We have applied bacteriophage display technology to construct and analyze the diversity of an IgG library of >1 × 108 clones from an adult sheep immunized against the hapten atrazine. We have identified eight new VH gene families (VH2–VH9) and five new Vκ gene families (VκV–VκIX). The heavy and κ light chain variable region gene loci were found to be far more diverse than previously thought.

The ability to display the entire functionally active Ab repertoire of a suitable host on the surface of filamentous bacteriophage has been successfully applied to the generation of mAbs without the need for B cell immortalization (1, 2, 3, 4). The majority of research has been conducted using libraries of human origin, but libraries have also been produced from rabbit, chicken, mouse, and cattle (5, 6, 7, 8, 9, 10), often with a view to producing Abs against Ags conserved in humans and/or mice or in mammals as a whole. The use of libraries circumvents the difficulties associated with generating stable heterohybridoma cell lines and also enables studies to be made of the immunology of the selected host species.

The diversity and organization of the variable region VH-D-JH heavy chain and VL-JL light chain genes of humans and mice are well understood, as are the mechanisms involved in generating the enormous primary Ab repertoire necessary to fulfil the immune system’s protective role (11, 12). In recent years much research has also been directed toward the study of domesticated animals of economic importance such as chickens and large farm animals, revealing variations in both the site of primary repertoire generation and the mechanisms used.

In humans and mice, B cell lymphopoiesis occurs in the bone marrow (13) and continues throughout life. In the chicken the bursa of Fabricius has been recognized as the site of B cell development for some time (14), and in rabbits the appendix functions as a bursal equivalent (15). More recently it has been shown that other gut-associated lymphoid tissues, i.e., the ileal Peyer’s patches, serve as sites of B cell diversification in sheep and cattle, although diversification is also seen in the bovine spleen (16, 17). Birds, together with a number of mammal species, have less extensive germline VH repertoires than mice and humans and/or exhibit a restricted expression of Ig VH genes and, perhaps as a consequence, use strategies of primary repertoire development that overcome this limitation (18). Sheep, cattle, and swine are all thought to express relatively few VH genes belonging to a single VH family, that of the former two being homologous to human VH4 and the latter to human VH3 (19, 20, 21, 22, 23). The sheep Ig light chain primary repertoire is diversified by extensive somatic hypermutation and is independent of Ag (16, 24), whereas cattle and possibly swine also use templated gene conversion by nonreciprocal recombination (17, 25, 26). Chicken and rabbit have more extensive VH loci in that each possess ∼100 genes, related to human VH3. However, VH gene usage is limited by restricted functionality or preferential expression, and gene conversion plays a significant role in Ab diversity (27, 28).

In this study, we demonstrate for the first time that phage display technology can be used to study diversity, that sheep possess and utilize a more diverse Ig germline gene pool than was previously thought, and that sequences derived from pseudogenes may contribute to an ongoing process of IgG diversification.

Phagemid vector was transformed into Escherichiacoli TG1 (supE thi-1 Δ(lac-proAB) Δ(mcrB-hsdSM)5(rKmK)(F′ traD36 proAB laqIqZΔM15)). Soluble expression was conducted in E. coli XL1-Blue (supE44, hsdR17, recA1, endA1, gyrA96, thi-1, relA1, lac(F′ proAB, lacIqZΔM15, tn10 (tetr))).

Total mRNA was isolated with the Quick-prep-mRNA purification kit (Pharmacia, Milton Keynes, U.K.) from a total of 400 mg spleen removed from a 10-year-old Welsh breed/Suffolk sheep that had been hyperimmunized against a hapten target (atrazine) conjugated to bovine-thyroglobulin (Guildhay, Surrey, U.K.). To prepare cDNA, 200 ng mRNA was made up to 25 μl with RNase-free water, and 25 pmol of FOR primer specific for sheep heavy, λ, or κ light chain constant regions was added. The mixture was heated to 70°C for 10 min and cooled to 42°C before adding 8 μl of 5× concentration first-strand buffer (Life Technologies, Paisley, U.K.), 4 μl 0.1 M DTT, and 1 μl dNTP mix (10 mM each). The mixture was incubated at 42°C for 2 min before adding 1 μl (200 U) SuperScript II (Life Technologies) reverse transcriptase, and incubation continued at 42°C for 50 min and then for 15 min at 70°C.

PCR reactions comprising 25 pmol each OvVHBACK and OvVHFOR primers, 1 μl dNTP mix (25 mM each), 5 μl 10× concentration Bioline reaction buffer (160 mM NH4SO4, 670 mM Tris-HCl (pH 8.8 at 25°C), 0.1% Tween 20), 2 μl 50 mM MgCl2, 1 μl heavy chain cDNA, and sterile water to 50 μl were prepared. The reactions were heated to 94°C for 5 min and held while 0.5 μl Bioline Taq DNA polymerase (5 U/μl) was added. They were then incubated for 30 temperature cycles of 94°C for 1 min, 60°C for 1 min, 72°C for 1 min, and then a final incubation of 72°C for 7 min. Separate sets of 10 PCR reactions were performed using each combination of OvVHBACK and OvJHFOR primers (Fig. 1). Both λ and κ light chains were amplified as above using OvVλBACK/OvJλFOR and OvVκBACK/OvJκFOR primer combinations with λ and κ cDNA templates, respectively. PCR products were purified by electrophoresis through a 1% TAE agarose gel, and bands of the correct size were excised. DNA was recovered using QIAquick columns (Qiagen, Surrey, U.K.) and eluted with 10 mM Tris-HCl (pH 8.5). Purified VH and VL (λ or κ) DNA was linked together by PCR. Approximately 10–20 ng each of heavy and light chain DNA was used per reaction for seven cycles as described above. Reactions were then heated to 94°C and held while 25 pmol each of OvVHBACKSfi and OvJλFORNot or OvJκFORNot pull-through primers were added. PCR was continued for an additional 30 cycles. Separate reactions were performed for each combination of pull-through primers with the appropriate VH and VL template DNA. Multiple reactions were performed as necessary. Linked heavy and light chains were purified by electrophoresis using low melting point SeaPlaque GTG agarose (FMC Bioproducts, Rockland, IL). DNA was recovered from gel slices with AgarACE (Promega, Southampton, U.K.) and then with phenol chloroform extraction and ethanol precipitation.

