We report a high-quality draft of the genome sequence of the grey, short-tailed opossum (Monodelphis domestica). As the first metatherian ('marsupial') species to be sequenced, the opossum provides a unique perspective on the organization and evolution of mammalian genomes. Distinctive features of the opossum chromosomes provide support for recent theories about genome evolution and function, including a strong influence of biased gene conversion on nucleotide sequence composition, and a relationship between chromosomal characteristics and X chromosome inactivation. Comparison of opossum and eutherian genomes also reveals a sharp difference in evolutionary innovation between protein-coding and non-coding functional elements. True innovation in protein-coding genes seems to be relatively rare, with lineage-specific differences being largely due to diversification and rapid turnover in gene families involved in environmental interactions. In contrast, about 20% of eutherian conserved non-coding elements (CNEs) are recent inventions that postdate the divergence of Eutheria and Metatheria. A substantial proportion of these eutherian-specific CNEs arose from sequence inserted by transposable elements, pointing to transposons as a major creative force in the evolution of mammalian gene regulation.

T cells recognize antigens by using T cell receptors (TCRs) encoded by gene segments, called variable (V), diversity (D), and joining (J), that undergo somatic recombination to create diverse binding specificities. Four TCR chains (␣, ␤, ␥, and ␦) have been identified to date, and, as T cells develop in the thymus, they express exclusively either an ␣␤TCR or a ␥␦TCR heterodimer. Here, we show that marsupials have an additional TCR (TCR) that has V, D, and J that are either somatically recombined, as in conventional TCRs, or are already prejoined in the germ-line DNA in a manner consistent with their creation by retrotransposition. TCR does not have a known homolog in eutherian mammals but has features analogous to a recently described TCR␦ isoform in sharks. TCR is expressed in at least two mRNA isoforms that appear capable of encoding a full-length protein, both of which are transcribed in the thymus and spleen. One contains two variable domains: a somatically recombined V and a prejoined V. This appears to be the dominant isoform in peripheral lymphoid tissue. The other isoform contains only the prejoined V and is structurally more similar to conventional TCR chains, however invariant. Unlike other TCRs, TCR uses prejoined gene segments and is likely present in all marsupials. Its similarity to a TCR isoform in sharks suggests that it, or something similar, may be present in other vertebrate lineages and, therefore, may represent an ancient receptor system. evolution ͉ immune system H allmarks of the adaptive immune systems in jawed vertebrates are cells (lymphocytes) that use somatic DNA recombination to assemble the genes that encode antigen receptors. This recombination provides the means to generate a large repertoire of receptors with diverse binding specificities (1). There are two classes of antigen receptors that are used by B and T cells, respectively: Ig and T cell receptor (TCR). Although there is variation in the isotypes of Ig present, to date, all jawed vertebrates appear to have the same four TCR homologs (2, 3): TCR␣, -␤, -␥, and -␦. All contain V domains that are encoded by the variable (V), diversity (D), and joining (J) gene segments; recombined V and J encode the V domain in TCR␣ and TCR␥, whereas the TCR␤ and TCR␦ chains use V, D, and J (4). A typical TCR locus is organized in a ''translocon'' arrangement in which an array of V segments is upstream of one or more D segments (in the case of TCR␤ and TCR␦), followed by one or more J segments (4). In developing T cells, these gene segments undergo so-called VDJ recombination that is mediated, in part, by the recombination activating gene (RAG) recombinase system to assemble the exon encoding the V domain (1, 5). To date, the V, D, and J gene segments in TCR loci have always been found in a nonrecombined state in the germ-line DNA and require somatic cell recombination for expression. After recombination, T cells are then selected in the thymus to eliminate those that bind self-antigens (contributing to self-tolerance) and, in the case of ␣␤TC...

Background: All jawed-vertebrates have four T cell receptor (TCR) chains: alpha (TRA), beta (TRB), gamma (TRG) and delta (TRD). Marsupials appear unique by having an additional TCR: mu (TRM). The evolutionary origin of TRM and its relationship to other TCR remain obscure, and is confounded by previous results that support TRM being a hybrid between a TCR and immunoglobulin locus. The availability of the first marsupial genome sequence allows investigation of these evolutionary relationships.

Summary The content and organization of the Xenopus tropicalis TCRα/δ locus was determined. This locus is highly conserved among tetrapods with the genes encoding the TCRδ chains embedded with those encoding TCRα. However, the frog TCRα/δ is unusual by containing V genes that appear indistinguishable from those in the IgH locus (VH). These V genes, termed VHδ, make up 70% of the V genes at the TCRδ locus and are expressed exclusively in TCRδ chains. Finding TCRδ chains that use antibody-like V domains in frogs is similar to shark TCRδ variants and TCRμ in marsupials. These results suggest that such unconventional TCR may be more widespread across vertebrate lineages and raise the possibility of previously unrealized subsets of T cells. We also revealed close linkage of TCRα/δ, IgH, and Igλ in Xenopus, which in combination with linkage analyses in other species is consistent with previous models for the emergence of these antigen receptor loci.

TCRμ is an unconventional TCR that was first discovered in marsupials and appears to be absent from placental mammals and non-mammals. Here we show that TCRμ is also present in the duckbill platypus, an egg-laying monotreme, consistent with TCRμ being ancient and present in the last common ancestor of all extant mammals. As in marsupials, platypus TCRμ is expressed in a form containing double V domains. These V domains more closely resemble antibody V than that of conventional TCR. Platypus TCRμ differs from its marsupial homologue by requiring two rounds of somatic DNA recombination to assemble both V exons and has a genomic organization resembling the likely ancestral form of the receptor genes. These results demonstrate that the ancestors of placental mammals would have had TCRμ but it has been lost from this lineage.