Current Genetics
CurrentGenetics (1982) 5:153-155
© Springer-Verlag 1982
Short Communication
Uptake of DNA by Fragile Mutants of Saccharomyces cerevisiae Pencho V. Venkov and Vesselin P. Ivanov Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Summary. When Saccharomyces cerevisiae SY15 rho ° mutant cells grown in media stabilized with 10% sorbitol were suspended in 2% sorbitol solutions, 60-70% of the population did not lyse and became permeable to native high molecular weight DNA. Maximal incorporation of DNA to DNase resistant state was measured after 60 min of incubation in presence of 5 ~g/ml DNA and 10 mM CaC12. These results suggest that the fragile mutants might be tested as hosts for transformation of whole yeast cells. Key words: Yeast - Mutant cell-wall - Permeability exponentialy
Yeast cell wall is an absolute barrier for the uptake of DNA and transformation with DNA (Hinnen et al. 1978) or fusion with mitochondria (Gunge and Sakaguchi 1979) have been achieved on yeast spheroplasts only. The isolation of osmotic dependent yeast mutants defective in the structure of the cell wall has been reported (Venkov et al. 1974; Reuter et al. 1979). In media supplemented with 10% sorbitol as osmotic stabilizer the mutant cells grow exponentialy in fragile form: suspended in water the cells break open and the cellular content flows out through holes in the cell wall and membrane (Mateeva et al. 1976). The fragile yeast mutants display sensitivity to antibiotics like rifampicin (Venkov et al. 1975), actinomycin D (Waltschewa et al. 1976), toyocamycin (Venkov et al. 1977) known to be without effect on wild type yeast strains, which suggests an increased permeability of the mutant cells. Here, we report results showing that under appropriate experimental conditions
Offprint requests to: P.V. Venkov
the fragile yeast cells became also permeable to high molecular weight DNA. One of the fragile mutants reported is Saccharomyces cerevisiae SY15 obtained from strain A364 (Venkov et al. 1974). A rho ° derivative of SY15 has been isolated by treatment with ethidium bromide (Golgring et al. 1970) and the complete absence of mitochondrial DNA was verified by centrifugation in caesium cloride density gradients. When exponentialy growing SY15 rho ° cells were suspended in media with lower concentrations of the osmotic stabilizer (1% to 5% sorbitol) part of the population did not lyse and without begining a new division cycle could survive for at least 2 h. I t was found that under such conditions the fragile cells became permeable to DNA isolated from different cells, including yeast. Because of difficulties to obtain highly labeled yeast DNA, in the following experiments DNA purified from B. subtilis cells labeled in vivo with either 32p. phosphate or 3H-thymidine was used. The highest DNA incorporation was measured after suspending the SY15 rho ° cells in 2% sorbitol solutions, which allows survival of 60-70% of the population. The concentration of 2% sorbitol is crytical for SY15 rho ° strain. The other fragile yeast mutants behave similarly to SY15 rho °, however due to the different extend of there fragility a specific concentration of the osmotic stabilizer for each mutant was found. No DNA incorporation was established in SY15 rho ° cells in sorbitol solutions of 6% or more, or in wild type yeast strains suspended in water or in sorbitol solutions with different concentrations. Therefore, the observed DNA incorporation is a pattern typical for the fragile yeast cells suspended in concentrations of the osmotic stabilizer just enough to prevent the lysis but not sufficient to support the cell-growth. The results presented on Table 1 show that at low concentrations the binding and the uptake of DNA in SY15 rho ° cells is proportional to the amount of DNA
o172-8o83/82/ooo5/o153/$Ol.OO
154
P.V. Venkov and V. P. Ivanov: Uptake of DNA by Fragile Mutants of S. eerevisiae
Table 1. Influence of DNA concentration on the reactions between SY15 rho° cells and DNA DNA conch ~g/ml
Incubation
Incubation
min
mixture
Interaction of DNA with 107 cells Bound cpm
0.5 1.0 2.0 3.0 4.0 5.0 7.0 9.0
Table 2. Kinetics of DNA-yeast cell interactions
1,050 4,200 18,600 37,800 60,000 94,500 121,800 145,800
DNase resistent % total DNA
cpm
0.7 1.4 3.1 4.2 6.0 6.3 5.8 5.4
60 270 960 2,160 3,600 5,500 6,090 6,750
%total DNA 0.04 0.09 0.16 0.24 0.30 0.37 0.29 0.25
Saccharomyces cerevisiae SY15 rho° was grown at 30 °C in YM5 medium (Hartweel 1967) supplemented with 10% sorbitol. At middle exponential phase (about 107 cells/ml) the culture was chilled on ice, centrifuged and the cells suspended in 2/3 the culture volume of 2% sorbitol solution and incubated at 30 °C for 20 min with gentle shaking. This will give a survival of 107 colony-forming units per 1 ml cell-suspention. 3H-labeled DNA (300,000 cpm/1 ~g and average molecular weight of 50 kb) purified from a B. subtilis t h y - strain was added to appropriate volumes of the cell-suspention and incubated at 30 °C. Samples were taken in duplicate at 0 min and at 60 rain, For determination of bound DNA the cells from 1 ml suspention were washed with 50 ml 10% sorbitol solution on glass fiber filters (Reeve angels, Clifton, New Jersey) and the radioactivity was measured. For determination of DNase resistent incorporation the cells from 2 ml samples were washed as described the cells suspended in 2 mlDNase buffer (pancreatic DNase 20 #g/ml; MgCt2 10 raM; sorbitol 10%) and after incubation for 30 min at 37 °C samples of 1 ml were washed with 50 ml 10% sorbltol an glass fiber filters and the radioactivity measured. The cpm in the 0 min samples were subtracted from the radioactivity in the 60 rain samples.
added. The maximal incorporation measured as percent from total DNA was found at DNA concentrations of 4 - 5 #g/ml, while somewhat lower values were established at higher DNA concentrations. Essentially the same resuits have been obtained in experiments where DNA uptake was measured in cells suspended in 2N sorbitol solutions containing 1 /ag/ml highly labeled DNA and increasing amounts of cold DNA. Such effect of inhibition at enhanced DNA concentration is typical for transformation of competent procaryotic cells (Hubavenkova and Markov 1975) and was explained with the existence of limited numb er of spots through which the DNA could enter the recipient cell. A similar explanation might be given for the fragile yeast ceils, since ultrastructural studies (Mateeva et al. 1976)have shown the appearance of only a few local defects in there cell wall and membranes after lowering the osmotic presure in the medium.
1 3 5 20 60 120 60 60 60
2% sorbitol 2% sorbitol 2% sorbitol 2% sorbitol 2% sorbitol 2% sorbitol 2% PEG + CaC12 2% PEG + CaC12 denatured DNA 2% PEG + CaC12 hydrolyzed DNA
Percent from total DNA Bound
DNase resistent
0.9 3.5 6.1 6.3 6.8 6.6 19.8
0.05 0.11 0.13 0.19 0.39 0.34 0.62
14.0
0.09
0.1
0.00
The experimental protocol described in the legend of Table 1 was used. Native DNA was used in concentration of 5 ~g/ml. DNA solutions to be denatured were heated,at 90 °C for 5 min and immersed in ice. Alternatively, hydrolyzed DNA was prepared by digestion of labeled native DNAwith DNase (100 gg/ml) for 30 rain at 37°C and heating for 15 min at 90°C. PEGis polyethylenglycol 4,000 (Sigma) and CaC12 was used in concentrations of 10 mM
Table 2 shows the kinetics of the cell-DNA reactions using ceils suspended in 2% sorbitol and native DNA at concentrations of 5 /~g/ml. The DNA-binding reaction is linear with time over the first 5 rain after which time the binding of DNA is essentially completed. Contrary to the situation with competent procaryotic cells (Ranhand 1980) the incorporation of DNA to DNase resistant state in the fragile yeast cells occured for an additional 60 min. Essentially the same results were obtained when polyethylen glycol was used instead of sorbitol. The presence of CaC12 did not change the kinetics of the reactions, however strongly increased the values obtained for both, the binding of DNA and the uptake of DNA to DNase resistent state. The uptake of DNA in the fragile yeast cells seems to be specific for double stranded molecules since heat denatured DNA binds b u t enters purely into the cells (Table 2). Deoxyribonucleotides neither bind, nor were incorporated into the fragile yeast cells suggesting that the measured DNA uptake is not due to labeled nucleotides present in the DNA preparations used. These results and others not presented here (for instance only cells of middle exponentials phase of growth could incorporate DNA) permit some analogy to be made between competent procaryotic cells and our fragile yeast mutants. However, it should be pointed out that the competence of procaryots appears as physiological state in there cellgrowth while the fragile yeast cells became premeable to native DNA only in a crytical concentration of the
P. V. Venkov and V. P. Ivanov: Uptake of DNA by Fragile Mutants of S. cerevisiae osmotic stabilizer. We are incline to think that the fragile yeast mutants might be tested as hosts in transformation experiments of whole yeast cells with purified DNA or cellular organelles. In support o f such opinion are the results o f preliminary experiments showing that circular DNA (plasmid or mitochondrial) are incorporated into the fragile yeast cells at amounts comparable to that o f linear native DNA.
