Note on the disruption of yeast cell walls for the isolation of yeast nucleic acids

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Note on the Disruption of Yeast Cell Walls for the Isolation of Yeast Nucleic Acids

For the isolation of nucleic acids or other substances from yeast it is always a problem to destroy the strong wall of the yeast cells in order to release their content for successive treatment. The widely employed grinding in a hand mortar with Carborundum, quartz sand, or glass powder (l-3) is hardly reproducible and very cumbersome. The use of ultrasound was found to be inadequate and the use of the hydrolytic enzymes of the snail He& pomatiu is not economical (4). Therefore a series of experiments was conducted to find optimal conditions for the disruption of the yeast cell wall for the isolation of nucleic acids. A cooled rotary-type cell homogenizer (cell homogenizer MSK made by B. Braun, Melsungen, Germany) was found to be satisfactory and easily operated and the results obtained were reproducible. The effect of this kind of rotary homogenizer on cells used for biological studies had already been reported in detail (5). Our aim was mainly not the effect of destruction on the cell but that of obtaining a good yield and purity of the isolated nucleic acids from the cell homogenate. Procedure. 10 gm of yeast (Saccharomyces cerevisiue) was washed twice with 0.9% NaCl solution and placed into the homogenizer bottle with 10 ml of NaCl (0.9%)-Na citrate (0.01 M) buffer (pH 6.6), 50 gm glass beads (ballotoni) or Carborundum, and another 10 ml buffer, and homogenized at different times and speeds, and with different sizes of glass beads. After homogenization, the homogenate was separated from the glass beads by filtering through a small mesh plastic tea sieve and washing with the buffer solution. Centrifugation can also be used but is slower. If the size of the Carborundum or glass beads was too small for separation from the yeast homogenate by centrifugation or filtration, the homogenized yeast was decanted with the buffer or was used together with the Carborundum, without separation, for the isolation. The RNA was isolated from the homogenate with sodium dodecyl sulfate, according to Crestfield et al. (6), and analyzed for purity (RNA content) and DNA and protein contents (7-9). Yield was also calculated. Almost all of the experiments were repeated three to four times. Results are given in Table 1. As can be seen, the optimal speed was 2000 rpm, optimal time 10-20 set, and glass bead size 0.17 mm or Carborundum 80400. The majority of the yeast cells were destroyed. The homogenate itself was very suitable without further fractionation for the isolation of

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the nucleic acids by using the optimal conditions. The importance of all the described factors must be emphasized for a change even in the amount of the used yeast or in the Carborundum/glass beads gives different results! ACKNOWLEDGMENT This work w&s supported by the Swiss National Mr. P. Herzog for technical help.

Foundation.

The author tha&

REFERENCES 1. OSAWA, S., Biochim. Biophys. Acta 42, 244 (1960). 2. Fu-CHUAN CHAO AND SCTIACHMAN, H. II., Arch. B&hem. Biophys. 3. OSAWA, S., Biochim. Biophys. Acta 43, 110 (1960). 4. KAMNA, E., Radial. C&a. Biol. 35, 18 (l&%3). 5. MEBKENSCHLAGER, M., SCHLOSSMANN, K., AND Kuaz, W., B&hem.

61,220

(1956).

2. 329, 332

(1957). 6. &ESTFIEZD, A. M., SMITH, K. C., AND ALLEN, F. W., J. Bid. (1955). 7. DRURY, H. F., Arch. B&hem. 19,455 (1948). 8. BURTON, K., B&hem. J. 62, 315 (1956). 9. FOLIN, O., AND CIOCALTEU, V., J. Biol. Chem. 73,68 (1927).

Chem. 216, 136

E. KATONA~ Radiobiological Institute University of Zurich Zurich, Switzerland Received August 29, 1966 ‘Present

address: 280 St. George St., Toronto

5, Ontario,

Canada.