Theodor-Boveri-Institut für Biowissenschaften
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Die Lamine gehören zu einer Familie von Proteinen, die als strukturelle Hauptelemente die Kernlamina ausbilden, einen wesentlichen Bestandteil der Kernhülle eukaryontischer Zellen. In Säugern exprimieren differenzierte somatische Zellen die Lamine A, C, B1 und B2. Die Kernhülle in Keimzellen unterscheidet sich in Bezug auf Struktur und Proteinzusammensetzung deutlich von der einer somatischen Zelle. So exprimieren Keimzellen Lamin B1 als einziges der somatischen Lamine und zwei kurze keimbahnspezifische Spleißvarianten, die Lamine C2 und B3. Die vorliegende Arbeit enthält eine detaillierte Analyse des Expressionsmusters und der zellulären Verteilung von Lamin B3 im Verlauf der Spermatogenese der Maus. Die Daten aus RT-PCR, Western Blot und Immunfluoreszenz belegen eindeutig, dass Lamin B3 ausschließlich in postmeiotischen Stadien während der Spermiogenese exprimiert wird. In runden Spermatiden konnte das Protein an der Kernhülle und überraschenderweise auch im Nukleoplasma nachgewiesen werden. Im weiteren Verlauf der Spermiogenese kommt es zu einer Umverteilung des Proteins, es konzentriert sich zunehmend am posterioren Pol des Spermatidenkerns. Damit ist die Lamina während der Säuger-Spermiogenese nur aus B-Typ-Laminen aufgebaut und Lamin B3 ist in Säugern das erste Beispiel für ein Lamin, das selektiv nur in postmeiotischen Stadien der Spermatogenese exprimiert wird. Die ektopische Expression von Lamin B3 in Kulturzellen führt zu einer Deformation der Zellkerne, die eine hakenförmige Gestalt annehmen. Mit Hilfe von Transfektionsexperimenten in COS-7-Zellen konnte eindeutig gezeigt werden, dass die auftretenden morphologischen Veränderungen der Kerne transfizierter Zellen auf die trunkierte zentrale Stäbchendomäne in Lamin B3 zurückzuführen ist. Darüber hinaus zeigte das Protein eine stark erhöhte Löslichkeit im Vergleich zu Lamin B2 und die Analyse transfizierter Kulturzellen mit „fluorescence recovery after photobleaching“ (FRAP) und „fluorescence loss in photobleaching“ (FLIP) ergab, dass ein erheblicher Anteil der Lamin-B3-Moleküle eine hohe Mobilität aufweist, die ebenfalls ausschließlich durch die kurze Stäbchendomäne begründet ist. Die Ergebnisse führen zu dem Schluss, dass Lamin B3 die Kernhülle in Keimzellen flexibler macht, was eine Voraussetzung für einige Vorgänge in der Spermiogenese sein könnte. Mit einem Fusionsprotein aus GST und dem 84 Aminosäuren umfassenden N-Terminus von Lamin B3 wurde über einen „Pull-Down-Assay“ nach möglichen Interaktionspartnern in Keimzellen gesucht. Mit MSY2, MSY2a und MSY4 wurden drei hoch interessante Kandidaten identifiziert. Sie gehören zu den Y-Box-Proteinen, DNA- und RNA-bindende Proteine, die bei der Speicherung und späteren Translation von mRNAs beteiligt sind, u.a. die mRNA von Protamin 1 (diese Form der Regulation von Genexpression hat in der Spermatogenese große Bedeutung). Die Interaktion von Lamin B3 mit diesen Proteinen muss noch überprüft werden, würde aber einen weiteren Bezug zwischen Kernhülle und Chromatinreorganisation in der Spermiogenese herstellen, wie es für die Kernhüllenproteine GCL und LBR bereits gezeigt werden konnte. Außerdem wäre es ein erster Hinweis auf eine funktionelle Bedeutung der N-terminalen Domäne von Lamin B3.
In this study pore forming proteins of the gram-negative bacteria B. burgdorferi, B. duttonii and E.coli were investigated. Therefore the study is subdivided into three parts. In the first part outer membrane preparation of three relapsing fever Borrelia were investigated. In the second part the putative TolC homologue BB0124 of B. burgdorferi, the Lyme borreliosis agent, was studied. In the last part the influence of point mutants within the greasy slide of the maltose specific porin (LamB) of E. coli were shown. In the first part of this study outer membrane preparations of three Borrelia relapsing fever strains have been studied for pore-forming activity in the black lipid bilayer assay. Histograms of conductance fluctuations were obtained from single-channel experiments with outer membrane preparations of B. hermsii, B. recurentis and B. duttonii. All strains had a different conductance fluctuation pattern with a broad range of single-channel conductance values varying from 0.5 nS – 11 nS. Common for all three strains was a high pore-forming activity at around 0.5 nS. Furthermore the proteins of the outer membrane of B. duttonii were separated by chromatographic methods. Some eluate fractions contained a channel-forming protein, which was forming stable channels with a single-channel conductance of 80 pS in 1 M KCl. Characterization of this channel showed that it is slightly anionic selective and voltage independent. The small single-channel conductance suggests that it is a specific pore. However, a substrate specificity could not be determined. In the second part, for the B. burgdorferi HB19 and p66 knock out strain HB19/K02, their outer membrane preparations were characterized in the black lipid bilayer assay. Comparing the histograms of single-channel conductions fluctuations of both strains showed no single-channel activity at 11.5 nS for the p66 knock out strain. This verifies earlier studies that P66 is a pore-forming protein in B. burgdorferi. Furthermore, one fraction obtained by anion exchange chromatography of the p66 knock out outer membrane protein preparation showed a uniform channel-forming activity with a single channel conductance of 300 pS. The electrophysically characterization of the 300 pS channel showed that it is not ionselective or voltage dependent. By mass spectrometry using peptide mass finger prints, BB0142 could be identified as the sole channel forming candidate in the active fraction. A BLAST search and a conserved domain search showed that BB0142 is a putative TolC homologue in B. burgdorferi. Furthermore the location of the bb0142 gene within the chromosome is in an operon encoding a multidrug efflux pump. In this study the expression of an outer membrane component of a putative drug efflux system of B. burgdorferi was shown for the first time. In the third part functional studies of the maltooligosaccharide-specific LamB channel were performed. The 3D-structure of LamB suggests that a number of aromatic residues (Y6, Y41, W74, F229, W358 and W420) within the channel lumen is involved in carbohydrate and ion transport. All aromatic residues were replaced by alanine (A) scanning mutagenesis. Furthermore, LamB mutants were created in which one, two, three, four and five aromatic residues were replaced to study their effects on ion and maltopentaose transport through LamB. The purified mutant proteins were reconstituted into lipid bilayer membranes and the single-channel conductance was studied. The results suggest that all aromatic residues provide some steric hindrance for ion transport through LamB. Highest impact is provided by Y6 and Y41, which are localized opposite to Y118, which forms the central constriction of the LamB channel. Stability constants for binding of maltopentaose to the mutant channels were measured using titration experiments with the carbohydrate. The mutation of one or several aromatic amino acids led to a substantial decrease of the stability constant of binding. The highest effect was observed when all aromatic amino acids were replaced by alanine because no binding of maltopentaose could be detected in this case. However, binding was again possible when Y118 was replaced by tryptophane (W). The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant LamB-channels. The analysis of the power density spectra of some of the mutants allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate binding to the binding-site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. For most mutants k1 decreased and k-1 increased.
