Heterogeneity and expression of the Plasmodium falciparum 5.8S ribosomal RNA genes

The number and expression of some of the large ribosomal RNA (rRNA) gene classes present in the human malaria parasite Plasmodium falciparum was determined. Southern blot analyses, using the 5.8S rRNA coding region as a marker, indicate that the P. falciparum genome contains at least 5 distinct subclasses of large rRNA genes. Dideoxy sequencing of the 5.8S rRNA domain and Northern blot analyses demonstrate that only one subclass is transcribed during the parasite's asexual erythrocytic life cycle.     

Characterization and complete nucleotide sequence of a 5.8S ribosomal RNA gene from Plasmodium falciparum

The 5.8S and 5S rRNA components from the FCR-3/The Gambia strain of Plasmodium falciparum have been identified and the complete nucleotide sequence of a 5.8S ribosomal RNA gene determined. Unlike the 5S rRNA species, the 5.8S is a single homogeneous population of molecules of 157 nucleotides. Comparison of its nucleotide sequence with previously reported 5.8S rRNA sequences indicates that it is homologous to these molecules, but distantly related to them. The sequence of the 5.8S rRNA coding region from the pfrib-2 recombinant of the HG13 Gambian isolate of P. falciparum is identical.     

Ribosomal RNA sequences of Sarcocystis muris, Theileria annulata and Crypthecodinium cohnii reveal evolutionary relationships among apicomplexans, dinoflagellates, and ciliates

Sarcocystis muris is a coccidium with a two-host life cycle involving the domestic cat and the mouse, Mus musculus. S. muris and Theileria annulata belong to the phylum Apicomplexa, but the latter organism is a tick-borne protozoon in the subclass Piroplasmea and causes tropical theileriosis in cattle. The small-subunit ribosomal RNA (16S-like rRNA) coding regions of these organisms as well as that of the free living dinoflagellate Crypthecodinium cohnii were amplified using polymerase chain reaction techniques and compared to 16S-like rRNA sequences from other eukaryotes. The 16S-like rRNA genes of S. muris and T. annulata are more similar to each other than either is to Plasmodium falciparum, the cause of malignant tertian malaria of humans or Plasmodium berghei, the agent of the commonly studied malaria of rodents. Evolutionary trees inferred from the rRNA sequence similarities support a close phylogenetic relationship between the Apicomplexa and Dinoflagellata as represented by Prorocentrum micans and C. cohnii. Apparently members of these related phyla arose from an ancestral stock that gave rise to the ciliated protozoa.     

Detection and quantification of Plasmodium falciparum in blood samples using quantitative nucleic acid sequence-based amplification

A quantitative nucleic acid sequence-based amplification (QT-NASBA) assay for the detection of Plasmodium parasites has been developed. Primers and probes were selected on the basis of the sequence of the small-subunit rRNA gene. Quantification was achieved by coamplification of the RNA in the sample with one modified in vitro RNA as a competitor in a single-tube NASBA reaction. Parasite densities ranging from 10 to 10(8) Plasmodium falciparum parasites per ml could be demonstrated and quantified in whole blood. This is approximately 1,000 times more sensitive than conventional microscopy analysis of thick blood smears. Comparison of the parasite densities obtained by microscopy and QT-NASBA with 120 blood samples from Kenyan patients with clinical malaria revealed that for 112 of 120 (93%) of the samples results were within a 1-log difference. QT-NASBA may be especially useful for the detection of low parasite levels in patients with early-stage malaria and for the monitoring of the efficacy of drug treatment.     

The complete sequence of a Plasmodium malariae SSUrRNA gene and its comparison to other plasmodial SSUrRNA genes

A gene encoding the small subunit rRNA (SSUrRNA) has been isolated from the human parasite, Plasmodium malariae. The gene has been sequenced. It contains conserved and variable regions which conform to patterns established for other eukaryotic SSUrRNA genes. Comparisons with other SSUrRNA genes from Plasmodium species reveal regions unique to P. malariae which could be used in specific diagnostic probes for this organism, and provide evidence that the gene is of the type expressed during asexual growth. In addition the '5.8S' gene has been cloned from P. malariae. The gene has been sequenced. It contains bases universally conserved in '5.8S' genes but there is considerable divergence between the P. malariae sequence and that of the P. falciparum gene.     

The 13p HSR in a human neuroblastoma cell line does not contain rRNA genes

We have studied a human neuroblastoma cell line containing a homogeneously staining region (HSR) on the short (p) arm of chromosome #13. The HSRs are believed to represent sites of gene amplification, containing multiple copies of one or a small number of genes. The 18S and 28S ribosomal RNA genes are the only structural genes so far assigned to the p arms of the human acrocentric chromosomes, which include number #13. Using two recombinant DNA probes specific for sequences in the 18S/28S ribosomal RNA (rRNA) gene complex for in situ hybridization, we have been able to establish that the 13pHSR in this line does not contain rRNA genes.     

