© Springer-Verlag 1984
Sensitivity to ethidium bromide during meiosis in Saccharomyces cerevisiae S. L. Kelly 1 and J. M. Parry Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK
Summary. Ethidium bromide was found to inhibit nuclear and mitochondrial DNA synthesis during meiosis which resulted in the inhibition of meiotic gene conversion and sporulation and was also lethal. Protection from the effects of ethidium bromide on meiotic gene conversion and survival was found to coincide with DNA synthesis, but it is possible that protection from sporulation inhibition occurs only later in meiosis. Key words: Yeast - Ethidium bromide - Meiosis
Introduction Cytoplasmic petite mutants contain defective mitochondrial DNA from which coding sequences are absent (Goldring et al. 1970) and they are unable to grow on non-fermentable carbon sources (Slonimski et al. 1978). These mutant cells are also unable to undergo meiosis and spore formation in sporulation medium which contains acetate as a carbon source. Cytoplasmic petites are effectively induced by the mutagen ethidium bromide which has been shown in yeast to bind selectively to mitochondrial DNA (Slonimski et al. 1968), to inhibit the replication of mitochondrial DNA and to cause the fragmentation of preexisting mitochondrial DNA molecules (Goldring et al. 1970; Perlman and Mahler 1971). The inability of petite mutants to sporulate and the inhibition of sporulation by erythromycin, an inhibitor of mitochondrial protein synthesis in yeast, prompted the suggestion that mitochondrial gene products were required for sporulation (Puglisi and Zennaro 1971). How-
1 Offprint requests to." S. L. Kelly, Wolfson Institute of Bio-
technology, University of Sheffield, Sheffield SIO 2TN UK
ever, Kuenzi et al. (1974) found that if the mitochondrial genome is mutagenised with ethidium bromide prior to sporulation, but after adaptation to the use of acetate as a carbon source for mitotic growth, then sporulation can occur. The spores produced by such a treatment with ethidium bromide were found by Kuenzi et al. (1974) to produce petite colonies and no mitochondrial DNA was detected in the sporulating culture by caesium chloride gradient analysis. These observations were taken by Kuenzi et al. (1974) to indicate that the mitochondrial genome was not required for sporulation, but only for adaptation to the use of acetate which is the carbon source in sporulation medium. Subsequently, Newlon and Hall (1978) reported that sporulation was however inhibited after treatment of acetate-adapted cultures with ethidium bromide at early times during meiosis. This result suggested that ethidium bromide may be affecting cells during meiosis by its action on oxidative metabolism or that its effect may be sporulation-specific. In this study the sensitivity of meiotic cells of Saccharomyces cerevisiae to treatment with ethidium bromide has been examined by continuous exposure in sporulation medium and by interrupting meiosis by plating cells back onto vegetative medium. Meiotic cells will still revert to mitotic growth during and after meiotic DNA synthesis, when they exhibit commitment to the execution of meiotic levels of recombination. Only at the time of first spindle pole body separation in prophase I do they become committed to the execution of meiotic cell division in vegetative medium (Esposito 1980).
Materials and methods Three yeast strains were employed in this study. These were the yeast strains D7 (Zimmermann et al. 1975), SK1 (Kane and Roth 1974) and a/c~wild type diploid.
70
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis The strain D 7 has the following genotype:
a
ilvl-92
trp5-12
ade2-40
cyhr2
a
ilvl-92
trp5-27
ade2-119
CYHS2
D 7 has been frequently used in mitotic studies to assay basepair substitution mutation to isoleucine independence, gene conversion to tryptophan independence and reciprocal recombination at the adenine locus. The products of mitotic reciprocal recombination are detected as red-pink, twin sectored colonies with the ade2-119 allele giving rise to a pink pigmentation and the ade2-40 allele being responsible for the red pigmentation. These alleles exhibit heteroallelic complementation giving rise to a white diploid colony. In meiotic studies dramatic increases in tryptophan convertants and coloured colonies are found in samples plated back onto vegetative medium during meiotic DNA synthesis. These colonies are the result of a commitment by the meiotic ceils to execute meiotic recombination followed by a return to mitotic growth. Only later in meiosis are haploid coloured colonies detected after plating meiotic ceils onto vegetative medium and these indicate a commitment to execute a meiotic reductional division rather than revert directly to mitosis (Olson and Zimmermann 1978).
Growth and sporulation. All strains were grown at 28 oC in acetate
sure survival and gene conversion. In this way the time of release from ethidium bromide sensitivity was investigated. II. Media shift experiments. The strain D7 was plated onto vegetative medium containing ethidium bromide at different times during meiosis. Other experiments involved the treatment of cells in sporulation medium with ethidium bromide followed by plating back onto vegetative medium at later times. In this way the timing of events leading to ethidium bromide sensitivity was investigated. The yeast strain SK1 was also treated at different times during meiosis for 15 rain in pH 7.0 phosphate buffer and plated back onto 1% glucose complete medium to assay survival and petite mutant induction using the tetrazolium overlay technique (Ogur et al. 1957).
Plating. Appropriately supplemented minimal plates were used for scoring gene conversion (Olson and Zimmermann 1978). These plates were scored after six days. Viability was assayed on YPG medium (Cox and Bevan 1962) after three days. These plates were examined for the presence of completely coloured and sectored colonies after six days. In all cases between 50-200 colonies per plate were scored on three replicate plates after appropriate dilutions.
DNA estimation. The total DNA present in samples was estimated
presporulation medium consisting of 10 g potassium acetate, 20 g peptone and 10 g yeast extract per litre (Fast 1973). Cells were harvested at 2 x 107 cells/ml and washed three times in saline. After resuspension in sporulation medium the cells were sonicated and made up to a final concentration of 2 x 107 cells/ ml. The sporulation medium ingredients were 8.2 g sodium acetate, 1.9 g potassium chloride, 1.2 g sodium chloride and 0.35 g magnesium sulphate (von Borstel 1978). For D7 the sporulation medium was supplemented with 25 mg/l adenine, tryptophan and isoleueine. The time of resuspension of yeast cells in sporulation medium constituted time zero in meiosis and only 150 ml of culture was inoculated into a litre flask. This enabled vigorous aeration to occur which is important in obtaining high levels of sporulation and synchrony of meiotic events. The strain D7 achieved high levels of sporulation under these conditions, but the wild-type diploid used in this study sporulated at a lower level. The homothallic strain SK1 underwent a rapid and synchronous sporulation and b y 10 h had reached almost 100% sporulation.
spectrophotometrically by diphenylamine reaction (Burton 1956) using the standard method (Haber and Halvorson 1975).
Chemicals. Ethidium bromide and oligomycin were obtained
sphaeroplasted for each caesium chloride gradient. Samples were resuspended in 0.1 M EDTA at pH 7.0 and left on ice for 30 min. After this time the samples were frozen in liquid nitrogen which was previously shown not to effect the profiles. These samples were thawed and resuspended in 0.5 M EDTA pH 7.0 with 25 #1/ ml 2-mercaptoethanol and incubated for 30 min on ice. Cells were then sphaeroplasted using zymolyase 5,000 (Kirin Breweries, Japan) at 10 mg/ml in 1 M sorbitol, 0.1 M EDTA, 0.1 M Tris at pH 7.5. Incubation was at 37 °C and sphaeroplasting was complete within 5 rain for samples early in meiosis although up to 15 min was required for samples at later times.
from Sigma London Chemical Co. Ltd.
