ACPFGvector Issue 9 2009 The magazine of the Australian Centre for Plant Functional Genomics What is this? ANSWER ON Page 2 ACPFG VECTOR The DNA puzzle it’s like working on a puzzle without a picture on the box. ACPFG doctoral student Mike Imelfort recently won a University of Queensland competition, in which students have three minutes to explain their thesis to a team of judges. Mike won the Three Minute Thesis competition by explaining his topic, The Development and Application of Bioinfomatics and Statistical Tools for the Assembly and Analysis of Plant Genome Sequence Data, without notes and with just one image, featured on the cover of this magazine. Inside 2 By Mike Imelfort I am primarily a mathematician and a programmer. I’ve always loved solving puzzles and the incredible amount of puzzle solving currently going on in bioinformatics is what has drawn me to this area. I began university studying electronic engineering but soon discovered that I was not an engineer. I transferred ‘temporarily’ to mathematics to buy time while deciding what to do next and I never really left. My work now is focused on assembling plant genome data from next generation sequencing (NGS) technology. This technology promises to revolutionise genomic sequencing, by producing large amounts of sequence data at very low costs. Older sequencing methods produce accurate sequence fragments that are long – between 700 and 1000 base pairs (bp). NGS technology produces many millions of very short (25–400bp), but error-prone sequence fragments called reads. I work with technology that typically produces ultra-short reads of up to 75bp. This type of technology has proved successful in resequencing projects, where the short reads are mapped to an already sequenced genome from the same or similar species. It would be more useful to Australian metabolomics services expanding Harvard researcher comes home Unravelling protein structures through molecular modelling States visit for stem strength knowledge Summer scholar numbers expand Scholarship leads to work Not just a lab lackey Congratulations Some recent publications New faces 4 6 8 10 11 12 14 16 17 18 ISSUE 9 – 2009 From left: UQ’s Pro-Vice-Chancellor of Research and Research Training, Professor Alan Lawson, second place winner Ashley Wilkinson, winner Michael Imelfort, and ABC science journalist Robyn Williams. Photo by Jeremy Patten. be able to assemble reads from genomes that haven’t been sequenced yet, but this is no easy task. A jigsaw puzzle is a fitting metaphor for this type of sequence assembly; many of the problems we face can be understood in terms of their puzzle counterparts. First the length of the individual reads is tiny in comparison to the length of the target DNA being sequenced. This leads to problems when resolving repetitive regions in the target DNA. Repetitive regions are like areas of the puzzle which contain exactly the same pieces; imagine trying to solve a puzzle with multiple plausible solutions! Paired end sequencing has been introduced to combat this problem. We can produce pairs of reads that we know are a given distance apart on the target genome. This is like being able to figure out if one piece of a puzzle is positioned correctly based on the relative positions of some other pieces. The next biggest problem is the abundance of errors in the reads. Each read is like one piece in a jigsaw puzzle. Reads with errors are like puzzle pieces which look like they might fit, but just don’t belong in the box. To guarantee there will be at least one correct copy of every puzzle piece in the box, NGS technologies produce a lot of extra reads, so we have to exclude the false ones as well as putting the right ones together. When we’re working on an unsequenced genome, it’s like working on a puzzle without a picture on the box. We have no ready means to separate the correct pieces of the puzzle from the extra ones! There are many other aspects of this puzzle which contribute to its difficulty. For example, each strand of DNA has a reverse compliment, this makes our puzzle double sided. Also, DNA consists of just four distinct letters, which makes a highly repetitive puzzle in which all the pieces look nearly identical. Finally, to reduce the overall cost we are pooling the sequencing of several bacterial artificial chromosomes. This is like mixing the pieces of some very similar puzzles together in the same box. We are currently developing software which can automatically solve these puzzles using a lot of maths, many pages of computer code, one large computer, far too much coffee and a pinch of luck. So next time you’re stuck on that 1000 piece puzzle and you feel like throwing in the towel, spare a thought for us. 3 ACPFG VECTOR Tony Bacic (right) and Ute Roessner explaining metabolomics to Gavin Jennings (centre). Australian metabolomics services expanding 4 ISSUE 9 – 2009 Ute Roessner at the launch. ACPFG researchers are leading Victoria’s node of Metabolomics Australia, launched in November. Ute Roessner and Tony Bacic at the University of Melbourne’s School of Botany were joined by Victorian Minister for the Environment, Climate Change and Innovation, Gavin Jennings, for the facility’s launch. “The Victorian node of MA fits with the core goals of our innovation statement – health, sustainability and productivity – and it is a great addition to the State’s solid biotechnology research infrastructure,” the Minister said. Metabolomics is the study of metabolites – the products of cellular metabolism. Metabolic reactions include the production of energy and the manufacture and breakdown of molecules, and are so specific that they can be used as indicators of the condition of the cell, or ‘biomarkers’. Biomarkers can indicate disease abnormalities such as cancer and determine responses to drugs or environmental effects. In plants, metabolomics can be used to monitor food quality and to discover molecules for crop varieties or biofuels. The launch of the Victorian node of MA has been six years in planning and is particularly significant for Ute Roessner, who moved to Australia from Germany in 2003 to establish a metabolomics platform for ACPFG. She now splits her time between the two organisations. “Personally it’s expanding my horizons, because I have a lot to do with other projects completely outside of plants, which is exciting. Research areas like human disease metabolomics are more advanced than plants in data analysis techniques, so it’s useful for ACPFG because I can employ the techniques I learn on other projects here,” she said. Tony and Ute lead the facility together with Malcolm McConville and Vladimir Likic from the Bio21 Molecular Science and Biotechnology Institute. “Research areas like human disease metabolomics are more advanced than plants in data analysis techniques, so it’s useful for ACPFG because I can employ the techniques I learn on other projects here .” “With the help of powerful computing and improved software it will be possible to map metabolites onto known metabolic pathways and also identify novel pathways and networks of responses that will lead to the identification of the function of proteins and genes as well as the discovery of new biomarkers,” Tony said. ACPFG CEO Peter Langridge is confident the development of MA will enhance ACPFG’s research. “Part of our brief was to build capability in Australia in plant genomics, including associated advances in transcriptomics, proteomics and metabolomics, so we are pleased to be closely associated with this new facility,” he said. The Victorian facility forms the hub of MA, with other nodes at the University of Queensland, the Australian Wine Research Institute based in Adelaide, and in Western Australia, both at the University of Western Australia and Murdoch University. The Victorian node is supported by $5.3 million of National Collaborative Research Infrastructure Strategy (NCRIS) funds and $2.65 million of Victorian Government funds to form the hub of MA. The University of Melbourne is contributing $1.65 million to the venture. An additional $2 million in NCRIS funding via the Australian Bioinformatics Facility of Bioplatforms Australia Ltd will support MA’s bioinformatics capability to manage the vast amounts of data being generated. For more information on MA services, go to www.metabolomics.net.au 5 ACPFG VECTOR Harvard researcher comes home Associate Professor Desmond Lun has returned to Australia from Boston to work for ACPFG and UniSA and direct the new Phenomics and Bioinformatics Research Centre. How did you end up working in bioinformatics? Can you explain for potential students in your lab what there is to be passionate about? It wasn’t exactly planned. In graduate school at the Massachusetts Institute of Technology (MIT), I took a few classes in computational biology – outside of my field at the time, information theory – just out of interest. I liked them. The applications were exciting and had the potential for significant impact. Fortunately Muriel Medard, my doctoral adviser, was very open-minded (which might contribute to some degree to her brilliance) and, in my final year, Muriel and I started up a collaborative project on DNA sequencing with the MIT/ Harvard Broad Institute. When graduation came, I was unsure what I wanted to do. I thought about something related to biology, but it seemed like a leap. Muriel, whose own research career spanned several areas, encouraged me that working on something completely different after graduation was not necessarily a bad thing, so I applied for a job as a computational biologist at the Broad, gave them a talk about communications networks, and, when they surprisingly made me an offer, I took it. Engineering life is a grand challenge for humanity. It is funny to think that we, living organisms ourselves, are in position to design and create other living organisms, but it’s true. And the technology that’s making this possible is becoming available as we speak. We have measurement technology that’s allowing us to understand the intricate workings of living organisms at unprecedented levels, and we have fabrication technology that’s allowing us to modify existing organisms at unprecedented levels – and even to create fundamentally new organisms. There’s tremendous potential to use all this technology to tackle fundamental problems facing humanity. I’m interested in training students who want to do this. 6 “It is funny to think that we, living organisms ourselves, are in position to design and create other living organisms, but it’s true. And the technology that’s making this possible is becoming available as we speak.” ISSUE 9 – 2009 Do you feel it makes much difference having agricultural aims from your work compared to what you’ve done in the past? Plants, of course, are more complicated than microbes, which have been the focus of my work so far. But the core aim of the ACPFG – of improving stress tolerance in cereals – is an immensely important one, in general and for Australia in particular. Ultimately, it’s the goal of the work I feel is most important, so I don’t feel the plant/microbe distinction makes that much difference. With the arrival of Desmond Lun, the PBRC is now actively engaged in enrolling students and recruiting staff for its activities, and is having an exciting year of growth in 2009. The year began with a kickoff meeting held on 22nd and 23rd January What do you think about Adelaide so far, compared to other places you’ve lived? at the University of Adelaide Waite Campus. The I’m originally a Melbournian, so I was always encouraged to think disparagingly of Adelaide. But now that I’m here, I rather like it. It’s a change from Boston (not least because I left Boston just as it was beginning to get seriously cold) and I’m still adjusting, but I could see myself being very happy in Adelaide. meeting was a thorough success, with 18 talks from What’s it like balancing being out at UniSA’s Mawson Lakes Campus, but spending time at the University of Adelaide’s Waite Campus and being part of ACPFG nationally? resulting in engaging discussion that is sure to keep I’m not a fan of commuting. But what I do like is that each location brings with it different expertise and a different culture. And I find it stimulating to switch back and forth. In my previous position, I split my time between the Broad Institute and Harvard Medical School, and I appreciated being able to choose which location I’d go to on a given day based, at least partially, on my mood. speakers based at UniSA and from ACPFG nodes all around Australia. There was lively exchange among mathematicians, bioinformaticians, and biologists, the PBRC well-equipped with ideas for 2009. Pictured above are researchers at the meeting, from left: Dave Edwards (University of Queensland), Bettina Berger, Mark Tester and Karthika Rajendran (University of Adelaide), Ute Roessner (University of Melbourne), What would you like to see happening in your lab in two years time? Rachel Burton (University of Adelaide) and Desmond I’d like to have a successful lab, which to me primarily means good science and good character. I’d like to have a happy lab, where people enjoy working and enjoy being, and where there is a strong sense of purpose. I’ve trained in some very happy labs, and that’s what I’d like to create. If I can achieve that, I’m less concerned about whether the lab has five people or 50. Lun (University of South Australia). 7 ACPFG VECTOR Unravelling protein structures through molecular modelling By Maria Hrmova Given we’re a centre for plant genomics it’s evident we work predominantly on understanding plants’ genes. But plant proteins are in the focus of the centre as well. Between the genetic blueprint and the resulting plant are the proteins that mediate catalysis or transport, which are the focus of my research. Understanding the structure of a protein allows us to learn the mechanisms of how it functions and fulfils its biological role. Proteins are built from sequences of amino acid residues, ordered by the corresponding genetic code. The linked amino acid residues bond in space to form a three-dimensional (3D) structure. Knowledge of the 3D structure allows us to understand how proteins work and what molecular mechanisms underpin their functions. Protein structure can be determined experimentally using X-ray crystallography, nuclear magnetic resonance spectroscopy and cryoelectron microscopy, but this is time-consuming. Predictive computer molecular modelling is a useful alternative, which has kept biochemists, computational chemists, physicists, mathematicians and computer scientists challenged for decades. Protein structures are guided by two sets of principles operating on vastly different time scales. The first set of principles is defined by the laws of physics, while the second set is directed by the theory of evolution. Each of these two sets of principles has led to the development of predictive methods for building 3D protein models. A rule of physics is that every system seeks to achieve a minimum of free energy. De novo or ab initio modelling techniques based on this rule assume that the 3D protein structure corresponds to the minimum free energy accessible during a lifespan of a protein. These techniques evaluate and assess many conceivable protein conformations based on the minimum free energy. At the moment this type of modelling is only reliable for proteins up to around 100 amino acid residues long, so much work remains to be done to refine these techniques. Computer modelling based on evolutionary principles relies on having a known protein structure with detectable sequence similarity to the protein we’re investigating. Once the structure of one protein in a family has been determined experimentally, the other related members of the family can also be modelled. Structural information for a particular class of homologous proteins can be seen as a valuable currency, with which the success of structural genomics is measured. This currency has enormous significance in protein molecular modelling, where programs are becoming more sophisticated and are likely to continue to improve, as they ‘educate themselves’ from new experimental protein structures deposited in the structural databases. How does comparative protein modelling actually work? Comparative modelling was first based on ‘fragment assembly’, which involved the construction of a complete model from conserved structural fragments. Later a ‘segment matching’ technique was introduced, where the target protein was subdivided into a series of short segments, each of which was matched to its own template fitted from the experimental 3D 8 The model of a barley boric acid transporter predicts how boric acid is excluded through its pore. The model of a barley UDP-glucose 4-epimerase suggests how substrates bind in the active site region. The model of a barley expansin forecasts its 3D conformation. ISSUE 9 – 2009 The conformation is the overall form and structure of a system, determined by the arrangement of its parts. Homologous proteins, genes or chromosomes, are those which have similar structural characteristics due to their shared ancestry. The secondary structure of a protein is the way the linked amino acid residues fold. The Protein Data Bank (PDB) is an internet repository for 3D structural data of large biological molecules, including proteins and nucleic acids, which is freely available to the public. The model of a barley xyloglucan xyloglucosyl transferase foresees how substrates bind in the catalytic cleft. The model of a barley multi-functional aquaporin predicts how water, silicic acid and glycerol are transported. The model of a wheat lipid-binding protein suggests its biological function and how lipoid molecules bind in the protein’s central cavity. structures deposited in Protein Data Bank. Currently, one