| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
ANIMAL GENETICS |
Cell Biology Group, Agriculture and Life Sciences Division, Lincoln University, Canterbury, New Zealand
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
; Ellis and Ballingall, 1999).
Despite extensive studies documenting gene structure and polymorphism in the DQA genes from sheep (Hickford et al., 2000, 2004; Zhou and Hickford, 2004) and cattle (Ballingall et al., 1997, 1998; Gelhaus et al., 1999), relatively little is known about their counterparts in goats, with only one DQA gene (DQA1) having been investigated recently (Amills et al., 2004).
In sheep, there are two DQA genes, called DQA1 and DQA2 (Scott et al., 1991). 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
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), 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., 2004), with comparison to caprine DQA1 sequences (Amills et al., 2004). 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).
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) two-parameter method, using MEGA v. 3.0 (Kumar et al., 2004; http://www.megasoftware.net/). The reliability of the trees was estimated by bootstrap confidence values (Felsenstein, 1985), 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 2), 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.
|
The assignment and naming of the caprine DQA2 alleles followed criteria proposed for the cattle MHC (Davies et al., 1997; http://www.projects.roslin.ac.uk/bola/bolahome.html), which have been used for sheep DQA genes (Hickford et al., 2004; Zhou and Hickford, 2004). 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
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 1). 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., 2004). This is consistent with the pattern in sheep (Hickford et al., 2004; Zhou and Hickford, 2004).
|
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 2
). 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 1 domain of DQA2, 38 (46%) were polymorphic; however, in the putative antigen-binding region (Reche and Reinherz, 2003), 14 out of the 19 AA sites (74%) were polymorphic (Figure 2). The most polymorphic sites were observed at 14 (D, E, V, T), 68 (I, K, T, A), and 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., 1997; Toussirot et al., 1999).
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 3). 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.
|
; Toussirot et al., 1999).
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., 2004). This argument is supported by the sequence data, where *0801 and *0901 have an isoleucine residue at 26, whereas the other sequences have a threonine. In sheep, a threonine at 26 is associated with DQA1 and DQA2 alleles, whereas DQA2-like alleles have an isoleucine at this position (see Figure 2).
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., 2000). 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 26, the threonine codon that is conserved in both DQA1 and DQA2 sequences has been converted to an isoleucine codon.
|
Implications |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
|
Footnotes |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionImplicationsLiterature Cited |
|---|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
and the bovine DQA4 sequences (Ballingall et al., 1998
. The putative DQA2-like sequences from goats and sheep are marked with 
.