Rapid identification of isometamidium-resistant stocks of Trypanosoma b. brucei by PCR-RFLP.
Analyses were made on the adenosine transporter-1 gene in Trypanosoma brucei (TbAT1), encoding a P2-like nucleoside transporter, from T. brucei brucei field stocks to investigate a possible link between the presence of mutations in this gene and isometamidium resistance. We have analysed the gene from 11 isometamidium-sensitive field stocks isolated from cattle in Uganda, two sensitive reference clones and two resistant reference clones. A sequence alignment showed that the isometamidium-sensitive T. b. brucei contained the wild-type sequence patterns. In contrast, the isometamidium-resistant T. b. brucei stocks showed the mutant-type sequence patterns with six point mutations that had previously been reported in a laboratory-derived arsenical-resistant T. brucei strain. To analyse the restriction fragment length polymorphism pattern of a fragment of TbAT1 (nucleotides 430-1108), the 677-bp polymerase chain reaction products from eight of the isometamidium-sensitive and two of the isometamidium-resistant T. b. brucei were subjected to digestion with Sfa NI. The results revealed two different banding patterns: the digest resulted in fragment sizes of 566 and 111 bp in the case of TbAT1 from isometamidium-sensitive stocks, whereas it produced fragment sizes of 435 and 242 bp in the case of TbAT1 from isometamidium-resistant stocks. Thus, the isometamidium-sensitive and isometamidium-resistant T. b. brucei could be successfully distinguished by digestion with the restriction endonuclease Sfa NI.
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Königsweg 67, 14163, Berlin, Germany.
Yohannes
Y
Afework
Pascal
P
Mäser
Benjamin
B
Etschmann
Georg
G
von Samson-Himmelstjerna
Karl-Hans
KH
Zessin
Peter-Henning
PH
Clausen
D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002417:Cattle; D016054:DNA, Protozoan / Q000235:genetics*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D004351:Drug Resistance / Q000235:genetics*; D020125:Mutation, Missense; D033703:Nucleoside Transport Proteins / Q000235:genetics; D010617:Phenanthridines / Q000494:pharmacology*; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D014344:Trypanocidal Agents / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000145:classification / Q000235:genetics* / Q000254:growth & development* / Q000302:isolation & purification; D014354:Trypanosomiasis, Bovine / Q000469:parasitology
D016054:DNA, Protozoan; D033703:Nucleoside Transport Proteins; C486446:P2 transporter, Trypanosoma brucei; D010617:Phenanthridines; D014344:Trypanocidal Agents; C000702:isometamidium chloride; D015252:Deoxyribonucleases, Type II Site-Specific; C065993:endodeoxyribonuclease SfaNI