3futation Research, 28 (1975) 163 173
© Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
163
M U T A T I O N S I N D U C E D B Y X - R A D I A T I O N IN T H E Y E A S T SCHIZOSACCHAROMYCES
POMBE*
N. LOPRIENO, R. BARALE, S. BARONCELLI, A. CAMMELLINI, MANUELA MELANI, R. NIERI, M. NOZZOLINI AND ANNA M. ROSSI Laboratorio di l~Iutagenesi e Differenziamento. C.N.R., and Istilulo di Genetica, University, Pisa (Italy)
(Received May I4th, I974) (Revision received January 2nd, 1975)
SUMMARY E x p e r i m e n t s on strains of y e a s t w i t h different genetic b a c k g r o u n d s were done to e v a l u a t e t h e kinetics of i n a c t i v a t i o n a n d m u t a t i o n i n d u c t i o n b y X - r a d i a t i o n s . A s y s t e m of f o r w a r d m u t a t i o n i n d u c t i o n in five loci was used a n d a specific m u t a t i o n r a t e of o.14' lO -8 × locus × r a d was e v a l u a t e d for t h e wild t y p e . F r o m a c o m p a r i s o n of o b s e r v a t i o n s with wild t y p e a n d r a d i a t i o n - s e n s i t i v e strains, it m a y be a s s u m e d t h a t , in this y e a s t , m u t a t i o n s are m a i n l y t h e result of a r e p a i r - a c t i v e process. T h e r a n g e of g e n o t y p i c a n d p h e n o t y p i c influence u p o n t h e specific locus m u t a tion r a t e was e v a l u a t e d with a p p r o p r i a t e biological m a t e r i a l a n d e x p e r i m e n t s .
INTRODUCTION As p a r t of a research p r o g r a m aiming at an e x t e n s i v e k n o w l e d g e of t h e r e l a t i o n ships b e t w e e n r a d i a t i o n doses a n d m o l e c u l a r t y p e s of m u t a t i o n , in this p a p e r we p r e s e n t results on m u t a t i o n i n d u c t i o n frequencies o b t a i n e d w i t h X - r a d i a t i o n s on t r e a t i n g two h a p l o i d strains of Schizosaccharomyces pombe h a v i n g different s e n s i t i v i t y to i n a c t i v a t i o n . F r o m t h e results it is possible to e v a l u a t e t h e kinetics of t h e m u t a t i o n i n d u c t i o n , a n d so to define t h e specific m u t a t i o n r a t e per t a d p e r locus in this o r g a n i s m for comp a r i s o n w i t h p u b l i s h e d data~, 14. T h e influence of genetic b a c k g r o u n d a n d s p o n t a n e o u s m u t a t i o n b a c k g r o u n d on t h e specific m u t a t i o n r a t e i n d u c e d b y X - r a y s m a y also be analyzed. For the present experiments both forward-mutation and back-mutation systems h a v e been tested. * Publication No. lO5 from the Laboratorio di Mutagenesi e Differenziamento, C.N.R., Via Cisanello 147, 56Ioo-Pisa (Italy).
164 MATERIAL
.~. LOPRIENO et al. AND
METHODS
Yeast strains F o r w a r d m u t a t i o n s were i n d u c e d in two ade6 isogenic purple m u t a n t s of S. pombe, b o t h c o n t a i n i n g a mis-sense m u t a t i o n in the locus ade 6 (ade 6- 6 o) ~; one of t h e m had, in addition, a m u t a t i o n in t h e r a d i o locus (radio-z98) which controls t h e i n d u c e d l e t h a l i t y b y several p h y s i c a l a n d chemical agentsS, l°-12 a n d the s p o n t a n e o u s overall m u t a b i l i t y of several lociS, ~. The i n d u c t i o n of gene-forward m u t a t i o n was assessed b y scoring a m o n g survivors white colonies r e p r e s e n t i n g t h e p r o g e n y of a cell in which a second gene m u t a t i o n h a d been i n d u c e d b y t r e a t m e n t in one of five more loci (adez, aden, ade4, ade5, ade9) controlling t h e b i o c h e m i c a l r e a c t i o n s preceding t h e purple p i g m e n t formationL The genetic n a t u r e (nuclear m.utation) of the m u t a n t s scored with this s y s t e m a n d the p o s s i b i l i t y of recovering all t y p e s of m o l e c u l a r a l t e r a t i o n i n d u c e d in the D N A h a v e p r e v i o u s l y been e x t e n s i v e l y d e m o n s t r a t e d L F o r these t~vo s t r a i n s an e v a l u a t i o n of the s p o n t a n e o u s reverse m u t a t i o n rates of the ade6-6o allele has been made. Reverse m u t a t i o n s i n d u c e d b y different dcses of X - r a y s have been also e v a l u a t e d in two isogenic strains of S. pombe b o t h c o n t a i n i n g t h e allele ade7-z52 (mis-sense m u t a t i o n ) s. In these e x p e r i m e n t s the influence of the allele radzo-x98, present in one strain, on the i n d u c e d m u t a b i l i t y was also e v a l u a t e d . X - R a y treatment S u s p e n d e d cells of S. pombe (IO s cell/ml in o.I5 M K H2PO,, p H 4.5) were i r r a d i a t e d at r o o m t e m p e r a t u r e b y a Siemens S t a b i l i p a n X - r a y m a c h i n e with 2 him A1 filter, o p e r a t i n g a t 200 K V a n d 2o mA. The e s t i m a t e d dose r a t e was 2500 R per rain, as determ i n e d b y a Siemens X - r a y dosimeter. O x y g e n was p r o v i d e d b y m a g n e t i c stirring of the cell suspension during t r e a t m e n t . All the results p r e s e n t e d were d e r i v e d front several i n d e p e n d e n t e x p e r i m e n t s a f t e r s t a t i s t i c a l t r e a t m e n t of t h e d a t a b y regression analysis *.
RESULTS Spontaneous mutation rates The r a t e s were e v a l u a t e d in t h e wild t y p e (rad +) a n d u n d e r t h e influence of the r a d i o - z 9 8 m u t a t i o n b y t h e m e t h o d of LURIA AND DELBR/OCK1~ in five i n d e p e n d e n t
TABLE [ SPONTANEOUS
MUTATION
RATE IN
Schizosaccharomyces pombe a
Experiment b No.
ade 6-6o/rad+/hmutation rate (. ~o-~ p.c,d.) Five loci One locus°
ade 6-60/radzo-z98/hmutation rate (.¢o 5 p.c.d.) Five loci One locus
I 2 3 4 5 Average(~ S.E.M.) e
0.27 o.4I 0.55 o.46 o.23 o.38 ± o.o59
i4.oo i2.5o 6.96 6.o6 3.io 8.52 ± 2.04
0.054 0.082 o.i1 o.o92 o.o46 o.o76]± o.o12
ant In (aNtC) (ref. 13). b For each experilnellt, io independent cultures were evaluated. e S.E.M., standard error of mean. d p.c.d., per cell division. a
r
--
2.80 2.50 1.39 1.2i o.62 1.3o ~= o.55
R A D I A T I O N M U T A T I O N IN Y E A S T TABLE
16 5
1[
SPECIFIC LOCUS SPONTANEOUS MUTATION RATES (FORWARD AND REVERSE) IN Schizosaccharomyces
pombe Strain (purple)
Forward mutations • ade 6 --~ ade 6 /ade x (purple) (white)
Back mutations • ade 6 --> ade + (purple) (white)
ade6-6o/racl ~/hade6-6o/rad;o-~:98[h-
0 . 7 6 ~ o . 1 2 . i o -~ p . c . d , 1 . 3 o ± 0 . 5 5 . i o ~ c . d . -~
o . 1 7 • i o ~ c . d . -1 o . 1 5 . IO - " c . d . ~
~ r = a n t In (aNtC) (ref, 13).
