Artifical mutant alleles have been made with the single mutant forms of dhfr in various systems. It seems the 51 single mutant form of dhfr does not confer resistance to the antifolates - and therefore would confer no advantage to the parasite carrying it, and it would not be selected. In addition the N51I mutation seems to affect the Kcat of the enzyme, which could affect the fitness of the parasite in the absence of antifolate drugs as well.

So we don't see the single mutant because although it could occur by random mutation, the parasite carrying the mutation would have no selective advantage in the presence of antifolates, and would also be worse off in the absence of drug. The parasite would therefore not outcompete wild-type (or other mutant forms), and would either remain at the very low frequency it started at, be lost by genetic drift, or may even not survive at all if the fitness cost in the absence of drug is significant.

These papers may be useful in providing the detail:

CHUSACULTANACHAI, S., THIENSATHIT, P., TARNCHOMPOO,

B., SIRAWARAPORN, W. & YUTHAVONG, Y. (2002). Novel

antifolate resistant mutations of Plasmodium falciparum

dihydrofolate reductase selected in Escherichia coli.

Molecular and Biochemical Parasitology 120, 61–72.

FERLAN, J. T., MOOKHERJEE, S., OKEZIE, I. N., FULGENCE, L.

& SIBLEY, C. H. (2001). Mutagenesis of dihydrofolate

reductase from Plasmodium falciparum: analysis in

Saccharomyces cerevisiae of triple mutant alleles

resistant to pyrimethamine or WR99210. Molecular and

Biochemical Parasitology 113, 139–150.

HANKINS, E. G., WARHURST, D. C. & SIBLEY, C. H. (2001).

Novel alleles of the Plasmodium falciparum dhfr highly

resistant to pyrimethamine and chlorcycloguanil, but

Malarial DHFR-TS as antifolate target 257

not WR99210. Molecular and Biochemical Parasitology

117, 91–102.

SIRAWARAPORN, W., SATHITKUL, T., SIRAWARAPORN, R.,

YUTHAVONG, Y. & SANTI, D. V. (1997b). Antifolateresistant

mutants of Plasmodium falciparum

dihydrofolate reductase. Proceedings of the National

Academy of Sciences, USA 94, 1124–1129.

Thank you very much for your helpful comment, Dr. Lisa. This means that if 51I mutation appeared while sequencing the P. falciparum dhfr without being accompanied by 108N, the possible cause could be a random event (not a true selection).

Best regards,

Rashad

Probably. If you amplify parasite DNA by PCR before sequencing, you can get mutations in this process, especially if you use a non-proof reading DNA polymerase such as Taq. However a polymerase error probably wouldn't happen in the same place twice, so repeating the amplification from the starting material with a proof-reading/hi fidelity DNA polymerase (e.g. Pfu) and re-sequencing a few times can help to eliminate that possibility, and would indicate if this mutation actually exists in the parasite, or is an artefact of the PCR/sequencing process.

Regards

Lisa

Thank you very much, Dr. Lisa. Your comments are very helpful.

Best regards