Derived amino acid sequences of NS4 genes. The amino acid sequences of most of the cloned NS4 genes shown in Fig. 2 are compared with R4. Dots indicate identity, dashes indicate gaps. R19 and S32 contain stop codons (asterisks), and one nucleotide was left out of CDR3 in R19 in order to obtain a reading frame that is comparable to the other sequences (indicated by a hyphen). R6 carried too many disruptions and is not shown.

Hypothetical scheme for evolution of Ig L chain genes. The precursor Ig gene underwent gene duplication, generating the V and C exons. Sometime in or before the early Devonian, >400 Myr BP, a mobile DNA sequence was inserted in the V exon, splitting it into V and J gene segments. Dark arrows on the DNA insert indicate the RSS. Gene duplication of the individual genes in tandem generated the overall L chain Ig gene arrangement in tetrapods, whereas duplication of V, J, and C genes produced the multiple cluster organization common in cartilaginous fishes. Based on diagram from reference 4.

Genomic Southern blot hybridized with probes to the coding and intervening regions of NS4. Top, erythrocyte DNA from shark Y was digested with EcoRI, electrophoresed on a 1.2% agarose gel, and transferred onto a nylon filter. The filters were hybridized with radiolabeled DNA sequences from the V region of NS4 (V) and the intervening sequence between V and J (I). Arrows in the EcoRI lane point to the location of V-hybridizing bands at 2.1, 2.4, and 4.6 kb that were not detected by the I probe. These were cloned and contained germline-rearranged NS4 genes. Arrow in the PstI lane points to a fragment of 727–744 bp containing joined genes R4, R7, R18, R6, and R19, predicted from DNA sequence in Fig. 3 (dots over positions 79 and ∼816); accordingly, this fragment does not hybridize to I probe. The slower migrating bands in the same lanes at ∼1,150, 1,250, and 1,400 bp correspond to sizes expected with the presence of an intervening sequence of ∼500 bp; these bands accordingly hybridized with both V and I probes. Molecular size markers from λ phage DNA digested with HindIII are shown. The PstI fragment sizes were calculated from another gel; not shown. Bottom, location of V and I probes from cloned split (S5) and joined (R4) genes.

Nucleotide sequences of NS4 genes. The sequences of cloned NS4 genes containing the leader, V, and J exons are compared with the first sequence, R4. Dots indicate identity, dashes indicate gaps. R4, R7, R18, RE18, R6, and R19 are germline rearranged as VJ. S1, S10, S31, and S32 are not joined, and their V gene segments are shown up to but not including the RSS. Slashes indicate that the intervening sequence and the RSS on either side are left out. These sequences recommence with the coding region of the J gene segments and the 3′ noncoding sequence. Regulatory signals as the octamer and splice sites are boldfaced, and their locations are based on homology with other vertebrate L chain genes. The location of the 3′ splice signal of J is derived from NS4 full-length cDNA sequence, where the amino acid preceding the first residue of the constant region is arginine (reference 15), so that the last nucleotide in J is adenyl. The coding region is underlined; the leader sequence is split by an intron. The beginning of the V region is indicated by an asterisk over the first base. The division of the V region into FR and CDR is indicated (reference 19). The two PstI sites (CTGCAG) referred to in the Fig. 2 legend and text are indicated by bold dots.

Derivation of the joints in NS4 germline-rearranged genes. The V and J flanks of split genes are shown without RSS. The possible processing events producing the joints of germline-joined R7, R4, RE18, R6, R19, and R18 are shown aligned with the most similar nonjoined NS4 coding ends isolated in these studies. Assuming that the split ancestor of R7 resembled S10, the R7 joint could have been produced by (a) cleavage at the RSS and relegation of blunt ends, or (b) cleavage at the RSS, formation of hairpin at V and J ends, single-stranded nick occurring two or more nucleotides from end, processing resulting on P region (underlined), and deletion of two bases from the J flank. Pathway R7 (a) does not preclude hairpin formation, but there is no processing evidence for it. Under cDNA, germline split S10 is compared with cDNA sequences obtained by RT-PCR from shark Y that share similar CDR3. The analysis of the cDNA joints suggest P region in both sequences. The sequences in the cDNA joints that do not appear to belong to either V or J may be N region. R4 is similar to R7. RE18 joint could have resulted from deletion in the V coding end (a), or J coding end (b), or both (c). The flanks of nonjoined ancestor of R6, R19, and R18 could have resembled S32, in which case deletion only from J end occurred during recombination (a). In all other possibilities, nucleotides were removed from either end. Examples of coding flanks from NS4 split genes are included for comparison; the RSS resemble those from other vertebrates. Clade I refers to the cluster of genes including R4, R7, and R18, clade II to that including R6, R19, and RE18 (see Fig. 6); clade III genes are characterized by divergent FR3 and CDR3 of 24 bp; complete sequences are not shown. Asterisk indicates split gene is pseudogene. S32 is shown in Fig. 3 and Fig. 4; S15 has TGA in FR2; S5 sequence is not shown but carries TGA in FR3, a GT–AT splice signal combination in leader intron, and noncanonical RSS heptamer sequences.

