Immunoglobulin Heavy Chain Exclusion in the Shark

The adaptive immune system depends on specific antigen receptors, immunoglobulins (Ig) in B lymphocytes and T cell receptors (TCR) in T lymphocytes. Adaptive responses to immune challenge are based on the expression of a single species of antigen receptor per cell; and in B cells, this is mediated in part by allelic exclusion at the Ig heavy (H) chain locus. How allelic exclusion is regulated is unclear; we considered that sharks, the oldest vertebrates possessing the Ig/TCR-based immune system, would yield insights not previously approachable and reveal the primordial basis of the regulation of allelic exclusion. Sharks have an IgH locus organization consisting of 15–200 independently rearranging miniloci (VH-D1-D2-JH-Cμ), a gene organization that is considered ancestral to the tetrapod and bony fish IgH locus. We found that rearrangement takes place only within a minilocus, and the recombining gene segments are assembled simultaneously and randomly. Only one or few H chain genes were fully rearranged in each shark B cell, whereas the other loci retained their germline configuration. In contrast, most IgH were partially rearranged in every thymocyte (developing T cell) examined, but no IgH transcripts were detected. The distinction between B and T cells in their IgH configurations and transcription reveals a heretofore unsuspected chromatin state permissive for rearrangement in precursor lymphocytes, and suggests that controlled limitation of B cell lineage-specific factors mediate regulated rearrangement and allelic exclusion. This regulation may be shared by higher vertebrates in which additional mechanistic and regulatory elements have evolved with their structurally complex IgH locus.


Introduction
The adaptive immune system in vertebrates is founded on lymphocytes expressing a vast, anticipatory repertoire of antigen receptors. Only a single species of immunoglobulin (B cells) or T cell receptor (T cells) is allowed per cell. This restriction is termed allelic exclusion, and it describes the requirements for monoallelic receptor gene expression in each cell (for a recent review, see [1]). Allelic exclusion is considered the basis of adaptive immune system function, but how this founding principle was established in evolution has been only a matter of conjecture [2]. In this report, we present data that clarify the basis of allelic exclusion in the shark, representative of the most primitive organism with an adaptive immune system shared by mice and human beings.
The diversity of antigen receptors in lymphocytes is somatically generated by a recombination process in which various gene segments are joined together [3]. The number of gene segments and their organization vary amongst species but are generally comparable among all vertebrate classes, with the exception of the cartilaginous fishes, sharks and skates [4,5]. Figure 1 illustrates the differences between the complex mouse IgH locus and the multiple, minimalist shark clusters [4]. Elucidating their divergent and shared regulatory processes will allow us to understand the basis for allelic exclusion, the phenomenon that ensures specific recognition and response to pathogen invasion.
Each mammalian B lymphocyte must express an immunoglobulin (Ig) antigen receptor with a single specificity, although there are three loci that potentially encode two heavy (H) chains and four light (L) chains. The mouse IgH consists of an array of 200 V H gene segments spaced over 2 Mb and located upstream from 10-13 D, four J H gene segments, and eight constant (C) region genes [6] (Figure 1). Initiated by the RAG recombinase, the joining of V H , D, and J H gene segments generates the ligand-binding V region that encodes the N-terminus of the H chain polypeptide [7]. Accessibility [8] of the gene segments to the recombinase is tissue-, developmental stage-, and gene-specific [9] and is associated with their transcription, although the nature of this connection is not entirely elucidated [10,11]. During B cell differentiation, chromatin domains encompassing the D, J, and Cl genes become activated, probably through the intronic enhancer [12,13], allowing recombination of one of the D genes to a J gene segment. Only in B cells does V H to DJ H rearrangement take place to form VDJ, and this stage not only requires lineage-specific regulation, but sets in motion the process resulting in monoallelic H chain expression at the IgH locus. The chromatin domains encompassing the V H become activated, and locus contraction is required to bring most of them into close proximity with the DJ [14,15]. After the completion of a productively rearranged VDJ, the H chain is expressed at the cell surface, and this initiates a feedback process by signaling the next step in differentiation [16,17]. Since the V to DJ step is asynchronous between the alleles, the first functional VDJ rearrangement will encode the antigen receptor.
Based on these mouse studies, a model for the regulation of allelic expression was developed. Recent work has shown that many factors at various levels-stage-specific expression of RAG [18], differentially activated chromatin domains [19], locus contraction and decontraction [14,20], and subnuclear relocation [15,21]-are involved. Because of the large distances between a rearranged DJ and the available V H gene segments in animals such as mouse and humans, the locus contraction mechanism would appear to be part and parcel of the rearrangement process as well as its regulation. Moreover, the model is based on only one locus, that is, distinguishing one gene from its allele.
In contrast, IgM H chain in cartilaginous fishes (sharks and skates) is encoded by multiple , independently rearranging IgH loci ( Figure 1). What is more, these miniloci may bypass the need for locus contraction, which seems to be a key regulatory step for monoallelic expression at a single IgH locus in the tetrapod model. The question of how allelic The VDJ entity generated through the process ''V(D)J rearrangement'' is transcribed with one of the downstream constant (C) region genes, here simplified as single units (blue box is Cl). The V H is represented by olive boxes, preceded by the leader sequence in dark green, and flanked by the recombination signal sequence (RSS; white triangle) at the 39 end, that consists of heptamer and nonamer motifs separated by a 23bp spacer sequence. The RSS are the sites recognized by the RAG recombinase enzymes that mediate V(D)J rearrangement. As indicated, the distance between the 39-most V H and the first functional D is 90 kb [6]. The D gene segments, in red, are flanked on both sides by RSS (black triangles) containing 12-bp spacers, and the J H gene segments (orange) with 23-bp spacer RSS. After D to J rearrangement: the first stage of rearrangement involves recombination between D and J H , with the intervening DNA excised. The DJ product is depicted as a fusion of the red and orange boxes, with the RSS flanking its 59 end. Rearranging V to DJ: locus contraction and looping of the DNA allows distant V H gene segments to approach and recombine with the DJ. The final VDJ product is shown as Rearranged VDJ. Germline shark Ig H chain loci: the IgM H chain genes in sharks and skates (cartilaginous fishes) are multiple ''clusters'' or miniloci, each consisting of V H , two D, one J H , and one Cl gene (blue box). The gene segments in any nurse shark IgH gene are located about 400 bp apart as shown but are distant (e.g., 6.3-6.8 kb) from the Cl1 exon. The physical relationships among the loci are not clear except for one instance, where the genes G2A and G5 (both part of this study) were found linked and spaced 120 kb apart [23]. Rearranged VDJ: as demonstrated in this study, the four gene segments rearrange within the minilocus to VDDJ (called VDJ). In mouse IgH, gene rearrangement takes place in a strict order (D to J H before V H to DJ), but what the rearrangement process entailed in the shark miniloci was unknown up until this study. doi: 10

Author Summary
Lymphocytes provide a limitless repertoire of antigen receptors, but each lymphocyte expresses only one kind of receptor per cell in order to provide specific recognition and response to pathogen invasion. The restriction, called allelic exclusion, operates in tetrapod vertebrates from frogs to human beings. In mouse, immunoglobulin (Ig) heavy chain (H) exclusion depends on ordered activation of component parts of the highly complex, three-megabase IgH locus in a process that differentiates between the two alleles. However, the regulation and mechanisms ensuring allelic exclusion remain uncertain. Sharks represent the earliest vertebrates with an adaptive immune system; their IgH organization, consisting of multiple miniloci, is considered primitive and ancestral to the classical IgH locus in other vertebrates. We show that allelic exclusion nonetheless exists in shark B lymphocytes, although attained by alternative means. Thus, major aspects of the complex pathway described for allelic exclusion in mammals evolved with their IgH organization. Elucidating shared and divergent regulatory processes allows us to gain insight into the basis and evolution of allelic exclusion, which provides the foundation for the functioning of the adaptive immune system.
exclusion is managed in sharks has thus been a long-standing puzzle.