Treatment. Yeast cells were treated under two different regimes: I. Inhibition of meiosis and sporulation. Cells were exposed to the chemicals 2 h after initial inoculation into sporulation medium except for the strain SK1 which was treated after 1 h. This allowed some time for the completion of mitosis. The duration of the exposure was 48 h, after which samples were washed and the ascus frequency determined before plating onto appropriate media to examine recombination and survival. Mitotic comparisons were made to samples treated immediately after removal from presporulation medium in pH 7.0 phosphate buffer for 48 h. In this way cells were treated during a mitotic cell cycle. For treatment at different stages of meiosis ceils were removed from sporulating cultures and resuspended in sporulation medium containing ethidium bromide. The treatments were for 48 h followed by determination of sporulation frequency and plating to mea-
Testing for ploidy. Ploidy was inferred by mating and sporulation tests. Colonies were classified as diploid if they gave rise to asci in sporulation medium after five days and did not give mating figures after mixing with haploid strains of either matingtype. Similarly colonies which did not give rise to asci after five days incubation in sporulation medium, but which gave mating figures on mixing with a or a strains, were inferred to be mainly haploid. Radioactive labelling o f DNA. The DNA was labelled with (6-3H) uracil and (2-14C) uracil obtained from Amersham International. The specific activity of (6-3H) uracil was approximately 25 Ci/ mMol and was added at 6 #Ci/ml under sporulation conditions. The specific activity of (2-14C) uracil was 58 Ci/mMol and this was added at 0.5 ~Ci/ml. Labelling under presporulation conditions was overnight and after 2 h during sporulation.
Preparation of sphaeroplasts. Samples of 5 x 108 cells were
L ysis of sphaeroplasts and caesium chloride gradients. The sphaeroplasts were centrifuged at 1,000 rpm for 10 rain and resuspended in 0.001 M EDTA, 0.04 M NaCI, 0.01 M Tris HC1 buffer at pH 8.0 containing 50 ttg/ml proteinase K. The samples were vortexed for 2 min to ensure lysis of sphaeroplasts and incubated at 35 °C for 4 h. 4 ml of the samples was then added to 4.8 g CsC1 in nitrocellulose tubes. Centrifugation was at 40,000 rpm for 40 h in a
S. L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis Completion of mitosis & entry into meiosis
Meiotic DNA
Spore formation
71
100-
synthesis Meiotic cell division
80 -6 :> 60 u0
,~ 5.0
o (9
5O.~_
o o_
g
40 20
LO 2
-7. 4o *¢ :7 C3
30
~30
20 10
2.0
od
~S -5 bq
0 o~ 0
2
Z,
6
8
10 12 1L h in SPM
16
18
20
22
2L
Fig. I shows the time course of various landmark events in the sporulation of the strain DT. These include meiotic DNA synthesis indicated by the DNA content of successive samples (* *), commitment to sporulation indicated by the appearance of haploid coloured colonies after plating sporulating cells onto YPG medium (-- -') and the ascus frequency in sporulating samples (_- _-)
c 100
.2 u
8O 60
a
o~ 40 o
o_ 20 0 80 > 50 o~~
40
20 0
C U3
100 O_~
>--~
10
0
1.0 log dose (tag/ml)
10
Fig. 2a-c shows the effect of ethidium bromide treatment on sporulation inhibition aand survival b in the strains SK1 (• []), D7 ( ~ ) and an a/o~ wild type diploid ( ~ ) . c shows the effect of ethidium bromide treatment on the inhibition of meiotic gene conversion in the strain D 7 (A ~)
Beckman 50 Ti rotor and the gradients were then collected by puncturing the bottom of the tube with a needle. Between 3035 fractions were collected and 0.2 ml 0.1 M NaOH was added to each and left overnight. The fractions were put onto chromatography paper strips and the DNA precipitated in 5% TCA for 12 h. Following two 30 min rinses in ethanol the chromatography paper strips were dried at 28 °C and counted in an LKB scintillation counter.
0
g
1.'0 log dose (gg/ml)
10
Fig. 3 shows the effect of ethidium bromide on the survival of logarithmic phase acetate grown cells of the strains D7 (~ ~), SK1 ( ~ ) and an a/c~ wild-type diploid ( ~ ) after treatment for 48 h in pH7 buffer
Results Figure 1 shows the time course of some of the landmark events in the sporulation of the strain DT. Meiotic DNA synthesis, as estimated by the diphenylamine reaction, occurred between 6 and 10 h and this is coincident with an increasing frequency of gene conversion found in samples plated onto vegetative medium at these times (see Fig. 4). The first appearance of haploid, coloured colonies is also shown for samples plated from sporulating cultures onto vegetative medium. These were first detected in samples taken at 12 h into sporulation and indicates the point of first commitment of cells to the completion of meiotic cell division after medium switch. Asci were first detected at about sixteen hours into sporulation and the percentage sporulation was found to increase to about 50% at 24 h and to between 7 0 - 8 0 % at 36h. The effects of exposure of sporulating cells to ethidium bromide is shown in Fig. 2. Treatment resulted in sporulation inhibition and in a cell lethality which was approximately proportional to the level of sporulation in the untreated cultures of the different strains examined. The supersporulating strain S K 1 exhibited the greatest sensitivity to the lethal effects of ethidium bromide, followed by the strain D 7 and the wild-type diploid strain respectively. The frequency of gene conversion found in treated samples of D 7 decreased to mitotic levels in those samples where ascus formation was totallyinhibited. Figure 3 shows the effects of treating acetate adapted exponential phase mitotic cells with ethidium bromide in pH 7.0 phosphate buffer for an equivalent period to the meiotic treatment regime. In this way the cells were treated during a mitotic cell cycle. The strain D 7 and the wild-type strain exhibited no lethality under these conditions after ethidium bromide treatment although considerable induction of petite mutation occurred. However, the supersporulating strain SK1 did exhibit an equiv-
72
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
Table 1. The effect of the mitochondrial ATPase inhibitor oligomycin on viability and ascus formation in sporulating cultures of the yeast strain D7 and an a/a wild type m1-1)
0
2.5
5
10
20
40
44.9 85.6 -+6.7
21.2 89.7 ± 7.8
4.5 84.7 ± 9.1
1 81.4 -+ 11.2
0 96.6 -+ 8.5
0 78.3 ± 5.9
71.0 123.7 ± 1 0 . 6
32.0 107.6 -+8.7
0 116.7 -+16.1
0 119.7 -+6.1
a/~ Percentage sporulation Plate counts ± S.D. D7 Percentage sporulation Plate counts ± S.D.
co
60
c3
z,O o_ (/1
o~ -8
._> > ~-
2O
8O
60
113
40 20 1000 0)
"E o
~--
100
o
~ .-&
10
o
~.v 0
0
i
;
8 1'o 12 h in SPM
~
IF--
1L
Fig. 4a-c shows the effect on the strain D7 of a sporulation inhibition after resuspension in both fresh sporulation medium (~) and sporulation medium containing 5 gg/ml ethidium bromide (A zx) at different times during meiosis and b lethality after these ethidium bromide treatments, c shows the frequency of gene conversion found after plating cells onto vegetative, selective medium at different times during meiosis (~ A) and the gene conversion frequencies found for samples taken at the same times and incubated for 48 h in fresh sporulation medium (~) and sporulation medium containing 5 ~g/ml ethidium bromide (o o)
alent lethality to that of treatment in sporulation medium, but examination of untreated samples revealed that the strain SK1 underwent almost 100% sporulation in pH 7.0 phosphate buffer under these conditions, i.e. these cells were undergoing meiosis in phosphate buffer unlike the other strains used.