TABLE
IiI
INACTIVATION AND FORWARD MUTATIONS INDUCED BY X-RAYS ON TWO STRAINS OF Schizosac-
charomyces pombe Dose (R)
Survival ( % ) (:~ S . E . M . )
N u m b e r of colonies analyzed
N u m b e r of mutants scored
Mutation frequency ( . i o ~ survivors ~_ S . E . 3 I . ) 5 loci • locus
5-43 i.oo 1.46 o.12
176141 160305 245283 200495 261538
3 21 65 162 28c~
o-17 1.31 2.65 8.08 11.o 5
• ~ =~ ~ =~
o.o4 o.07 o.15 0.85 1.37
o-o3 0.26 o.53 1.61 2.21
± ± ± ~= ±
o-oo3 o.oi 0.03 o.i 7 0.27
ioo.oo 66.52 ~ 7.08 1 8 . 4 2 -k 1.16 0 . 8 3 ~_ o . 1 6
62991 82395 6349~ i o i 54 °
51 lO 5 224 398
4.61 13.92 36.3 ° 4o.04
~-~ ~_ ±
1.62 2.46 3.62 3.23
0.94 2.78 7.26 8.Ol
=c ~ ± ±
0.32 0.49 0.72 0.64
ade 6-6o/rad +/hControl 32ooo 6900o i2oooo 160000
ioo.oo 84.75 62.00 lO.84 1.46
± ± ~_ =c
ade 6-6o [rad Io-~98 ~hControl 16000 48000 96000
experiments (Table I). As stated in the previous section, white colonies represent additional m u t a t i o n s in five loci. Moreover, t h e y might be the result of a reverse m u t a t i o n a l event occarring at the ade6-6o site. Therefore reverse m u t a t i o n rates of the ade6-60 allele in both strains were evaluated. The complete set of d a t a is reported in Table I I : the reverse m u t a t i o n rates were lO 3 to lO 5 times lower t h a n the forward m u t a t i o n rates and their influence can be completely ignored. The forward m u t a t i o n rate during mitosis was increased 17 times b y the presence of the radxo-I98 allele.
X-Ray-induced mutation The results obtained from seven experiments in which the induction of forward m u t a t i o n was evaluated are reported in Table I I I . The d a t a are presented in Figs. 1-4. The cell inactivation in the two strains is described b y the equation s log % survival = e ~*
(I)
where x is the dose ( × IOOO R) and fl the regression coefficient. As shown in Fig. I for b o t h strains, the inactivation (log% survival) was proportional to the square of the dose, and these relations indicate t h a t the survival was the result of repair events t h a t reduce the effects of the induced damage(alteration of D N A bases, D N A breaks, etc.). In strain radzo-~98 a very slow repair ability was still operating.
~ . L O P R I E N O et al.
166
10
i°°
u
9
J 0
J
J
J
J
Z
i 0.1 100 150 0 5O 100 1 0 X-Roy dose (x 1000 R) (x) X-Ray dose (xIOOOR) (x) F i g . i . I n a c t i v a t i o n c u r v e s of X - r a y t r e a t m e n t s w i t h t w o s t r a i n s of S. pombe: ©, rad +, [~ o . o o o o 7 2 - 1 o -2 (y = 2 . 0 4 3 0 . 0 0 0 0 7 2 x2; F 3 3 7 , 4 4 * * * ; r = 0 . 9 9 5 5 * * * ) ; O, radzo-z98, ~ -o . o o o 2 2 - 1 o -2 ( y : 1 . 8 9 2 3 - - - o . o o o 2 2 x2; F = 1 8 4 . 1 o 4 " * * ; r - - 0 . 9 9 4 6 * * * ) . (/~ = % of i n a c t i v a t i o n ,x~; x I o o o R.) F i g . 2. L o c u s s p e c i f i c m u t a t i o n f r e q u e n c y i n d u c e d b y X - r a y s i n t w o s t r a i n s of S. pombe : O, tad +, o - - o . o 1 4 2 . 1 o -4 s u r v i v o r s × l o c u s × i o o o R (y - - - - o . i 5 3 4 + o . o i 4 2 x; F -- 74.405**; r o . o 9 8 o 4 " * ) ; O, radzo-±98, / ~ - - o . I 3 O 9 X l O -4 s u r v i v o r s × l o c u s × i o o o R ( y - - o . . 5 9 + o . 1 3 o 9 x; F - - 4 7 . 7 7 * ; r - . 0 . 9 8 9 6 * ) . T h e s p e c i f i c m u t a t i o n r a t e p e r ] o c u s p e r r a d is: rad= o.i4.1o s ( n o r m a l i z e d v a l u e (n.v.) - - 1 . 8 o . lO :) ; rad::o-::98 o. t 3 × Io ~ ( n o r m a l i z e d v a l u e 1 . 6 7 . i o ~.)
~o
16 100
$~ ~ 12
J
g
~
°
~ ~
100
10 log ~/~ survival
, 1
t~ x ---1~0 :
~o~x~ e-aX (i + ~ - - ~
~, ra~+; ~, rad~o-I98.
0"10
X
~0 100 150 X - Roy dose (1000 R) F i g . 3. M u t a t i o n f r e q u e n c y a s a f u n c t i o n of l o g p e r c e n t a g e s of s u r v i v a l i n t h e t w o s t r a i n s of S. pombe: O, ra,.t*, ~ ~ 1 , 5 6 4 o . I o - 4 × 1 o g % s u r v i v a l (y = 3 . 2 4 ~ 9 ~ I . $ 6 4 o x ; F ~ i 7 ~ . 3 6 7 8 " * ; r ~ o.9943"*); o, ra~o-~98, ~ ~ 8.425I-iO-~1og % survival (y-- i7.6729--8.425i x; F-535-4731"*; r ~ o.9991"*.) I:ig. 4. S. pombe f o r w a r d m u t a t i o n s (a~e l o c u s ) . E f f e c t of X - r a y t r e a t m e n t s o n m u ~ t i o n i n d u c t i o n e x p r e s s e d b y t h e r a t i o K / t C o. T h e d a ~ h a v e b e e n t r e a t e d & c c o r d i n g ~o t h e r e l a t i o n (4). (x)
RADIATION
MUTATION
IN
167
YEAST
Fig. 2 shows t h e rates for m u t a t i o n i n d u c t i o n b y X - r a y s in b o t h strains after t r e a t m e n t of d a t a b y t h e regression analysis r e p r e s e n t e d b y e q u a t i o n (2)
(2)
y = a+~x
where y is t h e i n d u c e d m u t a t i o n t r e q u e n c y a n d x is t h e dose ( × IOOO R). The rad + s t r a i n showed a r a t e c o n s t a n t (8) of o.o142. IO-~ survivors × locus × IOOO R, w i t h a specific m u t a t i o n r a t e per locus per r a d of o.14. IO -8, whereas the radzo-±98 h a d a r a t e c o n s t a n t (fl) of o.13o 9- IO-~ survivors per locus per i o o o R, w i t h a specific m u t a tion r a t e per locus per r a d of o.13-IO 7, i.e. IO t i m e s higher t h a n the wild t y p e . This was not t h e result of the higher l e t h a l i t y i n d u c e d b y X - r a y s in the radro-±98 strain, since if we c o m p a r e the relationships b e t w e e n m u t a t i o n frequencies a n d s u r v i v a l s in the two s t r a i n s (Fig. 3), a higher i n d u c e d m u t a b i l i t y for t h e s t r a i n radzo ~98 is observed. ( F o r comparison, t h e m u t a t i o n f r e q u e n c y i n d u c e d a t l O % s u r v i v a l was 1.68- IO -~ × locus in t h e rad +, a n d 9 . 2 5 IO-~× locus in the radio-z98. ) The m u t a t i o n f r e q u e n c y (K,) per s u r v i v o r (S,) i n d u c e d b y a given dose of X r a y s (x) is expressed b y t h e e q u a t i o n :
K ~ _ K0 + ~x S~ So
(3)
where Ko is t h e s p o n t a n e o u s n m t a t i o n f r e q u e n c y a n d So is t h e s p o n t a n e o u s s u r v i v a l of t h e cell p o p u l a t i o n ; ~ r e p r e s e n t s t h e p r o b a b i l i t y of t h e i n d u c t i o n of m u t a t i o n per dose unit a n d p e r cell. Insertion of e q u a t i o n (I) into e q u a t i o n (3) p r o d u c e s the following relationS:
K, / So~x \ Ko - - e ~* I 1 + K ~ - - )
(4)
which expresses the real increase of t h e i n d u c e d m u t a t i o n over t h e s p o n t a n e o u s level for each X - r a y t r e a t m e n t , giving a certain p e r c e n t a g e of survival. Fig. 4 shows t h e K/Ko values o b t a i n e d b y t r e a t i n g t h e e x p e r i m e n t a l d a t a rep o r t e d in T a b l e I I I for b o t h rad + a n d radio-z98 strains. These d a t a were confirmed ~vhen reverse m u t a t i o n s occurring a t t h e ade7-z5~ allele in presence or absence of radxo-z98 m u t a t i o n were a n a l y z e d : t h e e x p e r i m e n t a l d a t a are a s s e m b l e d in T a b l e IV a n d t h e K/Ko values for b o t h s t r a i n s are shown in Fig. 5. T h e increase of the K/Ko values in b o t h s y s t e m s was u n d e r genetic control, TABLE IV INACTIVATION
AND REVERSE
MUTATIONS
INDUCED
BY X-RAYS
ON TWO STRAINS
Average data from 8 experiments.