of the most popular comparative modelling programs is MODELLER, which uses empirical spatial restraints and statistical analysis of the relationships between pairs of homologous structures. It is free for academics and was written by Andrej Sali and Thomas Blundell in Cambridge, UK. MODELLER works through the alignment of the target protein sequence with the template sequence of a known 3D structure. The alignment, together with the coordinates of the known protein structure, is ‘embedded’ in a computer script that instructs the computer constructing the models. The array of typically 20–1000 models produced by MODELLER can afterwards be evaluated through a jury of another set of computer programs that assess spatial quality and energy profiles of protein models. The jury deliberates and chooses the top protein models, which usually need to be optimised or refined to tune in loop and side chain conformations. The prerequisite for any homology modelling is generation of a reliable sequence alignment. Computer modelling programs use information about the amino acid type, distribution of secondary structures, positions of gaps and respective distances and solvent accessibility to produce the final alignment that is used for modelling. But errors can occur with automation so a human eye (and brain!) can help edit mistakes. The next stage where models typically go awry is during the building of loops. Loops are part of a protein’s secondary structure, connecting sheets and helices at the protein backbone. To make sure that loop building – but also overall model building and optimisation – proceed optimally, a sampling strategy and knowledge of energy function is used to guide the search through the conformational space, to avoid near-correctly folded decoy leads. After the optimised and verified model is calculated, it needs to be put into perspective with a biological function and tested to see if it is helpful in proposing and testing hypotheses in biology. For example, in our centre, the boric acid tolerance gene Bot1 was recently identified in a tolerant barley landrace Sahara. It was important to predict the 3D conformation of this α-helical membrane protein that consists of approximately 670 amino acid residues. With the help of molecular modelling, its 3D structural model was constructed (pictured, top left). It was predicted that the protein spans the membrane between 10 to 12 times and that the toxic boric acid could be excluded through a central pore that is formed by the protein’s helical bundle. The collection of cereal models pictured illustrates some of the examples where molecular modelling has helped predicting architectures of proteins and their biological functions. As we benefit from high-throughput data processing and automation in bioinformatics and molecular modelling, we are able to tackle larger and more complex biological systems. We are able to start studying the dynamics of proteins in conjunction within their environment and explaining and describing protein evolution in time and space. For more information see the publication: Hrmova M, Fincher GB (2009) Functional genomics and structural biology in the definition of gene function. In: Plant Genomics, Humana Press Inc, Totowa, NJ, USA (Somers D, Langridge P, Gustafson P, Eds). Methods in Molecular Biology 513, 175–198. 9 ACPFG VECTOR States visit for stem strength knowledge In Minneapolis, I discussed a collaboration between ACPFG’s Stem Strength Project and the University of Minnesota, with special reference to the protocols for harvesting barley stems, with strength testing on the Instron in mind. This has led to the arrival of approximately 1,000 of our barley samples at Pioneer in Des Moines, Iowa, where it is hoped I will return in the near future to conduct the analysis using Pioneer’s Instron instrument. Finally, it was on to Boston where I attended an Instron training course on material testing and software operation. My classmates at Instron included a composites engineer from Boeing; an aerospace engineer from the Florida Space Station and several mining engineers from Argentina! Imagine their smiles when we all had to introduce ourselves and describe our application of Instron technology. They all thought I was joking when I said “agricultural plant stems”, but I now have reinforced my understanding and appreciation of the capabilities of the software system and it’s manipulation in testing barley and maize stems, leaves and rind strength. By Jillian Taylor I have been working on stem strength in maize and barley at ACPFG as part of a collaboration with DuPont business Pioneer Hi-Bred for two years now. While the knowledge base for barley is very significant here at the University of Adelaide’s Waite Campus, the growth and maintenance of healthy maize lines was becoming a real issue. We couldn’t be sure if the phenotypic variations we saw across Mutator (Mu) insertion lines were actually related to the particular Mu insertion or a factor of inbreeding depression, nutritional deficiencies or poor insect pest management practices. At one of our early morning teleconferences with our collaborators at Pioneer, we were invited to visit them in Des Moines, Iowa, USA. So last June, I headed to Des Moines to visit our Pioneer collaborators, as well as to the University of Minnesota to visit Professor Brian Steffensen, followed by a trip to Boston, Massachusetts for a three day course at Instron headquarters. We use Instron software and hardware for our stem strength analysis. My hosts for the Pioneer visit were Ms Lynne Fallis and Dr Kanwarpal Dhugga, and I met several other people who were only too happy to share with me their enthusiasm and technical advice for maize plant growth and propagation. We are now applying that advice to the maize plant work we are doing for the Pioneer collaboration project here at ACPFG. 