FIGURE 1.

Primers were designed using published sheep antibody gene sequences (16192433343536 ) except OvJλ2FOR, which was derived from cDNAs amplified and sequenced as part of this study (data not shown), HuVH4aBACKSfi (37 ), and MuVHBACKSfi. Degenerate nucleotides are encoded as follows: M = A/C; R = A/G; W = A/T; S = G/C; Y = C/T; and K = G/T. Constant region primers anneal to the 5′ end of heavy IgG1/2, λ, and κ constant regions, respectively. “BACK” primers anneal to the 5′ end (FR 1) of heavy/light chain variable regions. Positions of “FOR” primers are indicated in Figs. 3, 5, and 7. “Sfi” and “Not” primers include extensions complementary to the PelB and gIII regions of the phagemid vector pDM1. Sequencing primers AH-1 and Fd seq1 are located in the phagemid vector PelB and gIII regions, respectively. Sfi1 and Not1 restriction endonuclease sites are underlined. Nco1 restriction endonuclease sites are in bold. Nucleotides encoding the flexible linker are in italics.

FIGURE 1.

Primers were designed using published sheep antibody gene sequences (16192433343536 ) except OvJλ2FOR, which was derived from cDNAs amplified and sequenced as part of this study (data not shown), HuVH4aBACKSfi (37 ), and MuVHBACKSfi. Degenerate nucleotides are encoded as follows: M = A/C; R = A/G; W = A/T; S = G/C; Y = C/T; and K = G/T. Constant region primers anneal to the 5′ end of heavy IgG1/2, λ, and κ constant regions, respectively. “BACK” primers anneal to the 5′ end (FR 1) of heavy/light chain variable regions. Positions of “FOR” primers are indicated in Figs. 3, 5, and 7. “Sfi” and “Not” primers include extensions complementary to the PelB and gIII regions of the phagemid vector pDM1. Sequencing primers AH-1 and Fd seq1 are located in the phagemid vector PelB and gIII regions, respectively. Sfi1 and Not1 restriction endonuclease sites are underlined. Nco1 restriction endonuclease sites are in bold. Nucleotides encoding the flexible linker are in italics.

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Purified VH-VL DNA was digested with 20 U of SfiI (Roche, Sussex, U.K.) per μg DNA for 10 h at 65°C, the buffer composition was altered, and 20 U per μg DNA NotI (Roche) was added. Incubation was continued at 37°C for 16 h. The phagemid vector (10 μg) pDM-1 (kindly provided by D. McGregor, Rowett Research Institute, Aberdeen, U.K.) was digested in the same way. The scFv fragments were ligated with vector (2 μg each) using 10 U T4 DNA ligase (Roche) for 16 h at 16°C. Ligated DNA was extracted with phenol/chloroform, ethanol precipitated, washed twice with 1.0 ml 70% ethanol, and redissolved in 20 μl sterile water. Transformation of E. coli TG1 (Stratagene, Cambridge, U.K.) was conducted by electroporation (29) using 2 μl ligation product/40 μl cells, and transformants were plated onto tryptone yeast extract (TYE) agar containing 1% glucose and 100 μg/ml ampicillin (TYE-Glu-Amp). PCR was performed on individual colonies using AH-1 and Fd seq1 primers to determine the proportion of clones containing a scFv fragment of the correct size (∼850 bp). After incubation overnight at 30°C, the colonies were scraped off into 2 ml of 2× TY-Amp-15% glycerol per plate and pooled. Aliquots were prepared and stored at −80°C. To rescue phage, 100 μl of glycerol stock (∼3 × 109 cells) was inoculated into 500 ml of 2× TY-Glu-Amp and incubated with shaking at 37°C to an OD600 of 0.6 (1–2 h). M13KO7 helper phage (Pharmacia) was added at 20× multiplicity to 50 ml of the culture that was incubated at 37°C without shaking for 30 min. Infected cells were pelleted, resuspended in 500 ml 2× TY-Amp-Kan-Glu, and incubated overnight with shaking at 30°C. Phage particles were concentrated from the culture supernatant by two successive precipitations with 1/5 volume PEG (20% polyethylene glycol weight to volume ratio, 2.5 M NaCl) as described by Griffiths et al. (30).

Ab VH and VL genes from clones selected at random from the original library glycerol stocks were PCR amplified using the AH-1 and Fd seq1 primers. PCR products were sequenced using the same primers on an ABI 377 automated DNA sequencer (Applied Biosystems, Foster City, CA) in both directions. Sequences were compared and dendograms were constructed using the GAP and PILEUP programs (Daresbury, U.K.). Comparisons were restricted to those parts of the rearranged genes encoded by the VH, Vλ, or Vκ gene segments.

The library was constructed from a sheep immunized with atrazine conjugated to BSA for the isolation high-affinity anti-atrazine Abs (to be described elsewhere). The sheep was sacrificed, and samples of the spleen were removed. Variable region genes were amplified by PCR, and the library was constructed as illustrated in Fig. 2. The linker sequence is a modification of that described by Chaudhary et al. (31). By using multiple electroporations, a library containing 1.1 × 109 clones was produced. PCR revealed that 85% contained inserts of the correct size, and digestion with the enzyme BstN1 indicated that 89% of these had unique restriction patterns (not shown). Therefore, the library was estimated to include 8.5 × 108 different clones.

FIGURE 2.

Outline of the steps used to construct the scFv library. The use of primers (JHFOR and VLBACK), which include two-thirds of the linker sequences (modification of Chaudhary et al. (31 )), enables all heavy and light chains to be joined without bias via the center 15 bases.

FIGURE 2.

Outline of the steps used to construct the scFv library. The use of primers (JHFOR and VLBACK), which include two-thirds of the linker sequences (modification of Chaudhary et al. (31 )), enables all heavy and light chains to be joined without bias via the center 15 bases.

