Gene amplification and drug resistance is an important phenomenon in the biology and chemotherapy of Leishmania, as both laboratory and clinical isolates can often develop resistance to drugs in clinical usage (pentavalent antimonials) or to agents under study for prospective chemotherapy. An understanding of the genes amplified and their mechanism of amplification is essential to our efforts to develop effective chemotherapy and inhibit the emergence of drug resistance. The H region amplification will be a major focus as this amplification encodes multiple drug resistance genes and has been observed in pathogenic species. Resistance genes encoded by this amplification will be characterized, including the protein products and function of 1) the P-glycoprotein gene family which mediates metal resistance but not classical multi-drug resistance. These studies will focus on the role of these genes in resistance to the clinically-utilized pentavalent antimonials; 2) the methotrexate resistance gene hmtx(r), which encodes a protein exhibiting substantial homology to a large family of aldoketoreductases. The protein will be isolated, and its enzymatic properties and role in methotrexate resistance and the unusual features of pteridine metabolism in Leishmania will be studied. Additionally, resistance genes within the H region for two other drugs of interest to prospective antileishmanial chemotherapy (a squalene epoxidase inhibitor and an 8-aminoquinoline) will be mapped, sequenced and the protein products characterized. The second major goal is to understand the mechanism of gene amplification and the ability of a given gene to confer resistance by this mechanism. The intrinsic ability of a given gene to confer resistance by amplification will be assessed by overexpression screening of Leishmania, using DNA transection approaches and multicopy shuttle vectors. Transfectants will be plated on drugs such as methotrexate that show differential ability to amplify in different species of Leishmania, or drugs that have never shown amplification of expected targets; the genes encoded on the transfecting DNA will be recovered and characterized. Secondly, a system designed to simplify the recovery and measurement of amplification frequencies will be used to study the effect of the chromosomal setting on the frequency and nature of amplifications of the H and R regions. Homologous gene targeting will be used to modify critical elements such as repetitive DNA elements shown previously to be key components in the formation of amplified DNAs. The effect of metabolic agents which inhibit DNA repair and recombination will be tested for their ability to decrease the frequency of gene amplification.
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