|Title:||Identification of novel chloroquine resistance genes by using yeast complementation system|
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Applied Biology and Chemical Technology|
|Pages:||xvi, 197 leaves : ill. (some col.) ; 30 cm|
|Abstract:||Malaria is one of the fatal diseases in the world and the situation has become more worst due to the presence of multidrug resistance. Several phenotypic changes are associated with chloroquine resistance in Plasmodium falciparum, including reduced chloroquine accumulation and the changes in the binding of chloroquine to ferriprotoporphyrin IX. In the past, several candidate genes have been proposed, including pfmdrl, NHE, cg2, and other putative mechanisms mediating the changes in ferriprotoporphyrin IX binding to chloroquine. The main reason why the molecular biology of this parasite is not the same as that of bacteria is the transfection of gene to P. falciparum is not well established. Therefore, the role of many candidate genes in resistance mechanism remains to be searched. We have used a yeast fhnctional complementation approach to identify novel chloroquine resistance genes. A 3D7 plasmodial cDNA library, constructed in a yeast expression vector, was used to transfect a chloroquine-sensitive yeast strain and plated on chloroquine-containing plates at a concentration which can kill the chloroquine-sensitive yeast strain. After primary and secondary screening, about 30 colonies, supposed to contain plasmodial drug resistance genes, were picked and analyzed. By using yeast drug assay, clones that confer resistance were chosen and sequenced. Finally, about eight clones were further characterized. Since the P. falciparum genome project is still in progress, the function of many genes remains to be searched. By searching the sequence of this eight genes from Genbank database, thee of them were found from plasmodial expressed sequence tags database, four of them can only be found from unfinished sequencing data of plasmodial database and one of them was a Plasmodium falciparum knob-associated histidine-rich protein. The resistance phenotype of those resistant clones were confirmed by drug assays and northern analysis shown that thy would express their mRNA. By using yeast complementation system, putative chloroquine resistance genes can be rapidly found out.|
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