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The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae
Mutation Research, 108 (1983) 109-120
109
Elsevier BiomedicalPress
The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae S.L.Kelly and James M. Parry Department of Genetics, University Collegeof Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)
(Received4 May 1982) (Revisionreceived27 August 1982) (Accepted9 September 1982)
Summary Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.
The yeast Saccharomyces cerevisiae is convenient organism for studies upon the meiotic and mitotic cell cycles (Esposito and Esposito, 1975; Hartwell, 1978). We have investigated the sensitivity of cells to UV during the two alternative cell cycles as part of a programme of investigation of the comparative effects of a wide range of environmental mutagens. Variation in sensitivity to ultraviolet irradiation has been reported for synchronised mitotic yeast cultures (Esposito, 1968; Parry and Cox, 1968; Chanet et al., 1973; Davies et al., 1978). The period of greatest cell sensitivity to the lethal effects of UV was observed in the G1 phase of growth followed by a period of greater resistance during the S and G2 phases, an effect which has been attributed to the activity of recombinational-repair mechanisms (Fabre, 1973). The G1 sensitivity observed corresponded with maximal induction of various genetic end-points. Meiosis may be induced in Saccharomyces cerevisiae by a media shift which results in the completion of mitosis, meiotic DNA synthesis, meiotic cell division and 0027-5107/83/0000-0000/$03.00 © ElsevierSciencePublishers
110
the formation of haploid spores in an ascus (Haber and Halvorson, 1975). In studies on meiosis it was found that cells plated back onto vegetative medium during meiosis become committed to meiotic levels of recombination before they become committed to the execution of the events leading to ascus formation (Sherman and Roman, 1963; Olson and Zimmerman, 1978). The process of commitment to recombination was shown to be intimately associated with the meiotic DNA synthesis (Silva-Lopez et al., 1975) while commitment to meiotic cell division and sporulation occurs at the time of the first spindle pole body separation (Esposito, 1980). An increase in the level of genetic recombination is the most dramatic difference observed between meiosis and mitosis, but it has also been reported that a 'meiotic effect' of enhanced spontaneous frameshift mutation exists in yeast (Von Borstel et al., 1964) and that certain agents may have a mutagenic effect ~i~ meiosis and an antimutagenic effect in mitosis (Puglisi, 1967). The effect of UV light on meiotic cells in yeast has been the subject of conflicting reports (Simchen et al., 1973; Hottinguer-de Margerie and Moustacchi, 1979; Machida and Nakai, 1980a, 1980b). Simchen et al. found an increased sensitivity for viability and for the ability to continue through sporulation to form asci at a stage comparable to meiotic prophase I. In a later paper (Salts et al., 1976), reduced recombination was reported after UV irradiation at the same stage. In their study Hottinguer-de Margerie and Moustacchi (1979) reported that meiotic cells exhibit increasing sensitivity until meiotic DNA synthesis begins, while Machida and Nakai (1980) found no evidence of cyclical variations in UV-induced lethality. We report here the effect of UV irradiation during meiosis in yeast on the strain D7 and on a supersporulating strainSK1.
Materials and methods Strains. The strains D7 (Zimmermann etal., 1975) and SK1 (Kane and Roth, 1974) were used in this investigation. The strain SK1 is homothallic and sporulates rapidly to a high level. The strain D7 has the following genotype:
a ilv 1-92 trp 5-12 ade 2-40 cyff-2 a ilvl-92 trp 5-27 ade2-119 CYHS.2 D7 has been frequently used in mitotic studies to assay base-pair substitution mutation to isoleucine independence, intragenic recombination to tryptophan independence and reciprocal intergenic recombination at the adenine locus. The product of mitotic intergenic recombination is detected as a red-pink, twin-sectored colony. The allele ade 2-119 is responsible for the pink pigmentation and the allele ade 2-40 is responsible for the red pigmentation. The combination of these alleles in a diploid results in heteroalleic complementation to give a white colony colour. D7 was used in this work to study base-pair substitution mutation, intragenic recombination, survival and intergenic recombination and chromosome segregation
during meiosis using the method of plating cells on vegetative medium from sporulating cultures. Confirmation of any variations in survival after irradiation was obtained by study of the response of the supersporulator SKI. Growth and sporulation. The strains were grown at 28°C in acetate presporulation medium consisting of 10 g potassium acetate, 20 g peptone and 10 g yeast extract per 1 (Fast, 1973). Cells were harvested at 2 X 10’ cells/ml, and washed 3 times ins saline. On resuspension in sporulation medium the cells were sonicated and made up to a final concentration of 2 X 10’ cells/ml. The sporulation medium chloride, 1.2 g sodium ingredients were 8.2 g sodium acetate, 1.9 g potassium chloride and 0.35 g magnesium sulphate per 1 (Von Borstel, 1978), supplemented with 25 mg/l adenine, tryptophan and isoleucine. The resuspension of cells in sporulation medium was taken as time zero in meiosis in the experiments and only 150 ml of culture was inoculated into a litre