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The effects of BC, 4CMB and 4HMB on meiosis in yeast cells
Mutation Research, 100 (1982) 173-177
173
Elsevier Biomedical Press
THE EFFECTS OF BC, 4CMB AND 4HMB ON MEIOSIS IN YEAST CELLS
S.L. KELLY and JAMES M. PARRY Department of Genetics, University College of Swansea, Swansea SA2 8PP (Great Britain)
(Received 12 April 1981) (Accepted 19 May 1981)
SUMMARY BC, 4HMB and 4CMB have been assayed for their effects upon meiotic cell division in yeast. All 3 compounds inhibited sporulation in strain D7. However, 4CMB showed activity at concentrations which did not induce significant lethality. This effect was also evident for the inhibition of meiotic gene conversion and for the production of coloured colonies.
The vast majority of the data in short-term mutagenicity tests are derived from the use of mitotically dividing cells. When estimating genetic risk it is important that the induction of genetic change in meiosis does not differ significantly from that induced in mitotic test systems. The yeast Saccharomyces cerevisiae provides a useful system for studying genetic change in meiosis as the meiotic cell cycle may be induced by media shift. The most obvious differences between meiosis and mitosis are the dramatic increases of both intergenic and intragenic recombination (Prakash et al., 1980). These increases are intimately associated with premeiotic DNA synthesis (Silva-Lopez et al., 1975). It has also been reported that a 'meiotic effect' of enhanced rates of spontaneous frameshift mutation exists in Saccharomyces cerevisiae (Von Borstal et al., 1964). We are currently evaluating the effects of genetically active compounds upon a variety of yeast strains during meiosis and have assayed the 3 trial compounds for their effects on meiosis in strain D7. This strain permits the assay of mutation, meiotic gene conversion and the production of pink and red colonies by recombination and segregation at the ade 2 locus.
0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
174 MATERIALS AND METHODS
Strain The genotype of D7 is: a c~
ilv 1-92 ilv 1-92
trp 5-12 trp 5-27
ade 2-40 ade 2-119
Growth and sporulation The strain was grown in acetate presporulation medium to a final concentration of 2 x 107 cells/ml. Cells were harvested, washed and resuspended in sporulation medium. Under control conditions premeiotic D N A synthesis and commitment to intragenic recombination begins at 6 h and lasts approximately 4 h. Asci appear at 16 h and rise to a final level of 60-65°7o. Treatment Cells were treated with the chemicals in sporulation medium after a period of 1.5 h had elapsed for the completion of mitosis. The chemicals were dissolved in DMSO and exposure was for 48 h.
100
v
t\ MAX. SPORULATION
~
0
0
, 50
B
C
~-HMB |
]00
DOSE(uglml)
Fig. 1. Inhibition of sporulation after the compounds 4CMB (11), 4HMB (V), BC (e) and MNNG (D).
175
Inhibition of sporulation This was assayed as the percentage asci as indicated by haemocytometer and was plotted as a percentage of the control level of sporulation.
Plating (i) Appropriate minimal plates were used for gene conversion and mutation (Zimmerman, 1973). These plates were scored after 6 days. (ii) Viability was assayed on complete media after 3 days. These plates were examined for the presence of completely coloured and sectored colonies after 6 days. RESULTS AND DISCUSSION The 3 compounds do not induce increases in mutation frequency at the ilv-1 locus. They do, however, inhibit sporulation from the control level of 63°7o but 4CMB is active at lower concentrations as shown in Fig. 1, and at doses that do not "induce significant lethality (as shown in Fig. 2). BC and 4HMB begin to inhibit the formation of asci at doses that induce lethality. The inhibition appears to be associated with a block of the entire meiotic cycle as the fall in percentage asci
lO0
%
SURVIVAL 50
o
i0
m
lOO
DOSE (ug/ml)
Fig. 2. Survival of the strain D7 after 48 h treatment with 4CMB (m), 4HMB (~), BC ( • ) and MNNG (C3).
