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Characterisation of a fourth immunoglobulin light chain isotype in the common carp
Fish & Shellfish Immunology Fish & Shellfish Immunology 16 (2004) 369–379 www.elsevier.com/locate/fsi
Characterisation of a fourth immunoglobulin light chain isotype in the common carp Jun Ishikawa a, Etsuou Imai a, Tadaaki Moritomo b, Miki Nakao c, Tomoki Yano c, Mitsuru Tomana a* a
Department of Applied Biological Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b Department of Veterinary Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan c Laboratory of Marine Biochemistry, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan Received 6 March 2003; received in revised form 3 June 2003; accepted 26 June 2003
Abstract Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp (Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription–polymerase chain reaction (RT–PCR) method including 5#- and 3#-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5#- and 3#-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences. 2003 Elsevier Ltd. All rights reserved. Keywords: Immunoglobulin light chain; Isotype; Sterile transcript; Common carp; evolution
1. Introduction Immunoglobulins (Ig) consist of heavy (H) and light (L) chains, and exert effector functions dependent on the IgH class defined by the constant (C) domains, whereas the antigen (Ag)-binding specificity of Ig is determined by both IgH and IgL variable (V) domains. Therefore, the diversification of VL should lead to * Corresponding author E-mail address: [email protected] (M. Tomana). 1050-4648/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2003.06.002
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Fig. 1. Position of degenerate primers for the isolation of carp IgL gene sequences.
the acquisition of diverse Ag-binding specificities. Although in birds only one IgL isotype, , has been characterised [1], in most major vertebrate groups two or three isotypes within a species have been found: and in mammals [2–5]; , , and in Xenopus laevis [6–8] (amphibians); type I, II, and III in Heterodontus francisci [9–11] (elasmobranchs); and L1, L2, and L3 in zebrafish (Danio rerio) [12], Atlantic salmon (Salmo salar L.) [13], and rainbow trout (Oncorhynchus mykiss) [14] (teleosts). With respect to genomic organisation, IgL locus could be divided into three major types, denoted as the translocon (as shown in mammals) [2,5], minimalistic (birds) [1], and clustered (elasmobranchs) type [11,15]. Teleost IgL loci studied to date have a cluster-like organisation as do cartilaginous fish [13,16–22]. In teleosts, IgL isotypes characterised so far could be largely classified into four types, based on both phylogenetic analyses and the CL amino acid similarity between each isotype, defined as possessing less than 40% similarity: type L1/G represented by rainbow trout L1 [16] and channel catfish (Ictalurus punctatus) G isotypes [17]; type L2 by trout [18] and zebrafish L2 [12]; type L3/F by zebrafish L3 [12] and catfish F [19]; and Atlantic cod (Gadus morhua) L2 (Wermenstam and Pilstro¨m, unpublished, GenBank accession AJ293807). As a remarkable feature of teleost IgL, abundance of sterile transcripts has been described [16,18,20], and this type of transcript has been characterised in detail, particularly in zebrafish L2 [12]. So far it has been revealed that common carp possesses three isotypes of IgL (classified into two types according to the criterion mentioned above), L1A and L1B, both in type L1/G, and L3 in type L3/F [21,23]. Therefore, this study is aimed to identify and characterise carp L2 isotype, one of the isotypes in previously unidentified IgL types in this species. 2. Materials and methods 2.1. Isolation of cDNA clones encoding partial VL and CL regions Total RNA was extracted from pronephros of a single common carp, according to the manufacturer’s instructions, using TRIzol reagent (Invitrogen, San Diego, USA). cDNA was synthesised with reverse transcriptase (SUPERSCRIPTe II, Invitrogen) using random primers (Invitrogen) and 2 µg of total RNA as template. Degenerate primers corresponding to conserved sequences in the 3#-end of the VL FR3 (forward) and within the second half of the CL (reverse) of L2 genes found in other teleosts were used in reverse transcription PCR (RT–PCR) (Fig. 1). The sequences of the primers were: VL (DSAVYYC): GAY/WSI/GCI/GTI/TAY/TAY/TG (Y=T or C; W=A or T; S=C or G; I=inosine); and CL (EW(N/D)(Q/ K)D): RTC/IYK/RTY/CCA/YTC (R=A or G; K=G or T). Forty cycles of amplification (95 (C for 0.5 min, 52.5 (C for 0.5 min, and 72 (C for 1 min) were performed in 25 µl reaction mixture. The amplified DNA (about 330 bp in length) was gel-purified and cloned into pCR2.1 vector using a TA cloning kit (Invitrogen). Several clones encoding partial VL and CL regions were isolated, represented by clone Cph1, with which 5#-RACE was performed.
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2.2. Isolation of cDNA clones encoding complete VL or CL region using RACE method Total RNA was extracted from pooled spleens of four carp as described above. Poly(A)+RNA was purified from the total RNA using an Oligotexe-dT30^Super&mRNA Purification kit (Takara, Shiga, Japan). Amplification of mRNA sequences encoding an entire VL or CL region was accomplished using a SMARTe RACE cDNA Amplification kit (Clontech, Palo Alto, USA). Carp spleen, but not pronephros, was used here for isolation of IgL cDNA clones with the RACE method, because RACE cDNA clones of the other carp IgL isotypes have been successfully isolated from spleen [21,23]. Gene specific primers corresponding to the sequences in the CL region of the pronephric clone Cph1 and the VL of the splenic clone Cpi1 were employed for 5#- or 3#-RACE, respectively. The sequences of the primers were: CL (NHMSGAFADV): C/ATC/AGC/AAA/AGC/CCC/AGA/CAT/GTG/A; or VL (TWDSSAKEYV): ACA/ TGG/GAC/AGC/TCT/GCT/AAA/GAG/TAC/G. RACE was performed according to the manufacturer’s instructions. The PCR products (550–700 bp in length in 5#-RACE, 650–850 bp in 3#-RACE) were gel-purified and cloned into pCR2.1-TOPO vector (Invitrogen). 2.3. DNA sequencing and sequence analysis The PCR products were sequenced in both strands by the dideoxy method [24]. The deduced amino acid sequences were analysed using the tfasta program [25] against the DDBJ sequence database. Multiple alignments were made using the clustal W program [26]. A phylogenetic tree was constructed by the neighbour-joining method [27]. The reliability of tree topologies was evaluated by the bootstrap test (1000 replications) [28], in which probability values were computed for each interior branch of the tree. The computer program TREEVIEW [29] was used to display the tree. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers: AB091110 (Cph1), AB091112 (Cpi1), AB091113 (Cpi2), AB091115 (Cpi4), AB091117 (Cpi6), AB103559–AB103564 (Cpi7–Cpi12, respectively), AB091118–AB091120 (Cpj1–Cpj3, respectively), and AB103558 (Cpj4). 2.4. Southern blot analysis Carp erythrocyte DNA was extracted [30] and digested with EcoRI or HindIII (Takara). The DNA (10 µg/lane) was separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, Uppsala, Sweden) by blotting in 0.4 M NaOH. The filter was fixed by UV cross-linking, prehybridised, and hybridised with 32P-labelled random-primed probes in a medium containing 0.5 M NaH2PO4/NaHPO4 (pH 7.2), 1 mM ethylenediaminetetraacetate (EDTA), and 7% (w/v) SDS at 65 (C overnight. Probes specific for the CL region of clone Cph1 and the VL of Cpi1 or Cpi11 were used. The filter was washed in 40 mM NaH2PO4/NaHPO4 and 1% SDS at 65 (C for 10 min, and the radioactivity was detected with a phosphoimager (Molecular Dynamics, Sunnyvale, USA). 3. Results 3.1. Isolation of carp L2 cDNA clones First, carp cDNA clones were obtained from total RNA isolated from carp pronephros by RT–PCR using primers, one in the 3#-end of the VL framework region (FR) 3 and another within the second half of the CL region of L2 genes identified in other teleosts. The PCR products of about 330 bp in length were then cloned and sequenced. Several clones examined contained partial CL region sequences, represented by clone Cph1, with 82% similarity to L2 CL region of zebrafish [12] (data not shown). Next, to obtain the full length VL region, 5#-RACE was employed for poly(A)+RNA from carp spleen, using the primer within the first half of the CL region in clone Cph1. This yielded 9 VL sequences, which
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Fig. 2. Nucleotide and deduced amino acid sequences of carp IgL chain cDNA clones Cpi1, Cpi2, and Cpi6. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen, except for a region upstream of FR4 in clone Cpi6. Leader peptide, putative FR/CDR, and CL regions, defined based on sequence comparisons with other teleost L2, are indicated above sequences. Recombination signal sequences (RSS), nonamer (9-mer) and heptamer (7-mer), in clone Cpi6 are boxed. The regions corresponding to primer sequences used to obtain clone Cph1 and entire CL-encoding clones, 3#-RACE products, are underlined in clones Cpi1 and Cpi2, respectively.
