Duplicated copies of the bovine JH locus contribute to the Ig repertoire
Arsalan Hosseini,
Gordon Campbell1,
Marko Prorocic1 and
Robert Aitken1
Department of Pathobiology, School of Veterinary Medicine, Shiraz University, Shiraz 71345, Iran 1 Division of Infection and Immunity, Institute of Biomedical and Life Sciences, Joseph Black Building, University of Glasgow, Glasgow G12 8QQ, UK
Correspondence to: R. Aitken; E-mail: r.aitken{at}bio.gla.ac.uk
Transmitting editor: D. Tarlinton
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Abstract
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We report the cloning and analysis of a bovine JH locus comprising a DQ52 segment, six JH segments and sequence to a 5' H chain intronic enhancer. The contig was mapped to BTA 11 and evidence was found for rearrangement of the sixth JH segment at a low but detectable frequency. In contrast, the fourth segment present at a second copy of the bovine JH locus mapping to BTA 21 was found to rearrange at high-frequency, forming FR4 in the majority of bovine Ig H chains. The data thus show that bovine H chains can be generated from segments at two distinct genomic locations. Further investigation should establish if rearrangement takes place at each locus or if the participating segments are brought together from different chromosomal locations by less conventional processes (for example by gene conversion or trans-chromosomal rearrangement).
Keywords: antibodies, J genes, joining, rearrangement
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Introduction
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Studies of the structure, function and genetics of Ig from livestock animals and other veterinary species have revealed many facets of immunology that could not have been predicted from the humanmurine paradigm. For example, the Igs of camels and llamas have evolved to functional independence from L chains (1) and veterinary immunologists have shown that post rearrangement processes can generate diverse H chain repertoires from small families of conserved segments or even single V genes (2).
The bovine Ig system has several properties that distinguish it from mice or humans. The H chain repertoire is founded upon the expression of a single gene family of modest size comprising segments of very limited diversity (35). In consequence, cattle are unable to generate Ig diversity through rearrangement, a process which underlies Ab formation in mice and humans (6). Humoral immunity is therefore reliant upon post-rearrangement diversification, the nature of which is presently obscure (7). The length of CDR3 is also distinctive. Bovine H chains possess CDR3 sequences that are frequently long (35) and sometimes in excess of 50 amino acids in length (8). This arises in foetal lymphoid tissue (3,9) indicating that it is created through rearrangement of long D segments (10) rather than from antigen-driven processes.
Here we report our characterization of the bovine JH system. The mammalian JH locus typically carries six or more segments that are utilized to varying degrees (11). For example, in humans the JH4 segment forms the fourth framework region (FR4) in
50% of Ig H chains (12). In cattle, bias towards a single JH segment is even more pronounced and a common FR4 sequence can be observed in a high proportion of bovine H chain cDNAs (35). Infrequent rearrangement of an alternative JH segment also appears possible (3). The aims of our study were therefore to define the number of JH segments present at the JH locus, to identify which segments undergo rearrangement and to seek an explanation for the bias apparent in this process. In tackling these objectives, we have identified a further distinctive property of the bovine Ig system.
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Methods
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Recovery of the bovine JH locus
Since bovine and ovine Igs are very similar, we used PCR to recover the main part of the JH locus with primer pairs designed from the ovine sequence (11). As template, bovine genomic DNA was prepared with a Wizard DNA purification kit (Promega, Southampton, UK) from fresh liver tissue obtained from a local slaughterhouse from several individual animals. The tissue was stored at 80°C prior to DNA isolation. One microgram of genomic DNA was used as template in PCR with a high-fidelity polymerase mixture (Expand HiFi, Roche, Lewes, UK) and homology primer 1 and downstream primer (Table 1). Homology primer 1 (Table 1) spanned the heptamer motif and adjacent regions of the 5' terminal segment at the ovine JH locus, whilst downstream primer annealed to sequences
80 bp downstream from the 3' terminal segment. Primers were used at final concentrations of 600 nM in a reaction buffer containing 5 mM magnesium chloride and 750 µM dNTPs. After initial denaturation at 93°C for 1.5 min, reactions were cycled 35 times through 93°C (30 sec), 55°C (1 min) and 68°C (2.5 min, extended by 5 sec for each block of 5 cycles). The 1.8 kb amplicon was isolated from agarose gels, blunted by reaction with the Klenow fragment of Escherichia coli DNA polymerase (Promega) and then ligated into pZErO2 (Invitrogen, Paisley, UK). Escherichia coli DH5
transformed with the ligation products were selected on Lauria agar plates containing 35 µg/ml kanamycin and 3 mM isopropyl ß-D-thiogalactopyranoside. Induction of the ccd gene borne on the plasmid provided efficient selection for vector containing inserts. After characterization of candidate clones by restriction analysis, inserts were sequenced using M13 forward and reverse primers and a primer-walking strategy with internal primers 7, 8 and 9 (Table 1). Conventional protocols for automated sequencing were used based upon Sanger chemistry (13), using Big Dye reagents (Applied Biosystems, Warrington, UK) and ABI 373 stretch and 377 instrumentation. Sequencing was carried out at the Molecular Biology Support Unit (IBLS, University of Glasgow).
