Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes

Developmental and Comparative Immunology 26 (2002) 635±647 www.elsevier.com/locate/devcompimm

Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes Stein Tore Solem*, Trond é. Jùrgensen Department of Marine Biotechnology, Norwegian College of Fishery Science, Breivika 9037 Tromsù, Norway Received 17 August 2001; received in revised form 29 January 2002; accepted 10 February 2002

Abstract By screening a cDNA library and analysis of DNA produced by a combined 3 0 RACE/5 0 -anchored PCR, we have isolated three isotypes of IgL in the Atlantic salmon. Two of the isotypes were homologous to rainbow trout IgL1 and L2 sequences, while the third represents a previously uncharacterised salmonid IgL. The novel type 3 CL region is homologous to spotted wolf®sh c1 and yellowtail sequences, while the VL region is more similar to channel cat®sh F class than to any other ®sh VL sequences. Southern analysis indicates that the gene segments of all three isotypes are organised in multiple clusters. In addition, the VL gene segments of type 3 are arranged in opposite orientation relative to the JL and CL segments, while gene segments in type 2 clusters are all in the same orientation. Although transcripts of type 1 and 3 were readily found in the spleen and head kidney, only minute amounts of type 2 transcripts were seen. The majority of type 3 messages were truncated, suggesting that spliced and full-length transcripts of this isotype probably are present at a low level compared to type 1 transcripts. The uniqueness of the type 3 VLJL sequences suggests that this isotype offers additional diversity to the antigen-binding site of Atlantic salmon immunoglobulins. q 2002 Elsevier Science Ltd. All rights reserved. Keywords: Atlantic salmon; IgL; Isotypes; CDR; Organisation; Transcription

1. Introduction Analysis of vertebrate immunoglobulin light chain (IgL) sequences has demonstrated the evolution of at least three different IgL lineages: (1) k-like, (2) l-like and (3) sequences with no relationship to either k or l. In mammals, IgL is classi®ed as k or l expressing distinctive serological properties and unambiguous sequence features [1]. Analysis of IgL genes and cDNAs of ectothermic vertebrates have revealed the existence of three isotypes in Xenopus laevis (r, s and type III) [2±4] and cartilaginous ®sh (type I±III) [5±7]. * Corresponding author. Tel.: 147-77-64-55-71; fax: 147-77-6460-20. E-mail address: [email protected] (S.T. Solem).

In teleost ®sh like rainbow trout (Oncorhynchus mykiss W.) [8,9], channel cat®sh (Ictalurus punctatus) [10,11], and Atlantic cod (Gadus morhua L.) [8,12] two IgL isotypes are characterised. In common carp (Cyprinus carpio L.) and sea bass (Dicentrarchus labrax L.) only one isotype is so far isolated [13,14], while in spotted wolf ®sh (Anarhichas minor) two related but distinctive IgL sequences were found [15]. In general, it is dif®cult to relate IgL sequences of ectothermic vertebrates to k or l, however, most of the teleost, Xenopus r and cartilaginous ®sh type III IgL sequences may be considered as k-like [12]. Cartilaginous ®sh type II and Atlantic cod L2 are rated as l-like [6,12], while rainbow trout L2, X. laevis s, and cartilaginous ®sh type I sequences are unclassi®able and constitute the aforementioned third IgL lineage [4,9].

0145-305X/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved. PII: S 0145-305 X(02)00 012-5

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Table 1 Primer sequence and speci®city Name

Sequence a

Site b

Gene region

S1 c S2 c S11 c S12 c S32 S33 S34 S35 S36 S41 S57 S58 d S62 S66 S67 S68 oligo(dt) anchor

5 0 -ACT GGY CYC CTG CTT CTG 5 0 -AGC TCC TCA ACT CCT TAT TTA C 5 0 -AGG GGA CCG CCA CTT TTC ACT GT 5 0 -TCG CTG GAC GGA GGG AGG ATG 5 0 -CAG AAC AGT CAG CCC TCC TCA 5 0 -AAC TCC TTA TTT ACT ATG CTA CAA CA 5 0 -GAG CTC CTG AAC TCC TAA TA 5 0 -CCC CAC CCT CAC CGT CCT G 5 0 -ACC ATC CTC CCT CCG TCC A 5 0 -TCA GGC TAT TCA GTG CTA CAT TCA 5 0 -GTG GCG CTA CCA AAA GTN 5 0 -GTN AAR CCN TCY GTG TMT CTR 5 0 -CCA GCC CAG CAT TAG ACC A 5 0 -CAC TGG GGC TCC TTG TTC A 5 0 -GTA ATC TCC TGC ATC TTC TTC CTG 5 0 -CAG CAG GGG AAG GCC ACT CTC AT 5 0 -TCT GAA TTC TCG CGT CGT CAT CTT (TTT)5 V 5 0 -TCT GAA TTC TCG CGT CGT CAT CTT

162 143 49 50 319 151 142 17 28 347 20 17 426 2 32 306 57

CL1 Onmy, rev VL1 Onmy, forw VL2 Onmy, forw CL2 Onmy, rev CL1 Sasa, rev VL1.III Sasa, forw VL1.I Sasa, forw CL1 Sasa, forw CL1 Sasa, rev CL2 Sasa, rev JL1 Sasa, rev CL DareL3/Icpu F, forw 3 0 UTR Sasa L3, rev leader Sasa L3, forw VL3 Sasa, rev CL3 Sasa, forw poly A w/adaptor adaptor

a b c d

IUB codes: N ˆ aNy base, M ˆ A/C, R ˆ A/G, V ˆ A/C/G, Y ˆ C/T. IMGT numbering for nucleotide sequences [24], see Fig. 1. Constructed using rainbow trout IgL sequences [8,9]. Based on zebra®sh and channel cat®sh IgL sequences [11,17].