10 The University of Minnesota, picture by Jillian Taylor ISSUE 9 – 2009 Pictured: Monia Ogierman (centre) with the 08/09 summer scholars during the information and nibbles session. Summer scholar numbers expand We received so many outstanding applicants for the 08/09 summer scholar program that we increased the number of scholars from the usual six to 10. This year scholars were from either Flinders University or the University of Adelaide, with majors in biotechnology, agricultural science or molecular biology. Once the students were assigned to their supervisors, they were paired with a ‘buddy’ – a person from outside their laboratory who acted as a friend and mentor. Mid-way through the program, an information and nibbles session was held for the scholars, their supervisors and ‘buddies’. The supervisors gave informal presentations, enabling scholars to see the breadth of ACPFG research, and hopefully encourage students to consider us for further studies. 11 ACPFG VECTOR Luke Holtham, right, with supervisor Charlotte Jorgensen Scholarship leads to work 12 ISSUE 9 – 2009 By Luke Holtham I decided to apply for a summer scholarship at the ACPFG when I met Education Manager Monica Ogierman at an AusBiotech Students Association meeting early in 2008. I told her what I was studying and that I was looking for somewhere to gain some practical experience and she told me that I was a perfect candidate for the summer scholarship programme at ACPFG. My family own a cereal cropping farm, so I have a strong interest in agricultural biotechnology and the focus that ACPFG has on Australian cereal crops is very relevant. As a result ACPFG sounded exactly what I was looking for so I wrote my application and applied on the first day the applications opened. I am currently in my fourth year of an honours degree in biotechnology at Flinders University. I first gained interest in biotechnology when I did an introductory elective topic in year 11 at high school. Then when I finished year 12 and decided to do tertiary study, I was looking at my options in the booklets and saw biotechnology, and straight away put that as my first preference. I’ve always had an interest in science and biology always seemed to simply make sense to me. When I began my summer scholarship at ACPFG I didn’t know what to expect. Immediately I was struck by the friendly nature of all staff and the real community spirit that echoed through the entire company. All of the staff were completely approachable at any time and were more than happy to answer questions and help out wherever possible. There was such an immense diversity of staff that specialise in all different areas of research under one roof, which meant that any question you had could be solved or anything you needed could be “Overall I think I truly learnt what it is to be a research scientist and realised that in science, things don’t always work according to plan and that’s why we are scientists, we have to work out what’s going on.” arranged. Although everyone was working in separate teams on different projects, it really felt as if everyone was working together towards the same goal. Working at ACPFG exceeded all my expectations, the amount of responsibility that I was given was both challenging and rewarding and as a result I gained so much from the experience. On my first day I was expecting doctors all suited up scrutinising everything I was doing, but the stereotypes of a scientist do not apply at ACPFG. Immediately I noticed the relaxed, casual nature of all staff and realised that I was far overdressed on my first day. The scariest part about doing a summer scholarship was simply applying. Following that was worrying about the expectations the supervisors may have, and that after three years of study I would now have to put into practice all the skills I had learnt. To my own surprise I was a lot more qualified than I thought and was able to be an effective member of the team. The best thing was that I was able to put into practice all the theory I had learnt at university from lab techniques and background knowledge to statistical analysis of data, as well as strengthening all the practical skills I already had. Also the contacts that I made when I was there have now led to me possibly doing my honours and PhD at ACPFG. The things I learnt from my experience are endless. I did extensive work in PCR genotyping plants, which gave me a real insight to troubleshooting the process, which you don’t get from university practicals. Also I was also involved in phenotyping plants and using statistical analysis techniques I had learnt at university to correlate data. I also did DNA and RNA extractions using a variety of methods including researching and developing the most suitable methods for our plant varieties. Overall I think I truly learnt what it is to be a research scientist and realised that in science, things don’t always work according to plan and that’s why we are scientists, we have to work out what’s going on. From here I will now be returning to ACPFG for a part time position in the nitrogen use efficiency team until I finish my undergraduate degree and am also considering doing my honours there. My advice to people who are thinking of applying is to simply just do it. It will be one of the most beneficial experiences of your degree, the lab skills you will learn are priceless, and the contacts and friends you will make may last a lifetime. It is the first step in beginning your career as a research scientist. 