Magister Scientiae - MSc (Biotechnology)

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A study of the problems and possibilities of using the marine intertidal zone for teaching principles of ecology in senior secondary schools: A survey of biology teachers in the western cape.
(University of the Western Cape, 1994) Reddy, Chris; Keats, D.W.
In this study I investigate the attitudes of a group biology teachers in the Western Cape, to using the marine intertidal zone for teaching principles of ecology in senior secondary schools, by exploring the problems experienced and solutions envisaged. The study investigates the problems perceived/experienced by means of a semistructured interview and seeks solutions via teacher workshops and an excursion to the seashore. Teachers mentioned many constraints and school based problems such as time-tab1ing, teacher attitudes, lack of ethos lack of funds, Iarge numbers in classes and collectively found useful answers which reduced the emphasis of many of the problems mentioned. Problems such ds, the lack of knowledge of the marine environment, limited experience of fieldwork technique and management, could only be solved by pre and in-service teacher education programmes. The workshops produced useful solutions and suggestions for implementation by teachers, education departments and governmental and non-governmental agencies that would assist in making this a reality. These include resource development, teacher networking, peer teaching, in-service and pre-service programmes with a marine emphasis, and funding of appropriate programmes
Human testis angiotensin-converting enzyme: crystal structure of a glycosylation mutant and investigation of a putative hinge-mechanism by normal mode analysis
(University of the Western Cape, 2004) Watermeyer, Jean Margaret; Sewell, Bryan Trevor; Sturrock, E.D.; Dept. of Biotechnology; Faculty of Science
Human angiotensin-converting enzyme (ACE) is a key enzyme in the regulation of blood pressure via the renin-angiotensin and kallikrein-kinin systems. A number of orally active drugs have been developed over the years that target somatic ACE, for the treatment of hypertension, myocardial infarction and congestive heart failure. Protein structural information about ACE is an important key for the understanding of the mechanism and substrate-specificity of the enzyme. However, this information has only begun to be elucidated in the past year, with the solution of crystal structures of human testis ACE (tACE), and homologues Drosophila AnCE and human ACE2. tACE is identical to the C-terminal domain of somatic ACE, which consists of two homologous domains, each having a slightly different substrate-specificity. This thesis describes the purification, crystallisation and X-ray crystal structure-determination of a glycosylation-deficient mutant of tACE, tACEG1,3, to 2.9 Å. The structure of tACE-G1,3 aligns closely with that of native tACE, indicating that the mutations did not alter the conformation. The ability to achieve minimal glycosylation of tACE for crystallisation purposes via mutation, rather than using expensive glycosidase inhibitors, iii should prove advantageous for further structural studies, such as the study of the binding of novel inhibitors. In all of the tACE structures thus far observed, the active site is closed off from the external medium in a deep cleft, so that it is unclear how a large substrate molecule could gain access. However, a hinge motion that opens this cleft has been observed in the structures of ACE2. Temperature factor and sequence comparison between tACE, tACE-G1,3, AnCE and ACE2 suggests the functional conservation of three flexible loop regions, as well as the sequence conservation of three constrained regions, involved in the hinge. Normal mode analysis reveals the intrinsic flexibility of tACE, and further suggests that a putative open form of tACE would behave similarly to the open form of ACE2. Based on these indications, a conservation of the ACE2 hinge-bending mechanism is proposed. Temperature factor analysis also reveals that subdomain II, containing bound chloride ions, is more structurally rigid than subdomain I, in all structures considered. Based on these results, lines of investigation are suggested that should yield insight into the mechanisms of action of ACE and its association with various substrates and inhibitors, ideally aiding in the development of novel drugs for the treatment of cardiac disease.
