TY - THES A1 - Mishra, Dushyant T1 - The content of olfactory memory in larval Drosophila T1 - Olfaktorisches Gedächtnis in der Drosophila Larve N2 - An animal depends heavily on its sense of smell and its ability to form olfactory associations as this is crucial for its survival. This thesis studies in two parts about such associative olfactory learning in larval Drosophila. The first part deals with different aspects of odour processing while the second part is concerned with aspects related to memory and learning. Chapter I.1 highlights how odour intensities could be integrated into the olfactory percept of larval Drosophila. I first describe the dose-effect curves of learnability across odour intensities for different odours and then choose odour intensities from these curves such that larvae are trained at intermediate odour intensity, but are tested for retention with either that trained intermediate odour intensity, or with respectively HIGHer or LOWer intensities. I observe a specificity of retention for the trained intensity for all the odours used. Further I compare these findings with the case of adult Drosophila and propose a circuit level model of how such intensity coding comes about. Such intensity specificity of learning adds to appreciate the richness in 'content' of olfactory memory traces, and to define the demands on computational models of olfaction and olfactory learning. Chapter I.2 provides a behaviour-based estimate of odour similarity using four different types of experiments to yield a combined, task-independent estimate of perceived difference between odour-pairs. Further comparison of these perceived differences to published measures of physico- chemical difference reveals a weak correlation. Notable exceptions to this correlation are 3-octanol and benzaldehyde. Chapter I.3 shows for two odours (3-octanol and 1-octene-3-ol) that perceptual differences between these odours can either be ignored after non-discriminative training (generalization), or accentuated by odour-specific reinforcement (discrimination). Anosmic Or83b1 mutants have lost these faculties, indicating that this adaptive adjustment is taking place downstream of Or83b expressing sensory neurons. Chapter II.1 of this thesis deals with food supplementation with dried roots of Rhodiola rosea. This dose-dependently improves odour- reward associative function in larval Drosophila. Supplementing fly food with commercially available tablets or extracts, however, does not have a 'cognitive enhancing' effect, potentially enabling us to differentiate between the effective substances in the root versus these preparations. Thus Drosophila as a genetically tractable study case should now allow accelerated analyses of the molecular mechanism(s) that underlie this 'cognitive enhancement' conveyed by Rhodiola rosea. Chapter II.2 describes the role of Synapsin, an evolutionarily conserved presynaptic phosphoprotein using a combined behavioural and genetic approach and asks where and how, this protein affects functions in associative plasticity of larval Drosophila. This study shows that a Synapsin-dependent memory trace can be pinpointed to the mushroom bodies, a 'cortical' brain region of the insects. On the molecular level, data in this study assign Synapsin as a behaviourally- relevant effector of the AC-cAMP-PKA cascade. N2 - Das Überleben von Tieren ist in hohem Maße abhängig von ihrer Fähigkeit zu riechen und olfaktorische Gedächtnisse zu bilden. Meine Arbeit besteht aus zwei Abschnitten, in denen ich solche Prozesse anhand von Drosophila Larven untersuche. Im ersten Abschnitt beschreibe ich verschiedene Aspekte der Geruchsprozessierung, der zweite Abschnitt betrifft Gedächtnis- und Lernprozesse. Kapitel I.1 handelt davon, wie Geruchsintensitäten in die olfaktorische Wahrnehmung von Drosophila-Larven integriert sein könnten. Zuerst beschreibe ich die Lernbarkeit verschiedener Duftstoffe abhängig von ihren Intensitäten. Anhand dieser Dosis-Wirkungs-Kurven wähle ich dann eine niedrige, eine mittlere, und eine hohe Duft-Intensität. Ich trainiere Larven mit der mittleren Duft-Intensität und teste sie entweder mit dieser