Browsing by Subject "Molecular and Cell Biology"

Now showing 1 - 20 of 157
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    Open Access
    The amino acid sequence of chicken histone F3
    (1973) Brandt, Wolf Friedrich; Von Holt, Claus
    Histone F3 (III) from chicken erythrocytes was isolated by selective extraction from nucleoprotein with ethanolic-HCl and purified by a single gel filtration step. This protein was found to be homogeneous by the following criteria: gel filtration, electrophoretic mobility, N- and C-terminal amino acid residues and amino acid analysis. The primary structure of this histone was established without resorting to the use of overlapping sequences. This has been achieved with specific chemical cleavages rather than enzymatic degradations chosen and applied, first to the original protein chain, and subsequently to the generated polypeptides, to yield sets of not more than 3 peptides in any single cleavage. Their relative position in the protein or polypeptides became evident after comparison of the N- and C-terminal amino acids in the cleavage products and the uncleaved starting material. The simplicity of the peptide mixture after each cleavage, resulting in easy separation of the peptides, together with the highly efficient Edman degradation of automatic sequencing, allowed a rapid and relatively nonlaborious primary structure determination. Finally, the amino acid sequence is compared with those of protamines and other histones. The evolution and the structure of this protein in relation to DNA is briefly considered.
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    An investigation into the role of cytosolic free Ca2+ in Salicylic acid mediation of disease resistance in Arabidopsis
    (2001) Petersen, Lindsay Natalie; Denby, Katherine J
    Salicylic acid (SA) accumulation upon pathogen attack is a fundamental requirement for the activation of numerous plant defence mechansms. Cytosolic free Ca2+ ([Ca2+]c) is a ubiquitous signalling molecule involved in a host of cellular processes. Using transgenic Arabidopsis thaliana seedlings expressing the Ca2+-sensitive photoprotein aequorin, we previously reported a rapid and transient increase in [Ca2+]c upon application of exogenous SA. We now investigated the characteristics of the SA-induced [Ca2+]c increase and report that the majority of the response is derived from internal stores. It appears likely that SA triggers Ca2+-induced Ca2+-release. Preliminary evidence suggests a role for the SA-induced [Ca2+]c increase in the regulation of NPR1 expression since modulation of the SA-induced [Ca2+]c response with the extracellular Ca2+ chelator BAPTA causes a reduction in NPR1 mRNA levels. We have isolated two putative mutants that are defective in their ability to produce a SA-induced [Ca2+]c increase. Characterisation of these mutants is underway and will prove invaluable in identifying the components or events that cause the SA-induced [Ca2+]c transient, thereby aiding in the understanding of the role of [Ca2+]c in SA-mediated signal transduction.
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    An investigation of the role of Arabidopsis thaliana plant natriuretic peptide in planta
    (2009) Donaldson, Lara Elizabeth; Denby, Katherine; Gehring, Chris; Ingle, Rob
    The sessile nature of plants demands that they respond appropriately to changes in their environment (stresses) in order to survive. Critical to survival is the maintenance of water and ion homeostasis. The mechanisms by which plants achieve this are poorly understood. Traditionally plant stress responses were thought to be communicated by five classical plant hormones - auxin, cytokine, gibberellic acid, absisic acid and ethylene. Nowadays a plethora of other molecules are known to fulfil this function including nitric oxide, salicylic acid, jasmonic acid, brassinosteroids and peptide hormones. Plant natriuretic peptides have been proposed to be peptide hormones involved in maintaining water and ion homeostasis in plants. Evidence for this has been provided by studies of plant responses to exogenous natriuretic peptide treatment, however a demonstration of their function in planta remains outstanding. This study was undertaken to gain insight into the mechanisms regulating water and ion homeostasis in Arabdopsis by examining second messenger responses to stresses that perturb water and ion homeostasis; characterization of an Arabidopsis thaliana plant natriuretic peptide (atpnp-a) mutant and transcriptome analysis of AtPNP-A, in order to establish whether AtPNP-A plays a role in maintaining water and ion homeostasis in planta. Results indicated that recombinant AtPNP-A induces second messenger responses reminiscent of the response to NaCl, suggesting that AtPNP-A may play a signalling role in response to disturbances in water and ion homeostasis. In support of this, characterization of an atpnp-a mutant revealed that AtPNP-A is likely to be involved in processes that require adjustments to water and ion homeostasis including cell expansion, stomatal opening and NaCl and osmotic stress responses, consistent with reported responses to natriuretic peptide treatment. Furthermore, the atpnp-a mutant revealed a role for AtPNP-A in the defence response. Evidence to support this came from the computational analysis of AtPNP-A expression which correlates with genes involved in the defence response. Additionally, the transcriptome response to recombinant AtPNP-A treatment further implicated the involvement of AtPNP-A in the defence response. Therefore AtPNP-A is hypothesized to play a role in growth, abiotic and biotic stress responses that enables the plant to mount an integrated response to the environment.
