Thesis (doctoral)--Georg-Augusts-Universität zu Göttingen, 1900. / Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains vii, 96 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 84-96).

Univ., Diss., 2009--Kassel.

No description available.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

Doctor of Philosophy (PhD) / The field of epigenetics emerged primarily from studies in Drosophila, and is now being studied intensively by mammalian biologists. In order to increase our knowledge of epigenetic gene control in the mouse, I have studied modifiers of epigenetic gene silencing. My main method of investigation involved the characterisation of mutants from a sensitised ENU mutagenesis screen performed previously in our laboratory. The screen was carried out in an FVB/NJ strain carrying a variegating GFP transgene expressed in erythrocytes. To date we have recovered 12 dominant (D) and seven recessive (R) mutant mouse lines from this screen that display altered transgene expression. We have named these Mommes (Modifiers of murine metastable epialleles). I investigated the phenotype and attempted to identify the underlying causative mutation of two of these Momme mutants. MommeD6 is a semi-dominant, homozygous lethal mutation that acts as a suppressor of variegation with respect to the GFP transgene. This mutation has a large effect on the level of expression of the transgene in expressing cells, but little effect on the percentage of cells expressing the transgene. MommeD6 is linked to a 2.5 Mbp interval on chromosome 14. MommeD9 is a semi-dominant, homozygous lethal mutation that acts as an enhancer of variegation with respect to the GFP transgene. Mutants have a tendency to become obese as they age, show abnormal haematology profiles, and females develop infertility. MommeD9 is linked to a 17.4 Mbp region on chromosome 7. I produced and studied a strain carrying the same GFP transgene but in a new strain background, C57BL/6J. This strain provided an opportunity to look for strain-specific modifiers of expression of the GFP transgene. Several regions were mapped to chromosomal locations. Further work will be needed to identify the genes involved. This mouse will be useful in future mutagenesis screens of this type.

Position Effect Variegation (PEV) is the mosaic expression of a gene that has been moved out of its optimal environment and into a different area on the chromosome. Changing a gene’s environment may have profound effects on its eligibility for proper expression, which is a complicated process regulated by many factors. The PEV phenomenon is used as an assay to study gene expression as regulated by chromatin structure. In this study, the Drosophila melanogaster white gene was used as a reporter to study the various effects of CHD1, a chromatin regulating factor, on PEV gene expression. Inspired by preliminary data generated by the Armstrong Lab where overexpression of CHD1 resulted in suppression of gene silencing of the brown gene and loss of CHD1 resulted in enhancement of gene silencing, this study uses PEV as an assay to examine whether loss of function chd1 mutant alleles function dominantly to enhance silencing of the white gene when it is placed in a repressive chromatin environment. Surprisingly, I found that a chd1 loss of function mutant allele dominantly suppressed gene silencing (meaning I saw an increase in gene expression), suggesting that the CHD1 protein is normally required for effective silencing. The results demonstrated that CHD1 is a dominant modifier of PEV gene expression. CHD1 significantly modifies gene expression by suppressing silencing of the white gene inserted into pericentric heterochromatin on the second and fourth chromosomes and an insertion into the medial region of the fourth chromosome, while it shows no significant modification of the white gene inserted into telomeric heterochromatin of the fourth chromosome. Together, these intriguing results regarding varying gene expression at different chromosomal sites show that PEV is a dynamic phenomenon meriting further research and studying the effects of CHD1 as a modifier of PEV may be influential to understanding the mechanism and characteristics of gene expression.

Die vorliegende Arbeit befasst sich mit züchtungsmethodischen Untersuchungen zur Schaffung blattvariegater Pelargonium x zonale-Hybriden auf tetraploidem Leistungsstand. Basierend auf dem Wirkstoff Trifluralin konnte eine effektive Behandlungsvariante zur somatischen Polyploidisierung periklinalchimärischer Pelargonium x zonale-Klone etabliert werden. Unter Ausnutzung biparentaler Erbgänge wurden fünf ausgewählte Plasmotypen an das Leistungsniveau moderner Sortimente herangeführt. Daneben erbrachten Kreuzungen innerhalb der Sektion Ciconium hybridvariegate F1-Pflanzen. Die Möglichkeit der Ausnutzung von Kern-Plasma-Wechselwirkungen in der Pelargonienzüchtung wird diskutiert. Im Hinblick auf den Aufbau eines Protoplastenregenerationssystems konnten Zellsuspensionskulturen etabliert werden. Im Anschluss an enzymatische Verdauungen wurde die Regeneration von Kallus beobachtet. Variegate Pflanzen aus Mutationsversuchen mit NMH (Nitroso-Methyl-Harnstoff), einer weiteren experimentellen Variante, erwiesen sich als steril, so dass eine weiterführende Züchtungsarbeit auf diesem Weg bisher noch nicht möglich war / The study analyzes breeding schemes concerning the development of variegated tetraploid Pelargonium x zonale-hybrids (Pelargonium x hortorum). With a focus on practical relevance breeding methods for periclinal chimeric leaf patterns are discussed. Trifluralin-induced tetraploid Pelargonium x zonale-hybrids were successfully crossed with modern cultivars. Via biparental mode of inheritance five defined plasmotypes were transfered to the karyological background of current high-performance Pelargonium series. In a crossing-program within the section Ciconium hybrid-variegation was detected. The possibility of using nucleo-plasmatic interactions in developing new Pelargonium cultivars is discussed. First steps concerning a biotechnological approach to create variegated plants included the establishment of cell-suspension-cultures as the base for a protoplast regeneration system. Following the enzymatic digestion of Pelargonium-liquid cultures up to now, callus regeneration was achieved. Variegated plants resulting from mutagenic treatments with NMU (Nitroso-methylurea) proved to be sterile.

