This thesis has focused on the regulation of the gene encoding the essential DNA replication protein Proliferating Cell Nuclear Antigen (PCNA). PCNA expression in <I>Plasmodium falciparum </I>had earlier been shown to be stage specific during development in the intraerythrocytic cycle. Here its expression was examined in more detail. In addition, a preliminary analysis of the regulation of the gene encoding DNA polymerase δ, of which PCNA is an auxiliary factor, was made. Antisera raised against <I>P. falciparum </I>PCNA (PfPCNA) and DNA polymerase δ (PfPolδ) have been used against extracts from synchronised parasites to show that both proteins accumulate in trophozoites and persist in schizonts. The steady-state transcripts from both PfPCNA and PfPolδ also accumulate at the trophozoite stage. However, nuclear run on analysis shows that, whereas PfPolδ promoter activity is absent in rings but present in trophozoites and schizonts, the PfPCNA promoter is active throughout the intraerythrocytic cycle. This suggests that mechanisms regulating the expression of these two genes may be different although their coordinated activity is required for DNA replication. A major transcription start site 960bp upstream of the translational start of the PfPCNA coding sequence was identified. A second, minor, site is situated a further 40bp upstream. These results were developed from a number of methods to reduce the possibility of ambiguities which may arise due to the extreme AT-richness of <I>P. falciparum </I>non-coding DNA. Consensus TATA boxes and an OCT-1 box were identified upstream of the putative transcription start sites. Analysis of cDNA clones identified a putative transcription stop site 250bp downstream of the stop codon. Intraerythrocytic stages were successfully transfected with constructs containing a firefly luciferase reporter gene under the transcriptional control of variously modified elements of the PfPCNA 5' flanking sequence to test the effect on promoter activity.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:652597 |
| Date | January 1996 |
| Creators | Horrocks, Paul Daniel |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/14098 |
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