Abstract
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 pyrimethamine-resistant 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 allele employed and the conditions of selection, 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 bringing the full power of molecular genetic analysis to bear on Toxoplasma, these studies should greatly facilitate the development of a model genetic system for Apicomplexan parasites.
Full text
PDF![11703](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5efb/48052/435a377a1211/pnas01531-0275.png)
![11704](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5efb/48052/cf4b66f5cd29/pnas01531-0276.png)
![11705](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5efb/48052/fcb39ab380f6/pnas01531-0277.png)
![11706](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5efb/48052/b1d98ef0b277/pnas01531-0278.png)
![11707](https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5efb/48052/a2113ecde0d2/pnas01531-0279.png)
Images in this article
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Cruz A., Beverley S. M. Gene replacement in parasitic protozoa. Nature. 1990 Nov 8;348(6297):171–173. doi: 10.1038/348171a0. [DOI] [PubMed] [Google Scholar]
- Howell E. E., Foster P. G., Foster L. M. Construction of a dihydrofolate reductase-deficient mutant of Escherichia coli by gene replacement. J Bacteriol. 1988 Jul;170(7):3040–3045. doi: 10.1128/jb.170.7.3040-3045.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kapler G. M., Coburn C. M., Beverley S. M. Stable transfection of the human parasite Leishmania major delineates a 30-kilobase region sufficient for extrachromosomal replication and expression. Mol Cell Biol. 1990 Mar;10(3):1084–1094. doi: 10.1128/mcb.10.3.1084. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laban A., Tobin J. F., Curotto de Lafaille M. A., Wirth D. F. Stable expression of the bacterial neor gene in Leishmania enriettii. Nature. 1990 Feb 8;343(6258):572–574. doi: 10.1038/343572a0. [DOI] [PubMed] [Google Scholar]
- Lee M. G., Van der Ploeg L. H. Homologous recombination and stable transfection in the parasitic protozoan Trypanosoma brucei. Science. 1990 Dec 14;250(4987):1583–1587. doi: 10.1126/science.2177225. [DOI] [PubMed] [Google Scholar]
- Leport C., Raffi F., Matheron S., Katlama C., Regnier B., Saimot A. G., Marche C., Vedrenne C., Vilde J. L. Treatment of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in 35 patients with the acquired immunodeficiency syndrome. Efficacy of long-term continuous therapy. Am J Med. 1988 Jan;84(1):94–100. doi: 10.1016/0002-9343(88)90014-9. [DOI] [PubMed] [Google Scholar]
- Luft B. J., Remington J. S. Toxoplasmic encephalitis in AIDS. Clin Infect Dis. 1992 Aug;15(2):211–222. doi: 10.1093/clinids/15.2.211. [DOI] [PubMed] [Google Scholar]
- McClary J. A., Witney F., Geisselsoder J. Efficient site-directed in vitro mutagenesis using phagemid vectors. Biotechniques. 1989 Mar;7(3):282–289. [PubMed] [Google Scholar]
- Peterson D. S., Walliker D., Wellems T. E. Evidence that a point mutation in dihydrofolate reductase-thymidylate synthase confers resistance to pyrimethamine in falciparum malaria. Proc Natl Acad Sci U S A. 1988 Dec;85(23):9114–9118. doi: 10.1073/pnas.85.23.9114. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Pfefferkorn E. R., Eckel M. E., McAdams E. Toxoplasma gondii: in vivo and in vitro studies of a mutant resistant to arprinocid-N-oxide. Exp Parasitol. 1988 Apr;65(2):282–289. doi: 10.1016/0014-4894(88)90133-6. [DOI] [PubMed] [Google Scholar]
- Pfefferkorn E. R., Pfefferkorn L. C. Specific labeling of intracellular Toxoplasma gondii with uracil. J Protozool. 1977 Aug;24(3):449–453. doi: 10.1111/j.1550-7408.1977.tb04774.x. [DOI] [PubMed] [Google Scholar]
- Pfefferkorn L. C., Pfefferkorn E. R. Toxoplasma gondii: genetic recombination between drug resistant mutants. Exp Parasitol. 1980 Dec;50(3):305–316. doi: 10.1016/0014-4894(80)90034-x. [DOI] [PubMed] [Google Scholar]
- Sibley L. D., Boothroyd J. C. Construction of a molecular karyotype for Toxoplasma gondii. Mol Biochem Parasitol. 1992 Apr;51(2):291–300. doi: 10.1016/0166-6851(92)90079-y. [DOI] [PubMed] [Google Scholar]
- Tanaka M., Gu H. M., Bzik D. J., Li W. B., Inselburg J. W. Dihydrofolate reductase mutations and chromosomal changes associated with pyrimethamine resistance of Plasmodium falciparum. Mol Biochem Parasitol. 1990 Feb;39(1):127–134. doi: 10.1016/0166-6851(90)90015-e. [DOI] [PubMed] [Google Scholar]
- Thaithong S., Chan S. W., Songsomboon S., Wilairat P., Seesod N., Sueblinwong T., Goman M., Ridley R., Beale G. Pyrimethamine resistant mutations in Plasmodium falciparum. Mol Biochem Parasitol. 1992 Jun;52(2):149–157. doi: 10.1016/0166-6851(92)90047-n. [DOI] [PubMed] [Google Scholar]
- Tobin J. F., Laban A., Wirth D. F. Homologous recombination in Leishmania enriettii. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):864–868. doi: 10.1073/pnas.88.3.864. [DOI] [PMC free article] [PubMed] [Google Scholar]
- ten Asbroek A. L., Ouellette M., Borst P. Targeted insertion of the neomycin phosphotransferase gene into the tubulin gene cluster of Trypanosoma brucei. Nature. 1990 Nov 8;348(6297):174–175. doi: 10.1038/348174a0. [DOI] [PubMed] [Google Scholar]
- van den Hoff M. J., Moorman A. F., Lamers W. H. Electroporation in 'intracellular' buffer increases cell survival. Nucleic Acids Res. 1992 Jun 11;20(11):2902–2902. doi: 10.1093/nar/20.11.2902. [DOI] [PMC free article] [PubMed] [Google Scholar]