The induction of gene conversion in yeast by herbicide preparations

Mutation Research, 21 (1973) 83-91

~) Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands

$3

T H E I N D U C T I O N OF G E N E CONVERSION IN YEAST BY H E R B I C I D E PREPARATIONS

JAMES M. PARRY Department of Genetics, University College of Swansea, Singleton Park, Swansea SA2 8PP (Great Britain)

(Received October 5th, 1972)

SUMMARY Three commercial herbicide preparations containing the active ingredients 2(4-chloro-2-methylphenoxy)propionic acid (mecoprop), I,I'-dimethyl-4,4'-bipyridylium (paraquat), and 2-(I-methyl-n-propyl)-4,6-dinitrophenol (dinoseb), were shown to induce mitotic gene conversion in yeast cultures heteroallelie at two loci. Paraquat and dinoseb induced conversion at high survival levels whereas mecoprop was active only at concentrations giving low viability. The concentrations of the herbicides which gave genetic activity were compatible with those used in commercial practice.

INTRODUCTION In living cells genetic change can occur by either mutation (the alteration or deletion of information) or by recombination (the rearrangement of information). In diploid cultures of the yeast Saccharomyces cerevisiae, it is possible to detect mitotic recombination both between (inter) and within (intra) genes6,15. The intragenic event occurs by gene conversion, a process characterised by its failure to yield reciprocal products during recombination. At the molecular level gene conversion involves the accurate transfer of information 2~ from one chromosome strand to another of DNA regions of I00 to 2oo nucleotides ~ resulting in homozygosis of small chromosome regions. In yeast cultures carrying two different non-competing alleles of an auxotrophic marker (heteroalleles) gene conversion results in the production of prototrophie recombinants which m a y be detected by plating upon appropriate selective media. Prototroph production in heteroallelic diploid cultures of yeast occurs at frequencies of up to I 0 0 0 × per nucleotide higher than that found in homoalMic diploids, thus indicating that reverse mutation at one of the m u t a n t sites will not account for the observed yield of prototrophs 12. The frequency of prototrophic recombinants produced by gene conversion has Abbreviations: Dinoseb, 2-(i-methyl-n-propyl)-4,6-dinitrophenol; DNOC, 2-methyl-4,6-dinitrophenol; mecoprop, 2-(4-chloro-2-methyl-phenoxy)propionic acid; paraquat, I,I'-dinlethyl-4,4'bipyridylium; ppm, parts per million.

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been shown to be increased by treatment with UV light 17, ionising radiations 11 and chemical mutagens such as ethyl methanesulphonate ~. An extensive study of induced conversion by ZIMMERMANN~3indicated that every tested mutagen active in yeast also produced a significant increase in the frequency of prototroph production by conversion and perhaps more important in the context of this paper the induction of conversion by chemical mutagens was non-specific. Irrespective of the mode of action of the mutagen used at the level of the DNA, increases in prototroph frequency produced by conversion may be detected. The two important properties of gene conversion, i.e. its lack of specificity and the high level of prototroph yield makes this process attractive as a screening system for environmental mutagens. The value of this system has been discussed fully in previous publications~3, 23 and has been used to demonstrate genetic activity amongst a number of commercial fungicides ~9 and the immunosuppressive drug, cyclophosphamide ~°. Chemical compounds with herbicidal properties have been screened for genetic activity by a number of workers using a range of organisms. Such studies include the demonstration of increased back mutation at the met3 locus of Aspergillus by 3',4'dichloropropionanilidelL the induction of respiratory deficiency in Saccharomyces cerevisiae by chloroprophan~L the induction of resistance to erythromycin, streptomycin and phage resistance in Rhizobium meliloti by dinoseb acetate and LinuronL and reductions in mitotic division in human lymphocyte cultures by propham and chloropropham 2°. KISSNE AND KISS ° have demonstrated the induction of genetic change for a number of characteristics such as spore formation and antibiotic production in Streptomyces globisporus after treatment with 2,4-D, DNOC and simazine. The results of these authors contrast with the lack of mutagenie activity shown by simazine in the work of KASZUBIAKs. Field studies include the demonstration of increased mutation rate in the spring onion Allium fistulosum 1 after treatment with paraquat and the production of inherited abnormalities in tomato 3, after 2,4-D treatment, potato ~ after mecoprop and Ist generation abnormalities of cereal crops 14. In an attempt to standardize the mutagenicity screening of herbicidal preparations in view of the wide range of systems previously used and the variability of the results, we have utilized the technique of prototrophic induction produced by gene conversion in the yeast Saccharomyces cerevisiae. The present paper describes the genetic activity of three commercial herbicides containing the agents paraquat, mecoprop and dinoseb. MATERIALS AND METHODS