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    Analysis of Actinobacterial Biodiversity in Marine Sediment from Gericke's Point (South Africa) and Screening of Isolates for Novel Antimycobacterial Compounds
    (2022) Dreyer, Ashleigh; Meyers, Paul
    Thirty-three (33) presumptive actinobacterial strains were isolated using traditional culturebased techniques from sediment taken from marine habitats (a subtidal zone, a rock pool and a beach area) at Gericke's Point (Garden Route National Park, Sedgefield, South Africa). Twenty-seven (27) of the 33 presumptive actinobacterial isolates were identified to the genus level: 26 Streptomyces strains and one Nocardia strain. The partial 16S-rRNA gene sequences obtained for each confirmed actinobacterial isolate were used to determine their phylogenetic positions within their respective genera. Further investigation of specific isolates was done utilising the gyrB gene to determine whether these isolates are clones. Metagenomic data generated from next-generation sequencing of 16S-rRNA amplicons were used to reveal the actinobacterial biodiversity of the Gericke's Point sediment that was not seen in the culture-dependent part of this study. A total of 1 541 544 actinobacterial partial 16S-rRNA gene sequences were identified using the SILVA 16S-rRNA gene database. Actinobacteria that could not be assigned to a class or order made up ~41% of the total actinobacterial strains found in the Gericke's Point sediment samples. The rest of the identified actinobacterial strains belonged to the orders Candidatus Microtrichales (~45%), Candidatus Actinomarinales (~9%), Propionibacteriales (~3%) and other actinobacterial orders that each made up less than one percent (<1%) of the actinobacterial strains found in Gericke's Point. The other actinobacterial orders include Bifidobacteriales, Euzebyales, Frankiales, Geodermatophilales, Micrococcales, Micromonosporales, Mycobacteriales, Pseudonocardiales, Streptomycetales and Streptosporangiales. This is one of the first detections of Frankiales strains in a marine environment. The majority (99%) of actinobacterial strains identified at Gericke's Point could not be assigned to a known genus. This represents an abundance of novel actinobacterial diversity that has yet to be revealed. Multidrug-resistance in Mycobacterium tuberculosis is a global threat to public health which has increased the need for new antibiotics to treat tuberculosis. In this study, all confirmed actinobacterial isolates and two presumptive actinobacterial isolates (29 strains in total) were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard agar overlay method. To investigate their spectrum of antibiotic activity, all isolates were also screened for activity against Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923. Twenty-one (21) isolates (20 Streptomyces strains and one unidentified strain) displayed strong to very strong antimycobacterial activity (defined as a zone of growth inhibition of over 2000 mm2 ). In addition, two Streptomyces strains displayed strong to very strong activity against S. aureus ATCC 25923. Compounds that displayed strong antimycobacterial activity were analysed using High Performance Liquid Chromatography-Mass Spectrometry and resulting mass spectra were compared to those of known compounds within the Global Natural Products Social Molecular Networking (GNPS) database. Eighteen (18) strains produced compounds with no matches in the GNPS database indicating these compounds could be novel. One strain produced a potential analogue of abyssomicin L (a rare antibiotic). Overall, the results obtained in this study emphasize the potential of marine environments as a source of novel actinobacteria and novel bioactive compounds.
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    Analysis of actinobacterial biodiversity in reservoir sediment and cave soil and screening of isolates for antimycobacterial activity
    (2020) Rakiep, Adeebah; Meyers, Paul
    A total of 56 presumptive actinobacterial strains was isolated from three different samples taken from the Silvermine Nature Reserve (Table Mountain National Park, Cape Town), namely, cave soil, the wall of the cave and sediment from the shallow waters of a reservoir. Twenty nine (29) isolates were successfully identified to the genus level by 16S-rRNA gene analysis: one Micrococcus strain, one Streptacidiphilus strain, one Micromonospora strain and 26 Streptomyces strains. The phylogenetic position of each identified strain within its genus was investigated by generating a phylogenetic tree based on its 16S-rRNA gene sequence. Further analysis of the Streptacidiphilus strain was conducted based on the gyrB gene. Metagenomic analysis was used to further analyse the actinobacterial diversity of the freshwater reservoir sediment from the Silvermine Nature Reserve. A total of 97 16S-rRNA gene clones was obtained from the reservoir sediment sample, RS1, using actinobacteriumspecific 16S-rRNA gene primers S-C-Act-0235-a-S-20-F and S-C-Act-0878-a-A-19-R and each clone was identified using the EzBioCloud database. Analysis based on unique phylotypes in the clone library revealed that 80% of the clone library was composed of actinobacterial strains belonging to the orders Acidimicrobiales, Streptomycetales, Streptosporangiales, Corynebacteriales, Sporichthyales and the family Jatrophihabitandaceae (the remaining 20% was identified as non-actinobacterial strains). The percentage composition of the actinobacterial clonal diversity for each order was as follows: Acidimicrobiales, 56%; Streptomycetales, 29%; Streptosporangiales, 9%; Corynebacteriales, 4%; Sporichthyales, 1% and family Jatrophihabitandaceae, 1%. Rarefaction analysis revealed that the total actinobacterial diversity of the sample was not represented in the clone library. Therefore, further sampling and analysis of the sample site would uncover greater actinobacterial diversity. Thirty seven (37) putative actinobacterial isolates of the 56 that were isolated from the Silvermine Nature Reserve were screened for antimycobacterial activity against the non-pathogenic Mycobacterium aurum strain A+ using a standard over-lay method. A total of five identified 2 actinobacterial isolates (Streptomyces strains RS6, RS7, RS9, RS13 and RS15) and an unidentified actinobacterium, strain RS4, demonstrated very strong antimycobacterial activity (zone of growth inhibition of over 3000 mm2 ). In addition, 15 of the 37 strains were active against Staphylococcus aureus ATCC 25923 and three were active against Escherichia coli ATCC 25922. Streptomyces strains CS1, CS3, CS12, CS18, CS19, CW5, RS3, RS6, RS9, RS13 and RS15, displaying varying strengths of antimycobacterial antimicrobial activity, were selected for antibiotic extraction from culture broths. The resulting crude extracts were subjected to spot bioautography to test for antibacterial activity. The organic compounds extracted from the cell mass of Streptomyces strain CS3 and the broth fraction of Streptomyces strain RS3 demonstrated strong activity against M. aurum strain A+. Furthermore, the crude extracts of 15 actinobacterial isolates (Micromonospora strain RS10 and Streptomyces strains CS1, CS3, CS12, CS18, CW2, CW5, RS3, RS6, RS7, RS9, RS13, RS15, RS18 and RS19) were additionally tested for antiplasmodial activity against Plasmodium falciparum strain NF54. Seven of these strains showed activity against Plasmodium namely, Streptomyces strains CW2, CW5, RS3, RS7, RS13, RS15 and RS19. Streptomyces strains CW2, CW5 and RS7 displayed the strongest activity against P. falciparum strain NF54 with IC50 values below the guideline threshold of 1000 ng/mL (strain CW2 culture broth crude extract: IC50 40 ng/mL, strain CW5 culture broth crude extract: IC50 128 ng/mL and strain RS7 culture broth crude extract: IC50 70 ng/mL).