The presence of more than two IgL isotypes in teleosts was initially suggested by a serological approach investigating the presence of structurally and antigenic different light chains in serum of rainbow trout [16]. This hypothesis was recently supported by the isolation of cDNAs representing a third IgL isotype in zebra®sh (Danio rerio) [17]. To test whether salmonid ®sh possess more than two IgL isotypes as well, sequence analysis of isolated Atlantic salmon (Salmo salar L.) cDNAs was undertaken. Thus, the aim of the present study was to isolate possible IgL isotypes in the Atlantic salmon and to study transcription, diversity and organisation of gene segments and genomic complexity of these IgL loci.

2. Materials and methods 2.1. Animals Atlantic salmon (S. salar L.) 100±150 g were kept in continues ¯owing freshwater and fed commercial diet (Skretting, Norway).

2.2. Isolation and preparation of total RNA, poly(A) 1 RNA, cDNA and gDNA Total RNA was extracted from Atlantic salmon tissues using the Trizole reagent (BRL, Gaithersburg, MD, USA), and poly (A) 1 RNA subsequently puri®ed by passage through a Oligotex column (Qiagen GmbH, Hilden, Germany). To generate cDNA, 10 mg of total RNA was reverse transcribed at 42 8C for 60 min by 200 U M-MLV RT (Promega, Madison, WI), 500 mM dNTP's (Amersham Pharmacia Biotech, Uppsala, Sweden) and 0.8 mM oligo(dt) primer (Table 1). Genomic DNA was isolated from the liver using standard protocols [18]. 2.3. Isolation of IgL cDNAs of Atlantic salmon An ampli®ed cDNA library was constructed in the UNI-Zap XR vector, according to the manufacturer's instructions (Stratagene, LaJolla, CA, USA), using poly(A) 1 RNA from the pronephros of one ®sh vaccinated with Apoject 2-fural (Alpharma Inc., Oslo, Norway). The library was screened as described

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elsewhere [19] using DNA probes generated by Plabelling [20] of an equimolar mixture of two gelpuri®ed (SeaKem GTG, FMC BioProducts, Denmark) PCR products ampli®ed by the pro®le: 30 cycles of 95 8C for 10 s; 50 8C for 30 s; 72 8C for 30 s, using Atlantic salmon cDNAs, 1 U DyNAzyme (Finnzymes, Finland), 200 mM dNTP's and 0.3 mM each of the S1/S2 and S11/S12 primers (Table 1). The PCR products showed sequence features related to rainbow trout L1 and L2 light chains. The 3 0 -end of an additional salmonid IgL isotype was ampli®ed by 3 0 RACE [18], using Atlantic salmon cDNAs, S58 combined with an anchor primer (Table 1), 0.3 mM each, 200 mM dNTP's and 1.5 U Pfu polymerase (Promega). The PCR conditions were as follows: 10 cycles of 94 8C for 30 s, 45 8C for 30 s and 72 8C for 90 s followed by 20 cycles of 94 8C for 30 s, 50 8C for 30 s and 72 8C for 90 s, with a ®nal incubation of 7 min at 72 8C. A gene speci®c reverse primer (S62, Table 1) located to the 3 0 UTR of the amplicons obtained by 3 0 RACE was synthesised, and the 5 0 -part of the molecule ampli®ed by anchored PCR for 30 cycles of 94 8C for 10 s, 60 8C for 10 s and 72 8C for 90 s followed by a 7 min extension at 72 8C, using 0.3 mM S62 and 0.9 mM T3 primers, 200 mM dNTP's, Pfu polymerase (1.5 U) and 5 ml of the cDNA library preincubated at 94 8C for 15 min.

10 min extension at 68 8C. The elongation time was increased by 20 s per cycle during the last 20 cycles. Ampli®ed products were analysed by 1% agarose gel (SeaKem GTG) electrophoresis (5 V/cm), puri®ed and ligated into the pGEM w-T Easy vector (Promega). Finally, recombinant plasmids were treated as described above.

2.4. Tailing and cloning of PCR products

2.7. Sequence data deposition

Amplicons obtained by 3 0 RACE and 5 0 -anchored PCR were gel puri®ed and A-overhangs added by 1 U DyNAzyme (Finnzymes) and 200 mM dATP (Amersham Pharmacia Biotech) at 72 8C for 30 min. Subsequently, the A-tailed cDNAs were ligated into the pGEM w-T Easy vector (Promega). Recombinant plasmids were transformed into XL1-blue [21], isolated and puri®ed using Qiaprep (Qiagen) or Wizard (Promega) columns.

