Our limited phenotypic screen for attenuated parasite growth confirmed the feasibility of such approaches inP. falciparumby identifying several genes and pathways critical for blood-stage development. One of the most severely affected mutant parasites identified in our screen is a knockout of MAL8P1.104 (clone F3), which is thePlasmodiumorthologue of yeastCaf1(CCR4-associated factor 1) selleck . In yeast, CAF1 is a component of CCR4-NOT complex that is a global regulator of gene expression, controlling chromatin remodelling, transcriptional regulation, mRNA stability and protein degradation . MEK phosphorylation Experimental protein interaction data indicates
a similar functional complex exists inP. falciparum and with a scarcity of known transcription factors or identifiable conserved regulatory elements inPlasmodium, deadenylation may be extremely significant in controlling gene expression through regulating mRNA
abundance by degradation . The significance of protein phosphorylation and dephosphorylation in regulating parasite cellular activities is also clearly ICG-001 demonstrated by the attenuated growth phenotype of our knockout of PFF0770c (clone A5), which encodes one of the 12 type 2C protein phosphatases (PP2C) found inPlasmodium. PP2Cs carry out a wide range of functions in higher eukaryotes including intracellular signalling and providing cell cycle and developmental check points [37–39]. Two PP2Cs, in Non-specific serine/threonine protein kinase the closely related apicomplexanToxoplasma,
were recently shown to be involved in parasite motility and host cell modulation [40,41]. Another mutant clone displaying attenuated growth was a knockout of PF10_0350 (clone E6) that codes for a hypothetical protein unique toPlasmodiumspecies and attests to the theory that such uniquePlasmodiumgenes need to be investigated further as antimalarial targets.piggyBacinsertion in the 5′ UTRs of PFC0271c and PFC0275w, coding for glutaredoxin and glycerol-3 phosphate dehydrogenase, respectively, resulted in increased levels of both transcripts in the mutant clone B7 as seen by quantitative RT-PCR (data not shown), indicating that optimal expression of genes is essential for normal parasite growth. Several other phenotypic screens such as those for virulence, drug resistance, gametocytogenesis and transmissibility of infection to mosquito hosts can now be accomplished inP. falciparumthat will contribute immensely to our current understanding of parasite biology. Apart from its application in whole-genome mutagenesis and phenotype screens,piggyBacis also a powerful tool for stable transgene expression inP. falciparumas any parasite strain or clone of interest can be transformed. We have confirmed the functionality ofpiggyBacsystem in three different strains ofP.