University, Diss., 2002--Frankfurt (Main).

Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: A number of proteins essential for the survival of a plant are encoded by the chloroplast genome. The characterization and sequencing of a number of algal and plant chloroplast genomes has facilitated researchers understanding of cellular functions and metabolism. Chloroplast DNA (cpDNA) has also been used to determine inter- and intraspecies evolutionary relationships and this organelle offers an alternative means of expressing foreign genes. Although a number of species' chloroplast genomes have been characterized and sequenced, no previous attempts of this kind have been made for a chloroplast genome of the family Vitaceae. In this study, attempts were made to characterize and partially sequence the chloroplast genome of Vilis vinifera. Chloroplast DNA was isolated from the Sultana and Sugra 1 cultivars and digested with restriction enzymes that produced cpDNA fragments of a suitable size for cloning. The fragments were shotgun-cloned into a plasmid vector and white colonies were screened by means of PCR and colony blotting. Three EcoRI-digested clones and one PstI-digested clone were obtained in this manner. Walking outwards from a previously sequenced grapevine rrn 16 gene region by means of PCR also allowed us to sequence a further -3310 bp region of the Sultana chloroplast genome. BAC clones containing V. vinifera cv L. Cabernet Sauvignon cpDNA inserts became available later in the project. It was decided to use these clones for further library construction instead of isolated cpDNA. The 5' and 3' end sequences of seven of the 24 BAC clones were obtained. These were compared to sequences found in the NCBI database to find - homologous chloroplast regions and determine the size of each BAC insert. One clone appeared to contain the entire grapevine chloroplast genome, apart from a 500 bp region. This clone was selected for further analysis. The BAC clone DNA was isolated and restriction-digested fragments were shotgun-cloned into a plasmid vector. White colonies were screened by isolating the plasmid DNA and digesting it with appropriate restriction enzy~es. The 5' and 3' ends of putative positive clones were sequenced and mapped onto the Atropa belladonna chloroplast genome. A total of 15 clones were obtained in this project. Five of these contain cpDNA isolated from grapevine leaves and 10 contain fragments sub-cloned from the BAC clone. The biggest problem encountered with both methods used for library construction was genomic DNA contamination. Genomic DNA either originated from the plant nuclear genome or from the bacterial host cells in which the BAC clones were maintained. Many of the clones screened contained genomic DNA, and these could only be identified and removed once the clones had been sequenced. Even when a commercial kit was used for BAC clone isolation, 31% of the clones screened contained genomic DNA. This kit was specifically designed for the isolation of genomic DNA-free large constructs. The clones obtained from the two strategies provided a good representation of the grapevine chloroplast genome. The only region not represented was the Small Single Copy (SSC) region. Approximately 40% of the grapevine chloroplast genome was covered by these clones. This provides a basis for further genome characterization, physical mapping and sequencing of the grapevine chloroplast genome. / AFRIKAANSE OPSOMMING: Die chloroplasgenoom kodeer VIr 'n hele aantal proteïene wat essensieel is VIr die voortbestaan van 'n plant. Die karakterisering en volgorde bepaling van 'n aantal alg en plant chloroplasgenome het dit. vir navorsers moontlik gemaak om sellulêre funksies en metabolisme van plante te ontrafel. Chloroplas DNA (cpDNA) is ook gebruik om intra- en interspecies evolusionêre verwantskappe vas te stel. Dié organel verskaf ook 'n alternatiewe manier vir die uitdrukking van transgene. Alhoewel die chloroplasgenome van 'n hele aantal species al gekarakteriseer is en die DNA volgorde daarvan bepaal is, is daar nog geen navorsing van bogenoemde aard op