The nucleotide sequence data reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers: AF273012±AF273021 (isotype 1), AF297518 (0114; isotype 2), AF406963 (II-10; isotype 2), AF406964 (II-41; isotype 2), AF406956±AF406962 (isotype 3), AF462234±AF462237 (isotype 3, gDNA).

2.5. Ampli®cation of genomic DNA Genomic DNA (180 ng) was ampli®ed by PCR using 2 U DyNAzymee EXT polymerase (Finnzymes) in the presence of 360 mM dNTP's, 2 mM MgCl2 and 0.6 mM of each primer (Fig. 2a, Table 1). After an initial 2 min denaturation at 94 8C, the reaction proceeded by 30 cycles of 94 8C for 30 s, 60 8C for 20 s and 68 8C for 2.5 min followed by a

2.6. Sequence analysis All clones were sequenced on both strands by the dideoxynucleotide chain-termination method [22] using automated sequencing technology (ABI Prism 310, PE, Foster City, CA, USA). Primers used for sequencing were T3, T7, and SP6, in addition to the gene speci®c primers; S32, S12, S41 and S62 (Table 1). The deduced amino acid sequences were aligned using the ClustalX program [23], and assigned into framework (FRs) and complementarity determining regions (CDRs) according to the IMGT scienti®c chart [24]. Leader cleavage sites were predicted by the SignalP V1.1 software [25]. Possible canonical structures in the CDRs were outlined by identi®cation of structurally determining residues (SDR) using the web site: http://www.bioinf.org.uk/abs/ [26]. Phylogenetic analysis were performed by the Neighborjoining (NJ) method [27] using ClustalX.

2.8. Northern analysis Total RNA and poly(A) 1 RNA were fractionated by 1% denaturing agarose gel (LE, Ambion, Austin, Texas, USA) electrophoresis at 4.5 V/cm, followed by transfer onto Hybond N 1 (Amersham Pharmacia Biotech) by downward capillary transfer using NorthernMaxe transfer buffer (Ambion). The RNA was immobilised by UV-crosslinking at 120 mJ/cm 2. Prehybridisation and hybridisation were performed at

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42 8C with NorthernMax buffer (Ambion) supplemented with 1% Blocking reagent (Roche Diagnostics GmbH, Mannheim, Germany), using alkaline labile digoxigenin (DIG) labelled DNA probes. The probes were generated by the PCR DIG probe synthesis kit (Roche Diagnostics GmbH) and the following primer combinations: S35/S32 (CL type 1), S36/S41 (CL type 2) and S68/S62 (CL type 3) (Table 1), while plasmids containing characterised Atlantic salmon IgL sequences served as templates. A b-actin probe was included as control. Subsequent washing steps were performed at low stringency (2 £ sodium chloride/sodium citrate (SSC), 0.1% sodium dodecyl sulphate (SDS), 25 8C) for 2 £ 5 min and then at high stringency (0.1 £ SSC, 0.1% SDS, 68 8C) for 2 £ 15 min. 2.9. Southern analysis Genomic DNA was digested using Eco RI and/or Hin dIII (Promega), followed by 0.7% agarose gel (SeaKem GTG) electrophoresis (2 V/cm), and subsequently blotted onto a Nytran nylon-membrane (Schleicher & Schull GmbH, Dassel, Germany) under 20 £ SSC. The transferred DNA was UVcrosslinked, prehybridised and hybridised at 50 8C using DIG-labelled DNA VL and CL probes. The VL probes were ampli®ed as above using the following primer combinations: S34/S57, S33/S57, S11/S12 and S66/S67 (Table 1). The membranes were subsequently washed for 2 £ 5 min at low stringency, followed by 2 £ 15 min at either moderate (1 £ SSC, 0.1% SDS, 56 8C) or high stringency. 2.10. Detection and stripping of hybridised DIGlabelled probes Detection of hybridised probes was done according to the DIG Wash and Block Buffer Set (Roche Diagnostics GmbH) in combination with alkaline phosphatase conjugated sheep a-DIG Fab fragments, CDP-Stare substrate (Tropix Inc., USA), and Lumi®lm (Roche Diagnostics GmbH) exposure. Subsequently, probes were stripped from Southern blots by 2 £ 15 min incubations in 0.2 M NaOH, 0.1% SDS at 45 8C. The membranes were neutralised by 2 £ SSC at room temperature, and eventually rehybridised.