Dose (R) ade 7-252 Irad+/hControl 32ooo 96ooo 160000
Survival (%) (~: S.E.M.) ioo.o 57.5 ~_ 11.2 9-4 • 1-7 0.2 ~ 0.05
Mutation frequency (.xo -9 S ± S.E.M.) 1.92 ± o.66
51.51 ± 9.9 ° 47-o5 ± lO.O7 24.50 ± 2.45
ade 7-;52/tad ~o-298 /hControl 16ooo 32000 48000 96000
IOO.O 49.1 ~ 6.37 21. 3 ~_ 8.07 7-7 -~ 0.76 0. 4 -~ 0.06
3.2o 23.79 40.23 71.44 182.54
± 1.46 ± 4.85 ~ 9.48 ~.- 1°'14 ~ 116.Ol
OF S.
po~be
N. LOPRIENO et al.
168
10C
l 1C
',
~
~ \
\x ~\ \
!
~.
1~[ [ ~ - ~
~
o~ g ~ ~,~,~,~,
"~"'..... " ' ' . ~
' 6~
~6o
X-Rey dose (xl03 R) Fig. 5- E g e c t of X - r a y t r e a t m e n t s on reversion i n d u c t i o n (ade- -~ ade +) for t h e locus ade6 in t w o strains of S. pombe, expressed b y t h e r a t i o ~ / K o. C~, f a d + ; ~, ggdzo-z98.
since a different behavior between md+ and radzo-zg8strains was observed. However, it seems an indirect control, because the spontaneous mutation frequency directly influenced the E/Eo. In our case this influence was higher in the md+ than in the mdzozgg strain. To evaluate a possible influence of phenotypic factors on the rate of mutation induction, we treated with different doses of X-rays independent clones of radzo-z98 cells showing a diEerent spontaneous mutation back~ound. Two clones with a spontaneous mutation frequency respectively of 4.08. IO -~ N locus (culture A) and o.6~. ~o -~ Nlocus (culture B) were analyzed for the induction of forward mutations. The data from 3 experiments are assembled in Table V. The inactivation curves of the two clones were s i ~ ] a r (Fig. 6), where-as the kinetics ot mutation induction digered greatly from each other (Fig. ?). TABLE
V
INACTIVATION
AND
FORWARD
MUTATIONS
INDUCED
sr~A[~" ade6-6o/mdzo-z98/h- o~ S. pombe w ~
BY X-RAYS
a ~F~N~
IN TWO
INDEPENDENT
FREQUENCY
Average data from 3 experiments.
Dose (R)
Survival (%)
Number ~[ mutants Number of colonies
Mutation frequency (. zo 4 survivors × locus)
Culture A Control ~2 500 25ooo IOOOOO
i oo.o 74.8 68.0 o. 4
34/I 6656 26/7986
4.08 6.51 5.27
Culture B Control 175 °o 5oooo iooooo
IOO.O 6o.6 I7.5 o.9
~ I/35228 35/36696 Io2/34619
34/I2789 78/27368
224/61432
5.7 o
o.62 i .90 5.89 7.29
CULTURES
OF
VA~V~ OF S P O X ~ a ~ O V S ~U~A~IO~
RADIATION
MUTATION
16 9
IN YEAST
100
~
10
/
1(
g
/
g g
5 ~0 X-Ray
dose
~ . 100 (×IO00R)
~ 150 (x)
~
0'
t
50 X-Rey
-
-
|
~
1O0 150 dose ( x l O 0 0 R) (x)
F i g . 6. Survival curves of two independent cultures of S. pombe (SP-I98 ade6-6o]radlo-I98, h-) after X - r a y treatments : ©, culture A , / 5 = o . o o o 2 3 . I O - Z ( y = 1 . 9 6 3 7 - - o . o o o 2 3 x~; F = 1 8 2 3 . 1 1 2 " * " r = 0.9994***); r = o . 9 9 1 9 " * * ).
O,
culture
B,
/~ ~
o.oooI9-io
-~ (y =
1.868o--o.ooo19
x~; F =
122.449"**;
F i g . 7- M~tation frequency induction in two independent cultures of the strain SP-z98 ade6-6o[ o f S pombe after treatments with X-rays. The cultures had different spontaneous mutation frequencies : ©, C u l t u r e A , / 5 = o . o o 6 9 - lO 4 s u r v i v o r s × locus × i o o o R ; specific mutation rate per locus per tad = 0 . 6 9 . lO -9 ( n . v . = o . 8 9 " I O 7) (data statistically not significant) ( y ~ 5.1515+o.oo69 x ; F ~ 0 . 2 0 n . s . ; r = 0 . 3 0 7 5 n . s . ) ; O , c u l t u r e B , ~5 ~ o o 6 8 9 . 1 o -4 s u r v i v o r s × l o c u s × i o o o R; specific mutation rate per locus per rad ~ o . 6 8 . 1 o * ( n . y . = o . 8 8 . I O -~) (y = i.o38+o 0689 x; F ~ 2o.5471"; r = 0.9546*).
radzo-z98, h-
Although the data from culture A are not statistically significant, we have used them for a better comparison : the data represent limit values due to higher spontaneous mutation frequency. Other cell clones with spontaneous mutation frequency between the values from culture A and culture B gave intermediate values. The late of locus mutation induction in culture B was ten times higher (0.0689 • lO-4) than in culture A (0.0069. lO4). Fig. 8 shows the relationships between survival and mutation frequency observed in the two cultures. The results of the treatment of the data according to equation (4) are shown in Fig. 9 : a mutation induction by X-rays over the spontaneous level was observed only for culture B. DISCUSSION
In S. pombe, as in other organisms, active repair processes to reduce damage produced in the D N A are operating: this is demonstrated by the isolation of mtltants which are no longer able to repair inactivating events 3, and also by the evaluation of the dose-effect relationships. In our experiments, when a wild-type strain was treated with X-rays its survi-
N. LOPRIENO et al.
17o
tn 16 um _o. 14
//
~.12
~
//
//
,~ 10
,~ 8 o ~ 6
/
///
o/
d,
g
8 ._ ~
S
~
i
~o
~oo
~
i
log % survival
(x)
I
o.~
~ i { . 8. M u t a t i o n frequency ~s ~ fmnction of ]o~ perceat~ges of s u r v i v M in Lbe two cultures o[ t~e strain SP-z98 ~6-6o/m~zo-]98, h- o[ S. 2 ~ : o, Cu]ture A, ~ ~ too.87, mo-~x ]o% % s u r v i v M (d~t~ statistically a o t significant) (y = ~6.o536 zo.87~ 5 ~; ~ = L6o98 n.s.; ~ ~ o.785~ ms.); ~, cu]taTe ~ , ~ = 6.99"~o -~X ]o~% s u r v i v M (y = z~.7253--6.9973 ~; ~" :- 3~5.29~o*; ~ = o.998~*). 10
~ol
0.1
)
0.01
o.oo~
~'o
~&--
1~0 I
X-Ray dose (xlO 3 R) Fig. 9. Effect of X - r a y t r e a t m e n t s on m u t a t i o n induction expressed by t h e ratio K/A"o in two cultures of t h e strain SP-z98 ade6-6o/radio-z98, h- of S. pombe with different s p o n t a n e o u s m u t a tion frequencies: O, culture A; e, culture B.
