In Silica analysis of available biological data is a powerful tool for not only the identification of new genes, but also to study evolutionary relationships and regulatory mechanisms. In this study, a number of bioinformatic tools and techniques were applied on the available sequence data of the malaria parasite, Plasmodium falciparum. In Silica techniques were used for the identification of a genomic sequence tag (GST) matching the facilitated glucose transporter family as assessed by BLAST. The open reading frame encoding the fUll-length glucose transporter gene was subsequently assembled from contig sequences of chromosome 2 of the malaria parasite. The frequency of occurrence of di-, tri- and tetranucleotide sequences in both the coding and non-coding regions of chromosome 2 of P. falciparum was also exhaustively analysed. The relative abundance (observed, compared to expected values) of these oligonucleotide sequences, normalised for the nucleotide base composition, was calculated as an odds ratio and compared to those of other organisms. These relative abundancies are referred to as the organism's genomic signature. The CC•GG and CG-dinucleotides exhibited the highest and the lowest odds ratios, respectively. These genome signatures were shown to be constrained by the codon preference and amino acid abundancies. A number of genes with genomic signatures differing significantly from the average signature were also identified and were deduced to be acquired by lateral transfer from unidentified sources. A definite association between interspaced TGCA tetranucleotides and polymorphic traits of the FC27 allele of merozoite surface antigen 2 (MSA-2) was shown. The observed switching and deletion of a limited number of identical nucleotide sequences of several alleles interspersed between direct repeats, provided clues to potential mechanisms employed by the parasite to affect antigenic polymorphism. The identification of a number of motifs for intragenic (homologous) recombination led us to propose a mechanism by which the parasite achieves antigenic variation in single copy genes. These results have profound implications for the design of candidate anti-malarial vaccines, microsatellite typing and characterisation of proteins mediating these recombination events. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2001. / Biochemistry / unrestricted

Malaria remains 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 chemotherapies. The need for novel therapeutic targets and drugs is therefore of the utmost importance and detailed knowledge of the biochemistry of the parasite is imperative. This study was directed at the biochemical characterisation of the polyamine metabolic pathway of P. falciparum in order to elucidate differences between the parasite and its human host that can be exploited in the design of novel antimalarials. The thesis focussed on the two rate-limiting enzymes in polyamine biosynthesis, S¬adenosylmethionine decarboxylase (AdoMetDC) and ornithine decarboxylase (ODC), which occur as a unique bifunctional complex in P. falciparum. The genomic structure of the bifunctional gene indicated a single, monocistronic transcript with large untranslated regions that were predicted to be involved in unique translational regulatory mechanisms. This gives rise to a bifunctional protein containing both decarboxylase activities on a single polypeptide forming a heterotetrameric complex. Activity of the decarboxylases decreases dramatically if these proteins are expressed in their monofunctional forms as homodimeric ODC and heterotetrameric AdoMetDC. The deduced amino acid sequence indicated that all the essential residues for catalysis are conserved and highlighted the presence of three parasite-specific insertions. The parasite-specific inserts were shown to be essential for the catalytic activity of the respective domains and also to influence the activity of the neighbouring domain, indicating that intramolecular communication exists in the heterotetrameric complex. The most structured and smallest insert was also shown to mediate protein-protein interactions between the two domains and to stabilise the complex. Further structure- functional characterisations of specifically the ODC domain were deduced from a comparative homology model. The model predicted an overall structure corresponding to those of other homologous proteins. The validity of the model is supported by mutagenesis results. However, certain parasite-specific properties were identified in the active site pocket and dimerisation interface. The former was exploited in the rational design of novel putative ODC inhibitors directed only against the P. falciparumprotein by in silico screening of chemical structure libraries. This study therefore describes the identification of certain parasite-specific properties in a unique bifunctional protein involved in regulation of polyamine metabolism of P. falciparum. Such discoveries are invaluable in strategies aimed at elucidating biochemical and metabolic differences between the parasite and its human host that could be exploited in the design of alternative, parasite-specific chemotherapies. Moreover, the thesis also contributed new knowledge on certain less well-understood biological phenomena characteristic of P. falciparum, the nature and origin of bifunctional proteins and the functional properties of parasite-specific inserts found in some proteins of the parasite. / Thesis (PhD (Biochemistry))--University of Pretoria, 2002. / Biochemistry / unrestricted