mittleren Intensität, oder mit der höheren, oder mit der niedrigen Duft-Intensität. Ich beobachte, dass der Gedächtnisabruf mit der trainierten Intensität für alle verwendeten Duftstoffe am besten ist. Außerdem vergleiche ich diese Ergebnisse mit denen von adulten Fruchtfliegen und schlage ein Schaltkreis-Modell vor, das erklärt, wie eine solche Kodierung der Intensität zustande kommen kann. Eine solche Spezifität für Intensitäten beim Lernen erweitert die bisher bekannte Fülle des ‚Inhalts’ von olfaktorischen Gedächtnisspuren und die Anforderungen an Computermodelle über Riechen und Geruchslernen. In Kapitel I.2 untersuche ich Ähnlichkeitsbeziehungen zwischen Duftpaaren anhand der Wahrnehmung von Larven. Ich verwende dazu vier verschiedene Typen von Lernexperimenten. Durch Kombination der Ergebnisse dieser vier Experimente erhalte ich eine aufgabenunabhängige Abschätzung der vom Tier wahrgenommenen Ähnlichkeiten zwischen Paaren von Duftstoffen. Ein Vergleich dieser wahrgenommenen Ähnlichkeiten mit veröffentlichten Messungen von physikalischen und chemischen Ähnlichkeiten ergibt eine schwache Korrelation. Eine erwähnenswerte Ausnahme zu dieser Korrelation ist das Duftpaar 3-Octanol und Benzaldehyd. Kapitel I.3 zeigt für zwei Duftstoffe (3-Octanol und 1-Octen-3-ol), dass die wahrgenommene Ähnlichkeit zwischen diesen beiden Duftstoffen abhängig ist von der Art des Trainings. Wenn die Tiere nicht-diskriminativ trainiert werden, werden die Düfte vom Tier generalisiert, während diskriminatives Training die wahrgenommene Unterschiede zwischen den Düften erhöht. Anosmische Or83b1-Mutanten haben diese Fähigkeiten verloren, was darauf hindeutet, das diese adaptive Anpassung in Nervenzellen stattfindet, die den Or83b-exprimierenden sensorischen Neuronen nachgeschaltet sind. In Kapitel II.1 untersuche ich die Auswirkung von Zugabe getrockneter Wurzeln der Pflanze Rhodiola rosea zum Fliegenfutter. Ich finde heraus, dass Rhodiola rosea dosisabhängig die olfaktorische Konditionierung von Drosophila-Larven verbessert. Die Zugabe von kommerziell verfügbaren Tabletten oder Extrakten zum Fliegenfutter hat keinen positiven Effekt auf solche „kognitiven“ Fähigkeiten, was uns möglicherweise erlaubt, zwischen den effektiven Substanzen der Wurzel und diesen Präparaten zu differenzieren. Drosophila als genetisch manipulierbarer Modellorganismus sollte uns nun weiterführende Analysen der molekularen Mechanismen erlauben, die dieser „kognitiven Verbesserung“ durch Rhodiola rosea zugrunde liegen. Kapitel II.2 beschreibe ich die Funktion von Synapsin, einem evolutionär konservierten präsynaptischen Phosphoprotein. Ich verwende dazu einen kombinierten verhaltensbasierten und genetischen Ansatz. Untersucht wird, wo und wie dieses Protein assoziative Plastizität im Gehirn von Drosophila-Larven beeinflusst. Diese Studie zeigt, dass eine Synapsin-abhängige Gedächtnisspur im Pilzkörper, einer „kortikalen“ Gehirnregion der Insekten, lokalisiert werden kann. Auf der molekularen Ebene zeigen die Ergebnisse dieser Studie Synapsin als einen im Verhalten wichtigen Effektor der AC-cAMP-Kaskade. * Many thanks to M. Schlayer, T. Niewalda and T. Saumweber for their help in this translation. KW - Drosophila KW - Insektenlarve KW - Geruchssinn KW - Lernen KW - Drosophila melanogaster KW - Olfaktion KW - Neurogenetik KW - Speicher KW - Neurogenetics KW - Drosophila melanogaster KW - Olfaction KW - Learning KW - Memory KW - Reinforcement Y1 - 2011 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-66316 ER - TY - THES A1 - Schmitt, Kathrin T1 - Identification and Characterization of GAS2L3 as a Novel Mitotic Regulator in Human Cells T1 - Die Identifizierung und Charakterisierung von GAS2L3 als neuer Regulator der Mitose in humanen Zellen N2 - Precise control of mitotic progression is vital for the maintenance of genomic integrity. Since the loss of genomic integrity is known to promote tumorigenesis, the identification of knew G2/M regulatory genes attracts great attention. LINC, a human multiprotein complex, is a transcriptional activator of a set of G2/M specific genes. By depleting LIN9 in MEFs, a core subunit of LINC, Gas2l3 was