The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Esteves, P. J.
Right arrow Articles by van der Loo, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Esteves, P. J.
Right arrow Articles by van der Loo, W.
Right arrowPubmed/NCBI databases
*Protein
The Journal of Immunology, 2004, 172: 1044-1053.
Copyright © 2004 by The American Association of Immunologists

Allelic Variation at the VHa Locus in Natural Populations of Rabbit (Oryctolagus cuniculus, L.)1

Pedro J. Esteves*,{dagger}, Dennis Lanning{ddagger}, Nuno Ferrand*, Katherine L. Knight{ddagger}, Shi-Kang Zhai{ddagger} and Wessel van der Loo2,*,{dagger}

* CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Campus Agrário de Vairão, Rua Padre Armando Quintas, Vairão, Portugal, and Departamento de Zoologia e Antropologia, Faculdade de Ciências da Universidade do Porto, Porto, Portugal; {dagger} Institute of Molecular Biology and Biotechnology, Vrije Universiteit Brussel, Brussels, Belgium; and {ddagger} Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL 60153


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The large interallelic distances between the three rabbit Ig VHa lineages, a1, a2 and a3, suggest that the persistence time of the VHa polymorphism could amount to 50 million years, which is much longer than that of MHC polymorphisms. Rabbit originated in the Iberian Peninsula where two subspecies coexist, one of which is confined to Southwestern Iberia (Oryctolagus cuniculus algirus). We studied the VH loci in the original species range to obtain a better understanding of the evolutionary history of this unusual polymorphism. Serological surveys revealed that sera from the subspecies algirus, when tested with VHa locus-specific alloantisera, showed either cross-reactivity ("a-positive" variants) or no reaction at all ("a-blank"). Using RT-PCR, we determined 120 sequences of rearranged VH genes expressed in seven algirus rabbits that were typed as either a-positive or a-blank. The data show that the VH genes transcribed in a-positive rabbits are closely related to the VH1 alleles of domestic rabbits. In contrast, a-blank rabbits were found to preferentially use VH genes that, although clearly related to the known VHa genes, define a new major allotypic lineage, designated a4. The a4 sequences have hallmark rabbit VHa residues together with a number of unprecedented amino acid changes in framework region 2 and 3. The net protein distances between the VHa4 and the VHa1, a2, and a3 lineages were 20, 29, and 21% respectively. We conclude that at least four distantly related lineages of the rabbit VHa locus exist, one of which seems to be endemic in the Iberian range.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In rabbit, the gene locus controlling the variable region of the Ig heavy chain (IGVH, or VH) is unusual by showing extensive allelic diversity. Initially, this was discovered by serological methods, which revealed that VH regions of nearly all rabbit Abs can be placed, on the basis of the allotypic motifs, into one of three groups, namely, a, x, or y (1, 2, 3). Three "a" allotypic lineages have been described, and their Mendelian inheritance was established by extensive breeding studies (1, 4, 5). These so called a1, a2, and a3 allotypes have characteristic amino acid sequence differences in framework region (FR)3 1 (Kabat positions 5, 8, 10, 12, 13, 16, and 17) and FR3 (positions 65, 67, 70, 71, 74, 84 and 85;6). These differences reflect allelic variation at the D-proximal VH gene, VH1, which accounts for ~80% of VDJ gene rearrangements (7). Unlike for other mammalian species, where VH1, VH2, VH3, etc. refer to gene "families" with pronounced sequence similarity, in rabbit, VH gene segments are numbered according to their relative position on the chromosome of a given allotype. (Note: for convenience "VH gene segments" will be written below "VH genes"). Thus, VH1-a1, is the 3'-most VH gene of the a1 haplotype; VH7-a2 is immediately 5' of the VH6-a2 gene of the a2 haplotype, etc. Only a small number of the >100 rabbit germline VH genes have been sequenced (8). Besides VH1, other VH genes have been identified that encode VH1-associated "allotypic" motifs (including VH4-a1, {varphi}VH2-a1, {varphi}VH3-a1, VH4-a2, {varphi}VH3-a2, VH7-a2, VH9-a2). They contribute to VDJ gene diversification by gene conversion (9) and evolved to some extent in concert with the neighboring VH1 gene, defining therefore a haplotype polymorphism. The group of these genes will be indicated as "VHa" or "a-positive," to be distinguished from VHn genes, which do not encode VH1-associated allotypic markers (VHx, VHy, and VHz, or "a-negative";10). The latter genes are situated >50 kb upstream of the VHa genes and contribute to <20% of rabbit Abs (11, 12). In this study, the concept of "VHa genes," which depends on serology, will be extended to "VH genes showing significant monophyletic clustering with one or more known VHa genes in phylogenetic inference analyses based upon DNA or protein sequences." Newly described VH genes sharing derived character states with the VHa genes can then be classified as such, independent of serology. VHa genes of the aj haplotype will be designated as VHa-aj, where j = 1, 2, 3, 4, etc.

Rabbit Ig allotypes are among the first protein polymorphisms ever described (13), but after 50 years of research, we still do not understand their raison d’être. The problem posed by the Mendelian inheritance of genetic markers at a multigene locus was resolved by the finding that rabbit preferentially uses the VH1 gene (7) and, further, by the mechanism of concerted evolution (14). However, neither preferential expression nor concerted evolution can explain how, or why, such large differences between allelic lineages have evolved (concerted evolution being a mechanism that tends to homogenize the gene pool). Large differences between alleles are believed to be the outcome of diversity enhancement selection, which can favor both prolonged allele persistence times and increased evolutionary rates. According to an evolutionary analysis by Su and Nei (15), the large genetic distances between the three VH1 alleles suggest allele persistence times of ~50 million years (My), which is an order of magnitude larger than average mammalian speciation times. Both prolonged persistence times and increased evolutionary rates imply that this population diversity must fulfill some crucial function (cf.16, 17, 18). However, the former hypothesis has specific and profound population genetic implications in regard to founder population sizes, which must always be large enough to contain each of the different alleles. In view of the importance of the questions raised by the rabbit IgH a-locus polymorphism for half a century, it might be surprising that data on the rabbit VH genes remain fragmentary and concern only a small number of domestic breeds. These breeds are recent genetic isolates (<200 years) of the subspecies Oryctolagus cuniculus cuniculus, while the genus originated some 4–6 My ago on the Iberian Peninsula (19, 20). Today, this area is inhabited by two subspecies, O. c. cuniculus and O. c. algirus, which, according to mitochondrial DNA data, could be separated by some 2 My (for details, see Refs.21 and 22).

Serological studies of Cazenave et al. (23) had already indicated that wild rabbits from the Iberian Peninsula can express VHa allotypes that differ from those occurring in domestic breeds. A large number of alleles would not be supportive of extremely long allele persistence times, because it is unlikely that many allelic lineages would be maintained throughout numerous speciation steps. However, the serological characterization suggested that the "wild-type" allotypes could represent more recent variations occurring within one of the three VHa lineages of domestic rabbits (for convenience referred to as "domestic" allotypes). This was also supported by partial amino acid sequence of one of these allotypes (a100), which showed a close relationship with the a3 lineage (i.e., 4 aa differences in the FR regions;24). When we analyzed VH allotypes in wild rabbits from the Iberian Peninsula, a large fraction of samples from the Southwestern areas did not show any cross-reactivity with VHa-specific alloantisera. As illustrated in Fig. 1, this phenotype was correlated with genetic markers characteristic of the subspecies, O. c. algirus, and in particular to the mitochondrial DNA type A (25, 26). The cytonuclear disequilibrium between the cytotype A and the "a-blank" allotype (nucleotype), when estimated according to Asmussen and Arnold (27), was highly significant (X21 = 36; data not shown). Among the possible explanations were 1) these rabbits preferentially use VHx or VHy genes, possibly because the D-proximal VH genes were reshuffled, damaged, or deleted (cf.7, 10), or 2) they express a VH1 allotype that differs markedly from those occurring in domestic breeds.



View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 1. Geographic distribution of VHa locus allotype frequencies. Determined for populations of European wild rabbit (O. cuniculus) from the Iberian Peninsula, France, and Belgium. The subspecies O. c. algirus occupies the Southwestern part of Iberia, which is indicated by A. Rabbits of the Northwestern areas of Iberia and of the rest of Europe (indicated by B) belong to the subspecies O. c. cuniculus. The contact zone between the two subspecies is indicated in gray. The coloring of the disks reflects the relative allele frequencies per locality, analyzed as a two-allele locus with a-blank (white) and a-positive (black) alleles.

 
We have sequenced the VH genes expressed in eight wild specimens of the subspecies O. c. algirus, four of which were typed a-blank. The data show that these latter rabbits preferentially express VH genes that define a fourth major lineage of VH1 allotypes (hypothesis 2). The lack of cross-reactivity with allotype-specific alloantisera can be explained by the observed amino acid changes. At the same time, the sequences obtained from a-positive O. c. algirus rabbits suggest that variations have also occurred within the major VH1 lineages.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
PCR amplification, cloning, and nucleotide sequencing of rearranged VDJ genes

Spleen and serum samples were obtained from adult wild rabbits (O. c. algirus) that were collected within the military domain of Alcochete (Ribatejo, Portugal). Serum samples were analyzed serologically by double immunodiffusion to determine a1, a2, and a3 VH allotypes. Total RNA was isolated from frozen spleen samples with TRIzol, following the manufacturer’s instructions (Life Technologies, Grand Island, NY). First-strand cDNA was synthesized from 3 µg of RNA using oligo(dT) as a primer (28). VDJ gene rearrangements were PCR-amplified using primers specific for conserved regions in the VH leader (29) and in the JH gene segments (JH-B: 5'-GAGCTCACCTGAGAGACGGTGACCA-3'). PCR amplifications proceeded for 30 cycles (each cycle: 94°C for 45 s, 60°C for 45 s, and 72°C for 60 s). PCR products ~500 bp in length were gel-purified and cloned into the pGEM-TEasy vector (Promega, Madison, WI). Nucleotide sequences of the cloned VDJ genes were determined using an automated ABI Prism 310 sequencer with Big Dye-labeled terminators (PerkinElmer Applied Biosystems, Foster City, CA).

Phylogenetic studies

The VH nucleotide sequences were aligned using the computer program Pileup (GCG© package, Wilkinson, as provided by the Belgian EMBnet Node (www.be.embnet.be) and Clustal W (30) as provided by the freeware DAMBE package (31 ; aix1.uottawa.ca/~xxia). The alignments were corrected upon visual inspection using Genedoc (32 ; www.psc.edu/biomed/genedoc). Phylogenetic analyses were performed using the MEGA 2.1 computer program (33 ; www.megasoftware.net). Evolutionary distances between nucleotide or protein sequences were estimated using uncorrected p-distance. For nucleotide sequences, we also show results using Kimura’s 2-parameter option. We prefer the former approach because it makes a minimal number of assumptions and, according to Nei and Kumar (34), it gives better results when large numbers of relatively short sequences are compared. Phylogenetic trees were constructed using the neighbor-joining (NJ) method (35). For the construction of the phylograms, we included a sample of published diversified rabbit VH gene sequences representing the three domestic allotypes (accession nos. AF264476, AF264478-81, AF264493, AF264496, AF264499, AF264528, AF264544, AF264545, AF264546, AF264571-73, AF264577, AF264583, AF264585, AF264587, AF264589, AF264592, AF264595-96). The reliability of the phylogenetic tree was tested by determining the values of bootstrap (36) and confidence (37, 38) probabilities, respectively. For sequence pairs under comparison, alignment gaps were excluded from analysis (pairwise deletion option in MEGA). The molecular clock was tested by the relative rate test (39), as provided in the MEGA package.