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Variations in the nomenclature used by researchers when discussing the organization and ancestry of heavy chain variable region genes can lead to confusion. In the context of this article and in reference to others works, the following system will be used: heavy chain gene families, where applicable, will be numbered using Arabic numerals; and the three major homologous groups will be referred to as “clans” and will be identified by Roman numerals using the classification of Kabat et al. (32), such that clan I includes human VH1, VH5, and VH7 families and clan II includes human VH2, VH4, and VH6 families.

A large number of clones were selected at random and the VH and VL chain genes were amplified by PCR with the AH-1 and Fd seq1 primers (Fig. 1). The sequences of 45 rearranged heavy chain genes are compared with the V5a germline sequence (19) and JH1 sequence (Ref. 38 and Fig. 3). A dendogram constructed through Daresbury using the PILEUP program demonstrates significant clustering (Fig. 4). The region from framework region 1 (FR1)3 to the end of FR3 was used for this analysis, including complementarity-determining regions (CDRs) 1 and 2.

FIGURE 3.

Sheep heavy chain VH-D-JH rearranged genes from clones selected at random from the sheep phage display library. The sequences were obtained by PCR from γ heavy chain cDNAs and are compared with the V5a (19 ) and JH1 (38 ) germline genes. Positions of CDRs and amino acid numbering are from Kabat et al. (32 ). At the D-JH junction, the two known functional JH segments are included, and the region encoded by PCR primers is indicated. Gaps in the sequence have been introduced to maximize homology. Dashes indicate nucleotide identity with the V5a and JH1 genes. Sequences are grouped according to family classifications as shown in Fig. 4. Motifs derived from pseudogenes (38 ) are in bold underlined type or boxed. ∗, Derived from the JH2 gene.

FIGURE 3.

Sheep heavy chain VH-D-JH rearranged genes from clones selected at random from the sheep phage display library. The sequences were obtained by PCR from γ heavy chain cDNAs and are compared with the V5a (19 ) and JH1 (38 ) germline genes. Positions of CDRs and amino acid numbering are from Kabat et al. (32 ). At the D-JH junction, the two known functional JH segments are included, and the region encoded by PCR primers is indicated. Gaps in the sequence have been introduced to maximize homology. Dashes indicate nucleotide identity with the V5a and JH1 genes. Sequences are grouped according to family classifications as shown in Fig. 4. Motifs derived from pseudogenes (38 ) are in bold underlined type or boxed. ∗, Derived from the JH2 gene.

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FIGURE 4.

Dendogram of rearranged sheep VH segments with the V5a germline gene (19 ) constructed using the PILEUP program. Alignment was made using FR1 to FR3 of rearranged genes. New families have been assigned according to <80% sequence homology (certain genes share <80% sequence identity with other members of the same family). The clans to which families belong are indicated with Roman numerals.

FIGURE 4.

Dendogram of rearranged sheep VH segments with the V5a germline gene (19 ) constructed using the PILEUP program. Alignment was made using FR1 to FR3 of rearranged genes. New families have been assigned according to <80% sequence homology (certain genes share <80% sequence identity with other members of the same family). The clans to which families belong are indicated with Roman numerals.

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According to Kabat et al. (32), members of the same family share ≥80% homology. Berman (39) and Matthyssens and Rabbitts (40) have shown that cDNA and genomic DNA from the same family differ by only a few nucleotides. This criterion has previously been applied to the analysis of both germline genes and rearranged IgM swine genes (26), IgM and IgG1 from cattle (22), and IgG from cattle (21). Therefore, it is assumed that the accumulation of somatic mutations during affinity maturation is not sufficient to affect the family classification of cDNAs. Seven groups can be identified with two or more members, and other individual clones (H6, H23, and H17) appear not to be closely related to any other sequences. Consensus sequences for the nine groups indicated in Fig. 4 (VH1–VH9) were subjected to pair-wise comparison using the GAP program (Daresbury). Homology between groups ranged from 52.7% for VH3 and VH5 to 77.7% for VH1 and VH7 (data not shown). It should be noted that degeneracy necessarily included in consensus sequences due to the small number of sequences in some groups would tend to increase the apparent homology between groups. The majority of comparisons gave <70% nucleotide identity.

A similar analysis was conducted on the members of VH1 together with the closely positioned sequences H6 and H23 (Table I). It is notable that many of the clones share >80% homology with some, including the V5a germline gene, and <80% with others. However, the dendogram suggests a clear phylogenetic relationship with the family VH1.

Table I.

Pair-wise comparison of rearranged sheep heavy chain genes belonging to the VH1 family and the divergent clones H6 and H23a

