School of BioSciences - Research Publications

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    Proteomics and Deep Sequencing Comparison of Seasonally Active Venom Glands in the Platypus Reveals Novel Venom Peptides and Distinct Expression Profiles
    Wong, ESW ; Morgenstern, D ; Mofiz, E ; Gombert, S ; Morris, KM ; Temple-Smith, P ; Renfree, MB ; Whittington, CM ; King, GF ; Warren, WC ; Papenfuss, AT ; Belov, K (AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2012-11)
    The platypus is a venomous monotreme. Male platypuses possess a spur on their hind legs that is connected to glands in the pelvic region. They produce venom only during the breeding season, presumably to fight off conspecifics. We have taken advantage of this unique seasonal production of venom to compare the transcriptomes of in- and out-of-season venom glands, in conjunction with proteomic analysis, to identify previously undiscovered venom genes. Comparison of the venom glands revealed distinct gene expression profiles that are consistent with changes in venom gland morphology and venom volumes in and out of the breeding season. Venom proteins were identified through shot-gun sequenced venom proteomes of three animals using RNA-seq-derived transcripts for peptide-spectral matching. 5,157 genes were expressed in the venom glands, 1,821 genes were up-regulated in the in-season gland, and 10 proteins were identified in the venom. New classes of platypus-venom proteins identified included antimicrobials, amide oxidase, serpin protease inhibitor, proteins associated with the mammalian stress response pathway, cytokines, and other immune molecules. Five putative toxins have only been identified in platypus venom: growth differentiation factor 15, nucleobindin-2, CD55, a CXC-chemokine, and corticotropin-releasing factor-binding protein. These novel venom proteins have potential biomedical and therapeutic applications and provide insights into venom evolution.
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    Unique small RNA signatures uncovered in the tammar wallaby genome
    Lindsay, J ; Carone, DM ; Brown, J ; Hall, L ; Qureshi, S ; Mitchell, SE ; Jannetty, N ; Hannon, G ; Renfree, M ; Pask, A ; O'Neill, M ; O'Neill, R (BIOMED CENTRAL LTD, 2012-10-17)
    BACKGROUND: Small RNAs have proven to be essential regulatory molecules encoded within eukaryotic genomes. These short RNAs participate in a diverse array of cellular processes including gene regulation, chromatin dynamics and genome defense. The tammar wallaby, a marsupial mammal, is a powerful comparative model for studying the evolution of regulatory networks. As part of the genome sequencing initiative for the tammar, we have explored the evolution of each of the major classes of mammalian small RNAs in an Australian marsupial for the first time, including the first genome-scale analysis of the newest class of small RNAs, centromere repeat associated short interacting RNAs (crasiRNAs). RESULTS: Using next generation sequencing, we have characterized the major classes of small RNAs, micro (mi) RNAs, piwi interacting (pi) RNAs, and the centromere repeat associated short interacting (crasi) RNAs in the tammar. We examined each of these small RNA classes with respect to the newly assembled tammar wallaby genome for gene and repeat features, salient features that define their canonical sequences, and the constitution of both highly conserved and species-specific members. Using a combination of miRNA hairpin predictions and co-mapping with miRBase entries, we identified a highly conserved cluster of miRNA genes on the X chromosome in the tammar and a total of 94 other predicted miRNA producing genes. Mapping all miRNAs to the tammar genome and comparing target genes among tammar, mouse and human, we identified 163 conserved target genes. An additional nine genes were identified in tammar that do not have an orthologous miRNA target in human and likely represent novel miRNA-regulated genes in the tammar. A survey of the tammar gonadal piRNAs shows that these small RNAs are enriched in retroelements and carry members from both marsupial and tammar-specific repeat classes. Lastly, this study includes the first in-depth analyses of the newly discovered crasiRNAs. These small RNAs are derived largely from centromere-enriched retroelements, including a novel SINE. CONCLUSIONS: This study encompasses the first analyses of the major classes of small RNAs for the newly completed tammar genome, validates preliminary annotations using deep sequencing and computational approaches, and provides a foundation for future work on tammar-specific as well as conserved, but previously unknown small RNA progenitors and targets identified herein. The characterization of new miRNA target genes and a unique profile for crasiRNAs has allowed for insight into multiple RNA mediated processes in the tammar, including gene regulation, species incompatibilities, centromere and chromosome function.