Identification of novel genetic variants

For discerning the germline VH genes from groups of diversified VH sequences, the algirus sequences were aligned with all known rabbit VH germline genes. The algirus sequences often show some systematic variation that differentiates them from sequences obtained from domestic rabbits. For the vast majority of sequences of a-blank rabbits, this "unprecedented" variation clearly indicates novel variants of VH genes in algirus rabbits. This is less obvious for some sequences of a-positive rabbits, where recurrent variation could either indicate "novel" genetic variants of the "domestic" allotypes or the consistent use, for gene conversion events, of donor VH genes present in both algirus and domestic rabbits. The latter possibility was evaluated by searching the GenBank database for rabbit sequences showing the same unexpected amino acid replacements. This was tested by submitting such variant sequences, or segments of them, to protein BLAST searches using the software provided by National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/blast). The alignments for the 1000 most similar rabbit VH gene sequences were edited under the "flat query-anchored with identities" option, and examined visually.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serological typing and sequence determination

cDNA was obtained from spleen and peripheral blood cells of seven adult wild rabbits: Rb07, Rb11, Rb27, Rb32, Rb36, Rb40, Rb43, and Rb60. According to their mitochondrial DNA (26), as well as to other genetic markers (protein and microsatellite DNA;21), these rabbits belong to the subspecies O. c. algirus (data not shown). When tested with conventional alloantisera, Rb43-typed "a1/a2" heterozygous, Rb07 and Rb36 "a1 only," and Rb11 "a3 only." Sera of Rb32 showed weak cross-reaction with a3-specific antisera (a3-variant 32). Sera of rabbits Rb27, Rb40, and Rb60 did not show any cross-reactivity (a-blank). Because of the prevalence of a-blank in the study area (>20%), the a-positive rabbits (Rb07, Rb36, Rb11, Rb32) have a reasonable chance of being heterozygous for the a-blank genotype.

A total of 120 VH sequences, comprising the entire VH exon, were obtained, of which nine were duplicate sequences that were obtained from a same rabbit. One hundred eleven nonindentical sequences were deposited in GenBank under accession numbers AY207941 to AY208048 and AY299401 to AY299403. Except AY208000 and AY208009 (both from Rb43), which differ by a synonymous C-T transition at the arginine codon of amino acid position 38, all submitted sequences encode unique amino acid sequences. Forty-four of them were expressed in rabbits typed as homozygous a-blank, while 11 more were obtained from a rabbit that appeared to be heterozygous for the a-blank allotype (Rb07). Table I lists, for each of the eight serotypes, the number of sequences leading to unique amino acid sequences (110 sequences, of which 52 were classified a-blank, 55 a-positive, and 3 a-negative). Unless stated otherwise, we present and discuss the results at the level of the protein sequence.


View this table:
[in this window]
[in a new window]
  		Table I. Number and allotypic classification of VDJ sequences obtained from eight rabbits

 
Inferring VH genes expressed in O. c. algirus

The VH regions of rearranged VDJ genes in adults are likely somatically diversified versions of the used genomic VH genes. Sequence comparisons can help to infer the likely sequences of these germline genes, as well as identify genes contributing to the VH gene diversification by gene conversion.

The O. c. algirus sequences were therefore aligned with all published rabbit genomic VH sequences. Rabbit VH sequences belong to the class C (40, 41). For this reason, we used, as outgroups in phylogenetic analysis, vertebrate VH sequences that are separated by a relatively short branch from the root of class C sequences (human VH3, camel VH, and duck VH). Fig. 2 displays the unique amino acid sequences inferred from the nucleotide data, grouped according to highest similarity to known rabbit germline genes. They are compared with a consensus sequence of rabbit VHa genes to highlight the differences among the VHa allotypes. Residues are numbered according to IMGT numberings (imgt.cines.fr;42) or to Kabat et al. (43), the latter numbering being used throughout the text below. For groups of VH sequences that show systematic differences from known genes (which is the case for the majority of sequences obtained from a-blank rabbits), consensus sequences are proposed. These likely represent the underlying germline sequence. The possibility that differences might be due to gene conversion with VH genes also present in domestic breeds was examined by BLAST searches outlined in Materials and Methods.



View larger version (96K):
[in this window]
[in a new window]
  		FIGURE 2. Alignment of unique protein sequences of VH regions. VH regions shown are inferred from VDJ genes obtained from eight wild Iberian rabbits and of published genomic rabbit VH genes. Sequence names of the former refer to the donor rabbits (Rb07, Rb11, Rb27, Rb32, Rb36, Rb40, Rb43, and Rb60). They are compared with a consensus sequence of rabbit VH1 gene segment (CONS-vh1) and grouped according to phylogenetic relationships, shown in Fig. 3. Allotypic marker positions are indicated by {blacktriangledown} and for the a1 and a2 allotypes, respectively, and corresponding matches are highlighted in bold. GenBank accession numbers of underlying nucleotide sequences are included. Each VDJ gene cluster is compared with germline VH gene(s) (marked GL-) of that cluster. For VDJ groups showing recurrent differences with known genomic genes, consensus sequences are proposed (indicated by CONS-). Sequence names of suspected crossing-over products are marked by x (a4.1 sequences are underlined and a4.2 sequences are enclosed in boxes). Potential gene conversions are marked by rc. Hypervariable parts of complementarity-determining regions are shaded. A, Sequences showing significant clustering with one or more known genomic sequences. B, Sequences clustering apart from known genomic sequences. C, Comparison of inferred consensus sequences with relevant genomic sequences of rabbit and with class C VH sequences used as outgroups in phylogenetic trees presented in Figs. 3 and 4.

 


View larger version (81K):
[in this window]
[in a new window]
  		FIGURE 2A. Continued.

 
VH genes used by a-positive algirus rabbits

VHa-a1 genes expressed by Rb07, Rb36, and Rb43. Rb07 appears heterozygous for the a1 allele and another allele that is also expressed in a-blank rabbits (see below). Whereas the a1 sequence of Rb07 conforms to a domestic VH1-a1 gene, those expressed by Rb36 and Rb43 consistently show at positions 17, 74, 82, and 82C the residues that distinguish VH4-a1 from VH1-a1. The second deletion in FR3 (position 74), which is characteristic of VH1-a1 and absent in VH4-a1, is also absent in all VHa-a1 sequences of these two rabbits, except for sequence Rb36_424. The latter sequence, however, shows more similarity with the pseudogene VH3-a1 and is likely a product of gene conversion (Fig. 2A). One sequence from Rb07 (Rb07_332; Fig. 2A) is clearly derived from a VHx-like gene, while one sequence from Rb43 (Rb_499) is clearly derived from a VHy-like gene.

