TY - JOUR A1 - Helmprobst, Frederik A1 - Kneitz, Susanne A1 - Klotz, Barbara A1 - Naville, Magali A1 - Dechaud, Corentin A1 - Volff, Jean-Nicolas A1 - Schartl, Manfred T1 - Differential expression of transposable elements in the medaka melanoma model JF - PLoS One N2 - Malignant melanoma incidence is rising worldwide. Its treatment in an advanced state is difficult, and the prognosis of this severe disease is still very poor. One major source of these difficulties is the high rate of metastasis and increased genomic instability leading to a high mutation rate and the development of resistance against therapeutic approaches. Here we investigate as one source of genomic instability the contribution of activation of transposable elements (TEs) within the tumor. We used the well-established medaka melanoma model and RNA-sequencing to investigate the differential expression of TEs in wildtype and transgenic fish carrying melanoma. We constructed a medaka-specific TE sequence library and identified TE sequences that were specifically upregulated in tumors. Validation by qRT- PCR confirmed a specific upregulation of a LINE and an LTR element in malignant melanomas of transgenic fish. KW - melanoma KW - genomics KW - transposable elements KW - cancer genomics KW - malignant tumors KW - gene prediction KW - human genomics KW - retrotransposons Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-260615 VL - 16 IS - 10 ER - TY - GEN A1 - Tort, Jose F. A1 - Mitreva, Makedonka A1 - Brehm, Klaus R. A1 - Rinaldi, Gabriel T1 - Editorial: Novel Frontiers in Helminth Genomics T2 - Frontiers in Genetics N2 - No abstract available. KW - flatworm KW - nematodes KW - genomics KW - helminths KW - neglected diseases Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-210209 SN - 1664-8021 VL - 11 IS - 791 ER - TY - THES A1 - Terhoeven, Niklas T1 - Genomics of carnivorous Droseraceae and Transcriptomics of Tobacco pollination as case studies for neofunctionalisation of plant defence mechanisms T1 - Genomik karnivorer Droseraceae und Transkriptomik der Befruchtung von Tabak als Fallstudien zur Umfunktionierung pflanzlicher Verteidigungsmechanismen N2 - Plants have evolved many mechanisms to defend against herbivores and pathogens. In many cases, these mechanisms took other duties. One example of such a neofunction- alisation would be carnivory. Carnivory evolved from the defence against herbivores. Instead of repelling the predator with a bitter taste, the plant kills it and absorbs its nutrients. A second example can be found in the pollination process. Many of the genes involved here were originally part of defence mechanisms against pathogens. In this thesis, I study these two examples on a genomic and transcriptomic level. The first project, Genomics of carnivorous Droseraceae, aims at obtaining annotated genome sequences of three carnivorous plants. I assembled the genome of Aldrovanda vesiculosa, annotated those of A. vesiculosa, Drosera spatulata and Dionaea muscipula and com- pared their genomic contents. Because of the high repetitiveness of the D. muscipula genome, I also developed reper, an assembly free method for detection, classification and quantification of repeats. With that method, we were able to study the repeats without the need of incorporating them into a genome assembly. The second large project investigates the role of DEFL (defensin-like) genes in pollen tube guidance in tobacco flowers. We sequenced the transcriptome of the SR1 strain in different stages of the pollination process. I assembled and annotated the transcriptome and searched for differentially expressed genes. We also used a method based on Hidden- Markov-Models (HMM) to find DEFLs, which I then analysed regarding their expression during the different stages of fertilisation. In total, this thesis results in annotated genome assemblies of three carnivorous Droser- aceae, which are used as a foundation for various analyses investigating the roots of car- nivory, insights into the role of DEFLs on a transcriptomic level in tobacco pollination and a new method for repeat identification in complex