Subject(s)
Adenine/analogs & derivatives , Antiparasitic Agents/pharmacology , DNA-Directed DNA Polymerase/genetics , Organophosphonates , Organophosphorus Compounds/pharmacology , Plasmodium falciparum/drug effects , Adenine/pharmacology , Amino Acid Sequence , Amino Acid Substitution/genetics , Animals , Binding Sites , DNA-Directed DNA Polymerase/chemistry , Drug Resistance/genetics , Humans , Molecular Sequence Data , Mutation , Plasmodium falciparum/genetics , Plasmodium falciparum/radiation effects , Sequence Alignment , Ultraviolet RaysABSTRACT
The gene encoding the primase small subunit was isolated from genomic DNA of strain K1 of the human malarial parasite Plasmodium falciparum. Isolation of a complete cDNA clone revealed the presence of 15 introns in the genomic sequence. This is unprecedented for Plasmodium genes, which usually contain no or only 1 or 2 introns. The gene is present as a single copy and the cDNA contains an open reading frame of 1356 nt encoding a protein of 452 amino acids. A single mRNA of 2.1 kb was identified by Northern blotting. Comparison of the amino acid sequence with five eukaryotic small primase subunits revealed the presence of eight conserved regions. Sequence alignments allowed the identification of putative motifs A, B and C that are essential features of the catalytic centre of DNA polymerases, RNA polymerases and reverse transcriptases. Also, similarity of a C-terminal region of approximately 100 amino acids to a conserved region in herpes virus primases, alpha-like DNA polymerases and RNA polymerase II was noted. The complete gene was expressed as a fusion product containing an N-terminal polyhistidine tag using a baculovirus expression vector. The protein was overproduced in insect cells and purified. Activity assays demonstrated the ability of the p53 subunit to initiate de novo primer formation.
Subject(s)
Genes, Protozoan/genetics , Plasmodium falciparum/enzymology , RNA Nucleotidyltransferases/chemistry , Amino Acid Sequence , Animals , Baculoviridae/genetics , Base Sequence , Blotting, Northern , Cloning, Molecular , DNA Primase , DNA, Complementary/chemistry , DNA-Directed DNA Polymerase/chemistry , DNA-Directed RNA Polymerases/chemistry , Electrophoresis, Polyacrylamide Gel , Gene Expression/genetics , Hydrogen-Ion Concentration , Introns/genetics , Molecular Sequence Data , Recombinant Proteins/genetics , Sequence Alignment , Sequence AnalysisABSTRACT
An in vitro test which quantifies drug inhibition of Plasmodium falciparum replication by measuring the fluorescence intensity of Hoechst 33258 dye bound to DNA is described. The procedure does not require expensive reagents or equipment and can be completed in less than 10 min. The assay was highly accurate and sensitive: cultures with as few as 0.4% schizont-infected erythrocytes could reliably be analyzed. The method was not biased by the actual parasite stage used; i.e., the amount of fluorescence detected in a sample of a culture of mature schizonts equaled the amount detected with the ring form culture derived from these schizonts. Even the presence of large proportions of free merozoites, which are easily neglected in microscopic estimates, did not bias the results. Furthermore, measurement of the chloroquine susceptibility of the multidrug-resistant K1 strain and the chloroquine-susceptible NF54 strain showed that the method is most suitable for quantifying the drug resistance of P. falciparum.
Subject(s)
Antimalarials/pharmacology , Chloroquine/pharmacology , DNA, Protozoan/metabolism , Plasmodium falciparum/drug effects , Animals , Bisbenzimidazole , Dose-Response Relationship, Drug , Fluorescent Dyes , Humans , Microbial Sensitivity Tests , Plasmodium falciparum/metabolismABSTRACT
The very effective (ID50 = 47 nM) and selective antimalarial compound (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) abruptly arrests Plasmodium falciparum-cultured schizonts at concentrations between 1 and 10 x ID50 as soon as their DNA content reaches 8 times that of the haploid ringform stage. Even very high HPMPA concentrations do not inhibit the first 2-3 rounds of schizogonic DNA replication. Also, in the presence of HPMPA, replication of the 6-kb mitochondrial and 35-kb chloroplast-like DNA proceeds normally and in close concert with each other, both to a 16-fold amount within 5 h during the trophozoite stage. Hence the in in vitro assays HPMPApp-sensitive plasmodial DNA polymerase gamma-like enzyme (IC50 = 1 microM)--assumed to be involved in mitochondrial DNA replication--is not the target of HPMPA in vivo (living parasites), nor seems to be the DNA polymerization activities of the--in vitro also HPMPA-sensitive (IC50 = 38 microM)--DNA polymerase alpha or of any other nuclear DNA polymerase of Plasmodium. In vitro assays demonstrated that HPMPApp does not act as an alternative substrate for plasmodial polymerases, contradicting the suggestion that the observed delayed inhibition of plasmodial schizogony might be the result of DNA strand breakage caused by HPMPApp incorporation. Neither do results support the idea that the HPMPA-induced arrest of DNA replication might be due to chain termination as a result of such incorporation. We investigated whether arrest of DNA replication by HPMPA in schizonts could be explained by inhibition of the DNA synthesis rate limiting ribonucleotide reductase enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)
