LepA is a highly conserved GTP-binding protein of unknown function. Its amino acid sequence reveals that it is a GTPase with homology to elongation factor G (EF-G). Previous data led to the hypothesis that LepA negatively regulates a posttranslational process such as protein folding. To examine this possibility, two sets of strains carrying mutated alleles encoding molecular chaperones in E. coli were transformed with a lepA expression vector. LepA had a dominant negative effect specifically in a dnaK25 strain whose product exhibits a 20-fold lower ATPase activity compared to wild-type DnaK. The expression of DnaK and other heat-shock proteins is repressed following temperature downshift. Aptly, it was found that temperature shift from 37 degrees Celcius to 15 degrees Celcius in cells harboring a lepA expression vector led to the induction of lepA and downstream lepB. Furthermore, like cold-shock genes, lepA and lepB are induced by sublethal doses of chloramphenicol, although it appears that lep operon induction is related to the antibiotic's action on the 50S ribosome. Due to LepA's insolubility, it could not be confirmed whether it interacts with DnaK, DnaJ or which other proteins it interacts with. Two-dimensional gel electrophoretic analysis revealed the absence of an isoform of OmpA in two lepA deletion strains. It is possible that LepA is involved in a folding pathway that is responsible for the conformation of this isoform. Phylogenetic analysis showed that while LepA is extremely well conserved and has been identified in all completed Bacterial and Eukaryal genomes, it is not present in the completed genomes of any Archaea. Sequence analysis revealed the existence of N-terminus mitochondrial import sequences in Eukaryal LepA orthologues. Additionally, A. thaliana contains a second LepA orthologue that clusters phylogenetically with Synechocystis LepA and has a chloroplastic import sequence. This indicates that plastidal LepA was acquired in A. thaliana (and probably in all plants) through endosymbiosis of an ancestral cyanobacterium. In constrast, mitochondrial LepA are not closely related to those of a- proteobacteria, believed to be the precursors of mitochondria. These findings imply that in sharp contrast to mitochondrial LepA, chloroplastic LepA is under strong evolutionary pressure to remain conserved.

Human telomeric DNA consists of tandem repeats of the sequence 5'-d(TTAGGG)-3' assembled into a nucleoprotein complex that functions to protect the ends of chromosomes. Such guanine-rich DNA is capable of forming a variety of G-quadruplexes, which in turn, can have varying functional consequences on telomere maintenance. G-quadruplex stabilizing ligands have been shown to induce chromosome end-to-end fusions, senescence and apoptosis, effects similar to the expression of a dominant-negative TTAGGG Repeat Factor 2 (TRF2). With this in mind, we analyzed the effect of sequence and length of human telomeric DNA, as well as cation conditions on G-quadruplex formation by native polyacrylamide gel electrophoresis and circular dichroism. We show that K+ and Sr2+ can induce human telomeric DNA to form both inter- and intramolecular structures. Circular dichroism results suggest that the structures in K+ were a mix of parallel and antiparallel G-quadruplexes, while Sr2+ induced only parallel-stranded structures. We also found that TRF2, a protein essential for telomere maintenance, affects G-quadruplex structure. These structures serve as useful models to study the effects of G-quadruplexes on the activities of telomeric proteins, like TRF2, from human cells. The G-strand overhang at the ends of telomeres may periodically adopt at least some of these quadruplex conformations, which could subsequently affect protein binding and telomere function. TRF2, a protein essential for telomere maintenance, is not known to bind single-strand (ss) DNA, work performed in the lab suggested that the type of 3'-overhang in telomeric DNA ss/ds-junctions affects TRF2-binding. Specifically, preventing G-quadruplex formation by changing the overhang sequence from 5'-d(TTAGGG)4-3', to 5'-dTTAGGG(TTAGAG)2TTAGGG-3', reduced TRF2 recruitment to the ss/ds-junction from HeLa cell extracts. Using the same techniques as above, we show that the N-terminal basic domain of TRF2 in K+ induces a switch from the mixed parallel/antiparallel-stranded G-quadruplexes usually stabilized by K+-alone, to parallel-stranded G-quadruplexes. Interestingly, it also promotes intermolecular parallel G-quadruplex formation on non-quadruplex, single-stranded intermediates, but will not induce a switch from an antiparallel to a parallel G-quadruplex in Na+. These results are the first to demonstrate specific TRF2 G-quadruplex interactions, suggesting a novel mechanism for TRF2 recognition of the ds/ss junction of telomeres.