2.3 114 +-9.9
0.2 127.7 _+6.7
The possibility that the action of ethidium bromide demonstrated with sporulating cells was by the inhibition of oxidative metabolism was examined by treating cells with oligomycin at concentrations which inhibit mitochondrial ATPase (Avner et al. 1973). Table 1 shows that oligomycin treatment produced no detectable lethal effects on sporulating cells at doses which totally inhibit ascus formation, i.e. with this drug there was a clear separation between cell lethality and the inhibition ofsporulation. However, the effects of oligomycin on respiration were not measured here. Figure 4 shows the effects of treating D7 in sporulation medium containing ethidium bromide at a concentration of 5 pg/ml at different times during sporulation followed by incubation for 48 h. Both ascus formation and meiotic gene conversion were inhibited in cultures treated prior to the time of meiotic DNA synthesis and these samples also exhibited lethality. However, reduced lethality and increasing levels of gene conversion were found in samples treated during meiotic DNA synthesis and this protection from the effects of ethidium bromide on lethality and on the inhibition of meiotic gene conversion appeared complete following meiotic DNA synthesis. The transition point for the effect of ethidium bromide on sporulation inhibition may however be later than for meiotic gene conversion and lethality. The increasing frequency of gene conversion in samples treated during meiotic gene conversion also closely paralleled the increases found in meiotic samples plated back onto vegetative medium to assay commitment to recombination at these times. This indicates that escape from the effects of ethidium bromide on meiotic gene conversion inhibition is closely correlated with the time of commitment to recombination, which is itself correlated with the period of meiotic DNA synthesis. Figure 5 shows a similar experiment carried out with the supersporulating strain SK1. Figure 5a shows the time of meiotic DNA synthesis, as assayed by the diphenylamine reaction, occurring between 2 - 4 h and that
S. L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
(3
80 c ._o
% 5.0 (9
% 4.0
60 ~ ~-
~s.o
2O o~
73
-6 100 ->
~ 75
40 o
Z
CI
g
~
50
b
=
:
:
25 o o o
~ 60 S~20
100
X3 >
% Q- 60
o o
40 0
, 2
,
, 4 6 h in 5PM
, 8
d_ 0
10
sporulation ( ~ ) at these different times; b shows the percentage sporulation ( ~ ) and survival (o o) of samples taken during the sporulation of SK1 and incubated for 48 h in sporulation medium containing 5 ~tg/ml ethidium bromide and c shows the percentage petite induction in samples taken during the sporulation of SKI and treated in pH 7.0 buffer for 15 min with 5 #g/rot ethidium bromide prior to plating
~80
o
100 ~o~
,7. o c
o_~
10
c5_.~
> 0- ------r O 2
/~
;
4
6
8
1'0
I'2 l'L
24
h in SPM
Fig. 5 a - c shows for the strain S K I a the DNA content ( ~ - ~ ) in successive samples of a sporulating culture and the percentage
100 ~- 9 0
II--
0 2
1;' h in SPM 8
1'2
. fr14 24
Fig. 6 a shows the effect on survival of plating onto vegetative medium containing 5 #g/ml ethidium bromide at different times of meiosis for the strain D 7 (~- ~-) and in b the frequency of gene conversion after media switch onto normal selective medium (-- e) and selective medium containing ethidium bromide at 5 ~g/ml (,~-~A) is shown
asci first appeared at 7 h and increased to almost 100% at 10 h. Figure 5b shows that for the strain SKI, as with D7, the lethal effects of ethidium bromide were reduced at the time of meiotic DNA synthesis and no lethal effects of ethidium bromide treatment were found after meiotic DNA synthesis. As with D7 protection from the
Fig. 7 shows a the percentage survival found for cultures of D 7 treated with 5 ~g/ml ethidium bromide at 2 h (-,) after inoculation into sporulation medium followed by plating onto vegetative medium at later times; b shows the gene conversion frequency at trp5 in D 7 found for untreated cultures ( ~ _ A ) , and cultures treated with 5 #g/ml ethidium bromide at 2 h (~---~) and 4 h ( - ~ ) after inoculation into sporulation medium, followed by plating at intervals onto vegetative, selective medium
effects of ethidium bromide on ascus formation in SK1 appeared later than for lethality. Figure 5c shows the results of a separate experiment involving the treatment of samples of a sporulating culture of the strain SKI with 5/~g/ml ethidium bromide in pH 7.0 phosphate buffer for 15 rain followed by plating on low glucose complete medium. This treatment regime did not produce cell lethality and Fig. 5c shows that some cyclic variation in petite mutant induction was found after treatment at the different times of meiosis. The induction of petites by ethidium bromide at the later times, after meiotic cells exhibit protection from the lethal effects of ethidium bromSde, indicates that the protection is not associated with an absolute permeability barrier against ethidium bromide. Figure 6 shows the effects of the interruption of meiosis in D7 followed by plating onto vegetative medium containing ethidium bromide at 5/~g/ml. In this experiment cells can revert directly to mitosis after meiotic DNA synthesis up to the point of commitment to sporulation, which occurs for the most advanced cells at about 12 h. No effect of this treatment on lethality, or on the kinetics of commitment to meiotic gene conversion, was detected even during meiotic DNA synthesis. This suggested that while protection from ethidium bromide induced lethality occurs at the time of meiotic DNA synthesis, continued incubation in sporulation medium might be necessary for the manifestation of the ethidium
74
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
3000
1500
~E (3_
~E 13..
ca
2000
1000 ca
1000
500
r CO
(.D ..t
J_A d
1500
3000
r
ca 2000
i
13-
1000 ca 7-
r
ca
500
1000
0
0 400
f
~E 13..
ca 200
J\ 01 Distance sedimented
Fig. 8a-f shows the caesium chloride gradients of samples of 5 x 108 cells of the strain D7 labelled with (3H-6) uracil (-- --) after 2 h in sporulation medium with and without treatment with 5 gg/ml ethidium bromide; a-d show the profiles of untreated samples taken at a 2 h, b 6 h, c 10 h and d 12 h into sporulation where the parental DNA is labelled using (2-14C) uracil (; ;); e-f show the profiles of samples taken at e 2 h and f 12 h after treatment with ethidium bromide and where the position of the nuclear and mitochondrial DNA is indicated by 14C labelled yeast marker DNA (-)
bromide induced effects and that cells could be rescued by reverting to mitotic growth. Figure 7 shows the effect of adding 5 #g/ml of ethidium bromide to sporulation medium at early times during meiosis (2 h and 4 h) in D7, followed by plating samples onto vegetative medium at subsequent times to investigate the timing of events resulting in ethidium bromide induced lethality. The time of meiotic DNA synthesis and commitment to meiotic gene conversion in
untreated cultures was found to be the time when increasing lethality resulted after plating ethidium bromide treated ceils back onto vegetative medium. Prior to the time of meiotic DNA synthesis in untreated cultures ethidium bromide treatment did not result in detectable lethality on plating back onto vegetative medium. Ethidium bromide treatment also totally inhibited meiotic gene conversion. In order to investigate the association of ethidium bromide sensitivity with meiotic DNA synthesis the effect of ethidium bromide treatment on the incorporation of DNA precursors into the nuclear and mitochondrial DNA in D7 was examined using caesium chloride gradient analysis (Fig. 8). This was undertaken by labelling sporulating cultures with (6-3 H) uracil 2 h after inoculation into sporulation medium and taking subsequent samples of 5 x 10 s cells for each caesium chloride gradient. Incorporation of aH label into the nuclear and mitochondrial DNA was detected through the period of meiotic DNA synthesis in untreated cultures (Fig. 8a-8d), where the parental DNA was labelled with (2J4c) uracil under presporulation conditions. No incorporation of 3H label was detected into the nuclear or mitochondrial DNA when ethidium bromide was added at 5 #g/ml at the time of addition of (6-3H) uracil, i.e. 2 h after resuspension of the culture in sporulation medium. The position of the nuclear and mitochondrial DNA in these ethidium bromide treated samples was indicated by ( 2 J 4 C ) marker yeast DNA (Fig. 8e-8f).
