The halophilic bacterium Vibrio parahaemolyticus is widely distributed as a natural inhabitant of marine and estuarine environments. Some strains cause gastroenteritis in humans. Clinical isolates are thought to possess virulence factors that are absent from the majority of environmental isolates. Studies were undertaken to differentiate clinical (virulent) and environmental (mainly avirulent) forms of Vibrio parahaemolyticus. Initially, identification and confirmation of a total of 55 V. parahaemolyticus isolates (23 clinical and 32 environmental) was carried out by using selective media, biochemical and nutritional tests. Identity was confirmed by specific polymerase chain reaction (PCR) targeting the toxR gene. In an attempt to differentiate between virulent and avirulent forms V. parahaemolyticus, potential virulence factors (enzyme activities), presence of plasmids and analyses of whole cell protein profiles and extracellular products (ECPs) by SDS-PAGE were performed. The results suggested that the presence of plasmids in isolates was not linked to virulence and SDS-PAGE profiles did not differentiate between virulent and avirulent forms, but a combination of enzyme activities may contribute to virulence. The ECPs of all 55 V. parahaemolyticus isolates were tested for their cytotoxicity towards two types of cell lines, the clinical isolates showed that 21 out of 23 (91%) and 2 out of 23 (8.69%) showed high and medium cytotoxicity, respectively. Amongst the environmental isolates 2 out of 32 (6.25%), 2 out of 32 (6.25%) and 28 out of 32 (87.5%) showed high, medium and low cytotoxicity, respectively. Randomly amplified polymorphic DNA (RAPD)-PCR was used to analyse the two groups of isolates. Firstly a 600 bp band was recognised in mainly clinical isolates. This DNA fragment was cloned and sequenced and found to code for an outer membrane protein (OMP). Two PCR primers were designed to specifically amplify a 200bp unique sequence from presumptive virulent strains (PCR-OMP); however, not all clinical isolates were positive (21 out of 23, 91%). A second RAPD-PCR identified a further unique band of approximately 310 bp in mostly clinical isolates. After cloning this band’s DNA, the DNA sequence revealed a hypothetical gene, htp, whose function is not known. Specific primers, VPHTP1 and VPHTP2 were developed for the detection of the htp sequence, but again not all clinical isolates were positive (19 out of 23, 82.6%). This led to the development of a multiplex M-PCR which detects all isolates of V. parahaemolyticus and differentiates them into potentially virulent and avirulent forms. The M-PCR works by targeting the toxR gene, and sequences for omp and htp. The M-PCR was performed on all V. parahaemolyticus isolates used in this study, as well as other Vibrio species and a selection of non-Vibrio species. The amplification of toxR gene 367 bp fragment was found in all V. parahaemolyticus tested; all clinical isolates (100%) showed amplification of omp and/or htp. This multiplex PCR detected 3 (9.37%) environment isolates, which may potentially be able to cause disease. No amplification was seen for the other species tested. Thus, the M-PCR could be used for identifying V. parahaemolyticus and detecting / differentiating potentially virulent and avirulent forms. This method should be

Human adenoviruses (HAdV) are non-enveloped viruses with an icosahedral capsid and a linear double-stranded DNA genome. These viruses belong to the family Adenoviridae and genus Mastadenovirus. An important property of the HAdV is that it is non-enveloped making it highly resistant to detergents and harsh environmental conditions. This virus is grouped in seven species (A-G) with more than 88 genotypes. These seven species are associated with several diseases, such as, respiratory infections, keratoconjunctivitis, urinary infections, hepatitis and gastrointestinal infections. The HAdV is one of the etiological causes of acute gastroenteritis, mainly caused by HAdV-F40 and HAdV-F41. The virus can be transmitted via the faecal-oral