Isolation and characterization of marsupial IL5 genes

The genomic nucleotide sequence and chromosomal position of the interleukin 5 (IL5) gene has been described for the model marsupial Macropus eugenii (tammar wallaby). A 272 base pair genomic IL5 polymerase chain reaction (PCR) product spanning exon 3, intron 3, and exon 4 was generated using stripe-faced dunnart (Sminthopsis macroura) DNA. This PCR product was used to isolate a genomic lambda clone containing the complete IL5 gene from a tammar wallaby EMBL3 lambda library. Sequencing revealed that the tammar wallaby IL5 gene consists of four exons separated by three introns. Comparison of the marsupial coding sequence with coding sequences from eutherian species revealed 61 to 69% identity at the nucleotide level and 48 to 63% identity at the amino acid (aa) level. A polymorphic complex compound microsatellite was identified within intron 2 of the tammar wallaby IL5 gene. This microsatellite was also found in other marsupials including the swamp wallaby, tree kangaroo, stripe-faced dunnart, South American opossum, brushtail possum, and koala. Fluorescence in situ hybridization using DNA from the IL5 clone on tammar wallaby chromosomes indicated that the IL5 gene is located on Chromosome 1.     

Pulmonary cytochrome P450 enzymes belonging to the CYP4B subfamily from an Australian marsupial,the tammar wallaby (Macropus eugenii)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported the cloning and characterisation of the koala CYP4A15, the first reported member of the CYP4 family from marsupials, and have demonstrated important species differences in CYP4A activity and tissue expression. In the present study, the cloning of CYP4B1 in the wallaby (Macropus eugenii) and their expression across marsupials is described. Rabbit anti-mouse CYP4B1 antibody detected immunoreactive proteins in lung and liver microsomes from all test marsupials, with relative weak signal detected from the koala, suggesting a species-specific expression. Microsomal 2-aminofluorene bio-activation (a CYP4B1 marker) in wallaby lung was comparable to that of rabbit, with significant higher activities detected in wallaby liver and kidneys compared to rabbit. A 1548 bp wallaby lung CYP4B complete cDNA, designated CYP4B1, which encodes a protein of 510 amino acids and shares 72% nucleotide and 69% amino acid sequence identity to human CYP4B1, was cloned by polymerase chain reaction approaches. The results demonstrate the presence of wallaby CYP4B1 that shares several common features with other published CYP4Bs; however the wallaby CYP4B1 cDNA contains four extra amino acid residues at the NH₂-terminal, a fundamentally conserved transmembrane anchor of all eukaryote CYPs.     

Identification of tammar wallaby SIRH12, derived from a marsupial-specific retrotransposition event

In humans and mice, there are 11 genes derived from sushi-ichi related retrotransposons, some of which are known to play essential roles in placental development. Interestingly, this family of retrotransposons was thought to exist only in eutherian mammals, indicating their significant contributions to the eutherian evolution, but at least one, PEG10, is conserved between marsupials and eutherians. Here we report a novel sushi-ichi retrotransposon-derived gene, SIRH12, in the tammar wallaby, an Australian marsupial species of the kangaroo family. SIRH12 encodes a protein highly homologous to the sushi-ichi retrotransposon Gag protein in the tammar wallaby, while SIRH12 in the South American short-tailed grey opossum is a pseudogene degenerated by accumulation of multiple nonsense mutations. This suggests that SIRH12 retrotransposition occurred only in the marsupial lineage but acquired and retained some as yet unidentified novel function, at least in the lineage of the tammar wallaby.     

Proteins of marsupial erythrocyte membranes

The proteins of erythrocyte membranes from the red kangaroo, western grey kangaroo, eastern grey wallaroo (euro), red-necked wallaby, Tammar wallaby, and brush-tail possum have been fractionated on acrylamide gels in the presence of sodium dodecyl sulfate. The pattern of proteins was remarkably similar between the different marsupial species. The pattern of Coomassie blue-staining proteins in the membranes of these species was also very similar to that of the human erythrocyte membrane. However, the glycoproteins in the marsupial erythrocyte membranes were markedly less conspicuous than those of the human erythrocyte membrane. Furthermore, the mobilities of the glycoproteins from the marsupials were different from those of the human erythrocyte membrane. The erythrocytes of the western grey kangaroo, the eastern wallaroo and the red-necked wallaby showed pronounced resistance to hypotonic lysis compared with those of the Tammar wallaby and the human. This effect seems to be related to the size of the erythrocytes rather than to differences in their protein composition.     

