Topoisomerase II of the malarial parasite

• Main Author: Cheesman, Sandra
• Published: University of Edinburgh 1998
• Subjects: 572.8
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.642774

The gene encoding topoisomerase II of <I>P. falciparum </I>(PfTopoII) was isolated and characterised in 1994. This study has focussed on heterologous expression of the gene to enable sufficient recombinant protein to be purified for biochemical analysis and drug screening. The species divergent C-terminal region of the protein was expressed in <I>E. coli</I>, and used to raise polyclonal antiserum in rabbits, which detects a protein of 160/175 kDa in extracts from <I>P. falciparum. </I>The same antiserum was used to monitor expression of recombinant PfTopoII in baculovirus. A triplet of proteins of 160/175 kDa was detected by the antiserum in cell extracts derived from baculovirus-infected insect cells, but in very small quantities. A modified version of the PfTopoII gene, in which the first four codons were replaced with the first five codons of the yeast topoisomerase II gene, was expressed in <I>S. cerevisiae. </I>A control plasmid expressed the human topoisomerase II α homologue to high levels, but no evidence of expression of recombinant PfTopoII was detected. Northern analysis of the mRNA revealed the presence of truncated transcripts, and RT-PCR mapped the sites of polyadenylation to 370 and 376 nucleotides from the AUG. An investigation into the developmental regulation of PfTopoII throughout the blood stages of parasite growth was also undertaken. Northern analysis indicates that three transcripts accumulate principally in the trophozoite stage, although interestingly, work conducted by Paul Horrocks indicates that the promoter is active in both ring and trophozoite/schizont stage parasites. Western analysis of protein extracts derived from synchronised populations of parasites shows that trophozoite and schizont stages express a triplet of proteins of 160/175 kDa in size, and at similar levels. Although protein extracts from trophozoite and schizont stage parasites are proficient at decatenation, activity was highest in schizonts, and could be inhibited completely after immunodepleting the parasite extracts.