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The demonstration ofthe chromosomal mode ofsex determinationvia genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the mostprimitive form of sex chromosornes. In many different taxa the evolutionary process involved in the differentiation ofadvanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hydridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparaus fishes ofthe family Poeciliidae. The hybridization pattern together with specific staining ofthe constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as weil as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.
Hierarchical structures among male indlviduals in a population are frequently reflected ln differences in aggressive and reproductive behavior and access to the females. In general, sodal dominance requires the Investments, which in turn then may have to be compensated for by high reproductive success. However, this hypothesls has so far only been sufficiently tested in small mating groups (one or two males with one or two females) due to the difficulties of determining paternity by conventional methods. DNA fingerprinting overcomes these problems by offering the possibility to determine genetic relationships and mating patterns within larger groups [Borke, T. (1989) Trends Ecol. Evol. 4, 139-144]. We show here that in the poecUiid fish Limia perugitu, in small matlng groups the dominant male has 8 mating success of 100%, whereas ln larger groups lts contribution to the offspring unexpectedly drops to zero.
Melanoma formation in the teleost Xiphophorus is caused by a dominant genetic locus, Tu. This locus includes the Xmrk oncogene, which encodes a receptor tyrosine kinase. Tumor induction is. suppressed in wild-type fish by a tumor suppressor locus, R. Molecular genetic analyses revealed that the Tu locus emerged by nonhomologaus recombination of the Xmrk proto-oncogene with a previously uncharacterized sequence, D. This event generated an additional copy of Xmrk with a new promoter. Suppression of the new Xmrk promoter by R in parental fish and its deregulation in hybrids explain the genetics of melanoma formation in Xiphophorus.
The silver carp (Hypophthalmichthys molitrix) growth hormone (GH) genewas isolated and sequenced following amplification from genomic DNA by the polymerase chain reaction. The gene spans a region of approx. 2.5 kb nucleotides (nt) and consists of five exons. The sequence predicts a polypeptide of 210 amino acids (aa) including a putative signal peptide of 22 hydrophobic aa residues. The arrangement of exons and introns is identical to the GH genes of common carp, grass carp, and very similar to mammals and birds, but quite different from that for the GH genes of tilapia and salmonids. The silver carp GH gene shares a high homology at the nt and aa Ievels with those of grass carp (95.3% nt, 99.5% aa) and of common carp (81% nt, 95.7% aa).
In order to develop all-fish expression vectors for microinjection into fertilized fish eggs, we have prepared the following cunstructs: rainbow trout metallothionein a/b and the gilthead seabream growth hormone cDNA (ptMTa-gbsGHcDNA, ptMTb-gsbGHcDNA), carp ß-actin gilthead seabream GH cDNA (pcAßgsbGHcDNA). The inducible metallothionein promoters a and b were cloned from rainbow trout, and the constitutive promoter ß-actin was isolated from carp. The metallothionein promoters were cloned by using the PCR technique. The tMTa contains 430 bp, while the tMTb contains 260 bp (Hong et al. 1992). These two promoters were introduced to pGEM-3Z containing the GH cDNA of Sparus aurata to form ptMTa-gsbGH and ptMTb-gsbGH, respectively. The carp cytoplasmic ß-actin gene was chosen as a source for isolating strong constitutive regulatory sequences. One of these regulatory sequences in pUC118 was Iigated to GH cDNA of S. aurata to form the pcAß-gsbGHcDNA. Expression of the constructs containing the metallothionein promoters was tested in fish cell culture and was found tobe induced effectively by zinc. The ptMTa gsb-GH cDNA construct was microinjected into fertilized carp eggs, and integration in the genome of carp was detected in the DNA isolated from fins at the age of two months.
Das angeborene Immunsystem von Insekten besteht aus einer humoralen Komponente, einer zellulären Komponente und dem Prophenoloxidase-aktivierenden System. Fast alle Erkenntnisse über das angeborene Immunsystem stammen von Arbeiten mit Modellorganismen wie z.B. Drosophila oder Anopheles gambiae. Wie genau das Immunsystem der Honigbiene (Apis mellifera) funktioniert, ist jedoch noch relativ unbekannt. In der vorliegenden Arbeit wurden die unterschiedlichen Immunreaktionen aller drei Entwicklungsstadien der Honigbiene nach artifizieller Infektion mit Gram-negativen und Gram-positiven Bakterien (Escherichia coli und Micrococcus flavus) und dem Akuten Bienen Paralyse Virus (ABPV) untersucht und verglichen. Eine E. coli-Injektion zeigt bei Larven und adulten Arbeiterinnen nur wenig Auswirkung auf das äußere Erscheinungsbild und die Überlebensrate. In beiden Entwicklungsstadien wird die humorale Immunantwort stark induziert, erkennbar an der Expression der antimikrobiellen Peptide (AMPs) Hymenoptaecin, Defensin1 und Abaecin. Zusätzlich werden allein in Jungbienen nach bakterieller Infektion vier weitere immunspezifische Proteine exprimiert. Unter anderem eine Carboxylesterase (CE1) und das Immune-Responsive Protein 30 (IRp30). Die Expression von CE1 und IRp30 zeigt dabei den gleichen zeitlichen Verlauf wie die der AMPs. In Jungbienen kommt es zudem nach E. coli-Injektion zu einer raschen Abnahme an lebenden Bakterien in der Hämolymphe, was auf eine Aktivierung der zellulären Immunantwort schließen lässt. Ältere Bienen und Winterbienen zeigen eine stärkere Immunkompetenz als Jungbienen. Selbst nicht-infizierte Winterbienen exprimieren geringe Mengen der immunspezifischen Proteine IRp30 und CE1. Die Expression von IRp30 kann dabei durch Verwundung oder Injektion von E. coli noch gesteigert werden. Eine weitere Besonderheit ist die im Vergleich zu Jungbienen raschere Abnahme an lebenden Bakterien in der Hämolymphe bis hin zur vollständigen Eliminierung. Die Reaktion von Puppen auf eine bakterielle Infektion war völlig unerwartet. Nach Injektion von E. coli-Zellen kommt es innerhalb von 24 h p.i. zu einem tödlichen Kollaps, der sich in einer Graufärbung des gesamten Puppenkörpers äußert. Da keine Expression von AMPs nachzuweisen war, wird die humorale Immunantwort offensichtlich nicht induziert. Auch die zelluläre Immunantwort scheint nicht aktiviert zu werden, denn es konnte keine Abnahme an lebenden E. coli-Zellen beobachtet werden. Aufgrund dieser fehlenden Immunreaktionen vermehrt sich E. coli im Hämocoel infizierter Puppen und scheint damit deren Tod herbeizuführen. Nach viraler Infektion wurden in allen drei Entwicklungsstadien der Honigbiene gänzlich andere Reaktionen beobachtet als nach bakterieller Infektion. Bei dem verwendeten Akuten Bienen Paralyse Virus (ABPV) handelt es sich um ein Picorna-ähnliches Virus, dessen Vermehrung in der Hämolymphe über die massive Synthese der Capsidproteine verfolgt werden kann. Eine Injektion von sehr wenigen ABPV-Partikeln ins Hämocoel hat dramatische Auswirkungen auf Larven. Nach Virusinjektion kommt es innerhalb weniger Stunden zu einer raschen Virusvermehrung und schon 24 h p.i. zum Tod, häufig begleitet von einer Schwarzfärbung der gesamten Larve. Kurz vor dem Ableben kommt es neben dem Abbau hochmolekularer Speicherproteine zur Expression zahlreicher Proteine, die u.a. an der Translation oder dem Schutz vor oxidativem Stress beteiligt sind. Auf Jungbienen hat eine ABPV-Infektion keine so dramatischen Auswirkungen wie auf Larven. Sie zeigen lediglich Zeichen von Paralyse, zudem überleben sie länger bei höheren injizierten Partikelzahlen, die Virusvermehrung ist langsamer und es kommt zu keiner starken Veränderung des Hämolymph-Proteinmusters. Es konnte gezeigt werden, dass es in ABPV-infizierten Larven oder adulten Bienen zu keiner erkennbaren Aktivierung des humoralen Immunsystems in Form von exprimierten AMPs kommt. Zudem scheint die humorale Immunantwort auch nicht unterdrückt zu werden, denn nach gleichzeitiger Injektion von E. coli und ABPV kommt es neben der Expression viraler Capsidproteine auch zur Expression von AMPs. Zusätzlich konnte in Jungbienen nach Infektion mit ABPV eine zelluläre Immunantwort in Form von Nodulation ausgeschlossen werden. Ältere Bienen scheinen nicht nur mit bakteriellen Infektionen, sondern auch mit einer ABPV-Infektion besser zurechtzukommen. Bei einer Menge an ABPV-Partikeln, die in Jungbienen spätestens 72 h p.i. zum Tod führt, ist in Winterbienen eine Virusvermehrung erst ab 96 h p.i. erkennbar und diese beeinträchtigt die Überlebensrate kaum. Puppen sind einer Virusinfektion genauso schutzlos ausgeliefert wie einer Bakterieninfektion. Es kommt zwar zu keiner starken Änderung des äußeren Erscheinungsbildes, jedoch bleiben Puppen in ihrer Entwicklung komplett stehen. Das Virus muss sich daher stark vermehren, allerdings nicht überwiegend - wie bei Larven und adulten Bienen - in der Hämolymphe.
