Abstract not available

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 purpose of this experiment was to determine if nucleic acid sequence based amplification (NASBA) is a suitable application for the differentiation of body fluids that might comprise a forensic evidence sample. NASBA is a sensitive RNA transcription based amplification system. NASBA could theorhetically be used for bodily fluid identification based upon amplification of tissue-specific mRNA transcripts present in a given forensic sample.Amplification of both Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and Matrix Metalloproteinase 1 1 (MMPmRNA transcripts were used to determine that NASBA could amplify body fluid transcripts and whether it could distinguish between menstrual and non-menstrual blood, respectively. GAPDH is a housekeeping gene that is constituently expressed and its mRNA transcripts could therefore be used to determine whether non-menstrual blood could be amplified using the NASBA procedure. MMP 11 is a menstrual cycle-specific gene associated with endometrial breakdown. Using the mRNA transcripts from MMP 11, NASBA could be utilized for menstrual blood identification. In this study, non-menstrual and menstrual blood samples were analyzed with NASBA both in the presence and absence of chemical contamination. Contaminants utilized ranged from commercial automotive wax, transmission fluid, brake fluid, artificial tears, hand soap, 10% bleach, and the luminol blood detecting reagent. Non-menstrual blood was aliquoted onto a 1 cm x 1 cm cotton cloth for contamination, while menstrual blood was provided on a 1 cm x 1 cm area of sterile menstrual pad. All samples underwent Tri reagent extraction to obtain RNA samples for NASBA amplification.With respect to NASBA amplification data, non-menstrual blood data (from extracted RNA and unextracted blood samples) revealed the highest levels of amplification as shown in relative fluorescence units (RFU). Uncontaminated menstrual blood revealed the second highest amplification data. In the presence of chemical contamination, high levels of amplification were observed when samples were contaminated with brake fluid and commercial hand soap. Moderately low amplification was observed with samples contaminated with transmission fluid, 10% bleach, and artificial tears. NASBA amplification was completely inhibited in the presence of automotive wax and luminol. Cycle threshold (CO values for each amplification result were also obtained from each reaction. Smaller Ct values correspond to a higher NASBAreaction efficiency and therefore larger amplification values. The Ct values obtained for each amplified sample correlate strongly with the amount of amplification observed from reaction. Based upon the results of this experiment, NASBA should be considered as a novel tool for forensic evidence analysis. / Department of Biology

Access to abstract permanently restricted to Ball State community only / Access to thesis permanently restricted to Ball State community only / Department of Biology

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.

There is a transition nationwide on the analysis of forensic evidentiary stains containing biological material from traditional serology to Polymerase Chain Reaction (PCR) methodologies. The increased sensitivity of PCR, the limited number of alleles at each locus, and the necessity of producing unambiguous data for entry into the FBI's Combined DNA Index System make this study of extraction procedures of utmost importance. A "single tube" extraction procedure for blood stains collected onto FTA™ paper and a modified differential nonorganic extraction method from spermatozoa containing mixed stains were analyzed and compared. The extraction success was evaluated by amplification and typing of the amplified fragment length polymorphism, D1S80. These modifications of the nonorganic method utilized gave an improved separation of the spermatozoa-containing mixed stains.

Shen, Jiayun. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 166-199). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