Shark Immunoglobulin Gene Organization
In the nurse shark, Ginglymostoma cirratum, there are about 15 IgM H chain loci per genome, and every functional gene contains one V H , two D, and one J H gene segments located within 2 kb ( [22]; Figure 1, bottom). These miniloci are located at least 120 kb apart, and aside from two IgH genes depicted in Figure 1, their linkage relationships are not known [23]. Among outbred individuals there can be 9-12 active IgH, classified into subfamilies called Groups 1-5. A detailed characterization of two functional loci [22][23][24] and 78 of their rearrangements show that V(D)J recombination took place within the minilocus ( [22,23] and V. Lee and E. Hsu, unpublished data). There do not seem to be longdistance recombination events between the widely separated IgH loci or, presumably, a major role for chromatin contraction in nurse shark IgH rearrangement.
To elucidate the rules for V(D)J recombination in the shark, we first investigated rearrangement patterns at the two defined shark H chain loci, asking whether differential V H , D, and J H activation existed in the short (;400 bp) intersegmental distances. We have found that all combinations are possible, and a completed VDJ is accomplished during one stage only, as if it were like the initial D to J step in mammals. Our results also confirmed that long-distance recombination between different IgH loci in B cells is rare, if it exists. Thus, two elements thought to be intrinsic to regulating the rearrangement process and resulting in allelic exclusion in mammals-ordered long distance recombination and chromatin contraction-are absent in sharks. Thus these findings tell us that certain mechanics of the rearrangement process can be dissociated from the phenomenon of allelic exclusion and that the two processes developed separately in evolution.
We investigated rearrangement in shark lymphocytes at the population and the single-cell level and established that H chain exclusion does occur in shark B cells, where only one or a few of its many IgH loci rearrange in any one cell. We also looked at IgH loci in shark thymocytes (precursor T cells, see Text S1). Although T cells do not express Ig, the IgH genes were extensively, although partially, rearranged; Ig transcripts were not detected. The differences between B cells and thymocytes demonstrated here suggest there exists in precursors to B and T cells an IgH chromatin state already permitting rearrangement, but in B cells it is further potentiated by lineage-specific factors, leading to efficient recombination at one or a few H chain genes and results in H chain exclusion. We propose that the molecular basis establishing allelic exclusion was achieved in the earliest vertebrates possessing Ig genes, and it is independent of the wide variation in Ig gene number observed in different species.

Results Overview
The experiments are summarized as follows. We first focused on how rearrangement takes place in one IgH subfamily, Group 2, in tissues and isolated cell populations. We demonstrated that partially and fully rearranged V H sequences can be amplified from lymphoid tissue DNA, but not from red blood cell (RBC) control DNA ( Figure 2). All anticipated genomic rearrangement configurations were obtained (Table 1). These data demonstrated that rearrangement in sharks is different from the ordered, two-stage process observed in mammals.
Lymphoid tissue can carry B cells, which express IgM, and T  cells, which do not. The initial experiments revealed the unexpected finding that somatic IgH recombination is also present in the thymus, an almost exclusively T cell-containing tissue. We further investigated this finding, using genomic Southern analyses. Compared to RBC and heart DNA, newsomatically rearranged-bands were observed in all lymphoid tissue DNA tested. These new bands were demonstrated to be V(D)J rearrangements mapping to predictable locations ( Figure 3). The recombined bands were identified by probes detecting all V H or only the Group 2 subfamily. A comparison of surface IgM-positive (sIgMþ) B cell DNA with thymus DNA showed that their respective patterns differed in the quantity and quality of the rearranged bands ( Figure 4). Thus, we discovered that V(D)J recombination is much more extensive but mostly incomplete in T cells. This result was confirmed through investigating the status of all functional IgH loci in thymocytes and in B cells by single-cell genomic PCR ( Figure  5). As predicted from the genomic Southern analysis results, only a few rearrangements could be obtained from single B cells, and these were fully rearranged VDJ; the other IgH loci were in the nonrearranged, or germline (GL), configuration ( Figure 6 and Table 3). Unlike in thymocytes, partial rearrangements ( Figure 7) were infrequent in B cells. These results show that in the developing B cell, there was a limited number of genes activated to rearrange, but once initiated, the recombination process went efficiently to completion.
In contrast, multiple and mostly incomplete Ig rearrange-ments were found in single thymocytes (Table 2), and neither Ig H chain transcripts nor L chain expression and rearrangement could be detected in the thymus ( Figure 8). This ability of DNA to act as substrate for RAG in the absence of transcription suggests a previously unknown state of chromatin activation. It was possible to detect this state only in an animal with multiple, independently rearranging sites, but such an observation signals that RAG may act on nontranscribing loci in other organisms as well. We propose that IgH in all shark precursor lymphocytes can be acted upon by RAG recombinase but that B lineage-specific factors are responsible for regulated rearrangement-and H chain exclusion-in the B cells.

V(D)J Rearrangements Amplified from Lymphoid Genomic DNA
PCR primers (Int/JH2; Figure 2) targeting the leader intron of Group 2 V H and the J H gene segment amplified DNA sequences of 1.6 kb from an individual shark (-JS) whole blood DNA ( Figure 2B, lane 2); this band contained the two functional Group 2 genes in the nonrearranged, or GL, configuration (see PCR and probes, [22]). The Int/JH2 primers amplified the same 1.6-kb fragment from erythrocyte DNA in other genetically unrelated individuals ( Figure 2B, lanes 4 and 6), demonstrating that Group 2 GL gene segments are in the organizational configuration depicted in Figure 1.
