YEAST
VOL. 6: 205-2
12 (1990)
Cell Fusion of Saccharomyces cerevisiae Fragile Mutants D. H. PHILIPOVA A N D P. V. VENKOV Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Received 22 September 1989; revised 4 December 1989
Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane. The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium. These mutants display a general increase in the permeability of the plasma membrane. We show here that fragile yeast cells of the same mating type can fuse without protoplast formation. The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts. Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products. The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes. KEY WORDS -Cell
fusion; Saccharomyces cerevisiae; fragile mutants
INTRODUCTION The phenotype of the fragile mutants of Saccharomyces cerevisiae (Venkov et al., 1974) is characterized by cell lysis in hypotonic media and increased permeability to substances unable to penetrate wildtype yeast. It has been shown that the fragile yeast cells are permeable to adenosine (Venkov et al., 1977), actinomycin D (Waltschewa et al., 1976), proteins (Venkov and Scheit, 1984), and high molecular weight DNA (Venkov and Ivanov, 1982; Philipova, 1985). Thus, their increased permeability is of a general nature which suggests a significantly altered plasma membrane structure in the fragile yeast mutants. Changes in membrane permeability or transport processes are prerequisites for membrane fusion (Lucy, 1986; Dimitrovand Jain, 1984). In thecaseof yeast, these changes may be imposed on protoplasts either by treatment with polyethylene glycol (PEG) or by electric forces: successful fusion of yeast has been reported for protoplasts only. The yeast cell wall is considered as a barrier for fusion because it prevents contact between the membrane bilayers and the breakdown of the membrane structure. In this communication we report that fragile cells of the same mating type can fuse without the need to convert them to protoplasts. The most likely explanation of this finding is the existence of local defects on the surface of the mutant cells where the cell wall 0749-503X190/0302054)8 $05.00 0 1990 by John Wiley & Sons Ltd
is thinner (or even missing), thus allowing a direct contact of two cells by means o f their structurally altered plasma membranes. MATERIALS AND METHODS Strains and media The S. cerevisiae strains used are listed in Table 1. VYl160 is the originally isolated fragile mutant (Venkov et al., 1974). The fragile cells are osmotic dependent and need a stabilizer (e.g. 10% sorbitol) for growth. Removal of the osmotic stabilizer leads to immediate cell lysis. Genetic analysis has revealed the presence of a single mutation, denotes srbl, which confers the fragile phenotype on W 1 1 6 0 cells (Kozhina e f al., 1979). To obtain other fragile clones, VY 1 160 was crossed to non-fragile ancestral strains which carried auxotrophic markers with very low reversion rates. Strains containing the srbl mutation in combination with various auxotrophic markers were isolated from the meiotic progeny of the diploids. Complementation analysis using appropriate tester strains was used to confirm the designation of each auxotrophic mutation. The leu2, his3 and ura3 markers in the strains are double mutations and reverted at a frequency less than I Spontaneous revertants to non-fragile phenotype (SRBI)were obtained by plating the srbl strains on media not supplemented with osmotic stabilizer. VY4/pRN15 is a transformant from the
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D. H. PHILIPOVA AND P. V. VENKOV
Table 1. Saccharornyces cerevisiae strains used Strains
Genotype
Source
Non-fragile AH2 15 FLlOO
MATa leu2-3 leu2-112 his3-11 his3-15 MATa ura3-160 ura3-288
A. Hinnen F. Lacroute
Fragile VY1160 VY4 VY4/pRN 15 VY7 VY8 VY25 VY26 VY26rho0 VY41
MATa leu2 trpl tyr- thr- ade- srbl MATa trpl leu2-3 leu2-112 ura3-160 ura3-288 srbl MATa trpl leu2-3 leu2-112 ura3-160 ura3-288 srbllURA3 M A Tu leu2-3 leu2- 1 12 ura3- 160 ura3-288 srbl M A Ta leu2-3 leu2- 1 12 srb 1 MATa leu2-3 leu2- 112 his3- 11 his3- 15 srbl MATa ura3-160 ura3-288 srbl MATa ura3-160 ura3-288 srbl [rho'] MATa ura3-I60 ura3-288 srbl
Venkov et al. (1974) Philipova (1985) This study Philipova (1 985) This study This study This study This study This study
fragile strain VY4 using the yeast shuttle episomal plasmid pRNI5 (Nebreda et a[., 1986) which consists of 2 pm DNA origin of replication, the URA3 gene as selection marker and pBR322 sequences. VY26[rho0]is a derivative of VY26 isolated by treatment with 10 pg/ml ethidium bromide (Slonimski et al., 1968). Its [rhoo]petite phenotype was evidenced by the absence of growth on YPG or YPDG media (Sherman et al., 1986) and by the lack of mitochondrial DNA in CsCl buoyant density gradients of DNA purified from VY26[rho0] cells. The rich YPD, minimal SD, presporulation and sporulation media were prepared according to Sherman et al. (1986). In the case of the fragile strains, the media were supplemented with sorbitol to a final concentration of 10% wjv. The selection of fusion hybrids was performed on SD medium containing 10% sorbitol for VY41 x VY8, VY26 x AH215 and on SD supplemented with leucine (30 pg/ml) and sorbitol for VY7 x VY25, VY7 x VYI 160 and VY4 x AH215. The medium SG (2% glycerol) with 10% sorbitol was used to select fusion hybrids from VY4/pRN15 x VY26[rhoD]. Protoplast formation, fusion and regeneration
Protoplast formation using Zymolyase IOOT (Kirin Brewery Co.) and regeneration of fusion hybrids were as described by Skala et al. (1988). PEG-mediated fusion was according to Maraz et al. (1978). The electrofusion was performed at 20°C in helical chambers (Schnettler and Zimmermann, 1985) with the BEST-1.0 equipment developed in
the Central Laboratory of Biophysics, Bulgarian Academy of Sciences. The cells (2 x 10') in a total volume of 120 pi were dielectrophoretically collected for 2 min by AC fields of frequency 2 MHz and intensity 250V/cm. Then three DC pulses of 10 ps duration and an intensity of 10 kV/cm were applied. The period between the pulses was 0.5 s. After electrofusion the cells were grown on selective media and the number of hybrids per chamber (i.e. per 1 x lo8 cells from each partner in the fusion experiment) was counted. Protoplast formation and regeneration of hybrids were omitted when PEG-mediated fusion or electrofusion was performed with cells instead of yeast protoplasts. For spontaneous fusion (without PEG or electric treatment) lo8 cells from a fragile strain, or the equivalent amount of regenerating protoplasts from a non-fragile strain, were washed and suspended in 50 p1 TES buffer (10 mM-Tris-HC1, 1 mM-EDTA, 10% w/v sorbitol, pH 8.0), with the same amount and volume of the second strain and incubated at 30°C for 120 min without shaking. The cells were centrifuged at 5000 rpm for 15 min and plated on selective media. The fusion hybrids were counted after 7-8 days of cultivation at 30°C. Staining of fusion products