VHa-a2 genes expressed by Rb43. The majority (14/16) of a2-like sequences from Rb43 encodes a glutamic acid residue at position 75 (E75; Fig. 2A), where VH1-a2 encodes a leucine residue (L75). E75 has also been observed in VDJ gene sequences from domestic a2 rabbits, although at much lower frequencies. As two tandem nucleotide are involved (GAG vs CTG), this variation is probably not due to somatic hypermutation, but more likely due to somatic gene conversion involving E75-encoding donor genes, such as VH7-a2 or VH9-a2 (cf.44). However, the high frequency of occurrence suggests that the VH1-a2 gene of Rb43 could represent an E75-encoding VH1-a2 variant allotype. In that case, the L75 codons of the two remaining Rb43 sequences (Rb43_008 and Rb43_011; Fig. 2A) may represent gene conversion events involving L75-encoding donor genes, such as VH4-a2.

VHa-a3 genes expressed in Rb11. The amino acid variation among Rb11 sequences is similar to that of domestic a3 rabbits (Fig. 2A). However, four sequences (Rb11_373, _374, _378, and _379) are atypical in containing arginine (R) residues at positions 64 and 71, where virtually all published a3 sequences (>99%) contain lysine (K). The sequence of Rb11_376 between positions 63 and 78 could be the product of gene conversion between VH1-a3 and a gene homologous, or identical, to domestic rabbit pseudogene VH3-a3.

VHa-a3 variant genes expressed in Rb32 (VH1-a3v32). Serological analysis shows that rabbit Rb32 expresses some, but not all, of the a3 determinants. The Rb32 sequences differ from those of the VH1-a3 allotype at nine FR positions: two in FR1 (D10, Q13), and seven in FR3 (Q66, D79, K81, T82, G85, H86, M87). The amino acid differences at positions 10 (D/G) and 13 (Q/P) of FR1 have also been observed in domestic a3 rabbits, although much less frequently, possibly due to gene conversion with VH6-a3 or a similar donor gene. In contrast, the substitutions in FR3 were never, or extremely rarely, observed in domestic rabbits. Among the 1000 rabbit VH sequences retrieved by a protein BLAST search using the FR3 region of Rb32_634 as query sequence (see Materials and Methods), none showed either the residues Q66 or H86 that are present in all but two of the R32 sequences. Residues T82, G85, and M87 each occurred at frequencies below 0.5%. D79 and K81 are frequently observed among a1 and a2 sequences, but not among a3 sequences. Rb32 undoubtedly expresses a genetic variant of the a3 lineage, which we call a3v32. Interestingly, residues G10, Q13, D79, and M87 have previously been reported in the partial amino acid sequence obtained by Tonelle et al. (24) from a wild rabbit typed a100 (Fig. 2A). It is possible that a100 and a3v32 are different names for the same allotype.

VH genes used by a-blank algirus rabbits define a fourth VHa allotypic lineage: VHa-a4

VH genes expressed in a-blank rabbits belong to the VHa group. Except for one single sequence (Rb60_822), which is clearly a product of an a-negative VHx-like gene, all VH genes expressed in the a-blank rabbits (Rb27, Rb40, Rb60) appear to be derived from very similar germline genes. Visual inspection of the inferred protein sequences (Fig. 2B) shows that they are clearly more related to the VHa than to VHx or VHy genes. This conclusion is supported by a variety of phylogenetic inference programs conducted at both the nucleotide and protein levels and is independent of the complementarity-determining regions.

For the construction of the phylogram shown in Fig. 3, we included a random sample of published diversified VH sequences of domestic rabbit (see Material and Methods). The a-blank sequences form a monophyletic cluster with the VH1-a genes, which clearly branches apart from the VHn genes (VHx, VHy, and VHz). At the protein level, the a-blank sequences share a number of apomorphic (or "derived") characters with one or more of the domestic VHa genes. Examples are the VHa hallmark peptides (18)LTLTCT(23) of FR1 and (62)WAK(64) of FR3. These are unique to rabbit VHa genes and do not occur in the VHx, VHy, or VHz sequences. The a-blank type shares a larger number of derived characters with the a1 and a2 allotypes than with the a3 allotype. The two deletions between positions 72 and 74, which are characteristic of the VH1-a1 allotype, are also present in each of the 49 a-blank sequences, where we observe either (71)K-T-ST(76) (as in VH1-a1) or (71)R-T-ST(76), confirming a close affiliation with the rabbit VH1 genes.



View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 3. Phylogenetic neighbor-joining tree of rabbit VH regions. Based upon nucleotide p-distances. Germline genes are labeled by dots and comprise: •, VHa genes (a1-vh(1,4), a2-vh(1,4,7,9), a3-vh(1,4)); , VHn genes (a2-vh6, a3-vh(3,6), x, y, z); , VHa pseudogenes (a1-vh(2,3)); {circ}, outgroup (Hum VH3, Camel VH, Duck VH). Squares, triangles, and diamonds indicate VH regions of RT-PCR-amplified VDJ genes obtained from wild rabbits of subspecies O. c. algirus: {blacktriangleup}, Rb07; , Rb11; {diamond}, Rb27; {square}, Rb32; , Rb36; , Rb40; , Rb43; {triangleup}, Rb60. VDJ gene sequences representing domestic rabbits of the a1, a2, and a3 allotypes are not labeled (accession numbers are displayed in Materials and Methods). Sequences reflecting possible crossing-over or gene conversions (marked by x and rc in Fig. 2) were excluded from the analysis. Confidence probability values (1000 replicates) are shown at branch nodes if larger than 85, except for the a4.2 clade (confidence probability = 73). The tree illustrates the fact that the vast majority of VDJ genes expressed in rabbits Rb07, Rb27, Rb40, and Rb60 form a well-defined cluster that is monophyletic and equidistant from the clades formed by the VHa genes expressed in domestic rabbits of the a1 and a2 allotypes.

 
VH a-blank defines a new VH1 allele. The a-blank VHa cluster is well separated from the three clusters defined by the sequences expressed in a1, a2, and a3 rabbits, respectively. Bootstrap and confidence probabilities are highest when a-blank sequences, or their consensus sequence, are solely compared with genomic VH genes (Fig. 4). Visual inspection of the protein sequences (Fig. 2B) reveals that the a-blank sequences, although clearly belonging to the VHa genes, have unique features in common that set them apart from the a1, a2, and a3 alleles. The most consistent marker residues are found in FR3, where, for each of 49 a-blank VHa sequences, the VHa consensus residue K/Q(81) is replaced by T(81). The VHa consensus peptide (83)TAA(85), which is TTE in the a1 allotype, is replaced by ELS, or by QPS in most a-blank sequences.



View larger version (16K):
[in this window]
[in a new window]
  		FIGURE 4. Phylogenetic neighbor-joining tree of rabbit VH regions. Based upon amino acid p-distances. The consensus sequences of the a4 allotype subtypes a4.1 and a4.2 were inferred from the data presented in Fig. 2B and compared with inferred amino acid sequences of published genomic rabbit VH genes. The tree is rooted on duck, camel, and human class C VH sequences. Bootstrap probability values were obtained with 1000 bootstrap replicates.

 
Although FR2 is highly conserved among vertebrate class C VH genes (including the rabbit VH1 genes), the a-blank sequences present some unprecedented changes. In the majority of them, the canonical (42)GK/NG(44) is replaced by either EEG or EER. Quite remarkable is the change of A(40) into VP(40), due to a 3-bp insertion before the second codon position of A40(gct->g[tg c]ct). This was observed in 17 sequences, obtained with three different rabbits. Assuming that, like domestic rabbits, a-blank rabbits preferentially use the D-proximal VH gene, we propose that the majority of the sequences obtained from a-blank rabbits are derived from a genuine allele of the VH1 gene, which we call VH1-a4. The phylograms in Figs. 3 and 4 compare the a4 sequences with the known functional genomic VH sequences of rabbits. The pairwise distances between the a4 sequence and the a1, a2, or a3 sequences were similar to those between the latter three (domestic) allotypes (Table II), confirming that the a4 lineage cannot be considered a subtype of one of the known allotypic lineages.


View this table:
[in this window]
[in a new window]
  		Table II. Estimation of genetic distances between major VHa allotypic lineagesa

 
Allelic diversity among VH1-a4 sequences? The degree of diversity among a4 sequences tended to be larger than currently observed among a-positive sequences of the same allotype in domestic rabbits (data not shown). This could have to do with the fact that our sample of a4 sequences was obtained from seven different haplotypes (one heterozygous and three homozygous a4 rabbits), i.e., cryptic allelic variation could account for some of this diversity. Phylogenetic clustering methods and visual inspection of the amino acid alignments indeed indicate that there might be at least two a4 variants, a4.1 and a4.2. The consensus sequences of these two variants are presented in Fig. 2B. Rb60 appears to be homozygous for one variant (a4.1). Rabbit Rb07, which is heterozygous a1/a4, appears to possess the a4.2 variant, whereas Rb27 and Rb40 appear to be heterozygous a4.1/a4.2. The phylogram in Fig. 4 highlights the position of the a4.1 and a4.2 consensus protein sequences among known genomic VH genes.

Gene conversion. There are strong indications that the putative a4 variants are diversified by gene conversion events involving VH donor genes that, in some cases, might be present in both lineages. At a number of positions, amino acid replacements requiring two or more nucleotide substitutions occur more often than expected if due to somatic hypermutation (examples are D/G10, 27ID/FS28, W/Y47, 71AK/VN72, N/A82b). Inspection of the sequence alignments indicates that in some cases variation could possibly be due to PCR crossing-over. Indeed, sequences Rb40_905, Rb40_901, and Rb27_561 unite characteristics of a4.1 and a4.2 sequences in the 5' and 3' regions, respectively, while the opposite is true for Rb40_908 (Fig. 2). These sequences were not included in the phylogenetic analysis shown in Fig. 3. As these sequences were obtained from rabbits that express both subtypes, PCR crossing-over, and/or gene conversion could be an additional source of variation. However, crossing-over was not observed between the a1 and a4 genes of Rb07 or between the a1 and a2 genes of Rb43. The presence or absence of the valine (position 39A) insertion in FR2 among the a4.1 sequences indicates that more than one VH gene contributes to this particular allotype. The sequence of Rb60_810 between position 1 and 17 could be a product of gene conversion of the putative a4.2 gene and a gene resembling the VH4-a1 gene of domestic rabbit. We conclude that the VHa-a4 sequences presented here are likely diversified by gene conversion. In Fig. 2B, we have indicated frequent alternates of the consensus sequences.

Gene usage. In sharp contrast with our first hypothesis, the a-blank rabbits do not preferentially use VHn genes. In fact, of the 43 sequences obtained from rabbits Rb27, Rb40, and Rb60, 42 had the VHa signature, while only one was VHx-like. If indeed the number of PCR clones reflects gene usage, VHn genes were used significantly less frequently in a-blank rabbits than was previously observed in domestic rabbits. This pronounced bias in gene usage is apparently not a characteristic linked to the a-blank genotype, because it was also observed for the a-positive rabbits of the same population (55 VHa sequences vs zero VHx and one VHy).

Allelic imbalance. Another characteristic of the rabbit VHa allotypes is "allelic imbalance" in gene expression or "pecking order" (45, 46, 47). This refers to the fact that the different VHa alleles are not used to the same extent in heterozygous animals. Thus, heterozygous a1/a2 rabbits consistently use a1 genes more often than a2 genes. We note that with Rb43, which typed a1/a2 heterozygous, the ratio a1/a2 was on the contrary 3:16. This could possibly have to do with the fact that the VH1-a1 of this rabbit differs from that in domestic breeds (see above). For rabbit Rb07, which is heterozygous a1/a4, the a1/a4 ratio was 1:7, indicating that allelic imbalance, if occurring at all, would favor the expression of the a4 allotype in the presence of the a1 allotype.

Absence of VHa serological markers. In Fig. 2, the VHa-allotypic residues (i.e., residues known to be associated with serological determinants of the allotype) are highlighted. The a4 sequences present most of the domestic allotypic marker residues of FR1. These marker residues however are apparently not presented in combinations required for serological cross-reactivity. For example, while the a2 marker residues K5, F12, and T17 (FR1 region) are frequently found among a4 molecules, they never occur together. The same is true for the a1-associated FR1 residues R10 and T13, except for two Rb60 sequences (Rb60_810, Rb60_878), where we would expect the phenotypic expression of an a1 determinant. The fact that rabbit Rb60 was typed a-blank suggests that the serum level of such molecules is too low for detection or, more likely, that a majority of the Abs of our a1-specific antisera are directed against determinants in the FR3 region. Indeed, none of the marker residues of the a1, a2, or a3 allotypes was observed in the FR3 region of the a4 sequences.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Proteins encoded by allelic genes show in general not >1–3% aa differences. Evolutionary theory offers two possible explanations for interallelic distances as large as those observed at the rabbit VHa locus: unusually long allele persistence times and increased mutant recruitment rates. The concept of a molecular clock is a powerful tool that evolutionists avoid abandoning. According to Su and Nei (15), the fact that alloantisera raised in rabbit distinguish three VHa allotypes in populations of Lepus americanus suggests that both species have inherited the same three lineages, which could explain the large interallelic distances without invoking acceleration of evolution rates.

The main objective of this study was to determine whether the study of natural populations in the original species range could contribute to a more accurate estimate of the genetic variation at this locus. We show here that there are at least four highly divergent lineages (Fig. 2). A correct estimation of the distance between the a4 lineage and the other VHa lineages is hampered by the fact that only diversified sequences of the putative VH1-a4 gene are available. However, the net distances between diversified VH sequences of the four lineages are similar (Table II). If we assume with Su and Nei (15) that the VHa genes of different haplotypes evolved at a similar rate as other proteins (i.e., 1.4 x 10-9 nucleotide substitutions per year), then not three but four lineages have been maintained over numerous speciation steps (40–60 My). However, the present geographical distribution of the four allotypes in the native species range shows that the a4 allele is associated with the maternal markers of the subspecies O. c. algirus and did not pass the Pyrenean Mountains (Fig. 1).

A detailed evolutionary analysis of the present data is beyond the scope of this paper. It is nevertheless interesting to compare the present observations with those made in a study of gene diversity at the IGKC1 or CK1 b locus in Iberian rabbit populations (48). The CK1 gene, which encodes the constant region of the Ig{kappa}1 L chain, is used in >95% of L chain rearrangements. Allelic differences at this locus are even larger than those at the VHa locus and contrast sharply with the very limited variation at the quasi-silent CK2 bas locus. The b4 and b5 CK1 alleles, which predominate in all populations of O. c. cuniculus, were less frequent in populations of O. c. algirus. These populations instead express genetic variants serologically related to the b4 and b5 alleles but differing from them at up to 15% of the amino acid residues. It was argued that the association of specific alleles with subspecies markers is in contradiction with extremely long allele persistence times.

The present results, showing that the VHa allotypes that prevail in O. c. cuniculus populations, are in part "replaced" in O. c. algirus populations by the related a4 allotype (Fig. 1), mirrors to some extent the situation described for the CK1 b locus. In the study mentioned, the molecular clock hypothesis was rejected for the CK1 lineages, and the data analysis furthermore indicated that, compared with the b9 lineage, the b4 and b5 lineages showed both higher allele turnover rates and higher evolutionary rates.

Our data suggest that evolutionary modes might also vary among VHa lineages. In Fig. 4., we show a phylogenetic tree reduced to the genomic sequences and the a4 consensus sequences. It strongly suggests that amino acid divergence was markedly increased in the VHa a1, a2, and a4 lineages in comparison to that in the a3 and VHn lineages. The statistical significance of these differences was confirmed by Tajima’s relative rate tests (Table III) and the Felsenstein maximum likelihood ratio test (data not shown). Because of the increased evolutionary rate in the VHa a1, a2, and a4 lineages, allele persistence time could be largely overestimated. A considerable fraction of the amino acid differences characterizing the a4 allele might indeed have accumulated after the separation of the subspecies, explaining its association with subspecies O. c. algirus.


View this table:
[in this window]
[in a new window]
  		Table III. Tajima’s relative rate test for rate constancy among rabbit VH genesa

 
In view of the evidence suggesting a role for superantigens in the expansion of B cell lineages that present VHa allotypic motifs (16, 17, 18), and of the gut microflora in Ab repertoire diversification (49, 50), another explanation for the correlation between the complex Ig allotypes (VHa and CK1b) and subspecies markers should be considered. By occupying different habitats, each subspecies might indeed acquire a different intestinal microflora, which could favor habitat-specific frequency distributions of (pre-existing) VHa allotypes. All 52 a4 sequences here obtained share highly unusual residues at H81-H85 (or H90-H97 in IMGT numbering), suggesting that this region is not affected by gene conversion, or/and that B cells presenting these motifs are preferentially stimulated to expand. X-ray crystallography shows that in human the VH3 domain residues at these positions interact with a protein A domain of Staphylococcus aureus (H81-H82b;51). Thus, the four VHa allotypes were perhaps present in the common ancestor species, but the expansion of the a4 allotypes was favored by selection only in Southwestern regions. Selection factors have indeed previously been invoked by Sehgal et al. (44) and Zhu et al. (29) for explaining the "reconstruction" the a2 allotypic motifs in VH1-a2 knockout rabbits (i.e., Alicia strain).

Unlike FR3, the FR2 region is not expected to interact with superantigens. In fact, the allelic variation at the FR2 of VHa-a4 proteins affects residues that interact with the VL domain and are highly conserved among vertebrate VH genes. The replacements of glycine residues at positions H42 and H44 by charged residues are surprising. It is noteworthy that in the camel and lama, amino acid replacements in this molecular region were observed for the VHH domains of the H chain Abs (including a G/E exchange at H44) and have been associated with the loss of L chains (52).

In conclusion, the presented data show that the study of wild specimens of the original species range can contribute significantly to our understanding of the evolution and biological meaning of the rabbit Ig polymorphisms.


