Báo cáo khoa học: A1-L1 Micromechanical studies of mitotic chromosome structure docx

Sunday, July 8, 2007 A1-L1 Micromechanical studies of mitotic chromosome structure J. F. Marko Northwestern University, Evanston, IL, USA I will discuss experiments which probe the large-scale organization of mitotic chromosomes, using a combination of chemical modifi- cations and micromechanical force measurements. Restriction nucleases cause dissolution of the chromosome, indicating that DNA itself is the contiguous structural element of the chromosome. Therefore the non-DNA (i.e., protein) components must be isolated from one another, suggesting that the mitotic chromosome is best thought of as a network or gel of chromatin. Experiments with proteases produce a different result: a gradual expansion of the chromosome occurs, consistent with gradual removal of network nodes (crosslinks). Studies using topoisomerases which suggest that DNA entanglements may play a role in compacting the chromo- some will also be discussed. A1-L2 Spindle checkpoint control by the chromosomal passenger complex G. Vader, C. Cruijsen, T. van Harn, M. Vromans, R. Medema and S. M. A. Lens University Medical Center Utrecht, Utrecht, THE NETHERLANDS During each round of cell division chromosomes need to be equally distributed over the two daughter cells in order to maintain a stable diploid genome. The spindle assembly checkpoint (SAC) ensures faithful chromosome segregation by monitoring the presence and quality of chromosome-microtubule interactions. As a weakened SAC may be one of the underlying mechanisms of the aneup- loidy observed in many solid tumors, it is important to understand how this checkpoint is controlled in normal and malignant cells. The chromosomal passenger complex (CPC: Aurora B, INCENP, Survivin and Borealin), becomes essential for SAC function when chromosomes have made connections to the microtubules of the mitotic spindle that are not bipolar and hence do not create ten- sion. Unlike the classical SAC proteins Mad2 and BubR1, the CPC does not directly inhibit the Anaphase Promoting Complex (APC). By destabilizing incorrect microtubule-chromosome attachments, the CPC can re-create unattached kinetochores that could subsequently propagate the APC inhibitory signal. Using a siRNA complementation approach, we found that a deletion mutant of INCENP (lacking the coiled-coil domain) is capable of correcting miss-attached microtubules, but unable to communicate a lack of tension to the SAC. This suggests that both functions of the CPC are not necessarily coupled as currently thought and thus opens up the possibility to identify CPC targets specifically involved in spindle checkpoint control. 6 A1-L3 Telomere dysfunction as a cause of genomic instability in Werner syndrome L. Crabbe 1,2 , A. Jauch 3 , C. Naeger 1 , H. Holtgreve-Grez 3 and J. Karlseder 1 1 The Salk Institute, La Jolla, CA, USA, 2 Institut de Ge ´ ne ´ tique Humaine, Montpellier, FRANCE, 3 Institute of Human Genetics, Heidelberg, GERMANY Werner Syndrome (WS) is a rare human premature-aging disease, caused by mutations in the gene encoding the RecQ helicase WRN. In addition to the aging features this disorder is marked by genomic instability, associated with an elevated incidence of cancer. Several lines of evidence suggest that telomere dysfunction is associated with the aging phenotype of the syndrome, however, the origin of the genomic instability observed in WS cells, and the reason for the high incidence of cancer in WS has not been established. We previously proposed that WRN helicase activity was essential to prevent dramatic telomere loss during DNA replication. Lack of WRN led to the loss of individual telomeres from a few chromosome ends, a phenomenon we termed Sister Telomere Loss. The missing telomeres were mostly the ones replicated by the lagging strand machinery, suggesting the requirement of WRN for the efficient replication of G rich sequences. Consequently, we set out to demonstrate that replication associated telomere loss is responsible for the fused chromosomes and genome instability frequent in WS fibroblasts. Moreover, using metaphase analysis we show that telomere elongation by telomerase can significantly reduce the appearance of new chromosomal aberrations in cells lacking WRN, similarly to complementation of WS cells with WRN. Our results suggest that the genome instability in WS cells is directly dependent on telomere dysfunction, linking chromo- some end maintenance to chromosomal aberrations in this disease. A1-L4 Cytoplasmic dynein is responsible for the rapid poleward motions seen during mitosis in animals Z. Yang 1 , U. S. Tulu 2 , P. Wadsworth 2 and C. L. Rieder 1 1 Wadsworth Center, Albany, NY, USA, 2 University of Massachusetts, Amherst, MA, USA As the nuclear envelope breaks down (NEB) to initiate mitosis in animal cells, the sister kinetochores on each chromosome begin to interact with dynamic microtubules growing from the separating centrosomes (spindle poles). Due to the random nature of this ‘search and capture’ process one kinetochore often acquires an attachment to a spindle pole before its sister does. When this occurs the chromo- some moves towards the pole at rates often exceeding 35-lm/min (Rieder CL, Alexander SP. J. Cell Biol, 1990; 110: 81–95). Although we hypothesized in 1990 that these rapid motions are mediated by cytoplasmic dynein there is currently no direct evidence that this is so. We have therefore tracked kinetochore motions in live human (U2OS) cells, expressing CENP-B-GFP and Centrin-GFP, after knock- ing down zw10 by siRNA. This approach allowed us to selectively deplete kinetochores of dynein, without disrupting bipolar spindle assembly. As expected, in untreated U2OS cells many kinetochores exhibit rapid translocations towards a centrosome during the first 5 min of spindle formation, some with rates >20 lm/min. However, after zw10 siRNA treatment the number of these motions, and their velocities, is greatly attenuated. In these cells the rate of anaphase A chromosome motion is also reduced by ~35%. Consistent with these observations, the rapid motion of chromosomes toward centrosome-associated microtubule arrays that form in LLC-Pk1 cells depleted of TPX2 using siRNA are also sensitive to antibody mediated inhibition of dynein. From these live cell observations we con- clude that during mitosis in animals, cytoplasmic dynein powers the initial rapid poleward motions of attaching chromosomes. A1-L5 F. Uhlmann London Research Institute, London, UK No abstract available. Sunday Symposia Lectures 7 B1-L1 N-linked protein glycosylation in pro- and eukaryotes M. Aebi ETH Zu ¨ rich, Zu ¨ rich, SWITZERLAND N-linked protein glycosylation is the most frequent protein modification in eukaryotic cells. This process initiates at the membrane of the endoplasmic reticulum, where an oligosaccharide, Man 5 GlcNAc 2 , is assembled on the lipid carrier, dolichylpyrophosphate, translo- cated across the membrane and completed to Glc 3 Man 9 glcNAc 2 . This oligosaccharide is then transferred to selected asparagine residues of nascent polypeptide chains. N-linked protein glycosylation does also take place in archaea and in bacteria. The recently discovered N-linked protein glycosylation process in Campylobacter jejuni was transferred into Escherichia coli, enabling a genetic and biochemical analysis of the prokaryotic pathway. The high sequence similarity of the bacterial oligosaccharyltransferase with one subunit of the euk- aryotic enzyme, the very similar protein acceptor sequence as well as the finding that oligosaccharides linked to isoprenoid lipids serve as substrates in the reactions suggest that the bacterial and the eukaryotic N-linked protein glycosylation are homologous processes. In con- trast to the bacterial process, N-linked glycosylation in eukaryotes occurs before folding of the protein, enabling the use of the N-linked glycan as a general signal that reflects the folding status of the protein. The direct comparison of the homologous process in pro- and eukaryotes made it possible to formulate hypotheses regarding the eukaryote-specific components of the pathway. In particular, the role of eukaryote-specific subunits of the oligosaccharyltransferase complex can be addressed. B1-L2 The TRIM-NHL proteins Brat and Mei-P26 regulate cell growth and proliferation in Drosophila stem cell lineages R. Neumueller, I. Poernbacher and J. A. Knoblich Institute of Molecular Biotechnology, Vienna, AUSTRIA Stem cells can generate self-renewing and differentiating daughter cells at the same time. We are using Drosophila as a model system to understand, how they control the balance between these two fundamentally different types of progeny. Drosophila neuroblasts and germ- line stem cells are two of the best characterized model system for stem cell biology. While neuroblasts control self-renewal by segregating the TRIM-NHL protein Brat into one of their