Chemical characterization of milk oligosaccharides of the koala (Phascolarctos cinereus)

Previous structural characterizations of marsupial milk oligosaccharides had been performed in only two macropod species, the tammar wallaby and the red kangaroo. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of the koala milk carbohydrate fraction were characterized in this study. Neutral and acidic oligosaccharides were separated from the carbohydrate fraction of milk of the koala, a non-macropod marsupial, and characterized by ¹H-nuclear magnetic resonance spectroscopy. The structures of the neutral saccharides were found to be Gal(β1-4)Glc (lactose), Gal(β1-3)Gal(β1-4)Glc (3′-galactosyllactose), Gal(β1-3)Gal(β1-3)Gal(β1-4)Glc (3′,3″-digalactosyllactose), Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (lacto-N-novopentaose I) and Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl lacto-N-novopentaose I), while those of the acidic saccharides were Neu5Ac(α2-3)Gal(β1-4)Glc (3′-SL), Neu5Ac(α2-3)Gal(β1-3)Gal(β1-4)Gal (sialyl 3′-galactosyllactose), Neu5Ac(α2-3)Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose a), Gal(β1-3)[Neu5Ac(α2-6)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose b), Gal(β1-3)[Neu5Ac(α2-3)Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc (sialyl lacto-N-novopentaose c), and Neu5Ac(α2-3)Gal(β1-3){Gal(β1-4)[Fuc(α1-3)]GlcNAc(β1-6)}Gal(β1-4)Glc (fucosyl sialyl lacto-N-novopentaose a). The neutral oligosaccharides, other than fucosyl lacto-N-novopentaose I, a novel hexasaccharide, had been found in milk of the tammar wallaby, a macropod marsupial, while the acidic oligosaccharides, other than fucosyl sialyl lacto-N-novopentaose a had been identified in milk carbohydrate of the red kangaroo. The presence of fucosyl oligosaccharides is a significant feature of koala milk, in which it differs from milk of the tammar wallaby and the red kangaroo.     

Testosterone dehydrogenase activity in koala liver: characterisation of cofactor and steroid substrate differences

We have studied the hepatic microsomal 17β-hydroxysteroid dehydrogenase (17β-HSD) capacity of koala (Phascolarctos cinereus) and tammar wallaby (Macropus eugenii). A detailed comparison of the activity in hepatic fractions from koala and rat was made. Hepatic microsomal NADP-supported 17β-HSD activity was significantly higher in koala (11.64±3.35 nmoles/mg protein/min), (mean±S.D.) than in tammar wallaby liver (1.52±0.79 nmoles/mg protein/min). However, when NAD was utilised as cofactor the activity was similar in both marsupial species (2.83±2.03 nmoles/mg protein/min, koala; 0.70±0.71 nmoles/mg protein/min, tammar wallaby). Data for rat indicated a cofactor preference for NAD rather than NADP (17.94±6.40 nmoles/mg protein/min, NAD; 2.18±1.04 nmoles/mg protein/min, NADP). Michaelis–Menten parameters for the kinetics of 17β-HSD testosterone oxidation by NADP and NAD were determined in the koala. The Km for testosterone was of the order of 10.0–24.0 μM (n=6) irrespective of the cofactor used, whilst the Km for NADP was 0.28–0.43 μM (n=2) and for NAD was 13.9–18.5 μM (n=2). 17β-estradiol was found to be an inhibitor of both NAD- and NADP- supported 17β-HSD activity. These findings indicate that NADP-mediated, but not NAD-mediated testosterone dehydrogenation is a major pathway of steroid biotransformation in koala liver; the reaction is less extensive in fractions from wallaby, human and rat. Such species-related differences in cofactor preference may contribute along with species differences in gene expression to observed rates of 17β-HSD activity in mammals.     