Ref
nces
Goldring ES, Gossman LI, Krupnick D, Cryer DR, Marmur J (1970) J Mol Bio152:323-335 Gunge N, Sakaguchi K (1979) Mol Gen Genet 170:243-247 Hartwell LH (1967) J Bacteriol 93:1662-1670 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933
155
Hubavenkova E, Markov K (1975) CR Acad Bulg Sci 28:15371540 Mateeva Z, Petrov P, Venkov P, Hadjiolov A (1976) J Microsc giol Cellul 26:73-75 Ranhand JM (1980) J Bacteriol 142:568-580 Reuter G, MS.rkischU, Hering V, Venkov P (1978) Z Allg Mikrobiol 19:414-418 Venkov PV, Hadjiolov AA, Battaner E, Schlessinger D (1974) Biochem Biophys Res Commun 56:599-604 Venkov P, Milchev G, Hadjiolov A (1975) Antimicrob Agents Chemother 8:627-632 Venkov PV, Stateva LI, Hadjiolov AA (1977) Biochim Biophys Acta 474:245-253 Waltschewa LW, Venkov PV, Stoyanova BB, Hadjiolov AA (1976) Arch Biochem Biophys 176:630-637 (1976)
Communicated by F. Kaudewitz Received January 25, 1982
CurrentGenetics (1982) 5:153-155
© Springer-Verlag 1982
Short Communication
Uptake of DNA by Fragile Mutants of Saccharomyces cerevisiae Pencho V. Venkov and Vesselin P. Ivanov Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Summary. When Saccharomyces cerevisiae SY15 rho ° mutant cells grown in media stabilized with 10% sorbitol were suspended in 2% sorbitol solutions, 60-70% of the population did not lyse and became permeable to native high molecular weight DNA. Maximal incorporation of DNA to DNase resistant state was measured after 60 min of incubation in presence of 5 ~g/ml DNA and 10 mM CaC12. These results suggest that the fragile mutants might be tested as hosts for transformation of whole yeast cells. Key words: Yeast - Mutant cell-wall - Permeability exponentialy
Yeast cell wall is an absolute barrier for the uptake of DNA and transformation with DNA (Hinnen et al. 1978) or fusion with mitochondria (Gunge and Sakaguchi 1979) have been achieved on yeast spheroplasts only. The isolation of osmotic dependent yeast mutants defective in the structure of the cell wall has been reported (Venkov et al. 1974; Reuter et al. 1979). In media supplemented with 10% sorbitol as osmotic stabilizer the mutant cells grow exponentialy in fragile form: suspended in water the cells break open and the cellular content flows out through holes in the cell wall and membrane (Mateeva et al. 1976). The fragile yeast mutants display sensitivity to antibiotics like rifampicin (Venkov et al. 1975), actinomycin D (Waltschewa et al. 1976), toyocamycin (Venkov et al. 1977) known to be without effect on wild type yeast strains, which suggests an increased permeability of the mutant cells. Here, we report results showing that under appropriate experimental conditions
Offprint requests to: P.V. Venkov
the fragile yeast cells became also permeable to high molecular weight DNA. One of the fragile mutants reported is Saccharomyces cerevisiae SY15 obtained from strain A364 (Venkov et al. 1974). A rho ° derivative of SY15 has been isolated by treatment with ethidium bromide (Golgring et al. 1970) and the complete absence of mitochondrial DNA was verified by centrifugation in caesium cloride density gradients. When exponentialy growing SY15 rho ° cells were suspended in media with lower concentrations of the osmotic stabilizer (1% to 5% sorbitol) part of the population did not lyse and without begining a new division cycle could survive for at least 2 h. I t was found that under such conditions the fragile cells became permeable to DNA isolated from different cells, including yeast. Because of difficulties to obtain highly labeled yeast DNA, in the following experiments DNA purified from B. subtilis cells labeled in vivo with either 32p. phosphate or 3H-thymidine was used. The highest DNA incorporation was measured after suspending