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    Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method

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    Author
    Tonkin, CJ; van Dooren, GG; Spurck, TP; Struck, NS; Good, RT; Handman, E; Cowman, AF; McFadden, GI
    Date
    2004-09-01
    Source Title
    MOLECULAR AND BIOCHEMICAL PARASITOLOGY
    Publisher
    ELSEVIER SCIENCE BV
    University of Melbourne Author/s
    Tonkin, Christopher; VAN DOOREN, GIEL; McFadden, Geoffrey; SPURCK, TIMOTHY; Cowman, Alan
    Affiliation
    Botany
    Metadata
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    Document Type
    Journal Article
    Citations
    Tonkin, C. J., van Dooren, G. G., Spurck, T. P., Struck, N. S., Good, R. T., Handman, E., Cowman, A. F. & McFadden, G. I. (2004). Localization of organellar proteins in Plasmodium falciparum using a novel set of transfection vectors and a new immunofluorescence fixation method. MOLECULAR AND BIOCHEMICAL PARASITOLOGY, 137 (1), pp.13-21. https://doi.org/10.1016/j.molbiopara.2004.05.009.
    Access Status
    This item is currently not available from this repository
    URI
    http://hdl.handle.net/11343/26618
    DOI
    10.1016/j.molbiopara.2004.05.009
    Description

    C1 - Journal Articles Refereed

    Abstract
    The apicoplast and mitochondrion of the malaria parasite Plasmodium falciparum are important intracellular organelles and targets of several anti-malarial drugs. In recent years, our group and others have begun to piece together the metabolic pathways of these organelles, with a view to understanding their functions and identifying further anti-malarial targets. This has involved localization of putative organellar proteins using fluorescent reporter proteins such as green fluorescent protein (GFP). A major limitation to such an approach is the difficulties associated with using existing plasmids to genetically modify P. falciparum. In this paper, we present a novel series of P. falciparum transfection vectors based around the Gateway recombinatorial cloning system. Our system makes it considerably easier to construct fluorescent reporter fusion proteins, as well as allowing the use of two selectable markers. Using this approach, we localize proteins involved in isoprenoid biosynthesis and the posttranslational processing of apicoplast-encoded proteins to the apicoplast, and a protein putatively involved in the citric acid cycle to the mitochondrion. To confirm the localization of these proteins, we have developed a new immunofluorescence assay (IFA) protocol using antibodies specific to the apicoplast and mitochondrion. In comparison with published IFA methods, we find that ours maintains considerably better structural preservation, while still allowing sufficient antibody binding as well as preserving reporter protein fluorescence. In summary, we present two important new tools that have enabled us to characterize some of the functions of the apicoplast and mitochondrion, and which will be of use to the wider malaria research community in elucidating the localization of other P. falciparum proteins.
    Keywords
    Protein Targeting and Signal Transduction; Genetic Technologies: Transformation; Site-directed Mutagenesis; etc.; Medical Parasitology ; Infectious Diseases

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