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Short communication

Organization and genomic complexity of bovine l-light chain gene locus

Yfke Pasman, Surinder S. Saini 1, Elspeth Smith, Azad K. Kaushik *

Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, Canada N1G 2W1

1. Introduction

The jawed vertebrates, with the exception of avianspecies (galliforms and anseriforms), principally expresstwo light chain isotypes (Edelman and Gally, 1962),kappa (k) and lambda (l), that associate with theimmunoglobulin heavy chain both in a stochastic(Kaushik et al., 1990) and non-stochastic (Knott et al.,1998; Yurovsky and Kelsoe, 1993) manner. Relativeexpression of k or l light chains in antibodies differsacross species, for example, k-light chains are mostlyexpressed in mice (95%) (Hood, 1969) and rabbits (90%)(Hole et al., 1991) whereas l-light chains are predomi-

nantly expressed in dogs, cats cattle, sheep and horses(Arun et al., 1996) [reviewed by (Butler, 1998)]. Inter-estingly, l-light chains are exclusively expressed inchickens (Reynaud et al., 1983), where a single light chainlocus exists (Ratcliffe, 2006; Reynaud et al., 1985). Someantibodies from camel (Hamers-Casterman et al., 1993)and nurse shark (Greenberg et al., 1995) are devoid oflight chains altogether. Differences in k:l ratio expressedacross species have led to suggest that these might be dueto: (a) genomic complexity dependent stochastic expres-sion (Cohn and Langman, 1990; McGuire and Vitetta,1981); (b) ordered rearrangement of k- and l-light chain(Rolink et al., 1991); (c) exogenous antigen selection(Haughton et al., 1978; Nishikawa et al., 1984) orendogenous counter selection (Knott et al., 1998); (d)recombination signal sequence dependent recombina-tion (Ramsden et al., 1994).

The bovine antibody repertoire is dominated by l lightchain genes with minimal k-light chain expression (Arunet al., 1996). The variable lambda genes (Vl) are classifiedinto three bovine gene families; Vl1, Vl2 and Vl3 (Saini

Veterinary Immunology and Immunopathology 135 (2010) 306–313

A R T I C L E I N F O

Article history:

Received 3 December 2009

Accepted 30 December 2009

Keywords:

Light chain

Antibody diversity

Bovine Vl

JlCl genes

A B S T R A C T

Complete characterization and physical mapping of bovine lambda (l) light chain locus,

spanning 412 kbp, on chromosome 17, has revealed twenty-five Vl genes, seventeen being

functional, organized in three sub-clusters 23.7 kbp 50 of the Jl-Cl units. Three Vl sub-

clusters are separated by two large introns of 126.8 and 138.3 kbp. The predominantly

expressed Vl1 genes are present in the two 50 sub-clusters, while Jl-proximal Vl sub-

cluster comprises rarely expressed Vl2 and Vl3 genes. The preferential expression of Vl1

genes in the bovine immunoglobulin repertoire is influenced by the composition of

recombination signal sequences (RSS). Of the Jl-Cl cluster, it is mainly Jl3-Cl3 unit that is

expressed in reading frame 2, though Jl2 and Jl3 have identical RSS. The predominant

expression of Jl3-Cl3 genes over Jl2-Cl2 is likely due to endogenous counter selection for

Jl2 encoded CDR3 and framework 4 regions. Differences in the genomic complexity of Vl

genes in Hereford and Holstein cattle are due to polymorphism at the l-light chain gene

locus. Despite more potential germline encoded combinatorial diversity, restricted Vl1-

Jl3-Cl3 recombinations encode the most l-light chain repertoire in cattle.

� 2010 Elsevier B.V. All rights reserved.

Abbreviations: Vl, variable lambda light chain gene; Jl, joining

lambda light chain gene; Cl, constant lambda light chain gene; RSS,

recombination signal sequence; RF, reading frame.

* Corresponding author. Tel.: +1 519 824 4120x54389.

E-mail address: akaushik@uoguelph.ca (A.K. Kaushik).1 Current address: Canadian Food Inspection Agency, 174 Stone Road

west, Guelph, Ontario, Canada N1G 4S9.

