Bridget Genevieve Crampton
University of Pretoria
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Featured researches published by Bridget Genevieve Crampton.
BioTechniques | 2004
Noëlani van den Berg; Bridget Genevieve Crampton; Ingo Hein; Paul R. J. Birch; Dave K. Berger
Efficient construction of cDNA libraries enriched for differentially expressed transcripts is an important first step in many biological investigations. We present a quantitative procedure for screening cDNA libraries constructed by suppression subtractive hybridization (SSH). The methodology was applied to two independent SSHs from pearl millet and banana. Following two-color cyanin dye labeling and hybridization of subtracted tester with either unsubtracted driver or unsubtracted tester cDNAs to the SSH libraries arrayed on glass slides, two values were calculated for each clone, an enrichment ratio 1 (ER1) and an enrichment ratio 2 (ER2). Graphical representation of ER1 and ER2 enabled the identification of clones that were likely to represent up-regulated transcripts. Normalization of each clone by the SSH process was determined from the ER2 values, thereby indicating whether clones represented rare or abundant transcripts. Differential expression of pearl millet and banana clones identified from both libraries by this quantitative approach was verified by inverse Northern blot analysis.
Malaria Journal | 2011
John Vw Becker; Marina M van der Merwe; Anna C van Brummelen; Pamisha Pillay; Bridget Genevieve Crampton; Edwin M. Mmutlane; Christopher J. Parkinson; Fanie R. van Heerden; Neil R. Crouch; Peter J. Smith; Dalu Mancama; Vinesh Maharaj
BackgroundAnti-malarial drug resistance threatens to undermine efforts to eliminate this deadly disease. The resulting omnipresent requirement for drugs with novel modes of action prompted a national consortium initiative to discover new anti-plasmodial agents from South African medicinal plants. One of the plants selected for investigation was Dicoma anomala subsp. gerrardii, based on its ethnomedicinal profile.MethodsStandard phytochemical analysis techniques, including solvent-solvent extraction, thin-layer- and column chromatography, were used to isolate the main active constituent of Dicoma anomala subsp. gerrardii. The crystallized pure compound was identified using nuclear magnetic resonance spectroscopy, mass spectrometry and X-ray crystallography. The compound was tested in vitro on Plasmodium falciparum cultures using the parasite lactate dehydrogenase (pLDH) assay and was found to have anti-malarial activity. To determine the functional groups responsible for the activity, a small collection of synthetic analogues was generated - the aim being to vary features proposed as likely to be related to the anti-malarial activity and to quantify the effect of the modifications in vitro using the pLDH assay. The effects of the pure compound on the P. falciparum transcriptome were subsequently investigated by treating ring-stage parasites (alongside untreated controls), followed by oligonucleotide microarray- and data analysis.ResultsThe main active constituent was identified as dehydrobrachylaenolide, a eudesmanolide-type sesquiterpene lactone. The compound demonstrated an in vitro IC50 of 1.865 μM against a chloroquine-sensitive strain (D10) of P. falciparum. Synthetic analogues of the compound confirmed an absolute requirement that the α-methylene lactone be present in the eudesmanolide before significant anti-malarial activity was observed. This feature is absent in the artemisinins and suggests a different mode of action. Microarray data analysis identified 572 unique genes that were differentially expressed as a result of the treatment and gene ontology analysis identified various biological processes and molecular functions that were significantly affected. Comparison of the dehydrobrachylaenolide treatment transcriptional dataset with a published artesunate (also a sesquiterpene lactone) dataset revealed little overlap. These results strengthen the notion that the isolated compound and the artemisinins have differentiated modes of action.ConclusionsThe novel mode of action of dehydrobrachylaenolide, detected during these studies, will play an ongoing role in advancing anti-plasmodial drug discovery efforts.
