HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 17/3/470 02-0565fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SCHLUTER, S. F. Articles by MARCHALONIS, J. J. Search for Related Content PubMed PubMed Citation Articles by SCHLUTER, S. F. Articles by MARCHALONIS, J. J.

Cloning of shark RAG2 and characterization of the RAG1/RAG2 gene locus¹

Department of Microbiology and Immunology, University of Arizona, Tucson, Arizona, USA

²Correspondence: University of Arizona, Microbiology and Immunology, College of Medicine, P.O. Box 24–5049, 1501 Nth Campbell Ave, Tucson, AZ, 85724, USA. E-mail: marchjj{at}email.arizona.edu

SPECIFIC AIMS

The products of the recombination-activating genes (RAG) are required for the formation of active antigen receptor genes and thus are essential for expression of the vertebrate immune system. To elucidate conserved features of the RAG genes, we cloned the RAG2 gene from the sandbar shark (Carcharhinus plumbeus) and characterized the entire RAG1/RAG2 locus. We compared the shark sequences with those of higher vertebrates to determine phylogenetic homology and assess conservation of residues and segments required for function.

PRINCIPAL FINDINGS

1. Cloning of the shark RAG locus
The RAG2 gene is in close proximity to the RAG1 gene in species from teleosts to humans. Since we had previously cloned the shark RAG1 gene, our approach for cloning the shark RAG2 gene was by chromosomal walking from RAG1. The shark RAG2 gene is 9.4 kb from the 3' end of the RAG1 gene coding region and contains a single 1560 bp open reading frame in the opposite transcriptional orientation. As with all other species, the shark RAG2 gene has no introns within the coding region.

2. Shark RAG2 sequence is conserved
The RAG2 sequence has been remarkably conserved during evolution. Shark RAG2 is 50% identical at the protein level with RAG2 from other species, ranging from 46% with trout to 52% with human and mouse. Matches between the human and shark sequences (Fig. 1 ) are essentially representative of the overall phylogenetic alignment of RAG2 sequences from a diverse range of species (see Fig. 3 ). It has recently been proposed that RAG2 is composed of a kelch domain that fits almost precisely to the RAG2 active core region. The high degree of sequence conservation between shark and human RAG2 allowed to us fit the shark sequence to the predicted RAG2 domain structure as shown in Fig. 1 . The shark RAG2 has essentially the same structure comprising a kelch domain, an acidic hinge region, and a globular zinc binding domain. A distinguishing feature of kelch superfamily molecules is a motif present in ß2 blocks consisting of four contiguous hydrophobic residues, followed by two glycine residues. This motif is also a property of the ß2 blocks present in shark RAG2. The positions of mutations in human and mouse RAG2 genes reported in the literature are shown in Fig. 1 . Residues critical for RAG2 function are conserved in the shark gene and distributed throughout the sequence. The zinc finger domain is not required for expression of RAG2 activity in in vitro cell assays. However, as expected from the fact that it is one of the most highly conserved regions of the RAG2 gene, natural mutations in the zinc finger domain cause immunodeficiency in humans.

View larger version (57K): [in this window] [in a new window]   Figure 1. Conservation of predicted 3D structure and critical functional residues in shark RAG2. Alignment of the protein sequences of shark and human RAG2 is taken from the overall alignment of sequences from 9 species including shark, teleost fish, chicken, toad, rabbit, mouse, and human as reflected by occasional gaps at the same position in both sequences. Residue numbering refers to the human sequence. Identical residues are shaded and similar residues are boxed. Positions that are absolutely conserved in the other 8 sequences but are different in the shark are shown in lowercase. The kelch domain, an acidic hinge region, and the globular zinc binding domain predicted by sequence alignment and homology modeling to comprise human RAG2 are indicated. The shark sequence can be precisely fitted to this model. The kelch domain consists of 6 repeated structural elements, each composed of 4 anti-parallel beta sheets (ß1 block through ß4 block), which form a 6-bladed propeller-like structure. Segments between the ß blocks form loops (e.g., loop 2–3 connects the ß2 and ß3 blocks) on each side of the propeller structure. The kelch domain is an example of a protein in which the structural repeat does not precisely match the sequence repeat. Thus, the first 10 amino-terminal residues actually form the ß4 block of the 6th propeller, as shown on line 6. A summary of the effects of various mutations on RAG2 function is shown. Filled circles represent the positions of natural mutations that occur in humans. Artificial mutations are difficult to summarize because of different experimental conditions and aims. The pound sign shows residues that can be mutated with little affect as measured by the ability to form coding joints. The plus sign indicates minimal change in activity as assayed by the ability to bind and cleave appropriate substrates in vitro, but the formation of coding joints was not assessed. Solid triangles indicate mutations for which joint formation is abrogated even though there is normal RAG1/RAG2 association accompanied by DNA recognition and cleavage. Symbols connected by a line are simultaneous mutations. Stars indicate residues whose mutation leads to abrogation of RAG1/RAG2 association. An asterisk indicates reduced activity correlated with reduced RAG association. Open triangles show conserved cysteines and histidines of the zinc finger. The sequences reported in this paper have been deposited in GenBank under accession no. AY172838.

View larger version (34K): [in this window] [in a new window]   Figure 3. Schematic diagram.

