Molecular characterisation of the tammar wallaby (Macropus eugenii) CD3 epsilon chain cDNA.

The cDNA encoding the epsilon chain of the tammar wallaby CD3 complex (CD3epsilon) was isolated by PCR. This is the first CD3 component to be cloned in a marsupial. The tammar wallaby cDNA coding region was 61.7 and 63.0% identical to the human and mouse cDNA coding sequences, respectively. Similarly, the predicted amino acid sequence was 56.5 and 52.9% identical to the human and mouse sequences. When compared with other known CD3epsilon peptide sequences, the most conserved region of the tammar wallaby CD3epsilon chain peptide was the cytoplasmic domain and the least conserved was the extracellular portion. Phylogenetic reconstruction based on the deduced amino acid sequence placed the tammar wallaby sequence in its expected position outside of all the eutherian mammals.     

Further characterization of T cell receptor chains of marsupials

cDNA clones encoding T cell receptor alpha (TCRalpha) and beta (TCRbeta) from the South American opossum, Monodelphis domestica were isolated and characterized. A single clone isolated encoding a TCRalpha chain was full length, containing the complete V (variable), J (joining) and C (constant) regions. Three partial cDNA clones were isolated for TCRbeta which contained complete C sequences. Phylogenetic analysis of the TCR Valpha revealed that the M. domestica sequence and a sequence from the Australian brushtail possum, Trichosurus vulpecula, belong to separate Valpha families and intersperse with sequences from eutherian mammals. Similar to results described for marsupial and eutherian light chains, diversity at the V region of the TCR is ancient and maintained. In contrast phylogenetic analysis of the TCR Calpha and Cbeta sequences from M. domestica, T. vulpecula, and other vertebrates revealed that the marsupial TCR C grouped together forming a sister group to eutherian mammals.     

Primary structure and variation of the T-cell receptor delta-chain from a marsupial,Macropus eugenii

Although gammadelta T-cells form only a small portion of circulating T-cells in mice and humans, they are more frequent in many other types of mammals and this has lead to speculation regarding their roles and the evolutionary significance of their relative abundance. Moreover, whilst clear homologues of four types of T-cell receptor (TCR) chains (alpha, beta, delta and gamma) have been identified in vertebrates as distantly related as eutherian mammals and cartilaginous fish, there are still many gaps in our knowledge of these TCR components from various taxa. Such knowledge would further illuminate the evolution and function of these receptors and of gammadelta T-cells. Here, we report the molecular cloning of a TCR-delta chain cDNA from the tammar wallaby (Macropus eugenii) which represents the first component of the gammadelta TCR to be characterised from a marsupial. A PCR-based survey of variable (V) segment usage in tammar wallaby mammary-associated lymph node indicated that, although gammadelta T-cells may be sparse in this type of tissue, this species has at least three subfamilies of V genes that have been broadly conserved across vertebrate evolution. Two V subfamilies found in the tammar wallaby were relatively similar and may have diverged more recently, an event that probably occurred at some point in the marsupial lineage.     

A cDNA encoding feline CD4 has a unique repeat sequence downstream of the V-like region.

We have cloned, sequenced and expressed a cDNA encoding the feline CD4 glycoprotein. This clone, termed FT121, contains a unique repeat sequence downstream of the V-like region which has not been reported in other mammalian CD4 cDNA. In addition, the cysteine residues at positions 45 and 175 have an unusual pattern. On the other hand, regions other than the unique sequence share significant homology with human CD4 and mouse L3T4 cDNA in the amino acid sequence deduced from the nucleotide sequence, especially in the putative cytoplasmic region. Here, we show that FT121 has a complete open reading frame and efficiently expresses feline CD4 molecules on COS cells. Further we found that the cytoplasmic components, lymphocyte-specific protein-tyrosine kinase p56lck binding sites and phosphorylation site (serine resides), are well conserved, though the extracellular region of the feline CD4 molecule deduced from FT121 is probably different from that of human CD4 and mouse L3T4.     

Cloning of the MHC class II DRB cDNA from the brushtail possum (Trichosurus vulpecula)

The cell-surface glycoproteins encoded by the major histocompatibility complex (MHC) bind to processed foreign antigens and present them to T lymphocytes. Two classes of MHC molecules and their corresponding gene sequences have been extensively studied in eutherian mammals and birds, but data on the marsupial MHC are limited. Marsupials split from eutherian mammals about 125 million years ago and represent a distinct branch in mammalian evolution. Here the cDNA cloning of MHC class II genes of the brushtail possums (Trichosurus vulpecula) is reported. The sequences obtained were found to be relatively conserved when compared to the red-necked wallaby (Macropus rufogriseus) and an South American marsupial, Monodelphis domestica. The T. vulpecula sequence shared an average overall sequence identity of 75.4% at the deduced amino acid level with M. rufogriseus and M. domestica, respectively.     