3. Results 3.1. Characterisation of Atlantic salmon IgL sequences Among the 20 cDNA clones isolated, three different kinds of putative IgL sequences were identi®ed. All were recognised as IgL according to the: (1) BLAST search option (http://www.ncbi.nlm.nih.gov/ blast/index.html), (2) possession of typical light chain residues in the VL region (Cys 23, the WYQQ and T/DYYC motifs), JL region (Phe 3) and CL region (Pro 7, Pro 13, Pro 14, Cys 29, Trp 43, Ser 73, Ser 74, Leu 76, Cys 91, and Cys 110), and 3) expected size of a full-length IgL cDNA sequence. A total of 10 type 1 sequences were isolated from the cDNA library. Nine were productive rearrangements (Fig. 1), two of which showed truncated leader peptides. The majority of type 1 cDNAs ranged from 1007 to 1036 bp. Clone 01-15 utilises an alternative polyadenylation signal and was extended to 1127 bp (not shown). The last clone (02-01, Fig. 1c) was a 1062 bp JLCL spliced germline transcript with a consensus [1] J nonamer (GGTTTTTGT) and heptamer (CACTGTG) separated by a Jk like 23 bp spacer. A single clone of type 2 (01-14) was isolated from the cDNA library. It was identi®ed as a 1293 bp JLCL spliced germline transcript with a truncated VL sequence 546 bp upstream, and in the same transcriptional orientation as the JLCL region (not shown). The deduced amino acid sequences of its exons, denoted v1-14, j1-14 and 01-14, are shown in Fig. 1. The recombination signal sequences (RSS) 5 0 of the JL region conformed to the consensus sequence, but was separated by a spacer of two nucleotides only. The V heptamer was mutated in the fourth and ®fth position (CACcaTG) and joined to a V nonamer expressing a single base deletion (ACAAAA-CC). Attempts to isolate type 2 cDNAs of productive rearrangements by anchored PCR failed. Messages of rearranged type 2 genes (clone II-10 and II-41) were ampli®ed by RT-PCR (Fig. 1b). However, by alignment of the II-10 VL region to a L2 cDNA of rainbow trout (not shown) three frameshift mutations were recognised: (1) deletion of the entire `F' b-strand, (2) an insertion of two bases upstream of the conserved JPhe codon, and (3) deletion of seven nucleotides of the

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Fig. 1. Alignment of the deduced amino acid IgL leader (a), VLJL (b) and CL (c) sequences isolated from the Atlantic salmon. Residues identical to clone 02-02 are indicated by dots. Gaps are designated by hyphens. Framework regions, CDRs, amino acid positions [24] and putative b-strands [37] are indicated above the sequences. The localisation of b-strands in the CL-region was adapted from mammalian Igk sequences. Conserved residues are marked by asterisks and frameshift mutations indicated by slashes. Numbers to the right indicate percentage of amino acids identical to clone 02-02. Gaps were estimated as mismatches.

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and CL gene segments was analysed by ampli®cation of genomic DNA using the primers shown in Fig. 2a. The ampli®ed products in lane 3 (Fig. 2b) were isolated and analysed. The second largest DNA fragment, represented by the clones g17 (2317 bp) and g20 (2315 bp), harboured leader and VL exons located in opposite orientation to the CL-3 0 UTR sequence. The VL and CL exons were separated by 1.2 kb, which represents the intercluster distance. Amino acid sequences of the exons are shown in Fig. 1. The V heptamer and nonamer RSS conformed to the consensus, and were separated by a Vk-like spacer of 12 bp (not shown). Two additional clones, g2 and g6, representing the smallest (1560 bp) and the largest (,2400 bp) PCR product, exhibited a CL or VL sequence only. Amino acid sequences of the exons are shown in Fig. 1b and c, respectively. 3.2. Features of the Atlantic salmon VL region

Fig. 2. Genomic PCR indicating the germline polarity of the unrearranged Atlantic salmon type 3 VL-gene segments relative to type 3 CL-gene segments. Primer locations (sequences are given in Table 1) and expected size of amplicons (a), and obtained gPCR products visualised by ethidium bromide (b). Lanes indicated in panel (a) conform to the lanes in panel (b). Lane 4 is the negative ampli®cation control. Molecular size (kp) is shown to the left.

JL segment. This suggests that some of these sequences probably represent non-functional proteins. An additional IgL isotype of the Atlantic salmon, type 3, was ampli®ed by 3 0 RACE using a degenerate primer corresponding to the conserved VKPSVSLL motif in the `A'-strand of the zebra®sh type III (AF246193), and channel cat®sh F (U25705) CL region. The ampli®ed fragment held 260 bp of a CL region (clone 8, Fig. 1c) and a complete 3 0 UTR followed by a poly(A) tail. Subsequently, 5 0 -anchored PCR yielded DNA fragments of approximately 400 and 900 bp. These fragments displayed partial 3 0 -ends and constituted transcripts of either truncated CL regions (clone 15, 28 and 30), or productive rearrangements with complete 5 0 -ends (clone III-1, III-4, and III-6) (Fig. 1). By assembling the overlapping sequences with complete 3 0 - (clone 8) and 5 0 -ends (clone 1-III, 4-III and 6-III) the full-length of type 3 sequences (1023± 1031 bp) was resolved. The orientation of type 3 VL