flask. This enabled vigorous aeration to occur which is important in maintaining high levels of sporulation and synchrony of meiotic events. Meiosis in the strain 07 has been previously studied (Olson and Zimmermannn, 1978) and achieves a high level of sporulation with good synchrony under the conditions described here. Treatment. Samples were taken at different times during meiosis and resuspended at 2 x 10’ cells/ml in 10 ml of pH 7.0 phosphate buffer. This was irradiated in a 9-cm petri dish with UV light at 254 nm from a Hanovia Ila low pressure mercury discharge tube monitored by the use of a calibrated photocell. All manipulations were performed in red light to avoid photoreactivation. Plating. Appropriate minimal plates were used for scoring intragenic recombination and mutation (Olson and Zimmermann, 1978). These plates were scored after 6 days. Viability was assayed on YPG medium (Cox and Bevan, 1962) after 3 days. These plates were examined for the presence of completely coloured and sectored colonies after 6 days. DNA estimation. The DNA present in samples was estimated spectrophotometrically by the diphenylamine reaction using the standard method (Haber and Halvorson, 1975). Testing for ploidy. Ploidy was inferred by a mating and sporulation test. Colonies were taken to be diploid if they gave rise to asci in sporulation medium after 5 days and did not give mating figures on mixing with haploid strains of either mating type. Similarly, colonies which did not give asci after 5 days’ incubation in sporulation medium but which gave mating figures on mixing with a or OLhaploid strains were inferred to be mainly haploid. Cell counts. Per cent sporulation and estimates of cell concentration were calculated after scoring a minimum of 500 cells on a haemocytometer slide. The results presented here are representative of experiments repeated several times. Results
Fig. 1 illustrates some of the landmark 07. The period of meiotic DNA synthesis
events which occur during sporulation in began at around 6 h after inoculation into
112
'Completion of mitosis and entry into the meiotic cycle
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sporulation medium. D N A synthesis was largely over after 10 h in sporulation medium and this has been confirmed by studies using a labelled D N A precursor (Kelly, unpublished). The first asci appeared after about 16 h in sporulation medium and this level increased to about 50% sporulation by 24 h. The final level of sporulation was between 70-80%. In the specific experiment whose data is presented here, the final level of sporulation was 79.6%. The percentage of red and pink colonies which were inferred to be haploid was first found to rise above zero in samples plated from sporulating cultures onto vegetative medium after 12 h. This indicates that cells start becoming committed to sporulation between 11 and 12 h after inoculation into sporulation medium and at times before this, cells revert to mitosis after plating onto vegetative medium. Fig. 2 shows the spontaneous mutation frequency as well as the induced mutation frequency and cell survival when samples were irradiated with 110 J / m 2 at various time during meiosis. The spontaneous mutation frequency was found to be unchanged throughout meiosis, but lethality and the induced mutation frequency were found to show cyclic variations. The cells were more sensitive to the effects of UV prior to their entry into meiotic D N A synthesis and this sensitivity corresponds to a 2-3-fold increase in the induction of mutation at the ilv 1-92 locus. Following this period of sensitivity, the
113
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cells showed increased resistance to the lethal effects of UV during meiotic D N A synthesis and for a period to just beyond the point of first commitment to sporulation. The induced mutation frequency was reduced as the cells enter this period of D N A synthesis and throughout meiotic prophase I. No evidence of an increased sensitivity of cells during meiotic prophase I was found. Cultures irradiated at a late time in sporulation, i.e. at 24 h, exhibited greater sensitivity to the lethal effects of UV irradiation and a higher mutation frequency was induced. This irradiation time corresponded to a point where sporulation had reached 50%.
114 In order to confirm the meiotic origin of these responses, the yeast strain SKI was also used. This strain undergoes rapid and synchronous sporulation as shown in Fig. 3. In SK1, DNA synthesis was found to begin between 2-2.5 h after inoculation into sporulation medium and the DNA content in successive samples increased until 4 h after inoculation. The first tetrads appeared after only 7.5 h incubation in sporulation medium and the percentage sproulation increased rapidly to almost 100% after 10 h in sporulation medium. The response of SK1 to irradiation during meiosis was found to confirm the data obtained for D7, with increased sensitivity to the lethal effects of UV irradiation in samples taken prior to the initiation of the meiotic DNA synthesis. Greater resistance was found in cultures undergoing meiotic DNA synthesis but, as cultures progressed to the latter stages of meiosis and spore formation, then the cells become more sensitive again.
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Fig. 3. Fig. 3 shows: (a) D N A content (ll) and percentage sporulation (0) at different times in the meiosis of SKI, and (b) the percentage survival after UV irradiation with 55 J / m 2 (v) and 110 J / m 2 (e).