die chloroplasgenoom van die Vitaceae familie gedoen rue. In hierdie studie is beoog om die chloroplasgenoom van Vitis vinifera te karakteriseer en gedeeltelike volgordebepaling daarvan te doen. Chloroplas DNA is geïsoleer vanaf Sultana en Sugra 1 kultivars en restriksie-ensiem vertering is gedoen met ensieme wat cpDNA fragmente, met geskikte grootte vir klonering, produseer. Dié fragmente is in 'n plasmiedvektor gekloneer met die haelgeweer-metode en wit kolonies is gesif deur middel van PKR en die kolonieklad metode. Op hierdie manier is drie EcoRI-verteerde klone en een PstI-verteerde kloon verkry. Deur uitwaarts te loop, deur middel van PKR, vanaf 'n druif rrnl6 geenstreek, waarvan die volgorde voorafbepaal is, was dit vir ons moontlik om ook die volgorde te bepaal van 'n verdere ~3310 bp streek van die Sultana chloroplasgenoom. BAC klone wat V. vinifera cv L. Cabernet Sauvignon cpDNA fragmente bevat, het later in die projek beskikbaar geraak. Daar is besluit om hierdie klone, i.p.v. die geïsoleerde cpDNA, te gebruik vir verdere biblioteek konstruksie. Die 5' en 3' entpuntvolgordes van sewe uit die 24 BAC ~lone is verkry. Hierdie volgordes is vergelyk met volgordes in die NCB Idatabasis om homoloë chloroplas streke te identifiseer, en die grootte van elke BAC fragment te bepaal. Die het geblyk dat die hele druif chloroplasgenoom in een van die klone vervat is, behalwe vir 'n 500 bp streek. Die BAC-kloon DNA is geïsoleer en die restriksie-verteerde fragmente is in 'n plasmiedvektor gekloon d.m.V. die haelgeweer-metode. Wit kolonies is gesif deur die isolering van plasmied DNA en die vertering daarvan met geskikte restriksie-ensieme. Die volgorde van die 5' en 3' entpunte van skynbare positiewe klone is bepaal en gekarteer op die Atropa belladonna chloroplasgenoom. In hierdie studie is 'n totaal van 15 klone verkry. Vyf hiervan bevat cpDNA wat vanaf druifblare geïsoleer is, en 10 bevat fragmente wat vanaf die BAC-klone gesubkloneer is. Genorniese DNA kontaminasie was die grootste probleem wat ondervind is tydens beide metodes wat gebruik is vir biblioteek konstruksie. Genomiese DNA was afkomstig vanaf óf die plant nukleêre genoom óf die bakteriële gasheerselle waarin die BAC-klone gehou is. Baie van die klone wat gesif is, het genomiese DNA bevat, en dit kon eers geïdentifiseer en verwyder word nadat die volgorde van die klone bepaal is. Selfs al is 'n kommersiële produk vir BAC-kloon isolasie gebruik, het 31% van die gesifde klone steeds genomiese DNA bevat. Dié kommersiële produk is spesifiek vir die isolasie van groot konstrukte, wat genomiese DNA vry is, ontwerp. Die klone wat deur die twee strategeë verkry is, het 'n goeie verteenwoordiging van die druif chloroplasgenoom gegee. Die enigste streek wat die verteenwoordig is nie, was die Klein Enkelkopie (SSC) streek. Ongeveer 40% van die druif chloroplasgenoom is deur hierdie klone gedek. Dit verskaf 'n basis vir verdere genoomkarakterisering, fisiese kartering en volgordebepaling van die druif chloroplasgenoom.

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While genetic improvement of susceptible crop species may enhance resistance to microbial pathogens and facilitate reduced pesticide load, the possibility for transmission of novel genes to wild relatives has hampered acceptance of GM crops in some markets. Chloroplast transformation presents an attractive alternative to nuclear transformation and offers the potential to ameliorate these environmental concerns. Most agronomically important species exhibit maternal inheritance of organellar genomes which eliminates the threat of transgene escape through pollen. Gene silencing is absent due to site directed, single copy insertion by homologous recombination. Foreign proteins can accumulate to high levels (up to 50% of total soluble protein) and are retained within the chloroplast envelope protecting them from degradation by host cytoplasmic proteases. A bacterial chloroperoxidase gene (cpo-p) was transformed into the tobacco chloroplast genome to test its efficacy against plant pathogens and the mycotoxin producing saprophyte Aspergillus flavus.