The intersegmental distances in Group 2 as well as other IgM genes are all about 400 bp ( Figure 1). A single, initial was performed with the vd2, dd2, dj2 probes in order to obtain the rare types, and V-D-DJ was isolated. These splenic clones are listed in Figure S1, with ''VD'' or ''DD'' as part of the clone name. b DNA was isolated from shark-GR PBL selected for sIgM expression. Colonies selected sequentially with dd2, vd2, dj2, and lastly, vh probes (see Figure 1, top) in order to isolate partial rearrangements. The rearrangement configuration was determined according to probe hybridization and EcoRI and AseI patterns (see footnote c); some clones were verified by sequencing. somatic rearrangement event (1R), such as joining of V to D1, would delete this interval and reduce the total V to J genomic span detected by the Int/JH2 primers to about 1,200 bp. Likewise, two rearrangement events (2R) would give rise to a PCR product of about 800 bp, and three rearrangements (3R) 400 bp. From lymphoid (spleen and peripheral blood leukocytes [PBL]) genomic DNA, a ladder of PCR products hybridizing to the vh probe ( Figure 2) can be detected, corresponding to the anticipated sizes of partial and completed genomic rearrangements ( Figure 2B, lanes 1, 3, and 5). The arrows at the left of lane 1 point to the fragments that were later cloned from all three sharks and identified as having one, two, or three rearrangements. Splenic lymphocytes and PBL from adult sharks do not express RAG recombinase ( [25]; W. Feng and E. Hsu, unpublished data), so that these rearrangement intermediates would be relics from earlier stages of lymphocyte differentiation.
No Strict Order in the Joining of Gene Segments PCR products obtained from shark-JS splenic DNA were cloned, and the insert sequences are classified by size in Table  1. Within each size group, different rearrangement combinations were found, but some are more frequent than others. The junctions ( Figure S1) show that each clone is unique, with the typical diversity generated by trimming and N/P region addition.
In order to determine which cells contained these rearrangements, surface IgM-expressing (sIgMþ) cells were isolated from PBL (see Figure S2). The PCR reactions performed on this population and the thymus both amplified sequences that showed higher frequency of V H to D1 joining (Table 1), but all combinations exist. The fully rearranged VDJ (i.e., VDDJ) from the sIgMþ cells tended to be in-frame, whereas in those from the spleen and thymus, the nonfunctional ones are in the majority (last two rows, Table 1). Thus, it appeared that IgH recombination had occurred in both precursor T and B lymphocytes. The pattern of rearrangement for Group 2, as demonstrated by the frequencies of the intermediate configurations, was similar in all samples.
Although the 59 primer is specific for Group 2, the 39 primer could target any IgH J H . If rearrangement occurred between Group 2 V H and another locus, it would have been possible to detect non-Group 2 intersegmental sequence in the partial configurations shown in Table 1. (All but one of the partially recombined clones had rearranged within Group 2 loci, as ascertained by sequencing or restriction enzyme analyses. In one thymus VDD-J clone, the V H originated from Group 2, but the D2, D2-J intersegmental sequence and J H were from Group 1; only four nucleotides belong to the D1 of either Group. If this sequence were a PCR artifact, the area of homology would have to have been in the N region sequence between V H and D1 or D1 and D2. We screened a total of 58 thymic VDD-J [unpublished data], but this was the only apparent instance of interlocus rearrangement outside these Group 2 IgH.) Two kinds of probes were used during screening, all of which had been derived from a GL Group 2 bacteriophage clone (Materials and Methods): the vh probe and the intersegmental probes, vd2, dd2, and dj2 ( Figure 2, top). . The predicted sizes of some 1R-3R fragments after rearrangement are immediately below (in blue). In both GL and rearranged configurations, these genes will resolve as DNA bands from approximately 3 kb to 630 bp and smaller after the double digestion. Group 7 is a pseudogene, as are Group 6 and 8 [23,24], whose vh-hybridizing fragments form the higher bands at greater than 4 kb. Rearrangements, if any, in the pseudogenes have not been observed by genomic Southern blotting. Center and right: Southern blot analyses of genomic DNA from shark-J PBL and RBC digested with BamHI and NcoI. Molecular size markers are shown on the left (k Hind III and 100-bp DNA ladder, NEB). The filter was hybridized with vh probe (center) after standardization with the ns3v probe (right). Band intensities were measured by phosphorimaging, followed by autoradiography with X-ray film. The 3.7-kb nonrearranging NS3 V region band (arrow) was used for standardizing transferred DNA (RBC lane ¼ 1, PBL lane ¼ 0.98) and verified by a comparison of TdT bands (RBC ¼ 1, PBL ¼ 1 [unpublished data]). The relative intensity of the 1.9-kb band in the vh probe panel was calculated as follows: (PBL vh probe score divided by 0.98) divided by RBC vh probe score. Using this calculation, the 1.9-kb PBL band is 77% of the 1.9kb RBC GL band. doi:10.1371/journal.pbio.0060157.g003 The latter were used to detect the infrequent recombination intermediates in these experiments (Table 1, footnote a; e.g., the Group 2 V-D-DJ configuration is vd2þ, dd2þ, dj2À). On genomic Southern blots, they proved to be specific for Group 2 only, whereas in contrast, the vh probe cross-hybridizes with nurse shark V gene segments from all subfamilies (see Figure  S3). In the following genomic Southern blotting experiments, these probes were used to detect rearrangement globally (vh probe) as well as specifically at three Group 2 IgH (vd2, dj2 probe) in lymphoid tissue DNA.

IgH Rearrangement Visible by Genomic Southern Blotting
All the genes encoding nurse shark IgM H chain have been cloned and the functional genes can be classified into five subfamilies, Groups 1-5 (see legend, Figure 3); the V H gene segments share .75% nucleotide identity [24]. The various vh-hybridizing bands in RBC DNA can be correlated with anticipated fragment sizes after BamHI/NcoI digestion . Compared to RBC DNA, the bands at 1,500 bp and 700 bp in PBL are more intense, and a new band appears at 1,100 bp. These three bands correspond to predicted configurations of rearranged DNA from the various IgH, but mostly from Groups 2 and 4 ( Figure 3, 1R-3R in blue). At the same time, the 1.9-kb band encompassing the Groups 2þ4 GL gene segments in PBL is ca. 23% less than that of the RBC counterpart (see Figure 3, legend), demonstrating loss of the GL band after acquisition of rearranged configurations.

Heavy Chain Loci Rearranged in Thymic Tissue
We observed that the relative amount of DNA rearranged was different between thymus (predominantly T cell) and sIgMþ cells (B cells from PBL). Although the images in Figure  4 are from X-ray films, phosphorimager analyses were performed for a quantitative analysis. We centered our analyses on depletion of the 1.9-kb band because it is a single GL configuration of known genes Group 2þ4, whereas ''gain'' measurements cannot be so clearly resolved. For instance, gain of signal in the 1.5-kb region means a combination of Group 2/4 1R plus nonrearranged GL Group 5, but minus an unknown amount of loss by Group 5 rearrangement.
To obtain a rough idea of the proportion of rearranged IgH in B cells only, the DNA from sIgMþ cells from shark-GR PBL was compared to DNA from its RBC ( Figure 4A). The ''flow-through'' sample is from the population mostly depleted of sIgMþ cells and consists of thrombocytes, (B) IgH rearrangement in thymus and spleen as detected by vh probe. Genomic DNA from shark-JS spleen, thymus, blood, and heart were digested with BamHI/NcoI and treated as described above. The blot was hybridized with vh probe (top) and with ns3v (3.7-kb band, bottom). The 3.7-kb ns3v signal was used as standard: RBC ¼ 1.0, spleen ¼ 1.1, and thymus ¼ 0.98. The 1.9-kb vh signals, calculated as described in Figure  granulocytes, and lymphocytes (T cells and some B cells that slipped through). An obvious difference between sIgMþ and the flow-through population is the greater intensity of the 700-bp band in the former ( Figure 4A). This band mostly contains 3R species, suggesting that most Group 2þ4 rearrangements in B cells are VDDJ.
