Zymolyase100t

To measure growth inhibition caused by either zymolyase or tunicamycin, cell cultures were inoculated from an overnight culture to an O.D. _{620 nm} = 0.025 in YPD medium supplemented with different amounts of either zymolyase100T (ICN Biomedicals, Inc., dissolved in Tris-HCl [pH 7.5]–5% glucose) or tunicamycin (Sigma-Aldrich, dissolved in DMSO). The assay was performed using duplicate rows of a 96-well plate and incubated overnight at 37°C. Graphs represented the percentage of growth estimated by O.D. _{620 nm} measures for each strain in YPD plus both compounds compared to similar untreated cells.

To prepare agarose plugs containing chromosomal DNA, cells were first washed in SP1 (50 mM citrate-phosphate [pH 5.6], 40 mM EDTA, 1.2 M sorbitol) and then incubated for ~1 h at 37 °C in SP1 with 0.6 mg per ml Zymolyase-100T (ICN Biomedicals). Cells were then pelleted and resuspended at 6–7 × 10⁸ cells per ml in TSE (10 mM Tris-HCl [pH 7.5], 0.9 M sorbitol, 45 mM EDTA). The cell suspension was warmed to 42 °C, and 1 volume of 1.1% low-melting agarose (BioRad) in TSE was added. Aliquots were dispensed into plug molds and allowed to solidify, and plugs were then incubated at 50 °C for 2 h in 0.25 M EDTA, 50 mM Tris-HCl (pH 7.5), 1% SDS and then 48 h in 1% lauryl sarcosine, 0.5 M EDTA, 10 mM Tris (pH 9.5), 1 mg per ml proteinase K. Plugs were washed 4× in T10 × E buffer (10 mM Tris [pH 7.5], 10 mM EDTA) and stored at 4 °C in T10xE buffer. NotI-digested chromosomal DNAs embedded in agarose plugs were separated on 1% agarose gel with 0.5 × TBE buffer at 14 °C, using the BioRad CHEF-DR III system at 6 V cm⁻¹ (200 V) and a pulse time of 60–120 s for 24 h. ³²P labeled probes specific for telomeric NotI fragments from chromosomes I and II (C, I, L, and M fragments as indicated in Fig. 3c) were used to analyze telomeric Not I fragments by Southern blot analysis^{13 (link),59 (link)}.

For immunoblotting, cells were grown to mid-log phase and cells were collected and resuspended in 5 ml of buffer E (50 mM sodium citrate, 100 mM sodium phosphate, pH 6.0, and 0.8 M sorbitol). Protoplasts were generated by incubation with Glucanex (1 mg/ml; Sigma-Aldrich) and Zymolyase-100 T (3 mg/ml; ICN Biomedicals) for 1 h. Then, the protoplasts were resuspended in lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Triton X-100, 1 mM DTT, 1 mM PMSF, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 1 μg/ml pepstatin). Protein concentrations were determined using the BioRad Protein Assay kit (BioRad). 50 μg of protein extracts were resolved by SDS–polyacrylamide gel electrophoresis (PAGE) on 10% gels and probed with anti-GFP (JL-8 Living Colors, Clontech), polyclonal DsRed (Living Colors, Clontech), anti-HA Rat monoclonal antibody (3F10, Roche) or anti-tubulin (Sigma 75168). Protein transfer, blotting, and chemiluminescence detection were performed using standard procedures ^{49 (link)}.