Transgene Pflanzen nehmen einen wachsenden Stellenwert bei der Produktion therapeutischer Proteine, besonders von Impfstoffen, ein. Einige dieser Proteine benötigen für ihre Funktionalität verschiedenste post-translationale Modifikation und es ist nicht bekannt, ob Pflanzen zu diesen Stoffwechselleistungen befähigt sind. In der vorliegenden Arbeit sollte anhand eines bakteriellen Antigens geprüft werden, ob in Chloroplasten von Nicotiana tabacum an einem rekombinanten Protein eine besondere Form der post-translationalen Modifikation, die Lipidierung, durchgeführt wird und ob somit ein alternatives Produktionssystem für einen Lipoprotein-Impfstoff entwickelt werden kann. Basis für diese Untersuchungen war das bakterielle Lipoprotein OspA (outer surface protein A) aus Borrelia burgdorferi. Dieses Protein wird zur Prävention von Lyme-Borreliose eingesetzt und frühere Untersuchungen zeigten, dass OspA sowohl in B. burgdorferi als auch nach heterologer Expression in E. coli einer post-translationalen Lipidierung am N-Terminus unterliegt. Dabei wird das Protein unter Mitwirkung dreier Enzyme (Lgt, Lsp und Lnt) am N-Terminus mit einer Pam3Cys-Struktur versehen und die N-terminale Signalsequenz abgespalten. In dieser Arbeit konnten nach der Etablierung eines Expressionssystems mehrere unabhängige transplastome OspA-Tabakpflanzen generiert werden. Der Anteil an rekombinantem, plastidärem OspA (rpOspA) am löslichen Gesamtprotein wurde mit ca. 1% bestimmt. Dabei lag rpOspA sowohl in einer lipidierten, membrangebundenen als auch in einer nicht-modifizierten, löslichen Form vor. Strukturelle Untersuchungen an rpOspA ergaben, dass in Chloroplasten eine Bakterien-ähnliche Lipidierung an dem Protein durchgeführt wurde. Dabei wurde am N-Terminus ein Diacylglycerin über eine Thioetherbindung an das einzige in der Sequenz vorkommende Cystein gebunden. Anders als in Bakterien wurde die Signalsequenz in Chloroplasten jedoch nicht abgespalten und infolgedessen keine dritte Fettsäure an das Cystein gebunden. Die plastidäre Modifikation an rekombinantem OspA resultierte daher in einer Pam2Cys-Struktur mit Signalsequenz. Untersuchungen zur Immunogenität des rpOspA in Mäusen zeigten eindeutig, dass das rekombinante Protein aus Tabak die Bildung spezifischer Antikörper induziert und damit in seiner Wirkung vergleichbar dem bakteriellen Protein ist. Um die Lipidierungsrate des akkumulierten OspA in Chloroplasten zu erhöhen, wurden weitere transplastome Tabakpflanzen generiert. Diese wiesen neben dem ospA-Gen auch die Gene (lgt, lsp und lnt) der drei modifizierenden Enzyme aus E. coli auf. Durch die simultane Bildung von OspA und den drei modifizierenden Enzyme konnte eine quantitative Lipidierung von rpOspA mit einer Pam2Cys-Struktur erlangt werden. Als Grundlage für vergleichende Untersuchungen zwischen plastidärer und cytoplasmatischer Lipidmodifikation in Tabak wurden ebenfalls Transformationen des Zellkerns mit ospA durchgeführt, wobei jedoch keine heterologe ospA-Expression erreicht werden konnte. Erst durch die Anwendung eines transienten, viralen Expressionssystems war es möglich, ospA im Zellkern zu exprimieren. In dieser Arbeit wurde gezeigt, dass in Chloroplasten Höherer Pflanzen eine Bakterien-ähnliche Modifikation an rekombinantem OspA durchgeführt wird und das resultierende Protein eine spezifische Immunantwort in Mäusen induzieren kann. Das Potential von