Development of experimental systems for studying the biology of Nudaurelia capensis ß virus
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
- Full Text:
- Authors: Walter, Cheryl Tracy
- Date: 2005
- Subjects: Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3948 , http://hdl.handle.net/10962/d1004007 , Imbrasia cytherea , Insects -- Viruses , RNA viruses , DNA
- Description: After 20 years, Nudaurelia ß virus (NßV) was re-isolated from a population of Nudaurelia capensis larvae exhibiting similar symptoms to those described in 1974 for a tetravirus infection. NßV is a member of the Tetraviridae, a family of positive sense insect RNA viruses that exclusively infect Lepidopteran insects. In addition to NbV, there was evidence that the insects were infected with another small RNA virus. SDS-PAGE and Western analysis revealed two proteins (p56 and p58), that cross-reacted with anti-NbV antibodies. Transmission Electron Microscopy (TEM) analysis showed the presence of particles exhibiting a morphology described for NbV and majority of particles of a diameter of 37 nm. In addition there was a second, minor population of particles with a diameter of 34 nm, which also exhibited the characteristic pitted surface of NßV, raising the possibility of two species of NßV in the N. capensis population. To further investigate this, cDNA corresponding to the 3` end of the replicase gene as well as the entire capsid gene of NbV was synthesized and sequenced. Alignments of the cDNA sequence showed a 99.46 % identity to the published sequence of NbV. Two amino acid substitutions were observed in the capsid coding sequence, one of which was a conservative substitution. Both of these substitutions were found in the b-sandwich domain of the capsid protein. Inspection of the capsid coding sequence showed a second methionine (Met50) at the VCAP amino terminus raising the possibility that p56 might arise from a translation product starting at this site. To investigate this, a full length and truncated capsid coding sequence starting at Met50, were expressed in a baculovirus expression system. VLPs were examined by TEM and Western analysis showed the presence of virus like particles with NßV morphology, but smaller in diameter than the wild-type with an average of 33.33 nm, similar to the smaller particles observed in the virus preparations of NßV. This result supported the hypothesis that NßV translates a smaller coat protein from the second in-frame methionine residue.
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Identification of cis-elements and transacting factors involved in the abiotic stress responses of plants
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Authors: Maclear, Athlee
- Date: 2005 , 2013-06-10
- Subjects: Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4074 , http://hdl.handle.net/10962/d1007236 , Plants -- Effect of stress on , Proteins -- Analysis , Bioinformatics , DNA , Plant genetics
- Description: Many stress situations limit plant growth, resulting in crop production difficulties. Population growth, limited availability and over-utilization of arable land, and intolerant crop species have resulted in tremendous strain being placed on agriculturalists to produce enough to sustain the world's population. An understanding of the principles involved in plant resistance to environmental stress will enable scientists to harness these mechanisms to create stress-tolerant crop species, thus increasing crop production, and enabling the farming of previously unproductive land. This research project uses computational and bioinformatics techniques to explore the promoter regions of genes, encoding proteins that are up- or down-regulated in response to specific abiotic stresses, with the aim of identifying common patterns in the cis-elements governing the regulation of these abiotic stress responsive genes. An initial dataset of fifty known genes encoding for proteins reported to be up- or down-regulated in response to plant stresses that result in water-deficit at the cellular level viz. drought, low temperature, and salinity, were identified, and a postgreSQL database created to store relevant information pertaining to these genes and the proteins encoded by them. The genomic DNA was obtained where possible, and the promoter and intron regions identified. The Neural Network Promoter Prediction (NNPP) software package was used to predict the transcription start signal (TSS) and the promoter searching software tool, TESS (Transcription Element Search Software) used to identify known and user-defined cis-elements within the promoter regions of these genes. Currently available promoter prediction software analysis tools are reported to predict one promoter per kilobase of DNA, whilst functional promoters are thought to only occur one in 30-40 kilobases, which indicates that a large perccntage of predictions are likely to be false positives (pedersen et. al., 1999). NNPP was chosen as it was rated as the highest performing promoter prediction software tool by Fickett and Hatzigeorgiou (1997) in a thorough review of eukaryotic promoter prediction algorithms, however results were less than promising as very few predicted TSS were identified in the area 50 bps up- and downstream of the gene start site, where biologically functional TSSs are known to occur (Reese, 2000; Fickett and Hatzigeorgiou, 1997). TESS results seemed to support the hypothesis that drought, low-temperature and high salinity plant stress response proteins have similar as-elements in their promoter regions, and suggested links to various other gene regulation mechanisms viz. gibberellin-, light-, auxin- and development-regulated gene expression, highlighting the vast complexity of plant stress response processes. Although far from conclusive, results provide a valuable basis for future comparative promoter studies that will attempt to deduce possible common transcriptional initiation of abiotic stress response genes. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