The deduced amino acid sequences of Atlantic salmon IgL cDNAs were aligned and organised into leader, VLJL and CL regions (Fig. 1). Type 1 VL regions were assigned into three different subgroups by amino acid identity: I, II, and III (Fig. 1b). Subgroup II and III were slightly more similar to each other than to subgroup I sequences. Some clones shared almost identical VLJL sequences (clone 01-15 and 02-03), however, substitutions in the CL (Fig. 1c) and 3 0 UTR (not shown) suggested a separate clonal origin. The three subgroups shared 72±79% identical amino acids in the VLJL region and were readily recognised by variability in the leader peptide, FR1, CDR1 and 3 (Fig. 1a and b). Notably, Gly 121 of the Phe-Gly-X-Gly-Ser/Thr motif in the `G' b-strand was mutated to Ala in type 1.III sequences. This substitution will probably promote a change in direction of the `G'-strand during formation of the b-bulge [28]. The VLJL sequence of type 2 diverged from both type 1 and type 3 sharing 18±19% identical amino acids (Fig. 1b), while an amino acid identity of 57.8± 60.2% was evident when the VLJL sequences of type 3 were compared to clone 02-02 (type 1). As described above, most substitutions were identi®ed in the leader peptide, FR1, CDR1 and CDR3. Analysis of nine additional type 3 clones did not reveal any additional VL families of this isotype (not shown). The number of amino acids in CDR1 varied both between the subgroups and isotypes. The CDR1 of

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Table 2 Assignment of possible main-chain conformations in LCDR-loops as determined by size and identi®cation of SDR Sequence identity

Number of clones

Canonical class a LCDR1

LCDR2

LCDR3

VL type 1.I

3

Class 4-like Thr 25 (Ser) c Val 29 (Leu)

Class 1

None b

VL type 1.II

3

Class 5l-like d Thr 25 (Gly) His 30 (Ile) Phe 87 (Ala)

Class 1

None

VL type 1.III

3

Class 3-like Val 2 (Ile) Thr 25 (Ser)

Class 1

None

None

None

None

Class 2

Class 1

Class 1

VL type 2

1±2

VL type 3

3

a b c d

Class assignment according to Ref. [26], using SDRs de®ned by Refs. [29±32]. None ˆ deviating SDRs and length compared to the de®ned canonical classes. Positions with residues other than the allowed SDRs. Allowed SDRs are shown within parenthesis. Canonical class 5l is derived from a l antibody, PDB-entry: 2fb4.

type 1.I held a consistency of 11 amino acids, type 1.II had eight, while 12 were found in type 1.III sequences. Both type 2 and 3 displayed six amino acids in the CDR1, although only one sequence, clone II-10, represented type 2 in this region (Fig. 1b). Type 1 and 3 showed three amino acids each in CDR2, while type 2 sequences revealed 10 or 15 amino acids in this region. The CDR3 varied from 9 to 13 amino acids in type 3 and 1 sequences, respectively. Interestingly, the section from the `F'-strand in FR3, through CDR3 and FR4 of clone 01-09 was 100% identical to the corresponding sequence of clone 05-02 (Fig. 1a). This indicates a low level of coding-end processing and also a conservation of CDR3 sequences. According to the size as well as the identi®cation of SDR at key positions, each subtype and isotype may adapt individual conformations in CDR1 (Table 2). Still, the SDRs of type 2 sequences could not be identi®ed as no full-length sequence of this isotype was found. Type 1 and 3 sequences expressed SDRs related to a single mammalian CDR2 conformation, while the size of CDR2 in type 2 sequences suggests the formation of an alternative structure in this region. In CDR3, only type 3 sequences showed SDRs related to a mammalian loop-structure. Although some deviations

from the allowed SDRs [29±32] were seen, the data suggests that considerable structural diversity are con®ned to the Atlantic salmon VL region, in particular to CDR1. 3.3. The CL region of the Atlantic salmon The Atlantic salmon type 1 CL sequences were assigned into two groups represented by ®ve clones each. The ®rst group was characterised by Met 28 and Ile 96 (Fig. 1c). Sequences within this group shared 94.3±100% identical amino acids (Fig. 1c). The absolute identity between clone 01-09 and 02-03 continued through the 3 0 UTR (not shown), although the VLJL sequences diverged (Fig. 1b).The second group, identi®ed by Thr 28 and Lys/Asn 96, displayed 90.5±93.3% amino acid identity when compared to clone 02-02 representing the ®rst group (Fig. 1c). Also, the 3 0 UTR segregated into these two groups (not shown). Both VL1.I and 1.III regions were accompanied by the ®rst group of CL regions, while the second group was associated to VL1.I and VL1.II sequences. Type 2 CL sequence shared 30.9% identical amino acids with the corresponding region of clone 02-02, while type 3 demonstrated 61.5±63.3% identity to this sequence. Moreover, insertions of two

