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* 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
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Although cartilaginous fish µ-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 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.
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Materials and Methods |
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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).
cDNA library screens, Northern blot analysis, and RT-PCR
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, 22, 23, 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 µg 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 µg/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.
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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 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; andIgXlong 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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 until directly demonstrated as membrane-associated proteins through biochemical characterization.
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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, 33, 34). The deduced IgWshort NC Sec tail in all species contains several Cys residues that could participate in either intra- or interchain disulfide bonds (or even J-chain association) and one potential Asn-linked glycosylation site.
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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 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.
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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, 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.
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/Ig
coreceptor association at specific positions (*, 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.
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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 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 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.
Individual nurse sharks express IgWshort mRNA differentially in secondary lymphoid tissue
Because IgWshort was believed not to exist in sharks in previous studies (2, 4, 7), we investigated Ig mRNA expression in the 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 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, 9, 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 µCH3 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 inteleosts. 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 µ 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 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 germline-joined 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 T-independent) 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 IgWshortwere 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 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 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 isolate all of the IgW gene clusters from individual animals to confirm the splicing hypothesis.
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Footnotes |
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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: Lynn.Rumfelt{at}sw.ca
3 Current address: Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, Research Triangle Park, NC, 27709-2233.
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.
Received for publication January 28, 2004. Accepted for publication May 14, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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light chains: implications for the evolution of light chains. Proc. Natl. Acad. Sci. USA 90:10603.
Globulins Little, Brown, and Co., Boston.
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