Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH

Mutation Research, 139 (1984) 189-192

189

Elsevier MRLett 0543

Studies of genetic effects in the D7 strain of Saccharomyces cerevisiae under different conditions of pH N i c l a N a n n i a, C a r l o B a u e r a, E n r i c o C u n d a r i a, C l a u d i o C o r s i a, R e n a t a Del C a r r a t o r e a, R i c c a r d o Nieri a, M o r e n o P a o l i n i a, J o h n C r e w s h a w b a n d G i o r g i o B r o n z e t t i a a istituto di Mutagenesi e Differenziamento CNR, Via Svezia, 10, 1 56100 Pisa (Italy) and b Georgia Institute of Technology, Atlanta (U.S.A.)

(Accepted 12 January 1984)

Summary The genetic effects of variation in pH in culture media and in suspension tests were examined in a diploid strain (D7) of the yeast, Saccharomyces cerevisiae. Deviation from the normal pH of 6.24 in the liquid culture medium, has a significant effect on cellular growth and on mitotic gene conversion at the trp5 locus. Frequencies of reversion at the ilv 1-92 locus and of mitotic crossing-over at the ade2 locus are not significantly influenced. Suspension tests, performed using phosphate buffer (pH 5.8), strongly confirm the original results. Our data suggest that the increase in mitotic gene conversion under various conditions of pH is due to alspecific effect of pH itself on the cells of S. cerevisiae. I n fact, increases were obtained using the same pH in both cellular growth and non-growth conditions. The maximum effect detected with both procedures was obtained at pH 5.8; in the growth test, at this pH, gene conversion frequency appeared to be most pronounced, being about 10 times higher than that of the control. These results suggest that pH exerts its specific action both on growing and non-growing yeast ceils, and the difference in induction of genetic effect between these two conditions is probably due to a time factor.

It is generally considered that pH is one of the factors that can influence cellular growth. Some investigations have shown that pH, together with certain mutagenic compounds, acts synergistically in the induction of a number of genetic effects. The influence of pH on formation and mutagenicity of N-nitroso compounds is well known (Singh and Kaul, 1978). The influence of pH on the effect of 2,4-D sodium salt on S. cerevisiae and 0165-7992/84/$ 03.00 © 1984Elsevier SciencePublishers B.V.

Salmonella t y p h i m u r i u m has been studied by Zet-

terberg et al. (1977). Other reports have appeared on the effects of pH on the mutagenicity of sodium azide in N e u r o s p o r a crassa and S. t y p h i m u r i u m (Tomlinson and Craig, 1980). In this study, we have analyzed the genetic effects that appear to be due to pH alone in strain D7 of S. cerevisiae.

190

Materials and methods

Yeast strain and media Saccharomyces cerevisiae strain D7 was obtained from Dr. F.K. Zimmermann. This strain can detect, simultaneously, mitotic gene conversion at the trp5 locus, point mutation of the mutant allele ilv 1-92 and mitotic recombination between the centromere and the ade2 locus. Mitotic crossingover can be detected visually as pink and red twin, sectored colonies which are due to the formation of homozygous cells of the genotype ade2-40/ade2-40 (deep red) and ade2-119/ade2119 (pink) from the originally heteroallelic condition, which forms white colonies. Mitotic gene conversion can be detected by the appearance of tryptophan-non-requiring colonies on selective media. The alleles involved are trp5-12 and trp5-27 derived from the widely used strain D4. Mutation induction can be followed by the appearance of isoleucine-non-requiring colonies on selective media. D7 is homoallelic ilv 1-92:ilv 1-92 (Zimmermann et al., 1975). In this work we consider mitotic gene conversion and reverse point mutation to follow the induction of genetic effects (Bronzetti et al., 1978). The composition of the complete liquid growth medium was: 1%0 yeast extract (Difco), 2% bacto peptone (Difco), 2O7oglucose. The composition of the selective synthetic medium plates was: 0.6% bacto yeast nitrogen base (without amino acids) (Difco), 2% glucose, 0.5% adenine, 1% Lisoleucine (on the selective medium for gene conversion) or 1% tryptophan (on the selective medium for point mutation). Method to detect effects of p H in conditions o f growth The pH of the medium is normally 6.24. Therefore, 0.1 M HCI or 0.1 M N a O H were added to obtain the desired pH conditions in the media. Into every flask containing 200 ml of such culture medium, 180 cells of strain D7 harvested from a stationary phase culture were inoculated. The flasks were shaken at 30°C for 40 h. Cellular counts for evaluation of the growth phase were

carried out. After appropriate dilution, each cellular suspension was plated on selective medium for enumeration of trp convertants and ilv revertants, which were expressed, respectively, as numbers of cells per 105 and 106 survivors, and on complete medium for survivor counts (Zimmermann, 1973).

Method to detect effects of p H in non-growth conditions An aliquot of cells was harvested from a stationary-phase culture (initial pH o f the medium was 6.24). These cells were centrifuged at 4000 rpm, washed and resuspended in deionized water. Into a series of flasks were placed 1.0 ml cell suspension (ca. 600 x 106 ml) and 3 ml of 0.1 M phosphate buffer with a range of predetermined pH values (5-5.8-6.2-7-8). The flasks were incubated at 37°C for 2 h on a roller drum, after which the suspensions were plated on selective and complete media as described above. Results

Specific pH conditions, both in the growth medium and in the incubation mixture for the suspension test, appeared to bring about an enhancement of the frequency of spontaneous gene conversion in strain D7 of S. cerevisiae (Tables 1 and 2), while they did not affect significantly frequencies of point mutation (data not shown). Table 1 reports the combined results of independent experiments carried out during cell growth at different initial pH values. At extreme pH values, cell growth was greatly inhibited. However, no significantly inhibitory effects were observed in the pH range 5.0-7.0. The cultures grown with an initial pH of 5.8 showed the highest gene conversion frequency, nearly 10 times greater than that of the control. Gene conversion frequencies at pH values other than 5.8 were considerably lower, the minimum being determined for cultures grown in the conventional non-pH-adjusted medium (pH 6.24). Strong confirmation of the above results was obtained from experiments carried out with non-growth conditions. Table 2

191 TABLE 1 INDUCTION OF GENE CONVERSION AND EFFECT ON GROWTH IN S. cerevisiae STRAIN D7 AT DIFFERENT pH VALUES IN LIQUID CULTURE MEDIUM Initial pH

3.80 4 5 5.8 6 7 Control 6.24

Number of cells/ml after Colonies counted 40 h of incubation in liquid medium

070 Survivors after plating

Locus trp5 Convertants counted

Convertants/105 survivors

1400 45 × 106 144 × 10 6 180 x 106 178 x 106 162 X 10 6

824 2310 7764 8128 8459 7635

9.7 27.3 91.9 96.2 100 90.4

243 761 3690 8963 2265 2169

2.9 3.2 4.7 11.0 2.6 2.8

180 × 106

8445

100

1175

TABLE 2 INDUCTION OF GENE CONVERSION IN S. cerevisiae STRAIN D7 AT DIFFERENT pH VALUES UNDER NONGROWTH CONDITIONS pH

Colonies counted

5 5.8 6.2 7 8

2905 2120 3010 3130 2685

Control 7.4

3685

°70 Survivors Locus trp5

78.8 57.5 81.6 84.9 72.8 100

Convertants counted

Convertants/105 survivors

262 322 264 220 327

0.9 1.5 0.8 0.7 1.2

258

0.7

shows the results o f 3 e x p e r i m e n t s in which yeast ceils, g r o w n o n c o n v e n t i o n a l m e d i u m , were r e s u s p e n d e d in 0.1 M p h o s p h a t e at d i f f e r e n t p H values. T h e m a x i m u m effect was o b s e r v e d , as in the earlier e x p e r i m e n t , at p H 5.8. Since the yeast is n o t a l l o w e d to g r o w u n d e r these e x p e r i m e n t a l conditions, it was possible to extend the p H range to p H 8.0, a c o n d i t i o n which does n o t p e r m i t g r o w t h . p H 8.0 was also f o u n d to increase g e n e - c o n v e r s i o n f r e q u e n c y significantly, b u t to a lesser extent t h a n p H 5.8. T h e e n h a n c e m e n t o f gene c o n v e r s i o n freq u e n c y in the n o n - g r o w t h c o n d i t i o n was not as p r o n o u n c e d as in the g r o w t h c o n d i t i o n . T h e results were, however, s u b j e c t e d to analysis ( f a c t o r i a l

1.39

A N O V A ) a n d it was f o u n d t h a t the frequencies o f gene c o n v e r t a n t s at p H 8.0 a n d at p H 7.4 (controls) were highly significantly d i f f e r e n t ( P < 0 . 0 1 ; F1,24 = 18.34) ( W i n e r , 1971). W h i l e there is also a significant d i f f e r e n c e between the v a r i o u s exp e r i m e n t s , the v a r i a n c e due to i n t e r a c t i o n is n o t significant ( P > 0 . 0 5 ; F2,24 = 3.14). C o m p a r i s o n s o f c o n v e r t a n t frequencies between p H 7.4 (control) a n d p H 5.8 were a n a l y z e d b y a c o m b i n a t i o n o f techniques. In Expts. II a n d III, there were equal n u m b e r s o f o b s e r v a t i o n s in each g r o u p a n d the results were also a n a l y z e d by fact o r i a l A N O V A . Differences in c o n v e r t a n t frequencies d u e to p H p r o v e d to be highly significant ( P < 0 . 0 1 ; K1,16 = 50.06). T h e r e was also a highly significant difference d u e to e x p e r i m e n t a t i o n T P < 0 . 0 1 ; F~,16 = 16.81), b u t i n t e r a c t i o n v a r i a n c e was n o t significant ( P > 0.5; F~,16 = 3.81). U n e q u a l n u m b e r s o f observ a t i o n s in E x p t . I were c o m p e n s a t e d for b y Stud e n t ' s t-test analysis, one-tailed. It was f o u n d t h a t the f r e q u e n c y o f gene c o n v e r t a n t s was significantly greater at p H 5.8 t h a n at p H 7.4 ( P < 0 . 0 5 ; t g = 1.87), thus s u p p o r t i n g the analysis o f v a r i a n c e results.

Discussion O u r d a t a f r o m the cellular g r o w t h test experim e n t clearly s h o w e d a c o r r e l a t i o n between p H a n d

192 mitotic gene conversion frequency. This observation could have been due to either or both of two factors: (1) possible interference of the media and the growth conditions; and (2) specific action of p H itself. To explore the latter possibility, we exposed the yeast to the same pH values as in the original experiment, but in completely different experimental conditions in the non-growth condition. The results obtained suggested that the induction of gene conversion is essentially due to a specific action of the pH itself. This action would be effective on resting cells, but apparently not during growth. This is indicated by the observation that, during growth, pH decreased considerably reaching, after 20 h of incubation, the final pH of 5.4, so that the cultures, the starting pH of which ranged between 7 and 6, went beyond the critical pH of 5.80 in the log-phase, but they did not show the significant increase detected in the culture starting at pH 5.80. The fact that gene conversion frequencies in the growth test with an initial pH of 5.8 were so much higher than those in the suspension test experiment, is probably due to a time factor. In the latter experiment, cells were treated for a 2-h period after which they were washed and plated. In the growth test, however, cells were maintained for a period of

40 h, during which, as indicated above, the pH did change. Even so, the length of time cells continued to be exposed at a pH of (about) 5.8 was probably considerably greater than 2 h.

References Bronzetti, G., E. Zeiger and D. Frezza (1978) Genetic activity of trichloroethylene in yeast, J. Environ. Pathol. Toxicol., 1, 411-418. Singh, C., and B.L. Kaul (1978) Role of pH in chemical mutagenesis, J. Sci. Indres, 37(8), 426-432. Tomlinson, C.R. (1980) Effect of pH on the mutagenicity of sodium azide in Neurospora crassa and Salmonella typhimurium, Mutation Res., 70, 179-192. Winer, B.J. (1971) Statistical Principles in Experimental Design, McGraw-Hill, New York, pp. 341-505. Zetterberg, G., L. Busk, R. Elouson, I. Starecnordenhammar and H. Ryttman (1977) The influence of pH on the effects of 2,4-D on Saccharomyces cerevisiae and Salmonella typhimurium, Mutation Res., 42(1), 3-17. Zimmermann, F.K. (1973) Detection of genetically active chemicals using various yeast system, in: A. Hollaender (Ed.), Chemical Mutagens: Principle and Methods for Their Detection, Vol. 3, Plenum, New York, pp. 209-239. Zimmermann, F.K., R. Kers and H. Rosemberger (1975) A yeast strain for simultaneous detection of induced mitotic crossing-over, mitotic gene conversionand reverse mutation, Mutation Res., 28, 381-388.