Affiliation:
1. Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California 94305-5124
Abstract
ABSTRACT
Within its intermediate host,
Toxoplasma gondii
switches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT).
T. gondii
was transfected with promoterless
HXGPRT
and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing
HXGPRT
. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using
Dolichos biflorus
agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had
Dolichos
immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the
Toxoplasma
family of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive
P36
gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.
Publisher
American Society for Microbiology
Subject
Cell Biology,Molecular Biology
Cited by
80 articles.
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