A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum - PubMed (original) (raw)

A set of independent selectable markers for transfection of the human malaria parasite Plasmodium falciparum

C B Mamoun et al. Proc Natl Acad Sci U S A. 1999.

Erratum in

Abstract

Genomic information is rapidly accumulating for the human malaria pathogen, Plasmodium falciparum. Our ability to perform genetic manipulations to understand Plasmodium gene function is limited. Dihydrofolate reductase is the only selectable marker presently available for transfection of P. falciparum. Additional markers are needed for complementation and for expression of mutated forms of essential genes. We tested parasite sensitivity to different drugs for which selectable markers are available. Two of these drugs that were very effective as antiplasmodial inhibitors in culture, blasticidin and geneticin (G418), were selected for further study. The genes BSD, encoding blasticidin S deaminase of Aspergillus terreus, and NEO, encoding neomycin phosphotransferase II from transposon Tn 5, were expressed under the histidine-rich protein III (HRPIII) gene promoter and tested for their ability to confer resistance to blasticidin or G418, respectively. After transfection, blasticidin and G418-resistant parasites tested positive for plasmid replication and BSD or NEO expression. Cross-resistance assays indicate that these markers are independent. The plasmid copy number and the enzymatic activity depended directly on the concentration of the drug used for selection. These markers set the stage for new methods of functional analysis of the P. falciparum genome.

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Figures

Figure 1

Figure 1

Maps of pCBM-BSD and pCBM-NEO vectors.

Figure 2

Figure 2

Inhibition of 3D7, HB3, W2, and Dd2 parasite clones as a function of blasticidin S (A) or G418 (B) concentrations.

Figure 3

Figure 3

(A) Detection of pCBM-BSD DNA from parasites selected on 1 μg/ml (BS1), 2 μg/ml (BS2), or 5 μg/ml (BS5) of blasticidin S. Genomic DNA (prepared 7 weeks posttransfection) and DNA from the original plasmid (pCBM-BSD) were restricted with _Sca_I (S, which cuts once in the vector) or _Sca_I + _Dpn_I (SD, with 15 pBluescript recognition sites), hybridized with a 32P-labeled pBluescript probe, and detected by autoradiography. A longer exposure of the autoradiograph is also shown. (B) Detection of pCBM-NEO DNA from parasites selected on 500 μg/ml G418 (G5). DNA was prepared as described for A. Untransfected 3D7 served as a control in both A and B.

Figure 4

Figure 4

Plasmid copy number in G3 and G5 clones (Upper) and BS1, BS2, and BS5 (Lower). By using the genome size of 3 × 107 bp, the amount of NEO (800 bp) or BSD (390 bp) signal that would represent one copy per P. falciparum genome was calculated; 0.5 μg of DNA from 3D7 and the various transformants were used for comparison.

Figure 5

Figure 5

Enzyme assays. (A) BSD activity. Crude cell extracts from 3D7 alone or 3D7 transfected parasites selected on 1 μg/ml (BS1), 2 μg/ml (BS2), or 5 μg/ml (BS5) of blasticidin S were added to substrate in the assay buffer. Samples were taken at 0, 10, 20, and 30 min after incubation. The differential UV absorbance between the substrate (blasticidin S) and the deaminated product was recorded. (B) Neomycin phosphotransferase II activity. Crude cell extracts from 3D7 alone or 3D7 transfected parasites selected on 300 μg/ml (G3) or 500 (G5) μg/ml G418 were incubated with [γ-32P]ATP and kanamycin sulfate in the assay buffer at 37°C for 60 min. The buffer alone was used as a control.

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