route, inhalation of respiratory droplets and direct contact with contaminated environments. The virus is known to be ubiquitous in environments where human contamination is likely to occur such as wastewater treatment plants. These human contaminations could occur through contaminated secretion and excretions within aqueous environments. There is currently a limited amount of information on the HAdV in water Molecular characterisation of human adenoviruses from environmental environments, particularly in Tshwane, Gauteng. Therefore, the aim of the study was to investigate the presence and genotypes of human adenovirus in environmental samples namely raw sewage and treated effluent, using molecular methods. For genotypic characterisation, Sanger sequencing was used on amplicons from 12 HAdV positive samples and next generation sequencing were used on all the amplicons from HAdV positive samples. A total of 150 environmental samples (75 raw sewage and 75 effluent) were collected from two wastewater treatment plants in Tshwane over the study period of 18 months. These environmental samples comprised of 1 L raw sewage and 10 L treated effluent samples. The primary viral recovery for the 1 L raw sewage and 10 L treated effluent samples were performed using skimmed milk flocculation procedure and glass wool adsorption elution technique, respectively. For secondary viral recovery, both environmental samples were subjected to polyethylene glycol/sodium chloride precipitation. Manual extraction was used to extract the nucleic acids from the virus concentrate with mengovirus (MV) used as an extraction control. For the quantification of HAdV, standard curves prepared from known dilutions of HAdV and MV were used. Human adenovirus was detected in 140/150 (93%) of the environmental samples comprising of 69/75 (92%) being raw sewage and 71/75 (95%) being effluent samples. The HAdV concentrations detected in wastewater treatment plant 1 (WWTP 1) ranged from 1.38x105 gc/L to 4.50 x 109 gc/L for raw sewage and 5.08x103 gc/l to 4.30x108 gc/L for effluent. The HAdV concentrations detected in WWTP 2 ranged from 6.84x104 gc/L to 1.69x1012 gc/L for raw sewage and 5.27x103 gc/L to 1.16x108 gc/L for effluent. The HAdV hexon amplification success rate from the nucleic acids was 43/140 (31%). Eighteen HAdV genotypes were successfully characterised using Sanger sequencing. The HAdV-D was the most predominant species in both WWTPs, follow by HAdV-B and HAdV-F. The HAdV-A and HAdV-E species were the least identified. Next generation sequencing identified four times as many genotypes as Sanger sequencing (77 different genotypes). The HAdV-D (types 8, 9, 13, 17, 19, 20, 23, 24, 28, 29, 32, 33, 36, 42, 44, 47, 49, 51, 56, 60, 62, 64, 67 and 81) and HAdV-B (types 2, 3, 7, 11 and 66) were the most predominant species followed by HAdV-F (types 40 and 41), HAdV-A (types 12 and 76), HAdV-E ( type 4) and HAdV-C (type 1). Testing wastewater treatment plants is advantageous as it allows for the detection and identification of HAdV types circulating in the surrounding communities. Due to the large number of species identified using NGS, it is the superior typing method and should be used for future studies. These include strains causing symptomatic and asymptomatic infections. Human adenovirus was detected at comparable frequencies in raw sewage and treated effluent wastewater, with slightly higher detection in effluent samples. However, the viability of these viruses is unknown and should be investigated in further studies. The detection of viruses in wastewater treatment plants are a public health concern as the treated effluent is discharged into rivers, which may be used by communities for domestic and recreational purposes. / Dissertation (MSc (Medical Virology))--University of Pretoria, 2020. / NRF, PRF / Medical Virology / MSc (Medical Virology) / Restricted