transgenen Pflanzen als alternatives Produktionssystem für Lipoprotein-Impfstoffe wird diskutiert. / Transgenic plants play an increasing role in the production of therapeutical proteins, especially vaccines. Some of those proteins need a posttranslational modification for their activity of immunogenicity and it is still ambiguous if plants are capable of performing these modifications. In the present study it was investigated by the use of a bacterial antigen if a particular form of posttranslational modification, the lipidation of proteins, is performed in chloroplasts of Nicotiana tabacum and if the resulting plants could be further developed into an alternative production system for lipoprotein-vaccines. For this investigation, the bacterial lipoprotein OspA (outer surface protein A) of Borrelia burgdorferi was chosen, a protein used as a vaccine against Lyme disease. Previous studies have been shown that OspA is lipidated on its N-terminus both in B. burgdorferi and when expressed in Escherichia coli. This modification is performed by three different enzymes (Lgt, Lsp and Lnt) which build up a Pam3Cys-structure at the N-terminus of the protein while the N-terminal signal sequence is cleaved. In the present study several independent transgenic plant lines could be generated by the use of a suitable expression system. Analysis showed that the fraction of recombinant plastidal OspA (rpOspA) in transplastomic plants reached approx. 1% of the total soluble protein. Moreover, a lipid modification could be detected in a fraction of the rpOspA which resulted in the membrane association of a portion of the recombinant protein. The structural analysis of rpOspA revealed that in chloroplasts, rpOspA is lipidated in a bacteria-like manner, including the attachment of a diacylglycerol moiety to the sole cysteine via a thioether linkage. Other than in bacteria, the signal peptide is not cleaved in chloroplasts; thus, no additional fatty acid is attached to the cysteine. The plastidal modification on recombinant OspA therefore resulted in a Pam2Cys-structure including the signal sequence. Furthermore, the rpOspA from tobacco showed similar immunogenic properties as the recombinant protein from bacteria when tested in mice, showing the potential of plants as a production system for lipoprotein-vaccines. To further increase and modify the lipidation rate and pattern of tobacco-derived OspA, a new set of transplastomic tobacco plants were generated. These plants not only carried the ospA gene, but also the genes encoding for the three modifying enzymes (lgt, lsp and lnt). This simultaneous expression shifted the ratio between lipidated and non-lipidated rpOspA almost quantitatively to the lipidated form. For further comparisons of the OspA-lipidation either in plastids or in the cytosol transgenic tobacco plants were established. However, after stable integration of ospA into the nuclear genome, no detectable heterologeous expression could be achieved. Only by using a transient viral expression system the nuclear ospA expression was feasible. In this study it has been shown, that in chloroplasts of higher plants a bacteria-like modification on recombinant OspA was performed and that the resulting protein induced a specific immune response in mice. The potential of transgenetic plants as an alternative production system for lipoprotein-vaccines is discussed.