115
Fig. 4 shows the frequency of spontaneous and UV-induced prototrophs produced by intragenic recombination (TRP ÷) in samples of D7 that were taken at different times during meiosis. The level of spontaneous intragenic recombination was found to increase at the time of meiotic DNA synthesis up to a level 500 × above the mitotic level. The level of UV-induced intragenic recombination increased at times approaching meiotic DNA synthesis, but after the initiation of DNA synthesis became indistinguishable from the spontaneous gene conversion frequency as this attained the full meiotic level. The percentage of colonies which were red-pink, twin-sectored on control and irradiated plates at different times of meiosis is shown in Fig. 5. These have been
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Fig. 4. Fig. 4 shows the induced (O) and spontaneous (m) levels of prototrophs resulting from intragenic recombination after UV irradiation of D7 at 110 J / m 2 at different times of meiosis.
116
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Fig. 5. Fig. 5 shows the percentage red-pink twin-sectoredcolonieson control plates (n) and on treated plates (O) after UV irradiation of D7 with 119 J/m 2.
taken to be the products of intergenic recombination in mitotic cultures where they occur spontaneously at a low frequency (Zimmermann et al., 1975). In the experiments described here, the red-pink, twin-sectored colonies were taken to represent the product of intergenic recombination until 12-h samples, when mating nonsporulating colonies were first detected. Fig. 5 shows that the percentage of red-pink, twin-sectored colonies increased at the same time as commitment to intragenic recombination at the trp5 locus. The level of induced red-pink, twin-sectored colonies increased during the period of commitment to recombination and remained above the spontaneous level, up to the point of first commitment to haploidization. After this time red-pink, twin-sectored colonies may be generated by segregation at the ade2 locus.
Discussion
Previous investigations upon the effect of UV irradiation of meiotically dividing yeast cells have reported a number of conflicting observations. Salts et al. (1976) reported an increase in cell sensitivity during prophase to UV-induced cell lethality, the ability to form spores and to induced recombination. In contrast, Hottinguer-de Margerie and Moustacchi (1979) described an increase in cellular sensitivity to the lethal effects of UV prior to meiotic DNA synthesis, whereas no evidence of any cyclic variation in either the lethal or mutagenic effect of UV were reported by
117
Machida and Nakai (1980a). However, Machida and Nakai (1980b) did observe some differences in the levels of induction of inter- and intragenic recombination after UV exposure. The results presented here are in broad agreement with the findings of Hottinguer-de Margerie and Moustacchi (1979). Meiotic cells of the yeast strains SK1 and D7 became more sensitive to the lethal effects of UV light as they entered a pre-replicative meiotic state after the completion of mitosis. This is similar to the situation in the mitotic cell cycle where G1 cells are more sensitive to the lethal effects of ultraviolet light, but the size of the difference in cellular sensitivity is greater in mitosis than that found here during meiosis (Davies et al., 1978). The importance of meiotic DNA synthesis in the recovery of cellular resistance from the lethal effects of UV light was shown by Hottinguer-de Margerie and Moustacchi (1979) who used a mutant strain blocked in meiotic DNA synthesis. The response of this strain was similar to that of a haploid strain and exhibited increased sensitivity to UV light as a result of transition from exponential to stationary phase (Davies and Parry, 1976). Sporulating cells have been inferred to enter meiosis from the G1 portion of mitotic cell cycle (Hirschberg and Simchen, 1977) and the folded chromosome confirmation of cells at early times of meiosis has been found to be similar to that of mitotic stationary-phase cells (Pifion, 1979). It is possible that meiotic cells at these early times also show GO stage-type sensitivity; a stage which is more resistant than the G1 stage of mitotic growth (Tippins and Parry, 1981). The increased sensitivity of yeast strains D7 and SK1 to irradiation at late times in meiosis probably reflects the increased sensitivity associated with haploid genomes in a prereplicative state (Saeki et al., 1980). The broad agreement in the response of the strains SK1 and D7 to UV treatment is in contrast to studies on the appearance of single-strand nicks during meiosis (Jacobson et al., 1975; Kassir and Simchen, 1960; Resnick et al., 1981). Hardly any detectable single-strand nicking was found during meiosis for the strain SK1 unlike other strains examined which exhibit increased single-strand nicking during the period of commitment to recombination. This might be taken to indicate an abnormal meiosis in SK1 but the data presented here suggests that at least in respect of the response to UV irradiation, meiosis in the strain SK1 is comparable to that of the strain D7. The frequency of spontaneous base-substitution mutation at the ilvl-92 locus in strain D7 was found not to change significantly during meiosis after plating upon selective medium. However, induced mutation frequencies after UV irradiation showed cyclic variations with higher levels of induction at times of greatest lethality. This result is contrary to the only other report on induced mutation in meiosis (Machida and Nakai, 1980a) where no cyclic variations for a variety of mutational lesions were found. The lower levels of sporulation (35% at 48 h), less synchrony of meiosis in their strains, or differences in the alleles tested, may explain this difference. The level of spontaneous intragenic and intergenic recombination in the strain D7 was found to increase dramatically during meiotic DNA synthesis, but the two
118
events could not be separated in time using our system. Previously, Sherman and Roman (1963) suggested that fixation of these two events was separable, but subsequent investigations have failed to demonstrate this clearly (Esposito and Esposito, 1974; Olson and Zimmermann, 1978; Machida and Nakai, 1980b). It is possible that any such differences result from the temporal order of replication of genes during meiotic DNA synthesis. After UV irradiation, increases in induced intragenic recombination were found through the period of commitment to recombination. This increase is often found before the level of spontaneous intragenic recombination has begun to rise at treatment times around 6 h and has been found for a number of other mutagens (Kelly, unpublished). It therefore appears that DNA damage either induces DNA metabolizing enzymes or depresses factors that result in premature meiotic recombination. This would produce a higher proportion of the culture having undergone commitment to recombination up to the point of full spontaneous commitment to intragenic recombination. Alternatively the damage induced or the resulting modification by DNA repair mechanisms, may produce directly prerecombinagenic intermediate structures at the time of DNA replication. The level of induced intragenic recombination at times after full commitment to intragenic recombination is not distinguishable from the spontaneous frequency of conversion even under the conditions of high levels of mitotic induction reported here. It is, however, possible that mitotic type increases in the level of intragenic recombination do occur, but are obscured by the level of meiotic recombination. Induced intergenic recombination was found to increase during commitment to recombination and to remain above the spontaneous level up to the point of first commitment to chromosome disjunction. This difference between intergenic and intragenic recombination is similar to that reported by Machida and Nakai (1980b) and may indicate that while intragenic recombination is at a maximum in meiosis, the levels of reciprocal recombination may still be enhanced.
Acknowledgements This work was supported in part by a grant from Euratom. During the course of the work one of us (S.L.K.) held a BP SRC Case Studentship. We would like to thank Dr. A.P. Regnier of B.P. Ltd. for his support for this work.
References Chanet, R., D.H. Williamson and E. Moustacchi (1973) Cyclic variation in killing and 'petite' mutagenesis induced by ultraviolet light in synchronised yeast strains, Biochim. Biophys. Acta, 324, 290-299. Cox, B.S., and E.A. Bevan (1962) Aneuploidy in yeast, New Phytol., 61. Davies, P.J., and J.M. Parry (1976) The induction of mitotic gene conversion by chemical and physical mutagens as a function of culture age in the yeast Saccharomyces cerevisiae, Molec. Gen. Genet., 148, 165-170. Davies, P.J., R.S. Tippins and J.M. Parry (1978) Cell-cycle variation in the induction of lethality and
119 mitotic recombination after treatment with UV and nitrous acid in the yeast, Saccharomyces cerevisiae, Mutation Res., 51,327-346. Esposito, M.S., and R.E. Esposito (1974) Genetic recombination and commitment to meiosis in Saccharomyces, Proc. Natl. Acad. Sci. (U.S.A.), 71, 3172-3176. Esposito, R.E. (1968) Genetic recombination in synchronized cultures of Saccharomyces cerevisiae, Genetics, 59, 445-459. Esposito, R.E. (1980) Workshop report on sporulation, in: Tenth International Conference on Yeast Genetics and Molecular Biology, pp. 19-25. Esposito, R.E., and M.S. Esposito (1975) Mutants of meiosis and ascospore formation, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. XI, Academic Press, New York, pp. 303-326. Fabre, F. (1973) The role of repair mechanisms in the variations of ultraviolet and "y-radiation sensitivity during the cell cycle of Schizosaccharomycespombe, Radiation Res., 56, 528-539. Fast, D. (1973) Sporulation synchrony of Saccharomyces cerevisiae grown in various carbon sources, J. Bacteriol., 116, 925-930. Haber, J.E., H.P. Halvorson (1975) Methods in sporulation and germination of yeasts, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. XI, Academic Press New York, pp. 45-69. Hartwell, L.H. (1978) Cell division from a genetic perspective, J. Cell Biol., 77, 627-637. Hirschberg, Y., and G. Simchen (1977) Commitment to the mitotic cell cycle in yeast in relation to meiosis, Exp. Cell Res., 105, 245-258. Hottinguer-de Margerie, H., and E. Moustacchi (1979) Abolition of the cyclic variations in radiosensitivity during meiosis in a sporulation mutant blocked in premeiotic DNA synthesis, Molec. Gen. Genet., 175, 259-265. Jacobson, G.K., R. Pifion, R.E. Esposito and M.S. Esposito (1975) Single strand scissions of chromosomal DNA during commitment to recombinant at meiosis, Proc. Natl. Acad. Sci. (U.S.A.), 72, 1887-1891. Kane, S.M., and R. Roth (1974) Carbohydrate metabolism during ascospore development in yeast,J. Bacteriol., 118, 8-14. Kassir, Y., and G. Simchen (1960) Single strand scissions of nuclear yeast DNA occur without meiotic recombination, Current Genet., 2, 70-85. Machida, I., S. Nakai (1980a) Induction of spontaneous and UV-induced mutation during commitment to meiosis in Saccharomyees cerevisiae, Mutation Res., 73, 59-68. Machida, I., and S. Nakai (1980b) Differential effect of UV irradiation on induction of intragenic and intergenic recombination during commitment to meiosis in Saceharomyces cerevisiae, Mutation Res., 73, 69-79. Olson, L.W., and F.K. Zimmermann (1978) Meiotic recombination and synaptonemal complexes in Saccharomyces eerevisiae, Molec. Gen. Genet., 166, 151 - 159. Parry, J.M., and B.S. Cox (1968) Mitotic reombination induced by ultraviolet light in synchronous cultures of yeast, Mutation Res., 5, 373-384. Pifion, R. (1979) Folded chromosomes of Saccharomyces eerevisiae, Comparison of response to sporulation medium, arrest at start and G O arrest, Chromosoma (Berlin), 81,379-391. Puglisi, P.P. (1967) Mutagenic and antimutagenic effects of acridinium salts in yeast, Mutation Res., 4, 289-294. Resnick, M.A., J.N. Kasimos, J.C. Game, R.S. Braun and R.M. Roth (1981) Changes in DNA during meiosis in a repair-deficient mutant (rad52) of yeast (Saccharomyces cerevisiae), Science, 212, 543-545. Saeki, T., I. Machida and S. Nakai (1980) Genetic control of diploid recovery after y-irradiation in the yeast Saccharomyces cerevisiae, Mutation Res., 73, 251-265. Salts, Y., G. Simchen and R. Pinon (1976) DNA degradation and reduced recombination following UV irradiation during meiosis in yeast (Saccharomyces cerevisiae), Molec. Gen. Genet., 146, 55-59. Sherman, F., and H. Roman (1963) Evidence for two types of allelic recombination in yeast, Genetics, 48, 255-261. Silva-Lopez, E., T.J. Zamb and R. Roth (1975) Role of premeiotic replication in gene conversion, Nature (London), 253, 212-214. Tippins, R.S., and J.M. Parry (1981) The influence of cell size on UV induced cell cycle variation in