Immediately after PEG treatment or electrofusion, aliquots were taken and the nuclei of the hybrids stained by incubation with Hoechst dye 33258 (5 pg/ml, Fluka) for 20 min (Williamson and
207
CELL FUSION OF SACCHAROMYCES CEREVISIAE FRAGILE MUTANTS
Table 2. Fusion of fragile yeast mutants Number of hybrids from 1O8 cells Strains
Spontaneous fusion
( A ) Cell x cell* V Y 7 (srbl) x VY25(srbl) V Y 7 ( S R B l )x VY25(SRBl) VY7(srbl)x V Y 1160(srbl) VY7(SRBI) x V Y I I60(SRB1) V Y 4 l ( s r b l )x VY8(srbl) V Y 4 1 ( S R B l )x VY8(SRBI) VY26(srbl) x AH21 5 (non-fragile)
PEG-mediated fusion
Electrofusion
2 0 2 0 3 0 0
30 0 24 0 20 0 0
90 0 78 0 85 0 0
( B ) Cell x protoplast VY26(srbl)x AH215 (non-fragile) VY26(SRBl) x AH215 (non-fragile) VY4(srbl)x AH215 (non-fragile) V Y 4 ( S R B l )x AH21 5 (non-fragile)
10 0 22 0
150
0 425 0
450 0 1400 0
(C)Protoplast x protoplast VY26(srbl)x AH215 (non-fragile) VY4(srbl)x AH215 (non-fragile)
54 58
4670 4100
10 260
ND
*The fragile phenotype of the strains is indicated by (srbl) and the spontaneous reversion to a non-fragile phenotype by (SRBl). Strains with the wild-type SRBl gene are marked by (non-fragile). ND, not determined.
Fennel], 1975). Observation was under a Leitz epifluorescence microscope using filter A. Determination of D N A content The DNA content of the fusion partners and fusion products was determined according to Ferenczy and Maraz (1977). Induction ofhaploidization with benomyl The source of benomyl used was Benlate (Du Pont Co.), a commercial fungicide containing 50% benomyl as the only active principle (Wood, 1982). To find out the optimal concentration of benomyl for induction of haploidization, each fusion hybrid was cultivated in YPD medium at 30°C in the presence of different concentrations of Benlate. The optimum was defined as the concentration of benomy1 which gave the maximal number of auxotrophs and had the minimal inhibitory effect on cell growth. Concentrations of Benlate between 150 and 220pg/ml were determined as optimal for the different hybrids. The genetic characteristics of the
auxotrophs obtained from benomyl-treated fusion hybrids were determined by replica plating on appropriate minimal media. Plasmid isolation Plasmid DNA from fusion hybrids and partner strains was isolated according to Perez-Ortin and Estruch (1988). RESULTS The results obtained by PEG mediated fusion and electrofusion are summarized in Table 2. Combinations of cells from different fragile strains of the same mating type (Table 2A) led to the production of hybrids. The number of hybrids obtained by electrofusion was higher than that from PEGmediated fusion. Single hybrids were produced even when intact fragile cells were mixed without PEG or electric treatments. Although the frequency of this spontaneous fusion is very low, hybrids were reproducibly obtained in all 10 repetitions of the experiment. However, hybrids were not formed spontaneously or induced by PEG or electric forces
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D. H. PHILIPOVA AND P. V. VENKOV
Table 3. Fusion of fragile yeast strains with chromosomal and cytoplasmic markers Number of hybrids from lo8cells Strains
Spontaneous fusion
PEG-mediated fusion
( A j Cell x cell VY26rho' x VY4/pRN 15
5
45
( B ) Cell x protoplust VY26rho' x VY4/pRNlS VY4/pRNI5 x VY26rho'
110 90
900 620
( C ) Protoplast x protoplust VY26rho' x VY4/pRNlS
180
5100
when revertants to non-fragile phenotype were used. The controls for spontaneous revertants of the auxotrophic markers were negative in all fusion experiments (data not shown), which is in agreement with the very low reversion rates (less than lop9) typical for the double mutations used in the selection of the fusion hybrids. These results suggested that the colonies appearing in the fusion experiments are not rare revertants. They also indicated that the observed cell x cell fusion is a characteristic related to the fragile (srbl) mutants only and is not due to some other factor in the genetic background. Since this is the first observation of fusion of intact yeast cells, we performed some experiments to study the mechanism of cell fusion. Negative results were obtained in experiments using a combination of one fragile and one non-fragile strain (VY26 x AH215, Table 2A) suggesting that successful cell fusion occurs only if both partners contain the srbl mutation. Removal of the cell wall of the non-fragile partner (AH215 strain) led to formation of fusion hybrids between the fragile cells and the protoplasts (Table 2B). The use of non-fragile revertants VY26(SRB1) and VY4(SRBI) gave negative results, demonstrating again the crucial role of the srbl mutation for cell fusion. Although increased when compared to the cell x cell fusion, the frequency of the cell x protoplast fusion was significantly lower than that observed for the protoplast x protoplast fusion (Table 2C). This suggests the existence of a limited number of places on the surface of the fragile cells where fusion events could take place. It should be noted that frequencies of lop6have been obtained for the illegitimate mating reported to occur in some
yeast strains (Inge-Vechtomov, 1967) or for the reversion of single mutations. Therefore, the existence of cell fusion between fragile yeast cells needs further proof. First, cell fusion was observed between fragile strains which in addition to chromosomal mutations contain cytoplasmically inherited markers (Table 3). VY26[rhoq is a double mutant in the URA3 gene and lacks the mitochondria1 DNA which confers a non-revertible 'petite' phenotype (Slonimski et al., 1968).VY4/pRNl5 is a trpl leu2-3 leu2-112 ura3-160 ura3-288 mutant transformed with the episomal plasmid pRN15 to Ura' phenotype. The selection of fusion products between the two fragile partners was carried out on minimal medium with glycerol as the only carbon source and sorbitol as osmotic stabilizer. Only the fusion hybrids containing both the chromosomal and the cytoplasmic genomes of the two partners could grow on this selection medium. The results in Table 3 show that fusion does occur at frequencies similar to those reported for other pairs of fragile strains (see Table 2). Although the fusion hybrids were selected on glycerol-containing medium, their rho+ phenotype was confirmed by several passages in ethanol-containing media. The presence of the p R N l5 plasmid in the fusion hybrids determining their Uraf phenotype was verified by the appearance of uracil-auxotrophic clones in mitotic segregation experiments and by isolation of plasmid DNA directly from part of the fusion products (Figure 1). Second, the fusion of intact fragile cells was observed microscopically. The micrographs (Figure
CELL FUSION OF SACCHAROMYCES CEREVISIAE FRAGILE MUTANTS
a b c d e f g h
Figure 1. Agarose gel electrophoresis of plasmid DNA. Plasmid DNA was isolated and analysed from Escherichiu coli HBlOl transformed with pRNI5 (lanes a and h), Succhuromyces cwevisiae strains VY26rho" (lane b) and VY4/pRN15 (lane c ) o r fusion hybrids VY26rho" x VY4/pPN15 (lanes d-g).