[Truncated abstract] The collection of buccal cells is common practise in the epidemiological and forensic science. Unlike venipuncture collection of blood; it is a safer, non-invasive method for collection of biological material. The methods by which these cells are collected from the inner cheek of an individual and stored are the key elements in preserving DNA. Typically, forensic samples require long term storage. Samples are commonly collected on cotton swabs and stored moist at low to ultra-low temperatures (less than -20oC). Although this is the method of choice in most forensic facilities, there are drawbacks. The samples are inherently contaminated with microflora within the oral cavity and the moisture allows a plethora of microorganisms to grow. As the time frame that has elapsed from collection to storage increases, there is an exponential increase in bacterial cells. Storage of containers containing swabs coated with cells at temperatures below 20oC is also costly due to requirements for large freezers which are running and monitored over 24 hours. In the pass 10 to 15 years, researchers have focussed on alternative ways to store buccal cells. The FTA card system by Whatman is one such development. The FTA card is unique in that it provides a means for the collection of buccal cells for storage at room temperature. DNA profiling from samples stored in this way for 11 years has been successfully achieved. The filter paper matrix of the FTA card binds and subsequently lyses cells. ... (2) The second component of this thesis describes a study which subjected cells on buccal swabs to various conditions of increased temperature over periods of time to establish if DNA could be amplified. The aim was to mimic exposure to the vigours of field conditions, particularly in the extreme local environments that prevail in the United Arab Emirates. a. Initially, buccal cells stored at -20oC over 360 days were used to mimic standard archiving procedures. The cells were subsequently transferred to FTA cards, amplified and profiled by using ABI AmpFLSTR Identifiler PCR Amplification Kit (Applied Biosystems, Foster City, CA). Complete STR profiles were successfully recovered from the archived swabs. In most cases 100% of alleles were recovered, suggesting that it is feasible to transfer DNA from properly archived buccal swabs to FTA cards. b. The second phase involved the storage of fresh swabs that had been artificially aged by using incubation temperatures ranging from 40oC to 100oC. Partial profiles resulted from artificially aged samples, indicating that the prevailing conditions prior to low temperature storage of the swabs plays an important role in ensuring cellular integrity and thus, DNA quality. Results from this study suggest that it is possible for biological samples stored under correct conditions to be transferred from swabs to FTA card. In combination, the two chapters presented in this study show that it is feasible to transfer achieved forensic biology samples from swabs to the FTA card system. However, it is necessary to ensure that the samples are treated in the correct manner so as to minimise contamination from external sources and to maintain the correct environmental state to maintain intact cells and usable DNA.

Maize (Zea mays L.) is an important agronomic crop with the maize industry forming an important component of the South African economy. Considerable effort has been directed towards the genetic improvement of maize through both conventional breeding and biotechnology. Genotype identification by DNA fingerprinting is becoming an important activity in plant breeding. A widely used molecular based and relatively inexpensive method for DNA fingerprinting is the randomly amplified polymorphic DNA (RAPD) technique. The RAPD technique was tested in this study for its potential use in maize breeding programmes. Initial results using the technique showed a low degree of reproducibility, therefore both the DNA isolation and RAPD protocols were extensively optimised. DNA quality and quantity, and choice of Taq polymerase buffer were three of the variables found to be influential in ensuring reproducibility. The ability of the RAPD technique to characterise seven maize genotypes was evaluated. Sixty random oligonucleotide primers were screened. Forty two primers scored a total of 233 fragments (an average of 5.5 per primer), but not all primers gave reproducible profiles. Eighteen primers scored a total of 110 loci for the presence (1) and absence (0) of DNA fragments. RAPD markers were able to distinguish between all seven genotypes with five primers producing specific fragments for four genotypes. Genetic similarity matrices were calculated using two software programmes i.e. Genstat 5™ release 4.1 (1993) and PAUP (Phylogenetic Analysis Using Parsimony) 4.0 beta version (Swafford, 1998). Cluster analysis was used to generate dendrograms to visualise the genetic relationships of the seven maize genotypes (only minor differences were observed between the Genstat or PAUP method of analysis). Genetic diversity ranged from 0.62 to 0.96. The estimation of genetic relationship was in accordance with the presumed pedigree of the genotypes showing that the RAPD technique demonstrates potential for genome analysis of maize. The applicability of the technique for marker assisted selection was also evaluated. Near-isogenic lines (NILs) for leaf blight (Helminthosporium spp.) were screened for polymorphisms using a total of 120 primers. Ten primers identified polymorphisms between the NILs. Four primers produced five polymorphic fragments present in the resistant inbred K0315Y and absent in the susceptible inbred D0940Y. A small F2 population of 14 individuals was produced by selfing the F1 of a cross between K0315Y and D0940Y. To speed up the generation time, the F1 and F2 plants were cultured by embryo rescue from 18d old harvested seed. One fragment of 627 base pairs produced by primer OPB-01 (5' GTTTCGCTCC 3') showed a 3: 1 segregation in the small F2 population and was considered putatively linked to the HtN gene for leaf blight resistance. This study shows that the RAPD technique does have application in maize breeding programmes. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

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.