Tumore der Nebennieren stellen häufige Tumore dar, welche bei mindestens 3 % der Population über 50-Jähriger vorkommen. Im Gegensatz dazu ist das Nebennierenrindenkarzinom mit einer Inzidenz von 1-2 Einwohner pro Million ein sehr seltener Tumor. Da seine Prognose allerdings ungünstig, und diese maßgeblich davon abhängt wie fortgeschritten der Tumor bei Diagnosestellung ist, ist es wichtig, dass die richtige Diagnose frühzeitig gestellt wird. Bis heute ist kein zuverlässiger immunhistochemischer Nebennierenrindenkarzinom-spezifischer Marker etabliert um das Nebennierenrindenkarzinom von anderen retroperitonealen Tumoren zu differenzieren. Sasano et al. schlug bereits 1995 erstmalig den Transkriptionsfaktor Steroidogenic Factor 1 (SF1) als Marker zur Differenzierung von Nebennierenrinden- und Nicht-Nebennierenrindentumoren vor. Allerdings wurde die diagnostische Wertigkeit bisher nur in sehr kleinen Fallserien mit insgesamt nur 17 Nebennierenrindenkarzinomen untersucht. In der vorliegenden Arbeit wurde die SF1 Protein-Expression bei 163 Nebennierenrindenkarzinomen, 52 Nebennierenrinden-Adenomen, 12 normalen steroidogenen Geweben (6 Nebennieren und 6 Ovare), sowie 73 Nicht-Steroidtumoren immunhistochemisch untersucht. Hierbei zeigte sich, das SF1 bei 158 von 161 evaluierbaren Nebennierenrindenkarzinomen und bei allen Proben von normalen und gutartigen Geweben (n=64) nachweisbar war. Im Gegensatz dazu war keine der 73 Nicht-Steroidgeweben SF1 positiv, so dass die diagnostische Genauigkeit extrem gut ist (Sensitivität: 98.6 %, Spezifität: 100 %, positive und negative predictive value jeweils 100 % und 97.3 %). In einem zweiten Schritt wurde untersucht ob die Protein-Expression von SF1 beim Nebennierenrindenkarzinom auch prognostische Bedeutung hat. Hierbei zeigte sich, dass Patienten mit Tumoren mit starker SF1 Färbung (30 %) ein deutlich schlechteres tumorstadium-adjustiertes Rezidiffreies- und Gesamt-Überleben haben als Patienten mit geringer SF1 Expression (hazard ratio: 2.45). Zusätzlich zu den immunhistochemischen Untersuchungen wurden FISH Analysen durchgeführt. Hierbei zeigte sich allerdings keine signifikante Korrelation zwischen SF1 Gendosis und der SF1 Protein-Expression, so dass zu vermuten ist, dass SF1 maßgeblich auf Transkriptions- und Translationsebene reguliert wird. In einem Versuch diese Frage zu beantworten wurden zwei mutmaßliche SF1 Interaktionspartner, FATE1 und DAX1, genauer immunhistochemisch untersucht. Hierbei wurde deutlich, dass FATE1 bei 62 von 141 evaluierbaren Nebenierenrindenkarzinomen und 12 von 62 normalen und gutartigen Geweben nachweisbar war. Im Gegensatz hierzu waren alle 9 Nicht-Steroidgewebe FATE1 negativ. Dies zeigt, das FATE1 nicht zur Diagnostik nutzbar ist (Sensitivität: 61 %, Spezifität: 100 %, positive und negative predictive value 100 % bzw. 14 %). Die DAX1 Analyse zeigte, dass alle 20 normalen und gutartigen Gewebe eine positive DAX1 Färbereaktion zeigten. Von 126 Nebennierenrindenkarzinomen waren 71 DAX1 positiv. Von den 8 untersuchten Nicht-Steroidgeweben waren 6 DAX1 positiv. Diese Ergebnisse belegen, dass auch DAX1 keine diagnostische Genauigkeit besitzt (Sensitivität: 56 %, Spezifität: 25 %, positive und negative predictive value 92 % bzw. 4 %). Die Untersuchung der prognostischen Fähigkeiten von FATE1 und DAX1 zeigte, dass Patienten mit Tumoren mit starker FATE1 Färbung (39 %) ein schlechteres tumorstadium-adjustiertes Gesamt- aber nicht Rezidiffreies-Überleben haben als Patienten mit niedriger FATE1 Protein-Expression (hazard ratio: 2.01). Weiterhin wurde deutlich, dass DAX1 keine deutlichen prognostischen Fähigkeiten besitzt. Zusammenfassend läßt sich aus der vorliegenden Arbeit folgern, das SF1 aktuell der beste diagnostische Marker zur Diagnose von Tumoren der Nebennierenrinde ist und damit Eingang in die histopathologische Routine-Diagnostik von Nebennierentumoren finden wird. Zusätzlich ist die SF1 Expression ein sehr guter prognostischer Marker beim Nebennierenrindenkarzinom, wobei sich die prognostische Aussage durch zusätzliche Färbung von FATE1 und DAX1 nur unwesentlich verbessern läßt.
Which home for coelacanth?
(1993)
Hereditary melanoma in Xiphophorus hybrids canying the melanoma·induclng Tu-Sd locus is caused by transcriptional activation of the Xmrk gene that resides at the Tu·Sd locus and encodes a novel member of receptor tyrosine kinases (RTK). ln this study, a total of 17 hereditary melanomas from various hybrid genotypes harbouring 7 different Tu alleles were also found to aver-express the correspondlng Xmrlc alleles. The Ievei of over-expression correlated with the degree of malignancy of the melanoma. ln addition, Xsrc expression was high ln many malignant melanomas. Expression pattems and Ieveis of the Xiphophorus EGF-receptor gene (Xerb B), the c-myc (Xmyc), and the PDGF (Xsls) gene(s) were not intriguing. Transcription of the ras gene(s) may be correlated to secondary events of melanoma progression. Expression pattems of Xfms, the Xiphophorus CSF-1 receptor homologue, can be explained by different contents of infiltrating macrophages in the tumors. ln carcinogen-induced tumors includlng one melanoma no significant expression of the Xmrk oncogene could be detected. Xsrc expression, however, was strikingly high. This indicates that activation of oncogenes other than Xmrk ls instrumental in tumorigenesls of neoplasia of non-hereditary origin.
