title: Cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1) of Toxoplasma gondii, related to genes encoding small heat-shock proteins of plants.
abstract: Stage conversion between the tachyzoite and bradyzoite forms of the protozoan parasite Toxoplasma gondii is an important aspect in the pathogenesis of toxoplasmosis. In an initial investigation of molecular regulation of stage conversion in T. gondii, we describe the cloning and characterization of a bradyzoite-specifically expressed gene (hsp30/bag1). Bradyzoite formation was induced in cell culture by alkaline pH, and this was followed by purification of this parasitic stage using magnetic cell sorting. A bradyzoite cDNA library was constructed by random amplification using the polymerase chain reaction. Screening with a bradyzoite-specific monoclonal antibody identified a reactive clone. The amino acid sequence derived from the 687 bp open reading frame showed similarity to the conserved C-terminal region of small heat-shock proteins from plants. Stage-specific expression of the naturally occurring 30 kDa antigen in bradyzoites was confirmed by polyclonal antisera generated against the recombinant antigen. Immunoelectron microscopy indicated a cytosolic location of this antigen in bradyzoites. The expression of HSP30/BAG1 seems to be regulated at the mRNA level, since reverse polymerase chain reaction using bradyzoite-specific primers amplified transcripts in bradyzoites only, not in tachyzoites.
authors.affiliation: Institute of Hygiene and Microbiology, University of Würzburg, Germany.
authors.forename: W
authors.initials: W
authors.lastname: Bohne
authors.forename: U
authors.initials: U
authors.lastname: Gross
authors.forename: D J
authors.initials: DJ
authors.lastname: Ferguson
authors.forename: J
authors.initials: J
authors.lastname: Heesemann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D017125:Genes, Protozoan; D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000737:chemistry / Q000235:genetics*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D010944:Plants / Q000737:chemistry / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014122:Toxoplasma / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D014124:Toxoplasmosis, Animal / Q000469:parasitology; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics*
chemical_list: C091732:BCL2-associated athanogene 1 protein; D004268:DNA-Binding Proteins; D050890:HSP30 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors