Medical Biology - Research Publications
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ItemNo Preview AvailableA newly discovered protein export machine in malaria parasites(NATURE PORTFOLIO, 2009-06-18)Several hundred malaria parasite proteins are exported beyond an encasing vacuole and into the cytosol of the host erythrocyte, a process that is central to the virulence and viability of the causative Plasmodium species. The trafficking machinery responsible for this export is unknown. Here we identify in Plasmodium falciparum a translocon of exported proteins (PTEX), which is located in the vacuole membrane. The PTEX complex is ATP-powered, and comprises heat shock protein 101 (HSP101; a ClpA/B-like ATPase from the AAA+ superfamily, of a type commonly associated with protein translocons), a novel protein termed PTEX150 and a known parasite protein, exported protein 2 (EXP2). EXP2 is the potential channel, as it is the membrane-associated component of the core PTEX complex. Two other proteins, a new protein PTEX88 and thioredoxin 2 (TRX2), were also identified as PTEX components. As a common portal for numerous crucial processes, this translocon offers a new avenue for therapeutic intervention.
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ItemMolecular genetics and comparative genomics reveal RNAi is not functional in malaria parasites(OXFORD UNIV PRESS, 2009-06)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.
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ItemA Novel Family of Apicomplexan Glideosome-associated Proteins with an Inner Membrane-anchoring Role(AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC, 2009-09-11)The phylum Apicomplexa are a group of obligate intracellular parasites responsible for a wide range of important diseases. Central to the lifecycle of these unicellular parasites is their ability to migrate through animal tissue and invade target host cells. Apicomplexan movement is generated by a unique system of gliding motility in which substrate adhesins and invasion-related proteins are pulled across the plasma membrane by an underlying actin-myosin motor. The myosins of this motor are inserted into a dual membrane layer called the inner membrane complex (IMC) that is sandwiched between the plasma membrane and an underlying cytoskeletal basket. Central to our understanding of gliding motility is the characterization of proteins residing within the IMC, but to date only a few proteins are known. We report here a novel family of six-pass transmembrane proteins, termed the GAPM family, which are highly conserved and specific to Apicomplexa. In Plasmodium falciparum and Toxoplasma gondii the GAPMs localize to the IMC where they form highly SDS-resistant oligomeric complexes. The GAPMs co-purify with the cytoskeletal alveolin proteins and also to some degree with the actin-myosin motor itself. Hence, these proteins are strong candidates for an IMC-anchoring role, either directly or indirectly tethering the motor to the cytoskeleton.
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ItemAnalysis of a set of Australian northern brown bandicoot expressed sequence tags with comparison to the genome sequence of the South American grey short tailed opossum(BMC, 2007-02-13)BACKGROUND: Expressed sequence tags (ESTs) have been used for rapid gene discovery in a variety of organisms and provide a valuable resource for whole genome annotation. Although the genome of one marsupial, the opossum Monodelphis domestica, has now been sequenced, no EST datasets have been reported from any marsupial species. In this study we describe an EST dataset from the bandicoot, Isoodon macrourus, providing information on the transcriptional profile of the bandicoot thymus and the opportunity for a genome wide comparison between the bandicoot and opossum, two distantly related marsupial species. RESULTS: A set of 1319 ESTs was generated from sequencing randomly chosen clones from a bandicoot thymus cDNA library. The nucleic acid and deduced amino acid sequences were compared with sequences both in GenBank and the recently completed whole genome sequence of M. domestica. This study provides information on the transcriptional profile of the bandicoot thymus with the identification of genes involved in a broad range of activities including protein metabolism (24%), transcription and/or nucleic acid metabolism (10%), metabolism/energy pathways (9%), immunity (5%), signal transduction (5%), cell growth and maintenance (3%), transport (3%), cell cycle (0.7%) and apoptosis (0.5%) and a proportion of genes whose function is unknown (5.8%). Thirty four percent of the bandicoot ESTs found no match with annotated sequences in any of the public databases. Clustering and assembly of the 1319 bandicoot ESTs resulted in a set of 949 unique sequences of which 375 were unannotated ESTs. Of these, seventy one unannotated ESTs aligned to non-coding regions in the opossum, human, or both genomes, and were identified as strong non-coding RNA candidates. Eighty-four percent of the 949 assembled ESTs aligned with the M. domestica genome sequence indicating a high level of conservation between these two distantly related marsupials. CONCLUSION: This study is among the first reported marsupial EST datasets with a significant inter-species genome comparison between marsupials, providing a valuable resource for transcriptional analyses in marsupials and for future annotation of marsupial whole genome sequences.
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ItemEvolution and comparative analysis of the MHC Class III inflammatory region(BMC, 2006-11-02)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.
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ItemIn silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals.(Springer Science and Business Media LLC, 2006-11-10)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-gamma, 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.
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ItemAnalysis of the platypus genome suggests a transposon origin for mammalian imprinting(BIOMED CENTRAL LTD, 2009)BACKGROUND: Genomic imprinting is an epigenetic phenomenon that results in monoallelic gene expression. Many hypotheses have been advanced to explain why genomic imprinting evolved in mammals, but few have examined how it arose. The host defence hypothesis suggests that imprinting evolved from existing mechanisms within the cell that act to silence foreign DNA elements that insert into the genome. However, the changes to the mammalian genome that accompanied the evolution of imprinting have been hard to define due to the absence of large scale genomic resources between all extant classes. The recent release of the platypus genome has provided the first opportunity to perform comparisons between prototherian (monotreme; which appear to lack imprinting) and therian (marsupial and eutherian; which have imprinting) mammals. RESULTS: We compared the distribution of repeat elements known to attract epigenetic silencing across the entire genome from monotremes and therian mammals, particularly focusing on the orthologous imprinted regions. There is a significant accumulation of certain repeat elements within imprinted regions of therian mammals compared to the platypus. CONCLUSIONS: Our analyses show that the platypus has significantly fewer repeats of certain classes in the regions of the genome that have become imprinted in therian mammals. The accumulation of repeats, especially long terminal repeats and DNA elements, in therian imprinted genes and gene clusters is coincident with, and may have been a potential driving force in, the development of mammalian genomic imprinting. These data provide strong support for the host defence hypothesis.