Digestion of yeast genomic DNA overnight at 37°C with 5 units each of BamHI and AscI (New England Biolabs, NEB, Beverly, MA, USA) produced either a 3.2 kb BamHI wild-type sequence fragment (Figure 1B), or a 2.9 kb mutant fragment, resulting from introduction of the additional AscI site (Figure 1C). RNAse cocktail (0.25 μl, Ambion, Inc., Austin, TX, USA) was included in the digests to remove contaminating RNA. DNA was fractionated on a 0.8% Seakem GTG agarose gel (BMA, Rockland, ME, USA) in 0.5X TBE (44.5 mM Tris, 44.5 mM boric acid, 1 mM EDTA) followed by capillary blotting onto positively charged ZetaProbe GT nylon membrane (Bio-Rad, Hercules, CA, USA) in 10X SSC (1.5 M NaCl, 0.15 M sodium citrate, pH 7.0). The blot was hybridized using a standard protocol with 1 × 106 cpm probe per ml of hybridization solution [10 ]. The probe was a 683 bp ScaI-SfiI fragment located upstream of 5'HS5, and was radiolabeled using a DecaPrime II Kit (Ambion) in the presence of α-32P-dCTP (Amersham Pharmacia Biotech, Pisscataway, NJ, USA). Following two 15-minute washes [5% SDS, 20 mM sodium phosphate (pH 7.2)] the blot was exposed on a phosphorimager screen for three hours.