In order to study the divergence of teleost sex chromosomes, subtractive cloning was carried out between genomic DNA ofmales and females ofthe rainbow trout (XX/XY) and of Leporinus elongatus (ZW /ZZ). Inserts cloned in a plasmid vector were individually tested on Southern blots of DNA of males and females for sex specificity. No sex-specific insert was obtained from trout, but two out of ten inserts cloned from L. elongatus showed sex-specific patterns in this species: one corresponds to a sequence present on both Z and W chromosomes, while the other is W specific. Sequences of these two inserts show neither clear homology with other known sequences, nor an open reading frame. They cross-hybridize with the genomic DNA of Leporinusfriderici, but without sex-specific patterns. Twenty-four L. elongatus adults were sexed by gonadal observation, chromosomed examination and Southern hybridization with one or the other insert. Ten males and 11 females had chromosomes and hybridization patterns typical of their sex. One ZW female was recognized as a male with the W-specific probe. This was also the case for two unusual ZW males, one having a male hybridization pattern with the other probe. These three atypical individuals may result from single genetic exchanges between four regions of the Z and the W, giving rise to three atypical W chromosomes. Finding males with such atypical heterochromosomes in a female heterogametic species may indicate that a gradual transition occurs between the heterogametic systems.
The male-polymorphic poeciliid fish, Limia perugiae, a small teleostean endemic to the southeast of the Caribbean island Hispafiola, consists of three male size morphs with uniform females. Large males differentiate at a size va:rying between 25 and 38 mm; intermediate males, between 21 and 25 mm. Under competition, !arge males exhibit an elaborate courtship display, whereas small males show only a sneak-chase behavior. Intermediate males adapt their tactics to the respective competitors. However, all malemorphs can switch from courtship display to sneak-chase behavior. In large mating groups with four males of different size and five or six virgin females, large dominant a-males as weil as small subordinate \(\delta\)-males did not produce any offspring. Unexpectedly, all progeny were sired exclusively by the intemediate subordinate ß- and \(\gamma\)-males. Breeding experiments with the three male morphs can best be explained by a model of Y -linked genes for small and !arge size which are both suspended by the activity of an autosomal recessive repressor responsible for the development of intermediate males. The dominant allele of the recessive repressor, in either its homoorits heterozygous state, activates the Y-chromosomal genes for !arge or small size, respectively. Accordingly, intermediate males may produce male offspring of all size classes, depending on the presence of either the Y-linked gene or the autosomal repressor.
According to a changing environment it is crucial for animals to make experience and learn about it. Sensing, integrating and learning to associate different kinds of modalities enables animals to expect future events and to adjust behavior in the way, expected as the most profitable. Complex processes as memory formation and storage make it necessary to investigate learning and memory on different levels. In this context Drosophila melanogaster represents a powerful model organism. As the adult brain of the fly is still quite complex, I chose the third instar larva as model - the more simple the system, the easier to isolate single, fundamental principles of learning. In this thesis I addressed several kinds of questions on different mechanism of olfactory associative and synaptic plasiticity in Drosophila larvae. I focused on short-term memory throughout my thesis. First, investigating larval learning on behavioral level, I developed a one-odor paradigm for olfactory associative conditioning. This enables to estimate the learnability of single odors, reduces the complexity of the task and simplify analyses of "learning mutants". It further allows to balance learnability of odors for generalization-type experiments to describe the olfactory "coding space". Furthermore I could show that innate attractiveness and learnability can be dissociated and found finally that paired presentation of a given odor with reward increase performance, whereas unpaired presentations of these two stimuli decrease performance, indicating that larva are able to learn about the presence as well as about the absence of a reward. Second, on behavioral level, together with Thomas Niewalda and colleagues we focussed on salt processing in the context of choice, feeding and learning. Salt is required in several physiological processes, but can neither be synthesized nor stored. Various salt concentrations shift the valence from attraction to repulsion in reflexive behaviour. Interestingly, the reinforcing effect of salt in learning is shifted by more than one order of magnitude toward higher concentrations. Thus, the input pathways for gustatory behavior appear to be more sensitive than the ones supporting gustatory reinforcement, which is may be due to the dissociation of the reflexive and the reinforcing signalling pathways of salt. Third, in cooperation with Michael Schleyer we performed a series of behavioral gustatory, olfactory preference tests and larval learning experiments. Based on the available neuroanatomical and behavioral data we propose a model regarding chemosensory processing, odor-tastant memory trace formation and the 'decision' like process. It incorporates putative sites of interaction between olfactory and gustatory pathways during the establishment as well as behavioral expression of odor-tastant memory. We claim that innate olfactory behavior is responsive in nature and suggest that associative conditioned behavior is not a simple substitution like process, but driven more likely by the expectation of its outcome. Fourth, together with Birgit Michels and colleagues we investigated the cellular site and molecular mode of Synapsin, an evolutionarily conserved, presynaptic vesicular phosphoprotein and its action in larval learning. We confirmed a previously described learning impairment upon loss of Synapsin. We localized this Synapsin dependent memory trace in the mushroom bodies, a third-order "cortical" brain region, and could further show on molecular level, that Synapsin is as a downstream element of the AC-cAMP-PKA signalling cascade. This study provides a comprehensive chain of explanation from the molecular level to an associative behavioral change. Fifth, in the main part of my thesis I focused on molecular level on another synaptic protein, the Synapse associated protein of 47kDa (Sap47) and its role in larval behavior. As a member of a phylogenetically conserved gene family of hitherto unknown function. It is localized throughout the whole neuropil of larval brains and associated with presynaptic vesicles. Upon loss of Sap47 larvae exhibit normal sensory detection of the to-be-associated stimuli as well as normal motor performance and basic synaptic transmission. Interestingly, short-term plasticity is distorted and odorant–tastant associative learning ability is reduced. This defect in associative function could be rescued by restoring Sap47 expression. Therefore, this report is the first to suggest a function for Sap47 and specifically argues that Sap47 is required for synaptic as well as for behavioral plasticity in Drosophila larva. This prompts the question whether its homologs are required for synaptic and behavioral plasticity also in other species. Further in the last part of my thesis I contributed to the study of Ayse Yarali. Her central topic was the role of the White protein in punishment and relief learning in adult flies. Whereas stimuli that precede shock during training are subsequently avoided as predictors for punishment, stimuli that follow shock during training are later on approached, as they predict relief. Concerning the loss of White we report that pain-relief learning as well as punishment learning is changed. My contribution was a comparison between wild type and the white1118 mutant larvae in odor-reward learning. It turned out that a loss of White has no effect on larval odorant-tastant learning. This study, regarding painrelief learning provides the very first hints concerning the genetic determinants of this form of learning.
DARWIN\(^1\) believed that sexual selection accounts for the evolution of exaggerated male ornaments, such as the sword-like caudal fin extensions of male fishes of the genus Xiphophorus, that appear detrimental to survival. Swordtails continue to feature prominently in empirical work and theories of sexual selection; the pre-existing bias hypothesis has been offered as an explanation for the evolution of swords in these fishes\(^{2,3}\). Based upon a largely morphological phylogeny, this hypothesis suggests that female preference to mate with sworded males arose in ancestrally swordless species, thus pre-dating the origin of the sword itself and directly driving its evolution. Here we present a molecular phylogeny (based on mitochondrial and nuclear DNA sequences) of Xiphophorus which differs from the traditional one: it indicates that the sword originated and was lost repeatedly. Our phylogeny suggests that the ancestor of the genus is more likely to have possessed a sword than not, thus questioning the applicability of the pre-existing bias hypothesis as an explanation for the cvolution of this sexually selected trait.