V5aH224H333H247H21H165H288H266H39H36H145H1H41H294H69H320H3H13H321H123H6H23
V5a — 92.4 87.3 92.1 88.7 88.0 84.5 84.5 78.7 86.3 90.4 82.8 81.8 86.3 86.9 84.5 84.9 85.6 80.4 88.0 78.7 72.5 
H224  — 91.4 93.8 90.7 88.7 83.5 84.9 79.7 81.8 86.3 81.8 83.5 86.9 88.0 84.2 84.5 87.0 81.1 86.3 79.0 73.5 
H333   — 89 86.3 85.2 79.7 79.7 78.0 78.7 83.5 79.7 80.4 84.9 85.2 81.4 82.1 82.1 77.7 83.8 75.9 70.8 
H247    — 90.4 88.7 83.8 83.8 79.4 82.1 86.6 82.1 82.1 84.9 86.6 83.8 82.8 83.9 80.1 85.6 77.3 72.5 
H21     — 83.5 80.8 81.1 78.4 80.1 84.9 79.7 79.7 82.8 86.3 81.4 80.4 82.8 81.4 85.2 74.9 70.4 
H165      — 80.8 79.7 77.0 79.0 82.5 78.4 77.7 81.4 83.2 82.5 79.4 80.7 77.0 83.2 75.9 69.8 
H288       — 78.4 75.9 79.0 82.5 80.4 77.3 78.7 80.4 77.3 78.4 78.6 75.3 81.4 73.2 70.8 
H266        — 73.9 75.3 79.7 75.3 74.9 81.1 78.4 76.6 76.6 76.5 73.5 79.0 74.2 68.0 
H39         — 72.9 75.9 74.2 74.2 75.9 77.0 76.3 75.3 73.0 73.2 78.0 71.1 62.5 
H36          — 81.1 78.0 74.2 79.4 79.7 77.7 77.7 77.2 71.8 81.8 75.6 68.7 
H145           — 79.0 77.3 83.8 83.5 81.4 80.4 80.4 77.3 86.3 75.9 70.8 
H1            — 81.1 85.2 84.9 80.8 81.1 78.6 75.9 82.1 74.6 68.0 
H41             — 81.1 82.8 78.7 80.8 78.6 72.5 81.8 72.9 70.4 
H294              — 87.3 84.2 82.1 82.8 78.0 84.2 78.0 70.4 
H69               — 84.5 82.5 84.6 79.0 86.6 78.4 72.9 
H320                — 82.8 82.1 77.3 82.8 74.2 68.7 
H3                 — 80.7 77.0 80.7 74.9 69.8 
H13                  — 77.9 84.2 69.4 69.5 
H321                   — 86.6 70.1 67.0 
H123                    — 77.7 71.8 
H6                     — 68.7 
H23                      — 
V5aH224H333H247H21H165H288H266H39H36H145H1H41H294H69H320H3H13H321H123H6H23
V5a — 92.4 87.3 92.1 88.7 88.0 84.5 84.5 78.7 86.3 90.4 82.8 81.8 86.3 86.9 84.5 84.9 85.6 80.4 88.0 78.7 72.5 
H224  — 91.4 93.8 90.7 88.7 83.5 84.9 79.7 81.8 86.3 81.8 83.5 86.9 88.0 84.2 84.5 87.0 81.1 86.3 79.0 73.5 
H333   — 89 86.3 85.2 79.7 79.7 78.0 78.7 83.5 79.7 80.4 84.9 85.2 81.4 82.1 82.1 77.7 83.8 75.9 70.8 
H247    — 90.4 88.7 83.8 83.8 79.4 82.1 86.6 82.1 82.1 84.9 86.6 83.8 82.8 83.9 80.1 85.6 77.3 72.5 
H21     — 83.5 80.8 81.1 78.4 80.1 84.9 79.7 79.7 82.8 86.3 81.4 80.4 82.8 81.4 85.2 74.9 70.4 
H165      — 80.8 79.7 77.0 79.0 82.5 78.4 77.7 81.4 83.2 82.5 79.4 80.7 77.0 83.2 75.9 69.8 
H288       — 78.4 75.9 79.0 82.5 80.4 77.3 78.7 80.4 77.3 78.4 78.6 75.3 81.4 73.2 70.8 
H266        — 73.9 75.3 79.7 75.3 74.9 81.1 78.4 76.6 76.6 76.5 73.5 79.0 74.2 68.0 
H39         — 72.9 75.9 74.2 74.2 75.9 77.0 76.3 75.3 73.0 73.2 78.0 71.1 62.5 
H36          — 81.1 78.0 74.2 79.4 79.7 77.7 77.7 77.2 71.8 81.8 75.6 68.7 
H145           — 79.0 77.3 83.8 83.5 81.4 80.4 80.4 77.3 86.3 75.9 70.8 
H1            — 81.1 85.2 84.9 80.8 81.1 78.6 75.9 82.1 74.6 68.0 
H41             — 81.1 82.8 78.7 80.8 78.6 72.5 81.8 72.9 70.4 
H294              — 87.3 84.2 82.1 82.8 78.0 84.2 78.0 70.4 
H69               — 84.5 82.5 84.6 79.0 86.6 78.4 72.9 
H320                — 82.8 82.1 77.3 82.8 74.2 68.7 
H3                 — 80.7 77.0 80.7 74.9 69.8 
H13                  — 77.9 84.2 69.4 69.5 
H321                   — 86.6 70.1 67.0 
H123                    — 77.7 71.8 
H6                     — 68.7 
H23                      — 
a

The regions encoded by DH and JH segments were not included in calculations. Homologies of >80% are in bold type; >90% are underlined; <70% are boxed.

The lengths of CDR3s we have observed in sheep heavy chains range from 23 aa in clone H257 down to 3 aa in clones H69 and H261. The JH locus has been characterized by Dufour and Nau (38), who identified two functional JH segments and four pseudogenes in a region 5 kb upstream of the Cmu gene and spanning 1.85 kb. Both functional genes have been included in Fig. 3, and the rearranged sequences are compared with JH1. As with the 5′ end of FR1, the terminal eight codons of FR4 are encoded by primers used in PCR, and so sequence variability should be ignored in this region. Of the sequences belonging to VH1, 3 of 20 have used the JH2 gene segment as determined by the presence of G in position 2 of codon 105 (Kabat numbering) encoding an arginine (CGA). The remaining 17 have a proline here (CCA). This preferential use of JH1 is in agreement with previous findings (38).

Examination of the JH-encoded regions of the remaining sequences belonging to families other than VH1 reveals some interesting features. All such sequences except H261 have either AG (19 clones) or AA (five clones) in positions 2 and 3 of codon 105, giving rise to a glutamine residue. The frequency with which this occurs is too great to be a result of convergent somatic mutation. Analysis of the published JH segments reveals that the pseudogenes JH-ps2, JH-ps3, and JH-ps4 all encode codons 103–105 in this way (5′-TGGGGCCAG-3′) (38). Moreover, clones H242, H225, H168, H9, and H297 include the motif 5′-TGCTTTTGA-3′ (boxed sequence in Fig. 3). Once again this sequence is found in the correct position in the pseudogene JH-ps3. A second motif, 5′-ACGG-3′ is found spanning the codons 2 and 3 aa upstream of position 101 (Fig. 3) in clones H3, H204, H11, H264, H217, H257, H15, and H23. This motif is found in JH2, and so its presence in H3 is not unexpected. However, the region encoding positions 101–102 in six of the other seven clones suggests that these rearrangements involved the use of JH1 and not JH2.