genomes. N2 - Im Laufe der Evolution haben Pflanzen viele Methoden entwickelt, um sich gegen Fress- feinde und Pathogene zu verteidgen. Viele dieser Methoden wurden im Laufe der Zeit umfunktioniert. Ein Beispiel hierfür ist die Karnivorie, welche aus der Verteidigung ge- gen Fressfeinde entstanden ist. Anstelle einen Angreifer durch bitteren Geschmack zu vertreiben, tötet die Pflanze das Tier und nimmt seine Nährstoffe auf. Ein weiteres Bei- spiel ist der Bestäubungs- und Befruchtungsprozess. Viele der Gene, die hier involviert sind, stammen ursprünglich aus Mechanismen zur Verteidigung gegen Pathogene. In dieser Arbeit untersuche ich diese beiden Beispiele auf genomischer und transkrip- tomischer Ebene. Die Zielsetzung des ersten Projekts, Genomik von karnivoren Dro- seraceaen, ist es, assemblierte und annotierte Genome von drei karnivoren Pflanzen zu generieren. Ich habe dazu das Genom von Aldrovanda vesiculosa assembliert und dieses, sowie die Genome von Drosera spatulata und Dionaea muscipula annotiert und mit- einander verglichen. Aufgrund des hohen Anteils repetitiver Elemente im D. muscipula Genom habe ich reper, eine Methode zum Detektieren, Klassifizieren und Quantifizieren von Repeats, entwickelt. Mit dieser Methode ist es nun möglich, repetitive Elemente zu untersuchen, ohne diese in einem Genomassembly integrieren zu müssen. Das zweite große Projekt untersucht die Rolle von DEFL (defensin-like) Genen im Pollenschlauchwachstum in Tabakblüten. Dazu haben wir das Transkriptom der SR1 Variante zu verschiedenen Zeitpunkten im Befruchtungsprozess sequenziert. Ich habe dieses Transkriptom assembliert und annotiert und darin nach differentiell exprimierten Genen gesucht. Zudem haben wir mit einer auf Hidden Markov Modellen (HMM) ba- sierten Methode nach DEFL Genen gesucht und ich habe die Expression dieser in den verschiedenen Stadien untersucht. Zusammenfassend beinhalten die Ergebnisse dieser Thesis annotierte Genomassemb- lies von drei karnivoren Droseraceaen, Erkenntnisse über die Rolle von DEFL Genen bei der Befruchtung auf einer transkriptomischen Ebene und eine neue Software zur Analyse von repetitiven Elementen in komplexen Genomen. KW - Droseraceae KW - Genom KW - Nicotiana tabacum KW - Transkriptomanalyse KW - Repeats KW - genomics KW - carnivorous plants KW - next generation sequencing Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-189712 ER - TY - JOUR A1 - Mehmood, Rashid A1 - Alsaleh, Alanoud A1 - Want, Muzamil Y. A1 - Ahmad, Ijaz A1 - Siraj, Sami A1 - Ishtiaq, Muhammad A1 - Alshehri, Faizah A. A1 - Naseem, Muhammad A1 - Yasuhara, Noriko T1 - Integrative molecular analysis of DNA methylation dynamics unveils molecules with prognostic potential in breast cancer JF - BioMedInformatics N2 - DNA methylation acts as a major epigenetic modification in mammals, characterized by the transfer of a methyl group to a cytosine. DNA methylation plays a pivotal role in regulating normal development, and misregulation in cells leads to an abnormal phenotype as is seen in several cancers. Any mutations or expression anomalies of genes encoding regulators of DNA methylation may lead to abnormal expression of critical molecules. A comprehensive genomic study encompassing all the genes related to DNA methylation regulation in relation to breast cancer is lacking. We used genomic and transcriptomic datasets from the Cancer Genome Atlas (TGCA) Pan-Cancer Atlas, Genotype-Tissue Expression (GTEx) and microarray platforms and conducted in silico analysis of all the genes related to DNA methylation with respect to writing, reading and erasing this epigenetic mark. Analysis of mutations was conducted using cBioportal, while Xena and KMPlot were utilized for expression changes and patient survival, respectively. Our study identified multiple mutations in the genes encoding regulators of DNA methylation. The expression profiling of these showed significant differences between normal and disease tissues. Moreover, deregulated expression of some of the genes, namely DNMT3B, MBD1, MBD6, BAZ2B, ZBTB38, KLF4, TET2 and TDG, was correlated with patient prognosis. The current