Effect of irradiation and mutagenic chemicals on the generation of ADH2-constitutive mutants in yeast

53

Mutation Research, 177 (1987) 53-60 Elsevier MTR 04302

Effect of irradiation and mutagenic chemicals on the generation of ADH2-constitutive mutants in yeast Significance for the inducibility of Ty transposition Cornelia Morawetz Gesellschaftf~r Strahlen- und Umweltforschung, lnstitut fftr Strahlenbtologle, 8042 Neuherberg (F.R. G.) (Received 27 June 1986) (Revision received 13 October 1986) (Accepted 15 October 1986)

Key words: Yeast; A DH2-constitutive mutants; Ty transposition, inducibility; Antimycin-A-resistantphenotype.

Summary Mutations caused by the insertion of a Ty element resulting in an antimycin-A-resistant phenotype in an adhl- strain of Saccharomyces cerevisiae were used as an assay for the quantitative detection of Ty transposition. Antimycin-A-resistant mutants were found to be inducible by ethyl methanesulfonate (EMS) as well as by 3'- and UV irradiation. DNA analysis of 3'-induced mutants showed an increase of the fraction of Ty insertions in the ADH2 locus with increasing dose.

The yeast Saccharomyces cerevisiae has 3 different isoenzymes of alcohol dehydrogenase, ADHI, ADHII and ADHIII, coded by 3 unlinked loci (Ciriacy, 1975). ADHI is the fermentative enzyme, ADHII is present when gluconeogenic pathways are used, and is repressed in the presence of fermentable carbon sources such as glucose. The role of the mitochondrial enzyme ADHIII is not known, null mutations in this gene have no detectable effect on the cell. Mutant strains lacking the ADHI enzyme cannot grow on glucose when respiration is blocked by antimycin A or by high glucose concentrations (Ciriacy, 1976). Strains which regenerated the ability to ferment glucose in spite of a defective ADH1 gene Correspondence: Dr. C. Morawetz, Gesellschaft ftlr Strahlenund Umweltforschung, Institut ftir Strahlenbiologie, 8042 Neuherberg (F.R.G.).

were isolated. Out of 9 cis-dominant mutants with constitutive expression of the ADH2 gene, 7 were shown to carry the yeast mobile element, Ty, in the promoter region of the ADH2 gene (Ciriacy and Williamson, 1981). This observation suggests the possibility of screening a population of irradiated or chemically treated cells for the fraction of Ty-induced ADH2-constitutive clones by the ability of an adhl- mutant to grow anaerobically on glucose when respiration is blocked by antimycin A (Paquin and Williamson, 1984). There are several possibilities for the time schedule of an inducible transposition event. It may occur (1) during or directly after mutagenic treatment, (2) during a postincubation in permissive medium, or (3) during growth on the selective medium. (4) Alternatively, transposition may not be affected by mutagenic processes, and may occur randomly.

0027-5107/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

54 Transposition events can be divided into several steps, depending on the mechanism of transposition. As the number of Ty elements per cell has been found to be stable, transposition events should be associated with deletion of a Ty element from its original site. Whether new transcription of T y - D N A is necessary or whether the high spontaneous level of Ty transcripts (up to 10% of the poly(A)-mRNA in the cell) can serve as a template is not yet known. According to the high constitutive levels of Ty-mRNA, regulation of transposition at the translational level may also be possible, i.e. the synthesis of the enzymes coded on the Ty element (Fulton et al., 1985) is necessary for the transposition event. Ballario et al. (1983) have shown the existence of covalently closed circular copies of T y - D N A in yeast cells, suggesting that these intermediates may act in the mechanism of Ty transposition. Boeke and coworkers (1985) have shown that Ty encodes reverse transcriptase activity and that transposition can take place via reverse transcription of Ty-mRNA. They have also described the inducibility of Ty transposition after overproduction of a single Ty element. The transcription of Ty elements is also influenced by mating type (Taguchi et al., 1984; Errede et al., 1985; Roeder et al., 1985), carbon source (Taguchi et al., 1984) and DNA-damaging agents (Rolfe, 1985; McClanahan and McEntee, 1984). The transposition of Ty elements is (in us and in trans) under the influence of a transcription of the Ty element (Boeke et al., 1985; Garfinkel et al., 1985) and, moreover, it has been shown to respond to temperature. Paquin and Williamson (1984, 1986) have shown a relationship between the number of new Ty insertions and the time and temperature of incubation. Their results are in agreement with the finding of Garfinkel and coworkers (1985) concerning the temperature optimum for the reverse transcriptase activity of the Ty element. To contribute to the understanding of cellular processes underlying the transposition of Ty elements, I have undertaken experiments to determine the inducibility of mutations caused by an insertion of Ty elements. The effects of UV and ),-irradiation and treatment with ethyl methanesulfonate (EMS) on the frequency of this mutation