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    Limited Genetic Diversity Preceded Extinction of the Tasmanian Tiger
    Menzies, BR ; Renfree, MB ; Heider, T ; Mayer, F ; Hildebrandt, TB ; Pask, AJ ; Orlando, L (PUBLIC LIBRARY SCIENCE, 2012-04-18)
    The Tasmanian tiger or thylacine was the largest carnivorous marsupial when Europeans first reached Australia. Sadly, the last known thylacine died in captivity in 1936. A recent analysis of the genome of the closely related and extant Tasmanian devil demonstrated limited genetic diversity between individuals. While a similar lack of diversity has been reported for the thylacine, this analysis was based on just two individuals. Here we report the sequencing of an additional 12 museum-archived specimens collected between 102 and 159 years ago. We examined a portion of the mitochondrial DNA hyper-variable control region and determined that all sequences were on average 99.5% identical at the nucleotide level. As a measure of accuracy we also sequenced mitochondrial DNA from a mother and two offspring. As expected, these samples were found to be 100% identical, validating our methods. We also used 454 sequencing to reconstruct 2.1 kilobases of the mitochondrial genome, which shared 99.91% identity with the two complete thylacine mitochondrial genomes published previously. Our thylacine genomic data also contained three highly divergent putative nuclear mitochondrial sequences, which grouped phylogenetically with the published thylacine mitochondrial homologs but contained 100-fold more polymorphisms than the conserved fragments. Together, our data suggest that the thylacine population in Tasmania had limited genetic diversity prior to its extinction, possibly as a result of their geographic isolation from mainland Australia approximately 10,000 years ago.
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    Promoter-Specific Expression and Imprint Status of Marsupial IGF2
    Stringer, JM ; Suzuki, S ; Pask, AJ ; Shaw, G ; Renfree, MB ; Thomas, T (PUBLIC LIBRARY SCIENCE, 2012-07-25)
    In mice and humans, IGF2 has multiple promoters to maintain its complex tissue- and developmental stage-specific imprinting and expression. IGF2 is also imprinted in marsupials, but little is known about its promoter region. In this study, three IGF2 transcripts were isolated from placental and liver samples of the tammar wallaby, Macropus eugenii. Each transcript contained a unique 5' untranslated region, orthologous to the non-coding exons derived from promoters P1-P3 in the human and mouse IGF2 locus. The expression of tammar IGF2 was predominantly from the P2 promoter, similar to humans. Expression of IGF2 was higher in pouch young than in the adult and imprinting was highly tissue and developmental-stage specific. Interestingly, while IGF2 was expressed throughout the placenta, imprinting seemed to be restricted to the vascular, trilaminar region. In addition, IGF2 was monoallelically expressed in the adult mammary gland while in the liver it switched from monoalleleic expression in the pouch young to biallelic in the adult. These data suggest a complex mode of IGF2 regulation in marsupials as seen in eutherian mammals. The conservation of the IGF2 promoters suggests they originated before the divergence of marsupials and eutherians, and have been selectively maintained for at least 160 million years.
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    Evolution of coding and non-coding genes in HOX clusters of a marsupial
    Yu, H ; Lindsay, J ; Feng, Z-P ; Frankenberg, S ; Hu, Y ; Carone, D ; Shaw, G ; Pask, AJ ; O'Neill, R ; Papenfuss, AT ; Renfree, MB (BMC, 2012-06-18)
    BACKGROUND: The HOX gene clusters are thought to be highly conserved amongst mammals and other vertebrates, but the long non-coding RNAs have only been studied in detail in human and mouse. The sequencing of the kangaroo genome provides an opportunity to use comparative analyses to compare the HOX clusters of a mammal with a distinct body plan to those of other mammals. RESULTS: Here we report a comparative analysis of HOX gene clusters between an Australian marsupial of the kangaroo family and the eutherians. There was a strikingly high level of conservation of HOX gene sequence and structure and non-protein coding genes including the microRNAs miR-196a, miR-196b, miR-10a and miR-10b and the long non-coding RNAs HOTAIR, HOTAIRM1 and HOXA11AS that play critical roles in regulating gene expression and controlling development. By microRNA deep sequencing and comparative genomic analyses, two conserved microRNAs (miR-10a and miR-10b) were identified and one new candidate microRNA with typical hairpin precursor structure that is expressed in both fibroblasts and testes was found. The prediction of microRNA target analysis showed that several known microRNA targets, such as miR-10, miR-414 and miR-464, were found in the tammar HOX clusters. In addition, several novel and putative miRNAs were identified that originated from elsewhere in the tammar genome and that target the tammar HOXB and HOXD clusters. CONCLUSIONS: This study confirms that the emergence of known long non-coding RNAs in the HOX clusters clearly predate the marsupial-eutherian divergence 160 Ma ago. It also identified a new potentially functional microRNA as well as conserved miRNAs. These non-coding RNAs may participate in the regulation of HOX genes to influence the body plan of this marsupial.