Banana bunchy top virus (BBTV) is a circular, single-stranded (css) DNA virus that belongs to the genus Babuvirus in the family Nanoviridae. BBTV is responsible for the most devastating virus disease of banana known as "bunchy top", for which conventional control measures are generally ineffective. Genetically engineered resistance appears to be the most promising strategy to generate BBTV-resistant bananas but the success of this strategy is largely dependent upon the molecular characterisation of the target virus and knowledge of the virus life cycle, particularly the replication strategy. This PhD study was aimed at the molecular characterisation of the intergenic regions of BBTV, in order to complement the molecular information currently available and to potentially contribute to the development of transgenic resistance strategies against BBTV in banana. Three putative iterative sequences (iterons; GGGAC) previously identified in the BBTV intergenic regions were initially characterised. In order to determine their role in the binding of the master BBTV replication initiation protein (M-Rep), the putative iterons (F1 and F2 in the virion sense, and R in the complementary sense) were independently mutated in a BBTV DNA-6 greater-than-genome-length clone (1.1 mer). The DNA-6 1.1 mers (native and mutants) and the M-Rep-encoding component (DNA-1) were co-bombarded into banana (Musa spp. cv."Lady finger") embryogenic suspension cells and transient replication was evaluated by Southern hybridisation. Analysis of the DNA-6 replicative forms showed a significant decrease of approximately 41% for the F1 iteron mutant and 61% for the R iteron mutant in comparison with native levels. However, the mutation in the F2 iteron caused the most dramatic effect, decreasing replication to levels barely detectable by Southern hybridisation. These results suggest that the three iterons all play a role in BBTV replication, most likely as recognition and binding sites for the M-Rep, but that the F2 iteron appears to be the most important in replication. Following the observation that all BBTV isolates sequenced to date have identical iteron sequences, the extent to which the M-Rep would recognise, bind and initiate replication of heterologous components from geographically diverse BBTV isolates (the South Pacific and the Asian groups) was evaluated. Cross replication assays revealed that heterologous M-Reps from Fiji, Hawaii (South Pacific group) and Vietnam (Asian group) were able to initiate replication of the coat protein-encoding component (DNA-3) from the Australian BBTV isolate (South Pacific group). However, replication of DNA-3 from the Vietnamese isolate was not initiated by heterologous M-Reps from the two South Pacific isolates tested (Australia and Hawaii). These results suggest that a broad-range transgenic resistance strategy based on replication using Australian BBTV intergenic regions may be successful as this region will be recognised by the M-Reps from both Asian and South Pacific BBTV isolates. However, a Rep protein-mediated resistance strategy will more likely be specific to geographical isolates and, therefore, less suitable as a broad-range control strategy. To further characterise the BBTV intergenic regions and to gain a better understanding of the BBTV transcription process, the 5' untranslated regions (UTRs) of the major open reading frames (ORFs) associated with each of the six BBTV DNA components were mapped. In all cases, the transcription start sites were located 3' of a putative TATA box and the 5' UTRs varied in length from 23 nucleotides (DNA-6) to 5 nucleotides (DNA-3). Two potential transcription start sites (nt 84 and 87) were mapped for DNA-1, but whether these represent the transcription start sites of the two genes associated with DNA-1 remains to be determined. Two start sites were also associated with DNA-2 which is thought to be monocistronic. Whether one of these start sites is an artefact or whether they are due to natural sequence variability of BBTV is unknown. These results now enable us to define the transcribed regions of each BBTV DNA component and accurately predict their promoter regions in an attempt to gain a fundamental understanding of BBTV gene expression patterns.

