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Hycanthone as a specific frameshift mutagen in Saccharomyces cerevisiae
397
Mutation Research, 72 (1980) 397--404 © Elsevier/North-Holland Biomedical Press
H Y C A N T H O N E AS A SPECIFIC F R A M E S H I F T M U T A G E N IN
Saccbaromyces cerevisiae GIOVANNA LUCCHINI, SILVIO SORA and LUCIA PANZERI
Istituto di Genetica, UniversiM di Milano, Milano (Italy) (Received 31 March 1980) (Accepted 21 April 1980)
Summary Reversion of mutations of different molecular nature was studied after treatment with hycanthone in mild conditions (0.05--0.4 mM, 4 h in the dark, pH 7.2). The mutagen had a very low reversion activity on 3 missense and 4 nonsense mutations (2 U A A and 2 UAG), although it was very active on 3 frameshift mutations. Our data on intragenic reversion and frameshift suppressors indicate that h y c a n t h o n e can induce both insertions and deletions.
The molecular specificity of mutagenic agents has only seldom been demonstrated in yeast. Among the base-substitution mutagens, 2-AP seems to induce practically only AT -* GC transitions (Sora et al., 1973), whereas others, such as EMS, DES, MNNG, are recognized to cause GC-~ AT transitions (Prakash and Sherman, 1973), b u t in rather restrictive experimental conditions, such as at low dosage, high survival and short treatment. Frameshifts have been reported for yeast, and the most specific inducer of such mutants seems to be ICR170 (Brusick, 1970), although it is known that this agent can also cause missense and nonsense mutations (Sherman et al., 1974). Even under ideal experimental conditions, n o t more than 50% of the mutants induced seems to be of the insertion-deletion t y p e (Culberston et al., 1977). H y c a n t h o n e (Ong, 1978) is claimed to be a specific frameshift mutagen in Salmonella typhimurium (Brusick and Zeiger, 1972; Hartman et al., 1973; Ray et al., 1975) and phage T4 (Hartman et al., 1971}. In yeast, treatment with hycanthone at pH 5.9 and 7.0 for long exposures causes reversion of frameshift, missense and nonsense mutations at almost equal rates (Meadows et al., 1973; Abbreviations: 2-AP, 2-aminopurine; EMS, ethyl methanesulphonate; DES, diethyl sulphate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; ICR170, 2-methoxy-6 chloro-9[3-(ethyl-2-chloroethyl)arninopropylamino ] aeridine-2-HCl.
398
yon Borstel and Igali, 1975); therefore it is claimed that this mutagen does not show any molecular specificity in Saccharomyces cerevisiae, even though, at pH 7.0, with the shortest time of treatment (4.5 h), a frameshift mutant (hom310) shows a much higher increase of the reversion rate than the 2 base substitutions. An investigation was carried o u t with the aim of finding whether, in particular treatment conditions, hycanthone also shows a real molecular specificity in yeast. It was f o u n d that, at pH 7.2, low dosages of hycanthone (between 0.05 and 0.4 mM), which cause not more than 70--80% death for short treatment times, showed an appreciable specificity in inducing reversion of frameshift mutations. Material and methods
Media YEPD, minimal medium 40 and sporulation medium VB, as previously described (Magni et al., 1977). Supplemented medium was obtained by adding, to minimal medium 40, adenine, histidine, arginine, leucine, phenylalanine, tyrosine, t r y p t o p h a n and uracil at 25 rag/l, methionine at 30 mgfl and threonine at 60 mg/1 (horn3-10 requires homoserine or m e t h i o n i n e + threonine). Omission media were used for the selection of revertants. Analysis o f nonsense suppressors The frequency of nonsense and frameshift suppressors was estimated in 2 ways: (a) co-reversion of 2 nonsense or frameshift mutants; and (b) segregation of suppressors in random spore analysis for single revertants. Hycanthone treatment Cells grown in YEPD medium to the stationary phase were collected, washed and resuspended in phosphate buffer 1/60 M, pH 7.2. Hycanthone, dissolved in the same buffer, was added at suitable concentrations. The final cell concentration was 5 × 108 cells/ml. The treatment was carried o u t at 37°C, in the dark, for 4 h, with gentle shaking, and stopped by centrifugation and washing with distilled water. The treated cells were regrown for 2 h in YEPD medium, in the dark, at 28°C, before seeding on to complete and selective media. Seeding was done under weak yellow light. The plates were incubated in the dark at 28°C, and colonies were scored after 4 days for survival and after 7--10 days for reversion. Strains 5853/4a 6143/2c 6198/12c 6110/11a 6126/16c 2268
a, his4-1, leu2-1, tyr7-1, trpl-1, ura3, can100-48 a, his4-519, horn3-10, ura4-10, arg4-18, ade2-1 a, his4-519, leu2-3, lys2-1 a, his4-519, horn3-10, ura4-10, arg4-18, ade2-1 a, adel-lO, arg4-27, tyr7-1, trpl-1, his4-1 a, leu2-3, his4-519, can1-101Fs
These strains were constructed by us; the alleles hom3-10 and adel-lO originated in our laboratory; his4-519, leu2-3 and can1-101 F S are gifts from Dr. G.R. Fink; all the other mutants were originally received from the Yeast Genetic Stock Center of Berkeley.