2) demonstrate the existence of multi-nuclear cells immediately after the PEG treatment or electrofusion. Such heterokarions have been shown to represent intermediate products in the fusion process (Svoboda, 1978). For the experiments reported here, the number of multi-nuclear cells was counted in approximately 10' cells. In the case of cellx cell fusion, only single binuclear products were observed (Figure 2A), suggesting that cell fusion is a rare event which takes place between two cells only. In cell x protoplast and protoplast x protoplast samples, the number of multi-nuclear cells increased up to lo-' and respectively. In addition, the trinuclear and tetranuclear cells represented 30% of the multi-nuclear cells, which suggests that fusion occurs more readily and could involve more than two cells (Figure 2B and C). The data of DNA estimation (Table 4) show that the fusion products obtained by cell x cell fusion (VY7 x VY25; VY7 x VY 1160) are diploids since the DNA content of the isolates is about two times that of the haploid partner cells. However, hybrids obtained by cell x protoplast fusion (VY4 x AH2 15) had higher DNA content, indicating that three cells had taken part in the fusion process. Third. the fusion products were characterized genetically. Contrary to the partners used in the experiments. the hybrids form large, stable and fastgrowing colonies. They are of the same mating type as the partners and do not sporulate. For this reason it was impossible to analyse the hybrids by conventional genetic methods and they were characterized
209
by the use of benomyl known to induce mitotic chromosomal loss and partial haploidization in S. cerevisiae (Wood, 1982; Maraz et al., 1978). Auxotrophic colonies were isolated from the progeny of benomyl-treated fusion products and the auxotrophies and the mating type was determined. Table 5 summarizes the data obtained with representatives of the cell x cell and cell x protoplast fusion experiments. Following benomyl treatment all fusion hybrids produced auxotrophs which were of either parental or recombinant type. Furthermore, all auxotrophs had the same mating type, which was the same as that of the two partners used in the experiment. These results supply genetic evidence for the fusion nature of the hybrids and excludes the possibility that the fusion products might be rare revertants of the partners.
DISCUSSION The fusion of yeast protoplasts to generate hybrids is successful when (i) the electric charges of the cell surface are neutralized and the protoplasts are brought into very close contact and (ii) breakdown of plasma membrane occurs at the contact sites (Lucy, 1986). These two requirements seem to be fulfilled with intact cells of the fragile mutants of S. cerevisiae. Studies on membrane transport (Kotyk et al., 1988) supply evidence for a very low electric charge at the cell surface and a lack of the protonmotive force across the plasma membrane of the fragile mutant cells. This would permit a very close contact between the fragile cells. Further, electron microscopic observations have shown the existence of local defects in the cell wall of the fragile cells (Mateeva et al., 1976). Recently, these defects were visualized by scanning electron microscopy as gaps in the cell wall which are limited in number to one to three per fragile cell (Schade and Venkov, in preparation). This would allow two fragile cells to make rare contacts with each other by their plasma membranes. The exact defect of the plasma membrane in the fragile mutants is unknown. However, a number of studies have demonstrated an increased permeability of the mutant cells. Changes in the transport processes or plasma membrane permeability are supposed to facilitate membrane breakdown and fusion (Lucy, 1986; Dimitrov and Jain, 1984). As a consequence of these alterations of the cell wall and membrane structures, one would expect that intact cells of the fragile mutants could fuse without removal of the cell wall by protoplasting. The data presented in this paper show that this is actually the
210
D. H. PHILIPOVA AND P. V. VENKOV
A
B
C
Figure 2. Multi-nuclear fusion products. (A) Binuclear hybrid from a cell x cell VY4l(srbl) x VY8(srbl), spontaneous fusion. (B) Trinuclear hybrid from a cell x protoplast VY4(srbl) x AH215, PEG-mediated fusion. (C) Tetranuclear hybrid from a protoplast x protoplast VY26(srbl) x AH21 5 , electrofusion.
Table 4.
DNA content
Strains
Haploid AH215 VY4 VY7 VY25
2 5 9 + 2.5 24.6 f2.8 22.8 f 1.9 23.5 f2.6
Fusion products VY7 x VY 1160 VY7 x VY25 VY4 x AH215
40.1 rfr2.6 41.2 k4.1 48.8 k 3.2
*Values are & standard error. Standard errors are based on three independent DNA determinations.
case. Yeast cells of different fragile clones having equal mating type were found to fuse. Microscopic observations, DNA estimations and genetic analysis of the fusion products supply evidence for the fusion nature of the hybrids and exclude experimental artefacts such as reversion or illegitimate mating. Because of the very low frequency of cell x cell fusion, the differentiation
between fusion and illegitimate mating becomes a problem. In an attempt to solve this problem we isolated spontaneous non-fragile revertants from all fragile strains and repeated the fusion experiments. The negative results obtained with all pairs of revertants (Table 2A and B) are in favour of the fusion nature of the hybrids obtained from the corresponding fragile strains and make it highly unlikely that diploids that are homoallelic or heteroallelic at the mating type locus are produced by illegitimate mating. Further, the genetic analysis of the hybrids by induction of partial haploidization (Table 5) shows that all segregants have the same mating type, thus excluding the participation of heteroallelic illegitimate mating in the formation of the hybrids. The very low frequency found for the cell x cell fusion could be explained by the limited number of surface gaps per fragile cell and the low probability with which two fragile cells meet each other with the local defects of their cell walls facing. In agreement with this interpretation, the frequency of fusion is increased significantly when the cells of one partner are converted to protoplasts (Table 2B). In this case every contact between a surface gap of the fragile cell and any part of the protoplast’s surface could create a possibility for formation of a fusion hybrid.