identified as a novel LINC target gene. The so far uncharacterized Gas2l3 gene encodes for a member of the family of growth arrest specific 2 (GAS2) proteins, which share a highly conserved putative actin binding CH and a putative microtubule binding GAS2 domain. In the present study GAS2L3 was identified as a LINC target gene also in human cells. Gene expression analysis revealed that GAS2L3 transcription, in contrast to all other GAS2 family members, is highly regulated during the cell cycle with highest expression in G2/M. The GAS2L3 protein showed a specific localization pattern during the M phase: In metaphase, GAS2L3 localized to the mitotic spindle, relocated to the spindle midzone microtubules in late anaphase and concentrated at the midbody in telophase where it persisted until the end of cytokinesis. Overexpression of a set of different GAS2L3 deletion mutants demonstrated that the localization to the mitotic microtubule network is dependent on the C-terminus, whereas the midbody localization is dependent on full length GAS2L3 protein. Additionally, exclusive overexpression of the CH domain induced the formation of actin stress fibers, suggesting that the CH domain is an actin binding domain. In contrast, the GAS2 domain was neither needed nor sufficient for microtubule binding, indicating that there must be an additional so far unknown microtubule binding domain in the C-terminus. Interestingly, immunoblot analysis also identified the C-terminus as the domain responsible for GAS2L3 protein instability, partially dependent on proteasomal degradation. Consistent with its specific localization pattern, GAS2L3 depletion by RNAi demonstrated its responsibility for proper mitosis and cytokinesis. GAS2L3 depletion in HeLa cells resulted in the accumulation of multinucleated cells, an indicator for chromosome mis-segregation during mitosis. Also the amount of cells in cytokinesis was enriched, indicating failures in completing the last step of cytokinesis, the abscission. Strikingly, treatment with microtubule poisons that lead to the activation of the spindle assembly checkpoint (SAC) indicated that the SAC was weakened in GAS2L3 depleted cells. Although the exact molecular mechanism is still unknown, fist experiments support the hypothesis that GAS2L3 might be a regulator of the SAC master kinase BUBR1. In conclusion, this study provides first evidence for GAS2L3 as a novel regulator of mitosis and cytokinesis and it might therefore be an important guardian against tumorigenesis. N2 - Der korrekte Verlauf durch die Mitose des Zellzyklus trägt entscheidend zur Aufrechterhaltung der genomischen Integrität bei. Da ein Verlust der genomischen Integrität die Tumorentstehung begünstigt, ist die Identifizierung neuer G2/M regulatorischer Gene ein Forschungsbereich, der großes Interesse weckt. Der humane Multiproteinkomplex LINC ist für die transkriptionelle Aktivierung einer Vielzahl G2/M spezifischer Gene verantwortlich. Durch die Depletion von LIN9 in MEFs, einer Kernkomponente von LINC, wurde Gas2l3 als ein neues Zielgen von LINC identifiziert. Das bisher uncharakterisierte Gas2l3 Gen codiert für ein der GAS2 (growth arrest specific 2) Familie zugehöriges Protein, deren Mitglieder sich durch eine hoch konservierte putative Aktin-bindende Domäne (CH) und eine putative Mikrotubuli-bindende Domäne (GAS2) auszeichnen. In der vorliegenden Arbeit konnte gezeigt werden, dass GAS2L3 auch in humanen Zellen ein Zielgen von LINC ist. Die Transkription von GAS2L3 wies, im Gegensatz zu allen anderen GAS2 Familienmitgliedern, eine starke Regulation während des Zellzyklus auf, wobei die höchste Genexpression in der G2/M Phase vorlag. Das GAS2L3 Protein zeigte eine spezifische Lokalisation während der M Phase: In der Metaphase findet sich GAS2L3 an der mitotischen Spindel, wandert von dort an die Mikrotubuli der zentralen Spindel der Anaphase und konzentriert sich in der Telophase am Midbody, wo es bis zum Ende der Zytokinese verweilt. Der Einsatz unterschiedlicher Deletionsmutanten demonstrierte, dass die Lokalisation an die mitotischen Mikrotubuli vom C-Terminus abhängig ist, wohingegen die Lokalisation am Midbody