Molecular Plant Pathology | 2009
Bridget Genevieve Crampton; Ingo Hein; Dave K. Berger
Studies were undertaken to assess the induction of defence response pathways in pearl millet (Pennisetum glaucum) in response to infection with the leaf rust fungus Puccinia substriata. Pretreatment of pearl millet with salicylic acid (SA) conferred resistance to a virulent isolate of the rust fungus, whereas methyl jasmonate (MeJA) did not significantly reduce infection levels. These results suggest that the SA defence pathway is involved in rust resistance. In order to identify pearl millet genes that are specifically regulated in response to SA and not MeJA, and thus could play a role in resistance to P. substriata, gene expression profiling was performed. Substantial overlap in gene expression responses between the treatments was observed, with MeJA and SA treatments exhibiting 17% co-regulated transcripts. However, 34% of transcripts were differentially expressed in response to SA treatment, but not in response to MeJA treatment. SA-responsive transcripts represented genes involved in SA metabolism, defence response, signal transduction, protection from oxidative stress and photosynthesis. The expression profiles of pearl millet plants after treatment with SA or MeJA were more similar to one another than to the response during a compatible infection with P. substriata. However, some SA-responsive genes were repressed during P. substriata infection, indicating possible manipulation of host responses by the pathogen.
BMC Genomics | 2010
John Vw Becker; Linda Mtwisha; Bridget Genevieve Crampton; Stoyan Stoychev; Anna C van Brummelen; S.B. Reeksting; Abraham I. Louw; Lyn-Marie Birkholtz; Dalu Mancama
BackgroundPlasmodium falciparum, the causative agent of severe human malaria, has evolved to become resistant to previously successful antimalarial chemotherapies, most notably chloroquine and the antifolates. The prevalence of resistant strains has necessitated the discovery and development of new chemical entities with novel modes-of-action. Although much effort has been invested in the creation of analogues based on existing drugs and the screening of chemical and natural compound libraries, a crucial shortcoming in current Plasmodial drug discovery efforts remains the lack of an extensive set of novel, validated drug targets. A requirement of these targets (or the pathways in which they function) is that they prove essential for parasite survival. The polyamine biosynthetic pathway, responsible for the metabolism of highly abundant amines crucial for parasite growth, proliferation and differentiation, is currently under investigation as an antimalarial target. Chemotherapeutic strategies targeting this pathway have been successfully utilized for the treatment of Trypanosomes causing West African sleeping sickness. In order to further evaluate polyamine depletion as possible antimalarial intervention, the consequences of inhibiting P. falciparum spermidine synthase (PfSpdSyn) were examined on a morphological, transcriptomic, proteomic and metabolic level.ResultsMorphological analysis of P. falciparum 3D7 following application of the PfSpdSyn inhibitor cyclohexylamine confirmed that parasite development was completely arrested at the early trophozoite stage. This is in contrast to untreated parasites which progressed to late trophozoites at comparable time points. Global gene expression analyses confirmed a transcriptional arrest in the parasite. Several of the differentially expressed genes mapped to the polyamine biosynthetic and associated metabolic pathways. Differential expression of corresponding parasite proteins involved in polyamine biosynthesis was also observed. Most notably, uridine phosphorylase, adenosine deaminase, lysine decarboxylase (LDC) and S-adenosylmethionine synthetase were differentially expressed at the transcript and/or protein level. Several genes in associated metabolic pathways (purine metabolism and various methyltransferases) were also affected. The specific nature of the perturbation was additionally reflected by changes in polyamine metabolite levels.ConclusionsThis study details the malaria parasites response to PfSpdSyn inhibition on the transcriptomic, proteomic and metabolic levels. The results corroborate and significantly expand previous functional genomics studies relating to polyamine depletion in this parasite. Moreover, they confirm the role of transcriptional regulation in P. falciparum, particularly in this pathway. The findings promote this essential pathway as a target for antimalarial chemotherapeutic intervention strategies.
European Journal of Plant Pathology | 2012
J. Korsman; B. Meisel; Frederik J. Kloppers; Bridget Genevieve Crampton; David Kenneth Berger
Grey leaf spot is an important maize foliar disease caused by the fungal pathogens Cercospora zeae-maydis and Cercospora zeina. Although methods exist to detect these Cercospora species in maize, current techniques do not allow quantification of the fungi in planta. We developed a real-time SYBR® Green PCR assay for quantification of grey leaf spot disease in maize based on the amplification of a fragment of a cytochrome P450 reductase (cpr1) gene. In planta fungal DNA content was normalised to a maize glutathione S-transferase III gene (gst3) to yield values of ng Cercospora DNA/mg maize DNA. The assay was specific to the two Cercospora spp., and we observed no amplification of the cpr1 fragment in non-target maize leaf pathogens or saprophytes. The assay was employed to quantify C. zeina in glasshouse inoculated maize plants and grey leaf spot infected field plants of resistant and susceptible maize lines. In both instances, C. zeina DNA content correlated with symptomatic leaf lesion area, and the susceptible maize line contained significantly more C. zeina DNA than the resistant line. Sequence differences between the C. zeina and C. zeae-maydis cpr1 amplicons enabled us to perform melt curve analyses to identify the Cercospora species causing grey leaf spot at a particular location. This assay has application in the early detection and quantification of Cercospora spp., both of which are important tools in grey leaf spot disease management and maize breeding programmes.