120 lethality and mitotic recombination in the yeast Saccharomyces cerevisiae, Int.J. Radiation Biol., 40, 327-331. Von Borstel, R.C. (1978) Measuring spontaneous mutation rates in yeast, in: D.M. Prescott (Ed.), Methods in Cell Biology, Vol. XX, Academic Press, New York, pp. 1-24. Von Borstel, R.C., M.J. Bond and C.M. Steinberg (1964) Spontaneous reversion rates of a super-suppressible mutant during mitosis and meiosis, Genetics, 50, 293. Zimmermann, F.K., R. Kern and H. Rasenberger (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversion and reverse mutation, Mutation Res., 28, 381-388.
109
Elsevier BiomedicalPress
The induction of mutation and recombination following UV irradiation during meiosis in Saccharomyces cerevisiae S.L.Kelly and James M. Parry Department of Genetics, University Collegeof Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)
(Received4 May 1982) (Revisionreceived27 August 1982) (Accepted9 September 1982)
Summary Irradiation of yeast cultures with ultraviolet light at discrete stages during meiosis produces cyclic variations in sensitivity, i.e. cells are more sensitive to the lethal effects of UV light prior to entry into the meiotic DNA synthesis, and this corresponds to a peak of induction of point mutation. Cells become more resistant to both induced point mutation and lethality as they enter meiotic DNA synthesis, but become more sensitive again during spore formation. The induced level of intragenic recombination rises during the period of commitment to recombination to a level indistinguishable from the full meiotic level of spontaneous intragenic recombination. Induced reciprocal recombination remains above the spontaneous level up to the point of commitment to sporulation.
The yeast Saccharomyces cerevisiae is convenient organism for studies upon the meiotic and mitotic cell cycles (Esposito and Esposito, 1975; Hartwell, 1978). We have investigated the sensitivity of cells to UV during the two alternative cell cycles as part of a programme of investigation of the comparative effects of a wide range of environmental mutagens. Variation in sensitivity to ultraviolet irradiation has been reported for synchronised mitotic yeast cultures (Esposito, 1968; Parry and Cox, 1968; Chanet et al., 1973; Davies et al., 1978). The period of greatest cell sensitivity to the lethal effects of UV was observed in the G1 phase of growth followed by a period of greater resistance during the S and G2 phases, an effect which has been attributed to the activity of recombinational-repair mechanisms (Fabre, 1973). The G1 sensitivity observed corresponded with maximal induction of various genetic end-points. Meiosis may be induced in Saccharomyces cerevisiae by a media shift which results in the completion of mitosis, meiotic DNA synthesis, meiotic cell division and 0027-5107/83/0000-0000/$03.00 © ElsevierSciencePublishers
110
the formation of haploid spores in an ascus (Haber and Halvorson, 1975). In studies on meiosis it was found that cells plated back onto vegetative medium during meiosis become committed to meiotic levels of recombination before they become committed to the execution of the events leading to ascus formation (Sherman and Roman, 1963; Olson and Zimmerman, 1978). The process of commitment to recombination was shown to be intimately associated with the meiotic DNA synthesis (Silva-Lopez et al., 1975) while commitment to meiotic cell division and sporulation occurs at the time of the first spindle pole body separation (Esposito, 1980). An increase in the level of genetic recombination is the most dramatic difference observed between meiosis and mitosis, but it has also been reported that a 'meiotic effect' of enhanced spontaneous frameshift mutation exists in yeast (Von Borstel et al., 1964) and that certain agents may have a mutagenic effect ~i~ meiosis and an antimutagenic effect in mitosis (Puglisi, 1967). The effect of UV light on meiotic cells in yeast has been the subject of conflicting reports (Simchen et al., 1973; Hottinguer-de Margerie and Moustacchi, 1979; Machida and Nakai, 1980a, 1980b). Simchen et al. found an increased sensitivity for viability and for the ability to continue through sporulation to form asci at a stage comparable to meiotic prophase I. In a later paper (Salts et al., 1976), reduced recombination was reported after UV irradiation at the same stage. In their study Hottinguer-de Margerie and Moustacchi (1979) reported that meiotic cells exhibit increasing sensitivity until meiotic DNA synthesis begins, while Machida and Nakai (1980) found no evidence of cyclical variations in UV-induced lethality. We report here the effect of UV irradiation during meiosis in yeast on the strain D7 and on a supersporulating strainSK1.
