Nitrogen Starvation Induces sme2 Expression

Diploid cells were cultured at 30 °C in YEA medium for 24 h and then transferred to EMM–N medium at 30 °C for nitrogen starvation. Cultures were collected at 0, 3, 6 and 9 h. Total RNA was isolated by incubating cells in phenol heated to 65 °C for 10 min, followed by three additional extractions using phenol–chloroform. The RNA was precipitated by sodium acetate and ethanol. Northern blots were performed as described previously^{19 (link)}. The RNA (5 μg) was resolved on a 1% formaldehyde–agarose denaturing gel, capillary transferred using NorthernMAX transfer buffer (Thermo Fisher) onto positively charged BrightStar-Plus nylon membrane (Ambion) and crosslinked using a UV Stratalinker 2400 (Stratagene). A T7 in vitro transcription kit (Promega) was used to generate α-P³²-UTP (PerkinElmer)-labelled RNA probes (sme2, 27–329) that were hybridized to the membrane overnight at 65 °C in ULTRAhyb buffer (Ambion). The membrane was exposed and scanned using a Typhoon FLA 9500 phosphor imager (GE Healthcare).