Recovery of the downstream flanking regions
The primer Eµ reverse (Table 1) was designed from aligned sequences of H chain 5' intronic enhancers including that of the sheep (GenBank accession number Z98207). FR4 primer (Table 1) carried a sequence commonly observed in the fourth framework region of bovine H chain cDNA. These primers were used in PCR with bovine genomic DNA. The 900 bp product was isolated from agarose gels, blunted and phosphorylated by concurrent reaction with Klenow and T4 polynucleotide kinase (Promega). The reaction was conducted at 37°C for 40 min in a 50 µl volume containing about 1 µg of amplicon, 1 mM ATP, 35 µM dNTPs, 20 mM magnesium chloride, 5 mM DTT and 80 µM Tris pH 7.6. Ten units of kinase and five units of Klenow were used. After heat inactivation and precipitation, the DNA was ligated into dephosphorylated SmaI-cut pUC18 (Amersham Biosciences, Little Chalfont, UK) and transformed into E. coli. Recombinant clones were identified by restriction analysis and plasmid DNA was sequenced using M13 forward and reverse primers.
Recovery of the upstream flanking regions
Initially, a primer was designed from alignment of human, mouse, rabbit and shrew DQ52 sequences (14). PCR with this oligonucleotide and primers for the bovine JH locus was unsuccessful. Therefore, a lambda clone carrying bovine Cµ exons [clone 15 (15)] was obtained from Professor K. Knight, Stritch School of Medicine, Loyola University, Chicago. Comparison of the published characterization of this clone with emerging sequence of the JH locus suggested that a limited upstream stretch might be recoverable. Once the orientation of the insert had been established, PCR was carried out with primers against the lambda right arm and the JH locus (
right reverse and primer 8 reverse, respectively; Table 1). The 1.2 kb amplicon was blunted and phosphorylated for ligation into pUC18 as described above. Sequencing was carried out with M13 forward and reverse primers, a lambda-specific primer designed to anneal close to the bovine insert (
right reverse 2) and primer 10 reverse (Table 1).
Chromosomal localization of a JH locus
It is known that a duplication of the IgM locus exists on BTA11 (16). PCR was carried out with primers Cµ forward and Cµ reverse (Table 1) using DNA from a lambda library prepared specifically from bovine chromosome 11 (17). This reaction was also carried out with BAC 944D11, a clone found to carry the JH locus isolated in our studies (see Results), and BAC 66R4C11, a clone carrying a second JH locus (18,19). Amplicons were cloned into pCR-TOPO (Invitrogen) following the manufacturers protocol. Several independent clones were sequenced with M13 forward and reverse primers and compared with each other and depositions at GenBank.
Identification of the dominant rearranging segment
Genomic DNA prepared from isolated peripheral B cells was provided by Dr S. Stephens, Institute of Animal Health, Compton, UK. B cell DNA was used in PCR with VHF and downstream primer (Table 1) to isolate rearranged segments and adjacent, downstream regions of the JH locus. VHF annealed to a leader sequence common to all members of the expressed VH gene family (5). The downstream primer was complementary to sequences
80 bp from the segment at the 3' terminus of the bovine JH locus. This strategy was free of assumptions regarding which JH segment(s) might undergo rearrangement. Four prominent products from the reaction were isolated from agarose gels, cloned separately into pCR4-TOPO. Three of the four products proved to be irrelevant, but an amplicon of 1.5 kb was informative. Following this analysis, further PCRs were conducted with genomic DNA from leukocytes of the same donor animal. The JH loci were recovered with the following primer pairs: internal primers 6 and 8; internal primer 11 and insert reverse; insert forward and downstream primer (see below for further explanation).
