Fig. 1.

[³H]hypoxanthine incorporation into DNA during microgametogenesis. (A) Incorporation of [³H]hypoxanthine over time of gametogenesis. Gametocytes were purified as described previously [10] with minor modifications. Female NMRI mice (Charles River) were pre-treated with 0.1 ml phenylhydrazine (25 mg/ml in PBS) and infected two days later with 0.5–2 × 10⁷P. berghei ANKA clone 2.34 parasites from frozen blood stocks. On day 4 p.i. 20 mg/ml sulfadiazine in drinking water was applied to kill asexual stages. On day 6 p.i., mice were bled by cardiac puncture, the blood washed in gametocyte maintenance buffer (GMB: 4 mM sodium bicarbonate, 20 mM glucose, 137 mM NaCl, 4 mM KCl, 1 mM MgCl₂, 1 mM CaCl₂, 20 mM Hepes pH 7.24–7.29, 0.1% BSA) and purified on a 48% Nycodenz/GMB gradient (Nycodenz stock solution: 27.6% (w/v) Nycodenz in 3 mM KCl, 0.3 mM EDTA, 5 mM Tris–HCl pH 7.2). Gametocytes were resuspended in GMB and kept at 20 °C. As determined by Giemsa stained blood film, gametocytes were enriched to approximately 95% with contaminants being late stage trophozoites (≈4%), few red blood cells and occasionally some very few white blood cells. Gametocytes were activated at room temperature (22–26 °C) by transferring them to gametocyte activation medium (GAM; RPMI 1640 with 20 mM Hepes, 4 mM sodium bicarbonate, adjusted to pH 8.0 and supplemented with 100 μM XA) while the medium for controls was adjusted to pH 7.0. GAM was supplemented with 0.5 μM [³H]hypoxanthine (previously evaporated from 52 μM in water/ethanol 1:1, specific activity of 1 mCi/ml, GE Healthcare). 100 μl aliquots containing (3–15) × 10⁶ gametocytes were removed at the indicated time points and shock frozen in 96-well microtiter plates on liquid N₂. Subsequently, macromolecules including DNA were recovered by filtering the lysates onto glass-fiber filter 96 plates (Perkin Elmer UniFilter 96 GF/C and Packard Filtermate Harvester). The membrane plate was washed 5 times by perfusion with H₂O, bleached with 10% H₂O₂, dried at 50 °C for 30 min, loaded with 30 μl/well PerkinElmer Microscint 0, and the radioactivity determined in a TopCount NXT microplate scintillation counter (Packard Instruments). Values are expressed as cpm per 10⁶ cells and are averages of duplicates in one experiment representative of five. (B) Radioactive labelling of nucleic acids over time with fixed amount of radioactive [³H]hypoxanthine (0.5 μCi/well, 260 nM) supplemented with the indicated concentrations of cold hypoxanthine. (C) Total hypoxanthine incorporation during gametogenesis calculated from data shown in (B). (D) Effect of pre-incubation of gametocytes with 260 nM [³H]hypoxanthine before activation. Activation was performed at the indicate time and cells incubated for 20 min. Results are shown with S.E.M, n = 3. All graphs and analyses were generated using GraphPad Prism software.