Molecular management targets the right molecules to be at the right place, at the right time and at the right price. It consists of molecular characterisation of refining streams, molecular modelling and optimisation of refining processes, as well as overall refinery optimisation integrating material processing system and utility system on the molecular level. The need to increase modelling details to a molecular level is not just a result of political regulations, which force refiners to managing the molecule properly, but also seems to be a very promising to increase the refining margin. In this work, four aspects of molecular management are investigated respectively. Molecular Type Homologous Series (MTHS) matrix framework is enhanced on both representation construction and transformation methodology. To improve the accuracy and adequacy of the representation model, different strategies are formulated separately to consider isomers for light and middle distillates. By introducing statistical distribution, which takes the composition distribution of molecules into account, the transformation approach is revolutionised to increase the usability, and tackle the challenge of possibly achieving significantly different compositions from the same bulk properties by the existing approaches. The methodology is also enhanced by applying extensive bulk properties. Case studies demonstrate the effectiveness and accuracy of the methodology. Based on the proposed characterisation method, refining processes are modelled on a molecular level, and then process level optimisation is preformed to have an insight view of economic performance. Three different processes, including gasoline blending, catalytic reforming, and diesel hydrotreating, are investigated respectively. Regarding gasoline blending, the property prediction of blending components, and the blending nonlinearity are discussed. To tightly control on the property giveaway, a molecular model of gasoline blending is developed, and then integrated into the recipe optimisation. As for the conversion processes, catalytic reforming and diesel hydrotreating, reactions and reactors are modelled separately, and then followed by the consideration of catalyst deactivation. A homogeneous rigorous molecular model of a semiregenerative catalytic reforming process, considering pressure drop, has been developed. In addition, a multi-period process optimisation model has been formulated. Regarding diesel hydrotreating, a molecular model of reactions with a three-phase trickle-bed reactor has been developed. The concept of reaction family is successfully applied. A structural contribution approach is used to obtain kinetics and adsorption parameters. A series of procedures are developed to solve the complex problem. Thereafter, a process optimisation model has been developed with the consideration of catalyst deactivation, with a new strategy on the division of catalyst life. Finally, a two-level decomposition optimisation method is extended to incorporate molecular modelling into the overall refinery optimisation, and then applied in two aspects. Firstly, with the integration of the process and the site-level models, a better perspective is obtained with regard to a material processing system. By molecular modelling of refining streams and processes, the integrated approach not only controls the molecules in products properly, but also increases the overall performance. In the second application, a framework integrating a hydrogen network with hydroprocesses is developed to target the maximum profit, rather than saving hydrogen. It allocates hydrogen on the hydrogen network level and utilise hydrogen efficiently on the process level by optimising operating conditions. Consequently, the extent of achieving the maximum profit could be fully exploited with optimal hydrogen utilisation.

Cassava (Manihot esculenta Crantz) is a basic staple food crop in Tanzania. Cassava mosaic disease (CMD) caused by cassava mosaic geminiviruses (CMGs) constitutes a major limiting factor to cassava production in the country. This study was undertaken to characterize the CMGs occurring in Tanzania using molecular techniques and to map their geographical distribution to generate information on which the formulation of control measures can be based. Using Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) for analysis of CMGs DNA-A genomes, different CMGs were found to be associated with CMD. Higher molecular diversity was observed among East African cassava mosaic viruses (EACMVs) than African cassava mosaic viruses (ACMVs), which was confirmed later by complete nucleotide sequence analysis. In addition to EACMV and ACMV isolates, two isolates of EACMV Cameroon virus (EACMCV) were found in Tanzania. These were confirmed to be strains of EACMCV Cameroon, originally described in Cameroon, West Africa and here named EACMCV- [TZ1] and EACMCV-[TZ7]. They had high (92%) overall DNA-A nucleotide sequence identity and EACMCV-[TZ1] was widespread in the southern part of the country. A subgenomic DNA form of CMG that appeared to be truncated was identified in a CMD-infected cassava plant. It was confirmed upon sequence analysis to be a defect of EACMV DNA-A and had a capacity of attenuating symptoms when coinoculated with wild-type EACMV. In addition, this study revealed for the first time the presence of two novel non-geminivirus single-stranded DNA (ssDNA) sub-genomic molecules associated with CMG infection. They were shown to be dependent on CMG for replication and movement within the plants, confirming their status as satellite molecules named here as satDNA-II and satDNA-III. When present in coinfection with CMGs, they enhance symptoms and can break high levels of resistance in a cassava landrace. Finally a simple, inexpensive technique is described of archiving, transporting and recovering plant DNA for downstream geminivirus characterisation. / Thesis (PhD)--University of Pretoria, 2007. / Microbiology and Plant Pathology / Unrestricted