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    Analysis of genes and enzymes involved in the degradation of hemicellulose and cellulose by Butyrivibrio fibrisolvens H17c
    (1992) Lin, Long-Liu; Thomson, Jennifer Ann
    B. fibrisolvens H17c is a Gram-positive obligate anaerobe which has been found in the rumen of most ruminants. Strains of B. fibrisol vens have been reported to exhibit activity toward cellulosic and hemicellulosic substrates. The aim of this thesis was to screen a genebank of B. fibrisolvens H17c DNA and to isolate genes expressing cellulase and xylanase activity. Two genes encoding β-1-4- glucosidase (BglA) and endo-β-1-4-xylanase (XynB) were cloned in E. coli.
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    Antiretroviral drugs differentially modulate glucocorticoid activity via the glucocorticoid receptor in vitro
    (2019) Kuipa, Michael; Hapgood, Janet; Moliki, Mosoko; Maritz, Michelle
    Concurrent use of anti-retroviral drugs (ARVs) and progestin-based hormonal contraceptives is widespread. During times of stress and during glucocorticoid (GC) therapy, intracellular ARVs are in the presence of high concentrations of GCs, which regulate all aspects of immunity and inflammation via the glucocorticoid receptor (GR). However, the reciprocal modulation of ARV and steroid intracellular functions is relatively unexplored. In this study, the effects of the ARVs tenofovir disoproxil fumarate (TDF), dapivirine (DPV), and maraviroc (MVC) on activation of the GR and GR-regulated mRNA expression were investigated, in the absence and presence of select GR ligands. The effects of TDF and DPV on GR protein levels and phosphorylation were also determined. The inhibitory activity of these ARVs on HIV-1 infection in the presence of the progestins medroxyprogesterone acetate (MPA) and levonorgestrel (LNG), and a GR agonist, dexamethasone (DEX) was also assessed. This study shows that (0.01 nM-10 µM) TDF, DPV and MVC do not transactivate reporter gene expression via the unliganded GR exogenously expressed in the steroid receptor-deficient U2OS human osteosarcoma cell line, or alter the reporter gene transcriptional activity of (100 nM) MPA or LNG via the GR in these cells. However, (1 µM) TDF and DPV modulate the reporter gene transcriptional efficacy of (0.01 nM-10 µM) DEX via the GR. In the U2OS cell line model, (1 µM) TDF, but not DPV significantly decreased (1µM and 10µM) DEX-induced mRNA expression of the anti-inflammatory glucocorticoid-induced leucine zipper (GILZ) gene. TDF also appeared to decrease (1 µM) cortisol (CORT)- and MPA-induced GILZ mRNA expression. This may be mediated by the apparent increase in (100 nM and 1µM) DEXinduced phosphorylation at Serine 226 on the GR, observed in the presence of (1µM) TDF in this study. DPV and TDF (at 1µM) did not significantly alter GR protein levels, or cell-viability in the absence and presence of (100 nM) DEX, CORT or MPA in U2OS cells. However, (1 µM) DPV and TDF alone, significantly altered cell viability in peripheral blood mononuclear cells (PBMCs). In PBMCs, (1 µM) TDF, MVC and DPV alone altered basal GILZ mRNA expression and had variable, donor-specific effects on interleukin (IL)-6, IL-8, and interferon (IFN)-γ gene expression. In PBMCs from some of the nine donors tested, these ARVs had proinflammatory effects which may undermine their efficacy at preventing HIV-1 acquisition in pre-exposure prophylaxis products. Moreover, the ARVs proinflammatory effects may negatively impact HIV-1 disease progression and increase the risk of non-AIDS mortality in individuals using the ARVs therapeutically. Neither (1 µM) DPV, TDF nor MVC significantly altered the effects of (100 nM) DEX on the immunomodulatory genes assessed in PBMCs. DEX, MPA and LNG (at 100 nM) did not affect the anti-HIV-1 activity of the ARVs (at 1 µM) in PBMCs from the majority of the three donors tested in this study. Taken together, the results show that ARVs can modulate GR activity in an ARV-, steroid-, gene- and cell-specific manner, while the steroids investigated did not modulate ARV anti-HIV-1 activity.