Materials and methods Strains. The strains D7 (Zimmermann etal., 1975) and SK1 (Kane and Roth, 1974) were used in this investigation. The strain SK1 is homothallic and sporulates rapidly to a high level. The strain D7 has the following genotype:
a ilv 1-92 trp 5-12 ade 2-40 cyff-2 a ilvl-92 trp 5-27 ade2-119 CYHS.2 D7 has been frequently used in mitotic studies to assay base-pair substitution mutation to isoleucine independence, intragenic recombination to tryptophan independence and reciprocal intergenic recombination at the adenine locus. The product of mitotic intergenic recombination is detected as a red-pink, twin-sectored colony. The allele ade 2-119 is responsible for the pink pigmentation and the allele ade 2-40 is responsible for the red pigmentation. The combination of these alleles in a diploid results in heteroalleic complementation to give a white colony colour. D7 was used in this work to study base-pair substitution mutation, intragenic recombination, survival and intergenic recombination and chromosome segregation
during meiosis using the method of plating cells on vegetative medium from sporulating cultures. Confirmation of any variations in survival after irradiation was obtained by study of the response of the supersporulator SKI. Growth and sporulation. The strains were grown at 28°C in acetate presporulation medium consisting of 10 g potassium acetate, 20 g peptone and 10 g yeast extract per 1 (Fast, 1973). Cells were harvested at 2 X 10’ cells/ml, and washed 3 times ins saline. On resuspension in sporulation medium the cells were sonicated and made up to a final concentration of 2 X 10’ cells/ml. The sporulation medium chloride, 1.2 g sodium ingredients were 8.2 g sodium acetate, 1.9 g potassium chloride and 0.35 g magnesium sulphate per 1 (Von Borstel, 1978), supplemented with 25 mg/l adenine, tryptophan and isoleucine. The resuspension of cells in sporulation medium was taken as time zero in meiosis in the experiments and only 150 ml of culture was inoculated into a litre flask. This enabled vigorous aeration to occur which is important in maintaining high levels of sporulation and synchrony of meiotic events. Meiosis in the strain 07 has been previously studied (Olson and Zimmermannn, 1978) and achieves a high level of sporulation with good synchrony under the conditions described here. Treatment. Samples were taken at different times during meiosis and resuspended at 2 x 10’ cells/ml in 10 ml of pH 7.0 phosphate buffer. This was irradiated in a 9-cm petri dish with UV light at 254 nm from a Hanovia Ila low pressure mercury discharge tube monitored by the use of a calibrated photocell. All manipulations were performed in red light to avoid photoreactivation. Plating. Appropriate minimal plates were used for scoring intragenic recombination and mutation (Olson and Zimmermann, 1978). These plates were scored after 6 days. Viability was assayed on YPG medium (Cox and Bevan, 1962) after 3 days. These plates were examined for the presence of completely coloured and sectored colonies after 6 days. DNA estimation. The DNA present in samples was estimated spectrophotometrically by the diphenylamine reaction using the standard method (Haber and Halvorson, 1975). Testing for ploidy. Ploidy was inferred by a mating and sporulation test. Colonies were taken to be diploid if they gave rise to asci in sporulation medium after 5 days and did not give mating figures on mixing with haploid strains of either mating type. Similarly, colonies which did not give asci after 5 days’ incubation in sporulation medium but which gave mating figures on mixing with a or OLhaploid strains were inferred to be mainly haploid. Cell counts. Per cent sporulation and estimates of cell concentration were calculated after scoring a minimum of 500 cells on a haemocytometer slide. The results presented here are representative of experiments repeated several times. Results
Fig. 1 illustrates some of the landmark 07. The period of meiotic DNA synthesis
events which occur during sporulation in began at around 6 h after inoculation into
112
'Completion of mitosis and entry into the meiotic cycle
j
Meiotic
DNA synthesis Meiotic
cell
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sporulation medium. D N A synthesis was largely over after 10 h in sporulation medium and this has been confirmed by studies using a labelled D N A precursor (Kelly, unpublished). The first asci appeared after about 16 h in sporulation medium and this level increased to about 50% sporulation by 24 h. The final level of sporulation was between 70-80%. In the specific experiment whose data is presented here, the final level of sporulation was 79.6%. The percentage of red and pink colonies which were inferred to be haploid was first found to rise above zero in samples plated from sporulating cultures onto vegetative medium after 12 h. This indicates that cells start becoming committed to sporulation between 11 and 12 h after inoculation into sporulation medium and at times before this, cells revert to mitosis after plating onto vegetative medium. Fig. 2 shows the spontaneous mutation frequency as well as the induced mutation frequency and cell survival when samples were irradiated with 110 J / m 2 at various time during meiosis. The spontaneous mutation frequency was found to be unchanged throughout meiosis, but lethality and the induced mutation frequency were found to show cyclic variations. The cells were more sensitive to the effects of UV prior to their entry into meiotic D N A synthesis and this sensitivity corresponds to a 2-3-fold increase in the induction of mutation at the ilv 1-92 locus. Following this period of sensitivity, the