val curve was proportional to the square of the dose; the same relationship was found for a strain almost completely lacking the repair ability (radxo-z98) 15, but the regression coefficient was higher t h a n t h a t of the wild type, thus indicating only a partial repair ability. This finding m a y also be confirmed by the fact t h a t a linear dose-effect relationship m a y be obtained by the same survival data of the radro-zg8, although here an F (~D~I~0)ll lower than the F reported in Fig. I ( P ( I } ~ ) was observed. In both strains the m u t a t i o n induction presented a linear relation with the dose : the radio-z98 strain showed a rate constant (fl) higher than t h a t shown b y the rad+ strain, also when similar survivals were compared. B y analyzing the ratio K/Ko as described in the results, a higher inducibility of m u t a t i o n in the rad + strain was observed expressed as an increase over the sponta-
N. LOPRIENO et al.
172 T A B L E VII FORWARD GENE MUTATION RATES IN S. pombe
Strain
Mutagen
Mutation rate per locus (A)
Normalized specific mutation per locus (A :7.75.2o-~) •
tad + (wt) rad±o-~98 rad + (wt) radio-z98 radlo-I98(A) b radzo-z98(B)b
Spontaneous Spontaneous X-Rays X-Rays X-Rays X-Rays
0.76. lO-6 (p.c.d.) 1.3o. io -~ (p.c.d.) o.14 • io 6 (p. tad) o.13 • io -~ (p. rad) 0.69. IO-~ (p. tad) 0.68. IO-s (p. rad)
0.98- IO-* (p.c.d.) 1.67 • IO-~ (p.c.d.) 1.8o- io ~ (p. tad) 1.67. lO-6 (p. tad) 0.89. IO-~ (p. tad) o.88. io 6 (p. tad)
a S. pombe haploid cells contain 0.0225 pg DNA per nucleus, only 7-75' IO-a times the amount of DNA as compared with h u m a n (2.9 pg of DNA per haploid nucleus). b Independent cultures of strain radxo-I98 with different spontaneous background mutation. p g b y ABBONDANDOLO, f r o m t h i s l a b o r a t o r y , as a n a v e r a g e of w i l d t y p e a n d s e v e r a l a u x o t r o p h s ~. F o r T a b l e V I I w e h a v e c a l c u l a t e d t h e n o r m a l i z e d specific m u t a t i o n r a t e p e r l o c u s i n t h e t w o s t r a i n s rad + a n d r a d x o - I 9 8 a n d i n d i f f e r e n t c l o n e s of t h e s a m e s t r a i n (radzo-I98) : for t h e w i l d t y p e a v a l u e of 1.8o. lO -7 × l o c u s × t a d w a s o b t a i n e d , a v a l u e v e r y close t o t h a t u s e d b y ABRAHAMSON. D e p e n d i n g o n t h e s t r a i n s t e s t e d ( r a d i a t i o n - s e n s i t i v e s t r a i n s ) or o n t h e s p o n t a n e o u s m u t a t i o n b a c k g r o u n d of t h e t r e a t e d cell p o p u l a t i o n s , t h e v a l u e s of specific m u t a t i o n r a t e s p e r l o c u s p e r t a d m a y v a r y f r o m 1.8o. IO -7 (rad +) t o 1.67 • IO -~ for a c l o n e of t h e r a d i o - x 9 8 s t r a i n . I n o u r o p i n i o n s u c h a f i n d i n g d o e s n o t i n d i c a t e a h i g h e r m u t a b i l i t y of S. pombe i n c o m p a r i s o n w i t h o t h e r y e a s t s a n d species, b u t r a t h e r i t reflects a general situation in relation to the genetic system used or to the strain treated with X-rays. Moreover, the different values found between the wild-type and t h e r a d i a t i o n - s e n s i t i v e s t r a i n of S. pombe m a y reflect g e n e r a l s i t u a t i o n s a m o n g differe n t g e n o t y p e s i n t h e s a m e species. ACKNOWLEDGEMENTS
P a r t of t h e s e i n v e s t i g a t i o n s h a s b e e n s u p p o r t e d b y g r a n t No. i i i 7 2 - 1 B I O I f r o m t h e C o m m i s s i o n of t h e E u r o p e a n C o m m u n i t i e s , B i o l o g y D i v i s i o n , t o t h e U n i v e r s i t y of P i s a . REFERENCES I ABBONDANDOLO, A., Mutation and nuclear stage in Schizosaccharomyces pombe, I. A genetic
2 3 4 5 6 7 8
system for studying the role of recombinational repair in m u t a t i o n induction, Mutation Res., 27(~975) 225 233. ABRAHAMSON, S., M. A. BENDER, A. D. CONGER AND S. WOLFF, Uniformity of radiationinduced mutation rates among different species, Nature (London), 245 (1973) 46o-462. AUERBACH, C., AND B. J. KILBEY, Mutation in eukaryotes, Ann. Rev. Genet., 5 (I97 I) I63-218. BAUTZ-FREESE, E., AND E. FREESE, Induction of reverse mutations and cross reactivation of nitrous acid-treated phage T4, Virology, 13 (I96I) 19-3 o. CONGER, A. D., IS DNA the only chromosomal component contributing to target size for mutation induction by ionizing radiation ?, Mutation Res., 25 (1974) I3I-I34CROW, J. F., AND M. KIMURA, An Introduction to Population Genetics Theory, Harper and Row, Nexv York, 197 o, pp. 486-493. GUTZ, H., H. HESLOT, U. LEUPOLD AND ~N'. LOPRIENO, Schizosaccharomyces pombe, in R. C. KING (Ed.), Survey of Genetics, Vol. 2, Plenum, New York, 1974. LOPRIENO, N., UV radiation-sensitivity and m u t a t o r activity in yeast S. pombe, Proc. I V Int. Congr. Radiat. Res., Evian, I (1971) 2 IO.
RADIATION
MUTATION
IN
171
YEAST
neous level. Since an active repair ability to reduce DNA damage operates in the wild type, the higher mutability of this strain m a y be assulned to result from the repair processes, which therefore are of the error-prone type 1~. Both forward and revelse mutational events are of this type. It is possible to postulate, therefore, that the mutations induced by X-rays in the radro-I98 strain are partly the result of direct damage to DNA, whereas in the wild type (rad +) they are the results of an error-prone repair. If this is so, then we should expect mutations of possible different molecular nature inducible by X-rays on treatments done with the two strains. Preliminary results obtained by studies on the molecular nature of mutations induced by X-rays in the ade6 and ade 7 loci of two strains of S. pombe (rad + and radzo x98 ) indicate that with the wild type the incidence of base-pair substitution mutations is higher (45%) than that of the same class of m u t a n t s obtained with the radlo ~9 ~' (2.2°/~)i°, 11. To decide whether the rate constant (/3) of mutation induction is under genetic or phenotypic control, attempts were made in the present study to compare two strains with gene-controlled different spontaneous mutabilities and two clones of the same strain with different spontaneous mutation frequencies. Our results indicate that in both attempts the spontaneous mutation background was strongly responsible for the slope of the mutation-induction curve. This finding illustrates the difficulty of comparing different experiments of mutation-induction done with cell populations containing different spontaneous mutation backgrounds. D a t a on mutation induction by X-ray treatment in yeast, as in other organisms, have been used by ABRAHAMSONel al. '2. to evaluate the correlation of radiation-induced mutation rates among different species. These data are of great interest, as for the first time they suggest a uniformity in the specific locus mutation rates in different organisms if the data are normalized for the amount of DNA (Table VI). Although a correlation with another possible structure (i.e. chromatin mass) has been postulated recently a, the data of ABRAH~,SISON et aI. require detailed analysis before their final confirmation. Irrom the data reported in the present paper it is possible to better define how the yeast S. pombe supports ABRAI~AMSONet al.'s data. A recent paper by NASIM has already reported a few data for S. pombeIL confirming the data obtained with the canavanine resistance mutational system used with Saccharo~,ces cerevisiae ~. In S. pombe, the content of DNA per cell (G2 stage) has been evaluated as o.o225 TABLE
VI
SPECIFIC LOCUS MUTATION RATE INDUCED
BY RADIATIONS a
Organism
Relative amount of D N A as compared with h u m a n ( ~ )
iVIutation frequency per locus per tad (B)
E. coli ~'. cerevisia~ N . crassa Drosophila melanogasler 3/Ius musculus H. vulgate Homo sapiens
4-5" lO 3 8.3 • IO -3
I . O . IO ~ i .6- io 9
2.2 i .9
1. 4 • i o - ~ 0.058
2. 7 • i o 9 1. 4 . I O s
1.9 2. 4
a From
ABRAHAMSON
Normalized value B / A ( . z o -~)
0.78
1. 7 . i o
7
2.2
3.45 i.o
i.o. IO -~ Average
2.9 2.2
et al. ~,
Inodified.