Auf dem Weg der genetischen Information stellt die Translation der RNA in eine Aminosäuresequenz den letzten Schritt dar. In Chloroplasten, den grünen Organellen der Pflanzenzellen, findet ein Großteil der Regulation der Genexpression auf Ebene der Initiation dieses Schrittes statt. Eine Vielzahl von Eigenschaften der RNA und von Faktoren, die an die RNA binden, entfalten einen Einfluss auf diesen Schritt. Bisher unvollständig aufgeklärt ist die Rolle einer konservierten Nukleotidsequenz in der untranslatierten Region der RNA -- der Shine-Dalgarno-Sequenz. Diese stellt in Bakterien, wie z.B. E. coli als Ribosomenbindestelle sicher, dass Ribosomen den Anfang der zu translatierenden Sequenz zuverlässig erkennen. Im Rahmen dieser Arbeit wurden diverse DNA-Konstrukte in Plastiden von Tabak eingebracht. Hierzu zählten Konstrukte, die sowohl eine erhöhte Anzahl von Ribosomenbindestellen enthielten als auch vermehrte Startpunkte der Translation. Zusätzlich wurden Konstrukte hergestellt, die die Situation von mehreren zu translatierenden Regionen in der RNA nachstellten. Es konnte festgestellt werden, dass plastidäre Ribosomen die strangaufwärts gelegenen Translationsstartpunkte bevorzugen -- im Gegensatz zu E. coli, wo alle Startpunkte gleichmäßig genutzt wurden. Hierdurch zeigten die prokaryotischen Ribosomen aus Chloroplasten, die sich aus bakteriellen Systemen ableiten, Eigenschaften von eukaryotischen Ribosomen. Ein zweites Teilprojekt dieser Arbeit beschäftigte sich mit der Inkompatibilität von Chloroplasten mit dem Kerngenom. In Kreuzungen von Arten der Gattung Pelargonium fielen Kombinationen auf, bei denen die Tochterpflanzen bleiche Blattbereiche bis hin zu vollständig weißen Pflanzen zeigten. Dieses Phänomen wird als Bastardbleichheit bezeichnet. In der Gattung Pelargonium werden Chloroplasten von beiden Elternteilen an die Tochterpflanzen vererbt. Da das Phänomen der Bastardbleichheit nur in einem der Plastiden vorkommt, nicht jedoch im anderen in der gleichen Pflanze, muss von einem Effekt ausgegangen werden, der von Plastiden ausgeht. Die Interaktionen zwischen Zellkern und Chloroplasten sind offensichtlich stark gestört. Zur detaillierten Untersuchung dieses Effekts wurde die Nukleotidsequenz von drei Chloroplastengenomen aufgeklärt. Es konnte eine Reihe von Sequenzunterschieden der Genome ermittelt werden. Aus diesen wurde eine Reihe von Unterschieden beobachtet, die einen solchen Effekt zur Folge haben können. Aus diesen Unterschieden wurde eine Reihe von potentiellen Kandidatengenen zusammengestellt, die in weiteren Arbeiten auf ihre Rolle in der Entstehung der Bastardbleichheit untersucht werden. / Chloroplasts are the green organelles of plants with a evolutionary prokaryotic background. During evolution chloroplasts established translation initiation as the major step in regulation of gene expression. A vast number of factors, e.g. sequence elements, secondary structures or RNA binding proteins, influences the regulation of translation initiation. A conserved sequence – Shine-Dalgarno sequence – can be identified both in prokaryotes as well as chloroplasts. In prokaryotes this sequence provides a faithful means for positioning of the ribosome to the start codon. Due to lower conservation of Shine-Dalgarno sequences the role of this sequence in translation initiation is not completely understood. We designed a series of constructs that contain different arrangements of these sequences in the 5’ UTR resulting in an increased number of potential ribosome binding sites or translation initiation sites. Additionally we constructed a series of 5’ UTRs that resembled polycistronic transcripts. The results showed a dramatic effect of the different constructs on the translation efficiency of the reporter protein. It could be shown that numerous translation initiation sites increase translation efficiency, whereas increased numbers of ribosome binding sites do not. Additionally it could be shown, that plastidic ribosomes preferentially initiate on 5’ translations initiation sites in contrast to prokaryotic ribosomes that recognize initiation sites equally. This illustrates that plastidic ribosomes in contrast to prokaryotic ribosomes show a scanning like mechanism. Hence plastidic ribosomes gained some eukaryotic properties during evolution. A second project was dealing with hybrid variegation. This phenomenon is based on plastid-nuclear genome incompatibility. Due to biparental plastid inheritance in Pelargonium hybrids may show chimeric phenotypes with bleached (incompatible) and green (compatible) sectors. This points to the plastome as cause for the hybrid variegation. To this end the nucleotide sequence of three plastid genomes was determined and an array of candidate genes causing the incompatibility could be compiled.

Chromatin structure is a fundamental determinant of eukaryotic gene expression and it is composed of two chromatin environments, euchromatin and heterochromatin. Euchromatin provides an accessible platform for transcription factors; hence it is permissive for gene expression. Heterochromatin on the other hand is highly compacted and inaccessible, which in most cases leads to transcriptional repression. A locus that is composed of both of these environments is the ribosomal DNA (rDNA). In eukaryotes the rDNA is composed of hundreds to thousands of tandemly repeated genes where maintaining both silent and active copies is fundamental for the stability of the genome. The aim of this research was to investigate the role of the rDNA in gene expression in Drosophila melanogaster. In D. melanogaster the rDNA loci are present on the X and Y chromosomes. This research used the Y-linked rDNA array to investigate the role of this locus on gene expression. A genetic and molecular strategy was designed to create and quantify specific, graded and isogenic Y- linked rDNA deletions. Then the deletions were used to address the effect of rDNA deletions on gene expression using reporter genes sensitive to Position Effect Variegation (PEV). In addition, the effect of the deletions in nucleolus size and structure as well as the effect of spontaneous rDNA deletions on gene expression were tested in this study. This research found that changes in rDNA size change the chromatin balance, which resulted in increased expression of the reporter genes, decreased nucleolus volume, and altered nucleolus structure. These findings prompted a further research question on whether this effect on gene expression occured globally in the genome. This was addressed by performing microarray analysis where the results showed that rDNA deletions affect about half of the genes on the genome. Presented in this dissertation is evidence that suggest a novel role for the rDNA is a global modulator of gene expression and also is a contributor to the gene expression variance observed in natural populations.