ABSTRACT
Mature exoerythrocytic (EE) forms of two cloned lines (3D7 and HB3) of Plasmodium falciparum were obtained in the livers of splenectomized chimpanzees. Sectioned preparations were examined by immunofluorescence (IFA) using mAbs that distinguished allelic variants of the blood-form antigen gp195 and mAbs that recognized multiple conserved epitopes of gp195. EE forms and blood schizonts exhibited identical IFA reactions for each respective clone, showing that the antigen was expressed identically in liver and blood-stage parasites. A third chimpanzee was infected with sporozoites derived from a mixture of 3D7 and HB3 gametocytes that had undergone cross-fertilization in the mosquitoes. IFAs on the EE forms in this animal showed that segregation of each gp195 allele had occurred earlier in the life cycle, providing evidence that the parasite is haploid for the whole of its mammalian development.
Subject(s)
Antigens, Protozoan/genetics , Liver/parasitology , Malaria/parasitology , Plasmodium falciparum/immunology , Alleles , Animals , Antigens, Protozoan/biosynthesis , Fluorescent Antibody Technique , Gene Expression Regulation , Genotype , Pan troglodytes , Plasmodium falciparum/genetics , Plasmodium falciparum/growth & developmentABSTRACT
Monoclonal antibodies against blood forms of Plasmodium falciparum were used to demonstrate considerable antigenic diversity in this species. Different isolates were distinguished by their ability to react with certain antibodies, and most of the antibodies reacted specifically with merozoites, schizonts, or both. The distribution of different antigenic types appeared not to be related to geographic origin. Serological typing with monoclonal antibodies extends the range of methods for identification of different strains of this malaria parasite.
Subject(s)
Antibodies, Monoclonal , Antigens/analysis , Epitopes/analysis , Plasmodium falciparum/immunology , Animals , Humans , Plasmodium falciparum/physiology , Species SpecificityABSTRACT
The dynamics of multiple Plasmodium infections in asymptomatic children living under intense malaria transmission pressure provide evidence for a density-dependent regulation that transcends species as well as genotype. This regulation, in combination with species- and genotype-specific immune responses, results in nonindependent, sequential episodes of infection with each species.
Subject(s)
Malaria/parasitology , Parasitemia/parasitology , Plasmodium/physiology , Adolescent , Animals , Child , Child, Preschool , Female , Genotype , Humans , Malaria/immunology , Malaria Vaccines , Male , Papua New Guinea , Plasmodium/genetics , Plasmodium falciparum/physiology , Plasmodium malariae/physiology , Plasmodium vivax/physiology , Species SpecificityABSTRACT
Malaria parasites are haploid for most of their life cycle, with zygote formation and meiosis occurring during the mosquito phase of development. The parasites can be analyzed genetically by transmitting mixtures of cloned parasites through mosquitoes to permit cross-fertilization of gametes to occur. A cross was made between two clones of Plasmodium falciparum differing in enzymes, drug sensitivity, antigens, and chromosome patterns. Parasites showing recombination between the parent clone markers were detected at a high frequency. Novel forms of certain chromosomes, detected by pulsed-field gradient gel electrophoresis, were produced readily, showing that extensive rearrangements occur in the parasite genome after cross-fertilization. Since patients are frequently infected with mixtures of genetically distinct parasites, mosquito transmission is likely to provide the principal mechanisms for generating parasites with novel genotypes.
Subject(s)
Plasmodium falciparum/genetics , Adenosine Deaminase/genetics , Animals , Anopheles/parasitology , Antigens, Protozoan/genetics , Chromosomes , Clone Cells , Crosses, Genetic , Insect Vectors , Malaria/parasitology , Pan troglodytes , Pyrimethamine/pharmacology , Recombination, GeneticABSTRACT
Merozoite surface antigen MSA-2 of the human parasite Plasmodium falciparum is being considered for the development of a malaria vaccine. The antigen is polymorphic, and specific monoclonal antibodies differentiate five serological variants of MSA-2 among 25 parasite isolates. The variants are grouped into two major serogroups, A and B. Genes encoding two different variants from serogroup A have been sequenced, and their DNA together with deduced amino acid sequences were compared with sequences encoded by other alleles. The comparison shows that the serological classification reflects differences in DNA sequences and deduced primary structure of MSA-2 variants and serogroups. Thus, the overall homologies of DNA and amino acid sequences are over 95% among variants in the same serogroup. In contrast, similarities between the group A variants and a group B variant are only 70 and 64% for DNA and amino acid sequences, respectively. We propose that the MSA-2 protein is encoded by two highly divergent groups of alleles, with limited additional polymorphism displayed within each group.
Subject(s)
Antigens, Protozoan/genetics , Antigens, Surface/genetics , Membrane Glycoproteins/genetics , Plasmodium falciparum/genetics , Polymorphism, Genetic , Protozoan Proteins , Amino Acid Sequence , Animals , Antibodies, Monoclonal , Base Sequence , Chromosome Deletion , Membrane Glycoproteins/immunology , Molecular Sequence Data , Molecular Weight , Oligonucleotide Probes , Plasmodium falciparum/classification , Plasmodium falciparum/immunology , Polymerase Chain Reaction , Sequence Homology, Nucleic AcidABSTRACT
It is well recognized that drug resistance is the most significant obstacle to gaining effective malaria control. Despite the enormous advances in the knowledge of the biochemistry and molecular biology of malaria parasites, only a few genes determining resistance to the commonly used drugs have been identified. The idea that rodent malaria parasites should be exploited more widely for such work, in view of the practical problems of studying this subject experimentally in human malaria, is presented.
Subject(s)
Antimalarials/pharmacology , Malaria/parasitology , Mutation , Plasmodium berghei/drug effects , Plasmodium chabaudi/drug effects , Animals , Disease Models, Animal , Drug Resistance/genetics , Plasmodium berghei/genetics , Plasmodium chabaudi/geneticsABSTRACT
The principal findings of the P. falciparum surveys are given below. Considerable diversity of enzymes, antigens, drug sensitivity and other characters is seen among P. falciparum isolates. Cloning studies show that certain isolates contain mixtures of parasites which may be diverse in one or more of these characters. No obvious regional distribution is seen in the enzymic and antigenic characters examined, although differences in the frequencies of certain enzymes appear to exist. Variations in drug sensitivity are seen among parasites from different regions, the occurrence of resistant forms usually being correlated with the extent of use of the drug concerned.
Subject(s)
Malaria/parasitology , Plasmodium/genetics , Animals , Antigens/genetics , Drug Resistance, Microbial , Enzymes/genetics , Genetic Variation , Humans , Plasmodium/enzymology , Plasmodium/immunology , Plasmodium falciparum/genetics , Rodent Diseases/parasitology , Rodentia/parasitologyABSTRACT
We report an in situ PCR technique for visualising amplified DNA of blood forms of Plasmodium falciparum on microscope slides by fluorescence microscopy. The method is used to assess the changes in frequency of different alleles of the MSP1 gene in cultures of the progeny of a cross. We show that parasites with a recombinant form of this protein possess an initial growth advantage before declining in numbers over the long-term.
Subject(s)
Genes, Protozoan , Merozoite Surface Protein 1/genetics , Plasmodium falciparum/genetics , Polymerase Chain Reaction/methods , Recombination, Genetic , Alleles , Animals , Gene Frequency , Plasmodium falciparum/growth & developmentABSTRACT
Two dimensional electrophoresis has been used to examine protein variation in clones of two Plasmodium falciparum isolates. Variant forms of 12 proteins were detected. Five genetically distinct parasite types were identified in one isolate, and two in the second isolate. Examination of uncloned parasites using this technique showed that the frequency of each genotype altered during six months of culture.
Subject(s)
Genetic Variation , Plasmodium falciparum/genetics , Proteins/genetics , Animals , Clone Cells , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Phenotype , Plasmodium falciparum/analysis , Proteins/analysis , ThailandABSTRACT
We have located a possible chloroquine resistance locus in the genome of the rodent malaria parasite Plasmodium chabaudi. Two genetically distinct clones of the parasite were grown in vivo and allowed to undergo genetic crossing. The clones differed from each other in their susceptibility to chloroquine; AS(3CQ) had been selected for a low level of resistance to the drug whereas AJ is chloroquine-sensitive. Independent recombinant progeny (20) were cloned from the products of two crosses, phenotyped for their susceptibility to chloroquine, and genotyped for their inheritance of 46 chromosome-specific markers. No association was found between chloroquine susceptibility and the inheritance of pcmdr1, the P. chabaudi homologue of the pfmdr1 multi-drug resistance gene of P. falciparum. Also, there was no association between chloroquine susceptibility and the inheritance of a marker linked to a putative chloroquine resistance locus in a P. falciparum cross. However, 16 of the progeny clones showed co-segregation of four linked markers on chromosome 11 with their resistance phenotype. This result suggests that a locus for chloroquine resistance exists on this chromosome in P. chabaudi.
Subject(s)
Antimalarials/pharmacology , Chloroquine/pharmacology , Plasmodium chabaudi/drug effects , Plasmodium chabaudi/genetics , Animals , Chromosome Mapping , Crosses, Genetic , Dose-Response Relationship, Drug , Drug Resistance, Multiple/genetics , Genes, Protozoan , Genetic Linkage , Genetic Markers , HSP90 Heat-Shock Proteins/genetics , Male , Meiosis , Mice , RatsABSTRACT
Intragenic recombination in the merozoite surface protein MSP-1 of Plasmodium falciparum has been demonstrated in a cross between two cloned lines (3D7 and HB3) of this species. Following passage of a mixture of the clones through mosquitoes, uncloned progeny were examined by PCR for molecules containing sequences of both parent MSP-1 alleles. A recombinant molecule possessing both 3D7 and HB3 sequences has been obtained. Such molecules were not obtained from artificial mixtures of the blood forms of each clone. It is concluded that the novel allele was formed by a recombination event during meiosis of a hybrid 3D7/HB3 zygote.
Subject(s)
Plasmodium falciparum/genetics , Protein Precursors/genetics , Protozoan Proteins/genetics , Recombination, Genetic , Alleles , Animals , Anopheles/parasitology , Antigens, Protozoan/genetics , Base Sequence , Crosses, Genetic , DNA Primers/genetics , DNA, Protozoan/genetics , Female , Male , Meiosis/genetics , Merozoite Surface Protein 1 , Molecular Sequence Data , Plasmodium falciparum/immunology , Polymerase Chain Reaction , Protein Precursors/immunology , Protozoan Proteins/immunologyABSTRACT
The number of chromosomes and the chromosomal location and linkage of more than 50 probes, mainly of genes, have been established in four species of Plasmodium which infect African murine rodents. We expected that the location and linkage of genes would not be conserved between these species of malaria parasites since extensive inter- and intraspecific size differences of the chromosomes existed and large scale internal rearrangements and chromosome translocations in parasites from laboratory lines had been reported. Our study showed that all four species contained 14 chromosomes, ranging in size between 0.5 and 3.5 Mb, which showed extensive size polymorphisms. The location and linkage of the genes on the polymorphic chromosomes, however, was conserved and nearly identical between these species. These results indicate that size polymorphisms of the chromosomes are more likely due to variation in non-coding (subtelomeric, repeat) sequences and show that a high plasticity of internal regions of chromosomes that may exist does not frequently affect chromosomal location and linkage of genes.
