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Der RAF-MEK-ERK-Signalweg steuert grundlegende, oftmals entgegengesetzte zelluläre Prozesse wie die Proliferation und Apoptose von Zellen. Die Dauer des vermittelten Signals wurde als entscheidener Faktor für die Steuerung dieser Prozesse identifiziert. Es ist jedoch nicht eindeutig geklärt, wie die verschiedenen früh und spät reagierenden Genexpressionsmodule kurze und lange Signale unterscheiden können und durch welche kinetischen Merkmale ihre Antwortzeit bestimmt wird. In der vorliegenden Arbeit wurden sowohl Proteinphosphorylierungsdaten als auch Genexpressionsdaten aus HEK293-Zellen gewonnen, die ein induzierbares Konstrukt des Proto-Onkogens RAF tragen. Hierbei wurde ein neues Genexpressionsmodul identifiziert, dass sich aus sofort induzierten aber spät antwortenden Genen zusammensetzt. Es unterscheidet sich in der Genexpressionsdynamik und Genfunktion von anderen Modulen, und wurde mit Hilfe mathematischer Modellierung experimenteller Daten identifiziert. Es wurde festgestellt, dass diese Gene aufgrund von langen Halbwertszeiten der vermitteltenden mRNA in der Lage sind spät auf das eingehende Signal zu reagieren und die Dauer des Signals in die Amplitude der Genantwort zu übersetzen. Trotz der langsamen Akkumulation und damit späten Antwortzeit, konnte aufgrund einer GC-reichen Promoterstruktur zunächst vermutet und mit Hilfe eines Markerverfahrens bestätigt werden, dass die Transkription dieser Gene instantan mit Beginn der ERK-Aktivierung startet. Eine vergleichende Analyse zeigte, dass das Prinzip der Signaldauer-Entschlüsselung in PC12-Zellen und MCF7-Zellen, zwei paradigmatischen Zellsystemen für die ERK-Signaldauer, konserviert ist. Insgesamt deuten die Ergebnisse der Untersuchung darauf hin, dass das neu identifizierte Genexpressionsmodul der Entschlüsselung der ERK-Signaldauer dient und das mRNA Halbwertszeiten sowohl hierfür, als auch für die zeitliche Abfolge der Genantwort eine entscheidende Rolle spielen. / The RAF-MEK-ERK signalling pathway controls fundamental, often opposing cellular processes such as proliferation and apoptosis. Signal duration has been identified to play a decisive role in these cell fate decisions. However, it remains unclear how the different early and late responding gene expression modules can discriminate short and long signals and what features govern their timing. Both protein phosphorylation and gene expression time course data was obtained from HEK293 cells carrying an inducible construct of the proto-oncogene RAF. A new gene expression module of immediate-late genes (ILGs) distinct in gene expression dynamics and function was identified by mathematical modelling. It was found that mRNA longevity enables these ILGs to respond late and thus translate ERK signal duration into response amplitude. Despite their late response, their GC-rich promoter structure suggested and metabolic labelling with 4SU confirmed that transcription of ILGs is induced immediately. A comparative analysis showed that the principle of duration decoding is conserved in PC12 cells and MCF7 cells, two paradigm cell systems for ERK signal duration. Altogether, the findings of this study indicate that ILGs decode ERK signal duration and that both decoding capacity and gene expression timing are governed by mRNA half-life.

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A cDNA clone encoding the entire amino acid sequence of rabbit brown adipose tissue uncoupling protein has been isolated and sequenced. The coding region of this cDNA is 80.6% identical to that of the rat uncoupling protein cDNA. In contrast to rat uncoupling protein for which there are two mRNAs of 1500 and 2000 nucleotides there is only one rabbit uncoupling protein mRNA of 2000 nucleotides. Whereas the rat cDNA hybridizes more strongly to the shorter rat uncoupling protein mRNA the rabbit cDNA hybridizes more strongly to the longer rat uncoupling protein mRNA. Primer extension and Northern blot analysis were performed to try to account for the difference of 430 ± 75 nucleotides between the two rat uncoupling protein mRNAs. Northern blot analysis indicated the presence of 355 more nucleotides in the 3'-untranslated region of the 2000 nucleotide long rat uncoupling protein mRNA than in the 1500 nucleotide long rat uncoupling protein mRNA. The two rat uncoupling protein mRNAs could therefore arise by differential processing. Primer extension revealed that the two rat uncoupling protein mRNAs have a 5'-untranslated region of approximately 186 nucleotides. The deduced amino acid sequence of rabbit UCP is 86% identical with both the rat and hamster proteins. Several regions are conserved in all three uncoupling proteins. The two longest regions of conservation are residues 52 to 69 and 82 to 100 of the mature proteins and correspond to two of several basic regions of the protein that have been suggested as possible targeting sequences. These conserved regions fall within amino acids 52 to 104 of the mature rat protein, which has been shown by others to target a passenger protein to mitochondria. Helical wheel diagrams that correspond to residues 52 to 68 and residues 72 to 92 reveal possible amphiphilic α-helical formations that may be involved in targeting. Regions corresponding to those conserved in the three UCPs are also conserved in three mammalian ADP/ATP carriers and may indicate a common role for these regions, perhaps including targeting. There is almost complete conservation of lysine, arginine, and cysteine residues that are thought to be involved in nucleotide binding and proton transport in the three UCPs. There is a threonine to alanine change at the carboxyl-terminus of the rabbit protein compared to the rat protein. This amino acid difference may explain the differential reactivities of rabbit and rat UCP with an antibody preparation against rat UCP. / Thesis / Master of Science (MSc)