Hepatitis A virus (HAV) is a non-enveloped, positively charged single stranded RNA hepatotropic agent from the family Picornaviridae, and the sole member of the genus Hepatovirus. There is only one HAV serotype but there are seven genotypes. Hepatitis A (HA) infection is usually self-limiting and the severity of the illness is age dependant. In children, infection with HAV is usually asymptomatic, while most adults and immunocompromised patients develop moderate to severe clinical disease. HA is endemic in South Africa (SA) with 100% of children from the lower socio-economic population acquiring immunity before the age of 10. With the current trends in urbanisation, a change in the epidemic vulnerability of the SA population can be expected. HAV is predominantly transmitted by the faecal-oral route and contaminated food and water are important sources of infection. However, the contribution of waterborne HAV to the burden of HA disease in SA is unknown. The aims of this investigation were to assess techniques for the recovery, isolation and detection of HAV from water sources. Thereafter these techniques were applied to estimate the potential risk of infection posed to communities using the water sources for recreational and domestic purposes. This would elucidate whether or not water plays a role in the spread of HAV infection in SA. An effective and sensitive concentration method is fundamental to the successful detection of HAV in water sources. Three primary recovery and two secondary concentration techniques were investigated in this study. An in-house modified glass wool technique, using 15g of glass wool and with the addition of three metal gauze grids at 5g intervals, proved to be the most sufficient and cost effective technique for the primary recovery of HAV from water sources. The packing density of the glass wool and positioning of the grids proved to be essential for efficient HAV recovery. A polyethylene glycol/sodium chloride secondary concentration technique proved to be more cost effective than commercial centrifugal devices. Combinations of cell cultures, propagation conditions, RNA extraction protocols and detection techniques were assessed for the isolation and detection of HAV. A combination of FRhK-4R cell culture propagation and reverse transcription-polymerase chain reaction-oligonucleotide probe assay was demonstrated to be the simple, most efficient technique for the detection of HAV. The nucleotide sequences of HAV strains from water sources and clinical specimens were compared to ascertain whether the strains from water were a potential source of infection. Although the majority of clinical strains clustered seperately from the water strains, one strain from an asymptomatic patient was identical to a number of strains from water. This suggests that HAV in the environment is a potential source of infection in SA. To assess the potential risk of infection constituted by HAV to persons using surface dam and river water for domestic and recreational purposes, a deterministic exponential risk assessment model which works with mean values and conservative assumptions was applied. Results indicated a minimal risk of infection to the higher socio-economic, non-immune population using the water for recreational purposes, if 100 ml of water was ingested per day. No risk was identified for the lower socio-economic, predominantly immune, population who uses the same water sources for domestic and drinking purposes. This study represents the first comprehensive data on risk of infection constituted by waterborne HAV in SA. / Dissertation (MSc)--University of Pretoria, 2008. / Medical Virology / unrestricted

Malaria is one of the most serious tropical infectious diseases affecting mankind. The prevention of the disease is hampered by the increasing resistance of the parasite to existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets and drugs is therefore enormous and the understanding of the biochemistry of the parasite is imperative. The aim of this study was the identification and molecular characterisation of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the human malaria parasite P. falciparum. The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique, RACE. No sequence data were available and the primers used were based on consensus areas identified in the protein sequences from other related organisms. The isolation and identification of the eDNA with degenerate primers was successful in 3' -RACE, but necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as starting material. The sequence obtained facilitated the application of 5' -RACE with ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC eDNA sequence was obtained by overlap-alignment of various segments. A novel suppression PCR technology was applied during the 5' -RACE in order to create an uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and translates to a large 939 amino acid protein. The protein contained large internal insertions and was extended by '""273 N-terminal residues compared to ODCs from other organisms. Several possible signature motifs were identified for phosphorylation, glycosylation and transamidation. The P. falciparum ODC protein seems to contain more hydrophilic and a-helix forming residues. These characteristics should be further investigated after expression of the recombinant protein. The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as an antimalarial target. / Dissertation (MSc)--University of Pretoria, 1998. / gm2014 / Biochemistry / unrestricted