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    Evolution of vertebrate interferon inducible transmembrane proteins
    Hickford, D ; Frankenberg, S ; Shaw, G ; Renfree, MB (BMC, 2012-04-26)
    BACKGROUND: Interferon inducible transmembrane proteins (IFITMs) have diverse roles, including the control of cell proliferation, promotion of homotypic cell adhesion, protection against viral infection, promotion of bone matrix maturation and mineralisation, and mediating germ cell development. Most IFITMs have been well characterised in human and mouse but little published data exists for other animals. This study characterised IFITMs in two distantly related marsupial species, the Australian tammar wallaby and the South American grey short-tailed opossum, and analysed the phylogeny of the IFITM family in vertebrates. RESULTS: Five IFITM paralogues were identified in both the tammar and opossum. As in eutherians, most marsupial IFITM genes exist within a cluster, contain two exons and encode proteins with two transmembrane domains. Only two IFITM genes, IFITM5 and IFITM10, have orthologues in both marsupials and eutherians. IFITM5 arose in bony fish and IFITM10 in tetrapods. The bone-specific expression of IFITM5 appears to be restricted to therian mammals, suggesting that its specialised role in bone production is a recent adaptation specific to mammals. IFITM10 is the most highly conserved IFITM, sharing at least 85% amino acid identity between birds, reptiles and mammals and suggesting an important role for this presently uncharacterised protein. CONCLUSIONS: Like eutherians, marsupials also have multiple IFITM genes that exist in a gene cluster. The differing expression patterns for many of the paralogues, together with poor sequence conservation between species, suggests that IFITM genes have acquired many different roles during vertebrate evolution.
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    The mammary gland-specific marsupial ELP and eutherian CTI share a common ancestral gene
    Pharo, EA ; De Leo, AA ; Renfree, MB ; Thomson, PC ; Lefevre, CM ; Nicholas, KR (BMC, 2012-06-08)
    BACKGROUND: The marsupial early lactation protein (ELP) gene is expressed in the mammary gland and the protein is secreted into milk during early lactation (Phase 2A). Mature ELP shares approximately 55.4% similarity with the colostrum-specific bovine colostrum trypsin inhibitor (CTI) protein. Although ELP and CTI both have a single bovine pancreatic trypsin inhibitor (BPTI)-Kunitz domain and are secreted only during the early lactation phases, their evolutionary history is yet to be investigated. RESULTS: Tammar ELP was isolated from a genomic library and the fat-tailed dunnart and Southern koala ELP genes cloned from genomic DNA. The tammar ELP gene was expressed only in the mammary gland during late pregnancy (Phase 1) and early lactation (Phase 2A). The opossum and fat-tailed dunnart ELP and cow CTI transcripts were cloned from RNA isolated from the mammary gland and dog CTI from cells in colostrum. The putative mature ELP and CTI peptides shared 44.6%-62.2% similarity. In silico analyses identified the ELP and CTI genes in the other species examined and provided compelling evidence that they evolved from a common ancestral gene. In addition, whilst the eutherian CTI gene was conserved in the Laurasiatherian orders Carnivora and Cetartiodactyla, it had become a pseudogene in others. These data suggest that bovine CTI may be the ancestral gene of the Artiodactyla-specific, rapidly evolving chromosome 13 pancreatic trypsin inhibitor (PTI), spleen trypsin inhibitor (STI) and the five placenta-specific trophoblast Kunitz domain protein (TKDP1-5) genes. CONCLUSIONS: Marsupial ELP and eutherian CTI evolved from an ancestral therian mammal gene before the divergence of marsupials and eutherians between 130 and 160 million years ago. The retention of the ELP gene in marsupials suggests that this early lactation-specific milk protein may have an important role in the immunologically naïve young of these species.