Discussion The ability of acetate adapted yeast cultures to sporulate after treatment with ethidium bromide prior to inoculation into sporulation medium (Kuenzi et al. 1974; Newlon and Hall 1978; Kelly 1982), indicates that the mitochondrial genome is not required for sporulation except to permit adaptation to growth on acetate as a non-fermentable carbon source. Newlon and Hall (1978) reported that treatment with ethidium bromide at early times of meiosis did however result in an inhibition o f sporulation. We have confirmed these findings and further demonstrated that ethidium bromide treatment of ceils early in meiosis inhibits nuclear and mitochondrial DNA synthesis which has the effect of blocking meiotic gene conversion and sporulation. This inhibitory effect of ethidium bromide is also correlated with a lethal effect which increases with the sporulation competance o f the strains used and suggests that the lethal effect may be associated with attempted sporutation. Support for a sporulation-specific action of ethidium bromide comes from the absence of lethality in acetateadapted, mitotic cells treated during the division prior to
S. L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis inoculation into sporulation medium (Kelly 1982), from the absence of lethality found for growing, acetate-adapted cells treated in buffer and from the sensitivity of the strain SKI in buffer where it can sporulate. The origin of the effect of ethidium bromide on meiotic cells may not be mitochondrially-associated, but may be associated with an effect on meiotic DNA synthesis. This is supported by the finding that oligomycin can inhibit sporulation in the absence of lethality indicating that there may be more to the action of ethidium bromide than by blocking oxidative metabolism. Similarly erythromycin, an inhibitor of mitochondrial protein synthesis, has also been reported to inhibit sporulation in the absence of lethality (Marmiroli et al. 1981), but in the sporulation conditions used here concentrations of erythromycin up to 2 mg/ml do not inhibit sporulation in the erythromycin-sensitive strain D 7 (Kelly 1982). This suggests that the absence of mitochondrial protein synthesis is insufficient to explain the lethal effect of ethidium bromide on meiotic cells. It has also been reported that the addition of glucose reverses the inhibition of sporulation by ethidium bromide (Tsuboi et al. 1974), which might suggest a mitochondrial origin for the effect of ethidium bromide. However, we have found no such reversal of the effects of ethidium bromide on sporulation, lethality, or meiotic gene conversion up to doses of glucose which themselves inhibit sporulation. Protection from the lethal effects of ethidium bromide treatment was found to coincide with the time of meiotic DNA synthesis in the strains SKI and DT. This suggests that the origin of the ethidium bromide sensitivity of meiotic cells may lie in the mechanism of inhibition of meiotic DNA synthesis. Further support for this hypothesis is derived from the similarity between the kinetics of commitment to meiotic gene conversion (an indication of the proportion of the culture having undergone or undergoing meiotic DNA synthesis) and the level of meiotic gene conversion found in ethidium bromide treated samples of D7. The successive sampling of cultures treated with ethidium bromide prior to meiotic DNA synthesis followed by plating onto vegetative medium also identified events surrounding meiotic DNA synthesis with ethidium bromide induced lethality. Increasing lethality was observed after plating cells from treated cultures back onto vegetative medium during the period of meiotic DNA synthesis in untreated cultures. Similar changes in survival have been observed for some recombination-deficient mutants of Saccharomyces cerevisiae which undergo meiotic DNA synthesis but give inviable products, probably as a result of failed recombination (Game et al. 1980). The effect of ethidium bromide on meiotic cells differs however, as mitochondrial and nuclear DNA synthesis were inhibited, but it is possible that the lethality observed here may result from some defective recombination event.
75
The media switch experiments, involving plating cells from untreated sporulating cultures onto vegetative medium containing ethidium bromide, did not reveal any effect of ethidium bromide on survival, or on the kinetics of commitment to meiotic gene conversion. This finding may still be reconciled with an effect of ethidium bromide on meiotic DNA synthesis if the sensitivity is the result of an effect prior to, or at the initiation point of meiotic DNA replication. Cells prior to this point would revert to mitosis directly after media switch having not encountered the ethidium bromide sensitive phase and cells undergoing meiotic DNA synthesis would have passed the ethidium bromide sensitive phase and exhibit commitment to meiotic recombination. This hypothesis would explain the absence of detectable lethality in samples of cells plated onto vegetative medium containing ethidium bromide during meiotic DNA synthesis. Protection from the inhibitory effects of ethidium bromide on sporulation for the strains studied appeared to be later than for protection from the effects on survival and meiotic gene conversion. This may indicate other effects of ethidium bromide on meiotic ceils, perhaps during prophase I. One such possible effect might be the induction of pachytene arrest as is found during elevated temperature stress during meiosis (Byers and Goetsch 1982), or perhaps on the DNA metabolism associated with meiotic prophase that has been shown in Lilium (Stern and Hotta 1973). The origin of the lethal effect of ethidium bromide on meiotic cells remains unclear, but the data presented indicates its action to be sporulation-specific, or due to an event coupled to sporulation which is correlated with meiotic DNA synthesis. The effect need not be associated with the mitochondria, which appear to be necessary in sporulation only as a site for oxidative metabolism, and it is possible that the lethal effect is associated with an effect of ethidium bromide on the nuclear genome. This might be the result of defective replication initiation during meiosis in the presence of ethidium bromide, but further work is required to distinguish between this and other possibilities, such as the occurrence of attempted recombination in the absence of DNA synthesis. Acknowledgements. This work was supported by Euratom and the Royal Society. During the course of the work one of us (SLK) held a B.P. SRC CASE studentship. We would like to thank Dr. Tony Regnier of B.P. Ltd. for his support for this work.
References Avner PR, Coen D, Dujon B, Sloniminski PO (1973) Mol Gen Genet 125:9-52 Burton K (1956) Biochem J 62:315-373 Byers B, Goetsch L (1982) Mol Gen Genet 187:47-53 Cox BS, Bevan EA (1962) New Phytologist 61:342-355
76
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
Esposito MS, Esposito RE (1974) Proc Natl Acad Sci USA 71: 3172-3176 Esposito RE (1980) Workshop report on sporulation in Tenth International Conference on Yeast Genetics and Molecular Biology, pp 1 9 - 2 5 Fast D (1973) J Bacteriol 116:925-30 Game JC, Zamb TJ, Braun RJ, Resnick M, Roth RM (1980) Genetics 9 4 : 5 1 - 6 8 Goldring ES, Grossmann LI, Knipnick D, Cryer DR, Marmur J (1970) J Mol Biol 52:323 Haber JE, Halvorson HO (1975) Methods in sporulation and germination of yeasts. In: Prescott DM (ed) Methods in Cell Biology, vol XI. Academic Press, pp 4 5 - 6 9 Kane SM, Roth R (1974) J Bacteriol 1 1 8 : 8 - 1 4 Kelly SL (1982) A comparative study of genetic change in meiotic and mitotic cells of Saecharomyces cerevisiae. Ph. D. thesis, University of Wales Kuenzi MT, Tingle MA, Halvorson HO (1974) J Bacteriol 117: 80-88 Marmiroli N, Tassi F, Bianchi L, Algeri AA, Puglisi PP, Esposito MS (1981) Curr Genet 4 : 5 1 - 6 2 Newlon MC, Hall BD (1978) Mol Gen Genet 165:113-114
Ogur M, St John R, Nagai S (1957) Science 125:928 Olson LW, Zimmermann FK (1978) Mol Gen Genet 1 6 6 : 1 5 1 159 Perlman PS, and Mahler HR (1971) Nature New Biol. 2 3 1 : 1 2 16 Puglisi PP, Zennaro E (1971) Experientia 27:963-964 Slonimski PP, Perrodin G, Croft HH (1968) Biochem Biophys Res Commun 30:232 Stern H, Hotta Y (1973) Ann Res Genet 7 : 3 7 - 6 6 Tsuboi M, Kona K, Yansishima W (1974) Arch Microbiol 99: 295 - 3 0 5 Von Borstel RC (1978) Measuring spontaneous mutation rates in yeast. In: Prescott D-M (ed) Methods in cell biology, vol XX. Academic Press, pp 1 - 2 4 Zimmermann FK, Kern R, Rosenberger H (1975) Mutat Res 38: 381-388