There is a 19% signal reduction of the 1.9-kb Group 2þ4 GL band in the sIgMþ lane. The ''flow-through'' DNA also contained few rearrangements, as assessed by both loss of GL (8%) and gain of rearranged bands. However, unlike the sIgMþ sample, the ''flow-through'' was a mixture of cell types, and lymphocytes in PBL can range from 5%-30%.
DNA from shark-JS spleen, thymus, whole blood, and heart were compared. There were rearranged Ig bands in spleen and thymus, but these were not detected in blood or heart DNA ( Figure 4B). The frequency of lymphocytes in whole blood is Figure 5. Single-Cell PCR of Nurse Shark Lymphocytes Diagram: A first PCR round was performed with degenerate primers targeting the leader intron (''GR'' series) and J H (JH5) sequences of Group 1-5 genes. Aliquots from the first round were amplified in a second round of PCR employing nested degenerate primers in V H (''VG'' series) and in J H (JH6) that also collectively targeted the same genes. To identify the rearranged genes amplified by the VG/JH6 primers, separate second PCR rounds were done with JH6 in combination with five nested primers (Fam1, Int, GR3N2, Fam4, and Fam5) targeting leader intron sequence downstream of GR and specific to each of the five Groups. Top and middle: nurse shark thymocytes depleted of surface L chain-positive cells from shark-PI were picked by hand. After every second thymocyte, a RBC was picked as a check for the purity of isolation and processing. Top: A first PCR round was performed with the GR/JH5 primers targeting all Groups 1-5 genes; the nested round of PCR with VG/JH6 is shown in this panel. The expected band sizes are: 1.6 kb (GL), 1.2 kb (one rearrangement, 1R), 0.8 kb (2R), 0.4 kb (3R). Middle: one of the Group-specific nested reactions (primer pair Int/JH6), that targeting Group 2 genes, is shown in the middle. The DNA fragments of nested Group-specific PCR are expected to be overall about 52 bp longer than those described for the nested VG/JH6 reactions. Rearranged Group 2 products are identified in Figure S4. Bottom: surface L chain-positive peripheral blood leukocytes (i.e., B cells) from shark-GR were picked alternating with RBC for purity controls. PCR reactants and conditions are identical to that described in top panel. Each sIgþ PBL is flanked by a RBC. The names of the cells are shown per lane and correspond to those in Table 3. doi:10.1371/journal.pbio.0060157.g005 very low (0.02%-0.12%, 1 PBL/250 RBC), and in shark-JS, the heart tissue was bled out. The amounts of DNA in the first three lanes in Figure 4B are similar, and a comparison of the intensities of the 1,500-bp, 1,100-bp, and 700-bp bands between the spleen and thymus samples in Figure 4B suggests that more Ig rearrangements were present in the thymus DNA. Indeed, upon calculation, 60% of the thymus vhhybridizing GL 1.9-kb Group 2þ4 band was depleted.  Figure 5, bottom) were selected. To ascertain the rearrangement status of all IgH in a cell, the first-round PCR sample was subjected to nested PCR reactions with Group-specific primers, five sets for ''A'' and five sets for ''B'' (diagram). Top: the A series (Fam1, Int, GR3N2, Fam4, or Fam5 with JH6) detecting rearrangement are shown with the locations they target in GL and recombined (3R) configuration. The B series (G1DF/G1JR, FD2/RD2, G3DF/G3JR, G4DF/DR34, and G5DF/G5JR) detect only nonrearranged IgH and are shown with the locations targeted in the V-D and D-J intersegmental regions. Bottom: the A rearrangement panels show that 1-3 VDJ (arrow) can be detected in each cell. Each 3R band was cloned and subject to the analyses detailed in Figure S9 and S11. The B panels show the IgH remaining in GL configuration. The method of identifying the bands, as well as individual members of the Group 2 and Group 4 subfamilies, is detailed in Figure S10. The summary of the identification of the GL and VDJ genes in each B cell is in Table 3. doi:10.1371/journal.pbio.0060157.g006 In summary, in one B cell-enriched sample (sIgMþ cells from PBL), 19% of Group 2þ4 genes were rearranged and mostly to VDDJ, whereas in thymus, 60% of Group 2þ4 genes were rearranged, mostly to intermediate configurations.
In order to analyze these blotted DNA samples in more detail, we performed hybridizations with probes that detect only Group 2 IgH (G2A, G2B, and pseudogene G2C). The resulting bands can be correlated with Group 2 rearrange- Summarized results, analyses shown in Figures S4-S7 b Cloned sequences from thymocyte 9:   There are ten IgH loci characterized in shark-GR (NCBI accession numbers EU627680-EU627683 and EU719629-EU719633), classified into Groups 1-5. The VDJ were detected by 59 groupspecific primers with a universal JH primer. In-frame VDJ rearrangements that are potentially functional (VDJþ) are distinguished from those with stops in CDR3 (VDJn); out-of-frame rearrangement (VDJø) is marked with a null symbol. The CDR3 sequences are shown in Figure S11. The unrearranged GL sequences were amplified using group-specific primers in the V-D and D-J regions. The GL IgH genes of donor shark-GR were distinguished by restriction enzymes sites, as shown in Figure S10. b G2A in shark-GR and some other sharks consists of alleles V1 and V2 [22]. In KS23 the G2A out-of-frame VDJ is the V2 allele; the G2A 2R species VDD-J is the V1 allele. doi:10.1371/journal.pbio.0060157.t003 ment intermediates characterized in Table 1 ( Figures 4C, vd2, and 4D, dj2). The 1,500-bp (VD-D-J/V-DD-J) and 1,100-bp (VDD-J) bands detected by dj2 probe in thymus appear to be as intense as the 1.9-kb Group 2 GL signal and reflect the high frequency of these events (Table 1). Again, using the GL band as an internal reference, the Group 2 vd2 signal demonstrates that other Group 2 configurations (V-DD-J/V-D-DJ at 1,500 bp and V-DDJ at 1,100 bp) do exist but are less frequent, consistent with results from Table 1.