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Marine biotechnology : evaluation and development of methods for the discovery of natural products from fungi
- Authors: Pather, Simisha
- Date: 2005 , 2013-06-18
- Subjects: Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3839 , http://hdl.handle.net/10962/d1007652 , Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Description: One of the major impediments in the development of marine natural products is the provision of biologically active natural products in sufficient quantity for complete pharmacological evaluation, clinical trials and eventual commercial production. Marine microorganisms show great promise in providing a renewable source of biologically active natural products. The main aim of this study was to develop and evaluate methods for the isolation, identification and cultivation of marine fungi from the South African marine environment for the production of biologically active secondary metabolites. Twenty-four species of fungi were isolated from marine algae collected from the intertidal zone near Port Alfred, South Africa. The fungi were cultivated in small-scale under static and agitated conditions and their crude intra- and extracellular organic extracts were screened by ¹H NMR and a series of bioassays. Using this as a basis, one isolate was selected for further study. By analyses of the lTS1 region of the ribosomal DNA, the fungal isolate was identified as a marine-derived isolate of Eurotium rubrum (Aspergillus ruber). Although E. rubrum has been isolated from the marine environment, no investigations have been undertaken to determine the adaptation of these isolates to the marine environment. In order to optimise productivity, creativity and incubation time, the fungus was cultivated in small-scale using a variety of carbon (glucose, fructose, lactose, sucrose, marmitol and maltose) and nitrogen sources (ammonium tartrate, urea, peptone and yeast extract). An HPLC-DAD method was developed to assess the metabolic creativity and productivity under different fermentation conditions. Distinctive variations in the range and yield of metabolites produced as well as morphology and growth time were observed. The crude extracts from all fermentations were combined and six known compounds were isolated by reversed-phase chromatography and their structures elucidated by spectroscopic techniques. The known compounds were fIavoglaucin, aspergin, isodihydroauroglaucin, isotetrahydroauroglaucin, neoechinuline A and physcion. Neoechinuline A, isodihydroauroglaucin and isotetrahydroauroglaucin showed activity against oesophageal and cervical cancer cell lines.
- Full Text:
- Authors: Pather, Simisha
- Date: 2005 , 2013-06-18
- Subjects: Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3839 , http://hdl.handle.net/10962/d1007652 , Marine biotechnology , Marine fungi -- South Africa , Natural products -- South Africa , Marine plants -- South Africa , Marine metabolites -- South Africa , Cancer -- Treatment , DNA
- Description: One of the major impediments in the development of marine natural products is the provision of biologically active natural products in sufficient quantity for complete pharmacological evaluation, clinical trials and eventual commercial production. Marine microorganisms show great promise in providing a renewable source of biologically active natural products. The main aim of this study was to develop and evaluate methods for the isolation, identification and cultivation of marine fungi from the South African marine environment for the production of biologically active secondary metabolites. Twenty-four species of fungi were isolated from marine algae collected from the intertidal zone near Port Alfred, South Africa. The fungi were cultivated in small-scale under static and agitated conditions and their crude intra- and extracellular organic extracts were screened by ¹H NMR and a series of bioassays. Using this as a basis, one isolate was selected for further study. By analyses of the lTS1 region of the ribosomal DNA, the fungal isolate was identified as a marine-derived isolate of Eurotium rubrum (Aspergillus ruber). Although E. rubrum has been isolated from the marine environment, no investigations have been undertaken to determine the adaptation of these isolates to the marine environment. In order to optimise productivity, creativity and incubation time, the fungus was cultivated in small-scale using a variety of carbon (glucose, fructose, lactose, sucrose, marmitol and maltose) and nitrogen sources (ammonium tartrate, urea, peptone and yeast extract). An HPLC-DAD method was developed to assess the metabolic creativity and productivity under different fermentation conditions. Distinctive variations in the range and yield of metabolites produced as well as morphology and growth time were observed. The crude extracts from all fermentations were combined and six known compounds were isolated by reversed-phase chromatography and their structures elucidated by spectroscopic techniques. The known compounds were fIavoglaucin, aspergin, isodihydroauroglaucin, isotetrahydroauroglaucin, neoechinuline A and physcion. Neoechinuline A, isodihydroauroglaucin and isotetrahydroauroglaucin showed activity against oesophageal and cervical cancer cell lines.