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    Selected imprinting of INS in the marsupial
    Stringer, JM ; Suzuki, S ; Pask, AJ ; Shaw, G ; Renfree, MB (BMC, 2012-08-28)
    BACKGROUND: In marsupials, growth and development of the young occur postnatally, regulated by milk that changes in composition throughout the long lactation. To initiate lactation in mammals, there is an absolute requirement for insulin (INS), a gene known to be imprinted in the placenta. We therefore examined whether INS is imprinted in the mammary gland of the marsupial tammar wallaby (Macropus eugenii) and compared its expression with that of insulin-like growth factor 2 (IGF2). RESULTS: INS was expressed in the mammary gland and significantly increased, while IGF2 decreased, during established milk production. Insulin and IGF2 were both detected in the mammary gland macrophage cells during early lactation and in the alveolar cells later in lactation. Surprisingly, INS, which was thought only to be imprinted in the therian yolk sac, was imprinted and paternally expressed in the liver of the developing young, monoallelically expressed in the tammar mammary gland and biallelic in the stomach and intestine. The INS transcription start site used in the liver and mammary gland was differentially methylated. CONCLUSIONS: This is the first study to identify tissue-specific INS imprinting outside the yolk sac. These data suggest that there may be an advantage of selective monoallelic expression in the mammary gland and that this may influence the growth of the postnatal young. These results are not consistent with the parental conflict hypothesis, but instead provide support for the maternal-infant co-adaptation hypothesis. Thus, imprinting in the mammary gland maybe as critical for postnatal growth and development in mammals as genomic imprinting in the placenta is prenatally.
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    HOXA13 and HOXD13 expression during development of the syndactylous digits in the marsupial Macropus eugenii
    Chew, KY ; Yu, H ; Pask, AJ ; Shaw, G ; Renfree, MB (BMC, 2012-01-11)
    BACKGROUND: Kangaroos and wallabies have specialised limbs that allow for their hopping mode of locomotion. The hindlimbs differentiate much later in development but become much larger than the forelimbs. The hindlimb autopod has only four digits, the fourth of which is greatly elongated, while digits two and three are syndactylous. We investigated the expression of two genes, HOXA13 and HOXD13, that are crucial for digit patterning in mice during formation of the limbs of the tammar wallaby. RESULTS: We describe the development of the tammar limbs at key stages before birth. There was marked heterochrony and the hindlimb developed more slowly than the forelimb. Both tammar HOXA13 and HOXD13 have two exons as in humans, mice and chickens. HOXA13 had an early and distal mRNA distribution in the tammar limb bud as in the mouse, but forelimb expression preceded that in the hindlimb. HOXD13 mRNA was expressed earlier in the forelimb than the hindlimb and was predominantly detected in the interdigital tissues of the forelimb. In contrast, the hindlimb had a more restricted expression pattern that appeared to be expressed at discrete points at both posterior and anterior margins of the limb bud, and was unlike expression seen in the mouse and the chicken. CONCLUSIONS: This is the first examination of HOXA and HOXD gene expression in a marsupial. The gene structure and predicted proteins were highly conserved with their eutherian orthologues. Interestingly, despite the morphological differences in hindlimb patterning, there were no modifications to the polyalanine tract of either HOXA13 or HOXD13 when compared to those of the mouse and bat but there was a marked difference between the tammar and the other mammals in the region of the first polyserine tract of HOXD13. There were also altered expression domains for both genes in the developing tammar limbs compared to the chicken and mouse. Together these findings suggest that the timing of HOX gene expression may contribute to the heterochrony of the forelimb and hindlimb and that alteration to HOX domains may influence phenotypic differences that lead to the development of marsupial syndactylous digits.