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Fig. 3. Relationship of deduced VL FRs 1, 2 and 3 (a), and CL (b) amino acid sequences of cartilaginous and teleost ®sh as suggested by the NJ method. Distances (p) are given as the corrected proportion of amino acid differences. Genomic sequences are indicated by asterisks. Species (abbreviation) and accession numbers are: Acipenser baeri (Acba): X90557; Anarhicas minor (Anmi): c1 ˆ AF137397, c10 ˆ AF137398; Carcharhinus plumbeus (Capl): U01657; Cyprinus carpio (Cyca): AB01509; Danio rerio (Dare): L1 ˆ AF246185 (clone 1-8), AF246187 (clone 1-1); L2 ˆ AF246179 (clone 101a), AF246183 (clone 9), AF246181; L3 ˆ AF246193; Dicentrarchus labrax (Dila): AJ00216; Gadus morhua (Gamo): L1 ˆ AF104898 p (10.3); L2 ˆ AJ2093807; Ginglymostoma cirratum (Gici): L16765; Heterodontus francisci (Hefr): I ˆ X15315 p; II ˆ L25560 p, L25559; III K4 ˆ L25561, III K5 ˆ L25562, III K6 ˆ L25563; Hydrolagus colliei (Hyco): L25551, L25555 p; I. punctatus (Icpu): G ˆ L25533; F ˆ U25705; Oncorhynchus mykiss (Onmy): L1 ˆ X65260 (rtSE), X68519 (rtSg3), AJ521651 p (E2); L2 ˆ AJ251648 p (1 and 4), AJ251647 p (2 and 3); Raja erinacea (Rare): I ˆ U19209 p, II ˆ L25566; Seriola quinqueradiata (Sequ): AB062633. Bootstrap values above 600 of 1000 replica samplings are indicated.

and four amino acids were seen in the sequence between the predicted `C' and `D' b-strands of type 2 and 3 sequences, respectively. Notable, substitution of the hydrophilic Glu 17/Asp 17 to a hydrophobic Val 17 in type 3 CL sequences may affect the ability of this isotype to associate with the IgH chain. Conserved sequence motifs con®ned primarily to the `A', `B' and `E' b-strands. 3.4. Phylogeny of the Atlantic salmon IgL sequences Phylogenetic analysis were performed using VL

sequences including the two carboxyl terminal amino acids of the `F' b-strand and CL sequences spanning Asn/Leu/Asp 4 to Cys 110. The CDRs and all positions in loop-structures postulating gaps, in particular those connecting the `D' and `E' strands in VL regions and the `C' and `D' strands in CL regions, were deleted and excluded from the analysis. Also the truncated Atlantic salmon type 2 VL sequences were left out of the analysis. Still, the type 1 (VL, CL) and 2 (CL) sequences were clearly homologous to rainbow trout L1 and L2 (Fig. 3a and b), sharing the phylogeny previously described

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Fig. 4. Southern hybridisations using VL and CL speci®c probes at either moderate or high stringency. The identity of the probes is given below each blot. Arrows highlight barely visible bands. Use of Eco RI (E), Hin dIII (H), or combination of the two (E 1 H) is given above each blot. Numbers to the left indicate the molecular size (kp) of the fragments shown in the adjacent panel.

[8,9,12]. Atlantic salmon VL type 1.I and 1.II sequences were 84.8±93.6% identical to the Rtsg3 and E sequences of rainbow trout, suggesting that at least two of the VL subgroups evolved before the Atlantic salmon and rainbow trout separated. As shown in Fig. 3b, the Atlantic salmon type 3 CL region is located to a relatively tight cluster harbouring k-like teleost L1 and G sequences. The salmon type 3 consensus CL sequence shared 78.7±83.8% amino acid identity to the homologous yellowtail and spotted wolf®sh c1 sequences, respectively. The similarity to other CL sequences within the cluster ranged from 54 to 64.8% identity, represented by zebra®sh L1 and spotted wolf®sh c10, respectively. Sequences outside the cluster showed 27% (zebra®sh L3) to 37% (cartilaginous type III) identity to the salmon CL type 3sequence. Salmonid VL3 and channel cat®sh F-class VL sequences formed a separate group outside the obvious cluster of teleost L1 and G sequences (Fig. 3a). Accordingly, salmonid type 3 VL and cat®sh F class sequences shared 65.5% identical amino acids. Still, the interior branch approaching the node separating type 3/F and L1/G sequences was not highly supported in the bootstrap analysis. Finally, the similarity between salmonid VL3 and cartilaginous ®sh type III sequences was comparable to that between salmonid VL3 and VL1 sequences, i.e. around 60% amino acid identity.

3.5. Southern blot analysis of the Atlantic salmon light chain genes As seen in Fig. 4, both VL and CL speci®c probes hybridised to numerous restriction fragments derived from each of the three IgL loci. This suggests the presence of multiple gene copies within each loci. At high stringency both type 2 and 3 VL probes hybridised to a single DNA fragment only. The additional VL gene segments of these isotypes, detected at moderate stringency, may correspond to extra VL families. However, most of the 3±8 additional bands detected by the type 3 VL probe at moderate stringency were detected by type 1 VL probes as well. Due to extensive crosshybridisation of type 1.II and 1.III VL probes at high stringency, only one of these blots is shown in Fig. 4. The type 1.I VL probe crosshybridised to gene segments recognised by the type 1.II/III probes as well, although distinctive signals from each of the blots were seen. Additional bands did not emerge by use of type 1 VL probes at moderate stringency (not shown). 3.6. Transcription of IgL in the Atlantic salmon Type 1 transcripts were expressed as messages of the expected size primarily in spleen and head kidney. Lower levels of message were seen in thymus and excretory kidney (Fig. 5). Type 2 transcripts were

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Fig. 6. The size variation of Atlantic salmon IgL type 2 and 3 transcripts from the head kidney (HK) and spleen (S) visualised by Northern analysis using ,3 mg poly (A) 1 RNA and CL speci®c probes. The type 2 and 3 blots were exposed for 60 and 10 min, respectively. Molecular sizes (bases) are shown to the left. The blot is a representative of four individuals.

