Members of the Plasmodium falciparum var gene family encode clonally variant adhesins, which play an important role in the pathogenicity of tropical malaria. Here we employ a selective panning protocol to generate isogenic P.falciparum populations with defined adhesive phenotypes for CD36, ICAM-1 and CSA, expressing single and distinct var gene variants. This technique has established the framework for examining var gene expression, its regulation and switching. It was found that var gene switching occurs in situ. Ubiquitous transcription of all var gene variants appears to occur in early ring stages. However, var gene expression is tightly regulated in trophozoites and is exerted through a silencing mechanism. Transcriptional control is mutually exclusive in parasites that express defined adhesive phenotypes. In situ var gene switching is apparently mediated at the level of transcriptional initiation, as demonstrated by nuclear run-on analyses. Our results suggest that an epigenetic mechanism(s) is involved in var gene regulation.

The hemoglobins S and C protect carriers from severe Plasmodium falciparum malaria. Here, we found that these hemoglobinopathies affected the trafficking system that directs parasite-encoded proteins to the surface of infected erythrocytes. Cryoelectron tomography revealed that the parasite generated a host-derived actin cytoskeleton within the cytoplasm of wild-type red blood cells that connected the Maurer's clefts with the host cell membrane and to which transport vesicles were attached. The actin cytoskeleton and the Maurer's clefts were aberrant in erythrocytes containing hemoglobin S or C. Hemoglobin oxidation products, enriched in hemoglobin S and C erythrocytes, inhibited actin polymerization in vitro and may account for the protective role in malaria.

Operator sequence and repressor protein regulate the activity of the lac promoter over a >1000-fold range. Combinations of the lac operator with other promoter sequences, however, differ vastly in the level of repression. The data presented show that the extent of repression is determined largely by the rates of complex formation of the competing systems operator-repressor and promoter-RNA polymerase and by the rate at which RNA polymerase clears the promoter. Moreover, up to 70-fold differences in the level of repression were found when the operator was placed in different positions within the promoter sequence. A kinetic model is proposed that explains the observed effects and that allows predictions on promoters controlled by negatively acting elements.The lac repressor, though present in only 10-20 copies per cell (1), is capable of reducing the expression of the Escherichia coli lac operon by a factor of %-1000 (2). When placed on a multicopy plasmid the lac promoter (Piac) can be controlled to the same extent, provided that the intracellular repressor concentration is adjusted properly (3, 4). Under identical conditions the lac promoter mutant PlacL8.UV5 (5), which is homologous to Plac throughout the entire operator region and which is about equally active in vivo (6), is repressed an order of magnitude less efficiently (4). Moreover, Ptac (7), a derivative Of PlacL8.UV5 and -3 times stronger in vivo than Plac, is reduced in its activity by a factor of only 50 by lac repressor (4). These observations indicate that the extent of repression is neither determined by the interaction between repressor and operator alone nor correlated with promoter strength in vivo. Additional parameters evidently play a significant role in a functionally optimized promoter/ operator system. We have studied various promoter/operator combinations by analyzing their repression in vivo as well as the kinetics of their interaction with repressor and RNA polymerase (RNAP), respectively. Our data show that the kinetic parameters of the RNAP-promoter interaction as well as the position of an operator within the promoter sequence drastically affect the occupancy of the operator by its repressor, which ultimately defines the efficiency of repression. Proteins. RNAP preparations were either prepared according to Burgess and Jendrisak (9) or purchased from Pharmacia Freiburg. lac repressor was purified as described (10). MATERIALS AND METHODSDissociation of lac Repressor-Operator Complexes. A mixture of end-labeled fragments carrying the promoter/ operator constructs was allowed to equilibrate with a stoichiometric amount of lac repressor (5 nM) for 10 min at 370C in 1 mM MgCl2/50 mM NaCI/0.1 mM EDTA/0.1 mM dithiothreitol/2.5% (vol/vol) dimethyl sulfoxide/10 mM Tris HCl, pH 7.6 (assay volume, 100,u1). A 100-fold excess of unlabeled operator DNA was added before aliquots were withdrawn at different times and subjected to nitrocellulose filtration. The adsorbed complexes were eluted and analyzed by PAGE and autoradiography (11). As...

Edited by Sergio Papa, Gianfranco Gilardi and Wilhelm JustKeywords: Drug resistance Antimalarial drug pfcrt pfmdr1 Plasmodium falciparum Plasmodium vivax a b s t r a c tResistance to antimalarial drugs has often threatened malaria elimination efforts and historically has led to the short-term resurgence of malaria incidences and deaths. With concentrated malaria eradication efforts currently underway, monitoring drug resistance in clinical settings complemented by in vitro drug susceptibility assays and analysis of resistance markers, becomes critical to the implementation of an effective antimalarial drug policy. Understanding of the factors, which lead to the development and spread of drug resistance, is necessary to design optimal prevention and treatment strategies. This review attempts to summarize the unique factors presented by malarial parasites that lead to the emergence and spread of drug resistance, and gives an overview of known resistance mechanisms to currently used antimalarial drugs.

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