dna methylation mapping of myelodysplastic syndrome
TRANSCRIPT
QMB Abstracts
Speaker Abstracts: Session1
Q1. Merging the power of visualization with the power of identification – strategies and developments in cryo-electron microscopy
Juergen M. Plitzko1, Andrew Leis2, Alexander Rigort1 and Wolfgang Baumeister1
1Max Planck Institute of Biochemistry, Dept. of Molecular Structural Biology, D-82152 Martinsried (near Munich), Germany; 2CSIRO Australian Animal Health Laboratory
East Geelong VIC 3219, Australia
Cryo-electron tomography (cryo-ET) of whole cells allows us to investigate the structure-function relationship of molecular complexes and supramolecular assemblies in their native environment [1]. It thus makes a fundamental change in the way we approach biochemical processes that underlie and orchestrate higher cellular functions. In the past, molecular interactions were studied mostly in a collective manner, whereas now we have the tools to visualize the interactions between individual molecules in their unperturbed functional environments. Although they share common underlying principles, no two cells or organelles are identical, owing to the inherent stochasticity of biochemical processes in cells as well as their functional diversity. Therefore, it will be a major challenge to extract generic features from the three dimensional maps, such as the modes of interaction between molecular species. However, the ultimate goal, the discovery of general rules that underlie cellular processes, has to go beyond observing qualitative features and has to be based on stringent analytical criteria combining the information gathered from different methods for a complete integrative analysis [2].
Merging the power of identification (e.g. by quantitative mass spectrometry or correlative light microscopy) with advanced preparation techniques (e.g. focused ion beam milling) while utilizing the power of visualization techniques (by cryo-ET/EM approaches) is definitely a ‘nouvelle route’ with a great potential in the field of molecular structural biology that promises to provide a non-invasive and deep insight into the functional organization of cellular proteomes [3,4]).
1. Leis, A., B. Rockel, L. Andrees, and W. Baumeister: Visualizing cells at the nanoscale. Trends Biochem Sci 2009, 34: 60-70.
2. Robinson CV, A. Sali, W. Baumeister: The molecular sociology of the cell. Nature 2007, 450:973-982 3. Plitzko, J.M., A. Rigort, and A. Leis: Correlative cryo-light microscopy and cryo-electron
tomography: from cellular territories to molecular landscapes. Curr Opin Biotechnol 2009, 20: 83-9. 4. Rigort, A, FJB. Baeuerlein, A. Leis, M. Gruska, C. Hoffmann, T. Laugks, U. Boehm, M. Eibauer, H.
Gnaegi, W. Baumeister and J.M. Plitzko: Micromachining tools and correlative approaches for cellular cryo-electron tomography. J Struct Biol 2010, article in press (doi:10.1016/j.jsb.2010.02.011).
Q2. Super-resolution Fluorescence Microscopy for Live Imaging
Peter Kner1* and Mats Gustafsson2
1Faculty of Engineering, University of Georgia, Athens GA, USA
2Howard Hughes Medical Institute, Janelia Farm Research Campus, Ashburn VA, USA
*email: [email protected]
The image of a point source in a fluorescent microscope is limited by diffraction to a disk with a diameter of , the Airy disk. This length scale, which is roughly 200nm for a modern high-resolution
microscope, limits the resolution of a microscope because two points which are much closer than this will appear to be one point in the image. Recently, several techniques have been developed which achieve resolutions better than , the diffraction limit, in fluorescence microscopy. The resolution achievable
with these techniques ranges from roughly 20nm for Photo-Activatable Localization Microscopy (PALM) to 100nm for linear Structured Illumination Microscopy (SIM). All these techniques were originally demonstrated on two-dimensional fixed samples, and currently there is a lot of interest in applying these techniques to live in vivo imaging. In this talk, I will give an overview of the different super-resolution techniques and discuss their applicability to live imaging. Specifically, I will discuss recent work done in the Gustafsson lab demonstrating live imaging of microtubule dynamics and kinesin movement at 100nm resolution using Structured Illumination Microscopy. I will conclude by discussing research into adaptive optics in microscopy which promises to improve the performance of super-resolution techniques for live imaging.
Q3. New optical super-resolution methods for nanometre resolution fluorescence imaging
Christian Soeller ([email protected]), Department of Physiology, University of Auckland, New Zealand
The distribution of proteins and their proximity at the nanometre scale critically determines cellular function but until recently such detail could not be observed with optical microscopy due to its limited resolution. Nevertheless, fluorescence imaging has become a major tool in biology due to the availability of highly specific markers and the sensitivity of photon detection. The resolution limitation has recently been overcome by optical super-resolution imaging which combines the strength of existing fluorescent labelling technology with greatly improved detail resolution as small as 10 nm. While several optical methods can now be used to improve resolution I will here report on practical results using localisation microscopy (related to techniques that have become known under the acronyms PALM and STORM) which involves the localisation of single molecule fluorescence with an accuracy of ~10 nm.
We have modified a total internal reflection fluorescence microscope for super-resolution microscopy and developed imaging protocols to achieve this high resolution with conventional commercial fluorochromes and fluorescent proteins. While the original demonstration of localisation microscopy achieved resolution improvement only in two dimensions and was limited to a single fluorescent label the approach has now been extended to high resolution in all three dimensions and the imaging of several spectrally distinct labels to allow for nanometre resolution co-localization studies. I will present results obtained with single cells, cultured cell monolayers and stained tissue sections that illustrate the dramatic improvement in resolution, the excellent spectral separation of markers and the novel biological insight arising from this new data. To date we have applied the technique to improve our understanding of cardiovascular physiology, neuroanatomy and neurophysiology and it should be widely applicable to many areas of biological molecular scale research.
Q4. Electron tomography and super-resolution optical imaging of malaria parasites
Tilley L1, Hanssen E2, Klonis N1, Dixon M1, McMillan P1, Abu-Bakar N1, Whitchurch C3
1La Trobe Institute for Molecular Science and Centre of Excellence for Coherent X-ray Science, La Trobe University, Melbourne 3086, VIC, Australia; 2Electron Microscopy Unit, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia; 3Institute for the Biotechnology of Infectious Diseases, University of Technology Sydney, NSW, 2007, Australia.
Imaging technologies have provided us with phenomenal insight into the micro- and nano-scopic domains and efforts to answer the major medical and biotechnology questions of the 21st century will be heavily reliant on the use of advanced imaging techniques. However there are limitations. Conventional light microscopy can be used with hydrated (in some cases, live) cells but has limited resolution, particularly for full-field imaging. Conventional electron microscopy offers very high resolution however the strong absorption of electrons by air and by the sample means that it can only be used with very thin, fixed, dehydrated samples. Imaging technologies that overcome some of the disadvantages of optical and electron microscopies are keenly sought.
We have used two “bridging” imaging modalities to explore sub-cellular topography. Three-dimensional structured illumination microscopy (3D-SIM) permits super-resolution fluorescence imaging of cells that are specifically labelled with fluorescent probes. Immunoelectron tomography offers high resolution imaging of individual ultrastructural features in a cellular context. Combined with serial sectioning and immunogold labeling it permits precise mapping of whole cell architecture.
The malaria parasite, Plasmodium falciparum, develops within human erythrocytes. As it grows the parasite establishes a membrane network outside its own limiting membrane in the cytoplasm of its host cell. These membrane structures play an important role in the trafficking of virulence proteins to the host cell surface, however their ultrastructure is only partly defined and there is on-going debate regarding their origin, organization and connectivity. Parasite endocytic processes are also poorly understood. The parasite consumes host haemoglobin in order to support its own growth. Small packets of haemoglobin are transferred from the host cell cytoplasm to a parasite digestive vacuole for haemoglobin digestion and heme detoxification however the precise mechanism for uptake is debated. Advanced imaging methods have provided novel insights into parasite cell architecture.
Q5. Using TIRF to understand better the mechanism of insulin secretion
Professor Tao Xu
Institute of Biophysics, Chinese Academy of Sciences, Beijing 100080, China
Speaker Abstracts: Session 2
Q6. Next-Generation Sequencing and its Impact.
Richard Michelmore, The Genome Center, The University of California, Davis.
We are in the middle of several technological revolutions. It has long been recognized that phenotype is the consequence of interactions of genotype with environmental factors; however, our ability to measure all three of these aspects is currently unprecedented. In particular, our ability to rapidly determine DNA sequences from large numbers of diverse organisms is revolutionizing biological research and will have profound impacts on society. I will review the current and imminent sequencing technologies and then consider several applications of massively parallel sequencing, including de novo resequencing genome sequencing, RNAseq, mapping by sequencing, BSAseq, and CHIPseq. This is resulting in large amounts of information that are challenging the way data are curated and analyzed. We are transitioning from a data-poor to a data-rich environment. I will discuss how we are meeting these challenges and opportunities at the UC Davis Genome Center for comparative functional genomics. Finally, I will consider the impacts that large amounts of DNA sequence data will have on agriculture and society.
Q7. New Developments in 454 Sequencing Technology.
Todd E. Arnold, Ph.D.
Vice President of Development, 454 Life Sciences, a Roche Company.
454 Sequencing remains the only long read, next-generation sequencing technology currently available to the market. The GS FLX System, coupled with the latest generation of GS FLX Titanium reagents delivers over one million high quality reads with 400 base Q20 read lengths; and, its utility has been proven in over 800 peer-reviewed publications to date. The introduction of the bench top GS Junior System provides the individual researcher access to high throughput sequencing with read lengths and data quality equivalent to the GS FLX System. Results from recent research projects will be presented, as well as an overview of current and future product development efforts.
Speaker Abstracts: Session 3
Q8. Differences downunder - macropods, methane and metagenomics
Mark Morrison, CSIRO Australia ([email protected]) The Australian macropods are nationally unique and evolved in geographic isolation from other placental mammals. As herbivores, they have coevolved with a microbiome that resides within a tubiform foregut that coordinates efficient plant biomass degradation. Our group has begun a metagenomic analysis of this microbiome using Australia’s “model” marsupial, the Tammar Wallaby (Macropus eugenii). With support from the US Department of Energy’s community sequencing program we produced Sanger sequence and 454 data from small insert libraries of community DNA and select fosmid clones, respectively. The rrs gene sequence data and the gene-centric analysis of community DNA indicate the Firmicutes and Bacteroidetes are predominant. The gamma-Proteobacteria were also in high abundance, but comprised solely of a "novel" operational taxonomic unit (WG-1). Sufficient genomic data for WG-1 was recovered from the datasets to support metabolic reconstruction in silico and this knowledge was ultimately used to design cultural conditions that resulted in the axenic cultivation of WG-1. This bacterium favors a fermentation scheme that produces succinate, and perhaps, an alternative route of fermentation in the wallaby foregut that is not directly linked to methanogenesis. We also observed that a small percentage of the fosmid clones of metagenomic DNA promoted autoaggregation and biofilm formation in the E. coli host strain. These fosmids have since been shown to bear a gene encoding a modular carbohydrate active enzyme, and that extracellular DNA release is responsible for the autoaggregation phenotype; providing new evidence for how biofilms of commensal bacteria may develop within gut microbiomes. In summation our metagenomic analysis of the Tammar foregut microbiome has revealed some interesting new directions of investigation for bacterial and archaeal physiology, plant biomass degradation, and fermentation in gut microbiomes.
Q9. Structural Genomics of the Expanding Protein Universe
Ian A. Wilson, The Joint Center for Structural Genomics (JCSG;www.jcsg.org), The Scripps Research Institute, La Jolla, CA 92037 USA. Email: [email protected]
Over the past decade, the overwhelming amount of novel sequence data that has been generated from genome sequencing projects has demanded a re-evaluation of the size and diversity of the protein universe. As one of the four large-scale centers of the NIH Protein Structure Initiative (http://www.nigms.nih.gov/Initiatives/PSI), the JCSG has aimed at increasing coverage of structure space and, in particular, of proteins of unknown function1. We have also explored structural coverage of a single organism, and recently completed a complete structural reconstruction of the metabolic network of Thermotoga maritima2. The advent of metagenomics has also been particularly exciting, where the human gut microbiome sequencing projects have already uncovered fascinating new families and expansions of known families that have enabled adaptation to particular environments. Analysis of what these structures have told us about the evolution and function of these protein families and to what extent structure by itself leads to plausible functional predictions will be discussed. The JCSG is located at The Scripps Research Institute, the Genomic Institute of the Novartis Research Foundation, U.C. San Diego, Sanford-Burnham Medical Research Institute, and SSRL/Stanford University, and supported by NIGMS PSI.
1 Jaroszewski et al. (2009) Exploration of uncharted regions of the protein universe. PLoS Biol. 7:e1000205.
2 Zhang, Y. et al. (2009) Three-dimensional structural view of the central metabolic network of Thermotoga maritima. Science 325:1544-1549
Q10. Mechanisms and patterns of post-transcriptional gene control
Thomas Preiss
Molecular Genetics Division, Victor Chang Cardiac Research Institute;
University of New South Wales
Eukaryotic gene expression is a multi-step process and intricately regulated. Control may be exerted not only at the level of gene transcription in the nucleus but also post-transcriptionally at the steps of mRNA processing, transport, translation and decay, and even at the posttranslational level. My Lab is focusing on the mRNA as the template for translation by the ribosome and how generic (5’ cap and 3’ poly(A) tail) as well as mRNA-specific features (e.g. cis-acting elements in the untranslated regions) affect this process. Projects in the lab are either geared towards detailed analysis of molecular mechanism using conventional molecular biology approaches, or use transcriptome-wide approaches to study global patterns of post-transcriptional gene regulation. The seminar will cover current work from both of these categories. Animal microRNAs (miRNAs) typically regulate gene expression by binding to partially complementary target sites in the 3’ untranslated region (UTR) of messenger RNA (mRNA) reducing its translation and stability. They also commonly induce shortening of the mRNA 3’ poly(A) tail, which contributes to their mRNA decay promoting function. Using transfection of reporter constructs into mammalian cells we observed rapid target mRNA deadenylation. A series of experiments furthermore allowed us to conclude that deadenylation, while not absolutely required, does augment miRNA-mediated translational repression in mammalian cells beyond stimulating mRNA decay. It is thus critically involved in establishing the quantitatively appropriate physiological response to miRNAs. Next Generation Sequencing is highly suited for the identification and ‘counting’ of RNA species in a transcriptome (RNASeq). We use an Applied Biosystems SOLiD™ 3 system for a range of RNASeq applications, which include small RNA as well as mRNA profiling. I will present our results with profiling small RNAs in a cardiomyocyte cell line as well as work on the development of a novel approach to experimentally identify miRNA targets based on transcriptome-wide measurements of mRNA polyadenylation state. The latter is based on the premise that targets for a given miRNA can be identified by virtue of their change from a short-tailed state when the miRNA is active, to a long-tailed state when it is inactive.
Speaker Abstracts: Session 4
Q11. Advancing Next Generation Biofuels
Blake A. Simmons, Ph.D.