Strains The diploid yeast culture used was of the following genotype: a/s, ade 2-o/ade 2-1, his 1-I/his 1- 7 A haploid wild type yeast, a mating type, was also used. The symbols a and ~ refer to the mating type locus, ade-2-o and ade-2 I are non-complementing alleles of the gene adenine-2, his-i-i and his-i- 7 are non-complementing alleles of the gene histidine-i. Thus, the diploid cultures require both

GENE CONVERSION IN YEAST BY HERBICIDES

85

adenine and histidine in the medium for growth. The presence of the gene adenine 2 results in the production of red colonies when grown on complete medium. Gene conversion at the adenine 2 and histidine I loci produces cells capable of growth upon minimal medium deficient in either adenine or histidine.

Media The complete and minimal media have been described in detail elsewhere (PARRyla).

Herbicide preparations (I) Fisons' Supersevtox, containing 185 g/1 of an amine formulation of dinoseb ;

(2) ICI Gramoxone W, containing IOO g/1 of paraquat dichloride ; (3) ICI Methoxane 2, containing 742 g/1 of mecoprop. The three herbicides were utilised as agricultural formulations and concentrations given in the text are expressed as ppm.

Method of treatment Late log phase cultures of yeast grown on solid medium were washed in sterile saline, shaken vigorously and suspended at a concentration of IoT/ml. Suitable dilutions of the herbicide preparations were then added to 2o-ml samples of yeast suspension. Each sample was aerated and incubated for 18 tl at 28 ° to allow for maximum activity of each preparation. After exposure, each culture was washed at least three times by centrifugation at 4000 g and suspended in 20 ml of sterile saline. The final suspension was diluted appropriately and o.i-ml samples plated upon complete and selective medium. Minimal medium plus adenine and minimal medium plus histidine detect the presence of histidine and adenine prototrophs, respectively. The plates were incubated for 5 days at 28 ° before scoring. At sufficiently high concentrations, all three herbicides result in reductions in the cell viability of the yeast suspensions. A number of preliminary experiments were performed to determine the concentration range of each herbicide which results in the induction of prototrophie convertants and the reduction of culture viability. Under the conditions of these experiments, the diluted herbicides gave pH values of 6.0 to 6.5 for dinoseb, 6.0 to 6.5 for mecoprop and 5.5 to 6.0 for paraquat dichloride, respectively. The preparation of mecoprop utilised here gave a pH of 6.5 in concentrated solution in contrast to the manufacturer's indicated pH of IO to II. All experiments were performed at least twice and representative results are presented here. RESULTS

Fig. I shows the effect of I8-h exposures to solutions of 742 to 6678 ppm of mecoprop upon the viability of both haploid and diploid cultures of yeast. The cultures show characteristic differences in their survival curves. In diploid cultures, a resistant shoulder is shown after exposures to solutions of up to 2968 ppm followed bv a period of exponential decline in survival down to o.I °/o viability at a concentration of 5194 ppm. In contrast, the survival curve of the haploid cultures shows no resistant

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Fig. I. T h e effects u p o n t h e cell v i a b i l i t y of h a p l o i d a n d diploid c u l t u r e s of y e a s t p r o d u c e d b y 742 to 6678 p p m m e c o p r o p solution. @, Diploid c u l t u r e s ; solid s y m b o l s indicate c u l t u r e s a u x o t r o p h i c for a d e n i n e a n d histidine, open s y m b o l s indicate a d e n i n e - i n d e p e n d e n t r e v e r t a n t s , m, h a p l o i d cultures.