176
corresponds with a reduction in the level of gene convertants and coloured colonies as shown in Figs. 3 and 4 respectively. This indicates that the cells are being inhibited from passing through premeiotic DNA synthesis. This is unlike treatment with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which produces the full meiotic level o f gene conversion in the absence of asci. The alkylating agents appear to be potent inducers of mitotic gene conversion but this level is reduced to spontaneous levels during commitment to meiosis (Kelly, unpublished). It appears that BC and 4HMB do not inhibit premeiotic DNA synthesis to the same extent as 4CMB as cells treated with these compounds still exhibit meiotic levels of gene conversion. The test compounds appear to act in a different manner to the alkylating agents we have previously studied. They block meiosis before premeiotic DNA synthesis unlike the alkylating agents which appear to block before the point of commitment to ascus formation. The compound 4CMB was most active of the 3. However, this is not a clear indication that it is most active genetically since sporulation may be blocked by mechanisms such as the repression of the enzymes of the tricarboxylic acid cycle or the presence of critical concentrations of nitrogenous compounds.
2.25-
2.0 < m
-
~
1.75
zY -I
•
1.5
A
d~ 1.25 0
1.0
0 DQ6E
|
v
50
100
(~uglml)
F!g. 3. Inhibition of meiotic gene conversion after 48 h treatment with 4CMB ( I ) , 4HMB (V), BC ( • ) and MNNG (D).
177 35
V
30
25 o o
St'= 2 0 m 0
o
SZ
15
m
10
I
o
0
p 50
i
100
OOSE(,ugtmO Fig. 4. Percentage of coloured colonies on viability plates after 48 h treatment with 4CMB ( I ) , 4HMB (V), BC ( I ).
ACKNOWLEDGEMENTS
Part of this work was supported by funds from the EEC Environmental Programme. One o f us (SLK) held an S.R.C. research studentship during the course o f the work. We would like to express his thanks to Dr. A. Regnier of BP for his interest in the work. REFERENCES Prakash, S., L. Prakash, W. Burke and B.A. Montclone (1980) Effects of the RAD52 gene on recombination in Saccharomyces cerevisiae, Genetics, 94, 31-50. Silva-Lopez, E., T.J. Zamb and R. Roth (1975) Role of premeiotic replication in gene conversion, Nature (London), 253, 212-214. Von Borstal, R.C., M.J. Bond and C.M. Steinberg (1964) Spontaneous reversion rates of a supersuppressible mutant during meiosis, Genetics, 50, 293. Zimmerman, F.K. (1973) Detection of genetically active chemicals using yeast systems, in: A. Hollaender (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. 3, Plenum, New York, pp. 209-239.
173
Elsevier Biomedical Press
THE EFFECTS OF BC, 4CMB AND 4HMB ON MEIOSIS IN YEAST CELLS
S.L. KELLY and JAMES M. PARRY Department of Genetics, University College of Swansea, Swansea SA2 8PP (Great Britain)
(Received 12 April 1981) (Accepted 19 May 1981)
SUMMARY BC, 4HMB and 4CMB have been assayed for their effects upon meiotic cell division in yeast. All 3 compounds inhibited sporulation in strain D7. However, 4CMB showed activity at concentrations which did not induce significant lethality. This effect was also evident for the inhibition of meiotic gene conversion and for the production of coloured colonies.
The vast majority of the data in short-term mutagenicity tests are derived from the use of mitotically dividing cells. When estimating genetic risk it is important that the induction of genetic change in meiosis does not differ significantly from that induced in mitotic test systems. The yeast Saccharomyces cerevisiae provides a useful system for studying genetic change in meiosis as the meiotic cell cycle may be induced by media shift. The most obvious differences between meiosis and mitosis are the dramatic increases of both intergenic and intragenic recombination (Prakash et al., 1980). These increases are intimately associated with premeiotic DNA synthesis (Silva-Lopez et al., 1975). It has also been reported that a 'meiotic effect' of enhanced rates of spontaneous frameshift mutation exists in Saccharomyces cerevisiae (Von Borstal et al., 1964). We are currently evaluating the effects of genetically active compounds upon a variety of yeast strains during meiosis and have assayed the 3 trial compounds for their effects on meiosis in strain D7. This strain permits the assay of mutation, meiotic gene conversion and the production of pink and red colonies by recombination and segregation at the ade 2 locus.
0165-1218/82/0000-0000/$02.75 © Elsevier Biomedical Press
174 MATERIALS AND METHODS
Strain The genotype of D7 is: a c~
ilv 1-92 ilv 1-92
trp 5-12 trp 5-27
ade 2-40 ade 2-119
Growth and sporulation The strain was grown in acetate presporulation medium to a final concentration of 2 x 107 cells/ml. Cells were harvested, washed and resuspended in sporulation medium. Under control conditions premeiotic D N A synthesis and commitment to intragenic recombination begins at 6 h and lasts approximately 4 h. Asci appear at 16 h and rise to a final level of 60-65°7o. Treatment Cells were treated with the chemicals in sporulation medium after a period of 1.5 h had elapsed for the completion of mitosis. The chemicals were dissolved in DMSO and exposure was for 48 h.