Vice-President, Deconstruction Division, Joint BioEnergy Institute, Emeryville, CA
Senior Manager, Biofuels and Biomaterials Science and Technology, Sandia National Laboratories, Livermore, CA
Today, carbon-rich fossil fuels, primarily oil, coal and natural gas, provide 85% of the energy consumed in the United States. Fossil fuel use increases CO2 emissions, increasing the concentration of greenhouse gases and raising the risk of global warming. The high energy content of liquid hydrocarbon fuels makes them the preferred energy source for all modes of transportation. In the US alone, transportation consumes around 13.8 million barrels of oil per day and generates over 0.5 gigatons of carbon per year. This has spurred research into alternative, non-fossil energy sources. Among the options (nuclear, concentrated solar thermal, geothermal, hydroelectric, wind, solar and biomass), only biomass has the potential to provide a high-energy-content transportation fuel. Biomass is a renewable resource that is potentially carbon-neutral.Plant-derived biomass contains cellulose, which is more difficult to convert to sugars. The development of cost-effective and energy-efficient processes to transform polysaccharides in biomass into fuels is hampered by significant roadblocks, including the lack of specifically developed energy crops, the difficulty in efficiently frctionating biomass components, low activity of enzymes used to deconstruct biomass, and the inhibitory effect of fuels and processing byproducts on organisms responsible for producing fuels from biomass monomers.
Pretreatment of biomass is essential for breaking apart highly ordered and crystalline plant cell walls and loosening the lignin and hemicellulose conjugation to cellulose microfibrils, thereby facilitating enzyme accessibility and adsorption and reducing costs of downstream saccharification processes. Recent reports have shown very high yields at very low enzyme loadings. However, pretreatment still remains one of the most costly steps in lignocellulosic biofuel production. Ionic liquids are solvents showing great promise for cellulose solubilization. Instant rejection of dissolved cellulose upon anti-solvent addition shows promise for recyclability in addition to other desired attributes like low volatility, non-flammability and thermal stability. Although shown to be very effective in cellulose solubilization, the disposition of hemicellulose and lignin are not fully understood. The aim of this ongoing work is to understand ionic liquid pretreatment by monitoring and analyzing process streams towards gaining better understanding of pretreatment process and development and selection of pretreatment conditions for selective depolymerization of either cellulose or lignin whereby fractionation of different cellulosics and lignin components could be better designed and manipulated.
Q12. Lignocellulosic Biomass to Biofuels and Coproducts; a New Zealand story
Elspeth MacRae
Scion, 49 Sala St, Rotorua 3010
To produce transport fuels a petroleum refinery massages the incoming oils to fractionate 90% to usable fuels and 10% to sustain a large chemical industry including the plastics industry. Lignocellulosic biomass, eg trees, offers an effective route for sustainable fuel production in a biorefinery that can parallel petroleum refinery operations. New Zealand has the potential to be self sustaining through development of pine plantations on low value land for use as a feedstock for biorefineries. While there is rapidly growing effort internationally to establish lignocellulosic biorefineries, there are still several roadblocks to be overcome. Not the least of these is biomass processing to become a sugar source for fermentation, and the establishment of valuable coproduct streams. Scion’s research on biomass processing will be profiled, alongside our focus on lignin and bioplastic co-product streams.
Q13. Gas fermentation: No such thing as waste
Dr. Sean Simpson Lanzatech 24 Balfour Road Parnell, Auckland 1052 [email protected]
The use of gases in place of sugars as the carbon and energy source for fermentation allows a broad spectrum of resources to be considered as feedstocks for product synthesis. LanzaTech has developed gas fermentation system for the production of alternative transport fuels and commodity chemicals. The company has focused initially on the use of industrial waste gases, and specifically gases produced as a by-product of steel manufacture, for fuel ethanol production. Today, 50% of the world steel is produced in China, where a rapidly growing economy has been coupled with dramatic increases in vehicle sales, an increasing dependence on foreign oil imports, and a commitment to minimising greenhouse gas emissions. Gas fermentation offers an efficient route to add much greater value to steel industry gas streams than established technologies, while also reducing CO2 emissions and providing a strategically important alternative to the use of food or farmed resources for domestic fuel production. LanzaTech has successfully run a pilot plant using its proprietary technology to produce ethanol from steel mill flue gases at NZ Steel at Glenbrook in Auckland since 2008, and is now poised to begin construction of a demonstration facility. A demonstration plant is the last stage before full commercial operation. However, the application of this technology for ethanol production is just a first step; the company has demonstrated the potential for a range of higher value commodity chemicals to be produced via this route.
Speaker Abstracts: Session 5
Q14. Copying and reprogramming of heterochromatin with RNAi.
Rob Martienssen Cold Spring Harbor Laboratory, Cold Spring Harbor NY11724 Heterochromatin is composed of transposable elements (TE) and related repeats which silence genes located nearby, and play a major role in epigenetic regulation of the genome. Far from being inert, heterochromatin is transcribed and small interfering RNA corresponding to heterochromatic sequences can be detected in plants, animals and fission yeast. In fission yeast, centromeric repeats are transcribed, but the transcripts are rapidly turned over by RNA interference, through the combined action of DNA dependent RNA polymerase, Argonaute and RNA dependent RNA polymerase, each of which is associated with heterochromatin. Histone H3 lysine-9 dimethylation (H3K9me2) depends on RNAi, mediated by the Rik1-Clr4 complex. We have found that heterochromatin is lost transiently during chromosomal replication, allowing heterochromatic transcripts to accumulate. Rapid processing of these transcripts into small RNA during S phase promotes restoration of heterochromatic modifications and the retention of cohesin in G2. These results explain how “silent” heterochromatin can be transcribed, and lead to a model for epigenetic inheritance during replication. In plants, small interfering RNA (siRNA) corresponding to some classes of TE depends on DNA methyltransferase MET1, the SWI/SNF ATPase, DDM1, or both, but not on the histone deacetylase SIL1/HDA6. All three genes are required for silencing transposons in the absence of siRNA, and we are exploring the roles of these complementary mechanisms in the inheritance of epigenetic silencing from generation to generation, and in dividing cells during development. In Arabidopsis, down regulation of DDM1 and MET1 in pollen companion cells leads to heterochromatin reprogramming, and the translocation of siRNA into neighboring sperm, where it promotes the silencing of transposons. A similar mechanism in ovules also influences differentiation of germ cells from the surrounding nucellus, and contributes to asexual reproduction, or apomixis.
Q15. Identity and functional properties of skeletal muscle stem cells
Shahragim Tajbakhsh
Stem Cells & Development, Dept. of Developmental Biology, CNRS URA 2578, Institut Pasteur, 25 rue du Dr Roux, Paris 75015, France.
Skeletal muscle development is characterised by the presence of multiple stem cell populations with different genetic requirements (1, 2). Pax3 acts in a pathway complementary to the core determination genes Myf5, and Mrf4, upstream of Myod in the body. However, in the head, Tbx1, but not Pax3, complements the Mrfs for pharyngeal muscle development (2). After embryonic development is complete, a pool of juvenile and adult satellite cells is allocated for further skeletal muscle growth and regeneration postnatally. Numerous studies have indicated that the satellite cell population is herteogeneous, comprised of a "stem-like" and committed cells.
After embryonic development is complete, a pool of juvenile and adult satellite cells is allocated for further skeletal muscle growth and regeneration postnatally. Numerous studies have indicated that the satellite cell population is herteogeneous, comprised of a "stem-like" and committed cells. Using a pulse-chase approach with BrdU, we identifed label retaining cells in skeletal muscle after a long chase period (3). These cells are clonogenic and a subpopulation can selectively segregate their DNA asymmetrically to one daughter cell after muscle injury and exit of satellite cells from the quiescence state. We observe that template DNA strands co-segregate to one daughter cell during cell division.
Non-random DNA segregation has been reported in different organisms, which indicates that this phenomenon is evolutionary ancient. The relevance to stem cells will be discussed. A major technical challenge is to identify daughter cells among the cells undergoing this process, either by capturing them in mitosis, or by identifying true daughters by videomicroscopy to investigate the mechanism that guides this phenomenon. These ongoing studies will be discussed in the context of presumed stem cell properties.
References:
1) Tajbakhsh, S. Stem cell: what's in a name? Nature Reports Stem Cells. June 25. 2) Tajbakhsh S. (2009). J Intern Med. 266:372. 3) Shinin et al. (2006) Nature Cell Biology 8:677.
Q16. Signal transduction and gene expression in a pathogenic bacterium
Iain L Lamont, Karla A Mettrick and Richard C Draper
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
Email: [email protected]
The bacterium Pseudomonas aeruginosa is commonly found in a wide range of environments and is also an opportunistic pathogen that infects patients suffering from a number of predisposing conditions. P. aeruginosa secretes an iron-scavenging compound (siderophore) called pyoverdine that is critical to its ability to cause infections in animal models of disease and is also made during infections of humans. As well as acquiring iron for the bacteria, pyoverdine is the trigger for a signalling pathway that spans both the inner and outer membranes of the cells and involves at least four proteins. This pathway controls production of the pyoverdine receptor protein, of pyoverdine itself, and of two secreted proteins that contribute to the infectious process. P. aeruginosa can also acquire iron by taking up siderophores made by other microorganisms and at least two of these trigger gene expression via signal transduction pathways that are analogous to the pyoverdine system. This talk will describe recent research on the mechanisms of signal transduction in these systems. A key step that occurs in all pathways is partial proteolytic degradation of a membrane-spanning protein, releasing an active component that controls the activity of a transcription factor. However, despite clear homology between different iron uptake systems, the ways in which transcription factor activity is controlled also have unexpected differences.
Invitrogen Young Scientist of the Year: Dr Justin O’Sullivan
Q17. Beyond the linear: sequencing genome architecture
C.R. Rodley1, G. Gruenert1,4, L.R. Gehlen1, J.A. Mullaney2, M.B. Jones3, B.H.A. Rehm2, J. Langowski4, and J.M. O’Sullivan1
1Institute of Natural Sciences, 3Institute of Information and Mathematical Sciences, Massey University, Auckland. 2Institute of Molecular Biosciences, Massey University, Palmertson North. 4DKFZ, Heidelberg, Germany. [email protected]
Recent work has highlighted the high degree of spatial organization and inter-chromosomal connections within the yeast and human genomes. While still in its infancy, the study of the spatial organization of genomes on a global scale is providing insights that enable us to study genome biology as a network of inter-linked systems and not simply as the isolated loci we were previously restricted to. High-throughput DNA sequencing is an integral and novel component of the systems biology approaches which are used to map and compare the spatial organization of genomes. We will present three-dimensional reconstructions of a eukaryotic and prokaryotic genome that are based on high-throughput DNA sequencing. We will discuss the generation of these models. Finally, we will outline how the eukaryotic and prokaryotic genomic organizations integrate into the over-all metabolic system of the organisms.
Speaker Abstracts: Session 6
Using TIRF microscopy in studies of cell migration
Dr Lilian Soon
University of Sydney
Speaker Abstracts: Session 7
Q18. Broad Neutralization of Influenza Virus and Implications for a Universal Flu Vaccine
Ian A. Wilson, Department of Molecular Biology & Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037 USA. Email: [email protected]
Influenza constitutes a perpetual threat to public health worldwide as it continuously evades the immune system through extensive variation in the highly exposed, non-functional regions of its surface glycoproteins, and through limited access to its functional sites. Understanding how the immune system can be induced to elicit antibodies against conserved epitopes so as to neutralize a wide range of influenza subtypes is critical for vaccine design. The major surface antigen, the hemagglutinin (HA), is the main target of neutralizing antibodies. However, most antibodies are strain-specific and protect only against highly related strains within the same subtype. To address this limitation, we are investigating a wide variety of flu antibodies to identify accessible conserved regions. We have determined co-crystal structures of broadly neutralizing antibodies that target a highly conserved site in the fusion domain of all type 1 influenza viruses1, including the human H1N1 1918 pandemic and H5N1 avian influenza. We have also structurally elucidated a common epitope in human 1918 H1N1 and swine 2009 H1N1 viruses2. Such studies provide new opportunities for rational design of vaccines that afford greater protection against the plethora of influenza A subtypes.
1 Ekiert, D. C., Bhabha, G., Elsliger, M. A., Friesen, R. H., Jongeneelen, M., Throsby, M., Goudsmit, J., and Wilson, I. A. (2009) Antibody recognition of a highly conserved influenza virus epitope. Science 324:246-251
2 Xu, R., Ekiert, D.C., Krause, J.C., Hai, R., Crowe, J.E. Jr, and Wilson, I.A. (2010) Structural basis of preexisting immunity to the 2009 H1N1 pandemic influenza virus. Science 328:357-360
Q19. Insect virus polyhedra, infectious protein crystals that contain virus particles.
Elaine Chiu and Peter Metcalf
School of Biological Sciences, University of Auckland, New Zealand
Larvae infected with insect polyhedrosis viruses become milky white, a result of the formation of masses of intracellular protein crystals. The crystals, or viral polyhedra, consist of a cubic lattice of viral polyhedrin molecules containing thousands of virus particles embedded in the crystalline lattice. Viral polyhedra are remarkably stable, and can remain infectious in soil for years for feeding larvae. Polyhedra survive conditions that would denature most protein molecules, but do dissolve at pH > 10.5 in the mid gut, releasing the embedded virus particles.
We are interested in learning why viral polyhedra are so stable, how they specifically incorporate virus particles inside cells and also in applications e.g. for polyhedra engineered to contain other proteins in place of the embedded virus particles. Working with C. Schulze-Briese (SLS) we developed micro X-ray crystallography techniques and determined the atomic structures of polyhedra produced by the dsRNA virus cypovirus(1) and the DNA virus baculovirus(2), the same virus used in the expression system familiar to molecular biologists. These are amongst the smallest protein crystals ever used for de-novo atomic structure determination. The talk will describe these results, and also our recent work with granulovirus, a type of baculovirus that forms tiny 400nm polyhedra with a crystalline polyhedrin layer only 7 unit cells thick surrounding a single virus particle.
1) The molecular organization of cypovirus polyhedra (2007) Coulibaly F, Chiu E, Ikeda K, Gutmann S, Haebel PW, Schulze-Briese C, Mori H, Metcalf P. Nature. 446, 97-101
2) The atomic structure of baculovirus polyhedra reveals the independent emergence of infectious crystals in DNA and RNA viruses (2009) Coulibaly F, Chiu E, Gutmann S, Rajendran C, Haebel PW, Ikeda K, Mori H, Ward VK, Schulze-Briese C, Metcalf P. Proc Natl Acad Sci U S A. 106, 22205-10
Q20. Engineered OB-fold domains as versatile scaffolds for molecular recognition
John D. Steemson1, Matthias Baake2, Jasna Rakonjac3 and Vickery L. Arcus1
1University of Waikato, 2University of Auckland, 3Massey University
OB-fold protein domains are found in all three kingdoms of life. They are small, five-stranded �-barrel protein domains with a common binding face. Over evolutionary time this binding face has been adapted to bind to a wide range of ligands including oligosaccarides, oligonucleotides, proteins and small molecules. In at least one case, enzymatic activity has also evolved at this common face. We have sought to mimic this adaptability in vitro and have developed a range of combinatorial protein libraries based on the OB-fold domain from an aspartyl-tRNA synthetase. We have used these libraries in phage display selection for binding back to the native ligand (asp-tRNA) as well as to a prototypic protein ligand (lysozyme). OB-fold variants obtained from selection against lysozyme have been characterised for binding and show moderate affinity (in the micromolar range). The three-dimensional structure has been determined for a single OB-fold variant in complex with lysozyme and this structure provides details of the protein-protein interface. Based on this structure, we have constructed new libraries and have improved the binding affinity by 30-fold. We have also determined the structures of these new OB-fold-lysozyme complexes. The combination of binding loops and a binding face presented by the OB-fold domain libraries (OBodies), which can be adapted for tailor-made molecular interactions, is a new avenue for exploration in seeking alternatives to antibodies in diagnostics and therapeutics.
Speaker Abstracts: Session 8
Q21. Bitter-sweet genetics of taste and aroma perception.
Danielle R Reed
Monell Institute, Philadelphia
Eating is dangerous. While food contains nutrients and calories that animals need to produce heat and energy, it may also contain harmful parasites, bacteria, or chemicals. To guide food selection, the senses of taste and smell have evolved to alert us to the bitter taste of poisons and the sour taste and off-putting smell of spoiled foods. These sensory systems help people and animals to eat defensively, and they provide the brake that helps them avoid ingesting foods that are harmful. But choices about which foods to eat are motivated by more than avoiding the bad; they are also motivated by seeking the good, such as fat and sugar. However, just as not everyone is equally capable of sensing toxins in food, not everyone is equally enthusiastic about consuming high-fat, high-sugar foods. Genetic studies in humans and experimental animals strongly suggest that the liking of sugar and fat is influenced by genotype; likewise, the abilities to detect bitterness and the malodors of rotting food are highly variable among individuals. Our research has uncovered genes involved in the detection of bitter stimuli such as phenylthiocarbamide (a thyroid poison; TAS2R38) and quinine (TAS2R19), the smell of asparagus metabolites (OR2M7), the odor associated with boar taint in ham (androstenone), and the sweet taste of sugar (TAS1R3). Understanding the exact genes and genetic differences that affect food intake may provide important clues in obesity treatment by allowing caregivers to tailor dietary recommendations to the chemosensory landscape of each person.
Q22. Variation in human ability to detect flavour compounds and its impact on food and beverage preferences: Gastronomics research down under
Sara R. Jaeger1, Jeremy F. McRae1, Richard D. Newcomb1,2,3
1. The New Zealand Institute for Plant & Food Research Limited 2. School of Biological Sciences, University of Auckland 3. The Allan Wilson Centre for Molecular Ecology and Evolution [email protected] Ever wondered why you like a particular wine and your friend doesn’t? Well, it may come down to genetics. The biology behind our sensory ability is complex, controlled by a multitude of interacting genetic and environmental factors. To date more is known about the environmental influences on sensory perception such as culture and branding, but the human genetics revolution has presented the opportunity to identify the genetic determinants of sensory ability, and by proxy, whether food preferences have a genetic basis. The Plant & Food Research Gastronomics programme seeks to examine the relationships among variation in the ability to detect various flavour compounds important to New Zealand’s food and beverage sector, human genetic variation, and food and beverage preference and liking. The ability to detect these various flavour compounds varies markedly among individuals. Individuals at the opposite ends of the spectrum of sensory abilities have markedly different flavour experiences, as judged by liking scores and the relative importance of terms that they use to describe the aroma. Genome-wide association using SNP arrays has been used to localize regions of the genome associated with sensory ability for these compounds, with a region on chromosome six containing a family of odorant receptor genes found to be associated with cis-3-hexenol perception. Finally, associations with sensory ability, genotype and liking are beginning to be tested in model food and beverage scenarios.
Q23. Yeast and Sauvignon blanc: Of grapefruit, passionfruit and cats pee
Richard Gardner Wine Science, University of Auckland; [email protected] Bakers’ yeast, Saccharomyces cerevisiae, is the best understood eukaryote on the planet. Functional data is now available for 4857 of its 5797 genes, with huge datasets for the yeast genome, transcriptome and proteome. Unparalleled genetic resources are available, including complete sets of strains with individual genes deleted, overexpressed, or tagged with epitopes, and the complete nucleotide sequence of over 50 different strains. In addition to its role as a model system for research, yeast is also an important industrial organism, with a role in bread-making and in the fermentation of beer, wine, spirits, and more recently bioethanol. Our lab is focusing on the genetic improvement of S. cerevisiae for winemaking, and specifically for improving the aroma characteristics of Sauvignon blanc wines. A major goal is to increase the yields of thiol and ester aroma compounds that are produced by yeast during fermentation and are key contributors to the ‘fruity’ aromas of wine. I will review work from our lab on a beta-lyase gene responsible for cleavage of thiol precursors to produce notes of ‘grapefruit’, ‘passionfruit’ and ‘cats pee’ in our national wine variety.
Q24. Detecting sequence and structural variation in grapevine: origin, structure and function of the dispensable genome.
Michele Morgante
Dipartimento di Scienze Agrarie ed Ambientali, Università di Udine, Via delle Scienze 208, 33100 Udine, Italy and Istituto di Genomica Applicata, Parco Scientifico e Tecnologico di Udine, Via Linussio 51, 33100 Udine, Italy; Email: [email protected]
The comparative sequencing of several plant genomes revealed that transposable elements are largely responsible for extensive variation in both intergenic and local genic content not only between closely related species but also among individuals within a species. In addition larger structural variants can be detected, similar to the copy number variants identified in the human genome and involving hundreds of Kbp of DNA and tens of genes. A single genome sequence may therefore not reflect the entire genomic complement of a species and prompted us to introduce in plants the concept of the pan-genome, which includes core genomic features common to all individuals and a dispensable genome composed of non-shared DNA elements that can be individual- or population-specific. The pan-genome model has recently been proposed also for the human genome, where, however, the size of the dispensable genome seems to be negligible when compared to that observed in some plant species. We will describe what has been learned so far about the dispensable genome in grapevine and compare it to another fruit tree species whose genome has been fully sequenced, peach. We will describe the variation that can be detected among genotypes not only as SNP but also and especially as structural variants due either to simple transposable element insertions or to insertions/deletions of large genomic regions using next generation sequencing methodologies. We will describe the size and composition of the dispensable genome as well as discuss possible molecular mechanisms leading to such variation. Uncovering the intriguing nature of the dispensable genome, namely its composition, origin and function, represents a step forward towards an understanding of the processes that generate genetic diversity and phenotypic variation.
Poster Abs tracts (by first author)
Q25. Hypermethylation of a silenced transgene in rederived bovine cell lines
L. Alonso-Gonzalez*, C. Couldrey, M. Meinhardt & G. Laible.
*Research performed at AgResearch, Ruakura Research Centre, Hamilton, New Zealand. E-mail: [email protected]
Cell-mediated transgenesis, based on somatic cell nuclear transfer (SCNT), provides us with the opportunity to shape the genetic make-up of cattle. Bovine primary fetal fibroblasts, commonly used cells for SCNT, suffer from a limited lifespan, and complex modifications that require more than one round of transfections can be challenging time and cost-wise. To overcome these limitations, SCNT is commonly used to rejuvenate the cell lines and restore their exhausted growth potential. We have designed a construct to be used in a 2-step cassette exchange experiment; our transgene contains a puromycin resistance marker gene and an EGFP expression cassette - both driven by a strong mammalian promoter – and is flanked by loxP sites and sequences from the bovine β-casein locus. Several cell lines were generated by random insertion of this construct into primary bovine cell lines. Two of the original cell lines, featuring different insertion sites, were selected based on their low transgene copy number, and subsequently rederived by SCNT. New primary cell lines, with the same genetic makeup as the original donor ones, were established from day 37 fetuses. Phenotypical expression of the transgene was assessed in all lines. The original cell lines are puromycin-resistant and have a characteristic EGFP expression profile. In contrast, all rejuvenated cell lines are sensitive to puromycin, and display a range of EGFP expression levels, indicative of various degrees of silencing. When the methylation state of individual CpGs within the transgene was analyzed, we found a dramatic increase in methylation in the analyzed transgene regions in rederived cell lines. Hypermethylation appears to be transgene sequence-specific and independent of insertion site. Unexpectedly, we found that hypermethylation is not restricted to xenogeneic DNA, but also affects some CpGs in the construct’s β-casein flanking sequences. If such differential methylation proves to be commonplace during nuclear reprogramming, it could hinder transgene expression in genetically-modified animals.
Q26. Comparison of urate transport via OAT2 in different species Andrew Bahn, [email protected] Department of Physiology, School of Medical Sciences, University of Otago, Dunedin, New Zealand
Plasma urate levels in humans are comparatively high due to a non-functional hepatic uricase and an active re-absorption of urate in the kidneys. In the last couple of years, urate has come back into clinical focus because of its impact on cardiovascular and neurodegenerative diseases. This has resulted in the identification of several urate transporters such as URAT1, OAT4, ABCG2 and GLUT9, which play a crucial role in plasma urate homeostasis. However, there are significant differences in the expression and functional characteristics of these urate transporters in the different species due to a functional uricase in lower mammals. This is especially true for the newly identified urate transporter, namely organic anion transporter 2 (OAT2), because of its expression in the apical (mouse and rat) or basolateral (pig, rabbit and human) membranes of the kidneys. In this study we compare urate transport characteristics of the pig orthologue of Oat2 (pOat2) with rat and human OAT2 to get an idea about its contribution to plasma urate homeostasis. OAT2 transport of urate was saturable with a Kt value of 75 µM (rat) and an IC50 value of 10 µM (human) suggesting a high affinity of OAT2 for urate. OAT2 mediated urate transport was inhibited by probenecid and nicotinate, whereas glutarate, succinate or lactate did not alter urate uptake at all. Trans-stimulation experiments providing glutarate, lactate, or succinate on one or the other side of the cell did not stimulate urate uptake or efflux, indicating that OAT2 operates in a different mode than the known OATs. Using p-aminohippurate (PAH) as a substrate pOat2 showed substantial pH dependence, suggesting an exchange mechanism PAH-/OH-. Our data demonstrate that OAT2 is a high affinity urate transporter in all tested species possibly driven by OH--exchange.
Q27. Spring1, an early flowering Medicago truncatula mutant with an altered response to vernalisation Martin Balcerowicz1, Chin-Chin Yeoh1, Lulu Zhang1, Pascal Ratet2 and Jo Putterill1
1Plant Molecular Sciences, School of Biological Sciences, University of Auckland, Auckland, New Zealand. 2CNRS, France. [email protected] Using mutants to understand the control of flowering time has yielded networks of genes that act either as repressors or promoters of flowering that are arranged into pathways that respond to different flowering signals. In the model plant Arabidopsis, these cues include seasonal signals such as changing photoperiod (day length) and vernalisation - exposure to extended period of cold as would be experienced in winter. We are studying flowering time control in the model eudicot Medicago truncatula (Medicago) in order to expand our knowledge of the genes that regulate flowering to other groups of plants, in this case the legumes. In particular, we are interested in vernalisation control in Medicago, as previous work suggests that the vernalisation circuitry is very different in the eudicot Arabidopsis and the monocot temperate cereals. Medicago flowering is promoted in long day conditions after the plants have received a vernalisation treatment. An early flowering mutant in the R108 background we named spring1 was identified in a screen. We have characterised its response to different growth environments, and correlated this with changes in expression of candidate flowering time genes. The spring1 mutant flowers rapidly in long day conditions, in the absence of vernalisation. This is associated with up regulation of the Medicago FTLa gene, which is likely to be the orthologue of the Arabidopsis FT gene which encodes a flower promoting hormone, florigen. However, photoperiodic control of flowering is maintained, with low levels of MtFT expression in spring1 and wild type in short day conditions. This suggests that the spring1 mutant has a constitutive vernalisation phenotype. Efforts are underway to map and clone the gene. We present the results of this analysis and a model for the regulation of flowering time in Medicago.
Q28. Targeted marker development in onion (Allium cepa)
S Baldwin1, M Pither-Joyce1, F Kenel1, M Fiers1, R Crowhurst2, J McCallum1
1 The New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch 8104,
NEW ZEALAND.
2 The New Zealand Institute for Plant & Food Research Limited, Private Bag 92 169, Auckland 1142,
NEW ZEALAND
Onion (Allium cepa) is an internationally important crop but has less genomic resources than other monocots such as the grasses. This is partly due to onion being biennial, with high levels of heterozygosity and a large genome. Traditional molecular marker development was laborious with limited capabilities but the use of next generation sequencing has allowed rapid large-scale marker development. Transcriptome sequencing of cDNA from parents of a mapping population has enabled the design of markers aimed specifically at putative polymorphic nucleotides in coding regions of the genome. Also, a skim sequence of genomic DNA from a doubled haploid was used to identify simple sequence repeats (SSRs) that could be used as neutral markers for determining population structure for application in future association mapping analysis. The SSRs were assessed on a diverse collection of onion germplasm including medium–long day or short day types and Indian land races. A. roylei and A. fistulosum were also included to test for interspecific amplification of the SSRs. All markers were assessed for validation regarding amplicon size, single locus amplification and polymorphism. The sequence information and the markers designed represent a valuable resource for Allium genetics.
Q29. MEIOTIC RECOMBINATION IN ARABIDOPSIS
Philippa Barrell, Michelle Gatehouse, Tony Conner
New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch 8140, New
Zealand
email [email protected]).
A key requirement for analysis of recombination is a convenient test system to measure changes in the frequency of genetic recombination. We use a seed-based assay for recombination in Arabidopsis thaliana that utilises green fluorescent protein (GFP) and red fluorescent protein (RFP) expressed under the seed-specific napin promoter from Brassica napus. This recombination ‘test-bed’ exists as homozygous lines with GFP and RFP markers linked on different chromosome arms at known genome locations in the A. thaliana ‘Columbia’ genetic background. Most importantly the same phenotypic markers can be used to assay recombination at different genome regions by simply using different tester lines with the markers on different chromosomes. The scoring of segregating progeny produced on heterozygous plants allows the frequency of meiotic recombination to be conveniently estimated soon after embryo formation. F2 progeny seeds segregate for RFP/GFP; RFP; GFP; and non-fluorescent alleles in a ratio of 9:3:3:1 if the RFP and GFP loci are unlinked. This ratio of RFP, GFP, or non-fluorescent seeds is distorted by linkage and the frequency of recombination between the loci. The frequency of recombination (r) can be determined by simply counting the proportion of only RFP and GFP seeds. We use a confocal microscope to independently examine the RFP and GFP expression in seeds. This allows the automated examination of several hundred seed in a short time. Lines where insert locations of the GFP and RFP markers could be confirmed by PCR have been selected, and are being used in investigations into genetic and environmental factors influencing meiotic recombination frequency.
Q30. Improved Method for Conditioning Cancer Killing T Cells
Anna E.S. Brooks1, Daniel J. Verdon1, Y. Joyce Ho1, P. Rod Dunbar1,2
1School of Biological Sciences, University of Auckland, Auckland, New Zealand
2The Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, Auckland, New Zealand
We have established new culture methods for expanding human white blood cells, called human cytotoxic T lymphocytes (CTL) that can recognise and kill cancer cells. This new culture method depends on the survival factor, IL-7, rather than the classically used growth factor, IL-2, suggesting a new approach to cancer immunotherapy. Our new method for expanding CTL avoids the propensity towards T cell exhaustion and sensitivity to death upon reactivation, inherent in IL-2-based cell culture systems. Adoptive cell transfer (ACT) is currently the most effective treatment for patients with metastatic melanoma, where human CTL are purified from patients, grown in the lab to increase their numbers, and infused back into patients. Current ACT methods involve cell culture conditions (high dose IL-2) that decrease the ability of the T cells to survive in the body after infusion as well as promoting a highly differentiated memory cell state. Accumulating evidence indicates that less differentiated, ‘early’ stage memory cells are likely to improve the effectiveness of the response in fighting tumour burden. We show that when substituting exogenous IL-2 with IL-7, naïve human CTL proliferate strongly and come to rest bearing classical hallmarks of ‘early’ memory T cells. In marked contrast to naïve human CTL cultured in IL-2, these rested IL-7-dependent cells do not die in response to re-stimulation. Finally we applied our new culture systems to the cloning of melanoma-specific human CTL, to generate resting IL-7-dependent T cell clones with characteristics of ‘early’ memory T cells. These clones were shown to not only have a desirable phenotype, but were also effective at killing melanoma cell lines in culture. The ability to generate such cancer-specific T cell lines, with phenotypic and functional properties that are desirable for use in patients, suggests our IL-7-based T cell culture may be useful for ACT in the treatment of cancer.
Q31. Diabetes mellitus alters protein content in amniotic fluid: a proteomic study
Christina Buchanan, Mirjana Stojkovic, Mia Jüllig, Ayesha Pinto do Rosario and Snezana Brankovic contact: [email protected] Research into diabetic pregnancy (DP) has great relevance to human health since both mother and baby are potentially compromised. Firstly, women with DP are more likely to develop severe health problems (high blood pressure, increased risk of developing type 2 diabetes later in life); secondly, if untreated or poorly controlled, DP can cause the baby to have excessive weight at birth (which in addition to complicating delivery often results in jaundice, respiratory distress syndrome) and increased risk of developing obesity and diabetes later in life. Amniotic fluid (AF) surrounds the fetus, and at 12 weeks primarily contains solutes that are derived from the fetus. AF also contains products from the mother, which have been transferred to the baby via the umbilicus and are subsequently metabolized and/or excreted by the fetus. AF samples therefore provide a snapshot of the health status of both mother and unborn child. Here we quantified the levels of major proteins present in the AF of women with DP compared to controls. The quantitative proteomic investigation using 8-plex iTRAQ technology is validated by various orthologous immunoassay techniques. Results are discussed in context of technology comparison and in relation to disease implication.
Q32. 454 resequencing of candidate genes for the development of genetic markers in an autotetraploid Margaret Carpenter, Mark Fiers, John McCallum, Gail Timmerman-Vaughan Plant and Food Research, Private Bag 4704, Christchurch, New Zealand [email protected] The development of genetic markers in the potato, an autotetraploid, is complicated by the possibility of four alleles at any locus. New sequencing technologies provide an efficient mechanism for allelic discovery in specific genes of interest. We are conducting an association mapping study to identify natural allelic variation associated with the degree of starch phosphorylation in potato – a characteristic of relevance for industrial and food uses of starch. To accelerate marker development, resequencing was carried out on eight candidate genes. These were the genes encoding starch synthases (SSI, SSII, SSIII, GBSS), starch branching enzymes (SBEI, SBEII) and (phospho)glucan water dikinases (GWD, PWD). The eight genes were amplified from genomic DNA from two high-phosphate and two low-phosphate potato lines, and used to make four separate bar-coded libraries for 454 sequencing. The resulting sequence reads revealed 1946 polymorphisms over 89 kb. The sequence reads were aligned to reference sequences using gsMapper and converted to a GFF file for import into Geneious Pro software, allowing the positions of polymorphic sites to be visualised on the reference sequence, together with positions of exons. The SNPs occurring in exons were examined to determine if they would cause amino acid substitutions. The frequencies of reference and variant alleles at each polymorphic site were determined for each bar-coded library. These data will facilitate optimisation of marker design such that markers are spread throughout the genes, non-synonymous substitutions are targeted, and SNPs which distinguish high- and low-phosphate potato lines are included.
Q33. Sequence divergence and functional conservation of genes at the parthenogenesis locus of Hieracium caespitosum support an historical evolutionary origin.
Andrew Catanach, Saira Wilson, Sylvia Erasmuson and Ross Bicknell.
The New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch 8140, New Zealand.
Parthenogenesis in Hieracium caespitosum is controlled by a single dominant locus that we have termed LOSS OF PARTHENOGENESIS (LOP). We have sequenced approximately 1Mb of LOP, all of which appears to lie within a region of suppressed recombination. Approximately 11 LOP genes have been identified but the molecular mechanism of parthenogenesis is yet to be elucidated. To provide clues towards identifying the key molecular determinants of parthenogenesis, we are using a co-linearity approach to reveal distinguishing features of LOP. Comparisons of sequences of LOP genes with variant forms elsewhere in the genome show that the LOP forms are highly diverged. Furthermore, coding sequence of an LOP gene, while diverged, shows only synonymous base substitutions indicating functional conservation. By contrast, a gene that is closely linked to LOP but not within the region of suppressed recombination shows no significant sequence divergence from other sequence variants. These data support recent contentions that apomixis genes are likely to have evolutionary origins that pre-date the apomictic complexes in which they are found today. The data are also consistent with findings of apomixis in other species being determined by hemizygous loci. What this means in terms of approaches toward identifying genetic determinants of parthenogenesis will be discussed.
Q34. Crystal Structure of Rv1346: An Acyl-ACP Dehydrogenase from the Mycobactin Biosynthetic Pathway in Mycobacterium tuberculosis
A. F. Chai, E. M. M. Bulloch, J. M. Johnston, J. S. Lott, E. N. Baker
School of Biological Sciences, University of Auckland, Auckland, New Zealand;
Maurice Wilkins Centre for Molecular Biodiscovery; Email: [email protected]
Mycobacterium tuberculosis produces iron-chelating siderophores called mycobactins for iron acquisition in the host macrophage. Two genetic loci, mbt and mtbt-2, have been identified which encode for mycobactin biosynthetic enzymes [1]. Here we focus on one of the gene products from mbt-2, Rv1346, which is annotated as a putative acyl-CoA dehydrogenase (FadE14) [2]. The structure of Rv1346 has very recently been solved to 2.3 Å resolution by molecular replacement. The protein is a homotetramer with a subunit mass of 41 kDa and crystallizes in the space group H32 with a = b = 139.29 Å and c = 253.18 Å. The overall polypeptide fold is similar to those of the same family, which is composed of an N-terminal α-helical domain, a middle β-sheet domain and a C-terminal α-helical domain. Initial analysis has shown an FAD derivative bound to the conserved active site in the structure. Studies to elucidate the biochemical mechanism of Rv1346 are ongoing.
1. Rodriguez, G. M. (2006). Trends Microbiol. 14(7):320-327.
2. Cole, S. T. et. al. (1998). Nature. 393:537-544.
Q35. Investigation of inositol regulation in tissues susceptible to diabetic complications
Hao-Han Chang1 & Kerry M Loomes1,2
Corresponding author [email protected] School of Biological Sciences, University of Auckland, 2 Maurice Wilkin’s Centre for Molecular Discovery. myo-inositol (MI) is a core component of phosphoinositides and an important intracellular osmolyte. However, during diabetes, depletion of MI is observed in tissues susceptible to diabetic complications. The biochemical mechanisms underlying this phenomenon remain unclear although MI depletion is implicated in the development and progression of nephropathy, neuropathy and diabetic eye disease.
Hence, to understand these processes in more detail, we investigated the expression of enzymes involved in MI homeostasis during diabetes using quantitative real-time PCR. STZ-induced diabetic rats were used as an animal model and tissue expression investigated at 4- and 8-week post-injection. The genes investigated include: (i) myo-inositol oxygenase (MIOX), a key enzyme that regulates the only known catabolic route of MI; (ii) sodium dependent myo-inositol transporter subtypes 1 and 2 (SMIT1 and 2) which facilitate MI uptake; (iii) myo-inositol phosphate synthase (MIPS) that synthesises MI from glucose. In 4-week diabetic kidneys, expression of MIOX, SMIT1 and SMIT2 were up-regulated when comparing to corresponding controls, whereas MIPS expression remained unaltered. In contrast, the expression of MIOX and SMIT1 in diabetic kidneys at 8-week declined compared to non-diabetic controls. A similar trend was also observed in livers. The expression of MIOX, SMIT1 and MIPS were unaltered in sciatic nerves at both 4- and 8-week time points and in the lens at 4-week. However, at 8-week, MIOX expression was mostly non-detectable in control lens but exclusively maintained in diabetic lens. These findings show that MIOX and SMIT expression are elevated in kidney and liver at 4-week diabetes suggesting enhanced transporter uptake and catabolic capacity but unaltered MI synthetic capacity. Other mechanisms must also operate which mediate the observed decrease in expression in these tissues at 8-week diabetes.
Q36. Genome-Scale DNA Methylation Mapping in Normal Population
Aniruddha Chatterjee, Euan J. Rodger, Ian M. Morison
Department of Pathology, Dunedin School of Medicine, University of Otago, New Zealand
Email : [email protected]
A detailed understanding of how epigenetic marks vary between individuals will determine their role in
altering gene expression, and affecting physiological, biochemical and behavioural phenotypes. We will
quantify the methylation status of almost 24,633 CpG islands (87% of all CpG islands in the genome;
18,500 of these are associated with promoters) across a normal human population, by using reduced
representation bisulfite sequencing (RRBS). We selected 40-220 bp fragments from MspI digested
genomic DNA (1.5% of the human genome and 6.7 Mb of sequence) for massively parallel sequencing.
Our reduced representational library consists of ~3 million CpG sites and will provide us with 30-fold
enrichment in terms of CpG islands. By using next generation sequencing (Illumina) we will obtain
around 10-fold coverage of each CpG site and we will measure the percentage of methylation for each
CpG island at single nucleotide resolution. Thereby we will use a small group of individuals to establish a
set of methylomes to detect candidate population ‘epipolymorphisms’. Then, by focussing on genes that
show inter-individual variation, we will explore specific cohorts to document epigenetic influences on
human phenotypes and disease.
Q37. β-catenin as a novel glucose-sensor in INS1E β-cells
Emmanuelle Cognard, Coralie Dargaville and Peter Shepherd
Department of Molecular Medicine and Pathology, University of Auckland
Canonical Wnt signaling is a key feature of growth, differentiation and metabolism. In β-cells, Wnt/β-catenin pathway induces proliferation and glucose-induced insulin secretion. Previously, we showed that changes in glucose levels can acutely regulate β-catenin levels in macrophage cell lines, via an autocrine activation of Wnt pathway. In our current study, we investigate the effect of glucose on Wnt/β-catenin pathway in glucose-responsive and insulin-secreting INS1E cells, a model of pancreatic β-cells. Our most significant finding is that glucose itself increases β-catenin levels in INS1E β-cells and this leads to β-catenin translocation to the nucleus. Wnt co-receptor LRP6 is also phosphorylated in response to glucose indicating an autocrine activation of Wnt pathway. Looking downstream of β-catenin, we found that the transcription of its target gene CyclinD1 is also up-regulated with glucose. Glucose-induced insulin secretion is not involved in the autocrine activation of Wnt/β-catenin as insulin itself has no effect, either on β-catenin level nor on phosphorylation of LRP6. Besides, inhibition of insulin secretion with octreotide or somatostatin does not change glucose-induced β-catenin stabilization or CyclinD1 mRNA level. Finally silencing β-catenin using specific siRNA inhibits glucose effect on CyclinD1 transcription implying that glucose is acting via the Wnt/β-catenin pathway. Thus in this study, we present a new mechanism for pancreatic β-cells to sense glucose levels. Glucose stabilizes β-catenin independently of insulin and induces the canonical Wnt pathway via autocrine activation. This provides a previously unreported mechanism by which pancreatic β-cells can respond to changes in glucose levels. One consequence of this is increased expression of the TCF7L2 target-gene CyclinD1, which is involved in β-cell proliferation. Besides TCF7L2 polymorphisms were identified as being strongly associated with type-2 diabetes. Therefore these data provides new insights into mechanisms by which glucose levels can regulate β-cell proliferation through the Wnt pathway.
Q38. Glucose regulates gene expression through the Wnt/β-catenin pathway in INS1E β-cells
Coralie Dargaville, Emmanuelle Cognard and Peter Shepherd
Department of Molecular Medicine, University of Auckland
The canonical Wnt/�-catenin signaling pathway plays roles in diverse aspects of metabolism, differentiation and growth. Several studies recently showed that polymorphisms of TCF7L2, the transcription factor downstream �-catenin, are strongly associated with type-2 diabetes. Of interest to the pathological states of diabetes and cancer, our recent studies have identified the Wnt/β–catenin signaling pathway as being responsive to changes in glucose concentrations. In several cell lines, cytosolic and nuclear β–catenin levels were seen to increase upon glucose treatment, leading to up-regulation of Wnt/�-catenin target-gene expression. Collaborative results have led to the hypothesis that upregulation of β-catenin could be one mechanism used by glucose to regulate gene expression. Following up our study in the pancreatic β-cell line INS1E, we use the Rat Wnt Signaling Pathway PCR Array to measure gene expression changes. Upregulation of the Wnt pathway was achieved using glucose, while siRNA targeting β-catenin mRNA was used to downregulate the Wnt pathway. Therefore we can distinguish genes regulated by glucose and β-catenin, those regulated by glucose independently of β-catenin and those regulated by solely β-catenin. Using the Wnt PCR Profiler Array we were able to detect quantitative changes in Wnt/ β–catenin target gene expression and other components on the Wnt pathway, including inhibitors or co-activators. We found that only a few genes of the Wnt pathway were regulated in our conditions and that Wnt4 is the most expressed among the Wnt family in INS1E β-cells. Looking at the target-genes we found that CyclinD1 was regulated through glucose and β-catenin, whereas Ccnd2 only through β-catenin. Wnt inhibitors Dkk1 and Wif1 were down-regulated when β-catenin is absent. Secreted frizzled-related proteins (sFRP) were regulated differently sFRP-1 and -3 were down-regulated with β-catenin siRNA, sFRP-4 with glucose whereas sFRP-5 was up-regulated with glucose. For co-activators of Wnt pathway, our results rather indicate that some are down-regulated with β-catenin silencing and other ones are up with glucose. Thus with our Wnt PCR Profiler Array performed in INS1E β-cells we confirmed former results found by real-time PCR and we identified new genes that glucose can regulate through the Wnt/β-catenin pathway or independently of it. These results give new insight of glucose functions in pancreatic β-cells.
Q39. Structural characterization of the M. tuberculosis TetR-type transcriptional regulator
Rv3557c/KstR2.
Stephanie Dawes1, Chen Gao1, Sharon Kendall2, Shaun Lott1.