Thirteen of the clones sequenced from the unselected phage library contained κ light chains (Fig. 5). Five new families can be identified (Table II), two of which include two genes and a further three each assigned from a single gene. The phylogenetic relationship of all of the sheep Vκ families is illustrated in the dendogram in Fig. 6. In a previous study (41), the amino acid at the 3′ end of CDR3 (Kabat position 97) was found to be either alanine or serine, corresponding to the use of the Jκ1 and Jκ2 gene segments, respectively. All of the clones we have identified have a threonine (ACT/ACG) in this position, the ACT codon being found in the pseudogene Jκ3. In three clones (K227, K13, and K321) the last two codons of CDR3 are 5′-TGG ACG-3′, which does not correspond with any of the three known Jκ gene segments.

FIGURE 5.

Sheep light chain Vκ–Jκ rearranged genes. Gaps in sequences have been introduced to maximize homology. Dashes indicate nucleotide identity to the Vκ 196 gene. Positions of CDRs and amino acid numbering are as Kabat et al. (32 ).

FIGURE 5.

Sheep light chain Vκ–Jκ rearranged genes. Gaps in sequences have been introduced to maximize homology. Dashes indicate nucleotide identity to the Vκ 196 gene. Positions of CDRs and amino acid numbering are as Kabat et al. (32 ).

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Table II.

Pair-wise comparison of rearranged sheep Vκ genesa

Vκ1Vκ2.1Vκ2.2Vκ3Vκ4K17K257K19K217K321K203K7K196K227K13K168K225K4Vκ Family
Vκ1 — 76.7 77.3 66.0 66.3 73.7 72.7 64.7 67.0 70.2 62.2 63.2 65.6 64.6 64.2 64.9 63.9 61.4 
Vκ2.1  — 85.3 61.0 64.9 76.0 72.0 66.0 66.7 67.7 59.0 63.2 65.3 62.5 64.6 63.5 63.5 60.7 II 
Vκ2.2   — 61.7 64.5 77.0 74.3 61.7 65.7 65.6 56.3 59.3 64.9 63.5 61.8 62.8 63.2 58.2 II 
Vκ3    — 64.9 59.7 62.1 58.6 63.2 65.6 59.3 62.1 64.6 63.9 64.2 65.3 64.2 58.2 III 
Vκ4     — 67.6 67.3 69.8 73.0 74.7 67.3 77.6b 81.1 80.9 80.1 81.5 80.4 69.8 IV 
K17      — 87.3 67.7 70.7 68.4 61.5 63.2 67.0 66.0 65.6 65.6 64.6 61.1 
K257       — 65.0 68.0 67.4 59.4 60.0 62.8 68.0 62.1 61.1 60.7 60.7 
K19        — 89.1 75.1 66.7 71.2 69.5 69.5 69.8 71.6 69.8 66.0 VI 
K217         — 79.3 68.8 72.6 72.3 73.3 71.6 75.1 74.7 68.1 VI 
K321          — 71.2 78.2 76.8 76.1 77.9 76.5 75.4 67.7 VII 
K203           — 68.1 69.1 68.4 68.4 70.5 67.7 64.2 VIII 
K7            — 89.1 86.3 87.4 86.0 83.9 73.0 IV 
K196             — 93.3 89.1 88.4 86.3 71.2 IV 
K227              — 87.4 87.0 85.6 71.6 IV 
K13               — 88.1 86.0 73.7 IV 
K168                — 95.4 74.4 IV 
K225                 — 74.7 IV 
K4                  — IX 
Vκ1Vκ2.1Vκ2.2Vκ3Vκ4K17K257K19K217K321K203K7K196K227K13K168K225K4Vκ Family
Vκ1 — 76.7 77.3 66.0 66.3 73.7 72.7 64.7 67.0 70.2 62.2 63.2 65.6 64.6 64.2 64.9 63.9 61.4 
Vκ2.1  — 85.3 61.0 64.9 76.0 72.0 66.0 66.7 67.7 59.0 63.2 65.3 62.5 64.6 63.5 63.5 60.7 II 
Vκ2.2   — 61.7 64.5 77.0 74.3 61.7 65.7 65.6 56.3 59.3 64.9 63.5 61.8 62.8 63.2 58.2 II 
Vκ3    — 64.9 59.7 62.1 58.6 63.2 65.6 59.3 62.1 64.6 63.9 64.2 65.3 64.2 58.2 III 
Vκ4     — 67.6 67.3 69.8 73.0 74.7 67.3 77.6b 81.1 80.9 80.1 81.5 80.4 69.8 IV 
K17      — 87.3 67.7 70.7 68.4 61.5 63.2 67.0 66.0 65.6 65.6 64.6 61.1 
K257       — 65.0 68.0 67.4 59.4 60.0 62.8 68.0 62.1 61.1 60.7 60.7 
K19        — 89.1 75.1 66.7 71.2 69.5 69.5 69.8 71.6 69.8 66.0 VI 
K217         — 79.3 68.8 72.6 72.3 73.3 71.6 75.1 74.7 68.1 VI 
K321          — 71.2 78.2 76.8 76.1 77.9 76.5 75.4 67.7 VII 
K203           — 68.1 69.1 68.4 68.4 70.5 67.7 64.2 VIII 
K7            — 89.1 86.3 87.4 86.0 83.9 73.0 IV 
K196             — 93.3 89.1 88.4 86.3 71.2 IV 
K227              — 87.4 87.0 85.6 71.6 IV 
K13               — 88.1 86.0 73.7 IV 
K168                — 95.4 74.4 IV 
K225                 — 74.7 IV 
K4                  — IX 
a

Data for Vκ1, 2.1, 2.2, 3, and 4 taken from Hein and Dudler (41). The region encoded by the Jκ segment was not included in calculations. Homologies of >80% are in bold.

b

The sequence of clone K7 shows only 77.6% homology to Vκ4 but >80% to all other members of the VκIV family and so has been assigned to this group.

FIGURE 6.

