Age-related neurodegenerative proteinophaties, including polyglutamine (polyQ) diseases such as Huntington’s disease, are a group of disorders in which a single protein or a set of proteins misfold and aggregate resulting in a progressive and selective loss of anatomically or physiologically related neuronal systems. Despite evidence showing a clear relationship between mitochondrial dysfunction, aging and neurodegenerative proteinophaties, the extent of the mitochondrial respiratory chain deficits, the involvement of mitochondrial dysfunction and the mechanisms responsible for these processes are largely unknown. Using yeast models of cellular aging and polyQ disorders we show that mitochondrial dysfunction is an important contributor to the process of aging and age-related neurodegenerative diseases. Preserving mitochondrial function is essential for standard wild-type aging. Enhancement of mitochondrial biogenesis ameliorates polyQ cytotoxicity and is a required component of interventions that retard the aging process.

Acyl-CoA:diacylglycerol acyltransferase (EC 2.3.1.20, DGAT or DAGAT) is a membrane protein found mainly in the endoplasmic reticulum (ER). It catalyzes the final step in the biosynthesis of triacylglyerol (TAG or TG), which is the principal repository of fatty acids for energy utilization and membrane formation. Several lines of evidence have indicated that DGAT has a substantial effect on carbon flux into TAG. DGAT has at least two discrete family members (DGAT1 and DGAT2) with different physiological roles. High-resolution structures of both DGATs, however, are absent due to difficulties in purification. In order to gain insight into structural and functional relationships of DGATs, a functional DGAT2 protein from the yeast Saccharomyces cerevisiae (ScDGAT2, also known as Dgalp) was selected. The structural and functional role of cysteine residues in ScDGAT2 was studied using site-directed mutagenesis (SDM) in combination with chemical modification. Although ScDGAT2 is susceptible to thiol-modifying reagents, none of the cysteines are essential for the catalytic activity or involved in structure support though disulfide linkages. Inhibition of DGAT activity by thiol-specific modification was localized to cysteine314, which is in the proximity of a highly conserved motif in DGAT2s. Thus, cysteine314 may reside in a crucial position near a possible active site or related to proper protein folding. The functional importance and topological orientation of signature motifs in ScDGAT2 were also studied using the same methods. Both the N- and C-termini of ScDGAT2 are oriented toward the cytosol. A highly conserved motif, 129YFP131, and a hydrophilic segment exclusive to ScDGAT2, reside in the ER and play essential roles in enzyme catalysis. In addition, the strongly conserved H195, which may be part of the active site of DGAT2, is likely embedded in the membrane. Although ScDGAT2 has a topology similar to that of murine DGAT2, there are striking differences which suggest that the topological organization of DGAT2 is not ubiquitously conserved. / Plant Science

Thesis (M.S.)--University of Hawaii at Manoa, 2006. / Includes bibliographical references (leaves 140-151). / xii, 151 leaves, bound ill., maps 29 cm

