Validation of a modified method for Bxb1 mycobacteriophage integrase-mediated recombination in Plasmodium falciparum by localization of the H-protein of the glycine cleavage complex to the mitochondrion

The Bxb1 integrase system was used to localize the Plasmodium falciparum H-protein to the mitochondrion. Modifications to the transfection method resulted in decreased selection times. The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5...

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Bibliographic Details
Published in:Molecular and biochemical parasitology 2010-08, Vol.172 (2), p.156-160
Main Authors: Spalding, Maroya D., Allary, Marina, Gallagher, John R., Prigge, Sean T.
Format: Article
Language:English
Subjects:
Bxb1 integrase
DNA, Mitochondrial - genetics
Genes, Reporter
Genetics, Microbial - methods
Glycine cleavage
Glycine Decarboxylase Complex - genetics
Green Fluorescent Proteins - genetics
H-protein
Integrases - genetics
Integrases - metabolism
Malaria
Mitochondrion
Mycobacteriophages - genetics
Plasmodium falciparum
Plasmodium falciparum - genetics
Recombinant Fusion Proteins - genetics
Recombination, Genetic
Viral Proteins - genetics
Viral Proteins - metabolism
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Summary:The Bxb1 integrase system was used to localize the Plasmodium falciparum H-protein to the mitochondrion. Modifications to the transfection method resulted in decreased selection times. The glycine cleavage complex (GCV) is a potential source of the one carbon donor 5,10-methylene-tetrahydrofolate (5,10-CH2-THF) in the malaria parasite Plasmodium falciparum. One carbon (C1) donor units are necessary for amino acid and nucleotide biosynthesis, and for the initiation of mitochondrial and plastid translation. In other organisms, GCV activity is closely coordinated with the activity of serine hydroxymethyltransferase (SHMT) enzymes. P. falciparum contains cytosolic and mitochondrial SHMT isoforms, and thus, the subcellular location of the GCV is an important indicator of its role in malaria metabolism. To determine the subcellular localization of the GCV, we used a modified version of the published method for mycobacteriophage integrase-mediated recombination in P. falciparum to generate cell lines containing one of the component proteins of the GCV, the H-protein, fused to GFP. Here, we demonstrate that this modification results in rapid generation of chromosomally integrated transgenic parasites, and we show that the H-protein localizes to the mitochondrion.
ISSN:0166-6851
1872-9428
DOI:10.1016/j.molbiopara.2010.04.005