Dendogram of rearranged sheep Vκ segments. Sequences comprise five genes previously reported (43 ) labeled Vκ 1, Vκ 2.1, Vκ 2.2, Vκ 3, and Vκ 4, together with 13 additional genes. Nine sheep Vκ families are identified based on >80% nucleotide homology and are labeled VκI–VκIX. ∗, Families described previously.

FIGURE 6.

Dendogram of rearranged sheep Vκ segments. Sequences comprise five genes previously reported (43 ) labeled Vκ 1, Vκ 2.1, Vκ 2.2, Vκ 3, and Vκ 4, together with 13 additional genes. Nine sheep Vκ families are identified based on >80% nucleotide homology and are labeled VκI–VκIX. ∗, Families described previously.

Close modal

Forty-seven rearranged Vλ sequences were aligned with the 5.1 germline gene described previously (Ref. 16 and Fig. 7). All were found to segregate with members of the Vλ I family described by Reynaud et al. (Ref. 16 and not shown). GAP analysis of λ light chains revealed a wide range of diversity between these sequences that belong to the same family (not shown). Twenty-five percent of the clones had less than 80% homology with the majority of other clones. Clones L123 and L21 have >80% identity with the 17 germline genes belonging to the closely related but distinct family IV described by Reynaud et al. (16). However, L123 has 91.6% identity with 16.1 and L21 has 94.9% identity with 4.1, and so these clones are clearly members of the Vλ I family.

FIGURE 7.

Sheep light chain Vλ-Jλ rearranged genes. The sequences were obtained by PCR from cDNAs and are compared with the 5.1 germline gene (16 ). Gaps in sequences have been introduced to maximize homology, and dashes indicate nucleotide identity. The region of FR4 encoded by JλFOR PCR primers is indicated. Positions of CDRs and amino acid numbering are as Kabat et al. (32 ).

FIGURE 7.

Sheep light chain Vλ-Jλ rearranged genes. The sequences were obtained by PCR from cDNAs and are compared with the 5.1 germline gene (16 ). Gaps in sequences have been introduced to maximize homology, and dashes indicate nucleotide identity. The region of FR4 encoded by JλFOR PCR primers is indicated. Positions of CDRs and amino acid numbering are as Kabat et al. (32 ).

Close modal

The preferential expression of λ light chains is a feature common to many domesticated species including sheep (42, 43). The fact that only a single Vλ family was identified despite the extent of sequence divergence observed validates the sequence analysis methods applied and supports the existence of the multiple VH and Vκ families we have shown.

By producing a phage display library, we potentially have access to the whole expressed IgG repertoire of the host animal. The primer sequence used to produce heavy chain cDNA (CH1FOR) is conserved in both IgG1 and IgG2 isotypes. In total, 45 heavy chains, 47 λ light chains, and 13 κ light chains were sequenced from clones selected at random. Sequences have been analyzed according to the established method of Kabat et al. (32 which assumes that sequences that diverge by greater than 80% are derived from different germline gene families. In some cases this analysis has provided only a single family member sequence and may be less decisive. However, in these cases homology with all other sequences is less than 70% with no clustering of sequence variation, and therefore it is unlikely due to PCR errors or artifacts such as template jumping during library construction. Because we have sequenced only 47 clones, a germline family providing 2% of the repertoire would be represented by a single clone.

All previously reported VH genes from sheep belong to a single family (19) that shows greatest homology to the single family expressed in cattle (22). When compared with human VH genes, both sheep and cattle are homologous to VH4, a member of clan II (38). The heavy chain genes of other species expressing a single family such as swine, rabbit, and chicken are more closely related to human VH3 (clan III) (23, 44, 45), which has been proposed as the ancestral VH gene family (46). We have identified nine heavy chain gene families in sheep, eight of which have not been previously reported (Fig. 4). Of the new families, VH5, VH7, VH8, and VH9 are homologues of clan VH II, together with the VH1 family already described. VH3, VH4, and VH6 are homologues of clan VH I, and VH2 is a homologue of clan VH III. The isolation of sheep genes related to clan VH III confirms the evidence obtained by Tutter and Riblet (46), who observed hybridization of probes derived from the murine S107 and 7183 gene families to sheep genomic DNA. The sheep families VH1, VH7, VH8, and VH9 are most similar to human VH4, and the sheep VH5 is most similar to human VH6 (not shown). Saini et al. (47) reported detecting homologues of murine VH11 in bovine genomic DNA by Southern blot. In view of the close homology between ovine and bovine Ig genes, the sequences reported in this study may prove valuable in a more extensive analysis of the bovine genome.

All of the sequences we have obtained were derived from cDNA and so were being expressed in the host animal. Greater than 55% of the VH genes belong to new sheep families. Previous detailed studies have not revealed the heavy chain diversity that we have seen. Selective breeding has not resulted in variations in the diversity of Ig light chain loci between sheep belonging to different breeds (35). A possible factor is the age of the donor sheep. Heavy chain genes sequenced by Patri and Nau (36), Dufour et al. (19), and Dufour and Nau (38) were derived from spleen obtained from a slaughterhouse or were from animals described as adult. Previous analyses of light chain genes have used material taken either from fetuses or lambs up to 4 mo after birth (16, 24) or from sheep of unspecified age (34, 35). We have used spleen from an animal that was more than 10 years old when sacrificed, leaving the possibility that sheep may utilize a greater diversity of germline heavy chain genes in later life than are expressed when young.

From our results it can be seen that members of the same gene family frequently share unusually low nucleotide identity, i.e., <80%. This is particularly noticeable in the sheep heavy chain family VH1 and the λ family Vλ I. A similar observation has been reported in the llama for both the conventional VH and the VHH homodimer cDNA sequences (37) and may be a result of the necessity to generate a diverse Ab repertoire from a gene pool of restricted diversity. Our data demonstrate that in contrast to the llama, the sheep heavy chain germline is more diverse than had been previously thought. In addition, from analysis of heavy chain CDR3 there is evidence of use of JH pseudogene segments, which has not previously been reported in sheep. If there are in fact only two functional JH segments in sheep, as described by Dufour and Nau (38), then the apparent use of pseudogenes has been restricted to genes belonging to families other than VH1. It is possible that the sheep genome families VH2–9 may include multiple highly homologous V genes that have originated during evolution by gene duplication and subsequent mutation. The clones with pseudogene sequences may represent further somatic mutation of these genes or gene conversion events.

