Merino, Corriedale, Borderdale, Romney, Awassi, and Finnish Landrace). These sheep were sired by 45 rams unrelated by at least one generation from seven farms. Genomic DNA utilized in PCR amplification was isolated from blood using a high salt procedure as described by Montgomery and Sise (1990), or extracted from blood collected on FTA Classic cards (Whatman BioScience, Newton, MA), following the manufacturer’s protocol.
PCR Amplification
Two PCR primers for amplifying the entire second exon (249 bp), flanked by 209 bp of intron 1 and 370 bp of intron 2 sequences of the ovine DQA2 gene, were designed based on two published ovine DQA2 sequences (M33305, Scott et al., 1991; Z28421, Wright and Ballingall, 1994) with comparison to a published DQA1 sequence (M33304, Scott et al., 1991). These primers were DQA2-up (5'-CACATGTTACAGTGCAAAARCAGC-3', where R means A or G) and DQA2-dn (5'-CCCTCYCACCAACGTTTCCCAG-3', where Y means C or T). Subsequently, internal PCR primers DQA2s-up (5'-ACTACCAATCTCATGGTCCCTCT-3') and DQA2s-dn (5'-GGAGTAGAATGGTGGACACTTACC-3') were designed to amplify the variable region of exon 2 for SSCP analysis based on the sequence information obtained in this study. Primers were synthesized by Proligo (Proligo LLC, Colorado, CA).
Amplifications for allele detection used a nonproofreading Taq DNA polymerase (Qiagen, Hilden, Germany), whereas amplifications for allele cloning and sequencing used the proofreading enzyme, ProofStart DNA polymerase (Qiagen), to decrease PCR-associated nucleotide substitutions. Each PCR was performed in a 20-µL reaction volume containing 50 ng of genomic DNA from whole blood or genomic DNA on one 1.2-mm punch of an FTA card, 0.25 µM each primer, 150 µM dNTP (ABgene, Surrey, U.K.), 1 U of DNA polymerase and 1 x reaction buffer supplied (containing 1.5 mM MgCl2). Amplification was carried out in an iCycler (Bio-Rad, Hercules, CA) and consisted of denaturation at 94°C for 2 min, followed by 32 cycles of 94°C for 30 s, 59°C (for DQA2-up and DQA2-dn) or 62°C (for DQA2s-up and DQA2s-dn) for 30 s, and 72°C for 50 s (for DQA2-up and DQA2-dn) or 30 s (for DQA2s-up and DQA2s-dn). This was followed by a final extension step at 72°C for 5 min. Amplimers were visualized by electrophoresis in 1% Seakem LE agarose (BioWhittaker Molecular Applications, Rockland, ME) gels using 1 x TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA), containing 200 ng/mL ethidium bromide.
Single-Strand Conformational Polymorphism Analysis
A 0.7-µL aliquot of each amplimer was mixed with 7 µL of loading dye (98% formamide, 10 mM EDTA, 0.025% bromophenol blue, 0.025% xylene-cyanol). After denaturation at 95°C for 5 min, samples were cooled rapidly on wet ice and then loaded on 16-cm x 18-cm, 14% acrylamide:bisacrylamide (37.5:1; Bio-Rad) gels. Electrophoresis was performed using Protean II xi cells (Bio-Rad), at 380 V for 18 h at 5°C in 0.5 x TBE buffer. Gels were silver-stained according to the method of Bassam et al. (1991).
Cloning of PCR Amplimers and Clone Screening
Sheep DNA representative of different SSCP patterns were selected for amplification, and each of the amplimers was subsequently cloned. Amplifications typically used the DQA2-up and DQA2-dn primers, but the DQA2s-up and DQA2s-dn primers were used if no amplimer was detected.
Amplimers were ligated to the pCR4 Blunt-TOPO vector (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. A 2-µL aliquot of the ligation mixture was used to transform competent Escherichia coli cells (One Shot INVF', Invitrogen), following the protocol recommend by the manufacturer. Between 10 and 15 insert positive colonies for each transformation were picked and incubated overnight in Terrific broth (Invitrogen) at 37°C, in a shaking rotary incubator (225 rpm).
DNA Sequencing
Plasmid was extracted from overnight cultures using a QIAprep Spin Miniprep kit (Qiagen) and was sequenced in both directions using the M13-forward and reverse primers by the Waikato DNA Sequencing Facility, University of Waikato, Hamilton, New Zealand. Identical sequences obtained from at least three separate clones from different sheep, or two independent PCR amplifications from the same sheep, were subjected to further sequence analysis.