the SY15 rho ° cells in 2% sorbitol solutions, which allows survival of 60-70% of the population. The concentration of 2% sorbitol is crytical for SY15 rho ° strain. The other fragile yeast mutants behave similarly to SY15 rho °, however due to the different extend of there fragility a specific concentration of the osmotic stabilizer for each mutant was found. No DNA incorporation was established in SY15 rho ° cells in sorbitol solutions of 6% or more, or in wild type yeast strains suspended in water or in sorbitol solutions with different concentrations. Therefore, the observed DNA incorporation is a pattern typical for the fragile yeast cells suspended in concentrations of the osmotic stabilizer just enough to prevent the lysis but not sufficient to support the cell-growth. The results presented on Table 1 show that at low concentrations the binding and the uptake of DNA in SY15 rho ° cells is proportional to the amount of DNA
o172-8o83/82/ooo5/o153/$Ol.OO
154
P.V. Venkov and V. P. Ivanov: Uptake of DNA by Fragile Mutants of S. eerevisiae
Table 1. Influence of DNA concentration on the reactions between SY15 rho° cells and DNA DNA conch ~g/ml
Incubation
Incubation
min
mixture
Interaction of DNA with 107 cells Bound cpm
0.5 1.0 2.0 3.0 4.0 5.0 7.0 9.0
Table 2. Kinetics of DNA-yeast cell interactions
1,050 4,200 18,600 37,800 60,000 94,500 121,800 145,800
DNase resistent % total DNA
cpm
0.7 1.4 3.1 4.2 6.0 6.3 5.8 5.4
60 270 960 2,160 3,600 5,500 6,090 6,750
%total DNA 0.04 0.09 0.16 0.24 0.30 0.37 0.29 0.25
Saccharomyces cerevisiae SY15 rho° was grown at 30 °C in YM5 medium (Hartweel 1967) supplemented with 10% sorbitol. At middle exponential phase (about 107 cells/ml) the culture was chilled on ice, centrifuged and the cells suspended in 2/3 the culture volume of 2% sorbitol solution and incubated at 30 °C for 20 min with gentle shaking. This will give a survival of 107 colony-forming units per 1 ml cell-suspention. 3H-labeled DNA (300,000 cpm/1 ~g and average molecular weight of 50 kb) purified from a B. subtilis t h y - strain was added to appropriate volumes of the cell-suspention and incubated at 30 °C. Samples were taken in duplicate at 0 min and at 60 rain, For determination of bound DNA the cells from 1 ml suspention were washed with 50 ml 10% sorbitol solution on glass fiber filters (Reeve angels, Clifton, New Jersey) and the radioactivity was measured. For determination of DNase resistent incorporation the cells from 2 ml samples were washed as described the cells suspended in 2 mlDNase buffer (pancreatic DNase 20 #g/ml; MgCt2 10 raM; sorbitol 10%) and after incubation for 30 min at 37 °C samples of 1 ml were washed with 50 ml 10% sorbltol an glass fiber filters and the radioactivity measured. The cpm in the 0 min samples were subtracted from the radioactivity in the 60 rain samples.
added. The maximal incorporation measured as percent from total DNA was found at DNA concentrations of 4 - 5 #g/ml, while somewhat lower values were established at higher DNA concentrations. Essentially the same resuits have been obtained in experiments where DNA uptake was measured in cells suspended in 2N sorbitol solutions containing 1 /ag/ml highly labeled DNA and increasing amounts of cold DNA. Such effect of inhibition at enhanced DNA concentration is typical for transformation of competent procaryotic cells (Hubavenkova and Markov 1975) and was explained with the existence of limited numb er of spots through which the DNA could enter the recipient cell. A similar explanation might be given for the fragile yeast ceils, since ultrastructural studies (Mateeva et al. 1976)have shown the appearance of only a few local defects in there cell wall and membranes after lowering the osmotic presure in the medium.
