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J. Anim. Sci. 2005. 83:963-968
© 2005 American Society of Animal Science

ANIMAL GENETICS

Polymorphism of the DQA2 gene in goats1

H. Zhou, J. G. H. Hickford2 and Q. Fang

Cell Biology Group, Agriculture and Life Sciences Division, Lincoln University, Canterbury, New Zealand


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
Variation in the caprine DQA2 gene was investigated using PCR–single-strand conformational polymorphism (SSCP) and DNA sequencing. Eleven DQA2 alleles were defined by SSCP patterns from 23 goats. All the caprine alleles shared high sequence homology to ovine DQA2 sequences, and exhibited a pattern of polymorphism similar to DQA2 alleles from sheep and cattle but different from caprine DQA1 sequences. Thirty-eight AA positions in the {alpha}1 domain of caprine DQA2 molecules were polymorphic, and a high degree of polymorphism was observed in the putative antigen-binding region, with 74% of the positions being polymorphic. Phylogenetic analysis of caprine, ovine, and bovine DQA sequences revealed that the caprine DQA2 sequences identified here grouped with ovine DQA2, bovine DQA2, DQA3, and DQA4 sequences but are separate from the group of caprine DQA1 alleles. Nine of the caprine DQA2 sequences were more similar to ovine DQA2 alleles, whereas the remaining two were more closely related to ovine DQA2-like and bovine DQA3 alleles. This finding suggests that the caprine DQA2 sequences may represent two loci, which probably arose by either gene duplication or gene conversion events. Allelic lineages were evident for both DQA2 and DQA2-like loci, supporting the trans-species mode of evolution of major histocompatibilitly complex genes. The high level of polymorphism and similarity between caprine and ovine DQA2 alleles suggests that the DQA2 gene may play an important role in immune responses to shared pathogens.

Key Words: DQA2 • Goat • Major Histocompatibility Complex • Polymorphism • Single-Strand Conformational Polymorphism


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 
The major histocompatibility complex (MHC) class II molecules are cellular glycoproteins involved in antigen presentation to CD4+ T cells. The genes encoding these molecules are polymorphic, and in sheep and cattle, high levels of polymorphism are observed in both the DRB and DQA genes (Escayg et al., 1996Go; Ellis and Ballingall, 1999Go).

Despite extensive studies documenting gene structure and polymorphism in the DQA genes from sheep (Hickford et al., 2000Go, 2004Go; Zhou and Hickford, 2004Go) and cattle (Ballingall et al., 1997Go, 1998Go; Gelhaus et al., 1999Go), relatively little is known about their counterparts in goats, with only one DQA gene (DQA1) having been investigated recently (Amills et al., 2004Go).

In sheep, there are two DQA genes, called DQA1 and DQA2 (Scott et al., 1991Go). As sheep and goats are closely related species, it might be predicted that a similar DQA gene structure exists in goats. Therefore, the purpose of this study was to identify the caprine DQA2 gene and to analyze any sequence variation found in exon 2 of this gene.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Implications
 Literature Cited
 
Blood and DNA Isolation
Blood samples were collected from 23 Boer goats from unrelated dams and sired by two different rams. Genomic DNA used in PCR amplification was isolated from blood using a high-salt procedure as described by Montgomery and Sise (1990)Go, or extracted from blood collected on FTA cards (Whatman BioScience, Middlesex, U.K.) following the manufacturer’s protocol.

Polymerase Chain Reaction Amplification
Two PCR primers were designed to amplify the entire of exon 2 and parts of the flanked intron sequences for the caprine DQA2 gene, based on the published ovine DQA2 sequences (Hickford et al., 2004Go), with comparison to caprine DQA1 sequences (Amills et al., 2004Go). These primers were C-DQA2-up (5'-CTTCCTGCTCCT CACCCTCAC-3') and C-DQA2-dn (5'-AAAGAGAAGT AGAATGGTGGACACTT). Primers were synthesized by Proligo, Boulder, CO.