Production of interleukin-1 in a South American opossum (Monodelphis domestica)

A homologous thymocyte costimulatory assay using thymocytes from a South American opossum (Monodelphis domestica) detected and measured interleukin-1 (IL-1). Opossum IL-1 was obtained from lipopolysac-charide-stimulated macrophage and skin cultures and its molecular weight was determined to be 15,000 to 17,000. Opossum IL-1 did not stimulate proliferation of murine thymocytes; conversely, neither human nor murine IL-1 stimulated opossum thymocytes. Anti-human IL-1 antibodies were also not reactive with opossum IL-1. These observations indicate that there is no serological and functional crossreactivity between the opossum (marsupial mammals) and human and mouse (eutherian mammals) in an IL-1/thymocyte system. This is unusual because such crossreactivity occurs between rodents and distant, nonmammalian species. M. domestica has been used in our laboratory as a model for photobiological research; studies on IL-1 may provide insight into the relationship of immunosuppression and tumorigenesis induced by ultraviolet radiation.     

Evolutionary history of novel genes on the tammar wallaby Y chromosome: Implications for sex chromosome evolution

We report here the isolation and sequencing of 10 Y-specific tammar wallaby (Macropus eugenii) BAC clones, revealing five hitherto undescribed tammar wallaby Y genes (in addition to the five genes already described) and several pseudogenes. Some genes on the wallaby Y display testis-specific expression, but most have low widespread expression. All have partners on the tammar X, along with homologs on the human X. Nonsynonymous and synonymous substitution ratios for nine of the tammar XY gene pairs indicate that they are each under purifying selection. All 10 were also identified as being on the Y in Tasmanian devil (Sarcophilus harrisii; a distantly related Australian marsupial); however, seven have been lost from the human Y. Maximum likelihood phylogenetic analyses of the wallaby YX genes, with respective homologs from other vertebrate representatives, revealed that three marsupial Y genes (HCFC1X/Y, MECP2X/Y, and HUWE1X/Y) were members of the ancestral therian pseudoautosomal region (PAR) at the time of the marsupial/eutherian split; three XY pairs (SOX3/SRY, RBMX/Y, and ATRX/Y) were isolated from each other before the marsupial/eutherian split, and the remaining three (RPL10X/Y, PHF6X/Y, and UBA1/UBE1Y) have a more complex evolutionary history. Thus, the small marsupial Y chromosome is surprisingly rich in ancient genes that are retained in at least Australian marsupials and evolved from testis-brain expressed genes on the X.     

Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE,IgG and IgA from the marsupial Monodelphis domestica

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.     

Isolation,X location and activity of the marsupial homologue of <Emphasis Type="Italic">SLC16A2</Emphasis>, an <Emphasis Type="Italic">XIST</Emphasis>-flanking gene in eutherian mammals

X chromosome inactivation (XCI) achieves dosage compensation between males and females for most X-linked genes in eutherian mammals. It is a whole-chromosome effect under the control of the XIST locus, although some genes escape inactivation. Marsupial XCI differs from the eutherian process, implying fundamental changes in the XCI mechanism during the evolution of the two lineages. There is no direct evidence for the existence of a marsupial XIST homologue. XCI has been studied for only a handful of genes in any marsupial, and none in the model kangaroo Macropus eugenii (the tammar wallaby). We have therefore studied the sequence, location and activity of a gene SLC16A2 (solute carrier, family 16, class A, member 2) that flanks XIST on the human and mouse X chromosomes. A BAC clone containing the marsupial SLC16A2 was mapped to the end of the long arm of the tammar X chromosome and used in RNA FISH experiments to determine whether one or both loci are transcribed in female cells. In male and female cells, only a single signal was found, indicating that the marsupial SLC16A2 gene is silenced on the inactivated X.     

Isolation and sequence of a cDNA coding for the heavy chain constant region of IgG from the Australian brushtail possum, Trichosurus vulpecula.

A brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened with a South American short-tailed opossum (Monodlelphis domestica) immunoglobulin gamma heavy chain constant region (Cgamma) probe, resulting in the isolation of a 1518 nucleotide cDNA clone. The sequence corresponds to exons 1-3 of Cgamma. The Australian marsupial (T. vulpeculla) sequence is 70% identical at the amino acid level with the American marsupial (M. domestica) sequence, but less similar to the eutherian mammals (45-50%). These data provide the opportunity to compare the evolution of IgG between orders of marsupials separated by at least 75 million years and confirm the appearance of IgG prior to the metatherian/eutherian divergence.     

Sequence of the bovine CD44 cDNA: comparison with human and mouse sequences

CD44 is a cell-surface glycoprotein involved in leukocyte adherence, T-cell activation and lymphocyte homing. We have isolated and sequenced a cDNA clone which encodes for bovine CD44. The predicted amino acid sequence of bovine CD44 has an overall high similarity with that of human and mouse CD44, 79.5 and 73.2%, respectively. In all three species, CD44 has a similar transmembrane region and cytoplasmic tail. In addition, all of the cysteine residues and a majority of the putative N-linked glycosylation sites in the extracytoplasmic domain are conserved between bovine, human and mouse. All three species have an area of low interspecies similarity within the extracytoplasmic domain. This area has a similarity of 34% between bovine and human, 27% between bovine and mouse, and 35% between human and mouse. The location of this area of low similarity is conserved between species.     