<p>Double-stranded DNA breaks are potentially lethal lesions that can be repaired in mitotic cells by either homologous recombination (HR) or non-homologous end- joining (NHEJ) pathways. In the HR pathway, the broken DNA molecule is repaired using either the sister chromatid or the homolog as a template. Mitotic recombination events involving the homolog often result in loss of heterozygosity (LOH) of markers located distal to the crossover. In humans that are heterozygous for a mutation in a tumor suppressor gene, mitotic recombination leading to LOH can be an early step in cancer development.</p><p> In my thesis research, I analyzed mitotic recombination in the yeast Saccharomyces cerevisiae using oligonucleotide-containing microarrays to detect LOH of single-nucleotide polymorphisms (SNPs). In analyzing cells treated with ionizing radiation, I performed the first whole-genome analysis of LOH events done in any organism (Chapter 2). I showed that irradiated cells had between two and three unselected LOH events. I also showed that crossovers were often associated with non- reciprocal exchanges of genetic information (gene conversion events) and that these conversion events were more complex than predicted by standard models of homologous recombination.</p><p> In Chapter 3, I describe my mapping of spontaneous crossovers in a 1.1 Mb region of yeast chromosome IV. This analysis is the first high-resolution mitotic recombination map of a substantial fraction (about 10%) of a eukaryotic genome. I demonstrated the existence of recombination "hotspots" and showed that some of these hotspots were homolog-specific. Two of the strongest hotspots were formed by closely- spaced inverted repeats of retrotransposons. I demonstrated that the hotspot activity was a consequence of a secondary DNA structure formed by these repeats. Additionally, the majority of spontaneous LOH events reflect DNA lesions induced in unreplicated chromosomes during G1 of the cell cycle, indicating that G1-initiated lesions threaten genome stability more than G2-initiated lesions.</p><p> In Chapter 4, I describe mitotic crossovers associated with DNA replication stress induced by hydroxyurea (HU) treatment. Surprisingly, most HU-induced crossovers had conversion tracts indicative of DNA lesions initiated in G1. Additionally, HU- induced recombination events were very significantly associated with solo delta elements, a 330 bp sequence that is repeated several hundred times in the yeast genome.</p> / Dissertation

Errata slip inserted on back end-paper. / Bibliography: leaves 106-125. / vii, 125 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 1999

Title page, table of contents and summary only. The complete thesis in print form is available from the University of Adelaide Library. / Assimilable nitrogen, which is typically lacking in grape juice, is an important nutritional requirement of Saccharomyces cerevisiae. As such, fermentations frequently become protracted, terminate prematurely or develop undesirable aroma profiles. Amino acids and ammonium are the main sources of assimilable nitrogen in grape juice. The amino acid proline often predominates. Proline uptake is mediated by a high affinity, proline-specific permease, Put4p, and a low affinity general amino acid permease, Gap1p. The expression and activity of these transporters is subject to nitrogen catabolite repression (NCR) and nitrogen catabolite inactivation (NCI). That is, in the presence of a preferred nitrogen source, the expression of PUT4 and GAP1 is repressed and the permeases are inactivated. For yeast to fully exploit proline, its transport must be derepressed by depletion of other (preferred) amino acids and molecular oxygen must be present to allow proline catabolism by proline oxidase. Consequently, as oxygen is typically depleted well before the other amino acids in grape juice are reduced to non-repressive concentrations, proline is largely un-utilised by yeast during oenological fermentation. This study aims to overcome these metabolic restrictions on proline utilisation. A preliminary study was conducted to determine the potential for proline transport-capable strains to utilise proline during the initial stages of fermentation when oxygen may be present, particularly in red grape must. Initially, the transcriptional regulation of the PUT4 gene was targeted to generate strains capable of proline transport under normally repressive conditions. In the first case, the URE2 gene, encoding a negative regulator involved in nitrogen discrimination, was deleted. In the second case, PUT4 was expressed from the constitutive TEF2 promoter. It was observed that both strains express PUT4 in the presence of a preferred nitrogen source. This expression led to Put4p activity during the initial stages of growth and fermentation, with Put4p activity declining over the course of the growth phase. Proline removal from the media, however, was limited to the initial stages of fermentation while oxygen was available. It seems that the rapid depletion of oxygen limits the amount of proline transported into the yeast cell. The two proline transport-capable mutants were analysed for growth and fermentation characteristics. It was found that the deletion of the URE2 gene led to a slow initial growth and the formation of a larger biomass. The ure2 delete strain also utilised significantly more nitrogen during fermentation than the wild type. Consequently, a ure2 delete strain would not be suitable for industrial use. The expression of PUT4 from a constitutive promoter did lead to an increase in nitrogen assimilation during fermentation when compared with the wild type. However, this observed increase was significantly less than that observed in the ure2 delete strain. In an effort to produce a proline transport-capable strain with potential industrial benefit, strains constitutive for PUT4 specifically were isolated using random, in vitro mutagenesis of the PUT4 promoter region. Four point mutations were identified that, when introduced singly into the PUT4 promoter, led to expression of PUT4 in the presence of a preferred nitrogen source. The rapid depletion of oxygen observed in the preliminary study will limit the potential usefulness of strains capable of proline transport. Micro-oxygenation is rapidly becoming an accepted practice during oenological fermentation. The potential benefit of the controlled addition of oxygen during fermentation is restricted by the timing of any oxygen addition. Oxygen additions made at the onset of the stationary phase are the most beneficial. During the preliminary study, it was noted that Put4p activity decreased during the growth phase to low levels at the onset of the stationary phase. To ensure that sufficient active Put4p is present at the onset of the stationary phase, the post-translational control of the Put4p was investigated. Site-directed mutagenesis was used to target residues in the carboxy-terminal region of Put4p that are potentially involved in the ammonia-induced down-regulation of the permease. The substitution, S605A, lead to the amelioration of ammonia-induced down-regulation of Put4p. The activity of the Put4p S605A variant decreased over the course of the growth phase, but not to the same extent observed in the wild type. Furthermore, a recovery seen after down-regulation restored a greater percentage of the original activity compared with the wild type. To determine whether such a strain proved better able to ferment media in the presence of micro-oxygenation, the fermentation kinetics of a strain constitutively expressing PUT4(S605A) were compared with the wild type. Micro-oxygenation of ferments did not result in an increase in fermentation rate nor a decrease in fermentation time in the mutant. However, the cell viability of the strain capable of proline transport was increased in comparison with the wild type, suggesting a role for proline in stress responses within the yeast cell. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1048932 / Thesis (Ph.D.) -- University of Adelaide, Dept. of Horticulture, Viticulture and Oenology, 2002