have been examined. The system studied was the Ty-induced ADH2 constitutivity described above, i.e. the antimycin A resistance obtained in an adhl adh3 tester strain in the presence of glucose and antimycin A.

(1) Materials and methods

(1.1) Strains Different haploid strains of Saccharomyces cerevisiae were used, having no detectable A D H I or ADHIII. Strain 43-2B (adhl-ll ADH2 adh3 ural his4 MATalpha) and D7-12.2A (adhl-del ADH2 adh3 trpl ura MATa) were kindly provided by M. Ciriacy (Universit~it Di~sseldorf, F.R.G.). (1.2) Media and growth conditions Standard yeast YPD medium was made from 1% yeast extract, 2% bacto peptone (Difco), supplemented with 2% glucose. In selective media, antimycin A (Serva) was added to a final concentration of 5 ppm. Plates were solidified by 2% agar (Difco). Cells were grown at 28°C. (1.3) Mutagenesis Cells were grown overnight and harvested at 1-2 × 10 7 cells/ml. After appropriate dilutions, cells were plated on non-selective medium (YPD) or selective antimycin-A-containing medium and irradiated directly on the plates. For UV exposure a transilluminator was used. The intensity was reduced using plastic wrap at a dose rate of 8.5 J/mZ/sec. For y-irradiation a 6°Co y-source was used at a dose rate of 82 G y / m i n . Chemical treatment was carried out in phosphate buffer at 4 ° C for 2 h using EMS (Serva) at the indicated concentrations before plating. For the determination of survival rates plates were incubated for 4 days, whereas antimycin-A-containing plates were incubated for 7 days at 28°C. For each dose 3 non-selective and 3 selective plates were scored. Each experiment was repeated 3 times at minimum. (1.4) Mathematical calculattons The mutant frequency, induced mutants per surviving fraction, has been calculated according

55 from Boehringer Mannheim; [a-32p]dCTP was from New England Nuclear.

to Haynes and Eckardt (1979).

M

N~(x) N(x)

(1)

(2) Results

To compare the dose dependence of mutant induction of various agents with different dosimetry, mutant yields were plotted over the "biological dose" of lethal hits (negative logarithm of surviving fraction, - I n S). To prove the dose dependence of mutant induction, mutant yield Y(x), induced mutants per cell treated at dose x, has been calculated according to Haynes and Eckardt (1979) as:

Y

N (x)

Nm(0)'S(x)

N0

N0

(2)

where NO is the number of treated cells, S(x) is the surviving fraction calculated as the ratio of the number of surviving cells after dose x to the number of treated cells N0, Nm(x ) is the number of mutants at dose x, Nm(0) is the number of mutants at dose zero (Haynes and Eckardt, 1979). The negative logarithm of the surviving fraction ( - I n S) is used in graphs where the action of different mutagenic agents is compared.

(1.5) DNA analysis Small-scale DNA was prepared as described by Struhl et al. (1979). DNA was electrophoresed in 0.7% agarose gel with 50 V for 20 h in Tris/borate/EDTA buffer. Southern transfer was done as described by Wahl et al. (1979) to GeneScreen membrane (NEN). [a-32p]dCTPnicktranslated (Rigby et al., 1977) plasmid ADR2Bsa kindly provided by M. Ciriacy (Williamson et al., 1981), was used for hybridization. Hybridization was in 2 × SCP, 250 p g / m l salmon sperm denatured DNA, 0.5% SDS for 20 h at 65°C (20 × SCP = 2 M NaC1/0.6 M Na2POJ10 mM EDTA, pH 7.5). To remove unbound DNA probe, filters were washed twice in 2 × SCP/I% SDS, twice in 2 × SCP and once in 0.4 × SCP for 15 min each at 45 o C. Autoradiographs were achieved by keeping the filters at - 7 0 ° C on Fuji RX film with CurixMR800 Enhancer sheets (from AGFA Gevaert). Plasmid DNA was prepared according to Birnboim and Doly (1979). All enzymes were