Single-Cell PCR
All the previous experiments were performed on mixed and purified cell populations, and although we can anticipate the general trend in T cells (many and partial rearrangements) and in B cells (few and completed rearrangements), this remains to be shown at the individual cell level. Singlecell analysis was made possible by previous studies in which all the GL IgH sequences in nurse shark have been characterized [22,23] so that degenerate, universal primers could be synthesized, targeting and detecting only the functional genes. Likewise, it was possible to devise primers specific for each Group, just as Int was specific for Group 2 genes. We first focused on thymocytes. We picked single thymocytes, performed single-cell PCR with the universal primers in a two-stage assay (Materials and Methods) and demonstrated the existence of multiple IgH rearrangements. For controls, an erythrocyte was picked after every two thymocytes. Out of 24 RBC, five failed to amplify and the remaining 19 showed only the GL bands. Of the 48 thymocytes, 44 had a variety of 1R, 2R, and 3R bands. Figure  5 (top) shows the results from the first 18 cells after the second round of PCR with nested universal primers. Other nested PCR was also performed with Group-specific primers to Group 2 ( Figure 5, middle panel, and Figure S4), Group 1 ( Figure S5), Group 4 ( Figure S6), and Group 5 ( Figure S7). The summary of these results is shown in Table 2. For the most part, little GL sequence can be detected, except in the RBC controls, suggesting either that most of the IgH had rearranged or that the many rearrangements caused the longer GL fragments to be out-competed. Either possibility is the result of widespread IgH rearrangement in the single thymocyte. The various anticipated rearrangements could be cloned from any thymocyte ( Table 2, footnote).
The thymocyte result is in contrast to what we obtained in B cells ( Figure 5, top and bottom, respectively). Using the identical PCR conditions and reagents, the PCR performed with the universal primers on surface L chain-positive B cells produced predominantly 3R bands. Moreover, GL bands were also present in almost every one of these samples. When several samples of B cell 3R fragments were analyzed on denaturing gels, they appeared to consist of only one or two species per sample ( Figure S8).

Heavy Chain Exclusion in Shark B Cells
We went on to identify the rearranged and nonrearranged IgH in single B cells. Using the Group-specific primers, we performed nested PCR on the first-round products of the single B cells (Figure 6, A amplifications) and found that each B cell carried one or only a few rearrangements. Each 3R band was cloned and the number of VDJ species determined per cell (detailed in Figure S9 and legend). We also amplified nonrearranged GL sequence from each cell by using Groupspecific primers directed to the intersegmental regions ( Figure 6, B amplifications) and identified the genes in each fragment by restriction enzyme sites. These tests were tailored for the donor, shark-GR, all of whose IgH were isolated and sequenced for this experiment ( Figure S10). Table 3 summarizes the results from 13 B cells. The CDR3 sequences of these VDJ are shown in Figure S11; the rearrangements in Table 3 are indicated as out-of-frame (VDJø) or in-frame (VDJþ) or nonfunctional (in-frame but containing stops, VDJn). All 13 B cells contained 3R rearrangements, and one cell (KS23) carried a 2R species as well (Figure 7).  [22]. The Group 2 subfamily contains two members, G2A and G2B. In the panel at the right, a nonrearranged Group 2 product found by the GL primers. This was, as expected, the G2B sequence which is distinguished from G2A by restriction enzyme analyses (supporting information Figure S10). doi:10.1371/journal.pbio.0060157.g007 We have shown a remarkable disparity between T cells and B cells in IgH gene configuration. In thymocytes, there are multiple and mostly partial IgH rearrangements per cell. Although we cannot claim to detect every VDJ rearrangement present in a B cell, the many IgH genes that remain in GL configuration support the observation that few IgH were rearranged in a single B cell. Many VDJ in Table 3 are out-offrame or contain stops, consistent with there being only one functional VDJ per cell.
In one cell, KM13, we found two VDJ that were both inframe (G1; G4CG) and carried no stops in CDR3 (Table 3), whereas the third VDJ is out-of-frame (G2A). One of the former (G4CG) encodes a CDR3 of 24 amino acids, an aberration among nurse shark cDNA CDR3, which range from 4-17 codons (average 11.6 codons, n ¼ 64) in one study [24] and 7-16 codons in another (adult G4 cDNA, average 11.3 codons, n ¼ 41, W. Feng and E. Hsu, unpublished data). However, the G1 VDJ not only contains a CDR3 of average size (11 codons) but is also the only one that has been hypermutated, and its mutations show evidence of positive selection (National Center for Biotechnology Information [NCBI; http://www.ncbi.nlm.nih.gov/] accession number EU719628). There are eight substitutions, with only those in the CDRs resulting in replacement changes. Three point mutation changes in FR2 and FR3 are synonymous, but the CDR1 point mutation (R to W), and the point mutation (Q to K) and 3-bp tandem mutation (S to R) in CDR2 all result in nonconserved changes. Tandem mutations are characteristic of the nurse shark hypermutation process [22], and the frequency of PCR-induced changes after 70 cycles in these studies is 0.14% (13/10,371 bp), or less than one change per 400-bp VDJ fragment. We do not know whether the VDJ with the 24-codon CDR3 encodes an IgM protein, but it is clearly not part of the selection process acting on this hypermutating B cell.
Perhaps, considering their very different CDR3 sizes, there is L chain preference for one polypeptide enabling its expression. We then ask, how often do two rearrangements result in similar CDR3? There are four cells (KM5, KM13, KS3, and KS23) in which more than one VDJ is present although most of these are nonfunctional. The junction sizes range widely. The number of nucleotides between TGT in the V H flank and TGG in the J H flank are 34 bp/45 bp in the KM5 VDJ, 39 bp/44 bp/78 bp in KM13, 24 bp/59 bp/72 bp in KS3, and 33 bp/41 bp in KS23 ( Figure S11). With six flanks trimmed and three sites for N region addition per VDJ, it seems unlikely that any two VDJ in a B cell, even if both are potentially functional, would have such similar CDR3 sequence content and loop sizes that they would combine equally well with the available L chain. Thus, constraints operating at two levels-the combination of the random nature of V(D)J rearrangement and L chain compatibilityserve to enforce H chain exclusion.
We propose that rearrangement ceases with the production of a successful H and L chain combination. There are few partially rearranged IgH present in B cells, as the 2R in KS23.
Here, the constellation of in-frame (presumed functional) G4 VDJ, the out-of-frame G2A VDJ, and the partially rearranged G2A 2R allele suggest that there was a signal for cessation of rearrangement for the G2A in VDD-J configuration once a viable l protein was generated.

Rearranged IgH Transcribed Only in B Cells
Ig transcripts from functional and nonfunctional rearrangements can be cloned from B cell-containing shark-JS lymphoid tissue using Int/JH2; we found that the use of a primer in leader intron selects for Ig transcripts unspliced in this region, the majority of which are from aberrant (out-offrame, partially rearranged) genes. The 3R (VDDJ) sequences were obtained from spleen cDNA, and many were mutated regardless of whether they were productive VDJ or not. Of the 17 2R events we cloned, two were VD-DJ, and one of them carried several mutations in the V region although not in the D-D intergenic sequence. Of 15 independent VDD-J clones, nine were mutants, of which seven contained substitutions Northern analyses were performed with total RNA (ca. up to 5 lg) from shark-JS thymus, epigonal organ, and spleen, and the blot hybridized with probes containing the sequences of: domains Cl3-Cl4, the transmembrane sequence of Cl, TCRb C region, NS3 C region and nucleotide diphosphate kinase (NDPK). Probes are described in Materials and Methods. These detected, respectively, transcripts of both the l H chain secreted and membrane form, l H chain membrane form alone, TCR b chain, NS3 L chain, and NDPK. There is considerably more of the secreted form of H chain than the membrane form, and an arrow shows the relative position of the latter transcript. Signal patterns and intensities from a second L chain probe, ns4c (not shown) were identical to those detected by ns3c. Signal intensities from vh probe (unpublished data) were similar to results from Cl membrane probe, mem. doi:10.1371/journal.pbio.0060157.g008 throughout V and the D-J intergenic sequence. The mutation patterns are typical of the type previously described in shark Ig, consisting of point and tandem mutations [26]. One such example, A36, is shown in Figure S12.