Phytopathology | 2016
M.F. Muller; Irene Barnes; N.T. Kunene; Bridget Genevieve Crampton; Burt H. Bluhm; Sonia M. Phillips; Nicholas Abraham Olivier; David Kenneth Berger
South Africa is one of the leading maize-producing countries in sub-Saharan Africa. Since the 1980s, Cercospora zeina, a causal agent of gray leaf spot of maize, has become endemic in South Africa, and is responsible for substantial yield reductions. To assess genetic diversity and population structure of C. zeina in South Africa, 369 isolates were collected from commercial maize farms in three provinces (KwaZulu-Natal, Mpumalanga, and North West). These isolates were evaluated with 14 microsatellite markers and species-specific mating type markers that were designed from draft genome sequences of C. zeina isolates from Africa (CMW 25467) and the United States (USPA-4). Sixty alleles were identified across 14 loci, and gene diversity values within each province ranged from 0.18 to 0.35. High levels of gene flow were observed (Nm = 5.51), and in a few cases, identical multilocus haplotypes were found in different provinces. Overall, 242 unique multilocus haplotypes were identified with a low clonal fraction of 34%. No distinct population clusters were identified using STRUCTURE, principal coordinate analysis, or Weirs theta θ statistic. The lack of population differentiation was supported by analysis of molecular variance tests, which indicated that only 2% of the variation was attributed to variability between populations from each province. Mating type ratios of MAT1-1 and MAT1-2 idiomorphs from 335 isolates were not significantly different from a 1:1 ratio in all provinces, which provided evidence for sexual reproduction. The draft genome of C. zeina CMW 25467 exhibited a complete genomic copy of the MAT1-1 idiomorph as well as exonic fragments of MAT genes from both idiomorphs. The high level of gene diversity, shared haplotypes at different geographical locations within South Africa, and presence of both MAT idiomorphs at all sites indicates widespread dispersal of C. zeina between maize fields in the country as well as evidence for sexual recombination. The outcomes of this genome-enabled study are important for disease management since the high diversity has implications for dispersal of fungicide resistance should it emerge and the need for diversified resistance breeding.
Phytotherapy Research | 2015
Namrita Lall; Navneet Kishore; Saeideh Momtaz; A.A. Hussein; Sanushka Naidoo; M. Nqephe; Bridget Genevieve Crampton
Skin hyper‐pigmentation is a condition initiated by the overproduction of melanin existing in the melanocytes. Melanin pigment is responsible for the colour of skin in humans. It is formed through a series of oxidative reactions involving the amino acid tyrosine in the presence of the key enzyme tyrosinase. In continuation with our efforts to identify tyrosinase inhibitors from plants sources, the methanol extract from leaf, bark and fruit of Ceratonia siliqua were screened for tyrosinase inhibition and diphenolase activity. The bark extract exhibited significant inhibition on mushroom tyrosinase using L‐tyrosine as a substrate and showed diphenolase activity. The extract further significantly lowered tyrosinase mRNA levels in B16‐F10 mouse melanocytes. Bioassay‐guided fractionation led to the isolation of six compounds. Compounds (−)‐epicatechin‐3‐O‐gallate, 1,2,3,6‐tetra‐O‐galloyl‐ß‐D‐glucose and gallocatechin‐3‐O‐gallate showed tyrosinase inhibitions with the IC50 values of 27.52, 83.30 and 28.30 µg/mL, respectively. These compounds also exhibited L‐DOPA activities with IC50 values of >200, 150 and 200 µg/mL, respectively. A clinical study was conducted using 20 volunteers in a patch testing trial for irritancy potential and skin depigmentation. The clinical results showed the sample to be non‐irritant with irritancy potential of −34.21 and depigmentation trial showed an improvement in the even skin tone of UV induced pigmentation at 3% after 28 days of application. Copyright
Molecular Plant-microbe Interactions | 2017
Velushka Swart; Bridget Genevieve Crampton; John B. Ridenour; Burt H. Bluhm; Nicholas Abraham Olivier; J.J. Marion Meyer; David Kenneth Berger