RADIATION MUTATION IN YEAST
173
9 LOPRIENO, ~X~., A m u t a t o r gene in the y e a s t Schizosaccharomyces pornbe, Genetics, Suppl. 73 (1973) 161-164. io LOPRIENO, ~7., A. ZkBBONDANDOLO, S. BARONCELLI, M. SIMILI AND R. ]~ARALE, Molecular n a t u r e of p o i n t m u t a t i o n s induced b y X - r a d i a t i o n s in S. pombe, Annual Report ~972. Programme Biology-Health Protection, 1973 , pp. 242-243. i i LOPRIENO, Iq., .A_. ABBONDANDOLO, S. BARONCELLI, ~1. SIMILI AND R. BARALE, Molecular n a t u r e of point m u t a t i o n s induced b y X - r a d i a t i o n s in S. pombe, Annual Report ~973. Programme Biology-Health Protection, 1974, pp. 276-277. 12 LOPRIENO, ~X~.,R. BARALE, C. ]~AUER, S. ]3ARONCELLI, G. BRONZETTI, .A,. CAMMELLINI, A. CINCI, G. CORm, C. LEPORINI, 1~. NIERI, M. NOZZOLINI AND CRISTINA SERRA, The use of different test s y s t e m s w i t h yeast for the evaluation of chemically induced ggne-conversions and genem u t a t i o n s , Mutation Res., 25 (1974) 197-217. 13 LURIA, S. ]~., AND •. DELBR~ICK, Mutations of bacteria from virus sensitivity to virus resistance, Genetics, 28 (1943) 491-511. 14 NASIM, A., Radiation-induced m u t a t i o n rate and D N A c o n t e n t in Schizosaccharomyces pombe, Mutation Res., 24 (1974) 211-212. 15 SCHUPBACH, *~/[.,The isolation a n d genetic classification of UV-sensitive m u t a n t s of Schizosaccharomyces pombe, Mutation Res., i i (1971) 361-371. 16 VqlTKIN, E. M., Ultraviolet-induced m u t a t i o n and D N A repair, Ann. Rev. Microbiol., 23 (1969) 487-514 .
© Elsevier Scientific Publishing Company, Amsterdam--Printed in The Netherlands
163
M U T A T I O N S I N D U C E D B Y X - R A D I A T I O N IN T H E Y E A S T SCHIZOSACCHAROMYCES
POMBE*
N. LOPRIENO, R. BARALE, S. BARONCELLI, A. CAMMELLINI, MANUELA MELANI, R. NIERI, M. NOZZOLINI AND ANNA M. ROSSI Laboratorio di l~Iutagenesi e Differenziamento. C.N.R., and Istilulo di Genetica, University, Pisa (Italy)
(Received May I4th, I974) (Revision received January 2nd, 1975)
SUMMARY E x p e r i m e n t s on strains of y e a s t w i t h different genetic b a c k g r o u n d s were done to e v a l u a t e t h e kinetics of i n a c t i v a t i o n a n d m u t a t i o n i n d u c t i o n b y X - r a d i a t i o n s . A s y s t e m of f o r w a r d m u t a t i o n i n d u c t i o n in five loci was used a n d a specific m u t a t i o n r a t e of o.14' lO -8 × locus × r a d was e v a l u a t e d for t h e wild t y p e . F r o m a c o m p a r i s o n of o b s e r v a t i o n s with wild t y p e a n d r a d i a t i o n - s e n s i t i v e strains, it m a y be a s s u m e d t h a t , in this y e a s t , m u t a t i o n s are m a i n l y t h e result of a r e p a i r - a c t i v e process. T h e r a n g e of g e n o t y p i c a n d p h e n o t y p i c influence u p o n t h e specific locus m u t a tion r a t e was e v a l u a t e d with a p p r o p r i a t e biological m a t e r i a l a n d e x p e r i m e n t s .
INTRODUCTION As p a r t of a research p r o g r a m aiming at an e x t e n s i v e k n o w l e d g e of t h e r e l a t i o n ships b e t w e e n r a d i a t i o n doses a n d m o l e c u l a r t y p e s of m u t a t i o n , in this p a p e r we p r e s e n t results on m u t a t i o n i n d u c t i o n frequencies o b t a i n e d w i t h X - r a d i a t i o n s on t r e a t i n g two h a p l o i d strains of Schizosaccharomyces pombe h a v i n g different s e n s i t i v i t y to i n a c t i v a t i o n . F r o m t h e results it is possible to e v a l u a t e t h e kinetics of t h e m u t a t i o n i n d u c t i o n , a n d so to define t h e specific m u t a t i o n r a t e per t a d p e r locus in this o r g a n i s m for comp a r i s o n w i t h p u b l i s h e d data~, 14. T h e influence of genetic b a c k g r o u n d a n d s p o n t a n e o u s m u t a t i o n b a c k g r o u n d on t h e specific m u t a t i o n r a t e i n d u c e d b y X - r a y s m a y also be analyzed. For the present experiments both forward-mutation and back-mutation systems h a v e been tested. * Publication No. lO5 from the Laboratorio di Mutagenesi e Differenziamento, C.N.R., Via Cisanello 147, 56Ioo-Pisa (Italy).
164 MATERIAL
.~. LOPRIENO et al. AND
METHODS
Yeast strains F o r w a r d m u t a t i o n s were i n d u c e d in two ade6 isogenic purple m u t a n t s of S. pombe, b o t h c o n t a i n i n g a mis-sense m u t a t i o n in the locus ade 6 (ade 6- 6 o) ~; one of t h e m had, in addition, a m u t a t i o n in t h e r a d i o locus (radio-z98) which controls t h e i n d u c e d l e t h a l i t y b y several p h y s i c a l a n d chemical agentsS, l°-12 a n d the s p o n t a n e o u s overall m u t a b i l i t y of several lociS, ~. The i n d u c t i o n of gene-forward m u t a t i o n was assessed b y scoring a m o n g survivors white colonies r e p r e s e n t i n g t h e p r o g e n y of a cell in which a second gene m u t a t i o n h a d been i n d u c e d b y t r e a t m e n t in one of five more loci (adez, aden, ade4, ade5, ade9) controlling t h e b i o c h e m i c a l r e a c t i o n s preceding t h e purple p i g m e n t formationL The genetic n a t u r e (nuclear m.utation) of the m u t a n t s scored with this s y s t e m a n d the p o s s i b i l i t y of recovering all t y p e s of m o l e c u l a r a l t e r a t i o n i n d u c e d in the D N A h a v e p r e v i o u s l y been e x t e n s i v e l y d e m o n s t r a t e d L F o r these t~vo s t r a i n s an e v a l u a t i o n of the s p o n t a n e o u s reverse m u t a t i o n rates of the ade6-6o allele has been made. Reverse m u t a t i o n s i n d u c e d b y different dcses of X - r a y s have been also e v a l u a t e d in two isogenic strains of S. pombe b o t h c o n t a i n i n g t h e allele ade7-z52 (mis-sense m u t a t i o n ) s. In these e x p e r i m e n t s the influence of the allele radzo-x98, present in one strain, on the i n d u c e d m u t a b i l i t y was also e v a l u a t e d . X - R a y treatment S u s p e n d e d cells of S. pombe (IO s cell/ml in o.I5 M K H2PO,, p H 4.5) were i r r a d i a t e d at r o o m t e m p e r a t u r e b y a Siemens S t a b i l i p a n X - r a y m a c h i n e with 2 him A1 filter, o p e r a t i n g a t 200 K V a n d 2o mA. The e s t i m a t e d dose r a t e was 2500 R per rain, as determ i n e d b y a Siemens X - r a y dosimeter. O x y g e n was p r o v i d e d b y m a g n e t i c stirring of the cell suspension during t r e a t m e n t . All the results p r e s e n t e d were d e r i v e d front several i n d e p e n d e n t e x p e r i m e n t s a f t e r s t a t i s t i c a l t r e a t m e n t of t h e d a t a b y regression analysis *.