Locus-specific PCR
To check if copies of the JH loci identified in these studies represented variants of the same allele, locus-specific reactions were designed using BAC clones 944D11 and 66R4C11 as control templates. PCR was carried out with primers lo forward and downstream primer, and with internal primer 11 and hi reverse. With an annealing temperature of 58°C, the former reaction was specific for the locus carried on 944D11 and the latter reaction only recovered sequence from 66R4C11. The reactions were then carried out with genomic DNA isolated from sperm from three individual animals from three breeds (South Devon, Belgian Blue, Limousin). Sperm was obtained from Lindsays AI, Carlisle, UK. DNA was isolated using QIAamp reagents and the manufacturers modified protocols (Qiagen, Crawley, UK).
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Results
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Recovery and sequencing of a bovine JH locus
Following preliminary work that showed close similarity between the JH loci of cattle and sheep, the majority of a bovine locus was recovered from liver genomic DNA as a single 1.8 kb amplicon by optimized PCR. The product was cloned and sequence was gathered from its termini using M13 forward and reverse primers. Internal primers were designed to complete the characterization of the insert. Our strategy of cloning by homology was extended to recover the downstream flanking region with a primer designed from alignment of H chain 5' intronic enhancer sequences. Once reaction conditions had been optimized, a single product of 900 bp was obtained which was cloned and sequenced. Attempts to extend the analysis upstream from the JH locus with a primer designed by alignment of DQ52 sequences failed. However, this region was successfully recovered on a 1.2 kb amplicon from a lambda clone provided by Professor K. Knight. Overall, the resulting data formed a contiguous sequence of 3282 bp starting about 70 bp upstream from a bovine DQ52 segment, through the JH locus to the 5' intronic enhancer region lying
570 bp downstream (GenBank accession number AY149283). Although the sequence of the locus was assembled in several sections from different sources, its existence as a contiguous sequence was confirmed by successful recovery from a BAC clone (see below) and bovine genomic DNA (data not shown).
The contig was analysed by comparison with the ovine JH locus (11), by searching for RSS and by checking for FR4 sequences observed in H chain cDNA. As predicted from preliminary studies, the overall homology between the bovine locus and its ovine homologue was high (89% identity). Nucleotide differences were scattered throughout the 2.7 kb overlapping region with four areas where insertion or deletion of between 11 and 27 nt could be detected. Analysis by BLAST (20) and NIX (http://www.hgmp.mrc.ac.uk) did not suggest that these features were of functional significance. For reasons that will become clear, this contig is designated the JHlow locus throughout the remainder of our report.
Identification of six JH segments
Analysis of the sequence of the JHlow locus revealed six segments with spacing and organization similar to that observed in the sheep. The segments were numbered according to their order from the DQ52 proximal to the distal regions of the contig. In summary, they spanned a region of
1.8 kb with inter-segment distances ranging from 130 to 500 bp. The nucleotide sequences of these segments are presented in Fig. 1. RSS motifs could be identified immediately upstream of each segment and matched to a greater or lesser extent the Ig consensus. Generally, nonamer motifs and the spacer regions were variable in sequence, the spacer ranging from 20 (JH5) to 23 bp (JH4). The heptamer sequences showed better match to the consensus, exceptions being those adjacent to the second and third segments. Potential reading frames carried on each segment were aligned and translated around the W codon which characteristically forms the first amino acid of FR4 in cattle and many other vertebrates, and the two S codons at the carboxy-terminus of this region. The amino acid sequences predicted by these criteria are also shown in Fig. 1.

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Fig. 1. Aligned nucleotide and protein coding sequences of bovine JH segments present at the JHlow locus. The nucleotide sequences of six bovine segments (JH1 to JH6) are aligned using features of the RSS indicated at the top, and the TGG codon for W that marks the start of FR4. Reading frames are shown over each nucleotide sequence, using the reading frame for the W codon. JH4 is aligned with a sequence commonly observed in bovine H chains cDNA (clone F27M) (3). JH4 is also aligned with its ovine homolog (ovine JH1) (11). JH6 is aligned with an alternative sequence detected in bovine H chain cDNA (clone F52M) (3) and its ovine homolog [ovine JH2; (11)].