(2.1) Induction of mutations that lead to constitutive expression of the alcohol dehydrogenase gene These mutants were isolated by selection for resistance to glucose repression of ADH2, i.e. resistance to antimycin A. A strain with the genotype adhl-del adh3 was used to generate these mutants. Fig. 1 shows the induction of antimycin-A-resistant mutants as well as the surviving fraction of treated cells and the lethal hits (see Materials and methods) after y-irradiation. The spontaneous mutant frequency is 4 - 7 . 1 0 -7 . No difference has been found between a strain adhl-ll (presumably a point mutation) and an adhl-del strain (which has a - 1400/+ 40 deletion in the ADH1 gene). Survival rates of the tester strain and isolated mutants after 6°Co y-irradiation have been determined demonstrating that there is no difference between survival rates of the original strain and the induced antimycin-A-resistant mutants (data not shown). From separate experiments, about 100 antimycin-A-resistant clones -- irradiated and spontaneously grown - - have been isolated. They were tested for dominance of the phenotype by crossing with an adhl- ADH2 tester strain and replica plating of the diploids on antimycin-A-containing

O,5 1

0,001

100

0

200

400

600

800

1000Gray

gamma dose

Fig. 1. Mutant frequency (A), surviving fraction and lethal hits (O) of a strain lacking ADHI after mutagenesis with 6°Co-y. For additional details see text and Materials and methods.

56 plates. All mutants were dominant in this test. The size of the colonies grown on the selective plates was found to be very heterogeneous. Between very small and medium-sized colonies, very large colonies also appeared. Clones that form large colonies on selective plates were found to grow much faster than cells from colonies with a smaller diameter. Therefore, the size of the colonies does not depend on the time of mutation fixation. The phenotype of some clones forming large colonies is highly unstable suggesting that the resistance to antimycin A is lost at a high rate during mitotic growth. Additionally it was found that from experiments with a postincubation phase some (1 out of 24) mutants were resistant to antimycin A and allyl alcohol at the same time. The reasons for these findings are not yet clear. As none of the mutants were resistant to antimycin A on glycerol medium, a mutation leading to a general resistance to antimycin A could be ruled out.

t

(2.3) Induction of mutants by UV irradiation and EMS After it had been shown that 7-irradiation induces antimycin-A-resistant mutants the question arose whether other mutagenic agents act in the same way. Fig. 3a and b show the induction of mutant frequency as well as the surviving fraction

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Q

5.0

o

2.9-

S

b

(2.2) Analysis of the DNA of antimycin-resistant mutants From 2 experiments, the D N A of spontaneous and -/-irradiation-induced mutant clones was isolated. After digestion with EcoRl, gel electrophoresis and Southern blotting, the D N A samples were hybridized to a probe containing the ADH2 structural gene together with 1.1 kb of the upstream promoter region. Samples were identified with an altered restriction pattern (Fig. 2), indicating mutants with an insertion with one or two EcoRI sites in the promoter region of the ADH2 gene. The results of this experiment are summarized in Table 1. These data show that the fraction of mutants carrying an insertion in the promoter region of the ADH2 gene increases with dose. This indicates that not only the total number of antimycin-A-resistant mutants rises by v-irradiation, but also the fraction of mutants with a Ty insertion.

1

E

B

'

t~.-.-.-.-.,zJ-zz/z/S-.,~

S

E

EE

i

E

E

I

1

m

Fig 2 (a) Hybridization pattern of antimycm-A-reslstant mutants after EcoRI digestion. Mutant lanes showingan insertion are indicated by an arrow. (b) Restriction patterns of the ADtI2 locus from antimycm-A-resistantmutants. E, EcoRI; B, BamHI; S, SphI; 10 bases next to the TATA box. The hatched area indicates the part of the locus homologous to the probe. the arrow indacates the reading frame Open boxes: the internal epsilon fragment of the Ty; shaded boxes: delta fragment; dotted boxes this part of the insertion does not hybridize under the conditions used

and the lethal hits after mutagenesis by UV irradiation and EMS treatment. Both agents obviously are able to induce the antimycin-A-resistant phenotype in an adhl adh3 tester strain. To compare the dose dependence of mutant yield induction for the 3 agents, the mutant yield was plotted over the negative logarithm of the surviving fraction, - l n S. So Fig. 4 shows the mutant yield in relation to cell inactivation. In this graph EMS appears as the strongest agent in terms of induction of mutant yield. UV and "y-rays

