Immunome database for marsupials and monotremes

Background: To understand the evolutionary origins of our own immune system, we need to characterise the immune system of our distant relatives, the marsupials and monotremes. The recent sequencing of the genomes of two marsupials (opossum and tammar wallaby) and a monotreme (platypus) provides an opportunity to characterise the immune gene repertoires of these model organisms. This was required as many genes involved in immunity evolve rapidly and fail to be detected by automated gene annotation pipelines.Description: We have developed a database of immune genes from the tammar wallaby, red-necked wallaby, northern brown bandicoot, brush-tail possum, opossum, echidna and platypus. The resource contains 2,235 newly identified sequences and 3,197 sequences which had been described previously. This comprehensive dataset was built from a variety of sources, including EST projects and expert-curated gene predictions generated through a variety of methods including chained-BLAST and sensitive HMMER searches. To facilitate systems-based research we have grouped sequences based on broad Gene Ontology categories as well as by specific functional immune groups. Sequences can be extracted by keyword, gene name, protein domain and organism name. Users can also search the database using BLAST.Conclusion: The Immunome Database for Marsupials and Monotremes (IDMM) is a comprehensive database of all known marsupial and monotreme immune genes. It provides a single point of reference for genomic and transcriptomic datasets. Data from other marsupial and monotreme species will be added to the database as it become available. This resource will be utilized by marsupial and monotreme immunologists as well as researchers interested in the evolution of mammalian immunity

In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals

BACKGROUND: Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals. RESULTS: The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations. CONCLUSION: The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals

Identification and analysis of divergent immune gene families within the Tasmanian devil genome

Background: The Tasmanian devil (Sarcophilus harrisii) is being threatened with extinction in the wild by a disease known as devil facial tumour disease (DFTD). In order to prevent the spread of this disease a thorough understanding of the Tasmanian devil immune system and its response to the disease is required. In 2011 and 2012 two genome sequencing projects of the Tasmania devil were released. This has provided us with the raw data required to begin to investigate the Tasmanian devil immunome in depth. In this study we characterise immune gene families of the Tasmanian devil. We focus on immunoglobulins, T cell receptors and cytokine families. Results: We identify and describe 119 cytokines including 40 interleukins, 39 chemokines, 8 interferons, 18 tumour necrosis family cytokines and 14 additional cytokines. Constant regions for immunoglobulins and T cell receptors were also identified. The repertoire of genes in these families was similar to the opossum, however devil specific duplications were seen and orthologs to eutherian genes not previously identified in any marsupial were also identified. Conclusions: By using multiple data sources as well as targeted search methods, highly divergent genes across the Tasmanian devil immune system were identified and characterised. This understanding will allow for the development of devil specific assays and reagents and allow for future studies into the immune response of the Tasmanian devil immune system to DFTD

Analysis of the platypus genome suggests a transposon origin for mammalian imprinting

Comparisons between the platypus and eutherian mammalian genomes provides new insights into how epigenetic imprinting may have evolved in mammalian genomes

Telomere dynamics and homeostasis in a transmissible cancer

Devil Facial Tumour Disease (DFTD) is a unique clonal cancer that threatens the world\u27s largest carnivorous marsupial, the Tasmanian devil (Sarcophilus harrisii) with extinction. This transmissible cancer is passed between individual devils by cell implantation during social interactions. The tumour arose in a Schwann cell of a single devil over 15 years ago and since then has expanded clonally, without showing signs of replicative senescence; in stark contrast to a somatic cell that displays a finite capacity for replication, known as the “Hayflick limit”

Devil Facial Tumor Disease, A Potential Model of the Cancer Stem-Cell Process?

Tasmanian devil facial tumor disease (DFTD) is a naturally occurring contagious cancer which is transmitted as a clonal cell line between devils. The malignant cell line evolved from a Schwann cell or precursor prior to 1996 and since then has undergone continuous division without exhausting its replicative potential, suggesting a profound capacity for self renewal. It is therefore important to elucidate whether DFTD may have a stem cell origin. Deciphering the pathways regulating DFT cell proliferation and survival could lead to increased understanding of this transimissible cancer and to the development of successful therapies to halt the disease. We investigated whether DFT cells have originated from transformed stem cells by measuring the expression levels of thirteen genes characteristic to embryonic stem and/or pluripotent germ cells. No differences in gene expression were observed between DFT cells and peripheral nerve controls, and therefore our results provide additional support for Schwann cell or peripheral nerve origin of DFTD. Although our dataset is preliminary, it does not suggest that DFTs have cancer stem cells (CSCs) origin. We provide details of further experiments needed to ultimately confirm the role of cancer stem cells in DFTD progression