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Segments JH1, JH2, JH3 and JH5 were judged likely to be pseudogenes. For JH1, the nonamer and splice site for RNA processing departed from consensus (21). For JH2, the splice site for RNA processing was also aberrant. The heptamer adjacent to JH3 departed markedly from consensus. The nonamer associated with JH5 departed from the consensus, the RSS spacer was short (20 nt) and the RNA splice site was defective. Functionality of the remaining two segments was assessed by comparison with the sheep JH locus and bovine FR4 sequences observed in Ig H chain cDNA.
Comparison of JH4 with its homolog at the ovine locus (termed JH1) revealed striking similarity (Fig. 1). In sheep, this segment is identical to the majority of FR4 sequences in ovine H chain cDNA suggesting that it undergoes high-frequency rearrangement and expression (11). Specifically, there were only three nucleotide differences in the RSS (two substitutions, one gap) and two differences 5' to the conserved W codon (one gap, one substitution) which would lead to amino acid substitutions in this region. Further differences between the bovine and ovine segments occurred in the main part of the reading frame where five nucleotide differences gave rise to three amino acid substitutions. In cattle, a single FR4 sequence is also predominant (Fig. 1); in one study, Berens et al. (3) isolated this sequence repeatedly from foetal splenic cDNA, suggesting minimal alteration by combinatorial imprecision or other processes of diversification. It is clear from Fig. 1 that the bovine JH4 segment at the JHlow locus could not form this FR4 sequence. Specifically, five nucleotide differences led to three amino acid changes upstream from the W codon, and six nucleotide differences created four amino acid substitutions in the main part of the reading frame. Focusing on the latter features, a CAA (Q) codon in the dominant sequence was represented in JH4 as CCA (P), and a CTC (L) codon was mismatched by ATC (I). Finally a run of four A residues in JH4 spanned Q and N codons (CAA AAC) whereas in IgH cDNA, TGGT contributed to L and V codons (CTG GTC).
Of the six segments present at the JHlow locus, the data indicated that just one was expressed directly: JH6 matched perfectly an alternative FR4 sequence identified by Berens et al. (3) in foetal and adult Ig cDNA (Fig. 1). JH6 was also near-identical to its equivalent at the ovine locus (Fig. 1).
A bovine DQ52 segment departs from the vertebrate consensus
A bovine DQ52 homologue was located upstream from the JHlow locus (Fig. 2). The coding sequence was flanked by RSS and, at 14 nt, was
27% longer than that observed in other species. Alignment with DQ52 segments from llama, humans, mouse, rabbit and house shrew (Suncus murinus) revealed marked differences, explaining the failure to recovery the segment from bovine genomic DNA with consensus primers. Although features of the bovine upstream RSS suggested potential functionality, translation of the coding sequence did not reveal the frequency of Y and C codons that are characteristic of bovine H chain CDR3 (35,810). It therefore seemed unlikely that the segment was utilized with significant frequency during foetal or adult life. This was reinforced by examination of the downstream RSS in which there was a marked departure from consensus in the heptamer motif (Fig. 2).

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Fig. 2. Nucleotide and protein coding sequence of the bovine DQ52 segment. Features of the RSS and translated sequences in the three forward reading frames are shown at the top of the figure. DQ52 segments from other species are also shown with their GenBank accession numbers. The sequences have been aligned with the bovine sequence, dots indicating identity, dashes the introduction of gaps to maximize alignment.