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    ASMT gene polymorphisms are associated with Autism Spectrum Disorder (ASD) symptom severity in a South African population
    (2016) De Waal, Margaretha; O'Ryan, Colleen; Roden, Laura
    Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterised by behavioural and social impairments. ASD shows evidence of a genetic aetiology, with a large body of research linking ASD to polymorphisms in several different genes and gene families, including those involved in circadian rhythm generation and melatonin biosynthesis. Sleep disorders are highly comorbid with ASD in both children and adults, and range from sleep onset delay, phase shift and sleep disruption. These parasomnias can have a significant impact on the quality of life for persons with ASD and their families, and sleep deprivation can feed into the behavioural deficits in ASD. Melatonin supplementation is often prescribed to assist in alleviating the above mentioned sleep dysfunction. Melatonin is a hormone in the circadian clock system, and is a biochemical signal for darkness to synchronise peripheral cells to the master oscillator. Clinical trials reported that melatonin supplementation at night assists in sleep initiation. However both the mode of action of supplemental melatonin, as well as whether melatonin deficiency is common in ASD, remains unclear. Furthermore, any research on ASD is often hamstrung by the heterogeneous nature of the disorder, necessitating clear phenotyping. This study examines single nucleotide polymorphisms (SNPs) in the gene acetylserotonin methyl transferase (ASMT), which encodes an enzyme in melatonin biosynthesis, in a South African ASD cohort (n=28) and controls (n=6). All participants completed and Autism Diagnostic Observation Schedule-2 assessment that allowed partitioning of the ASD individuals into ASD endophenotypes, to reduce phenotyping heterogeneity. This study found SNPs previously associated with ASD in the promoter and intronic region. Additionally, this study found novel SNPs, and a SNP in a putative transcription factor binding site not previously associated with ASD. The associations found between SNPs and ASD endophenotypes, together with the positions of the SNPs, suggest a potential link between ASMT polymorphisms and ASD symptom severity. Further research, using language assessment tools as well as quantitative measures of melatonin and sleep disruption, may establish the role of melatonin in language impairment in ASD.
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    Autism Spectrum Disorder and Mitochondrial Dysfunction: The Role of Mitochondrial Dynamics
    (2022) Buchanan, Erin; O'ryan, Colleen
    Numerous genes and biological pathways are implicated in the aetiology of the neurodevelopmental disorder, autism spectrum disorder (ASD). Our research group reported that mitochondrial dysfunction was associated with ASD in South African children diagnosed with ASD using differential methylation and metabolomics studies. Propionyl-CoA Carboxylase Subunit Beta (PCCB) was differentially methylated in our cohort ASD study, and its dysregulation can lead to the accumulation of propionic acid. This links PCCB's function to a well-established animal model that uses sodium propionate (NaP) to study ASD in rats. This thesis aimed to i) examine ASD-associated mitochondrial dysfunction in a neuronal-like cell model using NaP and to investigate its effects on mitochondrial dynamics and morphology; ii) investigate the application of this in a South African ASD cohort by measuring differential methylation of essential genes involved in mitochondrial dynamics. Undifferentiated, neuronal-like SH-SY5Y cells were treated with NaP at 1.5mM, 3mM and 5mM to induce mitochondrial stress. The effects of NaP mitochondrial-induced stress were quantified using MTT and ATP assays. Transmission electron microscopy (TEM), using cryogenic techniques, was used to examine mitochondrial morphology by measuring nine parameters: area, area2 , form factor, area-weighted form factor, aspect ratio, perimeter, circularity, Feret's diameter and roundness. TEM images were analysed using Fiji/Image J. Mitochondrial DNA (MT-DNA) copy number and STOML2 expression were measured using RTqPCR, and these data were compared to TEM data. STOML2 was the most differentially methylated gene in our ASD cohort in our previous study. Differential methylation of six essential genes (DRP1, FIS1, MFN1, MFN2, OPA1, STOML2) involved in mitochondrial fusion and fission were examined using targeted next-generation bisulfite sequencing (tNGBS) between ASD and control participants in a South African cohort. Significance for all experiments was determined using unpaired t-tests, one-way ANOVA and correlation analysis (p< 0.05). SH-SY5Y cells treated with NaP showed mitochondrial dysfunction as reflected in changes in ATP levels and no changes in cell viability were observed except at 9mM NaP. In addition, NaP treatments led to significant changes in mitochondrial morphology with significant decreases in mitochondrial area, perimeter, form factor and Feret's diameter between NaP treatments and control and a significant increase in circularity. These changes in morphological data were supported by a significant increase in MT-DNA copy number at 5mM and significant decreases in STOML2 expression at all concentrations. Together, the TEM and expression data highlight the balancing act between mitochondrial fusion and fission, with increasing levels of fission occurring with increasing NaP concentrations. In the South African cohort, there were significant differences in methylation between the ASD group compared to controls at two CpG sites in MFN2, two CpG sites in STOML2 and one CpG site in FIS1 (significant increases) and two CpG sites in OPA1 (significant decreases). The increase in FIS1 and decrease in OPA1 methylation are consistent with this balancing act between mitochondrial fusion and fission in mitochondrial dysfunction in this cohort. These results suggest a potential link between mitochondrial dysfunction and the fluctuation in mitochondrial dynamics and morphology. The inclusion of TEM demonstrates its unique ability to visualise mitochondrial ultrastructure and the effect changes in mitochondrial dynamics have on morphology as opposed to only examining these changes through gene expression and metabolic assays. Although the exact relationship between the TEM and gene expression data could not be fully explained in this study, it highlights the need to explore further the relationship between mitochondrial dynamics, biogenesis and mitophagy in the context of ASD aetiology and neuronal development.