113
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cells showed increased resistance to the lethal effects of UV during meiotic D N A synthesis and for a period to just beyond the point of first commitment to sporulation. The induced mutation frequency was reduced as the cells enter this period of D N A synthesis and throughout meiotic prophase I. No evidence of an increased sensitivity of cells during meiotic prophase I was found. Cultures irradiated at a late time in sporulation, i.e. at 24 h, exhibited greater sensitivity to the lethal effects of UV irradiation and a higher mutation frequency was induced. This irradiation time corresponded to a point where sporulation had reached 50%.
114 In order to confirm the meiotic origin of these responses, the yeast strain SKI was also used. This strain undergoes rapid and synchronous sporulation as shown in Fig. 3. In SK1, DNA synthesis was found to begin between 2-2.5 h after inoculation into sporulation medium and the DNA content in successive samples increased until 4 h after inoculation. The first tetrads appeared after only 7.5 h incubation in sporulation medium and the percentage sproulation increased rapidly to almost 100% after 10 h in sporulation medium. The response of SK1 to irradiation during meiosis was found to confirm the data obtained for D7, with increased sensitivity to the lethal effects of UV irradiation in samples taken prior to the initiation of the meiotic DNA synthesis. Greater resistance was found in cultures undergoing meiotic DNA synthesis but, as cultures progressed to the latter stages of meiosis and spore formation, then the cells become more sensitive again.
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Fig. 3. Fig. 3 shows: (a) D N A content (ll) and percentage sporulation (0) at different times in the meiosis of SKI, and (b) the percentage survival after UV irradiation with 55 J / m 2 (v) and 110 J / m 2 (e).
115
Fig. 4 shows the frequency of spontaneous and UV-induced prototrophs produced by intragenic recombination (TRP ÷) in samples of D7 that were taken at different times during meiosis. The level of spontaneous intragenic recombination was found to increase at the time of meiotic DNA synthesis up to a level 500 × above the mitotic level. The level of UV-induced intragenic recombination increased at times approaching meiotic DNA synthesis, but after the initiation of DNA synthesis became indistinguishable from the spontaneous gene conversion frequency as this attained the full meiotic level. The percentage of colonies which were red-pink, twin-sectored on control and irradiated plates at different times of meiosis is shown in Fig. 5. These have been
.-I
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Fig. 4. Fig. 4 shows the induced (O) and spontaneous (m) levels of prototrophs resulting from intragenic recombination after UV irradiation of D7 at 110 J / m 2 at different times of meiosis.
116
3.oL commitment
commitment
to r e c o m b i n a t i o n
to s p o r u l a t i o n
i
7
2.0
twin sectored
1'0'
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0
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,l/, 6
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// I0
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Fig. 5. Fig. 5 shows the percentage red-pink twin-sectoredcolonieson control plates (n) and on treated plates (O) after UV irradiation of D7 with 119 J/m 2.
taken to be the products of intergenic recombination in mitotic cultures where they occur spontaneously at a low frequency (Zimmermann et al., 1975). In the experiments described here, the red-pink, twin-sectored colonies were taken to represent the product of intergenic recombination until 12-h samples, when mating nonsporulating colonies were first detected. Fig. 5 shows that the percentage of red-pink, twin-sectored colonies increased at the same time as commitment to intragenic recombination at the trp5 locus. The level of induced red-pink, twin-sectored colonies increased during the period of commitment to recombination and remained above the spontaneous level, up to the point of first commitment to haploidization. After this time red-pink, twin-sectored colonies may be generated by segregation at the ade2 locus.