Contents lists available at ScienceDirect

Veterinary Immunology and Immunopathology

journal homepage: www.e lsev ier .com/ locate /vet imm

0165-2427/$ – see front matter � 2010 Elsevier B.V. All rights reserved.

doi:10.1016/j.vetimm.2009.12.012

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et al., 2003b; Sinclair et al., 1995). The Vl1 gene family ispredominantly expressed and comprises several subfa-milies based on serine signature (Saini et al., 2003b). Sincethe l-light chain gene locus is only partially characterized(Chen et al., 2008; Ekman et al., 2009), we have nowdeveloped a complete map of the bovine l-light chainlocus together with elucidation of genomic complexity,organization and expression of the Vl and Jl-Cl genes.Similar to humans but distinct from mice, bovine Vl genesare clustered together, approximately 23.7 kb 50 of the Jl-Cl cluster on chromosome 17. Such an organization,distinct from the Vl-Jl-Cl recombination restricted to acassette as in mice (Carson and Wu, 1989; Storb et al.,1989), permits relatively greater potential of combinator-ial diversity to the light chain variable-region where anyof Vl genes could recombine with either of the twofunctional Jl-Cl units. Despite such potential diversity,the Vl1-Jl3-Cl3 recombination mostly encodes thelambda light chains that predominate in the cattleantibody repertoire.

2. Materials and methods

2.1. Gene identification and mapping of lambda locus

Germline Vl (Parng et al., 1996; Saini et al., 2003b;Sinclair et al., 1995) (Table 1), Jl and Cl gene sequences(Chen et al., 2008) were used to analyze the bovine genomesequence (Btau_4.0 Hereford [cow build 4 genomedatabase] (reference assembly only); database name,gpipe/9913/ref_contig; 14,195 sequences) using BLASTN2.2.20+ (Zhang et al., 2000) and Geneious Pro version 4.6.4(Drummond et al., 2009). Sequence matches were eval-uated based on percent identity and unique structuralcharacteristics (Chen et al., 2008; Saini et al., 2003b) and,accordingly, various genes were identified and mappedonto the chromosome 17.

2.2. Gene expression analysis

Searches were performed on the Bos taurus ESTs(expressed sequence tags) database (p/9913.10708/bt_est[1,464,814 sequences]), utilizing BLASTN 2.2.20+ (Zhang etal., 2000). The matches with �97% identity [�33 out of38 bp] for Jl and �99% [�313 of 320 bp] for Cl genesformed the basis of analysis. The functionality of theannotated Jl and Cl genes was ensured by their in frametranslation using Geneious Pro version 4.6.4 (Drummondet al., 2009; Rozen and Skaletsky, 2000). Availableannotated Vl cDNA sequences (Parng et al., 1996; Sainiet al., 2003b; Sinclair et al., 1995) were taken intoconsideration for gene expression analysis.

2.3. Southern blot hybridization

Approximately 10–15 mg of genomic DNA, extractedfrom peripheral blood lymphocytes of Holstein Friesian,Jersey and Hereford cattle (Stratagene, LaJolla, CA, USA),was digested to completion with BamH I, EcoR I and Hind IIIrestriction enzymes, electrophoresed and transferred ontomaximum strength nytran membrane (Schleicher andSchuell Inc., Keene, NH, USA) using VacuGene (PharmaciaLKB Biotechnology, Uppsala, Sweden) as described (Saini etal., 1997). The membranes were pre-hybridized overnightat 42 8C followed by hybridization (50% formamide, 5�Denhardt’s solution, 5� SSPE, 0.5% SDS, 100 mg/mlsonicated and denatured salmon sperm DNA and 2.5–3.0� 106 dpm of [a-32P]dCTP radiolabeled cattle Vl1 genespecific BLV8C11 DNA probe as described (Saini et al.,2003a). The DNA probe was first excised by restrictiondigestion with Sal I and Xba I enzymes, purified and againdigested with Hinf I enzyme to obtain a 176 bp Vl1-specificDNA probe spanning nucleotides 12–188. The DNA probewas radiolabeled with [a-32P]dCTP (Amersham CanadaLtd., Oakville, ON, Canada) by random priming (Boehringer

Table 1

Percent expression of Vl genes in bovine B lymphocytes.