Telomeres comprise the physical ends of chromosomes. In the absence of telomerase, the enzyme responsible for replenishing telomeric DNA, telomeres progressively shorten due to the end replication problem. Eventually telomeres reach a length where they are recruited into end-to-end chromosome fusions. Through the use of novel PCR strategies, I followed the fate of telomeres in plants lacking telomerase as they progressed into dysfunction. I uncovered two distinct structural/functional length transitions. The first transition (~1 kb) marks the onset of telomere dysfunction, where telomeres are transiently uncapped and a subset of them engage in end-to-end fusions. The second transition (~300 bp) defines complete telomere dysfunction as telomeres below this length lack G-overhangs and the vast majority of the chromosome ends fuse. Thus, these two telomere lengths define architectural transitions that link structure and function. In addition, I uncovered a hierarchy of end-joining pathways that join dysfunctional telomeres in which the non-homologous end-joining (NHEJ) protein, KU predominates. In the absence of KU, telomeres are joined by a microhomologymediated end-joining pathway (MMEJ) that is dependent on Mre11. I also show that DNA ligase IV (LIG4) is the predominant enzyme that ligates dysfunctional telomeres as fusions are reduced in its absence. These studies highlight the importance of repairing DSBs and demonstrate that Arabidopsis possesses highly redundant means for processing dysfunctional telomeres. The G-overhang is an essential feature of the telomere that is required for proper telomere function. I employed methods to examine G-overhang status in various mutants known to contribute to telomere maintenance in Arabidopsis. My analysis revealed that the putative G-overhang binding proteins POT1a, POT1b and POT1c, make modest, but distinct contributions to the G-overhangs. Additionally, I uncovered a major role for the putative telomere capping protein, CIT1 in maintenance of the Goverhang. G-overhang signals obtained from cit1 mutants were grossly increased indicating that CIT1 is involved in either protecting the C-rich strand of the telomere from nuclease attack, or in controlling telomerase extension of the G-strand. Together, these data have provided new insight into factors that contribute to telomere integrity and have further developed Arabidopsis as a model for telomere biology.

Texte remanié de: Proefschrift--Brussel--Vrije universiteit, 2000. / Bibliogr. p. 653-695.

Thesis (Ph. D.)--University of Queensland, 2001. / Includes bibliographical references.

Doctor's dissertation at Amsterdam University. / eContent provider-neutral record in process. Description based on print version record.

Proefschrift--Groningen.

Proefschrift--Amsterdam.

Thesis (Ph. D.)--University of Queensland, 2002. / Includes bibliographical references.

At independence in 1964, Malta's economy remained entirely dependent upon the revenue and employment provided by the British and NATO fortress on the Islands. The implications of this dependence were serious as, in 1957, the British Ministry of Defence had announced its intention to run down, and eventually to terminate its military involvement in the Maltese Islands. Faced with the imminent loss of a substantial proportion of the economy's revenue and employment, successive Maltese Governments (of both the major political parties) adopted vigorous economic diversification strategies. It was generally accepted that domestically generated economic development was impossible, and that economic diversification could only be achieved through the deliberate attraction of overseas enterprise. Multinational Corporations (MNCs) were therefore invited to participate in, and develop the three crucial sectors of the Maltese economy - the dockyards, the new manufacturing sector, and tourism. Inevitably, the attraction of MNCs into the crucial sectors of the economy ensured that control of the economy was placed in the hands of overseas entrepreneurs, whose interests were not necessarily consistent with those of the Maltese Islands. The subsequent development of strongly dependent dual structures throughout key areas of the Maltese economy had by 1980 induced extreme economic instability. Given this background, the intention of this thesis has been to evaluate Malta's post-independence development record. One of the best means of achieving this aim was seen as to be the adoption and adaption of a number of themes contained within 'dependency theory'. This avenue of enquiry has proved to be rewarding in two ways. first, the various concepts of of dependence have helped to shed light upon the development experiences of Malta. Also important, however, is the fact that the Maltese case has itself raised many relevant questions concerning the application of dependency theory in empirical studies. The thesis itself can be divided into three distinct sections. Section One briefly discusses development, and then proceeds to examine the concept of dependence Of particular interest is the degree to which dependency theory is applicable to small countries, and the role of MNCs in the generation of dependence. Section Two provides the essential historical background to Malta's current position, and Section Three sets about examining in some detail the development of the Maltese economy, with particular reference to the growth of manufacturing industry and tourism. The conclusions of the thesis again fall into two distinct groups. With respect to dependency theory, it seems that dependency themes can be of substantial use in the evaluation of a country's development process. With respect to Malta, it is apparent that Malta in the 1980s is facing a crisis of dependence that is rather more severe than that which faced the Islands at independence in 1964. The problem has clearly been that of excess dependence upon MNC subsidiaries. On the one hand, MNCs are seen as essential due to the inability of the domestic economy to generate its own industry. On the other hand, the dependent structures that have ensued have proved to be unstable and unreliable. Clearly, a compromise situation is desirable in which Government intervention ensures that foreign industries are attracted to the Islands, but that they are attracted selectively, and that their less desirable activities are meticulously restricted by the Government.