Selection against the Dihydrofolate Reductase-Thymidylate Synthase (DHFR-TS) Locus as a Probe of Genetic Alterations in Leishmania major

Abstract

The genome of the trypanosomatid protozoan genus Leishmania has been shown to undergo a number of changes relevant to drug resistance and virulence, such as gene amplification, chromosomal rearrangement, and variation in ploidy. Experimental approaches to the study of genomic changes have in some cases been limited by the fact that Leishmania cells are asexual diploids, as are some other trypanosomatids, pathogenic fungi, 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. First, we show that DHFR-TS can function as a positive/negative marker. We used selection against DHFR-TS on a heterozygous line (؉/HYG) to generate colonies exhibiting both loss of heterozygosity and structural mutations in DHFR-TS, permitting the first measurement of mutation frequencies in this parasite. Loss of heterozygosity occurred at a frequency ranging from 10 ؊4 to 10 ؊6 and was elevated 24-fold by treatment with ␥-irradiation, while the frequency of other events was less than 10 ؊6 and was increased more than 1,000-fold by nitrosoguanidine treatment. The frequency of loss of heterozygosity relative to other processes such as mutation and gene replacement has important implications for genetic variability in natural Leishmania populations and the 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 negative 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 auxotrophic Leishmania cells show potential as an attenuated vaccine, and the methods developed here provide a new approach for manipulating and characterizing the plasticity of the Leishmania genome.

Transposon Mutagenesis of Trypanosoma brucei Identifies Glycosylation Mutants Resistant to Concanavalin A

“…The authors were unable to account for the large variation in frequency between experiments but were able to demonstrate that the LOH frequency could be increased by ␥-irradiation. As previously noted (48), an LOH rate at the upper end of this range could have important implications for genetic variability within this group of organisms.…”

“…These experiments will be reported in detail elsewhere, after they have been extended to additional loci. LOH has also been measured in L. major (21,48), where the rate ranged from 10 Ϫ4 to 10 Ϫ6 at the DHFR-TS locus. The authors were unable to account for the large variation in frequency between experiments but were able to demonstrate that the LOH frequency could be increased by ␥-irradiation.…”

An in vitro system for developmental and genetic studies of Leishmania donovani phosphoglycans

“…An LdB line lacking the LPG2 gene (termed lpg2 − ) was generated by standard gene replacement methods, and a loss-of-heterozygosity protocol was used to generate a homozygous knockout from a single heterozygous replacement [29,30,36]. Flow cytometry showed that these parasites lacked LPG, as judged by reactivity with ␤-Gal binding lectin ricin agglutinin, or the anti-PG repeat antibody CA7AE (Fig.…”

The LPG1 gene family of Leishmania major

“…To study the localization of LPG1L and LPG1R, L. major cells containing pX63HYG-LPG2-HA (a marker of the parasite Golgi apparatus; strain B1878 [22]) were transfected with pXG-LPG1L-GFP (B4400) or pXG-LPG1R-GFP (B4455) and selected with hygromycin and G418. The lpg1 − lpg1l − /lpg1r − triple knockout parasites were created using a loss-of-heterozygosity (LOH) approach [25]. Briefly, the first allele of LPG1 in the lpg1l − /lpg1r − double knockout was replaced by the BSD marker.…”