Bovine Vl gene CDR1L length (codon) Member

Family Subfamily cDNA(s) % Vl gene

expression

Vl1 Vl1a 14 BLV1F3, BLV3D11, BLV4B8, BLV6D10, BLV9C12, BLV9H3,

BLV7F8, BLV4H9, BLV3D7, BLV1G9, BLV7A8, BLV5H4, U32250

(Clone3), U32255 (Clone10), U32261 (Clone16), U32254

(Clone8), U31106, U32249 (Clone2), Clone C6, Clone V15

31.7

Vl1b 13 U32263 (Clone4), U32264 (Clone20), BLV10H2, U32259

(Clone14), CloneC7, Clone V8, Clone C2, Clone C3, U32257

(Clone5), U32251 (Clone9), U32253 (Clone7), BLV2C7,

Clone 32.5, Clone 32.6, Clone 32.7, Clone 32.16, Clone B4,

Clone 32.2, BLV11F3, BLV7B7, BLV7G1, BLV9B8, BLV7D7,

U32256 (Clone11), U32258 (Clone15), U32260 (Clone15),

U32262 (Clone17), Clone V2, U32252 (Clone 6)

46.0

Vl1c 13 Clone 15* 1.6

Vl1d 14 BLV8C11 1.6

Vl1e 13 BF1H1 1.6

Vl1x 13 BLV1H12, BLV5B8, BLV5D3 4.8

Vl1(Other) Clone 32.12, Clone 32.13, Clone 32.1, Clone 32.4, Clone 32.14 7.9

Vl2 – 14 Clone13* 1.6

Vl3 – 11 Clone V6, Clone C4 1.6

The sequences with prefix BLV are from our laboratory (Saini et al., 2003b) and those with prefix U and * are from Sinclair et al. (1995). Other sequences are

from Parng et al. (1996).

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Mannheim, Germany). The membranes were washedstringently in 0.1� SSC, 0.1% SDS at 65 8C for 10–15 minand exposed to XAR-5 films (Eastman Kodak Co., Roche-ster, NY, USA) for 2–5 days at �70 8C.

3. Results and discussion

3.1. Bovine lambda light chain locus organization permits

higher potential combinatorial diversity

Examination of the cow genome (Btau_4.0 Hereford) forbovine l-light chain locus led to identification of a5.65 Mbp region on chromosome 17 for analysis, wheretwenty-five Vl genes are identified to be clusteredtogether, spanning 367.9 kbp 50 to the Jl-Cl cluster(Supplementary Table 1). The Vl genes are organized inthree sub-clusters that are separated by two large sizedintrons, 128 kbp and 138.3 kbp, respectively (Fig. 1).Seventeen Vl1 genes are present in two sub-clusters thatlie 50 to the Jl-proximal Vl sub-cluster comprising the Vl2and Vl3 genes. The conserved leader gene (Fig. 2) sequence(LS) lies approximately 96–113 bp 50 of Vl1 genes (Fig. 1)followed by conserved recombination signal sequences(RSS) with a 21–24 bp spacer (Fig. 3) 30 of each Vl1 gene.The most 50 Vl1 gene sub-cluster comprises one Vl1d andtwo Vl1e pseudogenes, while the other Vl1 sub-clusterhas four Vl1e pseudogenes (Fig. 1). The Jl-proximal sub-

cluster (Fig. 1) comprising four Vl2 and Vl3 genes eachwith 50 group specific leader sequences (Fig. 2), andintervening introns ranging from 116 to 162 bp, arecharacteristically distinct from the two 50 sub-clusterscomprising Vl1 gene family. All the identified Vl genes fallinto either of three Vl families classified earlier (Saini et al.,2003b) and these correspond to three human and sheep Vl

