Abstract:
Malaria is one of the most serious tropical infectious diseases affecting mankind. The
prevention of the disease is hampered by the increasing resistance of the parasite to
existing chemotherapy and -prophylaxis drugs. The need for novel therapeutic targets
and drugs is therefore enormous and the understanding of the biochemistry of the parasite
is imperative. The aim of this study was the identification and molecular characterisation
of the eDNA of one such metabolic target protein, ornithine decarboxylase (ODC), in the
human malaria parasite P. falciparum.
The P. falciparum ODC eDNA was isolated by means of a modified RT-PCR technique,
RACE. No sequence data were available and the primers used were based on consensus
areas identified in the protein sequences from other related organisms. The isolation and
identification of the eDNA with degenerate primers was successful in 3' -RACE, but
necessitated the optimisation of the eDNA synthesis protocol and the use of total RNA as
starting material. The sequence obtained facilitated the application of 5' -RACE with
ODC-specific primers based on the 3' -RACE sequence data. The full-length ODC
eDNA sequence was obtained by overlap-alignment of various segments. A novel
suppression PCR technology was applied during the 5' -RACE in order to create an
uncloned eDNA library of amplified cDNAs representing only the mRNA population. The P. falciparum ODC eDNA contains an open reading frame of ---2847 bp and
translates to a large 939 amino acid protein. The protein contained large internal
insertions and was extended by '""273 N-terminal residues compared to ODCs from other
organisms. Several possible signature motifs were identified for phosphorylation,
glycosylation and transamidation. The P. falciparum ODC protein seems to contain more
hydrophilic and a-helix forming residues. These characteristics should be further
investigated after expression of the recombinant protein.
The isolation of the P. falciparum ODC eDNA facilitates the validation of this protein as
an antimalarial target.
