TY - THES A1 - Förster, Frank T1 - Making the most of phylogeny: Unique adaptations in tardigrades and 216374 internal transcribed spacer 2 structures T1 - Einzigartige Anpassungen in Tardigraden und 216374 "internal transcribed spacer 2" Strukturen N2 - The phylum Tardigrada consists of about 1000 described species to date. The animals live in habitats within marine, freshwater and terrestrial ecosystems allover the world. Tardigrades are polyextremophiles. They are capable to resist extreme temperature, pressure or radiation. In the event of desiccation, tardigrades enter a so-called tun stage. The reason for their great tolerance capabilities against extreme environmental conditions is not discovered yet. Our Funcrypta project aims at finding answers to the question what mechanisms underlie these adaption capabilities particularly with regard to the species Milnesium tardigradum. The first part of this thesis describes the establishment of expressed sequence tags (ESTs) libraries for different stages of M. tardigradum. From proteomics data we bioinformatically identified 144 proteins with a known function and additionally 36 proteins which seemed to be specific for M. tardigradum. The generation of a comprehensive web-based database allows us to merge the proteome and transcriptome data. Therefore we created an annotation pipeline for the functional annotation of the protein and nucleotide sequences. Additionally, we clustered the obtained proteome dataset and identified some tardigrade-specific proteins (TSPs) which did not show homology to known proteins. Moreover, we examined the heat shock proteins of M. tardigradum and their different expression levels depending on the actual state of the animals. In further bioinformatical analyses of the whole data set, we discovered promising proteins and pathways which are described to be correlated with the stress tolerance, e.g. late embryogenesis abundant (LEA) proteins. Besides, we compared the tardigrades with nematodes, rotifers, yeast and man to identify shared and tardigrade specific stress pathways. An analysis of the 50 and 30 untranslated regions (UTRs) demonstrates a strong usage of stabilising motifs like the 15-lipoxygenase differentiation control element (15-LOX-DICE) but also reveals a lack of other common UTR motifs normally used, e.g. AU rich elements. The second part of this thesis focuses on the relatedness between several cryptic species within the tardigrade genus Paramacrobiotus. Therefore for the first time, we used the sequence-structure information of the internal transcribed spacer 2 (ITS2) as a phylogenetic marker in tardigrades. This allowed the description of three new species which were indistinguishable using morphological characters or common molecular markers like the 18S ribosomal ribonucleic acid (rRNA) or the Cytochrome c oxidase subunit I (COI). In a large in silico simulation study we also succeeded to show the benefit for the phylogenetic tree reconstruction by adding structure information to the ITS2 sequence. Next to the genus Paramacrobiotus we used the ITS2 to corroborate a monophyletic DO-group (Sphaeropleales) within the Chlorophyceae. Additionally we redesigned another comprehensive database—the ITS2 database resulting in a doubled number of sequence-structure pairs of the ITS2. In conclusion, this thesis shows the first insights (6 first author publications and 4 coauthor publications) into the reasons for the enormous adaption capabilities of tardigrades and offers a solution to the debate on the phylogenetic relatedness within the tardigrade genus Paramacrobiotus. N2 - Der Tierstamm Tardigrada besteht aus derzeitig etwa 1000 beschriebenen Arten. Die Tiere leben in Habitaten in marinen, limnischen und terrestrischen Ökosystemen auf der ganzen Welt. Tardigraden sind polyextremophil. Sie können extremer Temperatur, Druck oder Strahlung widerstehen. Beim Austrocknen bilden sie ein so genanntes Tönnchenstadium. Der Grund für die hohe Toleranz gegenüber extremen Umweltbedingungen ist bis jetzt nicht aufgeklärt worden. Unser Funcrypta Projekt versucht Antworten darauf zu finden, was die hinter dieser Anpassungsfähigkeit liegenden Mechanismen sind. Dabei steht die Art Milnesium tardigradum im Mittelpunkt. Der erste Teil dieser Arbeit beschreibt die Etablierung einer expressed sequence tags (ESTs) Bibliothek für verschiedene Stadien von M. tardigradum. Aus unseren Proteomansatz konnten wir bislang 144 Proteine bioinformatisch identifizieren, denen eine Funktion zugeordnet werden konnte. Darüber hinaus wurden 36 Proteine gefunden, welche spezifisch für M. tardigradum zu sein scheinen. Die Erstellung einer umfassenden internetbasierenden Datenbank erlaubt uns die Verknüpfung der Proteom und Transkriptomdaten. Dafür wurde eine Annotations-Pipeline erstellt um den Sequenzen Funktionen zuordnen zu können. Außerdem wurden die erhaltenen Proteindaten von uns geclustert. Dabei konnten wir einige Tardigraden-spezifische Proteine (tardigrade-specific protein, TSP) identifizieren die keinerlei Homologie zu bekannten Proteinen zeigen. Außerdem