The melanoma·inducing gene of Xiphophorus fish encodes the Xmrk receptor tyrosine kinase. U sing a highly specific antiserum p~oduced against the recombinant receptor expressed with a baculovirus, it is shown that Xmrk is the most abundant phosphotyrosine protein in fish melanoma and thus highly activated in the tumors. Studies on a melanoma cellline revealed that these cells produce an activity that considerably stimulates receptor autophosphorylation. The stimulating activity induces receptor down-regulation and can be depleted from the melanoma cellsupernatant by the immobilized recombinant receptor protein. The fish melanoma cells can thus be considered autocrine tumor cells providing a source for future purification and characterization of the Xmrk ligand.
DURING vertebrale development, many neurons depend for survival and differentiation on their target cells\(^{1-3}\). The best documented mediator of such a retrograde trophic action is the neurotrophin nerve growth factor (NGF)\(^1\). NGF and the other known members of tbe neurotrophin family, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT -3) and neurotrophin-4/5 (NT -4/5) are conserved as distinct genes over large evolutionary distances\(^{4 -6}\). Here we report the cloning of neurotrophin-6 (NT -6), a new member of this family from the teleost fish Xiphophorus. NT -6 distinguishes itself from the other known neurotrophins in that it is not found as a soluble protein in the medium of producing cells. The addition of heparin (but not chondroitin) effects the release of NT -6 from cell surface and extracellular matrix molecules. Recombinant purified NT -6 has a spectrum of actions similar to NGF on chick sympathetic and sensory neurons, albeit with a lower potency. NT -6 is expressed in tbe embryonie valvulla cerebelli; expression persists in some adult tissues. The interaction of NT-6 with heparin-binding molecuJes may modulate its action in the nervous system .
Honeybees (Apis mellifera) forage on a great variety of plant species, navigate over large distances to crucial resources, and return to communicate the locations of food sources and potential new nest sites to nest mates using a symbolic dance language. In order to achieve this, honeybees have evolved a rich repertoire of adaptive behaviours, some of which were earlier believed to be restricted to vertebrates. In this thesis, I explore the mechanisms involved in honeybee learning, memory, numerical competence and navigation. The findings acquired in this thesis show that honeybees are not the simple reflex automats they were once believed to be. The level of sophistication I found in the bees’ memory, their learning ability, their time sense, their numerical competence and their navigational abilities are surprisingly similar to the results obtained in comparable experiments with vertebrates. Thus, we should reconsider the notion that a bigger brain automatically indicates higher intelligence.
All animals learn in order to cope with challenges imposed on them by their environment. This is true also for both larval and adult fruit flies as exemplified in pavlovian conditioning. The focus of this Thesis is on various aspects of the fruit flies learning ability. My main project deals with two types of learning which we call punishment-learning and pain-relief learning. Punishment learning happens when fruit flies are exposed to an odour which is followed by electric shock. After such training, flies have learned that that odour signals pain and consequently will avoid it in the future. If the sequence of the two stimuli is reversed such that odour follows shock, flies learn the odour as a signal for relief and will later on approach it. I first report a series of experiments investigating qualitative and parametric features of relief-learning; I find that (i) relief learning does result from true associative conditioning, (ii) it requires a relatively high number of training trials, (iii) context-shock training is ineffective for subsequent shock-odour learning. A further question is whether punishment-learning and pain-relief learning share genetic determinants. In terms of genetics, I test a synapsin mutant strain, which lacks all Synapsin protein, in punishment and relief-learning. Punishment learning is significantly reduced, and relief-learning is abolished. Pan-neuronal RNAi-mediated knock-down of Synapsin results in mutant-like phenotypes, confirming the attribution of the phenotype to lack of Synapsin. Also, a rescue of Synapsin in the mushroom body of syn97 mutants restores both punishment- and relief-learning fully, suggesting the sufficiency of Synapsin in the mushroom body for both these kinds of learning. I also elucidate the relationship between perception and physiology in adult fruit flies. I use odour-shock conditioning experiments to identify degrees of similarity between odours; I find that those similarity measures are consistent across generalization and discrimination tasks of diverse difficulty. Then, as collaborator of T. Völler and A. Fiala, I investigate how such behavioural similarity/dissimilarity is reflected at the physiological level. I combine the behaviour data with calcium imaging data obtained by measuring the activity patterns of those odours in either the sensory neurons or the projection neurons at the antennal lobe. Our interpretation of the results is that the odours perceptual similarity is organized by antennal lobe interneurons. In another project I investigate the effect of gustatory stimuli on reflexive behaviour as well as their role as reinforcer in larval learning. Drosophila larvae greatly alter their behaviour in presence of sodium chloride. Increasing salt concentration modulates choice behaviour from weakly appetitive to strongly aversive. A similar concentration-behaviour function is also found for feeding: larval feeding is slightly enhanced in presence of low salt concentrations, and strongly decreased in the presence of high salt concentrations. Regarding learning, relatively weak salt concentrations function as appetitive reinforcer, whereas high salt concentrations function as aversive reinforcer. Interestingly, the behaviour-concentration curves are shifted towards higher concentrations from reflexive behaviour (choice behaviour, feeding) as compared to associative learning. This dissociation may reflect a different sensitivity in the respective sensory-motor circuitry.
Trypanosomen sind Protozoen, die Krankheiten bei Mensch und Tier verursachen, die unbehandelt infaust verlaufen. Die Zellen sind hoch motil, angetrieben von einem einzelständigen Flagellum, welches entlang des Zellkörpers angeheftet ist. Selbst in Zellkultur hören Trypanosomen niemals auf sich zu bewegen und eine Ablation funktioneller Bestandteile des Flagellarapparates ist letal für Blutstromformen. Es wurde gezeigt, dass Motilität notwendig ist für die Zellteilung, Organellenpositionierung und Infektiosität. Dies macht Trypanosomen zu besonders geeigneten Modellorganismen für die Untersuchung der Motilität. Dennoch ist erstaunlich wenig über die Motilität bei Trypanosomen bekannt. Dies gilt auch noch genereller für die Protozoen. Unlängst ist dieses Gebiet allerdings in den Fokus vieler Arbeiten gerückt, was bereits erstaunliche, neue Erkenntnisse hervorgebracht hat. Doch Vieles ist noch nicht abschliessend geklärt, so z.B. wie der Flagellarschlag genau reguliert wird, oder wie sich der Schlag des Flagellums entlang des Zellkörpers ausbreitet. Die vorliegende Arbeit befasst sich besonders mit den Einflüssen, die die Mikroumgebung auf die Motilität von Blutstromform-Trypanosomen ausübt. In ihrem natürlichen Lebensraum finden sich Trypanosomen in einer hoch komplexen Umgebung wieder. Dies gilt sowohl für den Blutkreislauf, als auch für den Gewebezwischenraum in ihrem Säugerwirt. Die hohe Konzentration von Zellen, Gewebeverbänden und extrazellulären Netzwerken könnte man als Ansammlung von Hindernissen für die Fortbewegung auffassen. Diese Arbeit zeigt dagegen, dass der Mechanismus der Bewegung eine Adaptation an genau diese Umweltbedingungen darstellt, so z.B. an die Viskosität von Blut. Es wird auch ein Bewegungsmodell vorgestellt, das erläutert, worin diese Adaption besteht. Dies erklärt auch, warum die Mehrheit der Zellen einer Trypanosomenkultur eine ungerichtete Taumel-Bewegung aufweist in nieder-viskosem Medium, das keine solchen “Hindernisse” enthält. Die Zugabe von Methylcellulose in einer Konzentration von ca. 0,5% (w/v) erwies sich als geeigneter Ersatz von Blut, um optimale Bedingungen für gerichtetes Schwimmen von Blutstromform Trypanosomen zu erreichen. Zusätzlich wurden in dieser Arbeit unterschiedliche Arten von Hindernissen, wie Mikroperlen (Beads) oder molekulare Netzwerke, sowie artifizielle, geordnete Mikrostrukturen verwendet, um die Interaktion