1 3 5 20 60 120 60 60 60
2% sorbitol 2% sorbitol 2% sorbitol 2% sorbitol 2% sorbitol 2% sorbitol 2% PEG + CaC12 2% PEG + CaC12 denatured DNA 2% PEG + CaC12 hydrolyzed DNA
Percent from total DNA Bound
DNase resistent
0.9 3.5 6.1 6.3 6.8 6.6 19.8
0.05 0.11 0.13 0.19 0.39 0.34 0.62
14.0
0.09
0.1
0.00
The experimental protocol described in the legend of Table 1 was used. Native DNA was used in concentration of 5 ~g/ml. DNA solutions to be denatured were heated,at 90 °C for 5 min and immersed in ice. Alternatively, hydrolyzed DNA was prepared by digestion of labeled native DNAwith DNase (100 gg/ml) for 30 rain at 37°C and heating for 15 min at 90°C. PEGis polyethylenglycol 4,000 (Sigma) and CaC12 was used in concentrations of 10 mM
Table 2 shows the kinetics of the cell-DNA reactions using ceils suspended in 2% sorbitol and native DNA at concentrations of 5 /~g/ml. The DNA-binding reaction is linear with time over the first 5 rain after which time the binding of DNA is essentially completed. Contrary to the situation with competent procaryotic cells (Ranhand 1980) the incorporation of DNA to DNase resistant state in the fragile yeast cells occured for an additional 60 min. Essentially the same results were obtained when polyethylen glycol was used instead of sorbitol. The presence of CaC12 did not change the kinetics of the reactions, however strongly increased the values obtained for both, the binding of DNA and the uptake of DNA to DNase resistent state. The uptake of DNA in the fragile yeast cells seems to be specific for double stranded molecules since heat denatured DNA binds b u t enters purely into the cells (Table 2). Deoxyribonucleotides neither bind, nor were incorporated into the fragile yeast cells suggesting that the measured DNA uptake is not due to labeled nucleotides present in the DNA preparations used. These results and others not presented here (for instance only cells of middle exponentials phase of growth could incorporate DNA) permit some analogy to be made between competent procaryotic cells and our fragile yeast mutants. However, it should be pointed out that the competence of procaryots appears as physiological state in there cellgrowth while the fragile yeast cells became premeable to native DNA only in a crytical concentration of the
P. V. Venkov and V. P. Ivanov: Uptake of DNA by Fragile Mutants of S. cerevisiae osmotic stabilizer. We are incline to think that the fragile yeast mutants might be tested as hosts in transformation experiments of whole yeast cells with purified DNA or cellular organelles. In support o f such opinion are the results o f preliminary experiments showing that circular DNA (plasmid or mitochondrial) are incorporated into the fragile yeast cells at amounts comparable to that o f linear native DNA.
Ref
nces
Goldring ES, Gossman LI, Krupnick D, Cryer DR, Marmur J (1970) J Mol Bio152:323-335 Gunge N, Sakaguchi K (1979) Mol Gen Genet 170:243-247 Hartwell LH (1967) J Bacteriol 93:1662-1670 Hinnen A, Hicks JB, Fink GR (1978) Proc Natl Acad Sci USA 75:1929-1933
155
Hubavenkova E, Markov K (1975) CR Acad Bulg Sci 28:15371540 Mateeva Z, Petrov P, Venkov P, Hadjiolov A (1976) J Microsc giol Cellul 26:73-75 Ranhand JM (1980) J Bacteriol 142:568-580 Reuter G, MS.rkischU, Hering V, Venkov P (1978) Z Allg Mikrobiol 19:414-418 Venkov PV, Hadjiolov AA, Battaner E, Schlessinger D (1974) Biochem Biophys Res Commun 56:599-604 Venkov P, Milchev G, Hadjiolov A (1975) Antimicrob Agents Chemother 8:627-632 Venkov PV, Stateva LI, Hadjiolov AA (1977) Biochim Biophys Acta 474:245-253 Waltschewa LW, Venkov PV, Stoyanova BB, Hadjiolov AA (1976) Arch Biochem Biophys 176:630-637 (1976)
Communicated by F. Kaudewitz Received January 25, 1982