Subject(s)
Chromosome Mapping , Chromosomes/genetics , Genes, Protozoan/genetics , Malaria/parasitology , Plasmodium/genetics , Animals , Electrophoresis, Gel, Pulsed-Field , Gene Rearrangement , Genetic Linkage , Mice , Molecular Probes , Plasmodium berghei/genetics , Plasmodium chabaudi/genetics , Plasmodium yoelii/genetics , Polymorphism, GeneticABSTRACT
A cloned Plasmodium berghei (ANKA) isolate was syringe passaged repeatedly to generate a line that was non-infective to Anopheles stephensi. Ribosomal gene organisation of this non-infective line was then compared to its infective ancestor. DNA was also prepared from asexual parasites and gametocytes of P. chabaudi and the arrangement of the rRNA genes of this species was studied. Although macrogametocytes have many more ribosomes than microgametocytes, this increase does not appear to stem from an amplification of the rRNA genes, as no differences either in the quantity or the arrangement of the rDNA could be detected. Furthermore, the loss of infectivity of the P. berghei gametocytes does not seem to be due to a reduction or rearrangement of sequences coding for the rRNA genes. P. chabaudi and P. berghei DNA failed to show any homology to a repetitive DNA sequence cloned from P. falciparum. We conclude that this probe, PFH8rep20, is specific for P. falciparum.
Subject(s)
DNA, Ribosomal/genetics , Plasmodium/genetics , RNA, Ribosomal/genetics , Animals , Genes , Plasmodium/growth & development , Plasmodium berghei/genetics , Plasmodium berghei/growth & development , Rats , Repetitive Sequences, Nucleic Acid , ReproductionABSTRACT
Previous results, relating mosquito infectivity to percentage of repetitive DNA in the genome of Plasmodia, are re-examined in the light of the finding that a parasite line used in the previous studies and classified as Plasmodium berghei NK65, was a mixed infection, where the major component appeared to be Plasmodium yoelii. This conclusion was reached through cloning and isoenzyme typing of different clones. Isoenzyme typing alone is not sufficiently sensitive to reveal contamination amounting to less than 20% in a mixture. Attention is drawn to the risk inherent in work with uncloned lines, where the proportions of species or sub-species present may vary according to line history and gametocyte viability.
Subject(s)
Culicidae/parasitology , DNA/analysis , Plasmodium/classification , Animals , Glucose-6-Phosphate Isomerase/analysis , Isoenzymes/analysis , Malaria/parasitology , Malaria/veterinary , Mice , Plasmodium/enzymology , Plasmodium/genetics , Plasmodium/physiology , Repetitive Sequences, Nucleic Acid , Rodent Diseases/parasitologyABSTRACT
Individual oocysts from Plasmodium falciparum-infected Anopheles gambiae and Anopheles stephensi mosquitoes have been examined by the PCR technique, after their removal from the midgut. The DNA obtained from these oocysts has been amplified using oligonucleotide primers specific for part of the merozoite surface antigen MSA-1 gene. This technique distinguishes oocysts which are the products of self-fertilisation events from those which are the products of cross-fertilisation between different parasite clones.
Subject(s)
DNA, Protozoan/genetics , Hybridization, Genetic , Plasmodium falciparum/genetics , Animals , Anopheles , Antigens, Protozoan/genetics , Base Sequence , Merozoite Surface Protein 1 , Molecular Sequence Data , Plasmodium falciparum/immunology , Polymerase Chain Reaction , Protein Precursors/genetics , Protozoan Proteins/geneticsABSTRACT
We have attempted to establish the degree of linkage conservation between different species of the malaria parasite Plasmodium. Initially, the chromosome locations of 42 homologous genes were established in parasites from a rodent malaria species and the human malaria parasite P. falciparum. Of these genes, 26 appeared to be conserved within ten synteny groups between the two genomes. Several synteny groups were analysed further by long-range restriction mapping of digested chromosomes. Finally, a fine restriction map of one of the linkage groups was made from the rodent malaria parasites P. berghei and from P. falciparum and from the simian malaria parasite P. knowlesi. The fine-scale organisation of this linkage group appears to have remained intact among the three species, despite the evolutionary distance between them. This provides the first example of linkage conservation between the rodent, simian and human malaria species, which represent three different branches of the inferred phylogenetic tree of the genus Plasmodium.