shoulder. The final slope of the survival curve of both haploid and diploid cultures is identical with a concentration increase of lO4O ppm producing a viability reduction of 90%. The concentration range of 742 to 6678 ppm of mecoprop shows genetic activity as indicated by the induction of mitotic gene conversion at two separate loci, i.e. ade-2 and his-I. The increase in conversion frequency against the concentration of .o

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® 0.1 Fig. 3. The effects upon the cell viability of haploid and diploid cultures of yeast produced by too to 9oo ppm paraquat solution. @, Diploid cultures; solid symbols indicate cultures auxotrophic for adenine and histidine, open symbols indicate adenine-independent revertants, m, Haploid cultures. mecoprop is shown in Fig. 2. Prototrophic c o n v e r t a n t s are i n d u c e d at both loci with m a x i m a of I6OO 4:_ 8 o / I o 6 survivors a n d 353 :~ 3o/Io6 survivors in the case of the adenine a n d histidine loci, respectively. The m a x i m a of p r o t o t r o p h i n d u c t i o n occur at c o n c e n t r a t i o n s of 5936 p p m for the adenine locus a n d 2968 p p m for the histidine locus a n d at viabilities of o . o i 8 % a n d 5.6O/o, respectively. The effect of the second herbicide used, p a r a q u a t , upon the viability of diploid a n d haploid yeast cultures is shown in Fig. 3, using p a r a q u a t c o n c e n t r a t i o n s of ioo to 9oo ppm. The survival curve of the diploid culture shows a resistant shoulder after exposures to solutions of u p to 4oo p p m followed b y an e x p o n e n t i a l decline in survival,

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a concentration increase of 68o ppm producing a viability reduction of 9o0/0. After exposure to a solution of 900 ppm of paraquat, a reduction in viability to lO% of the original ceils is achieved. The haploid survival curve is characterised by the absence of a resistant shoulder and by a change in slope after a concentration of 420 ppm. At low concentrations, viability falls rapidly with a concentration increase of 360 ppm producing a viability reduction of 900/0. At concentrations greater than 420 ppm, a resistant tail can be seen. The final slope of the survival curve shows that a concentration increase of 680 ppm results in a viability reduction of 90%, identical to the final slope of the diploid survival curve. This effect was also seen above after treatment of haploid and diploid cultures after treatment with mecoprop. dinoseb 100

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As shown by the results in Fig. 4, the concentrations of paraquat producing viabilities of between ioo and lO% also result in the induction of gene conversion at both the ade-2 and his-I loci. The frequency of prototrophic adenine convertants rises to a maximum of 154 ° ± IlO/IO s survivors at paraquat concentrations of between 600 and 700 ppm, corresponding to viabilities of 32 % and 2I %. In contrast, the maximum of histidine convertants of 820 ~ 3o/Io e survivors is produced at concentrations of between 400 and 600 ppm corresponding to viabilities of 58% and 32%. The frequencies of both adenine and histidine prototrophs are reduced at concentrations of paraquat above 700 ppm. Figs. 5 and 6 show the effects produced by the third herbicide used, dinoseb, upon cell viability and induced gene conversion. Fig. 5 demonstrates the viability of diploid and haploid yeast cultures exposed to solutions containing from 185 to 1665 ppm of dinoseb. The diploid culture shows a resistant shoulder up to a concentration of 555 ppm. At higher concentrations, cell viability falls exponentially with the slope