study, to our best knowledge, is the first to provide a comprehensive molecular and genetic profile of DNA methylation machinery genes in breast cancer and identifies DNA methylation machinery as an important determinant of the disease progression. The findings of this study will advance our understanding of the etiology of the disease and may serve to identify alternative targets for novel therapeutic strategies in cancer. KW - DNA methylation KW - epigenetic modification KW - breast cancer KW - genomics KW - in silico analysis Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-321171 SN - 2673-7426 VL - 3 IS - 2 SP - 434 EP - 445 ER - TY - JOUR A1 - Sendell-Price, Ashley T. A1 - Tulenko, Frank J. A1 - Pettersson, Mats A1 - Kang, Du A1 - Montandon, Margo A1 - Winkler, Sylke A1 - Kulb, Kathleen A1 - Naylor, Gavin P. A1 - Phillippy, Adam A1 - Fedrigo, Olivier A1 - Mountcastle, Jacquelyn A1 - Balacco, Jennifer R. A1 - Dutra, Amalia A1 - Dale, Rebecca E. A1 - Haase, Bettina A1 - Jarvis, Erich D. A1 - Myers, Gene A1 - Burgess, Shawn M. A1 - Currie, Peter D. A1 - Andersson, Leif A1 - Schartl, Manfred T1 - Low mutation rate in epaulette sharks is consistent with a slow rate of evolution in sharks JF - Nature Communications N2 - Sharks occupy diverse ecological niches and play critical roles in marine ecosystems, often acting as apex predators. They are considered a slow-evolving lineage and have been suggested to exhibit exceptionally low cancer rates. These two features could be explained by a low nuclear mutation rate. Here, we provide a direct estimate of the nuclear mutation rate in the epaulette shark (Hemiscyllium ocellatum). We generate a high-quality reference genome, and resequence the whole genomes of parents and nine offspring to detect de novo mutations. Using stringent criteria, we estimate a mutation rate of 7×10\(^{−10}\) per base pair, per generation. This represents one of the lowest directly estimated mutation rates for any vertebrate clade, indicating that this basal vertebrate group is indeed a slowly evolving lineage whose ability to restore genetic diversity following a sustained population bottleneck may be hampered by a low mutation rate. KW - evolutionary genetics KW - genomics KW - molecular evolution Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-357827 VL - 14 ER - TY - THES A1 - Ankenbrand, Markus Johannes T1 - Squeezing more information out of biological data - development and application of bioinformatic tools for ecology, evolution and genomics T1 - Mehr aus biologischen Daten herausholen - Entwicklung und Anwendung bioinformatischer Programme für Ökologie, Evolution und Genomik N2 - New experimental methods have drastically accelerated the pace and quantity at which biological data is generated. High-throughput DNA sequencing is one of the pivotal new technologies. It offers a number of novel applications in various fields of biology, including ecology, evolution, and genomics. However, together with those opportunities many new challenges arise. Specialized algorithms and software are required to cope with the amount of data, often requiring substantial training in bioinformatic methods. Another way to make those data accessible to non-bioinformaticians is the development of programs with intuitive user interfaces. In my thesis I developed analyses and programs to tackle current problems with high-throughput data in biology. In the field of ecology this covers the establishment of the bioinformatic workflow for pollen DNA meta-barcoding. Furthermore, I developed an application that facilitates the analysis of ecological communities in the context of their traits. Information from multiple public databases have been aggregated and can now be mapped automatically to existing community tables for interactive inspection. In evolution the new data are used to reconstruct phylogenetic trees from multiple genes. I developed the tool bcgTree to automate this process for bacteria. Many plant genomes have been sequenced in current years. Sequencing reads of those projects also contain data from the chloroplasts. The tool chloroExtractor