Molecular studies of protein coding sequences on the plastid-like DNA of Plasmodium falciparum

In this laboratory, the discovery and sequencing of an extrachromosomal, 35 kilobase (35 kb) circular DNA from the apicomplexan parasites Plasmodium, Toxoplasma and Eimeria has led to the idea that this molecule is similar to plastid DNAs. However, at the onset of the project outlined here, only two thirds of the 35 kb molecule from P. falciparum had been sequenced. A large portion of the reported data in this thesis was concerned with establishing the sequence of a 5.3 kb section of the 35 kb circle. These data led to the identification of the following genes encoding: two tRNAs (decoding serine and glycine), two open reading frames (ORFs) (one of which bears similarity to an ORF in the cyanelle of the alga Cyanophora paradoxa), the rpoC2 subunit of a prokaryote-like RNA polymerase (RNAP) and the ribosomal protein rps 2. In addition, the coding regions of the already partially sequenced rpoC1 and clpC genes were completed. An analysis of these genes and their chloroplast-encoded counterparts is included within this thesis. Antibodies were made to the rpoB gene product encoded by the 35 kb circle. Two methods were used 1) generation in vitro of monoclonal antibodies to predicted synthetic peptides and 2) generation of rabbit polyclonal antibodies to a fusion protein. These antibodies successfully identified the β subunit of the 35 kb-encoded RNAP in parasite extracts, constituting the first direct evidence for functionality of the genome. In addition, by means of a polyclonal serum obtained against the E. coli RNAP, it was shown that the 35 kb-encoded RNAP shares epitopes with the corresponding polypeptides in E. coli. In an attempt to ascertain the function of an ORF (ORF470) encoded on the plastid-like DNA of Plasmodium, I have established that homologues occur in a variety of bacteria including Mycobacterium, Corynebacter and Synechocystis spp. However, the function of this ORF remains unknown. Finally, because of the anti-malarial activity of certain drugs directed against the elongation cycle of prokaryotic protein synthesis it became necessary to establish whether P. falciparum carries not only the elongation factor encoded by the 35 kb DNA but also a mitochondrial equivalent. Accordingly a search was made with the S. cerevisiae tufM gene, encoding the mitochondrial elongation factor Tu. Evidence for the presence of a nucleus encoded, P. falciparum tufM gene was determined in addition to the identification of the chromosome carrying it