   Footnotes
 
1 This work was supported by Direccçao Geral de Florestas, by a grant from the Foundation for Science and Technology-Portugal (Praxis XXI/BD/16207/98; to P.E.), grants from the National Institute of Health (NIH AI50260 and AI49458; to K.L.K. and D.L.), and by a grant from the Belgian Fonds voor Wetenschappelijk Onderzoek Vlaanderen (Krediet 1.5579.98-FWOKN35; to W.vdL.). Back

2 Address correspondence and reprint requests to Dr. Wessel van der Loo, CIBIO, ICETA-UP, Campus Agrário de Vairão, Rua Padre Armando Quintas, Vairão, Portugal. E-mail address: wvdloo{at}ben.vub.ac.be Back

3 Abbreviations used in this paper: FR, framework region; My, million years. Back

Received for publication August 11, 2003. Accepted for publication October 27, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kim, B. S., S. Dray. 1972. Identification and genetic control of allotypic specificities on two variable region subgroups of rabbit immunoglobulin heavy chains. Eur. J. Immunol. 2:509.[Medline]
  2. Kim, B. S., S. Dray. 1973. Expression of the a, x, and y variable region genes of heavy chains among IgG, IgM, and IgA molecules of normal and a locus allotype-suppressed rabbits. J. Immunol. 111:750.[Abstract/Free Full Text]
  3. Roux, K. H.. 1981. A fourth heavy chain variable region subgroup, w, with 2 variants defined by an induced auto-antiserum in the rabbit. J. Immunol. 127:626.[Abstract]
  4. Oudin, J.. 1960. L’allotypie de certains antigènes proteidiques du sérum. Relation immunochimiques et génétiques entre six des principaux allotypes observés dans le sérum de lapin. CR Acad. Sci. Paris 242:2489.
  5. Dray, S. G., O. Young, A. Nisonoff. 1963. Distribution of allotypic specificities among rabbit immunoglobulin {gamma}-globulin molecules genetically defined at two loci. Nature 199:52.[Medline]
  6. Mage, R. G., K. E. Bernstein, N. McCartney-Francis, C. B. Alexander, G. O. Young-Cooper, E. A. Padlan, G. H. Cohen. 1984. The structural and genetic basis for expression of normal and latent VHa allotypes of the rabbit. Mol. Immunol. 21:1067.[Medline]
  7. Knight, K. L., R. S. Becker. 1990. Molecular basis of the allelic inheritance of rabbit immunoglobulin VH allotypes: Implications for the generation of antibody diversity. Cell 60:963.[Medline]
  8. Gallarda, J. L., K. S. Gleason, K. L. Knight. 1985. Organization of rabbit immunoglobulin genes. I. Structure and multiplicity of germ-line VH genes. J. Immunol. 135:4222.[Abstract]
  9. Becker, R. S., K. L. Knight. 1990. Somatic diversification of immunoglobulin heavy chain VDJ genes: Evidence for somatic gene conversion in rabbits. Cell 63:987.[Medline]
  10. Kelus, A. S., S. Weiss. 1986. Mutation affecting the expression of immunoglobulin variable regions in the rabbit. Proc. Natl. Acad. Sci. USA 83:4883.[Abstract/Free Full Text]
  11. Di Pietro, L. A., J. A. Short, S. K. Zhai, A. S. Kelus, D. Meier, K. L. Knight. 1990. Limited number of immunoglobulin VH regions expressed in the mutant rabbit "Alicia". Eur. J. Immunol. 20:1401.[Medline]
  12. Short, J. A., P. Sethupathi, S. K. Zhai, K. L. Knight. 1991. VDJ genes in VHa2 allotype-suppressed rabbits. Limited germline VH gene usage and accumulation of somatic mutations in D regions. J. Immunol. 147:4014.[Abstract]
  13. Kelus, A. S., P. G. Gell. 1967. Immunoglobulin allotypes of experimental animals. Prog. Allergy 11:141.[Medline]
  14. Smith, G. P.. 1976. Evolution of repeated DNA sequences by unequal crossover. Science 191:528.[Abstract/Free Full Text]
  15. Su, C., M. Nei. 1999. Fifty-million-year-old polymorphism at an immunoglobulin variable region gene locus in the rabbit evolutionary lineage. Proc. Natl. Acad. Sci. USA 96:9710.[Abstract/Free Full Text]
  16. Raman, C., K. L. Knight. 1992. CD5+ B cells predominate in peripheral tissues of rabbit. J. Immunol. 149:3858.[Abstract]
  17. Pospisil, R., R. G. Mage. 1998. CD5 and other superantigens as ‘ticklers’ of the B-cell receptor. Immunol. Today 19:106.[Medline]
  18. Mage, R. G., R. Pospisil. 2000. CD5 and other superantigens may select and maintain rabbit self-renewing B-lymphocytes and human B-CLL cells. Curr. Top. Microbiol. Immunol. 252:87.[Medline]
  19. Lopez-Martinez, N. 1989. Revision sistematica y biostratigrafica de los lagomorphos (Mammalia) del Terciario y Cuaternario de España. Memorias del Museo Paleontologico de la Universidad de Zaragoza, n°3. Diputacion General de Aragon.
  20. Callou, C.. 1995. Modifications de l’aire de répartition du Lapin (Oryctolagus cuniculus) en France et en Espagne, du Pléistocène à l’époque actuelle. État de la question. Anthropozoologica 21:95.
  21. Branco, M., N. Ferrand, M. Monnerot. 2000. Phylogeography of the European rabbit (Oryctolagus cuniculus) in the Iberian Peninsula inferred from RFLP analysis of the cytochrome b gene. Heredity 85:307.
  22. Branco, M., M. Monnerot, N. Ferrand, A. R. Templeton. 2002. Postglacial dispersal of the European rabbit (Oryctolagus cuniculus) on the Iberian Peninsula reconstructed from nested clade and mismatch analyses of mitochondrial DNA genetic variation. Evolution 56:792.[Medline]
  23. Cazenave, P. A., A. Bennamar, J. A. Sogn, T. J. Kindt. 1987. Immunoglobulin genes in feral populations. S. Dubiski, ed. The Rabbit in Contemporary Immunological Research 148. Longman Scientific & Technical, New York.
  24. Tonnelle, C., P. A. Cazenave, C. Brezin, D. Moinier, M. Fougereau. 1983. Structural correlates to the rabbit immunoglobulin heavy chain a100 allotype. Mol. Immunol. 20:753.[Medline]
