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Fig. 3. Conditional removal of cyclins. (A) Levels of clb1 and clb2 mRNA in the conditional strains. Wild-type FB1 and conditional strains TAU41 (FB1 clb1^nar) and TAU42 (FB1 clb2^nar) were grown for 3 hours in permissive conditions [minimal medium with nitrate (NO₃)] or restrictive conditions [minimal medium with ammonium (NH₄), or rich medium, (YPD)] and then RNA was extracted and subjected to northern analysis, after loading 10 µg total RNA per lane. The filters were hybridized with probes for clb1 or clb2. A probe for 18s rRNA was used as loading control. (B) Growth of conditional strain in solid medium. Serial tenfold dilutions of FB1, TAU41 (FB1 clb1^nar) and TAU42 (FB1 clb2^nar) cultures were spotted on solid rich medium (YPD), and minimal medium amended with nitrate (NO₃) or ammonium (NH₄). YPD and ammonium plates were incubated for 2 days, while nitrate plates were incubated for 3 days. All incubations were at 28°C. (C) Flow cytometry analysis of wild-type, TAU41 and TAU42 cells grown in permissive and restrictive conditions. Cells grown in MM-NO₃ were centrifuged, washed twice in minimal medium without nitrogen, and resuspended in the appropriated medium. Samples were taken for FACS analysis at the indicated times. Because of the shorter generation time of cells in rich medium, samples were taken every hour for a total of 3 hours.

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