The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation

Goman, Michael; Langsley, Gordon; Hyde, John E.; Yankovsky, N. K.; Zolg, J. Werner; Scaife, John

doi:10.1016/0166-6851(82)90012-3

Cited by 145 publications

(35 citation statements)

References 31 publications

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“…As the transferase source, we used P. fulcipurum isolated by the saponin method (Goman et al, 1982), subsequently lysed in distilled water containing protease inhibitors for 5 min on ice, then readjusted to the assay buffer conditions. Alternatively, microsomes from secondary chicken embryo fibroblasts or a mixture of both were used.…”

Section: Transfer Of Dolichol-linked Oligosaccharidesmentioning

confidence: 99%

Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum

Dieckmann-Schuppert

Bender

Odenthal-Schnittler

et al. 1992

European Journal of Biochemistry

119

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This study investigates protein glycosylation in the asexual intraerythrocytic stage of the malaria parasite, Plasmodium ,fakiparum, and the presence in the infected erythrocyte of the respective precursors.In in vitro cultures, P. fakiparum can be metabolically labeled with radioactive sugars, and its multiplication can be affected by glycosylation inhibitors, suggesting the capability of the parasite to perform protein-glycosylation reactions. Gel-filtration analysis of sugar-labeled malarial proteins before and after specific cleavage of N-glycans or 0-glycans, respectively, revealed the majority of the protein-bound sugar label to be incorporated into 0-glycans, but only little (7-12% of the glucosamine label) or no N-glycans were found. Analysis of the nucleotide sugar and sugar-phosphate fraction showed that radioactive galactose, glucosamine, fucose and ethanolamine were converted to their activated derivatives required for incorporation into protein. Mannose was mainly recovered as a bisphosphate, whereas the level of radiolabeled GDP-mannose was below the detection limit. The analysis of organic-solvent extracts of sugar-labeled cultures showed no evidence for the formation by the parasite of dolichol cycle intermediates, the dedicated precursors in protein N-glycosylation. Consistently, the amount of UDP-N-acetylglucosamine formed did not seem to be affected by the presence of tunicamycin in the culture. Oligosaccharyl-transferase activity was not detectable in a lysate of P. fakiparum, using exogenous glycosyl donors and acceptors.Our studies show that 0-glycosylation is the major form of protein glycosylation in intrderythrocytic P. julciparum, whereas there is little or no protein N-glycosylation. A part of these studies has been published in abstract form [Dieckmann-Schuppert, A,, Hensel, J. and Schwarz, R. T. (1991) Biol. Chem. Hoppe-Seyler 372,6451.Plasmodium fakiparum is the causative agent of human malignant malaria tropica. Despite huge efforts in vaccine and chemotherapy development, today this disease still causes the death of several million people/year (World Health Organization, 1989). A more thorough understanding of the biochemistry and cell biology of this parasite is required in order to develop better chemotherapy and vaccination strategies. One of the neglected areas of malaria biochemistry is the glycobiology of the parasite. Very little is known to date about the biological significance of oligosaccharides in P. julcipurum, be they linked to lipids or to proteins. Glycolipids may be membrane components and as such be potential antigens, or be involved in the formation of glycoproteins, e. g. dolichol

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Section: Transfer Of Dolichol-linked Oligosaccharidesmentioning

confidence: 99%

Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum

Dieckmann-Schuppert

Bender

Odenthal-Schnittler

et al. 1992

European Journal of Biochemistry

119

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“…Cell pellets of bulk cultures of a P. falciparum isolate, Kl from Thailand (Thaithong & Beale, 1981) were harvested as previously described (Goman et al 1982) and stored at -70 °C until required. They were then extracted at 4 °C for 1 h with an extraction buffer (2°0 (w/v)) sodium deoxycholate, 1 °0 (w/v) Nonidet P40, 50 m.M Tris-HCl (pH 8-(;), 5 m\i ethylenediaminetetra-acetic acid (EDTA), 5 m.\i l,2-di(2-aminoethoxy)ethane-iV,iY,A r ',7V'-tetra-acetic acid (EGTA)) containing 5 mM-iodoacetamide, 0'2 m\i phenylmethylsulphonyl fluoride (PMSF) and 10/yg/ml each of the protease inhibitors pepstatin, chymostatin, antipain, leupeptin and soybean trypsin inhibitor (all from Sigma Chemical Co. Ltd, Poole, UK).…”

Section: Affinity-purification Of Rap-1 Antigenmentioning

confidence: 99%

“…Sodium R. G. Ridley and others 188 dodecyl sulphate polvacrvlamide gel electrophoresis (SDS-PAGE), using the method of Laemmli (1970), together with Western blotting analysis have also been previously described (Takacs & Staehli, 1987). P. falciparum in vitro culture was maintained according to the method of Trager & Jensen (1976), later modified by Zolg et al (1982).…”

Section: Standard Techniquesmentioning

confidence: 99%

A rhoptry antigen ofPlasmodium falciparumis protective inSaimirimonkeys

Ridley¹,

Takács

Etlinger

et al. 1990

Parasitology

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SUMMARYA non-polymorphic antigen associated with the rhoptry organelles of Plasmodium falciparum has been purified by immunoaffinity chromatography. The antigen, RAP-1 (rhoptry associated protein-1), which is defined by monoclonal antibodies which inhibit parasite growth in vitro, is a multi-component antigen consisting of four major proteins of 80, 65, 42 and 40 kDa and two minor proteins of 77 and 70 kDa. These proteins were electro-eluted from preparative sodium dodecyl sulphate polyacrylamide gels and protected Saimiri sciureus monkeys from a lethal blood-stage infection of P. falciparum malaria. Sera from the protected animals recognized only proteins of the RAP-1 antigen when used to probe a Western blot of total parasite protein extract, confirming that RAP-1 is responsible for eliciting the protective immune response.

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“…falciparum, strain K1, AZAP cDNA library [ l l ] (approximately 1 X lo5 independent clones) and a ANM 1149 Hind111 genomic DNA library [12] (approximately 2X 10s independent clones), kindly provided by the late J. Scaife (University of Edinburgh).…”

Section: Screening Of Librariesmentioning

confidence: 99%

Isolation of a Novel Plasmodium falciparum Gene Encoding a Protein Homologous to the Tat‐Binding Protein Family

Hirtzlin¹,

Färber²,

Franklin³

1994

European Journal of Biochemistry

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We have cloned a Plasmodium falciparum gene that belongs to the nuclear Tat-binding protein (TBP) gene family. This gene, PjTBP, is (A+T)-rich and encodes a 49.5-kDa protein. The predicted protein encoded by this gene has highest similarity to the slime mold protein DdTBPlO (86%) and to the yeast protein SUGl (81.8%), both of which belong to the Tat-binding protein family. In agreement with the characteristics of this family, PjTBP contains a highly conserved domain of approximately 200 amino acids, in which are found the motifs A and B of ATPases, and amino acid sequences characteristic of a large family of RNA or DNA helicases, suggesting a role in RNA or DNA unwinding. Like DdTBPlO, the PffBP protein has a heptad repeat of four leucine residues, reminiscent of a leucine zipper motif known to mediate dimerization. We have further characterized PflBP gene expression by Northern-blot analysis. This gene is expressed in a stage-specific manner, with higher expression in the late trophozoite stage. The recombinant PflBP gene has been expressed in Escherichia coli and a polyclonal antiserum has been raised in rabbits against the recombinant protein. This antibody has been used to study the protein in the parasite. The gene product is expressed in a stage-specific manner with higher expression in the late trophozoite and schizont stages, and is localized in the nucleus of the erythrocytic stage parasite. Thus the protein might have a function in DNA synthesis and/or in transcription, as is the case for other Tat-binding proteins.The protozoan Plasmodium falciparum is the causative agent of human tertian malaria. The completion of the complex life cycle requires invertebrate and vertebrate hosts. The parasite undergoes three cycles of development in its vertebrate host. These cycles include the hepatic or exoerythrocytic cycle, the asexual blood cycle, which includes the intraerythrocytic ring, trophozoite, and schizont stages, and the extracellular merozoite, and finally the development of gametocytes (sexual stage). Clinical symptoms and mortality accompanying P: falciparum infections are associated with the erythrocytic phase of its development. As indicated by the morphological changes and the accumulation of stagespecific transcripts within both hosts [l, 31, gene expression is regulated during the life cycle of the parasite. The regulation of gene expression can occur at the transcriptional or the post-transcriptional level. Recently, much attention has been focused on the transcription factors and their target DNA sequences (promoters and enhancers) since they play an important role in modulating the specificity and efficiency of Correspondence to R. M.

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The establishment of genomic DNA libraries for the human malaria parasite Plasmodium falciparum and identification of individual clones by hybridisation

Cited by 145 publications

References 31 publications

Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum

Apparent lack of N‐glycosylation in the asexual intraerythrocytic stage of Plasmodium falciparum

A rhoptry antigen ofPlasmodium falciparumis protective inSaimirimonkeys

Isolation of a Novel Plasmodium falciparum Gene Encoding a Protein Homologous to the Tat‐Binding Protein Family

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