von der gesamten Proteinsequenz abhängt. Die Ausbildung von Aktin-Streß-Filamenten nach alleiniger Überexpression der CH Domäne deutete darauf hin, dass die CH Domäne eine Aktin-bindende Domäne ist. Die GAS2 Domäne hingegen wurde weder für die Interaktion mit Mikrotubuli gebraucht, noch war sie alleine für diese ausreichend. Alle Daten weisen darauf hin, dass GAS2L3 eine bisher unbekannte Mikrotubuli-bindende Domäne im C-Terminus trägt. Interessanterweise ist der C-Terminus auch für die hohe Instabilität des GAS2L3 Proteins, die teilweise durch den Abbau im Proteasom verursacht wird, verantwortlich. Entsprechend der spezifischen Lokalisation zeigte die Depletion von GAS2L3 durch siRNA Transfektion dessen Wichtigkeit für den korrekten Verlauf der M Phase. GAS2L3 depletierte HeLa Zellen zeigten eine Anreicherung von multinukleären Zellen, welche ein Indikator für die fehlerhafte Verteilung der Chromosomen in der Mitose sind. Ein Hinweis auf Probleme im Beenden der Zytokinese stellte die erhöhte Anzahl von Zellen dar, die sich in der Zytokinese befanden. Eines der auffallendsten Merkmale war ein geschwächter mitotischer Spindelkontrollpunkt, den GAS2L3 depletierte Zellen nach der Behandlung mit den Kontrollpunkt aktivierenden Mikrotubuli-Giften aufwiesen. Auch wenn der exakte molekulare Mechanismus hierbei noch unbekannt ist, deuten erste Experimente darauf hin, dass GAS2L3 die Aktivität von BUBR1, einer essentiellen Kinase des mitotischen Spindelkontrollpunkts, beeinflusst. Alle Daten dieser Arbeit verdeutlichen die Wichtigkeit von GAS2L3 als einen neuen Regulator der Mitose und Zytokinese. Somit ist anzunehmen, dass die korrekte Funktion von GAS2L3 entscheidend zum Schutz vor Tumorentstehung beiträgt. KW - Mensch KW - Zelle KW - Mitose KW - Kernspindel KW - Kontrolle KW - Genregulation KW - Spindelkontrollpunkt KW - Zytokinese KW - Midbody KW - GAS2L3 KW - LIN9 KW - Zellzyklus KW - LIN9 KW - GAS2L3 KW - mitosis KW - cytokinesis KW - spindle assembly checkpoint Y1 - 2010 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-52704 ER - TY - JOUR A1 - Clauss, Gerd A1 - Winkler, Christoph A1 - Lohmeyer, Jürgen A1 - Anders, Fritz A1 - Schartl, Manfred T1 - Oncofetal antigen in Xiphophorus detected by monoclonal antibodies directed against melanoma-associated antigens N2 - Monoclonal antlbodies (MAbs) directed against Xiphophorus melanoma cells were deve(oped and tested by lndirect immunofluorescence and Immunoperoxidase staining for reactivity with a panel of I 5 allogeneic tissues and 12 allogeneic cell llnes. The reactivity of such MAbs was restricted to melanoma cells from tumor biopsies and melanoma-derived cell lines. ln addition, all embryonie cells of all histiotypes from developmental stages later than mld·organogenesis and from corresponding short term in vitro cultures reacted with these MAbs. ln contrast, normal tissues and organs from adult fish dlsplayed no reactivity, thus implying that the melanoma-associated antigens detected by the MAbs described are oncofetal antigens. KW - Physiologische Chemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61784 ER - TY - JOUR A1 - Friedenreich, Hildegard A1 - Schartl, Manfred T1 - Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells N2 - ln order to construct fish specific expression vectors for studies on gene regulation in vitro and in vivo a variety of heterologous enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial chloramphenicol acetyl transferase reporter gene in three teleost fish cell lines. Several viral enhancers were found to be constitutively active at high Ieveis. The human metallothionein promoter showed inducible expression in the presence of heavy metal Ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient splicing and Iack of translation were observed, pointing to limitations in the use of heterologous genes in gene transfer experiments. On the contrary, some heterologous promoters and enhancers functioned in fish c as weil as in their cell type of origin, indicating t at corresponding transcription factors are sufficient conserved between fish and human over a period of 900 million years of Independent evolution. KW - Physiologische Chemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61774 ER - TY - JOUR A1 - Dracopoli, Nicholas C. A1 - Feltquate, David M. A1 - Sam, Brigitta A1 - Schartl, Manfred T1 - Taql and Mspl RFLPs are detected by the human 2,3-biphosphoglycerate mutase (BPGM) cDNA N2 - No abstract available KW - Physiologische Chemie Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61763 ER - TY - JOUR A1 - Schartl, Manfred T1 - Homology of melanoma-inducing loci in the genus Xiphophorus N2 - Several species of the genus Xiphophorus are polymorphic for specific pigment patterns. Same of these give rise to malignant melanoma following the appropriate crossings. For one of these pattern Iod from the platyfish Xiphophorus maculatus the melanoma-inducing gene has been doned and found to encode a novel receptor tyrosine kinase, designated Xmrk. Using molecular probes from this gene in Southern blot analyses on single fish DNA preparations from 600 specimens of different populations of various species of the genus Xiphophorus and their hybrids, either with or without melanomapredisposing pattern, it was shown that all individuals contain the Xmrk gene as a proto-oncogene. It is located on the sex chromosome. All fish that carry a melanoma-predisposing locus which has been identified by Mendelian genetics contain an additional copy of Xmrk, closely linked to a specific melanophore pattern locus on the sex chromosome. The melanoma-inducing loci of the different species and populations are homologous. The additional copy of Xmrk obviously arose by a geneduplication event, thereby acquiring the oncogenic potential. The homology of the melanomainducing Iod points to a similar mechanism of tumor suppression in all feral fish populations of the different species of the genus Xiphophorus. KW - Physiologische Chemie KW - Xiphophorus Y1 - 1990 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61757 ER - TY - JOUR A1 - Winkler, Christoph A1 - Vielkind, Jürgen R. A1 - Schartl, Manfred T1 - Transient expression of foreign DNA during embryonic and larval development of the medaka fish (Oryzias latipes) N2 - Species of small fish are becoming useful tools for studies on vertebrate development. Wehave investigated the developing embryo of the Japanese medaka for its application as a transient expression system for the in vivo analysis of gene regulation and function. The temporaland spatial expression patterns ofbacterial chloramphenicol acetyltransferase and galactosidase reporter genes injected in supercoiled plasmid form into the cytoplasm of one cell of the two-cell stage embryo was promoter-specific. The transient expression was found to be mosaic within the tissue and organs reflecting the unequal distribution of extrachromosomal foreign DNA and the intensive cell mixing movements that occur in fish embryogenesis. The expression data are consistent with data on DNA fate. Foreign DNA persisted during embryogenesis and was still detectable in some 3- and 9-month-old adult fish; it was found in high molecular weight form as weil as in circular plasmid conformations. The DNA was replicated during early and late embryogenesis. Our data indicate that the developing medaka embryo is a powerful in vivo assay system for studies of gene regulation and function. KW - Physiologische Chemie KW - Medaka - Genetransfer - Transient expression - DNA fate - Fish developmental biology Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61743 ER - TY - JOUR A1 - Schartl, Manfred A1 - Schlupp, Ingo A1 - Schartl, Angelika A1 - Meyer, Manfred K. A1 - Nanda, Indrajit A1 - Schmid, Michael A1 - Epplen, Jörg T. A1 - Parzefall, Jakob T1 - On the stability of dispensable constituents of the eukaryotic genome: Stability of coding sequences versus truly hypervariable sequences in a clonal vertebrate, the amazon molly, Poecilia formosa N2 - In dooal unisexual vertebrales, the genes specifying the males become dispensable. To study tbe rate of such geoes the gynogeoetic all-female fisb Poecilillfonnolll was treated with androgens. Phenotypic males were obtained that exbibited the complete set of male cbaracteristics of dosely related gooocboristic species, induding body proportions, pigmentation, the extremely complex insemination apparatus of poecilüd fish, sexual bebavior, and spermatogeoesls. Tbe apparent stabllity of such genic structures, induding those involved in androgen regulation, is contrasted by high instability of noncoding sequeaces. Frequent mutations, thelr donal transmission, and at least two truly hypervariable Iod leading to individual difl'ereaces between these othenrise donal organisms were detected by DNA fingerprinting. These observations substantiate the concept that also in "ameiotic" vertebrates certain compartments of the genome are more prooe to mutatiooal alterations than others. KW - Physiologische Chemie KW - DNA fingerprinting KW - androgen-induced masculinization KW - gynogeaesls KW - simple repeat sequences Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61731 ER - TY - JOUR A1 - Förnzler, Dorothee A1 - Wittbrodt, Joachim A1 - Schartl, M.anfred T1 - Analysis of an esterase linked to a locus involved in the regulation of the melanoma oncogene and isolation of polymorphic marker sequences in Xiphophorus N2 - Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecu/ar identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confinn that ES 1 is a distant marker for R ( approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity Ievels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markersthat bridge the large distance between ESl and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding Iandmarks on a physical map of the R-containing chromosomal region. KW - Physiologische Chemie KW - Xiphophorus KW - melanoma ; oncogene regulation ; esterase ; molecular marker sequences Y1 - 1991 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61726 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Schartl, Manfred A1 - Feichtinger, Wolfgang A1 - Epplen, Jörg T. A1 - Schmid, Michael T1 - Early stages of sex chromosome differentiation in fish as analysed by simple repetitive DNA sequences N2 - Animal sex chromosome evolution has started on different occasions with a homologous pair of autosomes leading to morphologically differentiated gonosomes. In contrast to other vertebrate classes, among fishes cytologically dernonstrahle sex chromosomes are rare. In reptiles, certain motifs of simple tandemly repeated DNA sequences like (gata)\(_n\)/(gaca)\(_m\) are associated with the constitutive heterochromatin of sex chromosomes. In this study a panel of simple repetitive sequence probes was hybridized to restriction enzyme digested genomic DNA of poeciliid fishes. Apparent male heterogamety previously established by genetic experiments in Poecilia reticulata (guppy) was correlated with male-specific hybridization using the (GACA)\(_4\) probe. The (GATA)\(_4\) oligonucleotide identifies certain male guppies by a Y chromosomal polymorphism in the outbred population. In cantrast none of the genetically defined heterogametic situations in Xiphophorus could be verified consistently using the collection of simple repetitive sequence probes. Only individuals from particular populations produced sex-specific patterns of hybridization with (GATA)\(_4\). Additional poeciliid species (P. sphenops, P. velifera) harbour different sex-specifically organized simple repeat motifs. The observed sex-specific hybridization patterns were substantiated by banding analyses of the karyotypes and by in situ hybridization using the (GACA)\(_4\) probe. KW - Physiologische Chemie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61715 ER - TY - JOUR A1 - Götz, Rudolf A1 - Raulf, Friedrich A1 - Schartl, Manfrad T1 - Brain-derived neurotrophic factor is more highly conserved in structure and function than nerve growth factor during vertebrate evolution N2 - Mammalian nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) are members of a protein family with perfectly conserved domains arranged around the cysteine residues thought to stabilize an invariant three-dimensional scaffold in addition to distinct sequence motifs that convey different neuronal functions. To study their structural and functional conservation during