57 TABLE 1 NUMBER OF MUTANTS OUT OF 24 ANTIMYCIN-A-RESISTANT CLONES THAT HAVE AN INSERTION IN THE PROMOTER REGION OF THE A D H 2 GENE, AS DETERMINED BY SOUTHERN BLOT ANALYSIS (for the details see text) Dose (krad) 0

30

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Surviving fraction

Expt. 1 Expt. 2

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are also efficient in promoting mutant induction, but much less than EMS. 1

2

3

4

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lethalhits

(2.4) Influence of incubation time The question arose as to why EMS is so much (90(

Fig. 4. Mutant yield of antimycin resistance in a strain adhl-del after mutagenesis with different agents: e, EMS; A, UV; i , y-rays. The mutant yield is drawn as a function of lethal hits ( - l n S). For additional details see Materials and methods.

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more effective in generating this type of mutant. One possible explanation could be the difference in the length of time of the treatment. Whereas irradiation lasted only a few minutes, cells were exposed to EMS for 2 h. Therefore, the interval between the first exposure to the agents and the exposure to the selective medium was varied in a split-dose experiment. Cells grown overnight were harvested at a density of 2 × 10 7 cells/ml and irradiated with 8

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Fig. 3. Mutant frequency (A), surviving fraction and lethal hits (0) of antimycin A resistance in a strain adhl after mutagenesis with (a) EMS and (b) UV. The graph is drawn as a function of the dose.

Y (.lO-7) 100-

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80,6G~

11'5

40 20

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2 4 6 (h) mutantyield 0--'0survivingfraction Fig. 5. Plating efficiency (O) and induced mutant yield (I) after 6°Co y-irradiation and incubation at various times in YPD medium before plating on antimycin A plates.

58 Gy in liquid medium. At this dose no mutant induction was found. The cells were grown for 2 more hours at 28°C and then irradiated once more with 100 Gy, giving a surviving fraction of 0.6. The cells were resuspended in fresh YPD medium. Samples were taken immediately, then after 2, 4 and 6 h samples were plated for the determination of mutants and survival rate as described in Materials and methods. The results are shown in Fig. 5. After 2 h, the survival rate is lower than after direct plating, but later the cells start to recover. The number of surviving cells increases during the postincubation period, but the number of total cells, as counted in a Neubauer chamber, remains unchanged. From this it is concluded that no cell division takes place during the first 6 h after irradiation (data not shown). Despite this, there is a significant increase in the mutant yield during postincubation. The mutant yield rises very strongly in the first 4 h, and a saturation level is reached between 4 and 6 h. During this time the mutant yield increases to a level similar to that reached by the EMS treatment.

(3) Discussion Ionizing irradiation, as well as different chemical agents, is known to cause DNA damage and mutations. The D N A damage is recognized by specific proteins and enzymes and DNA-repair processes can lead to alterations in genomic information and structure. But all of these alterations have also been found to take place in the absence of DNA-damaging agents. Transposable elements are involved in spontaneous as well as induced mutations (for a review see Sankaranarayanan, 1986). In Drosophila, Fahmy and Fahmy (1983) report an influence of mutagenic agents on the reversion of a copiamediated white locus mutation to the wild-type allele. Retroviruses have been found to have moved to new places in the genome in osteosarcoma induced by irradiation (Schmidt et al., 1985). Rolfe (1985) and McClanahan and McEntee (1984) showed an increase of a Ty-mRNA after mutagenizing, however with variation of the length of transcript. In unirradiated cells, McClanahan and