by Edwina Po Sau Man. / The "0" & "1" in the title are subscripts. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (leaves 81-86). / Abstract --- p.II / Acknowledgment --- p.IV / Abbreviations --- p.V / List of Tables --- p.VIII / List of Figures --- p.IX / Table of Contents --- p.XII / Chapter Section 1 --- Introduction --- p.1 / Chapter 1.1 --- Photosynthesis --- p.1 / Chapter 1.2 --- Site of Photosynthesis --- p.3 / Chapter 1.3 --- The Structure of ATPase --- p.6 / Chapter 1.3.1 --- Functions of the Subunits of CF1 --- p.9 / Chapter 1.3.1.1 --- The ε - Subunit --- p.9 / Chapter 1.3.1.2 --- The δ - Subunit --- p.9 / Chapter 1.3.1.3 --- The γ- Subunit --- p.10 / Chapter 1.3.1.4 --- The α- and β- Subunits --- p.10 / Chapter 1.4 --- "Photosynthetic Electron Transport, Δ pH and Phosphorylation inside Chloroplasts" --- p.12 / Chapter 1.5 --- Pesticides --- p.16 / Chapter 1.5.1 --- Paraquat --- p.17 / Chapter 1.5.2 --- Carbamates --- p.20 / Chapter 1.6 --- Objectives of the Study --- p.21 / Chapter Section 2 --- Materials and Methods --- p.22 / Chapter 2.1 --- Apparatus --- p.22 / Chapter 2.2 --- Materials --- p.24 / Chapter 2.2.1 --- Reagents and Buffers for assay of Proton Transport --- p.25 / Chapter 2.2.2 --- Pesticides --- p.26 / Chapter 2.2.3 --- Buffers for SDS-PAGE --- p.27 / Chapter 2.2.4 --- Reagents of Bradford Protein Assay --- p.31 / Chapter 2.3 --- Methods --- p.32 / Chapter 2.3.1 --- Determination of ChlorophyllContent --- p.32 / Chapter 2.3.2 --- Determination of Protein Content in Chloroplast Thylakoids --- p.33 / Chapter 2.3.3 --- Measurement of Proton Transport --- p.34 / Chapter 2.3.3.1 --- Pesticide Concentration Study --- p.36 / Chapter 2.3.3.2 --- Time Course Study --- p.36 / Chapter 2.3.3.3 --- Kinetic Analysis of the Effects of Pesticides on Chloroplast Thylakoids Before and After Illumination --- p.37 / Chapter 2.3.3.4 --- Study of the Combined Effects of Two Pesticides --- p.37 / Chapter 2.3.4 --- Effect of Pesticides on Chloroplast Membranes by SDS-PAGE --- p.38 / Chapter Section 3 --- Results --- p.39 / Chapter 3.1 --- Pesticide Concentration Study --- p.39 / Chapter 3.1.1 --- Paraquat Dichloride --- p.39 / Chapter 3.1.2 --- Methyl Carbamate --- p.41 / Chapter 3.1.3 --- Ethyl Carbamate --- p.43 / Chapter 3.1.4 --- Pyridinol Carbamate --- p.45 / Chapter 3.1.5 --- Pyrrolidinedithiocarbamate --- p.47 / Chapter 3.1.6 --- Diethyldithiocarbamic Acid --- p.49 / Chapter 3.1.7 --- Summary of the Pesticides Concentration Study --- p.51 / Chapter 3.2 --- Time-course Study --- p.52 / Chapter 3.3 --- Kinetic Analysis of the Effects of Pesticides on Chloroplast Thylakoids Before and After Illumination --- p.53 / Chapter 3.3.1 --- Paraquat Dichloride --- p.53 / Chapter 3.3.2 --- Methyl Carbamate --- p.56 / Chapter 3.3.3 --- Ethyl Carbamate --- p.57 / Chapter 3.3.4 --- Pyridinol Carbamate --- p.58 / Chapter 3.3.5 --- Pyrrolidinedithiocarbamate --- p.59 / Chapter 3.3.6 --- Diethyldithiocarbamic Acid --- p.60 / Chapter 3.4 --- Combined Effects of Paraquat and Carbamates --- p.61 / Chapter 3.4.1 --- Paraquat and Methyl Carbamate --- p.61 / Chapter 3.4.2 --- Paraquat and Ethyl Carbamate --- p.64 / Chapter 3.4.3 --- Paraquat and Pyridinol Carbamate --- p.66 / Chapter 3.4.4 --- Paraquat and Pyrrolidinedithiocarbamate --- p.69 / Chapter 3.4.5 --- Paraquat and Diethyldithiocarbamic Acid --- p.71 / Chapter 3.5 --- Gel Electrophoresis --- p.73 / Chapter Section 4 --- Discussion --- p.75 / Chapter Section 5 --- Conclusion --- p.80 / Chapter Section 6 --- References --- p.81 / References --- p.81 / Appendix I Kinetic Analysis of Pesticides with Chloroplast Thylakoids upon Illumination --- p.87 / Appendix II Kinetic Analysis of Pesticides with Chloroplast Thylakoids in the Dark --- p.88 / Appendix III The Initial Rate of Proton Transport in Chloroplast Thylakoids with Different Pesticides --- p.89 / Appendix IV The Conversion of Equivalent Protons from pH Changes --- p.90 / Appendix V Calculation of Proton Transport (%) --- p.91 / Appendix VI Determination of Protein Content in Chloroplast Thylakoids --- p.92 / Appendix VII Calculaiton of Relative Mobility (Rf) --- p.93

