Graduation date: 1993

published_or_final_version / Pathology / Master / Master of Philosophy

For organisms undergoing a developmental process it is ideal that specific genes are induced and repressed at the correct time and to the correct level in a coordinated manner. The process of meiosis and spore formation (collectively known as sporulation) in Saccharomyces cerevisiae provides a convenient system to elucidate transcriptional mechanisms of gene repression and the contribution such repression mechanisms offer to cells capable of undergoing a developmental process. This thesis focuses on transcriptional repression of sporulation-specific genes during both vegetative/mitotic conditions and sporulation. The fitness contribution of transcriptional repressors that regulate sporulationspecific genes during vegetative growth were investigated considering the similarities between meiosis and mitosis such as DNA replication, chromosome segregation and cytokinesis. Well-characterised sporulation genes of different functions were expressed in vegetative cells and ectopic expression of these genes was found not to be lethal. It was ascertained through strain competition studies that ectopic expression of the genes IME1, SMK1, SPR3 and DIT1 during mitotic growth did not affect cellular fitness. The expression of NDT80 in vegetative cells, however, caused a marked reduction in fitness and cells were also further compromised in the absence of the Sum1p repressor that regulates NDT80 transcription. The role of NDT80 as a transcriptional activator of middle sporulation genes, rather than the over-expression of NDT80 as a protein, caused the reduction of cell viability. Transcriptional regulation of the middle sporulation-specific gene SPR3 by the meiosis-specific Set3p repressor complex was investigated using synchronous sporulation cultures of the W303a/?? strain commonly used for sporulation studies. In a mutant W303a/?? ??set3/??set3 strain, lacking a key component of the Set3p repression complex, the transcription of SPR3 was uncharacteristically expressed at higher levels and derepressed during late sporulation. This SPR3 expression was consistent for both SPR3 transcript and SPR3::lacZ reporter protein studies. This preliminary work will enable future studies, using SPR3 promoter deletions fused to a lacZ reporter, aimed at determining the region of the SPR3 promoter that the Set3p complex may interact with to transcriptionally repress the gene during sporulation.

For organisms undergoing a developmental process it is ideal that specific genes are induced and repressed at the correct time and to the correct level in a coordinated manner. The process of meiosis and spore formation (collectively known as sporulation) in Saccharomyces cerevisiae provides a convenient system to elucidate transcriptional mechanisms of gene repression and the contribution such repression mechanisms offer to cells capable of undergoing a developmental process. This thesis focuses on transcriptional repression of sporulation-specific genes during both vegetative/mitotic conditions and sporulation. The fitness contribution of transcriptional repressors that regulate sporulationspecific genes during vegetative growth were investigated considering the similarities between meiosis and mitosis such as DNA replication, chromosome segregation and cytokinesis. Well-characterised sporulation genes of different functions were expressed in vegetative cells and ectopic expression of these genes was found not to be lethal. It was ascertained through strain competition studies that ectopic expression of the genes IME1, SMK1, SPR3 and DIT1 during mitotic growth did not affect cellular fitness. The expression of NDT80 in vegetative cells, however, caused a marked reduction in fitness and cells were also further compromised in the absence of the Sum1p repressor that regulates NDT80 transcription. The role of NDT80 as a transcriptional activator of middle sporulation genes, rather than the over-expression of NDT80 as a protein, caused the reduction of cell viability. Transcriptional regulation of the middle sporulation-specific gene SPR3 by the meiosis-specific Set3p repressor complex was investigated using synchronous sporulation cultures of the W303a/?? strain commonly used for sporulation studies. In a mutant W303a/?? ??set3/??set3 strain, lacking a key component of the Set3p repression complex, the transcription of SPR3 was uncharacteristically expressed at higher levels and derepressed during late sporulation. This SPR3 expression was consistent for both SPR3 transcript and SPR3::lacZ reporter protein studies. This preliminary work will enable future studies, using SPR3 promoter deletions fused to a lacZ reporter, aimed at determining the region of the SPR3 promoter that the Set3p complex may interact with to transcriptionally repress the gene during sporulation.