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Chromosomal assignment of the JHlow locus
BAC clone 944D11 was isolated from a genomic library (22) and PCR used as described earlier to recover the JH locus present on the insert. Sequencing confirmed that 944D11 carried the JHlow locus as described above. Further analysis of 944D11 and BAC clone 355H4, a standard marker for the bovine Ig H chain locus on BTA21 (23,24), revealed overlapping, identical sequences unconnected with the Ig system. This suggested two possibilities: that the JHlow locus mapped to the main Ig H chain locus on chromosome 21; or that the insert on BAC 944D11 was derived from a large translocation of sequence from BTA21 to another region of the bovine genome. To investigate these ideas, part of the Cµ locus was recovered as an amplicon of
500 bp from a BTA11-specific library, BAC 944D11 and BAC 66R4C11. BAC 66R4C11 carries a second JH locus that can be unequivocally anchored to BTA21 through the presence of Ig constant region genes (18,19). The sequence of amplicons from 944D11 were essentially identical to those from the BTA11-specific library whereas products from 66R4C11 consistently differed at the 3' terminus of Cµ exon 1, through the intron and into Cµ exon 2 (Fig. 3). Interestingly, the amplicon from 944D11 closely matched GenBank file U63637 (Fig. 3), a deposition made from the original description of bovine IgM constant region sequences (25). The sequence deposited as U63637 was originally isolated from Knights lambda clone 15 (15), used in our study to recover flanking sequence upstream from the JHlow locus. Data recovered from 66R4C11 matched GenBank file AY230207, a result that would have been predicted as they have a common origin (18). This enabled the elimination of PCR error rate as a significant confounding factor. Taken together, the results anchor the JHlow locus and lambda clone 15 to BTA11.


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Fig. 3. Nucleotide sequences of duplicated IgM loci. The consensus sequence of amplicons from a BTA11-specific library is compared with products from BAC 944D11 (three independent clones as indicated in parentheses), BAC 66R4C11 (two independent clones as indicated in parentheses) and GenBank depositions U63637 and AY230207. Annealing sites for the primers used (Table 1) are indicated with intron and exon boundaries. Data have been aligned to the amplicon from BTA11 with dots to indicate identity, dashes the introduction of gaps to maximize alignment and N, a nucleotide that could not be resolved.
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Analysis of B cell genomic DNA
In order to identify the JH segment that undergoes frequent rearrangement, B cell genomic DNA was used in a PCR with primers against the VH segment and the region downstream of the JH locus. A 1.5 kb amplicon was obtained from the reaction, cloned and DNA from multiple transformants was sequenced. At one terminus of each insert, part of the VH leader could be detected, followed by the VH intron and sequence through FR1 into CDR1. At the other terminus, sequence was near-identical to the downstream flanking region at the JHlow locus. These features confirmed that the amplicons originated from rearranged genomic DNA (data not shown). In all clones, alignment of the sequence with the JHlow locus showed rearrangement of a segment similar to JH4 had taken place. Sequences downstream of the rearranged segment showed excellent matches to the inter-genic regions present at the JHlow locus, and segments similar but not identical to JH5 and JH6 were identified. Given that the B cells were isolated from an adult animal, it was not surprising that all clones were unique since antigen-driven processes would likely have introduced nucleotide substitutions during the development of individual B lymphocytes.
The regions encoding FR4 from six independent clones are shown in Fig. 4(A). C to A and A to C substitutions to the sequence observed at the JHlow locus were observed, modifying codons for P and I to Q and L. Adjacent, the AAAA motif noted earlier at the JHlow locus was replaced in B cell DNA with TGGT. This substituted codons for Q and N with L and V. These substitutions matched the sequence observed commonly in FR4 of Ig H chain cDNA. Additional substitutions were also detected. Some were silent, others generated amino acid substitutions at a G codon (Fig. 4A).

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Fig. 4. Nucleotide sequences recovered from bovine B lymphocytes. (A) Sequences of germline and rearranged JH segments. The sequence of JH4, JHlow locus, is compared with six independent clones recovered from B cells (B2 to B22) from the same animal. Dots indicate nucleotide identity, N shows bases that could not be resolved. Loss of alignment with JH4 was taken to indicate the start of CDR3 sequence. The dominant FR4 cDNA sequence (F27M) is included for comparison [see Fig. 1; (3)]. The figure also shows the sequence of the JH4 segment from JHlow, as originally isolated and deposited at GenBank (AY149283). The protein coding sequences are shown with the W residue taken as the start of FR4. In B cell amplicon B2, X indicates an amino acid which could not be predicted because of nucleotide ambiguity. (B) Sequences of inserts detected upstream of the JH5 segment in B lymphocytes. The alignment has been made against the corresponding region of JHlow locus isolated from the B cell donor. Dots indicate nucleotide identity, dashes the introduction of gaps. Other details as described for (A). For comparison, the equivalent regions of the ovine JH locus (11) and JHlow, as originally isolated and deposited at GenBank (AY149283) are shown. (C) Sequences of JH6 segments in the germline and B lymphocytes. The alignment has been made against the corresponding region of JHlow locus, as originally determined (see Fig. 1). Dots indicate nucleotide identity. Only three clones recovered from B cells (B12 to B22) were sequenced through this region [compare with parts (A) and (B) of this figure].