399 TABLE 1 PATTERN OF SUPPRESSIBILITY SHIFT SUPPRESSORS Marker
OF A SERIES OF YEAST MUTANTS
BY EXTERNAL
FRAME-
Suppressor a SUF5-1
SUF145
SUF41
9
SUF514
his4-519
+
+
+
leu2-3
+
+-
+
+
can1-101
+
+-
--
--
horn3-10
--
+-
+-
+
adel-lO
--
--
+
a E f f i c i e n c y of s u p p r e s s i o n w a s e v a l u a t e d b y c o m p a r i n g t h e g r o w t h on s e l e c t i v e m e d i a o f w i l d - t y p e s t r a i n s w i t h s t r a i n s c a r r y i n g t h e l i s t e d m a r k e r s t o g e t h e r w i t h t h e single s u p p r e s s o r m u t a t i o n s . +, v e r y e f f i c i e n t s u p p r e s s i o n . +, g o o d s u p p r e s s i o n . --, n o s u p p r e s s i o n .
Molecular nature o f mutations used for reversion studies The 3 mutants arg4-18, arg4-27 and ura4-10 were classified as missense on the basis of the following pieces of evidence: arg4-18 and arg4-27 show nonpolarized complementation (Mortimer, personal communication), ura4-10 was induced by 2-AP (Sora et al., 1973); t h e y are all non-suppressible by external suppressors and they all show a high reversion rate when exposed to base substitution mutagens, such as EMS, DES and MNNG, and react only weakly to ICR170. The mutants leu2-1 and his4-1 both contain UAA nonsense codons; tyr7-1 and trpl-1 both contain UAG nonsense codons as proved by their suppressibility by nonsense suppressors of the 2 types (Hawthorne and Leupold, 1974). The other 4 m u t a n t s used in this work are characterized as follows: his4-519, leu2-3, adel-lO were induced by ICR170; horn3-10 originated spontaneously during meiosis; all 4 showed a high increase of reversion rate with ICR170 and their reversibility by base-substitution mutagens (EMS, DES, MNNG) was low. None of these 4 markers was reversible by nonsense suppressors; their suppressibility by other external suppressors is shown in Table 1. Suppressor SUF5-1 is an ICR170-induced m u t a t i o n (Culberston et al., 1977), recognized as a suppressor of addition mutations. The other 3 suppressors are spontaneous mutations isolated by us: S U F 1 4 5 and S U F 4 1 9 as external suppressors of his4-519, and S U F 5 1 4 as an external suppressor of horn3-10. S U F 1 4 5 was identified to be allelic to SUF5-1 ; S U F 4 1 9 and S U F 5 1 4 are allelic to each other, but n o t to SUFS-1. None of these 4 suppressors suppresses either nonsense UAA or UAG mutations (Culberston et al., 1977; our unpublished data), and SUF5-1 does n o t suppress UAG mutations (Culberston et al., 1977). We are therefore confident that the 3 mutations his4-519, leu2-3 and h o m 3 - 1 0 are frameshift mutations, although n o t necessarily of the same type. The molecular nature of m u t a t i o n a d e l - l O as a frameshift has n o t y e t been satisfactorily proved.
400
Results
Reversion of frameshift and base-substitution mutants The large number of markers used for our reversion studies made it impossible to prepare a unique strain carrying all the mutations. Different strains were therefore constructed and their sensitivity to the lethal effects of h y c a n t h o n e tested. Survival data reported in Fig. 1 indicate that the 5 strains used show almost identical sensitivity to h y c a n t h o n e , at least at the lowest doses. In addition, m a n y markers were present in more than one strain, and no significant differences were observed in the spontaneous and induced reversions of the same m u t a t i o n in different genetic backgrounds. The frequencies of spontaneous revertants are quite different for the 11 markers used, as shown in Table 2. For the nonsense mutations it was confirmed, as is well known, t h a t the over-all frequency of suppressor mutations is almost one order of magnitude higher than that of true back mutations for the same genes. To compare the effect of the same mutagen on genes showing such great differences of spontaneous reversion frequencies, we believed that the most effective way of expressing our mutagenesis data was the use of the parameter T/C, i.e. the ratio between frequency of revertants per survivor after t r e a t m e n t and frequency of spontaneous revertants. Mutagenesis data are reported in Figs. 2, 3, 4 and 5. The 3 missense mutations showed a very low induced reversion rate, if any, as the maximal increase at 0.4 mM did n o t exceed a TIC value of 5 (Fig. 3). toc-
~0
~-50
i
m
Hycanthone
mM
Hycant hone
rnM
Fig. 1. Survival after h y c a n t h o n e in 4-h t r e a t m e n t s of strains: 5 8 5 3 / 4 a (D), 6 1 9 8 / 1 2 c (o); 6 2 4 3 / 2 c (~); 6 1 1 0 / 1 1 a (A); 6 1 2 6 / 1 6 c (m). Fig. 2. Reversion of frameshift m u t a n t s by h y c a n t h o n e : a d e l - 1 0 (w); his4-519 (o); h o m 3 - 1 0 (o); leu2-3
(o).