100
v
t\ MAX. SPORULATION
~
0
0
, 50
B
C
~-HMB |
]00
DOSE(uglml)
Fig. 1. Inhibition of sporulation after the compounds 4CMB (11), 4HMB (V), BC (e) and MNNG (D).
175
Inhibition of sporulation This was assayed as the percentage asci as indicated by haemocytometer and was plotted as a percentage of the control level of sporulation.
Plating (i) Appropriate minimal plates were used for gene conversion and mutation (Zimmerman, 1973). These plates were scored after 6 days. (ii) Viability was assayed on complete media after 3 days. These plates were examined for the presence of completely coloured and sectored colonies after 6 days. RESULTS AND DISCUSSION The 3 compounds do not induce increases in mutation frequency at the ilv-1 locus. They do, however, inhibit sporulation from the control level of 63°7o but 4CMB is active at lower concentrations as shown in Fig. 1, and at doses that do not "induce significant lethality (as shown in Fig. 2). BC and 4HMB begin to inhibit the formation of asci at doses that induce lethality. The inhibition appears to be associated with a block of the entire meiotic cycle as the fall in percentage asci
lO0
%
SURVIVAL 50
o
i0
m
lOO
DOSE (ug/ml)
Fig. 2. Survival of the strain D7 after 48 h treatment with 4CMB (m), 4HMB (~), BC ( • ) and MNNG (C3).
176
corresponds with a reduction in the level of gene convertants and coloured colonies as shown in Figs. 3 and 4 respectively. This indicates that the cells are being inhibited from passing through premeiotic DNA synthesis. This is unlike treatment with the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which produces the full meiotic level o f gene conversion in the absence of asci. The alkylating agents appear to be potent inducers of mitotic gene conversion but this level is reduced to spontaneous levels during commitment to meiosis (Kelly, unpublished). It appears that BC and 4HMB do not inhibit premeiotic DNA synthesis to the same extent as 4CMB as cells treated with these compounds still exhibit meiotic levels of gene conversion. The test compounds appear to act in a different manner to the alkylating agents we have previously studied. They block meiosis before premeiotic DNA synthesis unlike the alkylating agents which appear to block before the point of commitment to ascus formation. The compound 4CMB was most active of the 3. However, this is not a clear indication that it is most active genetically since sporulation may be blocked by mechanisms such as the repression of the enzymes of the tricarboxylic acid cycle or the presence of critical concentrations of nitrogenous compounds.
2.25-
2.0 < m
-
~
1.75
zY -I
•
1.5
A
d~ 1.25 0
1.0
0 DQ6E
|
v
50
100
(~uglml)
F!g. 3. Inhibition of meiotic gene conversion after 48 h treatment with 4CMB ( I ) , 4HMB (V), BC ( • ) and MNNG (D).
177 35
V
30
25 o o
St'= 2 0 m 0
o
SZ
15
m
10
I
o
0
p 50
i
100
OOSE(,ugtmO Fig. 4. Percentage of coloured colonies on viability plates after 48 h treatment with 4CMB ( I ) , 4HMB (V), BC ( I ).
ACKNOWLEDGEMENTS
Part of this work was supported by funds from the EEC Environmental Programme. One o f us (SLK) held an S.R.C. research studentship during the course o f the work. We would like to express his thanks to Dr. A. Regnier of BP for his interest in the work. REFERENCES Prakash, S., L. Prakash, W. Burke and B.A. Montclone (1980) Effects of the RAD52 gene on recombination in Saccharomyces cerevisiae, Genetics, 94, 31-50. Silva-Lopez, E., T.J. Zamb and R. Roth (1975) Role of premeiotic replication in gene conversion, Nature (London), 253, 212-214. Von Borstal, R.C., M.J. Bond and C.M. Steinberg (1964) Spontaneous reversion rates of a supersuppressible mutant during meiosis, Genetics, 50, 293. Zimmerman, F.K. (1973) Detection of genetically active chemicals using yeast systems, in: A. Hollaender (Ed.), Chemical Mutagens: Principles and Methods for their Detection, Vol. 3, Plenum, New York, pp. 209-239.