13 ACPFG VECTOR Not just a lab lackey by Nic Reid My summer scholarship at ACPFG was supervised by Sergiy Lopato and Tatiana Pyvovarenko. Their research identifies stressinducible promoters in grains such as wheat and barley. With Tatiana, my main objective was to use the yeast one hybrid system to identify new promoter genes. Doing plant transformation practicals in third year really stimulated my interest in agricultural biotechnology. I later attended an Ausbiotech careers night where I spoke to a representative from ACPFG, who told me about what ACPFG researches. I was particularly interested in the research on drought tolerant crops. Initially I was studying a Bachelor of Forensic and Analytical Chemistry at Flinders University; however after some time I decided that it was not what I had expected or enjoyed, so was looking for other alternatives within the science field. During my time in forensics I noticed my particular interest in biology and more specifically, DNA. At the time, a friend was studying biotechnology and explained the kinds of things taught within the degree. She gave me an idea of the fields it can lead to and the breadth of the industry. The variety of areas covered within the degree really appealed to me and I eventually transferred. I particularly found biotechnology so interesting because I am able to relate the topics with the world around me. I didn’t really have any expectations when I began my scholarship; however I was pleasantly surprised by how much I enjoyed it. I loved the relaxed atmosphere, friendly staff and getting involved in the research. I thought that due to my lack of experience I would be a lackey in the lab, but to my surprise my supervisors made sure I was directly involved with the research at most levels. It was great to see scientists with so many different backgrounds working so well together. None of the staff at ACPFG were arrogant or self absorbed which was one of my concerns about academics. In fact, all the people I met were 14 the complete opposite, and were very willing to assist or pass on their knowledge to students like myself. At first I found it scary travelling to a new place, somewhere I had no idea about where I was meeting new people that were experts in the biotechnology field. I was concerned that my scientific knowledge, or lack of it, would hinder me or make it harder to integrate with the people at ACPFG. These concerns quickly went away however upon starting my placement. The large number of students that work in ACPFG and summer scholars that started with me made it much easier to feel like I fit in. For me the best thing about doing my summer scholarship at ACPFG was meeting scientists outside of the undergraduate environment and working alongside them in their research. I received invaluable advice about possible career directions and learnt many skills that are not taught in undergraduate courses. During my time I was exposed to many new skills and techniques including yeast transformations, electroporation, yeast mating, harvesting, seed threshing, northern blotting, plant transformation, laboratory troubleshooting processes, general laboratory practices and RNA extractions. It was great to experience all these skills and techniques in an actual research environment rather than in a controlled classroom. This year I will be doing honours at Flinders University within the agricultural biotechnology field. Once completed, I hope to follow up with a PhD, and if all goes well plan to study at ACPFG. With a PhD I believe there will be many paths I can travel to further my career in biotechnology. If anyone else might be considering undertaking a summer scholarship at ACPFG, all I can say is to go for it! You have nothing to lose and so much to learn. You can still have time to enjoy your summer and you will return to study feeling confident and absolutely prepared. It is such a great learning experience and prepares you for further study in ways no university topic can. ISSUE 9 – 2009 Tatiana demonstrating clone selection to Nic in a laminar flowhood in the Plant Genomics Centre. “I thought that due to my lack of experience I would be a lackey in the lab, but to my surprise my supervisors made sure I was directly involved with the research at most levels.” From right, Nic Reid with supervisors Sergiy Lopato and Tatiana Pyvovarenko 15 ACPFG VECTOR Congratulations... To Bradleigh Hocking for being the inaugural winner of the Australian Centre for Plant Functional Genomics Prize in 2008. The prize of $1000 is awarded to the graduating student considered most outstanding by the Program Management Committee for the degree of the Masters of Biotechnology (Plant Biotechnology) at the University of Adelaide. To James Edwards (pictured above) who won first prize for his poster at the University of Adelaide's School of Agriculture, Food and Wine Research Day on the 5th of November. 16 To Konny Oldach, John Harris, Nicky Featherstone and Rohan Singh, (pictured above) who formed Team Transformania to take the gold at the ACPFG Olympic Christmas party. Picture by Ming Li. ISSUE 9 – 2009 Some recent publications Abstract Edwards D and Batley J (2008), Bioinformatics: Fundamentals and Applications in Plant Genetics, Mapping and Breeding. Principles and Practices of Plant Genomics. Eds. Kole C and Abbott AG. Science Publishers Inc, (USA), pp269–302. To identify integral and peripheral plasma membrane (PM) proteins from Oryza sativa (rice), highly enriched PM fractions from rice suspension cultured cells were analyzed using two complementary approaches. The PM was enriched using aqueous two-phase partitioning and high pH carbonate washing to remove soluble, contaminating proteins and characterized using enzymatic and immunological analyses. Proteins from the carbonate-washed PM (WPM) were analyzed by either one-dimensional gel electrophoresis (1D-SDS-PAGE) followed by tryptic proteolysis or proteolysis followed by strong cation exchange liquid chromatography (LC) with subsequent analysis of the tryptic peptides by LC-MS/MS (termed Gel-LC-MS/MS and 2D-LC-MS/MS, respectively). Combining the results of these two approaches, 438 proteins were identified on the basis of two or more matching peptides, and a further 367 proteins were identified on the basis of single peptide matches after data analysis with two independent search algorithms. Of these 805 proteins, 350 were predicted to be PM or PM-associated proteins. Four hundred and twenty-five proteins (53%) were predicted to be integrally associated with a membrane, via either one or many (up to 16) transmembrane domains, a GPI-anchor, or membrane-spanning β-barrels. Approximately 80% of the 805 identified proteins were assigned a predicted function, based on similarity to proteins of known function or the presence of functional domains. Proteins involved in PM-related activities such as signalling (21% of the 805 proteins), transporters and ATPases (14%), and cellular trafficking (8%), such as via vesicles involved in endo- and exocytosis, were identified. Proteins that are involved in cell wall biosynthesis were also identified (5%) and included three cellulose synthase (CESA) proteins, a cellulose synthase-like D (CSLD) protein, cellulases, and several callose synthases. Approximately 20% of the proteins identified in this study remained functionally unclassified despite being predicted to be membrane proteins. Hrmova M, Farkas V, Harvey AJ, Lahnstein J, Wischmann B, Kaewthai N, Ezcurra I, Teeri TT and Fincher GB (2009), Substrate Specificity and Catalytic Mechanism of a Xyloglucan Xyloglucosyl Transferase HvXET6 from Barley (Hordeum vulgare L.), Federation of European Biochemical Societies Journal, 2.76, pp437-456. John UP, Polotnianka RM, Sivakumaran KA, Chew O, Mackin L, Kuiper MJ, Talbot JP, Nugent GD, Mautord J, Schrauf GE and Spangenberg GC (2009), Ice recrystallization inhibition proteins (IRIPs) and freeze tolerance in the cryophilic Antarctic hair grass Deschampsia antarctica E. Desv. Plant, Cell and Environment, 32, pp336–348 doi: 10.1111/j.13653040.2009.01925.x Lewis D, Bacic A, Chandler PM and Newbigin EJ. (2009) Aberrant cell expansion in the elongation mutants of barley. Plant and Cell Physiology, 50, pp554-571. Natera S, Ford K, Cassin A, Patterson J, Newbigin E and Bacic A (2008), Analysis of the Oryza sativa plasma membrane proteome using combined protein and peptide fractionation approaches in conjunction with mass spectrometry. Journal of Proteome Research, 7, pp1159–1187. Vandeleur, RK, Mayo, G, Shelden, MC, Gilliham, M, Kaiser, BN, and SD Tyerman (2008), The role of PIP aquaporins in water transport through roots: diurnal and drought stress responses reveal different strategies between isohydric and anisohydric cultivars of grapevine. Plant Physiology Preview, 10.1104, pp.108.128645. 17 ACPFG VECTOR New faces AT ACPFG Diyana Azlan Helli Meinecke Mamoru Okamoto I graduated from Australian National University (ANU) with a Bachelor of Biotechnology. I’ve always been interested in molecular biology and during my undergraduate years I became very interested in plant genomics, particularly in development studies. Depleting world conditions are putting heavy stress on plants, and with society’s huge dependence on crops, I feel that biotechnology can really help to develop plants better suited to current environmental conditions and thus enhance crop yield. Before I came to ACPFG for honours, I did a summer research project at ANU for three months with the Genomic Interactions group, in the Research School of Biological Sciences. My summer project involved studies of the secretome (proteins exported from cells) important in plant root and nodule formation, using the model plant Medicogo truncatula. I felt so comfortable doing research that I decided to embark on honours at the University of Adelaide. I learnt a lot during my three years in ANU and now I’m looking forward to a new start at a new place surrounded by new people and I’m glad that I’ve been accepted into ACPFG. People here are nice and my supervisor Delphine Fleury makes me feel welcome. I will start on a project that involves mapping quantitative trait loci in the genome of wheat chromosome 7A, and hopefully identifying several important genes in wheat residing in that chromosome. I’m now part of ACPFG and I hope to achieve the best out of this project and gain as much experience as I can. Who knows I may have the chance to stay for more than a year here! I grew up in Germany’s port city of Bremen where I worked in the shipping and transport industry. In May 1993, our young family migrated to Australia, settling in beautiful Adelaide. I joined the University of Adelaide’s Supply Unit at Waite Campus in 1996, and was invited to join a university project group on North Terrace Campus in 1999, which had been established to implement the university’s finance software ‘PeopleSoft’. I spent the last eight years working as the Business Manager for both the ARC Special Research Centre for the Molecular Genetics of Development and the ARC/NHMRC Research Network in Genes and Environment in Development, within the School of Molecular and Biomedical Science. In September 2008, I joined the School of Agriculture, Food and Wine, as Business Manager for The Plant Accelerator, which is expected to commence operation in November next year. Whilst the building is under construction, I will be located in Mark Tester’s office at ACPFG. I really enjoy being back at the Waite and look forward to working on this exciting project. Although I grew up in the city of Tokyo surrounded by concrete, I was interested in agriculture. After completing a Master of Science at Tokyo University of Agriculture and Technology in Japan, I moved to Vancouver, Canada, to join Anthony Glass’s lab at the University of British Columbia for my PhD. My research there involved characterisation of nitrate transporters at the molecular and physiological levels, using Arabidopsis as a model. I then moved down the west coast to San Diego, California for my postdoctoral training with Nigel Crawford at UC San Diego. In the Crawford lab I was a part of projects such as gene discovery of nitrate regulatory pathways, nitric oxide synthesis in plants, and characterizations of the related mutants. Thanks to the University of Adelaide’s Brent Kaiser, who is my long time friend, I was introduced to a job opportunity at ACPFG. Now as a Research Fellow in the nitrogen use efficiency (NUE) group, I am thrilled to be in this challenging project. 