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    Baseline surveys and metal binding proteins as metal pollution indicators
    (1985) Hennig, Helmke Friedrich-Karl Otto; Orren, Michael J; Branch, George M; Brandt, Wolf F
    The field of metal determination as a part of pollution studies, has been critically examined and metal pollution may be defined in one simple statement: The presence of metal binding proteins confirms toxic metal pollution. It has been shown that current methods of metal determination in biological systems are of little use. This has been illustrated by both a review of metal concentration in Southern African coastal water, sediments and biotopes, and by a comparative baseline study of organisms from Gough Island and Mar ion Island. These showed that extrapolation of results from one geographical area to another are invalid and that this interpretation is made difficult by factors such as age, sex, size life stage of the organisms. Furthermore, it was shown that many reports on metal pollution do not even mention fundamental information such as the size or the sex of the animals. Metal pollution could be linked to metal binding protein through an independent pollution er i ter ia, for example, the out of season moulting of crayfish. The new definition of metal pollution has then been tested by application to five different organisms (crayfish, Jasus lalandii; hermit crab, Diogenes brevirostris; shrimp, Palaemon pacificus; black mussel, Choromytilus meridionalis and limpet, Patella granularis) kept under identical conditions and it was shown that a much more meaningful interpretation of the results could be made. The new definition was al so tested with two naturally occurring metal accumulating organisms (whelk, Bullia digitalis and "kikuyu" grass) and it was shown that dramatic increases in metal may not necessarily be toxic. It was concluded that less effort and time should be spent on metal analysis in determination of metal pollution and attention should rather be directed to the presence or absence of metal-binding proteins.
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    Beak and feather disease virus candidate vaccine development
    (2012) Duvenage, Lucian; Rybicki, Ed; Hitzeroth, Inga; Meyers, Ann
    [Fix supervisors field.] Psittacine beak and feather disease, caused by a circovirus known as beak and feather disease virus (BFDV), is a threat to both wild and captive psittacine species. There is currently no vaccine against BFDV and safe and affordable vaccine candidates are needed to alleviate the disease burden caused by this virus. Production of the BFDV's major antigenic determinant, the capsid protein (CP), in the inexpensive and highly scalable plant expression system, could satisfy these requirements as a potential subunit vaccine. In this work, truncated CP (ÄN40 CP) was first expressed in E. coli to successfully generate anti-CP polyclonal antibodies. ÄN40 CP and full-length CP transient expression in tobacco (Nicotiana benthamiana) was optimised as fusions to elastin-like polypeptide (ELP). Fusion of CP or ÄN40 CP to ELPs of different lengths was shown to increase yield relative to unfused CP/ÄN40 CP. Free ELP and a GFP-ELP fusion could be purified by inverse transition cycling (ITC), using centrifugation and membrane filtration methods. A ÄN40 CP-ELP fusion expressed in plants could be partially purified and represents low-cost vaccine candidate against BFDV. A candidate DNA vaccine expressing ÄN40 CP was also evaluated for expression of the antigen in vitro and may prove useful in a prime-boost regimen together with one of the plant-produced vaccine candidates.
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    Biological control of a plant pathogen and pest by expression of a cloned Serratia marcescens chiA gene and Bacillus thuringiensis cryIA(c) gene in endophytic bacteria
    (1997) Downing, Katrina Jo; Thomson, Jennifer Ann
    Protection of plants from pathogens and pests by introduced microorganisms provides an alternative to environmentally hazardous chemical pesticides and fungicides. Plant associated, free living, non-pathogenic bacteria found in the rhizosphere and phyllo plane have been the emphasis of research on biological control· agents. These regions however pose problems of competition between introduced microorganisms and native microflora and environmental extremes. Endophytic bacteria, present in the interior regions of healthy plants, offer a solution to these problems. Since little work has been done to date with endophytes, the work reported in this thesis comprises a novel approach to achieving biological control of a plant pest and pathogens .An endophytic strain of Pseudomonas fluorescens was isolated from micro propagated apple plantlets and shown to be present in the roots of beans at a level of 1.2 x 10⁵ CFU/g fresh weight 10 days after introduction. Generation of spontaneous rifampicin resistant mutants of this strain resulted in P. fluorescens Rifl. Isolates of P. fluorescens and Aeromonas caviae were recovered from the interior regions of surface sterilized bean seeds. In addition, two sugarcane endophytes, Acetobacter diazotrophicus and Herbaspirillum seropedicae, which has also been isolated from rice, maize and sorghum, as well as an endophytic Citrobacter sp. of pine seeds and bean plants were used in this work. The gene coding for the major chitinase of S. marcescens, chiA, was cloned under the control of the tac promoter by PCR amplification. The gene contained the endogenous Shine Dalgarno sequence 7 bases upstream of the ATG start codon in the plasmid pTC33. The plasmid ptacchiA had previously been constructed with a distance of 20 bases between the ribosome binding site of this vector and the A TG start codon of the gene. Gene expression of chiA carried on pTC33 was shown to be approximately 16-foldhigher than that of ptacchiA. This clearly illustrated that the distance between the Shine Dalgarno sequence and ATG start codon was critical and that the shorter distance of 7 bases significantly increased the translation efficiency of chiA. The first and second generation tacchiA cassettes from ptacchiA and pTC33 respectively were cloned into the broad host range plasmids pKT240, pDER405 and pML122 and into the integration vector pJfF350.These plasmids were introduced into the endophytes P.fluorescens Rift, H. seropedicae and Citrobacter sp.AJl by conjugative transfer and electroporation and were shown to express the gene at varying levels. pKTCl and pMTC33 carrying the 1 and 2nd generation tacchiA cassettes on pKT240 and pML122respectively were not stably maintained in P. fluorescens Rift or H. seropedicae.
