Genetic analysis of drug resistance in Trypanosoma brucei

Taylor, Sonya Dorothy Anne (2004) Genetic analysis of drug resistance in Trypanosoma brucei. PhD thesis, University of Glasgow.

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Genetic mapping, positional cloning and reverse genetics provide an alternative to the biochemical and molecular approaches used to date, to determine the basis of arsenical resistance in Trypanosoma brucei. Genetic mapping of loci determining phenotypes of relevance to diseases has proved to be a powerful approach in a number of organisms including humans and Plasmodium falciparum, particularly when coupled with a full genome sequence. In this thesis, this approach has been established in T. brucei by determining the genetic basis of naturally occurring arsenical resistance and undertaking linkage analysis using our recently developed genetic map. I adapted a simple, verified screening assay for assessing drug sensitivity based on the use of AlamarBlue. Three stocks used as parents in genetic crosses differed in drug sensitivity; STIB 247-Sensitive, STIB 386-Resistant and TREU 927-Resistant. Genetic linkage analysis using 101 polymorphic markers Identified from the extensive sequence available from the genome project was then used to examine inheritance of the drug resistance phenotype in T. brucei. From this, the co-segregation (into F1 progeny) of markers and the resistance phenotype was determined using crosses, 247 x 386 and 247 x 927. Inheritance of resistance in both crosses was compatible with a simple single locus genetic model with one dominant allele determining resistance. Linkage analysis showed that the locus conferring resistance lay within an ~25 kilobase region on Chromosome II, which contains 6 open reading frames (ORFs). A reverse genetic approach was then used to disrupt alleles for each of the six ORFs in turn. An allele of one gene, Tb927.2.2380, was shown to determine resistance and this was confirmed by transfecting the resistance allele into a drug sensitive stock to generate an arsenical resistant line. This gene also determines cross-resistance to the major veterinary trypanocide, diminazene aceturate and has been named the arsenical and diamidine (ard) resistance gene.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Mike, Prof. Turner and Any, Prof. Tait
Date of Award: 2004
Depositing User: Mrs Monika Milewska-Fiertek
Unique ID: glathesis:2004-30898
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 12 Oct 2018 13:53
Last Modified: 12 Oct 2018 13:53
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