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.     

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.     

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 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.     

Evidence for an early appearance of modern post-switch immunoglobulin isotypes in mammalian evolution (II); cloning of IgE,IgG1 and IgG2 from a monotreme,the duck-billed platypus,Ornithorhynchus anatinus

To trace the emergence of the modern post-switch immunoglobulin (Ig) isotypes in vertebrate evolution we have studied Ig expression in mammals distantly related to eutherians. We here present an analysis of the Ig expression in an egg-laying mammal, a monotreme, the duck-billed platypus (Ornithorhynchus anatinus). Fragments of platypus IgG and IgE cDNA were obtained by a PCR-based screening using degenerate primers. The fragments obtained were used as probes to isolate full-length cDNA clones of three platypus post-switch isotypes, IgG1, IgG2, and IgE. Comparative amino acid sequence analysis against IgY, IgE and IgG from various animal species revealed that platypus IgE and IgG form branches that are clearly separated from those of their eutherian (placental) counterparts. However, the platypus IgE and IgG still conform to the general structure displayed by the respective Ig isotypes of eutherian and marsupial mammals. According to our findings, all of the major evolutionary changes in the expression array and basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the separation of monotremes from marsupial and placental mammals. Hence, our results indicate that the modern post-switch isotypes appeared very early in the mammalian lineage, possibly already 310-330 million years ago.     

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.     

Postnatal lung and metabolic development in two marsupial and four eutherian species

Two marsupial species (Monodelphis domestica, Macropus eugenii) and four eutherian species (Mesocricetus auratus, Suncus murinus, Tupaia belangeri and Cavia aperea) were examined to compare and contrast the timing of lung and metabolic development during the postnatal maturation of the mammalian respiratory apparatus. Using light, scanning and transmission electron microscopy, the lung structural changes were correlated with indirect calorimetry to track the metabolic development. Marsupial and eutherian species followed the same pattern of mammalian lung development, but differed in the developmental pace. In the two newborn marsupial species, the lung parenchyma was at the early terminal sac stage, with large terminal air sacs, and the lung developed slowly. In contrast, the newborn eutherian species had more advanced lungs at the late terminal sac stage in altricial species (M. auratus, S. murinus) and at the alveolar stage in precocial species (T. belangeri, C. aperea). Postnatal lung development proceeded rapidly in eutherian species. The marsupial species had a low metabolic rate at birth and achieved adult metabolism late in postnatal development. In contrast, newborn eutherian species had high metabolic rates and reached adult metabolism during the first week of life. The time course of the metabolic development is thus tightly linked to the structural differentiation of the lungs and the timing of postnatal lung development. These differences in the neonatal lung structure and the timing of postnatal lung maturation between marsupial and eutherian species reflect their differing reproductive strategies.     

Uterine remodelling during pregnancy and pseudopregnancy in the brushtail possum (Trichosurus vulpecula; Phalangeridae)

The formation of a placenta is critical for successful mammalian pregnancy and requires remodelling of the uterine epithelium. In eutherian mammals, remodelling involves specific morphological changes that often correlate with the mode of embryonic attachment. Given the differences between marsupial and eutherian placentae, formation of a marsupial placenta may involve patterns of uterine remodelling that are different from those in eutherians. Here we present a detailed morphological study of the uterus of the brushtail possum (Trichosurus vulpecula; Phalangeridae) throughout pregnancy, using both scanning and transmission electron microscopy, to identify whether uterine changes in marsupials correlate with mode of embryonic attachment as they do in eutherian mammals. The uterine remodelling of T. vulpecula is similar to that of eutherian mammals with the same mode of embryonic attachment (non‐invasive, epitheliochorial placentation). The morphological similarities include development of large apical projections, and a decrease in the diffusion distance for haemotrophes around the period of embryonic attachment. Importantly, remodelling of the uterus in T. vulpecula during pregnancy differs from that of a marsupial species with non‐invasive attachment (Macropus eugenii; Macropodidae) but is similar to that of a marsupial with invasive attachment (Monodelphis domestica; Didelphidae). We conclude that modes of embryonic attachment may not be typified by a particular suite of uterine changes in marsupials, as is the case for eutherian mammals, and that uterine remodelling may instead reflect phylogenetic relationships between marsupial lineages.     

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.     

Characterization of the Three Immunoglobulin G Subclasses of Macaques

Southern blot experiments with genomic DNA samples of rhesus monkeys and crab-eating macaques and human C gamma-specific probes indicated that the two macaque species studied here possessed three C gamma genes per haploid genome. By amplifying the cDNA from macaque-mouse hybridomas, the coding sequences of two different rhesus monkey immunoglobulin (Ig)G subclasses, IgG1rh (Cgamma1rh) and IgG2rh (Cgamma2rh), and one crab-eating macaque IgG subclass IgG1mafa (Cgamma1mafa), were characterized. None of the 16 rhesus monkey-mouse hybridomas studied here secreted IgG of the third subclass IgG3rh (Cgamma3rh). The Cgamma3rh gene was partly characterized at the genomic level. The cDNA of the Cgamma3rh gene was amplified from mRNA of rhesus monkey peripheral blood mononuclear cells (PBMC). The results are analysed in terms of phylogenesis of the C gamma genes. The cDNA sequences coding for the Cmu and the Ckappa domains of rhesus monkey Ig were established and compared to their human and non-human primate counterparts.     

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.     

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.     

Cellular immune response of a marsupial, Monodelphis domestica.

Marsupials are interesting subjects for studies of comparative and developmental immunology because they separated from eutherian mammals over 100 million years ago and because the newborns are still in a fetal state. We studied cellular immunity in a fully pedigreed colony of the marsupial, M. domestica (commonly called the gray short-tailed opossum). Peripheral blood lymphocytes were separated on nylon wool columns into adherent cells bearing surface immunoglobulin (B cells) and nonadherent cells (T cells) recovered in the ratio of 1:3. Peripheral blood lymphocytes responded by proliferation to Con A and other mitogens. Nonadherent cells were responsive to Con A, but adherent cells were not. Peripheral blood lymphocytes were stimulated weakly or not at all by allogeneic or xenogeneic (mouse) cells in mixed lymphocyte culture. Despite the weak MLC response, which was not due to genetic homogeneity, allogeneic and xenogeneic tail skin grafts were rejected promptly. These data suggest that the cellular immune response of M. domestica is similar to that of eutherian mammals with the notable exception of weak MLC responses.     

Characterisation of echidna IgM provides insights into the time of divergence of extant mammals

The immunobiology of monotremes is poorly understood. In this paper, we describe the characterisation of the heavy chain of IgM from Tachyglossus aculeatus, the short-beaked echidna. The echidna heavy chain constant region of IgM (Cμ) was isolated from a spleen cDNA library using a Trichosurus vulpecula probe. It has approximately 46.5% amino acid identity to marsupial and eutherian Cμs, and approximately 30% amino acid identity with Cμs from birds and reptiles. Phylogenetic analysis of mammalian Cμ provides strong support for the Theria hypothesis, with a sister grouping of the eutherians and marsupials to the exclusion of the monotremes. Cμ sequences suggest that monotremes and therians separated approximately 170 million years ago (mya), marsupials and eutherians separated approximately 130 mya, and Australian and American marsupials separated approximately 65 mya.     

Immune response of a marsupial (Monodelphis domestica) to sheep red blood cells

A study of the humoral immune response of a marsupial, Monodelphis domestica, was performed using sheep red blood cells as the immunogen and a hemolytic titration as the assay. The primary response to a single intramuscular injection was similar to that observed in other mammalian species, except that the titers remained high for as long as 37 weeks after the initial injection. The secondary response was weaker than the primary response and diminished as early as 15 weeks after the initial injection (and only 11 weeks after the single booster injection). The hyperimmune response was similar to the secondary response, except that the titers remained consistent for up to 10 weeks and then fell to very low levels at 35 weeks. The data support the view that the primary humoral response of this marsupial is similar to that of eutherian mammals. However, the secondary and hyperimmune responses appear to be very weak, if present at all.     

Molecular characterization and evolution of X and Y-borne <Emphasis Type="Italic">ATRX</Emphasis> homologues in American marsupials

In eutherians, the sex-reversing ATRX gene on the X has no homologue on the Y chromosome. However, testis-specific and ubiquitously expressed X-borne genes have been identified in Australian marsupials. We studied nucleotide sequence and chromosomal location of ATRX homologues in two American marsupials, the opossums Didelphis virginiana and Monodelphis domestica. A PCR fragment of M. domestica ATRX was used to probe Southern blots and to screen male genomic libraries. Southern analysis demonstrated ATRX homologues on both X and Y in D. virginiana, and two clones were isolated which hybridized to a single position on the Y chromosome in male-derived cells but to multiple sites of the X in female cells. In M. domestica, there was a single clone that mapped to the X but not to the Y, suggesting that it represents the M. domestica ATRX. However a male-specific band was detected in Southern blots probed with the D. virginiana ATRY and with a mouse ATRX clone, which implies that the Y copy in M. domestica has diverged further from other ATRX homologues. Thus there appears to be a Y-borne copy of ATRY in American, as well as Australian marsupials, although it has diverged in sequence, as have other Y genes that are testis-specific in both eutherian and marsupial lineages.     

The opossum genome: insights and opportunities from an alternative mammal

The strategic importance of the genome sequence of the gray, short-tailed opossum, Monodelphis domestica, accrues from both the unique phylogenetic position of metatherian (marsupial) mammals and the fundamental biologic characteristics of metatherians that distinguish them from other mammalian species. Metatherian and eutherian (placental) mammals are more closely related to one another than to other vertebrate groups, and owing to this close relationship they share fundamentally similar genetic structures and molecular processes. However, during their long evolutionary separation these alternative mammals have developed distinctive anatomical, physiologic, and genetic features that hold tremendous potential for examining relationships between the molecular structures of mammalian genomes and the functional attributes of their components. Comparative analyses using the opossum genome have already provided a wealth of new evidence regarding the importance of noncoding elements in the evolution of mammalian genomes, the role of transposable elements in driving genomic innovation, and the relationships between recombination rate, nucleotide composition, and the genomic distributions of repetitive elements. The genome sequence is also beginning to enlarge our understanding of the evolution and function of the vertebrate immune system, and it provides an alternative model for investigating mechanisms of genomic imprinting. Equally important, availability of the genome sequence is fostering the development of new research tools for physical and functional genomic analyses of M. domestica that are expanding its versatility as an experimental system for a broad range of research applications in basic biology and biomedically oriented research.