C o m m u n i c a t e d b y B. S. Cox Received April 20 / August 15, 1983
Sensitivity to ethidium bromide during meiosis in Saccharomyces cerevisiae S. L. Kelly 1 and J. M. Parry Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP, UK
Summary. Ethidium bromide was found to inhibit nuclear and mitochondrial DNA synthesis during meiosis which resulted in the inhibition of meiotic gene conversion and sporulation and was also lethal. Protection from the effects of ethidium bromide on meiotic gene conversion and survival was found to coincide with DNA synthesis, but it is possible that protection from sporulation inhibition occurs only later in meiosis. Key words: Yeast - Ethidium bromide - Meiosis
Introduction Cytoplasmic petite mutants contain defective mitochondrial DNA from which coding sequences are absent (Goldring et al. 1970) and they are unable to grow on non-fermentable carbon sources (Slonimski et al. 1978). These mutant cells are also unable to undergo meiosis and spore formation in sporulation medium which contains acetate as a carbon source. Cytoplasmic petites are effectively induced by the mutagen ethidium bromide which has been shown in yeast to bind selectively to mitochondrial DNA (Slonimski et al. 1968), to inhibit the replication of mitochondrial DNA and to cause the fragmentation of preexisting mitochondrial DNA molecules (Goldring et al. 1970; Perlman and Mahler 1971). The inability of petite mutants to sporulate and the inhibition of sporulation by erythromycin, an inhibitor of mitochondrial protein synthesis in yeast, prompted the suggestion that mitochondrial gene products were required for sporulation (Puglisi and Zennaro 1971). How-
1 Offprint requests to." S. L. Kelly, Wolfson Institute of Bio-
technology, University of Sheffield, Sheffield SIO 2TN UK
ever, Kuenzi et al. (1974) found that if the mitochondrial genome is mutagenised with ethidium bromide prior to sporulation, but after adaptation to the use of acetate as a carbon source for mitotic growth, then sporulation can occur. The spores produced by such a treatment with ethidium bromide were found by Kuenzi et al. (1974) to produce petite colonies and no mitochondrial DNA was detected in the sporulating culture by caesium chloride gradient analysis. These observations were taken by Kuenzi et al. (1974) to indicate that the mitochondrial genome was not required for sporulation, but only for adaptation to the use of acetate which is the carbon source in sporulation medium. Subsequently, Newlon and Hall (1978) reported that sporulation was however inhibited after treatment of acetate-adapted cultures with ethidium bromide at early times during meiosis. This result suggested that ethidium bromide may be affecting cells during meiosis by its action on oxidative metabolism or that its effect may be sporulation-specific. In this study the sensitivity of meiotic cells of Saccharomyces cerevisiae to treatment with ethidium bromide has been examined by continuous exposure in sporulation medium and by interrupting meiosis by plating cells back onto vegetative medium. Meiotic cells will still revert to mitotic growth during and after meiotic DNA synthesis, when they exhibit commitment to the execution of meiotic levels of recombination. Only at the time of first spindle pole body separation in prophase I do they become committed to the execution of meiotic cell division in vegetative medium (Esposito 1980).
Materials and methods Three yeast strains were employed in this study. These were the yeast strains D7 (Zimmermann et al. 1975), SK1 (Kane and Roth 1974) and a/c~wild type diploid.
70
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis The strain D 7 has the following genotype:
a
ilvl-92
trp5-12
ade2-40
cyhr2
a
ilvl-92
trp5-27
ade2-119
CYHS2
D 7 has been frequently used in mitotic studies to assay basepair substitution mutation to isoleucine independence, gene conversion to tryptophan independence and reciprocal recombination at the adenine locus. The products of mitotic reciprocal recombination are detected as red-pink, twin sectored colonies with the ade2-119 allele giving rise to a pink pigmentation and the ade2-40 allele being responsible for the red pigmentation. These alleles exhibit heteroallelic complementation giving rise to a white diploid colony. In meiotic studies dramatic increases in tryptophan convertants and coloured colonies are found in samples plated back onto vegetative medium during meiotic DNA synthesis. These colonies are the result of a commitment by the meiotic ceils to execute meiotic recombination followed by a return to mitotic growth. Only later in meiosis are haploid coloured colonies detected after plating meiotic ceils onto vegetative medium and these indicate a commitment to execute a meiotic reductional division rather than revert directly to mitosis (Olson and Zimmermann 1978).
Growth and sporulation. All strains were grown at 28 oC in acetate
sure survival and gene conversion. In this way the time of release from ethidium bromide sensitivity was investigated. II. Media shift experiments. The strain D7 was plated onto vegetative medium containing ethidium bromide at different times during meiosis. Other experiments involved the treatment of cells in sporulation medium with ethidium bromide followed by plating back onto vegetative medium at later times. In this way the timing of events leading to ethidium bromide sensitivity was investigated. The yeast strain SK1 was also treated at different times during meiosis for 15 rain in pH 7.0 phosphate buffer and plated back onto 1% glucose complete medium to assay survival and petite mutant induction using the tetrazolium overlay technique (Ogur et al. 1957).
Plating. Appropriately supplemented minimal plates were used for scoring gene conversion (Olson and Zimmermann 1978). These plates were scored after six days. Viability was assayed on YPG medium (Cox and Bevan 1962) after three days. These plates were examined for the presence of completely coloured and sectored colonies after six days. In all cases between 50-200 colonies per plate were scored on three replicate plates after appropriate dilutions.
DNA estimation. The total DNA present in samples was estimated
presporulation medium consisting of 10 g potassium acetate, 20 g peptone and 10 g yeast extract per litre (Fast 1973). Cells were harvested at 2 x 107 cells/ml and washed three times in saline. After resuspension in sporulation medium the cells were sonicated and made up to a final concentration of 2 x 107 cells/ ml. The sporulation medium ingredients were 8.2 g sodium acetate, 1.9 g potassium chloride, 1.2 g sodium chloride and 0.35 g magnesium sulphate (von Borstel 1978). For D7 the sporulation medium was supplemented with 25 mg/l adenine, tryptophan and isoleueine. The time of resuspension of yeast cells in sporulation medium constituted time zero in meiosis and only 150 ml of culture was inoculated into a litre flask. This enabled vigorous aeration to occur which is important in obtaining high levels of sporulation and synchrony of meiotic events. The strain D7 achieved high levels of sporulation under these conditions, but the wild-type diploid used in this study sporulated at a lower level. The homothallic strain SK1 underwent a rapid and synchronous sporulation and b y 10 h had reached almost 100% sporulation.
spectrophotometrically by diphenylamine reaction (Burton 1956) using the standard method (Haber and Halvorson 1975).
Chemicals. Ethidium bromide and oligomycin were obtained
sphaeroplasted for each caesium chloride gradient. Samples were resuspended in 0.1 M EDTA at pH 7.0 and left on ice for 30 min. After this time the samples were frozen in liquid nitrogen which was previously shown not to effect the profiles. These samples were thawed and resuspended in 0.5 M EDTA pH 7.0 with 25 #1/ ml 2-mercaptoethanol and incubated for 30 min on ice. Cells were then sphaeroplasted using zymolyase 5,000 (Kirin Breweries, Japan) at 10 mg/ml in 1 M sorbitol, 0.1 M EDTA, 0.1 M Tris at pH 7.5. Incubation was at 37 °C and sphaeroplasting was complete within 5 rain for samples early in meiosis although up to 15 min was required for samples at later times.
from Sigma London Chemical Co. Ltd.
Treatment. Yeast cells were treated under two different regimes: I. Inhibition of meiosis and sporulation. Cells were exposed to the chemicals 2 h after initial inoculation into sporulation medium except for the strain SK1 which was treated after 1 h. This allowed some time for the completion of mitosis. The duration of the exposure was 48 h, after which samples were washed and the ascus frequency determined before plating onto appropriate media to examine recombination and survival. Mitotic comparisons were made to samples treated immediately after removal from presporulation medium in pH 7.0 phosphate buffer for 48 h. In this way cells were treated during a mitotic cell cycle. For treatment at different stages of meiosis ceils were removed from sporulating cultures and resuspended in sporulation medium containing ethidium bromide. The treatments were for 48 h followed by determination of sporulation frequency and plating to mea-
Testing for ploidy. Ploidy was inferred by mating and sporulation tests. Colonies were classified as diploid if they gave rise to asci in sporulation medium after five days and did not give mating figures after mixing with haploid strains of either matingtype. Similarly colonies which did not give rise to asci after five days incubation in sporulation medium, but which gave mating figures on mixing with a or a strains, were inferred to be mainly haploid. Radioactive labelling o f DNA. The DNA was labelled with (6-3H) uracil and (2-14C) uracil obtained from Amersham International. The specific activity of (6-3H) uracil was approximately 25 Ci/ mMol and was added at 6 #Ci/ml under sporulation conditions. The specific activity of (2-14C) uracil was 58 Ci/mMol and this was added at 0.5 ~Ci/ml. Labelling under presporulation conditions was overnight and after 2 h during sporulation.
Preparation of sphaeroplasts. Samples of 5 x 108 cells were
L ysis of sphaeroplasts and caesium chloride gradients. The sphaeroplasts were centrifuged at 1,000 rpm for 10 rain and resuspended in 0.001 M EDTA, 0.04 M NaCI, 0.01 M Tris HC1 buffer at pH 8.0 containing 50 ttg/ml proteinase K. The samples were vortexed for 2 min to ensure lysis of sphaeroplasts and incubated at 35 °C for 4 h. 4 ml of the samples was then added to 4.8 g CsC1 in nitrocellulose tubes. Centrifugation was at 40,000 rpm for 40 h in a
S. L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis Completion of mitosis & entry into meiosis
Meiotic DNA
Spore formation
71
100-
synthesis Meiotic cell division
80 -6 :> 60 u0
,~ 5.0
o (9
5O.~_
o o_
g
40 20
LO 2
-7. 4o *¢ :7 C3
30
~30
20 10
2.0
od
~S -5 bq
0 o~ 0
2
Z,
6
8
10 12 1L h in SPM
16
18
20
22
2L
Fig. I shows the time course of various landmark events in the sporulation of the strain DT. These include meiotic DNA synthesis indicated by the DNA content of successive samples (* *), commitment to sporulation indicated by the appearance of haploid coloured colonies after plating sporulating cells onto YPG medium (-- -') and the ascus frequency in sporulating samples (_- _-)
c 100
.2 u
8O 60
a
o~ 40 o
o_ 20 0 80 > 50 o~~
40
20 0
C U3
100 O_~
>--~
10
0
1.0 log dose (tag/ml)
10
Fig. 2a-c shows the effect of ethidium bromide treatment on sporulation inhibition aand survival b in the strains SK1 (• []), D7 ( ~ ) and an a/o~ wild type diploid ( ~ ) . c shows the effect of ethidium bromide treatment on the inhibition of meiotic gene conversion in the strain D 7 (A ~)
Beckman 50 Ti rotor and the gradients were then collected by puncturing the bottom of the tube with a needle. Between 3035 fractions were collected and 0.2 ml 0.1 M NaOH was added to each and left overnight. The fractions were put onto chromatography paper strips and the DNA precipitated in 5% TCA for 12 h. Following two 30 min rinses in ethanol the chromatography paper strips were dried at 28 °C and counted in an LKB scintillation counter.
0
g
1.'0 log dose (gg/ml)
10
Fig. 3 shows the effect of ethidium bromide on the survival of logarithmic phase acetate grown cells of the strains D7 (~ ~), SK1 ( ~ ) and an a/c~ wild-type diploid ( ~ ) after treatment for 48 h in pH7 buffer
Results Figure 1 shows the time course of some of the landmark events in the sporulation of the strain DT. Meiotic DNA synthesis, as estimated by the diphenylamine reaction, occurred between 6 and 10 h and this is coincident with an increasing frequency of gene conversion found in samples plated onto vegetative medium at these times (see Fig. 4). The first appearance of haploid, coloured colonies is also shown for samples plated from sporulating cultures onto vegetative medium. These were first detected in samples taken at 12 h into sporulation and indicates the point of first commitment of cells to the completion of meiotic cell division after medium switch. Asci were first detected at about sixteen hours into sporulation and the percentage sporulation was found to increase to about 50% at 24 h and to between 7 0 - 8 0 % at 36h. The effects of exposure of sporulating cells to ethidium bromide is shown in Fig. 2. Treatment resulted in sporulation inhibition and in a cell lethality which was approximately proportional to the level of sporulation in the untreated cultures of the different strains examined. The supersporulating strain S K 1 exhibited the greatest sensitivity to the lethal effects of ethidium bromide, followed by the strain D 7 and the wild-type diploid strain respectively. The frequency of gene conversion found in treated samples of D 7 decreased to mitotic levels in those samples where ascus formation was totallyinhibited. Figure 3 shows the effects of treating acetate adapted exponential phase mitotic cells with ethidium bromide in pH 7.0 phosphate buffer for an equivalent period to the meiotic treatment regime. In this way the cells were treated during a mitotic cell cycle. The strain D 7 and the wild-type strain exhibited no lethality under these conditions after ethidium bromide treatment although considerable induction of petite mutation occurred. However, the supersporulating strain SK1 did exhibit an equiv-
72
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
Table 1. The effect of the mitochondrial ATPase inhibitor oligomycin on viability and ascus formation in sporulating cultures of the yeast strain D7 and an a/a wild type m1-1)
0
2.5
5
10
20
40
44.9 85.6 -+6.7
21.2 89.7 ± 7.8
4.5 84.7 ± 9.1
1 81.4 -+ 11.2
0 96.6 -+ 8.5
0 78.3 ± 5.9
71.0 123.7 ± 1 0 . 6
32.0 107.6 -+8.7
0 116.7 -+16.1
0 119.7 -+6.1
a/~ Percentage sporulation Plate counts ± S.D. D7 Percentage sporulation Plate counts ± S.D.
co
60
c3
z,O o_ (/1
o~ -8
._> > ~-
2O
8O
60
113
40 20 1000 0)
"E o
~--
100
o
~ .-&
10
o
~.v 0
0
i
;
8 1'o 12 h in SPM
~
IF--
1L
Fig. 4a-c shows the effect on the strain D7 of a sporulation inhibition after resuspension in both fresh sporulation medium (~) and sporulation medium containing 5 gg/ml ethidium bromide (A zx) at different times during meiosis and b lethality after these ethidium bromide treatments, c shows the frequency of gene conversion found after plating cells onto vegetative, selective medium at different times during meiosis (~ A) and the gene conversion frequencies found for samples taken at the same times and incubated for 48 h in fresh sporulation medium (~) and sporulation medium containing 5 ~g/ml ethidium bromide (o o)
alent lethality to that of treatment in sporulation medium, but examination of untreated samples revealed that the strain SK1 underwent almost 100% sporulation in pH 7.0 phosphate buffer under these conditions, i.e. these cells were undergoing meiosis in phosphate buffer unlike the other strains used.
