title: Inducible knockdown of Plasmodium gene expression using the glmS ribozyme.
abstract: Conventional reverse genetic approaches for study of Plasmodium malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control P. falciparum gene expression in which target genes bearing a glmS ribozyme in the 3' untranslated region are efficiently knocked down in transgenic P. falciparum parasites in response to glucosamine inducer. Using reporter genes, we show that the glmS ribozyme cleaves reporter mRNA in vivo leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The glmS ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (PfDHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of PfDHFR-TS mRNA and protein. PfDHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The glmS ribozyme may thus be a tool for study of essential genes in P. falciparum and other parasite species amenable to transfection.
authors.affiliation: National Center for Genetic Engineering and Biotechnology (BIOTEC), Khlong Nueng, Khlong Luang, Pathum Thani, Thailand.
authors.forename: Parichat
authors.initials: P
authors.lastname: Prommana
authors.forename: Chairat
authors.initials: C
authors.lastname: Uthaipibull
authors.forename: Chayaphat
authors.initials: C
authors.lastname: Wongsombat
authors.forename: Sumalee
authors.initials: S
authors.lastname: Kamchonwongpaisan
authors.forename: Yongyuth
authors.initials: Y
authors.lastname: Yuthavong
authors.forename: Ellen
authors.initials: E
authors.lastname: Knuepfer
authors.forename: Anthony A
authors.initials: AA
authors.lastname: Holder
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Shaw
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D005944:Glucosamine / Q000494:pharmacology; D006801:Humans; D009097:Multienzyme Complexes / Q000235:genetics; D010641:Phenotype; D010963:Plasmodium falciparum / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D016337:RNA, Catalytic / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D059386:Reverse Genetics; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
chemical_list: D001426:Bacterial Proteins; D005944:Glucosamine; D009097:Multienzyme Complexes; D016337:RNA, Catalytic; D012333:RNA, Messenger; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase; C056930:thymidylate synthase-dihydrofolate reductase; C000731191:component S, glutamate mutase protein, Bacteria; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing)
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