Evolution and comparative analysis of the MHC Class III inflammatory region

BACKGROUND: The Major Histocompatibility Complex (MHC) is essential for immune function. Historically, it has been subdivided into three regions (Class I, II, and III), but a cluster of functionally related genes within the Class III region has also been referred to as the Class IV region or "inflammatory region". This group of genes is involved in the inflammatory response, and includes members of the tumour necrosis family. Here we report the sequencing, annotation and comparative analysis of a tammar wallaby BAC containing the inflammatory region. We also discuss the extent of sequence conservation across the entire region and identify elements conserved in evolution. RESULTS: Fourteen Class III genes from the tammar wallaby inflammatory region were characterised and compared to their orthologues in other vertebrates. The organisation and sequence of genes in the inflammatory region of both the wallaby and South American opossum are highly conserved compared to known genes from eutherian ("placental") mammals. Some minor differences separate the two marsupial species. Eight genes within the inflammatory region have remained tightly clustered for at least 360 million years, predating the divergence of the amphibian lineage. Analysis of sequence conservation identified 354 elements that are conserved. These range in size from 7 to 431 bases and cover 15.6% of the inflammatory region, representing approximately a 4-fold increase compared to the average for vertebrate genomes. About 5.5% of this conserved sequence is marsupial-specific, including three cases of marsupial-specific repeats. Highly Conserved Elements were also characterised. CONCLUSION: Using comparative analysis, we show that a cluster of MHC genes involved in inflammation, including TNF, LTA (or its putative teleost homolog TNF-N), APOM, and BAT3 have remained together for over 450 million years, predating the divergence of mammals from fish. The observed enrichment in conserved sequences within the inflammatory region suggests conservation at the transcriptional regulatory level, in addition to the functional level

Molecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites

Techniques for targeted genetic disruption in Plasmodium, the causative agent of malaria, are currently intractable for those genes that are essential for blood stage development. The ability to use RNA interference (RNAi) to silence gene expression would provide a powerful means to gain valuable insight into the pathogenic blood stages but its functionality in Plasmodium remains controversial. Here we have used various RNA-based gene silencing approaches to test the utility of RNAi in malaria parasites and have undertaken an extensive comparative genomics search using profile hidden Markov models to clarify whether RNAi machinery exists in malaria. These investigative approaches revealed that Plasmodium lacks the enzymology required for RNAi-based ablation of gene expression and indeed no experimental evidence for RNAi was observed. In its absence, the most likely explanations for previously reported RNAi-mediated knockdown are either the general toxicity of introduced RNA (with global down-regulation of gene expression) or a specific antisense effect mechanistically distinct from RNAi, which will need systematic analysis if it is to be of use as a molecular genetic tool for malaria parasites

Targeting of the Tumor Suppressor GRHL3 by a miR-21-Dependent Proto-Oncogenic Network Results in PTEN Loss and Tumorigenesis

SummaryDespite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC

Novel venom gene discovery in the platypus

Background: To date, few peptides in the complex mixture of platypus venom have been identified and sequenced, in part due to the limited amounts of platypus venom available to study. We have constructed and sequenced a cDNA library from an active platypus venom gland to identify the remaining components.Results: We identified 83 novel putative platypus venom genes from 13 toxin families, which are homologous to known toxins from a wide range of vertebrates (fish, reptiles, insectivores) and invertebrates (spiders, sea anemones, starfish). A number of these are expressed in tissues other than the venom gland, and at least three of these families (those with homology to toxins from distant invertebrates) may play non-toxin roles. Thus, further functional testing is required to confirm venom activity. However, the presence of similar putative toxins in such widely divergent species provides further evidence for the hypothesis that there are certain protein families that are selected preferentially during evolution to become venom peptides. We have also used homology with known proteins to speculate on the contributions of each venom component to the symptoms of platypus envenomation.Conclusions: This study represents a step towards fully characterizing the first mammal venom transcriptome. We have found similarities between putative platypus toxins and those of a number of unrelated species, providing insight into the evolution of mammalian venom