Hepatic cytochrome P450 enzymes belonging to the CYP2C subfamily from an Australian marsupial, the koala (Phascolarctos cinereus)

Cytochromes P450 (CYPs) are critically important in the oxidative metabolism of a diverse array of xenobiotics and endogenous substrates. We have previously reported that the obligate Eucalyptus feeder koala (Phascolarctos cinereus) exhibits a higher hepatic CYP2C activity as compared to non-Eucalyptus feeders human or rat, with stimulation of CYP2C activity by cineole. In the present study, we examine CYP2C expression by immunohistochemistry and describe the identification and cloning of koala CYP2Cs. Utilising anti-rat CYP2C6 antibody, the expression of CYP2C was found to be uniform across the hepatic sections, being consistent with that observed in human and rat. Two 1647 and 1638 bp koala liver CYP2C complete cDNAs, designated CYP2C47 and CYP2C48 respectively, were cloned by cDNA library screening. The koala CYP2C cDNAs encode a protein of 495 amino acids. Three additional partial CYP2C sequences were also identified from the koala, indicating the multiplicity of the CYP2C subfamily in this unique marsupial species. The results of this study demonstrate the presence of koala hepatic CYP2Cs that share several common features with other published CYP2Cs; however CYP2C47 and CYP2C48 contain four extra amino acid residues at the NH2-terminal, a transmembrane anchor which was reported being a fundamentally conserved structure core of all eukaryote CYP enzymes.     

Artificial insemination in marsupials

Assisted breeding technology (ART), including artificial insemination (AI), has the potential to advance the conservation and welfare of marsupials. Many of the challenges facing AI and ART for marsupials are shared with other wild species. However, the marsupial mode of reproduction and development also poses unique challenges and opportunities. For the vast majority of marsupials, there is a dearth of knowledge regarding basic reproductive biology to guide an AI strategy. For threatened or endangered species, only the most basic reproductive information is available in most cases, if at all. Artificial insemination has been used to produce viable young in two marsupial species, the koala and tammar wallaby. However, in these species the timing of ovulation can be predicted with considerably more confidence than in any other marsupial. In a limited number of other marsupials, such precise timing of ovulation has only been achieved using hormonal treatment leading to conception but not live young. A unique marsupial ART strategy which has been shown to have promise is cross-fostering; the transfer of pouch young of a threatened species to the pouches of foster mothers of a common related species as a means to increase productivity. For the foreseeable future, except for a few highly iconic or well studied species, there is unlikely to be sufficient reproductive information on which to base AI. However, if more generic approaches can be developed; such as ICSI (to generate embryos) and female synchronization (to provide oocyte donors or embryo recipients), then the prospects for broader application of AI/ART to marsupials are promising.     

Chromosome evolution in kangaroos (Marsupialia: Macropodidae): cross species chromosome painting between the tammar wallaby and rock wallaby spp. with the 2n = 22 ancestral macropodid karyotype.

Marsupial mammals show extraordinary karyotype stability, with 2n = 14 considered ancestral. However, macropodid marsupials (kangaroos and wallabies) exhibit a considerable variety of karyotypes, with a hypothesised ancestral karyotype of 2n = 22. Speciation and karyotypic diversity in rock wallabies (Petrogale) is exceptional. We used cross species chromosome painting to examine the chromosome evolution between the tammar wallaby (2n = 16) and three 2n = 22 rock wallaby species groups with the putative ancestral karyotype. Hybridization of chromosome paints prepared from flow sorted chromosomes of the tammar wallaby to Petrogale spp., showed that this ancestral karyotype is largely conserved among 2n = 22 rock wallaby species, and confirmed the identity of ancestral chromosomes which fused to produce the bi-armed chromosomes of the 2n = 16 tammar wallaby. These results illustrate the fission-fusion process of karyotype evolution characteristic of the kangaroo group.     

Enzymes of galactose metabolism in livers of suckling and adult tammar wallabies (Macropus eugenii) and other marsupials

The activities of galactokinase, hexose-1-phosphate uridylyl transferase and UDPglucose 4-epimerase in homogenates of livers of two adult and 20 suckling tammar wallabies aged from 6 to 50 weeks were investigated. The activities of all three enzymes were high until 24-30 weeks post partum, after which they declined to low levels. The activities of the three liver enzymes were high in pouch young of six other species of marsupial. Comparison of the activities of the three liver enzymes in suckling tammar wallabies with those in suckling rats showed no difference between the two species in regard to galactokinase and uridylyl transferase, but the UDPglucose 4-epimerase activity in tammar wallabies was approximately double than found in rats. This may be related to the high galactose content of tammar wallaby milk compared with rat milk. In suckling tammar wallabies, the liver had higher enzyme activities than other tissues studied. It is concluded that, contrary to the suggestion of Stephens et al. (1975), pouch young marsupials are not deficient in their ability to metabolize galactose.     