supports the targeted extraction and analysis of the chloroplast genome. To compare the structure of multiple genomes specialized software is required for calculation and visualization of the relationships. I developed AliTV to address this. In contrast to existing programs for this task it allows interactive adjustments of produced graphics. Thus, facilitating the discovery of biologically relevant information. Another application I developed helps to analyze transcriptomes even if no reference genome is present. This is achieved by aggregating the different pieces of information, like functional annotation and expression level, for each transcript in a web platform. Scientists can then search, filter, subset, and visualize the transcriptome. Together the methods and tools expedite insights into biological systems that were not possible before. N2 - Neue experimentelle Methoden haben die Geschwindigkeit und Masse, in der biologische Daten generiert werden, in den letzten Jahren enorm gesteigert. Eine zentrale neue Technologie ist die Hochdurchsatzsequenzierung von DNA. Diese Technik eröffnet eine ganze Reihe Anwendungsmöglichkeiten in vielen Bereichen der Biologie, einschließlich der Ökologie, Evolution und Genomik. Neben den neuen Möglichkeiten treten jedoch auch neue Herausforderungen auf. So bedarf es spezialisierter Algorithmen und Computerprogramme, um mit der Masse an Daten umgehen zu können. Diese erfordern in der Regel ein fundiertes Training in bioinformatischen Methoden. Ein Weg, die Daten auch Wissenschaftlern ohne diesen Hintergrund zugänglich zu machen ist die Entwicklung von Programmen, die sich intuitiv bedienen lassen. In meiner Doktorarbeit habe ich Analysen und Programme entwickelt, um einige aktuelle Probleme mit Hochdurchsatzdaten in der Biologie zu lösen. Im Bereich der Ökologie umfasst das die Etablierung der bioinformatischen Methode, um Pollen DNA Metabarcoding durchzuführen. Darüberhinaus habe ich eine Anwendung entwickelt, die es ermöglicht Artgemeinschaften im Kontext ihrer Eigenschaften zu erforschen. Dazu wurden Informationen aus diversen öffentlichen Datenbanken zusammen getragen. Diese können nun automatisch auf bestehende Projekte übertragen und interaktiv analysiert werden. Im Bereich der Evolution ermöglichen die neuen Daten phylogenetische Berechnungen mit multiplen Genen durchzuführen. Um dies für Bakterien zu automatisieren habe ich das Programm bcgTree entwickelt. In den letzten Jahren wurden viele pflanzliche Genome sequenziert. Die Sequenzdaten des pflanzlichen Genoms enthalten auch die des Chloroplasten. Das Programm chloroExtractor unterstützt die gezielte Analyse des Chloroplasten Genoms. Um jedoch die Struktur mehrerer Genome miteinander vergleichen zu können, wird spezielle Software benötigt, die den Vergleich berechnen und visuell darstellen kann. Daher habe ich das Programm AliTV entwickelt. Im Gegensatz zu bestehenden Programmen erlaubt AliTV interaktive Anpassungen der erzeugten Grafik. Das erleichtert es die relevanten Informationen zu finden. Ein weiteres von mir entwickeltes Programm hilft dabei Transkriptom Daten zu analysieren, auch wenn kein Referenzgenom vorliegt. Dazu werden Informationen zu jedem Transkript, z.B. Funktion und Expressionslevel, in einer Webanwendung aggregiert. Forscher können diese durchsuchen, filtern und graphisch darstellen. Zusammen eröffnen die entwickelten Methoden und Programme die Möglichkeit, Erkenntnisse über biologische Systeme zu erlangen, die bislang nicht möglich waren. KW - bioinformatics KW - research software KW - ecology KW - evolution KW - genomics Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-156344 ER - TY - JOUR A1 - Esken, Jens A1 - Goris, Tobias A1 - Gadkari, Jennifer A1 - Bischler, Thorsten A1 - Förstner, Konrad U. A1 - Sharma, Cynthia M. A1 - Diekert, Gabriele A1 - Schubert, Torsten T1 - Tetrachloroethene respiration in Sulfurospirillum species is regulated by a two‐component system as unraveled by comparative genomics, transcriptomics, and regulator binding studies JF - MicrobiologyOpen N2 - Energy conservation via organohalide respiration (OHR) in dehalogenating Sulfurospirillum species is an inducible process. However, the gene products involved in tetrachloroethene (PCE) sensing and signal transduction have not been unambiguously identified. Here, genome sequencing of Sulfurospirillum strains defective in PCE respiration and comparative genomics, which included the PCE‐respiring representatives of the genus, uncovered the genetic inactivation of a two‐component system (TCS) in the OHR gene region of the natural mutants. The assumption that the TCS gene products serve as a PCE sensor that initiates gene transcription was supported by the constitutive low‐level expression of the TCS operon in fumarate‐adapted cells of Sulfurospirillum multivorans. Via RNA sequencing, eight transcriptional units were identified in the OHR gene region, which includes the TCS operon, the PCE reductive dehalogenase operon, the gene cluster for norcobamide biosynthesis, and putative accessory genes with unknown functions. The OmpR‐family response regulator (RR) encoded in the TCS operon was functionally characterized by promoter‐binding assays. The RR bound a cis‐regulatory element that contained a consensus sequence of a direct repeat (CTATW) separated by 17 bp. Its location either overlapping the −35 box or 50 bp further upstream indicated different regulatory mechanisms. Sequence variations in the regulator binding sites identified in the OHR gene region were in accordance with differences in the transcript levels of the respective gene clusters forming the PCE regulon. The results indicate the presence of a fine‐tuned regulatory network controlling PCE metabolism in dehalogenating Sulfurospirillum species, a group of metabolically versatile organohalide‐respiring bacteria. KW - genomics KW - organohalide respiration KW - RNA sequencing KW - tetrachloroethene KW - transcriptomics KW - two‐component system Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-225754 VL - 9 IS - 12 ER - TY - JOUR A1 - Schartl, Manfred A1 - Walter, Ronald B. A1 - Shen, Yingjia A1 - Garcia, Tzintzuni A1 - Catchen, Julian A1 - Amores, Angel A1 - Braasch, Ingo A1 - Chalopin, Domitille A1 - Volff, Jean-Nicolas A1 - Lesch, Klaus-Peter A1 - Bisazza, Angelo A1 - Minx, Pat A1 - Hillier, LaDeana A1 - Wilson, Richard K. A1 - Fürstenberg, Susan A1 - Boore, Jeffrey A1 - Searle, Steve A1 - Postlethwait, John H. A1 - Warren, Wesley C. T1 - The genome of the platyfish, Xiphophorus maculatus, provides insights into evolutionary adaptation and several complex traits JF - Nature Genetics N2 - Several attributes intuitively considered to be typical mammalian features, such as complex behavior, live birth and malignant disease such as cancer, also appeared several times independently in lower vertebrates. The genetic mechanisms underlying the evolution of these elaborate traits are poorly understood. The platyfish, X. maculatus, offers a unique model to better understand the molecular biology of such traits. We report here the sequencing of the platyfish genome. Integrating genome assembly with extensive genetic maps identified an unexpected evolutionary stability of chromosomes in fish, in contrast to in mammals. Genes associated with viviparity show signatures of positive selection, identifying new putative functional domains and rare cases of parallel evolution. We also find that genes implicated in cognition show an unexpectedly high rate of duplicate gene retention after the teleost genome duplication event, suggesting a hypothesis for the evolution of the behavioral complexity in fish, which exceeds that found in amphibians and reptiles. KW - genomics KW - genomic analysis KW - evolutionary biology Y1 - 2013 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-132152 VL - 45 IS - 5 ER - TY - JOUR A1 - Höhne, Christin A1 - Prokopov, Dmitry A1 - Kuhl, Heiner A1 - Du, Kang A1 - Klopp, Christophe A1 - Wuertz, Sven A1 - Trifonov, Vladimir A1 - Stöck, Matthias T1 - The immune system of sturgeons and paddlefish (Acipenseriformes): a review with new data from a chromosome‐scale sturgeon genome JF - Reviews in Aquaculture N2 - Sturgeon immunity is relevant for basic