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This study examined the role of <I>PYKI </I>coding sequences in the expression of <I>PYK1::lacZ </I>gene fusions in <I>Saccharomyces cerevisiae. </I>Further aims were to examine the effects of the vector system upon the mRNA levels from these gene fusions the effect that these gene fusions have upon the yeast cell in general. Analysis of the <I>PYK1::lacZ </I>gene fusions revealed that <I>PYK1 </I>coding sequences were responsible for elevating mRNA levels. This elevation was not due to a single element within the coding region of the <I>PYK1 </I>gene as had been previously proposed (Purvis <I>et al.,</I> 1987a; Lithgow, 1989). Models for the stimulatory action of the <I>PYK1 </I>coding region upon the transcription of the <I>PYK1::lacZ</I> gene fusion were presented. <I>PYKI </I>coding region fragments in the <I>PYK1::lacZ</I> gene fusions stabilized the mRNA, but the data presented here were not consistent with a stability element within the <I>PYK1 </I>coding region. An alternative model was presented whereby the translation rate of the mRNA can influence its decay. The effect of expression of these gene fusions upon the yeast cell in general was monitored by examining the mRNA level of two chromosomal loci, <I>PYK1 </I>and <I>PGK1, </I>and by measuring the generation time. In contrast to previous findings, <I>PYK1 </I>and <I>PGK1 </I>mRNA levels were found not to change and so it was concluded that expression of these gene fusions had no general effect upon transcription or mRNA decay. However expression of these gene fusions did lead to an increase in generation time, and it was proposed that this might be due to a general effect upon translation brought about by a reduction in the intracellular pools of tRNAs for non-preferred codons.

A dissertation submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Master of Science. July, 2017 / Eukaryotic protein synthesis occurs in three phases: initiation, elongation and termination. The elongation phase is mediated by elongation factors. Elongation factors are divided into elongation factor 1 (eEF1) and elongation factor 2 (eEF2). Elongation factor 1 complex are proteins that mediates the extension of growing polypeptide chains by adding one amino acid residue at a time. The eEF-1 complex comprises of four subunits, eEF1α, eEF1β, eEF1γ and eEF1δ. The β-subunit of elongation factor 1 complex (eEF1) plays a central role in the elongation step of eukaryotic protein biosynthesis, which essentially involves interaction with the α-subunits (eEF1α) and γ-subunits (eEF1γ). To biophysically characterise heEF1β, three E. coli expression vector systems was constructed for recombinant expression of the full length (FL-heEF1β), amino terminus (NT-heEF1β) and the carboxyl terminus (CT-heEF1β) regions of the protein. NT-heEF1β was created from the FL-heEF1β by site-directed mutagenesis using mutagenic forward and reverse primers. The results suggest that heEF1β is predominantly alpha-helical and possesses an accessible hydrophobic cavity in the CT-heEF1β. Both FL-heEF1β and NT-heEF1β forms dimers of size 62 kDa and 30 kDa, respectively, but the CT-heEF1β is monomeric. FL-heEF1β interacts with the N-terminus GST-like domain of heEF1γ (NT-heEF1γ) to form a 195 kDa complex, or a 230 kDa complex in the presence of oxidised glutathione. On the other hand, NT-heEF1β forms a 170 kDa complex with NT-heEF1γ and a high molecular weight aggregate of size greater than 670 kDa. This study affirms that the interaction between heEF1β and heEF1γ subunits occurs at the N-terminus regions of both proteins, also the N-terminus region of heEF1β is responsible for its dimerisation and the C-terminus region of heEF1β controls the formation of an ordered eEF1β-γ oligomer, a structure that may be essential in the elongation step of eukaryotic protein biosynthesis. / MT 2018

Maintenance of genome integrity requires that chromosomes be accurately and faithfully replicated. We are using Tetrahymena thermophila as a model system for studying the initiation and regulation of eukaryotic DNA replication. This organism contains a diploid micronucleus and polyploid macronucleus. During macronuclear development, the five diploid chromosomes of the micronucleus are fragmented into 280 macronuclear minichromosomes that are subsequently replicated to ~45 copies. In stark contrast, the 21 kb ribosomal DNA minichromosome (rDNA) is amplified from 2 to 10,000 copies in the same nucleus. Previous characterization of the rDNA replicon has led to the localization of its origin and the cis-acting regulatory determinants to the 1.9 kb 5'non-transcribed spacer region. The objective of this study was to identify and characterize non-rDNA origins of replication in Tetrahymena. This will help determine the underlying basis for differential regulation of rDNA and non-rDNA origins during development, as well as provide a better understanding of the organization of eukaryotic replicons. To this effect, I developed a DNA transformation assay that I used to isolate new Tetrahymena replication origins. A 6.7 kb non-rDNA fragment, designated TtARS1, was shown to support stable autonomous replication of circular plasmids in Tetrahymena. Genetic dissection revealed that TtARS1 contains two independent replicons, TtARS1-A and TtARS1-B. Full TtARS1-A function requires a minimal sequence of 700 bp, and two small regions in this fragment have been shown to be essential for origin function. TtARS1-B replicon function was localized to a 1.2 kb intergenic segment that contains little sequence similarity to TtARS1-A. Both non-rDNA replicons lack sequence similarity to the rDNA 5' NTS, suggesting that each replicon interact with a different set of regulatory proteins. This study indicates that the rDNA and the non-rDNA replicons have a modular organization, containing discrete, cis-acting replication determinants.

Thesis (Ph. D.)--University of Texas at Austin, 2001. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Supervisors: Josef A. Käs and Tess J. Moon. Vita. Includes bibliographical references. Also available from UMI.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

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