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Two other features of amplicons from bovine B cell DNA were significant. The first was the appearance of an insert of 21 bp immediately upstream of the JH5 segment (Fig. 4B). This was well conserved and very similar to a sequence from the equivalent region of the ovine JH locus. The second feature was a series of nucleotide substitutions in the JH6 segment that gave rise to changes in the coding sequence (Fig. 4C). The most significant of these alterations was a G to C substitution that created a radical W to C alteration at the start of the coding sequence for FR4.
We addressed the possibility that the genotype of the B cell donor differed in some fundamental way from animals previously sampled. To do this, the majority of the JHlow locus spanning segments JH3, JH4 and JH5 was recovered from leukocytic DNA of the donor animal by PCR with internal primers 8 and 6. No significant differences with the JHlow locus were detectable (Fig. 4A and B).
Duplication of the bovine JH locus
To resolve the mismatch between the JHlow locus and rearrangement products recovered from B cells, we sought evidence for a second JH locus that was selected for high-frequency rearrangement. We designated this JHhigh. Primers were designed against the insertion noted upstream of JH5 in rearranged DNA but absent from the JHlow locus (Fig. 4B; Table 1). PCR was carried out on liver genomic DNA with internal primer 11 and insert reverse to recover a product of
480 bp. A fragment of 750 bp was generated with insert forward and downstream primer. Multiple clones were sequenced (Fig. 5) and compared with the JHlow locus (Fig. 1), the bovine JH locus recovered from BAC 66R4C11 by Zhao et al. (18) (GenBank accession number AY158087), and B cell amplicons recovered earlier (Fig. 4). Alignment of the JH4 segments was particularly striking (Fig. 5A). Limited differences between JHlow and JHhigh were seen in the RSS; the substitution of G for T improved the match of the nonamer motif with consensus. In the reading frame for the JH4 segment, a nucleotide insertion and 10 substitutions were observed at JHhigh compared with JHlow. Rearrangement of JH4 from JHhigh would therefore create directly the FR4 sequence recovered from B cell genomic DNA and commonly observed in bovine Ig H chains (35). At the JHhigh locus, the sequence from the JH4 segment to the insertion matched precisely the corresponding region of AY158087, the JH locus recovered from BAC 66R4C11 by Zhao et al. (18) (Fig. 5A).



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Fig. 5. Comparison of the JHlow and JHhigh loci. (A) Sequence upstream from the JH5-proximal insertion. Five identical clones were sequenced of which one (JHhigh locus) is presented. Its sequence is aligned to the JHlow locus (Fig. 1; GenBank accession number AY149283), dots indicating identity, dashes the introduction of gaps. For comparison, data from Zhao et al. (18,19) (GenBank accession number AY158087) is also shown. Primer binding sites, features of the RSS and location of the JH4 segment are marked. The region spanning the JH4 segment also shows three clones recovered from bovine B cells (B12 to B22; see Fig. 4A) and the dominant FR4-coding sequence (see Fig. 1) (3). (B) Sequence downstream from the JH5-proximal insertion. Three clones were characterized which differed only slightly in sequence. A representative (JHhigh locus) is presented. Its sequence is aligned to JHlow locus (Fig. 1; GenBank accession number AY149283), dots indicating identity, dashes the introduction of gaps. For comparison, data from Zhao et al. (18,19) (GenBank accession number AY158087) is also shown. Primer binding sites, features of the RSS and the location of the JH5 and JH6 segments are marked. The region around the JH6 segment also shows sequences of three clones recovered from bovine B cells (B12 to B22; see Fig. 4C).
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Downstream from the JH4 segment, a copy of the JH5 segment was present at the JHhigh locus that was almost identical in sequence to that at JHlow (Fig. 5B). A 20 nt deletion was detected
350 bp downstream from this feature. Beyond, a JH6 segment was present. RSS sequences appeared identical at the two loci but there were significant differences in the reading frame at JHhigh: notably, the W codon was replaced with TGC (C). Comparison again showed that bases present in JH6 at JHhigh consistently appeared in sequences recovered from B cells. Throughout, the match to the sequence characterized by Zhao et al. (18) was striking.