Insights into the evolution of protein domains give rise to improvements of function prediction
(2005)
The growing number of uncharacterised sequences in public databases has turned the prediction of protein function into a challenging research field. Traditional annotation methods are often error-prone due to the small subset of proteins with experimentally verified function. Goal of this thesis was to analyse the function and evolution of protein domains in order to understand molecular processes in the cell. The focus was on signalling domains of little understood function, as well as on functional sites of protein domains in general. Glucosaminidases (GlcNAcases) represent key enzymes in signal transduction pathways. Together with glucosamine transferases, they serve as molecular switches, similar to kinases and phosphatases. Little was known about the molecular function and structure of the GlcNAcases. In this thesis, the GlcNAcases were identified as remote homologues of N-acetyltransferases. By comparing the homologous sequences, I was able to predict functional sites of the GlcNAcase family and to identify the GlcNAcases as the first family member of the acetyltransferase superfamily with a distinct catalytic mechanism, which is not involved in the transfer of acetyl groups. In a similar approach, the sensor domain of a plant hormone receptor was studied. I was able to predict putative ligand-binding sites by comparing evolutionary constraints in functionally diverged subfamilies. Most of the putative ligand-binding sites have been experimentally confirmed in the meantime. Due to the importance of enzymes involved in cellular signalling, it seems impossible to find substitutions of catalytic amino acids that turn them catalytically inactive. Nevertheless, by scanning catalytic positions of the protein tyrosine phosphatase families, I found many inactive domains among single domain and tandem domain phosphatases in metazoan proteomes. In addition, I found that inactive phosphatases are conserved throughout evolution, which led to the question about the function of these catalytically inactive phosphatase domains. An analysis of evolutionary site rates of amino acid substitutions revealed a cluster of conserved residues in the apparently redundant domain of tandem phosphatases. This putative regulatory center might be responsible for the experimentally verified dimerization of the active and inactive domain in order to control the catalytic activity of the active phosphatase domain. Moreover, I detected a subgroup of inactive phosphatases, which presumably functions in substrate recognition, based on different evolutionary site rates within the phosphatase family. The characterization of these new regulatory modules in the phosphatase family raised the question whether inactivation of enzymes is a more general evolutionary mechanism to enlarge signalling pathways and whether inactive domains are also found in other enzyme families. A large-scale analysis of substitutions at catalytic positions of enzymatic domains was performed in this work. I identified many domains with inactivating substitutions in various enzyme families. Signalling domains harbour a particular high occurrence of catalytically inactive domains indicating that these domains have evolved to modulate existing regulatory pathways. Furthermore, it was shown that inactivation of enzymes by single substitutions happened multiple times independently in evolution. The surprising variability of amino acids at catalytic positions was decisive for a subsequent analysis of the diversity of functional sites in general. Using functional residues extracted from structural complexes I could show that functional sites of protein domains do not only vary in their type of amino acid but also in their structural location within the domain. In the process of evolution, protein domains have arisen from duplication events and subsequently adapted to new binding partners and developed new functions, which is reflected in the high variability of functional sites. However, great differences exist between domain families. The analysis demonstrated that functional sites of nuclear domains are more conserved than functional sites of extracellular domains. Furthermore, the type of ligand influences the degree of conservation, for example ion binding sites are more conserved than peptide binding sites. The work presented in this thesis has led to the detection of functional sites in various protein domains involved in signalling pathways and it has resulted in insights into the molecular function of those domains. In addition, properties of functional sites of protein domains were revealed. This knowledge can be used in the future to improve the prediction of protein function and to identify functional sites of proteins.
Listeria monocytogenes ist ein fakultativ humanpathogenes Bakterium und aufgrund seiner Fähigkeit, in Zellen des Wirtes einzudringen und sich im Zytoplasma der befallenen Wirtszelle zu vermehren, ein attraktiver Träger, um heterolog exprimierte Antigene in den MHC-I- und -II-Präsentationsweg antigenpräsentierender Zellen (APC) einzuschleusen und so eine effektive zelluläre Immunreaktion zu erzeugen. Dabei hat die Art und Weise der Antigenexpression einen wesentlichen Einfluss auf die Erzeugung der antigenspezifischen Immunität. So konnte unter Verwendung extrazellulärer Trägerbakterien gezeigt werden, dass insbesondere die Verankerung von Antigenen auf der Zelloberfläche der Bakterien zu einer effektiven Induktion einer humoralen Immunantwort führt. Mit dem Ziel, mit einem derartigen Ansatz auch eine zelluläre Immunität zu erzeugen, wurde ein Plasmidsystem für die Expression von heterologen Proteinen in der Zellwand von L. monocytogenes entwickelt. Dabei gelang die Verankerung zahlreicher Proteine eukaryontischer wie auch prokaryontischer Herkunft über das LPXTG-Ankermotiv von Internalin A. Die so erzielte starke Expression in der Zellwand setzte aber sowohl die Fitness der Bakterien als auch deren Invasivität in vitro deutlich herab und verhinderte damit eine effektive MHC-I-Präsentation des verwendeten Modellantigens. Alternativ wurde L. monocytogenes bereits erfolgreich zur Übertragung von DNA-Vakzinen eingesetzt, um so auch die Synthese gegebenenfalls posttranslationell modifizierter Antigene in ihrer korrekten Konformation durch die infizierte Wirtszelle zu erzielen. Allerdings erfolgte die Expression des als Reporterprotein verwendeten EGFP insbesondere in APC sehr langsam und war von geringer Effizienz. Dabei konnte in der vorliegenden Arbeit erstmals gezeigt werden, dass nach bakterieller Übertragung von DNA-Vakzinen der Import der Plasmidmoleküle in den Kern insbesondere sich nicht teilender Zellen einen der wichtigsten Engpässe für eine möglichst frühzeitige und effektive Reportergenexpression darstellt. Einen Ausweg bietet die bakterielle Übertragung codierender mRNA, die unmittelbar nach der Freisetzung aus der Bakterienzelle im Zytoplasma der infizierten Wirtszelle zur Translation zu Verfügung steht. Dazu wurde das 5’-Ende der EGFP-codierenden Sequenz mit dem IRES-Element des Encephalomyocarditisvirus genetisch fusioniert. Um eine möglichst hohe Syntheserate zu erzielen und damit dem Abbau der mRNA in der Bakterienzelle entgegenzuwirken, erfolgte die Synthese der mRNA in L. monocytogenes mit Hilfe eines T7-RNA-Polymerase-basierten Transkriptionssystems. Im Gegensatz zur bakteriellen Übertragung von Plasmid-DNA konnte so bereits 4 h nach Infektion sowohl in epithelialen als auch in APC wie Makrophagen und humanen dendritischen Zellen eine deutliche EGFP-Expression nachgewiesen werden sowie bei Verwendung von Ovalbumin als Reporterprotein eine effektive MHC-I-Präsentation in vitro. Damit stellt die bakterielle Übertragung von mRNA einen vielversprechenden neuartigen Ansatz dar zur Erzeugung einer zellulären und gegebenenfalls auch humoralen Immunantwort gegen posttranslational modifizierte Antigene.
Honigbienen (Apis mellifera carnica) regulieren die Temperatur ihrer Brut in einem sehr engen Temperaturfenster, da vor allem die gedeckelte Brut sehr temperaturempfindlich reagiert (Groh et al. 2004). Die Thermoregulation ist nicht – wie lange angenommen – Beiprodukt von alltäglichen Arbeiten der Bienen im Brutbereich, sondern eine aktive und Energie- und Zeitaufwändige eigene Tätigkeit. Arbeiterinnen ziehen sich mit ihren Beinen an die Brutoberfläche, drücken ihren warmen Thorax auf die Brutdeckel und verharren so für einige Minuten um mit der eigenen Körperwärme die Brut zu temperieren (Bujok et al. 2002). Wie erwartet korrelierte die Thoraxtemperatur einer Arbeiterin mit der Frequenz der abdominalen Atembewegungen, bei sehr hohen Thoraxtemperaturen (über 40°C) erreichten die Bienen Atemfrequenzen von über 8Hz. Eine weitere Methode die Brut effektiv zu wärmen übten Bienen aus, die leere Zellen im gedeckelten Brutbereich besuchen (Kleinhenz et al. 2003). Arbeiterinnen gingen dabei bevorzugt in Zellen, die von möglichst vielen gedeckelten Zellen umgeben waren. Sowohl die Dauer der Zellbesuche, als auch die mittlere Thoraxtemperatur bei Ein- und Austritt der Zelle korrelierten mit der Anzahl der benachbarten Brutzellen – je mehr Brutzellen eine leere Zelle in ihrer direkten Nachbarschaft hatte umso länger dauerte der Besuch einer Biene und umso höher ist die Ein- bzw. Austrittstemperatur der Biene. Mindestes 48 Stunden alte Bienen unterschieden sich signifikant in ihrem Wärmeverhalten von jüngeren Bienen. Tote gedeckelte Brut wurde in manchen Fällen über viele Tage (durchgehend bis 10 Tage) gewärmt, sie unterschied sich in ihrer Temperatur nicht von unbehandelter gedeckelter Brut. In weiteren Versuchen lag die Bruttemperatur von toter Brut zwar unter der eines Kontrollbereiches, die Temperatur lag aber weiterhin im optimalen Bereich von 33,5 bis 35°C (Groh et al. 2004). In diesen Versuchen wurde die tote Brut vor dem Einsetzen in den Beobachtungsstock wieder auf 35°C erwärmt. Wachskegel in gedeckelten Zellen wurden erkannt und ausgeräumt. Aktive Signale, die von der Brut ausgehen scheinen also nicht notwendig für die effektive Bruttemperaturregulierung zu sein. Untersuchungen mittels Laser-Doppler-Vibrometrie zeigten auch keine Hinweise auf eine mechanische Kommunikation zwischen den Puppen und den Arbeiterinnen. Das Brutwärmen scheint eine Aktion zu sein, die von den Bienen nur in Gemeinschaft sinnvoll durchgeführt werden kann. In einigen Fällen kam es während der Puppenphase zu unerklärlichen Abfällen in der Bruttemperatur, die nur durch einen positiven Rückkopplungseffekt seitens der Arbeiterinnen erklärt werden kann. Beim Brutwärmen spielen die Antennen der Arbeiterinnen wahrscheinlich eine wichtige Rolle. Während sich die Bienen beim aktiven Brutwärmen den Brutdeckel annähern sind die Antennenspitzen immer auf die Brutdeckel gerichtet. Fehlen den Arbeiterinnen die Antennen, dann ist die Thermoregulation eingeschränkt oder unzureichend. Die Bruttemperatur korreliert mit der Anzahl der abgetrennten Antennensegmente, je mehr Antennensegmente fehlen, desto weniger gut wird die Temperatur im Brutbereich hoch und konstant gehalten. Zusätzlich scheint es eine Lateralität in der Antennenfunktion zu geben, wurde die rechte Antenne gekürzt wärmten die Bienen die Brut signifikant schlechter, als beim Kürzen der linken Antenne. Durch das Kürzen der Antennen änderte sich auch das Verhalten der Tiere: Kontrollbienen verharrten ruhig im Brutbereich, während Bienen mit gekürzten Antennen teilweise ähnlich warm waren, aber nicht mehr das oben beschriebene aktive Brutwärmeverhalten zeigten.
HMG-Proteine sind Architekturelemente des Chromatins und regulieren durch ihre Bindung an das Chromatin auf verschiedene Weise DNA-abhängige Prozesse wie Replikation, Transkription und DNA-Reparatur. Um zu verstehen, wie HMG-Proteine ihre vielfältigen Funktionen erfüllen können, wurde mit Hilfe von EGFP- und DsRed2-Fusionsproteinen ihre Funktion in vivo untersucht. Im Wesentlichen wurde dabei mit Hilfe von Bleichtechniken ihr dynamisches Verhalten charakterisiert. Daneben wurde für die HMGN-Proteine ihr bislang unbekanntes Expressionsverhalten in Tumorzellen bestimmt. So konnte für die HMGN-Proteine gezeigt werden, dass bestimmte Tumorzelllinien (HT-29, FTC-133, MCF-7, RPMI 8226, 697, Ishikawa, LNCap) eine relativ erhöhte Expression von HMGN2 aufweisen, die mit der Tumordifferenzierung korreliert. Eine relativ verringerte Expression von HMGN1 steht dagegen in Mammakarzinomen und Non-Hodgkin-Lymphomen in direktem Zusammenhang mit der Aggressivität der Tumore. Somit kann die HMGN-Expression bei diesen Tumoren als diagnostischer Marker verwendet werden. FRAP-Analysen mit EGFP-Fusionsproteinen führten zu der Erkenntnis, dass HMGN1, HMGN2, HMGA1a, HMGA1b und HMGB1 sich sehr schnell durch den Zellkern bewegen und nur transient an das Chromatin gebunden sind. Es konnte gezeigt werden, dass die spezifischen DNA/Chromatin-Bindungsmotive im Wesentlichen entscheiden, wo die Bindung der HMG-Proteine in vivo erfolgt, ihre Verweildauer im Euchromatin, Heterochromatin und zellzyklusabhängig dann aber durch Modifikationen (Phosphorylierungen, Acetylierungen) reguliert wird. Dies wurde beispielhaft durch punktmutierte und deletierte Fusionsproteine, sowie durch Inkubation der Zellen mit spezifischen Drogen für die HMGA1a-Proteine gezeigt. FRAP-Analysen haben außerdem gezeigt, dass die Spleißvarianten hHMGA1a und hHMGA1b unterschiedliche kinetische Parameter besitzen. Dies zeigt, dass beiden Varianten unterschiedliche Funktionen zugesprochen werden können. Die gefundenen spezifischen, transienten Verweildauern der einzelnen HMG-Proteine führen zu einem Modell eines dynamischen Chromatin-Netzwerkes, wobei alle HMG-Proteine in Wechselwirkungen innerhalb eines dynamischen Chromatinprotein-Cocktails DNA-abhängige Prozesse regulieren können. Die jeweiligen, wie hier gezeigt, durch Modifikationen regulierten Verweildauern der HMG-Proteine bestimmen darüber, welche anderen Chromatinproteine wie lange am Chromatin verbleiben und bestimmte Funktionen, wie beispielsweise die Modifikation der Core-Histone, übernehmen können. Die dynamischen Parameter einzelner HMG-Proteine erklären so, wie diese Proteine ihre vielfältigen Funktionen als Architekturelemente und bei der Regulation DNA-abhängiger Prozesse erfüllen können. Einige Vertreter, wie die HMGB1-Proteine, bewegen sich so schnell durch den Zellkern, dass ihre kinetischen Parameter durch das beschränkte zeitliche Auflösungsvermögen konfokaler Mikroskope der älteren Generation nicht erfassbar sind. Die Bestimmung von Dosis-Wirkungs-Beziehungen von Drogen, welche die kinetischen Parameter von HMGB1-Proteinen beeinflussen können, ist inzwischen mit Mikroskopen der neuen Generation möglich. Im Verlaufe der Arbeit zeigte sich, dass andere verwendete Fluorophore wie DsRed2 die kinetischen Eigenschaften von HMG-Fusionsproteinen beeinflussen können. Durch eine erhöhte Verweildauer können auch sehr transiente Interaktionen sichtbar gemacht werden. Wie gezeigt wurde, kann eine erhöhte Verweildauer aber auch zur Verdrängung anderer Proteine führen, die die gleichen Bindungsstellen benutzen und so eine Modulation des Chromatins bewirken. Die Nutzung von DsRed-Fluorophoren ermöglicht interessante neue Erkenntnisse. Diese müssen aber stets vor dem Hintergrund eines veränderten dynamischen Verhaltens der Fusionsproteine interpretiert werden. Zusammengenommen liefern die hier vorgestellten Ergebnisse zur Dynamik der HMG-Proteine grundlegende Informationen, die zur Klärung ihrer Funktion bei Chromatinmodulationen, etwa bei Differenzierungsprozessen oder der Entstehung von Tumorzellen entscheidend beitragen. Die Erkenntnis, dass diese Proteine lediglich transiente Interaktionen mit ihren Bindungspartnern eingehen können, sind im Hinblick auf die Behandlung von Tumoren, bei denen HMG-Proteine im Vergleich zu Normalgewebe häufig überexprimiert sind, von großer Bedeutung.
It has been known for a long time that Drosophila can learn to discriminate not only between different odorants but also between different concentrations of the same odor. Olfactory associative learning has been described as a pairing between odorant and electric shock and since then, most of the experiments conducted in this respect have largely neglected the dual properties of odors: quality and intensity. For odorant-coupled short-term memory, a biochemical model has been proposed that mainly relies on the known cAMP signaling pathway. Mushroom bodies (MB) have been shown to be necessary and sufficient for this type of memory, and the MB-model of odor learning and short-term memory was established. Yet, theoretically, based on the MB-model, flies should not be able to learn concentrations if trained to the lower of the two concentrations in the test. In this thesis, I investigate the role of concentration-dependent learning, establishment of a concentration-dependent memory and their correlation to the standard two-odor learning as described by the MB-model. In order to highlight the difference between learning of quality and learning of intensity of the same odor I have tried to characterize the nature of the stimulus that is actually learned by the flies, leading to the conclusion that during the training flies learn all possible cues that are presented at the time. The type of the following test seems to govern the usage of the information available. This revealed a distinction between what flies learned and what is actually measured. Furthermore, I have shown that learning of concentration is associative and that it is symmetrical between high and low concentrations. I have also shown how the subjective quality perception of an odor changes with changing intensity, suggesting that one odor can have more than one scent. There is no proof that flies perceive a range of concentrations of one odorant as one (odor) quality. Flies display a certain level of concentration invariance that is limited and related to the particular concentration. Learning of concentration is relevant only to a limited range of concentrations within the boundaries of concentration invariance. Moreover, under certain conditions, two chemically distinct odorants could smell sufficiently similarly such, that they can be generalized between each other like if they would be of the same quality. Therefore, the abilities of the fly to identify the difference in quality or in intensity of the stimuli need to be distinguished. The way how the stimulus is analyzed and processed speaks in favor of a concept postulating the existence of two separated memories. To follow this concept, I have proposed a new form of memory called odor intensity memory (OIM), characterized it and compared it to other olfactory memories. OIM is independent of some members of the known cAMP signaling pathway and very likely forms the rutabaga-independent component of the standard two-odor memory. The rutabaga-dependent odor memory requires qualitatively different olfactory stimuli. OIM is revealed within the limits of concentration invariance where the memory test gives only sub-optimal performance for the concentration differences but discrimination of odor quality is not possible at all. Based on the available experimental tools, OIM seems to require the mushroom bodies the same as odor-quality memory but its properties are different. Flies can memorize the quality of several odorants at a given time but a newly formed memory of one odor interferes with the OIM stored before. In addition, the OIM lasts only 1 to 3 hours - much shorter than the odor-quality memory.