could be grouped into two distinct VL families, designated VL2-1 and VL2-2, sharing less than 70% homology at the amino acid level (Fig. 2). Four and five sequences were identified within families VL2-2 and VL2-1, respectively, and the amino acid sequence similarity within each family ranged from 94 to 99% (VL2-1) and 79 to 98% (VL2-2) (Fig. 3). As shown in Fig. 3, when compared between the two VL families, the VL sequences showed marked differences in each FR and complementarity-determining region (CDR) except for FR4, particularly in CDR1. The sequences of these clones well matched the clone Cph1 (data not shown). When the amino acid sequences of carp VL segments were compared with sequences in the DDBJ databank using the tfasta comparison program, they were most similar to type L2 sequences in trout or zebrafish: clones Cpi1 (VL2-1) and Cpi2 (VL2-2) shared 68% similarity with trout VL2 sequence, and 78% with zebrafish VL2, respectively. But they did not show such a high similarity to any other IgL isotype in carp reported to date, L1A, L1B, and L3 [21,23] (data not shown). In the course of 5#-RACE, those amplification products represented by clone Cpi6 were obtained which contain a JL recombination signal sequence (RSS) comprised of a nonamer (GGTTTTTGT), a 23-bp spacer, and a heptamer (CACTGTG), followed by a JL segment (FR4) appropriately spliced to a CL segment, thus these products representing IgL sterile transcripts (Fig. 2). The JL RSS was well conserved within this type of clone obtained in this study (data not shown). As shown in Fig. 2, both the JL and CL sequences of Cpi6 were highly similar with Cpi1 and Cpi2 sequences. As for JL segments analysed, corresponding to FR4, sequence variations appear to be confined to a few positions (Fig. 3). To obtain the full-length CL region, 3#-RACE was subsequently employed using the sense primer based on the 5#-RACE products. Four clones with the complete CL region were then obtained, and the entire
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Fig. 3. Multiple alignment of the amino acid sequences of the carp L2 VL region. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen. Putative FR/CDR regions are indicated above sequences. Cysteine residues involved in intradomain interaction are indicated by *. Percentages of FR1-CDR3 amino acid similarity between clone Cpi7 and the others, and clone Cpi11 and the others are indicated.
nucleotide and amino acid sequences are shown in Fig. 4. The CL sequences of clones Cpj2, Cpj3, and Cpj4 were highly similar to each other, but only Cpj1 CL were slightly different from the others, as well as the 3#-untranslated region. The CL regions contained characteristic three cysteines (Cys-28, Cys-87, and Cys-105), probably forming an intradomain disulphide bridge (Cys-28 and Cys-87) or an interchain bridge (Cys-105). The J and partial CL amino acid sequences described above, represented by clones Cph1 and Cpi1, well matched these 3#-RACE clones (data not shown). In clone Cpj3, the following intergenic sequences (174 bp in the total length) were identified within the region 5# of the JC: a putative VL RSS (28 bp) with a 12-bp spacer; 107 nucleotides of an inter-RSS sequence; and a JL RSS (39 bp) which is identical to that of clone Cpi6 representing a sterile transcript. This intergenic sequence except for the JL RSS, however, did not show any significant similarity to the corresponding region in Cpi6, upstream of the JC. Thus, the clone Cpj3 is regarded as another representative of sterile transcripts. Next, the CL amino acid sequence of clone Cpj1 was compared with sequences in the DDBJ databank using the tfasta comparison programme. Cpj1 was most similar to zebrafish L2 (80% similarity), and so considered as carp L2 isotype (Fig. 5). But the amino acid sequence similarity between Cpj1 and representatives of the other carp IgL isotypes, Cpb4 (L1A isotype, AB015905), Cpc2 (L1B, AB035729), or Cpd1 (L3, AB035730) were as low as 33%, 29%, or 24%, respectively (Fig. 5). For comparison of carp L2 sequence with type L2 in other teleosts, they were aligned using clustal W (Fig. 5), showing a close relationship between carp and zebrafish L2. Genomic blots were hybridised with probes specific for the carp CL2, VL2-1, and VL2-2 sequences (Fig. 6). Hybridisation patterns observed with them were similar to each other, particularly between the CL2 and VL2-1, suggesting that the locus of L2 has a cluster-like organisation.
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Fig. 4. Nucleotide and deduced amino acid sequences of the JL and CL regions in carp cDNA clones Cpj1–4. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen. JL and CL regions are indicated above sequences. The stop codon are indicated by *. The polyadenylation signals are in bold. Recombination signal sequences (RSS), nonamer (9-mer) and heptamer (7-mer), in clone Cpj3 are boxed. The regions corresponding to primer sequences used to obtain clone Cph1 and entire VL-encoding clones, 5#-RACE products, are underlined in clones Cpj3 and Cpj2, respectively. It should be noted that at the 5#-end of JL in clones Cpj1, -2, and -4, a putative first codon is missing because it is 3#-end of the primer sequence used.