evolution, we have compared NGF and BDNF from a lower vertebrate, the teleost fi.sh Xiphophorus, with the mammalian homlogues. Genomic clones encoding fish NGF and BDNF were isolated by cross-hybridization using probes from the cloned mammalian factors. Fish NGF and BDNF were expressed by means of recombinant vaccinia viruses, purified, and their neuronal survival specificities for different classes of neurons were found to mirror those of the mammalian factors. The half-maximal survival concentration for chick sensory neurons was 60 pg/ml for both fish and mammalian purifi.ed recombinant BDNF. However, the activity ofrecombinant fish NGF on both chick sensory and sympathetic neurons was 6 ng,lml, 75-fold lower than that of mouse NGF. The different functional conservation of NGF and BDNF is also reflected in their structures. The DNA-deduced amino acid sequences of processed mature fish NGF and BDNF showed, compared to mouse, 63% and 90% identity, respectively, indicating that NGF bad reached an optimized structure later than BDNF. The retrograde extrapolation of these data indicates that NGF and BDNF evolved at strikingly different rates ftom a common ancestral gene about 600 million years ago. By RNA gel blot anaJysis NGF mRNA was detected during late embryonie development; BDNF was present in adult brain. KW - Physiologische Chemie KW - Brain-derived neurotrophic factor KW - Nerve growth factor KW - Fish KW - Recombinant protein expression KW - Neuronal survival Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61703 ER - TY - JOUR A1 - Wittbrodt, Joachim A1 - Lammers, Reiner A1 - Malitschek, Barbara A1 - Ullrich, Axel A1 - Schartl, Manfred T1 - Xmrk receptor tyrosine kinase is activated in Xiphophorus malignant melanoma N2 - Xmrk encodes a putative transmembrane glycoprotein of the tyrosine kinase family and is a melanoma-inducing gene in Xiphophorus. We attempted to investigate the biological function of the putative Xmrk receptor by characterizing its signalling properties. Since a potential Iigand for Xmrk has not yet been identified, it has been difficult to analyse the biochemical properlies and biological function of this cell surface protein. In an approach towards such analyses, the Xmrk extracellular domain was replaced by the closely related Iigand-binding domain sequences of the human epidennal growth factor receptor (HER) and the ligand-induced activity of the chimeric HER-Xmrk proteinwas examined. We show that the Xmrk protein is a functional receptor tyrosine kinase, is highly active in malignant melanoma and displays a constitutive autophosphorylation activity possibly due to an activating mutation in its extracellular or transmembrane domain. In the focus formation assay the HER-Xmrk chimera is a potent transfonning protein equivalent to other tyrosine kinase oncoproteins. KW - Physiologische Chemie KW - chimeric RTKs KW - melanoma KW - RTK KW - Xiphophorus Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61699 ER - TY - JOUR A1 - Schlupp, Ingo A1 - Parzefall, Jakob A1 - Epplen, Jörg T. A1 - Nanda, Indrajit A1 - Schmid, Michael A1 - Schartl, Manfred T1 - Pseudomale behaviour and spontaneous masculinization in the all-female teleost Poecilia formosa (Teleostei: Poeciliidae) N2 - Pseudosexual behaviour is a rare phenomenon associated with unisexuality in vertebrates. In the gynogenetic, all-female teleost Poecilia formosa, rare individuals occur that resemble males of closely related gonochoristic species both in behaviour and external morphology. These masculinized gynogens and normal gynogens are members of the same clone, as demonstrated by DNA-fingerprinting. The behaviour of these masculinized gynogens is described and compared to the behaviour of the gonochoristic species Poecilia mexicana, P. latipinna and their hybrid as weil as androgen-treated individuals of P. formosa. No statistically significant difTerences were found between masculinized gynogens and hormonetreated individuals nor between the gonochoristic P. mexicana and P. latipinna males. Differences exist between gonochoristic and unisexual species. Passihle causes and effects of masculinized gynogens are