Laboratory for Structural Biology, University of Auckland, Auckland, NZ and 2Pathology & Infectious Diseases, The Royal Veterinary College, London, UK. [email protected]
Cholesterol metabolism appears to be important for the persistence of the human pathogen Mycobacterium tuberculosis and thus the elucidation and disruption of this pathway is a focus for rational drug design. A cluster of 58 genes involved in the uptake and degradation of cholesterol has been identified, which also contains two TetR-type transcriptional repressors responsible for the regulation of cholesterol catabolic enzymes. One of these regulators, Rv3557c (Ketosteroid Regulator 2/KstR2), was expressed as recombinant protein in E. coli and gave crystals that diffracted to 1.86Å. The crystal structure of KstR2 was solved by molecular replacement and showed typical features of TetR-type family members, viz a homodimeric protein in which each monomer comprises a N-terminal DNA binding domain and C-terminal region responsible for ligand binding and dimer formation. Currently attempts are being made to determine which metabolic intermediate of cholesterol catabolism binds to KstR2 to facilitate transcription of its 15-gene regulon.
Q40. Effect of cold temperature fermentation on the production of aroma compounds by wine yeast
Rebecca C. Deed and Richard C. Gardner
Department of Wine Science, School of Biological Sciences, University of Auckland, Private Bag 92019, Auckland, 1142, New Zealand. [email protected]
Winemakers generally consider that white wines fermented at cold temperatures (10-18°C) are fruitier, a view primarily ascribed to changes in the balance of yeast-derived esters. However, there are costs associated with the use of low temperatures, including slower fermentation rates, greater risk of stuck fermentations and increased financial costs.
This research examined the effect of cold fermentation on aroma production and gene expression of a commercial wine strain, Enoferm M2, comparing Sauvignon blanc grape juice at 12.5°C and 25°C. Cold-fermented wines had lower concentrations of aroma compound (thiols, esters, higher alcohols and fatty acids), with decreases in specific acetate and ethyl esters being proportional to that of their corresponding precursor higher alcohols and fatty acids. Analysis of known aroma pathway genes showed down-regulation of only one transcript (~2-fold), encoding an ester synthase (EEB1), at mid/late cold fermentation. In contrast, there was differential expression in genes upstream of the aroma biosynthetic pathway, suggesting that the aroma differences in cold temperature are the result of changes in upstream substrate concentrations via shifts in central metabolism, rather than differential expression of ester biosynthetic genes.
Genes associated with low temperature fermentation were targeted to minimise cold-fermentation costs. A hybrid between two sequenced strains, RM11 (wine) and S288c (laboratory), was used to obtain cold fermentation phenotypes (maximal fermentation rate = Vmax) at 12.5°C for 123 random progeny, previously mapped using microarrays for 2957 markers. A major quantitative trait locus (QTL) for cold fermentation rate was mapped to a region on Chromosome I (LOD>3). Two candidate genes within located within this region: FLO1 (flocculation); and SWH1 (oxysterol binding). Fermentation rate data for RM11 and S288c wild-type and respective FLO1 and SWH1 gene knockouts demonstrated a 50% reduction in fermentation rate of the S288c FLO1 knockout at 12.5°C. Therefore, we have identified FLO1 as a candidate gene important for cold fermentation of yeast.
Q41. GENE REGULATION BY DRUGS USED TO TREAT MOOD DISORDERS
Xiaoyan Deng1, Kit Doudney1, Patrick C. McHugh1, Peter R. Joyce2 and Martin A. Kennedy1
Departments of Pathology1and Psychological Medicine2, UOC
Antidepressants and mood stabilizers are the main treatments for mood disorders including major depressive disorder and bipolar disorder. Although widely used, the mechanisms of action of these drugs are not well understood. Our first attempts to establish a model cell culture system involved pretreatment of RN46A cells for 14 days, with various drugs, followed by transfection with luciferase reporter constructs, in order to monitor gene expression effects. Although occasional significant differences in expression were noted between drug-exposed and control cell cultures, these proved not to be robust or reproducible despite considerable care in experimental design and execution.
Our second approach was to use quantitative PCR analysis of candidate genes after exposure of RN46A cells to various drugs for 72 hours. Our most striking results to date are: (1) a gene called sepiapterin reductase (SPR), which encodes a key enzyme in neurotransmitter synthesis, is massively (>1700-fold, p=0.001) and specifically up-regulated by sodium valproate (VPA); (2) the serotonin receptor 2A (HTR2A) is significantly down-regulated by paroxetine and citalopram, the two selective serotonin reuptake inhibitor antidepressants (p=.001 to .003); and (3) the histone deacetylase HDAC6 gene is upregulated significantly by VPA (6.031±1.400, p=0.016).
In addition, Western blot was used to validate the findings on SPR gene upregulation. It was found that VPA-treated RN46A cells showed higher level of SPR protein expression than the untreated samples, which provided support for the findings at the mRNA level. Multi-kinase ELISA array was used to determine if any signalling pathways are affected by VPA exposure or involved in SPR upregulation. No significant changes in phosphorylation levels of the six kinases tested (Akt2, Erk1, Erk2, GSK3α, GSK3ß, and p38α) were detected.
Q42. Functional Conservation Of Axillary Branching Control: Petunia Genes Orthologous To MAX1 And MAX2 Influence The Production Of Branches
Revel SM Drummond1, Hester Sheehan1, 2, Joanne L Simons1, N Marcela Martínez-Sánchez1, Rebecca M Turner1, Joanna Putterill2, Kimberley C Snowden1
1The New Zealand Institute for Plant & Food Research Ltd. 2Plant Molecular Sciences, School of Biological Sciences, University of Auckland. Email: [email protected] Branching is a fundamental habit of most land plants. The molecular control of this developmental process is likely to have evolved early in the history of land plants and be highly conserved, although the huge diversity of plant form must also be explained. The highly branched dad/max/rms/d mutants (of petunia, Arabidopsis, pea and rice, respectively) all appear to be products of mutations in the same strigolactone-mediated system that inhibits axillary branch outgrowth. We have cloned three genes from petunia, one orthologous to MAX1 (PhMAX1) and two orthologous to MAX2 (PhMAX2A and PhMAX2B). The identification of these genes further supports the conservation of this developmental control system. The PhMAX1 and PhMAX2A genes complement the max1 and max2 mutants of Arabidopsis, respectively, showing that the function of the genes is conserved. The pattern of transcript accumulation for the three genes in wild-type petunia plants is broadly similar to that reported in other species, although some differences in the pattern between PhMAX2A and PhMAX2B are apparent. The discovery of two MAX2 paralogues with significant sequence divergence and different expression profiles suggests the possibility of divergent functions. The transcript abundance of PhMAX1 increases in most tissues over development, PhMAX2A and PhCCD8 (another gene known to be involved in branching) show a similar change, although in a smaller number of tissues, suggesting the involvement of the strigolactone pathway in the control of branching during development.
Q43. Confirming function using co-expression.
Genevieve L. Evans1, Jodie M. Johnston1, J. Shaun Lott2, and Edward N. Baker1
1 Laboratory for Structural Biology, School of Biological Sciences, University of Auckland, Private Bag 92-019, Auckland, New Zealand 2 AgResearch Structural Biology Laboratory, School of Biological Sciences,University of Auckland,
Private Bag 92-019,Auckland,New Zealand contact: [email protected]
The enzymes of the tryptophan biosynthetic pathway represent potential drug targets given the pathways crucial role in survival of Mycobacterium tuberculosis. However several of these enzymes have yet to be conclusively identified. The first committed step of the pathway is catalysed by anthranilate synthase (AS), which in almost all microbial species is composed of two non-identical subunits. The α-subunit is encoded by the trpE gene, but for the β-subunit the situation is more complex. The β-subunit can sometimes can include functionality from other enzymes in the tryptophan pathway or be bifunctional; participating in folate as well as tryptophan biosynthesis. In the M. tuberculosis, Rv0013 has been annotated as the β-subunit of AS (trpG), although it was previously annotated as being involved in folate synthesis (pabA). Previous work expressing the gene in E. coli resulted in insoluble protein. This work outlines progress towards co-expression of Rv0013 with its putative partner trpE (Rv1609).
Q44. California poppy as a model system to search for regulatory genes controlling carotenoid biosynthesis
Gatehouse, M., Conner T. & Barrell P.
The New Zealand Institute for Plant & Food Research Limited, Private Bag 4704 Christchurch, New Zealand.
California poppy (Eschscholzia californica Cham.) typically has intense orange petals and orange pollen due to pigmentation by carotenoids, however flower colour variants ranging from white to yellow and orange also occur. The inheritance of the petal and pollen colour of twenty four yellow, pale yellow and white flower colour variants was investigated through complementation analysis and these variants were resolved into five loci (b, s, f, i and x). Each of the mutations is recessive at the genetic level. Biochemical assays showed reduced carotenoid levels in petal tissue of four yellow s, f, i and x loci, and a virtual absence of carotenoids in the white b locus flowers. Confocal analysis of plastids in petal tissue showed that chromoplasts are present in white and yellow petal tissue, indicating that the mutations causing the yellow and white phenotypes are not caused by an absence of chromoplasts. The mutation causing the white phenotype is hypothesised to be a regulatory gene controlling the initiation of carotenoid biosynthesis. We have generated a mapping population by crossing white variant b locus plants to wild type orange flowered plants. Heterozygous orange (Bb) flowers have half the carotenoid content of wild type (BB) flowers. Homozygous wild type orange flowered F2 individuals were identified in the mapping population via carotenoid assays. RNA was extracted from bb and BB individuals in the F2 population. Pooled RNA samples are currently being analysed by high throughput transcriptome sequencing using the Roche/454 GS FLX facility at Otago University.
Q45. Aurora B Phosphorylates Histone H1 During Mitosis
Tracy Hale and Sarah Bond
Chromatin Research Group, Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North 4442, New Zealand
The failure to properly fold and condense our chromosomes during cell division has catastrophic consequences. However the mechanisms that govern the dramatic changes in chromosome architecture that occur during mitosis remain an enigma. The linker histone H1 is a major structural component essential for condensing mitotic chromosomes, although its role in controlling this process remains elusive. H1 becomes hyper-phosphorylated in a cdc2-dependent manner as cells enter mitosis. It is proposed this phosphorylation causes structural changes within H1 allowing it to compact the chromatin fibres. Histone H1 then loses this phosphorylation as cells exit mitosis, resulting in rapid decondensation.
Using immunofluorescence and confocal microscopy we have identified a novel mitotic specific phosphorylated residue on the N-terminal tail of the abundant linker histone H1.4 variant. Interestingly, this residue is part of a bivalent mark on H1.4 that is similar to the well-studied core histone H3 ‘methK9 phosS10’ switch and the distribution of H1.4 during mitosis is dependent on the combination of post-translational modifications present on this bivalent mark. We demonstrate that like histone H3, the bivalent mark on H1.4 is phosphorylated by the Aurora B kinase. Since Aurora B orchestrates vital events in mitosis including chromosome condensation, we are exploring how this impacts on the role of histone H1 in controlling the window of chromosome condensation during mitosis.
Q46. Therapeutic reactivation of mutant p53 protein
In Young Hwang ([email protected]), Hamish S. Sutherland, Elaine S Marshall, Emma L Richardson, Bruce C. Baguley
Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand
The human tumour suppressor protein p53 is an ideal target for cancer therapy as it is inactivated in half of human tumours. This provides a novel target for the development of new anticancer drugs that reactivate mutant p53 protein to restore p53-dependent apoptosis and growth inhibitory activity. Several compounds, including the quinazoline derivative CP-31398 have been proposed to restore p53 activity in mutant cells.
A programme at the Auckland Cancer Society Research Centre has resulted in the synthesis of the novel compound (2-benzofuranyl) quinazoline derivative (SN-29885), chemically more stable than CP-31398, and of a series of analogues. p53 function was determined by examining the cell cycle arrest and the p21 expression induced by the SN-29885 and its analogues alone or in combination with DNA damaging agents such as radiation and cytotoxic drugs. Furthermore, the expression of Hsp90, a known chaperone of p53 protein maturation, was investigated.
The results show that restoring mutant p53 protein by the SN-29985 series can induce significant cell cycle arrest in human tumour cell lines and provide strategies for investigating the in vivo effects of these drugs. These drugs were also found to potentiate cell cycle arrest in response to two chemotherapeutic drugs, the antimetabolite 5-fluorouracil and the topoisomerase I poison camptothecin. Since Hsp90 is thought to be part of a complex that facilitates the formation of the active conformation of wild-type p53, SN-29885 and its analogues may function by stabilising an active form of the mutant p53 that binds to DNA and promotes transcription of p53 products.
Q47. Expanding on the reference potato genome sequence.
Jeanne Jacobs, Susan Thomson, Mark Fiers
The New Zealand Institute for Plant & Food Research Ltd., Private Bag 4704, Christchurch 8104, New Zealand
Plant & Food Research is a member of the international Potato Genome Sequencing Consortium (PGSC), which has released an advanced draft of the potato genome sequence (www.potatogenome.net). The genotype sequenced is a homozygous doubled monoploid with little genetic or commercial relevance. With the reference genome sequence available, we have started the genomic re-sequencing of four highly heterozygous autotetraploid potato cultivars and breeding lines, with the aim to capture the allelic variation of genes underpinning traits of interest in commercially meaningful material. Some initial results derived from our resequencing efforts will be presented including quality and quantity of data and methods for determining genetic (SNP) variation. The main issues surrounding resequencing are mapping back to the reference genome, quality assurance, identifying genetic variation and knowing how much data is enough. Application of an in-house developed method for quickly gaining insight into small scale variation of re-sequencing data will be presented.
Q48. Ten Years of TB Structural Genomics at the University of Auckland
J. M. Johnston1, J. S. Lott2, E. N. Baker1 and the Auckland Structural Biology group*
1Laboratory of Structural Biology, School of Biological Sciences, University of Auckland
[email protected] 2AgResearch Structural Biology Laboratory, School of Biological Sciences, University of Auckland
*due to the number of members involved in the project over the last 10 years ( >50) all contributors will be specified on the poster
Mycobacterium tuberculosis (Mtb) is the primary causative agent of the human form of tuberculosis (TB) which kills an estimated 1.8 million people worldwide per year. In 2000, as part of the International TB Structural Genomics Consortium, our laboratory started work on a number of gene targets from Mycobacterium tuberculosis with the hope of both understanding more about the bacterium and providing structures for use in structure based drug-design. All targets were taken through a structural genomics pipeline which commenced with bioinformatics analysis followed by cloning, protein expression, purification, crystallisation and X-ray crystallographic analysis, with attrition at each step. In the past 10 years we have worked on over 230 targets and solved 23 structures. Along the way we have developed a number of higher throughput technologies and new techniques (e.g. GATEWAY cloning, M. smegmatis expression, autoinduction for both E. coli and M. smegmatis expression, and robotic crystallization) which has improved our success rate and benefited our laboratory as a whole. In recent years our focus has changed from a broader approach to working with targets that are highly likely to be good drug design candidates. Indeed a number of our successful structures such as those for TrpD and MbtI are currently being used in drug design efforts.
Q49. An examination of protein changes associated with invasion and metastasis for colorectal cancer.