G E N E C O N V E R S I O N I N Y E A S T BY H E R B I C I D E S

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Fig. 6. Increase in adenine and histidine convertant frequency induced by 18 5 to 1665 t)pm dinoseb solution. The 95% confidence limits of each point are shown. &, Increase in adenine prototrophs. e, Increase in histidine prototrophs. of the survival curve, showing a 9o% reduction in viability produced by a concentration increase of 833 p p m falling to 2.8% viability at a concentration of 1665 ppm. In contrast, the survival curve of the haploid culture lacks the resistant shoulder, but is characterised by a change in slope at higher concentrations. At low concentrations, up to 314 ppm, the survival curve shows a steep initial slope where a concentration increase of 296 p p m produced at 9o% reduction in viability. At concentrations greater t h a n 314 ppm, the slope of the survival curve is reduced to where a concentration increase of 833 p p m produced a 9 o % reduction in viability. As with mecoprop and paraquat, the final slopes of haploid and diploid survival curves are identical. As shown in Fig. 6, the concentration range of 185 to 1665 p p m of dinoseb is effective in inducing gene conversion at both the ade-2 and hist-I loci. The increase in adenine prototrophs shows a m a x i m u m at the higher concentration of 1295 ppm, this m a x i m u m value of 55 ~ 3.o/lo6 survivors was produced at a viability of 7.0%I n order to determine the effects of the three herbicidal preparations upon the viability of the prototrophs produced by gene conversion, the sensitivity of such cells to all three agents was tested. Samples of 5o individual adenine prototrophs were taken from control plates and exposed to preparations of mecoprop, p a r a q u a t and dinoseb under the same conditions as those for auxotrophic cultures. The prototrophic cultures showed little or no variation in response to all three herbicides when compared with the auxotrophic test cultures. These results are shown in Figs. I, 3 and 5 and suggest t h a t the observed increases in prototroph production are not the result of selection in the presence of the herbicide preparations. I)ISCU SSION

Tile results presented here indicate t h a t the three herbicidal preparations containing the chemicals mecoprop, p a r a q u a t and dinoseb, show genetic activity as shown b y the induction of gene conversion at two loci in the yeast, Saccharomyces cerevisiae. In all cases, the frequency of induction of adenine prototrophs was signifi-

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j.M. PARRY

cantly higher than the frequency of histidine prototrophs. The ratio of the maximum frequency of adenine to histidine prototrophs was variable with values of 4.5 : I, 1. 9 : I and I I : I for mecoprop, paraquat and dinoseb respectively. The viability levels at which conversion induction may be detected varies with the three herbicide preparations. Both paraquat and dinoseb induce gene conversion at high survival levels whereas mecoprop induces conversion only at low survival levels. The concentration range of the three herbicides which resulted in the induction of gene conversion correlates with that used for spray purposes. For example, paraquat and mecoprop show activity in the concentration ranges of ioo to iooo ppm and 742 to 7420 ppm, respectively, and are used in agricultural practice at maximum concentrations of 500 ppm and 7400 ppm. The concentration of paraquat on food crops after proper spraying has been calculated to be IO ppm (ref. 4), a concentration IO times less than that giving genetic activity in these studies. The concentration range for genetic activity in the presence of paraquat, i.e. > IOO ppm, is similar to that shown to be mutagenic in the previous study of ALEKPEREW1. The relationship of results such as those presented here to the general problem of "mutagen exposure" of the human population remains debatable. The implications of the use of microbial systems in the determination of genetic activity has been discussed by BRIDGES~. The advantages and disadvantages of the use of conversion induction in yeast has been discussed in some depth by ZIMMERMANN~3 and PARRYla. A relevant factor that emerges from both this and other related studies is that we can positively say that a number of commercial herbicides are capable of inducing genetic change in laboratory cultures of bacteria and fungi. It would require very little extension of this present work to predict that such compounds may be capable of inducing genetic change in microbes of agricultural importance. In particular, characteristics such as the virulence of fungal pathogens and fungicide resistance may be influenced by induced mutation and recombination produced by herbicidal sprays. Clearly, if such genetic changes occur in the field they may be of considerable economic importance. ACKNOWLEDGEMENTS

I express my thanks to my wife, Dr. ELIZABETH M. PARRY, for reading and commenting upon the manuscript, and Mrs. SHEILA TERNAN for her skilled technical assistance. This work was supported by a grant from the Science Research Council.

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2 BRIDGES, B. A., Screening for environmental agents causing genetic damage, Lab. Pract., 21

(1972) 411 416.