21 1
CELL FUSION OF SACCHAROMYCES CEREVISIAE FRAGILE MUTANTS
Table 5. Genetic analysis of fusion hybrids
Hybrid ( A ) Cell x cell VY7 x VY25 (Spontaneous fusion) ( B ) Cell x protoplast VY26 x AH2 15 (PEG-mediated fusion) VY4 x AH215 (Electrofusion)
Per cent auxotrophs after benomyl treatment
Auxotrophs
Number
Parental type
Recombinants
a
a
4
24
9
15
0
24
3
18
7
11
18
0
4
29
11
18
29
0
Mating type
ACKNOWLEDGEMENTS T h a n k s are due to D. Dimitrov (Central Laboratory of Biophysics, Bulgarian Academy o f Sciences) f o r providing the equipment f o r electrofusion and to I. Tsoneva and P. D o i n o v f o r help i n electrofusion experiments.
REFERENCES Dimitrov, D. S. and Jain, R. K. (1984). Membrane stability. Biochem. Biophys. Acta 779,437-468. Ferenczy, L. and Maraz, A. (1977). Transfer of mitochondria by protoplast fusion in S . cerevisiae. Nature 268(5620), 524525. Inge-Vechtomov, S. G. (1967). Illegitimate copulation and polyploidy in yeast S. cerevisiae. Genetika 11, IO(L109. Kotyk, A , , Venkov, P. and Dvorakova, M. (1988). Membrane transport in an osmotically fragile mutant of S . cerevisiae. Yeast 4,241-247. Kozhina. T., Stateva, L. and Venkov, P. (1979). Genetic analysis of an osmotic sensitive S. cerevisiae mutant VY 1160. Molec. Gen. Genet. 170,351-354. Lucy, L. A. (1986). Salient features of artificially induced cell fusion. Biochem. Society Transactions 14,25&25 1. Maraz, A., Kiss, M. and Ferenczy, L. (1978). Protoplast fusion in S. cerevisiae strains of identical and opposite mating types. FEMS Microbiol. Letters 3,3 19-322. Mateeva, Z., Petrov, P., Venkov, P. and Madjiolov, A. A. (1976). Electron microscopic study of the lysis of an osmotic sensitive yeast mutant. J . Microsc. Biol. Cellulaire, 26,73-74. Nebreda, A. R., Villa, T. G., Villanueva, J. R. and Rey, del F. (1986). Cloning of genes related to exo-pglucanase production in S. cerevisiae: characterization
of an exo-p-glucanase structural gene. Gene 47, 245-259. Perez-Ortin, J. E. and Estruch, F. (1988). A rapid method for the screening of plasmids in transformed yeast strains. Curr. Microbiol. 17, 19-22. Philipova, D. H. (1985). PhD Thesis, Institute of Molecular Biology, Bulgarian Academy of Sciences. Schnettler, R. and Zimmermann, U. (1985). Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids. FEMS Microbiol. Letters 27,195-198. Sherman, R., Fink, G . ,and Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory, New York. Skala, J., Luty, J. and Kotylak, Z. (1988). Interspecific protoplast fusion between yeasts S. cerevisiae and S . fermentati. Curr. Genet. 13, 101-104. Slonimski, P. P., Perrodin, G. and Croft, J. K. (1968). Ethidium bromide-induced mutation of yeast mitochondria: Complete transformation of cells into respiratory deficient nonchromosomal “petites”. Biochem. Biophys. Res. Commun. 30,232-239. Svoboda, A. (1978). Fusion of yeast protoplasts induced by polyethylene glycol. J. Gen. Microbiol. 109,169-1 75. Venkov, P. V., Hadjiolov, A. A,, Battaner, E. and Schlessinger, D. (1 974). Saccharomyces cerevisiae sorbitol dependent fragile mutants. Biochem. Biophys. Res. Commun. 56,559-604. Venkov, P. V., Stateva, L. I. and Hadjiolov, A. A. (1977). Toyocamycin inhibition of ribosomal ribonucleic acid processing in an osmotic sensitive adenosine utilizing S . cerevisiae mutant. Biochem. Biophys. Acta 474, 245-253. Venkov, P. V. and Ivanov, V. P. (1982). Uptake of D N A by fragile mutants of S . cerevisiae. Curr. Genet. 5, 153-1 55.
212 Venkov, P. and Scheit, K.-H. (1984). Effect of seminal plasmin on rRNA synthesis in S. cerevisiae. FEBS Letters 172,21-24. Waltschewa, L. W., Venkov, P. V., Stoyanova, B. B. and Hadjiolov, A. A. (1976). Degradation of ribosomal precursor and polyadenylic acid containing ribonucleic acids in S. cerevisiae caused by actinomycin D.Arch. Biochem. Biophys. 176,630-637.
D. H. PHILIPOVA AND P. V. VENKOV
Williamson, D. H. and Fennell, D. J. (1975). The use of fluorescent D N A binding agents for detecting and separating yeast mitochondria1 DNA. Methods Cell Biol. 17,335-351. Wood, J. S. (1982). Genetic effects of methyl benzimidazole-2-yl-carbamate on S. cerevisiae. Mol. Cell Biol. 2, 1064-1079.