2.3 114 +-9.9
0.2 127.7 _+6.7
The possibility that the action of ethidium bromide demonstrated with sporulating cells was by the inhibition of oxidative metabolism was examined by treating cells with oligomycin at concentrations which inhibit mitochondrial ATPase (Avner et al. 1973). Table 1 shows that oligomycin treatment produced no detectable lethal effects on sporulating cells at doses which totally inhibit ascus formation, i.e. with this drug there was a clear separation between cell lethality and the inhibition ofsporulation. However, the effects of oligomycin on respiration were not measured here. Figure 4 shows the effects of treating D7 in sporulation medium containing ethidium bromide at a concentration of 5 pg/ml at different times during sporulation followed by incubation for 48 h. Both ascus formation and meiotic gene conversion were inhibited in cultures treated prior to the time of meiotic DNA synthesis and these samples also exhibited lethality. However, reduced lethality and increasing levels of gene conversion were found in samples treated during meiotic DNA synthesis and this protection from the effects of ethidium bromide on lethality and on the inhibition of meiotic gene conversion appeared complete following meiotic DNA synthesis. The transition point for the effect of ethidium bromide on sporulation inhibition may however be later than for meiotic gene conversion and lethality. The increasing frequency of gene conversion in samples treated during meiotic gene conversion also closely paralleled the increases found in meiotic samples plated back onto vegetative medium to assay commitment to recombination at these times. This indicates that escape from the effects of ethidium bromide on meiotic gene conversion inhibition is closely correlated with the time of commitment to recombination, which is itself correlated with the period of meiotic DNA synthesis. Figure 5 shows a similar experiment carried out with the supersporulating strain SK1. Figure 5a shows the time of meiotic DNA synthesis, as assayed by the diphenylamine reaction, occurring between 2 - 4 h and that
S. L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
(3
80 c ._o
% 5.0 (9
% 4.0
60 ~ ~-
~s.o
2O o~
73
-6 100 ->
~ 75
40 o
Z
CI
g
~
50
b
=
:
:
25 o o o
~ 60 S~20
100
X3 >
% Q- 60
o o
40 0
, 2
,
, 4 6 h in 5PM
, 8
d_ 0
10
sporulation ( ~ ) at these different times; b shows the percentage sporulation ( ~ ) and survival (o o) of samples taken during the sporulation of SK1 and incubated for 48 h in sporulation medium containing 5 ~tg/ml ethidium bromide and c shows the percentage petite induction in samples taken during the sporulation of SKI and treated in pH 7.0 buffer for 15 min with 5 #g/rot ethidium bromide prior to plating
~80
o
100 ~o~
,7. o c
o_~
10
c5_.~
> 0- ------r O 2
/~
;
4
6
8
1'0
I'2 l'L
24
h in SPM
Fig. 5 a - c shows for the strain S K I a the DNA content ( ~ - ~ ) in successive samples of a sporulating culture and the percentage
100 ~- 9 0
II--
0 2
1;' h in SPM 8
1'2
. fr14 24
Fig. 6 a shows the effect on survival of plating onto vegetative medium containing 5 #g/ml ethidium bromide at different times of meiosis for the strain D 7 (~- ~-) and in b the frequency of gene conversion after media switch onto normal selective medium (-- e) and selective medium containing ethidium bromide at 5 ~g/ml (,~-~A) is shown
asci first appeared at 7 h and increased to almost 100% at 10 h. Figure 5b shows that for the strain SKI, as with D7, the lethal effects of ethidium bromide were reduced at the time of meiotic DNA synthesis and no lethal effects of ethidium bromide treatment were found after meiotic DNA synthesis. As with D7 protection from the
Fig. 7 shows a the percentage survival found for cultures of D 7 treated with 5 ~g/ml ethidium bromide at 2 h (-,) after inoculation into sporulation medium followed by plating onto vegetative medium at later times; b shows the gene conversion frequency at trp5 in D 7 found for untreated cultures ( ~ _ A ) , and cultures treated with 5 #g/ml ethidium bromide at 2 h (~---~) and 4 h ( - ~ ) after inoculation into sporulation medium, followed by plating at intervals onto vegetative, selective medium
effects of ethidium bromide on ascus formation in SK1 appeared later than for lethality. Figure 5c shows the results of a separate experiment involving the treatment of samples of a sporulating culture of the strain SKI with 5/~g/ml ethidium bromide in pH 7.0 phosphate buffer for 15 rain followed by plating on low glucose complete medium. This treatment regime did not produce cell lethality and Fig. 5c shows that some cyclic variation in petite mutant induction was found after treatment at the different times of meiosis. The induction of petites by ethidium bromide at the later times, after meiotic cells exhibit protection from the lethal effects of ethidium bromSde, indicates that the protection is not associated with an absolute permeability barrier against ethidium bromide. Figure 6 shows the effects of the interruption of meiosis in D7 followed by plating onto vegetative medium containing ethidium bromide at 5/~g/ml. In this experiment cells can revert directly to mitosis after meiotic DNA synthesis up to the point of commitment to sporulation, which occurs for the most advanced cells at about 12 h. No effect of this treatment on lethality, or on the kinetics of commitment to meiotic gene conversion, was detected even during meiotic DNA synthesis. This suggested that while protection from ethidium bromide induced lethality occurs at the time of meiotic DNA synthesis, continued incubation in sporulation medium might be necessary for the manifestation of the ethidium
74
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
3000
1500
~E (3_
~E 13..
ca
2000
1000 ca
1000
500
r CO
(.D ..t
J_A d
1500
3000
r
ca 2000
i
13-
1000 ca 7-
r
ca
500
1000
0
0 400
f
~E 13..
ca 200
J\ 01 Distance sedimented
Fig. 8a-f shows the caesium chloride gradients of samples of 5 x 108 cells of the strain D7 labelled with (3H-6) uracil (-- --) after 2 h in sporulation medium with and without treatment with 5 gg/ml ethidium bromide; a-d show the profiles of untreated samples taken at a 2 h, b 6 h, c 10 h and d 12 h into sporulation where the parental DNA is labelled using (2-14C) uracil (; ;); e-f show the profiles of samples taken at e 2 h and f 12 h after treatment with ethidium bromide and where the position of the nuclear and mitochondrial DNA is indicated by 14C labelled yeast marker DNA (-)
bromide induced effects and that cells could be rescued by reverting to mitotic growth. Figure 7 shows the effect of adding 5 #g/ml of ethidium bromide to sporulation medium at early times during meiosis (2 h and 4 h) in D7, followed by plating samples onto vegetative medium at subsequent times to investigate the timing of events resulting in ethidium bromide induced lethality. The time of meiotic DNA synthesis and commitment to meiotic gene conversion in
untreated cultures was found to be the time when increasing lethality resulted after plating ethidium bromide treated ceils back onto vegetative medium. Prior to the time of meiotic DNA synthesis in untreated cultures ethidium bromide treatment did not result in detectable lethality on plating back onto vegetative medium. Ethidium bromide treatment also totally inhibited meiotic gene conversion. In order to investigate the association of ethidium bromide sensitivity with meiotic DNA synthesis the effect of ethidium bromide treatment on the incorporation of DNA precursors into the nuclear and mitochondrial DNA in D7 was examined using caesium chloride gradient analysis (Fig. 8). This was undertaken by labelling sporulating cultures with (6-3 H) uracil 2 h after inoculation into sporulation medium and taking subsequent samples of 5 x 10 s cells for each caesium chloride gradient. Incorporation of aH label into the nuclear and mitochondrial DNA was detected through the period of meiotic DNA synthesis in untreated cultures (Fig. 8a-8d), where the parental DNA was labelled with (2J4c) uracil under presporulation conditions. No incorporation of 3H label was detected into the nuclear or mitochondrial DNA when ethidium bromide was added at 5 #g/ml at the time of addition of (6-3H) uracil, i.e. 2 h after resuspension of the culture in sporulation medium. The position of the nuclear and mitochondrial DNA in these ethidium bromide treated samples was indicated by ( 2 J 4 C ) marker yeast DNA (Fig. 8e-8f).