discussed. KW - Physiologische Chemie Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61688 ER - TY - JOUR A1 - Hong, Yunhan A1 - Schartl, Manfred T1 - Structure of the rainbow trout metallothionein A gene N2 - To investigate the regulation of metallothionein-encoding genes in fish, we have isolated and sequenced the rainbow trout metallothionein-A-encoding gene (tMT-A) by polymerase chain reaction. This gene spans about 1.1 kb, consists of three exons and two introns, and has an A+ T-rieb 5' -region which contains a TATAAA signal, and two metal responsive elements (MREs). The transcription start point is centered around an A residue 81 nt upstream of the ATG codon. KW - Physiologische Chemie KW - Recombinant DNA ; polymerase chain reaction ; metallothionein gene ; rainbow trout ; fish Y1 - 1992 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61679 ER - TY - JOUR A1 - Hong, Yunhan A1 - Winkler, Christoph A1 - Brem, Gottfried A1 - Schartl, Manfred T1 - Development of a heavy metal-inducible fish-specific expression vector for gene transfer in vitro and in vivo N2 - The promoter of the rainbow trout metallothionein B gene ( tMTb) was isolated from genomic DNA by the polymerase chain reaction (PCR ), fused to the bacterial chloramphenicol acetyltransferase (CAT) genein an expression vector, and functionally analyzed in one human cellline and four fish celllines. This promoter exhibited an extremely low basal expression in all celllines and was zincand cadmium-inducible except in the fish melanoma cell line where the promoter was completely inactive. The metal-induced expression patterns were cellline-specific. In general the fish promoter was more species- and cell type-specific than its human counterpart. In a transient assay it was functional in developing embryos of the medaka ( Oryzias /atipes). These properties make this promoter suitable for inducible, tissue-specific expression of transgenes and for in vivo studies of gene function and regulation. KW - Physiologische Chemie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61666 ER - TY - JOUR A1 - Nanda, Indrajit A1 - Schartl, Manfred A1 - Epplen, Jörg T. A1 - Feichtinger, Wolfgang A1 - Schmid, Michael T1 - Primitive sex chromosomes in poeciliid fishes harbor simple repetitive DNA sequences N2 - 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. KW - Physiologische Chemie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61659 ER - TY - JOUR A1 - Schartl, Manfred A1 - Erbelding-Denk, Claudia A1 - Holter, Sabine A1 - Nanda, Indrajit A1 - Schmid, Michael A1 - Schroder, Johannes H. A1 - Epplen, Jörg T. T1 - Reproductive failure of dominant males in the poeciliid fish Limia perugiae determined by DNA fingerprinting N2 - 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. KW - Physiologische Chemie KW - reproductive success KW - sexual selection KW - size polymorpbism KW - sodal domlnance KW - simple repetitive sequences Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61643 ER - TY - JOUR A1 - Adam, Dieter A1 - Dimitrijevic, Nicola A1 - Schartl, Manfred T1 - Tumor suppression in Xiphophorus by an accidentally acquired promoter N2 - 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. KW - Physiologische Chemie Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61630 ER - TY - JOUR A1 - Hong, Yunhan A1 - Schartl, Manfred T1 - Sequence of the growth hormone (GH) gene from the silver carp (Hypophthalmichthys molitrix) and evolution of GH genes in vertebrates N2 - 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). KW - Physiologische Chemie KW - Recombinant DNA ; Growth hormone gene ; PCR; Silver carp ; Fish Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61620 ER - TY - JOUR A1 - Cavari, Benzion A1 - Hong, Yunhan A1 - Funkenstein, Bruria A1 - Moav, Boaz A1 - Schartl, Manfred T1 - All-fish gene constructs for growth hormone gene transfer in fish N2 - 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. KW - Physiologische Chemie KW - growth hormone gene KW - all-fish genes KW - transgenic fish KW - cellline transfection KW - Sparus aurata Y1 - 1993 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-61612 ER -