The focus of this research project is the complex of infraspecific taxa that make up the crisp-leaf buckwheat species Eriogonum corymbosum (Polygonaceae), which is distributed widely across southwestern North America. This complex provides an ideal taxonomic group for research into population relationships and speciation. To avoid unnecessary debates about taxonomic validity or contentious issues regarding appropriate species definitions, the historical evolution of the species concept is first reviewed in detail, demythologizing an often-assumed species problem. Following that review, the E. corymbosum complex is examined specifically. Although eight varieties of E. corymbosum are currently recognized based on morphological characters, this group of large, woody shrubs has a history of revisions that demonstrates the uncertainty inherent in circumscriptions based on morphology alone. The apparent rarity of some E. corymbosum varieties also presents conservation and management challenges, demonstrating the need for taxonomic verification. To bring greater resolution to this group, I genetically tested samples from populations of six of the eight varieties of E. corymbosum, as well as a number of related buckwheat species. With 103 AFLP loci and chloroplast sequence data from 397 samples, I found strong support for the designation of the recently named E. corymbosum var. nilesii. This predominantly yellow-flowered variety had previously been considered part of a more common variety, and thus its management had not been of particular concern. But as a separate variety, its known distribution is quite limited, and management for this rare plant is now advised. An examination of the biogeography of the E. corymbosum complex provides further support for the apparent rarity of var. nilesii, as well as var. aureum. Both taxa are found at the periphery of the complex, and both may represent insipient species. While all other varieties appear more closely related to each other than to varieties aureum and nilesii, with overlapping ranges confined mostly to the Colorado Plateau, both var. aureum and var. nilesii appear to have allopatric ranges largely off the Colorado Plateau. It appears these two peripheral varieties may each entail a separate center of origin for two new taxa.

The transport of phosphate (Pi) between subcellular compartments is central to metabolic regulation. Although some of the transporters involved in controlling the intracellular distribution of Pi have been identified in plants, others are predicted from genetic and biochemical studies. The Arabidopsis thaliana genome encodes a family of six proteins that share similarity with SLC17/type I Pi transporters, a diverse group of animal proteins involved in the transport of Pi, organic anions and chloride. Heterologous expression in yeast, and gene expression and localization studies in plants were used to characterize all six members of this Arabidopsis family, which we have named PHT4. All of the PHT4 proteins mediate Pi transport in yeast with high specificity. Bioinformatic analysis and localization of PHT4-GFP fusion proteins indicate that five of the proteins are targeted to the plastid inner envelope membrane, and the sixth resides in the Golgi apparatus. PHT4 genes are expressed in both roots and leaves although two of the genes are expressed predominantly in leaves and one mostly in roots. These expression patterns, together with Pi transport activities and subcellular locations, suggest roles for PHT4 proteins in the transport of Pi between the cytosol and chloroplasts, heterotrophic plastids and the Golgi apparatus.