Das E. coli EIIMtl gehört zu den PTSs (PEP-abhängiges Kohlenhydrat : Phosphotransferasesystem) und katalysiert den gekoppelten Transport von D-Mannitol in die Zelle. Es lässt sich in drei funktionelle Domänen unterteilen. Die membranüberspannende EIIC-Domäne mit der Substratbindestelle und der Transporterfunktion und die für die Übertragung der Phosphorylgruppe essentiellen hydrophilen Domänen EIIB und EIIA. Die exakte Topologie der EIIC-Domäne und deren Funktion bei Transport und Phosphorylierung sind bisher noch unklar. Zur Untersuchung der Funktion wurden Selektionssysteme konstruiert, um Mutanten aus Escherichia coli K-12 zu isolieren und charakterisieren, in denen der Transport von der Phosphorylierung entkoppelt ist. Diese Mutationen (E218A, E218V und H256P) wurden alle in der stark konservierten Schleife 5 des membrangebundenen Transporters EIIC gefunden, zwei davon in dem dort befindlichen GIH256E-Motiv. Die Suche nach Suppressormutationen ergab ebenfalls Mutationen in der Aminosäure 256 des GIHE-Motivs (P256A, P256Q und P256S). Die Kombination der verschiedenen Mutationen durch ortspezifische Mutagenese ergab unterschiedliche Effekte auf den Transport und der Phosphorylierung von D-Mannitol (Otte, S., Scholle, A., Turgut, S., and Lengeler, J.W. 2003. Mutations Which Uncouple Transport and Phosphorylation in the D-Mannitol Phosphotransferase System of Escherichia coli K-12 and Klebsiella pneumoniae 1033-5P14. J. Bacteriol. 185(7): 2267-2276). Bei der Untersuchung der Sekundärstruktur wurde die Lokalisation verschiedener Aminosäuren im EIIMtl durch die Methode des Cystein-Scanning überprüft. Mit Hilfe weiterer Sequenzuntersuchungen und unter Berücksichtigung weiterer Ergebnisse, konnte ein alternatives Sekundärstrukturmodell des EIICMtl vorgestellt werden, bei dem die Schleife 5 in die Membran gelegt wurde.

Developmental biology adresses how cells are organised into functional structures and eventually into a whole organism. It is crucial to understand the molecular basis for processes in development, by studying the expression and function of relevant genes and their relationship to each other. A gene function can be studied be creating loss-of-function situations, in which the change in developmental processes is examined in the absense of a functional gene product, or in gain-of-function studies, where a gene product is either intrinsically overproduced or ectopically upregulated. One approach for a loss-of-function situation is the creation of specific mutants in single genes, and the zebrafish (Danio rerio) has proven to be an excellent model organism for this purpose. In this thesis, I report on two forward genetic screens performed to find new mutants affecting brain development, in particular mutants defective in development and function of the midbrain-hindbrain boundary (MHB), an organiser region that patterns the adjacent brain regions of the midbrain and the hindbrain. In the first screen, I could identify 10 specific mutants based on morphology and the analysis of the expression patterns of lim1 and fgf8, genes functioning as early neuronal markers and as a patterning gene, respectively. Three of these mutants lacked an MHB, and by complementation studies, I identified these mutants as being defective in the spg locus. The second screen produced 35 new mutants by screening morphologically and with antibodies against acetylated Tubulin, which marks all axonal scaffolds, and anti-Opsin, which is a marker for photoreceptors in the pineal gland. According to their phenotype, I distributed the mutant lines into 4 phenotypic subgroups, of which the brain morphology group with 18 mutant lines was studied most intensively. In the last part of my thesis, I characterise one of these brain morphology mutants, broken heart. This mutant is defective in axonal outgrowth and locomotion, and shows a striking reduction of serotonergic neurons in the epiphysis and in the raphe nuclei in the hindbrain, structures involved in serotonin and melatonin production. Studies in other model organisms suggested a role of factors from the floor plate and the MHB in induction of the serotonergic neurons in the hindbrain, and using broken heart, I show that Fgf molecules such as Fgf4 and Fgf8 can restore partially the loss of serotonergic neurons in the mutant. I conclude that forward genetic screens are an invaluable tool to generate a pool of mutations in specific genes, which can be used to dissect complex processes in development such as brain development.

Developmental biology adresses how cells are organised into functional structures and eventually into a whole organism. It is crucial to understand the molecular basis for processes in development, by studying the expression and function of relevant genes and their relationship to each other. A gene function can be studied be creating loss-of-function situations, in which the change in developmental processes is examined in the absense of a functional gene product, or in gain-of-function studies, where a gene product is either intrinsically overproduced or ectopically upregulated. One approach for a loss-of-function situation is the creation of specific mutants in single genes, and the zebrafish (Danio rerio) has proven to be an excellent model organism for this purpose. In this thesis, I report on two forward genetic screens performed to find new mutants affecting brain development, in particular mutants defective in development and function of the midbrain-hindbrain boundary (MHB), an organiser region that patterns the adjacent brain regions of the midbrain and the hindbrain. In the first screen, I could identify 10 specific mutants based on morphology and the analysis of the expression patterns of lim1 and fgf8, genes functioning as early neuronal markers and as a patterning gene, respectively. Three of these mutants lacked an MHB, and by complementation studies, I identified these mutants as being defective in the spg locus. The second screen produced 35 new mutants by screening morphologically and with antibodies against acetylated Tubulin, which marks all axonal scaffolds, and anti-Opsin, which is a marker for photoreceptors in the pineal gland. According to their phenotype, I distributed the mutant lines into 4 phenotypic subgroups, of which the brain morphology group with 18 mutant lines was studied most intensively. In the last part of my thesis, I characterise one of these brain morphology mutants, broken heart. This mutant is defective in axonal outgrowth and locomotion, and shows a striking reduction of serotonergic neurons in the epiphysis and in the raphe nuclei in the hindbrain, structures involved in serotonin and melatonin production. Studies in other model organisms suggested a role of factors from the floor plate and the MHB in induction of the serotonergic neurons in the hindbrain, and using broken heart, I show that Fgf molecules such as Fgf4 and Fgf8 can restore partially the loss of serotonergic neurons in the mutant. I conclude that forward genetic screens are an invaluable tool to generate a pool of mutations in specific genes, which can be used to dissect complex processes in development such as brain development.

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