The κ genes represent six separate families. There is evidence that sheep preferentially express certain κ gene families at different stages of development (41), with VκIV dominating during the final stages of gestation. The single published gene isolated from adult tissue (35) belongs to this family. That this group is a major contributor to the adult Vκ repertoire is confirmed by our data in that six of the 13 genes we have identified are VκIV. However, phylogenetic comparison (Fig. 6) suggests that they form a separate subgroup to the Vκ4 gene. The Jκ-encoded region of our clones does not closely match the distinctive regions of any of the three known Jκ segments and may indicate that sheep possess a more extensive genomic Jκ region than that sequenced to date.

The sheep Vλ repertoire is known to be diverse, including at least six different families (16). Only genes from Vλ I, II, and VI have been identified from cDNA and so are known to be expressed (41). All such rearranged genes were isolated from fetal material. The 47 unselected sequences described in this study belong to the Vλ I family, which leads us to suggest that in contrast to the VH and Vκ repertoires, Vλ gene usage is restricted in adult sheep. We have found that expressed Vλ I genes are frequently highly divergent from known germline sequences. The extent of the observed divergence is such that two genes, L2 and L222, have >80% sequence identity only with each other, and a further two, L21 and L123, have >80% identity not only with Vλ I germline genes but also with the “17” germline gene belonging to the related but separate family Vλ II.

In conclusion, ruminants are thought to possess a smaller and less diverse gene pool than humans and mice and to utilize different mechanisms for generating their primary immune repertoire. Our studies indicate a greater level of functional diversity than previously described in sheep, though this does not necessarily imply a larger gene pool. Ab diversity and repertoire development are important components in animal health and understanding of disease processes. The studies we describe demonstrate the value of sheep Ab phage display libraries and provide a powerful new tool for such research.

We thank Elaine Durward for technical assistance and Alison McCaig for advice with sequence analysis software. We thank Dr. W. R. Hein (Basel Institute for Immunology) for providing us with six cDNA sheep VH segment sequences.

1

This work was supported by the United Kingdom Biotechnology and Biological Sciences Research Council’s Biological Treatment of Soil and Water LINK program.

3

Abbreviations used in this paper: FR, framework region; CDR, complementarity-determining region.