Thus, there was no evidence to indicate the JH4 segment present at JHlow and mapped to BTA11 underwent rearrangement, although rearrangement of JH6 could be detected. Rather, the majority of bovine Ig H chains originated from rearrangement of the fourth segments at the JHhigh locus located on BTA21. It seemed that the only feature of the sixth segment at this location to exclude it from rearrangement was the radical change that it would bring to the sequence of FR4. As would be predicted from their chromosomal assignments, JHlow and JHhigh could be recovered from bovine genomic DNA by locus specific PCR with internal primer 11 and hi reverse, and lo forward and downstream primer respectively. This result was obtained with samples from three individual animals from the breeds South Devon, Belgian Blue and Limousin (data not shown), excluding the possibility that the loci were variants of the same allele.
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Discussion
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The Ig H chains of cattle and sheep are very similar at the nucleotide and protein levels, both being founded upon the rearrangement and expression of single VH gene families and dominant JH segments (35,11,26). This study exploited these similarities to recover and characterize a copy of bovine JH locus (GenBank accession number AY149283) that we have termed JHlow since it undergoes rearrangement with low but detectable frequency. The sixth JH segment present at the locus formed the substrate for this process. The large majority of bovine Ig H chains carry a FR4 sequence derived from a second JH segment (35,8,9) but this could not be detected at the JHlow locus. This observation marks an important distinction between the Ig systems of cattle and sheep, animals that are otherwise strikingly similar in immunological terms.
This finding was unexpected and we have checked carefully that it did not arise from an artefact of the isolation strategy. Using PCR, the JHlow locus could be isolated reproducibly from multiple individual animals of different breeds, arguing against allelic variation within the bovine population or the existence of multiple allotypes. It was also recovered from a lambda clone carrying sequence to beyond the IgM constant region exons (15) and from a BAC clone carrying Cµ exons. Linkage with a Cµ locus has enabled assignment of JHlow to bovine BTA11, another important finding since the main bovine Ig H chain locus is located on chromosome 21 (23). Although trans-chromosomal switching of Ig class has been documented in rabbits (2729) and mice (30), translocation of the antigen-receptor loci to other chromosomes is more often associated with their exclusion from the rearrangement process (3133). In some cases, duplication on the same chromosome can have the same effect (34). The ability of the bovine Ig system to recruit the JHlow locus for rearrangement, albeit at low frequency, therefore seems a highly unusual property.
By recovering the rearranged H chain locus from B cell genomic DNA, it was apparent that the JH segment selected for rearrangement lay on a JH locus similar but not identical to JHlow. This was designated JHhigh and was found to be identical to the JH locus recently reported by Zhao et al. (18,19). These authors isolated the locus from BAC 66R4C11 that carries Cµ, C
, C
3 and C
1 constant region genes and through overlap with other clones carrying the remainder of the bovine Ig constant region locus, can be unequivocally mapped to BTA21.
Formation of the bovine H chain would thus seem possible by two pathways. The first pathway establishes the majority of the bovine H chain repertoire using the JHhigh locus on BTA21 and the VH, D and constant region exons known to be present at this chromosome (23,24,35). The second involves low-frequency rearrangement of the the sixth segment at the JHlow locus on BTA11. Ig H chain products of this pathway carry the FR4 sequence typified by foetal clone F52M (3) in which R residues substitute for the more commonly observed residues Q and L. For the moment, it remains unknown if VH and D segments are present on this chromosome. If they prove to be absent, less conventional mechanisms [e.g. trans chromosomal rearrangement (2729)] may have to be sought.
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Abbreviations
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CDRcomplementarity-determining region
FRframework region
RSSrecombination signal sequence
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Acknowledgements
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The authors are grateful to Professor K. Knight (Loyola University, Chicago), Professor A. Ponce de León (University of Minnesota), Dr S. Stephens (Institute for Animal Health, Compton, UK) and Dr F. Piumi (INRA, Jouy-en-Josas, France) for their support, advice and provision of materials. This work was supported by a scholarship to A.H. from the Ministry of Science, Research and Technology, Islamic Republic of Iran, and funds from the Research Committee, Institute of Biomedical and Life Sciences, University of Glasgow.
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