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    Characterisation of a maize mutant deficient in antifungal kauralexin accumulation
    (2017) Wighard, Sara; Murray, Shane L; Korsman, Jeanne
    Fusarium verticillioides and Cercospora zeina are two economically important fungal pathogens of maize in Southern Africa. Phytoalexins are low molecular weight anti-microbial compounds produced in plants in response to pathogen infection. In maize, two classes of non-volatile terpenoid phytoalexins, viz. kauralexins and zealexins, play a role in fungal resistance. It has previously been shown that maize lines inoculated with either F. verticillioides or C. zeina induces kauralexin and zealexin accumulation. In addition, kauralexin metabolite accumulation and candidate kauralexin biosynthetic gene expression were highly correlated. In this study a mutant line with a Dissociation transposon element inserted into An2 was identified with the goal of stopping An2 from being expressed. The mutants were maintained in an inbred W22 maize line. Gene expression was compared between transposon-insertion mutants and wild type W22 at the seedling stage. A F. verticillioides and C. zeina inoculation assay was carried out on a segregating knock-down line. Phytoalexin accumulation, gene expression and disease susceptibility were subsequently examined in the mutants and wild type. F. verticillioides-inoculated mutants displayed significantly decreased kauralexin and zealexins accumulation and An2 gene expression. Fungal load and symptoms was greater in mutants than wild type controls. Kauralexin accumulation and An2 expression were negatively correlated with the quantified fungal load. C. zeina-inoculated mutants did not display significantly reduced kauralexin accumulation and An2 expression as An2 did not appear to be upregulated in the W22 maize line in response to C. zeina. This is likely due to a genetically-controlled leaf flecking phenotype in W22 leading to broad-spectrum resistance, as well as potentially impacting the jasmonic acid pathway. Lastly, an attempt was made to clone An2 towards A. thaliana transformation for overexpression analysis. Only a truncated section of An2 was able to be cloned into the expression vector.
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    Characterisation of auxin and auxin-related genes in the response of Arabidopsis thaliana to salt stress
    (2019) Cackett, Lee; Donaldson, Lara; Ingle, Robert
    Soil salinization is prominent in agricultural land and has detrimental effects on plant growth and yield. Salt imposes both an osmotic and ionic stress on plants, and the aim of this study was to investigate the molecular basis of the plant response to the ionic component of salinity stress. To do so, transcriptome profiling using microarrays was performed at two developmental stages (two and four weeks post-germination) exposing plants to either NaCl (which imposes an osmotic and ionic stress) or iso-osmolar sorbitol (which imposes osmotic stress only). Clear transcriptomic differences in the plant response to NaCl and sorbitol were observed, allowing the identification of genes that may be involved specifically in the response to the ionic component of salinity stress. Differences in salt-responsive gene expression were also observed between early and later development as well as root and shoot tissues, indicating that there may be both developmental and tissue specific responses to NaCl. ‘Response to auxin’ was a highly enriched gene ontology term associated with genes that are significantly induced specifically in response to ionic stress, suggesting a potential role for this plant growth hormone in ionic stress tolerance. This hypothesis was supported by mass spectrometry analysis which demonstrated that active IAA levels show a significantly greater increase in response to NaCl than to sorbitol, demonstrating an ionic specific auxin response. In planta quantification and spatial distribution of IAA was further analysed in salt stressed plants using two auxin reporter lines, DR5::GUS and DII::VENUS. These analyses showed that IAA levels increased in the shoot and root tip in response to NaCl. Also, local IAA maxima distributed along the primary root of salt stressed seedlings were decreased compared to the untreated control, which may explain the decrease in lateral root number and primary root bending observed in salt stressed plants. There are several different pathways in the tryptophandependent auxin biosynthesis process, each with unique biosynthetic intermediates and ratelimiting enzymes. For this reason, changes in the levels of the different IAA biosynthetic intermediates were analysed in NaCl and sorbitol stressed Arabidopsis to identify a predominant pathway responsible for increasing IAA during ionic stress. The changes in IAN (the IAA biosynthetic intermediate of the IAOx pathway) in response to NaCl and sorbitol mirrored those observed for IAA. Additionally, mRNA levels of Nitrilase 2 (which converts IAN to IAA) were significantly increased in response to NaCl but not sorbitol. Taken together, these results suggest that IAA biosynthesis is increased in response to NaCl via the hydrolysis of IAN to IAA by NIT2. In agreement with this, a Nit2 overexpressing line (35s::Nit2) displayed both improved survival and growth in the presence of NaCl. Furthermore, the Nit2 overexpressor had increased IAA but deceased IAN compared to wild type plants in response to salt stress. Together these results indicate that Nit2 is likely involved in the salt stress response through altering IAA levels in planta. GH3.12 and ILL6, two genes involved in IAA storage and/or degradation via hydrolysis and conjugation to amino acids, were also differentially expressed in response to NaCl at both developmental stages tested. However, mutants for ill6 and gh3.12 were not altered in their salt tolerance, suggesting that IAA storage and/or degradation may not play an important role in modulating active IAA levels in response to salt stress. Finally, preliminary evidence was obtained that the expression of sorghum Nit2 homologs is also increased in response to salt stress, implying that auxin might play a role in salt tolerance across a phylogenetically broad range of plants, making this a potential route to improve salt tolerance in crop plants. Overall, this study provides novel information indicating that IAA levels are altered specifically in response to the ionic component of salinity stress which may contribute to the altered plant growth observed under these conditions, as well as the identification of an auxin-related candidate gene which can improve salt tolerance in plants when overexpressed.
