Browsing by Author "Venter, Mauritz"

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    Identification of a novel functional deletion variant in the 5'-UTR of the DJ-1 gene
    (BioMed Central, 2009-10) Keyser, Rowena J.; Van der Merwe, Lize; Venter, Mauritz; Kinnear, Craig; Warnich, Louise; Carr, Jonathan; Bardien, Soraya
    Background: DJ-1 forms part of the neuronal cellular defence mechanism against oxidative insults, due to its ability to undergo self-oxidation. Oxidative stress has been implicated in the pathogenesis of central nervous system damage in different neurodegenerative disorders including Alzheimer's disease and Parkinson's disease (PD). Various mutations in the DJ-1 (PARK7) gene have been shown to cause the autosomal recessive form of PD. In the present study South African PD patients were screened for mutations in DJ-1 and we aimed to investigate the functional significance of a novel 16 bp deletion variant identified in one patient. Methods: The possible effect of the deletion on promoter activity was investigated using a Dual- Luciferase Reporter assay. The DJ-1 5'-UTR region containing the sequence flanking the 16 bp deletion was cloned into a pGL4.10-Basic luciferase-reporter vector and transfected into HEK293 and BE(2)-M17 neuroblastoma cells. Promoter activity under hydrogen peroxide-induced oxidative stress conditions was also investigated. Computational (in silico) cis-regulatory analysis of DJ-1 promoter sequence was performed using the transcription factor-binding site database, TRANSFAC via the PATCH™ and rVISTA platforms. Results: A novel 16 bp deletion variant (g.-6_+10del) was identified in DJ-1 which spans the transcription start site and is situated 93 bp 3' from a Sp1 site. The deletion caused a reduction in luciferase activity of approximately 47% in HEK293 cells and 60% in BE(2)-M17 cells compared to the wild-type (P < 0.0001), indicating the importance of the 16 bp sequence in transcription regulation. The activity of both constructs was up-regulated during oxidative stress. Bioinformatic analysis revealed putative binding sites for three transcription factors AhR, ARNT, HIF-1 within the 16 bp sequence. The frequency of the g.-6_+10del variant was determined to be 0.7% in South African PD patients (2 heterozygotes in 148 individuals). Conclusion: This is the first report of a functional DJ-1 promoter variant, which has the potential to influence transcript stability or translation efficiency. Further work is necessary to determine the extent to which the g.-6_+10del variant affects the normal function of the DJ-1 promoter and whether this variant confers a risk for PD.
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    Isolation of grapevine promoters with special emphasis on the vacuolar pyrophosphatase
    (Stellenbosch : University of Stellenbosch, 2004-04) Venter, Mauritz; Botha, F. C.; Potier, Bernard; University of Stellenbosch. Faculty of Science. Dept. of Botany and Zoology.
    ENGLISH ABSTRACT: Understanding the complex nature of grapevine molecular biology is of great importance for viticulturists. Progress in the elucidation of key events on a genetic level could provide further insight into the underlying cues responsible for the precise control of physiological and metabolic changes during a specific condition such as fruit development. The use and analysis of molecular ‘tools’, such as promoters controlling the site and level of gene activity, could assist in the understanding of grapevine biology and serve as a platform for the future design and development of recombinant DNA protocols and strategies for Vitis vinifera L. A high-throughput gene expression system, cDNA-AFLPs, was successfully used to analyse large-scale transcriptional activity during berry ripening. Candidate cDNA fragments were selected on the basis of desired expression patterns and/or known gene function for subsequent promoter isolation. From three candidate cDNAs selected, the promoter of a gene encoding vacuolar pyrophosphatase (V-PPase) was isolated for computational and comparative analyses. Promoter activity was evaluated on a transient level using the green fluorescent protein (GFP) reporter gene. Comparative integration has allowed for putative correlation of cis-elements, acting as receptors within promoter regions, to regulate V-PPase gene expression in response to development, environmental stress and tissue-specificity. In this study, integration of genetic data have advanced the understanding and transcriptional role of a key enzyme (V-PPase) during grape ripening. Although never a replacement for experimental verification, this integrative strategy of combining gene expression profiles with bioinformatics and regulatory data will greatly assist in further elucidation of various other key components and regulatory cues associated with grapevine molecular biology. This study has allowed us to use molecular tools that could assist in gaining further insight into genetic complexities and could serve as a platform for a more refined genetic manipulation strategy in Vitis vinifera L.