Isolation of a shark immunoglobulin light chain cDNA clone encoding a protein resembling mammalian K light chains : Implications for the evolution of light chains ( cartilaginous fish / A light chains / phylogenetic trees )

Content Provider	Semantic Scholar
Author	Greenberg, Andrew S. Steiner, Lisa M. Kasahara, Masanori Flajnik, Martin F.
Copyright Year	2005
Abstract	The time of emergence of immunoglobulin K and A light (L) chains in evolution is unknown. An L chain cDNA clone was isolated from the nurse shark (Ginglymostoma cirratum), a cartilaginous fish, whose predicted variable (V) region amino acid sequence has up to 60% sequence identity to mammalian V, domains. Genomic analyses suggest a clustertpe gene organization for this L chain locus, similar to the shark A-like immunoglobulin L chain loci rather than mammalian K loci. We propose that divergence of the ancestral L chain into isotypes likely occurred before the emergence of elasmobranchs 400-450 million years ago. Similarities in gene organization between the two isotypes in sharks may reflect the gene organization utilized by the ancestral L chain. Immunoglobulins are the central effector proteins in the humoral adaptive immune system and are composed of two heavy (H) and two light (L) chains. In mammals, immunoglobulin L chains are designated as K or A, which are believed, on the basis of sequence similarities, identical domain structures, and a common function, to have arisen from an ancestral L chain gene (1). However, the timing in evolution of divergence into K and A isotypes is unknown, since L chains in nonmammalian vertebrates have not been studied extensively. Chickens have L chains apparently only of the A isotype (2). Two L chains have been characterized in the amphibian Xenopus laevis; one appears K-like (3, 4), and the other is not more related to mammalian K or A (5). The L chain constant (C) domain of another amphibian, Rana catesbeiana, cannot be clearly classified as CK or CA (6). A-like L chains are present in two species of sharks (7-9). K rearranging gene segments in mammals are arranged in clusters of variable (V) and joining (J) segments upstream of a single C (reviewed in ref. 10), while A loci in BALB/c mice and humans have either multiple or single V segments upstream of J-C clusters (11-14). However, not all L chain loci of nonmammalian vertebrates are organized as are those of mammals. For example, chicken B cells have single V and J segments, with diversity achieved by gene conversion events employing V pseudogenes upstream of the functional V (15, 16). Shark A-like L chain loci are organized in clusters of V-J-C, with the rearranging segments linked tightly within each cluster (8, 17). The finding of only A-like L chains in sharks led to the hypothesis that only one L chain isotype would be found in representatives of primitive vertebrate classes (7). Furthermore, the presence of A-like L chains in a chondricthyian led to the suggestion that A may have been the first isotype to emerge in evolution (9). However, the presence of other isotypes in sharks was not ruled out (9, 18), and phylogenetic The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. trees of all described K and A suggested that the ancestor of sharks and all other vertebrates possessed both isotypes (4). We report here the isolation of an L chain cDNA clone from the nurse shark, Ginglymostoma cirratum.¶ The predicted V region amino acid sequence is similar to mammalian VK (60% identity). This L chain differs from all other K described in that it has a cluster-type gene organization similar to the shark H (19) and A-like loci (8, 17). MATERIALS AND METHODS Oligonucleotides and PCR. The following three oligonucleotides were used for amplification of nurse shark cDNA: the immunoglobulin-superfamily-specific CB2 primer (described in Results; 5'-GCGAATTCAARGCNACNCTBGTNTG-3', where N represents A, C, G, or T, R is A or G, and B is G, T, or C), an adapter primer (5'-GACTCGAGTCGACATCG3') (20), and a (dT)17 adapter primer (5'-GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3') (20). The first eight bases of CB2 contain an EcoRI recognition sequence. Preparation of poly(A)+ RNA and its reverse transcription with the (dT)17 adapter primer were done as previously described (21). The PCR mixture (50 ,ul) contained 3 Al of cDNA, 200 ,uM dNTPs, 0.5 ,uM CB2 primer, 0.5 ,uM adapter primer, S ul of lOx reaction buffer (Stratagene), and 2.5 units of Taq DNA polymerase (Stratagene). The amplification protocol used was three cycles of 1 min at 94°C, 2 min at 37°C, and 3 min at 72°C with a ramp time of 4 sec/°C as suggested in ref. 22; followed by 30 cycles of 1 min at 94°C, 2 min at 55°C, and 3 min at 72°C with a final extension of 10 min at 72°C. Second-round PCR was performed on the original PCR product, and bands which reamplified with both CB2 and adapter primers were gel purified and then treated with Geneclean (Bio 101) and subcloned in the EcoRV site of pBluescript II SK (+) (Stratagene). V (nt 106-342 in Fig. 1) and C region-specific (nt 418-732) probes were also generated by PCR, and oligonucleotides present in the V segment (nt 106-123) and complementary to the J segment (nt 379396) were used to amplify the fragment separating V and J segments in the genomic clones. Southerm Blotting andcDNA Library Screening. For Southern blot analyses (23), 10 ,ug of genomic DNA, isolated from erythrocytes according to a standard protocol (ref. 24, pp. 9.16-9.19), was subjected to restriction enzyme digestion (BoehringerMannheim) and electrophoresed on a0.8% agarose gel. Prehybridization and hybridization were performed at 42°C in the same solution [50%o (vol/vol) formamide/6x SSC/5x Denhardt's solution/0.3% SDS, and denatured salmon sperm Abbreviations: V, variable; C, constant; J, joining; L, light; H, heavy; PIR, Protein Identification Resource. tTo whom reprint requests should be addressed. ¶The sequence reported in this paper has been deposited in the GenBank data base (accession no. L16765).
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