Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii
Introduction
The protozoan parasite Toxoplasma gondii is a ubiquitous pathogen of humans and other animals. Classically known as a source of congenital neurological birth defects, and more recently as a prominent opportunistic infection associated with immunosuppressive treatments and diseases [1], this parasite is also of some concern as a potential bioterrorism agent (it has been recently categorized by the CDC as a Category B agent of bioterrorism). T. gondii lacks many of the enzymes necessary for pyrimidine salvage [2] and therefore is particularly dependent on de novo biosynthetic pathways that consume reduced folate molecules. This requires that rapidly dividing parasites maintain an abundant folate pool, and enzymes of the folate metabolic pathway therefore provide an important target for chemotherapy of parasite infections [3]. A key enzymatic step in folate metabolism is the one catalyzed by the enzyme dihydrofolate reductase (DHFR), which is fused to thymidylate synthase (TS) in T. gondii. Clinical treatment of toxoplasmosis (and other infectious diseases) often exploits a combination of folate analogs to inhibit DHFR, with sulfonamides to inhibit a prior step in folate biosynthesis. Genomic and cDNA sequences coding for the Toxoplasma DHFR-TS gene have been cloned [4], and sequences derived from this gene have been used to produce vectors suitable for stable molecular transformation of T. gondii [5]. This enzyme has been expressed and characterized as recombinant protein [6], [7] and various mutations observed in drug-resistant Plasmodium falciparum (the causative agent of malaria) have been engineered into the T. gondii DHFR-TS for studies both in vitro [8] and in vivo [9].
To further our understanding of DHFR-TS expression and the regulation of housekeeping genes in T. gondii, we have examined the ends of DHFR-TS mRNAs, and tested the ability of flanking sequence domains to drive expression of chloramphenicol acetyl transferase (CAT) and firefly luciferase (LUC) reporters in transient transformation assays. These signals are likely to prove useful in furthering the development of transgenic expression systems [5], [10], in addition to enhancing our understanding of molecular regulation in Toxoplasma (particularly for key pharmacological targets). It will also be interesting to contrast the regulation of DHFR-TS gene expression in Toxoplasma with the expression of orthologous genes in host cells [11], [12], [13], [14] and other parasites [15].
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Nucleotide sequences and analysis
Genomic clones covering the DHFR-TS gene have previously been described [4]. Flanking region sequences were subsequently extended to provide 8.4 kb of contiguous DNA sequence from a genomic Hind III site 1.4 kb upstream of the protein initiation codon to a genomic Not I site 1.0 kb downstream of the termination codon (Genbank accession #L08489). A map of the DHFR-TS locus is provided in Fig. 1A.
Parasites growth and transfection
T. gondii tachyzoites (RH strain) were maintained by serial passage in primary cultures of human
The 5′ noncoding region of T. gondii DHFR-TS
The 5′ ends of T. gondii DHFR-TS mRNAs were mapped by RNase protection using several antisense in vitro run-off transcripts covering 262 nt of coding sequence (upstream of the Sph I site in Fig. 1A) and 282–1394 nt of noncoding genomic sequence. As shown in Fig. 1B, two prominent fragments were protected by total parasite RNA. Both bands were seen in several independent experiments, although the relative ratios varied somewhat. Assuming comparable migration rates for the protected RNA and the
Discussion
The accessibility of Toxoplasma gondii to molecular genetic manipulation has made this parasite an attractive system for study, but our understanding of transcriptional regulation is fragmentary. Genomic analysis of promoter regions reveals no TATAA box or other classical promoter motifs in most genes. Although a divergent TATA-binding protein has been described in the related parasite Plasmodium falciparum [27], function has not been demonstrated, and TATA boxes have never been functionally
Acknowledgements
We wish to thank members of the Roos laboratory for helpful comments and suggestions, and Alexandra Levitt for discussions regarding polyadenylation signals for the P. berghei CS gene. This work was supported by grants from the NIH.
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