Amplification was performed in a 20-µL reaction containing 50 ng of genomic DNA, or genomic DNA on one 1.2-mm punch of FTA paper, 0.25 µM of each primer, 150 µM of nucleotides (dNTP; Eppendorf, Hamburg, Germany), 2.5 mM of Mg2+, 0.5 U of DNA polymerase, and 1x reaction buffer supplied. Amplifications for cloning and sequencing used the proofreading enzyme ProofStart DNA polymerase (Qiagen, Hilden, Germany); otherwise, a nonproofreading Taq DNA polymerase (Qiagen) was used.

The thermal profile consisted of 2 min at 94°C, followed by 32 cycles of 30 s at 94°C, 30 s at 58°C, and 30 s at 72°C, with a final extension of 5 min at 72°C. Amplification was carried out in an iCycler (Bio-Rad Laboratories, Hercules, CA).

Amplimers were visualized by electrophoresis in 1% Seakem LE agarose (BioWhittaker Molecular Applications, Rochland, ME) gels, with 1x TBE buffer (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA) containing 200 ng/mL of 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, and 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 Laboratories) gels. Electrophoresis was performed using Protean II xi cells (Bio-Rad Laboratories), at 380 V for 18 h at 5°C in 0.5x TBE buffer. Gels were silver-stained according to the method of Bassam et al. (1991)Go.

Cloning of PCR Amplimers and Clone Screening
Goat DNA samples representative of different single-strand conformational polymorphism (SSCP) patterns were selected for amplification and the amplimers were subsequently cloned.

After the addition of an A overhang to the blunt-ended PCR products using an A-Addition kit (Qiagen), amplimers were ligated to the pDrive Cloning vector (Qiagen) according to the manufacturer’s instructions. A 2-µL aliquot of the ligation mixture was used to transform competent Escherichia coli cells. Between 10 and 15 insert-positive colonies for each transformation were picked and incubated overnight in Terrific broth (Invitrogen, Carlsbad, CA) at 37°C, in a shaking rotary incubator (225 rpm).

Plasmids were recovered from bacterial cells by boiling for 10 min in 0.8% (vol/vol) Triton X-100 solution and 1 µL of the supernatant fraction was used as a template for PCR under the conditions described previously. Amplimers from clones and the corresponding genomic DNA were run adjacent to each other on SSCP gels for comparison of the binding patterns, and only those clones for which the patterns matched those of the corresponding genomic DNA templates were selected for subsequent DNA sequencing.

DNA Sequencing
Plasmids were extracted from overnight cultures using a QIAprep Spin Miniprep kit (Qiagen) and were sequenced in both directions using the M13-forward and reverse primers at the Waikato DNA Sequencing Facility, University of Waikato, Hamilton, New Zealand. Identical sequences obtained from at least three separate clones from different goats, or two independent PCR amplifications from the same goat, were subjected to further sequence analysis.

Sequence Analysis
Sequence alignments, translations, and comparisons were carried out using DNAMAN (v. 4.0, Lynnon Bio-Soft, Vaudreuil, Canada). The BLAST algorithm was used to search the NCBI GenBank (http://www.ncbi.nlm.nih.gov/) databases for homologous sequences.

A neighbor-joining phylogenetic tree was constructed on the basis of genetic distances, estimated by the Kimura (1980)Go two-parameter method, using MEGA v. 3.0 (Kumar et al., 2004Go; http://www.megasoftware.net/). The reliability of the trees was estimated by bootstrap confidence values (Felsenstein, 1985Go), and 1,000 bootstrap replications were used.

The following caprine (Cahi), ovine (OLA), and bovine (BoLA) DQA sequences, together with the caprine DQA2 sequences identified in this study (see Figure 2Go), were used in the phylogenetic analysis: seven Cahi-DQA1 sequences (GenBank Accession No. AY464654 to AY464657, AY665664 to AY665666), 23 OLA-DQA2 sequences (GenBank Accession No. AY312375 to AY312397), and 22 BoLA-DQA sequences (GenBank Accession No. D50045, D50454, M30117, U80857, U80859 to U80862, U80865, U80866 to U80868, Y07820, Y14020 to Y14022, Z48185 to Z48196, Z79507, Z79514 to Z79516, Z79518, Z79519, Z79522, Z79525, and Z79526). All sequences were trimmed to a similar length corresponding to the same region of exon 2 before generating the tree.