Daniel Kay1*, Chandra Kirana2, Hong-Jun Shi2, Sarah Rothlisberger2, Sonal Sawhney2, Teresa Ward2, Janice Royds3, Noelyn Hung3, T.William Jordan4, Pisana Rawson4, Richard Stubbs2, Donia Macartney-Coxson1, Kylie Hood2,5.
1. Environmental Science and Research Ltd, Wellington, NZ 2. Wakefield Biomedical Research Unit, University of Otago, Wellington, NZ 3. Department of Pathology, University of Otago, Dunedin, NZ 4. Centre for Biodiscovery and School of Biological Sciences, Victoria University, Wellington, NZ 5. Arotec Diagnostics Ltd, Petone, Wellington , NZ
* corresponding author: [email protected]
Metastasis is the major negative prognostic indicator for colorectal cancer (CRC) survival. Twenty to thirty percent of CRC patients present with metastases at initial diagnosis, with a further forty percent developing metastases after bowel resection. The prognosis for all patients with metastases is poor, with only a five-ten percent five-year survival rate.
Improved understanding of the key factors driving metastasis will aid in the development of targeted therapeutics to prevent or treat metastatic spread. Successful metastasis requires tumour cells to detach from the primary tumour, migrate, access the blood or lymphatic vessels, disseminate and finally establish and proliferate at a distant site. Evidence suggests that cells in the invasive area of colorectal tumours dedifferentiate (an epithelial to mesenchymal transition) in order to invade and disseminate, and that establishment in the liver involves redifferentiation (a mesenchymal-epithelial transition). Thus, comparisons of the main tumour body, invasive front and liver metastases should reveal differences pertinent to tumour progression.
While many studies have compared the transcriptome of different grades of and cell types in colorectal tumours, far fewer have sought to compare the proteome. We previously performed saturation labeled 2D-DIGE (two-dimensional difference gel electrophoresis) on protein extracts isolated from laser capture micro-dissected tissue to identify differentially abundant proteins between the invasive front, main tumour body and matched liver metastases of five patients. A total of 38 protein spots were upregulated and 23 downregulated during tumour progression (≥1.5 fold difference in relative abundance, p<0.05 paired t-test). Protein spots showing the most interesting differences in abundance between invasive front, main tumour body and/or liver metastases were targeted for further analysis. These were identified by mass spectrometry
We present here immuno-histochemical validation of our proteomic analysis, discuss technical challenges and illustrate the potential of this approach to both increase our understanding of cancer biology, and as a biomarker discovery tool.
Q50. Modified CAROTENOID CLEAVAGE DIOXYGENASE8 expression correlates with altered branching in kiwifruit (Actinidia chinensis).
Susan E. Ledger, Bart J. Janssen, Sakuntala Karunairetnam, Tianchi Wang and Kimberley C. Snowden. New Zealand Institute for Plant & Food Research Limited, e-mail [email protected]
• CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants including Arabidopsis, pea, petunia and rice.
• Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8.
• Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence.
• The CCD pathway for control of branch development is conserved across a wide range of species including kiwifruit, a woody perennial.
Q51. Using zebrafish models to study the role of TES in childhood leukaemia (ALL)
G. Le Mée, R.Weeks, I. Morison Department of Pathology, University of Otago [email protected] Acute Lymphoblastic Leukaemia (ALL) is the most common childhood cancer. A key feature of ALL is gene rearrangement leading to the expression of fusion oncogenes, TEL/AML1 being the most prominent. However, only 1% of the children expressing TEL/AML1 at birth develop ALL; thus modification of other key factors is necessary to develop leukaemia.
We have discovered that TES is inappropriately switched off in nearly all cases of ALL (Weeks et al, Mol Cancer. 2010). Little is known about TES function. It is a highly conserved LIM-domain containing protein present in focal adhesion complexes. It is known that normal haematopoiesis requires coordinated interaction, adhesion and movement of the developing blood cells within the bone marrow. We propose that silencing of TES disrupts these interactions and contributes to the development of ALL.
Despite obvious differences between fish and humans, the molecular pathways controlling blood cell development are highly conserved. Zebrafish have provided a surprisingly reliable system to investigate the development of the blood system and therefore leukaemia. Indeed, transgenic zebrafish that express the most common fusion protein TEL/AML1 develop leukaemia (Sabaawy et al, PNAS 2006). Interestingly, only 3% of the transgenic fish develop leukaemia suggesting, as in humans, that expression of this oncogenic protein is not sufficient to cause leukaemia.
Our aim is to access the role of TES in leukaemia using this zebrafish model. Our work will be carried out in two parts, first the role of TES in zebrafish development and then the role of TES in normal haematopoiesis and leukaemogenesis. For this project, we have developed an antibody against zTES and an antisense morpholino technique allowing us to block TES expression in early development. Our preliminary results show that TES is essential for zebrafish early development, with the morphants having defects that may result from apoptosis problems during morphogenesis. Interestingly, TES protein size switches from the expected size of 57 kDa to an unexpected size of 100 kDa between day 4 and 5 post fertilization, the age at which haematopoietic activity appears in the kidney, the main haematopoietic organ. The molecular mechanisms that underlie these results will be investigated.
Q52. Expression of multi-domain PhoH2 from mycobacteria
Emma Littlejohn, Joanna McKenzie, Ray Cursons, and Vic Arcus
University of Waikato
The Mycobacterium tuberculosis genome has a strikingly high number of PIN domain proteins compared with the number found in other bacterial genomes. The biochemical function of these proteins is hypothesised to be linked to the persistence of M. tuberculosis. Each of the 45 PIN domain proteins is found with an associated inhibitor or antitoxin that under normal growth conditions, forms a benign protein complex. An extensive bioinformatics survey indicates that proteins from the AAA+ ATPase protein family are associated with these toxin-antitoxin modules, and this association poses a question as to the functional connection between these proteins (antitoxin, toxin, and AAA+ ATPase). The protein encoded by Rv1095 (PhoH2) is annotated as a PIN domain fused with a AAA+ ATPase/DExDc domain protein family member. This gene is induced under nutrient starvation in C. glutamicum, and currently no associated inhibitor or antitoxin is annotated. This protein serves as a model to study the relationship between PIN domain proteins and AAA+ ATPase protein family members in M. tuberculosis. We hypothesise based on the biochemical function of these protein domain families that the protein encoded by Rv1095 unwinds and cleaves DNA or RNA using energy from the hydrolysis of ATP under nutrient starvation. The homologous gene in M. bovis BCG str. Pasteur 1173P2, BCG_1155 that is identical to Rv1095 from M. tuberculosis has been cloned from its annotated start site, as separate domains, and with an upstream sequence to an alternative start site. BCG_1155 expresses within the soluble fraction when cloned with the upstream sequence, and insolubly when cloned from its annotated start site and as separate domains. These proteins have been expressed in M. smegmatis and E. coli.
Q53. Role of RING domain E3 ubiquitin ligases in regulating ENaC activity
Ly, K. McDonald, FJ.
Department of Physiology, University of Otago
The delta Epithelial Sodium Channel (δENaC) subunit is related to the α-, β-, γENaC subunits that are essential for sodium homeostasis/blood pressure regulation via mediating Na+ transport in the collecting ducts of the kidneys. Although the δ subunit can form a functional channel with β-, and γENaC the in-vivo role of δENaC is yet to be established. Down-regulation of surface proteins via the ubiquitination pathway is a well known phenomenon, where the conjugation of the ubiquitin protein to lysine residue(s) of a target protein (aided by a HECT (homologous to E6 AP C-terminus) or RING (really interesting new gene) domain E3 ligase) triggers a chain of events resulting in the endocytosis or degradation of that protein. Selection of proteins to be tagged with ubiquitin relies on association with E3 ubiquitin ligases of the HECT or RING families. Here we show that XIAP, a RING E3 ligase, and Nedd8, a ubiquitin-like protein that regulates RING E3s, have opposing effects on the δENaC protein, with XIAP exerting a positive and Nedd8 a negative effect on δENaC protein activity and expression pattern.
The short circuit current (Isc) generated by δENaC expressed in FRT cells was recorded with/without XIAP/Nedd8, and showed that XIAP increased the Isc of δENaC and Nedd8 decreased Isc. In support of this data XIAP increased δENaC suface expression, decreased δENaC ubiquitination, while Nedd8 had the opposite effects. We hypothesized that these RING E3 ligases work by regulating upstream effectors of δENaC, and we are currently investigating a number of candidiates.
Q54. Towards personalized medicine – genomic tools for prediction of clinical outcome
Donia Macartney-Coxson1*, David Hall 1,2, Bushra Nasir 3
Javed Fowdar3, Lyn Griffiths3, and Rod Lea1,3
1. Environmental Science and Research Ltd, NZ
2. Victoria University of Wellington, NZ
3. Genomics Research Centre, School of Medical Sciences, Griffith University, Australia
* Corresponding author [email protected]
The ability to accurately predict an individual’s disease risk and/or drug response is a major goal of clinical medicine. The advent of high-throughput genotyping technologies has created the ability to generate huge amounts of genetic information on thousands of individual samples. With this ability came the promise that increased genetic knowledge would translate to better tools for the treatment and prevention of disease. Has such promise been realized? Many novel loci for many complex diseases have been identified which have, in turn, provided new insights into disease etiology. However, the effect size of the vast majority of these variants when considered alone is small, and as such their predictive power is limited.
We are developing a novel bioinformatic approach for analyzing Genome Wide Association Data to identify SNPs (single nucleotide polymorphisms) predictive of disease. We call this genome signature analysis (GSA) and its function is two-fold: 1. to identify a genome signature which is predictive of disease 2. to identify variants which provide insights into disease biology and potential novel avenue(s) for therapeutic intervention.
We are using The Welcome Trust Case Control Consortium (WTCCC) data to develop and test our algorithms. We present data for both bipolar disorder and hypertension, two complex diseases for which the original WTCCC paper found 14 and 6 SNPs respectively associated with disease (p<1x10-5). Our GSA analyses found multi-SNP signatures which were significantly associated with disease risk (p<1x10-
8) and had significant predictive accuracy, AUC >0.75. This AUC is regarded as the threshold for a clinically useful diagnostic classifier when applied to a sample which is considered to be at risk. We discuss the need to develop tools of clinical utility, and how our approach might be useful for developing such diagnostic tests.
Q55. Growth and Pathogenicity Modulation in Candida by Essential Oil of Ocimum sanctum Amber Khan, Aijaz Ahmad, Luqman A Khan, Nikhat Manzoor* Department of Biosciences, Jamia Millia Islamia, New Delhi-110025. India. Email: [email protected]
*Present Address: Molecular Microbiology Laboratory, Department of Oral Sciences, School of Dentistry, University of Otago, P.O. Box 647, Dunedin, New Zealand-9054.
The increased incidence of drug resistant pathogens and host toxicity has drawn attention to the efficacy of natural products as antifungals in mucocutaneous infections and in combinational therapies. The composition and antifungal activity of the essential oil obtained from Ocimum sanctum (OSEO) was studied. GC-MS analysis showed OSEO had a high content of methyl chavicol (44.63%), and linalool (21.84%). Antifungal activity of OSEO and these main constituents were determined against sixty clinical and five standard laboratory isolates of Candida. The OSEO and its two lead components were fungicidal for all the strains tested. Their mechanism action was assessed using flow cytometry, confocal imaging and ergosterol biosynthesis. Propidium iodide rapidly penetrated the majority of yeast cells when they were treated with OSEO concentrations just above the MICs, implying that the fungicidal effect resulted from extensive lesions of the cell membrane. OSEO and its components also caused considerable reduction in the amount of ergosterol, a specific fungal cell membrane component. OSEO, methyl chavicol and linalool have considerable antifungal activity against Candida, including azole-resistant strains, suggesting a need for further investigations aimed at their clinical application in the treatment of fungal infections.
Q56. Osteoblast growth and differentiation in three-dimensional cultures Brya G Matthews1, Karen E Callon1, Rachel M Locklin2, Philippa A Hulley2, Dorit Naot1 and Jill Cornish1
1Department of Medicine, University of Auckland 2Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford Culturing bone-forming osteoblastic cells on plastic provides useful information about their biology. However 2D plastic surfaces poorly mimic the complex 3D microenvironment where osteoblasts operate in vivo. Type I collagen, which sets as a gel at appropriate temperature and pH, can be used as a scaffold for 3D culture of cells. This study aimed to compare growth and differentiation of osteoblasts cultured in collagen gels with cells cultured on plastic. Primary rat osteoblasts were cultured in parallel in plastic dishes and in 3mg/ml collagen 50μL minigels. Cell proliferation was measured by thymidine incorporation and differentiation was assessed by staining for mineralisation using von Kossa or alizarin red stains. Gene expression during differentiation was examined by qRT-PCR. Cells cultured in 3D responded to anabolic factors like TGFβ, PDGF and lactoferrin over a similar concentration range as in 2D, but the magnitude of the response increased in the gels. For example, 10pM TGFβ increased thymidine incorporation 1.6±0.06x in 2D and 3.3±0.19x in 3D. The targeted tyrosine kinase inhibitor imatinib, whose targets include the PDGF receptor, inhibited cell proliferation in both systems, with inhibition at concentrations 10x lower in 3D. Effects of 3D culture on differentiation included up-regulated expression of osteoblast differentiation marker genes alkaline phosphatase (20x), bone sialoprotein (40x) and osteocalcin (250x) in the first week of the assay and mineralisation occurred earlier in 3D than 2D. Collagen minigels are simple to set-up and produce reproducible results. 3D culture of rat osteoblasts appears to amplify the effect of selected anabolic agents and promote osteoblast differentiation. Presenting author: Brya Matthews, [email protected]
Q57. Insulin stimulation of the epithelial sodium channel is inhibited by COMMD1
McDonald, FJ., Swart, M., Ke, Y.
The epithelial sodium channel (ENaC) transports Na+ into epithelial cells located in the distal nephron, lung, and colon. Liddle’s syndrome, a rare early-onset form of hypertension, is caused by mutations in the genes coding for ENaC subunits. Under normal conditions active channel number at the cell surface is regulated by ubiquitin modification of channel subunits and endocytosis. Insulin, aldosterone and vasopressin enhance Na+ transport via the serum and glucocorticoid regulated kinase (SGK1) that inactivates the ubiquitin ligase Nedd4-2 that normally downregulates ENaC through ubiquitin tagging. Previously we reported that COMMD1 (copper metabolism Murr1 domain containing protein 1) also inhibits ENaC amiloride-sensitive current.
Here the objective was to determine if COMMD1 and ENaC are co-expressed in kidney, and to study the mechanism by which COMMD1 inhibits ENaC amiloride-sensitive current.
Using immunohistochemistry COMMD1 was found to be widely expressed in the kidney, and to co-localise with ENaC. Biochemical and electrophysiological assays showed that COMMD1 increased ubiquitin modification of ENaC and decreased its cell surface expression. COMMD1 abolished insulin-stimulated amiloride-sensitive current. COMMD1 co-immunoprecipitated with SGK1 and Akt1, but not Nedd4-2. Knock-down of COMMD1 enhanced the effect of SGK1 and Akt1 on amiloride-sensitive current, but did not change the effect of Nedd4-2.
In conclusion, COMMD1 appears to be an in vivo regulator of ENaC cell surface expression through interaction with the SGK-Nedd4-2 pathway to achieve downregulation of ENaC.
Support from the Marsden Fund Council from Government funding administered by the Royal Society of New Zealand
Q58. The Role of VapBC Proteins in Mycobacteria
Joanna McKenzie1, Jennifer Robson2, Gregory Cook2, Ray Cursons1, Vic Arcus1
1 Department of Biological Sciences, University of Waikato - [email protected]
2Department of Microbiology, University of Otago
Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) in humans, is a devastating infectious organism, which kills approximately 2 million people annually. The current suite of antibiotics used to treat TB face two main difficulties: (1) the emergence of multidrug resistant (MDR) strains of M. tuberculosis and (2) the persistent state of the bacterium which is less susceptible to antibiotics and dictates very long treatment regimes. M. tuberculosis has a surprisingly large repertoire of 45 VapBC proteins, VapBC proteins belong to a large family of proteins with homologues in a diverse range of bacteria. M. smegmatis is a model organism for M. tuberculosis and contains only one VapBC operon, thereby making it an ideal for system for uncovering the role of VapBC proteins in mycobacteria. VapC proteins from the thermophilic bacterium Pyrobaculum aerophilum which are homologues of those from mycobacteria and VapC from Mycobacterium smegmatis display Mg2+/Mn2+ dependent, sequence specific RNase activity against a variety of substrates. RNase activity is inhibited when the VapC is bound to VapB. Under conditions were VapC is released from the VapBC complex it cleaves mRNA and inhibits translation thereby inhibiting protein synthesis. The VapBC proteins from M. smegmatis have been shown to form a tight complex in a 1:1 VapB to VapC ratio and interactions between the proteins result in a tetramer of VapBC heterodimers that is auto-regulatory at the transcriptional level. VapB is susceptible to proteolytic degradation when not bound to DNA thus hinting at a mechanism for VapC activation.
Q59. X-linked genetic polymorphisms as predictors of survival in acute coronary syndromes.
B.R. Palmer,1 M.D Jarvis,1 A.P. Pilbrow,1 K.L. Ellis,1 C.M. Frampton,1 L Skelton,1 N. Davis,1 T.G. Yandle,1 R.N. Doughty,2 G.A. Whalley,2 C.J. Ellis, 2 R.W. Troughton,1 A.M.Richards1 and V.A. Cameron1
1Christchurch Cardioendocrine Research Group, Department of Medicine, University of Otago, Christchurch and 2Department of Medicine, Faculty of Medicine and Health Sciences, University of Auckland, Auckland, New Zealand. [email protected]
Genetic polymorphisms located on the X-chromosome are present in a single copy in males and two copies in females. Several X-linked genes have been shown to play a role in cardiovascular pathophysiology. We tested the hypothesis that the rare alleles of polymorphisms in two of these genes, ACE2 and KCNE5, were associated with adverse outcome following acute coronary syndromes (ACS) events. ACS cover the range of conditions that reflect an acute reduction in perfusion to a segment of the coronary circulation resulting in unstable angina or heart attack. Patients from the Coronary Disease Cohort Study (CDCS) were genotyped for the ACE2 A1075G (rs1978124) (n=1042) and the KCNE5 rs697829 (n=1740) SNPs. These genetic markers were tested for association with baseline measurements, echocardiographic and electrocardiographic measurements and clinical outcome, when followed for up to 7.7 years. As these genes are X-linked, analyses were initially performed separately for males and females. The A1075 allele was associated with covariate-adjusted poorer survival across both genders in the CDCS cohort (A/AA v G/GG, p=0.009). Male patients had higher covariate-adjusted mortality (p=0.047) and E/E’, an echocardiographic index of left ventricular diastolic function and filling pressure, was higher in males in the A1075 group (p=0.024). The G1075 allele was significantly more frequent in CDCS patients of Maori compared to European ancestry (p=0.035). Analysis of baseline ECG data revealed QTc interval was associated with KCNE5 rs697829 in male, but not female, patients, being extended in the G genotype group (p=0.005). Covariate-adjusted survival was poorest in G genotype patients in Cox proportional hazard modelling of mortality data of both gender groups from the CDCS cohort (poverall=0.023). Male patients with G genotype had a hazard ratio for death of 1.61 (1.11-2.33) when compared to the reference (A genotype) patient group of males (p=0.013) after adjustment for relevant covariates.
Q60. Investigation of the role of region 87-89 in human RAMPs 1 and 3 in receptor responses to CGRP and AM
Tao Qi, John Simms, David R Poyner, Debbie L Hay.
Corresponding author: [email protected]
School of Biological Sciences, University of Auckland, New Zealand (TQ, DLH);
Department of Pharmacology, University of Monash, Clayton 3800 (JS);
School of Life and Health Sciences, Aston University, Birmingham B4 7ET, U.K. (DRP)
Both calcitonin gene-related peptide (CGRP) and adrenomedullin (AM) belong to the calcitonin peptide family. Receptors for these two peptides comprise calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein (RAMP) complexes. CGRP receptors are formed when CLR associates with RAMP1, whereas AM receptors are CLR/RAMP2 or CLR/RAMP3 complexes. It is well known that RAMPs are required for receptor trafficking and ligand binding, however, how each RAMP differentially modulates receptor pharmacology is not fully understood. We exploited sequence differences between RAMPs 1 and 3 to identify residues/regions capable of altering receptor pharmacology.
Alignment of human RAMPs revealed three consecutive residues (87-89) that are conserved between RAMPs 2 and 3 but different in RAMP1, suggesting the potential importance of this region. Individual amino acid substitutions at these positions were carried out in RAMP1 by introducing the corresponding RAMP3 residues. Mutant or wild-type RAMPs were transfected into Cos 7 cells with CLR. Cell-surface expression of CLR and agonist-stimulated cAMP production were measured. None of these mutations significantly affected αCGRP or AM response. Further characterisation was performed with a RAMP1 triple mutant containing the RAMP3 residues at region 87-89. Interestingly, significant reductions in both αCGRP and AM potency were observed in the absence of an altered receptor cell-surface expression. This reduction was not seen with βCGRP. On the other hand, the reciprocal RAMP3 mutant containing the RAMP1 residues at region 87-89 did not affect any of αCGRP, AM or βCGRP response. The current work implies that region 87-89 of RAMP1 may be a contributor to αCGRP and AM potency.
Q61. Progress towards structural characterisation of Rv3262, a glutamyl ligase involved in coenzyme F420 production from Mycobacterium tuberculosis
Aisyah M. Rehan, Christopher J. Squire and Edward N. Baker
School of Biological Sciences, The University of Auckland, Auckland, New Zealand. ([email protected]) In bacteria, coenzyme F420 is present in certain members of the Actinobacteria phylum, including Mycobacterium tuberculosis (Mtb). This coenzyme has been hypothesized to be associated with Mtb viability in anaerobic conditions and in persistence [1]. Coenzyme F420 also converts NO2 to NO, which might protect Mtb from nitrosative damage [2]. We aim to explore F420 biosynthesis using biophysical techniques. At least three genes are involved in the biosynthesis of F420: Rv3261 (FbiA), Rv3262 (FbiB) and Rv1173 (FbiC). FbiB, which catalyses the synthesis of the variable polyglutamate tail of F420, is the protein most amenable to analysis amongst the three proteins. It is a multidomain protein from the F420 ligase (N-domain) and nitro-FMN-reductase (C-domain) superfamilies. FbiB is soluble as both His-tagged and GST-tagged constructs in E. coli. To further improve the protein yield and homogeneity, the protein was expressed in M. smegmatis. Thermal shift assay also showed proper protein folding of FbiB expressed in M. smegmatis. Though needle-shaped crystals are found in several robot screen conditions by the limited in-situ proteolysis method, they could not be optimized and not reproducible. Thus, efforts to purify and crystallize the N- and C-terminal domains of FbiB separately are currently being undertaken. Here we will present our recent progress in characterizing this enzyme using biophysical techniques.
1. Boshoff, H.I.M. and C.E. Barry Iii, Nature Reviews Microbiology, 2005. 3(1): p. 70-80. 2. Purwantini, E. and B. Mukhopadhyay, PNAS, 2009. 106(15): p. 6333-6338
Q62. DNA Methylation Mapping of Myelodysplastic Syndrome
Euan Rodger, Aniruddha Chatterjee, Ian M Morison
Department of Pathology, University of Otago, Dunedin
Myelodysplastic syndrome (MDS) is a chronic preleukaemic blood disorder that is highly prevalent in the
elderly. It is characterised by ineffective haematopoiesis, hyperplastic bone marrow, and an increased risk
of progression to acute myeloid leukaemia. Although MDS is common, it is difficult to diagnose and the
molecular changes that drive the disease phenotype are poorly understood. The limited success of several
demethylating drugs has implicated DNA hypermethylation as a potential mechanism in the development
and progression of MDS. Our aim is to quantify the methylation status of more than 24,000 CpG islands
(87% of all CpG islands in the human genome; 18,500 of these are associated with promoters) in 16 MDS
patients compared to normal, by using reduced representation bisulfite sequencing. Genomic DNA was
isolated from peripheral blood neutrophils and digested with MspI restriction enzyme to enrich for CpG-
rich regions. The 40-220 bp fragments (1.5% of the human genome and 6.7 Mb of sequence) were
selected to create a bisulfite-converted DNA library for massively parallel sequencing. Our reduced
representational library consists of ~3 million CpG sites and will provide us with 30-fold enrichment of
CpG islands. Illumina next generation sequencing technology will be used to obtain about 10-fold
coverage of each CpG site, measuring the percentage of methylation for each CpG island at single
nucleotide resolution. Regions showing high methylation in MDS but low methylation in normal blood
will then be assessed in a larger cohort of patients to develop a diagnostic test for this common disorder.
The identification of high frequency methylation differences between MDS compared to normal will aid
diagnosis, improve prediction of prognosis, and inform subsequent studies on the biology of MDS.
Q63. Gene expression and association in Bipolar Disorder
Catherine Sears, David Markie and Alison Fitches
The pathology of bipolar disorder is not fully understood, but shows strong evidence of a genetic component, most likely involving several genes of small effect. The mechanism of action by which mood stabilising drugs bring about their clinical effects is also unclear. One possibility is the generation of long-term neural changes mediated by differences in gene regulation.
In this study, two cellular models were used to determine the effect of drug treatment on both expression levels of BDNF, RAB7A, MDK and YWHAG. Neuroblastoma and lymphoblastoma cell lines were exposed to therapeutic concentrations of mood stabilising drugs for 28 days. Alterations in gene expression were quantitated using RT-PCR. The expression of BDNF was consistently up-regulated across all neuroblastoma cell lines with all drugs, and preliminary results suggest a predominance of up-regulation of BDNF across the lymphocyte cell lines.
Individual differences in gene sequence may also contribute to the pathology of bipolar disorder. To investigate this in our candidate genes, 21 single nucleotide polymorphisms were genotyped across the three genes BDNF, RAB7A and YWHAG. Family based association testing was used to look for association with bipolar disorder in the South Island Bipolar Study cohort. Significant association (p<0.01) was found in five BDNF SNPs and the rs13081864 SNP of RAB7A.
These results suggest that BDNF may have a role in the pathology of bipolar disorder, and perhaps a role in the mechanism of action of mood stabilising drugs.
Q64. MicroRNA regulation of Paternally Expressed Gene 10 (PEG10) Yosuke Shimaki, Michael Clark, Caillan Crowe-McAuliffe, Nathalie Saurat, Warren Tate and Elizabeth Poole Email address: [email protected] Paternally Expressed Gene 10 (PEG10) is an unusual mammalian gene that is derived from a retrotransposon. PEG10 expression into protein is restricted to embryonic tissues (amniotic membrane, embryo and especially placenta)1. By contrast, PEG10 mRNA has been detected in many tissues including brain, ovary and testis2. The presence of a 3′ long untranslated region (UTR) is suggestive of microRNA(s) (miRNA) regulation of this gene at the translational level. To validate the hypothesis experimentally, candidate miRNA(s) were first identified by bioinformatics, and two miRNAs (miR-34a and miR-182) that potentially regulate PEG10 expression were identified3. MiR-34a has one highly conserved and one poorly conserved binding site, while there is one highly conserved and three poorly conserved sites for miR-182. To test the effect of each miRNA individually and together for additive or synergetic effects on PEG10 translational repression, two truncated (Part A and B) as well as the full-length 3′ UTR were fused with a luciferase reporter gene4. The truncated versions contain one highly conserved binding site for either miR-34a (Part A) or miR-182 (Part B). The reporter assay results showed that the conserved binding site was sufficient to suppress PEG10 expression for miR-34a. All four binding sites were required for miR-182 to exert any translational suppression. Furthermore, there were no co-operative effects of these two miRNAs. This study provided the first supportive evidence for miRNA regulation, but the normalising control gene (a different luciferase) expression was somewhat affected by expressing the miRNAs. Another reporter gene, based on Green Fluorescent Protein (GFP), is being constructed for the future experiments. We wish to determine now whether miR-34a and miR-182 are physiologically relevant to PEG10 regulation in vivo, in tissues where there is no protein expression, and in a tissue like placenta where PEG10 protein is synthesised.
1. Clark, M. B. et al. (2007) Journal of Biological Chemistry, 282: 37359-37369 2. Ono, R. et al. (2001) Genomics, 73: 232-237 3. Crowe-McAuliffe, C. (2008/2009) Summer Studentship, University of Otago. 4. Saurat, N. (2009) Honours dissertation, University of Otago.
Q65. Multi-allelic knockout of CYPOR in human tumour cell lines using zinc finger nucleases
Jiechuang Su, Christopher P. Guise, Adam V. Patterson and William R. Wilson
Auckland Cancer Society Research Centre, The University of Auckland, Auckland, New Zealand.
Background: Zinc finger nucleases (ZFN) are designed to recognize a unique sequence in the genome, leading to site-specific DNA breakage and mutation at high frequency. NADPH:Cytochrome P450 oxidoreductase (CYPOR) catalyses one-electron reduction of various substrates including cytochrome P450s (CYPs) and hypoxia-activated bioreductive prodrugs including tirapazamine and PR-104. However the role of CYPOR relative to other oxidoreductases in the activation of these prodrugs is poorly understood. Aim: To characterise the role of CYPOR in the hypoxia-dependent activation of bioreductive prodrugs in human tumour cell lines. Method: CYPOR knockout cells were generated in HCT116 (CYPOR copy number 2) and SiHa (copy number 4) cells using custom-designed ZFN targeting a 33bp region of CYPOR. These cells were co-transfected with ZFN and a GFP plasmid using optimized transfection protocols for each line (Lipofectamine LTX and Amaxa Nucleofection respectively), sorted by flow cytometry, cloned by limiting dilution and screened for CYPOR expression by western blotting. Confirmation of CYPOR knockout was obtained by sequencing across the ZFN cut site. CYPOR enzyme activity was determined as cyanide-inhibitable NADPH-dependent cytochrome c reduction. Sensitivity of the knockout cells to a panel of bioreductive prodrugs under aerobic and anoxic conditions was compared to parental cells using antiproliferative (IC50) assays. Results: Frequency of CYPOR knockout clones was higher for HCT116 (3/14) than SiHa (0/46). A second ZFN transfection of two SiHa apparent partial-knockout clones gave 3/120 full CYPOR knockouts. The growth rate and morphology for these clones was the same as the parent lines. A range of mutations was found at the ZFN cut site with small insertions/deletions most common. Loss of cytochrome c reductase activity of the clones was confirmed, although residual activity indicated that this assay is not strictly specific for CYPOR. There was no effect on aerobic or anoxic IC50 values in HCT116 (not yet determined for SiHa). Conclusion: Surprisingly, bi-allelic knockout of CYPOR in HCT116 had no effect on sensitivity to any bioreductive prodrug indicating that activation (in HCT116 at least) is due to oxidoreductases other than CYPOR.