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    The characterisation of Ornithogalum mosaic virus
    (1991) Burger, Johan Theodorus; Von Wechmar, M Barbara
    Ornithogalum mosaic virus (OMV) is the most serious pathogen of commercially grown Ornithogalum and Lachenalia species in South Africa. Although omithogalum mosaic disease was first reported as early as 1940, attempts to purify or characterise the virus(es) were not successful. The extremely mucilaginous nature of omithogalum and lachenalia plant extracts severely hampered virus purification from these hosts. No alternative propagation host for OMV is known: a virus purification protocol for systemically infected ornithogalum and lachenalia was therefore developed. This method eliminated the mucilage in leaf extracts by hemicellulase digestion. Physicochemical characterisation of purified particles suggested that a single virus was present: it had elongated, filamentous particles with a modal length in the range 720- 760 nm; a single major coat protein of Mᵣ30 000, and a single genomic ssRNA of Mᵣ2.90 x 10⁶ daltons. Oligo(dT)-cellulose chromatography confirmed that the genomic RNA was polyadenylated.
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    Characterisation of phytoalexin accumulation in maize inoculated with Cercospora zeina, the causal organism of grey leaf spot disease
    (2016) Ntuli, Jean Felistas; Murray, Shane; Ingle, Robert A
    Grey Leaf Spot (GLS) is a fungal disease of Zea mays (maize) that is caused by Cercospora zeina. It thrives in sub-tropical climates and causes devastating crop losses of up to 60% in southern Africa where maize is grown as a staple food source. Phytoalexins are low molecular weight anti-microbial bio-chemicals that are synthesised in planta in response to biotic stress. Related studies have characterised many phytoalexins produced in various plants against several diseases. In maize, phytoalexins fall into to two terpenoid groups: kauralexins and zealexins. To date no studies have been carried out that examine the accumulation in maize of phytoalexins in response to C. zeina. This research project found that in maize samples inoculated with C. zeina, kauralexin accumulation significantly increased with disease development stages (T0 - 0 days post inoculation, T1 - 17 dpi, T2 - 18 dpi and T3 - 24 dpi) while zealexins did not change. Gene expression of the phytoalexin biosynthesis genes TPS6 and TPS11 (both encoding the protein terpene synthase 6/11, specific for zealexins) and CPPS2 (encoding ent-copalyl diphosphate synthase 2, specific for kauralexins) increased significantly at each time point, reaching a maximum level at T2. Infiltration of maize leaves with a chitosan elicitor to mimic fungal pathogen associated molecular pattern (PAMP), and a subsequent callose assay showed positive induction of a callose defence response. However, gene expression and phytoalexin accumulation did not change following chitosan treatment, although zealexin accumulation was higher than kauralexins. Previous studies have shown that phytoalexins accumulate transiently in seedlings. Six diverse Southern African maize lines were compared for phytoalexin accumulation at seedling stage. Zealexin accumulation was generally higher than kauralexins and there were significant differences in both zealexin and kauralexin accumulation in different lines. Gene expression analysis using Genevestigator looked at microarray files and found that expression of TPS6/11 (zealexin biosynthesis) and CPPS2 (kauralexin biosynthesis) genes to be largely co-regulated and highly expressed in response to fungal pathogens, nematodes, insect pests and abiotic stresses; Ustilago maydis, Phytophthora cinnamomi, Fusarium moniliforme, Colletotrichum graminicola, Sporisorium reilianum, Meloidogyne incognita, Ostrinia nubilalis, waterlogging and drought stress. Finally promoter region analysis showed similar cis-acting regulatory elements in the 1kb region upstream of the promoter of both genes and defence specific elements. Thus kauralexin phytoalexins are produced in response to C. zeina inoculation, chitin is not likely to be the key PAMP leading to phytoalexin accumulation, phytoalexin accumulation in seedlings is genotype-dependent and phytoalexin biosynthesis genes are expressed under different conditions suggesting a wider range of action beyond repelling fungal pathogens.
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    Characterisation of putative metal transport proteins in the nickel hyperaccumulator Senecio coronatus: investigating candidate genes for nickel tolerance and accumulation
    (2017) Cowlin, Ross Martin; Ingle, Robert A
    The accumulation of exceptionally high concentrations of heavy metals in plant tissues is an extreme phenotypic trait that has evolved independently in multiple plant taxa. The majority of research undertaken in this area has been performed on zinc/cadmium hyperaccumulators and comparatively little is known about the molecular mechanisms behind nickel accumulation. This is despite the fact that nickel hyperaccumulators constitute more than 75% of all known hyperaccumulator species. One such species is Senecio coronatus (Asteraceae), which is a useful model to study nickel hyperaccumulation - as both hyperaccumulator and non-accumulator populations have been identified on nickel-rich serpentine soils in South Africa. The nickel-transporting abilities of three proteins (ScMATE, ScVIT and ScCOP), previously shown to be constitutively over-expressed in shoot tissues of hyperaccumulating populations of S. coronatus, were investigated in order to determine if they play a role in nickel hyperaccumulation. The RNA-Seq derived nucleotide sequences of these genes were confirmed by reverse transcriptase PCR, and computational analysis suggested that the proteins encoded by these genes display identical topology to their homologues in Arabidopsis thaliana. Heterologous expression of these proteins in a metal-sensitive yeast strain was performed to determine whether they are capable of transporting nickel. Although a minor reduction in nickel sensitivity was observed in yeast expressing ScMATE, and a minor increase in ScCOP-expressing yeast, no marked changes in sensitivity to nickel were observed. C-terminal EYFP-tagged MATE and VIT fusion proteins were transiently expressed in live onion cells to determine the subcellular localization of these proteins in planta. Fluorescence microscopy indicated that MATE localises to the nucleus and VIT to the tonoplast or plasma membrane.