The genus Enterococcus comprises of a group of commensal organisms of the human gut which has been associated with cases of endocarditis and urinary tract infections. In the present study, 12 Enterococcus isolates were obtained from clinical specimens and characterized using genotyping techniques that have become an integral part of clinical research. There were three different genotyping methods used to identify the enterococci to species level and to determine the level of genetic diversity among the selected strains. These techniques were, randomly amplified polymorphic DNA-PCR (RAPD-PCR), 16S rDNA ribotyping analysis and pulse field gel electrophoresis (PFGE) respectively. The minimum inhibitory concentration (MIC) to penicillin and vancomycin were also determined using a disc diffusion assay and a microtitre plate dilution assay. All twelve strains were found to be vancomycin resistant enterococci (VRE) at a MIC value greater than 100μg/ml. Penicillin growth inhibition based on MIC values were categorized into three groups, susceptible (< 0.25 μg/ml), intermediate (≤ 3μg/ml) and resistant (≥ 4μg/ml) respectively. RAPD-PCR was performed using four random primers. Primers yielding the highest discriminative power were used for phylogenetic analysis. The phylogenetic analysis indicated that all 12 strains yielded clonal dissemination, therefore a low genetic diversity between them. The 16S rDNA of all strains were used to identify the enterococci at species level. The rDNA were sequenced and analysed using the NCBI BLAST algorithm and found to belong to three species of Enterococcus. These were E.faecalis, E.faecium and E.durans. PFGE analysis was performed by restriction of all 12 strain’s genomic DNA with the restriction enzyme SmaI. The PFGE patterns were divided into two groups with low genetic diversity. Compared with the RAPD PCR patterns PFGE gives a higher discriminatory power as a higher dissimilarity between the strains was observed. Similar penicillin MICs for each of the strains in the three categories are grouped together in the phylogenetic trees for both PFGE and RAPD-PCR. RAPDPCR is a sensitive, faster, specific and cost effective technique, PFGE analysis has given a higher discriminatory power, higher reproducibility of the results and the polymorphism seen in the patterns suggest that PFGE has a potential of being an essential tool in clinical diagnostics. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2007.

Indiana University-Purdue University Indianapolis (IUPUI) / The class IV alcohol dehydrogenase (ADH7, µ-ADH, σ-ADH) efficiently metabolizes ethanol and retinol. ADH7 is expressed mainly in the upper gastrointestinal tract with no expression in the liver unlike the other ADHs, and is implicated in various diseases including alcoholism, cancer and fetal alcohol syndrome. Genome wide studies have identified significant associations between ADH7 variants and alcoholism and cancer, but the causative variants have not been identified. Due to its association with two important metabolic pathways and various diseases, this dissertation is focused on studying ADH7 regulation and the effects of variants on this regulation using cell systems that replicate endogenous ADH7 expression. We identified elements regulating ADH7 transcription and observed differences in the effects of variants on gene expression. A7P-G and A7P-A, two promoter haplotypes differing in a single nucleotide at rs2851028, had different transcriptional activities and interacted with variants further upstream. A sequence located 12.5 kb upstream (7P10) can function as an enhancer. These complex interactions indicate that the effects of variants in the ADH7 regulatory elements depend on both sequence and cellular context, and should be considered in interpretation of the association of variants with alcoholism and cancer. The mechanisms governing the tissue-specific expression of ADH7 remain unexplained however. We identified an intergenic region (iA1C), located between ADH7 and ADH1C, having enhancer blocking activity in liver-derived HepG2 cells. This enhancer blocking function was cell- and position- dependent with no activity seen in CP-A esophageal cells. iA1C had a similar effect on the ectopic SV40 enhancer. The CCCTC-binding factor (CTCF) bound iA1C in HepG2 cells but not in CP-A cells. Our results suggest that in liver-derived cells, iA1C blocks the effects of downstream ADH enhancers and thereby contributes to the cell specificity of ADH7 expression. Thus, while genetic factors determine level of ADH7 transcriptional activity, iA1C helps determine the cell specificity of transcription.