Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii

David Bermudes, Jean François Dubremetz, Abderrahim Achbarou, Keith A Joiner

Research output: Contribution to journalArticle

59 Citations (Scopus)

Abstract

GRA3 is a 30-kDa protein located inside the dense granules of Toxoplasma gondii. Following invasion and exocytosis of dense granules within the parasitophorous vacuole, GRA3 becomes associated with the parasitophorous vacuolar membrane (PVM) and extensions of the PVM which protrude into the cytoplasm. A partial cDNA encoding GRA3 was isolated from a Toxoplasma gondii expression library using polyclonal and monoclonal antibodies to the mature GRA3 protein of tachyzoites. Antibodies affinity purified using the cloned fusion protein reacted with a 30-kDa band on immunoblots and recognized dense granules, the PVM, and PVM extensions by immunofluorescence staining of infected cells. Northern blot analysis indicated the major transcript was of a slightly larger size, and the complete cDNA encoding GRA3 was subsequently obtained. Southern blot analysis suggests that GRA3 is present as a single copy. The cDNA encodes two methionines at the N-terminus followed by an open reading frame with a hydrophobic region of 22 amino acids flanked by charged residues consistent with a signal sequence. Four shorter hydrophobic regions occur but are insufficient to span the membrane. No significant homology was detected to other proteins, including other dense granule proteins. In vitro translation of RNA generated from the cDNA containing either one or two of the N-terminal methionines yielded particles with apparent Mr of 35 000 and 37 000 respectively. Translation of RNA from the cDNA containing only the second initiation site in the presence of dog pancreas microsomes resulted in reduction of 4 kDa, sufficient to account for removal of the putative signal sequence.

Original languageEnglish (US)
Pages (from-to)247-257
Number of pages11
JournalMolecular and Biochemical Parasitology
Volume68
Issue number2
DOIs
StatePublished - 1994
Externally publishedYes

Fingerprint

Organism Cloning
Complementary DNA
Membranes
Toxoplasma
Protein Sorting Signals
Proteins
Methionine
RNA
Antibody Affinity
Exocytosis
Microsomes
Southern Blotting
Vacuoles
Northern Blotting
Open Reading Frames
Fluorescent Antibody Technique
Pancreas
Cytoplasm
Monoclonal Antibodies
Toxoplasma gondii GRA3 protein

Keywords

  • Dense granule
  • GRA3
  • Parasitophorous vacuole membrane
  • Toxoplasma gondii

ASJC Scopus subject areas

  • Molecular Biology
  • Parasitology

Cite this

Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii. / Bermudes, David; Dubremetz, Jean François; Achbarou, Abderrahim; Joiner, Keith A.

In: Molecular and Biochemical Parasitology, Vol. 68, No. 2, 1994, p. 247-257.

Research output: Contribution to journalArticle

Bermudes, David ; Dubremetz, Jean François ; Achbarou, Abderrahim ; Joiner, Keith A. / Cloning of a cDNA encoding the dense granule protein GRA3 from Toxoplasma gondii. In: Molecular and Biochemical Parasitology. 1994 ; Vol. 68, No. 2. pp. 247-257.
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