evolutionary and applied research, including caviar‐ and meat‐producing aquaculture, protection of wild sturgeons and their re‐introduction through conservation aquaculture. Starting from a comprehensive overview of immune organs, we discuss pathways of innate and adaptive immune systems in a vertebrate phylogenetic and genomic context. The thymus as a key organ of adaptive immunity in sturgeons requires future molecular studies. Likewise, data on immune functions of sturgeon‐specific pericardial and meningeal tissues are largely missing. Integrating immunological and endocrine functions, the sturgeon head kidney resembles that of teleosts. Recently identified pattern recognition receptors in sturgeon require research on downstream regulation. We review first acipenseriform data on Toll‐like receptors (TLRs), type I transmembrane glycoproteins expressed in membranes and endosomes, initiating inflammation and host defence by molecular pattern‐induced activation. Retinoic acid‐inducible gene‐I‐like (RIG‐like) receptors of sturgeons present RNA and key sensors of virus infections in most cell types. Sturgeons and teleosts share major components of the adaptive immune system, including B cells, immunoglobulins, major histocompatibility complex and the adaptive cellular response by T cells. The ontogeny of the sturgeon innate and onset of adaptive immune genes in different organs remain understudied. In a genomics perspective, our new data on 100 key immune genes exemplify a multitude of evolutionary trajectories after the sturgeon‐specific genome duplication, where some single‐copy genes contrast with many duplications, allowing tissue specialization, sub‐functionalization or both. Our preliminary conclusion should be tested by future evolutionary bioinformatics, involving all >1000 immunity genes. This knowledge update about the acipenseriform immune system identifies several important research gaps and presents a basis for future applications. KW - evolution KW - genomics KW - immune genes KW - immune organs KW - immune system KW - sturgeon Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239865 VL - 13 IS - 3 SP - 1709 EP - 1729 ER - TY - THES A1 - Liang, Chunguang T1 - Tools for functional genomics applied to Staphylococci, Listeriae, Vaccinia virus and other organisms N2 - Genome sequence analysis A combination of genome analysis application has been established here during this project. This offers an efficient platform to interactively compare similar genome regions and reveal loci differences. The genes and operons can be rapidly analyzed and local collinear blocks (LCBs) categorized according to their function. The features of interests are parsed, recognized, and clustered into reports. Phylogenetic relationships can be readily examined such as the evolution of critical factors or a certain highly-conserved region. The resulting platform-independent software packages (GENOVA and inGeno), have been proven to be efficient and easy to handle in a number of projects. The capabilities of the software allowed the investigation of virulence factors, e.g., rsbU, strains’ biological design, and in particular pathogenicity feature storage and management. We have successfully investigated the genomes of Staphylococcus aureus strains (COL, N315, 8325, RN1HG, Newman), Listeria spp. (welshimeri, innocua and monocytogenes), E.coli strains (O157:H7 and MG1655) and Vaccinia strains (WR, Copenhagen, Lister, LIVP, GLV-1h68 and parental strains). Metabolic network analysis Our YANAsquare package offers a workbench to rapidly establish the metabolic network of such as Staphylococcous aureus bacteria in genome-scale size as well as metabolic networks of interest such as the murine phagosome lipid signalling network. YANAsquare recruits reactions from online databases using an integrated KEGG browser. This reduces the efforts in building large metabolic networks. The involved calculation routines (METATOOL-derived wrapper or native Java implementation) readily obtain all possible flux modes (EM/EP) for metabolite fluxes within the network. Advanced layout algorithms visualize the