groups (Table 2). The Vl1 genes identified in the Herefordgenome belong to either of the five Vl1 subfamilies (Sainiet al., 2003b; Sinclair et al., 1995) taking into considerationVl1c and Vl1x as a single subgroup (Saini et al., 2003b).The most predominantly expressed Vl1a and Vl1bsubfamilies (Table 1) comprise 3 and 2 members,respectively, distributed in both the most 50 Vl sub-clusters. Of the eight Vl1e genes identified, two Vl1e genesare functional. The leader sequence of one of the six Vl1epsuedogenes (LS13 Vl1e.5) differs in the first codon, wherethe conserved methionine is replaced by threonine. TwoVl1d genes exist in most 50 sub-cluster of which only one isfunctional. A single member of Vl1x is identified in themost 50 Vl1 sub-cluster. Four Vl2 genes are identified inthe Jl proximal sub-cluster where one of them is apseudogene. Most Jl-proximal genes are, however, mem-bers of the Vl3 family and these are all functional butrarely expressed. Variations have been noted in theconserved leader sequences of Vl1 and Vl2 genes wherethe codons encoding ‘‘MAW’’ (Fig. 2) are conserved with

Fig. 1. Complete map of bovine l-lambda light chain locus, spanning 412 kbp, on chromosome 17, modified from Jl-Cl units (Chen et al., 2008) and Hereford

cattle genome (version Btau 4.0). Note three sub-clusters of Vl genes where most Jl-proximal cluster comprises Vl2 and Vl3 genes while 50 two sub-

clusters comprise Vl1 genes. Asterisk indicates pseudogene.

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few exceptions (Vl1e.5C, Vl2.2C and Vl2.4). Leadergenes of each Vl family are characteristically conserved atthe 30 end that encode unique amino acids (Vl1: GDWM;Vl2: GEAS; Vl3: GAAP). Thus, a total of twenty-five Vl

genes of which seventeen are functional, are identified onthe l-light chain locus on chromosome 17 of the Herefordgenome that falls into three phylogenetic groups with

most divergence within the Vl1 family (Fig. 3). The numberof Vl genes identified on chromosome 17 differs fromthose reported by (Ekman et al., 2009) where thirty-one Vl

genes are identified on chromosome seventeen and thirty-two Vl genes on yet to be identified chromosomes. Suchdifferences across cattle breeds may be due to polymorph-ism at the l-light chain locus. Indeed, two distinct RFLP

Fig. 2. Nucleotide sequence of leader gene 50 to Vl genes with deduced amino acid sequence and phylogenetic relationship. Note distinct phylogenetic

relationships between leader genes specific to each Vl gene family.

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patterns are observed in the genomic DNA from HolsteinFriesian, Jersey and Hereford cattle following hybridizationwith a bovine Vl1-specific DNA probe where 9–15 bovineVl1-hybridizing restriction fragments are noted in threecattle breeds (Fig. 4). Similarly, allelic polymorphism at theVl-locus has been noted in other species, e.g., humans(Taub et al., 1983) and sheep Vl-loci (Foley and Beh, 1992).The presence of Vl pseudogenes on chromosome 17 offersthe possibility of gene conversion based variable-regiondiversification as suggested earlier (Parng et al., 1996).Unlike mice, where Vl, Jl and Cl genes exist as restrictedrecombination units (Carson and Wu, 1989; Storb et al.,1989), the organization of bovine Vl genes permitsrelatively higher potential for variable light chain diversity.The organization of the light chain locus in cattle suggeststhat various Vl genes originated by gene duplicationevents. All the bovine Vl genes are organized in correct

transcriptional orientation that would permit deletion ofintervening DNA during Vl and Jl-Cl recombination.

The Jl-Cl cluster lies 23.7 kbp 30 of Vl genes (Fig. 1) withtwo functional Jl-Cl cassettes (Jl2-Cl2, Jl3-Cl3) since Cl1and Cl4 are pseudogenes (Chen et al., 2008). Whilecomplete Jl-Cl cluster from Holstein cattle breed has beensequenced and annotated (Chen et al., 2008), somedifferences are noted with regard to Jl-Cl cluster onchromosome 17 in Hereford cattle genome (Btau_4.0)where two copies of Cl2 gene exist 50 of Jl2-Cl2 unit. Wehave, however, developed the refined map taking intoconsideration Jl-Cl analysis in Holstein cattle (Chen et al.,2008). Such an ambiguity between Hereford and Holsteincattle is likely to be confirmed once refined annotation ofthe cow genome is confirmed and differences, such aspolymorphism, among various cattle breeds elucidated.The Jl2 and Jl3 genes have 86% sequence identity but are

Fig. 3. Phylogenetic relationships among Vl germline genes that group into three Vl gene families with considerable divergence within Vl1 gene family.

