Polyamines play an important role in DNA, RNA and protein synthesis as well as a variety of other biological processes (cell division, differentiation and death) as outlined in Chapter 1. Assaraf and co-workers (1984) demonstrated that treatment with DFMO resulted in the inhibition of polyamine biosynthesis as well as schizogony arrest in P. falciparum. However, they did not elaborate on any other consequences that polyamine depletion could exert on the parasite. This dissertation aims to elucidate the significance of the inhibition of polyamine biosynthesis within P. falciparum by using differential transcriptome profiling. Suppression subtractive hybridisation generated transcripts which were potentially up-and down-regulated due to endogenous polyamine depletion within the human malaria parasite P. falciparum. The resulting transcripts were subjected to a restriction enzyme analysis and those with unique digestion profiles were selected and sequenced. The sequences were analysed using PlasmoDB to identify the genomic sequences to which they were best matched. To confirm that the selected transcripts were indeed differentially expressed a reverse virtual Northern dot blot was performed. Transcripts for proteins involved in protein processing, methionine and polyamine metabolism, various transporters, proteins involved in cellular differentiation and signal transduction were found to be upregulated in the absences of polyamines. This could be suggestive of a metabolic response induced by the parasite in order to overcome this deficiency. Polyamines seem to influence protein synthesis and haemoglobin degradation as well since depletion of endogenous polyamines within the parasite seems to result in increased food vacuole acidification, haemoglobin degradation, transport of proteins to the cytoplasm and protein synthesis and stabilisation. The majority of downregulated transcripts were found to be involved in cell-cell adhesion and erythrocyte invasion, protein processing and transport indicating that these processes are dependent on polyamines. Further validation of these findings by microarray as well as proteomic analysis will need to be undertaken. These results validate that polyamines do play an essential role in the cellular biology of the parasite. They also confirm that the inhibition of polyamine biosynthesis is a viable route to undertake in the search for new and improved antimalarial targets. This would be especially useful if it was combined with other antimalarials and their synergistic effects were investigated by transcriptomic, proteomic and bioinformatic analysis / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / MSc / unrestricted

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In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.

In the late 1990s, a series of landmark publications described RNA interference (RNAi) and related RNA silencing phenomena in nematodes, plants and fungi. By manipulating RNA silencing, biologists have been able to create tools for specifically inactivating genes. In organisms from trypanosomes to insects, RNA silencing is now indispensible for studying gene function. RNA silencing has been used in a project aimed at systematically knocking out all genes in the model plant Arabidopsis thaliana. RNA silencing has a natural role in defending eukaryotic cells against virus replication. By assembling virus DNA sequences in a form that triggers RNA silencing, biologists have created plants resistant to specific viruses. In this study, we set out to test if a similar approach would protect plants against infection by the agriculturally important Brassica pathogen, Plasmodiophora brassicae. P. brassicae is an obligate intracellular biotroph, from the little studied eukaryotic supergroup, the Rhizaria. To identify the gene sequences that would be starting material for P. brassicae RNA silencing, new P. brassicae genes were gathered by cDNA cloning or genomic PCR-walking. Using suppression subtractive hybridisation (SSH) and oligo-capping cloning of full-length cDNAs, 76 new gene sequences were identified. A large proportion of the cDNAs were predicted to contain signal peptides for ER translocation. In addition to the new cDNA identified here, partial sequences for the P. brassicae actin and TPS genes were published by other researchers close to the beginning of this study. Using PCR-walking, full-length genomic DNA sequences from both genes were obtained. Later, genomic DNA sequences spanning or flanking a total of 24 P. brassicae genes were obtained. The P. brassicae genes were rich in typical eukaryotic spliceosomal introns. Transcription of P. brassicae genes also appears likely to begin from initiator elements rather than TATA-box-containing promoters. A segment of the P. brassicae actin gene was assembled in hairpin format and transformed into Arabidopsis thaliana. Observation of simultaneous knockdown of the GUS marker gene as well as detection of siRNAs indicated that the hpRNA sequences induced RNA silencing. However, inoculation of these plants with P. brassicae resulted in heavy club root infection. We were unable to detect decreases in actin gene expression in the infecting P. brassicae, at either early or late stages of infection. We conclude that, within the limits of the techniques used here, there is no evidence for induction of RNA silencing in P. brassicae by in planta produced siRNAs.