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  		Figure 2. Alignment of predicted {alpha}1 domain sequences of caprine (Cahi) and ovine (OLA) DQA2 alleles together with caprine DQA1 alleles. Amino acids are presented in one-letter code and the numbering above the aligned sequences refers to the human DQ{alpha} chain. Dashes represent residues identical to the AAs in the top sequence, and dots have been introduced to improve the alignment. Putative peptide binding positions as proposed for human DQ{alpha} chain (Reche and Reinherz, 2003Go) are shaded. The GenBank accession numbers of the allelic sequences refer to Figure 3Go.

 
Nomenclature
The assignment and naming of the caprine DQA2 alleles followed criteria proposed for the cattle MHC (Davies et al., 1997Go; http://www.projects.roslin.ac.uk/bola/bolahome.html), which have been used for sheep DQA genes (Hickford et al., 2004Go; Zhou and Hickford, 2004Go). These 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 AA sequences and consist of four or five digits, with the first two digits indicating the major type, the third and fourth digits indicating the subtype, and the fifth digit (if present) indicating unexpressed variation (silent substitutions). Alleles that differ by less than five AA in the first domain are assigned as subtypes within a single major type.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Implications
 Literature Cited
 
Identification of the Caprine DQA2 Polymorphism by PCR-SSCP
Using the primers C-DQA2-up and C-DQA2-dn, amplimers of approximately 305 bp were obtained with all of the goat genomic DNA samples. These amplimers exhibited polymorphism on SSCP analysis, and in total, 11 alleles could be identified from the 23 goats based on SSCP patterns (Figure 1Go). As more goats from different sires and different breeds are analyzed, more alleles may be identified. This indicates that the caprine DQA2 gene is highly polymorphic, and the level of polymorphism is higher than that reported for the DQA1 gene (Amills et al., 2004Go). This is consistent with the pattern in sheep (Hickford et al., 2004Go; Zhou and Hickford, 2004Go).



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  		Figure 1. Single-strand conformational polymorphism (SSCP) of the caprine DQA2 gene. Eleven unique SSCP banding patterns, corresponding with alleles *0101 (a), *1002 (b), *0201 (c), *03011 (d), *03012 (e), *0401 (f), *0501 (g), *0601 (h), *0701 (i), *0801 (k), and *0901 (l), were detected in this study. Goat DNA samples from which three DQA2 sequences were isolated were marked with #, demonstrating that these DQA2 sequences may be from two loci.

 
Characterization of the Caprine DQA2 Polymorphism by DNA Sequencing
Cloning and sequencing of PCR amplimers representative of the unique SSCP patterns followed by DNA sequencing revealed 11 novel DNA sequences. Because these sequences were based on clones derived from PCR amplification, caution was exercised in assigning these sequences. The possibility that some of the sequence polymorphism detected here might be the result of PCR and sequencing errors was carefully considered. All sequences reported here met the criteria used by the human and cattle nomenclature committees for the assignment of MHC alleles, and also were confirmed by comparison of the SSCP patterns generated by amplimers derived from clones and genomic DNA from which the clone sequences were isolated. This ensured that these sequences represented genuine caprine sequences and not PCR or sequencing artifacts.

The caprine DQA2 sequences were most similar to the ovine DQA2 sequences. The alignment of the predicted AA sequences from caprine DQA2 with ovine DQA2 and caprine DQA1 gene sequences revealed that caprine DQA2 alleles exhibited a pattern of conserved and variable AA sequences similar to ovine DQA2, but different from caprine DQA1 (Figure 2Go). This supports the contention that these allelic sequences represent the DQA2 gene and not the DQA1 gene. Based on the similarities between the sequences, all of the caprine DQA2 alleles were named and the sequences were deposited into the NCBI GenBank with the Accession No. AY829349 to AY829359.

