Abstract
BackgroundThe Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates. Among the nuclear markers currently used for phylogenetic purposes, Rag1 has especially enjoyed enormous popularity, since it successfully contributed to elucidating the relationships among and within a large variety of vertebrate lineages. We here report on a comparative investigation of the genetic variation, base composition, presence of indels, and selection in Rag1 in different vertebrate lineages (Actinopterygii, Amphibia, Aves, Chondrichthyes, Crocodylia, Lepidosauria, Mammalia, and Testudines) through the analysis of 582 sequences obtained from Genbank. We also analyze possible differences between distinct parts of the gene with different type of protein functions.ResultsIn the vertebrate lineages studied, Rag1 is over 3 kb long. We observed a high level of heterogeneity in base composition at the 3rd codon position in some of the studied vertebrate lineages and in some specific taxa. This result is also paralleled by taxonomic differences in the GC content at the same codon position. Moreover, positive selection occurs at some sites in Aves, Lepidosauria and Testudines. Indels, which are often used as phylogenetic characters, are more informative across vertebrates in the 5' than in the 3'-end of the gene. When the entire gene is considered, the use of indels as phylogenetic character only recovers one major vertebrate clade, the Actinopterygii. However, in numerous cases insertions or deletions are specific to a monophyletic group.ConclusionsRag1 is a phylogenetic marker of undoubted quality. Our study points to the need of carrying out a preliminary investigation on the base composition and the possible existence of sites under selection of this gene within the groups studied to avoid misleading resolution. The gene shows highly heterogeneous base composition, which affects some taxa in particular and contains sites under positive selection in some vertebrate lineages in the 5'-end. The first part of the gene (5'-end) is more variable than the second (3'-end), and less affected by a heterogeneous base composition. However, in some vertebrate lineages the 5'-end of the gene is not yet widely used for phylogenetic studies.
Highlights
The Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates
Our study points to the need of carrying out a preliminary investigation on the base composition and the possible existence of sites under selection of this gene within the groups studied to avoid misleading resolution
Amphibia and Lepidosauria have the highest percentage of variable amino acid sites
Summary
The Recombination Activating Proteins, RAG1 and RAG2, play a crucial role in the immune response in vertebrates. Several studies have focused on specific vertebrate groups (e.g., birds, turtles, sharks and amphibians [8,12,13,14], but see [10]) and have highlighted the characteristics of this gene in relation to its phylogenetic utility Some of these potentially useful characteristics of Rag include its existence as a single copy gene (except in polyploidy taxa such as Xenopus, [15]), uninterrupted exon (except in ray-finned fish where it has one or two introns [16]), the conserved nature of certain regions of the gene, especially its second half (3' end), which facilitates the design of degenerate "universal" primers for PCR, the presence of numerous sequences from a variety of taxa in public databases, and an overall lack of saturation [10]
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