The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase (MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that participates in the fourth step of the leucine catabolism is defective. Tandem mass spectrometry (MS/MS) based screening programmes in North America, Europe and Australia, showed that MCC deficiency is the most frequent organic aciduria detected, with an average frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering from mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3- hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine. Several groups are currently working on various aspects of this emerging disease with the focus on the molecular characterisation of MCC deficiency. In the RSA no molecular based diagnostic method which complements MS/MS screening programmes have yet been implemented. Therefore, the aim of this study was to implement the necessary techniques for the molecular characterisation of MCC deficiency, the determination of the sequence of the open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are present in the South African MCC deficient patient. For the implementation of the molecular characterisation, a two-pronged approached was used to characterize MCC of a MCC non-deficient individual (CFC). This approach included the reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected (genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with MCC deficiency in Caucasians. The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al., 2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or described mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5, mccB6 and mccB5-6. The implemented molecular approach was used to characterise MCC of our MCC deficient patient (HGS). The patient did not have any mutation in the four selected exons mccA8, mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of both ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes all 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17). The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficient individual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS. The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and 9-17 was sequenced but no described or novel mutations were identified. The lack of sequence data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency in HGS. In conclusion, the basic methods and techniques for the molecular characterisation of MCC deficient patients have been implemented locally. A few additional sequencing primers need to be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to be further refined to ensure better specificity. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.

Clavicipitaceous fungal endophytes of the genera Epichloë and Neotyphodium form symbioses with grasses of the family Pooideae in which they can synthesise an array of bioprotective alkaloids. Some strains produce the ergot alkaloid ergovaline, which is implicated in livestock toxicoses caused by ingestion of endophyteinfected grasses. Cloning and analysis of a plant-induced non-ribosomal peptide synthetase (NRPS) gene from Neotyphodium lolii and analysis of the E. festucae E2368 genome sequence revealed a complex gene cluster for ergot alkaloid biosynthesis. The EAS cluster contained a single-module NRPS gene, lpsB, and other genes orthologous to genes in the ergopeptine gene cluster of Claviceps purpurea and the clavine cluster of Aspergillus fumigatus. Functional analysis of lpsB confirmed its role in ergovaline synthesis and bioassays with the lpsB mutant unexpectedly suggested that ergovaline was not required for black beetle (Heteronychus arator) feeding deterrence from epichloë-infected grasses. Southern analysis showed the cluster was linked with previously identified ergot alkaloid biosynthetic genes, dmaW and lpsA, at a subtelomeric location. The ergovaline genes are closely associated with transposon relics, including retrotransposons, autonomous DNA transposons and miniature inverted-repeat transposable elements (MITEs), which are very rare in other fungi. All genes in the cluster were highly expressed in planta but expression was very low or undetectable in mycelia from axenic culture, including under nitrogen-, carbonor phosphate-limited conditions. Comparative analysis of the EAS gene cluster in four different epichloë strains showed marked differences in gene expression and ergot alkaloid synthesis. Gene order is conserved in each strain although evidence for recombination between two MITEs and expansion or reduction of a simple sequence repeat (SSR) at a single intergenic region was observed. Heterologous expression of a candidate regulatory gene, laeA, from Aspergillus nidulans, which is a global regulator of secondary metabolism in aspergilli, did not affect eas gene expression. This, along with phylogeny and microsynteny analysis, suggests there is not an orthologue of this gene in epichloë. This work provides a genetic foundation for elucidating biochemical steps in the ergovaline pathway, the ecological role of individual ergot alkaloid compounds, and the regulation of their synthesis in planta.