Discussion The ability of acetate adapted yeast cultures to sporulate after treatment with ethidium bromide prior to inoculation into sporulation medium (Kuenzi et al. 1974; Newlon and Hall 1978; Kelly 1982), indicates that the mitochondrial genome is not required for sporulation except to permit adaptation to growth on acetate as a non-fermentable carbon source. Newlon and Hall (1978) reported that treatment with ethidium bromide at early times of meiosis did however result in an inhibition o f sporulation. We have confirmed these findings and further demonstrated that ethidium bromide treatment of ceils early in meiosis inhibits nuclear and mitochondrial DNA synthesis which has the effect of blocking meiotic gene conversion and sporulation. This inhibitory effect of ethidium bromide is also correlated with a lethal effect which increases with the sporulation competance o f the strains used and suggests that the lethal effect may be associated with attempted sporutation. Support for a sporulation-specific action of ethidium bromide comes from the absence of lethality in acetateadapted, mitotic cells treated during the division prior to
S. L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis inoculation into sporulation medium (Kelly 1982), from the absence of lethality found for growing, acetate-adapted cells treated in buffer and from the sensitivity of the strain SKI in buffer where it can sporulate. The origin of the effect of ethidium bromide on meiotic cells may not be mitochondrially-associated, but may be associated with an effect on meiotic DNA synthesis. This is supported by the finding that oligomycin can inhibit sporulation in the absence of lethality indicating that there may be more to the action of ethidium bromide than by blocking oxidative metabolism. Similarly erythromycin, an inhibitor of mitochondrial protein synthesis, has also been reported to inhibit sporulation in the absence of lethality (Marmiroli et al. 1981), but in the sporulation conditions used here concentrations of erythromycin up to 2 mg/ml do not inhibit sporulation in the erythromycin-sensitive strain D 7 (Kelly 1982). This suggests that the absence of mitochondrial protein synthesis is insufficient to explain the lethal effect of ethidium bromide on meiotic cells. It has also been reported that the addition of glucose reverses the inhibition of sporulation by ethidium bromide (Tsuboi et al. 1974), which might suggest a mitochondrial origin for the effect of ethidium bromide. However, we have found no such reversal of the effects of ethidium bromide on sporulation, lethality, or meiotic gene conversion up to doses of glucose which themselves inhibit sporulation. Protection from the lethal effects of ethidium bromide treatment was found to coincide with the time of meiotic DNA synthesis in the strains SKI and DT. This suggests that the origin of the ethidium bromide sensitivity of meiotic cells may lie in the mechanism of inhibition of meiotic DNA synthesis. Further support for this hypothesis is derived from the similarity between the kinetics of commitment to meiotic gene conversion (an indication of the proportion of the culture having undergone or undergoing meiotic DNA synthesis) and the level of meiotic gene conversion found in ethidium bromide treated samples of D7. The successive sampling of cultures treated with ethidium bromide prior to meiotic DNA synthesis followed by plating onto vegetative medium also identified events surrounding meiotic DNA synthesis with ethidium bromide induced lethality. Increasing lethality was observed after plating cells from treated cultures back onto vegetative medium during the period of meiotic DNA synthesis in untreated cultures. Similar changes in survival have been observed for some recombination-deficient mutants of Saccharomyces cerevisiae which undergo meiotic DNA synthesis but give inviable products, probably as a result of failed recombination (Game et al. 1980). The effect of ethidium bromide on meiotic cells differs however, as mitochondrial and nuclear DNA synthesis were inhibited, but it is possible that the lethality observed here may result from some defective recombination event.
75
The media switch experiments, involving plating cells from untreated sporulating cultures onto vegetative medium containing ethidium bromide, did not reveal any effect of ethidium bromide on survival, or on the kinetics of commitment to meiotic gene conversion. This finding may still be reconciled with an effect of ethidium bromide on meiotic DNA synthesis if the sensitivity is the result of an effect prior to, or at the initiation point of meiotic DNA replication. Cells prior to this point would revert to mitosis directly after media switch having not encountered the ethidium bromide sensitive phase and cells undergoing meiotic DNA synthesis would have passed the ethidium bromide sensitive phase and exhibit commitment to meiotic recombination. This hypothesis would explain the absence of detectable lethality in samples of cells plated onto vegetative medium containing ethidium bromide during meiotic DNA synthesis. Protection from the inhibitory effects of ethidium bromide on sporulation for the strains studied appeared to be later than for protection from the effects on survival and meiotic gene conversion. This may indicate other effects of ethidium bromide on meiotic ceils, perhaps during prophase I. One such possible effect might be the induction of pachytene arrest as is found during elevated temperature stress during meiosis (Byers and Goetsch 1982), or perhaps on the DNA metabolism associated with meiotic prophase that has been shown in Lilium (Stern and Hotta 1973). The origin of the lethal effect of ethidium bromide on meiotic cells remains unclear, but the data presented indicates its action to be sporulation-specific, or due to an event coupled to sporulation which is correlated with meiotic DNA synthesis. The effect need not be associated with the mitochondria, which appear to be necessary in sporulation only as a site for oxidative metabolism, and it is possible that the lethal effect is associated with an effect of ethidium bromide on the nuclear genome. This might be the result of defective replication initiation during meiosis in the presence of ethidium bromide, but further work is required to distinguish between this and other possibilities, such as the occurrence of attempted recombination in the absence of DNA synthesis. Acknowledgements. This work was supported by Euratom and the Royal Society. During the course of the work one of us (SLK) held a B.P. SRC CASE studentship. We would like to thank Dr. Tony Regnier of B.P. Ltd. for his support for this work.
References Avner PR, Coen D, Dujon B, Sloniminski PO (1973) Mol Gen Genet 125:9-52 Burton K (1956) Biochem J 62:315-373 Byers B, Goetsch L (1982) Mol Gen Genet 187:47-53 Cox BS, Bevan EA (1962) New Phytologist 61:342-355
76
S.L. Kelly and J. M. Parry: Sensitivity to ethidium bromide during yeast meiosis
Esposito MS, Esposito RE (1974) Proc Natl Acad Sci USA 71: 3172-3176 Esposito RE (1980) Workshop report on sporulation in Tenth International Conference on Yeast Genetics and Molecular Biology, pp 1 9 - 2 5 Fast D (1973) J Bacteriol 116:925-30 Game JC, Zamb TJ, Braun RJ, Resnick M, Roth RM (1980) Genetics 9 4 : 5 1 - 6 8 Goldring ES, Grossmann LI, Knipnick D, Cryer DR, Marmur J (1970) J Mol Biol 52:323 Haber JE, Halvorson HO (1975) Methods in sporulation and germination of yeasts. In: Prescott DM (ed) Methods in Cell Biology, vol XI. Academic Press, pp 4 5 - 6 9 Kane SM, Roth R (1974) J Bacteriol 1 1 8 : 8 - 1 4 Kelly SL (1982) A comparative study of genetic change in meiotic and mitotic cells of Saecharomyces cerevisiae. Ph. D. thesis, University of Wales Kuenzi MT, Tingle MA, Halvorson HO (1974) J Bacteriol 117: 80-88 Marmiroli N, Tassi F, Bianchi L, Algeri AA, Puglisi PP, Esposito MS (1981) Curr Genet 4 : 5 1 - 6 2 Newlon MC, Hall BD (1978) Mol Gen Genet 165:113-114
Ogur M, St John R, Nagai S (1957) Science 125:928 Olson LW, Zimmermann FK (1978) Mol Gen Genet 1 6 6 : 1 5 1 159 Perlman PS, and Mahler HR (1971) Nature New Biol. 2 3 1 : 1 2 16 Puglisi PP, Zennaro E (1971) Experientia 27:963-964 Slonimski PP, Perrodin G, Croft HH (1968) Biochem Biophys Res Commun 30:232 Stern H, Hotta Y (1973) Ann Res Genet 7 : 3 7 - 6 6 Tsuboi M, Kona K, Yansishima W (1974) Arch Microbiol 99: 295 - 3 0 5 Von Borstel RC (1978) Measuring spontaneous mutation rates in yeast. In: Prescott D-M (ed) Methods in cell biology, vol XX. Academic Press, pp 1 - 2 4 Zimmermann FK, Kern R, Rosenberger H (1975) Mutat Res 38: 381-388
C o m m u n i c a t e d b y B. S. Cox Received April 20 / August 15, 1983