Subject(s)
Chromosome Mapping , Genes, Protozoan , Genetic Linkage , Plasmodium chabaudi/genetics , Plasmodium falciparum/genetics , Plasmodium/genetics , Protein Kinases , Animals , Humans , Peptide Elongation Factor 1 , Peptide Elongation Factors/genetics , Protozoan Proteins/genetics , Restriction MappingABSTRACT
Pulsed field gradient gel electrophoresis has been used to identify at least 10 large DNA fragments in the genome of the rodent malaria species Plasmodium chabaudi. The fragments range in size from approximately 650 to 5000 kb. All the fragments contain sequences homologous to a P. berghei telomere probe, suggesting that they represent intact chromosomes. Ribosomal RNA genes and P. chabaudi cDNA sequences have been mapped to specific fragments. The fragments vary in size in different cloned isolates of the parasite. In a cross between two cloned parasites differing in the sizes of chromosomes 4 and 5, independent segregation of each chromosome occurred during meiosis.
Subject(s)
Chromosomes/ultrastructure , DNA/analysis , Plasmodium/genetics , Animals , Cloning, Molecular , DNA/genetics , Electrophoresis, Agar Gel , Ethidium , Karyotyping , Meiosis , Nucleic Acid Hybridization , Polymorphism, Genetic , RNA, Ribosomal/genetics , Sequence Homology, Nucleic AcidABSTRACT
Glycoproteins of the asexual blood stages of Plasmodium falciparum were labelled with radioactive glucosamine and analysed by two-dimensional electrophoresis. Four major glycoproteins were detected in all eight parasite isolates studied. Two of the glycoproteins, designated GP2 and GP4, were invariant among the isolates, while the other two GP1 and GP3 were found to be polymorphic in both their biochemical and antigenic properties. By immunoblotting and immunoprecipitation with specific monoclonal antibodies, the two polymorphic glycoproteins were identified as surface antigens of merozoites.
Subject(s)
Antigens, Protozoan/analysis , Glycoproteins/analysis , Plasmodium falciparum/immunology , Polymorphism, Genetic , Animals , Antigens, Surface/analysis , Cloning, Molecular , Electrophoresis, Gel, Two-Dimensional , Fluorescent Antibody Technique , Immunoblotting , Plasmodium falciparum/genetics , Precipitin TestsABSTRACT
A repetitive DNA fragment cloned from the malaria parasite, Plasmodium falciparum, has been analysed. It contains a 21 base pair sequence which occurs in multiple tandem repeats. Two clusters of the same repeat are found in opposite orientations on the same DNA fragment. The repetitive DNA provides an additional way to distinguish between different strains of parasite by hybridisation to genomic blots and may serve as a species-specific probe for diagnosis.
Subject(s)
DNA/analysis , Plasmodium falciparum/genetics , Repetitive Sequences, Nucleic Acid , Animals , Bacteriophage lambda/genetics , Cloning, Molecular , DNA/isolation & purification , Nucleic Acid HybridizationABSTRACT
A range of 22 mouse anti-P. falciparum monoclonal antibodies have been characterized by indirect immunofluorescence and immunoprecipitation. On the basis of these studies, 5 groups of antibodies and 6 classes of antigen were defined. Group I antibodies give, bright, uniform, generalised staining of all blood stages including gametocytes. Three of these antibodies precipitate a metabolically labelled molecule(s) of 35 kDa. One precipitates a 50 kDa antigen. Group II antibodies, which give strong localised immunofluorescence in merozoites, and a weak diffuse pattern in earlier stages, precipitate biosynthetically labelled molecules of 160 kDa. Group III antibodies react with all asexual stages. With merozoites they produce intense staining around the perimeter, both in fixed and unfixed preparations. They precipitate biosynthetic molecules of 190 kDa. Group IV antibodies are identical to Group III except they are stage restricted to schizonts and merozoites. They also precipitate 190 kDa antigens. These, however, in contrast to group III, are readily accessible to 125I-lactoperoxidase labelling. One antibody also precipitates a set of smaller peptides. Finally, Group V antibodies produce very bright ill-defined staining of pigment-containing parasites, as well as of inclusions in the red cell. They precipitate a series of molecules of 160, 60 and 35 kDa which are readily accessible to 125I. The 160 kDa molecule is also labelled by [35S]methionine. These results are discussed in the context of the development of a malaria vaccine and immunodiagnostic tests.