A simple RNA analysis method shows var and rif multigene family expression patterns in Plasmodium falciparum

Kyes, Sue; Pinches, Robert; Newbold, Chris

doi:10.1016/s0166-6851(99)00193-0

Cited by 248 publications

(220 citation statements)

References 17 publications

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“…The VMOs at 1 μM and PPMOs at 1 and 5 μM were added to ring-stage synchronized cultures at 10% parasitemia (2% hematocrit), and the cultures were incubated for 6 h at 37°C. Total RNA extraction from untreated and treated parasite cultures was performed as previously described (44), and the concentration of RNA was determined using nanodrop. RNA samples were treated with 1 unit of RQ1 DNase (Promega), and the absence of DNA contamination was checked by real-time PCR.…”

Section: Methodsmentioning

confidence: 99%

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

Garg

Wesolowski

Alonso

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Identification and genetic validation of new targets from available genome sequences are critical steps toward the development of new potent and selective antimalarials. However, no methods are currently available for large-scale functional analysis of the Plasmodium falciparum genome. Here we present evidence for successful use of morpholino oligomers (MO) to mediate degradation of target mRNAs or to inhibit RNA splicing or translation of several genes of P. falciparum involved in chloroquine transport, apicoplast biogenesis, and phospholipid biosynthesis. Consistent with their role in the parasite life cycle, down-regulation of these essential genes resulted in inhibition of parasite development. We show that a MO conjugate that targets the chloroquine-resistant transporter PfCRT is effective against chloroquine-sensitive and -resistant parasites, causes enlarged digestive vacuoles, and renders chloroquine-resistant strains more sensitive to chloroquine. Similarly, we show that a MO conjugate that targets the PfDXR involved in apicoplast biogenesis inhibits parasite growth and that this defect can be rescued by addition of isopentenyl pyrophosphate. MO-based gene regulation is a viable alternative approach to functional analysis of the P. falciparum genome. malaria | intraerythrocytic development | peptide conjugated morpholino oligomer | vivo morpholino oligomer | gene expression

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Section: Methodsmentioning

confidence: 99%

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

Garg

Wesolowski

Alonso

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…Total RNA was isolated, size-fractionated, and blotted by using a standard P. falciparum protocol (24). DNA probes were sequentially hybridized to the nylon filters and washed to an appropriate stringency for the probe used (21).…”

Section: Methodsmentioning

confidence: 99%

Variable var transition rates underlie antigenic variation in malaria

Horrocks

Pinches

Christodoulou

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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133

148

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Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) is expressed on the surface of infected erythrocytes where it plays a central role in both infected erythrocytes cytoadhesion and immune evasion. Switches in clonal expression of PfEMP1 result in antigenic variation that facilitates long-term chronic infection of the host. The var gene family encodes PfEMP1 variants, with transcriptional switching between different var variants providing the molecular basis for antigenic variation. Despite the importance of var transcriptional switching in the evasion of the immune response, little is known about the way in which this process is regulated. Here we report the measurement of transition on and off rates for a series of var gene variants. We find (i) that on and off rates for a given variant are dissimilar, (ii) that these rates vary dramatically among different variants, and (iii) that in isogenic clones expressing the same var gene, both on and off rates are constant and appear to be an intrinsic property of that particular gene. These data would suggest that the information that determines the probability of the activation or silencing of var genes is present in their surrounding DNA. Furthermore, some transitions appear to be disallowed depending on the recent variant antigen expression history of the parasite clone. These findings have important implications for both the underlying molecular mechanisms of antigenic variation and the processes that promote chronicity of infection in vivo.

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“…This was repeated several times until RBC lysis was complete. After all supernatant was removed and 1 mL TRIzol was added per original 5 mL infected RBCs; this was processed for total RNA as the method of Kyes et al (2000). Briefly, 0.2 mL CHCl 3 was added per 1 mL TRIzol, followed by a 13,200 rpm spin, 25 min at 4°C.…”

Section: Rna Extraction and Size-fractionationmentioning

confidence: 99%

“…The aqueous layer (0.6 mL) was precipitated in 0.5 mL of isopropanol for 2 h on ice, vortexed, and then centrifuged 30 min at 4°C, 13,200 rpm. The pellets were resuspended in 0.2 mL of DEPCwater and checked for quality and concentration by agarose gel analysis (Kyes et al 2000) and by measuring OD 260 . To size-fractionate RNA samples for microarrays, RNA was electrophoresed on gels with a 4.5% acrylamide/7 M urea/1‫ן‬ TBE stacker layered on a 15% acrylamide/7 M urea/1‫ן‬ TBE running gel ‫ן1(‬ TBE: 0.089 M Tris, 0.089 M boric acid, 2 mM EDTA).…”

Section: Rna Extraction and Size-fractionationmentioning

confidence: 99%

“…For total RNA microarray samples, 20 µg total RNA (from saponin-lysed parasites) was treated with Turbo DNase, as per manufacturer's instructions (Ambion) and then ethanol precipitated. For Northern blot samples tested only by oligonucleotide hybridization, total RNA was prepared from ring and pigmented trophozoite stages as described by Kyes et al (2000), with no saponin-lysis step.…”

Section: Rna Extraction and Size-fractionationmentioning

confidence: 99%

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Genome-wide discovery and verification of novel structured RNAs inPlasmodium falciparum

et al. 2007

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We undertook a genome-wide search for novel noncoding RNAs (ncRNA) in the malaria parasite Plasmodium falciparum. We used the RNAz program to predict structures in the noncoding regions of the P. falciparum 3D7 genome that were conserved with at least one of seven other Plasmodium spp. genome sequences. By using Northern blot analysis for 76 high-scoring predictions and microarray analysis for the majority of candidates, we have verified the expression of 33 novel ncRNA transcripts including four members of a ncRNA family in the asexual blood stage. These transcripts represent novel structured ncRNAs in P. falciparum and are not represented in any RNA databases. We provide supporting evidence for purifying selection acting on the experimentally verified ncRNAs by comparing the nucleotide substitutions in the predicted ncRNA candidate structures in P. falciparum with the closely related chimp malaria parasite P. reichenowi. The high confirmation rate within a single parasite life cycle stage suggests that many more of the predictions may be expressed in other stages of the organism's life cycle.

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A simple RNA analysis method shows var and rif multigene family expression patterns in Plasmodium falciparum

Cited by 248 publications

References 17 publications

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

Targeting protein translation, RNA splicing, and degradation by morpholino-based conjugates in Plasmodium falciparum

Variable var transition rates underlie antigenic variation in malaria

Genome-wide discovery and verification of novel structured RNAs inPlasmodium falciparum

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