VOL. 6: 205-2
12 (1990)
Cell Fusion of Saccharomyces cerevisiae Fragile Mutants D. H. PHILIPOVA A N D P. V. VENKOV Institute of Molecular Biology, Bulgarian Academy of Sciences, 1113 Sofia, Bulgaria
Received 22 September 1989; revised 4 December 1989
Fragile mutants of Saccharomyces cerevisiae are defective in the structure of the cell wall and plasma membrane. The mutant cells lyse in hypotonic solutions but grow exponentially when osmotic stabilizer is included in the medium. These mutants display a general increase in the permeability of the plasma membrane. We show here that fragile yeast cells of the same mating type can fuse without protoplast formation. The frequency of cell x cell fusion is lower than that observed for protoplast x protoplast fusion and can be significantly increased if the cells of one partner are converted to protoplasts. Microscopic observations and genetic analysis demonstrate that the hybrids obtained are fusion products. The fusion between fragile cells is explained in terms of the existence of local defects on their surface where the cell wall is thinner (or even missing), thus allowing a direct contact of cells by means of their plasma membranes. KEY WORDS -Cell
fusion; Saccharomyces cerevisiae; fragile mutants
INTRODUCTION The phenotype of the fragile mutants of Saccharomyces cerevisiae (Venkov et al., 1974) is characterized by cell lysis in hypotonic media and increased permeability to substances unable to penetrate wildtype yeast. It has been shown that the fragile yeast cells are permeable to adenosine (Venkov et al., 1977), actinomycin D (Waltschewa et al., 1976), proteins (Venkov and Scheit, 1984), and high molecular weight DNA (Venkov and Ivanov, 1982; Philipova, 1985). Thus, their increased permeability is of a general nature which suggests a significantly altered plasma membrane structure in the fragile yeast mutants. Changes in membrane permeability or transport processes are prerequisites for membrane fusion (Lucy, 1986; Dimitrovand Jain, 1984). In thecaseof yeast, these changes may be imposed on protoplasts either by treatment with polyethylene glycol (PEG) or by electric forces: successful fusion of yeast has been reported for protoplasts only. The yeast cell wall is considered as a barrier for fusion because it prevents contact between the membrane bilayers and the breakdown of the membrane structure. In this communication we report that fragile cells of the same mating type can fuse without the need to convert them to protoplasts. The most likely explanation of this finding is the existence of local defects on the surface of the mutant cells where the cell wall 0749-503X190/0302054)8 $05.00 0 1990 by John Wiley & Sons Ltd
is thinner (or even missing), thus allowing a direct contact of two cells by means o f their structurally altered plasma membranes. MATERIALS AND METHODS Strains and media The S. cerevisiae strains used are listed in Table 1. VYl160 is the originally isolated fragile mutant (Venkov et al., 1974). The fragile cells are osmotic dependent and need a stabilizer (e.g. 10% sorbitol) for growth. Removal of the osmotic stabilizer leads to immediate cell lysis. Genetic analysis has revealed the presence of a single mutation, denotes srbl, which confers the fragile phenotype on W 1 1 6 0 cells (Kozhina e f al., 1979). To obtain other fragile clones, VY 1 160 was crossed to non-fragile ancestral strains which carried auxotrophic markers with very low reversion rates. Strains containing the srbl mutation in combination with various auxotrophic markers were isolated from the meiotic progeny of the diploids. Complementation analysis using appropriate tester strains was used to confirm the designation of each auxotrophic mutation. The leu2, his3 and ura3 markers in the strains are double mutations and reverted at a frequency less than I Spontaneous revertants to non-fragile phenotype (SRBI)were obtained by plating the srbl strains on media not supplemented with osmotic stabilizer. VY4/pRN15 is a transformant from the
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D. H. PHILIPOVA AND P. V. VENKOV
Table 1. Saccharornyces cerevisiae strains used Strains
Genotype
Source
Non-fragile AH2 15 FLlOO
MATa leu2-3 leu2-112 his3-11 his3-15 MATa ura3-160 ura3-288
A. Hinnen F. Lacroute
Fragile VY1160 VY4 VY4/pRN 15 VY7 VY8 VY25 VY26 VY26rho0 VY41
MATa leu2 trpl tyr- thr- ade- srbl MATa trpl leu2-3 leu2-112 ura3-160 ura3-288 srbl MATa trpl leu2-3 leu2-112 ura3-160 ura3-288 srbllURA3 M A Tu leu2-3 leu2- 1 12 ura3- 160 ura3-288 srbl M A Ta leu2-3 leu2- 1 12 srb 1 MATa leu2-3 leu2- 112 his3- 11 his3- 15 srbl MATa ura3-160 ura3-288 srbl MATa ura3-160 ura3-288 srbl [rho'] MATa ura3-I60 ura3-288 srbl
Venkov et al. (1974) Philipova (1985) This study Philipova (1 985) This study This study This study This study This study
fragile strain VY4 using the yeast shuttle episomal plasmid pRNI5 (Nebreda et a[., 1986) which consists of 2 pm DNA origin of replication, the URA3 gene as selection marker and pBR322 sequences. VY26[rho0]is a derivative of VY26 isolated by treatment with 10 pg/ml ethidium bromide (Slonimski et al., 1968). Its [rhoo]petite phenotype was evidenced by the absence of growth on YPG or YPDG media (Sherman et al., 1986) and by the lack of mitochondrial DNA in CsCl buoyant density gradients of DNA purified from VY26[rho0] cells. The rich YPD, minimal SD, presporulation and sporulation media were prepared according to Sherman et al. (1986). In the case of the fragile strains, the media were supplemented with sorbitol to a final concentration of 10% wjv. The selection of fusion hybrids was performed on SD medium containing 10% sorbitol for VY41 x VY8, VY26 x AH215 and on SD supplemented with leucine (30 pg/ml) and sorbitol for VY7 x VY25, VY7 x VYI 160 and VY4 x AH215. The medium SG (2% glycerol) with 10% sorbitol was used to select fusion hybrids from VY4/pRN15 x VY26[rhoD]. Protoplast formation, fusion and regeneration
Protoplast formation using Zymolyase IOOT (Kirin Brewery Co.) and regeneration of fusion hybrids were as described by Skala et al. (1988). PEG-mediated fusion was according to Maraz et al. (1978). The electrofusion was performed at 20°C in helical chambers (Schnettler and Zimmermann, 1985) with the BEST-1.0 equipment developed in
the Central Laboratory of Biophysics, Bulgarian Academy of Sciences. The cells (2 x 10') in a total volume of 120 pi were dielectrophoretically collected for 2 min by AC fields of frequency 2 MHz and intensity 250V/cm. Then three DC pulses of 10 ps duration and an intensity of 10 kV/cm were applied. The period between the pulses was 0.5 s. After electrofusion the cells were grown on selective media and the number of hybrids per chamber (i.e. per 1 x lo8 cells from each partner in the fusion experiment) was counted. Protoplast formation and regeneration of hybrids were omitted when PEG-mediated fusion or electrofusion was performed with cells instead of yeast protoplasts. For spontaneous fusion (without PEG or electric treatment) lo8 cells from a fragile strain, or the equivalent amount of regenerating protoplasts from a non-fragile strain, were washed and suspended in 50 p1 TES buffer (10 mM-Tris-HC1, 1 mM-EDTA, 10% w/v sorbitol, pH 8.0), with the same amount and volume of the second strain and incubated at 30°C for 120 min without shaking. The cells were centrifuged at 5000 rpm for 15 min and plated on selective media. The fusion hybrids were counted after 7-8 days of cultivation at 30°C. Staining of fusion products
