Current Genetics (1981) 4:85-90
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© Springer-Verlag 1981
Rapid Isolation of Yeast Nuclei Gregory J. Ide and Court A. Saunders Department of Biochemistryand Biophysics,OregonState University,Corvallis,Oregon97331, USA
Summary. A procedure has been developed for the rapid isolation of yeast nuclei in high yield using Percoll gradients. The nuclei are substantially free of cytoplasmic contamination as measured by alcohol dehydrogenase activities, have the typical chromatin digestion pattern when digested with nucleases, are useful for isolation of nuclear proteins and for in vitro transcription experiments. Key words: Saccharomyces cerevisiae - Nuclear isolation - Percoll - in vitro Transcription
Introduction
Published methods for isolation of yeast nuclei are of two major types. One is through lysis of normal yeast cells using various pressure methods (Tekamp et al. 1979; Bhargava and Halvorson 1971; Sajdel-Sulkowska et al. 1974); the other is to produce yeast protoplasts by enzymatic digestion of the yeast cell wall and subsequently lyse the protoplast by osmotic shock or detergent lysis of the cytoplasmic membrane, releasing the nuclei (May, 1971; Wintersberger et al. 1973; Rozijn and Tonino 1964; Schultz 1978; Tekamp et al. 1979; Groner and Phillips 1975). The former method has the advantage that the nuclei are from physiologically normal cells; however, the yield is quite low and the microscopic appearance of the nuclei indicates they are damaged during isolation. The method of nuclear isolation by detergent lysis of protoplasts gives high yields of yeast nuclei; however, the nutritional state of the nuclei during the protoplasting, possible artifacts introduced by detergent treatment, and the relatively long isolation Offprint requests to: G. J. Ide
procedure may adversely affect the physiological state of these nuclei. Almost all yeast nuclei used for biochemical studies involve production of yeast protoplasts intermediate to nuclear isolation. The method of nuclear isolation used previously in this laboratory and others for chromatin experiments (the differential centrifugation method) is an adaptation of the method of Wintersberger (1973) and was designed b y D. Lohr (1975, 1977a). It gives very clean nuclei, but suffers from the fact that many tedious and timeconsuming centrifugations are required. A method to very rapidly isolate nuclei from the yeast S. pombeii by centrifuging lysed spheroplasts through 3M sorbitol onto a cushion of 4M sorbitol has been reported (Duffus t969). This method has two problems: (1) the nuclei are contaminated with unbroken cells, cell walls and membranes; (2) these levels of sorbitol are hyperosmotic and tend to shrink the nuclei. Colloidal silica is a centrifugation medium which has a very low osmolality and allows one to attain relatively high densities without introducing hyperosmotic shrinkage. It has been used widely for the isolation of cells and subcellular organelles [see reviews by Schmitt and Hermann (1977)and Wolff (1975)], viruses (Pertoft 1970a, 1970b; Klingeborn and Pertoft 1972), plant cell nuclei (Hendriks 1972), spermatid nuclei (Loft and Wyrobek 1972), chloroplasts (Takabe et al. 1979), and pancreatic islets (Buitrago et al. 1977). Viability of several cell types is maintained after isolation on colloidal silica gradients (Pertoft et al. 1977). Since colloidal silica has been used for the isolation of such a wide range of biologically interesting particles, it was a logical choice for isolation of yeast nuclei. A technical problem arises, however, in that colloidal silica (Ludox) is an inhibitor of transcription. A sohltion to this problem proved to be the use of the polyvinylpyrollidone-coated colloidal silica (Percoll). Although O172-8083/81/0004/0085/$ 01.40
86
G.J. Ide and C. A. Saunders: Rapid Isolation of Yeast Nuclei similar in physical properties, Percoll does not inhibit transcription as does Ludox. The preparative technique tested in these experiments is to layer the 18% Ficoll protoplast lysate (or some dilution of this lysate) from the nuclear isolation procedure of Lohr and Ide (1979) on Percoll gradients of varying compositions and spin these gradients in Sorvall HB4 rotor. Nuclei are isolated directly from the gradients. Yeast nuclei are known to be very stable to lysis in this osmotically stabilized buffer. Gradient densities were varied b y changing the ratio of Percoll to water as the solvent for the gradient solutions. Using the gradient system we hoped to avoid the previously discussed problems of low yield, long isolation times, mechanical damage to the nuclei,, and the presence of detergents which have been implicated in artifacts due to nucleosome sliding. The nuclei isolated by this technique were evaluated by four criteria: (1) cytoplasmic contamination, (2) metabolic activity (transcription), (3) recovery of undegraded nuclear proteins, and (4) characteristic nucleosome patterns as assayed by nuclease digestion products.
Materials and Methods
Fig. 1 a-c. Photographsofyeast cells, a protoplasts, b and protoplasts lysed in 18% Ficoll, c to show relative sizes. Magnification is 960X
--
nuclei
Fig. 2. A typical Percoll gradient is shown after centrifugation
The initial stages of this isolation procedure were adapted from the nuclear isolation technique of Lohr and Ide (1979). Cells were harvested by centrifugation at 2,500 x g for 2 rain in a Sorvall GS3 rotor and washed once with distilled water, stationary phase cells were then treated for 15 rain at 35 oC in 100 mM EDTA (pH 8.0), 0.5% j3-mercaptoethanol, using 4 ml of this medium per gram (wet weight) of yeast cells. This step is not needed for logarithmic phase cells which are susceptible to protoplasting without it. Cells were then pelleted and suspended in S buffer (1.1 M sorbitol, 20 mM KH2PO4 (pH 6:5), 0.5 mM CaC12, 0.5% & mercaptoethanol), using 4 ml per gram (wet weight) of cells. Zymolyase 5000 (Kirin Breweries) which had been previously dissolved at 50 mg/ml in S buffer was then added to a final concentration of 1.5 mg/ml. Protoplasting was then carried out at 35 °C until cells were completely devoid of buds and spherical as seen in the light microscope. The time for protoplasting is highly strain dependent, varying from 15 to 180 min. During protoplasting, the mixture should be rocked gently to keep the cells suspended, but vigorous shaking, vChieh may prematurely lyse some protoplasts, must be avoided. Monitor the protoplasting microscopically. Protoplasts which are sufficiently treated are shown in Fig. lb. The protoplasts were then harvested by centrifugation at 4,000 x g for 5 rain in a Sorvall SS-34 rotor. All further manipulations were at 4 °C using buffers containing 0.5 mM phenylmethylsulfonylfluoride, which was added slowly from stock 0.1 M PMSF in isopropanol. The pelleted protoplasts were resuspended in 0.1 ml of 18% Ficoll 400 (Pharmacia; dissolved in 20 mM KH2PO4 at pH 6.5 and 0.5 mM CaC12) per gram of cells using a glass rod. Cells were then diluted with 3 ml of 18% Ficoll per gram of cells and homogenized with a loose fitting Potter-Elvehjem Teflon homogenizer. This step lyses the cytoplasmic membrane, releasing the nuclei. Release of the nuclei is monitored microscopically. A photograph of properly lysed protoplasts is shown in Fig. lc.
G. J. Ide and C. A. Saunders: Rapid Isolation of Yeast Nuclei
87
1) Cytoplasmic Contamination
Fig. 3. Photograph of nuclei after isolation from the gradient by the wash step. Magnification is 960X. Yields of nuclei, as assayed by DNA content, are routinely greater than 80%
Table 1. Alcohol dehydrogenase assay for cytoplasmic contamination
Cytoplasm (load fraction) Nuclei (direct from gradient) Washed nuclei
Preparation #1
Preparation #2
Preparation #3
1.08
1.18
0.87
0.10
0.06
0.06
~
~
~
© Springer-Verlag 1981
Rapid Isolation of Yeast Nuclei Gregory J. Ide and Court A. Saunders Department of Biochemistryand Biophysics,OregonState University,Corvallis,Oregon97331, USA
Summary. A procedure has been developed for the rapid isolation of yeast nuclei in high yield using Percoll gradients. The nuclei are substantially free of cytoplasmic contamination as measured by alcohol dehydrogenase activities, have the typical chromatin digestion pattern when digested with nucleases, are useful for isolation of nuclear proteins and for in vitro transcription experiments. Key words: Saccharomyces cerevisiae - Nuclear isolation - Percoll - in vitro Transcription
Introduction
Published methods for isolation of yeast nuclei are of two major types. One is through lysis of normal yeast cells using various pressure methods (Tekamp et al. 1979; Bhargava and Halvorson 1971; Sajdel-Sulkowska et al. 1974); the other is to produce yeast protoplasts by enzymatic digestion of the yeast cell wall and subsequently lyse the protoplast by osmotic shock or detergent lysis of the cytoplasmic membrane, releasing the nuclei (May, 1971; Wintersberger et al. 1973; Rozijn and Tonino 1964; Schultz 1978; Tekamp et al. 1979; Groner and Phillips 1975). The former method has the advantage that the nuclei are from physiologically normal cells; however, the yield is quite low and the microscopic appearance of the nuclei indicates they are damaged during isolation. The method of nuclear isolation by detergent lysis of protoplasts gives high yields of yeast nuclei; however, the nutritional state of the nuclei during the protoplasting, possible artifacts introduced by detergent treatment, and the relatively long isolation Offprint requests to: G. J. Ide
procedure may adversely affect the physiological state of these nuclei. Almost all yeast nuclei used for biochemical studies involve production of yeast protoplasts intermediate to nuclear isolation. The method of nuclear isolation used previously in this laboratory and others for chromatin experiments (the differential centrifugation method) is an adaptation of the method of Wintersberger (1973) and was designed b y D. Lohr (1975, 1977a). It gives very clean nuclei, but suffers from the fact that many tedious and timeconsuming centrifugations are required. A method to very rapidly isolate nuclei from the yeast S. pombeii by centrifuging lysed spheroplasts through 3M sorbitol onto a cushion of 4M sorbitol has been reported (Duffus t969). This method has two problems: (1) the nuclei are contaminated with unbroken cells, cell walls and membranes; (2) these levels of sorbitol are hyperosmotic and tend to shrink the nuclei. Colloidal silica is a centrifugation medium which has a very low osmolality and allows one to attain relatively high densities without introducing hyperosmotic shrinkage. It has been used widely for the isolation of cells and subcellular organelles [see reviews by Schmitt and Hermann (1977)and Wolff (1975)], viruses (Pertoft 1970a, 1970b; Klingeborn and Pertoft 1972), plant cell nuclei (Hendriks 1972), spermatid nuclei (Loft and Wyrobek 1972), chloroplasts (Takabe et al. 1979), and pancreatic islets (Buitrago et al. 1977). Viability of several cell types is maintained after isolation on colloidal silica gradients (Pertoft et al. 1977). Since colloidal silica has been used for the isolation of such a wide range of biologically interesting particles, it was a logical choice for isolation of yeast nuclei. A technical problem arises, however, in that colloidal silica (Ludox) is an inhibitor of transcription. A sohltion to this problem proved to be the use of the polyvinylpyrollidone-coated colloidal silica (Percoll). Although O172-8083/81/0004/0085/$ 01.40
86
G.J. Ide and C. A. Saunders: Rapid Isolation of Yeast Nuclei similar in physical properties, Percoll does not inhibit transcription as does Ludox. The preparative technique tested in these experiments is to layer the 18% Ficoll protoplast lysate (or some dilution of this lysate) from the nuclear isolation procedure of Lohr and Ide (1979) on Percoll gradients of varying compositions and spin these gradients in Sorvall HB4 rotor. Nuclei are isolated directly from the gradients. Yeast nuclei are known to be very stable to lysis in this osmotically stabilized buffer. Gradient densities were varied b y changing the ratio of Percoll to water as the solvent for the gradient solutions. Using the gradient system we hoped to avoid the previously discussed problems of low yield, long isolation times, mechanical damage to the nuclei,, and the presence of detergents which have been implicated in artifacts due to nucleosome sliding. The nuclei isolated by this technique were evaluated by four criteria: (1) cytoplasmic contamination, (2) metabolic activity (transcription), (3) recovery of undegraded nuclear proteins, and (4) characteristic nucleosome patterns as assayed by nuclease digestion products.