RESULTS Spontaneous mutation rates The r a t e s were e v a l u a t e d in t h e wild t y p e (rad +) a n d u n d e r t h e influence of the r a d i o - z 9 8 m u t a t i o n b y t h e m e t h o d of LURIA AND DELBR/OCK1~ in five i n d e p e n d e n t
TABLE [ SPONTANEOUS
MUTATION
RATE IN
Schizosaccharomyces pombe a
Experiment b No.
ade 6-6o/rad+/hmutation rate (. ~o-~ p.c,d.) Five loci One locus°
ade 6-60/radzo-z98/hmutation rate (.¢o 5 p.c.d.) Five loci One locus
I 2 3 4 5 Average(~ S.E.M.) e
0.27 o.4I 0.55 o.46 o.23 o.38 ± o.o59
i4.oo i2.5o 6.96 6.o6 3.io 8.52 ± 2.04
0.054 0.082 o.i1 o.o92 o.o46 o.o76]± o.o12
ant In (aNtC) (ref. 13). b For each experilnellt, io independent cultures were evaluated. e S.E.M., standard error of mean. d p.c.d., per cell division. a
r
--
2.80 2.50 1.39 1.2i o.62 1.3o ~= o.55
R A D I A T I O N M U T A T I O N IN Y E A S T TABLE
16 5
1[
SPECIFIC LOCUS SPONTANEOUS MUTATION RATES (FORWARD AND REVERSE) IN Schizosaccharomyces
pombe Strain (purple)
Forward mutations • ade 6 --~ ade 6 /ade x (purple) (white)
Back mutations • ade 6 --> ade + (purple) (white)
ade6-6o/racl ~/hade6-6o/rad;o-~:98[h-
0 . 7 6 ~ o . 1 2 . i o -~ p . c . d , 1 . 3 o ± 0 . 5 5 . i o ~ c . d . -~
o . 1 7 • i o ~ c . d . -1 o . 1 5 . IO - " c . d . ~
~ r = a n t In (aNtC) (ref, 13).
TABLE
IiI
INACTIVATION AND FORWARD MUTATIONS INDUCED BY X-RAYS ON TWO STRAINS OF Schizosac-
charomyces pombe Dose (R)
Survival ( % ) (:~ S . E . M . )
N u m b e r of colonies analyzed
N u m b e r of mutants scored
Mutation frequency ( . i o ~ survivors ~_ S . E . 3 I . ) 5 loci • locus
5-43 i.oo 1.46 o.12
176141 160305 245283 200495 261538
3 21 65 162 28c~
o-17 1.31 2.65 8.08 11.o 5
• ~ =~ ~ =~
o.o4 o.07 o.15 0.85 1.37
o-o3 0.26 o.53 1.61 2.21
± ± ± ~= ±
o-oo3 o.oi 0.03 o.i 7 0.27
ioo.oo 66.52 ~ 7.08 1 8 . 4 2 -k 1.16 0 . 8 3 ~_ o . 1 6
62991 82395 6349~ i o i 54 °
51 lO 5 224 398
4.61 13.92 36.3 ° 4o.04
~-~ ~_ ±
1.62 2.46 3.62 3.23
0.94 2.78 7.26 8.Ol
=c ~ ± ±
0.32 0.49 0.72 0.64
ade 6-6o/rad +/hControl 32ooo 6900o i2oooo 160000
ioo.oo 84.75 62.00 lO.84 1.46
± ± ~_ =c
ade 6-6o [rad Io-~98 ~hControl 16000 48000 96000
experiments (Table I). As stated in the previous section, white colonies represent additional m u t a t i o n s in five loci. Moreover, t h e y might be the result of a reverse m u t a t i o n a l event occarring at the ade6-6o site. Therefore reverse m u t a t i o n rates of the ade6-60 allele in both strains were evaluated. The complete set of d a t a is reported in Table I I : the reverse m u t a t i o n rates were lO 3 to lO 5 times lower t h a n the forward m u t a t i o n rates and their influence can be completely ignored. The forward m u t a t i o n rate during mitosis was increased 17 times b y the presence of the radxo-I98 allele.
X-Ray-induced mutation The results obtained from seven experiments in which the induction of forward m u t a t i o n was evaluated are reported in Table I I I . The d a t a are presented in Figs. 1-4. The cell inactivation in the two strains is described b y the equation s log % survival = e ~*
(I)
where x is the dose ( × IOOO R) and fl the regression coefficient. As shown in Fig. I for b o t h strains, the inactivation (log% survival) was proportional to the square of the dose, and these relations indicate t h a t the survival was the result of repair events t h a t reduce the effects of the induced damage(alteration of D N A bases, D N A breaks, etc.). In strain radzo-~98 a very slow repair ability was still operating.
~ . L O P R I E N O et al.
166
10
i°°
u
9
J 0
J
J
J
J
Z
i 0.1 100 150 0 5O 100 1 0 X-Roy dose (x 1000 R) (x) X-Ray dose (xIOOOR) (x) F i g . i . I n a c t i v a t i o n c u r v e s of X - r a y t r e a t m e n t s w i t h t w o s t r a i n s of S. pombe: ©, rad +, [~ o . o o o o 7 2 - 1 o -2 (y = 2 . 0 4 3 0 . 0 0 0 0 7 2 x2; F 3 3 7 , 4 4 * * * ; r = 0 . 9 9 5 5 * * * ) ; O, radzo-z98, ~ -o . o o o 2 2 - 1 o -2 ( y : 1 . 8 9 2 3 - - - o . o o o 2 2 x2; F = 1 8 4 . 1 o 4 " * * ; r - - 0 . 9 9 4 6 * * * ) . (/~ = % of i n a c t i v a t i o n ,x~; x I o o o R.) F i g . 2. L o c u s s p e c i f i c m u t a t i o n f r e q u e n c y i n d u c e d b y X - r a y s i n t w o s t r a i n s of S. pombe : O, tad +, o - - o . o 1 4 2 . 1 o -4 s u r v i v o r s × l o c u s × i o o o R (y - - - - o . i 5 3 4 + o . o i 4 2 x; F -- 74.405**; r o . o 9 8 o 4 " * ) ; O, radzo-±98, / ~ - - o . I 3 O 9 X l O -4 s u r v i v o r s × l o c u s × i o o o R ( y - - o . . 5 9 + o . 1 3 o 9 x; F - - 4 7 . 7 7 * ; r - . 0 . 9 8 9 6 * ) . T h e s p e c i f i c m u t a t i o n r a t e p e r ] o c u s p e r r a d is: rad= o.i4.1o s ( n o r m a l i z e d v a l u e (n.v.) - - 1 . 8 o . lO :) ; rad::o-::98 o. t 3 × Io ~ ( n o r m a l i z e d v a l u e 1 . 6 7 . i o ~.)
~o
16 100
$~ ~ 12
J
g
~
°
~ ~
100
10 log ~/~ survival
, 1
t~ x ---1~0 :
~o~x~ e-aX (i + ~ - - ~
~, ra~+; ~, rad~o-I98.
0"10
X
~0 100 150 X - Roy dose (1000 R) F i g . 3. M u t a t i o n f r e q u e n c y a s a f u n c t i o n of l o g p e r c e n t a g e s of s u r v i v a l i n t h e t w o s t r a i n s of S. pombe: O, ra,.t*, ~ ~ 1 , 5 6 4 o . I o - 4 × 1 o g % s u r v i v a l (y = 3 . 2 4 ~ 9 ~ I . $ 6 4 o x ; F ~ i 7 ~ . 3 6 7 8 " * ; r ~ o.9943"*); o, ra~o-~98, ~ ~ 8.425I-iO-~1og % survival (y-- i7.6729--8.425i x; F-535-4731"*; r ~ o.9991"*.) I:ig. 4. S. pombe f o r w a r d m u t a t i o n s (a~e l o c u s ) . E f f e c t of X - r a y t r e a t m e n t s o n m u ~ t i o n i n d u c t i o n e x p r e s s e d b y t h e r a t i o K / t C o. T h e d a ~ h a v e b e e n t r e a t e d & c c o r d i n g ~o t h e r e l a t i o n (4). (x)
RADIATION
MUTATION
IN
167
YEAST
Fig. 2 shows t h e rates for m u t a t i o n i n d u c t i o n b y X - r a y s in b o t h strains after t r e a t m e n t of d a t a b y t h e regression analysis r e p r e s e n t e d b y e q u a t i o n (2)
(2)
y = a+~x
where y is t h e i n d u c e d m u t a t i o n t r e q u e n c y a n d x is t h e dose ( × IOOO R). The rad + s t r a i n showed a r a t e c o n s t a n t (8) of o.o142. IO-~ survivors × locus × IOOO R, w i t h a specific m u t a t i o n r a t e per locus per r a d of o.14. IO -8, whereas the radzo-±98 h a d a r a t e c o n s t a n t (fl) of o.13o 9- IO-~ survivors per locus per i o o o R, w i t h a specific m u t a tion r a t e per locus per r a d of o.13-IO 7, i.e. IO t i m e s higher t h a n the wild t y p e . This was not t h e result of the higher l e t h a l i t y i n d u c e d b y X - r a y s in the radro-±98 strain, since if we c o m p a r e the relationships b e t w e e n m u t a t i o n frequencies a n d s u r v i v a l s in the two s t r a i n s (Fig. 3), a higher i n d u c e d m u t a b i l i t y for t h e s t r a i n radzo ~98 is observed. ( F o r comparison, t h e m u t a t i o n f r e q u e n c y i n d u c e d a t l O % s u r v i v a l was 1.68- IO -~ × locus in t h e rad +, a n d 9 . 2 5 IO-~× locus in the radio-z98. ) The m u t a t i o n f r e q u e n c y (K,) per s u r v i v o r (S,) i n d u c e d b y a given dose of X r a y s (x) is expressed b y t h e e q u a t i o n :
K ~ _ K0 + ~x S~ So
(3)
where Ko is t h e s p o n t a n e o u s n m t a t i o n f r e q u e n c y a n d So is t h e s p o n t a n e o u s s u r v i v a l of t h e cell p o p u l a t i o n ; ~ r e p r e s e n t s t h e p r o b a b i l i t y of t h e i n d u c t i o n of m u t a t i o n per dose unit a n d p e r cell. Insertion of e q u a t i o n (I) into e q u a t i o n (3) p r o d u c e s the following relationS:
K, / So~x \ Ko - - e ~* I 1 + K ~ - - )
(4)
which expresses the real increase of t h e i n d u c e d m u t a t i o n over t h e s p o n t a n e o u s level for each X - r a y t r e a t m e n t , giving a certain p e r c e n t a g e of survival. Fig. 4 shows t h e K/Ko values o b t a i n e d b y t r e a t i n g t h e e x p e r i m e n t a l d a t a rep o r t e d in T a b l e I I I for b o t h rad + a n d radio-z98 strains. These d a t a were confirmed ~vhen reverse m u t a t i o n s occurring a t t h e ade7-z5~ allele in presence or absence of radxo-z98 m u t a t i o n were a n a l y z e d : t h e e x p e r i m e n t a l d a t a are a s s e m b l e d in T a b l e IV a n d t h e K/Ko values for b o t h s t r a i n s are shown in Fig. 5. T h e increase of the K/Ko values in b o t h s y s t e m s was u n d e r genetic control, TABLE IV INACTIVATION
AND REVERSE
MUTATIONS
INDUCED
BY X-RAYS
ON TWO STRAINS
Average data from 8 experiments.