Investigation into the role of DWNN in cell death
(University of the Western Cape, 2004) Seameco, Tumelo; Rees, D.J.G.; Dept. of Biotechnology; Faculty of Science
Many genes are activated to influence the self-destruction programme of the cell. This programme entails synchronised instigation and implementation of numerous subprograms. The arrival of gene targeting aided in the determining of the functions of novel genes. Such genes may have been sequenced, but not functionally characterised. The fulfillment of this requirement through gene targeting technology has swiftly developed. The mode by which DWNN operate in organisms in which it is thought to be covalently linked to some other proteins, which have a definite role in apoptosis, is not yet unraveled. This study attempted the functional characterisation of DWNN in light to the hypothesis that it may be involved in Cytotoxic T lymphocyte killing and apoptosis.
The prevalence and survival of Campylobacter, Salmonella and Listeria species in poultry processing plant
(University of the Western Cape, 2004) Mabogo, Rudzani David Lesly; Gouws, P.A.; Dept. of Biotechnology; Faculty of Science
The organisms in this study were chosen due to their associations with foods and their potential as food borne pathogens. Food borne diseases are an import public health problem in most countries. Bacteria of the genera Campylobacter, Salmonella and Listeria can be transported by poultry and poultry products to humans. Gastroenteritis, typhoid fever, diarrhea, dysentery may originate from the infection. This study was undertaken to determine the incidence of pathogens in a poultry processing plant using polymerase chain reaction and conventional tests and to determine the formation and survival of biofilm cells of food pathogens in trisodium phosphate.
Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies
(University of the Western Cape, 2004) Ndabambi, Nonkululeko; Pugh, David J.R.; Dept. of Biotechnology; Faculty of Science
This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 1l of culture, which make it feasible to express 15N and 12C labelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of poly-lysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.
Ancient Genes in Cancer Gene Expression?
(University of the Western Cape, 2004) Panji, Sumir; Hide, Winston
Backsround: The Cancer/testis (CT) antigens are a division of germ cell specific genes not expressed in somatic cells, exceptions being placental cells and 20Vo - 4OVo of cancer types. The aptitude of CT antigens to elicit humoral immune responses, their restricted expression profile, absence of major histocompatability complex expression in male germline cells have contributed to the emergent attraction of CT antigens as ideal, prospective cancer vaccination candidates. Motivation: Presently there are M CT gene families containing a total of 97 gene products and isoforms. Due to the promulgation in sensitivity and specificity of rapid serological immunodetection assays e.g. serial analysis of recombinant cDNA expression libraries (SEREX), the magnitude of novel CT genes and gene families will increase. Hence, characteization of this unique subset of CT genes is fundamental to our erudition of this rapidly emerging novel subset of genes. Obiectives: The sequencing of the human genome provides a useful biological framework for the categoization and systematization of rapidly accumulating biological information. A genomic approach was used to ascertain the locations of the CT genes in the human genome and determine if the genomic locations of the CT genes is nonrandom. An in-silico expression study was conducted for the CT genes with the aim of establishing if CT gene expression is restricted to the testis. A portion of the human genome housing the largest proportion of the CT genes was selected for analysis in order to determine if the surrounding genomic architecture influences CT gene expression. A comparative genomics approach was used in determining if the CT genes are "ancient genes.
Microbial diversity of Antarctic Dry Valley mineral soil
(University of the Western Cape, 2004) Moodley, Kamini; Cowan, D.; Dept. of Biotechnology; Faculty of Science
Antarctica provides some of the most extreme environments on earth. Low temperatures, low water availability and nutrient deficiency are contributing factors to the limited colonisation of Antarctic biotopes, particularly in the continental Dry Valleys. The survival of microorganisms in this harsh continent provides the basis for the significance of this study. This study aimed to explore microbial phylotypic diversity across a 500 m altitudinal transect in the Miers Dry Valley, Ross Desert, East Antarctica. The study also attempted to infer from phylogenetic data, the possible presence of indicative phenotypes which might contribute to a functional microbial community.
