SELECTION AGAINST THE DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE (DHFR-TS) LOCUS AS A PROBE OF GENETIC ALTERATIONS IN LEISHMANIA-MAJOR

The genome of the trypanosomatid protozoan genus Leishmania has been s hown to undergo a number of changes relevant to drug resistance and vi rulence, such as gene amplification, chromosomal rearrangement, and va riation in ploidy, Experimental approaches to the study of genomic cha nges have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic f ungi, and cultured mammalian cells, Here we report upon a system which permits the measurement of several types of genomic change occurring at the dihydrofolate reductase-thymidylate synthase (DHFR-TS) locus, F irst, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (+/HYG) to g enerate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation fr equencies in this parasite, Loss of heterozygosity occurred at a frequ ency ranging from 10(-4) to 10(-6) and was elevated 24-fold by treatme nt with gamma-irradiation, while the frequency of other events was les s than 10(-6) and was increased more than 1,000-fold by nitrosoguanidi ne treatment, The frequency of loss of heterozygosity relative to othe r processes such as mutation and gene replacement has important implic ations for genetic variability in natural Leishmania populations and t he generation of both targeted and random mutations, We also developed a protocol for null targeting of diploid cells, in which transfection of a DHFR-TS deletion construct into Leishmania cells followed by neg ative selection yielded parasites lacking DHFR-TS or foreign sequences , The null-targeting method can be applied to any diploid cell, at any locus for which a negative selection exists, Such marker-free auxotro phic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and ch aracterizing the plasticity of the Leishmania genome.