Immediately after PEG treatment or electrofusion, aliquots were taken and the nuclei of the hybrids stained by incubation with Hoechst dye 33258 (5 pg/ml, Fluka) for 20 min (Williamson and
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CELL FUSION OF SACCHAROMYCES CEREVISIAE FRAGILE MUTANTS
Table 2. Fusion of fragile yeast mutants Number of hybrids from 1O8 cells Strains
Spontaneous fusion
( A ) Cell x cell* V Y 7 (srbl) x VY25(srbl) V Y 7 ( S R B l )x VY25(SRBl) VY7(srbl)x V Y 1160(srbl) VY7(SRBI) x V Y I I60(SRB1) V Y 4 l ( s r b l )x VY8(srbl) V Y 4 1 ( S R B l )x VY8(SRBI) VY26(srbl) x AH21 5 (non-fragile)
PEG-mediated fusion
Electrofusion
2 0 2 0 3 0 0
30 0 24 0 20 0 0
90 0 78 0 85 0 0
( B ) Cell x protoplast VY26(srbl)x AH215 (non-fragile) VY26(SRBl) x AH215 (non-fragile) VY4(srbl)x AH215 (non-fragile) V Y 4 ( S R B l )x AH21 5 (non-fragile)
10 0 22 0
150
0 425 0
450 0 1400 0
(C)Protoplast x protoplast VY26(srbl)x AH215 (non-fragile) VY4(srbl)x AH215 (non-fragile)
54 58
4670 4100
10 260
ND
*The fragile phenotype of the strains is indicated by (srbl) and the spontaneous reversion to a non-fragile phenotype by (SRBl). Strains with the wild-type SRBl gene are marked by (non-fragile). ND, not determined.
Fennel], 1975). Observation was under a Leitz epifluorescence microscope using filter A. Determination of D N A content The DNA content of the fusion partners and fusion products was determined according to Ferenczy and Maraz (1977). Induction ofhaploidization with benomyl The source of benomyl used was Benlate (Du Pont Co.), a commercial fungicide containing 50% benomyl as the only active principle (Wood, 1982). To find out the optimal concentration of benomyl for induction of haploidization, each fusion hybrid was cultivated in YPD medium at 30°C in the presence of different concentrations of Benlate. The optimum was defined as the concentration of benomy1 which gave the maximal number of auxotrophs and had the minimal inhibitory effect on cell growth. Concentrations of Benlate between 150 and 220pg/ml were determined as optimal for the different hybrids. The genetic characteristics of the
auxotrophs obtained from benomyl-treated fusion hybrids were determined by replica plating on appropriate minimal media. Plasmid isolation Plasmid DNA from fusion hybrids and partner strains was isolated according to Perez-Ortin and Estruch (1988). RESULTS The results obtained by PEG mediated fusion and electrofusion are summarized in Table 2. Combinations of cells from different fragile strains of the same mating type (Table 2A) led to the production of hybrids. The number of hybrids obtained by electrofusion was higher than that from PEGmediated fusion. Single hybrids were produced even when intact fragile cells were mixed without PEG or electric treatments. Although the frequency of this spontaneous fusion is very low, hybrids were reproducibly obtained in all 10 repetitions of the experiment. However, hybrids were not formed spontaneously or induced by PEG or electric forces
208
D. H. PHILIPOVA AND P. V. VENKOV
Table 3. Fusion of fragile yeast strains with chromosomal and cytoplasmic markers Number of hybrids from lo8cells Strains
Spontaneous fusion
PEG-mediated fusion
( A j Cell x cell VY26rho' x VY4/pRN 15
5
45
( B ) Cell x protoplust VY26rho' x VY4/pRNlS VY4/pRNI5 x VY26rho'
110 90
900 620
( C ) Protoplast x protoplust VY26rho' x VY4/pRNlS
180
5100
when revertants to non-fragile phenotype were used. The controls for spontaneous revertants of the auxotrophic markers were negative in all fusion experiments (data not shown), which is in agreement with the very low reversion rates (less than lop9) typical for the double mutations used in the selection of the fusion hybrids. These results suggested that the colonies appearing in the fusion experiments are not rare revertants. They also indicated that the observed cell x cell fusion is a characteristic related to the fragile (srbl) mutants only and is not due to some other factor in the genetic background. Since this is the first observation of fusion of intact yeast cells, we performed some experiments to study the mechanism of cell fusion. Negative results were obtained in experiments using a combination of one fragile and one non-fragile strain (VY26 x AH215, Table 2A) suggesting that successful cell fusion occurs only if both partners contain the srbl mutation. Removal of the cell wall of the non-fragile partner (AH215 strain) led to formation of fusion hybrids between the fragile cells and the protoplasts (Table 2B). The use of non-fragile revertants VY26(SRB1) and VY4(SRBI) gave negative results, demonstrating again the crucial role of the srbl mutation for cell fusion. Although increased when compared to the cell x cell fusion, the frequency of the cell x protoplast fusion was significantly lower than that observed for the protoplast x protoplast fusion (Table 2C). This suggests the existence of a limited number of places on the surface of the fragile cells where fusion events could take place. It should be noted that frequencies of lop6have been obtained for the illegitimate mating reported to occur in some
yeast strains (Inge-Vechtomov, 1967) or for the reversion of single mutations. Therefore, the existence of cell fusion between fragile yeast cells needs further proof. First, cell fusion was observed between fragile strains which in addition to chromosomal mutations contain cytoplasmically inherited markers (Table 3). VY26[rhoq is a double mutant in the URA3 gene and lacks the mitochondria1 DNA which confers a non-revertible 'petite' phenotype (Slonimski et al., 1968).VY4/pRNl5 is a trpl leu2-3 leu2-112 ura3-160 ura3-288 mutant transformed with the episomal plasmid pRN15 to Ura' phenotype. The selection of fusion products between the two fragile partners was carried out on minimal medium with glycerol as the only carbon source and sorbitol as osmotic stabilizer. Only the fusion hybrids containing both the chromosomal and the cytoplasmic genomes of the two partners could grow on this selection medium. The results in Table 3 show that fusion does occur at frequencies similar to those reported for other pairs of fragile strains (see Table 2). Although the fusion hybrids were selected on glycerol-containing medium, their rho+ phenotype was confirmed by several passages in ethanol-containing media. The presence of the p R N l5 plasmid in the fusion hybrids determining their Uraf phenotype was verified by the appearance of uracil-auxotrophic clones in mitotic segregation experiments and by isolation of plasmid DNA directly from part of the fusion products (Figure 1). Second, the fusion of intact fragile cells was observed microscopically. The micrographs (Figure
CELL FUSION OF SACCHAROMYCES CEREVISIAE FRAGILE MUTANTS
a b c d e f g h
Figure 1. Agarose gel electrophoresis of plasmid DNA. Plasmid DNA was isolated and analysed from Escherichiu coli HBlOl transformed with pRNI5 (lanes a and h), Succhuromyces cwevisiae strains VY26rho" (lane b) and VY4/pRN15 (lane c ) o r fusion hybrids VY26rho" x VY4/pPN15 (lanes d-g).
