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Title: The cloning and characterisation of the topoisomerase I gene from the malarial parasite, Plasmodium falciparum
Author: Tosh, Kerrie
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1997
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The gene encoding topoisomerase I (PfTopoI) from the malarial parasite, Plasmodium falciparum was isolated using two methods. (a) PCR of a cDNA library using oligonucleotides modelled on highly conserved regions of sequences from other species, isolated a 900bp fragment of the gene. (b) The entire PfTopoI open reading frame was obtained from a HinDIII/EcoRI genomic library after screening with a random-labelled heterologous TopoI probe from Saccharomyces cerevisiae. DNA sequence analysis indicated an open reading frame of 2520 nucleotides, encoding a deduced protein of 839 amino acids with no detectable introns. Two large amino acid inserts were present in the PfTopoI gene sequence which were present in both the genomic and cDNA indicating that they were not introns. The predicted amino acid sequence was compared with homologous sequences of other type I topoisomerases. On average, PfTopoI showed around 40% identity with all the sequences examined, with the TopoI sequence from man showing the highest level of homology. The gene is present as a single copy on chromosome 5 and Northern analysis identified a transcript of 3.8kb. The PfTopoI open reading frame was cloned into prokaryotic expression vectors in order to express, purify and assay the recombinant protein for TopoI relaxation activity. Both the full length PfTopoI protein and fragments were expressed, although no activity could be detected. However TopoI and II activity was observed in both crude and nuclear parasite extracts. Inhibition of the TopoI relaxation activity was observed when the type I topoisomerase specific inhibitor, camptothecin, was added to the assay. Stage specific expression of PfTopoI during the intraerythrocytic stages of the Plasmodium falciparum life cycle was examined using synchronised parasites. Nuclear run on assays indicate that the promoter is inactive during the early ring stage and is active only in the later mixed trophozoite and schizont stages.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available