mit einer festen Matrix zu untersuchen. In deren Anwesenheit war sowohl die Schwimmgeschwindigkeit, als auch der Anteil an persistent schwimmenden Trypanosomen erhöht. Zellen, die frei schwimmend in Flüssigkeiten vorkommen (wie Euglena oder Chlamydomonas), werden effizient durch einen planaren Schlag des Flagellums angetrieben. Trypanosomen hingegen mussten sich evolutionär an eine komplexe Umgebung anpassen, die mit einer zu raumgreifenden Welle interferieren würde. Der dreidimensionale Flagellarschlag des, an die Zelloberfläche angehefteten, Flagellums erlaubt den Trypanosomen eine effiziente Fortbewegung durch die Interaktion mit Objekten in jedweder Richtung gleichermassen. Trypanosomen erreichen dies durch eine hydrodynamisch verursachte Rotation ihres Zellkörpers entlang ihrer Längsachse, entgegen dem Uhrzeigersinn. Der Einfluss der Mikroumgebung wurde in früheren Untersuchungen bisher vernachlässigt, ist zum Verständnis der Motilität von T. brucei jedoch unerlässlich. Ein weiterer, bisher nicht untersuchter Aspekt der Beeinflussung der Motilität durch die Umwelt sind hydrodynamische Strömungseffekte, denen Trypanosomen im kardiovaskulären System ausgesetzt sind. Diese wurden in dieser Arbeit mittels Mikrofluidik untersucht. Um unser Verständnis der Motilität von Trypanosomen von 2D, wie üblich in der Motilitätsanalyse mittels Lebend-Zell-Mikroskopie, auf drei Dimensionen auszudehnen, wurde als bildgebendes Verfahren auch die Holographie eingesetzt. Mikrofluidik und Holographie sind beides aufkommende Techniken mit großem Anwendungspotential in der Biologie, die zuvor noch nie für die Motilitätsanalyse von Trypanosomen eingesetzt worden waren. Dies erforderte daher interdisziplinäre Kooperationen. Zusätzlich wurde in dieser Arbeit auch ein vollständig automatisiertes und Software-gesteuertes Fluoreszenzmikroskopiesystem entwickelt, das in der Lage ist, einzelne Zellen durch entsprechende Steuerung des Mikroskoptisches autonom zu verfolgen und somit eine Bewegungsanalyse in Echtzeit ermöglicht, ohne weitere Benutzerinteraktion. Letztendlich konnte dadurch auch die Bewegung der schlagenden Flagelle und des gesamten Zellkörpers mit hoher zeitlicher und räumlicher Auflösung mittels Hochgeschwindigkeits-Fluoreszenzmikroskopie aufgeklärt werden.
Bees are subject to permanent threat from predators such as ants. Their nests with large quantities of brood, pollen and honey represent lucrative targets for attacks whereas foragers have to face rivalry at food sources. This thesis focused on the role of stingless bees as third party interactor on ant-aphid-associations as well as on the predatory potential represented by ants and defense mechanisms against this threat. Regular observations of an aphid infested Podocarpus for approaching stingless bees yielded no results. Another aim of this thesis was the observation of foraging habits of four native and one introduced ant species for assessment of their predatory potential to stingless bees. All species turned out to be dietary balanced generalists with one mostly carnivorous species and four species predominantly collecting nectar roughly according to optimal foraging theory. Two of the species monitored, Rhytidoponera metallica and Iridomyrmex rufoniger were considered potential nest robbers. As the name implies, stingless bees lack the powerful weapon of their distant relatives; hence they specialized on other defense strategies. Resin is an important, multipurpose resource for stingless bees that is used as material for nest construction, antibiotic and for defensive means. For the latter purpose highly viscous resin is either directly used to stick down aggressors or its terpenic compounds are included in the bees cuticular surface. In a feeding choice experiment, three ant species were confronted with the choice between two native bee species - Tetragonula carbonaria and Austroplebeia australis - with different cuticular profiles and resin collection habits. Two of the ant species, especially the introduced Tetramorium bicarinatum did not show any preferences. The carnivorous R. metallica predominantly took the less resinous A. australis as prey. The reluctance towards T. carbonaria disappeared when the resinous compounds on its cuticle had been washed off with hexane. To test whether the repulsive reactions were related to the stickiness of the resinous surface or to chemical substances, hexane extracts of bees’ cuticles, propolis and three natural tree resins were prepared. In the following assay responses of ants towards extract treated surfaces were observed. Except for one of the resin extracts, all tested substances had repellent effects to the ants. Efficacy varied with the type of extract and species. Especially to the introduced T. bicarinatum the cuticular extract had no effect. GCMS-analyses showed that some of the resinous compounds were also found in the cuticular profile of T. carbonaria which featured reasonable analogies to the resin of Corymbia torelliana that is highly attractive for stingless bees. The results showed that repellent effects were only partially related to the sticky quality of resin but were rather caused by chemical substances, presumably sesqui- and diterpenes. Despite its efficacy this defense strategy only provides short time repellent effects sufficient for escape and warning of nest mates to initiate further preventive measures.
Epimutations in Germ-Cell and Embryo Development: Possible Consequences for Assisted Reproduction
(2011)
Assisted reproductive technologies (ART) emerged in the late 1970’s as a therapy for human infertility. Up till now more than 3 million babies have been conceived through ART, demonstrating the safety and efficiency of the technique. Published reports showed an increase in the rate of imprinting disorders (Beckwith Wiedemann Syndrome, Angelman Syndrome, etc.) in babies born after ART. What are the effects imposed through ART and should researchers reassess its safety and implications on the future offspring? Throughout this thesis, I analyzed the methylation patterns of germ cells and embryos to determine whether in vitro maturation and in vitro fertilization have a negative impact on the epigenetic patterns. Furthermore, DNA methylation was compared between sperm of infertile and presumably fertile controls in order to understand whether epigenetic disturbances lead to infertility at the first place. The occurrence of methylation aberrations in germ cells of infertile patients could be transmitted to new-borns and then cause epigenetic disorders. In order to elucidate the imprinting status within single cells, I developed a new technique based on limiting dilution where bisulfite treated DNA is distributed across several wells before amplification. This allowed methylation measurement at the single allele level as well parent of origin detection. In a total of 141 sperm samples from couples undergoing in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) including 106 with male factor or combined infertility and 28 with female infertility, I detected a significant correlation between lower quality of semen parameters (sperm count, percentage of abnormal sperm, and percentage of motile sperm) and the rate of imprinting errors. ALU repeats displayed a higher methylation in sperm DNA of patients leading to a pregnancy and live birth, compared to patients in which pregnancy was not achieved or a spontaneous abortion occurred. A discriminant analysis based on ALU methylation allowed correct classification of >70% of cases. Preliminary data from illumina methylation arrays where more than 27,000 CpGs were analyzed determined that only a single CpG site from the open reading frame C14orf93 was significantly different between the infertile and presumably fertile control group. However, further improvements on data normalization might permit detection of other differentially methylated regions. Comparison of embryos after natural conception, in vitro fertilized embryos from superovulated oocytes, and embryos achieved through fertilization of in vitro cultured oocytes revealed no dramatic effect on the imprinting patterns of Igf2r, H19, and Snrpn. Oocyte cryotop vitrification did not result in a dramatic increase of imprinting mutations in oocytes even though the rate of sporadic methylation errors in single Snrpn CpGs were higher within the in-vitrified group. Collectively, the results I will present within this thesis suggest an increase in the rate of imprinting errors within the germ cells of infertile patients, in addition to a decrease in genome wide methylation of ALU repetitive elements. I did not observe a detrimental effect on the methylation patterns of oocytes and the resulting embryos using in vitro maturation of oocytes and/or standard IVF with in vivo grown superovulated oocytes.
