Mutation rates in the dihydrofolate reductase gene of Plasmodium falciparum
A new method has been established to define the limits on a spontaneous mutation rate for a gene in Plasmodium falciparum. The method combines mathematical modelling and large-scale in vitro culturing and calculates the difference in mutant frequencies at 2 separate time-points. We measured the mutation rate at 2 positions in the dihydrofolate reductase (DHFR) gene of 3D7, a pyrimethamine-sensitive line of P. falciparum. This line was re-cloned and an effectively large population was treated with a selective pyrimethamine concentration of 40 nM. We detected point mutations at codon-46 (TTA to TCA) and codon-108 (AGC to AAC), resulting in serine replacing leucine and asparagine replacing serine respectively in the corresponding gene product. The substitutions caused a decrease in pyrimethamine sensitivity. By mathematical modelling we determined that the mutation rate at a given position in DHFR was low and occurred at less than 2.5×10−9 mutations/DHFR gene/replication. This result has important implications for Plasmodium genetic diversity and anti-malarial drug therapy by demonstrating that even with low mutation rates anti-malarial resistance will inevitably arise when mutant alleles are selected under drug pressure.(Received August 8 2000)
(Revised November 17 2000)
(Accepted November 28 2000)
Key Words: dihydrofolate reductase; mutation rate; malaria; mathematical modelling; Plasmodium falciparum.
c1 Corresponding author: Malaria and Arbovirus Unit, The Queensland Institute of Medical Research, QLD 4029, Australia. Tel: +61-7-33620420. Fax: +61-7-33620104. E-mail: sarahP@qimr.edu.au