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    Characterisation of two desiccation-linked dehydrins from Xerophyta humilis
    (2016) Fan, Cynthia; Farrant, Jill M; Rafudeen, Suhail
    In response to abiotic stresses, organisms throughout the plant kingdom, as well as microorganisms and micro-animals such as nematodes or tardigrades, have been observed to express Late Embryogenesis Abundant (LEA) proteins as protective mechanisms. However, despite two decades of research, little is understood about their physiological functions and this has led to extensive nomenclature, with a large amount of redundancy. The primary reason for this lack of insight into LEA protein functions is their highly hydrophilic and intrinsically disordered nature. Intrinsically disordered proteins (IDPs) cannot be studied using conventional methods of structural analyses such as X-ray crystallography and, therefore, alternative techniques are required. A combination of transgenic and in vitro studies have also shown that LEA proteins are most likely to behave as molecular chaperones by binding water and ions, preventing macromolecular aggregation and protecting enzymatic activity during dehydration. This study characterized two dehydrins that were expressed during dehydration in the desiccation tolerant plant, Xerophyta humilis. From a transcriptome analyses on X. humilis, cDNA for the two dehydrins were obtained. These sequences were first analysed using various in silico tools in order to identify putative dehydrin-specific characteristics. Subsequently, these two dehydrins were cloned and expressed for production of recombinant dehydrin protein. These proteins were then analysed in terms of structural and functional characteristics. Structurally, through the use of circular dichroism in an in vitro system, both dehydrins demonstrated the shift towards being increasingly alpha-helical when placed in environments of decreasing water content. The role of these two dehydrins in stabilizing enzymes during dehydration was subsequently investigated using citrate synthase (CS) and lactate dehydrogenase (LDH). The preservation of enzyme activity was observed in both CS and LDH. This preservation of enzyme activity was further maintained by the presence of trehalose. Anti-aggregation roles were also investigated, however, neither dehydrin demonstrated significant ability to minimize the aggregation of LDH. This study hopes to establish a pipeline for characterizing LEA proteins using structural and functional assays in order to provide alternative means of LEA protein classification.
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    Characterization of an alkalophilic Bacillus brevis isolate with respect to its endo-(1,3-1,4)-β-glucanase gene, protein hyperproduction and the degS-degU operon
    (1994) Louw, Maureen Elizabeth; Reid, Sharon J
    Bacillus brevis Alk 36 was isolated from soil during a screening programme for the selection of extracellular enzyme producing strains. A gene coding for an endo(1,3- 1,4 )-.8-glucanase (or lichenase) was cloned from B. brevis Alk 36 and expressed in Escherichia coli. The nucleotide sequence of this gene was determined and found to encode a protein of 252 amino acid residues. The amino acid sequence of the B. brevis lichenase gene showed only a 50% similarity to previously published data for Bacillus endo-(1,3-1,4)-β-glucanases. The enzyme exhibited some unique properties. The optimum temperature and pH for enzyme activity were 65-70°C and 8-10, respectively. When held at 75°C for 1 h, 75% residual activity was measured. The molecular mass was estimated to be 29 kDa and the enzyme was found to be resistant to sodium dodecyl sulphate (SDS). B. brevis Alk 36 was evaluated as a potential host strain for the efficient production and secretion of foreign proteins and was found to grow optimally between pH 8.0 and pH 9.5 and between 42°C and 52°C. B. brevis was successfully transformed using vector DNA and was found to produce relatively low levels of protease. In addition, it was evaluated as a possible protein hyper-secreting strain. However, using PCR technology, the highly conserved cell wall protein genes could not be positively identified in B. brevis Alk 36.
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    Characterization of endoglucanase cela from the rumen bacterium Clostridium longisporum
    (1994) Mittendorf, Volker; Thomson, Jennifer Ann
    Cellulose is the most abundant organic compound on earth and offers great potential as a source of renewable energy and other chemicals. Cellulases are being studied to elucidate the enzymatic degradation of cellulose. We are interested in the molecular mechanisms of microbial degradation of cellulose in the rumen. The long-term aim is the potential genetic modification of lignocellulolytic activities in the rumen. · Clostridium longisporum was obtained from Varel (1989) because oxygen-resistant endospores might be suitable "vectors" for the introduction of genetically modified enzyme systems into the rumen via animal feeds. It is a sporadically occurring rumen bacterium and its role in ruminal cellulolysis is unclear. The aim of this project was the initial characterization of cellulases produced by C. longisporum. The celA gene was obtained by screening a library of C. longisporum genomic DNA in Escherichia colifor clones expressing CMCase activity. Approximately 38 CMCase-positive clones were obtained and the plasmid pCM4 was isolated from the clone expressing the highest activity. Southern analysis indicated that another plasmid, pCM64, contained a larger insert including the insert of pCM4. A total of 3620 bp were sequenced and a 1548-bp open reading frame, termed celA, was found. This gene showed homology with other endo-B-1,4-glucanases from family 5 (Henrissat & Bairoch, 1993). Plasmid pCM64 was found to contain the whole celA gene encoding endoglucanase CelA, while pCM4 has a 5'-truncated gene, termed celM5', which encodes a fusion protein, CelMN', that was initiated from an ATG codon in the vector. Sequence analysis of celA revealed the presence of a type I cellulose-binding domain (Beguin & Aubert, 1994) at the COOH-terminus of CelA.