2) demonstrate the existence of multi-nuclear cells immediately after the PEG treatment or electrofusion. Such heterokarions have been shown to represent intermediate products in the fusion process (Svoboda, 1978). For the experiments reported here, the number of multi-nuclear cells was counted in approximately 10' cells. In the case of cellx cell fusion, only single binuclear products were observed (Figure 2A), suggesting that cell fusion is a rare event which takes place between two cells only. In cell x protoplast and protoplast x protoplast samples, the number of multi-nuclear cells increased up to lo-' and respectively. In addition, the trinuclear and tetranuclear cells represented 30% of the multi-nuclear cells, which suggests that fusion occurs more readily and could involve more than two cells (Figure 2B and C). The data of DNA estimation (Table 4) show that the fusion products obtained by cell x cell fusion (VY7 x VY25; VY7 x VY 1160) are diploids since the DNA content of the isolates is about two times that of the haploid partner cells. However, hybrids obtained by cell x protoplast fusion (VY4 x AH2 15) had higher DNA content, indicating that three cells had taken part in the fusion process. Third. the fusion products were characterized genetically. Contrary to the partners used in the experiments. the hybrids form large, stable and fastgrowing colonies. They are of the same mating type as the partners and do not sporulate. For this reason it was impossible to analyse the hybrids by conventional genetic methods and they were characterized
209
by the use of benomyl known to induce mitotic chromosomal loss and partial haploidization in S. cerevisiae (Wood, 1982; Maraz et al., 1978). Auxotrophic colonies were isolated from the progeny of benomyl-treated fusion products and the auxotrophies and the mating type was determined. Table 5 summarizes the data obtained with representatives of the cell x cell and cell x protoplast fusion experiments. Following benomyl treatment all fusion hybrids produced auxotrophs which were of either parental or recombinant type. Furthermore, all auxotrophs had the same mating type, which was the same as that of the two partners used in the experiment. These results supply genetic evidence for the fusion nature of the hybrids and excludes the possibility that the fusion products might be rare revertants of the partners.
DISCUSSION The fusion of yeast protoplasts to generate hybrids is successful when (i) the electric charges of the cell surface are neutralized and the protoplasts are brought into very close contact and (ii) breakdown of plasma membrane occurs at the contact sites (Lucy, 1986). These two requirements seem to be fulfilled with intact cells of the fragile mutants of S. cerevisiae. Studies on membrane transport (Kotyk et al., 1988) supply evidence for a very low electric charge at the cell surface and a lack of the protonmotive force across the plasma membrane of the fragile mutant cells. This would permit a very close contact between the fragile cells. Further, electron microscopic observations have shown the existence of local defects in the cell wall of the fragile cells (Mateeva et al., 1976). Recently, these defects were visualized by scanning electron microscopy as gaps in the cell wall which are limited in number to one to three per fragile cell (Schade and Venkov, in preparation). This would allow two fragile cells to make rare contacts with each other by their plasma membranes. The exact defect of the plasma membrane in the fragile mutants is unknown. However, a number of studies have demonstrated an increased permeability of the mutant cells. Changes in the transport processes or plasma membrane permeability are supposed to facilitate membrane breakdown and fusion (Lucy, 1986; Dimitrov and Jain, 1984). As a consequence of these alterations of the cell wall and membrane structures, one would expect that intact cells of the fragile mutants could fuse without removal of the cell wall by protoplasting. The data presented in this paper show that this is actually the
210
D. H. PHILIPOVA AND P. V. VENKOV
A
B
C
Figure 2. Multi-nuclear fusion products. (A) Binuclear hybrid from a cell x cell VY4l(srbl) x VY8(srbl), spontaneous fusion. (B) Trinuclear hybrid from a cell x protoplast VY4(srbl) x AH215, PEG-mediated fusion. (C) Tetranuclear hybrid from a protoplast x protoplast VY26(srbl) x AH21 5 , electrofusion.
Table 4.
