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.

The plastid genomes of land plants are generally highly conserved in gene content and order, genome organization, and rates of sequence evolution; however, a few groups have experienced genomic change. The previously published sequence of Pelargonium X hortorum (Geraniaceae) reveals the largest, most rearranged plastid genome among land plants, and rate heterogeneity and genomic change have been documented in the monocot family Poaceae. Three initiatives were taken to characterize plastid genome evolution better in these groups. First, I estimate and compare genome-wide rates of sequence evolution in Geraniaceae genes relative to other angiosperms. An analysis of nucleotide substitutions for 72 plastid genes from 47 angiosperms, including nine Geraniaceae, shows that values of dN are accelerated in ribosomal protein and RNA polymerase genes. dN/dS, an indicator of selection, is significantly elevated in the same two classes of genes and ATPase genes. Second, I sequenced three additional Geraniaceae plastid genomes (Erodium texanum, Geranium palmatum, and Monsonia speciosa) and compare these sequences to each other, P. X hortorum, and other rosids. Geraniaceae plastid genomes are highly variable in size, gene content and order, and base composition. The genome of M. speciosa is among the smallest land plant plastid genomes, and one copy of the IR region in E. texanum has been lost. Gene/intron loss and gene duplication are rampant in Geraniaceae plastid genomes, and a number of losses are phylogenetically inconsistent. To explain the unusual rates and patterns of genome evolution in Geraniaceae, I propose a model of aberrant DNA repair coupled with altered gene expression. Lastly, I characterize genome evolution in the family Poaceae and order Poales. There has been a recent surge in the availability of Poaceae sequences, but a comprehensive analysis of genome evolution had not been performed that included any non-grass Poales taxa. I present the sequence of Typha latifolia (Typhaceae), the first non-grass Poales sequenced to date, and I show that Poaceae plastid genomes exhibit increased genomic rearrangements and nucleotide substitutions. These analyses show the extent of lineage-specific rate acceleration on the branch leading to Poaceae and deceleration during the diversification of the family. / text

Plastid division, sustained by the equilibrium expression and coordination of plastid division genes is vital for the maintenance of plastid populations in dividing plant cells. Macrochloroplasts (MCP), the occurrence of one or a few chloroplasts per cell is due to the imbalance in the expression of plastid division genes. Because of the MCP size and number it was proposed that they may provide better targets for the plastid transformation than the normal (WT) chloroplasts and result in better plastid transformation frequencies. The objective of this research was to produce transgenic plants containing macrochloroplasts by nuclear transformation and then to use these plants as a model for the development of plastid transformation of crop species. By using AtFtsZ1-1 and AtMinD1 as query sequences in the TIGR (U.S.A) and ASTRA (Australia) Brassica oleracea EST databases, this project resulted in the isolation of cauliflower FtsZ1-1 (EU684588) and MinD (EU684589) genes. In addition, AtFtsZ1-1 was used as a control gene for comparison to the cauliflower FtsZ1-1. Binary vectors were constructed to express these genes in tobacco and cauliflower either by Agrobacterium tumefaciens-mediated or PEG-mediated transformation methods. Transgenic tobacco and cauliflower plants with abnormal chloroplasts (MCP, minichloroplasts, honeycomb or doughnut shaped chloroplasts, uneven surface membrane chloroplasts) were developed. Furthermore, the transgenic tobacco and cauliflower plants were examined by PCR, RT-PCR and Southern blotting. In addition, th ese plants were also analysed for the different abnormal chloroplast phenotypes by fluorescence microscopy. This project also generated the first plastid transformants from macrochloroplast bearing tobacco plants via biolistics. After one round of regeneration homoplasmic plastid transformants were obtained from both WT chloroplast and MCP tobacco plants. The homoplasmic nature of plastid transformants were confirmed by PCR and Southern blotting. Plastid expression of GFP in WT and MCP was confirmed by fluorescence/confocal microscopy and western blot analysis. This project showed for the first time the characterisation of cauliflower FtsZ1-1 and MinD plastid division genes in homologous and heterologous systems (cauliflower and tobacco). Moreover, obtaining homoplasmic plastid transformant shoots from one round of regeneration from the MCP containing tobacco plants is reported for the first time in this study. In addition this study explored the effect of transgene expression level on the chloroplast abnormality, highlighting the importance of analysing transgenic tobacco and cauliflower plants at the protein lev el specifically with regard to plastid division genes. The maintenance of MCP phenotype in the regenerated shoots and the requirement of standardisation of MCP containing plants via biolistics for increasing the plastid transformation frequency were also examined.