Table 2

Percent nucleotide identities of bovine Vl genes with the corresponding sheep and human Vl gene families.

Bovine Vl gene

family (Gene)

Sheep Vl genes Human Vl genes

VlI

(Clone 5.3)

VlII

(Clone 6.1)

VlIII

(Clone 3.1)

Unclassified

(Clone Vl 6b)

Vl I

(DPL 8)

Vl II

(HSLV 2066)

Vl III

(LBR 1104)

Vl1 (Clone10) 92.6 69.3 77.2 56.3 74.8 68.1 60.5

Vl2 (Clone 13) 75.2 88.9 68.5 58.2 72.6 79.3 63.6

Vl3 (Clone C4) 64.2 60.0 60.8 75.0 62.3 63.5 73.8

Clone 10 and Clone 13 (Sinclair et al., 1995); Clone C4 (Parng et al., 1996); Clone 5.3, Clone 6.1, and Clone 3.1 (Reynaud et al., 1997); Clone Vl6b (GenBank:

AF038146); Clone DPL 8 (Williams and Winter, 1993); Clone HSLV 2066 (Brockly et al., 1989); and Clone LBR 1104 (Fang et al., 1994). Maximum nucleotide

similarities are shown in bold.

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phylogenetically distant from the Jl1 and Jl4 genes(Supplementary Figure 1a). Each Jl gene has a classicalRSS with a 12 bp spacer. The Jl2 and Jl3 gene RSSs areidentical but these phylogenitically diverge from those ofthe Jl1 and Jl4 genes (Supplementary Figure 1b). The Cl

genes share 95–97% sequence identity between them-selves with Cl1 and Cl4 being pseudogenes. A ‘C’nucleotide deletion at position 214 of Cl1 gene and a ‘G’to ‘A’ substitution at position 125 of Cl4 gene generates astop codon (Supplementary Figure 2a). Further a nucleo-tide deletion at position 314 is noted for the Cl4 gene. Themost expressed Cl3 gene is, however, phylogeneticallydistant from other Cl genes (Supplementary Figure 2b).

3.2. Differences in recombinant signal sequence influence

Vl gene expression

The Vl2 and Vl3 gene families that comprise three andfour functional genes are rarely expressed in contrast toforemost expression of four Vl1a and two functional Vl1bgenes in B-cells (Table 1). An inspection of recombinationsignal sequences revealed that while the heptamer isconserved in all RSSs, differences exist in the nonamersequences. The nucleotide ‘‘C’’ is conserved at position 34in RSS of Vl2 genes compared to ‘‘A’’ in nonamersassociated with all the Vl1 genes, in addition to few otherdifferences (Fig. 5). Similarly, conserved ‘‘C’’ nucleotide isnoted at position 35 of the nonamer of RSS of Vl3 genes incontrast to ‘‘A’’ nucleotide in Vl1 genes. It is likely that such

differences in the nonamer influence the recombinationfrequencies of various Vl genes due to varying affinity forrecombinases.

The expression of Jl1 and Jl4 genes is affected due tothe corresponding Cl1 and Cl4 being pseudogenes. Clearly,Jl3 preferential expression, in contrast to Jl2 gene, is notinfluenced by RSSs as these are identical. But it may be dueto either endogenous counter selection or differences inthe splice site (Chen et al., 2008). The possibility ofendogenous counter selection seems likely because ofsignificant differences in the Jl3 encoded codons (1, 2, 8and 9) of framework 4 region in the preferred RF2 ascompared to Jl2 gene (Supplementary Figure 1a)