Fig. 5. Tissue speci®c transcription of IgL in the Atlantic salmon revealed by Northern hybridisation using 10 mg total RNA and CL type 1, 2, and 3, or b-actin speci®c probes as indicated to the right. Exposure: 10 min. Molecular sizes (bases) are indicated to the left. T ˆ thymus, S ˆ spleen, HK ˆ head kidney, K ˆ kidney, L ˆ liver, I ˆ intestine, G ˆ gills. The blot is a representative of four individuals.

not detected in any of the tissues when total RNA was analysed, although the probe gave a strong signal when hybridised to picogram quantities of a plasmid containing CL2 sequences (not shown). However, when a large amount of poly (A) 1 RNA was analysed transcripts related to this isotype were detected as four or ®ve messages of approximately 450 to ,1500 bases in both head kidney and spleen (Fig. 6). It was not determined if any of these messages corresponded to spliced full-length mRNA. Thus, it is unclear whether type 2 gene segments are expressed as functional proteins or not. Type 3 transcripts of ,400 and 600 bases were clearly detected in spleen and head kidney (Fig. 5). Additionally, a transcript of ,1 kb, was visible in both spleen and head kidney, either upon overexposure or by hybridisation with a large

amount of poly (A) 1 RNA (Fig. 6). This transcript corresponds, by predicted size, to the full-length message of type 3 genes. As judged by densitometric analysis of the exposed ®lm the ,1 kb messages constituted around 5% of the type 3 transcripts in both head kidney and spleen. The ,400 bases message corresponded by size to the cDNA of clones 15, 28 and 30 in Fig. 1. The identity of the 600 bases transcript is unknown. Transcripts of the structural gene b-actin showed that the quality of the analysed RNA was comparable in all tissues, while the relative quantity varied. Taken together, the data suggest that the bulk of IgL full-length and spliced IgL transcripts was dominated by type 1. 4. Discussion Evidence for the presence of at least three distinctive IgL isotypes in the Atlantic salmon was obtained by isolation and characterisation of 20 light chain cDNAs. Although, isotype 1 and 2 shared homology to rainbow trout L1 [8] and L2 [9], the third isotype represents a previously uncharacterised salmonid IgL sequence. Phylogenetic analysis of teleost and cartilaginous ®sh immunoglobulin CL sequences suggests that the salmonid IgL type 3 isotype is homologous to the spotted wolf®sh c1 and yellowtail sequences (Fig. 3b). This isotype most likely evolved by duplication and divergence of a common type 1 and type 3 precursor

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before the salmonids and Perciformes (spotted wolf®sh and yellowtail) separated. Thus, it is likely that this IgL variant exists in additional salmonids and Perciformes, as well as in other species that radiated after the salmonids, i.e. the Atlantic cod (Gadiformes) [33]. Alternatively, the type 3 gene(s) may have been lost in these species similar to the loss (or gain) of IgL gene segments indicated by a single genomic restriction fragment harbouring VL type 2 gene segments in the Atlantic salmon (Fig. 4), and numerous hybridisation bands with homologous sequences seen in the rainbow trout [9,34]. The NJ-tree of VL sequences (Fig. 3a) showed a different topology, although the major conclusions made on evolution of the CL gene segments apply also for the VL sequences. The transcriptional polarity of the Atlantic salmon type 1 gene segments was not determined. However, these VL gene segments are probably directed in opposite orientation compared to the CL. This anticipation is based on homology to the L1 sequences of the closely related rainbow trout (Fig. 3a), and the organisation of gene segments in the L1 locus of this species [34]. The Atlantic salmon type 2 VL, JL and CL gene segments are all in the same orientation, as observed in isolated germline transcripts (not shown). A similar organisation of the homologous L2 genes in rainbow trout is reported [34], supporting our anticipation of a similar organisation of homologous IgL gene segments. Analysis of PCR ampli®ed genomic DNA showed that the VL gene segments of salmonid IgL isotype 3 are in opposite orientation relative to the CL gene segments (Fig. 2). This indicates that an inverted arrangement of the VL and JL±CL gene segments was established before the type 1 and 3 genes diverged, and also suggests that all k-like sequences in the L1/G cluster (Fig. 3a and b) share a similar organisation. This is reasonable since this arrangement was probably established prior to radiation of the Siluriformes, as the VL is directed in opposite orientation to JL and CL gene segments in both the F and G class loci of the channel cat®sh as well [10,11]. Moreover, this is further supported by the opposite orientation of the L1 VL gene segments compared to the JL±CL in the Atlantic cod [35]. A distance of 1.2 kb separating the clusters of Atlantic salmon type 3 gene segments is noticeable shorter than the intercluster distances of 2.1±10.5 kb seen in other teleost IgL loci [10,11,34,35]. Although,