It is estimated that over 200,000 Australians have problem or chronic wounds at any one time (Australian Wound Management Association, 2008). Over the past 4 decades while there has been significant advancement in wound care, a high proportion of wounds become chronic. Despite the availability of wound care resources and specialist services, there remains an inconsistency in the management of chronic wounds that impacts both on the quality of life of individuals with chronic wounds and the health care budget (Harding 2002). Using a Grounded theory approach, the aim of this study was to explore and describe the impact of living with a chronic wound and findings indicate that individuals living with a chronic wound are receiving conflicting or inappropriate advice and care. Individuals living with a chronic wound experience a life of uncertainty related to the struggle to endure a wounded body and the layers of professional care they receive. When they are provided with conflicting or inappropriate advice and treatment, inconsistencies of care and poor coordination of care, layers of unnecessary burden are added to their experience. The uncertainty and dissonance individuals are faced with, leads them to question their care, themselves and the expertise and professionalism of their treating health professionals. As a result, they experienced a loss of respect and trust for their treating health professionals and a loss of confidence in their care. Chronic wounds impose of individuals, an intense burden of physical suffering, cause major disruption to the normality of their lives, and often entail a constant personal struggle to secure appropriate care and understanding from their treating health professionals. In order to enable individuals living with chronic wounds to develop appropriate coping strategies, it is essential that health professionals: understand the burden of suffering associated with living with a chronic wound; ensure that they develop and maintain a high level of knowledge with regards to contemporary wound care practices; ensure that their clientele are provided with high quality care information that is based on the best available evidence; ensure continuity of care; and foster quality professional-client relationships that negates the need for individuals to have to constantly question their care.

Sequenced diatoms Thalassiosira pseudonana and Phaeodactylum tricornutum belong to the chromist algae harboring secondary plastids, which display distinct evolutionary history when compared to photosynthetic organelles from rhodophytes, green algae and plants. Via secondary endosymbiosis, heterotrophic eukaryotic ancestor of diatoms engulfed red alga, and in addition to the new organelle, it obtained fitness increasing peculiarities in the chimerical cell metabolism and lifestyle. We examined phylogeny and in silico localization of the nuclear-encoded but plastid located enzymes of tryptophan biosynthesis. We suggest that the diatom tryptophan pathway represents an extreme in the trend of plastid (cyanobacterial) enzymes to be replaced by eukaryotic isoforms. In addition, the gene napped during the endosymbiotic gene transfer from the diatom plastid genome to the diatom nucleus (psb28) was described.

No description available.