two daughter cells, germline stem cells use a niche mechanism to control self renewal. Here, we describe Mei-P26, another TRIM-NHL protein that is highly related to Brat. Our data indicate that Mei-P26 is a key regulator of cell growth acting downstream of niche signalling in the ovarian stem cell lineage. In mei-P26 mutants, cells outside the niche increase ribosome biogenesis and grow abnormally large. They proliferate indefinitely and fail to differentiate leading to the formation of ovarian tumors. Although Mei-P26 is not required for proliferation control in the brain, it can inhibit proliferation when ectopically expressed in neuroblasts and might act through the same pathway as Brat. Our data establish TRIM-NHL proteins are important regulators of stem cell proliferation. Preliminary analysis of Brat and Mei-P26 homologs suggests that this function might be conserved in vertebrates as well. Symposia Lectures Sunday 8 B1-L3 Emerging fluorescence technologies for the analysis of protein localization and organelle dynamics J. Lippincott-Schwartz National Institute of Health, Bethesda, MD, USA The development of fluorescent proteins as molecular tags over the past decade has splurred a revolution by allowing complex biochemical processes to be correlated with the functioning of proteins in livings cells. Fluorescent proteins such as green fluorescent protein (GFP) from the jelly fish Aequorea Victoria and its variants can be fused to virtually any protein of interest to analyze protein geography, move- ment and chemistry in living cells. As such, they have provided an important new tool for understanding protein function, filling an urgent need now that the genome sequence of many organisms is complete. Among the new fluorescent proteins are those that can be photoacti- vated. These molecules are invisible until activated by a specific wavelength of light, at which point they become brightly fluorescent. In this talk, I will discuss results from two new techniques employing photoactivatable fluorescent proteins: in cellula pulse-chase analysis and photoactivated localization microscopy (PALM). Results using in cellula pulse-chase analysis have permitted the visualization of per- oxisome biogenesis in living cells, addressing the origin and mechanism of proliferation of these key lipid-metabolizing organelles. Results using PALM-which uses serial photoactivation and subsequent bleaching of numerous sparse subsets of photactivated fluorescent protein molecules to determine the centers of fluorescent emission of individual molecules have permitted fluorescently-tagged proteins within cells to be optically resolved at mean separations of a few nanometers, overcoming the diffraction barrier in fluorescence microscopy. B1-L4 Novel mechanisms of transport of soluble proteins through the Golgi complex: a role for diffusion via inter-cisternal continuities A. Luini Consorzio Mario Negri Sud, S. Maria Imbaro, ITALY Intracellular membrane trafficking has been proposed to occur by compartment maturation-progression, a process by which exocytic and endocytic trafficking organelles move gradually forward through the transport pathways while at the same time changing their com- position and identity (e.g. from a medial- to a trans-Golgi cisterna). This mechanism applies to supramolecular cargo, such as procolla- gen aggregates. Different proteins, however, might be transported by other mechanisms. Prompted by our recent observation that Golgi cisternae are connected by tubules, we have here examined whether soluble diffusable cargo proteins might traverse the Golgi compart- ments by flowing through these connections. Soluble secreted proteins represent a significant fraction of the genome and include mem- bers of great functional importance. We show that soluble cargoes (e.g. albumin) traverse the Golgi by diffusing within seconds through the Golgi cisternae via tubular continuities of about 50 nm in diameter and then concentrate in a pH-dependent fashion in trans-Golgi network exit sites, from where they leave for the plasma membrane. Thus, two mechanisms (diffusion-concentration and maturation-pro- gression) appear to operate in the Golgi to promote the transport of major protein classes with different physico-chemical properties. Sunday Symposia Lectures 9 B1-L5 Biogenesis of the Golgi apparatus G. Warren MFPL, Vienna, AUSTRIA The Golgi receives the output of newly-synthesized proteins and lipids from the ER, processes them in the stack, and sorts them in the TGN to their final destinations. During the cell cycle, the Golgi undergoes biogenesis as part of the process that helps to ensure propa- gation through successive generations. Duplication during interphase is followed by partitioning during mitosis, the latter having been extensively studied in mammalian cells. The process of Golgi duplication has been much harder to study in mammals because there are multiple copies subsumed within a ribbon-like structure near to the nucleus. Simpler organisms have, therefore, been employed, most notably the protozoan parasites, such Trypanosoma brucei, the causative agent of sleeping sickness. The anatomical simplicity coupled with genetic manipulability allows detailed study of Golgi biogenesis. Assembly of a new Golgi requires contributions from the old Golgi and is a highly ordered process that requires a centrin-based template. C1-L1 G. Ju ¨ rgens University of Tu ¨ bingen, Tu ¨ bingen, GERMANY No abstract available. Symposia Lectures Sunday 10 C1-L2 Genetic dissection of Drosophila courtship behaviour B. J. Dickson, L. Tirian and G. Dietzl IMP, Vienna, AUSTRIA Animal instincts are specified during development by genetic programs that preconfigure the appropriate neural circuitry. Genetic dissec- tion in model organisms thus has the potential to reveal the molecules, neurons, circuits and principles underlying animal instincts. With this goal in mind, we are studying the male courtship ritual of Drosophila melanogaster. This instinctive behaviour is specified during development by the male-specific products of the fruitless (fru) gene, Fru M , which are expressed in ~2000 cells in the male nervous sys- tem. Forced expression of Fru M in females is sufficient to program the male courtship instinct into the female nervous system. Genetic data suggest that distinct subsets of Fru M neurons are likely to have distinct functions in the courtship ritual. These distinct functions are in turn likely to have distinct genetic requirements. We are using a genome-wide transgenic RNAi screens to systematically identify the genes required to configure these neurons for male behaviour. We anticipate that these approaches will help to define the functions of the genes and neurons that together shape this complex innate behaviour. C1-L3 A combination of ephrins and neural activity are required for visual system mapping D. A. Feldheim UC Santa Cruz, Santa Cruz, CA, USA The generation of precise connectivity in the brain is thought to use a combination of inborn genetic cues (nature) and neural-activity dependent mechanisms (nurture). In this talk I will present our data that shows how these two mechanisms interact in visual system development. Visual information is processed in multiple areas of the mammalian brain, most notably the superior colliculus (SC) in the midbrain, the dorsal lateral geniculate nucleus (dLGN) in the thalamus, and the primary visual areas of the cortex (V1). All of these areas receive inputs that are topographic in nature. Topographic maps are the general rule of nervous system connections and are thought to keep the spatial information of a stimulus intact as it is transferred from one brain region to another. Here I will share our work on the study of how topographic maps develop in each of these visual regions. We have analyzed the topographic maps of each of these regions in various populations of mice: ephrin-A mutant mice, mice defective in correlated retinal activity (mice mutant in the b2 subunit of the nACh receptor), and mice defective for both ephrin-As and correlated neural activity. We analyzed the nature of the visual connections in these mice both anatomically, using axon tracing techniques, and functionally, using intrinsic optical imaging. We find that both ephrin-As and b2 are required for topographic mapping to all visual structures, but that the relat- ive contribution of each is different between the SC and V1. The analysis of these mice leads us to present a model whereby a combi- nation of topographic mapping molecules and neural activity dependent events act in parallel together to create the stereotypical connectivity patterns in the primary visual system. Sunday Symposia Lectures 11 C1-L4 Nuclear responses to WNT signaling R. Grosschedl, C. Sustmann, A. Gru ¨ nder and W. Roth Max Planck Institute of Immunobiology, Freiburg, GERMANY LEF1/TCF proteins are nuclear mediators of WNT signaling that activate transcription in association with beta-catenin. Transcrip- tional activation in response to Wnt signals can be enhanced by the interaction of beta-catenin with the proteins Bcl9 and Pygopus. Bcl9 protein is expressed in all cell types examined. We found that the function of Bcl9 displays cell type specificity and involves a transcriptional activation domain that synergizes with the transcriptional activation domain of beta-catenin. We identified protein complexes that interact with the transactivation domain of beta-catenin. Transcriptional activation by beta-catenin can be antagonized by interaction of LEF1 with PIASy, which mediates SUMO modification of LEF1 and sequesters LEF1 into nuclear bodies. In the mouse, LEF1 is required for the formation of organs that involve inductive interactions between epithelial and mesenchymal tissues. To examine which activities of LEF1 are mediated by its interaction with beta-catenin, we generated a mutant allele of Lef1 that car- ries point mutations in the beta-catenin-interaction domain. The mutant mice display some, but not all phenotypes observed in mice carrying a null mutation, indicating that LEF1 regulates gene expression by both Wnt-dependent and Wnt-independent mechanisms. C1-L5 Mechanism(s) of Toll receptor activation M. Gangloff University of Cambridge, Cambridge, UK Toll and Toll-like receptors (TLRs) provide the first line of defence against invading microbial pathogens. These receptors consist of an extracellular ligand-binding domain, a single transmembrane region and an intracellular signalling domain. TLRs are activated by var- ious stimuli that differ considerably in size, shape and chemical nature and range from a protein ligand called Spa ¨ tzle for drosophila Toll, to lipopolysaccharide (LPS) for TLR4 and nucleic acids for TLR3. Ligand binding is mediated by leucine-rich repeats, a versatile structural motif, which adopts a ‘sickle’ shape and lacks internal cavities. The mechanism of ligand recognition is diverse and sometimes involves accessory molecules. For example, drosophila Toll functions as a cytokine receptor for Spa ¨ tzle, which is produced as an inactive pro-protein, that is subsequently activated by endoproteolytic cleavage. Recently, we have shown that the N-terminal pro-domain is released upon receptor binding. There is no Spa ¨ tzle homologue in mammals and, in contrast to Toll, TLRs are thought to be true patho- gen-recognition receptors. Based on modelling studies, we have suggested a role for the TLR4 cofactor MD-2 in LPS recognition. Both the active C-terminal Spa ¨ tzle fragment and the MD-2-LPS complex are thought to trigger a series of events starting with receptor bind- ing, followed by dimerization and concerted conformational changes critical for the recruitment of intracellular adapter proteins. Symposia Lectures Sunday 12 D1-L1 Adaptation to extreme environments: the metabolism of halophilic archaea D. Oesterhelt Max Planck Institut fu ¨ r Biochemie, Martinsried, GERMANY Halophilic archaea thrive in brines that are exposed to a plethora of harsh conditions: low availability of water, high alkalinity, high temperatures, low oxygen tension, low availability of carbon or nitrogen sources and phosphate starvation, to name the most important ones. We made the genomic information for three representative halophilic archaea available for annotation and detailed investigation of their metabolism. All proteins are adapted to high ionic strength of their cytoplasm by covering their surfaces with acidic residues to keep water shells around the proteins. The general metabolism relies on amino acids as carbon sources and for Halobacterium salinarum a metabolic model was created from 800 reactions to describe the central metabolism and growth features. In a systems biological approach the model was verified by growth experiments and analysis of the medium constituents. Special conditions, such as alkaline environment, are met by Natronomonas pharaonis which developed sophisticated systems for nitrogen uptake and specific archaeal lipid anchors to avoid alkaline extraction of peripheral proteins. The square shaped Haloquadratum walsbyi finally is the world champion to fight low water activities as it grows in saturated sodium chloride solutions containing up to 2 M magnesium chloride. It developed a phosphonate metabolism and runs its energy metabolism with the help of two bacteriorhodopsins. As a clue to fight low water activity H. walsbyi expresses halomucin, the closest homologue of which is human mucin. This longest archaeal protein known of 9200 amino acids covers like a slime shell the cell and apparently serves a water-binding function. D1-L2 Single cell stable isotope probing with FISH-Raman spectroscopy for deciphering the ecophysiology of uncultured bacteria M. Wagner 1 and A. S. Whiteley 2 1 Department of Microbial Ecology, University of Vienna, Vienna, AUSTRIA, 2 Molecular Microbial Ecology Section, CEH Oxford, UK Millions of microbial species thrive on Earth, but till today microbiologists have only succeeded in isolating a few thousand bacterial and archaeal species. Uncultured microbes are essential for the health of the planet and provide, due to their enormous genetic diversity, a potentially rich source for new biotechnologically relevant products. However, for most uncultured microbes no information on their physiological capabilities is available. Thus, there is an obvious need for the development of methods which allow to simultaneously identifying and investigating the physiology of microorganisms within their natural habitat. For this purpose, we have coupled fluores- cence in situ hybridisation (FISH) with Raman microscopy for simultaneous cultivation-independent identification and determination of 13C incorporation into microbial cells. Cells which were 13C labelled through anabolic incorporation of the isotope exhibited key red- shifted spectral peaks in the highly resolved Raman confocal spectra, the calculated ‘red shift ratio’ being highly correlated with the 13C-content of the cells. Subsequently, Raman instrumentation and FISH protocols were optimized to allow combined epifluorescence and Raman imaging of probe-labeled microbial populations at the single cell level. In order to demonstrate the suitability of this tech- nology for structure-function analyses in complex microbial communities, Raman-FISH was deployed to show the importance of Pseu- domonas populations during naphthalene degradation in groundwater microcosms. Sunday Symposia Lectures 13 D1-L3 Multiple facets of synthesi s and breakdown of fatty acids in yeast K. Hiltunen, T. Glumoff and A. J. Kastaniotis University of Oulu, Oulu, FINLAND Lipid metabolism in yeast, as well as in all other eukaryotes, is a compartmentalized process. An example for metabolic compartmental- ization is fatty acid synthesis (FAS) occurring in at least two subcellular compartments in eukaryotes: in cytosol (FAS type 1) and in mitochondria (also in chloroplasts of plant cells; FAS type 2). Yeast cells deficient in mitochondrial FAS are respiratory deficient, which can partly, but not completely, be explained by a role for FAS type 2 in the synthesis of lipoic acid. We constructed a yeast strain carry- ing a genomic replacement of the ETR1 mitochondrial enoyl reductase gene by a chimeric construct expressing mitochondrially targeted FabIp enoyl reductase from Escherichia coli. This strain is respiratory competent, but complementation introduces sensitivity to the drug triclosan, a known inhibitor of FabIp. This modified yeast strain provides a novel model for assessment of metabolic interactions between mitochondrial FAS and other cellular processes. Our understanding of the b-oxidation cycle has increased significantly in the last years, particularly through the use of genetics and structural biology. Of particular interest is the structural organization of peroxi- somal multifunctional enzymes. The structures of individual domains of both mammalian and fungal MFE type 2 have been determined recently, shedding light on their assembly and function. The major future challenge will be the elucidation of molecular mechanisms transferring solutes and cofactors across the peroxisomal membrane. D1-L4 Chaperone-assisted protein folding in the cytosol F. Hartl Max Planck Institute of Biochemistry, Martinsried, GERMANY The efficient folding of many newly-synthesized proteins depends on assistance by molecular chaperones, which act to prevent protein misfolding and aggregation in the crowded environment of the cell. Nascent chain-binding chaperones, including trigger factor (TF) and DnaK (Hsp70), stabilize elongating chains on ribosomes in a non-aggregated state. Folding in the cytosol is achieved either on con- trolled chain release from these factors or following transfer of newly-synthesized proteins to downstream chaperones, such as the cylin- drical chaperonin GroEL. GroEL and its cofactor GroES of E. coli promote protein folding by sequestering non-native polypeptide in a cage-like structure. Around 250 different proteins interact with GroEL in wild-type cells and this number nearly doubles in cells lacking TF and DnaK. However, obligate chaperonin dependence is limited to only ~85 substrates. These proteins, including a number of essen- tial gene products, are stabilized by DnaK against aggregation but reach native state only upon transfer into the physical environment of the GroEL/GroES cage. Proteins with (ba 8 ) TIM barrel domains are enriched among the obligate GroEL substrates. The fact that the number of GroEL-dependent proteins is rather limited suggests a high degree of robustness for the E. coli proteome in terms of pro- tein folding. On the other hand, the chaperonin system may have facilitated the structural evolution of domain folds, such as the TIM barrel, that are highly represented among its obligate substrates. Symposia Lectures Sunday 14 D1-L5 Life inside cereals: genomic insights into the lifestyle of the mutualistic, N2-fixing endophyte Azoarcus sp. strain BH72 Barbara Reinhold, Andrea Krause, Melanie Bo ¨ hm, Frauke Friedrich, Sabrina Gemmer, Lucie Miche ´ , Janina Oetjen, Abhijit Sarkar and Thomas Hurek Laboratory of General Microbiology, University of Bremen, Bremen, GERMANY Azoarcus sp. strain BH72, a mutualistic endophyte of rice and other grasses, is of agro-biotechnological interest because it supplies fixed nitrogen to its host and colonises plants in remarkably high numbers without eliciting disease symptoms. Recently the complete genome was sequenced (1) for the first time for grass endophytes. It is 4 376 040 bp long and contains 3992 predicted protein-coding sequences. Genome comparison with the Azoarcus-related soil bacterium strain EbN1 revealed a surprisingly low degree of synteny. CDS involved in the synthesis of surface components potentially important for plant-microbe interactions were more closely related to those of plant- associated bacteria. Strain BH72 appears to be ‘disarmed’ compared to plant pathogens, having only few plant-cell-wall-degrading enzymes; it lacks type III and IV secretion systems, related toxins, and an AHL-based communication system. The genome sequence revealed also that this bacterium appears to be highly adapted to conditions of now nitrogen abundance, which will be described in more detail. Based on the genome sequences of the host plant rice and strain BH72, functional genomic analyses were carried out. Our results suggest that plant defense responses involving JA may contribute to restricting endophytic colonization in grasses. However in a compat- ible interaction, JA-induced stress responses or pathogenesis-related proteins are apparently not relevant. Proteome studies revealed remarkable changes in the rice root proteome upon infection, indicating that metabolic adaptations occur in association with endo- phytes. Reference 1. Krause et al. Genomic insights into the lifestyle of the mutualistic, N2-fixing grass endophyte Azoarcus sp. strain BH72. Nature Bio- technol 2006; 24: 1385–1391. E1-L1 The human genome as an RNA machine J. S. Mattick, L. J. Croft, M. E. Dinger, M. Pheasant, I. V. Makunin, M. Askarian Amiri, T. R. Mercer, K. C. Pang, C. Simons and R. J. Taft University of Queensland, Brisbane, AUSTRALIA It appears that the genetic programming of humans and other higher organisms has been fundamentally misunderstood for the past 50 years, because of the presumption – largely true in prokaryotes, but not in complex eukaryotes – that most genetic information is expressed as and transacted by proteins. The majority of the mammalian genome is in fact transcribed, apparently in a developmentally regulated manner, and most complex genetic phenomena in the higher organisms are RNA-directed. Evidence will be presented (i) that there are thousands of non-protein-coding transcripts in mouse that are dynamically expressed during differentiation and development, including in embryonal stem cell, muscle, genital ridge, macrophage, T-cell and neuronal cell differentiation, among others, many of which show precise expression patterns and subcellular localization in different regions of the brain; (ii) that there are millions of small RNAs expressed from the mouse and human genomes, the latter showing a massive expansion in the brain; and (iii) that much, if not most, of the mammalian genome is not evolving neutrally, but comprises different types of sequences evolving at different rates under different selection pressures and different structure-function constraints. These observations suggest that the majority of the human genome and those of other complex organisms is devoted to a hidden RNA regulatory system that directs the trajectories of differentiation and development by controlling chromatin archi- tecture and epigenetic memory, transcription, splicing, RNA modification and editing, mRNA translation and RNA stability. Sunday Symposia Lectures 15 [...]