untersuchten wir die Hitze-Schock-Proteine von M. tardigradum und deren differenzielle Expression in Abhängigkeit vom Stadium der Tiere. In weiteren bioinformatischen Analysen konnten wir viel versprechende Proteine und Stoffwechselwege entdecken für die beschrieben ist, dass sie mit Stressreaktionen in Verbindung stehen, beispielsweise late embryogenesis abundant (LEA) Proteine. Des Weiteren verglichen wir Tardigraden mit Nematoden, Rotatorien, Hefe und dem Menschen, um gemeinsame und Tardigraden-spezifische Stoffwechselwege identifizieren zu können. Analysen der 50 und 30 untranslatierten Bereiche zeigen eine verstärkte Nutzung von stabilisierenden Motiven, wie dem 15-lipoxygenase differentiation control element (LEA). Im Gegensatz dazu werden häufig benutzte Motive, wie beispielsweise AU-reiche Bereiche, gar nicht gefunden. Der zweite Teil der Doktorarbeit beschäftigt sich mit den Verwandtschaftsverhältnissen einiger kryptischer Arten in der Tardigradengattung Paramacrobiotus. Hierfür haben wir, zum ersten Mal in Tardigraden, die Sequenz-Struktur-Informationen der internal transcribed spacer 2 Region als phylogenetischen Marker verwendet. Dies erlaubte uns die Beschreibung von drei neuen Arten, welche mit klassischen morphologischen Merkmalen oder anderen molekularen Markern wie 18S ribosomaler RNA oder Cytochrome c oxidase subunit I (COI) nicht unterschieden werden konnten. In einer umfangreichen in silico Simulationsstudie zeigten wir den Vorteil der bei der Rekonstruktion phylogenetischer Bäume unter der Hinzunahme der Strukturinformationen zur Sequenz der ITS2 entsteht. ITS2 Sequenz-Struktur-Informationen wurden außerdem auch dazu benutzt, eine monophyletische DO-Gruppe (Sphaeropleales) in den Chlorophyceae zu bestätigen. Zusätzlich haben wir eine umfassende Datenbank, die ITS2-Datenbank, überarbeitet. Dadurch konnten die Sequenz-Struktur-Informationen verdoppelt werden, die in dieser Datenbank verfügbar sind. Die vorliegende Doktorarbeit zeigt erste Einblicke (6 Erstautor- und 4 Koautor-Publikationen) in die Ursachen für die hervorragende Anpassungsfähigkeit der Tardigraden und beschreibt die erfolgreiche Aufklärung der Verwandtschaftsverhältnisse in der Tardigradengattung Paramacrobiotus. KW - Phylogenie KW - Bioinformatik KW - Würzburg / Universität / Lehrstuhl für Bioinformatik KW - Anpassung KW - Datenbank KW - ITS2 KW - Marker KW - Tardigraden KW - Bärtierchen KW - ITS2 KW - Marker KW - Tardigrades KW - Waterbear Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-51466 ER - TY - JOUR A1 - Hoppe, J. A1 - Sebald, Walter T1 - Amino acid sequence of the proteolipid subunit of the proton-translocating ATPase complex from the thermophilic bacterium PS-3 N2 - No abstract available KW - Biochemie Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62754 ER - TY - JOUR A1 - Bollazzi, Martin A1 - Roces, Flavio T1 - Information Needs at the Beginning of Foraging: Grass-Cutting Ants Trade Off Load Size for a Faster Return to the Nest N2 - Background: Acquisition of information about food sources is essential for animals that forage collectively like social insects. Foragers deliver two commodities to the nest, food and information, and they may favor the delivery of one at the expenses of the other. We predict that information needs should be particularly high at the beginning of foraging: the decision to return faster to the nest will motivate a grass-cutting ant worker to reduce its loading time, and so to leave the source with a partial load. Principal Findings: Field results showed that at the initial foraging phase, most grass-cutting ant foragers (Acromyrmex heyeri) returned unladen to the nest, and experienced head-on encounters with outgoing workers. Ant encounters were not simply collisions in a probabilistic sense: outgoing workers contacted in average 70% of the returning foragers at the initial foraging phase, and only 20% at the established phase. At the initial foraging phase, workers cut fragments that were shorter, narrower, lighter and tenderer than those harvested at the established one. Foragers walked at the initial phase significantly faster than expected for the observed temperatures, yet not at the established phase. Moreover, when controlling for differences in the fragment-size carried, workers still walked faster at the initial phase. Despite the higher speed, their individual transport rate of vegetable tissue was lower than that of similarly-sized workers foraging later at the same patch. Conclusions/Significance: At the initial foraging phase, workers compromised their individual transport rates of material in order to return faster to the colony. We suggest that the observed flexible cutting rules and the selection of partial loads at the beginning of foraging are driven by the need of information transfer, crucial for the establishment and maintenance of a foraging process to monopolize a discovered resource. KW - Blattschneiderameisen Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68940 ER - TY - JOUR A1 - Karunakaran, Karthika A1 - Mehlitz, Adrian A1 - Rudel, Thomas T1 - Evolutionary conservation of infection-induced cell death inhibition among Chlamydiales N2 - Control of host cell death is of paramount importance for the survival and replication of obligate intracellular bacteria. Among these, human pathogenic Chlamydia induces the inhibition of apoptosis in a variety of different host cells by directly interfering with cell death signaling. However, the evolutionary conservation of cell death regulation has not been investigated in the order Chlamydiales, which also includes Chlamydia-like organisms with a broader host spectrum. Here, we investigated the apoptotic response of human cells infected with the Chlamydia-like organism Simkania negevensis (Sn). Simkania infected cells exhibited strong resistance to apoptosis induced by intrinsic stress or by the activation of cell death receptors. Apoptotic signaling was blocked upstream of mitochondria since Bax translocation, Bax and Bak oligomerisation and cytochrome c release were absent in these cells. Infected cells turned on pro-survival pathways like cellular Inhibitor of Apoptosis Protein 2 (cIAP-2) and the Akt/PI3K pathway. Blocking any of these inhibitory pathways sensitized infected host cell towards apoptosis induction, demonstrating their role in infection-induced apoptosis resistance. Our data support the hypothesis of evolutionary conserved signaling pathways to apoptosis resistance as common denominators in the order Chlamydiales. KW - Chlamydiales Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68978 ER - TY - JOUR A1 - Pahl, Mario A1 - Zhu, Hong A1 - Tautz, Jürgen A1 - Zhang, Shaowu T1 - Large Scale Homing in Honeybees N2 - Honeybee foragers frequently fly several kilometres to and from vital resources, and communicate those locations to their nest mates by a symbolic dance language. Research has shown that they achieve this feat by memorizing landmarks and the skyline panorama, using the sun and polarized skylight as compasses and by integrating their outbound flight paths. In order to investigate the capacity of the honeybees’ homing abilities, we artificially displaced foragers to novel release spots at various distances up to 13 km in the four cardinal directions. Returning bees were individually registered by a radio frequency identification (RFID) system at the hive entrance. We found that homing rate, homing speed and the maximum homing distance depend on the release direction. Bees released in the east were more likely to find their way back home, and returned faster than bees released in any other direction, due to the familiarity of global landmarks seen from the hive. Our findings suggest that such large scale homing is facilitated by global landmarks acting as beacons, and possibly the entire skyline panorama. KW - Biene Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68985 ER - TY - THES A1 - Schramm, Sabine T1 - SYCE3, ein neues Synaptonemalkomplexprotein: Expression, funktionelle Analyse und Bindungspartner T1 - SYCE3, a novel synaptonemal complex protein:Expression, functional analysis and binding partners N2 - Der Synaptonemalkomplex ist eine evolutionär hoch konservierte Struktur. Er wird spezifisch während der Prophase I der Meiose ausgebildet und ist essentiell für die Segregation der homologen Chromosomen während der Meiose und auch für die Entstehung genetischer Vielfalt. Der Synaptonemalkomplex ist eine proteinöse Struktur, deren Aufbau dem einer Leiter ähnelt. Dabei werden die Leiterholme als Lateralelemente bezeichnet. Sie bestehen unter anderem aus den Proteinen SYCP2 und SYCP3 und assoziieren mit dem Chromatin der homologen Chromosomen. Die Stufen der Leiter bestehen hingegen aus Transversalfilamenten, deren Hauptkomponente parallele Homodimere des meiosespezifische Proteins SYCP1 sind. Dabei wird ein SYCP1 Dimer mit seinem C-Terminus in den Lateralelementen verankert und kann über seine N-terminale Domäne eine schwache Interaktion mit der N-terminalen Domäne eines gegenüberliegenden SYCP1 Dimers eingehen. Um diese Bindung zu stabilisieren werden Proteine des Zentralelements des Synaptonemalkomplexes benötigt: Während SYCE1 durch seine Interaktion mit SYCP1 die N-terminale Assoziation zweier gegenüberliegender SYCP1 Dimere stabilisiert, verknüpfen die zwei anderen zentralelementspezifischen Proteine SYCE2 und Tex12 lateral benachbarte SYCP1 Filamente und breiten so das SYCP1 Netzwerk entlang der chromosomalen Achsen aus. Dieser Prozess wird als Synapse bezeichnet und stellt eines der Schlüsselereignisse der Meiose dar. Fehler während dieses Prozesses führen meist zu Aneuploidie der entstehenden Gameten oder zum Abbruch der Meiose und somit zu Infertilität des betroffenen Organismus. In dieser Arbeit wurde mit SYCE3 ein neues Protein des murinen Synaptonemalkomplexes charakterisiert. Es konnte gezeigt werden, dass SYCE3 meiosespezifisch in Männchen und Weibchen exprimiert wird und Bestandteil des Zentralelements des Synaptonemalkomplexes ist. Hierbei zeigt es dasselbe Verteilungsmuster wie SYCP1 und SYCE1 und kann mit beiden Proteinen interagieren. Eine zusätzliche Interaktion konnte zwischen SYCE3 und SYCE2 nachgewiesen werden. Durch Untersuchungen an entsprechenden Knockout Mausmodellen konnte in dieser Arbeit außerdem gezeigt werden, dass SYCE3 in Abwesenheit von SYCP1 nicht an die chromosomalen Achsen rekrutiert werden kann. Die Ausbildung der Lateralelemente und auch die Anwesenheit der anderen zentralelementspezifischen Proteine SYCE1 und SYCE2 sind hingegen für die Anlagerung von SYCE3 an die chromosomalen Achsen nicht essentiell. Somit steht SYCE3 hinsichtlich seiner Bedeutung für die Paarung und die Synapse der homologen Chromosomen hierarchisch offenbar über den bisher beschriebenen Zentralelementproteinen SYCE1, SYCE2 und Tex12. Die funktionelle Bedeutung von SYCE3 für die Synapse der homologen Chromosomen und für den korrekten Ablauf der homologen Rekombination wurde im Rahmen dieser Arbeit durch die Herstellung und die Charakterisierung einer Syce3-/- Maus detailliert untersucht: Dabei führte der Knockout von SYCE3 zur Infertilität in beiden Geschlechtern, die gleichzeitig mit einer signifikanten Reduktion der Größe der entsprechenden Hoden und Ovarien im Vergleich zum Wildtyp einherging. Weitere Untersuchungen ergaben zudem, dass es in Syce3 defizienten Tieren zu einem Abbruch der Meiose kommt. Dabei hatte das Fehlen von SYCE3 keinen Einfluss auf die Ausbildung der Axialelemente. Die Initiation der Synapse hingegen war sowohl in Oocyten als auch in Spermatocyten in Abwesenheit von SYCE3 stark gestört. Darüber hinaus konnte in der vorliegenden Arbeit nachgewiesen werden, dass das Fehlen von SYCE3 Einfluss auf die homologe Rekombination nimmt: Zwar können sich frühe (DNA Doppelstrangbrüche) und intermediäre (Transitionsknoten) Rekombinationsereignisse in der Abwesenheit von SYCE3 ausbilden, die Prozessierung zu späten Rekombinationsstrukturen (Rekombinationsknoten) und die damit einhergehende Ausbildung von Crossing-over Strukturen fand jedoch nicht statt. Zusammengefasst wurde in dieser Arbeit gezeigt, dass das neue Synaptonemalkomplexprotein SYCE3 essentiell für die Fertilität von Mäusen ist. Durch den Knockout von Syce3 kann die Synapse zwischen den Homoligen nicht initiiert werden und es findet kein Crossing-over statt. Im Assembly Prozess des Synaptonemalkomplexes agiert SYCE3 oberhalb der anderen zentralelementspezifischen Proteine und unterhalb von SYCP1. N2 - The synaptonemal complex is an evolutionary highly conserved structure. It assembles specifically during prophase I of meiosis and is essential for the segregation of homologous chromosomes and thus represents a major determinant of the genetic diversity of sexually reproducing organisms. The synaptonemal complex is a proteinacious, ladder-like structure. The ladder beams are termed lateral elements and are composed of the meiosis-specific proteins SYCP2 and SYCP3 which are associated with the chromatin of the homologs. The rungs are made up of transverse filaments mainly consisting of the meiosis-specific protein SYCP1. SYCP1 forms parallel homodimers that are anchored via their C-termini to the lateral elements and interact in a head-to-head fashion with an opposing SYCP1 homodimer. For stabilizing this interaction additional proteins are essential. These are components of the so-called central element of the synaptonemal complex: while SYCE1 stabilizes the N-terminal association of opposing SYCP1 homodimers, the two other central element specific proteins SYCE2 and Tex12 connect adjoined SYCP1 filaments and thus elongate the SYCP1 network along the homologs. This process is termed synapsis and is a key feature of meiosis. Errors occurring during this process frequently lead to aneuploidy of the resulting gametes or cause meiotic arrest and infertility. Within the scope of this study a novel protein of the murine synaptonemal complex, we named SYCE3, was characterized. SYCE3 is exclusively expressed during male and female meiosis and is a component of the central element. Its expression pattern resembles that of SYCP1 and SYCE1 and it is able to interact with both of these proteins. Additionally, an interaction between SYCE3 and SYCE2 could be verified. In the context of this dissertation it was found that loading of SYCE3 to the chromosomal axis requires SYCP1. In contrast, chromosome loading of SYCE3 was independent of lateral element assembly and of the presence of the other central element specific proteins, SYCE1, SYCE2 and Tex12. The second thematic complex addressed in this thesis was the relevance of SYCE3 for synapsis and homologous recombination. To this end a Syce3-/- mouse was generated. Syce3-/- mice are infertile and both testes and ovaries are characterized by a significant reduction in size compared to wild-type littermates. Furthermore, depletion of SYCE3 had no influence on the assembly of axial elements and in males alignment of homologs was not affected. However, Syce3-/- oocytes and spermatocytes were unable to initiate synapsis between homologous chromosomes. In addition, homologous recombination was analyzed in the scope of this study and the obtained data strongly points to a central role of SYCE3 during this process: while early (DNA double-strand breaks) and intermediate (transition nodules) recombination events could take place in the absence of SYCE3, structures indicating late recombination events (recombination nodules) and sites of homologous recombination (crossovers) failed to develop. Taken together, this thesis clearly demonstrates that the novel synaptonemal complex protein SYCE3 is essential for fertility in mice. Deletion of Syce3 blocks initiation of synapsis and formation of crossovers. During synaptonemal complex assembly, SYCE3 acts downstream of SYCP1, but upstream of other central element proteins (SYCE1, SYCE2 and Tex12). KW - Meiose KW - Molekularbiologie KW - Fertilität KW - Synaptonemalkomplex KW - Meiosis KW - Synaptonemal complex KW - fertility Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70903 ER - TY - JOUR A1 - Hoppe, J. A1 - Gatti, D. A1 - Weber, H. A1 - Sebald, Walter T1 - Labeling of individual amino acid residues in the membrane-embedded F\(_0\) part of the F\(_1\) F\(_0\) ATP synthase from Neurospora crassa. Influence of oligomycin and dicyclohexylcarbodiimide N2 - Three F0 subunits and the F\(_1\) subunit P of the ATP synthase from Neurospora crassa were labeled with the lipophilic photoactivatable reagent 3-(trifluoromethyl)-3-(m-[\(^{125}\)I]iodophenyl)diazirine ([\(^{125}\)I]TID). In the proteolipid subunit which was the most heavily labeled polypeptide labeling was confmed to five residues at the NH2-terminus and five residues at the C-terminus ofthe protein. Labeling occurred at similar positions compared with the homologaus protein (subunit c) in the ATP synthase from Escherichia coli, indicating a similar structure of the proteolipid subunits in their respective organisms. The inhibitors oligomycin and dicyclohexylcarbodiimide did not change the pattern of accessible surface residues in the proteolipid, suggesting that neither inhibitor induces gross conformational changes. However, in the presence of oligomycin, the extent oflabeling in some residues was reduced. Apparently, these residues provide part of the binding site for the inhibitor. After reaction with dicyclohexylcarbodiimide an additional labeled amino acid was found at position 65 corresponding to the invariant carbodümide-binding glutamic acid. These results and previous observations indicate that the carboxyl side chain of Glu-65 is located at the protein-lipid interphase. The idea is discussed that proton translocation occurs at the interphase between different types if F\(_0\) subunits. Dicyclohexylcarbodiimide or oligomycin might disturb this essential interaction between the F\(_0\) subunits. KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62598 ER - TY - JOUR A1 - Hoppe, J. A1 - Sebald, Walter T1 - Topological studies suggest that the pathway of the protons through F\(_0\) is provided by amino acid residues accessible from the lipid phase N2 - The structure of the F0 part of ATP synthases from E. coli and Neurospora crassa was analyzed by hydrophobic surface labeling with [125I]TID. In the E. co/i F0 all three subunits were freely accessible to the reagent, suggesting that these subunits are independently integrated in the membrane. Labeted amino acid residues were identified by Edman degradation of the dicyclohexylcarbodiimide binding (DCCD) proteins from E. coli and Neurospora crassa. The very similar patterns obtained with the two homologaus proteins suggested the existence of tightly packed cx-helices. The oligomeric structure of the DCCD binding protein appeared to be very rigid since little, if any, change in the labeling patternwas observed upon addition of oligomycin or DCCD to membranes from Neurospora crassa. When membrancs were pretrcated with DCCD prior to the reaction with [125I]TID an additionally labeled amino acid appeared at the position of Glu·65 which binds DCCD covalently, indicating the Jocation of this inhibitor on the outside of the oligomer. It is suggested that proton conduction occurs at the surface of the oligomer of the DCCD binding protein. Possibly this oligomer rotates against the subunit a or b and thus enables proton translocation. Conserved residues in subunit a, probably located in the Iipid bilayer, might participate in the pro· ton translocation mechanism. N2 - La structure de la partie F0 de l'ATP synthase a ete analysee au moyen de marquage par /e reactif hydrophobe TJD[125 I]. Les trois sous-unites de E. coli F0 sont accessibles au reactif ce qui semble indiquer que ces sous-unites sont integrees dans Ia membrane defaron independante. Les amino-acides marques ont ete identifies par Ia degradation d'Edman des profeines d'E. coli et de Neurospora associees au dicyclohexylcarbodiimide (DCCD). L 'analogie des courbes obtenues pour /es deux proteines homologues suggere l'existence d'a-helices rangees de faron serree. La structure oligomerique de Ia proreine associee au DCCD semble etre tres rigide puisque pratiquement aucun changement dans /e marquage n 'a ete observe par addition d'oligomycine ou de DCCD aux membranes de Neurospora crassa. Quand /es membranes sont traitees avec /e DCCD avant Ia reaction avec TJD[125 I], un amino-acide additionnellement marque apparait a Ia position Glu·65 et forme avec le DCCD une Iiaison covalente. Ce dernier resu/tat indique Ia localisation de cet inhibiteur a /'exterieur de J'oligo· mere. II semble donc que Ia conduction des protons ait lieu a Ia surface de /'oligomere de Ia proteine associee au DCCD. II serait possib/e que l'o/igomere se retourne contre Ia sous-unite a ou b, permettunt de ce fait Ia translocation des protons. Les residus conserves de Ia sous-unite a, probab/ement Jocalises dans Ia double couche lipidique, pourraient participer au mecanisme de translocation des protons. KW - Biochemie KW - conduction de protons KW - proteines membranaires KW - carbenes KW - proton conduction KW - membrane proteins KW - carbenes Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62602 ER - TY - JOUR A1 - Gabellini, N. A1 - Sebald, Walter T1 - Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Evaluation of the deduced amino acid sequences of the FeS protein, cytochrome b and cytochrome c\(_1\) N2 - No abstract available KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62615 ER - TY - JOUR A1 - McCarthy, J. E. A1 - Sebald, Walter A1 - Gross, G. A1 - Lammers, R. T1 - Enhancement of translational efficiency by the Escherichia coli atpE translational initiation region: its fusion with two human genes N2 - No abstract available KW - Biochemie Y1 - 1986 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62626 ER - TY - JOUR A1 - Harnisch, U. A1 - Weiss, H. A1 - Sebald, Walter T1 - The primary structure of the iron-sulfur subunit of ubiquinol-cytochrome c reductase from Neurospora, determined by cDNA and gene sequencing N2 - No abstract available KW - Biochemie Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62631 ER - TY - JOUR A1 - Gabellini, N. A1 - Harnisch, U. A1 - McCarthy, J. E. A1 - Hauska, G. A1 - Sebald, Walter T1 - Cloning and expression of the fbc operon encoding the FeS protein, cytochrome b and cytochrome c\(_1\) from the Rhodopseudomonas sphaeroides b/c\(_1\) complex N2 - The gene for the FeS protein of the Rhodopseudomonas sphaeroides b/c1 complex was identified by means of crosshybridization with a segment of the gene encoding the corresponding FeS protein of Neurospora crassa. Plasmids (pRSF1-14) containing the cross-hybridizing region, covering in total 13.5 kb of chromosomal DNA, were expressed in vitro in a homologous system. One RSF plasmid directed the synthesis of all three main polypeptides of the R. sphaeroides blc1 complex: the FeS protein, cytochrome b and cytochrome c1• The FeS protein and cytochrome c1 were apparently synthesized as precursor fonns. None of the pRSF plasmids directed the synthesis of the 10-kd polypeptide found in b/c1 complex preparations. Partial sequencing of the cloned region was performed. Several sites of strong homology between R. sphaeroides and eukaryotic polypeptides of the b/c1 complex were identified. The genes encode the three b/c1 polypeptides in the order: (5') FeS protein, cytochrome b, cytochrome c1• The three genes are transcribed to give a polycistronic mRNA of 2.9 kb. This transcriptional unit has been designated the jbc operon; its coding capacity corresponds to the size of the polycistronic mRNA assuming that only the genes for the FeS protein (jbcF), cytochrome b (jbcß) and cytochrome c1 (jbcC) are present. This could indicate that these three subunits constitute the minimal catalytic unit of the b/c1 complex from photosynthetic membranes. KW - Biochemie KW - R. sphaeroidesl KW - b/c1 complex KW - gene KW - cloning KW - in vitro expression KW - polycistronic mRNA Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62642 ER - TY - JOUR A1 - McCarthy, J. E. A1 - Schairer, H. U. A1 - Sebald, Walter T1 - Translational initiation frequency of atp genes from Escherichia coli: identification of an intercistronic sequence that enhances translation N2 - The c, b and ö subunit genes of the Escherichia coli atp operon were cloned individually in an expression vector between the tac fusion promoter and the galK gene. The relative rates of subunit synthesis directed by the cloned genes were similar in vitro andin vivo and compared favourably with the subunit stoichiometry of the assembled proton-translocating A TP synthase of E. coli in vivo. The rate of synthesis of subunit c was at least six times that of subunit b and 18 times that of subunit ö. Progressive shortening of the long intercistronic sequence lying upstream of the subunit c gene showed that maximal expression of this gene is dependent upon the presence of a sequence stretching > 20 bp upstream of the Shine-Dalgarno site. This sequence thus acts to enhance the rate of translational initiation. The possibility that similar sequences might perform the same function in other operons of E. coli and bacteriophage A is also discussed. Translation of the subunit b cistron is partially coupled to translation of the preceding subunit c cistron. In conclusion, the expression of all the atp operon genes could be adjusted to accommodate the subunit requirements of A TP synthase assembly primarily by means of mechanisms which control the efficiency of translational initiation and re-initiation at the respective cistron start codons. KW - Biochemie KW - E. coli atp operon KW - subunit stoichiometry KW - in vitro and in vivo expression KW - translational initiation Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62657 ER - TY - JOUR A1 - Lindenmaier, W. A1 - Dittmar, K. E. A1 - Hauser, H. A1 - Necker, A. A1 - Sebald, Walter T1 - Isolation of a functional human interleukin 2 gene from a cosmid library by recombination in vivo N2 - A method has been developed that allows the isolation of genomic clones from a cosmid library by homologaus recombination in vivo. This method was used to isolate a human genomic interleukin 2 (IL2) gene. The genomic cosmid library was packaged in vivo into A. phage particles. A recombination-proficient host strain carrying IL2 cDNA sequences in a non-homologaus plasmid vector was infected by the packaged cosmid library. After in vivo packaging and reinfection, recombinants carrying the antibiotic resistance genes of both vectors were selected. From a recombinant cosmid clone the chromosomal IL2 genewas restored. After DNA mediated gene transfer into mouse Ltk- cells human IL2 was expressed constitutively. KW - Biochemie KW - Recombinant DNA KW - DNA mediated gene transfer KW - expression plasmid KW - screening KW - packaging KW - bacteriophage lambda Y1 - 1985 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62662 ER - TY - JOUR A1 - Schmidt, B. A1 - Wachter, E. A1 - Sebald, Walter A1 - Neupert, W. T1 - Processing peptidase of Neurospora mitochondria. Two-step cleavage of imported ATPase subunit 9 N2 - Subunit 9 (dicyclohexylcarbodümide binding protein, 'proteolipid') of the mitochondrial F 1F0-ATPase is a nuclearly coded protein in Neurospora crassa. lt is synthesized on free cytoplasmic ribosomes as a larger precursor with an NH2-terminal peptide extension. The peptide extension is cleaved ofT after transport of the protein into the mitochondria. A processing activity referred to as processing peptidase that cleaves the precursor to subunit 9 and other mitochondrial proteins is described and characterized using a cell-free system. Precursor synthesized in vitro was incubated with extracts of mitochondria. Processing peptidase required Mn2 + for its activity. Localization studies suggested that it is a soluble component of the mitochondrial matrix. The precursor was cleaved in two sequential steps via an intermediate-sized polypeptide. The intermediate form in the processing of subunit 9 was also seen in vivo and upon import of the precursor into isolated mitochondria in vitro. The two dcavage sites in the precursor molecule were determined. The data indicate that: {a) the correct NH2-terminus of the mature protein was generated, (b) the NH2-terminal amino acid of the intermediate-sized polypeptide is isoleueine in position -31. The cleavage sites show similarity ofprimary structure. It is concluded that processing peptidase removes the peptide extension from the precursor to subunit 9 (and probably other precursors) after translocation of these polypeptides (or the NHrterminal part of these polypeptides) into the matrix space of mitochondria. KW - Biochemie Y1 - 1984 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62674 ER - TY - JOUR A1 - Hoppe, J. A1 - Schairer, H. U. A1 - Sebald, Walter T1 - The proteolipid of a mutant ATPase from Escherichia coli defective in H\(^+\)-conduction contains a glycine instead of the carbodiimide-reactive aspartyl residue N2 - No abstract available KW - Biochemie Y1 - 1980 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62769 ER - TY - JOUR A1 - Sebald, Walter A1 - Wachter, E. A1 - Tzagoloff, A. T1 - Identification of amino acid substitutions in the dicyclohexylcarbodiimide-binding subunit of the mitochondrial ATPase complex from oligomycin-resistant mutants of Saccharomyces cerevisiae N2 - No abstract available KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62770 ER - TY - THES A1 - Alcantarino Menescal, Luciana T1 - In vivo characterization of genetic factors involved in Xmrk driven melanoma formation in Medaka (Oryzias latipes): a closer look at braf, Stat5 and c-myc T1 - In vivo Charakterisierung genetischer Faktoren mit Einfluss auf Xmrk induzierte Melanome in Medaka (Oryzias latipes): Untersuchung von braf, Stat5 und c-myc. N2 - Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens. N2 - Melanome entstehen durch die krankhafte Transformation von Melanozyten und sind eine der aggressivsten Krebsarten beim Menschen. In Fischen der Gattung Xiphophorus können, wenn auch sehr selten, spontan Melanome entstehen oder durch spezielle Artenkreuzungen induziert werden. Grundlage für das Entstehen der Melanome in diesen Fischen ist die Rezeptortyrosinkinase Xmrk. Da alle Xiphophorus-Arten lebendgebärend sind und keine Manipulationen an Embryonen vorgenommen werden können, wurde ein Xmrk Melanommodel für Medaka (Oryzias latipes) etabliert. Die Expression