Emery-Dreifuss muscular dystrophy (EDMD) is a rare genetic disorder characterised by early contractures of the elbows, Achilles tendons and spine, slowly progressive muscle wasting and cardiomyopathy associated with cardiac conduction defect. The autosomal dominant form is caused by mutations in the LMNA gene which gives rise to lamin A and lamin C proteins by alternative splicing. These A-type lamins, together with B-type lamins, form the nuclear lamina, a network of intermediate filament proteins underlining the nuclear envelope. In order to ascertain the role lamin A and C separately contribute to the molecular phenotype, we analysed ten LMNA mutations and one single nucleotide polymorphism (SNP) in transfection studies in COS7 fibroblasts and, partially, in C2C12 myoblasts. The EGFP or DsRed2 tagged lamins were exogenously expressed either individually or both A-types together and examined by light and electron microscopy. The protein mobility of lamin A mutants was determined by FRAP analysis. Additionally, a co-immunoprecipitation binding assay of in vitro synthesised A-type lamins and emerin was performed.Eight of the LMNA mutations (R50S, R133P, E358K, E358K+C<T1698, E361K, R527P, L530P, R541S and G602S) and the SNP C<T1698, when expressed in lamin A, exhibited a range of nuclear mis-localisation patterns from a wild type phenotype to the formation of nuclear aggregates. Two mutations (T150P and delQ355) led to the severe mis-localisation of the exogenous protein and additionally affected nuclear envelope reassembly and mid-body protein composition after mitosis. Exogenously expressed DsRed2 tagged wild type and mutant lamin C was only inserted into the nuclear lamina if co-expressed with the equivalent EGFP tagged lamin A construct, except for the T150P mutation which prevented either lamin from reaching the nuclear lamina. The T150P, R527P and L530P mutations reduced the ability of lamin A, but not lamin C from binding to emerin. These data indicate that mutations in the rod domain of lamin A mainly impair its function as a structural protein, whereas mutations of the globular tail domain appear to disrupt protein-protein interactions important for gene regulation and signal transduction processes. In addition, our results suggest specific functional roles for the emerin-lamin A and emerin-lamin C containing protein complexes; this is the first report to propose that the A-type lamin mutations may be differentially dysfunctional for the same LMNA mutation.
In this thesis, I introduce the Virtual Brain Protocol, which facilitates applications of the Standard Brain of Drosophila melanogaster. By providing reliable and extensible tools for the handling of neuroanatomical data, this protocol simplifies and organizes the recurring tasks involved in these applications. It is demonstrated that this protocol can also be used to generate average brains, i.e. to combine recordings of several brains with the same features such that the common features are emphasized. One of the most important steps of the Virtual Insect Protocol is the aligning of newly recorded data sets with the Standard Brain. After presenting methods commonly applied in a biological or medical context to align two different recordings, it is evaluated to what extent this alignment can be automated. To that end, existing Image Processing techniques are assessed. I demonstrate that these techniques do not satisfy the requirements needed to guarantee sensible alignments between two brains. Then, I analyze what needs to be taken into account in order to formulate an algorithm which satisfies the needs of the protocol. In the last chapter, I derive such an algorithm using methods from Information Theory, which bases the technique on a solid mathematical foundation. I show how Bayesian Inference can be applied to enhance the results further. It is demonstrated that this approach yields good results on very noisy images, detecting apparent boundaries between structures. The same approach can be extended to take additional knowledge into account, e.g. the relative position of the anatomical structures and their shape. It is shown how this extension can be utilized to segment a newly recorded brain automatically.
Das fakultativ intrazelluläre Bakterium Listeria monocytogenes besitzt die Fähigkeit, eukaryotische Wirtszellen zu penetrieren, sich in diesen zu vermehren, fortzubewegen und zwischen den Zellen auszubreiten. Im Zuge des intrazellulären Lebenszyklus gehen Listerien Wechselwirkungen mit ver-schiedenen zellulären Proteinen ein. Als eines dieser Proteine konnte das zelluläre Phosphoprotein Stathmin identifiziert werden. Dieses Protein bindet an Untereinheiten des Tubulins und destabilisiert dadurch Mikrotubuli (MT). Es wird durch Phosphorylierung von vier spezifischen Serinresten in seiner MT-destabilisierenden Aktivität reguliert. Da Stathmin als Antwort auf externe Signale zelluläre Funktionen, z. B. Zell-Proliferation und Differenzierung reguliert, vermutet man seine Funktion in einer Art Relais welches verschiedene Signale aus dem Umfeld der Zelle integriert. In mit L. monocytogenes infizierten Wirtszellen wird Stathmin an die Oberfläche intrazellulärer Bakterien rekrutiert. Inwiefern diese Rekrutierung das Phosphorylierungsmuster von Stathmin und damit dessen Aktivität beeinflusst, konnte im Rahmen dieser Arbeit nicht geklärt werden. Stathmin knock-out Mäuse sollten sich gut eignen, um die Rolle von Stathmin während einer Infektion mit L. monocytogenes EGD in vitro und in vivo zu untersuchen. Es stellte sich heraus, dass in Stathmin(-/-)-Makrophagen der intrazelluläre Lebenszyklus der Listerien nicht signifikant beeinflusst ist. Nach intravenöser Verabreichung von 5x103 L. monocytogenes waren drei Tage nach der Infektion in Leber und Milz der knock-out Mäuse allerdings signifikant mehr Listerien nachzuweisen, als in den Organen wildtypischer Mäuse. Mittels Immunfluoreszenzmikroskopie und einem anti-Stathmin-Antiersum konnte an mit verschiedenen L. monocytogenes-Mutanten infizierten Zellen gezeigt werden, dass Stathmin mit der Oberfläche intrazellulärer Listerien kolokalisiert. Allerdings konnten dabei die Angaben in der Literatur nicht bestätigt werden, wonach für diese Kolokalisation die Expression von ActA notwendig ist. Die Ergebnisse dieser Arbeit sprechen im Gegensatz zu den publizierten Daten dafür, dass Stathmin über einen bisher noch unbekannten Mechanismus ActA-unabhängig an intrazelluläre Listerien rekrutiert wird. Zweikomponentensysteme ermöglichen Bakterien eine rasche Anpassung an sich verändernde Umweltbedingungen, da sie extra- und intrazelluläre Stimuli in zelluläre Signale umwandeln. Um die 16 in der Genomsequenz von L. mono-cytogenes EGDe identifizierten Zweikomponentensysteme charakterisieren zu können, wurden individuelle Mutanten konstruiert, in denen individuelle Response Regulatorgene deletiert sind. Die erhaltenen Mutanten wurden in vitro und in vivo auf ihr Wachstumsverhalten hin untersucht. Es zeigte sich, dass unter den angewandten Kultur- und Versuchsbedingungen keines der Zweikomponentensysteme eine signifikante Rolle bei der Anpassung an Temperatur, sowie an oxidativen oder osmotischen Stress spielt. Die Zugabe von 5 % Ethanol hatte einen stark hemmenden Effekt auf das Wachstum von 4 Mutanten, wohingegen zwei andere Mutanten in Gegenwart des Alkohols deutlich besser wuchsen. Unter anaeroben Bedingungen konnte kein Unterschied im Wachstum beobachtet werden. Die Expression wichtiger Virulenzgene war in keiner der untersuchten Mutanten im Vergleich zum Ausgangsstamm verändert. Die intrazelluläre Replikation sowie intrazelluläre Bewegung und Ausbreitung im Zellrasen waren durch die Deletion der Response Regulatorgene nicht beeinträchtigt. Abgesehen von geringen Unterschieden in der Invasivität einiger Deletionsmutanten für Cos-1 und Caco-2 Zellen zeigte sich keiner der Response Regulatoren für den intrazellulären Lebenszyklus von L. monocytogenes erforderlich. Es zeigte sich, dass der in der vorliegenden Arbeit verwendete L. monocyto-genes-Wildstamm auch bei der für die Flagellenexpression normalerweise nicht-permissiven Temperatur von 37° C noch beweglich ist. Die L. monocytogenes ΔdegU-Mutante war dagegen auf Weichagar temperaturunabhängig unbeweglich. Die elektronenmikroskopische Analyse ergab, dass dieser Stamm im Gegensatz zum Wildtyp auch bei 24° C keine Flagellen ausbildet. Durch vergleichende Proteomanalysen konnte gezeigt werden, dass L. monocytogenes ΔdegU bei 24° C wesentliche Proteine des Flagellenapparates nicht synthetisiert. Mittels Transkriptomanalysen konnten die Ergebnisse der Proteomanalysen bestätigt werden. Es wurden neben Genen, die für Proteine der Flagellenbiosynthese und Chemotaxis codieren, noch weitere Gene identifiziert, die offensichtlich unter der transkriptionellen Kontrolle des Response Regulators DegU stehen. Die Ergebnisse der in vivo Studien zeigten, dass L. monocytogenes ΔdegU deutlich virulenzattenuiert ist. Für die restlichen L. monocytogenes ΔTCS-Mutanten waren im Vergleich zum Wildtyp die Unterschiede in Leber und Milz nur leicht verändert und statistisch nicht signifikant.
