Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii
ABSTRACT Comparing the steady-state expression levels of recombinant proteins in Toxoplasma gondii parasites indicates considerable variability, and this has sometimes caused difficulties in the engineering of transgenic parasites. Anecdotal observations suggested that alteration of the N-terminus, e.g. by engineering as a fusion protein, permits stable expression of various transgenes that were previously difficult to express in their native form. We have exploited the sensitivity and quantitative nature of fire-fly luciferase (LUC) to examine expression levels in further detail. Fusing the 26 N-terminal residues derived from chloramphenicol acetyl transferase (DeltaCAT) to LUC permits efficient transient or stable luciferase expression in transgenic parasite tachyzoites, providing a useful reporter for studies in T. gondii. Site-directed mutagenesis was used to alter the second codon of DeltaCAT-LUC to encode all 20 possible amino acids, and these constructs showed that changes in the second amino acid can have dramatic effects on luciferase activity, with Ala, Glu, and Asp codons yielding the highest expression levels. Similar results were observed for the expression of both GFP and the T. gondii HXGPRT gene, demonstrating the generality of this effect.
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ABSTRACT: EtMIC4 is a microneme protein of Eimeria tenella, an intracellular apicomplexan protozoan that can cause severe enteritis in chickens. The EtMIC4 gene has been partially characterised, and in this study, we used a combined strategy of rapid amplification of cDNA ends (5'RACE) and reverse transcription-polymerase chain reaction to identify the authentic 5' end of the transcribed sequence (accession number AJ306453.2). Comparison of the predicted EtMIC4 transcription start site with predicted start sites for EtMIC1, 2 and 3 genes identified comparable initiator regions that each conform to the consensus sequence for a transcriptional initiator element. The EtMIC4 gene is organised over 11 exons and analysis of the full-length predicted protein identified a new N-terminal region that comprises a hydrophobic signal peptide followed by four thrombospondin-type 1 modules that are similar to those previously described further downstream in the protein. Best-fit analysis shows that EtMIC4 shares high homology with the Eimeria maxima protein EmTFP250 and with TgMIC12, a predicted Toxoplasma gondii microneme protein. EtMIC4 and EmTFP250 share 70% amino acid identity and all predicted structural domains are conserved between the two. EtMIC4 and TgMIC12 share 48% identity and they have very similar domain organisation and conservation of intron/exon boundaries.Parasitology Research 01/2009; 104(3):717-21. DOI:10.1007/s00436-008-1301-0 · 2.33 Impact Factor
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ABSTRACT: Experimental evidence suggests that apicomplexan parasites possess bipartite promoters with basal and regulated cis-elements similar to other eukaryotes. Using a dual luciferase model adapted for recombinational cloning and use in Toxoplasma gondii, we show that genomic regions flanking 16 parasite genes, which encompass examples of constitutive and tachyzoite- and bradyzoite-specific genes, are able to reproduce the appropriate developmental stage expression in a transient luciferase assay. Mapping of cis-acting elements in several bradyzoite promoters led to the identification of short sequence spans that are involved in control of bradyzoite gene expression in multiple strains and under different bradyzoite induction conditions. Promoters that regulate the heat shock protein BAG1 and a novel bradyzoite-specific NTPase during bradyzoite development were fine mapped to a 6-8 bp resolution and these minimal cis-elements were capable of converting a constitutive promoter to one that is induced by bradyzoite conditions. Gel-shift experiments show that mapped cis-elements are bound by parasite protein factors with the appropriate functional sequence specificity. These studies are the first to identify the minimal sequence elements that are required and sufficient for bradyzoite gene expression and to show that bradyzoite promoters are maintained in a 'poised' chromatin state throughout the intermediate host life cycle in low passage strains. Together, these data demonstrate that conventional eukaryotic promoter mechanisms work with epigenetic processes to regulate developmental gene expression during tissue cyst formation.Molecular Microbiology 07/2008; 68(6):1502-18. DOI:10.1111/j.1365-2958.2008.06249.x · 5.03 Impact Factor
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ABSTRACT: The protozoan phylum Apicomplexa encompasses approximately 5000 species of obligate intracellular parasites, including those responsible for malaria and toxoplasmosis. Rather than dividing by binary fission, apicomplexans use a remarkable mechanism for replication, assembling daughters de novo within the cytoplasm. Here, we exploit time-lapse microscopy of fluorescent markers targeted to various subcellular structures in Toxoplasma gondii tachyzoites to determine how these unicellular eukaryotes efficiently package a complete set of organelles, maintaining the highly polarized organization necessary for host cell invasion and pathogenesis. Golgi division and elongation of the apicoplast are among the first morphologically observable events, associated with an unusual pattern of centriolar migration. Daughter parasites are assembled on cytoskeletal scaffolding, whose growth proceeds from the apical end, first encapsulating the divided Golgi. Further extension of the cytoskeletal scaffold results in partitioning of the apicoplast, nucleus, endoplasmic reticulum, and finally the mitochondrion, which enters the developing daughters rapidly, but only very late during the division cycle. The specialized secretory organelles (micronemes and rhoptries) form de novo. This distinctive pattern of replication -- in which organellar segregation spans approximately 75% of the cell cycle, completely encompassing S phase -- suggests an unusual mechanism of cell cycle regulation.Journal of Cell Science 06/2008; 121(Pt 9):1559-68. DOI:10.1242/jcs.021089 · 5.33 Impact Factor