Law, Anna Elizabeth; (2000) A functional study of bacterial orthologues of the malarial plastid gene, ycf 24. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Malaria parasites have two DNA containing organelles, a mitochondrion and a plastid. Most of the plastid genes are involved in gene expression however there are a number of open reading frames (ORFs) of unknown function. In Plasmodium falciparum, ORF470 is particularly interesting as it is highly conserved with an orthologue (ycf 24) in both algae and bacteria. Some annotations describe ycf 24's product as an ABC transporter subunit but the level of significance is low. To investigate the function of ycf 24, the cyanobacterium Synechocystis, strain PCC6803, was transformed with a disrupted version of the gene and allowed to undergo several rounds of replication before selection with kanamycin. Synechocystis 6803 was chosen as it carries up to 10 copies of its genome per cell, allowing the support and selection of lethal genes. Primary transformants formed "ragged" colonies attributable to the death of homoplasmic transformants (complete "knockouts"). This indicates that loss of the gene is lethal. After selection, transformants were shown to be heteroplasmic (containing both disrupted and wild type ycf 24). Furthermore, heteroplasmic cells plated in the absence of kanamycin resumed WT growth rate and colony morphology indicating that the presence of the disrupted gene was deleterious and/or that the gene product is required in stoichiometric amounts. When the gross morphology of cells from ragged colonies was observed by scanning electron microscopy, a statistically significant accumulation of cells in the last stages of cell division was apparent. This suggests that disruption of ycf 24 leads to a defect in cytokinesis. Upon relaxation of kanamycin selection, the proportion of dividing cells returned to one resembling WT. Attempts were made to knock out the single copy of ycf 24 in E. coli. One approach made use of the temperature sensitive gene replacement vector pKO3 that contains genes for chloramphenicol resistance and levansucrase, which is lethal to E. coli in the presence of sucrose. This protocol allowed both positive and negative selection of recombination at the ycf 24 locus. However, no viable knockouts were obtained. Over-expression of ycf 24 in E. coli using the pMAL-c2 vector resulted in moderate filamentation, the fusion protein localizing as a band on either side of the nucleoid, sometimes extending along the membrane to the cell poles. Unlike some other partition mutants, FtsZ ring formation was inhibited in such cells and the first division of nucleoids following induction resulted in their accumulation transverse to the cell's length rather than longitudinally in the usual way. It is proposed that ycf 24 is an essential prokaryotic gene involved in cell division and/or DNA segregation.

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