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Title: The DNA replication apparatus in Plasmodium falciparum gametocytes
Author: Kariuki, Michael Muchiri
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2002
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DNA synthesis in gametocytes takes place at two points; at the onset of gametocytogenesis where the nucleic acid content increases from lc to 1.8c prior to the formation of stage I gametocytes, and again 10 to 12 days later at the onset of gametogenesis when the mature male gametocyte (stage V) replicates its genome three times leading to the formation of eight haploid male gametes in less than 10 minutes. The aim of this study was to evaluate the status of the P. falciparum DNA replication apparatus during gametocytogenesis, most of which no significant DNA synthesis takes place, and gametogenesis in which a sudden burst of DNA replication takes place. The proteins studied in this project were DNA topoisomerase 1 and 11 (Topol and II), Replication factor C (Rfc) and Proliferating cell nuclear antigen (Pcna) of which P. falciparum homologous have been previously identified, isolated and characterised. Standard indirect immunofluorescence assays (IFA) carried out on unsynchronised in vitro cultivated P. falciparum (3D7A) using rabbit polyclonal antiserum raised against recombinant PfRfcl, PfRfc2, PfRfc3, PfPcna and PfTopoII showed that all five proteins are present throughout gametocytogenesis. All five proteins appear to be predominantly located within the nuclear region and at significantly higher levels in stage I and V gametocytes. However PfRfc2 levels appeared to be significantly higher only in stage I gametocytes and was distinctly absent from the nucleus of stage V gametocytes. Western blot analysis showed no significant changes in the levels of these proteins occurred during gametogenesis, with the exception of PfRfc2, which appear to increase immediately after activation and then gradually decrease as gametogenesis progressed. RT-PCR detected the presence of PfRFC2, PARFC3, PfPCNA, PfDNA POL5 and PiTOPO 1 transcripts in mature gametocytes before and after activation. However, PfRFCl and PfTOPO II transcripts were not detected in mature gametocytes either before or after activation. Similar results in protein and RNA analysis were obtained whether gametocytes were grown in AlbuMax or serum supplemented medium. In situ hybridisation using fluorescein-labelled PfRFCl, PfRFC2, PfDNA POL8, PfTOPO 1 and PfTOPO II gene fragments showed pockets of fluorescence on the peripheral regions of schizonts away from the nuclear region stained by DAPI. PfRFC3 and PfPCNA probes appeared to show fluorescence emanating from the nuclear region of schizonts. In conclusion, the localisation and unique expression pattern of PfRfc2 observed before and during gametogenesis, from that of the other DNA replication proteins and in particular, PfRfcl and PfRfc3, appears to imply a significant role for PfRfc2. Further studies need to be carried out in order to get to a better understanding of the role of PfRfc2 during gametogenesis.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available