401 TABLE 2 S P O N T A N E O U S R E V E R S I O N F R E Q U E N C I E S O F T H E M U T A N T A L L E L E S U S E D IN T H I S S T U D Y Mutation
Reversion frequency/10 8 cells a
Total
Site r e v e r s i o n
his4-519 leu2-3 horn3-10 adel-lO
0.2 1.3 0.45 0.21
arg4-18 ura4-10 arg4-27
0.05 0.12 0.09
his4-1 leu 2-1 tyr7-1 trp l-1
44 37.5 32 21.8
2.1 0.3 0.9 0.62
a All v a l u e s are t h e a v e r a g e s f r o m a t least 3 i n d e p e n d e n t E x p t s .
The total reversion of nonsense mutations (due for more than 90% to external suppressors events) is practically unaffected by h y c a n t h o n e (Fig. 4), whereas their site reversion seems to be a little more sensitive to the mutagen. However, the results must be considered with caution, as the frequency of the event is very low and therefore the data are based on few colonies per dose, so
/
I /
/
/
3O-
t/
/
z
/
m c I
/
®20-
/
/
I ~v
/
o
I
/
/
to-
/
/
/ .
Hycanthone
mM
/
Hycanthone
mM
Fig. 3. R e v e r s i o n o f m i s s e n s e m u t a n t s b y h y c a n t h o n e : a r g 4 - 1 8 (o); a r g 4 - 2 7 (o), u r a 4 - 1 0 (o). Fig. 4. T o t a l r e v e r s i o n o f n o n s e n s e m u t a n t s b y h y c a n t h o n e (= b a c k m u t a t i o n s + s u p p r e s s o r m u t a t i o n s ) : his4-1 (4); l e u 2 - 1 (o); t r p l - 1 (o); t y r 7 - 1 (o).
402
/ /
/
c
-
/
b.
|
/
Iv
/ /
Hycanthone
mM
Fig. 5. Site reversion o f nonsense mutants b y hycanthone: his4-1 (A); leu2-1 ( e ) ; t r p l - 1 (=); t y r 7 - 1 (o).
t h a t the estimate may be biased by r a n d o m fluctuations. The increment of TIC never exceeded a value of 10 (Fig. 5). On the contrary, the 3 definite frameshift mutations and also adel-lO showed an appreciable increase of reversion due to h y c a n t h o n e that was already evident at 0.2 mM and with good dose--effect curves always reached T/C values above 50 at 0.4 mM {Fig. 2).
Does hycanthone induce both insertions and deletions? The above results indicate that .hycanthone is endowed with good specificity in inducing frameshift mutations, but t h e y do n o t allow any conclusion about its molecular specificity or whether this mutagen preferentially induces insertions or deletions or both. Some data on the reversion of hycanthone-induced mutations seem to support the hypothesis t h a t this mutagen can cause base losses and insertions. 2
TABLE 3 R E V E R S I O N BY H Y C A N T H O N E O F M U T A T I O N S I N D U C E D BY T H E S A M E M U T A G E N Hycanthone (raM)
0 0.1 0.2 0.3 0.4
R e v e r t a n t s X 109 adeH3
aroH6
0.67 3.3 7.4 13.0 26.0
9.4 78.0 330.0 600.0 530.0
403 TABLE 4 REVERSION Hycanthone (raM)
0 0.1 0.2 0.3
O F h i s 4 . 5 1 9 I N D U C E D BY H Y C A N T H O N E Intragenic mutations
Suppressor mutations
N ( X l 0 9)
T/C
N ( X l 0 9)
T/C
1.9 29.2 70.5 118
15.36 37.1 62.1
0.08 0.84 3.5 5.8
10.5 43.75 72.5
auxotrophic mutants, adeH3 and atoll6, obtained after t r e a t m e n t by hycanthone, were analyzed by our standard reversion procedure. Both mutations were highly sensitive to the mutagenic action of h y c a n t h o n e {Table 3). The m u t a n t adeH3 evidently reverted only by intragenic events, as no external suppressor was isolated among spontaneous or induced revertants. On the other hand, aroH6 was phenotypically reverted by external suppressors, whose frequency a m o u n t e d to about 50% among spontaneous revertants, and among hycanthone-induced revertants it can be as high as 90%. More convincing evidence was obtained with the m u t a t i o n his4-519, whose molecular nature is far better known. The frequency of spontaneous and hycanthone-induced reversions was carefully determined for both intragenic events and external suppressors. Table 4 indicates t h a t the mutagen was equally effective in inducing intragenic events and the molecularly opposite p h e n o m e n o n at the suppressor genes.
Discussion From the above results it is evident t h a t h y c a n t h o n e , in suitable experimental conditions, consisting essentially of a mild t r e a t m e n t in the dark at neutral pH, was much more effective at inducing frameshift reversions, than base-substitution mutations. That h y c a n t h o n e , in our conditions, induced only few, if any, base substitutions, was proved n o t only by the scanty reactivity of 3 missenses but also by the results on nonsense mutations: the 4 nonsense markers used for these experiments can easily be reverted by any type of base substitution, such as AT -* GC and GC -~ AT transitions and transversions. Mutant adel-lO was considered as a potential frameshift. The present data seem to substantiate this hypothesis considering its high reversibility by hycanthone. As for the specificity in inducing either insertions or deletions, or both, our data indicate that h y c a n t h o n e can determine both types of mutational event. The findings on the reversion of hycanthone-induced mutations prove t h a t h y c a n t h o n e can revert mutations induced by itself. The frameshift nature of such mutations can be presumed with good probability although n o t y e t with absolute certainty; t h e y are n o t suppressible by nonsense (UAA) suppressors and react to h y c a n t h o n e much more than any base-substitution m u t a n t so far tested. Our data on the induced reversion of his4-519 seem to be more conclusive.
404
The almost identical sensitivity to hycanthone of intragenic events and suppressor mutations indicates that this mutagen can induce, with equal efficiency, insertions and deletions, and for frameshift mutations, internal and external suppression must be mostly due to opposite events. Acknowledgement This work was partially supported by a grant of C.N.R. (Roma), Progetto Finalizzato: Promozione qualit~ dell'ambiente. References Brusick, D.G. (1970) The mutagenic activity of I C R 1 7 0 in Saccharomyces cerevisiae, Mutation Res., 10, 11--19. Brusick, D.G., and E. Zeiger (1972) A c o m p a r i s o n of chemically i nduc e d reversion pa t t e rns of Salmonella typhimurium and Saccharomyces cerevisiae, using in vitro plate tests, Mut a t i on Res., 14, 271--275. Culberston, M.R., L. Charnas, M.T. J o h n s o n and G.R. Fink (1977) Frameshifts and frameshift suppressors in Saccharomyces cerevzsiae, Genetics, 8 6 , 7 4 5 - - 7 5 6 . Hartman, P.E., K. Levine, Z. H a r t m a n and H. Berger (1971) H y c a n t h o n e : A frameshift mutagen, Science, 172, 1058--1060. Har tman , P.E., H. Berger and Z. H a r t m a n (1973) Comparison of h y c a n t h o n e (Etrenol), some h y c a n t h o n e analogs, m y x i n and 4 - n i t r o q u i n o l i n e - l - o x i d e as frameshift mutagens, J. Pharmacol. Exp. Ther., 186, 390--398. Hawthorne, D.C., and U. Leupold (1974) in: Current Topics in Microbiology and I m m u n o l o g y : Suppressor Mutations in Yeast, Springer, Berlin, Vol. 64, pp. 1--47. Magni, G.E., L. Panzeri and S. Sora (1977) Molecular specificity of X-radiation and its repair in Saccharomyces cerevisiae, M u t a t i o n Res., 42, 223--234. Meadows, M.G., S. Quah and R.C. yon Borstel (1973) Mutagenic action of h y c a n t h o n e and 1A-4 on yeast, J. Pharmacol. Exp. Ther., 187, 444--450. Ong, T. (1978) Genetic activity of h y c a n t h o n e and other antischistosomal drugs, Mutation Res., 55, 43-70. Prakash, L., and F. Sherman (1973) Mutagenic specificity: reversion of i s o - l - c y t o c h x o m e c m u t a n t s of yeast, J. Mol. Biol., 79, 65--82. Ray, V.A., H.E. Holden, J.H. Hellis Jr. and M.L. H y n e e k (1975) A comparative s t u d y on the genetic effects of h y c a n t h o n e and o x a m i q u i n e , J. Toxicol. Environ. Health, 1 , 2 1 1 - - 2 2 7 . Sherman, F., J.W. Stewart, M. Jackson, R. Gilmore and J. Parker (1974) Mutants of yeast defective in iso1-cytochrome c, Genetics, 7 7 , 2 5 5 - - 2 8 4 . Sofa, S., L. Panzeri, and G.E. Magni (1973) Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae, Mutation Res., 20, 207--213: yon Borstel, R.C., and S. Igali (1975) Mutagenicity testing of antischistosomal t h i o x a n t h e n o n e s and indazoles on yeast, J. Toxicol. Environ. Health, 1 , 2 8 1 - - 2 9 1 .
Mutation Research, 72 (1980) 397--404 © Elsevier/North-Holland Biomedical Press
H Y C A N T H O N E AS A SPECIFIC F R A M E S H I F T M U T A G E N IN
Saccbaromyces cerevisiae GIOVANNA LUCCHINI, SILVIO SORA and LUCIA PANZERI
Istituto di Genetica, UniversiM di Milano, Milano (Italy) (Received 31 March 1980) (Accepted 21 April 1980)
Summary Reversion of mutations of different molecular nature was studied after treatment with hycanthone in mild conditions (0.05--0.4 mM, 4 h in the dark, pH 7.2). The mutagen had a very low reversion activity on 3 missense and 4 nonsense mutations (2 U A A and 2 UAG), although it was very active on 3 frameshift mutations. Our data on intragenic reversion and frameshift suppressors indicate that h y c a n t h o n e can induce both insertions and deletions.
The molecular specificity of mutagenic agents has only seldom been demonstrated in yeast. Among the base-substitution mutagens, 2-AP seems to induce practically only AT -* GC transitions (Sora et al., 1973), whereas others, such as EMS, DES, MNNG, are recognized to cause GC-~ AT transitions (Prakash and Sherman, 1973), b u t in rather restrictive experimental conditions, such as at low dosage, high survival and short treatment. Frameshifts have been reported for yeast, and the most specific inducer of such mutants seems to be ICR170 (Brusick, 1970), although it is known that this agent can also cause missense and nonsense mutations (Sherman et al., 1974). Even under ideal experimental conditions, n o t more than 50% of the mutants induced seems to be of the insertion-deletion t y p e (Culberston et al., 1977). H y c a n t h o n e (Ong, 1978) is claimed to be a specific frameshift mutagen in Salmonella typhimurium (Brusick and Zeiger, 1972; Hartman et al., 1973; Ray et al., 1975) and phage T4 (Hartman et al., 1971}. In yeast, treatment with hycanthone at pH 5.9 and 7.0 for long exposures causes reversion of frameshift, missense and nonsense mutations at almost equal rates (Meadows et al., 1973; Abbreviations: 2-AP, 2-aminopurine; EMS, ethyl methanesulphonate; DES, diethyl sulphate; MNNG, N-methyl-N'-nitro-N-nitrosoguanidine; ICR170, 2-methoxy-6 chloro-9[3-(ethyl-2-chloroethyl)arninopropylamino ] aeridine-2-HCl.
398
yon Borstel and Igali, 1975); therefore it is claimed that this mutagen does not show any molecular specificity in Saccharomyces cerevisiae, even though, at pH 7.0, with the shortest time of treatment (4.5 h), a frameshift mutant (hom310) shows a much higher increase of the reversion rate than the 2 base substitutions. An investigation was carried o u t with the aim of finding whether, in particular treatment conditions, hycanthone also shows a real molecular specificity in yeast. It was f o u n d that, at pH 7.2, low dosages of hycanthone (between 0.05 and 0.4 mM), which cause not more than 70--80% death for short treatment times, showed an appreciable specificity in inducing reversion of frameshift mutations. Material and methods
Media YEPD, minimal medium 40 and sporulation medium VB, as previously described (Magni et al., 1977). Supplemented medium was obtained by adding, to minimal medium 40, adenine, histidine, arginine, leucine, phenylalanine, tyrosine, t r y p t o p h a n and uracil at 25 rag/l, methionine at 30 mgfl and threonine at 60 mg/1 (horn3-10 requires homoserine or m e t h i o n i n e + threonine). Omission media were used for the selection of revertants. Analysis o f nonsense suppressors The frequency of nonsense and frameshift suppressors was estimated in 2 ways: (a) co-reversion of 2 nonsense or frameshift mutants; and (b) segregation of suppressors in random spore analysis for single revertants. Hycanthone treatment Cells grown in YEPD medium to the stationary phase were collected, washed and resuspended in phosphate buffer 1/60 M, pH 7.2. Hycanthone, dissolved in the same buffer, was added at suitable concentrations. The final cell concentration was 5 × 108 cells/ml. The treatment was carried o u t at 37°C, in the dark, for 4 h, with gentle shaking, and stopped by centrifugation and washing with distilled water. The treated cells were regrown for 2 h in YEPD medium, in the dark, at 28°C, before seeding on to complete and selective media. Seeding was done under weak yellow light. The plates were incubated in the dark at 28°C, and colonies were scored after 4 days for survival and after 7--10 days for reversion. Strains 5853/4a 6143/2c 6198/12c 6110/11a 6126/16c 2268
a, his4-1, leu2-1, tyr7-1, trpl-1, ura3, can100-48 a, his4-519, horn3-10, ura4-10, arg4-18, ade2-1 a, his4-519, leu2-3, lys2-1 a, his4-519, horn3-10, ura4-10, arg4-18, ade2-1 a, adel-lO, arg4-27, tyr7-1, trpl-1, his4-1 a, leu2-3, his4-519, can1-101Fs
These strains were constructed by us; the alleles hom3-10 and adel-lO originated in our laboratory; his4-519, leu2-3 and can1-101 F S are gifts from Dr. G.R. Fink; all the other mutants were originally received from the Yeast Genetic Stock Center of Berkeley.
399 TABLE 1 PATTERN OF SUPPRESSIBILITY SHIFT SUPPRESSORS Marker
OF A SERIES OF YEAST MUTANTS
BY EXTERNAL
FRAME-
Suppressor a SUF5-1
SUF145
SUF41
9
SUF514
his4-519
+
+
+
leu2-3
+
+-
+
+
can1-101
+
+-
--
--
horn3-10
--
+-
+-
+
adel-lO
--
--
+
a E f f i c i e n c y of s u p p r e s s i o n w a s e v a l u a t e d b y c o m p a r i n g t h e g r o w t h on s e l e c t i v e m e d i a o f w i l d - t y p e s t r a i n s w i t h s t r a i n s c a r r y i n g t h e l i s t e d m a r k e r s t o g e t h e r w i t h t h e single s u p p r e s s o r m u t a t i o n s . +, v e r y e f f i c i e n t s u p p r e s s i o n . +, g o o d s u p p r e s s i o n . --, n o s u p p r e s s i o n .
Molecular nature o f mutations used for reversion studies The 3 mutants arg4-18, arg4-27 and ura4-10 were classified as missense on the basis of the following pieces of evidence: arg4-18 and arg4-27 show nonpolarized complementation (Mortimer, personal communication), ura4-10 was induced by 2-AP (Sora et al., 1973); t h e y are all non-suppressible by external suppressors and they all show a high reversion rate when exposed to base substitution mutagens, such as EMS, DES and MNNG, and react only weakly to ICR170. The mutants leu2-1 and his4-1 both contain UAA nonsense codons; tyr7-1 and trpl-1 both contain UAG nonsense codons as proved by their suppressibility by nonsense suppressors of the 2 types (Hawthorne and Leupold, 1974). The other 4 m u t a n t s used in this work are characterized as follows: his4-519, leu2-3, adel-lO were induced by ICR170; horn3-10 originated spontaneously during meiosis; all 4 showed a high increase of reversion rate with ICR170 and their reversibility by base-substitution mutagens (EMS, DES, MNNG) was low. None of these 4 markers was reversible by nonsense suppressors; their suppressibility by other external suppressors is shown in Table 1. Suppressor SUF5-1 is an ICR170-induced m u t a t i o n (Culberston et al., 1977), recognized as a suppressor of addition mutations. The other 3 suppressors are spontaneous mutations isolated by us: S U F 1 4 5 and S U F 4 1 9 as external suppressors of his4-519, and S U F 5 1 4 as an external suppressor of horn3-10. S U F 1 4 5 was identified to be allelic to SUF5-1 ; S U F 4 1 9 and S U F 5 1 4 are allelic to each other, but n o t to SUFS-1. None of these 4 suppressors suppresses either nonsense UAA or UAG mutations (Culberston et al., 1977; our unpublished data), and SUF5-1 does n o t suppress UAG mutations (Culberston et al., 1977). We are therefore confident that the 3 mutations his4-519, leu2-3 and h o m 3 - 1 0 are frameshift mutations, although n o t necessarily of the same type. The molecular nature of m u t a t i o n a d e l - l O as a frameshift has n o t y e t been satisfactorily proved.
400
Results
Reversion of frameshift and base-substitution mutants The large number of markers used for our reversion studies made it impossible to prepare a unique strain carrying all the mutations. Different strains were therefore constructed and their sensitivity to the lethal effects of h y c a n t h o n e tested. Survival data reported in Fig. 1 indicate that the 5 strains used show almost identical sensitivity to h y c a n t h o n e , at least at the lowest doses. In addition, m a n y markers were present in more than one strain, and no significant differences were observed in the spontaneous and induced reversions of the same m u t a t i o n in different genetic backgrounds. The frequencies of spontaneous revertants are quite different for the 11 markers used, as shown in Table 2. For the nonsense mutations it was confirmed, as is well known, t h a t the over-all frequency of suppressor mutations is almost one order of magnitude higher than that of true back mutations for the same genes. To compare the effect of the same mutagen on genes showing such great differences of spontaneous reversion frequencies, we believed that the most effective way of expressing our mutagenesis data was the use of the parameter T/C, i.e. the ratio between frequency of revertants per survivor after t r e a t m e n t and frequency of spontaneous revertants. Mutagenesis data are reported in Figs. 2, 3, 4 and 5. The 3 missense mutations showed a very low induced reversion rate, if any, as the maximal increase at 0.4 mM did n o t exceed a TIC value of 5 (Fig. 3). toc-
~0
~-50
i
m
Hycanthone
mM
Hycant hone
rnM
Fig. 1. Survival after h y c a n t h o n e in 4-h t r e a t m e n t s of strains: 5 8 5 3 / 4 a (D), 6 1 9 8 / 1 2 c (o); 6 2 4 3 / 2 c (~); 6 1 1 0 / 1 1 a (A); 6 1 2 6 / 1 6 c (m). Fig. 2. Reversion of frameshift m u t a n t s by h y c a n t h o n e : a d e l - 1 0 (w); his4-519 (o); h o m 3 - 1 0 (o); leu2-3
(o).
401 TABLE 2 S P O N T A N E O U S R E V E R S I O N F R E Q U E N C I E S O F T H E M U T A N T A L L E L E S U S E D IN T H I S S T U D Y Mutation
Reversion frequency/10 8 cells a
Total
Site r e v e r s i o n
his4-519 leu2-3 horn3-10 adel-lO
0.2 1.3 0.45 0.21
arg4-18 ura4-10 arg4-27
0.05 0.12 0.09
his4-1 leu 2-1 tyr7-1 trp l-1
44 37.5 32 21.8
2.1 0.3 0.9 0.62
a All v a l u e s are t h e a v e r a g e s f r o m a t least 3 i n d e p e n d e n t E x p t s .
The total reversion of nonsense mutations (due for more than 90% to external suppressors events) is practically unaffected by h y c a n t h o n e (Fig. 4), whereas their site reversion seems to be a little more sensitive to the mutagen. However, the results must be considered with caution, as the frequency of the event is very low and therefore the data are based on few colonies per dose, so
/
I /
/
/
3O-
t/
/
z
/
m c I
/
®20-
/
/
I ~v
/
o
I
/
/
to-
/
/
/ .
Hycanthone
mM
/
Hycanthone
mM
Fig. 3. R e v e r s i o n o f m i s s e n s e m u t a n t s b y h y c a n t h o n e : a r g 4 - 1 8 (o); a r g 4 - 2 7 (o), u r a 4 - 1 0 (o). Fig. 4. T o t a l r e v e r s i o n o f n o n s e n s e m u t a n t s b y h y c a n t h o n e (= b a c k m u t a t i o n s + s u p p r e s s o r m u t a t i o n s ) : his4-1 (4); l e u 2 - 1 (o); t r p l - 1 (o); t y r 7 - 1 (o).
402
/ /
/
c
-
/
b.
|
/
Iv
/ /
Hycanthone
mM
Fig. 5. Site reversion o f nonsense mutants b y hycanthone: his4-1 (A); leu2-1 ( e ) ; t r p l - 1 (=); t y r 7 - 1 (o).
t h a t the estimate may be biased by r a n d o m fluctuations. The increment of TIC never exceeded a value of 10 (Fig. 5). On the contrary, the 3 definite frameshift mutations and also adel-lO showed an appreciable increase of reversion due to h y c a n t h o n e that was already evident at 0.2 mM and with good dose--effect curves always reached T/C values above 50 at 0.4 mM {Fig. 2).
Does hycanthone induce both insertions and deletions? The above results indicate that .hycanthone is endowed with good specificity in inducing frameshift mutations, but t h e y do n o t allow any conclusion about its molecular specificity or whether this mutagen preferentially induces insertions or deletions or both. Some data on the reversion of hycanthone-induced mutations seem to support the hypothesis t h a t this mutagen can cause base losses and insertions. 2
TABLE 3 R E V E R S I O N BY H Y C A N T H O N E O F M U T A T I O N S I N D U C E D BY T H E S A M E M U T A G E N Hycanthone (raM)
0 0.1 0.2 0.3 0.4
R e v e r t a n t s X 109 adeH3
aroH6
0.67 3.3 7.4 13.0 26.0
9.4 78.0 330.0 600.0 530.0
403 TABLE 4 REVERSION Hycanthone (raM)
0 0.1 0.2 0.3
O F h i s 4 . 5 1 9 I N D U C E D BY H Y C A N T H O N E Intragenic mutations
Suppressor mutations
N ( X l 0 9)
T/C
N ( X l 0 9)
T/C
1.9 29.2 70.5 118
15.36 37.1 62.1
0.08 0.84 3.5 5.8
10.5 43.75 72.5
auxotrophic mutants, adeH3 and atoll6, obtained after t r e a t m e n t by hycanthone, were analyzed by our standard reversion procedure. Both mutations were highly sensitive to the mutagenic action of h y c a n t h o n e {Table 3). The m u t a n t adeH3 evidently reverted only by intragenic events, as no external suppressor was isolated among spontaneous or induced revertants. On the other hand, aroH6 was phenotypically reverted by external suppressors, whose frequency a m o u n t e d to about 50% among spontaneous revertants, and among hycanthone-induced revertants it can be as high as 90%. More convincing evidence was obtained with the m u t a t i o n his4-519, whose molecular nature is far better known. The frequency of spontaneous and hycanthone-induced reversions was carefully determined for both intragenic events and external suppressors. Table 4 indicates t h a t the mutagen was equally effective in inducing intragenic events and the molecularly opposite p h e n o m e n o n at the suppressor genes.