DNA content
Strains
Haploid AH215 VY4 VY7 VY25
2 5 9 + 2.5 24.6 f2.8 22.8 f 1.9 23.5 f2.6
Fusion products VY7 x VY 1160 VY7 x VY25 VY4 x AH215
40.1 rfr2.6 41.2 k4.1 48.8 k 3.2
*Values are & standard error. Standard errors are based on three independent DNA determinations.
case. Yeast cells of different fragile clones having equal mating type were found to fuse. Microscopic observations, DNA estimations and genetic analysis of the fusion products supply evidence for the fusion nature of the hybrids and exclude experimental artefacts such as reversion or illegitimate mating. Because of the very low frequency of cell x cell fusion, the differentiation
between fusion and illegitimate mating becomes a problem. In an attempt to solve this problem we isolated spontaneous non-fragile revertants from all fragile strains and repeated the fusion experiments. The negative results obtained with all pairs of revertants (Table 2A and B) are in favour of the fusion nature of the hybrids obtained from the corresponding fragile strains and make it highly unlikely that diploids that are homoallelic or heteroallelic at the mating type locus are produced by illegitimate mating. Further, the genetic analysis of the hybrids by induction of partial haploidization (Table 5) shows that all segregants have the same mating type, thus excluding the participation of heteroallelic illegitimate mating in the formation of the hybrids. The very low frequency found for the cell x cell fusion could be explained by the limited number of surface gaps per fragile cell and the low probability with which two fragile cells meet each other with the local defects of their cell walls facing. In agreement with this interpretation, the frequency of fusion is increased significantly when the cells of one partner are converted to protoplasts (Table 2B). In this case every contact between a surface gap of the fragile cell and any part of the protoplast’s surface could create a possibility for formation of a fusion hybrid.
21 1
CELL FUSION OF SACCHAROMYCES CEREVISIAE FRAGILE MUTANTS
Table 5. Genetic analysis of fusion hybrids
Hybrid ( A ) Cell x cell VY7 x VY25 (Spontaneous fusion) ( B ) Cell x protoplast VY26 x AH2 15 (PEG-mediated fusion) VY4 x AH215 (Electrofusion)
Per cent auxotrophs after benomyl treatment
Auxotrophs
Number
Parental type
Recombinants
a
a
4
24
9
15
0
24
3
18
7
11
18
0
4
29
11
18
29
0
Mating type
ACKNOWLEDGEMENTS T h a n k s are due to D. Dimitrov (Central Laboratory of Biophysics, Bulgarian Academy o f Sciences) f o r providing the equipment f o r electrofusion and to I. Tsoneva and P. D o i n o v f o r help i n electrofusion experiments.
REFERENCES Dimitrov, D. S. and Jain, R. K. (1984). Membrane stability. Biochem. Biophys. Acta 779,437-468. Ferenczy, L. and Maraz, A. (1977). Transfer of mitochondria by protoplast fusion in S . cerevisiae. Nature 268(5620), 524525. Inge-Vechtomov, S. G. (1967). Illegitimate copulation and polyploidy in yeast S. cerevisiae. Genetika 11, IO(L109. Kotyk, A , , Venkov, P. and Dvorakova, M. (1988). Membrane transport in an osmotically fragile mutant of S . cerevisiae. Yeast 4,241-247. Kozhina. T., Stateva, L. and Venkov, P. (1979). Genetic analysis of an osmotic sensitive S. cerevisiae mutant VY 1160. Molec. Gen. Genet. 170,351-354. Lucy, L. A. (1986). Salient features of artificially induced cell fusion. Biochem. Society Transactions 14,25&25 1. Maraz, A., Kiss, M. and Ferenczy, L. (1978). Protoplast fusion in S. cerevisiae strains of identical and opposite mating types. FEMS Microbiol. Letters 3,3 19-322. Mateeva, Z., Petrov, P., Venkov, P. and Madjiolov, A. A. (1976). Electron microscopic study of the lysis of an osmotic sensitive yeast mutant. J . Microsc. Biol. Cellulaire, 26,73-74. Nebreda, A. R., Villa, T. G., Villanueva, J. R. and Rey, del F. (1986). Cloning of genes related to exo-pglucanase production in S. cerevisiae: characterization
of an exo-p-glucanase structural gene. Gene 47, 245-259. Perez-Ortin, J. E. and Estruch, F. (1988). A rapid method for the screening of plasmids in transformed yeast strains. Curr. Microbiol. 17, 19-22. Philipova, D. H. (1985). PhD Thesis, Institute of Molecular Biology, Bulgarian Academy of Sciences. Schnettler, R. and Zimmermann, U. (1985). Influence of the composition of the fusion medium on the yield of electrofused yeast hybrids. FEMS Microbiol. Letters 27,195-198. Sherman, R., Fink, G . ,and Hicks, J. B. (1986). Methods in Yeast Genetics. Cold Spring Harbor Laboratory, New York. Skala, J., Luty, J. and Kotylak, Z. (1988). Interspecific protoplast fusion between yeasts S. cerevisiae and S . fermentati. Curr. Genet. 13, 101-104. Slonimski, P. P., Perrodin, G. and Croft, J. K. (1968). Ethidium bromide-induced mutation of yeast mitochondria: Complete transformation of cells into respiratory deficient nonchromosomal “petites”. Biochem. Biophys. Res. Commun. 30,232-239. Svoboda, A. (1978). Fusion of yeast protoplasts induced by polyethylene glycol. J. Gen. Microbiol. 109,169-1 75. Venkov, P. V., Hadjiolov, A. A,, Battaner, E. and Schlessinger, D. (1 974). Saccharomyces cerevisiae sorbitol dependent fragile mutants. Biochem. Biophys. Res. Commun. 56,559-604. Venkov, P. V., Stateva, L. I. and Hadjiolov, A. A. (1977). Toyocamycin inhibition of ribosomal ribonucleic acid processing in an osmotic sensitive adenosine utilizing S . cerevisiae mutant. Biochem. Biophys. Acta 474, 245-253. Venkov, P. V. and Ivanov, V. P. (1982). Uptake of D N A by fragile mutants of S . cerevisiae. Curr. Genet. 5, 153-1 55.
212 Venkov, P. and Scheit, K.-H. (1984). Effect of seminal plasmin on rRNA synthesis in S. cerevisiae. FEBS Letters 172,21-24. Waltschewa, L. W., Venkov, P. V., Stoyanova, B. B. and Hadjiolov, A. A. (1976). Degradation of ribosomal precursor and polyadenylic acid containing ribonucleic acids in S. cerevisiae caused by actinomycin D.Arch. Biochem. Biophys. 176,630-637.
D. H. PHILIPOVA AND P. V. VENKOV
Williamson, D. H. and Fennell, D. J. (1975). The use of fluorescent D N A binding agents for detecting and separating yeast mitochondria1 DNA. Methods Cell Biol. 17,335-351. Wood, J. S. (1982). Genetic effects of methyl benzimidazole-2-yl-carbamate on S. cerevisiae. Mol. Cell Biol. 2, 1064-1079.