3.2. Phylogenetic analysis To clarify the relationships among carp L2 and other fish IgL isotypes, phylogenetic trees were constructed from their amino acid sequences based on the neighbor-joining method (Fig. 7). The results showed that carp L2 CL sequence tightly clustered with type L2 of other teleost species, including zebrafish, trout, salmon, and Japanese flounder (Paralichthys olivaceus) (Fig. 7a). The VL phylogenetic tree, using only FRs 1, 2, and 3 sequences, demonstrated that carp L2 also highly clustered with other teleost L2, in addition, with horned shark and little skate type 1. It should be noted that carp VL2-1 and VL2-2 sequences clustered with representatives of the two VL families in zebrafish (Fig. 7b).
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Fig. 5. Multiple alignment of the CL amino acid sequences of the carp and other teleost species L2, and the carp other isotypes. The same residues as the carp sequence shown at the top are denoted by dots, and gaps introduced to maximise homology are indicated by hyphens. Characteristic cysteine residues are numbered. Percentages of amino acid similarity between clone Cpj1 and others are indicated. Accession numbers are: zebrafish L2 (AF246162); trout L2 (U69987); salmon L2 (AF297518); flounder (AU090352); carp L1A (AB015905), L1B (AB035729), and L3 (AB035730).
Fig. 6. Southern blot analyses of carp genomic DNA hybridised with the VL2-1, VL2-2, and CL2 specific probes. The restriction enzymes used are EcoRI (E) and HindIII (H), and the position of size markers (in kb) are shown on the left-hand side.
4. Discussion To date, type L2 IgL chain genes in teleost species have been identified in rainbow trout [18], zebrafish [12], Atlantic salmon [13], and Japanese flounder [31]. It has been reported that carp has three isotypes of
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IgL, L1A, L1B, and L3 [21,23], and L2 isotype was identified in current study, demonstrating that carp possesses at least four IgL isotypes. According to the comparison of the carp L2 CL amino acid sequence with sequences in DDBJ database, it was most similar to L2 in zebrafish, a species phylogenetically close to carp (both species belonging to a Cyprinidae family), followed by trout and salmon L2 (Fig. 5), in agreement with the result of CL phylogenetic analysis (Fig. 7a). During both 5#- and 3#-RACE, those sterile transcripts were identified which contain intergenic sequences 5# of JL, and in zebrafish an excessive number of such L2 sterile transcripts has been reported in detail as one of unusual features in bony fish IgL, compared with low expression of sterile transcripts in other taxa [12]. For example, one of the zebrafish sterile transcripts contained a VL segment followed by 517 nucleotides of intergenic sequence including VL- and JL-RSS at the 5#- and 3#-ends, respectively, and joined JC. Accordingly, clone Cpj3 with a putative VL-RSS might be derived from such a VL-possessing sterile transcripts, but in fact, the sequence began downstream of primer sequence in the 3#-end of VL CDR3 (Fig. 4). On the other hand, another clone Cpi6 was derived from sterile JC transcripts, but this type of L2 clone has not yet been reported in zebrafish. When compared with the nucleotide length of the zebrafish clone intergenic sequence (517 bp), that of Cpj3 (174 bp) was much shorter, suggesting that a VL2 segment is located very close to JL in the carp genome, thus likely close to CL2 as well. This assumption was supported by the Southern blot result, showing that at least VL2-1 and CL region are adjacent to one another, judging from the nearly identical hybridisation patterns observed (Fig. 6). The two VL families, represented by Cpi1 and Cpi2 VL, were identified in carp (Fig. 2), and could be assigned to the two VL families in zebrafish by the phylogenetic study (Fig. 7b). On the other hand, trout L2 has been reported to possess four VL families to date [20], although more VL families in carp and zebrafish may be found in future. As for VL families of the other IgL isotypes in carp, it is suggested that L1A, L1B, and L3 hold at least three, two, and two families, respectively, with the same degree as L2 [21,23]. In the previous work, some VL sequences derived from carp L1A and L3 were highly similar to each other [21], but such a close relationship could not be found between carp L2 and any other isotype (data not shown), consistent with the VL phylogenetic result demonstrating close relationships among type L2 sequences from various species including carp, segregated from the other types such as L1/G or L3/F (Fig. 7b). Thus, the VL sequences identified here should add further diversity to a carp IgL repertoire. Southern blot analyses using VL- and CL-specific probes suggested that the genomic organisation of carp L2 locus is a cluster-like (Fig. 6) as well as trout [18] and salmon [13]. With respect to other isotypes of carp IgL, it has been suggested that L1B has a cluster-like organisation as well, while those of L3 and L1A remain unclear [21]. Until now three isotypes of IgL chain within a species have been characterised in detail in X. laevis [6–8], H. francisci [9–11], and three teleost species, D. rerio [12], S. salar L [13], and O. mykiss [14]. In this
Fig. 7. Neighbor-joining trees of CL (a) and VL (b) amino acid sequences from various fish species. Only FR1, FR2, and FR3 were used in (b). The bootstrap values from a 1000 replications are indicated at the nodes, but only the values >500 are shown. Genetic distances are given under each tree. Accession numbers in (a) are: Acipenser (X90557); Hydrolagus (L25551); Heterodontus I (M64307), II (L25559), and III (L25561); Raja 1 (L25568) and 2 (L25566); Gynglymostoma (L16765); Gadus L1 (X68515) and L2 (GMO293807); Ictalurus G (L25531) and F (U25705); Cyprinus L1A (AB015905), L1B (AB035729), L2 (clone Cpj2, AB091119), and L3 (AB035730); Danio L1 (AF246185), L2 (AF246162), and L3 (AF246193); Paralichthys (AU090352); Oncorhynchus L1 (X68521) and L2 (U69987); Salmo L1 (AF273017), L2 (AF297518), and L3 (AF406956); Dicentrarchus (DLA400216); Anarhichas L1 (AF137397) and L2 (AF137398); Seriola (1) (AB062664), (2) (AB062653), and (3) (AB062667). Accession numbers in (b) are: Heterodontus I (HFL141) and III (HEFIGCVE); Raja 1 (SKTIGCVE) and 2 (SKTIGCVC); Oncorhynchus L1 (X68517), L2 (1) (OMU69987), L2 (2) and L2 (3) (OMY251647), and L2 (4) (OMY251650); Cyprinus L1A (AB073328), L1B (AB073331), L2 (1) (clone Cpi1, AB091112), L2 (2) (clone Cpi2, AB091113), and L3 (AB073342); Danio L1 (AF246185), L2 (1) (AF246162), L2 (2) (AF246179), and L3 (AF246189); Carcharhinus (CPLIGL); Gadus L1 (X76517) and L2 (GMO293807); Seriola (1) (AB062650), (2) (AB062653), and (3) (AB062647); Hydrolagus (HDLIGCVC); Dicentrarchus (DLA400216); Anarhichas L1 (AF137397) and L2 (AF137398); Acipenser (ABIGLCST5); Ictalurus G (ICTIGLSTD) and F (IPU25705); Salmo L1 (AF273017) and L3 (AF406956); Gynglymostoma (GINIGL).