Email address: [email protected]
Q66. The DNA-binding protein Lsr2 from Mycobacterium tuberculosis
Emma Summers1, Roberto Colangeli2, Ray Cursons1 and Vickery Arcus1
1Department of Biological Sciences, The University of Waikato, Hamilton, New Zealand. Email: [email protected], 2Department of Medicine, University of Medicine and Dentistry of New Jersey, New Jersey, USA.
Lsr2 is a small, basic protein that is highly conserved in mycobacteria and related actinomycetes (1). Previous studies have shown that Lsr2 is involved in down-regulating genes involved in cell wall synthesis and metabolic functions (2). Knock-out mutants in M. smegmatis show altered colony morphology and reduced biofilm formation (3). Research by Colangeli et al (2) has established that Lsr2 represses an operon that confers multi-drug tolerance in M. tuberculosis. Further microarray studies have also shown that Lsr2 is up-regulated at high temperatures and after nutrient starvation (4).
Lsr2 is proposed to be a DNA-binding protein that likely influences the organisation of bacterial chromatin and gene regulation (5). Due to its low molecular weight and basic nature, it is suggested that Lsr2 is related to histone-like proteins (1) however it has very low amino acid sequence identity and different predicted secondary structure from histone-like proteins. Analysis by atomic force microscopy (5) reveals that Lsr2 has the ability to bridge DNA and we have shown, by electron microscopy, that Lsr2 can physically protect DNA by binding and shielding it from degradation (6). Further studies in our lab show that site directed mutagenesis of the proposed C-terminal DNA-binding domain residues reduces the DNA-binding ability of Lsr2 and it is observed that these mutant expression cultures grow more rapidly. We have observed atypical culture flocculation in expression strains and abnormal cell division when several basic residues in a predicted α-helical region of Lsr2 are mutated.
An improved understanding of the role of Lsr2 may provide insights into the way that mycobacteria adapt to stresses such as antibiotic treatment. Lsr2 is an essential gene in M. tuberculosis under lab conditions, therefore understanding it’s biological function is of central importance
(1) Gordon, B.R., et al (2008). Journal of Bacteriology 190:7052-7059. (2) Colangeli, R., et al (2007). PLoS Pathogens 3 (6): e87-1-14. (3) Arora, K., et al (2008). Journal of Bacteriology 190:4291-4300. (4) Chen, J.M., et al (2006). Journal of Bacteriology 188: 633-641. (5) Chen, J.M., et al (2008). Nucleic Acids Research 36:2123-2135. (6) Colangeli, R., et al (2009). PNAS 106:4414-4418.
Q67. Genome-wide CHiP-SEQ Analysis of Histone 3 Lysine 27 Trimethylation During Ovine Skeletal Muscle Development Highlights Chromosome Organisation and Regulation of Gene Expression
Ross L. Tellam1, Keren Byrne1, Sean McWilliam1, Tony Vuocolo1, Noelle Cockett2, Cedric Gondro3
1CSIRO Livestock Industries, 306 Carmody Rd., St Lucia, QLD 4067, Australia. 2ADVS Department, University of Utah, Logan, Utah, USA. 3Department of Animal Science, University of New England, Armidale, 2351, NSW, Australia.
Corresponding author: [email protected]
Ruminant skeletal muscle undergoes a major switch in its gene expression program and metabolism during the fetal to post-natal developmental transition. We hypothesized that epigenetic modifications of the genome underpin coordinated changes in gene expression which prepare skeletal muscle for movement, support against gravity and altered metabolism in the post-natal environment. Using Illumina GAII sequencing (65 bp reads; ~6 Gb) we performed genome-wide mapping of Histone H3 lysine 27 trimethylation modifications (H3K27me3 CHiP-SEQ) for skeletal muscle samples taken at late fetal development (100 days; n=3) and 12 weeks postpartum (n=3). The sequences were mapped onto the bovine genome and processed using CisGenome. The H3K27me3 peaks fell into two major categories, local and regional peaks, with the latter being predominant. H3K27me3 peaks were associated with genes, transcriptional start sites and CpG islands. Gene-associated H3K27me3 was negatively correlated with gene expression from autosomes. There were strong associations between genes with promoters enriched for H3K27me3 and several gene ontology terms common to both biological states. Gene-associated H3K27me3 peaks present in the lamb samples but absent in the fetal samples were associated with the TGF-β and WNT signaling pathways. Homeobox genes showed strong regional enrichments for H3K27me3 in both biological states. H3K27me3 was substantially enriched on the X chromosome of females but not males, thereby implicating this epigenetic mark in X chromosome inactivation. These analyses revealed remarkable modifications of the epigenome that comment on many aspects of chromosomal organisation and gene activity in the context of development.
Q68. Microarray Analysis of Bovine Mammary Gene Expression in Holstein-Friesian and Jersey Breeds
Ward H, Berry S, Burrett A, Ankersmit-Udy A, Verkerk G, Thomas N, Smith A, Adams L, Beattie E, Davis S, Snell R, Lehnert K.
ViaLactia Biosciences Ltd
Holstein-Friesian and Jersey are the two predominant breeds of dairy cow that are of importance to the dairy industry in New Zealand. There are marked differences in the milk composition profiles between these breeds. For example, Jersey cows produce milk with a greater concentration of beta carotene in the milk fat. To determine whether such differences could be associated with differential gene expression, a microarray study was undertaken. Mammary biopsy samples were taken from eight Holstein-Friesian and eight Jersey animals at mid (November) and late (March) time points during lactation. Samples were hybridized on microarrays in random pairings. Analysis of differential gene expression between breeds revealed specific genes involved in fatty acid synthesis, fatty acid desaturation, mammary lipid uptake, lipid droplet formation, ketone metabolism and milk fat colour. SCARB1 was found to be 2-fold over-expressed in Jersey cows and is known to be involved in beta carotene transport. Genes involved in lactose synthesis did not appear to be differentially expressed (all P>0.05). These results will facilitate an understanding of the key genes associated with the milk composition differences between the two breeds.
Q69. Improving Nitrogen Use Efficiency in Perennial Ryegrass Dave Whittaker, Sathish Puthigae, Dharani Sontam, Kerry Templeton, Craigh Jones, Catherine Bryant, Geoff Gill and Zac Hanley. Pastoral Genomics, c/o ViaLactia Biosciences, P O Box 109185, Newmarket 1149, Auckland, New Zealand Email: [email protected] Perennial ryegrass (Lolium perenne L.) is the most important pasture grass for meat, dairy and wool production in New Zealand, covering more than 7 million hectares. In New Zealand pastures ryegrass is most commonly grown together with the nitrogen-fixing legume white clover (Trifolium repens) as a mixed sward, with supplemental nitrogen applied as urea. Nitrogen is a major factor limiting pasture productivity, and nitrogen fertilizer costs have increased considerably in the past decade. Increased nitrogen use efficiency (NUE) in ryegrass would allow high pasture yields to be maintained with lower inputs of synthetic nitrogen fertilizers and could help reduce both nitrogen leaching and N2O emissions. Experiments performed at the University of Alberta have demonstrated that over-expression of a barley AlaAT (alanine aminotransferase) gene using a tissue-specific promoter can substantially increase NUE in canola and rice. This technology also appears effective in both Arabidopsis and tobacco. We are evaluating the utility of this approach in perennial ryegrass improvement. As an initial step we have transformed ryegrass with a barley AlaAT construct. Subsequent experiments will evaluate the effectiveness of ryegrass genes and promoters for increasing NUE. Ultimately we aim to generate Cisgenic® plants with improved NUE for the benefit of pasture-based farming in New Zealand, increasing our environmental and economic advantages over alternative grain-based agricultural systems.
Q70. A New Zealand-Based Virtual Institute of Statistical Genetics
PL Wilcox1, RD Ball1, E. Macrae1, G. Timmerman-Vaughan2, T. Merriman3, M. Black3, P. Fisher4, K. Dodds4, B. Auvray4, D. McCartney-Cox5, B. Barrett5, G. Gill6, N. Jones7, J. McCallum2, N. DeSilva2 and D. Chagné2
1. Scion, 49 Sala Street, Glenholme, Rotorua
2. Plant and Food Ltd 120 Mt Albert Road, Sandringham, Auckland 1025
3. University of Otago, 364 Leith Walk, Dunedin 9016
4. AgResearch Ltd, Invermay Agricultural Centre, Puddle Alley, Private Bag 50034, Mosgiel 9053
5. ESR Ltd, 34 Kenepuru Drive, Porirua, Wellington
6. ViaLactia Biosciences Ltd, P O Box 109185, Newmarket, Auckland 1031
7. University of Auckland Private Bag 92019, Auckland 1142
A collaborative NZ-based ‘Virtual Institute for Statistical Genetics’ (VISG) has recently been established and involves geneticists and statisticians from seven research organisations: four CRIs, two universities and a private company. The aim is to establish a world class capability that addresses complex statistical challenges associated with the massive databanks generated by modern genomics technologies – in an applied NZ context. Research consists of several project areas each undertaken by teams where statisticians are assisted by geneticists to develop and test new analytical methods. These areas include (1) prediction of phenotype from large data sets using Bayesian methods, and data modelling of copy number variants from multiple whole genome sequences, (2) QTL mapping of allopolyploids in a model selection framework using various methods to account for uncertainty in marker genotype, and (3) use of Bayesian methods to design association sufficiently powerful but cost-effective association genetics experiments. A fourth project will be initiated in 2011. Outputs from the VISG will include publication of the methods and accompanying software to enable end-user geneticists and statisticians to analyse genomic data. The VISG uses shared NZ-based eResearch capabilities such as the KAREN infrastructure (www.karen.net.nz) and BeSTGRID (see https://www.bestgrid.org), as well as accessing supercomputing facilities at the University of Canterbury. The project is led by Scion, but is governed by a project governance group which includes senior geneticists from various NZ organisations as well as two world-leading statistical geneticists, Profs Bruce Weir and Peter Visscher. The need for this project arose from the cross-sector MapNet network of gene mapping researchers (see https://mapnet.agresearch.co.nz/mediawiki/index.php/MapNet:About) which identified gaps in international statistical genetics research that are relevant to NZ needs.
Q71. A study of human congenital disorders of glycosylation type Ic (CDG-Ic) in yeast using a chemical genetic approach
Manivannan Yegambaram ([email protected]), David Maass, Paul Atkinson
Congenital Disorders of Glycosylation (CDG) are a group of inherited disorders characterized by abnormal glycosylation in cells affecting many cellular functions. One type of CDG (CDG-Ic) is characterized by an accumulation of dolichyl pyrophosphate-linked Man9GlcNAc2 within the cells of affected patients owing to lack of an active alpha-1, 3 glucosyltransferase. A mutation at position C998T causing an amino acid substitution (A333V) on the asparagines linked glycosylation gene (ALG6) has been shown to cause CDG-Ic. The yeast homologous enzyme, alpha-1, 3 glucosyltransferase, is encoded by the yeast ALG6 gene and an analogous accumulation occurs with this gene knockout. We constructed series of alg6 variants expressed in yeast. The effect of the mutated alpha-1, 3 glucosyltransferase was analysed by observation of glycosylation of the yeast vacuolar protein Carboxypeptidase Y (CPY) which has four N-linked oligosaccharide chains but in alg6 mutant strains, it lacks one or two of these chains readily detected on SDS-PAGE. To determine the subcellular phenotype associated with CDG-Ic we created a whole-genome green fluorescent protein tagged set of strains on the alg6 (CDG-Ic) genetic background using a synthetic genetic array mass-mating technique visualising GFP fused with 4156 yeast individual proteins utilising the high throughput confocal microscope. As a biochemical marker, growth of the CDG-Ic and alg6 null mutant strains were seen to be inhibited by low levels of tunicamycin compared to wildtype. CDG-Ic strains that presented measurable phenotypes resulting from alg6 mis-glycosylation were screened against the Library of 1280 Pharmacologically Active Compounds where each strain was examined in the presence of individual compounds for reversion to normal growth. Six compounds were identified based on significant growth difference between CDG-Ic strains with and without compounds. We are investigating if any of these compounds can cause reversion of the CDG-Ic phenotype to normal subcellular protein localization.
Q72. Annotation of Wool Keratin Genes and Their Contribution to Fibre Formation
Zhidong Yu1, Nauman Maqbool2, John McEwan3, Paul Maclean2, Rudiger Brauning2,3, Jeff Plowman4, Jolon Dyer4, Janet Wildermoth1, Allan Nixon1 and Allan Pearson4 1 Animal Bioscience Section; 2 Bioinformatics, Mathematics and Statistics Section; AgResearch Ruakura, Private Bag 3123, Hamilton 3240, New Zealand
3 Animal Improvement Section; AgResearch Invermay, Private Bag 50034, Mosgiel 9074, New Zealand 4 Bio-based Products and Textile Section, AgResearch Lincoln Private Bag 4749, Cnr Springs Road and Gerald Street, Christchurch 8140, New Zealand
Email: [email protected]
Approximately 95% of proteins in hair and wool are produced by two extensive classes of genes: keratin
intermediate filament proteins (KIF) and keratin associated proteins (KAP). Sixty KIF genes and close to
90 KAP genes have been annotated in the human genome. A striking feature is that they all reside in a
few clusters representing a small portion of the human genome. Until recently relatively few KIF and
KAP genes in sheep had been identified and even less was known on how keratin gene expression
contributes to the formation of unique characteristics in wool and hair. The sequence from the first draft
of the sheep genome had indicated poor assembly of the keratin-rich regions. In order to annotate
unknown wool keratin genes and facilitate comparative studies, we sequenced six Bacterial Artificial
Chromosomes (BACs) containing, on average, 200 kb sheep genomic DNA using Titanium 454 paired-
end technology. This produced an average of 113,398 sequence reads with a median length of 392 bases
across all BACs sequenced, resulting in ~166X mean coverage. Comparisons with the current sheep
genome assembly and with the bovine UMD3 genome assembly indicated reliable assemblies of all the
BACs. Subsequent filling of some small gaps has allowed annotation of 27 KIF genes and 28 KAP genes,
including species-specific genes and others not previously identified. In situ hybridisation of these and
known genes revealed their patterns of expression in the wool follicles. Their sites of expression in the
presumptive structures in wool and human hair and the difference between the two species have provided
new insights into fibre attribute formation. These were further supported by correlations between the
quantitative expression of certain KIF and KAP genes and specific wool attributes.