No description available.

No description available.

No description available.

The centromere is the site of kinetochore assembly that ensures proper chromosome segregation. Active centromeres are formed at chromosomal locations that do not appear to share homology between different species; this and other analyses has lead to the conclusion that centromeres are epigenetically determined. In all organisms, centromere location is specified by the assembly of unusual nucleosomes containing the histone H3 variant CENP-A in place of H3. However, an apparent paradox is that CENP-A in most organisms generally occurs on certain preferred sequences. The analyses presented focuses on the influence of DNA sequence on the selection of the locus where CENP-A chromatin are formed and whether there are any particular DNA features that promote CENP-ACnp1 assembly at centromeres in Schizosaccharomyces pombe. S. pombe provides an excellent model to study the structure and function of a complex eukaryotic centromere as it possesses epigenetically regulated centromeres that are structurally related to those of metazoa. Furthermore, plasmid-based circular minichromosomes provide a useful tool for studying these centromeres and the inactivation of minichromosome centromeres does not affect cell viability. The main aim of this study was to identify those features that enable centromeric sequences to assemble CENP-A chromatin. Manipulated circular minichromosomes were utilised to investigate the minimal central core sequence requirement for establishment of CENP-ACnp1 chromatin. These analyses showed that a minimal 2kb region from the central core of cen2 could form a functional centromere. A second aim was to analyse the DNA sequence requirements for centromere function on this minimal 2 kb region. To facilitate this, the endogenous central core region of endogenous cen2 was replaced with the central core region of cen1. This modified strain allows the structural and biological properties associated with plasmid borne central core 2 sequences to be analysed. Transcription of central domain sequences has been proposed to play a role in CENP-A establishment and/or maintenance. To explore the contribution of transcription potential promoters were mapped within the minimal 2 kb sequence and their regulatory elements investigated. Mutation of the minimal DNA element impedes its ability to assemble CENP-A chromatin. Therefore the primary DNA sequence of fission yeast centromeres is important for establishing functional centromeres and thus centromere location not entirely epigenetically regulated. It remains to be determined if the characteristics associated with theses sequences, and their mode of action, are conserved at other centromeres.

<p>The genomes of many strains of baker’s yeast, Saccharomyces cerevisiae, contain multiple repeats of the copper-binding protein Cup1. Cup1 is a member of the metallothionein family, and is found in a tandem array on chromosome VIII. In this thesis, I describe studies that characterized these tandem arrays and their mechanism of formation across diverse strains of yeast. I show that CUP1 arrays are an illuminating model system for observing recombination in eukaryotes, and describe insights derived from these observations. </p><p>In our first study, we analyzed 101 natural isolates of S. cerevisiae in order to examine the diversity of CUP1-containing repeats across different strains. We identified five distinct classes of repeats that contain CUP1. We also showed that some strains have only a single copy of CUP1. By comparing the sequences of all the strains, we were able to elucidate the mechanism of formation of the CUP1 tandem arrays, which involved unequal non-homologous recombination events starting from a strain that had only a single CUP1 gene. Our observation of CUP1 repeat formation allows more general insights about the formation of tandem repeats from single-copy genes in eukaryotes, which is one of the most important mechanisms by which organisms evolve.</p><p>In our second study, we delved deeper into our mechanistic investigations by measuring the relative rates of inter-homolog and intra-/inter-sister chromatid recombination in CUP1 tandem arrays. We used a diploid strain that is heterozygous both for insertion of a selectable marker (URA3) inside the tandem array, and also for markers at either end of the array. The intra-/inter-sister chromatid recombination rate turned out to be more than ten-fold greater than the inter-homolog rate. Moreover, we found that loss of the proteins Rad51 and Rad52, which are required for most inter-homolog recombination, did not greatly reduce recombination in the CUP1 tandem repeats. Additionally, we investigated the effects of elevated copper levels on the rate of each type of recombination at the CUP1 locus. Both types of recombination are increased at high concentrations of copper (as is known to be the case for CUP1 transcription). Furthermore, the inter-homolog recombination rate at the CUP1 locus is higher than the average over the genome during mitosis, but is lower than the average during meiosis. </p><p>The research described in Chapter 2 is published in 2014.</p> / Dissertation