3. Phylogenetic analysis of RAG2
We constructed rooted and unrooted trees using the complete RAG2 sequence of shark, teleost fish, amphibians, birds, and mammals (Fig. 2 ). Since we expect the shark to be the phylogenetic ancestor of this group, we used the shark sequence to root the tree in Fig. 2A . The topology of the trees is as expected. The mammals form a well-defined group with birds and amphibians on the same tetrapod branch and teleost fish on a second branch.

View larger version (18K): [in this window] [in a new window]   Figure 2. Phylogenetic relationships of RAG2 proteins. A) Unrooted dendrogram. B) Rooted tree using the shark sequence as the out group. Bootstrap scores of 1000 repetitions are noted.

4. The intergenic region between RAG1 and RAG2 contains retroposon repeat elements
We identified four SINE (short interspersed repetitive element) and three LINE (long interspersed repetitive element, or non-LTR retroposon) retroposon elements in the region between the RAG genes. The sandbar shark SINEs identified here are members of the VSINE superfamily and thus were designated SbSVS (sandbar shark VSINEs). VSINEs are highly conserved and are found in lampreys, elasmobranchs, teleost fishes, and amphibians. Although the LINE fragments are severely 5' truncated, they can be identified as HER1 family members; we therefore termed them SbSR1 (sandbar shark HER1 family LINEs). HER1 retroposons are found in numerous elasmobranch species and belong to the CR1 LINE family that is distributed throughout many eukaryotic phyla.

CONCLUSIONS AND SIGNIFICANCE

Comparisons of the shark RAG 1 and 2 sequences and the overall RAG gene organization show that the RAG locus has been highly conserved during vertebrate evolution. Shark RAG2 is 52% identical to the human molecule, and many sequence motifs are absolutely conserved in species ranging from sharks to humans (Fig. 3 ). The same is true for RAG1, where the conservation is even more striking (Fig. 3) , with the human and shark proteins being 64% identical. Shark RAG2 apparently has the same domain structure as the human molecule, consisting of a kelch region with sixfold symmetry, an acidic hinge region, and a zinc finger domain. Residues identified in mutagenesis experiments as critical for activity are conserved in shark RAG2. Thus, it appears that the ancestor of the vertebrate RAG2 had essentially the same structure and activity as the modern form. The genomic organization of the shark genes is the same as found in all vertebrates, with the RAG1 and RAG2 genes being in close juxtaposition and in the opposite transcriptional orientation. Shark RAG genes (and most other vertebrate RAG genes) do not contain introns within the coding region.

A prokaryotic origin for the RAG genes was first suggested by these genomic properties. It is now accepted that they arose from a transposase introduced by the horizontal transfer of a prokaryote transposon. The basic catalytic mechanism of recombination mediated by RAG proteins is similar to transposition, and the RAG proteins can be induced to catalyze transposition reactions under the appropriate conditions. However, a more detailed model for the evolution of the RAG genes to identify a possible transposon ancestral form presents a perplexing problem. Although a combination of approaches establishes homology for RAG1 with the core catalytic domain of many transposons and retroviral integrases, no such relationship for RAG2 can be discerned. For RAG2, homology with the kelch superfamily of proteins is indicated. None of the cofactors and accessory proteins associated with transposon systems required for complete activity and specificity has been shown to contain kelch domains, and none appear in homology analyses for RAG2.

Despite much effort, homologs of RAG, or indeed any other molecular component of the vertebrate combinatorial/adaptive immune system, have not been detected in agnathans. This is puzzling, since based on the degree of RAG sequence identity within the gnathostomes, cyclostomes would be predicted to have genes with > 40% identity and so be relatively easy to detect. Basic features of the vertebrate combinatorial immune system essentially are fully present in the sharks. It is now hypothesized that integration of the RAG transposon occurred in the gnathostome ancestor of cartilaginous fishes, and this event triggered the rapid evolution (the "Big Bang") of the immune system. The time of the integration of the RAG genes remains unclear. Given that this event was crucial for the evolution of the vertebrate immune system, it is important to try to clarify this point by continuing the search in cyclostomes such as lampreys for gene fragments that may be remnants of the ancestral RAG/transposon. With the derivation of the shark sequences and a better understanding of functional regions, we should now be able to develop a rational approach to probe the lamprey genome.

SINEs and LINEs are a significant component of almost all eukaryotic genomes. They appear to be a major proportion of the sandbar shark genome, since we found seven of these elements in the short space between the RAG genes. Retroposon elements are stable and powerful evolutionary markers, and their presence in the shark RAG intergenic region should prove quite useful for characterizing the evolution of the RAG locus in elasmobranchs.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0565fje; to cite this article, use FASEB J. (January 22, 2003) 10.1096/fj.02-0565fje

This article has been cited by other articles:

				S. D. Fugmann, C. Messier, L. A. Novack, R. A. Cameron, and J. P. Rast An ancient evolutionary origin of the Rag1/2 gene locus PNAS, March 7, 2006; 103(10): 3728 - 3733. [Abstract] [Full Text] [PDF]

				M. E. DeVries, A. A. Kelvin, L. Xu, L. Ran, J. Robinson, and D. J. Kelvin Defining the Origins and Evolution of the Chemokine/Chemokine Receptor System J. Immunol., January 1, 2006; 176(1): 401 - 415. [Abstract] [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 17/3/470 02-0565fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SCHLUTER, S. F. Articles by MARCHALONIS, J. J. Search for Related Content PubMed PubMed Citation Articles by SCHLUTER, S. F. Articles by MARCHALONIS, J. J.