Dose (R) ade 7-252 Irad+/hControl 32ooo 96ooo 160000
Survival (%) (~: S.E.M.) ioo.o 57.5 ~_ 11.2 9-4 • 1-7 0.2 ~ 0.05
Mutation frequency (.xo -9 S ± S.E.M.) 1.92 ± o.66
51.51 ± 9.9 ° 47-o5 ± lO.O7 24.50 ± 2.45
ade 7-;52/tad ~o-298 /hControl 16ooo 32000 48000 96000
IOO.O 49.1 ~ 6.37 21. 3 ~_ 8.07 7-7 -~ 0.76 0. 4 -~ 0.06
3.2o 23.79 40.23 71.44 182.54
± 1.46 ± 4.85 ~ 9.48 ~.- 1°'14 ~ 116.Ol
OF S.
po~be
N. LOPRIENO et al.
168
10C
l 1C
',
~
~ \
\x ~\ \
!
~.
1~[ [ ~ - ~
~
o~ g ~ ~,~,~,~,
"~"'..... " ' ' . ~
' 6~
~6o
X-Rey dose (xl03 R) Fig. 5- E g e c t of X - r a y t r e a t m e n t s on reversion i n d u c t i o n (ade- -~ ade +) for t h e locus ade6 in t w o strains of S. pombe, expressed b y t h e r a t i o ~ / K o. C~, f a d + ; ~, ggdzo-z98.
since a different behavior between md+ and radzo-zg8strains was observed. However, it seems an indirect control, because the spontaneous mutation frequency directly influenced the E/Eo. In our case this influence was higher in the md+ than in the mdzozgg strain. To evaluate a possible influence of phenotypic factors on the rate of mutation induction, we treated with different doses of X-rays independent clones of radzo-z98 cells showing a diEerent spontaneous mutation back~ound. Two clones with a spontaneous mutation frequency respectively of 4.08. IO -~ N locus (culture A) and o.6~. ~o -~ Nlocus (culture B) were analyzed for the induction of forward mutations. The data from 3 experiments are assembled in Table V. The inactivation curves of the two clones were s i ~ ] a r (Fig. 6), where-as the kinetics ot mutation induction digered greatly from each other (Fig. ?). TABLE
V
INACTIVATION
AND
FORWARD
MUTATIONS
INDUCED
sr~A[~" ade6-6o/mdzo-z98/h- o~ S. pombe w ~
BY X-RAYS
a ~F~N~
IN TWO
INDEPENDENT
FREQUENCY
Average data from 3 experiments.
Dose (R)
Survival (%)
Number ~[ mutants Number of colonies
Mutation frequency (. zo 4 survivors × locus)
Culture A Control ~2 500 25ooo IOOOOO
i oo.o 74.8 68.0 o. 4
34/I 6656 26/7986
4.08 6.51 5.27
Culture B Control 175 °o 5oooo iooooo
IOO.O 6o.6 I7.5 o.9
~ I/35228 35/36696 Io2/34619
34/I2789 78/27368
224/61432
5.7 o
o.62 i .90 5.89 7.29
CULTURES
OF
VA~V~ OF S P O X ~ a ~ O V S ~U~A~IO~
RADIATION
MUTATION
16 9
IN YEAST
100
~
10
/
1(
g
/
g g
5 ~0 X-Ray
dose
~ . 100 (×IO00R)
~ 150 (x)
~
0'
t
50 X-Rey
-
-
|
~
1O0 150 dose ( x l O 0 0 R) (x)
F i g . 6. Survival curves of two independent cultures of S. pombe (SP-I98 ade6-6o]radlo-I98, h-) after X - r a y treatments : ©, culture A , / 5 = o . o o o 2 3 . I O - Z ( y = 1 . 9 6 3 7 - - o . o o o 2 3 x~; F = 1 8 2 3 . 1 1 2 " * " r = 0.9994***); r = o . 9 9 1 9 " * * ).
O,
culture
B,
/~ ~
o.oooI9-io
-~ (y =
1.868o--o.ooo19
x~; F =
122.449"**;
F i g . 7- M~tation frequency induction in two independent cultures of the strain SP-z98 ade6-6o[ o f S pombe after treatments with X-rays. The cultures had different spontaneous mutation frequencies : ©, C u l t u r e A , / 5 = o . o o 6 9 - lO 4 s u r v i v o r s × locus × i o o o R ; specific mutation rate per locus per tad = 0 . 6 9 . lO -9 ( n . v . = o . 8 9 " I O 7) (data statistically not significant) ( y ~ 5.1515+o.oo69 x ; F ~ 0 . 2 0 n . s . ; r = 0 . 3 0 7 5 n . s . ) ; O , c u l t u r e B , ~5 ~ o o 6 8 9 . 1 o -4 s u r v i v o r s × l o c u s × i o o o R; specific mutation rate per locus per rad ~ o . 6 8 . 1 o * ( n . y . = o . 8 8 . I O -~) (y = i.o38+o 0689 x; F ~ 2o.5471"; r = 0.9546*).
radzo-z98, h-
Although the data from culture A are not statistically significant, we have used them for a better comparison : the data represent limit values due to higher spontaneous mutation frequency. Other cell clones with spontaneous mutation frequency between the values from culture A and culture B gave intermediate values. The late of locus mutation induction in culture B was ten times higher (0.0689 • lO-4) than in culture A (0.0069. lO4). Fig. 8 shows the relationships between survival and mutation frequency observed in the two cultures. The results of the treatment of the data according to equation (4) are shown in Fig. 9 : a mutation induction by X-rays over the spontaneous level was observed only for culture B. DISCUSSION
In S. pombe, as in other organisms, active repair processes to reduce damage produced in the D N A are operating: this is demonstrated by the isolation of mtltants which are no longer able to repair inactivating events 3, and also by the evaluation of the dose-effect relationships. In our experiments, when a wild-type strain was treated with X-rays its survi-
N. LOPRIENO et al.
17o
tn 16 um _o. 14
//
~.12
~
//
//
,~ 10
,~ 8 o ~ 6
/
///
o/
d,
g
8 ._ ~
S
~
i
~o
~oo
~
i
log % survival
(x)
I
o.~
~ i { . 8. M u t a t i o n frequency ~s ~ fmnction of ]o~ perceat~ges of s u r v i v M in Lbe two cultures o[ t~e strain SP-z98 ~6-6o/m~zo-]98, h- o[ S. 2 ~ : o, Cu]ture A, ~ ~ too.87, mo-~x ]o% % s u r v i v M (d~t~ statistically a o t significant) (y = ~6.o536 zo.87~ 5 ~; ~ = L6o98 n.s.; ~ ~ o.785~ ms.); ~, cu]taTe ~ , ~ = 6.99"~o -~X ]o~% s u r v i v M (y = z~.7253--6.9973 ~; ~" :- 3~5.29~o*; ~ = o.998~*). 10
~ol
0.1
)
0.01
o.oo~
~'o
~&--
1~0 I
X-Ray dose (xlO 3 R) Fig. 9. Effect of X - r a y t r e a t m e n t s on m u t a t i o n induction expressed by t h e ratio K/A"o in two cultures of t h e strain SP-z98 ade6-6o/radio-z98, h- of S. pombe with different s p o n t a n e o u s m u t a tion frequencies: O, culture A; e, culture B.