In contrast, there is very little Ig mRNA in shark-JS thymus, as observed by northern blotting (Figure 8), whereas these and other probes for nurse shark L chain isotypes detect abundant mRNA in spleen and epigonal organ. TCR b chain is abundant in thymus RNA. Reverse transcriptase PCR (RT-PCR) experiments using Int/JH2 to detect Group 2 2R in thymus cDNA were negative (unpublished data). Given the extent of thymic IgH rearrangement described in the preceding section, we conclude that if Ig transcription does occur in precursor T cells, the RNA species are at extremely low levels.

Light Chain Rearrangement in Spleen and Thymus
As the IgH rearrangements in thymus were a surprising observation, we investigated whether Ig L chain genes were also active in any way. The nurse shark L chains are encoded by three isotypes, NS3, NS4, and NS5 [27]. NS4 is most abundant (about 60 to 70 IgL), consists of both rearranging and germline-joined loci, and contributes about 90% of the L chain cDNA clones; neither NS4 nor the germline-joined NS3 could be detected in thymus RNA (Figure 8). In NS5, there are four genes, two of which can rearrange; they each consist of one V L and one J L gene segment and one C exon. Whereas somatically rearranged NS5 genomic sequences can be amplified from any source that contains B cells, none was observed in the shark-JS thymus DNA sample ( Figure S13). The rearranged NS5 band in the control spleen sample was visually apparent in ethidium gels.
In thymus, few if any NS5 genes somatically rearrange, and certainly not on the scale of the IgH. Thus, like in mouse, Ig rearrangements in thymocytes involve only the H chain loci.

Discussion
The mechanisms that contribute to generating H chain exclusion-differential chromatin domain activation, locus contraction-have evolved with and are a consequence of the complex mammalian Ig organization. In this study, we have shown that these processes are not necessary to effect H chain exclusion in all vertebrates. Our model, the nurse shark, provides a naturally minimalist IgH locus with four rearranging gene segments. Because rearrangement can be initiated by any gene segment pair, it seems unlikely that the spatially close V, D, and J elements are regulated separately from each other or subject to different chromatin accessibility constraints. Preliminary data from non-Group 2 subfamilies show that rearrangement patterns can vary considerably; for instance, in Group 5, V-DDJ is a prominent configuration that is rarely observed for Group 2 (Tables 1 and 2). Such observations suggest that, once the gene is accessible to recombinase, a preferred order of rearrangement is probably governed by locus-specific factors, for instance, the relative recombination efficiency of particular RSS pairs.
With one possible exception, the 97 1R/2R rearrangements isolated in this study (Table 1) occurred within the minilocus, supporting conclusions drawn from cDNA observations. Long-distance recombination events and sequential chromatin activation do not occur during the shark IgH V(D)J recombination process, demonstrating that in the absence of major aspects of the complex pathways described for mouse allelic exclusion, H chain exclusion will still be managed by limitation of rearrangement.
We established in this report that IgM receptors appear to be clonally expressed in nurse shark and likely all elasmobranch fishes. In one study in the clearnose skate [28] one to three different CDR3 l junctions were obtained by RT-PCR from single cells. Unfortunately, most of the 100-200 clearnose skate IgH are not characterized, and a number of them are germline-joined VDJ, which make these results difficult to evaluate. We have classified all nurse shark Ig H chain genes in a BAC library and determined those that are functional [23]. Our PCR primers target these genes only. We found ten functional H chain genes in the individual shark-GR and detected in its B lymphocytes one to three VDJ rearrangements per cell. At best, only one VDJ per cell was potentially functional. The other IgH were nonproductive VDJ or in GL configuration.
We believe that most elasmobranch B cells express one dominant H chain mRNA and one IgM receptor. Eason and coworkers [28] hypothesized that one gene is activated at a time, like in the multigene olfactory receptor system. A mechanistic connection seems unlikely, in the absence of an evolutionary relationship between genes encoding Ig superfamily and seven transmembrane domain proteins. From our studies, it appears that either a few IgH loci are rearranging at the same time in the pro-B cell or there are sequential ''tries'' before a viable H chain protein is generated. The answer is possibly in between.
Partially rearranged 1R and 2R configurations do exist in B cells as best demonstrated by cloning of mutated cDNA ( Figure S12) and the 2R species detected in B cell KS23 (Figure 7 and Table 3). The relic incomplete rearrangement configurations in B cells might suggest a feedback mechanism that functions with staggered initiation of rearrangement among loci. Alternatively, a few IgH are fully activated to rearrange, more or less simultaneously; hence the infrequent laggard 2R in the population. In such a scenario, there would be a limited but clear possibility for allelic inclusion. That we do not find many such examples suggests that the probability for two viable rearrangements is low, and as illustrated in the case of KM13, L chain preference DNA might permit only one H chain polypeptide for the receptor. As in mammals, ongoing shark IgH rearrangement probably ceases with the formation of a functional VDJ and expression of the IgM receptor. If L chain rearrangement occurs subsequently, the H chain loci might be transiently inactivated, as occurs with the non-expressed allele in mouse [20]. We have speculated that H and L chain rearrangement occur simultaneously in shark [29], and all rearrangement ceases with the formation of a viable cell surface receptor. However, there currently is no experimental evidence favoring either possibility.
The question remains, how are 15 or 100 IgH loci to be regulated if more than one gene can be activated per cell?
In point of fact, genetically manipulated model systems with more than two H chain genes have been studied. In interspecies hybrid tetraploid and triploid Xenopus [30] and in mice triallelic for IgH [31] allelic exclusion of H chain was observed, despite the increased number of potentially competing genes. There is no reason to believe that in these animals Ig expression is regulated any differently than their diploid version. If that is the case, H chain exclusion is initiated by nonsynchronously occurring rearrangement, and it does not matter how many available genes there are. It is generally accepted that the crucial step differentiating two alleles or multiple genes should be at V to DJ stage [32]. However, in the shark, there is no such second stage; the asynchrony must occur at the initiating step of rearrangement.
Liang and coworkers [33] inserted a GFP reporter into the kappa locus to mark its activation and found that the gene was transcribed at an unexpectedly low frequency in pre-B cells. They suggested that allelic exclusion at the kappa locus is based on probabilistic enhancer activation. Possibly a predetermined allele preference [34,35] contributes to the initial choice, but it was also suggested [33] that a competition for transcription factors would forestall activation of the second gene.