3 DARK, S. O. S., A genetic effect on the progeny of tomato plants sprayed with 2,4-D, J. Natl. Inst. Agric. Botany, ii (1967) 199 204. 4 FLETCHER, K., Production and viability of eggs from hens treated with paraquat, Nature, 215

(1967) 14o7-14o8.

5 FOGEL, S., ANDR. I~. MORTIMER, Informational transfer in meiotic gene conversion, Proc. Natl. Acad. Sci. (U.S.), 62 (1969) 96-1o3. 6 JAMES, A. P., ANDB. LEE-WHITING, Radiation induced genetic segregations in vegetative ceils of yeast, Genetics, 4° (1955) 826-831.

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7 KABIESCH, W., Carry-over effects of m e c o p r o p in p o t a t o e s in the i s t and 2nd generation, Z. Pflanzenkrankh. Pflanzenpathol. Pflanzenschutz, 4 (1968) 137. 8 KASZUBIAK, H., The effects of herbicides on R h i z o b i u m 3- Influence of herbicides on m u t a t i o n , Acta Microbiol., Pol., 17 (1968) 51-579 KlSSNE, M., AND N. KISS, I n v e s t i g a t i n g the genotypic effects of herbicides on individual species of Streptomyces, Agrartud. Egyet. K6zlemen. (G6d6ll~'), (1966) ioo--i ~4lO MARQUARDT, H., AND D. SIEBERT, Ein neuer h o s t - m e d i a t e d assay (Urinversuch) Nachweis m u t a g e n e r Stoffe mit Saccharomyces cerevisiae, Naturwissenschaften, 58 (i 97I) 568i i MANNEY, R. T., AND R. K. MORTIMER, Allelic m a p p i n g in yeast b y X - r a y induced reversiou, Science, i43 (1964) 581 583 . J 2 PARRY, J. M., Comparison of the effects of UV light and ethyl m e t h a n e s u l p h a t e upon the frequency of mitotic r e c o m b i n a t i o n in yeast, Mol. Gen. Genet., lO6 (1969) 66 72. 13 PARRY, J . . ~ . , Mitotic r e c o m b i n a t i o n in yeast as a test of genetic damage, Lab. Pract., 2~ {1972) 417-42o. 14 PETERSEN, 1!. J., Chemical control of weeds in w i n t e r cereals, Ttdsskr. Planteavl., 69 (1965) 41o 417 . 15 PRASAD, I., Mutagenic effects of the herbicide 3',4'-dichloropropionanilide and its degradation products, Can. J. Microbiol., 16 (197 o) 369 372. 16 ROMAN, H., A s y s t e m selective for m u t a t i o n s affecting the synthesis of adenine ill yeast, Compt. Rend., Tray. Lab. Carlsberg, 26 (I956) 299-314. i7 ROMAN, H., AND F. JACOB, Effect de la lumi~re ultraviolette sur la recombination g6n6tique entre all~des chez la levure, Compt. Rend., 245 (1957) IO32-~o3418 SCHUBERT, A., Studies on the induction of respiratory-deficient yeast m u t a t i o n s with pesticides, Z. Mllg. Microbiol., 9 (1969) 483-485 . I9 SlEBERT, D., F. l(. ZIMMERMAN AND E. LEMPERLE, Genetic effects of fungicides, Mutation Res., io (197 o) 533-54320 TIMAN, J., Effect of the herbicide p r o p h a m and c h l o r o p r o p h a m on the rate o[ mitosis in h u m a n l y m p h o c y t e s in culture, Pesticide Sci., i (197 o) 191-192. 21 YORST, H. T., R. S. CHALEFF AND J . P. EINERTY, I n d u c t i o n and mitotic recombiuation in Saccharomyces cerevisiae b y ethyl m e t h a n e s u l p h o n a t e , Nature, 215 (1967) 660 661. 22 ZIMMERMANN, V. K., E n z y m e studies on the p r o d u c t s of mitotic gene conversion in Naccharomyces cerevisiae, Mol. Gen. Genet., lO2 (1968) 171-184. 23 ZIMMERMANN, F. K., I n d u c t i o n of mitotic gene conversion by mutagens, Mutation Res., r r (r97 t) 327 337-