Materials and Methods
Fig. 1 a-c. Photographsofyeast cells, a protoplasts, b and protoplasts lysed in 18% Ficoll, c to show relative sizes. Magnification is 960X
--
nuclei
Fig. 2. A typical Percoll gradient is shown after centrifugation
The initial stages of this isolation procedure were adapted from the nuclear isolation technique of Lohr and Ide (1979). Cells were harvested by centrifugation at 2,500 x g for 2 rain in a Sorvall GS3 rotor and washed once with distilled water, stationary phase cells were then treated for 15 rain at 35 oC in 100 mM EDTA (pH 8.0), 0.5% j3-mercaptoethanol, using 4 ml of this medium per gram (wet weight) of yeast cells. This step is not needed for logarithmic phase cells which are susceptible to protoplasting without it. Cells were then pelleted and suspended in S buffer (1.1 M sorbitol, 20 mM KH2PO4 (pH 6:5), 0.5 mM CaC12, 0.5% & mercaptoethanol), using 4 ml per gram (wet weight) of cells. Zymolyase 5000 (Kirin Breweries) which had been previously dissolved at 50 mg/ml in S buffer was then added to a final concentration of 1.5 mg/ml. Protoplasting was then carried out at 35 °C until cells were completely devoid of buds and spherical as seen in the light microscope. The time for protoplasting is highly strain dependent, varying from 15 to 180 min. During protoplasting, the mixture should be rocked gently to keep the cells suspended, but vigorous shaking, vChieh may prematurely lyse some protoplasts, must be avoided. Monitor the protoplasting microscopically. Protoplasts which are sufficiently treated are shown in Fig. lb. The protoplasts were then harvested by centrifugation at 4,000 x g for 5 rain in a Sorvall SS-34 rotor. All further manipulations were at 4 °C using buffers containing 0.5 mM phenylmethylsulfonylfluoride, which was added slowly from stock 0.1 M PMSF in isopropanol. The pelleted protoplasts were resuspended in 0.1 ml of 18% Ficoll 400 (Pharmacia; dissolved in 20 mM KH2PO4 at pH 6.5 and 0.5 mM CaC12) per gram of cells using a glass rod. Cells were then diluted with 3 ml of 18% Ficoll per gram of cells and homogenized with a loose fitting Potter-Elvehjem Teflon homogenizer. This step lyses the cytoplasmic membrane, releasing the nuclei. Release of the nuclei is monitored microscopically. A photograph of properly lysed protoplasts is shown in Fig. lc.
G. J. Ide and C. A. Saunders: Rapid Isolation of Yeast Nuclei
87
1) Cytoplasmic Contamination
Fig. 3. Photograph of nuclei after isolation from the gradient by the wash step. Magnification is 960X. Yields of nuclei, as assayed by DNA content, are routinely greater than 80%
Table 1. Alcohol dehydrogenase assay for cytoplasmic contamination
Cytoplasm (load fraction) Nuclei (direct from gradient) Washed nuclei
Preparation #1
Preparation #2
Preparation #3
1.08
1.18
0.87
0.10
0.06
0.06