Characterization of the opossum immune genome provides insights into the evolution of the mammalian immune system

The availability of the first marsupial genome sequence has allowed us to characterize the immunome of the gray short-tailed opossum (Monodelphis domestica). Here we report the identification of key immune genes, including the highly divergent chemokines, defensins, cathelicidins, and Natural Killer cell receptors. It appears that the increase in complexity of the mammalian immune system occurred prior to the divergence of the marsupial and eutherian lineages approximately 180 million years ago. Genomes of ancestral mammals most likely contained all of the key mammalian immune gene families, with evolution on different continents, in the presence of different pathogens leading to lineage specific expansions and contractions, resulting in some minor differences in gene number and composition between different mammalian lineages. Gene expansion and extensive heterogeneity in opossum antimicrobial peptide genes may have evolved as a consequence of the newborn young needing to survive without an adaptive immune system in a pathogen laden environment. Given the similarities in the genomic architecture of the marsupial and eutherian immune systems, we propose that marsupials are ideal model organisms for the study of developmental immunology.     

The region homologous to the X-chromosome inactivation centre has been disrupted in marsupial and monotreme mammals

Summary Marsupial, as well as eutherian, mammals are subject to X chromosome inactivation in the somatic cells of females, although the phenotype and the molecular mechanism differ in important respects. Monotreme mammals appear to subscribe at least to a form of dosage compensation of X-borne genes. An important question is whether inactivation in these non-eutherian mammals involves co-ordination by a control locus homologous to the XIST gene and neighbouring genes, which play a key regulatory role in human and mouse X inactivation. We mapped BACs containing several orthologues of protein-coding genes that flank human and mouse XIST and genes that lie in the homologous region in chicken and frog. We found that these genes map to two distant locations on the opossum X, and also to different locations on a platypus autosome. We failed to find any trace of an XIST orthologue in any marsupial or monotreme or on any flanking BAC, confirming the conclusion from recent work that non-eutherian mammals lack XIST. We propose the region homologous to the human and mouse X-inactivation centre expanded in early mammals, and this unstable region was disrupted independently in marsupial and monotreme lineages. In the eutherian lineage, inserted and existing sequences provided the starting material for the non-translated RNAs of the X-inactivation centre, including XIST. Electronic supplementary material The online version of this article (doi: ) contains supplementary material, which is available to authorized users.     

Immunohistochemistry of the lymphoid tissues of the tammar wallaby,Macropus eugenii

The lymphoid tissues of the metatherian mammal, the adult tammar wallaby, Macropus eugenii, were investigated using immunohistochemical techniques. Five cross-reactive antibodies previously shown to recognize surface markers in marsupial tissues and five previously untested antibodies were used. The distribution of T-cells in the tissue beds of spleen, lymph node, thymus, gut-associated lymphoid tissue (GALT) and bronchus-associated lymphoid tissue (BALT) was documented using antibodies to CD3 and CD5. Similarly, B-cells were identified in the same tissues using anti-CD79b. Antibodies to CD8, CD31, CD79a and CD68 failed to recognize cells in these tissue beds. In general the pattern of cellular distribution identified using these antibodies was similar to that observed in other marsupial and eutherian lymphoid tissues. This study provides further information on the commonality of lymphoid tissue structure in the two major groups of extant mammals, metatherians and eutherians.     

Cloning and structural analysis of IgM (mu chain) and the heavy chain V region repertoire in the marsupial Monodelphis domestica

To address the question of the Ig isotype repertoire of non placental mammals, we have examined the Ig expression in the marsupial Monodelphis domestica (grey short tailed opossum). Screening of an opossum spleen cDNA library has previously led to the isolation of full length clones for opossum IgG (γ chain), IgE ( chain) and IgA ( chain). We now present the isolation of several cDNA clones encoding the entire constant regions of the opossum IgM (μ chain). A comparative analysis of the amino acid sequences for IgM from various animal species showed that opossum IgM, within the various animals studied, is the most divergent member of its Ig class. However, it still conforms to the general structure of IgM in other vertebrates. Four Ig classes have now been identified in opossum and only one isotype is apparently present within each Ig class, IgM, IgG, IgA and IgE. Opossum has previously been shown to have a limited VH region diversity, with only two V gene families. Both of these belong to the group III of mammalian VH sequences. This limitation in variability is to some extent compensated for by a large variation in D, P and N regions, both in size and in sequence. However, evidence for the expression of only two functional J segments has so far been detected, which indicates a rather limited diversity also of the J segments in the opossum.