Bifunctional recombinant plasmids were constructed, comprised of the E. coli vectors pBR322, pBR325 and pACYC184 and different plasmids from Gram-positive bacteria, e.g. pBSU161-1 of B. subtilis and pUB110 and pC221 of S. aureus. The beta-lactamase (bla) gene and the chloramphenicol acetyltransferase (cat) gene from the E. coli plasmids were not transcribed and therefore not expressed in B. subtilis. However, tetracycline resistance from the E. coli plasmids was expressed in B. subtilis. Transcription of the tetracycline resistance gene(s) started in B. subtilis at or near the original E. coli promoter, the sequence of which is almost identical with the sequence recognized by σ<sup>55</sup> of B. subtilis RNA polymerase.
From a cosmid gene bank of Bacillus cereus GP4 in Escherichia coli we isolated clones which, after several days of incubation, formed hemolysis zones on erythrocyte agar plates. These clones contained recombinant cosmids with B. cereus DNA insertions of varying lengths which shared some common restriction fragments. The smallest insertionwas recloned as aPstl fragment into pJKK3-1, a shuttle vector which repücates in Bacillus subtilis and E. coli. When this recombinant plasmid (pJKK3-1 hly-1) was transformed into E. coli, it caused hemolysis on erythrocyte agar plates, but in liquid assays no extemal or intemal hemolytic activity could be detected with the E. coli transformants. B. subtilis carrying the same plasmid exhibited hemolytic activity at Ievels comparable to those ofthe B. cereus donor strain. The hemolysin produced in B. subtilis seemed to be indistinguishable from cereolysin in its sensitivity to cholesterol, activation by dithiothreitol, and inactivation by antibodies raised against cereolysin. When the recombinant DNA carrying the cereolysin gene was used as a probe in hybridization experiments with chromosomal DNA from a streptolysin 0-producing strain of Streptococcus pyogenes or from üsteriolysin-producing strains of Usteria monoeytogenes, no positive hybridization signals were obtained. These data soggest that the genes for these three SH-activated cytolysins do not have extended sequence homology.
A cloned cytolytic determinant from the genome of Bacillus cereus GP-4 has been characterized at the molecular Ievel. Nucleotide sequence determination revealed the presence of two open reading frames. 8oth open reading frames were found by deletion and complementation analysis to be necessary for expression of the hemolytic phenotype by Bacillus subtilis and Escherichia coli hosts. The 5' open reading frame was found to be nearly identical to a recently reported phospholipase C gene derived from a mutant B. cereus strain which overexpresses the respective protein, and it conferred a lecithinase-positive phenotype to the B. subtilis host. The 3' open reading frame encoded a sphingomyelinase. The two tandemly encoded activities, phospholipase C and sphingomyelinase, constitute a biologically functional cytolytic determinant of B. cereus termed cereolysin AB.
During an investigation into the substrate specificity and processing of subtilisin Carlsberg from Bacillus licheniformis, two major independent findings were made: (i) as has been shown previously, a stretch of five amino acids (residues 97-101 of the mature enzyme) that loops out into the binding cleft is involved in substrate binding by subtilisin Carlsberg. In order to see whether this loop element also determines substrate specificity, the coding region for these five amino acids was deleted from the cloned gene for subtilisin Carlsberg by site-directed mutagenesis. Unexpectedly the resulting mutant preproenzyme (P42c, M<sub>r</sub>=42 kDa) was not processed to the mature form (M<sub>r</sub> = 30 kDa) and was not released into the medium by a proteasedeficient B. subtilis host strain; rather, it accumulated in the cell membrane. This result demonstrates that the integrity of this loop element, which is very distant from the processing cleavage sites in the preproenzyme, is required for secretion of subtilisin Carlsberg. (ii) In culture supernatants from B. subtilis harbouring the cloned wild-type subtilisin Carlsberg gene the transient appearance (at 0-3 h after onset of stationary phase) of a processing intermediate (P38c, M<sub>r</sub> = 38 kDa) oftbis protease could be demonstrated. P38c very probably represents a genuine proform of subtilisin Carlsberg.
In culture supematants of both Listeria ivanovii and Listeria monocytogenes Sv4b, for the first time a hemolysin of molecular weight 58 kDa was identified, which had all the characteristics of an SH-activated cytolysin, and which was therefore identified as Iisteriolysin 0 (LLO). In the case of L. ivanovii a second major supematant protein of molecular weight 24 kDa co-purified with LLO. However, the function of this protein has to be determined. In culture supematants of L. ivanovii a sphingomyelinase and a Iecithinase activity could be detected, both enzymatic activities together contributing to the pronounced hemolysis caused by L. ivanovii. The N-tenninal amino acid sequences of LLO and the 24 kDa from L. ivanovii are shown.
Synaptonemal Komplexe (SC) sind evolutionär konservierte, meiosespezifische, proteinöse Strukturen, die maßgeblich an Synapsis, Rekombination und Segregation der homologen Chromosomen beteiligt sind. Sie zeigen eine dreigliedrige strickleiter-artige Organisation, die sich aus i) zwei Lateralelementen (LE), an die das Chromatin der Homologen angelagert ist, ii) zahlreichen Transversalfilamenten (TF), welche die LE in einer reißverschlussartigen Weise miteinander verknüpfen, und iii) einem zentralen Element (CE) zusammensetzt. Die Hauptproteinkomponenten der Säuger-SC sind das Transversalfilamentprotein SYCP1 und die Lateralelementproteine SYCP2 und SYCP3. Wie sich die SC-Struktur zusammenfügt war bisher nur wenig verstanden; es war nicht bekannt wie die TF innerhalb der LE-Strukturen verankert sind und dabei die homologen Chromosomen verknüpfen. Aufgrund dessen wurde die Interaktion zwischen den Proteinen SYCP1 und SYCP2 untersucht. Mit der Hilfe verschiedenster Interaktionssysteme konnte gezeigt werden, dass der C-Terminus von SYCP1 mit SYCP2 interagieren kann. Aufgrund der Bindungsfähigkeit zu beiden Proteinen, SYCP1 und SYCP3, kann angenommen werden, dass SYCP2 als Linker zwischen diesen Proteinen fungiert und somit möglicherweise das fehlende Bindungsglied zwischen den Lateralelementen und Transversalfilamenten darstellt. Obwohl die SC-Struktur in der Evolution hochkonserviert ist, schien dies nicht für seine Protein-Untereinheiten zuzutreffen. Um die Struktur und Funktion des SC besser verstehen zu können, wurde ein Vergleich zwischen den orthologen SYCP1 Proteinen der evolutionär entfernten Spezies Ratte und Medaka erstellt. Abgesehen von den erheblichen Sequenzunterschieden die sich in 450 Millionen Jahren der Evolution angehäuft haben, traten zwei bisher nicht identifizierte Sequenzmotive hervor, CM1 und CM2, die hochgradig konserviert sind. Anhand dieser Motive konnte in Datenbankanalysen erstmals ein Protein in Hydra vulgaris nachgewiesen werden, bei dem es sich um das orthologe Protein von SYCP1 handeln könnte. Im Vergleich mit dem SYCP1 der Ratte zeigten die Proteine aus Medaka und Hydra, neben den hoch konservierten CM1 und CM2, vergleichbare Domänenorganisationen und im heterologen System zudem sehr ähnliche Polymerisationseigenschaften. Diese Ergebnisse sprechen für eine evolutionäre Konservierung von SYCP1.