The genome of a Gondwanan mammal

Australia is thought of as the home of marsupials, but South America has 60 or so species of these interesting mammals. The genome of one of these, the South American grey short-tailed opossum, Monodelphis domestica, has just been sequenced and published in June.1 The high quality 6x coverage is the first marsupial genome completed, pipping the 2x coverage of the Australian tammar wallaby at the post by half a year. The opossum genome has an unusual structure with fewer chromosomes than the human genome (9 pairs versus 23 pairs) but a longer total length (3.4 billion versus 3 billion bases). The opossum autosomes, like those of all marsupials, are extremely large but, in contrast, the X chromosome is only 76 Mb long. The opossum genome has turned up several surprises and provided critical new information on the evolution of mammalian genomes.     

Isolation of major histocompatibility complex Class I genes from the tammar wallaby (Macropus eugenii)

The major histocompatibility complex (MHC) plays an essential role in the adaptive immune system of vertebrates through antigen recognition. Although MHC genes are found in all vertebrates, the MHC region is dynamic and has changed throughout vertebrate evolution, making it an important tool for comparative genomics. Marsupials occupy an important position in mammalian phylogeny, yet the MHC of few marsupials has been studied in detail. We report the isolation and analysis of expressed MHC Class I genes from the tammar wallaby, a model marsupial used extensively for the study of mammalian reproduction, genetics, and immunology. We determined that there are at least 11 Class I loci in the tammar genome and isolated six expressed Class I sequences from spleen and testes cDNA libraries, representing at least four loci. Two of the Class I sequences contain substitutions at sites known to be important for antigen binding, perhaps impacting their ability to bind peptides, or the types of peptide to which they bind. Phylogenetic analysis of tammar wallaby Class I sequences and other mammalian Class I sequences suggests that some tammar wallaby and red-necked wallaby loci evolved from common ancestral genes.     

1H NMR spectroscopic survey of plasma and erythrocytes from selected marsupials and domestic animals of Australia.

1. 1H NMR spectra were acquired from whole plasma, intact erythrocytes, and ultrafiltrates of erythrocytes from nine native and eight introduced (domestic) Australian animals; single-pulse, spin-echo and 2-dimensional spectra were obtained. The aim was to detect and at least semi-quantify metabolites in the samples and compare the profiles amongst the species. 2. The Australian natives that were studied were all marsupials: greater brown bandicoot; bettong; eastern grey kangaroo; red kangaroo; koala; possum; red necked pademelon; Tammar wallaby; and wombat. The introduced mammals that were studied were: cat; cattle; dog; goat; horse; pig; rabbit; and sheep. 3. Because of the range of habitats and diets amongst the animals, it was postulated that the concentrations of the common metabolites in the blood would show marked differences and that there would also be some metabolites that were peculiar to a given animal. There were several major differences in the spectra: in the spectra of plasma, the glycoprotein and lipoprotein resonances showed the largest inter-species variation, whereas the most dramatic finding from the spectra of erythrocytes was a very high concentration of lysine in the cells from the Tammar wallaby.     

Oral Disease in Animals: The Australian Perspective. Isolation and Characterisation of Black-Pigmented Bacteria from the Oral Cavity of Marsupials

A wide range of animals suffer from periodontal disease. However, there is very little reported on disease and oral micro-biota of Australian animals. Therefore, the oral cavity of 90 marsupials was examined for oral health status. Plaque samples were collected from the subgingival margins using curettes or swabs. Plaque samples were plated onto non-selective trypticase soy agar plates, selective trypticase soy agar, non-selective and selective Wilkens Chalgrens Agar. Plates were incubated in an anaerobic atmosphere and examined after 7–14 days for the presence of black–brown-pigmented colonies. A combination of morphological and biochemical tests were used (colonial morphology, pigmentation, aerobic growth, Gram reaction, fluorescence under long-wave UV light (360 nm), production of catalase, enzymatic activity with fluorogenic substrates and haemagglutination of sheep red cells) to identify these organisms. Black-pigmented bacteria were cultivated from the plaque of 32 animals including six eastern grey kangaroos, a musky rat kangaroo, a whiptail and a red-necked wallaby, 18 koalas, a bandicoot and five brushtail possums. No black-pigmented colonies were cultivated from squirrel or sugar gliders or quokkas or from marsupial mice. The majority of isolates were identified as Porphyromonas gingivalis -like species with the higher prevalence of isolation from the oral cavity of macropods (the kangaroos and wallabies). Oral diseases, such as gingivitis can be found in native Australian animals with older koalas having an increase in disease indicators and black-pigmented bacteria. Non-selective Wilkens Chalgren Agar was the medium of choice for the isolation of black-pigmented bacteria.