  25. Hardy, C, J. D. Vigne, D. Casane, N. Dennebouy, J. C. Mounoulou, M. Monnerot. 1994. Origin of European rabbit (Oryctolagus cuniculus) in a Mediterranean island: Zooarchaeology and ancient DNA examination. J. Evol. Biol. 7:217.
  26. Monnerot, M., J. D. Vigne, C. Biju-Duval, D. Casane, C. Callou, C. Hardy, F. Mougel, R. Soriguer, N. Dennebouy, J. C. Mounolou. 1994. Rabbit and man: Genetic and historic approach. Genet. Sel. Evol. 26:167.
  27. Asmussen, M. A., J. Arnold. 1991. The effects of admixture and population subdivision on cytonuclear disequilibria. Theor. Popul. Biol. 39:273.[Medline]
  28. Krug, M. S., S. L. Berger. 1987. First-strand cDNA synthesis primed with oligo(dT). Methods Enzymol. 152:316.[Medline]
  29. Zhu, X., A. Boonthum, S.-K. Zhai, K. L. Knight. 1999. B lymphocyte selection and age-related changes in VH gene usage in mutant Alicia rabbits. J. Immunol. 163:3313.[Abstract/Free Full Text]
  30. Thompson, J. D., D. G. Higgins, T. J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673.[Abstract/Free Full Text]
  31. Xia, X., Z. Xie. 2001. DAMBE: Software package for data analysis in molecular biology and evolution. J. Hered. 92:371.[Abstract/Free Full Text]
  32. Nicholas, K. B., H. B. Nicholas, Jr, D. W. Deerfield, II. 1997. GeneDoc: Analysis and visualization of genetic variation. EMBNEW News 4:14.
  33. Kumar, S., K. Tamura, I. B. Jakobsen, M. Nei. 2001. MEGA2: Molecular evolutionary genetics analysis software. Bioinformatics 17:1244.[Abstract/Free Full Text]
  34. Nei, M., S. Kumar. 2000. Molecular Evolution and Phylogenetics Oxford University Press, Oxford.
  35. Saitou, N., M. Nei. 1987. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406.[Abstract]
  36. Felsenstein, J.. 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39:783.
  37. Rzhetsky, A., M. Nei. 1992. Statistical properties of the ordinary least-squares, generalized least-squares, and minimum-evolution methods of phylogenetic inference. J. Mol. Evol. 35:367.[Medline]
  38. Sitnikova, T.. 1996. Bootstrap method of interior-branch test for phylogenetic trees. Mol. Biol. Evol. 13:605.[Abstract]
  39. Tajima, F.. 1993. Simple methods for testing the molecular evolutionary clock hypothesis. Genetics. 135:599.[Abstract]
  40. Tutter, A., R. Riblet. 1989. Evolution of the immunoglobulin heavy chain variable region (Igh-V) locus in the genus Mus. Immunogenetics 30:315.[Medline]
  41. Ota, T., M. Nei. 1994. Divergent evolution and evolution by the birth-and-death process in the immunoglobulin VH gene family. Mol. Biol. Evol. 11:469.[Abstract]
  42. Lefranc, M.-P.. 2001. IMGT, the international ImmunoGeneTics database. Nucleic Acids Res. 29:207.[Abstract/Free Full Text]
  43. Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, C. Foeller. 1991. Sequences of Proteins of Immunologic Interest U.S. Department of Health and Human Services, Public Health service, National Institutes of Health, Bethesda, MD.
  44. Sehgal, D., R. G. Mage, E. Schiaffella. 1998. VH mutant rabbits lacking the VH1a2 gene develop a2+ B cells in the appendix by gene conversion-like alteration of a rearranged VH4 gene. J. Immunol. 160:1246.[Abstract/Free Full Text]
  45. Akimenko, M. A., B. Mariame, F. Rougeon. 1986. Evolution of the immunoglobulin {kappa} light chain locus in the rabbit: Evidence for differential gene conversion events. Proc. Natl. Acad. Sci. USA 83:5180.[Abstract/Free Full Text]
  46. Lummus, Z., J. J. Cebra, R. G. Mage. 1967. Correspondence of the relative cellular distribution and serum concentration of allelic allotypic markers in normal and allotype suppressed heterozygous rabbits. J. Immunol. 99:737.[Abstract/Free Full Text]
  47. Mage, R. G.. 1967. Quantitative studies on the regulation of expression of genes for immunoglobulin allotypes in heterozygote rabbits. Cold Spring Harbor Symp. Quant. Biol. 32:203.[Abstract/Free Full Text]
  48. van der Loo, W., F. Mougel, M. S. Sanchez, C. Bouton, E. Castien, A. Fonseca, N. Ferrand, R. Soriguer, M. Monnerot. 1999. Cytonuclear disequilibria in wild populations of rabbit (Oryctolagus cuniculus L.) suggest unequal allele turnover rates at the b locus (IGKC1). Immunogenetics 49:629.[Medline]
  49. Cooper, M. D., D. Y. Perey, A. E. Gabrielsen, D. E. Sutherland, M. F. McKneally, R. A Good. 1968. Production of an antibody deficiency syndrome in rabbits by neonatal removal of organized intestinal lymphoid tissues. Int. Arch. Allergy Appl. Immunol. 33:65.[Medline]
  50. Lanning, D., X. Zhu, S. K. Zhai, K. L. Knight. 2000. Development of the antibody repertoire in rabbit: Gut-associated lymphoid tissue, microbes, and selection. Immunol. Rev. 175:214.[Medline]
  51. Graille, M., E. A. Stura, A. L. Corper, B. J. Sutton, M. J. Taussig, J. B. Charbonnier, G. J. Silverman. 2000. Crystal structure of a Staphylococcus aureus protein A domain complexed with the Fab fragment of a human IgM antibody: Structural basis for recognition of B-cell receptors and superantigen activity. Proc. Natl. Acad. Sci. USA 97:5399.[Abstract/Free Full Text]
  52. Nguyen, V. K., C. Su, S. Muyldermans, W. van der Loo. 2002. Heavy-chain antibodies in Camelidae: A case of evolutionary innovation. Immunogenetics 54:39.[Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Esteves, P. J.
Right arrow Articles by van der Loo, W.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Esteves, P. J.
Right arrow Articles by van der Loo, W.
Right arrowPubmed/NCBI databases
*Protein

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS