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Unprecedented Multiplicity of Ig Transmembrane and Secretory mRNA Forms in the Cartilaginous Fish This information is current as of June 18, 2017. Lynn L. Rumfelt, Marilyn Diaz, Rebecca L. Lohr, Evonne Mochon and Martin F. Flajnik J Immunol 2004; 173:1129-1139; ; doi: 10.4049/jimmunol.173.2.1129 http://www.jimmunol.org/content/173/2/1129 Subscription Permissions Email Alerts This article cites 41 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/173/2/1129.full#ref-list-1 Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 References The Journal of Immunology Unprecedented Multiplicity of Ig Transmembrane and Secretory mRNA Forms in the Cartilaginous Fish1 Lynn L. Rumfelt,2*† Marilyn Diaz,3† Rebecca L. Lohr,† Evonne Mochon,† and Martin F. Flajnik† I n the animal kingdom, only the jawed vertebrates have an adaptive immune system grounded upon somatically generated Ag receptors (1, 2). All jawed vertebrates also have multiple Ig H chain isotypes (1). Whereas IgM is present in all jawed vertebrates, with one exception (a form of IgD in teleosts, Ref. 3), the other mammalian isotypes are not found in ectotherms, with amphibians having IgX and IgY, and cartilaginous fish having two distinct isotypes, IgW and IgNAR (1, 2). Based on phylogenetic analysis, IgW and IgNAR previously were believed to be present only in cartilaginous fish (4); however, recently IgW was isolated from lungfish (5), showing that both IgW and IgM ancestral genes were present in the common ancestor of all extant jawed vertebrates. The IgW H chain long form is composed of seven domains in all cartilaginous fish so far studied (6 – 8) and of eight domains in the lungfish (5). An IgWshort form having three domains also has been described at the cDNA and genomic levels in skates (previously called IgX), presumably a splice variant of the long form (8, 9); in addition, a protein identified as a second Ig isotype besides IgM in batoids (rays and skates) and frill sharks is postulated to be this short form of IgW (10, 11). The long form of the IgW protein *Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL 33101; and †Department of Microbiology and Immunology, University of Maryland, Baltimore, MD 21201 Received for publication January 28, 2004. Accepted for publication May 14, 2004. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 M.D. was a postdoctoral fellow supported by Burroughs Wellcome Fund Fellow of the Life Sciences Research Foundation. M.F.F. was supported by National Institutes of Health Grant RR06603. 2 Address correspondence and reprint requests to Dr. Lynn L. Rumfelt at the current address: Department of Immunology, University of Toronto, Sunnybrook and Women’s Health Sciences Centre, 2075 Bayview Avenue, Room A331, Toronto, Ontario M4N 3M5 Canada. E-mail address: [email protected] 3 Current address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709-2233. Copyright © 2004 by The American Association of Immunologists, Inc. has also been isolated from metabolically labeled nurse shark splenocytes (6). To date, IgNAR has been described only in elasmobranchs (sharks, skates, and rays), and it is an unusual Ig composed of an H chain dimer that does not associate with L chains (12). The IgNAR V recognizes Ag as a single domain (see Fig. 8) and its gene mutates to very high levels, suggesting that it is important for the shark adaptive immune system (12–14). It has been known for many years that the B cell Ag receptor exists both in a transmembrane (Tm)4 form that acts to activate/ tolerize lymphocytes and as a secretory (Sec) form that binds to Ag and is responsible for the well-known effector functions of the humoral immune system (15). The Tm and Sec forms of all Ig isotypes studied to date are derived from mRNAs that are alternatively spliced. In almost all species, the membrane-proximal constant (CH) domain is contiguous with a Sec segment, and this CH domain and Sec piece are invariably encoded within one exon (16). Upstream of the nucleotides encoding the Sec tail is an “alternative” or cryptic splice site, which is used to splice the C exon onto Tm exons. In the teleost fish, instead of splicing the Tm exons into the ultimate C domain exon, Tm exons are rather spliced onto the penultimate (CH3) exon (3). The function of this form is not known, but it is a universal feature of IgM Tm mRNA forms in this vertebrate class. In other bony fish more primitive than the teleosts (i.e., the holosteans), there can be multiple spliced forms of the IgM Tm mRNA (17). Although cartilaginous fish -chain mRNA is spliced in the typical (human/mouse) fashion, as described, previous studies showed that skate IgW H chain cDNA had two secretory forms, one with two predicted CH domains and another with six (8). Recent lungfish work demonstrated two IgW Sec cDNA forms as well (5), although the short cDNA form had not been found previously in 4 Abbreviations used in this paper: Tm, transmembrane; Sec, secretory; FR, framework region of V domain; NC, noncanonical; VH, IgW H chain variable domain; VH, IgNAR H chain variable domain; CART, conserved Ag receptor motif; Igsf, Ig superfamily; UT, untranslated region. 0022-1767/04/$02.00 Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 In most jawed vertebrates including cartilaginous fish, membrane-bound IgM is expressed as a five Ig superfamily (Igsf)-domain H chain attached to a transmembrane (Tm) region. Heretofore, bony fish IgM was the one exception with IgM mRNA spliced to produce a four-domain Tm H chain. We now demonstrate that the Tm and secretory (Sec) mRNAs of the novel cartilaginous fish Ig isotypes, IgW and IgNAR, are present in multiple forms, most likely generated by alternative splicing. In the nurse shark, Ginglymostoma cirratum, and horn shark, Heterodontus francisci, alternative splicing of Tm exons to the second or the fourth constant (CH) exons produces two distinct IgW Tm cDNAs. Although the seven-domain IgW Sec cDNA form contains a canonical secretory tail shared with IgM, IgNAR, and IgA, we report a three-domain cDNA form of shark IgW (IgWshort) having an unusual Sec tail, which is orthologous to skate IgXshort cDNA. The IgW and IgWshort Sec transcripts are restricted in their tissue distribution and expression levels vary among individual sharks, with all forms expressed early in ontogeny. IgNAR mRNA is alternatively spliced to produce a truncated four-domain Tm cDNA and a second Tm cDNA is expressed identical in Igsf domains as the Sec form. PBL is enriched in the Tm cDNA of these Igs. These molecular data suggest that cartilaginous fish have augmented their humoral immune repertoire by diversifying the sizes of their Ig isotypes. Furthermore, these Tm cDNAs are prototypical and the truncated variants may translate as more stable protein at the cell surface. The Journal of Immunology, 2004, 173: 1129 –1139. 1130 DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING sharks until this report. The Tm cDNA forms of IgW have not been described in any species, but we assumed that they would be spliced to the ultimate CH domain, like the IgM (18) and IgNAR (14, 19) Tm cDNA previously identified. In this paper we re-examined this problem and found that, like teleost IgM, there are multiple-spliced Tm cDNA forms of IgW and IgNAR, possibly selected over evolutionary time to limit proteolysis of the putative cell surface proteins. In addition, our findings reveal that each elasmobranch group, as well as the lungfish, expresses the long and short Sec IgW mRNA forms, and their expression levels in the nurse shark varies among individuals with no apparent pattern. Materials and Methods Animals Nurse shark pups (Ginglymostoma cirratum) were delivered by Caesarean section from a female shark as described (20). The age of pups, feeding and caretaking were performed as described (21). Adult nurse sharks were captured off Little Torch Key, FL and horn sharks were obtained from Pacific Biomarine (Monterey, CA). The animals were sacrificed with an overdose of tricane methyl sulfonate (MS-222) anesthesia (20). Tissues were dissected from adult nurse and horn sharks, and from nurse shark pups, and total RNA was isolated as described (21). The newborn pup spleen and epigonal cDNA libraries, and adult horn shark (Heterodontus francisci) and nurse spleen and PBL cDNA libraries were constructed as described previously (12, 21–24). Probes for library screens and Figs. 6 and 7 are listed in Table I and were labeled with 32P[dCTP] as described (21). Spleen, epigonal organ, and PBL oligo(dT)-primed cDNA was made from 5 g of mRNA as described and used as templates for PCR amplification (21). Primers for RT-PCR are shown in Table II. Expected sizes for PCR products are listed in Table III. Positive controls to verify correct PCR amplification conditions were: IgW Sec clone 14S, IgW Tm clone 1E (GenBank accession no. AY524297), IgNAR Sec clone 3-4 (GenBank accession no. U18701; Ref. 12), and full-length IgNAR Tm clone 7A (GenBank accession no. U18721; Ref. 12). Nurse shark total RNA was used (20 g/lane) for Northern analyses. The labeled probes were hybridized with membranes for 20 h minimum at 42°C, then washed under high stringency conditions (25). Nurse shark NDPK RNA loading control was a gift from M. Kasahara (Graduate University of Advanced Studies (Sokendai), Hayama, Japan) (26). To identify horn shark and nurse shark Tm IgW and IgWshort, horn shark and nurse shark adult spleen cDNA libraries were plated and lifted as described using two membranes per plate (22) and hybridized to IgW VH and CH6 probes separately. Isolated clones were selected based on VH⫹ and CH6⫺ thereby enriching for the short forms. Alignments and phylogenetic trees IgW Sec tail cDNA sequences from library screenings were aligned with Sec tails from sequences with the following Swiss-Prot and GenBank accession numbers: Hf IgWsh1 P83742; Hf IgWsh2 P83743; Hf IgWsh3 P83744; Hf IgM X0778; Gc IgWshort clone 2-4 AY524287; Gc IgWshort clone 1-2 AY524282; Gc IgWshort clone 1-3 AY524289; Gc IgWshort pnc P83984; Gc IgWshort spl P83985; little skate, Raja erinacea, IgXshort clone 20 AAA49546; nurse shark IgW U51450; nurse shark IgM M92851; sandbar shark, Carcharhinus plumbeus, IgW 1117935; little skate IgM M29677; horn shark IgM X0778; ratfish, Hydrolagus colliei, IgM Results cDNA analysis reveals that IgW Tm and one form of IgNAR Tm are smaller than their secreted forms, and shark IgWshort is orthologous to skate IgWshort While screening neonatal nurse shark spleen and epigonal organ cDNA libraries with an IgW VH probe to examine the early VH gene repertoire, we isolated IgW Tm cDNA clones. In retrospect, this was not unexpected because these mRNA sources from young animals are highly enriched for IgM Tm mRNAs (21). The deduced proteins encoded by most of the cDNA clones unexpectedly were found to have the Tm region contiguous with the CH4 rather than the CH6 domain, the C-terminal domain in IgW Sec cDNA forms (see Fig. 8). Subsequently, additional cDNA clones were isolated from the adult nurse shark PBL cDNA library screened with an IgW VH probe and from adult nurse shark and horn shark spleen cDNA libraries screened differentially with IgW VH and CH6 probes (positive for the former and negative for the latter), thereby selecting for the smaller sizes of IgW cDNA. These screenings resulted in identification of more nurse shark IgW Tm (five domains) and Sec (seven domains) cDNA clones; unpredictably, three-domain Tm and Sec cDNA clones also were found that we designate IgWshort Tm and Sec (Fig. 1, see Fig. 8). These short Sec and Tm cDNA forms were unanticipated in sharks (4), and previously were believed to be present only in the skate as Sec cDNA (2, 4). The models we present here for the different sized Ig obviously H chain Tms derived from the cDNA data are tentative Table I. Probesa Name NS NS NS NS HS NS NS NS NS a IgW Tm IgM VH IgNAR VH IgW VH IgM VH IgM TM NDPK IgW CH6 IgNAR Tm Forward and Reverse Primers Template TM1E For ⫹ 3⬘ UT TM1E Rev VH FR1 For ⫹ VH FR4 (50S) Rev FR1VH (3⬘-4) For ⫹ FR3VH (3-4) Rev NEW12A FR1 For ⫹ 12A CH1 Rev HS VH For ⫹ HS VH Rev TM For (35S) ⫹ 3⬘ UT TM Rev (35S) NS NDPK For ⫹ NS NDPK Rev CH6 For (38E) ⫹ CH6 Rev (38E) TM (7A) For ⫹ 3⬘ UT TM (7A) Rev 1E (NS pup epig org lib) 50S (NS pup spln lib) 3– 4 (Accession no. U18701) 12A (NS spln lib) 3–5A (HS spln lib) 35S (NS pup spln lib) Accession no. M63964 38E (NS pup epig org lib) 7A (U18721) For, forward; Rev, reverse. Anneal 63°C 55°C 64°C 52°C 62°C 53°C 64°C 57°C 58°C 30 30 30 70 30 30 30 30 30 s s s s s s s s s Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 cDNA library screens, Northern blot analysis, and RT-PCR AAC12920; guitarfish, Rhinobatus productus IgNAR AY524298; Siberian sturgeon, Acipenser baerii (Ab) IgM Y13253; bowfin, Amia calva (Ac) IgM U12456; marbled lungfish, Protopterus aethiopicus (Pae) IgM AF437724; human, Homo sapiens IgM 4467842; mouse, Mus musculus, IgA AAB59662; human IgA2 546799; lungfish IgW AF437727; and IgXlong clone 9 (8), using Clustal X version 1.8 (27). Phylogenetic tree analysis was performed as described (28) using Clustal X version 1.8 and 1000 bootstrap cycles. The phylogram was drawn in Treeview version 1.6.6 (29) and labeled in Canvas version 9.0 (ACD Deneba Software, Miami, FL). Horn shark and nurse shark IgW Tm cDNA sequences were translated into proteins using the EXPASY translate tool and aligned in Clustal W version 1.8 (30) with Ig Tms from other vertebrate species using GenBank accession numbers referenced in Campbell et al. (31) and as follows: human IgM X17115; mouse IgM P01873; Xenopus IgM X90768; Xenopus IgY X90767; horn shark IgM X07781; Xenopus IgX CAA62293; channel catfish IgD AF363448; mouse IgG1 P01869; mouse IgG3 P03987; mouse IgG2a P01865; mouse IgG2b P01867; human IgG1 X52847; human IgE A46485; mouse IgE X03624; Atlantic cod IgM X58871; channel catfish IgM X52617; duck IgA CAC43282; human IgA2 M60194; human IgA1 AAA52745; mouse IgA K00691; horn shark IgW Tm2T7 P83978; nurse shark IgW Tm1T3 P83981; nurse shark IgWshort clone 2-5 AY524296; nurse shark IgW Tm clone 1E AY524297; nurse shark IgWshort clone 25E AY531553-AY531554; nurse shark IgNAR clone 7A U18721; nurse shark IgNAR NEP83977; horn shark IgW Tm7T7 P83979; horn shark IgW Tm3T3 P83980; nurse shark IgM Tm 1E AY609247; nurse shark IgW Tm6T3 P83982; nurse shark IgW Tm3C4 P83983. The Journal of Immunology 1131 Table II. Primersa Ig Class Sequence Priming Site Anneal Temp CH3 For CH6 For TM 1E For TM 1E Rev ⬘ UT TM 1E Rev SEC Rev 3⬘ UT SEC Rev NEW12A FR1 For 12A CH1 Rev CH6 For (38E) CH6 Rev (38E) HS FR1 For HS CH1 Rev CH2 (3– 4) For CH5 (3– 4) For TM (7A) Rev 3⬘ UT TM (7A) Rev SEC (3– 4) Rev 3⬘ UTSEC(3– 4) Rev FR1 VH (3– 4) For FR3 VH (3– 4) Rev HS VH For (3–5A) HS VH Rev (3–5A) VH FR1 For VH FR4 (50S) Rev CH1 Rev TM For (35S) 3⬘ UT TMRev(35S) IgW IgW IgW IgW IgW IgW IgW IgW IgW IgW IgW IgW IgW IgNAR IgNAR IgNAR IgNAR IgNAR IgNAR IgNAR IgNAR IgM IgM IgM IgM IgM IgM IgM 5⬘-ACAGCAGTCTGTTCTGATCCCAGC-3⬘ 5⬘-GGAGGCTGGAACTCGGGCAGT-3⬘ 5⬘-GTGGTCCCCCCAAATGTGAAA-3⬘ 5⬘-GGCCGGAACTGTTGGCGCGTT-3⬘ 5⬘-CACAAGACCTAAGGAATCAGTC-3⬘ 5⬘-GGAGTTAAAGCTTTCAG-3⬘ 5⬘-TCGCACATGATCAGGGACACG-3⬘ 5⬘-CCGAGTCAGTTGTGAAAAAGCC-3⬘ 5⬘-TTAGAGCTTGTGTCACCG-3⬘ 5⬘-GAGGTTAAGACTCACCCTCAA-3⬘ 5⬘-GCTGTGGGATTTATTAATGCT-3⬘ 5⬘-AATATCGTGTTGACCCAGCCC-3⬘ 5⬘-CCTGCCTTGCAGTGGTAGAC-3⬘ 5⬘-CAAATGGAACCAACTAAAATG-3⬘ 5⬘-AAGGGGAGTGGTTCCAGCTTCGTT-3⬘ 5⬘-AGTCATGATGGATAATGAACT-3⬘ 5⬘-TCAGCTTGTAGCAGTTAAGTG-3⬘ 5⬘-GGATTTAACAGTGTCGC-3⬘ 5⬘-ATGTGACTATTGCGTGCAATGA-3⬘ 5⬘-GAGCGAGTGGACCAAACGCC-3⬘ 5⬘-ACCGCAACGATACGTGCCAC-3⬘ 5⬘-GATGTCGTGTTGACTCAGCCA-3⬘ 5⬘-AGTCACCGTCACCATGGTCCC-3⬘ 5⬘-GAGATTACTTTGATTCAACCA-3⬘ 5⬘-TGTAGTCACGGTCACCATGGT-3⬘ 5⬘-ACGTCGGGGGAATAGTCCATCG-3⬘ 5⬘-TCGATAGATCACACTTGGATT-3⬘ 5⬘-TCGTAATTACCTCAATGATAT-3⬘ TAVCSDPS GGWNSGS VVPPNVK NAPTVPA 64 64 64 64 64 48 66 52 52 56 56 64 64 56 64 58 58 50 62 64 64 62 62 50 56 64 53 53 NS NDPK For NS NDPK Rev NDPK NDPK 5⬘-GGTAACAAGGAACGAACCTTC-3⬘ 5⬘-CTCATAGATCCAGTCTTGGGC-3⬘ a TESFNS ESVVKK GDTSSN EVKTHPQ SINKSHS NIVLTQ VYHCKAG QMEPTKM KGSGSSFV SSLSIMT SDTVKS ERVDQTP GGTYRCG DVVLTQP EITLIQP TMVTTT AMDYSPD SIDHTWI GNKERTF AQDWIYE 64 64 For, forward; Rev, reverse. until directly demonstrated as membrane-associated proteins through biochemical characterization. Inspection of cDNA clones from one animal revealed that IgW mRNA is transcribed from at least two different loci in nurse sharks, which are most divergent in the deduced CH2 domain (Fig. 1). The deduced amino acid identity between these two shark loci for V (framework region of V domain (FR)1-FR3), CH1, and CH2 are 72, 61, and 46%, respectively. Skate IgWshort (previously called IgX) is ⬃50% identical (amino acid) to either shark IgW locus for each VH, CH1, and CH2 domain (data not shown). This implies that the two loci diverged not long after the divergence of sharks and batoids (skate) 220 million years ago. The deduced AA identities for CH3-CH6 between the two shark IgW loci are much higher (Fig. 1), indicating that those domains have been under more stringent negative selection. Importantly, all cDNA forms of IgW, including IgW Tm, IgW Sec, IgWshort Tm, and IgWshort Sec, were encoded by each locus (or very closely related loci), implying they are products of alternative splicing rather than from separate loci encoding each form. Alignment of the horn shark IgWshort Sec cDNA deduced protein sequences, isolated by differential screening of the horn shark cDNA library, with the nurse shark IgWshort Sec cDNA-deduced protein sequences identified the shark transcripts as orthologs to the previously identified three-domain skate IgWshort cDNA having an unusual noncanonical (NC) Cys-rich Sec tail (9) (data not shown and Fig. 2A). We detected transcription of only one horn shark locus (or several closely related loci) with this NC Sec tail, which contains seven Cys and is somewhat longer than the nurse shark and skate NC Sec tails. Consistent with the presence of two very different nurse shark IgW loci, two divergent NC Sec tail cDNA sequences were identified that differ in length and Cys content. The NC Sec tail is dramatically distinct from canonical IgW/ IgNAR/IgM/IgA Sec tails, which are small, uniform in size, and contain an invariant Asn-linked glycosylation site and only one Cys in the penultimate position. In mammalian IgM and IgA both the Cys and the glycosylation site are required for J-chain association (32–34). The deduced IgWshort NC Sec tail in all species contains several Cys residues that could participate in either intraor interchain disulfide bonds (or even J-chain association) and one potential Asn-linked glycosylation site. Phylogenetic analysis demonstrates that the NC Sec tails diverge rapidly over evolutionary time (Fig. 2B). As described (Fig. 2A), nurse sharks (Gc) have two divergent loci for this NC tail, whereas the horn shark (Hf) may have only one; note that the divergence between the two nurse shark NC Sec tails is greater than that between the canonical Sec tails of nurse shark and skate (Re). Together, these results prove that IgWshort and its NC Sec tail were present in the elasmobranch common ancestor. Unfortunately, IgWshort in the lungfish was only detected by Northern blotting (5) and no cDNA sequence of its Sec tail was reported for inclusion in this phylogenetic analysis. Previously our laboratory identified H chain cDNA for both the IgNAR Tm and Sec forms having six Ig superfamily (Igsf) domains through screening of an adult splenic cDNA library (19). However, in another previous study examining IgNAR CDR3 diversification, serendipitously we identified Tm cDNA by RT-PCR containing only three putative CH domains, suggesting that IgNAR, like IgW, underwent alternative splicing of its Tm mRNA (12, 14). Thus, we further investigated the expression of IgW and IgNAR Tm mRNA by RT-PCR of nurse shark epigonal organ, spleen, and PBL (Fig. 3). Our strategy was to confirm: 1) IgNAR Tm mRNA exists in two forms (CH3-Tm and CH5-Tm), and 2) there is no Tm mRNA form of IgW having the same number of Igsf domains as the Sec mRNA (CH6-Tm). IgW forward primers Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 Primers 1132 DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING Table III. RT-PCR products IgW IgNAR Priming site Full-length (bp) Truncated (bp) Fig. 3A row CH3-Tm CH3–3⬘ UT Tm CH3-Sec CH6-Sec 1281 1592 1245 183 636 947 trunc CH3-Tm trunc CH3–3⬘ UT Tm Full-length CH3-Sec Full-length CH6-Sec Cartilaginous fish IgW, IgNAR, and IgM Tms contain the conserved Ag receptor motif (CART) The deduced Tm regions of horn shark and nurse shark IgW, IgWshort, IgNAR, and IgM cDNA clones were aligned with each other and with other vertebrate Ig Tms (Fig. 4). Shark Tms conform to a highly conserved motif in Ag-receptor phylogeny, CH2-Tm CH2–3⬘ UT CH5-Tm CH5–3⬘ UT CH2-Sec CH2–3⬘ UT CH5-Sec CH5–3⬘ UT Tm Tm Sec Sec Full-length (bp) Truncated (bp) Fig. 3B lanes 1257 1585 243 569 1170 1300 183 307 586 914 1–2 3–4 5–6 7–8 9–10 11–12 13–14 15–16 consisting of three components: an extracellular acid-rich spacer region, a hydrophobic membrane-spanning region, and a short cytoplasmic tail (18, 31). The extracellular regions of the IgW and IgNAR Tm forms are acidic, but as is true of Tm forms in other vertebrates, the sequence and length of this region are variable (31). In contrast, the Tm hydrophobic region is well conserved in sequence and size throughout phylogeny. The short cytoplasmic tail is also conserved, as was shown previously for horn shark IgM Tm cDNA (18), and now for IgW and IgNAR Tm cDNAs. The putative cytoplasmic tail of IgNAR Tm cDNA retains a polar residue as the last amino acid, substituting Asn for the more typical Lys. The putative IgW Tm cytoplasmic tail is composed of four amino acids, with a substitution of two Gln for the ultimate Lys. In mammalian Ig, the Lys residues are thought to tether the receptor at the plasma membrane, and are important for signaling, Ag presentation, and internalization of the Ig receptor (31); the terminal residues in IgW and IgNAR cytoplasmic tails are somewhat more similar to mammalian IgE and IgA than to IgM, suggesting a mechanism for differential signaling. Deduced nurse shark IgM Tm precisely conserves the residues shown to be important in the mammalian hydrophobic region. Within this region, Thr, Ser, and Tyr residues are required for mammalian Ig␣/Ig coreceptor association at specific positions (ⴱ, Fig. 4) (31). These residues are conserved in all surmised shark Ig Tms suggesting they could interact with these essential coreceptors. Residues in the Tm region form ␣-helical secondary structures having hydrophilic and hydrophobic portions. Fig. 5 compares the shark IgNAR and IgW Tm regions to the CART using helical wheel plots (refer to Fig. 4) (31). All shark Ig isotypes contain deduced residues in this Tm region that match the conserved motif in both their positions in the helix and residue qualities. Together, these data demonstrate that the surmised shark IgW, IgNAR, and IgM Tms preserve the conserved Ag receptor Tm motif, and thus their putative proteins may associate with an Ig␣/Ig-like coreceptor and signal through their C-terminal regions. Additionally, these deduced shark Ig proteins present a conserved ␣-helical conformation at the membrane interface with features shown to be important for structure and signaling in mammals. IgW and IgNAR Tm mRNAs are highly expressed in the spleen and PBL, and enriched in PBL The tissue distribution of IgW and IgNAR mRNA in an adult nurse shark was examined by Northern analyses with probes specific for either the Tm forms or all forms (Fig. 6, refer to Table I for probes). Note that the Tm forms are generally found at much lower levels than the Sec forms (15), and thus the Tm forms are difficult to detect with probes that hybridize to both forms (e.g., compare the PBL lane for IgW Tm and IgW Sec, Fig. 6, A and B: the Tm form at 2.3 kb (band 2 in Fig. 6A) is hardly visible in Fig. 6B using Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 were designed for the deduced CH3 or CH6 domains and reverse primers for the deduced Tm, 3⬘ untranslated region (UT) Tm, or Sec regions (Fig. 3A, refer to Table II for primers; expected PCR sizes in Table III). Only PCR products of 636 bp (CH3/Tm, Fig. 3A, first row) or 947 bp (CH3/3⬘ UT Tm, Fig. 3A, second row) for IgW Tm cDNA were produced under various PCR conditions with several individuals, representing the CH4-Tm spliced product (note that the PCR fragments were confirmed by sequencing). Other primer sets that could amplify the CH6 and Tm or 3⬘ UT Tm mRNA produced no bands (Fig. 3A, rows 5 and 6), confirming the absence of an IgW Tm transcript with seven putative Igsf domains. Other controls showed that only full-length products (encoding seven domains) of 1245 and 183 bp were amplified with CH3 and Sec primers, and CH6 and Sec primers, respectively (Fig. 3A, rows 3 and 4). Thus, we confirmed that there is no detectable mRNA encoding an IgW Tm form with seven-Igsf domains in primary and secondary lymphoid tissues. RT-PCR of the same tissue mRNA was done for the IgNAR Tm study (Fig. 3B). As mentioned, previous data suggested that IgNAR Tm mRNA occurred in two sizes, one with five putative constant domains and another lacking the two C-terminal domains. Thus, two bands were expected to be amplified using the primer pairs CH2/Tm (Fig. 3B, lane 1) or CH2/3⬘ UT Tm (Fig. 3B, lane 3, refer to Table III). Indeed, two bands were amplified with these primer sets, representing the six and four domain-encoding cDNAs, respectively (Fig. 3B, black arrows in rows 1 and 3). The band representing the smaller form was always more intense, which either suggests that it is preferentially amplified or is truly expressed at higher levels; the fact that an intense band representing the high m.w. product in Fig. 3B, lanes 1 and 3, was amplified with CH5/3⬘ UT Tm primers favors the former possibility (Fig. 3B, lane 5). Tm exons for the mRNA encoding the deduced five-CH domain form are spliced to the cryptic splice site within the exon encoding CH5, while the form with three CH domains is spliced to the Tm exons at the canonical splice site that flanks the exon (Fig. 3C). Primer pairs for CH2 or CH5 and Sec or 3⬘ UT Sec verified that IgNAR Sec mRNA is present only as a six-domain form, consistent with previous molecular and biochemical data (Ref. 12, Fig. 3B, lanes 9 –16). In summary, IgNAR Tm mRNA is found in two forms in primary and secondary lymphoid tissues, one form having the same number of putative CH domains as the Sec form (unlike IgW Tm); furthermore, unlike IgW Sec, we find mRNA of only one size encoding the deduced IgNAR Sec. Priming site The Journal of Immunology Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 1. IgW and IgWshort Tm and Sec cDNA forms are probably alternatively spliced transcripts. The various forms of IgW were aligned from cDNA clones (eight clones for locus 1 and eight clones for locus 2) for IgW Sec (LS), IgW Tm (LTM), IgWshort Sec (SS), and IgWshort Tm (STM) obtained from adult nurse shark (Y) and compared with the original adult nurse shark IgW Sec (U51450; Ref. 6); note that the original IgW clone was not derived from the same shark. The major amino acid differences between the VH, CH1, and CH2 domains indicate that at least two separate IgW loci are expressed. The few amino acid differences detected within the same family could either be derived from somatic changes or multiple closely related IgW loci. Refer to Fig. 8 for diagrammatic views of the various putative forms of IgW. GenBank accession numbers are as follows: clone 1-1 AY524294, clone 1-2 AY524282, clone 1-3 AY524289, clone 1-4 AY524279, clone 1-5 AY524277(5⬘end), AY524278 (3⬘end), clone 1-6 AY524286, clone 1-7 AY524280, clone 1-8 AY524283, clone 2-1 AY524291, clone 2-2 AY524288, clone 2-3 AY524284, clone 2-4 AY524287, clone 2-5 AY524296, clone 2-6 AY524285, clone 2-7 AY524281, clone 2-8 AY524290. 1133 1134 DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 2. Comparisons of the IgWshort Sec tails among elasmobranch species. A, IgWshort NC Sec tails from horn shark (Hf) and nurse shark (Gc) were aligned with that of skate (Re) IgWshort. Canonical Sec tails from cartilaginous fish IgNAR, IgW, IgM, and lobe-finned fish IgM and IgW were also aligned with mammalian IgM and IgA. A putative N-linked glycosylation site (bold asterisk and underlined) and multiple Cys residues (bold) are present in all of the NC Sec tails. The canonical tails for IgW and IgM are highly conserved in cartilaginous fish; the invariant glycosylation site and Cys are noted (bold asterisk and underlined). Slashes (/) indicate gaps added for alignment purposes (Hf, H. francisci; Gc, G. cirratum; Re, R. eglanteria; Rp, R. productus; Ab, A. baerii; Hc, H. colliei; Ac, A. calva; Pae, P. aethiopicus; Mm, M. musculus; Hs, H. sapiens. B, Phylogenetic trees for the Sec tails derived from the sequences in A. The trees were rooted using the canonical tail from human (H. sapiens, Hs) IgM. Abbreviations are the same as in A. the probe that recognizes both forms). IgW and IgWshort Tm mRNAs were expressed in a restricted pattern, present only in the spleen, pancreas, PBL, and at low levels in epigonal organ (Fig. 6A). Bands 2 and 3 correspond to the putative five and three domain IgW Tm forms, respectively; band 1 has not been detected in cDNA library screens or by RT-PCR. IgW Sec expression was also restricted, with high expression found in the spleen, epigonal organ, and testes. Low levels of IgWshort were observed in multiple tissues, including the liver, gill, kidney, esophagus, and testes and enriched expression in the pancreas of this animal. A higher m.w. IgW Sec transcript (3.6 kb-band 1, Fig. 6, A and B) was detected mainly in the spleen; this transcript has never been identified in multiple library screenings, and may be unspliced heterogeneous RNA as seen in nurse shark IgM expression (M. F. Flajnik, unpublished results), or perhaps another form of IgW mRNA resistant to cloning. Previously, we have shown that the epigonal organ, a bone marrow The Journal of Immunology 1135 equivalent in cartilaginous fish, functions as a reservoir for Sec Igs (21, 35), a situation similar to that of plasma cells residing in mammalian bone marrow. Thus, we expected to find IgW Sec mRNA expression there as well. As expected, based on the RT-PCR experiment (Fig. 3B) and previous biochemical experiments (12), IgNAR Sec was present as a single mRNA species and transcribed in most tissues (Fig. 6D), an expression pattern largely overlapping with IgM Sec (Fig. 6E); only IgM Tm was expressed in the thymus. In summary, Tm mRNA of all isotypes is enriched in the spleen and especially in the PBL (Fig. 6, A, C, and E). IgNAR and IgM Sec mRNAs have a similar, broad tissue distribution. In contrast, IgWshort Sec mRNA is expressed primarily in the spleen, pancreas, epigonal organ, and at low levels in several other tissues; IgW Sec mRNA expression is seen in these same tissues as well as in the testes. spleens of six adults and five pups to determine whether expression of this isotype is universal. Among the six adults investigated, IgWshort mRNA was strongly expressed in four animals and hardly at all in the others (Figs. 6 and 7, data not shown). Among very young animals, IgW and IgWshort mRNA expression varied between individuals ranging from high to very low. We conclude that both forms of IgW mRNA can be expressed early in ontogeny, at least during late embryonic development and at birth. We also conclude that, for unknown reasons, IgWshort can be expressed at very different levels in individual nurse sharks, providing an explanation for not detecting this form of IgW mRNA previously in sharks. Individual nurse sharks express IgWshort mRNA differentially in secondary lymphoid tissue Multiple forms of Ig isotypes in the cartilaginous fish Because IgWshort was believed not to exist in sharks in previous studies (2, 4, 7), we investigated Ig mRNA expression in the Discussion Fig. 8 shows our current understanding of cartilaginous fish Ig at the cDNA and/or protein levels. The five-domain IgM is clearly the most abundant serum Ig, present in 19S and 7S forms at nearly Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 3. IgW and IgNAR Tms are alternatively spliced in primary and secondary lymphoid tissues and in PBL. The epigonal organ, spleen, and PBL of an adult nurse shark were examined for IgW and IgNAR Tm and Sec expression by RT-PCR; primers for PCR noted in Table II and expected sizes of PCR products in Table III. A, IgW Tm is alternatively spliced. PCR positive controls were amplified products using plasmid cDNA templates. In row 1, primer pair CH3/Tm amplified only the alternatively spliced transcript of 636 bp in each tissue, i.e., the five-domain form. In row 2, primer pair CH3/3⬘ UT Tm gave the same results of a truncated PCR product of 947 bp. Rows 3 and 4 that demonstrated IgW Sec is expressed as a full-length transcript, i.e., the seven-domain form, using primer pairs CH3/Sec and CH6/Sec, which amplified 1245- and 183-bp products, respectively. Rows 5 and 6 show that the primer pairs CH6/Tm and CH6/3⬘UT Tm amplified no bands of the expected size, confirming that there is no expressed IgW Tm form with seven Igsf domains. B, IgNAR Tm is alternatively spliced to produce four- and six-domain Tm forms. Amplified products from tissue RNA are shown in the odd lanes and positive controls (cDNA inserts in plasmids) in the even lanes. In lane 1, two PCR products (arrows) were amplified using primer pair CH2/Tm that represent the full-length (1257 bp) and truncated (586 bp) IgNAR Tms (arrows). The same results were obtained in lane 3 using primer pair CH2/3⬘UT Tm. In lanes 5 and 7, using primer pairs CH5/Tm and CH5/3⬘UT Tm full-length, i.e., six-domain, IgNAR Tm expression was verified. IgNAR Sec is expressed only as a full-length form, shown in lanes 9 (CH3-Sec), 11 (CH3-3⬘UTSec), 13 (CH5-Sec), and 15 (CH5-3⬘UTSec). C, Sequences at the splice junctions of the four-domain (CH3/Tm) and six-domain (CH5/Tm) IgNAR Tm products. The Tm exon splices to the cryptic splice site in the CH5 exon and to the canonical splice site in the CH3 exon (3). 1136 DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING equivalent levels in adults (36). The deduced Tm form of the IgM H chain has five domains, as is seen in most vertebrates. Many IgM loci exist in all cartilaginous fish, and one gene cluster diversified through formation of a germline-joined H chain locus encoding the four-domain IgM1gj, so far found only in nurse sharks (2, 21). IgM1gj protein may have a function in early development as it is secreted in high amounts at birth. No Tm cDNA has been identified for this isotype and one may not exist. Results reported in this paper, together with previous data in batoids (8 –10), the frill shark (11), and lungfish (5) reveal that IgW has been selected to diversify its size using alternative mRNA splicing for two shortened Tm forms and a short Sec form, IgWshort. In contrast to IgW, only the IgNAR Tm form can be present in a truncated form and there are no multiple Sec forms. The structures, tissue distribution, and levels of somatic mutation differ among these three major Ig classes, strongly suggesting that each isotype has a distinct effector function, as shown for mammalian Ig classes (37). Thus, these ancient animals have a much greater complexity of Ig than previously believed, and it is predicted that the isotypes, and all their different forms, fulfill particular needs for their adaptive immune systems. Teleology for the short IgNAR and IgW Tm mRNA forms As described, bony fish splice the putative TM1 exon to the CH3 donor splice site producing a deduced membrane-bound IgM that is smaller by one domain (3). Tm IgW and IgNAR cDNAs are encoded by alternatively spliced products that result in putative truncation by two domains at the C terminus (and a second form of IgW Tm with only three domains, Fig. 8). It is difficult to fathom the functional reason(s) for the shortened Tm cDNA forms in teleosts. Teleost IgM Sec is unique among vertebrates in that it is tetrameric and assembles its quaternary protein structure in diverse ways through variable disulfide bonding between the monomers and half-mers (38). Perhaps a truncated IgM Tm on the bony fish B cell surface reduces or inhibits this structural diversity to ensure proper BCR signaling. For IgNAR, and by inference IgW, we propose a simple explanation for the alternative splicing suggested by our previous molecular and structural studies. Our previous work found IgW cDNA is homologous to IgNAR cDNA in the last four putative constant domains, i.e., these two isotypes shared a common ancestor ⬃220 million years ago, before the batoid (skate, ray) and shark divergence (6). Our structural study of IgNAR Sec proteins isolated by a specific mAb identified a highly flexible region between the third and fourth CH domains that were shown to bend up to 90° by immunoelectron microscopy (Ref. 13; inset, Fig. 8). In the putative IgNAR Tm form with three CH domains (Tm-2, Fig. 8) the alternative splicing would result in the removal of this bend region. The cDNA homology at their C termini between the two isotypes makes it likely that the same bend will be found in IgW Sec and alternative splicing to produce the truncated IgW Tm cDNA (VH-CH4) would result in its removal. We have preliminary evidence that this “bend” in IgNAR Sec proteins may be sensitive to proteolysis because Western analysis of IgNAR under denaturing conditions produces a smaller Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 4. IgW and IgNAR Tms are canonical. Horn shark and nurse shark IgW, IgWshort, and IgM, and nurse shark IgNAR Tm deduced residues were compared with Tms from all classes of vertebrate Igs and aligned in ClustalW. Shark IgW and IgNAR Tms contain the CART, composed of an acidic extracellular spacer region, a Tm region consisting of hydrophobic and polar residues, and a short cytoplasmic tail important for receptor signaling (31). All of the Tms contain the essential polar residues threonine (T), serine (S), and tyrosine (Y) (bold asterisks) in positions required for Ig ␣ association. Slashes (/) denote gaps added for alignment purposes and dashes (-) indicate residues identical to human IgM. The Journal of Immunology 1137 band the size of two CH domains recognized by the anti-IgNAR tail region specific mAb (H. Dooley and M. F. Flajnik, unpublished observations) which suggests it may be disadvantageous to express the “full-length” molecules at the cell surface. These preliminary data requires protein sequencing to verify the Western results. Evidence to date suggests that, despite their many clusters of Ig genes, each elasmobranch B cell expresses only one IgH locus (35, 39). Thus, if IgM, IgW, and IgNAR Tm are expressed clonally in each B cell, the shorter forms may limit proteolysis and permit signaling to occur in a similar fashion for all isotypes (at least regarding cross-linking of the receptor). Differential signaling among the isotypes may be, in part, indicated by amino acid differences in the cytosolic tails of IgW and IgNAR Tm, as compared to IgM (refer to Fig. 4). In contrast, Sec forms are composed of seven (IgW), six (IgNAR), or five (IgM) Igsf domains and this size may impact the type of effector responses generated when all three isotypes are induced in an Ag-specific response, i.e., if FcRs exist for all three isotypes on one type of hemopoietic cell, the longest one might “win” in the induction of a particular effector outcome. Role of IgWshort Sec in immune responses It has been more than a decade since skate IgWshort cDNA (formerly called IgXshort) was discovered and we now report that its ortholog in sharks is IgWshort cDNA, a miniature version of IgW. To date, IgWshort proteins have not been isolated in skates or sharks thus this discussion is based on mRNA molecular data. Like the deduced skate IgWshort, the shark deduced IgWshort consists of a VH-CH1-CH2-NC Sec tail. It can be expressed in late-term gestation nurse shark pups, just before birth and thereafter during ontogeny, and thus it does not appear to be developmentally regulated as seen for IgNAR and 7S IgM (21, 35). IgWshort is most highly expressed in the spleen, the only true secondary lymphoid tissue in cartilaginous fish. The high IgW levels in the spleen are of interest because that tissue is immature in newborn pups, colonized by IgM-bearing B cells yet lacking T cell zones and dendritic cells (35); consequently neonatal spleen lacks the environment necessary for Ag-specific responses. One possibility is that the IgW expressed by young animals is encoded by germlinejoined loci, yet this is unlikely as no germline-joined IgW locus has been discovered in nurse sharks after extensive library screens of adult and (especially) neonatal libraries. A second possibility is that IgW-expressing B cells develop earlier than B cells expressing other Ig isotypes in utero and receive the necessary (perhaps Tindependent) signals to become activated and secrete. This second choice can be tested once specific mAbs have been made that will identify the Tm and Sec forms of IgW and IgWshort. Variable IgWshort and IgW expression between individuals may be indicative of the amount or type of Ag to which these individuals have been exposed, and because IgW is secreted in significantly lower amounts than IgM and IgNAR, changes in its expression levels may be easier to detect (6, 21). In unimmunized adults (exposed to native ocean pathogens), IgWshort Tm and Sec forms are expressed in a novel site, the pancreas. As in mammals, the shark pancreas has exocrine (digestive enzymes) and endocrine (regulation of carbohydrate, protein, and lipid metabolism) functions (40, 41). Thus, in addition to secretion of digestive enzymes into the lumen of the intestine, sharks may secrete IgWshort as a GALT-type protection. Besides the pancreas, low levels of IgWshort were observed in multiple tissues; perhaps due to its small size and potential access to extracellular spaces, IgWshort-secreting cells may be enriched in these areas. There is a precedent in other vertebrates for expression of a truncated form of Ig: in ducks a short form of IgY exists that is derived by alternative splicing (42). It has Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 5. Hydrophobic and hydrophilic features of conserved Tm regions are maintained in IgW and IgNAR Tms. The Ig Tm region is based on conserved residues identified among vertebrate Ig classes (31). The IgNAR and IgW Tm residues starting after the tryptophan (W) (refer to Fig. 4) and ending one residue before the cytoplasmic tail were compared with the conserved Tm motif using a helical wheel, which displays ␣-helix amino acid residues at 100° intervals or 3.6 residues per 360°. Note that the amino acid characteristics in the shark Ig Tms form a conserved ␣-helical structure. 