3.3. Vl leader sequences differ between Vl gene families

Analysis of twenty-five leader sequences (LS) acrossthree Vl gene families fell into three phylogenetic groups(Fig. 2). The codons TGG and GGT, encoding tryptophan andglycine, are conserved in the leader gene of all Vl genes, atpositions 3 and 16, respectively. However, codons TCC, GAC,TGG and ATG, encoding serine, aspartate, tryptophan andmethionine, are conserved in the LS 50 of all Vl1 genefamilies at position 4, 17, 18 and 19, respectively (Fig. 2). Theresidues lysine, glutamine, glycine, glutamate, alanine andserine encoded by codons CTG, CAG, GGC, GAG, GCC, TCC, areprotein signatures for the LS 50 to Vl2 genes at positions 5,13, 14, 17, 18 and 19, respectively. Similarly, codons CCC,GCG, GCC and CCC encoding proline, alanine, alanine andproline are conserved at positions 9, 17, 18 and 19,respectively in the LS of Vl3 genes. Whether thesedifferences influence translation of Vl2 and Vl3 geneencoded proteins is yet to be understood.

3.4. Vl and Jl Cl gene expression

Unlike mice, where Vl genes are restricted to expres-sion within a given Vl-Jl-Cl cassette (Carson and Wu,1989; Storb et al., 1989), sub-clustering of Vl genes withseparate Jl-Cl gene units permits relatively more potentialrecombinations leading to more l-light chain variable-region diversity. Interestingly, most J-proximal Vl3 genesare either not expressed or, if at all, these are expressed at alow frequency. The predominantly expressed, Vl1a andVl1b (Saini et al., 2003b), genes are located in the middle ofthe Vl gene cluster, however. The Vl1x, Vl1d and Vl1egenes that are strictly noted to be expressed in light chainsin antibodies with exceptionally long CDR3H (Saini et al.,2003b) are scattered throughout the two 50 Vl1 sub-clusters. Clearly, predominant expression of Vl1a andVl1b genes is not related either to their genomiccomplexity or accessibility of the locus by recombinasesbut likely due to composition of RSSs that influence theaffinity to recombinases. The BLASTN search of Bos Taurus

EST database revealed most hits for Jl3 (499 hits E-value�2e-09, identity �97%, �33 out of 38 bp, �87% of Jsequence) as compared to Jl2 (13 hits E-value �2e-09,identity �97%, �33 out of 38 bp) genes. This is consistentwith higher Cl3 (284 hits with E-value �4e-163, identity�99% for sequence length �313 bp) hits as compared toCl2 (10 hits, E-value �4e-163, �99% identity, sequence

Fig. 4. Restricted fragment length polymorphism analysis of BamH I, EcoR

I and Hind III restriction digested genomic DNA from Holstein Friesian (1),

Jersey (2) and Hereford (3) cattle hybridized Vl1-specific DNA probe

(pBLV8C11). The arrows indicate polymorphism.

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length �313 bp) gene. High cut off rates were used todetermine conservative estimate of Jl-Cl gene expressiongiven high similarity between Cl exons (95–97%) (Sup-plementary Figure 2a). By contrast, no hits were noted forJl1, Jl2, Cl1 and Cl4 genes in the EST database consistentwith the observation that Cl1 and Cl4 are pseudogenes(Chen et al., 2008). Among the remaining Jl-Cl clusters, Jl3alone encodes the AVFGSGTTLTVL in reading frame 2(Supplementary Figure 1a) that is predominantly noted infetal VJ recombinations (Saini and Kaushik, 2002). Thus,Jl3-Cl3 genes are predominantly expressed in the lightchains of bovine antibodies with a minor role for Jl2-Cl2genes (Chen et al., 2008). Overall, these observationssuggest that a restricted Vl1-Jl3-Cl3 recombinationencodes most of lambda light chain antibody repertoirein cattle despite higher potential for germline encodedcombinatorial diversity.

Acknowledgement

This work was supported by NSERC Canada researchgrant to Dr. Azad K. Kaushik.

Appendix A. Supplementary data

Supplementary data associated with this article can be

found, in the online version, at doi:10.1016/j.vetimm.2009.

12.012.

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