Conjugation is a fundamentally important mechanism of horizontal DNA transfer between bacteria, bacteria x archea, and bacteria x eukaryotes. This work has concentrated on conjugation between bacteria x eukaryotes, specifically Escherichia coli x Saccharomyces cerevisiae. Four hypotheses were tested, investigating the barriers to this particular form of DNA transfer. The first investigated if a mutation that altered the cell-surface of the recipient S. cerevisiae could inhibit DNA transfer. The final three utilised a recombination-dependent-conjugation assay to investigate the barrier to DNA transmission through recombination. The hypotheses tested if the frequency of recombination, in this recombination-dependent-conjugation assay, differed when using similar or diverged DNA substrates, if a mismatch repair mutation within the recipient could affect the frequencies of recombination observed, and if the position on the plasmid of the gene of interest affected the frequency of transmission. Transmission of the Ura3 DNA sequence in the recipient S. cerevisiae was used to test all four hypotheses. The cell wall mutants mnn9, knr4, fks1 and kre6 were utilised to investigate if the cell-surface of the recipient could affect the frequency of transmission. The similar and diverged substrates utilised in the investigation of the affect of sequence similarity on recombination were the DNA sequences of ura3 from S. cerevisiae and Saccharomyces carlsbergensis, respectively and the MMR mutants utilised were msh2, pms1 and pol30-52. Cell wall mutants were not found to limit the frequency of transfer once donor-recipient contact was induced through the solid surface mating procedure. Sequence similarity, MMR and the relative position of the ura3 DNA sequence on the conjugative plasmids were shown to have little effect on the frequency of transmission in S. cerevisiae. This suggests that any DNA that enters the nucleus of S. cerevisiae (eukaryotes) can recombine with the chromosome and alter it to the same extent. However, trends within the data also suggest that DNA is transferred into the recipient and then transported to the nucleus to recombine with the chromosome as a single-stranded DNA molecule.

Thesis (Ph. D.)--University of Missouri-Columbia, 2007. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from title screen of research.pdf file (viewed on March 4, 2008) Vita. Includes bibliographical references.

Thesis (Ph.D) -- McMaster University, 1997 / Includes bibliographical references Also available via World Wide Web.

Thesis (Ph. D.)--University of Adelaide, Dept. of Plant Science, 1997? / Includes bibliographical references.