A gene coding for catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase; EC 1.11.1.6) of the grain-positive bacterium Listeria seeligeri was cloned from a plasmid library of EcoRI-digested chromosomal DNA, with Escherichia coli DHSa as a host. The recombinant catalase was expressed in E. coli to an enzymatic activity approximately SO times that of the combined E. coli catalases. The nucleutide sequence was determined, and the deduced amino acid sequence revealed 43.2% amino acid sequence identity between bovine liver catalase and L. seeligeri catalase. Most of the amino acid residues which are involved in catalytic activity, the formation of the active center accession channel, and heme binding in bovine liver catalase were also present in L. seeligeri catalase at the corresponding positions. The recombinant protein contained 488 amino acid residues and had a calculated molecular weight of 55,869. The predicted isoelectric point was 5.0. Enzymatic and genetic analyses showed that there is most probably a single catalase of this type in L. seeligeri. A perfect 21-bp inverted repeat, which was highly homologous to previously reported binding sequences of the Fur (ferric uptake regulon) protein of E. coli, was detected next to the putative promoter region of tbe L. seeligeri catalase gene.
The completc DNA scqucnccs coding for thc thiol-activated cytolysins from Listeria ivanovii, ivanolysin 0 (ILO) and for sccligerolysin 0 (LSO) from Listeria seeligeri have been dctermined. Thc deduced amino acid scquences revealed that: (i) the primary translation products comprise 528 (ILO) and 530 (LSO) amino acids. respectively. (ii) ILO contains two cysteines. LSO has a substitution in the conserved cysteine motif.
A gene (Imsod) encoding superoxide dismutase (SOD; EC 1.15.1.1) of the facultative intracellular pathogen, Listeria monocytogenes, was cloned by functional complementation of an SOD-deficient Escherichia coli mutant. The nucleotide sequence was determined and the deduced amino acid (aa) sequence (202 aa) showed close similarity to manganese-containing SOD's from other organisms. Subunits of the recombinant L. monocytogenes SOD (re-SOD) and of both E. coli SODs formed enzymatically active hybrid enzymes in vivo. DNA/DNA-hybridization experiments showed that this type of recombinant re-sod gene is conserved within the genus Listeria.
The cellular onc-genes c-src and c-yes are expressed very differently during chicken embryonic development. The c-src mRNA and its translational product are detectable at high levels in brain extracts of chicken embryos and adult chickens, whereas muscle extracts show an age-dependent decrease in the amounts of c-src-specific mRNA and pp60<sup>c-src</sup> kinase activity. In contrast, the Ievels of c-yes mRNA in brain, heart, and muscle are relatively low in early embryonic stages and increase later on to values comparable to those found for liver, while in adult animals the pattern of c-yes expression is similar to that of the c-src gene. From the close correlation between the Ievels of pp60<sup>c-src</sup>, its enzymatic activity, and its corresponding mRNA at a given stage of development and in given tissues, it appears that the expression of pp60<sup>c-src</sup> is primarily controlled at the level of transcription. It is suggested that because of the different patterns of expression, the two cellular oncogenes, c-src and c-yes, play different roles in cell proliferation during early embryonic stages as weil as in ensuing differentiation processes.
Activation of the pp60\(^{c-src}\) kinase during differentiation of monomyelocytic cells in vitro
(1986)
Tbe proto-oncogene c-src, the cellular homolog of the Rous sarcoma virus (RSV) transforming gene v-src, is expressed in a tissue-specific and age-dependent manner. Its physiological function, although still unknown, appears to be more closely related to differentiation processes than to proliferation processes. To obtain more information about the physiological role of the c-src gene in cells, we have studied differentiation-dependent alterations using the human HL-60 leukaemia cell line as a model system. Induction of monocytic and granulocytic differentiation of HL-60 cells by 12-0-tetradecanoylphorbol-13-acetate (TPA) and dimethylsulfoxide (DMSO) is associated with an activation of the pp60<sup>c-src</sup> tyrosine kinase, but not with increased c-src gene expression. Control experiments exclude an interaction of TPA and DMSO themselves with the pp60<sup>c-src</sup> kinase.
Chromosome 11p13 is frequently rearranged in individuals with the WAGR syndrome (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) or parts of this syndrome. To map the cytogenetic aberrations molecularly, we screened DNA from cell Unes with known WAGR-related chromosome abnormalities for rearrangements with pulsed fleld gel (PFG) analysis using probes deleted from one chromosome 11 homolog of a WAGR patient. The first alteration was detected in a cell line from an individual with aniridia, genitourinary anomalies, mental retardation, and a deletion described as 11p14.1-p13. We have located one breakpoint close to probe HU11-164B and we have cloned both breakpoint sites as well as the junctional fragment. The breakpoints subdivide current intervals on the genetic map, and the probes for both sides will serve as important additional markers for a long-range restriction map of this region. Further characterization and sequencing of the breakpoints may yield insight into the mechanisms by which these deletions occur.
The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.
A long-range restriction map of part of the short arm of ehromosome 11 including the WAGR region has been constructed using pulsed-field gel electrophoresis and a number of infrequently cutting restriction enzymes. A total of 15.4 Mbp has been mapped in detall, extending from proximal 11p14 to the distal part of 11p12. The map localizes 35 different DNA probes and reveals at least nine areas with features eharaeteristle of BTF islands, some of which may be candidates for the different loci underlying the phenotype of the WAGR syndrome. This map will furthermore allow screening of DNA from individuals with WAGR-related phenotypes and from Wilms tumors for associated chromosomal rearrangements.
The human gene encoding the myogenic determination factor myf3 (mouse MyoD1) has been mapped to the short arm of chromosome 11. Analysis of several somatic cell hybrids containing various derivatives with deletions or translocations revealed that the human MyoD (MYF3) gene is not associated with the WAGR locus at chromosomal band 11pl3 nor with the loss of the heterozygosity region at 11p15.5 related to the Beckwith-Wiedemann syndrome. Subregional mapping by in situ hybridization with an myf3 specific probe shows that the gene resides at the chromosomal band llp14, possibly at llp14.3.
The Wilms tumor gene WTl, a proposed tumor suppressor gene, has been identifled based on its location within a homozygous deletion found in tumor tissue. The gene encodes a putative transcription factor containing a Cys/His zinc finger domain. The critical homozygous deletions, however, are rarely seen, suggesting that in many cases the gene may be inactivated by more subtle alterations. To facilitate the seareh for smaller deletions and point mutations we have established the genomic organization of the WTl gene and have determined the sequence of all 10 exons and flanking intron DNA. The pattern of alternative splicing in two regions has been characterized in detail. These results will form the basis for future studies of mutant alleles at this locus.
Transiently activating (A-type) potassium (K) channels are important regulators of action potential and action potential firing frequencies. HK1 designates the firsthuman cDNA that is highly homologous to the rat RCK4 cDNA that codes for an A-type K-channel. The HK1 channel is expressed in heart. By somatic cell hybrid analysis, the HK1 gene has been assigned to human chromosome 11p13-pl4, the WAGR deletion region (Wilms tumor, aniridia, genito-urinary abnormalities and mental retardation). Subsequent pulsed field gel (PFG) analysis and comparison with the well-established PFG map of this region localized the gene to 11p14, 200-600 kb telomeric to the FSHB gene.
The direct involvment of the Wilm's tumor suppressor gene (WTl) in Denys-Drash syndrome through mutations within exons 8 or 9 has recently been established. The absence of such alterations in three patients with Frasier syndrome provides a molecular basis for distinguishing these two syndromes that are associated with streak gonads, pseudohermaphroditism and renal failure.
