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.     

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.     

Molecular characterisation and expression of CD4 in two distantly related marsupials: the gray short-tailed opossum (Monodelphis domestica) and tammar wallaby (Macropus eugenii)

The gene and corresponding cDNA for CD4 in the gray short-tailed opossum, Monodelphis domestica, and the cDNA sequence for CD4 in the tammar wallaby, Macropus eugenii, have been characterised. The opossum CD4 homolog reveals conserved synteny, preserved genomic organisation and analogous structural arrangement to human and mouse CD4. Opossum and tammar CD4 exhibit typical eutherian CD4 features including the highly conserved p56(lck) binding motif in the cytoplasmic region and the invariant cysteine residues in extracellular domains 1 and 4. Interestingly, the marsupial CD4 sequences substitute a tryptophan for the first cysteine in domain 2 negating the formation of a disulphide bond as seen in other eutherian CD4 sequences except human and mouse. Overall the marsupial CD4 sequences share amino acid identity of 59% to each other and 37-41% with eutherian mammals. However, in contrast to eutherian homologs, the marsupial CD4 sequences were found to be truncated at the terminal end of the cytoplasmic tail. This is the first report confirming the presence of CD4 in a marsupial and describing its key features.     

Molecular cloning of the cDNA encoding the constant region of the immunoglobulin A heavy chain (Cα) from a marsupial: Trichosurus vulpecula (common brushtail possum)

A cDNA encoding the brushtail possum immunoglobulin A heavy chain constant region (Cα) was isolated by screening a mesenteric lymph node cDNA library with a porcine Cα exon 3 probe. The larger of the two positive clones isolated (Tv4a) consisted of 1325 bp of possum cDNA that included an open reading frame of 1191 bp. Its deduced amino acid sequence had a high degree of sequence identity with known eutherian Cα sequences. This clone appears to encode the entire possum IgA heavy chain constant region. The possum Cα sequence had a nucleotide sequence identity of 57.7% with porcine Cα, 51% with mouse Cα, 46.7% with dog Cα and 45.9% with human Cα2. The corresponding amino acid identities were 46.7, 45.6, 49.4 and 49%, respectively.     

Molecular identification of interleukin-2 in the lymphoid tissues of the common brushtail possum, Trichosurus vulpecula

The common brushtail possum (Trichosurus vulpecula) is an Australian marsupial. Here we describe the identification of possum interleukin-2 in mitogen-stimulated lymph node cells. We used a strategy of Rapid amplification of cDNA ends using probes designed from recently-sequenced marsupial genomes to identify the IL2 gene and then confirmed that IL-2 expression in possum immune tissue occurs in a similar manner to that in their eutherian counterparts. The predictive possum IL-2 peptide showed 28% and 35% amino acid sequence homology with the mouse and human IL-2 molecules, respectively, consistent with the divergence found within this cytokine family. Despite this low sequence identity, possum IL-2 still possessed the characteristic hallmarks of mammalian IL-2, such as a predicted signal peptide and conserved family motifs.     

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.     

Molecular cloning of the brushtail possum (Trichosurus vulpecula) immunglobulin E heavy chain constant region

The immunobiology of marsupial IgE is poorly understood. As a first step towards the development of immunological reagents for marsupials and to obtain a further understanding of immunoglobulin evolution, a brushtail possum (Trichosurus vulpecula) mesenteric lymph node cDNA library was screened for the heavy chain constant region of IgE (Cε), using a partial Cε probe from the American marsupial, Monodelphis domestica. The cDNA sequence for T. vulpecula Cε was determined and found to be most similar to the M. domestica Cε sequence [(76%) at the amino acid level]. T. vulpecula Cε has amino acid sequence similarities ranging from 43–52% with various eutherian Cε sequences. The secondary structure of T. vulpecula Cε, based on loops formed by internal disulfide bonds, more closely resembles rodent Cε than the American marsupial sequence.     

Molecular confirmation of an adenovirus in brushtail possums (Trichosurus vulpecula)

Partial genome characterisation of a non-cultivable marsupial adenovirus is described. Adenovirus-like particles were found by electron microscopy (EM) in the intestinal contents of brushtail possums (Trichosurus vulpecula) in New Zealand. Using degenerate PCR primers complementary to the most conserved genome regions of adenoviruses, the complete nucleotide sequence of the penton base gene, and partial nucleotide sequences of the DNA polymerase, hexon, and pVII genes were obtained. Phylogenetic analysis of the penton base gene strongly suggested that the brushtail possum adenovirus (candidate PoAdV-1) belongs to the recently proposed genus Atadenovirus. Sequence analysis of the PCR products amplified from the intestinal contents of brushtail possums originating from different geographical regions of New Zealand identified a single genotype. This is the first report of molecular confirmation of an adenovirus in a marsupial.     

Marsupial genome analysis suggests that satellite DNA formation from walb endogenous retrovirus is an event specific to the red-necked wallaby

We recently identified walbRep, a satellite DNA residing in the genome of the red-necked wallaby Notamacropus rufogriseus. It originates from the walb endogenous retrovirus and is organized in a manner in which the provirus structure is retained. The walbRep repeat units feature an average pairwise nucleotide identity as high as 99.5%, raising the possibility of a recent origin. The tammar wallaby N. eugenii is a species estimated to have diverged from the red-necked wallaby 2–3 million years ago. In PCR analyses of these two and other related species, walbRep-specific fragment amplification was observed only in the red-necked wallaby. Sequence database searches for the tammar wallaby resulted in sequence alignment lists that were sufficiently powerful to exclude the possibility of walbRep existence. These results suggested that the walbRep formation occurred in the red-necked wallaby lineage after its divergence from the tammar wallaby lineage, thus in a time span of maximum 3 million years.     