Les plastes primaires proviennent d'une cyanobactérie qui a établi une relationendosymbiotique avec un hôte eucaryote. Cet événement a donné naissance au super-groupeArchaeplastida qui inclut les Viridiplantae (algues vertes et plantes terrestres), les Rhodophyta (alguesrouges) et les Glaucophyta. Suite à l'endosymbiose primaire, les algues rouges et vertes ont étendu lacapacité de photosynthèse à d'autres lignées eucaryotes via des endosymbioses secondaires. Bien quedes progrès considérables aient été réalisés dans la compréhension de l'évolution des eucaryotesphotosynthétiques, d'importantes questions sont restées ouvertes, telles que l’identité de la lignéecyanobactérienne la plus proche des plastes primaires ainsi que le nombre et l'identité des partenairesdans les endosymbioses secondaires.Ma thèse a consisté à étudier l'origine et l'évolution précoce des eucaryotes photosynthétiques enutilisant des approches phylogénétiques et phylogénomiques. Je montre par mon travail que les plastesprimaires ont évolué à partir d'un symbiote phylogénétiquement proche de Gloeomargarita lithophora,une cyanobactérie représentant un clade s’étant diversifié précocement et qui a été détectéeuniquement dans les milieux terrestres. Ce résultat fournit des pistes nouvelles sur le contexteécologique dans lequel l'endosymbiose primaire a probablement eu lieu. En ce qui concerne l'évolutiondes lignées eucaryotes avec des plastes secondaires, je montre que les génomes nucléaires deschlorarachniophytes et des euglénophytes, deux lignées photosynthétiques avec des plastes dérivésd'algues vertes, encodent un grand nombre de gènes acquis par transferts depuis des algues rouges.Enfin, je mets en évidence que SELMA, la machinerie de translocation des protéines à travers laseconde membrane externe des plastes rouges secondaires à quatre membranes, a une histoireétonnamment compliquée aux implications évolutives importantes : les cryptophytes ont recruté unensemble de composants de SELMA différent de ceux des haptophytes, straménopiles et alvéolés.Ainsi, ma thèse a permis d’identifier pour la première fois la lignée cyanobactérienne la plus proche desplastes primaires et apporte de nouvelles pistes pour éclaircir les événements complexes qui ontjalonné l’évolution des eucaryotes photosynthétiques secondaires. / Primary plastids derive from a cyanobacterium that entered into an endosymbioticrelationship with a eukaryotic host. This event gave rise to the supergroup Archaeplastida whichcomprises Viridiplantae (green algae and land plants), Rhodophyta (red algae) and Glaucophyta. Afterprimary endosymbiosis, red and green algae spread the ability to photosynthesize to other eukaryoticlineages via secondary endosymbioses. Although considerable progress has been made in theunderstanding of the evolution of photosynthetic eukaryotes, important questions remained debatedsuch as the present-day closest cyanobacterial lineage to primary plastids as well as the number andidentity of partners in secondary endosymbioses.The main objectives of my PhD were to study the origin and evolution of plastid-bearing eukaryotesusing phylogenetic and phylogenomic approaches to shed some light on how primary and secondaryendosymbioses occurred. In this work, I show that primary plastids evolved from a close relative ofGloeomargarita lithophora, a recently sequenced early-branching cyanobacterium that has been onlydetected in terrestrial environments. This result provide interesting hints on the ecological setting whereprimary endosymbiosis likely took place. Regarding the evolution of eukaryotic lineages with secondaryplastids, I show that the nuclear genomes of chlorarachniophytes and euglenids, two photosyntheticlineages with green alga-derived plastids, encode for a large number of genes acquired by transfersfrom red algae. Finally, I highlight that SELMA, the translocation machinery putatively used to importproteins across the second outermost membrane of secondary red plastids with four membranes, has asurprisingly complex history with strong evolutionary implications: cryptophytes have recruited a set ofSELMA components different from those present in haptophytes, stramenopiles and alveolates.In conclusion, during my PhD I identified for the first time the closest living cyanobacterium to primaryplastids and provided new insights on the complex evolution that have undergone secondary plastid-bearing eukaryotes

The plastid is an important organelle that allows eukaryotes to photosynthesize. The endosymbiotic event that led to the development of a primary plastid occurred more than one billion years ago. Since then the organelle has undergone a significant genome reduction, losing a large portion of non-essential genes whose function is covered by the eukaryotic host. The majority of the proteins needed for essential plastid function are transcribed in the nucleus and translated in the cytosol. These proteins are then translocated across the inner and outer plastid membranes. The difference in where proteins are translated and transported can be used to study plastid evolution, this can be done by examining what proteins are present in the red algae plastid and comparing it to proteins found in other algae groups. In this project, the exact placement of the proteins, mainly plastid proteins is of interest. In order to localize and identify proteins in the algal cell, an efficient method of cell lysis, both total and incomplete, where the preservation of organelles is essential needs to be developed. This thesis examines a set of different methods of cell lysis, both complete and incomplete, coupled with organelle fractionation, to achieve the isolation of proteins belonging to the different cellular compartments. I show that complete cell lysis with bead milling using 0.1-0.5 mm silica beads is more efficient than a method using a Dounce homogenizer. For incomplete lysis, I show that nitrogen cavitation at 750 psi for 15 min and 1,000 psi for 30 min provides the same level of cellular lysis, indicating that the nitrogen gas equilibrates within 15 minutes. Organelle fractionation with OptiprepTM gradient showed that the majority of the sample did not travel through the gradient, staying in the first layer, which also prevented revelation of the protein pattern on an SDS-PAGE gel, indicating insufficient centrifugation. A great deal of optimization is still required to make these methods as efficient as possible. The step that requires the most optimization is sample preparation for nitrogen cavitation and the use of an ultracentrifuge.

Garry oak (Quercus garryana Douglas ex. Hook) is a white oak (Quercus sect. Quercus) with a geographic range extending from southwestern BC to south-central California. It is the only native white oak in BC and Washington, and is the northernmost species of the California Floristic Province-Pacific Northwest white oak clade. I used molecular methods to address the following questions: 1) What are the patterns of genetic variation within Garry oak? 2) How do these patterns vary geographically, and how did the spatial distribution of the gene lineages come to occupy its current geographical range? 3) Does Garry oak show evidence of genetic interaction with other white oak species in western North America? 4) Is there morphological or genetic evidence to support the three described varieties of Garry oak? I obtained samples of Garry oak from 117 localities over its geographic range, as well as samples of two other California white oaks (Q. lobata and Q. douglasii) and a Rocky Mountain species (Q. gambelii). Analyses of DNA sequence data from four plastid DNA regions revealed 24 distinct molecular variants (haplotypes) in Garry oak. These show a strong south-to-north decrease in genetic diversity, consistent with post-glacial northward expansion. Haplotypes present in the northern part of the range provide evidence of two separate northward migrations, only one of which reached the northern range limit of Garry oak in BC. I found that Garry oak shared plastid DNA haplotypes with two other white oak species, indicating that it hybridizes with other oaks in the southern part of its range. The nuclear ribosomal ITS phylogeny showed poor resolution, but both cpDNA and nrDNA may indicate that Q. garryana is more closely related to the white oaks of central North America than was previously thought. My findings also suggest that the three currently recognized varieties of Garry oak (var. garryana, breweri and semota) are not well differentiated genetically, but show morphological variation at the regional level. This study shows the phylogeographic patterns within Q. garryana. In addition, it contributes to conservation efforts in Garry oak ecosystems by indicating regions of high genetic diversity in Garry oak, including genetically unique populations that may be especially worthy of preservation. / Graduate

In mature chloroplasts, the DNA (cpDNA) is surrounded by a potentially genotoxic environment that would make the mitochondrial DNA milieu look like a “nadree” (picnic). And yet, the slower evolution of cpDNA compared to other cellular genomes suggests that this organelle must have efficient mechanisms for repairing DNA. Unfortunately, those mechanisms have been barely noted, much less studied. This dissertation describes a novel approach that was developed to study how chloroplasts of Arabidopsis repair the most severe form of DNA damage, a double-strand break (described in Chapter 2). The success with this approach also prompted the development of a new method for site-specific modification of tobacco cpDNA that is described in Chapter 3. To study the consequences and repair of a break in the circular plastid genome, we developed an inducible system based on a psbA-intron endonuclease from Chlamydomonas (I-CreII) that specifically cleaves the psbA gene of Arabidopsis. The protein was targeted to the chloroplast using the rbcS1 transit peptide, and activation of the nuclear gene was made dependent on an exogenous inducer (β-estradiol). In Chlamydomonas, I-CreII cleavage at psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) that are abundant in that genome. By comparison, Arabidopsis cpDNA is very repeat-poor. Nonetheless, phenotypically strong and weak transgenic lines were obtained, and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of psbA, but not cpDNA as a whole, in the strongly-expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site that were indicative of repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) or no homology. The results provide evidence of alternative repair pathways in the Arabidopsis chloroplast that resemble the nuclear microhomology-mediated and nonhomologous end-joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, plus other considerations, the data suggests that an evolutionary relationship may exist between the repeat structure of cpDNA and the organelle’s ability to repair broken chromosomes. Taking advantage of the inducible I-CreII system, I developed a method to delete defined regions of cpDNA in tobacco, which was named DREEM (for direct repeat and endonuclease mediated). Chloroplast transformation was used to introduce an I-CreII cleavage site adjacent to an aadA:gfp marker and flanked by a direct repeat of 84 bp. When chloroplast-targeted I-CreII was induced with β-estradiol during germination, complete loss of the aadA:gfp marker occurred by SSA-type repair involving the 84-bp direct repeat. I obtained additional evidence for DREEM effectiveness by deleting 3.5 kb of native cpDNA that included part of the large ycf1 gene. DREEM can be used for other modifications besides gene deletions, partly because it is seamless and leaves no trace of introduced DNA. Since expression of the endonuclease is controlled by steroid application (and concentration), and the deleted cpDNA is probably destroyed during the SSA process, this inducible gene-ablation technique could enable the study of essential chloroplast genes in vivo. / text