topological structure of the network. In addition, the generated structure can be dynamically modified in the graphic interface. The generated network as well as the manipulated layout can be validated and stored (XML file: scheme of SBML level-2). This format can be further parsed and analyzed by other systems biology software, such as CellDesigner. Moreover, the integrated robustness-evaluation routine is able to examine the synthesis rates affected by each single mutation throughout the whole network. We have successfully applied the method to simulate single and multiple gene knockouts, and the affected fluxes are comprehensively revealed. Recently we applied the method to proteomic data and extra-cellular metabolite data of Staphylococci, the physiological changes regarding the flux distribution are studied. Calculations at different time points, including different conditions such as hypoxia or stress, show a good fit to experimental data. Moreover, using the proteomic data (enzyme amounts) calculated from 2D-Gel-EP experiments our study provides a way to compare the fluxome and the enzyme expression. Oncolytic vaccinia virus (VACV) We investigated the genetic differences between the de novo sequence of the recombinant oncolytic GLV-1h68 and other related VACVs, including function predictions for all found genome differences. Our phylogenetic analysis indicates that GLV-1h68 is closest to Lister strains but has lost several ORFs present in its parental LIVP strain, including genes encoding CrmE and a viral Golgi anti-apoptotic protein, v-GAAP. Functions of viral genes were either strain-specific, tissue-specific or host-specific comparing viral genes in the Lister, WR and COP strains. This helps to rationally design more optimized oncolytic virus strains to benefit cancer therapy in human patients. Identified differences from the comparison in open reading frames (ORFs) include genes for host-range selection, virulence and immune modulation proteins, e.g. ankyrin-like proteins, serine proteinase inhibitor SPI-2/CrmA, tumor necrosis factor (TNF) receptor homolog CrmC, semaphorin-like and interleukin-1 receptor homolog proteins. The contribution of foreign gene expression cassettes in the therapeutic and oncolytic virus GLV-1h68 was studied, including the F14.5L, J2R and A56R loci. The contribution of F14.5L inactivation to the reduced virulence is demonstrated by comparing the virulence data of GLV-1h68 with its F14.5L-null and revertant viruses. The comparison suggests that insertion of a foreign gene expression cassette in a nonessential locus in the viral genome is a practical way to attenuate VACVs, especially if the nonessential locus itself contains a virulence gene. This reduces the virulence of the virus without compromising too much the replication competency of the virus, the key to its oncolytic activity. The reduced pathogenicity of GLV-1h68 was confirmed by our experimental collaboration partners in male mice bearing C6 rat glioma and in immunocompetent mice bearing B16-F10 murine melanoma. In conclusion, bioinformatics and experimental data show that GLV-1h68 is a promising engineered VACV variant for anticancer therapy with tumor-specific replication, reduced pathogenicity and benign tissue tropism. N2 - Genom Sequenz Analyse Im Zuge der vorliegenden Doktorarbeit wurden verschiedene Programme zur Genomanalyse kombiniert, um eine effiziente Plattform zum interaktiven Vergleich lokaler Ähnlichkeiten bzw. Unterschiede in Genomen bereitzustellen. Damit können Gene und Operons schnell untersucht und “local collinear blocks” entsprechend ihrer Funktion kategorisiert werden. Phylogenetische Beziehungen, wie beispielsweise die Evolution spezifischer Elemente oder stark konservierter Regionen können leicht überprüft werden. Die hierfür entwickelte plattformunabhängige Software (GENOVA und inGeno) hat sich in mehreren Projekten als effizient und leicht handhabbar bewährt. Die Programme erlauben die Untersuchung von Virulenzfaktoren auf Sequenz- oder Annotationsebene. Während der vorliegenden Doktorarbeit konnten so die Genome von verschiedenen Staphylococcus aureus, Listeria spp., Escherichia coli und Vaccinia Stämmen untersucht werden. Metabolische Netzwerk Analyse Unser “YANAsquare” Programmpaket bietet eine Oberfläche um schnell metabolische Netzwerke vom genomweiten Anzatz bis hinunter zum Einzelnetzwerk zu analysieren. Dafür greift YANA mit Hilfe des integrierten KEGG-Browsers auf Onlinedatenbanken zu, um die notwendigen Informationen zum metabolischen Reaktionsweg bereitzustellen und reduziert so maßgeblich den Arbeitsaufwand beim Beschreiben von Netzwerke. Die implementierten Methoden zur Berechnung (METATOOL, eigene Implementation in Java) des Netzwerkes liefern exakt alle die möglichen Elementarmoden (EM/EP) für die Metabolite zurück. Durch den Einsatz von fortgeschrittenen Layout Algorithmen wird anschliessend die Darstellung der Netzwerktopologie möglich. Außerdem kann in der grafischen Darstellung das generierte Netzwerklayout dynamisch verändert werden. Das Speichern der Daten erfolgt im XML (SBML level-2) Format und erlaubt so die Weiterverwendung in anderen systembiologischen Programmen, wie dem “CellDesigner”. Mit Hilfe einer gen-Knockout Simulations Methode kann der Einfluss von einzelnen Mutationen im gesamten Netzwerk auf die Syntheseraten untersucht werden. Wir konnten mit dieser Methode Einzel- sowie Mehrfachgenknockouts und deren Effekte auf die Elementarmoden analysieren. Die Methode wurde ebenfalls auf Proteomdaten und extrazelluläre Metabolite von Staphylokokken angewandt, um Änderungen bezüglich der Flussverteilung zu untersuchen. Die Simulationen zu verschieden Zeitpunkten und unter verschiedenen Stessbedingungen zeigen große Übereinstimmung mit experimentell erhobenen Daten. Onkolytischer Vaccinia Virus (VACV) Wir haben die genetischen Unterschiede zwischen der de novo Sequenz des rekombinanten onkolytischen Virus GLV-1h68 und anderen VACVs untersucht und gefundene Unterschiede funktionell charakterisiert. Die phylogenetische Analyse zeigt das GLV-1h68 mit dem Lister Stamm am nächsten verwandt ist. Auffällig ist dabei der Verlust von einigen open reading frames (ORFs), die noch im Eltern LIVP Stamm vorhanden sind (CrmE, v-GAAP). Beim Vergleich der Funktion viraler Gene aus Lister, WR und COP Stämmen treten stamm-, gewebe- und wirtsspezifische Gene auf. Diese Tatsache ermöglicht die Optimierung der onkolytischen Virusstämme für den Einsatz bei humanen Krebstherapien. Die beim Vergleich identifizierten Unterschiede zwischen den ORFs enthalten Gene für die Wirtsselektion, Virulenz und immunmodulierende Proteine (Ankyrin ähnliche Proteine, Serine-Proteinasen Inhibitor SPI-2/CrmA, Tumor Nekrose Faktor (TNF) Rezeptorhomolog CrmC, semaphorinähnliche und Interleukin-1 rezeptorhomologe Proteine). An den Loki F14.5L, J2R und A56R des GLV-1h68 Virus wurden die Vorteile der eingesetzten fremden Genexpressionskassetten untersucht. So zeigt GLV-1h68 mit F14.5L-Inaktivierung gegenüber der F14.5L-Revertanten Viren eine reduzierte Virulenz. Das erlaubt die Schlussfolgerung, dass die Insertion von fremden Genexpressionskassetten in nicht-essentielle Loki zur Verminderung der Virulenz von VACVs führt, besonders, wenn der nicht-essentielle Lokus selbst ein Virulenzgen enthält. Das Replikationsvermögen, welches ausschlaggebend für die onkolytische Aktivität des Virus ist, wird trotz der verminderten Virulenz nicht eingeschränkt. Die reduzierte Pathogenität des GLV-1h68 Virus wurde durch experimentelle Daten unserer Kollaborationspartner in männlichen Mäusen mit Ratten C6 Gliom und in immunokompetenten Mäusen mit B16-F10 Mausmelanom nachgewiesen. Zusammenfassend zeigen experimentelle und bioinformatisch gewonnene Daten, dass GLV-1h68 eine vielversprechende VACV Variante für die Krebstherapie mit tumorspezifischer Replikation, verringerter Pathogenität und hoher Gewebsspezifität ist. KW - Genanalyse KW - Bioinformatik KW - Systembiologie KW - bacterial KW - virulence KW - systems biologie KW - genomic KW - algorithm KW - metabolic KW - network KW - pathway KW - flux KW - Bacterial KW - genomics KW - algorithm KW - tool KW - metabolic Y1 - 2009 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-48051 ER -