Isolation and identification of PNP-A homologues from Xerophyta viscosa Baker
(University of the Western Cape, 2004) Mabuto, S; Gehring, C
Maintenance of growth, development and normal physiological processes remains crucial for the survival of plants. Plant hormones are described as integrators of external signal and internal developmental process in plants. They also attribute as mediators of physiological and developmental adaptation throughout the plant life cycle (Wilkinson et al.,1997). It has been well over 50 years since Went and Thimann (1937) published their classical book Phytohorrnones. At that time, the term phytohorrnone was synonymous to auxin, although compounds like cell division factors were anticipated to be phytohormones based on physiological experiments. Since then a number of plant hormones were discovered and a series of studies concluded that plant hormones are at least partly responsible for plant growth, development, cell elongation, cell division, differentiation and stomatal movement in response to environmental stimuli. Plant hormones are categorized into three groups, known as classical, non-classical and peptide hormones. Here, Plant Natriuretic Peptide (PNP) will be reviewed as a proposed additional peptide hormone.
Recombinant expression of the pRb- and p53-interacting domains from the human RBBP6 protein for in vitro binding studies
(University of the Western Cape, 2004) Ndabambi, Nonkululeko; Pugh, David
This thesis describes the cloning and recombinant expression of domains from the human RBBP6 protein for future in vitro binding studies with pRb and p53. RBBP6 is a splicing-associated protein that is known to interact with both p53 and the Retinoblastoma gene product (pRb), and has recently been shown to be highly upregulated in oesophageal cancer. The pRb binding domain (RbBD) and the p53 binding domain (p53BD) were each expressed using the glutathione-S-transferase (GST) tag affinity system, and affinity purified using a glutathione-linked agarose column. Purified fusion proteins were cleaved to separate the target protein from GST using PreScission ™ Protease, for which there is a recognition sequence located immediately upstream of the multiple cloning site on the pGEX-6P series of plasmids. The pRb binding and p53 binding domains were further purified using cation exchange chromatography. Mass spectrometry confirmed that the RbBD was expressed as a single species of the expected molecular weight. However preliminary NMR analysis suggested that the domain was not fully folded. A total yield of 8 mg of protein was achieved from 11 of culture, which make it feasible to express 15Nand 12Clabelled samples for NMR. The p53BD was found to be expressed at lower levels and subject to C-terminal degradation, which suggest that the C-terminus is unstructured most likely due to the presence of polylysine tail. Human pRb protein was also successfully expressed and purified using the GST affinity system. Human p53 protein was expressed but was found to be insoluble and attempts to purify it were not pursued. Attempts to confirm the interactions between human RBBP6 and p53 and pRb proteins are on-going but fall outside the scope of this thesis. Expression constructs for the RING and zinc finger domains from human RBBP6 were also cloned into the pGEX system for future structural studies using NMR. Both domains were found to be expressed as soluble fusion proteins in preliminary expression studies.
An investigation of the plants used medicinally In self-care in the Bredasdorp / Elim region
(University of the Western Cape, 2004) Thring, T; Weitz, F
Much of the traditional medicinal plant knowledge is to a large extent known by the older generations. This knowledge is at risk of disappearing due to not being passed down to younger members of the respective families and communities. The Bredasdorp / Elim area in the Southern Overberg has many individuals who possess such knowledge. The aims of this study were to identify what plants were in use in the area, to document this knowledge and to choose certain plants to test in antimicrobial bioassays.
The genease activity of mung bean nuclease: fact or fiction?