von Xmrk in Pigmentzellen dieser Fischart resultiert mit 100%iger Penetranz in Melanomen. Das Xmrk ist ein Ortholog des menschlichen „epidermal growth factor“ (EGFR) und aktiviert verschiedene nachgeschaltete Signalwege. Beispiele für diese Aktivierungen sind die Phosphorylierung von BRAF, Stat5 und die erhöhte Expression von c-myc. BRAF ist eine Serin-Threoninkinase, welche oft in malignen Melanomen mutiert ist. Stat5 ist ein Transkriptionsfaktor, welcher dauerhaft in Fischtumoren aktiviert ist. C-myc ist ein Transkriptionsfaktor, welcher etwa 15% aller menschlichen Gene sowie die Entstehung vieler menschlicher Tumore reguliert. Um neue Einsichten in die Funktion der Kanidatengene im Prozess der Melanomentstehung in vivo zu erlangen, habe ich Orthologe von BRAF, Stat5 und C-myc bei Medaka identifiziert und analysiert. Die Domänen des BRAF Proteins sind hoch konserviert in allen Vertebraten. Weiterhin deuten die Ergebnisse meiner Arbeit auf eine Beibehaltung der Funktionen im MAPK Signalweg hin. Transgene Medakalinien, welche eine dauerhaft aktive Version des BRAF Gens (V614E) exprimieren, weisen einerseits eine stärkere Hautpigmentierung auf. Weiterhin treten in diesen Fischen Veränderungen der Augen auf. In einem weiteren Projekt meiner Arbeit gelang es mir, zwei Kopien des Stat5 Gens im Medaka zu identifizieren, Stat5ab/a und Stat5ab/b. Sequenzanalysen zeigten eine höhere Übereinstimmung zwischen den beiden Genkopien, als zwischen denen von Medaka und Menschen. Dieses Ergebnis deutet darauf hin, dass die beiden Medaka Gene durch eine unabhängige Duplikation entstanden. In meiner Arbeit habe ich beide Gene des Medakas kloniert und jeweils eine konstitutiv aktive und eine dominant negative Version der Gene hergestellt. Weiterhin konnte ich erfolgreich für jede Genversion eine transgene Medakalinie etablieren, welche die verschiedenen Genvarianten unter der Kontrolle des pigmentzellspezifischen Promoters des mitf Gens exprimieren. Diese Linien werden in Zukunft helfen, den Einfluss von Stat5 Signalen auf den Prozess der Melanomverbreitung und dessen Invasivität zu erklären. In einem dritten Projekt meiner Doktorarbeit untersuchte ich das Vorkommen und die Funktion der C-myc Gene des Medakas. Ich konnte zwei Genkopien identifizieren, c-myc17 und c-myc20, welche auf unterschiedlichen Chromosomen lokalisiert sind. Ich konnte induzierbare, stabil transgene Linien herstellen, welche ein Fusionsprotein aus C-myc17 und der Hormonbindungsdomäne des Östrogenrezeptors von Maus exprimiert. Diese Linie ermöglichte eine induzierbare Aktivität des Transgens. Vergleichbar zum menschlichen MYC ist C-myc17 fähig, nach Aktivierung Proliferation und Apoptose in vivo auszulösen. Dauerhafte Aktivierung über einen längeren Zeitraum führt in diesen Linien zu Hyperplasie in Leber. Die verschiedenen Fischmodelle, die während dieser Arbeit generiert wurden, werden essentiell sein, um neue Einsichten in die Rolle diese Faktoren während der Krebsentwicklung in vivo zu erlangen. Weiterhin ermöglichen diese transgenen Linien durch einfaches Auskreuzen auf Xmrk Linien, deren Einfluss auf die Verbreitung von Melanomen zu untersuchen. Letztendlich sind mit diesen Linien auch Untersuchungen der Entwicklung von Pigmentzellen über Zeit möglich, da die Embryonen transparent sind und sich für chemisches Hochdurchsatz-Screening eignen. KW - Japankärpfling KW - Melanom KW - Myc KW - Molekulargenetik KW - melanoma KW - medaka KW - BRAF KW - Stat5 KW - c-myc KW - melanoma KW - medaka KW - BRAF KW - Stat5 KW - c-myc Y1 - 2012 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-70762 ER - TY - JOUR A1 - Kraeussling, Michael A1 - Wagner, Toni Ulrich A1 - Schartl, Manfred T1 - Highly Asynchronous and Asymmetric Cleavage Divisions Accompany Early Transcriptional Activity in Pre-Blastula Medaka Embryos N2 - In the initial phase of development of fish embryos, a prominent and critical event is the midblastula transition (MBT). Before MBT cell cycle is rapid, highly synchronous and zygotic gene transcription is turned off. Only during MBT the cell cycle desynchronizes and transcription is activated. Multiple mechanisms, primarily the nucleocytoplasmic ratio, are supposed to control MBT activation. Unexpectedly, we find in the small teleost fish medaka (Oryzias latipes) that at very early stages, well before midblastula, cell division becomes asynchronous and cell volumes diverge. Furthermore, zygotic transcription is extensively activated already after the 64-cell stage. Thus, at least in medaka, the transition from maternal to zygotic transcription is uncoupled from the midblastula stage and not solely controlled by the nucleocytoplasmic ratio. KW - Fische KW - Embryo Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-68906 ER - TY - JOUR A1 - Michel, R. A1 - Wachter, E. A1 - Sebald, Walter T1 - Synthesis of a larger precursor for the proteolipid subunit of the mitochondrial ATPase complex of Neurospora crassa in a cell-free wheat germ system N2 - No abstract available KW - Biochemie Y1 - 1979 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-62789 ER -