Studies on the capacity of growing and non-growing cells to be induced to grow in different morphological forms suggested that metabolically active cells are better able to undergo dimorphism than metabolically quiescent cells. Accelerated growth kinetics requiring de novo RNA and or protein biosynthesis but not DNA synthesis were observed in metabolically active cells undergoing a yeast to hyphal transition. The roles that divalent cation calcium plays in eukaryotic growth and morphogenesis are illustrated in this study with regard to C. albicans. Free calcium was required for germ tube emergence from stationary phase yeast cells but not for subsequent growth or hyphal extension. The galvanotropic response of C. albicans hyphae was investigated and was shown to be quantitatively dependent on the concentration of available calcium in the medium and also on the integrity of calcium dependent second messenger systems. The mechanism of galvanotropism of C. albicans may be due to the electrophoretic movement of charged proteins, possibly calcium transport channels, in the plasma membrane as shown by the dependence of cathodotropic growth on external pH. Although the yeast to hyphal transition can be easily controlled and studied in vitro, attempts to use such methods to control morphological development, with a view to comparing the relative infectivity of each morphology, are complicated in the environment in vivo. Yeast cells were introduced into the rat vagina, or systemically into mice, along with chemical effectors which had been shown to prevent the yeast to mycelial transition in vitro without affecting the growth rate of the cells per se. It was concluded tentatively from this study, that, in the rat vaginal model of candidosis, the hyphal form of growth is better adapted and more pathogenic than the yeast form.

No description available.

Using two related dominant negative screens, galactose-negative cDNAs were isolated that contained C. albicans TUB2 (beta-tubulin), NHP6 (non-histone protein 6), CIT2 (citrate synthase) and MRPS9 (mitochondrial ribosomal protein S9) sequences. CaTUB2 encodes beta-tubulin, a component of the microtubule system of the cytoskeleton. Overexpression of this sequence in S. cerevisiae resulted in lethality associated with cell shape changes characteristics of an arrest in the G2/M phase of the cell division cycle. However, overexpression of an ADH1-CaTUB2 fusion in C. albicans did not affect cell shape. CaNHP6 encodes a protein that shows a high level of sequence identity to ScNhp6A and ScNhp6B, and is closely related to High Mobility Group proteins from other eukaryotes. CaNHP6 overexpression in S. cerevisiae cells caused cell cycle arrest. Expression of an ADH1-CaNHP6 fusion in C. albicans did not affect growth rate, but affected both of cell and colony morphogenesis. These effects were not dependent upon the PKC pathway. CaMRPS9 encodes a protein of the small mitochondrial ribosome subunit, and overexpression of this protein caused the slow development of galactose lethality in S. cerevisiae. The development of lethality correlated with the emergence of petite mutants, indicating that the overexpression of CaMRPS9 interferes with S. cerevisiae mitochondrial function, thereby preventing growth on galactose. Similarly, CaCIT2 expression was presumed to block growth of S. cerevisiae on galactose by interfering with respiratory metabolism. Separate approaches were taken to isolate CaGCN4. This gene was of interest because (a) hypha-specific promoters contain GCRE-like sequences [Gcn4 Response Element], (b) amino acid starvation promotes yeast-to-hypha morphogenesis, and (c) GCN-like responses are thought to occur in C. albicans. The 5'-truncated C. albicans GCN4 cDNA was isolated by functional complementation of a S. cerevisiae Delta gcn4 mutation. The 5'-region of the CaGCN4 ORF, including 624 bp of 5'-untranslated region, was isolated by PCR. The sequences of the overlapping 5' and 3'fragments revealed a major ORF capable of encoding a 323 aa protein with significant homology in its C-terminal bZIP domain to ScGcn4 and other fungal Gcn4-like proteins. The existence of this ORF in the C. albicans genome was confirmed by PCR. The CaGCN4 cDNA contained two upstream ORFs and was encoded by a single exon. The contribution of C. albicans Gcn4 to yeast-to-hypa morphogenesis remains to be verified.

Call number: LD2668 .T4 1958 L42 / Master of Science

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