Sequence Analysis
Sequence alignments, translations, and comparisons were carried out using DNAMAN (Version 4.0, Lynnon BioSoft, Vaudreuil, Quebec, Canada). The BLAST algorithm was used to search the NCBI GenBank (http://www.ncbi.nlm.hih.gov/) databases for homologous sequences.
The individual information contents (Ri, bits) of the splice sites were determined using an information theory-based approach incorporating information weight matrices derived from approximately 2,000 published human donor and acceptor sites (Schneider, 1997). The magnitude of the information content indicates how well conserved a base is in a natural splice junction binding site, with approximately 2.4 bits being the reported minimal functional value (Rogan et al., 1998). These analyses can be performed at http://www.lecb.ncifcrf.gov/~toms/delilaserver.html.
Neighbor-joining trees (Saitou and Nei, 1987) were constructed on the basis of genetic distances, estimated by Kimura’s (1980) two-parameter method, using MEGA version 2.1 (Kumar et al., 2001; http://www.megasoftware.net/). The reliability of the trees was estimated by bootstrap confidence values (Felsenstein, 1985) and 500 bootstrap replications were used.
The cattle DQA sequences used to construct the neighbor-joining tree were (with a NCBI GenBank accession number in brackets): BoLA-DQA*0101 (Z48185), *0102 (Z48194), *0103 (Z48187), *0202 (Z48190), *0204 (Z48188), *0301 (Z48195), *0401 (Z48196), *0801 (Z48186), *1001 (Z48191), *1201 (Z48193), *1301 (Z48192), *1401 (Z48189), *1202 (D50454), *1203 (M30117), *1302 (Z79507), *2001 (Z79515), *2002 (U80866), *2101 (Y07820), *2201 (Y07820), *22021 (D50045), *22022 (Z79518), *22023 (U80862), *22031 (U80861), *22032 (Z79514), *2204 (U80865), *2205 (Z79516), *2301 (Z79522), *2401 (U80868), *25011 (U80857), *25012 (Y14020), *2502 (U80859), *2601 (Z79519), *2602 (Y14021), *2603 (Z79526), *27011 (U80860), *27012 (Y14022), *2702 (Z79525) and *2801 (Z48197).
Nomenclature
As no workshop for naming of sheep MHC genes and alleles has been held, the criteria for assigning new alleles and the rules for naming alleles for the ovine DQA2 gene were based on those proposed for the cattle MHC (Davies et al., 1997; http://www.projects.roslin.ac.uk/bola/bolahome.html). The criteria are that when using DNA cloning and sequencing there have to be at least three identical cloned sequences. Names are based on the predicted amino acid sequences and consist of four or five digits, where the first two digits indicate the major type, the third and fourth digits indicate the subtype, and the fifth digit (if present) indicates unexpressed variation (silent substitutions). Alleles that differ by less than five amino acids in the first domain are assigned as subtypes within a single major type.
The sequences described in this study were submitted to the NCBI GenBank and assigned the accession numbers AY312375 to AY312397.
Results
Single-Strand Conformational Polymorphism of the Ovine DQA2 Gene
Amplimers of approximately 828 bp were obtained with sheep genomic DNA using PCR primers DQA2-up and DQA2-dn. The internal primers, DQA2s-up and DQA2s-dn, generated smaller DNA fragments of approximately 242 bp. These 242-bp amplimers exhibited polymorphism upon SSCP analysis and 20 haplotype-specific SSCP patterns could be detected. Ten of these patterns exhibited one or two major bands, whereas the others had three to five major bands (Figure 1).
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). Twenty-three different sequences were identified from the animals investigated (Figure 2), with between one and four sequences isolated from individual sheep.
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The predicted amino acid sequences of the DQA2 sequences that were identified shared a pattern of conserved and variable regions different from that of the ovine DQA1 sequences (Figure 3). Based on the identities and similarities between sequences, all the ovine DQA2 sequences were named, and this is summarized in Table 1 along with any previous designation.
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Variation in the Information Content of Splice Sites
The intronic sequences at the donor splice junctions were conserved, with all of the alleles, except *1401, containing a canonical GT dinucleotide. The sequence of *1401 had a noncanonical GC dinucleotide at the donor site (Figure 2). Splice site information analysis indicated that the substitution of a GC instead of a GT at the donor site leads to a dramatic decrease in Ri value and might activate a cryptic donor site 362 bp downstream of the natural donor site (Table 2).
