A vestigial, nonphotosynthetic plastid has been identified recently in protozoan parasites of the phylum Apicomplexa. The apicomplexan plastid, or ''apicoplast,'' is indispensable, but the complete sequence of both the Plasmodium falciparum and Toxoplasma gondii apicoplast genomes has offered no clue as to what essential metabolic function(s) this organelle might perform in parasites. To investigate possible functions of the apicoplast, we sought to identify nuclear-encoded genes whose products are targeted to the apicoplast in Plasmodium and Toxoplasma. We describe here nuclear genes encoding ribosomal proteins S9 and L28 and the fatty acid biosynthetic enzymes acyl carrier protein (ACP), ␤-ketoacyl-ACP synthase III (FabH), and ␤-hydroxyacyl-ACP dehydratase (FabZ). These genes show high similarity to plastid homologues, and immunolocalization of S9 and ACP verifies that the proteins accumulate in the plastid. All the putatively apicoplast-targeted proteins bear N-terminal presequences consistent with plastid targeting, and the ACP presequence is shown to be sufficient to target a recombinant green f luorescent protein reporter to the apicoplast in transgenic T. gondii. Localization of ACP, and very probably FabH and FabZ, in the apicoplast implicates fatty acid biosynthesis as a likely function of the apicoplast. Moreover, inhibition of P. falciparum growth by thiolactomycin, an inhibitor of FabH, indicates a vital role for apicoplast fatty acid biosynthesis. Because the fatty acid biosynthesis genes identified here are of a plastid͞bacterial type, and distinct from those of the equivalent pathway in animals, fatty acid biosynthesis is potentially an excellent target for therapeutics directed against malaria, toxoplasmosis, and other apicomplexan-mediated diseases.

A nonhomologous integration vector was used to identify the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyl transferase (HXGPRT) gene by insertional mutagenesis. Parasite mutants resistant to 6-thioxanthine arose at a frequency of approximately3 x 10(-7). Genomic DNA flanking the insertion sites was retrieved by marker rescue and used to identify molecular clones exhibiting unambiguous homology to H(X)GPRT genes from other species. Sequence analysis of vector/genome junction sites reveals that integration of the linearized vector occurred with minimal rearrangement of either vector or target sequences, although the addition of filler DNA and small duplications or deletions of genomic sequences at the transgene termini was observed. Two differentially spliced classes of cDNA clones were identified, both of which complement hpt and gpt mutations in Escherichia coli. Kinetic analysis of purified recombinant enzyme revealed no significant differences between the two isoforms. Internally deleted clones spanning the genomic locus were used to create "knock-out" parasites, which lack all detectable HXGPRT activity. Complete activity could be restored to these knock-out mutants by transient transformation with either genomic DNA or cDNA-derived minigenes encoding both enzyme isoforms. Stable HXGPRT+ transformants were isolated under selection with mycophenolic acid, demonstrating the feasibility of HXGPRT as both a positive and negative selectable marker for stable transformation of T. gondii.

To facilitate genetic analysis of the protozoan parasite Toxoplasma gondii, sequences derived from the parasite's fused dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene have been used to produce vectors suitable for stable molecular transformation. Mutations introduced into the DHFR coding region by analogy with pyrimethamineresistant malaria confer drug resistance to Toxoplasma, providing useful information on the structure of fused DHFR-TS enzymes and a powerful selectable marker for molecular genetic studies. Depending on the particular drug-resistance aflele employed and the conditions ofselection, stable resistance can be generated either by single copy nonhomologous insertion into chromosomal DNA or by massively amplified transgenes. Frequencies of integration are independent of selection, and transgenes are stable without continued selection. Cointegration of a reporter gene adjacent to the selectable marker (under the control of an independent promoter) shows no loss of the cointegrated sequences over many parasite generations. By brinnging the fuil power of molecular genetic analysis to bear on Toxoplasma, these studies should greatly facilitate the development of a model genetic system for Apicomplexan parasites.