Gene amplification is an important phenomenon in the biology and chemotherapy of Leishmania, as natural isolates of Leishmania exhibit gene amplification and laboratory isolates can develop amplification-mediated resistance to at least five different drugs (some of which are relevant to the development of anti- leishmanial chemotherapy). Once acquired, amplified DNA mediating drug resistance can be successfully maintained throughout the infectious cycle. We will study three amplifications: that of the R region, encoding the bifunctional dihydrofolate reductase-thymidylate synthetase gene; the H region, which appears to mediate resistance to several drugs of interest to chemotherapeutic strategies and which is amplified in several natural isolates of Leishmania; and the T region, first identified in a natural isolate of Leishmania and currently of unknown function. Our first objective is to characterize the protein products and the mechanism of resistance encoded by the H region, using a combination of molecular genetic approaches and protein biochemistry. The second goal is to understand the chromosomal basis of gene amplification, as this affects the stability of amplified DNA sequences which is crucial to their maintenance and spread in natural populations. Studies are proposed concerning a series of methotrexate-resistant lines, which exhibit extra-chromosomal circular amplification of R and H region DNA, chromosomal amplification of R region DNA, or extra-chromosomal linear amplifications of H region DNA. Our third goal is to continue to develop and test models for the mechanism of gene amplification, as a mechanistic understanding is essential in preventing the emergence of amplification- mediated drug resistance. We will show that homologous recombination between repeated DNA sequences determines both the location of DNA rearrangements necessary for amplification, as well as the final structure of the amplified unit (direct vs. inverted repetition). Our fourth goal is to examine in detail one of the repeated DNA sequences which appears to be a member of a family of dispersed repeats and may represent a transposable element linked to amplification. Our final goal is to survey other Leishmania isolates, both drug-resistant and natural, for the occurrence of DNA amplifications.
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