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). The Ri values were predicted to vary between 11.5 and 5.8 bits. The possible activation of cryptic splice sites was also suggested for alleles *1301, *1601, and *1701 (Table 2).
Frequencies of the Ovine DQA2 Alleles
The frequencies of the DQA2 alleles or haplotypes detected in this study are shown in Table 3. The most commonly found alleles were DQA2* 1401, *0101, *1201, *0602, and *0103, whereas the less common alleles were *0701, *1301, *0401, and *1001.
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). For example, DQA2*1301, *1401, *1501, *1601, and *1701 belonged to the same cluster in the tree based on exon 2, whereas they were clustered with different alleles in the trees based on analysis of introns 1 and 2 (Figure 4).
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). Within clusters, some of the sheep sequences were more similar to cattle sequences than to other sheep sequences. There were no sheep sequences that grouped with the cattle DQA5 sequence (Gelhaus et al., 1999).
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The diverse nature of the ovine DQA2 gene has been demonstrated using PCR-SSCP and sequence analyses. Twenty-three sequences, including 13 newly identified alleles, were isolated in this study. This, together with the varied numbers of sequences per haplotype retrieved from individual sheep, confirms the diversity of the ovine DQA2 gene.
The possibility that some of sequence polymorphism detected here might be the result of sequencing or PCR errors was carefully considered when analyzing the sequences isolated. All the ovine DQA2 sequences reported here met the criteria used by the human and cattle nomenclature committees for the assignment of MHC allele names (Marsh et al., 2002; http://www.projects.roslin.ac.uk/bola/bolahome.html). They are therefore believed to represent genuine DQA2 allelic sequences in sheep rather than PCR or sequencing artifacts.
The isolation of three or four DQA2 sequences from a single sheep suggests the existence of up to two DQA2 loci in sheep. This is supported by phylogenetic analysis that revealed that some of the DQA2 sequences were more closely related to cattle DQA3 or DQA4 than to the rest of the sheep DQA2 sequences. A phylogenetic tree based on the exon 2 region revealed these sequences to be grouped together and were located in a cluster differing from that of the other DQA2 sequences (Figure 4B). These sequences probably represent another locus of the DQA2 gene and were named DQA2-like in order to differentiate them from the other DQA2 sequences. The presence of two loci of DQA2 is consistent with the observation that these DQA2-like sequences are always coinherited with particular DQA2 sequences and are only found in haplotypes that lack a DQA1 gene. These results support the findings of Hickford et al. (2000). In cattle, variation in the number of DQA loci has also been described (Ellis and Ballingall, 1999).
The clustering of sheep sequences with similar cattle sequences was observed for both DQA2 and DQA2-like loci (Figure 5). This observation underlies the so-called trans-species hypothesis (Klein, 1987). Trans-species evolution has also been reported in the ovine DQA1 gene (Zhou and Hickford, 2004). This suggests that it may be common for the MHC genes. The clusters of sheep and cattle sequences may be derived from primordial sequences that were present in a common ancestor and have persisted in the sheep and cattle populations since their divergence. This pattern of evolution suggests the action of prolonged natural selection on the DQA genes because neutral polymorphism is not expected to persist very long in a population (Hughes and Yeager, 1998). Pathogen recognition may provide selection pressure for maintaining particular MHC sequences, and the observation that sheep and cattle share similar allelic sequences may be evidence of the need for a specific immune response to a common pathogen.
A comparison of the phylogenetic trees for different regions of the ovine DQA2 gene suggests that genetic exchanges of segments may have occurred between ancestral alleles. This was supported by the sharing of common sequence between alleles, despite other significant sequence variation. Recombination events may contribute to the generation of polymorphism in ovine DQA2 gene, but this is expected to be an essentially random process. It does not fit the observation that there is a higher level of polymorphism in exon 2 than in the flanking introns (Figure 2). The nucleotide sequences also indicate point mutation occurred more frequently in the exon than in the flanking introns, and this also suggests that selection pressures are exerted on the DQA2 gene. Point mutation appears to be of importance for the generation of polymorphism.