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investigation it was demonstrated that carp has at least four IgL isotypes classified into three types, L1A and L1B in type L1/G, L3 in type L3/F, and L2 in type L2, and thus this considerable diversity of IgL as seen in carp is likely to contribute to the complexity of the carp Ig molecule as well as IgH [32].
Acknowledgement This work was supported by a grant from the Ministry of Education, Science, Sports and Culture to promote advanced scientific research.
References [1] Raynaud CA, Dahan A, Weill JC. Complete sequence of a chicken light chain immunoglobulin derived from the nucleotide sequence of its mRNA. Proceedings of National Academy of Science USA 1983;80:4099–103. [2] Zachau HG. Immunoglobulin light-chain genes of the type in man and mouse. In: Honjo T, Alt FW, Rabbitts TH, editors. Immunoglobulin genes. London: Academic Press; 1989, pp. 91–109. [3] Bauser TR Jr, Blomberg B. The human L chain Ig locus. Characterization of JCl6 and identification of functional JCl7. Journal of Immunology 1991;146:2813–20. [4] Home WA, Ford JE, Gibson DM. L chain isotype regulation in horse. Journal of Immunology 1992;149:3927–36. [5] Kirschbaum T, Jaenichen R, Zachau HG. The mouse immunoglobulin locus contains about 140 variable gene segments. European Journal of Immunology 1996;26:1613–20. [6] Schwager J, Burckert N, Schwager M, Wilson M. Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity. EMBO Journal 1991;10:505–11. [7] Zezza DJ, Mikoryak CA, Schwager J, Steiner LA. Sequence of C region of L chains from Xenopus laevis Ig. Journal of Immunology 1991;146:4041–7. [8] Haire RN, Ota T, Rast JP, Litman RT, Chan FY, Zon LI et al. A third light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian genes. Journal of Immunology 1996;157:1544–50. [9] Shamblott MJ, Litman GW. Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes. Proceedings of National Academy of Science USA 1989;86:4684–8. [10] Shamblott MJ, Litman GW. Genomoic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization. EMBO Journal 1989;8:3733–9. [11] Rast JP, Anderson MK, Ota T, Litman RT, Margittai M, Shamblott MJ et al. Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny. Immunogenetics 1994;40:83–99. [12] Haire RN, Rast JP, Litman RT, Litman GW. Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish. Immunogenetics 2000;51:915–23. [13] Solem ST, Jorgensen T. Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes. Developmental and Comparative Immunology 2002;26:635–47. [14] Timmusk S, Stromberg S, Pilstrom L. Light chain promoter regions in rainbow trout Oncorhynchus mykiss: function of a classical and an atypical Ig promoter. Developmental and Comparative Immunology 2002;26:785–96. [15] Hohman VS, Schluter SF, Marchalonis JJ. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: Gene organization and implications for the evolution of light chains. Proceedings of National Academy of Science USA 1992;89:276–80. [16] Daggfeldt A, Bengte´n E, Pilstro¨m L. A cluster type organization of the loci of the immunoglobulin light chain in Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss Walbaum) indicated by nucleotide sequences of cDNAs and hybridization analysis. Immunogenetics 1993;38:199–209. [17] Ghaffari S, Lobb CJ. Structure and genomic organization of immunoglobulin light chain in the channel catfish. An unusual genomic organizational pattern of segmental genes. Journal of Immunology 1993;151:6900–12. [18] Partula S, Schwager J, Timmusk S, Pilstro¨m L, Charlemagne J. A second immunoglobulin light chain isotype in the rainbow trout. Immunogenetics 1996;45:44–51. [19] Ghaffari S, Lobb CJ. Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish. Journal of Immunology 1997;159:250–8. [20] Timmusk S, Partula S, Pilstro¨m L. Different genomic organization and expression of immunoglobulin light-chain isotypes in the rainbow trout. Immunogenetics 2000;51:905–14.
J. Ishikawa et al. / Fish & Shellfish Immunology 16 (2004) 369–379
379
[21] Tomana M, Ishikawa J, Imai E, Moritomo T, Nakao M, Yano T. Characterization of immunoglobulin light chain isotypes in the common carp. Immunogenetics 2002;54:120–9. [22] Okamoto K, Ikemura H, Savan R, Sakai M. Cloning, sequence and variability analysis of expressed immunoglobulin light chain genes from yellowtail Seriola quinqueradiata. Fish & Shellfish Immunology 2003;14:55–70. [23] Tomana M, Nakao M, Moritomo T, Fujiki K, Yano T. Isolation of cDNA encoding immunoglobulin light chain from common carp (Cyprinus carpio L.). Fish & Shellfish Immunology 1999;9:71–80. [24] Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proceedings of National Academy of Science USA 1977;74:5463–7. [25] Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proceedings of National Academy of Science USA 1988;85:2444–8. [26] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 1994;22:4673–80. [27] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 1987;4:406–25. [28] Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783–91. [29] Page RDM. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 1996;12:357–8. [30] Sambrook J, Frisch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd edn Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. [31] Kono T, Sakai M. The analysis of expressed genes in the kidney of Japanese flounder, Paralichthys olivaceus, injected with the immunostimulant peptidoglycan. Fish & Shellfish Immunology 2001;11:357–66. [32] Nakao M, Moritomo T, Tomana M, Fujiki K, Yano T. Isolation of cDNA encoding constant region of the immunoglobulin heavy-chain from common carp (Cyprinus carpio L.). Fish & Shellfish Immunology 1998;8:425–34.
Characterisation of a fourth immunoglobulin light chain isotype in the common carp Jun Ishikawa a, Etsuou Imai a, Tadaaki Moritomo b, Miki Nakao c, Tomoki Yano c, Mitsuru Tomana a* a
Department of Applied Biological Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan b Department of Veterinary Science, Nihon University, 1866 Kameino, Fujisawa, Kanagawa 252-8510, Japan c Laboratory of Marine Biochemistry, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, Hakozaki, Fukuoka 812-8581, Japan Received 6 March 2003; received in revised form 3 June 2003; accepted 26 June 2003
Abstract Three isotypes of immunoglobulin (Ig) light (L) chain, designated L1A, L1B, and L3, have been characterised in the common carp (Cyprinus carpio L.) to date. In this paper the molecular cloning of a fourth IgL isotype in carp, designated L2, is described. A reverse transcription–polymerase chain reaction (RT–PCR) method including 5#- and 3#-RACE was used to isolate carp L2 cDNA clones. The VL sequences could be divided into two distinct VL families, designated VL2-1 and VL2-2, most similar to rainbow trout (68% similarity) and zebrafish (78%) VL2 amino acid sequences, respectively. The CL amino acid sequences showed the highest similarity to zebrafish L2 (80%), and contained the characteristic cysteines necessary for intradomain or interchain disulphide bridges as did the VL sequences. Neither the VL nor CL sequences demonstrated such a high similarity to the other carp IgL isotypes, L1A, L1B, and L3. For JL segments, sequence variations appeared to be confined to a few positions. In the course of 5#- and 3#-RACE, cDNA clones containing recombination signal sequence (RSS), representatives of IgL sterile transcripts, were obtained. Southern blot analyses suggested that the locus of carp L2 has a cluster-like organisation. Phylogenetic analyses showed that both carp VL2 and CL2 amino acid sequences highly clustered with other teleost L2 sequences. 2003 Elsevier Ltd. All rights reserved. Keywords: Immunoglobulin light chain; Isotype; Sterile transcript; Common carp; evolution
1. Introduction Immunoglobulins (Ig) consist of heavy (H) and light (L) chains, and exert effector functions dependent on the IgH class defined by the constant (C) domains, whereas the antigen (Ag)-binding specificity of Ig is determined by both IgH and IgL variable (V) domains. Therefore, the diversification of VL should lead to * Corresponding author E-mail address: [email protected] (M. Tomana). 1050-4648/04/$ - see front matter 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2003.06.002
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Fig. 1. Position of degenerate primers for the isolation of carp IgL gene sequences.
the acquisition of diverse Ag-binding specificities. Although in birds only one IgL isotype, , has been characterised [1], in most major vertebrate groups two or three isotypes within a species have been found: and in mammals [2–5]; , , and in Xenopus laevis [6–8] (amphibians); type I, II, and III in Heterodontus francisci [9–11] (elasmobranchs); and L1, L2, and L3 in zebrafish (Danio rerio) [12], Atlantic salmon (Salmo salar L.) [13], and rainbow trout (Oncorhynchus mykiss) [14] (teleosts). With respect to genomic organisation, IgL locus could be divided into three major types, denoted as the translocon (as shown in mammals) [2,5], minimalistic (birds) [1], and clustered (elasmobranchs) type [11,15]. Teleost IgL loci studied to date have a cluster-like organisation as do cartilaginous fish [13,16–22]. In teleosts, IgL isotypes characterised so far could be largely classified into four types, based on both phylogenetic analyses and the CL amino acid similarity between each isotype, defined as possessing less than 40% similarity: type L1/G represented by rainbow trout L1 [16] and channel catfish (Ictalurus punctatus) G isotypes [17]; type L2 by trout [18] and zebrafish L2 [12]; type L3/F by zebrafish L3 [12] and catfish F [19]; and Atlantic cod (Gadus morhua) L2 (Wermenstam and Pilstro¨m, unpublished, GenBank accession AJ293807). As a remarkable feature of teleost IgL, abundance of sterile transcripts has been described [16,18,20], and this type of transcript has been characterised in detail, particularly in zebrafish L2 [12]. So far it has been revealed that common carp possesses three isotypes of IgL (classified into two types according to the criterion mentioned above), L1A and L1B, both in type L1/G, and L3 in type L3/F [21,23]. Therefore, this study is aimed to identify and characterise carp L2 isotype, one of the isotypes in previously unidentified IgL types in this species. 2. Materials and methods 2.1. Isolation of cDNA clones encoding partial VL and CL regions Total RNA was extracted from pronephros of a single common carp, according to the manufacturer’s instructions, using TRIzol reagent (Invitrogen, San Diego, USA). cDNA was synthesised with reverse transcriptase (SUPERSCRIPTe II, Invitrogen) using random primers (Invitrogen) and 2 µg of total RNA as template. Degenerate primers corresponding to conserved sequences in the 3#-end of the VL FR3 (forward) and within the second half of the CL (reverse) of L2 genes found in other teleosts were used in reverse transcription PCR (RT–PCR) (Fig. 1). The sequences of the primers were: VL (DSAVYYC): GAY/WSI/GCI/GTI/TAY/TAY/TG (Y=T or C; W=A or T; S=C or G; I=inosine); and CL (EW(N/D)(Q/ K)D): RTC/IYK/RTY/CCA/YTC (R=A or G; K=G or T). Forty cycles of amplification (95 (C for 0.5 min, 52.5 (C for 0.5 min, and 72 (C for 1 min) were performed in 25 µl reaction mixture. The amplified DNA (about 330 bp in length) was gel-purified and cloned into pCR2.1 vector using a TA cloning kit (Invitrogen). Several clones encoding partial VL and CL regions were isolated, represented by clone Cph1, with which 5#-RACE was performed.
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2.2. Isolation of cDNA clones encoding complete VL or CL region using RACE method Total RNA was extracted from pooled spleens of four carp as described above. Poly(A)+RNA was purified from the total RNA using an Oligotexe-dT30^Super&mRNA Purification kit (Takara, Shiga, Japan). Amplification of mRNA sequences encoding an entire VL or CL region was accomplished using a SMARTe RACE cDNA Amplification kit (Clontech, Palo Alto, USA). Carp spleen, but not pronephros, was used here for isolation of IgL cDNA clones with the RACE method, because RACE cDNA clones of the other carp IgL isotypes have been successfully isolated from spleen [21,23]. Gene specific primers corresponding to the sequences in the CL region of the pronephric clone Cph1 and the VL of the splenic clone Cpi1 were employed for 5#- or 3#-RACE, respectively. The sequences of the primers were: CL (NHMSGAFADV): C/ATC/AGC/AAA/AGC/CCC/AGA/CAT/GTG/A; or VL (TWDSSAKEYV): ACA/ TGG/GAC/AGC/TCT/GCT/AAA/GAG/TAC/G. RACE was performed according to the manufacturer’s instructions. The PCR products (550–700 bp in length in 5#-RACE, 650–850 bp in 3#-RACE) were gel-purified and cloned into pCR2.1-TOPO vector (Invitrogen). 2.3. DNA sequencing and sequence analysis The PCR products were sequenced in both strands by the dideoxy method [24]. The deduced amino acid sequences were analysed using the tfasta program [25] against the DDBJ sequence database. Multiple alignments were made using the clustal W program [26]. A phylogenetic tree was constructed by the neighbour-joining method [27]. The reliability of tree topologies was evaluated by the bootstrap test (1000 replications) [28], in which probability values were computed for each interior branch of the tree. The computer program TREEVIEW [29] was used to display the tree. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank nucleotide sequence databases and have been assigned the accession numbers: AB091110 (Cph1), AB091112 (Cpi1), AB091113 (Cpi2), AB091115 (Cpi4), AB091117 (Cpi6), AB103559–AB103564 (Cpi7–Cpi12, respectively), AB091118–AB091120 (Cpj1–Cpj3, respectively), and AB103558 (Cpj4). 2.4. Southern blot analysis Carp erythrocyte DNA was extracted [30] and digested with EcoRI or HindIII (Takara). The DNA (10 µg/lane) was separated on a 0.8% agarose gel and transferred to a nylon membrane (Hybond-N+, Amersham Pharmacia Biotech, Uppsala, Sweden) by blotting in 0.4 M NaOH. The filter was fixed by UV cross-linking, prehybridised, and hybridised with 32P-labelled random-primed probes in a medium containing 0.5 M NaH2PO4/NaHPO4 (pH 7.2), 1 mM ethylenediaminetetraacetate (EDTA), and 7% (w/v) SDS at 65 (C overnight. Probes specific for the CL region of clone Cph1 and the VL of Cpi1 or Cpi11 were used. The filter was washed in 40 mM NaH2PO4/NaHPO4 and 1% SDS at 65 (C for 10 min, and the radioactivity was detected with a phosphoimager (Molecular Dynamics, Sunnyvale, USA). 3. Results 3.1. Isolation of carp L2 cDNA clones First, carp cDNA clones were obtained from total RNA isolated from carp pronephros by RT–PCR using primers, one in the 3#-end of the VL framework region (FR) 3 and another within the second half of the CL region of L2 genes identified in other teleosts. The PCR products of about 330 bp in length were then cloned and sequenced. Several clones examined contained partial CL region sequences, represented by clone Cph1, with 82% similarity to L2 CL region of zebrafish [12] (data not shown). Next, to obtain the full length VL region, 5#-RACE was employed for poly(A)+RNA from carp spleen, using the primer within the first half of the CL region in clone Cph1. This yielded 9 VL sequences, which
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Fig. 2. Nucleotide and deduced amino acid sequences of carp IgL chain cDNA clones Cpi1, Cpi2, and Cpi6. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen, except for a region upstream of FR4 in clone Cpi6. Leader peptide, putative FR/CDR, and CL regions, defined based on sequence comparisons with other teleost L2, are indicated above sequences. Recombination signal sequences (RSS), nonamer (9-mer) and heptamer (7-mer), in clone Cpi6 are boxed. The regions corresponding to primer sequences used to obtain clone Cph1 and entire CL-encoding clones, 3#-RACE products, are underlined in clones Cpi1 and Cpi2, respectively.
could be grouped into two distinct VL families, designated VL2-1 and VL2-2, sharing less than 70% homology at the amino acid level (Fig. 2). Four and five sequences were identified within families VL2-2 and VL2-1, respectively, and the amino acid sequence similarity within each family ranged from 94 to 99% (VL2-1) and 79 to 98% (VL2-2) (Fig. 3). As shown in Fig. 3, when compared between the two VL families, the VL sequences showed marked differences in each FR and complementarity-determining region (CDR) except for FR4, particularly in CDR1. The sequences of these clones well matched the clone Cph1 (data not shown). When the amino acid sequences of carp VL segments were compared with sequences in the DDBJ databank using the tfasta comparison program, they were most similar to type L2 sequences in trout or zebrafish: clones Cpi1 (VL2-1) and Cpi2 (VL2-2) shared 68% similarity with trout VL2 sequence, and 78% with zebrafish VL2, respectively. But they did not show such a high similarity to any other IgL isotype in carp reported to date, L1A, L1B, and L3 [21,23] (data not shown). In the course of 5#-RACE, those amplification products represented by clone Cpi6 were obtained which contain a JL recombination signal sequence (RSS) comprised of a nonamer (GGTTTTTGT), a 23-bp spacer, and a heptamer (CACTGTG), followed by a JL segment (FR4) appropriately spliced to a CL segment, thus these products representing IgL sterile transcripts (Fig. 2). The JL RSS was well conserved within this type of clone obtained in this study (data not shown). As shown in Fig. 2, both the JL and CL sequences of Cpi6 were highly similar with Cpi1 and Cpi2 sequences. As for JL segments analysed, corresponding to FR4, sequence variations appear to be confined to a few positions (Fig. 3). To obtain the full-length CL region, 3#-RACE was subsequently employed using the sense primer based on the 5#-RACE products. Four clones with the complete CL region were then obtained, and the entire
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Fig. 3. Multiple alignment of the amino acid sequences of the carp L2 VL region. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen. Putative FR/CDR regions are indicated above sequences. Cysteine residues involved in intradomain interaction are indicated by *. Percentages of FR1-CDR3 amino acid similarity between clone Cpi7 and the others, and clone Cpi11 and the others are indicated.
nucleotide and amino acid sequences are shown in Fig. 4. The CL sequences of clones Cpj2, Cpj3, and Cpj4 were highly similar to each other, but only Cpj1 CL were slightly different from the others, as well as the 3#-untranslated region. The CL regions contained characteristic three cysteines (Cys-28, Cys-87, and Cys-105), probably forming an intradomain disulphide bridge (Cys-28 and Cys-87) or an interchain bridge (Cys-105). The J and partial CL amino acid sequences described above, represented by clones Cph1 and Cpi1, well matched these 3#-RACE clones (data not shown). In clone Cpj3, the following intergenic sequences (174 bp in the total length) were identified within the region 5# of the JC: a putative VL RSS (28 bp) with a 12-bp spacer; 107 nucleotides of an inter-RSS sequence; and a JL RSS (39 bp) which is identical to that of clone Cpi6 representing a sterile transcript. This intergenic sequence except for the JL RSS, however, did not show any significant similarity to the corresponding region in Cpi6, upstream of the JC. Thus, the clone Cpj3 is regarded as another representative of sterile transcripts. Next, the CL amino acid sequence of clone Cpj1 was compared with sequences in the DDBJ databank using the tfasta comparison programme. Cpj1 was most similar to zebrafish L2 (80% similarity), and so considered as carp L2 isotype (Fig. 5). But the amino acid sequence similarity between Cpj1 and representatives of the other carp IgL isotypes, Cpb4 (L1A isotype, AB015905), Cpc2 (L1B, AB035729), or Cpd1 (L3, AB035730) were as low as 33%, 29%, or 24%, respectively (Fig. 5). For comparison of carp L2 sequence with type L2 in other teleosts, they were aligned using clustal W (Fig. 5), showing a close relationship between carp and zebrafish L2. Genomic blots were hybridised with probes specific for the carp CL2, VL2-1, and VL2-2 sequences (Fig. 6). Hybridisation patterns observed with them were similar to each other, particularly between the CL2 and VL2-1, suggesting that the locus of L2 has a cluster-like organisation.
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Fig. 4. Nucleotide and deduced amino acid sequences of the JL and CL regions in carp cDNA clones Cpj1–4. The same residues as the sequence shown at the top are denoted by dots, and a gap introduced to maximise homology is indicated by a hyphen. JL and CL regions are indicated above sequences. The stop codon are indicated by *. The polyadenylation signals are in bold. Recombination signal sequences (RSS), nonamer (9-mer) and heptamer (7-mer), in clone Cpj3 are boxed. The regions corresponding to primer sequences used to obtain clone Cph1 and entire VL-encoding clones, 5#-RACE products, are underlined in clones Cpj3 and Cpj2, respectively. It should be noted that at the 5#-end of JL in clones Cpj1, -2, and -4, a putative first codon is missing because it is 3#-end of the primer sequence used.
3.2. Phylogenetic analysis To clarify the relationships among carp L2 and other fish IgL isotypes, phylogenetic trees were constructed from their amino acid sequences based on the neighbor-joining method (Fig. 7). The results showed that carp L2 CL sequence tightly clustered with type L2 of other teleost species, including zebrafish, trout, salmon, and Japanese flounder (Paralichthys olivaceus) (Fig. 7a). The VL phylogenetic tree, using only FRs 1, 2, and 3 sequences, demonstrated that carp L2 also highly clustered with other teleost L2, in addition, with horned shark and little skate type 1. It should be noted that carp VL2-1 and VL2-2 sequences clustered with representatives of the two VL families in zebrafish (Fig. 7b).
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Fig. 5. Multiple alignment of the CL amino acid sequences of the carp and other teleost species L2, and the carp other isotypes. The same residues as the carp sequence shown at the top are denoted by dots, and gaps introduced to maximise homology are indicated by hyphens. Characteristic cysteine residues are numbered. Percentages of amino acid similarity between clone Cpj1 and others are indicated. Accession numbers are: zebrafish L2 (AF246162); trout L2 (U69987); salmon L2 (AF297518); flounder (AU090352); carp L1A (AB015905), L1B (AB035729), and L3 (AB035730).
Fig. 6. Southern blot analyses of carp genomic DNA hybridised with the VL2-1, VL2-2, and CL2 specific probes. The restriction enzymes used are EcoRI (E) and HindIII (H), and the position of size markers (in kb) are shown on the left-hand side.
4. Discussion To date, type L2 IgL chain genes in teleost species have been identified in rainbow trout [18], zebrafish [12], Atlantic salmon [13], and Japanese flounder [31]. It has been reported that carp has three isotypes of
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IgL, L1A, L1B, and L3 [21,23], and L2 isotype was identified in current study, demonstrating that carp possesses at least four IgL isotypes. According to the comparison of the carp L2 CL amino acid sequence with sequences in DDBJ database, it was most similar to L2 in zebrafish, a species phylogenetically close to carp (both species belonging to a Cyprinidae family), followed by trout and salmon L2 (Fig. 5), in agreement with the result of CL phylogenetic analysis (Fig. 7a). During both 5#- and 3#-RACE, those sterile transcripts were identified which contain intergenic sequences 5# of JL, and in zebrafish an excessive number of such L2 sterile transcripts has been reported in detail as one of unusual features in bony fish IgL, compared with low expression of sterile transcripts in other taxa [12]. For example, one of the zebrafish sterile transcripts contained a VL segment followed by 517 nucleotides of intergenic sequence including VL- and JL-RSS at the 5#- and 3#-ends, respectively, and joined JC. Accordingly, clone Cpj3 with a putative VL-RSS might be derived from such a VL-possessing sterile transcripts, but in fact, the sequence began downstream of primer sequence in the 3#-end of VL CDR3 (Fig. 4). On the other hand, another clone Cpi6 was derived from sterile JC transcripts, but this type of L2 clone has not yet been reported in zebrafish. When compared with the nucleotide length of the zebrafish clone intergenic sequence (517 bp), that of Cpj3 (174 bp) was much shorter, suggesting that a VL2 segment is located very close to JL in the carp genome, thus likely close to CL2 as well. This assumption was supported by the Southern blot result, showing that at least VL2-1 and CL region are adjacent to one another, judging from the nearly identical hybridisation patterns observed (Fig. 6). The two VL families, represented by Cpi1 and Cpi2 VL, were identified in carp (Fig. 2), and could be assigned to the two VL families in zebrafish by the phylogenetic study (Fig. 7b). On the other hand, trout L2 has been reported to possess four VL families to date [20], although more VL families in carp and zebrafish may be found in future. As for VL families of the other IgL isotypes in carp, it is suggested that L1A, L1B, and L3 hold at least three, two, and two families, respectively, with the same degree as L2 [21,23]. In the previous work, some VL sequences derived from carp L1A and L3 were highly similar to each other [21], but such a close relationship could not be found between carp L2 and any other isotype (data not shown), consistent with the VL phylogenetic result demonstrating close relationships among type L2 sequences from various species including carp, segregated from the other types such as L1/G or L3/F (Fig. 7b). Thus, the VL sequences identified here should add further diversity to a carp IgL repertoire. Southern blot analyses using VL- and CL-specific probes suggested that the genomic organisation of carp L2 locus is a cluster-like (Fig. 6) as well as trout [18] and salmon [13]. With respect to other isotypes of carp IgL, it has been suggested that L1B has a cluster-like organisation as well, while those of L3 and L1A remain unclear [21]. Until now three isotypes of IgL chain within a species have been characterised in detail in X. laevis [6–8], H. francisci [9–11], and three teleost species, D. rerio [12], S. salar L [13], and O. mykiss [14]. In this
Fig. 7. Neighbor-joining trees of CL (a) and VL (b) amino acid sequences from various fish species. Only FR1, FR2, and FR3 were used in (b). The bootstrap values from a 1000 replications are indicated at the nodes, but only the values >500 are shown. Genetic distances are given under each tree. Accession numbers in (a) are: Acipenser (X90557); Hydrolagus (L25551); Heterodontus I (M64307), II (L25559), and III (L25561); Raja 1 (L25568) and 2 (L25566); Gynglymostoma (L16765); Gadus L1 (X68515) and L2 (GMO293807); Ictalurus G (L25531) and F (U25705); Cyprinus L1A (AB015905), L1B (AB035729), L2 (clone Cpj2, AB091119), and L3 (AB035730); Danio L1 (AF246185), L2 (AF246162), and L3 (AF246193); Paralichthys (AU090352); Oncorhynchus L1 (X68521) and L2 (U69987); Salmo L1 (AF273017), L2 (AF297518), and L3 (AF406956); Dicentrarchus (DLA400216); Anarhichas L1 (AF137397) and L2 (AF137398); Seriola (1) (AB062664), (2) (AB062653), and (3) (AB062667). Accession numbers in (b) are: Heterodontus I (HFL141) and III (HEFIGCVE); Raja 1 (SKTIGCVE) and 2 (SKTIGCVC); Oncorhynchus L1 (X68517), L2 (1) (OMU69987), L2 (2) and L2 (3) (OMY251647), and L2 (4) (OMY251650); Cyprinus L1A (AB073328), L1B (AB073331), L2 (1) (clone Cpi1, AB091112), L2 (2) (clone Cpi2, AB091113), and L3 (AB073342); Danio L1 (AF246185), L2 (1) (AF246162), L2 (2) (AF246179), and L3 (AF246189); Carcharhinus (CPLIGL); Gadus L1 (X76517) and L2 (GMO293807); Seriola (1) (AB062650), (2) (AB062653), and (3) (AB062647); Hydrolagus (HDLIGCVC); Dicentrarchus (DLA400216); Anarhichas L1 (AF137397) and L2 (AF137398); Acipenser (ABIGLCST5); Ictalurus G (ICTIGLSTD) and F (IPU25705); Salmo L1 (AF273017) and L3 (AF406956); Gynglymostoma (GINIGL).
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investigation it was demonstrated that carp has at least four IgL isotypes classified into three types, L1A and L1B in type L1/G, L3 in type L3/F, and L2 in type L2, and thus this considerable diversity of IgL as seen in carp is likely to contribute to the complexity of the carp Ig molecule as well as IgH [32].
Acknowledgement This work was supported by a grant from the Ministry of Education, Science, Sports and Culture to promote advanced scientific research.
References [1] Raynaud CA, Dahan A, Weill JC. Complete sequence of a chicken light chain immunoglobulin derived from the nucleotide sequence of its mRNA. Proceedings of National Academy of Science USA 1983;80:4099–103. [2] Zachau HG. Immunoglobulin light-chain genes of the type in man and mouse. In: Honjo T, Alt FW, Rabbitts TH, editors. Immunoglobulin genes. London: Academic Press; 1989, pp. 91–109. [3] Bauser TR Jr, Blomberg B. The human L chain Ig locus. Characterization of JCl6 and identification of functional JCl7. Journal of Immunology 1991;146:2813–20. [4] Home WA, Ford JE, Gibson DM. L chain isotype regulation in horse. Journal of Immunology 1992;149:3927–36. [5] Kirschbaum T, Jaenichen R, Zachau HG. The mouse immunoglobulin locus contains about 140 variable gene segments. European Journal of Immunology 1996;26:1613–20. [6] Schwager J, Burckert N, Schwager M, Wilson M. Evolution of immunoglobulin light chain genes: analysis of Xenopus IgL isotypes and their contribution to antibody diversity. EMBO Journal 1991;10:505–11. [7] Zezza DJ, Mikoryak CA, Schwager J, Steiner LA. Sequence of C region of L chains from Xenopus laevis Ig. Journal of Immunology 1991;146:4041–7. [8] Haire RN, Ota T, Rast JP, Litman RT, Chan FY, Zon LI et al. A third light chain gene isotype in Xenopus laevis consists of six distinct VL families and is related to mammalian genes. Journal of Immunology 1996;157:1544–50. [9] Shamblott MJ, Litman GW. Complete nucleotide sequence of primitive vertebrate immunoglobulin light chain genes. Proceedings of National Academy of Science USA 1989;86:4684–8. [10] Shamblott MJ, Litman GW. Genomoic organization and sequences of immunoglobulin light chain genes in a primitive vertebrate suggest coevolution of immunoglobulin gene organization. EMBO Journal 1989;8:3733–9. [11] Rast JP, Anderson MK, Ota T, Litman RT, Margittai M, Shamblott MJ et al. Immunoglobulin light chain class multiplicity and alternative organizational forms in early vertebrate phylogeny. Immunogenetics 1994;40:83–99. [12] Haire RN, Rast JP, Litman RT, Litman GW. Characterization of three isotypes of immunoglobulin light chains and T-cell antigen receptor alpha in zebrafish. Immunogenetics 2000;51:915–23. [13] Solem ST, Jorgensen T. Characterisation of immunoglobulin light chain cDNAs of the Atlantic salmon, Salmo salar L.; evidence for three IgL isotypes. Developmental and Comparative Immunology 2002;26:635–47. [14] Timmusk S, Stromberg S, Pilstrom L. Light chain promoter regions in rainbow trout Oncorhynchus mykiss: function of a classical and an atypical Ig promoter. Developmental and Comparative Immunology 2002;26:785–96. [15] Hohman VS, Schluter SF, Marchalonis JJ. Complete sequence of a cDNA clone specifying sandbar shark immunoglobulin light chain: Gene organization and implications for the evolution of light chains. Proceedings of National Academy of Science USA 1992;89:276–80. [16] Daggfeldt A, Bengte´n E, Pilstro¨m L. A cluster type organization of the loci of the immunoglobulin light chain in Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss Walbaum) indicated by nucleotide sequences of cDNAs and hybridization analysis. Immunogenetics 1993;38:199–209. [17] Ghaffari S, Lobb CJ. Structure and genomic organization of immunoglobulin light chain in the channel catfish. An unusual genomic organizational pattern of segmental genes. Journal of Immunology 1993;151:6900–12. [18] Partula S, Schwager J, Timmusk S, Pilstro¨m L, Charlemagne J. A second immunoglobulin light chain isotype in the rainbow trout. Immunogenetics 1996;45:44–51. [19] Ghaffari S, Lobb CJ. Structure and genomic organization of a second class of immunoglobulin light chain genes in the channel catfish. Journal of Immunology 1997;159:250–8. [20] Timmusk S, Partula S, Pilstro¨m L. Different genomic organization and expression of immunoglobulin light-chain isotypes in the rainbow trout. Immunogenetics 2000;51:905–14.
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[21] Tomana M, Ishikawa J, Imai E, Moritomo T, Nakao M, Yano T. Characterization of immunoglobulin light chain isotypes in the common carp. Immunogenetics 2002;54:120–9. [22] Okamoto K, Ikemura H, Savan R, Sakai M. Cloning, sequence and variability analysis of expressed immunoglobulin light chain genes from yellowtail Seriola quinqueradiata. Fish & Shellfish Immunology 2003;14:55–70. [23] Tomana M, Nakao M, Moritomo T, Fujiki K, Yano T. Isolation of cDNA encoding immunoglobulin light chain from common carp (Cyprinus carpio L.). Fish & Shellfish Immunology 1999;9:71–80. [24] Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proceedings of National Academy of Science USA 1977;74:5463–7. [25] Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proceedings of National Academy of Science USA 1988;85:2444–8. [26] Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 1994;22:4673–80. [27] Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 1987;4:406–25. [28] Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 1985;39:783–91. [29] Page RDM. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 1996;12:357–8. [30] Sambrook J, Frisch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd edn Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 1989. [31] Kono T, Sakai M. The analysis of expressed genes in the kidney of Japanese flounder, Paralichthys olivaceus, injected with the immunostimulant peptidoglycan. Fish & Shellfish Immunology 2001;11:357–66. [32] Nakao M, Moritomo T, Tomana M, Fujiki K, Yano T. Isolation of cDNA encoding constant region of the immunoglobulin heavy-chain from common carp (Cyprinus carpio L.). Fish & Shellfish Immunology 1998;8:425–34.