Discussion
Previous investigations upon the effect of UV irradiation of meiotically dividing yeast cells have reported a number of conflicting observations. Salts et al. (1976) reported an increase in cell sensitivity during prophase to UV-induced cell lethality, the ability to form spores and to induced recombination. In contrast, Hottinguer-de Margerie and Moustacchi (1979) described an increase in cellular sensitivity to the lethal effects of UV prior to meiotic DNA synthesis, whereas no evidence of any cyclic variation in either the lethal or mutagenic effect of UV were reported by
117
Machida and Nakai (1980a). However, Machida and Nakai (1980b) did observe some differences in the levels of induction of inter- and intragenic recombination after UV exposure. The results presented here are in broad agreement with the findings of Hottinguer-de Margerie and Moustacchi (1979). Meiotic cells of the yeast strains SK1 and D7 became more sensitive to the lethal effects of UV light as they entered a pre-replicative meiotic state after the completion of mitosis. This is similar to the situation in the mitotic cell cycle where G1 cells are more sensitive to the lethal effects of ultraviolet light, but the size of the difference in cellular sensitivity is greater in mitosis than that found here during meiosis (Davies et al., 1978). The importance of meiotic DNA synthesis in the recovery of cellular resistance from the lethal effects of UV light was shown by Hottinguer-de Margerie and Moustacchi (1979) who used a mutant strain blocked in meiotic DNA synthesis. The response of this strain was similar to that of a haploid strain and exhibited increased sensitivity to UV light as a result of transition from exponential to stationary phase (Davies and Parry, 1976). Sporulating cells have been inferred to enter meiosis from the G1 portion of mitotic cell cycle (Hirschberg and Simchen, 1977) and the folded chromosome confirmation of cells at early times of meiosis has been found to be similar to that of mitotic stationary-phase cells (Pifion, 1979). It is possible that meiotic cells at these early times also show GO stage-type sensitivity; a stage which is more resistant than the G1 stage of mitotic growth (Tippins and Parry, 1981). The increased sensitivity of yeast strains D7 and SK1 to irradiation at late times in meiosis probably reflects the increased sensitivity associated with haploid genomes in a prereplicative state (Saeki et al., 1980). The broad agreement in the response of the strains SK1 and D7 to UV treatment is in contrast to studies on the appearance of single-strand nicks during meiosis (Jacobson et al., 1975; Kassir and Simchen, 1960; Resnick et al., 1981). Hardly any detectable single-strand nicking was found during meiosis for the strain SK1 unlike other strains examined which exhibit increased single-strand nicking during the period of commitment to recombination. This might be taken to indicate an abnormal meiosis in SK1 but the data presented here suggests that at least in respect of the response to UV irradiation, meiosis in the strain SK1 is comparable to that of the strain D7. The frequency of spontaneous base-substitution mutation at the ilvl-92 locus in strain D7 was found not to change significantly during meiosis after plating upon selective medium. However, induced mutation frequencies after UV irradiation showed cyclic variations with higher levels of induction at times of greatest lethality. This result is contrary to the only other report on induced mutation in meiosis (Machida and Nakai, 1980a) where no cyclic variations for a variety of mutational lesions were found. The lower levels of sporulation (35% at 48 h), less synchrony of meiosis in their strains, or differences in the alleles tested, may explain this difference. The level of spontaneous intragenic and intergenic recombination in the strain D7 was found to increase dramatically during meiotic DNA synthesis, but the two
118
events could not be separated in time using our system. Previously, Sherman and Roman (1963) suggested that fixation of these two events was separable, but subsequent investigations have failed to demonstrate this clearly (Esposito and Esposito, 1974; Olson and Zimmermann, 1978; Machida and Nakai, 1980b). It is possible that any such differences result from the temporal order of replication of genes during meiotic DNA synthesis. After UV irradiation, increases in induced intragenic recombination were found through the period of commitment to recombination. This increase is often found before the level of spontaneous intragenic recombination has begun to rise at treatment times around 6 h and has been found for a number of other mutagens (Kelly, unpublished). It therefore appears that DNA damage either induces DNA metabolizing enzymes or depresses factors that result in premature meiotic recombination. This would produce a higher proportion of the culture having undergone commitment to recombination up to the point of full spontaneous commitment to intragenic recombination. Alternatively the damage induced or the resulting modification by DNA repair mechanisms, may produce directly prerecombinagenic intermediate structures at the time of DNA replication. The level of induced intragenic recombination at times after full commitment to intragenic recombination is not distinguishable from the spontaneous frequency of conversion even under the conditions of high levels of mitotic induction reported here. It is, however, possible that mitotic type increases in the level of intragenic recombination do occur, but are obscured by the level of meiotic recombination. Induced intergenic recombination was found to increase during commitment to recombination and to remain above the spontaneous level up to the point of first commitment to chromosome disjunction. This difference between intergenic and intragenic recombination is similar to that reported by Machida and Nakai (1980b) and may indicate that while intragenic recombination is at a maximum in meiosis, the levels of reciprocal recombination may still be enhanced.
Acknowledgements This work was supported in part by a grant from Euratom. During the course of the work one of us (S.L.K.) held a BP SRC Case Studentship. We would like to thank Dr. A.P. Regnier of B.P. Ltd. for his support for this work.
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