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the spacers separating type 3 JL and leader gene segments are of unknown length, our data strongly suggest that the Atlantic salmon type 3 locus is more compact than the other teleost IgL loci reported so far. However, the functionality of the type 3 gene segments analysed is unknown. A compact locus with VL gene segments in opposite orientation relative to JL and CL is believed to promote intercluster recombination [35]. Examination of Atlantic salmon IgL type 1 and 3 sequences neither supported nor contradicted this hypothesis. Nevertheless, no recombination between type 1 and 3 was observed when assayed by a PCR approach (not shown). Southern blot analysis (Fig. 4) indicated that each of the three IgL loci of the Atlantic salmon contained multiple copies of related VL and CL gene segments. The exact number of gene segments within each loci was not determined. Anyway, the result is consistent with an organisation of gene segments in multiple clusters of VL±JL±CL. Indeed, sequencing of genomic DNA have shown that L1 genes of Atlantic cod [35] and both L1 and L2 genes of rainbow trout [34] are arranged in multiple clusters. There are no available data on genes homologous to the salmonid type 3 sequence. However, the inferred origin of this isotype (Fig. 3) supports an organisation in multiple clusters of these gene segments as well. Our data imply that CDR1 of the Atlantic salmon IgL display considerable structural diversity (Table 2). This was suggested by relating the size of the CDR-loops and the identity of certain SDRs to similar features de®ning the different CDR-conformations in mammals [29±32]. With the exception of His 30 and Phe 87 found in type 1.II sequences (Fig. 1), the SDRs seen in salmon IgL chains (Table 2) shared their volume and hydrophobicity with the mammalian SDRs allowed in that position. In spite of that the inferred structures of salmon CDRs may deviate from the canonical CDR-conformations seen in mammals. Nevertheless, the conserved framework of immunoglobulin sequences suggests that CDR structures primarily depend on length of the loops [26], supporting the assumption that the CDR1 in salmonid IgL isotypes and subgroups adopt individual structures. This also suggests that the novel type 3 sequences probably offer an additional CDR1 structure compared to the other IgL sequences characterised. Although different variants of CDR2 were

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identi®ed in Atlantic salmon type 2 sequences (Fig. 1) the structural diversity of this region is probably restricted (Table 2), as seen in mammalian IgL chains [26,29,32]. The variability of the CDR3 in Atlantic salmon IgL sequences is comparable to that of CDR1 but the structural diversity remains unclear. As CDR-structures in both human and mouse are VL family and clan speci®c [36], we probed for additional VL families in the salmon genome. The results obtained by Southern blot analysis under moderate stringency (Fig. 4), indicates the existence of additional VL type 2 and 3 families. In line with this observation, four VL2 families were identi®ed in rainbow trout [34], while the type 2 sequences in zebra®sh were assigned into two VL families [2]. However, it is unclear whether the additional hybridisation bands detected under moderate stringency by the VL type 3 probe (Fig. 4) represent uncharacterised type 3VL gene segments. Most of these restriction fragments hybridised to the VL1 probes at high stringency as well, and may represent VL1 gene segments. Indeed, at moderate stringency a DNA VH probe hybridised to VH sequences sharing less than 60% identical nucleotides [19]. Eventually, the type 1 and 3 VL gene segments may have co-migrated on the same restriction fragment. Northern analysis showed that full-length and spliced transcripts only of type 1 were readily detected in haematopoietic tissues (Fig. 5). A lower amount of type 2 transcripts (Fig. 6), is consistent with the 80/ 20% relationship of L1 and L2 transcripts seen in rainbow trout [34]. This relationship is probably even more biased against type 1 in salmon. In fact, we were not able to isolate full-length transcripts of type 2 at all. Also, type 3 transcripts were dominated by shorter variants (Fig. 6), while full-length messages of this isotype were detected at a low level compared to type 1 (Fig. 5). Although a variety of sterile transcripts have been detected in other teleost ®sh [8±10,17], the short type 3 transcripts are unique in that the transcription started within the CL exon (Fig. 1c). Still, the identity of the ,600 bases message of this isotype is not known, and may constitute a conventional sterile JLCL spliced germline transcript. Substitution of the hydrophilic Glu 17 to a hydrophobic Val 17 in the CL region of type 3 sequences (Fig. 1a) may partially explain the low level of full-length and spliced type 3 transcripts

detected, as two neighbouring Glu residues at this position in mammalian light chains are involved in the association with the CH1 domain [37]. Impaired association of IgH and IgL may inhibit export of immunoglobulins to the surface [38], and consequently compromise B-cell development [39] and survival [40]. As channel cat®sh antibodies directed against dinitrophenyl preferentially expressed the F isotype (90%) [41], while a relative ratio of 60/40 (F/G) was seen in sera of unimmunised ®sh [42], antigen selection of B-cell clones may have contributed to the observed bias of IgL messages in our study, but this remains to be proved.

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