Posterior pituitary of the newborn marsupial possum,Trichosurus vulpecula

The fetal anterior pituitary-adrenal axis is thought to be involved in the initiation of birth in both eutherian and marsupial mammals. Little is known about the structure and function of the posterior pituitary at birth in the marsupial. Immunocytochemistry, high pressure liquid chromatography, and radioimmunoassay were used to identify vasopressin and mesotocin in the posterior pituitary of a newborn marsupial, the brushtail possum, Trichosurus vulpecula. The concentrations of vasopressin and mesotocin in the head of the newborn possum were 0.34 and 0.28 ng, respectively. The concentration of vasopressin was always greater than that of mesotocin, and the amounts of neuropeptides present in the head increased as the possum developed. © 1993 Wiley-Liss Inc.     

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.     

The common gamma chain cytokine interleukin‐21 is expressed by activated lymphocytes from two macropod marsupials,Macropus eugenii and Onychogalea fraenata

In mammals, interleukin‐21 is a member of the common gamma chain cytokine family that also includes IL‐2, IL‐4, IL‐7, IL‐9 and IL‐15. IL‐21 has pleiotropic effects on both myeloid and lymphoid immune cells and as a consequence, the biological actions of IL‐21 are broad: regulating both innate and adaptive immune responses and playing a pivotal role in antiviral, inflammatory and antitumour cellular responses. While IL‐21 genes have been characterized in mammals, birds, fish and amphibians, there are no reports for any marsupial species to date. We characterized the expressed IL‐21 gene from immune tissues of two macropod species, the tammar wallaby (Macropus eugenii), a model macropod, and the closely related endangered bridled nailtail wallaby (Onychogalea fraenata). The open reading frame of macropod IL‐21 is 462 nucleotides in length and encodes a 153‐mer putative protein that has 46% identity with human IL‐21. Despite the somewhat low amino acid conservation with other mammals, structural elements and residues essential for IL‐21 conformation and receptor association were conserved in the macropod IL‐21 predicted peptides. The detection of IL‐21 gene expression in T‐cell‐enriched tissues, combined with analysis of the promotor region of the tammar wallaby gene, suggests that macropod IL‐21 is expressed in stimulated T cells but is not readily detected in other cells and tissues. The similarity of gene expression profile and functionally important amino acid residues to eutherian IL‐21 makes it unlikely that the differences in B‐ and T‐cell responses that are reported for some marsupial species are due to a lack of important functional residues or IL‐21 gene expression in this group of 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.     

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.     

Isolation and comparison of the IgM heavy chain constant regions from Australian (Trichosurus vulpecula) and American (Monodelphis domestica) marsupials

cDNAs encoding IgM heavy chain constant region (Cμ) were isolated from two metatherians (marsupials) — the Australian common brushtail possum (Trichosurus vulpecula) and the South American grey short-tailed opossum (Monodelphis domestica). Analysis of the sequences suggested that they correspond to the secreted form of Cμ in both species. The domain size and structure of the marsupial Cμ sequences were compared with other Cμ sequences and a high degree of conservation throughout vertebrate evolution was observed. Amino acid sequence comparisons revealed a marked level of sequence similarity between the two marsupial sequences (79%), relatively high similarity between the marsupials and eutherians (63%), and lower similarities between marsupials and birds (45%), marsupials and amphibians (47%), marsupials and reptiles (45%) and marsupials and fish (37%). These data allow the incorporation of metatherians into the study of mammalian IgM evolution.     

Cloning, sequencing and expression in Escherichia coli of Pha a 1 and four isoforms of Pha a 5, the major allergens of canary grass pollen

Background: The pollen of canary grass, which was introduced as a pasture grass from Europe, is a major allergen source in the external environment of southern Australia. This study was performed to characterize the major recombinant allergens of canary grass pollen. It is anticipated that recombinant allergens may be useful in diagnosis and immunotherapy of grass pollen induced allergies. Objective: To clone major canary grass pollen allergens and assess their nucleotide and amino acid sequence homologies with other grass pollen allergens. This sequence information may then be useful in T and B cell epitope mapping studies. Methods: A canary grass pollen λgtl 1 cDNA expression library was constructed and screened with sera of grass-pollen-sensitive patients. IgE-reactive clones were isolated, sub-cloned into Escherichia coli, sequenced and, along with the deduced amino acid sequences, compared with other sequences in nucleotide and amino acid databases. Results: One of the clones encoded the group 1 allergen of canary grass pollen, Pha a 1, with a deduced amino acid sequence identity of 88.8% with Lol p 1, from rye-grass pollen, 88.1% with Hol 1 1, from velvet grass pollen and 86.6% with Phi p 1, from timothy grass pollen. The other clones (e.g. clones, 5, 14, 28, 29) encoded polymorphic forms of Pha a 5. These polymorphic forms showed between 60.6–95.5% nucleotide and 40.1–81.7% deduced amino acid sequence identities with each other. Moreover, they shared significant sequence identity with other group 5 allergens from rye-grass, timothy and Kentucky bluegrass pollens. Conclusions: Group 1 and four isoforms of group 5 allergens of canary grass pollen have been cloned and upon sequencing demonstrated strong nucleotide and amino acid sequence identities with other group 1 and 5 grass pollen allergens.