Der Notch Signalweg spielt während der Embryonalentwicklung eine zentrale Rolle in der Spezifizierung des Zellschicksales, der Proliferation und der Kommunikation benachbarter Zellen. Die Hey bHLH Transkriptionsfaktoren sind Zielgene des Notch-Signalweges und besitzen wichtige Funktionen in der kardiovaskulären Entwicklung. Hey2 Knockout (KO) Mäuse und Hey1/HeyL Doppelknockout-Mäuse (DKO) sind gekennzeichnet durch eine fehlerhafte Ausbildung der Herzscheidewand und der Herzklappen und durch eine unzureichende Differenzierung während der Blutgefäßentwicklung. Ziel dieser Arbeit war es, neue Zielgene der Hey Proteine zu finden, um ihre Funktion in der Organentwicklung und die Ausprägung der Hey KO Maus-Phänotypen besser verstehen zu können. Dazu wurde als Methode eine Kombination aus Microarray-Analyse und Chromatinimmunpräzipitation (ChIP) gewählt, um gleichzeitig einen Überblick über die regulierten Zielgene und der direkt gebundenen Promotoren zu gewinnen. Als Zellkulturmodell wurden HEK293-Zellen genutzt, die doxyzyklin-induzierbar Flag-markiertes Hey1, bzw. Hey2 Protein überexprimieren. Eine Microarray-Analyse nach Überexpression von Hey1, bzw. Hey2 ergab insgesamt ca. 100 bis zu 5-fach herunterregulierte Zielgene und nur für Hey2 15 Gene, die stärker als 2-fach hochreguliert waren. Eine ChIP mit αFlag-Antikörper zeigte eine direkte DNA-Bindung von Hey1, bzw. Hey2, im proximalen Promotorbereich von 4 herunterregulierten Zielgenen (HEY1, BMP2, KLF10 und FOXC1). Ist jedoch die DNA-bindende basische Domäne des Hey1-Proteins deletiert, bzw. durch Aminosäureaustausche (3 Arginine zu 3 Lysine) vermutlich nicht mehr DNA-bindend, kann eine Herunterregulation der Zielgene nach Überexpression der Hey1-Mutanten nicht mehr festgestellt werden. Ebenso kann eine Bindung der Hey1-Mutanten an die ausgewählten Promotoren von HEY1, BMP2, KLF10 oder FOXC1 mit ChIP nicht mehr nachgewiesen werden. Dies deutet darauf hin, dass die basische Domäne essentiell für die DNA-Bindung und für die Funktion der Hey Proteine ist. Mit ChIP-PET und anschließender Hochdurchsatz-Sequenzierung wurde ein genomweiter Screen der Hey1- und der Hey2-Bindungsstellen in HEK293-Zellen durchgeführt. Für Hey1 wurden 1453 Zielgene, für Hey2 4288 Zielgene bestimmt, wobei 1147 Gene gemeinsame Zielgene von Hey1 und Hey2 waren. Obwohl die Bindungsstellen in 5'- und 3'-Richtung von kodierenden Sequenzen und auch in Exons und Introns lokalisiert waren, waren 55 %, bzw. 49 % aller Bindungsstellen für Hey1, bzw. Hey2 im proximalen Promotorbereich von -0,5 kb und im ersten Exon lokalisiert. Eine in silico Analyse des Bindemotivs deutete auf eine repetitive GC-haltige Sequenz hin, die vermutlich in CpG Inseln lokalisiert ist. Diese Ergebnisse weisen auf eine direkte Regulation der Transkriptionsmaschinerie durch die Hey Proteine hin. Ein Vergleich der Zielgene aus den Microarray-Analysen mit den ChIP-PET Daten zeigte einen hohen Anteil an herunterregulierten Genen mit Bindestellen, die direkt von Hey gebunden waren. Während 60 % der herunterregulierten Hey2 Zielgene in der ChIP-PET Analyse eine direkte DNA-Bindung zeigen, weisen nur 20 % der hochregulierten Gene Bindestellen für Hey2 auf. Dies spricht für eine überwiegende Repressorfunktion der Hey Proteine. Um zu überprüfen, inwieweit die Hey Proteine zelltypspezifisch verschiedene Zielgene regulieren, wurden embryonale Stammzellen (ES-Zellen) generiert, die ebenfalls doxyzyklin-induzierbar Hey1, bzw. Hey2 überexprimieren. Diese ES-Zellen konnten effektiv zu Kardiomyozyten differenziert werden, so dass auch in diesen Zellen eine Hey Überexpression induziert und somit eine Genexpressionsanalyse durchgeführt werden konnte. Microarray Analysen der ES-Zellen und Kardiomyozyten ergaben mehr hoch- als herunterregulierte Gene im Vergleich zu HEK293-Zellen. Die Überlappung an gemeinsam regulierten Zielgenen in HEK293, ES-Zellen und Kardiomyozyten war sehr gering. Nur zwei Hey2-Zielgene wurden gleichzeitig in HEK293 und ES-Zellen stärker als 2-fach reguliert (Hes1, Zic2). Diese geringe Überlappung deutet auf ein enges zelltypspezifische Regulationspotential hin. Eine Genontologie-Analyse aller Zielgene zeigte Interaktionen der Hey Proteine mit verschiedenen Signalwegen (z.B. TGFβ-, Id- oder Wnt-Signalweg), die alle unersetzlich in frühen Entwicklungsprozessen sind. Diese Ergebnisse deuten darauf hin, dass die Hey Proteine zelltypspezifisch die Expression von Genen aus verschiedenen Signalwegen beeinflussen und modulieren können. Weiterhin eröffnen diese Daten neue Möglichkeiten für zukünftige Forschung, um die Rolle der Hey Proteine in der frühen Organentwicklung genauer ergründen.
Characterisation of Metalloprotease-mediated EGFR Signal Transactivation after GPCR Stimulation
(2011)
In the context of metalloprotease-mediated transactivation of the epidermal growth factor receptor, different monoclonal antibodies against ADAM17 / TACE were characterized for their ability to block the sheddase. Activity of some of them was observed at doses between 2µg/mL and 10µg/mL. Kinetic analyses showed their activity starting at around 30 minutes. In cellular assays performed with the antibodies, especially upon treatment of cells with sphingosine-1-phosphate a reduction in proliferation was observed with some candidates. Moreover this study provides potential new roles for ß-Arrestins. Their involvement in the triple membrane-passing signal pathway of EGFR transactivation was shown. Furthermore, in overexpressing cellular model systems, an interaction between ADAM17 and ß-Arrestin1 could be observed. Detailed analysis discovered that phosphorylation of ß-Arrestin1 is crucial for this interaction. Additionally, the novel mechanism of UV-induced EGFR transactivation was extended to squamous cell carcinoma. The mechanism happens in a dose dependent manner and requires a metalloprotease to shed the proligand Amphiregulin. The involvement of both ADAM9 and ADAM17, being the metalloproteases responsible for this cleavage, was shown for SCC9 cells.
Pericentric intrachromosomal insertion responsible for recurrence of del(11)(p13p14) in a family
(1993)
The combined use of qualitative and quantitative analysis of I I p I 3 polymorphic markers tagether with chromosomal in situ suppression hybridization (CISS) with biotin labeled probes mapping to I I p allowed us to characterize a complex rearrangement segregating in a family. We detected a pericentric intrachromosomal insertion responsible (or recurrence of del( I I )(p 13p 14) in the family: an insertion of band I I p 13-p 14 carrying the genes for predisposition to Wilms' tumor, WT I, and for aniridia, AN2, into the long arm of chromosome I I in II q 13-q 1<4. Asymptomatic balanced carriers were observed over three generations. Classical cytogenetics had failed to detect this anomaly in the balanced carriers, who were first considered to be somatic mosaics for del( II )(p 13). Two of these women gave birth to children carrying a deleted chromosome II. most likely resulting from the loss of the I I p 13 band inserted in I I q. Although in both cases the deletion encompassed exactly the same maternally inherited markers, there was a wide Variation in clinical expression. One child, with the karyotype 46,XY,del(ll)(pllpl4), presented the full-blown WAGR syndrome with anlridia, mental retardation, Wilms' tumor, and pseudohermaphroditism, but also had proteinuria and glomerular sclerosis reminiscent of Drash syndrome. In contrast, the other one, a girl with the karyotype 46,XX,del( I I )(p I 3), only had aniridia. Although a specific set of mutational sites has been observed in Drash patients, these findings suggest that the loss of one copy of the WTI gene can result in similar genital and kidney abnormalities.