The ovine DQA2 molecules exhibit considerable variation at the positions that have been considered as being important for peptide backbone binding and pocket formation in human DQA molecules (Paliakasis et al., 1996). Most of the alleles reported here possess different sequence motifs at these positions (Figure 3). Substitution at these positions may impact on the antigen-binding groove and antigenic-peptide binding ability and hence the peptide specificity. The importance of specific residues within the antigen-binding groove and how antigenic-peptide binding ability can be altered with only one or two amino acid changes in some alleles has been illustrated in humans (Seidl et al., 1997; Toussirot et al., 1999). However, sequence polymorphism was also observed at other positions (65, 72, and 79) that are invariant in human DQA molecules and deemed crucial for forming hydrogen bonds to the backbone of a foreign peptide. This suggests that the peptide binding groove of sheep DQA molecules may be structurally different from that of the human. Extrapolation from studies of human DQ and DR molecules may therefore be of limited use.
Sequence variation in the splice junctions may impact on RNA processing and consequently the functional activity of alleles. The effect of the splice sequences on RNA processing can be accurately and comprehensively evaluated with computational approaches based on information theory (Rogan et al., 1998). Information theory-based models have not yet been established for sheep or other small ruminants, but the similarity of human and murine models (P. K. Rogan, Children’s Mercy Hospital and Clinics, Kansas City, MO, personal communication) suggests that models established for one species may be applicable, to some extent, for other species. Using the human splice-site analysis model (Schneider, 1997), both DQA2 and DQA2-like loci exhibited variation in the information content of the exon 2 splice sites, but quite considerably low information content was observed for the DQA2-like loci. Four out of the five DQA2-like sequences had Ri values between 3.5 to 6.6 bits compared with between 8.5 to 11.5 bits for the other DQA2 sequences. In addition, the activation of cryptic splice sites was also predicted for some of these alleles (Table 2). These alleles may therefore be nonfunctional or have low functional activity. Further investigations are required to confirm this. Analyses using real-time RT-PCR may provide information on the levels of expression for these alleles in sheep.
The decrease in information content for the exon 2 splice donor site of DQA2-like allele *1401 was due to the absence of a canonical dinucleotide GT at the donor splice site. Interestingly, *1401 was the most common allele detected in the animals analyzed, and it was found in five haplotypes for which the DQA2 sequences belonged to different allelic lineages (Table 3; Figure 5). The preservation of this allele may occur for a number of reasons. First, the context of the splice site may allow its correct and efficient processing despite the noncanonical junction sequence. The observation of sequence conservation in *1401 isolated from different haplotypes supports the contention that natural selection has preserved this allele and that the substitution of the expected GC at the donor site offers an advantage. However, because sequences identical or similar to *1401 have not yet been found in cattle, this allele would seem to have arisen after the speciation of sheep and cattle and has therefore been preserved by some "sheep-specific" selective pressure. Second, the *1401 allele has accumulated a recent splice-site mutation and this has not yet been selected for correction or removal. This seems to be unlikely because of the large number of haplotypes containing *1401 and the frequency of this allele. Thirdly, the *1401 allele may not be expressed, but is instead maintained in several haplotypes for some other reasons (e.g., the linked DQB2-like gene is strongly selected as a partner in hybrid DQA2-DQB2-like functional pairs).
Another explanation for the conserved *1401 allele in sheep is that a recent gene conversion event may have occurred between DQA2 and DQA2-like loci in sheep only and therefore *1401 has not yet had time to diverge following this conversion. This is unlikely given the low Ri value at the donor splice site.
Finally, if *1401 is an inactive "pseudo-allele," it may not be subject to selection. This would appear to be unlikely, given that the other alleles that may be subject to selective pressure are highly polymorphic as a result of point mutation, and therefore this would be expected in allele *1401 as well.
Implications
Extensive polymorphism of the ovine DQA2 gene makes it a potential marker for the study of disease susceptibility. This can be assisted by the availability of a rapid typing mechanism using the single-strand conformational polymorphism of the second exon. Information on sequence variation in the ovine DQA2 can also be used to study the evolutionary history of the major histocompatibility complex.
Footnotes
1 We thank A. Hogan and R. Forrest for their technical assistance, C. Logan and M. Ridgway for sheep blood collection, and J. Rellis and T. Scheider (National Institutes of Health, MD) for the help with splice sites junction analyses. This research was supported by FRST (LIN X002) and MeatNZ (LU 165). 
2 Correspondence: P.O. Box 84 (phone: +64-3-325 2811; fax: +64-3-325 3851; e-mail: hickford{at}lincoln.ac.nz).
Received for publication December 3, 2003. Accepted for publication February 17, 2004.
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and those to the putative BoLA-DQA4 locus (Ballingall et al., 1998
. The putative ovine DQA2-like sequences are marked with 
.