1138 DIVERSITY OF CARTILAGINOUS FISH Ig GENE RNA PROCESSING been suggested that this truncated form has been selected to limit inflammatory reactions and thus may be more of a neutralizing Ab (43). This is likely to be true in the cartilaginous fish as well, and the hit-and-miss nature of finding IgWshort Sec forms in different individuals provides an explanation for the previous proposal that sharks only had the long form of IgW Sec. Like in ducks, we suggest that the presence/absence of IgWshort is a consequence of the types of ongoing immune responses at the time of analysis. Have all of the cartilaginous fish isotypes and their different forms been discovered? Fig. 8 shows that the cartilaginous fish have a large assortment of Ig isotypes, however, there may be more. We were unable to clone two forms of IgW mRNA (one Tm and one Sec) that may encode high m.w. proteins that we have detected from time to time in immunoprecipitation assays (M. F. Flajnik and H. Dooley, unpublished data). Thus, we must remain open to the possibility that there are other types or forms of shark Ig. Interestingly, despite the FIGURE 7. IgWshort is differentially expressed among nurse shark individuals. Northern analysis of IgWlong and IgWshort splenic expression in various aged nurse shark individuals; adult (A) and neonate pup (P). A, An IgW VH probe reveals that IgWlong is expressed by all individuals, regardless of age, with lower levels observed in young animals. IgWshort expression is more variable, ranging from high in adults 3 and 4, to very weak in adult 1 and pup 4. Autoradiogram exposure at 3 days. B, An IgNAR VH probe shows that IgNAR is expressed abundantly in the mature individuals, and poorly in immature pups, as previously described (21, 35). Autoradiogram exposure at 1 day. C, IgM expression revealed with an IgM VH probe. Note that adult 3 has lower levels of IgM, yet is enriched for IgW and IgWshort expression (refer to A). Autoradiogram exposure at 4 h. D, NDPK probe-NDPK loading control. Autoradiogram exposure at 4 h. E, An IgW VH probe reveals further that all individuals regardless of age had highest expression of IgW in the spleen. Pups preferentially express IgWshort. Note that the three pups in E were from a different family than the two pups in A–D. Epi, epigonal organ; Spl, spleen. great evolutionary distance between different groups of elasmobranchs (up to 220 million years, two to three times as long ago as the last common ancestor of placental mammals); all of the isotypes except IgM1gj have been identified to date in all of the different species so far examined. Finally, are the different Tm and Sec forms truly derived by alternative splicing? The evidence presented here and from previous work in the skate (8) strongly suggest that all of the different forms can be encoded by a single locus. However, especially for IgW, the cDNA clones that we have isolated may have been derived from very similar loci, and future studies are required to Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017 FIGURE 6. Expression of IgM, IgW and IgNAR. Northern analyses of one adult nurse shark to examine the tissue distribution of IgW/IgWshort Tm and Sec, and IgNAR Tm and Sec. Refer to Table I for probes. A, An IgW Tm probe reveals three bands; the two bottom bands (nos. 2 and 3) at 2.6 and 1.3 kb represent the five and three Igsf domain forms, and the upper band (no. 1) has been resistant to cloning. Autoradiogram exposure 13 days. B, An IgW VH probe reveals four bands of IgW Sec. The band at 2.6 kb (no. 2) represents the seven-domain form (6) and the 1.2-kb band (no. 4) the three-domain form. No. 3 indicates cross-hybridization of the IgW V probe with IgM Sec, which is present in very high amounts in the spleen (see E). The highest m.w. band (no. 1) has been resistant to cloning. Autoradiogram exposure at 10 days. C, An IgNAR Tm probe reveals two bands in spleen and PBL. Autoradiogram exposure at 9 days. D, An IgNAR VH probe reveals that IgNAR Sec mRNA is a single transcript with broad tissue distribution. Film exposure at 6 days. E, An IgM VH probe reveals very high expression in spleen and a broad tissue distribution. IgM Tm can be seen as a 2.6-kb band in PBL and thymus (swamped out in the spleen by the Sec form). Autoradiogram exposure at 1 day. F, The housekeeping gene nucleoside diphosphate kinase (NDPK) was used as an RNA loading control. Autoradiogram exposure at 1 day. Hrt, heart; Sto, stomach; Mus, muscle; Spl, spleen; Int, intestine; Pan, pancreas; Liv, liver; Gil, gill; Rec, rectal gland; Thy, thymus; Kid, kidney; Brn, brain; Epi, epigonal organ; PBL; Eso, esophagus; Tes, testes; Olf, olfactory bulb. The Journal of Immunology isolate all of the IgW gene clusters from individual animals to confirm the splicing hypothesis. References 1. Flajnik, M. F. 2002. Comparative analyses of immunoglobulin genes: surprises and portents. Nat. Rev. Immunol. 2:688. 2. Litman, G. W., M. K. Anderson, and J. P. Rast. 1999. Evolution of antigen binding receptors. Annu. Rev. Immunol. 17:109. 3. Wilson, M. R., A. Marcuz, F. van Ginkel, N. W. Miller, L. W. Clem, D. Middleton, and G. W. Warr. 1990. 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