STABLE MOLECULAR-TRANSFORMATION OF TOXOPLASMA-GONDII - A SELECTABLE DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE MARKER BASED ON DRUG-RESISTANCE MUTATIONS IN MALARIA

To facilitate genetic analysis of the protozoan parasite Toxoplasma go ndii, sequences derived from the parasite's fused dihydrofolate reduct ase-thymidylate synthase (DHFR-TS) gene have been used to produce vect ors suitable for stable molecular transformation. Mutations introduced into the DHFR coding region by analogy with pyrimethamine-resistant m alaria confer drug resistance to Toxoplasma, providing useful informat ion on the structure of fused DHFR-TS enzymes and a powerful selectabl e marker for molecular genetic studies. Depending on the particular dr ug-resistance allele employed and the conditions of selection, stable resistance can be generated either by single copy nonhomologous insert ion into chromosomal DNA or by massively amplified transgenes. Frequen cies of integration are independent of selection, and transgenes are s table without continued selection. Cointegration of a reporter gene ad jacent to the selectable marker (under the control of an independent p romoter) shows no loss of the cointegrated sequences over many parasit e generations. By bringing the full power of molecular genetic analysi s to bear on Toxoplasma, these studies should greatly facilitate the d evelopment of a model genetic system for Apicomplexan parasites.