McEntee found no Ty-mRNA signal. This could mean, that a special Ty element is involved and induced by mutagenic processes. In this study I have addressed the question of whether mutagenic agents are capable of promoting Ty insertions in new loci in the yeast genome. First, the mutant induction via the phenotypical change, i.e., the occurrence of antimycin-A-resistant mutants was measured. In a dose-dependent manner this mutation can be induced by UV light, y-irradiation and EMS. In these studies it became obvious, that after correcting for cell killing, EMS was the most potent agent in promoting this type of mutant. Another question was whether the induction of antimycin-A-resistant mutants is correlated to an increase in Ty-mediated mutants. There are different possibilities for this mutation: (1) elongation of a poly(dA)2 s tract to poly(dA)54 (Russell et al., 1983), (2) mutations in genes regulating repression or derepression of the ADH2 gene (Ciriacy, 1979; Denis, 1985); (3) Ty insertion in the 5'-region of the ADH2 gene; or (4) reversion of the Adhl-ll mutant to the wild-type allele. There is a silent gene ADH4 in the genome of Saccharomyces cerevisme, where an insertion of a Ty element leads to an antimycin-A-resistant phenotype (Paquin and Williamson, 1984, 1986). A comparison of spontaneous with induced mutants on the base of DNA analysis demonstrated an increase of Ty-mediated mutations after y-irradiation. This would mean that under the conditions used Ty transposition is induced at a higher rate than other mutations leading to an antimycin-A-resistant phenotype. Otherwise the ratio of Ty insertions and other mutations leading to the new phenotype would have been constant. Garfinkel and coworkers (1985) have found a correlation between the overproduction of Tym R N A and increased transposition rate. So it is evident that Ty transposition should increase after mutagenesis. The finding of a relative increase of Ty transposition in relation to all mutations (cf. Table 1) could be explained by the fact that in these experiments the induction of the transposition system is more pronounced than other mutagenic processes. A split-dose irradiation with relatively low doses of y-irradiation (Fig. 4) showed, that the time between the first exposure to a mutagenic agent

59

and the necessity to exhibit the new phenotype is important for the fixation of the mutation. This finding is not new, but it points to events which might be important concerning the movement of Ty elements. The increase in mutant yield starts soon after irradiation, it is clearly measurable after 2 h, but still increases until 4 h after mutagenization. Then saturation is reached. It appears that all possible mutations, including the Ty transpositions, are completed at this time. Rolfe (1985) and McClanahan and McEntee (1984) have recently found an increase in Ty-coded mRNA 30 min after UV irradiation. This level persisted for 5 h, and then decreased. Although the fraction of Ty-mediated mutations has not yet been determined, the time course of mutant yield induction in the experiments is in agreement with the findings described by Rolfe (1985). As it has been found by Paquin and Williamson (1986) that the Ty transposition rate can be increased by incubating the cells at 15°C it would be interesting to look for the increase of mutation rates and mutant yield after a postincubation at this temperature. However, in addition to the determination of the mutant frequency and mutant yield the transposition rate should be determined by DNA analysis.

Acknowledgments I would like to thank M. Ciriacy for providing plasmids and yeast strains as well as U. Hagen, H. Backhaus, F. Eckardt and M.E. Lambert for critical reading of the manuscript and C. Schippel for technical assistance and drawing of the graphs. This work was done during a postdoctoral fellowship and supported by the European Community (BI-0085-D (B)).

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60 Young (1983) DNA sequences of two promotor-up mutants, Nature (London), 304, 625-654 Sankaranarayanan, K. (1986) Transposable genetic elements, spontaneous mutations and the doubling-dose method of radiation, genetic risk evaluation in man, Mutation Res, 160, 73-86. Schmidt, J., V. Erfle and W.A. Miiller (1985) Activation of endogenous C-type retroviral genomes by internal alphairradiation of mice with 224Radium, Radlat. Environ. Biophys., 24, 17-25. Struhl, K., D.T. Stinchcomb, S. Scherer and R.W. Davis (1979) High frequency transformation of yeast: Autonomous replication of hybrid DNA molecules, Proc. Natl. Acad. Sci (U.S.A.), 76, 1035-1039.

Taguctu, A.K.W., M. Ciriacy and E.T. Young (1984) Carbon source dependence of transposable element-associated gene activation in Saccharomyces cerevlsiae, Mol Cell. Biol., 4, 61-68 Wahl, G.M., M Stern and G.R. Stark (1979) Efficient transfer of large DNA fragments from agarose gels to dlazobenzyloxymethyl-paper, Proc Natl. Acad. Sci. (U.S.A.), 76, 3683-3687 Williamson, V M., E.T. Young and M, Ciriacy (1981) Transposable elements associated with constitutive expression of yeast alcohol dehydrogenase II, Cell, 23, 605-614