T-Zellimmunantworten werden normalerweise durch folgenden Weg initiiert: unreife dendritische Zellen nehmen Antigen in der Peripherie auf, wandern in die sekundären lymphatischen Organe, wobei sie auf ihrem Weg sowohl reifen als auch das Antigen prozessieren. In den sekundären lymphatischen Organen angekommen, präsentieren sie als reife dendritische Zellen den T-Zellen die Antigene in Form von Peptiden zusammen mit kostimulierenden Molekülen. Dadurch rufen sie eine spezifische T-Zellantwort hervor. In der vorliegenden Arbeit wurde untersucht, ob nicht Situationen herbeigeführt werden können, die ein T-Zell priming außerhalb der sekundären lymphatischen Organe erlauben. Dazu wurden ein murines Modell, bei dem das Zytokin Lymphotoxin-alpha spezifisch am Tumor angereichert wurde, und ein humanes Modell, bei dem reife, antigenbeladene DC intradermal appliziert wurden, untersucht. Im murinen Modell zeigte sich, dass die gerichtete Anreicherung von Lymphotoxin-alpha am Tumor zu dessen Zerstörung führte, welche durch T-Zellen vermittelt wurde, und mit der Induktion eines tertiären lymphatischen Gewebes am Tumor assoziiert war. Dieses tertiäre lymphatische Gewebe war durch die Kompartimentalisierung von T- und B-Zellen und der Präsenz von high endothelial venules charakterisiert und besaß zudem mit dendritischen Zellen und naïven T-Zellen alle Voraussetzungen für ein in loco priming. Dementsprechend konnte in der Folge der gerichteten Lymphotoxin-alpa Therapie im Tumor ein Anstieg am T-Zellinfiltrat, welches sich oligoklonal zusammensetzte, beobachtet werden. In vitro Experimente verdeutlichte die Tumorspezifität der Therapie-induzierten T-Zellantwort, da die T-Zellen auf ein Tumorantigen mit der Ausschüttung von Interferon gamma reagierten und die Tumorzellen lysierten. Im humanen Modell wurden Hautbiopsien von Melanompatienten untersucht, denen im Rahmen einer klinischen Studie autologe, in vitro generierte und antigenbeladene DC intradermal appliziert wurden. Die Patienten erlaubten die Entnahme von Hautbiopsien aus den Injektionsstellen für wissenschaftliche Untersuchungen. Eine Induktion bzw. Verstärkung einer spezifischen T-Zellantwort durch die Vakzinierung mit antigenbeladenen dendritischen Zellen konnte bereits in zahlreichen Arbeiten und auch in dem in dieser Arbeit untersuchten Patientenkollektiv gezeigt werden. Bei der Analyse der Injektionsstellen zeigt sich, dass ein großer Teil der injizierten dendritischen Zellen in der Vakzinierungsstelle verharren und dass diese unabhängig von einer Beladung mit Antigen zu einer Induktion von high endothelial venules Charakteristika führte. Waren die dendritischen Zellen mit Antigen beladen, so führte dies zu einem stärkeren T-Zellinfiltrat in den Injektionsstellen, wobei sowohl naïve als auch central memory T-Zellen nachgewiesen wurde. Diese Zellen wurden vermutlich durch die Überexpression der DC CK1 und SDF1 Chemokinen in den Injektionsstellen, die chemotaktisch auf T-Zellen wirken, angezogen. Das Infiltrat in den Injektionsstellen war oligoklonal und wies tumorspezifische T-Zellen auf. Nachdem diese T-Zellklone im Blut der Patienten vor der Vakzinierung nicht nachweisbar waren, müssen sie zumindest in den Injektionsstellen expandiert sein. Interessanterweise konnte einer dieser Klone in Metastasen nachgewiesen werden, die nach der Vakzinierung dem Patienten entfernt wurden. In beiden Modellen wurde also durch die Manipulation des Mikromilieus, d.h. Lymphotoxin-alpa Anreicherung am Tumor bzw. Injektion von reifen dendritischen Zellen in die Haut, Strukturen wie z.B. high endothelial venules induziert, die ein in loco priming ermöglichen sollten. Dementsprechend riefen diese Veränderungen ein Tumorantigen-spezifisches Infiltrat hervor. Diese Ergebnisse deuten darauf hin, dass T-Zell priming auch außerhalb sekundärer lymphatischer Organe erfolgen kann. Prinzipiell scheint also nur der Kontakt von reifen, antigenbeladenen dendritischen Zellen mit den entsprechenden antigenspezifischen, naïven T-Zellen entscheiden zu sein. Die Möglichkeit des in vitro primings bekräftigt diese These. In vivo erfolgt dieses Aufeinandertreffen normalerweise in den sekundären lymphatischen Organen, doch konnte in der vorliegenden Arbeit gezeigt werden, dass Veränderungen des Mikromilieus diesen Kontakt auch in anderen Geweben ermöglicht.
Chemical neurotransmission is a complex process of central importance for nervous system function. It is thought to be mediated by the orchestration of hundreds of proteins for its successful execution. Several synaptic proteins have been shown to be relevant for neurotransmission and many of them are highly conserved during evolution- suggesting a universal mechanism for neurotransmission. This process has checkpoints at various places like, neurotransmitter uptake into the vesicles, relocation of the vesicles to the vicinity of calcium channels in order to facilitate Ca2+ induced release thereby modulating the fusion probability, formation of a fusion pore to release the neurotransmitter and finally reuptake of the vesicles by endocytosis. Each of these checkpoints has now become a special area of study and maintains its own importance for the understanding of the overall process. Ca2+ induced release occurs at specialized membrane structures at the synapse known as the active zones. These are highly ordered electron dense grids and are composed of several proteins which assist the synaptic vesicles in relocating in the vicinity of Ca2+ channels thereby increasing their fusion probability and then bringing about the vesicular fusion itself. All the protein modules needed for these processes are thought to be held in tight arrays at the active zones, and the functions of a few have been characterized so far at the vertebrate active zones. Our group is primarily interested in characterizing the molecular architecture of the Drosophila synapse. Due to its powerful genetics and well-established behavioural assays Drosophila is an excellent system to investigate neuronal functioning. Monoclonal antibodies (MABs) from a hybridoma library against Drosophila brain are routinely used to detect novel proteins in the brain in a reverse genetic approach. Upon identification of the protein its encoding genetic locus is characterized and a detailed investigation of its function is initiated. This approach has been particularly useful to detect synaptic proteins, which may go undetected in a forward genetic approach due to lack of an observable phenotype. Proteins like CSP, Synapsin and Sap47 have been identified and characterized using this approach so far. MAB nc82 has been one of the shortlisted antibodies from the same library and is widely used as a general neuropil marker due to the relative transparency of immunohistochemical whole mount staining obtained with this antibody. A careful observation of double stainings at the larval neuromuscular junctions with MAB nc82 and other pre and post-synaptic markers strongly suggested an active zone localization of the nc82 antigen. Synaptic architecture is well characterized in Drosophila at the ultrastructural level. However, molecular details for many synaptic components and especially for the active zone are almost entirely unknown. A possible localization at the active zone for the nc82 antigen served as the motivation to initiate its biochemical characterization and the identification of the encoding gene. In the present thesis it is shown by 2-D gel analysis and mass spectrometry that the nc82 antigen is a novel active zone protein encoded by a complex genetic locus on chromosome 2R. By RT-PCR exons from three open reading frames previously annotated as separate genes are demonstrated to give rise to a transcript of at least 5.5 kb. Northern blots produce a prominent signal of 11 kb and a weak signal of 2 kb. The protein encoded by the 5.5 kb transcript is highly conserved amongst insects and has at its N-terminus significant homology to the previously described vertebrate active zone protein ELKS/ERC/CAST. Bioinformatic analysis predicts coiled-coil domains spread all over the sequence and strongly suggest a function involved in organizing or maintaining the structure of the active zone. The large C-terminal region is highly conserved amongst the insects but has no clear homologues in veretebrates. For a functional analysis of this protein transgenic flies expressing RNAi constructs under the control of the Gal4 regulated enhancer UAS were kindly provided by the collaborating group of S.Sigrist (Gِttingen). A strong pan-neuronal knockdown of the nc82 antigen by transgenic RNAi expression leads to embryonic lethality. A relatively weaker RNAi expression results in behavioural deficits in adult flies including unstable flight and impaired walking behavior. Due to this peculiar phenotype as observed in the first knockdown studies the gene was named “bruchpilot” (brp) encoding the protein “Bruchpilot (BRP)” (German for crash pilot). A pan-neuronal as well as retina specific downregulation of this protein results in loss of ON and OFF transients in ERG recordings indicating dysfunctional synapses. Retina specific downregulation also shows severely impaired optomotor behaviour. Finally, at an ultrastructural level BRP downregulation seems to impair the formation of the characteristic T-shaped synaptic ribbons at the active zones without significantly altering the overall synaptic architecture (in collaboration with E.Asan). Vertebrate active zone protein Bassoon is known to be involved in attaching the synaptic ribbons to the active zones as an adapter between active zone proteins RIBEYE and ERC/CAST. A mutation in Bassoon results in a floating synaptic ribbon phenotype. No protein homologous to Bassoon has been observed in Drosophila. BRP downregulation also results in absence of attached synaptic ribbons at the active zones. This invites the speculation of an adapter like function for BRP in Drosophila. However, while Bassoon mutant mice are viable, BRP deficit in addition to the structural phenotype also results in severe behavioural and physiological anomalies and even stronger downregulation causes embryonic lethality. This therefore suggests an additional and even more important role for BRP in development and normal functioning of synapses in Drosophila and also in other insects. However, how BRP regulates synaptic transmission and which other proteins are involved in this BRP dependant pathway remains to be investigated. Such studies certainly will attract prominent attention in the future.
The development of ethanol tolerance is due to changes in synaptic plasticity. Since the mechanisms mediating synaptic plasticity are probably defective in the mutant hangAE10, it was a goal of the present study to find out how HANG contributes to synaptic plasticity. In particular, it was important to clarify in which neuronal process HANG plays a role. Antibody stainings against HANG revealed that the protein is localized in all neuronal nuclei of larval and adult brains; the staining is absent in hangAE10, thus confirming that this P-element insertion stock is a protein null for HANG. Detailed analysis of the subnuclear distribution of HANG showed that HANG immunoreactivity is enriched at distinct spots in the nucleus in a speckled pattern; these speckles are found at the inside of the nuclear membrane and do not colocalize with chromatin nor with the nucleolus; thus, HANG is probably involved in the stabilization, processing or export of RNAs. As synaptic plasticity can be studied in single neurons at the larval neuromuscular junction, the morphology of the synaptic terminals of hangAE10 mutants was analyzed at muscle 6/7, segment A4. These studies revealed that hangAE10 mutants display a 40 % increase in bouton number and axonal branch length; in addition, some boutons have an abnormal hourglass-like shape, suggesting that they are arrested in a semi-separated state following the initiation of bouton division. The increase in bouton number of hang mutants is mainly due to an increase in numbers of type Ib boutons. The analysis of the distribution of several synaptic markers in hang mutants did not show abnormalities. The presynaptic expression of HANG in hang mutants rescues the increase in bouton number and axonal branch length, thus proving that the phenotypes seen in the P-element insertion hangAE10 are attributable to the lack of HANG rather than to effects of the P-element marker rosy or to a secondary hit on the same chromsome during mutagensis. This finding is further supported by the fact that postsynaptic expression of HANG does not rescue the abnormal NMJ morphology of hangAE10. Alterations in cAMP levels regulate the number of boutons; since hang mutants display an increase in bouton number, the questions was whether this morphological abnormality was due to defects in cAMP signalling. To test this hypothesis, hangAE10 NMJs were compared to those of the hypomorphic allele dnc1 that has a defective cAMP cascade. Some aspects of the NMJ phenotype (e.g. the increase in bouton number and the unaltered ratio of active zones per bouton area) are similar in hangAE10 and dnc1, other differ. Expression of a UAS-dnc transgene in hangAE10 mutants does not modify the phenotype. In summary, the results of this study indicate that nuclear protein HANG might be involved in isoform-specific splicing of genes required for synaptic plasticity at the NMJ.
Somites are repeated epithelial segments that are generated in a rhythmic manner from the presomitic mesoderm (PSM) in the embryonic tailbud. Later, they differentiate into skeletal muscle, cartilage and dermis. Somitogenesis is regulated by a complex interplay of different pathways. Notch/Delta signaling is one of the pathways well characterized in zebrafish through mutants affected in its different components. Previous work in mouse, chicken and zebrafish has shown that also additional components are required during somitogenesis, most importantly through an FGF and Retinoic acid (RA) gradient, as well as Wnt signaling. However, no zebrafish mutants with defects in these pathways showing specific somite malformations are described. This was explained by functional redundancies among related genes that have resulted from a whole genome duplication which occurred in a teleost fish ancestor 350 million years ago. As distinct duplicates exist in different teleost species, a large scale mutagenesis screen in the medaka (Oryzias latipes) has been performed successfully in Kyoto, Japan. I analyzed nine of the isolated medaka mutants that show variable aspects of somitic phenotypes. This includes a complete or partial loss of somite boundaries (e.g. bms and sne), somites with irregular sizes and shapes (e.g. krz and fsl) or partially fused and enlarged somites (e.g. dpk). Although some of these medaka mutants share characteristics with previously described zebrafish somite mutants, most of the mutants represent unique phenotypes, not obtained in the zebrafish screens. In-situ hybridization analyses with marker genes implicated in the segmentation clock (e.g. her7), establishment of anterior-posterior (A-P) polarity (e.g. mesp) and differentiation of somites (e.g. myf5, lfng) revealed that the medaka mutants can be separated into two classes. Class I shows defects in tailbud formation and PSM prepatterning, and lateron somite boundary formation was impaired in these mutants. A unique member of this class with a novel phenotype is the doppelkorn (dpk) mutant that has single fused or enlarged somites. This phenotype has not been reported till now in zebrafish somite mutants. In-situ analyses on dpk showed that stabilization of the cyclically expressed somitogenesis clock genes must be affected in this mutant. This is accompanied by a disrupted regulation of A-P polarity genes like mesp. This suggests that dpk is a mutant deficient in the wave front, which is necessary for the down-regulation of oscillating genes in the anterior PSM. Furthermore, as the initiation of oscillation of all three cyclic her genes was unaffected in dpk embryos, I could exclude that this mutant in affected in the Notch/Delta pathway. Another mutant that belongs to this class is the samidare (sam) mutant. Morphologically, sam mutants are similar to zebrafish after eight (aei). In both cases, the first 7-9 somites are formed properly, but after this somite formation ceases. Different to the situation in aei, sam mutant embryos presented an additional defect in the mid-hindbrain boundary (MHB) region. Similar MHB defects were described in the zebrafish fgf8 mutant acerebellar (ace). In ace zebrafish mutant, somites were only slightly defective, although FGF signaling has been shown to be important for somite formation in chicken, mouse and zebrafish. This was explained by functional redundancy between fgf8 and fgf24 ligands in the tailbud of zebrafish. Thus, it is interesting to suggest that the sam mutant, based on the parallel defects in somites and MHB, is a potential member of the FGF signaling pathway muatnts. It was shown that FGF plays a crucial role during MHB formation in medaka. In addition, I showed that fgf8 acts non-redundantly during tailbud formation and somitogenesis in medaka. Furthermore, I showed that FGF signaling regulates somite size also in medaka and that fgfr1 is the only FGF receptor expressed in the tailbud and somites. In class II medaka somite mutants, PSM prepatterning appears normal, whereas A-P polarity, boundary formation, epithelialization or the later differentiation of somites appears to be affected. Such mutants have not been isolated so far in zebrafish, mice or chicken. Therefore, medaka class II somite mutants seem to be a novel group of mutants that opens new perspectives to analyze A-P polarity regulation, determination and boundary formation in the presence of a normally functioning clock in the PSM. Identifying the encoding genes for all analyzed medaka somite mutants will contribute to the understanding of the molecular interactions of different signaling pathways involved during somitogenesis, and is expected to result in the identification of new components.
The genetics of species differences is an outstanding question in evolutionary biology. How do species evolve to become phenotypically distinct and how is the genetic architecture organized that underlie species differences? Phenotypic diverged traits are supposed to be frequently involved in prezygotic isolation, i.e. they prevent the formation of hybrids, whereas postzygotic isolation occurs when hybrids experience a fitness reduction. The parasitic wasp genus Nasonia represents an appropriate model system to investigate the genetics of species differences as well as the genetics of postzygotic isolation. The genus consists of three species N. vitripennis, N. longicornis and N. giraulti that differ particularly in male traits that are assumed to posses an adaptive significance: courtship behaviour and wing size differences. The courtship behaviour consists of cyclically repeated series of head nods that are separated by pauses. The stereotypic performance allowed to split up the display into distinct courtship components. Males of N. vitripennis bear vestigial forewings and are incapable of flight, whereas N. longicornis wear intermediate sized wings and N. giraulti is fully capable of flying. Nasonia species can produce interspecific hybrids after removing Wolbachia bacteria induced hybrid incompatibilities with antibiotics. Postzygotic isolation occurs to different extent and is asymmetric among reciprocal crosses, e.g. inviability is stronger in the N. vitripennis (♀) x N. longicornis (♂) cross than in the N. longicornis (♀) x N. vitripennis (♂) cross. The formation of hybrids allow to study the genetic of species differences in QTL (quantitative trait locus) analyses as well as the genetics of postzygotic isolation causing hybrid inviability. The aim of the study was to investigate the genetic architecture of differences in courtship behaviour and wing size between N. vitripennis and N. longicornis and to assess the genetics of postzygotic isolation to gain clues about the evolutionary processes underlying trait divergence and establishment of reproductive isolation between taxa. In a QTL analysis based on 94 F2-hybrid individuals of an LV cross only few QTL for wing size differences have been found with relatively large effects, although a large proportion of the phenotypic variance remained unexplained. The QTL on courtship behaviour analysis based on 94-F2 hybrid males revealed a complex genetic architecture of courtship behaviour with QTL of large phenotypic effects that explained more than 40 % of the phenotypic variance in one case. Additionally, an epistatic analysis (non-additive interlocus interaction) of courtship QTL revealed frequent genetic interchromsomal relations leading in some instances to hybrid specific effects, e.g. reversion of phenotypic effects or the transgression of phenotypes. A QTL analysis based on a threefold sample size revealed, however, an overestimation of QTL effects in the analysis based on smaller sample size pointing towards a genetic architecture of many loci with small effects governing the phenotypic differences in courtship behaviour. Furthermore, the the study comprised the analysis of postzygotic isolation in the reciprocal crosses N. vitripennis (♀) x N. longicornis (♂) versus N. longicornis (♀) x N. vitripennis (♂) located several loci distributed over different chromosomes that are involved in hybrid incompatibility. The mapping of hybrid incompatibility regions reproduced for the first time the observed asymmetries in the strength of postzygotic isolation in reciprocal crosses of between the more distant related taxa within the genus Nasonia. Stronger postzygotic incompatibilities in the VL cross are supposed to result from the superposition of nuclear-nuclear incompatibilities with nuclear-cytoplasmic incompatibilities, whereas the coincidences of these to types of incompatibilities were found to be much weaker in the reciprocal LV cross.
The genus Pogonomyrmex is predisposed for analyzing the evolution of ant colony characteristics in general and the sociogenetic structure in particular, due to the renowned biology of several species and the diversity of mating frequency and queen number. This variation in the sociogenetic structure of colonies produces a high variance in intracolonial relatedness which can be a major component driving the evolution of various colony characteristics. To exactly determine the variability of the intracolonial relatedness in the genus Pogonomyrmex both were analyzed, the number of matrilines and patrilines, in selected members of Pogonomyrmex, namely P. (sensu stricto) rugosus, P. (sensu stricto) badius and P. (Ephebomyrmex) pima using DNA fingerprint techniques. The evolution of these colony characteristics were tried to be explained within a phylogenetic framework. For that purpose we constructed a gene-tree of 39 species of the genus Pogonomyrmex. The taxon sampling covered about 83 % of the North American species and 43 % of the South American species. Effective multiple mating of queens was confirmed for P. rugosus (me=4.1) and P. badius (me=6.7). Additionally, both species are monogynous. These results corroborate behavioral observations of multiple mating for these species. Multiple mating is now known from 9 Pogonomyrmex species (behavioral evidence for 3 species – genetic evidence for 6 species). However, in P. (E.) pima all queens that were analyzed were single mated (me=1.0). Therefore, multiple mating may have either evolved early during the evolution of the genus Pogonomyrmex and has subsequently been lost in the subgenus Ephebomyrmex (plesiomorphic hypothesis), or it has first been evolved in the subgenus Pogonomyrmex sensu stricto (apomorphic hypothesis). In P. huachucanus, a species basal to the North- American sensu stricto complex, smaller effective mating number of queens compared to its sensu stricto relatives (J. Gadau and C.-P. Strehl, unpublished) probably do mirror a change from monandry to polyandry during the evolution of more advanced sensu stricto species, which would support the apomorphic hypothesis. The intracolonial relatedness in P. (E.) pima is however rather low. This is probably the result of multiple reproducing queens (polygyny). Polygyny is also documented for at least four other species of the subgenus Ephebomyrex, but so far P. (E.) pima is the only species with genetic evidence. It might be that there was an evolutionary trade-off within the subgenus Ephebomyrmex between polyandry and polygyny. Therefore, both subgenera retained a high intracolonial genetic diversity. This high genetic diversity might be one cause for the success and radiation of the genus Pogonomyrmex in arid environments. Evolution might have favored high genetic diversity of Pogonomyrmex colonies, because it helps colonies to improve their colonial organization and efficiency in performing external tasks. At least in P. badius a link between patrilines and physical polyethism was found, indicative of an improvement of colonial organization via polyandry. Furthermore, the documented extreme levels of polyandry might help P. badius females to overcome the possibility of inbreeding due to restricted dispersal. Restricted dispersal is also found in P. (E.) pima due to wingless, intermorphic queens. However, in P. (E.) pima inbreeding is probably prevented by outcrossing via males because no significant inbreeding is found. In the presented gene trees the subgenus Pogonomyrmex Ephebomyrmex was separated from the subgenus Pogonomyrmex sensu stricto. Therefore, P. Ephebomyrmex might be elevated to generic status, also due to its distinct morphological and life history characters. Nevertheless, for a precise taxonomic revision a broader complement of species has to be applied. Regularly a low number of unrelated workers was found in P. rugosus colonies, which probably stem from brood raids between mature and founding colonies. It is well known that most founding colonies are destroyed by neighboring conspecific mature colonies, but so far it was assumed that the brood of these colonies was also destroyed. This often neglected aspect might be an important fitness token for mature colonies.
Corynebacterium glutamicum is together with C. callunae and C. efficiens a member of the diverse group of mycolic-acid containing actinomycetes, the mycolata. These bacteria are potent producer of glutamate, lysine and other amino acids on industrial scale. The cell walls of most actinomycetes contain besides an arabinogalactan-peptidoglycan complex large amounts of mycolic acids. This three-layer envelope is called MAP (mycolyl-arabinogalactan-peptidoglycan) complex and it represents a second permeability barrier beside the cytoplasmic membrane similar to the outer membrane of Gram-negative bacteria. In analogy to the situation in the outer membrane of Gram-negative bacteria, channels are present in the mycolic acid layer of the mycobacterial cell wall for the passage of hydrophilic solutes. Molecular studies have provided far-reaching findings on the amino acid flux and its balance in C. glutamicum in general, but the L-glutamate export still remains unknown. The properties of the outer layers, typical of mycolata, seem to be of major importance in this process, and diffusion seems to play a key role for this part of the cell wall. The major aim of this thesis was to identify and study novel channel-forming proteins of the amino acid producers C. glutamicum, C. callunae and C. efficiens. Cell wall extracts of the organisms were investigated and a novel pore-forming protein, named PorH, that is homologue in all three organisms, was detected and characterized. PorHC.glut was isolated from C. glutamicum cells cultivated in minimal medium. The protein was identified in lipid bilayer experiments and purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column. The purified protein forms cation-selective channels with a diameter of about 2.2 nm and an average single-channel conductance of about 2.5 nS in 1 M KCl in the lipid bilayer assay. Organic solvent extracts were used to study the permeability properties of the cell wall of C. callunae and C.efficiens. The cell extracts contained channel-forming activity, the corresponding proteins were purified to homogeneity by fast-protein liquid chromatography across a HiTrap-Q column and named PorHC.call and PorHC.eff. Channels formed by PorHC.call are cation-selective with a diameter of about 2.2 nm and an average single-channel conductance of 3 nS, whereas PorHC.eff forms slightly anion selective channels with an average single-channel conductance of 2.3 nS in 1 M KCl in the lipid bilayer assay. The PorH proteins were partially sequenced and the corresponding genes, which were designated as porH, were identified in the published genome sequence of C. glutamicum and C. efficiens. The chromosome of C. callunae is not sequenced, but PorHC.call shows a high homology to PorHC.eff and PorHC.glut. The proteins have no N-terminal extension, only the inducer methionine, which suggests that secretion of the proteins could be very similar to that of PorAC.glut of C. glutamicum. PorHC.glut is coded in the bacterial chromosome by a gene that is localized in the vincinity of the porAC.glut gene, within a putative operon formed by 13 genes that are encoded by the minus strand. Both porins are cotranscribed and coexist in the cell wall, which was demonstrated in RT-PCR and immunological detection experiments. The arrangement of porHC.glut and porAC.glut on the chromosome is similar to that of porBC.glut and porCC.glut and it was found that PorAC.glut, PorHC.glut, PorBC.glut and PorCC.glut coexist in the cell wall of C. glutamicum. The molecular mass of about 6 kDa of the PorH channel forming proteins is rather small and suggests that the cell wall channels are formed by oligomers. A possibly hexameric form was demonstrated for PorHC.glut in Western blot analysis with anti- PorHC.glut antibodies. Secondary structure predictions for PorHC.glut, PorHC.call and PorHC.eff predict that a stretch of about 42 amino acids of PorHC.glut and 28 amino acids of PorHC.call and PorHC.eff forms amphipathic -helices with a total length of 6.3 nm and 4.2 nm respectively. This should be sufficient to cross the mycolic acid layer. Another objective of this work was to establish an heterologous expression system for corynebacterial channel-forming proteins, to investigate the channel-forming properties of the up to now only hypothetical porins PorA, PorB, PorC from C. efficiens and PorC from C. glutamicum. We could demonstrate with recombinant expression experiments in E. coli that porBC.eff and porCC.eff encode for channel-forming proteins. They are, like PorBC.glut, anion-selective with a similar single-channel conductance of 1 nS in 1 M KCl.
