title: var gene transcription dynamics in Plasmodium falciparum patient isolates.
abstract: A major feature of Plasmodium falciparum parasitized red blood cells (pRBC) is their capacity to sequester in the microcirculation. The binding is mediated by PfEMP1 (P. falciparum erythrocyte membrane protein 1), a variable protein encoded by the var gene family. P. falciparum avoids the host antibody response generated against previously used variants by switching the expression of PfEMP1, which may affect the disease outcome. We have here studied var gene transcription over time within the life cycle of the parasite by semi-quantitative PCR and sequencing by employing three sets of degenerate primers to the 5-prime end of the var genes (corresponding to the DBL1alpha-domain). To accurately determine transcript levels, subsequent in-depth analysis was made by amplifying the 10 most frequently expressed var sequences identified in each developmental stage by quantitative PCR (Q-PCR). The maximum peak in var gene transcription seems to vary in time among parasites. In five out of seven parasites, var gene transcription was found to be higher or equal at 22-26h post-invasion compared to 4-10h post-invasion. Our data indicate that the intra-isolate var gene transcription dominance order may change between different developmental stages. The transcription of var genes in field isolates is more complex than in laboratory strains and often changes after in vitro adaption of the parasite. By using semi-quantitative PCR employing degenerate primers combined with quantitative-PCR using specific primers it is possible to monitor var gene transcription in detail during the life cycle of the parasite. The work presented here suggests that trophozoite pRBC is likely to be the optimal source of RNA for predicting the translated var gene species.
authors.affiliation: Department of Microbiology, Tumor and Cell Biology (MTC), Karolinska Institutet, Stockholm, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Blomqvist
authors.forename: Johan
authors.initials: J
authors.lastname: Normark
authors.forename: Daniel
authors.initials: D
authors.lastname: Nilsson
authors.forename: Ulf
authors.initials: U
authors.lastname: Ribacke
authors.forename: Judy
authors.initials: J
authors.lastname: Orikiriza
authors.forename: Petter
authors.initials: P
authors.lastname: Trillkott
authors.forename: Justus
authors.initials: J
authors.lastname: Byarugaba
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Egwang
authors.forename: Fred
authors.initials: F
authors.lastname: Kironde
authors.forename: Björn
authors.initials: B
authors.lastname: Andersson
authors.forename: Mats
authors.initials: M
authors.lastname: Wahlgren
mesh_terms: D000818:Animals; D002675:Child, Preschool; D017931:DNA Primers / Q000235:genetics; D020869:Gene Expression Profiling; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D016778:Malaria, Falciparum / Q000469:parasitology*; D010963:Plasmodium falciparum / Q000235:genetics* / Q000302:isolation & purification*; D015800:Protozoan Proteins / Q000096:biosynthesis* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D015800:Protozoan Proteins; C085731:erythrocyte membrane protein 1, Plasmodium falciparum