Gene transcription analysis of Saccharomyces cerevisiae exposed to neocarzinostatin protein- chromophore complex reveals evidence of DNA damage, a potential mechanism of resistance, and consequences of prolonged exposure -- Data Supplement - Supporting Information -- Proceedings of the National Academy of Sciences

Supporting information for Schaus et al. (September 18, 2001) Proc. Natl. Acad. Sci. USA, 10.1073/pnas.191340698.

Supporting Materials and Methods
Neocarzinostatin (NCS).
NCS protein-chromophore complex, generously provided by Kayaku, was stored as a dry powder in the dark at –80°C. The commercial powder was determined to be a mixture of NCS and apo-NCS (2:1, respectively). The mixture was separated into its pure components by ion-exchange chromatography using a Mono-Q 515 FPLC column (Amersham Pharmacia) and eluting with 20 mM aqueous ammonium acetate solution (pH 5.0) with a linear gradient of sodium chloride (0–1 M). Pure apo-NCS eluted from the column first. Like fractions were combined, and the pooled solutions of pure apo-NCS or NCS were separately dialyzed against doubly distilled water. The dialyzed protein solutions were then lyophilized, providing NCS or apo-NCS in pure form, each as a dry white powder (stored in the dark at –80°C). Synthesis of Fluorescently Labeled NCS (NCS-Fl).
A solution of purified NCS protein-chromophore complex (powder, 2.5 mg, 210 nmol) in sterile water (450 µl) at 23°C was brought to pH 9.0 by the addition of aqueous sodium bicarbonate solution (50 µl, 1 M NaHCO₃, final concentration NCS, 0.42 mM), then was transferred by syringe to a 1.5-ml Eppendorf tube containing the commercial fluorescence-tagging reagent Alexa Fluor 350 (1 mg, 2.4 mmol, Molecular Probes). The conjugation reaction was allowed to proceed for 2 h at 23°C before the reaction solution was neutralized (pH 7.4) by the addition of Hepes buffer solution (100 µl, 1 M). The fluorescently labeled product, NCS-Fl, was purified by ion-exchange chromatography using a Mono-Q 515 FPLC column (Amersham Pharmacia) eluting with 20 mM aqueous ammonium acetate solution (pH 5.0) with a linear gradient of sodium chloride (0–1 M). Fractions containing NCS-Fl were identified by their UV absorbance at 353 nM and were pooled. The solution of combined fractions was dialyzed against phosphate-buffered saline, then doubly distilled water. Lyophilization after dialysis provided pure NCS-Fl as a dry white powder, stored in the dark at
–20°C. Fluorescence Microscopy of Saccharomyces cerevisiae Treated with NCS-Fl.
A single colony of S. cerevisiae strain BY4741 [MATa his3D1 leu2D1 met15D0 ura3D0] was used to inoculate 50 ml of YPD medium (2% glucose/2% peptone/1% yeast extract). The cells were grown at 30°C on a shaker (225 rpm) overnight to a density of 1 ´ 10⁸ cells per ml, the growth medium was diluted 50-fold with YPD medium prewarmed to 30°C, and incubation was continued to a density of 1 ´ 10⁷ cells per ml (A₆₀₀ = 1.0), at which point a solution of NCS-Fl in sterile water (final concentration NCS-Fl, 50 µg/ml) was added. A control incubation was conducted in parallel by using sterile water in lieu of NCS-Fl solution. After 1 h, cells from both control and treated solutions were fixed by the addition of 37% aqueous formaldehyde solution (final concentration, 1%) with continued incubation for 15 min at 30°C. Cells from each incubation were then pelleted by centrifugation (3,000 ´ g) for 5 min at 20°C, and each pellet was washed with PBS (pH 7.4). The cells were pelleted again by centrifugation (3,000 ´ g) for 5 min at 20°C, and the pellet was washed with PBS (pH 7.4). This process was repeated once more before resuspension in PBS (pH 7.4) for viewing. Fluorescence microscopy was performed using a Laborlux S fluorescence microscope (Leitz) with a charge-coupled device camera (Apogee Instruments, Tucson, AZ). Imaging was conducted at ´1,000 magnification by using fluorescence excitation between 340 and 380 nm and a 400-nm emission filter.