Zymolyase 20t

Mononucleosomes were obtained as described before (1 (link),11 (link),19 (link)), and the resulting DNA was analysed by qPCR as described previously (1 (link),20 (link)). Briefly, strains were cultured in 250 ml of YE5S medium to an OD₆₀₀ of 0.5 and then cross-linked with formaldehyde (final concentration of 0.5% (v/v)) for 20 min at 25°C. Cells were then digested with Zymolyase 20T (Amsbio), and spheroplasts were treated with increasing concentrations of micrococcal nuclease (MNase; Sigma). Purified DNA was separated electrophoretically, and samples displaying 80–90% mononucleosomal DNA without subnucleosomal fragments (faster migration than mononucleosomes in the electrophoresis) were further analysed by qPCR with a set of overlapping primer pairs (see Supplementary Table S2). For each primer pair, numbers in Y-axis correspond to the relative value to the input, which was obtained using as a template DNA from cells not treated with MNase, and received a value of 1.

Genomic DNA was prepared using a modified protocol obtained from Dr. Juan Lucas Argueso (personal communication), in which approximately 2 × 10⁸ cells (1 ml) from each stationary phase culture were embedded in agarose plugs. Cells were resuspended in 180 µl of 0.5% low-melt SeaPlaque agarose (in 100 mM EDTA) with 12 µl of 25 mg/ml Zymolyase 20-T (Amsbio). The agarose mixture was then pipetted into two Bio-Rad plug molds to solidify at 4° for 30 min. Plugs were extruded from the plug mold into 24-well plates, two plugs per well, and incubated in 2 ml of 500 mM EDTA + 10 mM Tris at 37° overnight. 400 µl of 5 mg/ml proteinase K (Roche) in 500 mM EDTA were then added into each well and incubated at 50° for 5 hr. Plugs were subjected to four 1 hr washes with TE (10 mM Tris, 1 mM EDTA, pH 8.0) before being stored in TE at 4°. For restriction enzyme digests, plugs were washed eight times with TE before two 30 min washes in restriction buffer + BSA.

Flavonoids were either purchased from Alkemist labs (Costa Mesa, CA, USA) or MilliporeSigma (St. Louis, MO, USA). [glycine-2-³H]GSH was purchased from American Radiolabeled Chemicals (St. Louis, MO, USA). Zymolyase 20 T was purchased from AMS Biotechnology (Abingdon, UK). All other chemicals were purchased from MilliporeSigma or Fisher Scientific (Pittsburgh, PA, USA).

The relative abundance of 25S ribosomal RNA was measured by quantitative hybrid Southern/northern blotting. The total nucleic acid content of cells was extracted using a version of the “Smash-and-grab” DNA isolation protocol [93 (link)] with the modification that cell walls were enzymatically disrupted using Zymolyase - 20T (Amsbio) instead of glass beads. Nucleic acids were resuspended in TE pH 8 and then separated by electrophoresis in a 1.5% LE agarose gel with ethidium bromide (0.3 μg/ml). After the ribosomal RNA was separated away from genomic DNA, the gel was photographed and then cut to separate the two portions containing genomic DNA and rRNA. Subsequently, the two different portions of the gel were blotted following standard Southern (genomic DNA) and northern (rRNA) blotting protocols. The Southern blot was probed for ACT1 as a single copy control and the northern blot was probed for 25S rRNA sequence. Because the amount of rRNA on the blot could be in excess of the probe, hybridization of the northern blot was limited to 2 hours to ensure that the hybridization signal was proportional to the amount of target sequence. The hybridization signals were analyzed using a Bio-Rad Personal Molecular Imager and Quantity One software. The 25S rRNA hybridization signal was first normalized to ACT1 and then relative to wild type.