Of the 82 AA sites within the {alpha}1 domain of DQA2, 38 (46%) were polymorphic; however, in the putative antigen-binding region (Reche and Reinherz, 2003Go), 14 out of the 19 AA sites (74%) were polymorphic (Figure 2Go). The most polymorphic sites were observed at {alpha}14 (D, E, V, T), {alpha}68 (I, K, T, A), and {alpha}79 (L, R, H, W), which are all included in the putative antigen-binding region. Variation in this region may affect the antigen-binding groove and antigenic-peptide binding ability, and hence 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 AA changes, has been illustrated in humans (Seidl et al., 1997Go; Toussirot et al., 1999Go).

Analysis of the Evolutionary Relationship Between the Caprine DQA2 Alleles and Their Ovine and Bovine Counterparts
A phylogenetic tree constructed from exon 2 of the caprine DQA2 sequences and the reported caprine DQA1, ovine DQA2, bovine DQA2, DQA3, and DQA4 sequences, revealed two main groups of DQA sequences. The caprine DQA2 sequences grouped with ovine DQA2, bovine DQA2, DQA3, and DQA4 sequences, whereas the caprine DQA1 sequences formed the other group (Figure 3Go). With the caprine DQA2, ovine DQA2, bovine DQA2, DQA3, and DQA4, clustering of certain alleles from different species was evident (e.g., Cahi-DQA2*03011, Cahi-DQA2*03012, OLA-DQA2*1201, and BoLA-DQA*22032). This suggests the action of common selective pressures on the DQA2 gene.



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  		Figure 3. A neighbor-joining phylogenetic tree constructed from exon 2 sequences of DQA genes from goats, sheep and cattle. Two human DQA sequences were used as an outgroup. The GenBank accession numbers are shown between parentheses and the caprine DQA2 sequences are underlined. The numbers at the forks indicate the bootstrap confidence values and branch lengths are proportional to genetic distance. The bovine DQA3 sequences (Ballingall et al., 1997Go) are labeled with {clubsuit} and the bovine DQA4 sequences (Ballingall et al., 1998Go) are with {heartsuit}. The putative DQA2-like sequences from goats and sheep are marked with {diamondsuit}.

 
Goat alleles were more closely related to sheep alleles than to cattle alleles. Given the contention that DQA2 is instrumental in sheep immune presentation, it also could be argued that the same is true of goats. Furthermore, over the divergence of goat and sheep species, the same selective pressures may have acted on sheep and goats, although this argument must be balanced against the observation that closely related sequences can have quite different peptide-binding specificity (Seidl et al., 1997Go; Toussirot et al., 1999Go).

Of the 11 caprine DQA2 sequences, two sequences (Cahi-DQA2*0801 and *0901) clustered with ovine DQA2-like and bovine DQA3 sequences and not with the other caprine DQA2 sequences, although the bootstrap values defining this clustering were low. These sequences may represent another locus of the DQA2 gene and have been named DQA2-like sequences in sheep (Hickford et al., 2004Go). This argument is supported by the sequence data, where *0801 and *0901 have an isoleucine residue at {alpha}26, whereas the other sequences have a threonine. In sheep, a threonine at {alpha}26 is associated with DQA1 and DQA2 alleles, whereas DQA2-like alleles have an isoleucine at this position (see Figure 2Go).

The possibility of two loci of goat DQA2 is consistent with the observations that up to three sequences have been isolated from individual goats investigated in this study. The presence of haplotypes containing one DQA2 and one DQA2-like sequence has been documented in some sheep, and this seems to be exclusively associated with a DQA1-null allele, retaining the pattern of two DQA loci per haplotype (Hickford et al., 2000Go). Although the presence of a DQA1-null allele in goats has not been confirmed, the presence of DQA2-like sequences suggests that gene duplication has occurred at the goat DQA2 gene, or DQA1 has been converted to a DQA2-like sequence by a gene conversion event. However, if a gene duplication or conversion event has occurred to produce DQA2-like sequences, then at {alpha}26, the threonine codon that is conserved in both DQA1 and DQA2 sequences has been converted to an isoleucine codon.


   Implications
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
Literature Cited
 
The identification of 11 alleles from a limited number of goats indicates a high level of polymorphism in the caprine DQA2 gene, and it might be expected that more alleles may be found as other goats from diverse breeds are studied. The role of this variation in defining susceptibility to diseases remains to be identified.


   Footnotes
 
1 We thank W.-K. Lin and Y.-S. Lin for technical assistance and H. Ridgway for the goat blood samples. Back

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 12, 2004. Accepted for publication January 24, 2005.


   Literature Cited
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Implications
 Literature Cited
 


Amills, M., C. Sulas, A. Sanchez, G. Bertoni, R. Zanoni, and G. Obexer-Ruff. 2004. Nucleotide sequence and polymorphism of the caprine major histocompatibility complex class II DQA1 (Cahi-DQA1) gene. Mol. Immunol. 42:375–379.

Ballingall, K. T., A. Luyai, and D. J. McKeever. 1997. Analysis of genetic diversity at the DQA loci in African cattle: Evidence for a BoLA-DQA3 locus. Immunogenetics 46:237–244.[Medline]

Ballingall, K.T., B. S. Marasa, A. Luyai, and D. J. McKeever. 1998. Identification of diverse BoLA DQA3 genes consistent with non-allelic sequences. Anim. Genet. 29:123–129.[Medline]

Bassam, B. J., G. Caetano-Anolles, and P. M. Gresshoff. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 196:80–83.[Medline]

Davies, C. J., L. Andersson, S. A. Ellis, E. J. Hensen, H. A. Lewin, S. Mikko, N. E. Muggli-Cockett, J. J. van der Poel, and G. C. Russell. 1997. Nomenclature for factors of the BoLA system, 1996. Report of the ISAG BoLA Nomenclature Committee. Anim. Genet. 28:159–168.

Ellis, S. A., and K. T. Ballingall. 1999. Cattle MHC: Evolution in action? Immunol. Rev. 167:159–168.[Medline]

Escayg, A. P., J. G. Hickford, G. W. Montgomery, K. G. Dodds, and D. W. Bullock. 1996. Polymorphism at the ovine major histocompatibility complex class II loci. Anim. Genet. 27:305–312.[Medline]

Felsenstein, J. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783–791.

Gelhaus, A., B. Forster, C. Wippern, and R. D. Horstmann. 1999. Evidence for an additional cattle DQA locus, BoLA-DQA5. Immunogenetics 49:321–327.[Medline]

Hickford, J. G. H., H. J. Ridgway, and A. P. Escayg. 2000. Evolution of the ovine MHC DQA region. Anim. Genet. 31:200–205.[Medline]

Hickford, J. G. H., H. Zhou, and Q. Fang. 2004. Diversity of the ovine DQA2 gene. J. Anim. Sci. 82:1553–1563.[Abstract/Free Full Text]

Kimura, M. A. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16:111–120.[Medline]

Kumar, S., K. Tamura, and M. Nei. 2004. MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 5:150–163.[Abstract/Free Full Text]

Montgomery, G. W., and J. A. Sise. 1990. Extraction of DNA from sheep white blood cells. N. Z. J. Agric. Res. 33:437–441.

Reche, P. A., and E. L. Reinherz. 2003. Sequence variability analysis of human class I and class II MHC molecules: Functional and structural correlates of amino acid polymorphisms. J. Mol. Biol. 331:623–641.[Medline]

Scott, P. C., K. J. Gogolin-Ewens, T. E. Adams, and M. R. Brandon. 1991. Nucleotide sequence, polymorphism, and evolution of ovine MHC class II DQA genes. Immunogenetics 34:69–79.[Medline]

Seidl, C., U. Koch, G. Brunnler, T. Buhleier, R. Frank, B. Moller, E. Markert, G. Koller-Wagner, E. Seifried, and J. P. Kaltwasser. 1997. HLA-DR/DQ/DP interactions in rheumatoid arthritis. Eur. J. Immunogenet. 24:365–376.[Medline]

Toussirot, E., B. Auge, P. Tiberghien, J. Chabod, J. P. Cedoz, and D. Wendling. 1999. HLA-DRB1 alleles and shared amino acid sequences in disease susceptibility and severity in patients from eastern France with rheumatoid arthritis. J. Rheumatol. 26:1446–1451.[Medline]

Zhou, H., and J. G. H. Hickford. 2004. Allelic polymorphism in the ovine DQA1 gene. J. Anim. Sci. 82:8–16.[Abstract/Free Full Text]



This Article
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