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.
Afework
Yohannes
Y
Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Königsweg 67, 14163, Berlin, Germany.
Mäser
Pascal
P
Etschmann
Benjamin
B
von Samson-Himmelstjerna
Georg
G
Zessin
Karl-Hans
KH
Clausen
Peter-Henning
PH
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