Discussion From the above results it is evident t h a t h y c a n t h o n e , in suitable experimental conditions, consisting essentially of a mild t r e a t m e n t in the dark at neutral pH, was much more effective at inducing frameshift reversions, than base-substitution mutations. That h y c a n t h o n e , in our conditions, induced only few, if any, base substitutions, was proved n o t only by the scanty reactivity of 3 missenses but also by the results on nonsense mutations: the 4 nonsense markers used for these experiments can easily be reverted by any type of base substitution, such as AT -* GC and GC -~ AT transitions and transversions. Mutant adel-lO was considered as a potential frameshift. The present data seem to substantiate this hypothesis considering its high reversibility by hycanthone. As for the specificity in inducing either insertions or deletions, or both, our data indicate that h y c a n t h o n e can determine both types of mutational event. The findings on the reversion of hycanthone-induced mutations prove t h a t h y c a n t h o n e can revert mutations induced by itself. The frameshift nature of such mutations can be presumed with good probability although n o t y e t with absolute certainty; t h e y are n o t suppressible by nonsense (UAA) suppressors and react to h y c a n t h o n e much more than any base-substitution m u t a n t so far tested. Our data on the induced reversion of his4-519 seem to be more conclusive.
404
The almost identical sensitivity to hycanthone of intragenic events and suppressor mutations indicates that this mutagen can induce, with equal efficiency, insertions and deletions, and for frameshift mutations, internal and external suppression must be mostly due to opposite events. Acknowledgement This work was partially supported by a grant of C.N.R. (Roma), Progetto Finalizzato: Promozione qualit~ dell'ambiente. References Brusick, D.G. (1970) The mutagenic activity of I C R 1 7 0 in Saccharomyces cerevisiae, Mutation Res., 10, 11--19. Brusick, D.G., and E. Zeiger (1972) A c o m p a r i s o n of chemically i nduc e d reversion pa t t e rns of Salmonella typhimurium and Saccharomyces cerevisiae, using in vitro plate tests, Mut a t i on Res., 14, 271--275. Culberston, M.R., L. Charnas, M.T. J o h n s o n and G.R. Fink (1977) Frameshifts and frameshift suppressors in Saccharomyces cerevzsiae, Genetics, 8 6 , 7 4 5 - - 7 5 6 . Hartman, P.E., K. Levine, Z. H a r t m a n and H. Berger (1971) H y c a n t h o n e : A frameshift mutagen, Science, 172, 1058--1060. Har tman , P.E., H. Berger and Z. H a r t m a n (1973) Comparison of h y c a n t h o n e (Etrenol), some h y c a n t h o n e analogs, m y x i n and 4 - n i t r o q u i n o l i n e - l - o x i d e as frameshift mutagens, J. Pharmacol. Exp. Ther., 186, 390--398. Hawthorne, D.C., and U. Leupold (1974) in: Current Topics in Microbiology and I m m u n o l o g y : Suppressor Mutations in Yeast, Springer, Berlin, Vol. 64, pp. 1--47. Magni, G.E., L. Panzeri and S. Sora (1977) Molecular specificity of X-radiation and its repair in Saccharomyces cerevisiae, M u t a t i o n Res., 42, 223--234. Meadows, M.G., S. Quah and R.C. yon Borstel (1973) Mutagenic action of h y c a n t h o n e and 1A-4 on yeast, J. Pharmacol. Exp. Ther., 187, 444--450. Ong, T. (1978) Genetic activity of h y c a n t h o n e and other antischistosomal drugs, Mutation Res., 55, 43-70. Prakash, L., and F. Sherman (1973) Mutagenic specificity: reversion of i s o - l - c y t o c h x o m e c m u t a n t s of yeast, J. Mol. Biol., 79, 65--82. Ray, V.A., H.E. Holden, J.H. Hellis Jr. and M.L. H y n e e k (1975) A comparative s t u d y on the genetic effects of h y c a n t h o n e and o x a m i q u i n e , J. Toxicol. Environ. Health, 1 , 2 1 1 - - 2 2 7 . Sherman, F., J.W. Stewart, M. Jackson, R. Gilmore and J. Parker (1974) Mutants of yeast defective in iso1-cytochrome c, Genetics, 7 7 , 2 5 5 - - 2 8 4 . Sofa, S., L. Panzeri, and G.E. Magni (1973) Molecular specificity of 2-aminopurine in Saccharomyces cerevisiae, Mutation Res., 20, 207--213: yon Borstel, R.C., and S. Igali (1975) Mutagenicity testing of antischistosomal t h i o x a n t h e n o n e s and indazoles on yeast, J. Toxicol. Environ. Health, 1 , 2 8 1 - - 2 9 1 .