Molecular karyotyping of the rodent malarias Plasmodium chabaudi, Plasmodium berghei and Plasmodium vinckei

The molecular karyotypes of four isolates of Plasmodium chabaudi, three of the subspecies P. chabaudi adami and one P. chabaudi chabaudi, as well as P. berghei and P. vinckei were studied by means of pulsed field gradient (PFG) gel electrophoresis. Each species appears to have 14 chromosomes, ranging in size from approximately 730 kb to greater than 2000 kb. The three P. chabaudi adami isolates did not appear any more similar to each other than to the P. c. chabaudi isolate. The chromosome locations of genes for a heat shock protein (hsp) 70, ribosomal RNA (rRNA), the precursor to the major merozoite surface proteins, dihydrofolate reductase and P. falciparum antigen 352 as well as four cloned DNA markers and a telomere probe were determined. However, a number of probes representing cloned P. falciparum antigens failed to hybridize to P. chabaudi DNA. Hence genes for malaria antigens appear to be much more divergent than genes for housekeeping functions.     

The role of polymorphisms in the spliced leader addition domain in determining promoter activity in Brugia malayi

The antimalarial activity of the antibiotic thiostrepton has long been attributed to inhibition of apicoplast protein synthesis through binding of apicoplast ribosomal RNA. However, the kinetics of parasite death upon thiostrepton treatment differ from those seen for other inhibitors of apicoplast housekeeping functions. We have analysed global changes in gene expression of the malaria parasite, Plasmodium falciparum, in an attempt to shed light on the responses of the parasite to this drug. Our results indicate a delay in gene expression profiles of thiostrepton-treated parasites. A small number of genes appear to be regulated outside of this trend; our data suggest a response from genes encoding components of the mitochondrial translational machinery, while little response is seen from genes encoding apicoplast-targeted proteins. Our findings are consistent with an effect of thiostrepton on mitochondrial protein synthesis, and thus warrant a re-evaluation of the target of thiostrepton in Plasmodium. They also provide some suggestion of mitochondrion-nucleus signalling in the parasite.     

Species-specific PCR detection of malaria parasites by microtiter plate hybridization: clinical study with malaria patients.

A simple and convenient PCR method that amplifies the 18S rRNA genes has been developed for the purpose of detecting and differentiating four species causing malaria in humans. The advantage of the assay is that the biotinylated PCR product is visualized following hybridization with specific probes which are immobilized on plate wells (microtiter plate hybridization). This method has been previously evaluated in a field study and was found to be sensitive and specific for the detection of Plasmodium falciparum and Plasmodium vivax. In the current study, the microtiter plate hybridization PCR method was evaluated by using blood specimens from malaria patients. All of 36 cases of falciparum malaria, 26 of 27 cases of vivax malaria, all of 11 cases of ovale malaria, and 2 cases of malariae malaria were diagnosed species specifically by the PCR method. There were four smear-negative, PCR-positive cases that seemed to correspond to the convalescent stage of malaria. In contrast, 30 cases for which the diagnosis of malaria has been excluded on the basis of microscopy and clinical courses showed negative PCR results. By comparing parasite densities and PCR results following antimalarial treatment of some patients, it was revealed that the PCR results largely paralleled the parasite densities and that PCR could detect as few as 10 parasites per microliter of blood. We conclude that this PCR method is highly sensitive and specific for the detection of all four parasite species and can serve as a useful supplement to microscopy for the clinical management of malaria.     

A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.     

Ribosomal RNA-based diagnosis of Plasmodium falciparum malaria

We have identified useful target sites for the diagnosis of malaria infections by oligonucleotide hybridization on the small subunit RNA of Plasmodium falciparum. Acetic acid works as effectively as formaldehyde or methyl mercuric hydroxide in procedures designed to apply RNA to filters. We have confirmed the findings of others that the stability of ribosomal RNA suffices for its use as a target for diagnosis. We have achieved a detection level of at least 0.00046% parasitemia and suggest that detection of a single parasite is well within reach of this technology.     

Age-related increase in methylation of ribosomal genes and inactivation of chromosome-specific rRNA gene clusters in mouse

An age-related increase in DNA methylation of the multicopy 18S and 28S ribosomal RNA genes was found in CBA/Ca mice beginning between 6 and 18 months of age at the 5′ end of these genes in liver, brain and spleen. The highest level of age-associated hypermethylation was mapped to the proximal 5′ spacer domain. Silver staining of actively transcribing ribosomal genes in metaphase chromosomes from stimulated spleen cells provided cytological evidence that these mice have 3 rRNA cistrons located on chromosomes 15, 16, and 18. The ribosomal gene cluster located on chromosome 16 was preferentially inactivated in older animals. Exposure of spleen cells from older individuals to 5-azacytidine appeared to both reactivate ribosomal gene clusters and reduce rRNA gene methylation.     