... Institute of Molecular Pathology, Vienna, AUSTRIA, 4Osaka University, Osaka, JAPAN, 5Tokyo Metropolitan Institute of Medical Science, Tokyo, JAPAN The precise duplication of all chromosomes in each round of the cell cycle is required in order to maintain the integrity of the genome To understand the molecular mechanism that guarantees the genome integrity, it is essential to study the process of chromosome. .. tell us how molecular requirements for the maintenance of chromosome are variable depending on size and complexity of the chromosome I will also discuss the technical limitation and problems of ChIP-chip analyses 16 Sunday Symposia Lectures E1-L4 Molecular interactions of rhodopsin – from the proteome inventory of the outer segment towards analysis of functional protein networks M Ueffing1,2, M Swiatek-de... maintenance of genomes I will introduce our recent attempt for applying ChIP-chip for the analyses of complex human genome and present our most recent data of the molecular basis of functional elements on eukaryotic chromosomes (replication origins, cohesin sites, and so on.) in three eukaryotic species (S cerevisiae, S pombe and H sapiens) at the whole genome level Comparison of genomic structures... Mapping of the Yeast Genetic Interaction Network C Boone University of Toronto, Toronto, ON, CANADA No abstract available E1-L3 Analyses of eukaryotic chromosome structure and function by array based genomic approach T Itoh1, K Yoshida2, K S Went3, M Bando2, B K Koch3, Y Katou2, H Masukata4, H Masai5, J Peters3 and K Shirahige2 1 Mitsubishi Research Institute Inc., Tokyo, JAPAN, 2Tokyo Institute of Technology,... Health, Institute of Human Genetics, Munich-Neuherberg, GERMANY, 2Technical University, Munich, GERMANY, 3Department of Biology, University of Rome Tor Vergata, Rome, ITALY marius.ueffing@ gsf.de Mutations in the light receptive GPCR rhodopsin, and in proteins linked to its downstream signalling account for numerous blinding diseases The mechanistic interpretation of the majority of rhodopsin mutations... the isolated (super-) complexes and present an interaction map of rhodopsin based on the analysis of protein complexes recovered in their native form Out of all interactions, 101 from our dataset are putatively novel The authenticity of isolated protein interactions was independently cross-validated by either data-mining or experimental proof via a second alternative method By using the genome wide protein... our approach thereby gaining information on the topology of the complexes This analysis has also offered a view of the peripheral links between the main rhodopsin pathway and the rest of the cell functional pathways The dataset suggests that in addition to its role as a light transducing GPCR, rhodopsin plays a role in regulating photoreceptor structure and polarity and is likely to act as a multifunctional... approach Genetic and biochemical approaches have so far identified hundreds of proteins that function in some aspects of chromosome dynamics Now, genomic approach called ChIP-chip (Chromatin immuno-precipitation combined with high resolution tiling DNA chip) is able to show us how these proteins are integrated in the process of whole chromosomal dynamics, that is, how each elemental process is connected... some of them may affect its function indirectly by altering the correct wiring of a large and still poorly understood protein network With the aim of establishing a protein interaction map centred on rhodopsin, we systematically investigated protein interactions from isolated photoreceptor outer segment discs We combined biochemical native separation methods with mass spectrometric identification of the . Sunday, July 8, 2007 A1-L1 Micromechanical studies of mitotic chromosome structure J. F. Marko Northwestern University, Evanston,. which probe the large-scale organization of mitotic chromosomes, using a combination of chemical modifi- cations and micromechanical force measurements. Restriction