The perception is that inborn errors of metabolism (IEM) are rare, but the reality is that more than 600 lEMs are now recognized. The organic aciduria, 3-methylcrotonyl-CoA carboxylase (MCC) deficiency arises when 3-methylcrotonyl-Coenzyme A (CoA) carboxylase that participates in the fourth step of the leucine catabolism is defective. Tandem mass spectrometry (MS/MS) based screening programmes in North America, Europe and Australia, showed that MCC deficiency is the most frequent organic aciduria detected, with an average frequency of 1:50 000. Therefore MCC deficiency is considered an emerging disease in these regions. The incidence of MCC deficiency in the Republic of South Africa (RSA) is not yet known. However, one 48 year old male Caucasian individual (HGS) was diagnosed suffering from mild MCC deficiency, since elevated levels of 3-hydroxyisovaleric acid, 3- hydroxyisovalerylcarnitine, 3-methylcrotonylglycine was present in his urine. Several groups are currently working on various aspects of this emerging disease with the focus on the molecular characterisation of MCC deficiency. In the RSA no molecular based diagnostic method which complements MS/MS screening programmes have yet been implemented. Therefore, the aim of this study was to implement the necessary techniques for the molecular characterisation of MCC deficiency, the determination of the sequence of the open reading frame (ORF) of mccA and mccB subunits to determine which mutation(s) are present in the South African MCC deficient patient. For the implementation of the molecular characterisation, a two-pronged approached was used to characterize MCC of a MCC non-deficient individual (CFC). This approach included the reverse transcriptase polymerase chain reaction (RT-PCR) amplification of the ORFs of the associated genes [mccA (19 exons) and mccB (17 exons] and the PCR amplification of selected (genomic deoxyribonucleic acid (gDNA) regions (exons mccA8, mccA11 , mccB5, mccB6 and mccB5-intron 5-6 exon 6 (mccB5-6) which have been found to have mutations associated with MCC deficiency in Caucasians. The sequence analyses produced surprising results of the amplified ORFs (CFCmccA and CFCmccB) of the MCC non-deficient individual CFC. A non-synonymous single nucleotide polymorphism (SNP) (1391C→A, H464P) associated with MCC deficiency (Gallardo et al., 2001) was identified in the CFCmccA subunit. Another SNP (1368G→A, A456A) recently listed in GenBank was observed in the amplified CFCmccB ORF. No significant novel variations or described mutations were identified in the amplified genomic regions mccA8, mccA11 ,mccB5, mccB6 and mccB5-6. The implemented molecular approach was used to characterise MCC of our MCC deficient patient (HGS). The patient did not have any mutation in the four selected exons mccA8, mccA11, mccB5, mccB6 or the genomic region mccB5-6. The RT-PCR amplification of both ORFs (HGSmccA and HGSmccB) resulted in multiple amplicons. Gel extracted amplicons of the expected size were sequenced. Of the 36 exons, 34 exons were sequenced. This includes all 19 exons of HGSmccA and 15 of 17 exons of HGSmccB (exons 1-6 and exons 9-17). The non-synonymous SNP (1391C→A, H464P) detected in CFCmccA (MCC non-deficient individual), seems to be present in the HGSmccA subunit of the MCC deficient individual, HGS. The HGSmccB amplicons could not be entirely sequenced. However, the region exon 1-6 and 9-17 was sequenced but no described or novel mutations were identified. The lack of sequence data of region exon 7-8 led to an incomplete molecular characterisation of the MCC deficiency in HGS. In conclusion, the basic methods and techniques for the molecular characterisation of MCC deficient patients have been implemented locally. A few additional sequencing primers need to be designed to cover mccB7 and mccB8 as well as the entire coding and non-coding strands of each MCC gene (mccA and mccB). The primers for RT-PCR of both mccA and mccB need to be further refined to ensure better specificity. / Thesis (M.Sc. (Biochemistry))--North-West University, Potchefstroom Campus, 2007.