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Phylogeography and comparative ecophysiology of Chrysanthemoides Turn. Ex Medik. (Tribe Calenduleae)
- Authors: Howis, Seranne
- Date: 2005
- Subjects: Chrysanthemoides , Phylogeny , Ecophysiology , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4256 , http://hdl.handle.net/10962/d1008189
- Description: Chrysanthem Oides is a common Southern African shrub that grows in a variety of habitats. From coastal shrubland and fynbos to mountainous areas as far north as Kenya. The genus has two species and 8 subspecies. The diagnoses and delimitation of which have been based almost exclusively on morphological characteristics. This project aims to investigate, with the use of phylogenetic species concepts. The validity of these subspecies. Unlike biological species concepts that rely on reproductive isolation as a defining character of a species. Phylogenetic species concepts (PSC) are concerned with delimiting evolutionary significant units (ESUs). ESUs are evolutionarily isolated lineages, and under the PSC a species is an aggregation of organisms consistently diagnosable by a fixed character or combination of characters. This project therefore searched for genetic and physiological characters by which to delimit ESUs within the Cill), samhemoides genus. DNA sequencing was used to investigate the genetic characters, while gas exchange studies were used to investigate the ecophysiological characters. DNA sequence analysis indicated that the ESUs can be diagnosed by genetic means and that one species may be of hybrid origin. Field studies of three disparate genetically identifiable ESUs from three disparate climates found that there are noticeable differences in ecophysiological responses of these ESUs in the field. Plants from each ESU were transferred to a greenhouse and grown under identical conditions for several months and compared to determine if these traits are inherent, or elastic in relation to environmental conditions. Under simulated high rainfall conditions. There does not appear to be a significant difference in the photosynthetic traits.
- Full Text:
Phylogeography and comparative ecophysiology of Chrysanthemoides Turn. Ex Medik. (Tribe Calenduleae)
- Authors: Howis, Seranne
- Date: 2005
- Subjects: Chrysanthemoides , Phylogeny , Ecophysiology , DNA , Plant genetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4256 , http://hdl.handle.net/10962/d1008189
- Description: Chrysanthem Oides is a common Southern African shrub that grows in a variety of habitats. From coastal shrubland and fynbos to mountainous areas as far north as Kenya. The genus has two species and 8 subspecies. The diagnoses and delimitation of which have been based almost exclusively on morphological characteristics. This project aims to investigate, with the use of phylogenetic species concepts. The validity of these subspecies. Unlike biological species concepts that rely on reproductive isolation as a defining character of a species. Phylogenetic species concepts (PSC) are concerned with delimiting evolutionary significant units (ESUs). ESUs are evolutionarily isolated lineages, and under the PSC a species is an aggregation of organisms consistently diagnosable by a fixed character or combination of characters. This project therefore searched for genetic and physiological characters by which to delimit ESUs within the Cill), samhemoides genus. DNA sequencing was used to investigate the genetic characters, while gas exchange studies were used to investigate the ecophysiological characters. DNA sequence analysis indicated that the ESUs can be diagnosed by genetic means and that one species may be of hybrid origin. Field studies of three disparate genetically identifiable ESUs from three disparate climates found that there are noticeable differences in ecophysiological responses of these ESUs in the field. Plants from each ESU were transferred to a greenhouse and grown under identical conditions for several months and compared to determine if these traits are inherent, or elastic in relation to environmental conditions. Under simulated high rainfall conditions. There does not appear to be a significant difference in the photosynthetic traits.
- Full Text:
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