We observed few but mostly fully recombined IgH per shark B cell and propose that there are limiting amounts of trans-factors that target a gene for highly efficient, processive rearrangement, such that however many IgH genes are in the genome, recombination in B cells does not commence at the same time at more than one location. The focused activity at a few IgH also may have the effect of draining other components from general use. Since shark IgH genes lack the usually well-conserved upstream octamer motif [22,36], their trans-factors must differ from and are not competed for by L chain genes if they rearrange at the same time. The first compatible and viable H and L chain combination forming a receptor will generate the feedback signal. If by chance more than one viable H chain is produced at the same time, they may be differentiated by their ability to pair with the available L chain.
The surprising finding in these studies is V(D)J recombination at multiple IgH loci in every thymocyte, and despite the numerous H chain rearrangements present, Ig transcripts are not detected. The majority of thymic IgH are left incomplete as 1R or 2R, further underlining the difference of their estate from that in B cells. Since these IgH genes are not transcribed as in B cells, despite the extensive rearrangement, and are mostly not fully recombined, essential components are obviously lacking in thymocytes. Taken altogether, we propose that in those thymocytes which are in the process of actively recombining their TCR genes also harbor IgH in a rearrangement-permissive state, and this is possibly a prelude to full activation of the chromatin, which can only be achieved in the presence of B lineage-specific components that would include IgH transcription factors. Since cDNAs of rare, aberrant rearrangements of Ig V gene segments to TCRc have been observed in a shark thymocyte cDNA library (M. Criscitiello and M. Flajnik, unpublished data), we conclude that factors capable of binding the Ig promoter (and perhaps eliciting local chromatin remodeling [37]) could be present in thymocytes.
Most recently, transcription has been shown correlated with rearrangement competence and induction of chromatin changes [11]. One commentary [38] speculated on the connection between transcription, chromatin remodeling, and recruitment of RAG, pointing out that RAG2 contains a methyl lysine-binding region that may act as a reader for the histone code of the chromatin and thus may act differentially depending upon the pattern of the histone modifications. It is currently thought that the formation of a Ig/TCR promoterenhancer holocomplex, consisting of a complex of nuclear factors-DNA interaction, directs the chromatin remodeling and DNA modifications that promote chromatin interaction with RAG [39,40]. We propose that the limited number of rearranged IgH per shark B cell is a result of infrequent formation of the holocomplex, which contains lineagespecific factors. These ideas are summarized in Figure 9.
In the absence of B cell-specific factors participating in this holocomplex, IgH in shark precursor lymphocytes may still achieve alternative states of accessibility that are not optimal but not prohibiting for unregulated rearrangement. We propose that a quasi-activated level of chromatin accessibility can exist, supports interaction with RAG, and has distinguishable characteristics.

Materials and Methods
Animals. Shark-JS, -GR, -J, -Y, -BL, and -PI (G. cirratum) were captured off the coast of the Florida Keys and maintained in artificial seawater at approximately 28 8C in large indoor tanks at the National Aquarium at Baltimore. Shark-GR was 7 y of age at the time of bleeding. Whole blood was obtained from the caudal sinus and passed through a Ficoll gradient to separate PBL from RBC. Shark-JS was about 5-6 y of age when sacrificed, and its organs were harvested and frozen. Shark-PI was 3-4 y of age. The thymus was dissociated, passed through a cell strainer mesh (Falcon 2235), and subjected to magnetic cell sorting (see below). DNA and RNA were obtained from PBL and frozen tissues using routine procedures. DNA can be extracted from the RBC, which are nucleated.
The blots were subjected to autoradiography, and signal intensities of bands were quantified using a Storm 860 phosphorimaging system with ImageQuant software (Molecular Dynamics). Separation of membrane-bound IgM-positive cells by magnetic cell sorting. For IgMþ selection, the buffy coat was resuspended in a mixture of shark IgM-specific mAbs (CB5, CB11, and CB16; [43]), and then with goat-anti-mouse IgG Microbeads (Miltenyi Biotec). Approximately 1.5-5 3 10 7 cells were collected after two rounds of column purification (Miltenyi Biotec). The negative population was collected as the ''flow-through'' from the first round of magnetic activated cell sorting (MACS). The positive cells were small and round (lymphocyte-like) cells, whereas the negative population contained cells of different shapes and sizes. Thymocytes were mixed in medium containing a mAb specific for nurse shark NS4 L chain C region (LK14; [44]; E. Hsu, unpublished data), and the L chain-negative cells were collected as ''flow-through'' from the MACS LS column.
Single-cell PCR. Cells were collected after magnetic cell sorting, and RBC were obtained from the same individual for negative controls. Single cells were picked by hand under an inverted microscope with a finely drawn microcapillary pipette (Fisherbrand, #21-164-2G). MAC-sorted lymphocytes were picked, alternating with RBC from another dish; the pipette was rinsed three times in between. The cell was deposited in a 1-1.5-ll volume in shark PBS; 5 ll of lysis solution (13 PCR buffer, 10 mM DTT, 0.5% NP40) was added, topped by mineral oil, and the tubes were heated at 65 8C for one minute to break the nuclear membrane. The tubes were stored at À20 8C until needed. One hundred microliters of 13 PCR solution with dNTP, 0.5 units AmpliTaq (Roche) and primers targeting the V H and J H sequences of Groups 1-5 (two 59 primers: 20% GR1, 59-G T T T C T C T A C C T C A G C A A T -3 9 a n d 8 0 % G R 2 -5 , 5 9-GTTAGTCTMCCTCTGGAAT-39 with the 39 primer JH5, 59-TCA-CIGTCACCATGGT-39) were added and the reactions run for 39 cycles at 95 8C 1 min, 58 8C 1 min, 72 8C 1 min, and in the 40th cycle the elongation step was prolonged to 15 min. In the nested reaction, one microliter of the PCR products was added to 50 ll of a second mixture containing two 59 primers (20% VG1, 59-AAGGTGTC-CAATCGCAA-39 and 80% VG2-5, 59-AAGGTGTCCAGTCGGAG 39) with the 39 primer JH6 (59-TCACCATGGTYCCTTGT-39), and this reaction was run for 20-30 cycles at 95 8C 1 min, 54 8C, 1 min, 72 8C 1 min; again the elongation step was prolonged in the last cycle. The DNA patterns were identical for 20, 25, and 30 cycles. The universal 59 primers used in the first PCR round are located in the leader intron whereas the nested universal 59 primers are in FR1 of the V H , about 60 bp downstream.
