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Summer Research Fellowship Programme of India's Science Academies

Generation of Plasmodium berghei Topoisomerase III knockout cassette

Maithili Sriram

University of Mysore, Krishnaraja Boulevard Road, K.G Koppal, Mysuru, Karnataka

Dr. Sunanda Bhattacharyya

Assistant Professor, Department of Biotechnology and Bioinformatics, School of Life Sciences, University of Hyderabad, CUC, Prof C. R. Rao Road, P O Central University, Gachibowli, Hyderabad, Telangana 500046

ABSTRACT

Malaria is a disease caused by Plasmodium parasite and is transmitted by mosquitoes. About 198 million cases of malaria are recorded every year, with the annual deaths of 5,84,000 worldwide. Out of 7.2 billion of the world population, 3.2 billion people are at risk of this disease. Till now there is no effective vaccine against malaria and antigenic variation leads to resistance to anti- malaria drugs. These factors have made finding a cure for malaria an urgent public health priority. It is necessary to discover new targets which are crucial for the parasites to survive. These parasites need a mosquito host (sexual cycle) and a human host (asexual cycle) to complete their life cycle. In the mosquito midgut, gametocytes mature and undergo meiosis which is one of the most crucial steps for sexual reproduction. During meiosis I, the diploid zygote undergoes genetic recombination which results in generation of genetic diversity. It has been established in other organisms that topoisomerase III plays an indispensible role during meiosis I. It is essential for dissolution of Holliday junction created by DNA helicases at the time of meiotic recombination. Studies in yeast have shown that ∆topoIII strains are defective in meiotic replication and sporulation. We speculate that Plasmodium topoisomerase III might also play a crucial role in meiosis. If proved to be important in meiosis, it can block sporozoite formation and hence would qualify as a drug target. To establish the role of PbTopoIII during meiosis, we are trying to knockout Topoisomerase III gene from Plasmodium berghei. To that end, we are generating a knockout vector where regions 600 bp upstream and downstream of PbTopoIII will be cloned on both sides of the marker gene (TgDHFR) present in the pKO vector. Later, this cassette will be used for homologous recombination mediated knock out of PbTopoIII gene from the genomic locus.

Keywords: Reductional division, Homologous recombination, Meiosis, gene knockout.

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