(University of the Western Cape, 2004) Kula, Nothemba; Hide, Winston; Dept. of Biotechnology; Faculty of Science
The action of Mung Bean Nuclease (MBN) on DNA makes it possible to clone intact gene fragments from genes of the malaria parasite, Plasmodium. This “genease” activity has provided a foundation for further investigation of the coding elements of the Plasmodium genome. MBN has been reported to cleave genomic DNA of Plasmodium preferentially at positions before and after genes, but not within gene coding regions. This mechanism has overcome the difficulty encountered in obtaining genes with low expression levels because the cleavage mechanism of the enzyme yields sequences of genes from genomic DNA rather than mRNA. However, as potentially useful as MBN may be, evidence to support its genease activity comes from analysis of a limited number of genes. It is not clear whether this mechanism is specific to certain genes or species of Plasmodia or whether it is a general cleavage mechanism for Plasmodium DNA .There have also been some projects (Nomura et al., 2001;van Lin, Janse, and Waters, 2000) which have identified MBN generated fragments which contain fragments of genes with both introns and exons, rather than the intact genes expected from MBN-digestion of genomic DNA, which raises concerns about the efficiency of the MBN mechanism in generating complete genes.Using a large-scale, whole genome mapping approach, 7242 MBN generated genome survey sequences (GSSs) have been mapped to determine their position relative to coding sequences within the complete genome sequences of the human malaria parasite Plasmodium falciparum and the incomplete genome of a rodent malaria parasite Plasmodium berghei. The location of MBN cleavage sites was determined with respect to coding regions in orthologous genes, non-coding intergenic regions and exon-intron boundaries in these two species of Plasmodium. The survey illustrates that for P. falciparum 79% of GSSs had at least one terminal mapping within an ortholog coding sequence and 85% of GSSs which overlapped coding sequence boundaries mapped within 50 bp of the start or end of the gene. Similarly, despite the partial nature of P.berghei genome sequence information, 73% of P.berghei GSSs had at least one terminal mapping within an ortholog coding sequence and 37% of these mapped between 0-50 bp of the start or end of the gene. This indicates that a larger percentage of cleavage sites in both P.falciparum and P.berghei were found proximal to coding regions. Furthermore, 86% of P.falciparum GSSs had at least one terminal mapping within a coding exon and 85% of GSSs which overlapped exon-intron boundaries mapped within 50bp of the exon start and end site. The fact that 11% of GSSs mapped completely to intronic regions, suggests that some introns contain specific cleavage sites sensitive to cleavage and this also indicates that MBN cleavage of Plasmodium DNA does not always yield complete exons. Finally, the results presented herein were obtained from analysis of several thousand Plasmodium genes which have different coding sequences, in different locations on individual chromosomes/contigs in two different species of Plasmodium. Therefore it appears that the MBN mechanism is neither species specific nor is it limited to specific genes.
The evaluation of Y-STR loci for use in forensics
(University of the Western Cape, 2005) Ehrenreich, Liezle Suzette; Leat, Neil; Davison, Sean; Dept. of Biotechnology; Faculty of Science
The aim of this study was to investigate the forensic usefulness of various Y-chromosome short tandem repeat loci among South African sub-populations. Three different sets of Y-chromosome short tandem repeat loci were chosen for investigation.
