Browsing by Author "McCutchan, T.F."
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Item Genetic analysis of the human malaria parasite plasmodium falciparum(Science, 1987) Walliker, D.; Quakyi, I.A.; Wellems, T.E.; McCutchan, T.F.; Szarfman, A.; London, W.T.; Carter, R.Malaria parasites are haploid for most of their life cycle, with zygote formation and meiosis occurring during the mosquito phase of development. The parasites can be analyzed genetically by transmitting mixtures of cloned parasites through mosquitoes to permit cross-fertilization of gametes to occur. A cross was made between two clones of Plasmodium falciparum differing in enzymes, drug sensitivity, antigens, and chromosome patterns. Parasites showing recombination between the parent clone markers were detected at a high frequency. Novel forms of certain chromosomes, detected by pulsed-field gradient gel electrophoresis, were produced readily, showing that extensive rearrangements occur in the parasite genome after cross-fertilization. Since patients are frequently infected with mixtures of genetically distinct parasites, mosquito transmission is likely to provide the principal mechanisms for generating parasites with novel genotypes.Item Mechanism of pyrimethamine resistance in recent isolates of plasmodium falciparum(Antimicrobial Agents and Chemotherapy, 1984) McCutchan, T.F.; Welsh, J.A.; Dame, J.B.; Quakyi, I.A.; Graves, P.M.; Drake, J.C.; Allegra, C.J.Clones of Plasmodium falciparum prepared from recent isolates of infected blood were studied to determine the molecular mechanism of naturally occurring pyrimethamine resistance. Total DNA, as well as thymidylate synthetase and dihydrofolate reductase activities, were characterized from these lines. Restriction analysis of DNA from pyrimethamine-susceptible and -resistant lines of the parasite showed no obvious amplification of any DNA fragment. Further, analysis of DNA from resistant and susceptible lines by centrifugation in cesium chloride-ethidium bromide revealed no extrachromosomal amplification in the resistant line. Comparison of the dihydrofolate reductase enzyme activity in the two lines revealed similar K(m)s for substrate but a large difference in the inhibition constant for pyrimethamine. Additionally, the enzyme from the resistant line was considerably more stable in vitro than the corresponding enzyme from the susceptible line. The thymidylate synthetase activity in the two lines was similar and unaffected by pyrimethamine. The mechanism of drug resistance in this isolate involves altered properties of the dihydrofolate reductase conferring both a different affinity for the drug and increased stability.