Germline origin of rearranged NS4 genes. (A) EcoRI-digested DNA from seven wild nurse sharks hybridized with V and I probes. Lanes 1–3, DNA from RBC fraction of ficolled blood of three individuals; lanes 4 and 5, DNA from whole blood (WB) of two sharks; lanes 6 and 7, DNA from erythrocyte (RBCs) fraction (lane 6) and PBL fraction (lane 7) of ficolled whole blood from shark Y. The genomic DNA libraries were generated from these samples (see Materials and Methods). Lane 8, DNA from erythrocytes from shark B. The DNA samples were digested with EcoRI, electrophoresed together on one 1% agarose gel, and transferred to a nylon filter; the filter was cut in two and one half was hybridized to probe V, the other to probe I (see legend to Fig. 2). Arrows indicate bands at 4.6, 2.4, and 2.1 kb that hybridize to V but not to I. The band at 5.4 kb is variable in hybridization to I probe among individuals. When the V and I films were superimposed, the bands at 2.4 kb detected by V probe are distinct from the faint bands at ∼2.5 kb detected by I probe. (B) Identification of R4/R7 sequences in genomic DNA. The DNA from shark Y obtained from erythrocytes (lanes 1 and 2) and from PBLs (lanes 3 and 4) was digested with EcoRI and ClaI (lanes 2 and 4) or EcoRI alone (lanes 1 and 3). Hybridization with V probe showed that some of the EcoRI fragments containing NS4 V genes carried ClaI sites as well. Arrows point to ClaI and EcoRI-digested fragments that hybridize with V probe at 1,550, 1,450, 1,150, and 980 bp. The markers used are λ HindIII and φX174 HaeIII, at 4.36, 2.3, 2.0, 1.35, 1.08, and 0.872 kb. The interpretation of the digestion patterns is shown in the diagram. Top, split NS4 genes at 2.6 and 2.7 kb such as S1 and S31 with ClaI site in CDR2. Bottom, joined NS4 genes at 2.1 kb such as R4 and R7 with ClaI site in CDR2. Joined NS4 genes such R6 and R19 at 2.1 kb are not expected to be affected by ClaI, so the 2.1-kb signal in lanes 2 and 4 is reduced but not abolished.

Phylogenetic tree of shark L chain V sequences. The relationships among the nurse shark NS4 family of genes are emphasized by bold branches. Other L chain types from nurse shark (NS3 and NS5) and horned shark (types I–III) were included in the analysis and used to root the tree. Branch lengths were estimated by likelihood using the HKY85+I+Γ model (reference 23), correcting for invariance and rate variation across sites as detailed in Materials and Methods. Nodes representing presumed speciation events are indicated as circles; e.g., node B corresponds to the divergence between all nurse shark NS4 genes and all horned shark type III genes. Nodes representing the most recent common ancestor of all members within a particular gene family are designated by ♦; e.g., all the NS4 genes derived from a common ancestral gene at node A and all the type III genes derived from a common ancestral gene at node C. Arrows designate branches in which germline rearrangements in NS4 genes most likely occurred. Lengths of internal branches (numbered segments) are approximately proportional to the estimated accumulation of nucleotide substitutions per site (scale at bottom), except for branches 13 and 15, which are ∼10 times as long and are truncated in the figure. The most strongly supported branches are 1, 4, 7, 8, 10, 12, 13, 14, and 15. For example, in one of many parsimony jackknife analyses of only the coding regions (assuming transversions were weighted twice that of transitions, ignoring alignment gaps, with 2,000 replications of a random selection of 50% of the nucleotide characters), each branch appeared in the following percentages of replicates (heuristic searches with 1,000 random taxon additions each): 1, 88%; 2, 61%; 3, 71%; 4, 82%; 5, 66%; 6, 51%; 7, 97%; 8, 86%; 9, 61%; 10, 95%; 11, 31%; 12, 98%; 13, 100%; 14, 100%; and 15, 100%. In a similar analysis that included noncoding regions (and excluded taxa for which these regions were not sequenced), jackknife values were (2,000 replications of exhaustive searches): 1+2, 37%; 3, 47%; 4, 75%; 5, 50%; 6, 98%; and 7+8, 94%. Horned shark cDNAs h4, h5, and h6 (available from EMBL/GenBank/DDBJ under accession nos. L25561, L25562, and L25563, respectively) were isolated by cross-hybridization with NS4 sequence. Based on separate phylogenetic analyses of V and C regions, type III was identified as a homologue of NS4 (reference 13); this conclusion is also supported by the current phylogram. All available type III sequences grouped strongly together, as did all the representative NS4 sequences (germline-rearranged R7, R4, R18, R6, R19, and RE18, and split S1, S10, S31, S32, and SE7), suggesting gene diversification after the species diverged. Pseudogenes are indicated. Other sequences are from different L chain types in horned shark (type I, X15316; type II, L25560) and nurse shark (NS3 [reference 16], NS5 [reference 39]). SE7 is the most basally divergent member of the NS4 gene family identified to date, and its sequence is not shown in Fig. 3.