The last few years have seen dramatic advances in genomics, including the discovery of a large number of non-coding and antisense transcripts. This has revolutionised our understanding of multifaceted transcript structures found within gene loci and their roles in the regulation of development, neurogenesis and other complex processes. The recent and continuing surge of knowledge has prompted researchers to reassess and further dissect gene loci. The ghrelin gene (GHRL) gives rise to preproghrelin, which in turn produces ghrelin, a 28 amino acid peptide hormone that acts via the ghrelin receptor (growth hormone secretagogue receptor/GHSR 1a). Ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH) release, appetite regulation, and cancer development. A truncated receptor splice variant, GHSR 1b, does not bind ghrelin, but dimerises with GHSR 1a, and may act as a dominant negative receptor. The gene products of ghrelin and its receptor are frequently overexpressed in human cancer While it is well known that the ghrelin axis (ghrelin and its receptor) plays a range of important functional roles, little is known about the molecular structure and regulation of the ghrelin gene (GHRL) and ghrelin receptor gene (GHSR). This thesis reports the re-annotation of the ghrelin gene, discovery of alternative 5’ exons and transcription start sites, as well as the description of a number of novel splice variants, including isoforms with a putative signal peptide. We also describe the discovery and characterisation of a ghrelin antisense gene (GHRLOS), and the discovery and expression of a ghrelin receptor (growth hormone secretagogue receptor/GHSR) antisense gene (GHSR-OS). We have identified numerous ghrelin-derived transcripts, including variants with extended 5' untranslated regions and putative secreted obestatin and C-ghrelin transcripts. These transcripts initiate from novel first exons, exon -1, exon 0 and a 5' extended 1, with multiple transcription start sites. We used comparative genomics to identify, and RT-PCR to experimentally verify, that the proximal exon 0 and 5' extended exon 1 are transcribed in the mouse ghrelin gene, which suggests the mouse and human proximal first exon architecture is conserved. We have identified numerous novel antisense transcripts in the ghrelin locus. A candidate non-coding endogenous natural antisense gene (GHRLOS) was cloned and demonstrates very low expression levels in the stomach and high levels in the thymus, testis and brain - all major tissues of non-coding RNA expression. Next, we examined if transcription occurs in the antisense orientation to the ghrelin receptor gene, GHSR. A novel gene (GHSR-OS) on the opposite strand of intron 1 of the GHSR gene was identified and characterised using strand-specific RT-PCR and rapid amplification of cDNA ends (RACE). GHSR-OS is differentially expressed and a candidate non-coding RNA gene. In summary, this study has characterised the ghrelin and ghrelin receptor loci and demonstrated natural antisense transcripts to ghrelin and its receptor. Our preliminary work shows that the ghrelin axis generates a broad and complex transcriptional repertoire. This study provides the basis for detailed functional studies of the the ghrelin and GHSR loci and future studies will be needed to further unravel the function, diagnostic and therapeutic potential of the ghrelin axis.

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Die Technologie der Oligonukleotid-Arrays erlaubt es, tausende von Genen parallel auf ihre Expression hin zu untersuchen. Die Firma metaGen, bei der diese Doktorarbeit entstand, setzt die Genexpressionsanalyse zur Identifikation von Targetmolekülen für die Therapie solider Tumoren ein. Im Zuge dieser Arbeit gelang die Entwicklung eines robusten Verfahrens zur Datenanalyse für Oligonukleotid-Arrays. Gerade für die Untersuchung humaner Proben ist die Robustheit von großem Interesse, da das Gewebematerial oft nur in sehr begrenzten Mengen und mit Qualitätsschwankungen behaftet vorliegt. Anhand eines eingeschränkten Sets an Kontrollversuchen konnte gezeigt werden, dass die vorgeschlagene Methode besser die Erwartungen an das System erfüllt als herkömmliche Verfahren. Ein weiterer Teil der Arbeit bestand im Aufbau einer relationalen Datenbank und in der schrittweisen Automatisierung der Auswertung. Stellvertretend für andere Krebserkrankungen wurde eine detaillierte Analyse zweier publizierter Expressionsdatensätze zum Bronchialkarzinom vorgenommen. Es konnten zwar in beiden Datensätzen zwischen Tumor- und Normalgewebe differenziell exprimierte Gene identifiziert werden, aber die Gegenüberstellung der Ergebnisse zeigte auch einen deutlichen Einfluss der unterschiedlichen Array-Technologien auf die gemessenen Intensitäten. Der spezielle Aufbau des verwendeten Oligonukleotid-Arrays gestattete die Entdeckung putativer Antisense-Transkripte. Die Koexpression einiger Sense- und Antisense-Sonden ließen sich durch Northern-Blot-Experimente bestätigen. Das unterstreicht das Anwendungspotenzial dieser Technologie für die Genomannotation. In einer Untersuchung der Transkriptome der Bäckerhefe und der Fruchtfliege konnte darüber hinaus ein Zusammenhang zwischen den Längen von Introns und Exons und der mittleren Expression von Genen hergestellt werden. Die Vielfalt der Anwendungen und die Ausbaumöglichkeiten verdeutlichen die Bedeutung und das Potenzial der Array-Technologie für die Genexpressionsanalyse. Eine wichtige Aufgabe bleibt deshalb die weitere Verbesserung der Qualitätskontrolle der Experimente und der Datenanalyse. / Oligonucleotide arrays represent a modern technology for the investigation of the expression of thounsands of genes in parallel. The theses were worked out at the company metaGen that uses gene expression analysis for the identification of target molecules for the therapy of solid tumors. One major achievement was the developement of a robust method for oligonucleotide array data analysis. It turned out that for the investigation of human tissue samples the robustness is crutial because the material is often very limited and of variing quality. Using a restricted set of control experiments the superiority of the method over standard procedures could be demonstrated. A further important part of the work was the construction of a relational database and the automation of the analysis process. To demonstrate the applicability of the methods in cancer research two publicly available lung cancer data sets were analysed. A list of differentially expressed genes was identified. But the comparison also revealed that the expression signals are strongly distorted by technical factors. The special array used at metaGen allowed the discorvery of putative antisense transcripts. Three of the candidates had been validated by Northern-blot analysis. This clearly shows the applicability of the array technology to genome annotations. An analysis of the transcriptoms of the bakers yeast and the fruit fly revealed a relationship between the average gene expression and the lengths of introns and exons. The manifold applications and extentions illustrate the inportance and the potential of the array technology. So that the improvement of the technology and of the data analysis will remain a major concern.