Exploring diversity and ecology of nonarchaea in hydrothermal biotopes
(University of the Western Cape, 2005) Galada, Ncebakazi; Cowan, Donald A.; Dept. of Biotechnology; Faculty of Science
The Nanoarchaeota were proposed as the fourth archaeal sub-division in 2002, and the only fully characterized nanoarchaeon was found to exist in a symbiotic association with the crenarchaeote, Ignicoccus sp. This nanoarchaeote, named Nanoarchaeum equitans could not be detected with “universal” archaeal 16S PCR primers and could only be amplified using specifically designed primers. In order to identify and access a wide diversity of archaeal phylotypes a new set of “universal” archaeal primers A571F (5’-GCYTAA AGSRIC CGT AGC-3’) and UA1204R (5’-TTM GGG GCA TRCIKA CCT-3’) was designed, that could amplify the 16S rRNA genes of all four archaeal sub-divisions. Using these primers community DNA was amplified from Chinese and New Zealand hydrothermaystems. Upon sequencing of amplicons it was discovered that Chinese and. New Zealandsamples contained novel nanoarchaeal phylotypes. The preliminary nanoarchaeal phylotypes were used to design nanoarchaeal-specific primer N989R (5'-GGT TTC CGG TGT CAG TTC-3'), which was coupled with A571F and used in screening of nanoarchaeotes. The nanoarchaeal phylotypes identified with these primers were further screened by amplified ribosomal DNA restriction analysis (ARDRA), which was used to explore the diversity of these phylotypes. The novel nanoarchaeotes cluster into 9 cosely related clades which may represent separate species. Three of the New Zealand phylotypes form one separate clade which is closely related to the published nanoarchaeotes. The following nanoarchaeal sequences were submitted to the GenBank, TC9F (AY572420), TC11-5 (AY571283), TC11-B6 (AY727890), TC11-B7 (AY727887), TC11-C4 (AY727886), TC11-C6 (AY727889), TC11-C8 (AY727888), AND TC11-D4 (AY727891). Fluorescence in situhybridization was also used to simultaneously visualize, identify and localize nanoarchaeotes.
Monitoring of heavy metals in the bottelary river using typha capensis and phragmites australis
(2005) Ma, Ying; Raitt, Lincoln
The aim of this study was to use plants to determine the degree of heavy metal contamination in water and sediments in order to effectively monitor and provide possible recommendation to improve the water quality in the aquatic ecosystem of the Bottelary River.
Microbiology of fly ash-acid mine drainage co-disposal processes
(University of the Western Cape, 2005) Kuhn, Eloise M. R.; Cowan, D. A.
The waste products acid mine drainage (AMD), formed during coal mining and fly ash (FA) from coal burning power generation, pose substantial environmental and economic problems for South Africa. Eskom has developed a remediation system employing alkaline FA to neutralize and precipitate heavy metals from toxic acidic AMD streams. The aim of this study was to assess the microbial diversity in and microbial impact on this remediation system. The total microbial diversity was assessed by well-established molecular phylogenetic analyses using 16S rDNA gene sequences. The results obtained from the AMD confirmed the presence of acidophilic organisms, such as Acidithiobacillus ferrooxidans (At. ferrooxidans). After co-disposal of FA and AMD, microbial cell growth was not detected and microbial genomic DNA could not be extracted. The absence of microbial communities in the co-disposal phase is beneficial to the continuation of the development of such a co-disposal process. Results of this project will assist in the effective implementation of FA-AMD co-disposal systems, which may improve water quality in effected regions of South-Africa.
The microbial composition of a natural methanogenic consortium
(University of the Western Cape, 2005) Mashaphu, Nthabiseng; Cowan, Donald A.; Dept. of Biotechnology; Faculty of Science
Wetlands account for approximately 20% of annual global methane emissions. Many wetlands receive inputs of organic matter, nutrients, metals and various toxic compounds from adjacent agricultural and industrial areas. The present study aimed to investigate the microbial composition of a natural methanogenic consortium. A consortium-based molecular approach to study diversity of methanogenic microbial communities in a natural wetland at the primary inflow was used. Key microorganisms of a nethane producing consortium were identified. Extracted high molecular mss DNA ws analysed by PCR combined with denaturing gradient gel electrophoresis and subsequent sequencing of 16S rDNA. This study was also aimed to identify syntrophic microorganisms in the wetland system. The data obtained suggest a well established syntrophic relationship within the wetland.
Recombinant expression and full backbone assignment of the human DWNN using heteronuclear NMR
(University of the Western Cape, 2005) Faro, Andrew; Pugh, David J.R.; Dept. of Biotechnology; Faculty of Science
The cellular levels of a number of proteins have been found to be regulated by the ubiquitin-proteasome pathway. In this pathway, proteins are covalently tagged (“ubiquitinated”) by ubiquitin, which acts as a signal for degradation by the proteasome. A number of key cellular processes, including cell-cycle progression, transcription and DNA repair, are regulated in this way. In recent years a number of cellular proteins resembling ubiquitin in structure or function, the so-called ubiquitin-like proteins, have been identified. Ubiquitin-like proteins can be divided into two classes-the so-called “ubiquitin-like modifiers”, which consist of a single domain that structurally resembles ubiquitin, and “ubiquitin-domain” proteins, which are multi-domain proteins, which include domains that resemble ubiquitin. This thesis describes the recombinant expression, purification and full backbone assignment of the human DWNN domain, a novel ubiquitin-like domain. The DWNN domain occurs at the N-terminus of RBBP6, a protein that has been shown to interact with p53 and Rb as well as to be involved in mRNA processing and apoptosis. A bacterial expression system was used to overexpress the DWNN domain as a GST fusion protein. The domain was labelled with 15N and 13C to perform triple-resonance heteronuclear NMR experiments, from which full backbone assignments were obtained. Although full structure determination of the DWNN domain falls outside the scope of this thesis, the backbone assignments formed the basis for the subsequent structure determination, which confirmed that the DWNN domain is indeed a novel ubiquitin-like domain. The RBBP6 protein may therefore represent a novel E3 ubiquitin ligase that plays a role in regulating the cellular levels of p53 and Rb.
Analysis of ammonia-oxidizing bacteria associated with the roots of Proteaceae plant species in soils of Fynbos ecosystem
(University of the Western Cape, 2005) Cowan, D. A.
Molecular methods were used to investigate the microbial diversity and community structure of ammonia-oxidizing bacteria (AOB) associated with the roots of the Proteaceae plant family. The identification of ammonia oxidizing bacteria in this ecosystem is of particular interest since Proteaceae are adapted to acidic, low nutrient (e.g. nitrogen) soils. The ammonia monooxygenase operon was used as a molecular marker to identify ammonia-oxidizing bacteria associated with the proteoid roots of the three Proteaceae members and compared to non-plant associated soil. PCR amplification using primer sets targeting the ammonia monooxygenase gene (amoA subunits) were used to construct a clone library. Sequence diversity was determined by RFLP analysis of amoA to identify major groups of AOB of the ~-subclass of Proteobacteria in total community DNA, and DNA sequencing and phylogenetic analysis were also applied. DGGE analysis was performed to determine the community structure and distribution of ammonia-oxidizing bacteria in plant-associated and non-plant associated soils. The AOB genotypic diversity was similar in the plant-associated samples and non-plant associated soil. All AOB phylotypes belonged to Nitrosospira species and clustered with Nitrosospira cluster 3. The abundance of the amoA was quantified to be approximately 4.2 x 107 copies/g of dry soil, using a real-time PCR assay. These data suggest that the Nitrosospira species are the dominant phylotypes in that environment. This investigation provides new insights into the relationships between plants and ammonia-oxidizing bacteria in natural Fynbos ecosystems.
Developing a generic hazard analysis critical control point (HACCP) system for the wheat milling industry
(University of the Western Cape, 2005) Gillion, Lauren; Gouws, P.A; Dept. of Biotechnology; Faculty of Science
In South Africa there is a need within the milling industry for controlling food safety especially due to customer's demands and government's regulations. The best way to ensure food safety is with the implementation of a HACCP based food safety system. Therefore, the principal aim of this study was to develop a generic HACCP model for the flour milling industry. Afterwards this generic model can then be adapted for each specific mill and its needs.
Analysis of ammonia-oxidizing bacteria associated with the roots of Proteaceae plant species in soils of Fynbos ecosystem
(University of the Western Cape, 2005) Lako, Joseph; Cowan, Don A.; Dept. of Biotechnology; Faculty of Science
The major objective of this study was to investigate soil ammonia-oxidizing bacterial diversity and composition associated with plant roots of Proteaceae plants and to compare it with non-plant associated soil.

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