1
McCafferty, J., A. D. Griffiths, G. Winter, D. J. Chiswell.
1990
. Phage antibodies: filamentous phage displaying antibody variable domains.
Nature
348
:
552
2
Winter, G., A. D. Griffiths, R. E. Hawkins, H. R. Hoogenboom.
1994
. Making antibodies by phage display technology.
Annu. Rev. Immunol.
12
:
433
3
Vaughan, T. J., A. J. Williams, K. Pritchard, J. K. Osbourn, A. R. Pope, J. C. Earnshaw, J. McCafferty, R. A. Hodits, J. Wilton, K. S. Johnson.
1996
. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.
Nat. Biotechnol.
14
:
309
4
Sawyer, C., J. Embleton, C. Dean.
1997
. Methodology for selection of human antibodies to membrane proteins from a phage-display library.
J. Immunol. Methods
204
:
193
5
Ridder, R., R. Schmitz, F. Legay, H. Gram.
1995
. Generation of rabbit monoclonal antibody fragments from a combinatorial phage display library and their production in the yeast Pichia pastoris.
Bio/Technology
13
:
255
6
Davies, E. L., J. S. Smith, C. R. Birkett, J. M. Manser, D. V. Anderson-Dear, J. R. Young.
1995
. Selection of specific phage display antibodies using libraries derived from chicken immunoglobulin genes.
J. Immunol. Methods
186
:
125
7
Yamanaka, H. I., T. Inoue, O. Ikeda-Tanaka.
1996
. Chicken monoclonal antibody isolated by a phage display system.
J. Immunol.
157
:
1156
8
Clackson, T., H. R. Hoogenboom, A. D. Griffiths, G. Winter.
1991
. Making antibody fragments using phage display libraries.
Nature
352
:
624
9
Huse, W. D., L. Sastry, S. A. Iverson, A. S. Kang, M. Alting-Mees, D. R. Burton, S. J. Benkovic, R. A. Lerner.
1989
. Generation of a large combinatorial library of the immunoglobulin repertoire in phage λ.
Science
246
:
1275
10
O’Brien, P. M., R. Aitken, B. W. O’Neil, M. S. Campo.
1999
. Generation of native bovine mAbs by phage display.
Proc. Natl. Acad. Sci. USA
96
:
640
11
Cook, G. P., I. M. Tomlinson.
1995
. The human immunoglobulin VH repertoire.
Immunol. Today
16
:
237
12
Desiderio, S. V., G. D. Yancopoulos, M. Paskind, E. Thomas, M. A. Boss, N. Landau, F. W. Alt, D. Baltimore.
1984
. Insertion of N regions into heavy chain genes is correlated with expression of terminal deoxynucleotidyl transferase in B cells.
Nature
311
:
752
13
Claman, H. N., E. A. Chaperon, R. F. Triplett.
1966
. Thymus-marrow cell combination: synergisms in antibody production.
Proc. Soc. Exp. Biol. Med.
122
:
1167
14
Cooper, M. D., R. D. A. Peterson, R. A. Good.
1965
. Delineation of the thymic and bursal lymphoid systems in the chicken.
Nature
205
:
143
15
Archer, O. K., D. E. R. Sutherland, R. A. Good.
1963
. Appendix of the rabbit: a homologue of the bursa in chicken.
Nature
200
:
337
16
Reynaud, C. A., C. R. Mackay, R. G. Muller, J. C. Weill.
1991
. Somatic generation of diversity in a mammalian primary lymphoid organ: the sheep ileal Peyer’s patches.
Cell
64
:
995
17
Lucier, M. R., R. E. Thompson, J. Waire, A. W. Lin, B. A. Osborne, R. A. Goldsby.
1998
. Multiple sites of VL diversification in cattle.
J. Immunol.
161
:
5438
18
Weill, J. C., C.-A. Reynaud, O. Lassila, J. R. L. Pink.
1986
. Rearrangement of chicken immunoglobulin genes is not an ongoing process in the embryonic bursa of Fabricius.
Proc. Natl. Acad. Sci. USA
83
:
3336
19
Dufour, V., S. Malinge, F. Nau.
1996
. The sheep Ig variable region repertoire consists of a single VH family.
J. Immunol.
156
:
2163
20
Berens, S. J., D. E. Wylie, O. J. Lopez.
1997
. Use of a single VH family and long CDRs in the variable region of cattle Ig heavy chains.
Int. Immunol.
9
:
189
21
Sinclair, M. C., J. Gilchrist, R. Aitken.
1997
. Bovine IgG repertoire is dominated by a single diversified VH gene family.
J. Immunol.
159
:
3883
22
Saini, S. S., W. R. Hein, A. Kaushik.
1997
. A single predominantly expressed polymorphic immunoglobulin VH gene family, related to mammalian group I, clan II, is identified in cattle.
Mol. Immunol.
34
:
641
23
Sun, J., I. Kacskovics, W. R. Crown, J. E. Butler.
1994
. Expressed swine VH genes belong to a small VH family homologous to human VHIII.
J. Immunol.
153
:
5618
24
Reynaud, C. A., C. Garcia, W. R. Hein, J. C. Weill.
1995
. Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process.
Cell
80
:
115
25
Parng, C., S. Hansal, R. A. Goldsby, B. A. Osborne.
1996
. Gene conversion contributes to Ig light chain diversity in cattle.
J. Immunol.
157
:
5478
26
Sun, J., J. E. Butler.
1996
. Molecular characterization of VDJ transcripts from a newborn piglet.
Immunology
88
:
331
27
Reynaud, C. A., A. Dahan, V. Anquenz, J. C. Weill.
1989
. Somatic hyperconversion diversifies the single VH gene of the chicken with a high incidence in the D region.
Cell
59
:
171
28
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
29
Dower, W. J..
1989
. High efficiency transformation of E. coli by high voltage electroporation.
Nucleic Acids Res.
16
:
6127
30
Griffiths, A. D., M. Malmqvist, J. D. Marks, J. M. Bye, M. J. Embleton, J. McCafferty, M. Baier, K. P. Holliger, B. D. Gorick, N. C. Hughes-Jones, et al
1993
. Human anti-self antibodies with high specificity from phage display libraries.
EMBO J.
12
:
725
31
Chaudhary, V. K., J. K. Batra, M. G. Gallo, M. C. Willingham, D. J. Fitzgerald, I. Pastan.
1990
. A rapid method of cloning functional variable-region antibody genes in Escherichia coli as single-chain immunotoxins.
Proc. Natl. Acad. Sci. USA
87
:
1066
32
Kabat, E. A., T. T. Wu, H. M. Perry, K. S. Gottesman, C. Foeller.
1991
.
Sequences of Proteins of Immunological Interest
5th Ed. U.S. Department of Health and Human Services, Washington, DC.
33
Clarkson, C. A..
1993
. Sequence of ovine IgG2 constant region heavy chain cDNA and molecular modelling of ruminent IgG isotypes.
Mol. Immunol.
30
:
1195
34
Foley, R. C., K. J. Beh.
1989
. Isolation and sequence of sheep IgH and L-chain cDNA.
J. Immunol.
142
:
708
35
Foley, R. C., K. J. Beh.
1992
. Analysis of immunoglobulin light chain loci in sheep.
Anim. Genet.
23
:
31
36
Patri, S., F. Nau.
1992
. Isolation and sequence of a cDNA coding for the immunoglobulin μ chain of the sheep.
Mol. Immunol.
29
:
829
37
Vu, K. B..
1997
. Comparison of llama VH sequences from conventional and heavy antibodies.
Mol. Immunol.
34
:
1121
38
Dufour, V., F. Nau.
1997
. Genomic organization of the sheep immunoglobulin JH segments and their contribution to heavy chain variable region diversity.
Immunogenetics
46
:
283
39
Berman, J. E..
1988
. Content and organization of the human Ig locus: definition of three new VH families and linkage to the Ig CH locus.
EMBO J.
7
:
727
40
Matthyssens, G., T. H. Rabbitts.
1980
. Structure and multiplicity of genes for the human immunoglobulin heavy chain variable region.
Proc. Natl. Acad. Sci. USA
77
:
6561
41
Hein, W. R., L. Dudler.
1998
. Diversity of Ig light chain variable region gene expression in fetal lambs.
Int. Immunol.
10
:
1251
42
Hood, L. E., W. R. Gray, W. J. Dreyer.
1996
. On the mechanism of antibody synthesis: a species comparison of L-chains.
Proc. Natl. Acad. Sci. USA
55
:
826
43
Reynaud, C. A., V. Dufour, J. C. Weill.
1997
. Generation of diversity in mammalian gut-associated lymphoid tissues.
J. Immunol.
159
:
3093
44
Parvari, R., A. Avivi, F. Lentner, E. Ziv, S. Tel-Or, Y. Burstein, I. Schechter.
1988
. Chicken immunoglobulin γ-heavy chains: limited VH gene repertoire, combinatorial diversification by D gene segments and evolution of the heavy chain locus.
EMBO J.
7
:
739
45
Currier, S. J., J. L. Gallarda, K. L. Knight.
1988
. Partial molecular genetic map of the rabbit VH chromosomal region.
J. Immunol.
140
:
1651
46
Tutter, A., R. Riblet.
1989
. Conservation of an immunoglobulin variable-region gene familiy indicates a specific, noncoding function.
Proc. Natl. Acad. Sci. USA
86
:
7460
47
Saini, S. S., K. Teo, A. Nangpal, B. A. Mallard, A. Kaushik.
1996
. Homologues of murine Vh11 gene are conserved during evolution.
Exp. Clin. Immunogenet.
13
:
154