Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii

https://doi.org/10.1016/j.molbiopara.2003.12.015Get rights and content

Abstract

The fused dihydrofolate reductase/thymidylate synthase gene of Toxoplasma gondii contains ten exons spanning ~8 kb of genomic DNA. We have examined the ends of DHFR-TS transcripts within this gene, and find a complex pattern including two discrete 5′ termini and multiple polyadenylation sites. No TATAA box or other classical promoter motif is evident in 1.4 kb of genomic DNA upstream of the coding region, but transcript mapping by RNase protection and primer extension reveals two prominent 5′ ends at positions −369 and −341 nt relative to the ATG initiation codon. Upstream genomic sequences include GC-rich regions and the (opposite strand) WGAGACG motif previously identified in other T. gondii promoters. Mutagenesis of recombinant reporter plasmids demonstrates that this region is essential for efficient transgene expression. Sequencing the 3′ ends from multiple independent mRNA clones demonstrates numerous polyadenylation sites, distributed over >650 nt of genomic sequence beginning ~250 nt downstream of the stop codon. Within this region, certain sites seem to be preferred: 14 different positions were found among the 32 polyadenylated transcripts examined, but ~40% of the transcripts map to two loci. The 3′ noncoding region is rich in A and T nucleotides, and contains an imperfect 50 nt direct repeat, but no obvious poly(A) addition signal was identified.

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].

Section snippets

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.

References (50)

  • A.G. Hook et al.

    Localization and sequence analysis of poly(A) sites generating multiple dihydrofolate reductase mRNAs

    J. Biol. Chem.

    (1988)
  • V. Noe et al.

    Effect of differential polyadenylation and cell growth phase on dihydrofolate reductase mRNA stability

    J. Biol. Chem.

    (1999)
  • S.C. Guru et al.

    Characterization of a MEN1 ortholog from Drosophila melanogaster

    Gene

    (2001)
  • R.M. O’Connor et al.

    Expression of the highly polymorphic Cryptosporidium parvum Cpgp40/15 gene in genotype I and II isolates

    Mol. Biochem. Parasitol.

    (2002)
  • V. Ruvolo et al.

    The transcript encoding the circumsporozoite antigen of Plasmodium berghei utilizes heterogeneous polyadenylation sites

    Mol. Biochem. Parasitol.

    (1993)
  • S.D. Nagel et al.

    The a- and b-tubulins of Toxoplasma gondii are encoded by single copy genes encoding multiple introns

    Mol. Biochem. Parasitol.

    (1988)
  • J.B. Prince et al.

    Cloning expression and cDNA sequence of surface antigen P22 from Toxoplasma gondii

    Mol. Biochem. Parasitol.

    (1990)
  • A.M. Johnson et al.

    Cloning of Toxoplasma gondii gene fragments encoding diagnostic antigens

    Gene

    (1991)
  • S.F. Parmley et al.

    Genomic and corrected cDNA sequence of the P28 gene from Toxoplasma gondii

    Mol. Biochem. Parasitol.

    (1993)
  • B.J. Luft et al.

    Toxoplasmic encephalitis in AIDS

    Clin. Infect. Dis.

    (1992)
  • J.D. Schwartzman et al.

    Pyrimidine synthesis in intracellular Toxoplasma gondii

    J. Parasitol.

    (1981)
  • R.G. Brooks et al.

    Drugs used in treatment of Toxoplasmosis

    Antimicr. Agents Annu.

    (1987)
  • R.G. Donald et al.

    Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria

    Proc. Natl. Acad. Sci. U.S.A.

    (1993)
  • M. Trujillo et al.

    Heterologous expression and characterization of the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of Toxoplasma gondii

    Biochemistry

    (1996)
  • M.G. Reynolds et al.

    In vitro generation of novel pyrimethamine resistance mutations in the Toxoplasma gondii dihydrofolate reductase

    Antimicrob. Agents Chemother.

    (2001)
  • Cited by (33)

    • APDDD: Animal parasitic diseases and drugs database

      2024, Comparative Immunology, Microbiology and Infectious Diseases
    • TSS-seq of Toxoplasma gondii sporozoites revealed a novel motif in stage-specific promoters

      2022, Infection, Genetics and Evolution
      Citation Excerpt :

      While in Tz, substantial specific stage regulator has not yet been reported, the other motifs that control transcription were reported. For example, WGAGACG is identified upstream of SAG1 (Soldati and Boothroyd, 1995), GRA genes (Mercier et al., 1996), and DHFR-TS (Matrajt et al., 2004), and they localize just upstream of each TSS. Furthermore, the localization of CATGC is also observed around the −100 nts region from TSS in both Tz and Bz-specific genes (Markus et al., 2021).

    • Genetic manipulation of Toxoplasma gondii

      2020, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods
    • Regulation of gene expression in Toxoplasma gondii

      2020, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods
    • Epigenetic and Genetic Factors that Regulate Gene Expression in Toxoplasma gondii

      2013, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods: Second Edition
    • Genetic Manipulation of Toxoplasma gondii

      2013, Toxoplasma Gondii: The Model Apicomplexan - Perspectives and Methods: Second Edition
    View all citing articles on Scopus
    View full text