Rhodococcus equi is a Gram-positive intracellular pathogen which can cause severe bronchopneumonia in foals. In recent years, the role of this bacterium as human pathogen has been noted, as R.equi infections in humans have increase in frequency. This increase is associated with the rise in immunosupressed individuals, specially AIDS patients, where infection leads to symptoms and pathology similar to those seen in foals with a high mortality rate. Due to its capability to survive and multiply in murine and equine macrophages, R.equi has been classified as a facultative intracellular bacterium. R.equi is found frequently in macrophages in alveolar infiltrate from infected animals. The pathogenicity of R.equi depends on its ability to exist and multiply inside macrophages and has been associated with the presence of virulence plasmids. It has been observed that, inside foal alveolar macrophages, R.equi-containing vacuoles (RCVs) do not mature into phagolysosomes. However, most of the intracellular events during R.equi infection have not been investigated in detail. The aim of this study was to elucidate the intracellular compartmentation of R.equi and the mechanism by which the bacteria avoid destruction in host macrophages. The importance of the virulence-associated plasmids of R.equi for the establishment of RCVs was also evaluated. Furthermore, the intracellular fate of viable and non-viable R.equi was compared in order to study whether viability of R.equi influeciantes the establishment of RCVs. In this study, the RCV was characterized by using a variety of endocytic markers to follow the path of the bacteria trhough murine macropages. Transmission electron microscopy-base analysis showed that R.equi was found equally frequently in phagosomes with loosely or thightly apposed membranes, and RCV often contains numerous membranous vesicles. Laser scanning microscopy of infected macrophages showed that the majority of phagosomes containing R.equi acquired transiently the early endosomal markers Rab5, Ptlns3P, and EEA-1, suggesting initially undisturbed phagosome maturation. Although the RCV acquired some late endosomal markers, such as Rab7, LAMP-1, and Lamp-2, they did not acquired vATPase, did not interact with pre-labeled lysosomes, and failed to acidify. These data clearly suggest that the RCV is a compartment which has left vacuoles that resemble multivesicular body compartments (MVB), which are transport intermediates between early and late endosomes and display internal vesicles very similar to the ones observed within RCVs. Analyisis of several R.equi strains containing either VapA- or VapB-expressing plasmids or neither demonstrated that the possession of the virulence-associated plasmids does not affect phagosome trafficking over a two hour period of infection. The finding that non-viable R.equi was still able to inhibit phagosome maturation (although not to the same extent as viable R.equi did) suggests that heat-insensitive factors, such as cell periphery lipids, may play a major role in inhibition of phagosome maturation, although heat-sensitive factors may also be involved.
A large variety of sex determination systems have been described in fish. However, almost no information is available about sex determination in the classical fish models, the zebrafish Danio rerio and the pufferfish Takifugu rubripes. A DNA-binding protein gene called dmrt1bY (or DMY) has been recently described as an outstanding candidate for the primary sex-determining gene in the medaka fish Oryzias latipes. But this gene is not the universal master sex-determining gene in teleost fish, since dmrt1bY is not found in most other fishes. Hence, other fish models need to be examined including the platyfish Xiphophorus maculatus. Xiphophorus maculatus has three types of sex chromosomes (X, Y and W; females are XX, WX or WY; males are XY or YY). Its gonosomes are at an early stage of differentiation. The sex-determining locus on the sex chromosomes is flanked by two receptor tyrosine kinase genes, the Xmrk oncogene and its protooncogenic progenitor gene egfrb, which both delimit a region of about 0.6 centiMorgans. This situation should allow the positional cloning of the sex-determining gene (SD) of the platyfish. For this purpose, Bacterial Artificial Chromosome (BAC) contigs were assembled from a BAC library of XY males constructed in our laboratory, using the oncogene Xmrk, egfrb, as well as a Y-specific pseudogene called ps-criptY as starting points. The ps-criptY sequence was found to be closely linked to the SD gene, since no recombination was observed between SD and ps-criptY in more than 400 individuals tested. Two major BAC contigs for the X chromosome (about 2.5 Mb) and three major BAC contigs for the Y chromosome (about 3.5 Mb) were built up and analyzed by strategic sequencing. These are some of the largest contigs ever assembled for the sex chromosomes of a non-mammalian vertebrate species. The molecular analysis of the ps-criptY contig was the major objective of this work. The Y-specific ps-criptY contig has been extended over 1 Mb in this work with 58 identified molecular markers. Approximatively 700 kb of non-redundant sequences has been obtained from this contig by strategic sequencing. Numerous Y-linked markers from the contig including ps-criptY were also detected on the X chromosome. Nevertheless, major structural differences were observed between the X and Y chromosomes. Particularly, a large region, which is present at one copy on the X chromosome and contains several candidate genes, was found to be duplicated on the Y chromosome. Evidence for an inversion in the sex-determining region and for the Y-specific accumulation of a repeated sequence called XIR was also obtained. Such events might correspond to an initiation of differentiation between both types of gonosomes. Accumulation of transposable elements was also observed in the ps-criptY contig. A DNA transposable element, helitron, was isolated from the sex-determining region of X. maculatus. Three copies of helitron are located on the ps-criptY contig and one copy on the X-linked contig (helitron has roughly 15 copies per haploid genome). No in-frame stop codon, truncation or intron was found in these four copies, which present high nucleotide identities to each other. This suggests that helitron elements might be active or have been recently active in X. maculatus. A consensus open reading frame of helitron was also assembled from medaka (Oryzias latipes) genomic sequences. Two candidate genes from the ps-criptY contig are also located on the W chromosome in the X. maculatus Usumacinta strain (heterogamety). These markers show the relationship between the different types of gonosomes and allow to compare the male and female heterogameties in the platyfish. Several gene candidates were identified in the ps-criptY contig. However, some of them such as msh2, cript, igd and acr probably correspond to pseudogenes. Interestingly, a novel gene, called swimy, is exclusively expressed in spermatogonia of the adult testis. Swimy is a gene encoding a DNA-binding protein with several putative DNA-binding domains. The data suggest that swimy is a very promising candidate for the master SD gene. Another novel gene, which is called fredi and encodes a novel helix-turn-helix protein, is predominately expressed in the adult testis and currently under scrutiny. There is no doubt that the master SD gene of X. maculatus will be identified by positional cloning. Further molecular analysis of the contigs built in this work will shed new light on the molecular mechanism of sex determination and the evolution of sex chromosomes in fish.
Cloning and functional characterization of novel genes expressed preferentially in the human retina
(2005)
The human retina is a multi-layered neuronal tissue specialized for the reception and processing of visual information. The retina is composed of a great diversity of neuronal cell types including rod and cone photoreceptors, bipolar cells, ganglion cells, amacrine cells, horizontal cells and Müller glia. In response to light, a coordinated series of molecular events, the so-called phototransduction cascade, is triggered in photoreceptor cells and the signals from the photoreceptors are further processed by the bipolar and ganglion cells to the higher centers of the brain. The retina as highly complex system may be greatly susceptible to genetic defects which can lead to a wide range of disease phenotypes. Therefore, isolation and characterisation of the genes active in the human retina will facilitate our deeper understanding of retinal physiology and mechanisms underlying retinal degeneration and provide novel candidates for the retinal disease genes. To identify novel genes that are specifically or predominantly expressed in the human retina, a cDNA library enriched for retina specific transcripts was generated using suppression subtractive hybridization (SSH) technique. In total, 1113 clones were randomly isolated from the retina SSH cDNA library and partially sequenced. On the basis of BLASTN algorithm analysis these clones were classified into four categories including those with I) significant homology to known human genes (766/1113), II) significant homology to partial transcripts and hypothetical gene predictions (162/1113), III) no homology to known mRNAs (149/1113), and IV) vector sequences and clones derived from mitochondrial genes (36/1113). After correcting for redundancy, category I represented 234 known human genes and category II a total of 92unknown transcripts. Clones from category I, were selected for expression analysis by RT-PCR in a great number of human tissues. This resulted in the identification of 16 genes which were expressed exclusively in the retina, 13 which were highly expressed in the retina compared to other tissues, 12 genes which were specifically expressed in neuronal tissues and 48 ubiquitously expressed genes. Thus, our expression analysis resulted in the identification of 29 genes exclusively or abundantly transcribed in the human retina. Of those, retina specific genes L25,L33, L35, L37, L38 and L40 were selected for further analysis. To characterize the complete mRNA sequences of these transcripts a full-length human retina cDNA library was constructed. The analysis of the L25 gene revealed three splicing variants of the ABCC5 gene, consequently named ABCC5_SV1 (SV1), ABCC5_SV2 (SV2) and ABCC5_SV3 (SV3).These isoforms comprise the first five exons of ABCC5 and additional novel exons named 5a, 5b and 5c, generated by differential exon usage. The determined lengths of the three transcripts are 2039 bp, 1962 bp, and 1887 bp in size, respectively. RT-PCR, real-time PCR and Northern blot analysis of ABCC5 as well as the isoforms SV1, SV2 and SV3demonstrated high levels of expression for all transcripts in the retina compared to other tissues. Analysis of their nucleotide sequences revealed that inclusion of exon 5a in splicing variant SV1 produced a frame shift and premature termination codon (PTC). Our data show that this splice variant is the target of nonsense mediated mRNA decay (NMD). This was shown by inhibition of protein synthesis with antibiotics puromycin and anisomycin in human cell lines A-RPE 19 and Y79. Our analysis resulted in an increase of the PTC containing transcript and a decrease of the ABCC5 transcript. Conversely, the amount of both transcripts (SV1 and ABCC5) returned to pre-treatment levels after removal of the inhibitors. Together, our results suggest that alternative splicing of the ubiquitously expressed ABCC5 gene in addition to NMD is involved in retina-specific transcriptional regulation of the mRNA level of ABCC5. In contrast, additional experiments demonstrated that the levels of expression ofSV2 and SV3 isoforms do not appear to influence ABCC5 transcription. Several of the cloned genes were selected for additional genotyping of single nucleotide polymorphisms (SNPs) in order to construct their SNP maps which are going to be used for future association studies of complex disease AMD. Thus, identification of novel retinal genes and their functional characterization will further our elucidation of retinal physiology in general and in the diseased state in particular, by providing candidate retinal disease genes.