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. Author manuscript; available in PMC: 2009 Jun 8.
Published in final edited form as: Mol Microbiol. 2009 Jan 16;71(6):1386–1401. doi: 10.1111/j.1365-2958.2009.06610.x

FIG. 5. Plasmid segregational tests of DHCH1 essentiality.

FIG. 5

A. Starting dhch1−/+pXNG4-DHCH1 transfectant. The structure of one of the lines generated as described in Fig. 3A is shown, additionally showing the TK (ganciclovir sensitivity), GFP and SAT resistance markers encoded on the pXNG4-DHCH1 vector. B. Forced loss of pXNG4-DHCH1 by negative selection and sorting. The line depicted in Panel A was grown for 24 hr in the presence of ganciclovir but in the absence of nourseothricin. Fluorescent (bright) and non-fluorescent (dim) parasites were assigned using the M1 and M2 gates shown, and single cells were sorted into 96-well plates containing M199 medium and/or various supplements (see methods). With ‘bright’ cells, 478/672 (71 %) of the wells yielded growth. In contrast, only 48/2016 of wells inoculated with ‘dim’ cells (2.4 %) showed growth; all of these continued to grow in the presence of nourseothricin (SAT resistance) establishing retention of the pXNG-DHCH1 episome. C. Retention of pXNG4-DHCH1 in the absence of ganciclovir selection. The line shown in panel A was grown 24 hr without ganciclovir prior to GFP flow cytometry. D. WT L. major subjected to GFP flow cytometry.