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).     

Ovine theileriosis in China: a new look at an old story

A fatal disease of sheep and goats in the northwestern part of China has in the past been reported to be due to Theileria lestoquardi. However, some characteristics of the causative agent are not in accordance with attributes ascribed to this parasite. We therefore determined the nucleotide sequence of the 18 small subunit ribosomal RNA (18S rRNA) gene of T. lestoquardi and the parasite identified in China and compared it with that of other Theileria and Babesia species. In the inferred phylogenetic tree, the 18S rRNA sequence of the Chinese parasite falls inside the clade consisting of Theileria species evidencing that it belongs to this genus. The 18S rRNA sequence of the Chinese parasite was found to be most closely related to Theileria buffeli and clearly divergent to T. lestoquardi, suggesting that it was a yet unrecognized Theileria species. The phylogenetic relationship of Theileria species infecting sheep and goats on the basis of their 18S rRNA gene structure was addressed. We report on the existence of at least two additional ovine and caprine piroplasm species, designated T. luwenshuni and T. uilenbergi.     

Applied genomics: data mining reveals species-specific malaria diagnostic targets more sensitive than 18S rRNA

Accurate and rapid diagnosis of malaria infections is crucial for implementing species-appropriate treatment and saving lives. Molecular diagnostic tools are the most accurate and sensitive method of detecting Plasmodium, differentiating between Plasmodium species, and detecting subclinical infections. Despite available whole-genome sequence data for Plasmodium falciparum and P. vivax, the majority of PCR-based methods still rely on the 18S rRNA gene targets. Historically, this gene has served as the best target for diagnostic assays. However, it is limited in its ability to detect mixed infections in multiplex assay platforms without the use of nested PCR. New diagnostic targets are needed. Ideal targets will be species specific, highly sensitive, and amenable to both single-step and multiplex PCRs. We have mined the genomes of P. falciparum and P. vivax to identify species-specific, repetitive sequences that serve as new PCR targets for the detection of malaria. We show that these targets (Pvr47 and Pfr364) exist in 14 to 41 copies and are more sensitive than 18S rRNA when utilized in a single-step PCR. Parasites are routinely detected at levels of 1 to 10 parasites/μl. The reaction can be multiplexed to detect both species in a single reaction. We have examined 7 P. falciparum strains and 91 P. falciparum clinical isolates from Tanzania and 10 P. vivax strains and 96 P. vivax clinical isolates from Venezuela, and we have verified a sensitivity and specificity of ～100% for both targets compared with a nested 18S rRNA approach. We show that bioinformatics approaches can be successfully applied to identify novel diagnostic targets and improve molecular methods for pathogen detection. These novel targets provide a powerful alternative molecular diagnostic method for the detection of P. falciparum and P. vivax in conventional or multiplex PCR platforms.     

Quantification of Plasmodium falciparum gametocytes in differential stages of development by quantitative nucleic acid sequence-based amplification

Two quantitative nucleic acid sequence-based amplification assays (QT-NASBA) based on Pfs16 and Pfs25, have been developed to quantify sexual stage commitment and mature gametocytes of Plasmodium falciparum. Pfs16 mRNA is expressed in all sexual forms including sexually committed ring stages while expression of Pfs25 mRNA is restricted to late stage gametocytes. Both assays showed a sensitivity of one sexual stage parasite/microl of blood. Blood samples from experimentally infected non-immune human volunteers were tested for Plasmodium falciparum by standard microscopy, a previously developed asexual 18S rRNA QT-NASBA, Pfs16 and Pfs25 mRNA QT-NASBA. Pfs16 QT-NASBA was positive in 9 out of 10 volunteers within 48 h after first detection of 18S rRNA, mostly before or at the day of positive microscopy. In contrast, the Pfs25 mRNA QT-NASBA was negative during the 28 days of follow-up, but consistently positive in gametocyte samples from naturally infected Kenyan patients. These data suggest that sexual stage commitment can occur early in the blood-stage infection without successful maturation into infectious gametocytes. In conclusion, Pfs16 and Pfs25 QT-NASBA assays in combination with a previously developed asexual stage QT-NASBA allow for the separate quantification of all developmental stages present in the circulation. The application of sexual stage QT-NASBA assays may contribute to a better understanding of the biology and epidemiology of malaria transmission.