There are two sets of nested Group-specific reactions used to analyze the B cells, one set to identify the 3R fragments observed above, the other to ascertain which IgH remained in GL configuration. For both, the first-round PCR samples were subjected to ExoSAP-IT (USB) to remove remaining ''GR'' primers. Six microliters of the PCR sample was incubated with 2 ll of ExoSAP-IT for 15 min at 37 8C, followed by inactivation for 15 min at 80 8C. One microliter of the product was used in a 50-ll PCR reaction. For nested reactions to ascertain VDJ identity, the 59 primers targeted unique, Groupspecific sequences in the leader intron, up to 15 Figure S7. The dj5 probe was generated from Group 5 GL sequence and the primers DJF-2 (59-TCAGTGTKTACTTTTAC-39) and DJR-2 (59-ATCAMGAYA-WAYCTTCA-39). The first lane (vh probe, HincII digest) is from [23].       Figure 4, bottom). NcoI leaves a recessed 39 end that can be filled in with 32 P-CTP. The end-labeled samples were denatured and loaded on a sequencing gel along with the sequence of M13mp18 (lanes marked GATC). The blue dot at the bottom marks the C position at 339 bases of the phage, sequenced with the À40 primer (Sequenase Version 2.0, USB). There is some labeling in the absence of NcoI digestion (first and last lanes, L7 and K5). Lanes L7, L17, K8, and K5 show one band, whereas K11 show two (cloned as VDJ from Groups 4 and 5). Lane N11 was an artifact at 450 bp (determined by sequencing). The technique had been devised as a method of distinguishing Ig sequences utilizing the variability at CDR3 [45]. Found at doi:10.1371/journal.pbio.0060157.sg008 (1.54 MB TIF). Figure S9. Testing the Number of VDJ Species per 3R Band Every non-GL band found in the single-cell reactions (Figures 5-7) was cloned into pGEM vector after excising the band from an agarose gel and eluting the DNA using Qiagen columns. Usually the same sequence was obtained repeatedly in three to five clones. We determined whether there was one or more VDJ in the 3R band the following way. (1) Single bacterial colonies from the transformation would be suspended in LB medium and an aliquot subjected to PCR using the T7 and Sp6 primers to amplify the inserts. The 15-40 such PCR products would be digested with restriction enzymes, using a site in CDR3 that was found in the original sequence (listed in Figure  S11). Every nondigested band was directly sequenced. (2) The original PCR product (both the universal primer product as in Figure 5 and the Group-specific product in Figures 6 and 7) would digested with the same restriction enzyme to ascertain whether all components of the band were digested. This is illustrated in the Panel KM3/G2 where the PCR product raised by Group 2-specific 59 primer Int (and JH6) was completely digested with Mwo I. In the panel KM5/G4 the first sequenced plasmids contained a VDJ with an EcoRV site. The 3R fragment did not digest completely (as shown in lane 2), which suggested that a second, EcoRV-negative VDJ was present. Among 45 bacterial colonies, 18 carried an EcoRV site and 27 did not. The latter clones were grown up and sequenced, showing VDJ that carried a HaeIII site. Thus the cell sample KM5 carried two rearrangements of the G4 subfamily. Sometimes a GL sequence was amplified along with the 3R band as in panel KM15/G5. The VDJ carried two MseI sites, one in the V H and the second in CDR3, as diagrammed below the photograph of the gel. The 441-bp VDJ is expected to be digested by MseI into three fragments, 264 bp, 110 bp, and 67 bp. The 110-bp fragment is diagnostic and indicated with an arrow. There are multiple sites in the GL fragment, but these are present at a fraction of the VDJ and do not interfere with the interpretation. In summary, all the VDJ listed in Figure S11, except in cell KM5, were the only species present in the 3R band, and the results appeared as shown for sample KM3 or KM15. The enzymes used are listed in Figure S11.   1,224 bp). I. Group 1 GL contains unique PvuII site in the V-D region, which can be detected as shown in agarose gel at right. GL sequences were amplified from single cells, which were confirmed to be G1 by presence of PvuII site. C is control, no enzyme. II. Group 2A GL sequence can be distinguished from Group 2B by two sites, NdeI and EcoRI, which are absent in G2B. In the gels, sample 1 carries G2B but not G2A; and sample 2 carries G2A but not G2B; the rest (3-5) carry both G2A and G2B. III. Group 3 GL contains an ApaLI site in the D-D region (as shown) but is negative for PvuII, EcoRI, and EcoRV (unpublished). IV. The four Group 4 GL sequences can be distinguished by a series of restriction enzyme digestions. A combination of SspI and EcoRI demonstrates whether G4A (607, 273, and 265 bp) are components of the PCR products. The bolded fragment, 607 bp, is indicated by the blue bar in panel IV, G4A, and is present only (arrows) in samples 1 and 4 in the first gel at left. In this photograph, the marker lane was transposed closer to the four sample lanes than it was in the original gel. To distinguish whether the G4 samples whose PCR products contain G4D or G4A or both, they were incubated with SalI (G4D: 796, 348 bp, G4A: 1019, 126 bp). The diagnostic band in the SalI gel are marked with arrows. C is control, no enzyme. The presence of G4CG and G4E involve somewhat more complex digestion patterns but the diagnostic bands are distinct. Digestions of the GL PCR product with ScaI and EcoRI provide only G4CG with a 186-bp fragment   Table 3 are aligned under the GL flanks of the V H and the J H gene segments and the coding regions of D1 and D2. The trimmed positions are shown with dashes, for gaps, and other sequences are assigned as N or P nucleotides. The GL sequences were cloned from shark-GR, so that mutated positions could be identified clearly (indicated as lower case). ''þ'' indicates in-frame potentially functional sequence, ''non'' is in-frame CDR3 with stops (underlined), null symbol indicates outof-frame sequence. The VDD-J in KS23 has undergone only two rearrangement events. The CDR3-based restriction enzymes listed at the right were used to detect the number of VDJ species per 3R band. Found at doi:10.1371/journal.pbio.0060157.sg011 (36 KB DOC). Figure S12. Transcripts from Partially Rearranged Heavy Chain Genes Show Evidence of Hypermutation Top, diagram showing positions of two of the PCR primer pairs used and the transcripts detected relative to the GL genes. RT-PCR was performed on shark-JS spleen RNA with various PCR primer combinations described in Materials and Methods, and the products were cloned and sequenced. Bottom, representative mutated sequences are aligned to the GL gene G2-V2, whose V H , D1, D2, and J H gene segments are labeled. The CDR1 and CDR2 in the V H are underlined; RSS are bolded, as is the leader intron acceptor splice site. Clone A36: Int/JH2 primer pair, two-rearrangement VDD-J sequence (accession number: DQ857392). Clone LVD1-3: Int/RSSD1 primer pair, VD1 sequence, derived from VD-DJ or VD-D-J transcript. Clone DDC4: IntDD-F/V18C2-39 primer pair, DJC sequence (accession number: DQ857391), derived from transcript carrying VD-DJ or V-D-DJ; its Group 2 C region sequence is not shown. These clones were chosen for the presence of mutations throughout the sequence; no portion was shared with any non-Group 2 sequences. The tandem substitutions are typical of hypermutated shark Ig [26]. Dots denote identity with the reference sequence, dashes gaps. Substitutions are shown in capital letters, N region in lower case in front of the D or J H sequence. Insertions are marked with arrow. Sites of the PCR primers are indicated in brackets. Found at doi:10.1371/journal.pbio.0060157.sg012 (856 KB TIF). Text S1. Glossary of Terms Found at doi:10.1371/journal.pbio.0060157.sd001 (82 KB DOC).