Mapping and characterization of the pfmdr1 5' upstream region, and analysis of the transcriptional response to chloroquine in Plasmodium falciparum

Malaria is a preventable parasitic infection that kills over 2 million people each year. The morbidity and mortality attributed to this disease is worsening as a result of the spread of drug resistance within the most lethal species of malaria parasite, Plasmodium falciparum . Delineating the molecular mechanisms underlying this resistance is crucial to the development of novel therapies to combat this disease.;The work presented in this thesis addresses the role of transcriptional regulation in the drug response of P. falciparum. This is achieved through a detailed molecular analysis of the P. falciparum multidrug resistance gene, pfmdr1; and through a whole genome analysis of the chloroquine response in susceptible parasites. We prove that pfmdr1 transcript levels are specifically increased in chloroquine sensitive parasites treated with quinoline antimalarial drugs. In an effort to determine if cis-regulatory elements played a role in the control of gene expression, we mapped the 5' end of the pfmdr1 transcript to an adenine residue 1.94kb upstream of the start methionine. Functional analysis of upstream sequence identified a 381bp region 5' of the transcription start point as the putative promoter region for this gene. Evaluation of the genetic diversity of the 5' upstream region demonstrated an unusually low level of polymorphism, suggesting that this region is under negative selection. We tested the hypothesis that polymorphisms might be associated with chloroquine resistance in a sample of field isolates from Senegal; and found that one single nucleotide polymorphism was prevalent in the majority of field isolates, irrespective of chloroquine sensitivity.;An oligonucleotide array was utilized to study the global transcriptional response of mixed asexual stage parasites to two different concentrations of chloroquine for two different time periods. After stringent filtering of two independent experiments, it was found that a total of six hundred genes were differentially expressed in response to chloroquine. Only thirty-six of the genes on this master list were common to both experiments. Expression profiling analysis led to the identification of a novel gene family, and four putative regulatory motifs in clusters of coordinately expressed genes.