Ellis, Katherine Elizabeth Sleigh; (2001) Nuclear genes involved in maintenance of the malarial plastid. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Plasmodium species (the causative agents of malaria), and related parasites of the phylum Apicomplexa such as Toxoplasma gondii, have a plastid organelle of endosymbiotic origin. This organelle is regarded as a potential new drug target. It contains its own genome encoding many of the proteins and RNAs required for protein synthesis, but its function is unclear. Nucleus-encoded proteins are likely to control the primary function and biogenesis of the plastid and I have studied two of these: FtsZ (involved in bacterial and plastid division), and Ycfl6 (a protein of unknown function that may interact with Ycf24, encoded on the plastid genome). First I set up a screen to check that any genes I identified were malarial in origin, and not from the mycoplasma that contaminate our cultures. Searches for ftsZ by degenerate PCR, database analysis and low-stringency hybridisation, were unsuccessful. The possibility remains that the apicomplexan plastid divides by some other mechanism, as mitochondria and some bacteria do, for example using a dynamin-related protein. A dynamin-like sequence was identified on chromosome 14 in P. falciparum, and its transcription at the appropriate stage of the erythrocytic cycle was confirmed. However, this protein could be involved in mitochondrial division. AycfI6-like sequence was found on chromosome 14 of P. falciparum and expressed in Escherichia coli for antibody production. This sequence encodes a putative plastid-targeting amino-terminal peptide; reporter protein and immunofluorescence studies will confirm whether it is localised in the plastid. To study the function of ycfl6,1 disrupted an orthologous version in the cyanobacterium Synechocystis and found it is an essential gene whose partial loss was deleterious. The δycfl6 mutant cyanobacteria were arrested in cytokinesis and resembled those found previously in our laboratory for δycf24 mutants, but the effects were less severe. I confirmed that ycf24 and ycfl6 are co-transcribed in Synechocystis, and perhaps form part of the same operon. These findings add weight to the hypothesis that Ycfl6 and Ycf24 act in the same pathway, and that the imported Ycfl6 and the endogenous plastid protein Ycf24 are both essential for plastid maintenance. Based on recent additions to the literature, I propose that Ycfl6 is involved in maturation of Fe-S cluster-containing proteins required for resistance to oxidative stress and the provision of reducing power for biosynthetic pathways that occur in the plastid of apicomplexans.

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