18 ISSUE 9 – 2009 Monique Shearer John Toubia Jenny Washington Last year I completed a Bachelor of Biotechnology (honours) at Flinders University. A summer studentship at CSIRO Plant Industry in Canberra first established my interest in plant biotechnology. I then went on to complete my Honours year under the supervision of Chris Franco, looking at a proteomic approach to characterise the interaction between endophytic actinobacteria and Arabidopsis. This year I have started a PhD at ACPFG, working with Mark Tester and Darren Plett to characterise a group of Arabidopsis genes, which are potentially responsible for the initial entry of salt into plant roots. This project has plenty of potential and I am excited to be involved. My recent completion of a Bachelor of Science Degree with an Extended Major in Bioinformatics from Flinders University in South Australia, has given me the privilege to join the Adelaide bioinformatics team at ACPFG. As the new Bioinformatics Help Desk Officer, I am excited about applying, extending and further developing my practical and theoretical skills in this internationally renowned environment. Before my tertiary studies I spent more than five years as a self-employed small business operator in the hospitality industry. After a very long period of early mornings (2am starts) and late nights, I commenced tertiary study; the first step in fulfilling the lifelong dream of becoming a specialist in my chosen field. In 2007, I completed a 10 week summer scholarship at The South Australian Partnership for Advanced Computing (now known as eResearch SA). My project involved creating a web-based interface for a commonly used phylogenetic program, integrating existing programs and those I produced. In 2008, I completed a mini honours project at Flinders University under Leigh Burgoyne and Mike Schwartz on ‘The Darwinistic selection forces operating in amplifying trace DNA’. The core aim of this project was to understand the molecular realities of working with anonymous sequences at the limit of detection as in palaeontological or archaeological material or trace DNA in explosives or drugs (the latter being of most concern). I thrive on challenges, enjoy bouncing ideas around with my peers and have the self-motivation and drive to nut out complex problems. I am looking forward to commencing honours later this year. My interests outside of work include sports of any kind (especially cycling and tennis), open water diving, reading, and most of all, spending time with my family. Since completing a science degree at the University of Adelaide I have worked in marsupial and rodent reproduction and genetics, cancer and embryonic stem cell research and food research. In 1998 I began work with the barley breeding group at the Waite Campus to investigate alternative end uses of barley. In this project I worked closely with industry to identify barley quality traits for Japanese Shochu, an alcoholic beverage that is made predominantly from Australian grown barley. In this capacity I studied grain quality traits associated with pearling and endosperm hardness. Also, in this position I worked alongside the hulless barley breeder, Amanda Box, to investigate food uses of hulless barley. One of the quality traits of interest was high beta-glucan for human nutritional benefit. After leaving this position in 2005, I returned to work in embryonic stem cell research with Peter Rathjen within the School of Molecular and Biomedical Science and more recently with Bob Gibson in the School of Agriculture, Food and Wine, to help establish the University of Adelaide’s Foodplus centre. In late 2008 I was delighted to accept a research associate position at ACPFG with the Fincher group, funded by ABB Grain, to investigate the genes associated with betaglucan deposition in barley grain. The aim of this project is to lower beta-glucan in malting varieties in order to reduce steeping times and water consumption in the malt-house. 19 ACPFG 2009 Symposium salinity tolerance, from genomics to field 16–18 November 2009 Field and yield – physiology and trait dissection – genes and machines Internationally recognised researchers will gather in Adelaide to discuss plant responses to salinity stress and research strategies for more robust crops in the context of global climate change. The conference will span molecular, biochemical and physiological approaches through to whole plant studies. Speakers will discuss the genetic and biochemical mechanisms and morphological and physiological processes used by model and crop plant species to sense and trigger adaptive responses to salinity and associated environmental problems. Abstract submission and registration information available soon through the ACPFG website. Contributions, comments and queries are welcome. If you have any information on ACPFG news, events, research or international travel that should be included in the next issue, please contact: Cobi Smith, Communications Manager Email: [email protected] Phone: (08) 8303 7230 Fax: (08) 8303 7102 For more ACPFG news and information, go to: www.acpfg.com.au ACPFG gives no warranty and makes no representation that the information in this document is suitable for any purpose or is free from error. ACPFG accepts no responsibility for any person acting or relying on the information contained in this document, and disclaim all liability for any loss, cost or expense incurred by reason of any person using or relying on the information contained in this document or by reason of any error, omission, defect, or mis-statement contained therein. Australian Centre for Plant Functional Genomics PMB 1, Glen Osmond, South Australia 5064 To subscribe to Vector, send an email to [email protected] with your address and ‘subscribe to Vector’ in the subject, or post to the address above.
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