Transkriptionskontrolle des nuoA-N Operons (NADH-Dehydrogenase I) von Escherichia coli

Das Elektronentransportsystem von E. coli enthält zwei verschiedene NADH-Dehydrogenasen. Die NADH-DehydrogenaseI (nuoA-N) koppelt im Gegensatz zur NADH-DehydrogenaseII die Oxidation von NADH an eine Protonentranslokation und trägt zur Energiekonservierung bei. Die NADH-DehydrogenaseI wird über die Promotoren P1 und P2 exprimiert und besitzt mehrere Bindestellen für verschiedene Regulatoren.Die separate Klonierung der Promotoren, lacZ-Fusionen, Inaktivierung von Transkriptionsfaktoren, sowie die Nutzung mutierter Regulatorbindestellen in vivo zeigen, dass P1 im wesentlichen die Expressionshöhe bestimmt und ist unter aeroben und anaeroben Bedingungen aktiv. P2 trägt in wesentlich geringerem Maße als P1 zur Expression des Enzyms bei. Er ist stark abhängig von ArcA und IHF. Beide Promotoren wirken nicht additiv.Unter anaeroben Bedingungen wird die Transkription von nuo durch das Zweikomponenten-System ArcB/A reprimiert. ArcA bindet unabhängig und mit unterschiedlicher Affinität an die beiden Bindestellen arc1 und arc2. Von den 8 ArcA-Konsensussequenzen führen nur Mutationen der Konsensussequenzen arc1ab in vitro zu verminderter Bindungsaffinität von ArcA an die Bindestelle arc1. Dieselben führen in vivo unter anaeroben Bedingungen zur Derepression des Promotors P1 bzw. P1+P2. Unter aeroben Bedingungen zeigen nur Mutationen in arc2 eine Derepression, die nicht durch ArcA vermittelt wird. Der veröffentliche ArcA-Konsensus scheint deshalb hier in dieser einfachen Form nicht gültig zu sein.

Detailed analyses of mutations affecting structural formation of neuromuscular synapses in Drosophila melanogaster

The establishment of appropriate synapses between neurons and their target cells is an essential requirement for the formation of functional neuronal circuits. However, there is very little insight into the mechanisms underlying de novo formation of synapses and synaptic terminals. To identify novel genes involved in signalling or structural aspects of these processes I capitalised on possibilities provided by the model organism Drosophila. Thus, I contributed to a screen of a collection of third chromosomal mutations (Salzberg et al., 1997, Genetics 147, 1723ff.) selecting those mutant strains displaying structural defects of Drosophila neuromuscular junctions (NMJ). Carrying out genetic mapping experiments, I could assign 7 genes to interesting candidate mutations. All 7 mutations selected in this process cause size alterations of the embryonic NMJ, and one shows additional disturbances in the distribution of synaptic markers. 4 of these turned out to be transcription factors, not falling into the remit of this project. Only for one of these, the neuronal transcription factor Castor, I could show that its overgrown mutant NMJ phenotype is due to an increase in the number of motorneurons. The remaining genes encode a potential nitrophenylphosphatase, the translation initiation factor eIF4AIII, and a novel protein Waharan. Unfortunately, the nitophenylphosphatase gene was identified too late to carry out functional studies in the context of this project, but potential roles are discussed. eIF4AIII promotes NMJ size tempting to speculate that local translation at the NMJ is affected. I found that the synaptic scaffolding molecule Discs large (Dlg; orthologue of PSD95) is upregulated at eIF4AIII mutant NMJs. Targeted upregulation of Dlg can not mimic the eIF4AIII mutant phenotype, but dlg mutations suppress it. Therefore, Dlg function is required but not sufficient in this context. My findings are discussed in detail, pointing out future directions. The main focus of this work is the completely novel gene waharan (wah), an orthologue of the human gene KIAA1267 encoding a big brain protein of likewise unknown structure and function. My studies show that mutations or RNAi knock-down of wah cause NMJ overgrowth and reveal additional crucial roles in the patterning of wing imginal discs. RNAi studies suggest Wah to be required pre- and postsynaptically at NMJs and, consistently, wah is transcribed in the nervous system and muscles. Anti-Wah antisera were produced but could no longer be tested here, but preliminary studies with newly generated HA-targeted constructs suggest that Wah localises at NMJs and in neuronal nuclei. In silico analyses predict Wah to be structurally related to the Rad23-family of proteins, likely to target ubiquitinated proteins to the proteasome for degradation (Chen et al., 2002, Mol Cell Biol 22, 4902ff.) . In agreement with this prediction, poly-ubiquitinated proteins were found to accumulate in the absence of wah function, and wah-like mutant phenotypes were induced in NMJs and wing discs by knocking down proteasome function. My analysis further revealed that poly-ubiquitinated proteins are reduced in nuclei of wah mutant neurons and muscles, suggesting that Wah may play additional roles in ubiquitin-mediated nuclear import. Taken together, this study has uncovered a number of interesting candidate genes required for the de novo formation of Drosophila NMJs. 3 of these genes fell into the focus of this project. As discussed in detail, discovery of these genes and insights gained into their function have high potential to be translatable into vertebrate systems.