Gray leaf spot (GLS), caused by the sibling species Cercospora zeina or Cercospora zeae-maydis, is cited as one of the most important diseases threatening global maize production. C. zeina fails to produce cercosporin in vitro and, in most cases, causes large coalescing lesions during maize infection, a symptom generally absent from cercosporin-deficient mutants in other Cercospora spp. Here, we describe the C. zeina cercosporin toxin biosynthetic (CTB) gene cluster. The oxidoreductase gene CTB7 contained several insertions and deletions as compared with the C. zeae-maydis ortholog. We set out to determine whether complementing the defective CTB7 gene with the full-length gene from C. zeae-maydis could confer in vitro cercosporin production. C. zeina transformants containing C. zeae-maydis CTB7 were generated by Agrobacterium tumefaciens-mediated transformation and were evaluated for in vitro cercosporin production. When grown on nitrogen-limited medium in the light-conditions conducive to cercosporin production in other Cercospora spp.-one transformant accumulated a red pigment that was confirmed to be cercosporin by the KOH assay, thin-layer chromatography, and ultra performance liquid chromatography-quadrupole-time-of-flight mass spectrometry. Our results indicated that C. zeina has a defective CTB7, but all other necessary machinery required for synthesizing cercosporin-like molecules and, thus, C. zeina may produce a structural variant of cercosporin during maize infection.
IMA FUNGUS | 2017
Brenda D. Wingfield; Dave K. Berger; Emma Theodora Steenkamp; Hye-Jin Lim; Tuan A. Duong; Burton H Bluhm; Z. Wilhelm de Beer; Lieschen De Vos; Gerda Fourie; Kershney Naidoo; Nicky Olivier; Yao-Cheng Lin; Yves Van de Peer; Fourie Joubert; Bridget Genevieve Crampton; Velushka Swart; Nicole Soal; Catherine Tatham; Magriet A. van der Nest; Nicolaas Albertus Van der Merwe; Stephanie van Wyk; P. Markus Wilken; Michael J. Wingfield
The genomes of Cercospora zeina, Fusarium pininemorale, Hawksworthiomyces lignivorus, Huntiella decipiens, and Ophiostoma ips are presented in this genome announcement. Three of these genomes are from plant pathogens and otherwise economically important fungal species. Fusarium pininemorale and H. decipiens are not known to cause significant disease but are closely related to species of economic importance. The genome sizes range from 25.99 Mb in the case of O. ips to 4.82 Mb for H. lignivorus. These genomes include the first reports of a genome from the genus Hawksworthiomyces. The availability of these genome data will allow the resolution of longstanding questions regarding the taxonomy of these species. In addition these genome sequences through comparative studies with closely related organisms will increase our understanding of how these species or close relatives cause disease.
Phytopathology | 2016
Maria Petronella Human; Irene Barnes; Maryke Craven; Bridget Genevieve Crampton
Exserohilum turcicum is the causal agent of northern corn leaf blight, a destructive foliar disease of maize that results in yield losses worldwide. In South Africa, typical yield losses range from 15 to 30%. Previous studies found high haplotypic diversity with evidence for sexual recombination in E. turcicum populations from tropical climates such as Kenya. However, the population genetic structure and method of reproduction of E. turcicum in South Africa is unknown and, therefore, was investigated. Twelve polymorphic microsatellite markers were screened on 258 E. turcicum isolates from maize collected during 2012 and 2013 from three maize fields in South Africa. A multiplex polymerase chain reaction assay amplifying both mating type idiomorphs was applied to investigate the distribution of mating types. No distinct genetic clusters were observed. Shared haplotypes were identified between isolates separated by distances of up to 762 km, which provided evidence of migration. High haplotypic diversity indicated that sexual reproduction is occurring among E. turcicum isolates, although mating type ratios and linkage disequilibrium analyses did not support the hypothesis of random mating. The population genetic structure of E. turcicum in South Africa is likely due to the direct movement and spread of isolates undergoing a mixed reproductive lifecycle.