Plasmodium falciparum is the most important causative agent of human malaria. The present study focuses on the identification and characterization of pfgcn20, a novel-member of the ATP-binding cassette protein gene family in P. falciparum. In a PCR-based cloning strategy, I employed redundant oligonucleotides directed against sequences encoding the highly conserved Walker motifs within the ATP-binding cassette, and the resulting amplified DNA fragments were used for library screening and gene walking experiments that resulted in the molecular isolation of the pfgcn20 gem. The pfgcn20 gene spans 2448 nucleotides and was predicted to encode a 95.5 kilo-daltons hydrophilic protein, PFGCN20, with two conserved ATP-binding sites but no predicted membrane spanning domains. Pfgcn20 a localized on chromosome 11 and was shown to be expressed as a 3.3 kilobase mRNA transcript. The predicted PFGCN20 protein shows significant sequence homology to a subgroup of ATP-binding canette proteins related to the Saccharomyces cerevisiae translational regulator Gcn20p, to yeast and fungi EF-3 elongation factors, and to a subfamily of ATP-binding subunits of bacterial ATP-binding cassette transporters. However, no clear function for PFGCN20 could be inferred from these homology searches. / A PFGCN20 specific polyclonal antibody was generated in rabbits and used to reveal that PFGCN20 is expressed as a 95 kilo-dalton protein throughout the entire cycle of the erythrocytic stage of P. falciparum. Localization studies showed that during the ring stage, PFGCN20 is present mainly in the parasite cell either in the cytoplasm and/or in discontinuous electron dense compartments at the cell periphery. In contrast, during the trophozoite and schizont stages, PFGCN20 was found to be transported into the host cell cytoplasm where it was associated with membranes of the tubovesicular membrane system and the erythrocytic plasma membrane. In addition, a fraction of PFGCN20 appeared in the red cell cytoplasm in a membrane-free form. These results suggest that at least part of PFGCN20 function is related to physiological processes in the cytoplasm of infected red cells. / In the final chapter of my thesis, I have analyzed the functional homology between PFGCN20 and Gcn20p, by expression of PFGCN20 in the B2511 S. cerevisiae GCN20 deletion strain. Similar to Gcn20p, overproduction of PFGCN20 in H2511 (Deltagcn20) partially restored yeast cell tolerance to, an inhibitor of histidine biosynthesis, 3-amino-1,2,4-triazole. Expression of truncated pfgcn20 and chimeric pfgcn20/GCN20 constructs revealed that the N-terminal segment (211 acids) of PFGCN20 is sufficient for the restoration of partial 3-amino-.1,2,4-triazole tolerance of H2511 (Deltagcn20 ) cells. From my combined results, I have derived the working hypothesis that PFGCN20 may function as translational regulator within the plasmodial cell and fulfills a second possibly transport-related, function in the cytoplasm of infected red blood cells. However, whether PFGCN20 truly represents an example of a bi-functional ATP-binding protein must be evaluated by additional experimental work.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.35680 |
| Date | January 1998 |
| Creators | Bozdech, Zbynek. |
| Contributors | Schurn, Erwin (advisor) |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Division of Experimental Medicine.) |
| Rights | All items in eScholarship@McGill are protected by copyright with all rights reserved unless otherwise indicated. |
| Relation | alephsysno: 001650671, proquestno: NQ50117, Theses scanned by UMI/ProQuest. |
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