val curve was proportional to the square of the dose; the same relationship was found for a strain almost completely lacking the repair ability (radxo-z98) 15, but the regression coefficient was higher t h a n t h a t of the wild type, thus indicating only a partial repair ability. This finding m a y also be confirmed by the fact t h a t a linear dose-effect relationship m a y be obtained by the same survival data of the radro-zg8, although here an F (~D~I~0)ll lower than the F reported in Fig. I ( P ( I } ~ ) was observed. In both strains the m u t a t i o n induction presented a linear relation with the dose : the radio-z98 strain showed a rate constant (fl) higher than t h a t shown b y the rad+ strain, also when similar survivals were compared. B y analyzing the ratio K/Ko as described in the results, a higher inducibility of m u t a t i o n in the rad + strain was observed expressed as an increase over the sponta-
N. LOPRIENO et al.
172 T A B L E VII FORWARD GENE MUTATION RATES IN S. pombe
Strain
Mutagen
Mutation rate per locus (A)
Normalized specific mutation per locus (A :7.75.2o-~) •
tad + (wt) rad±o-~98 rad + (wt) radio-z98 radlo-I98(A) b radzo-z98(B)b
Spontaneous Spontaneous X-Rays X-Rays X-Rays X-Rays
0.76. lO-6 (p.c.d.) 1.3o. io -~ (p.c.d.) o.14 • io 6 (p. tad) o.13 • io -~ (p. rad) 0.69. IO-~ (p. tad) 0.68. IO-s (p. rad)
0.98- IO-* (p.c.d.) 1.67 • IO-~ (p.c.d.) 1.8o- io ~ (p. tad) 1.67. lO-6 (p. tad) 0.89. IO-~ (p. tad) o.88. io 6 (p. tad)
a S. pombe haploid cells contain 0.0225 pg DNA per nucleus, only 7-75' IO-a times the amount of DNA as compared with h u m a n (2.9 pg of DNA per haploid nucleus). b Independent cultures of strain radxo-I98 with different spontaneous background mutation. p g b y ABBONDANDOLO, f r o m t h i s l a b o r a t o r y , as a n a v e r a g e of w i l d t y p e a n d s e v e r a l a u x o t r o p h s ~. F o r T a b l e V I I w e h a v e c a l c u l a t e d t h e n o r m a l i z e d specific m u t a t i o n r a t e p e r l o c u s i n t h e t w o s t r a i n s rad + a n d r a d x o - I 9 8 a n d i n d i f f e r e n t c l o n e s of t h e s a m e s t r a i n (radzo-I98) : for t h e w i l d t y p e a v a l u e of 1.8o. lO -7 × l o c u s × t a d w a s o b t a i n e d , a v a l u e v e r y close t o t h a t u s e d b y ABRAHAMSON. D e p e n d i n g o n t h e s t r a i n s t e s t e d ( r a d i a t i o n - s e n s i t i v e s t r a i n s ) or o n t h e s p o n t a n e o u s m u t a t i o n b a c k g r o u n d of t h e t r e a t e d cell p o p u l a t i o n s , t h e v a l u e s of specific m u t a t i o n r a t e s p e r l o c u s p e r t a d m a y v a r y f r o m 1.8o. IO -7 (rad +) t o 1.67 • IO -~ for a c l o n e of t h e r a d i o - x 9 8 s t r a i n . I n o u r o p i n i o n s u c h a f i n d i n g d o e s n o t i n d i c a t e a h i g h e r m u t a b i l i t y of S. pombe i n c o m p a r i s o n w i t h o t h e r y e a s t s a n d species, b u t r a t h e r i t reflects a general situation in relation to the genetic system used or to the strain treated with X-rays. Moreover, the different values found between the wild-type and t h e r a d i a t i o n - s e n s i t i v e s t r a i n of S. pombe m a y reflect g e n e r a l s i t u a t i o n s a m o n g differe n t g e n o t y p e s i n t h e s a m e species. ACKNOWLEDGEMENTS
P a r t of t h e s e i n v e s t i g a t i o n s h a s b e e n s u p p o r t e d b y g r a n t No. i i i 7 2 - 1 B I O I f r o m t h e C o m m i s s i o n of t h e E u r o p e a n C o m m u n i t i e s , B i o l o g y D i v i s i o n , t o t h e U n i v e r s i t y of P i s a . REFERENCES I ABBONDANDOLO, A., Mutation and nuclear stage in Schizosaccharomyces pombe, I. A genetic
2 3 4 5 6 7 8
system for studying the role of recombinational repair in m u t a t i o n induction, Mutation Res., 27(~975) 225 233. ABRAHAMSON, S., M. A. BENDER, A. D. CONGER AND S. WOLFF, Uniformity of radiationinduced mutation rates among different species, Nature (London), 245 (1973) 46o-462. AUERBACH, C., AND B. J. KILBEY, Mutation in eukaryotes, Ann. Rev. Genet., 5 (I97 I) I63-218. BAUTZ-FREESE, E., AND E. FREESE, Induction of reverse mutations and cross reactivation of nitrous acid-treated phage T4, Virology, 13 (I96I) 19-3 o. CONGER, A. D., IS DNA the only chromosomal component contributing to target size for mutation induction by ionizing radiation ?, Mutation Res., 25 (1974) I3I-I34CROW, J. F., AND M. KIMURA, An Introduction to Population Genetics Theory, Harper and Row, Nexv York, 197 o, pp. 486-493. GUTZ, H., H. HESLOT, U. LEUPOLD AND ~N'. LOPRIENO, Schizosaccharomyces pombe, in R. C. KING (Ed.), Survey of Genetics, Vol. 2, Plenum, New York, 1974. LOPRIENO, N., UV radiation-sensitivity and m u t a t o r activity in yeast S. pombe, Proc. I V Int. Congr. Radiat. Res., Evian, I (1971) 2 IO.
RADIATION
MUTATION
IN
171
YEAST
neous level. Since an active repair ability to reduce DNA damage operates in the wild type, the higher mutability of this strain m a y be assulned to result from the repair processes, which therefore are of the error-prone type 1~. Both forward and revelse mutational events are of this type. It is possible to postulate, therefore, that the mutations induced by X-rays in the radro-I98 strain are partly the result of direct damage to DNA, whereas in the wild type (rad +) they are the results of an error-prone repair. If this is so, then we should expect mutations of possible different molecular nature inducible by X-rays on treatments done with the two strains. Preliminary results obtained by studies on the molecular nature of mutations induced by X-rays in the ade6 and ade 7 loci of two strains of S. pombe (rad + and radzo x98 ) indicate that with the wild type the incidence of base-pair substitution mutations is higher (45%) than that of the same class of m u t a n t s obtained with the radlo ~9 ~' (2.2°/~)i°, 11. To decide whether the rate constant (/3) of mutation induction is under genetic or phenotypic control, attempts were made in the present study to compare two strains with gene-controlled different spontaneous mutabilities and two clones of the same strain with different spontaneous mutation frequencies. Our results indicate that in both attempts the spontaneous mutation background was strongly responsible for the slope of the mutation-induction curve. This finding illustrates the difficulty of comparing different experiments of mutation-induction done with cell populations containing different spontaneous mutation backgrounds. D a t a on mutation induction by X-ray treatment in yeast, as in other organisms, have been used by ABRAHAMSONel al. '2. to evaluate the correlation of radiation-induced mutation rates among different species. These data are of great interest, as for the first time they suggest a uniformity in the specific locus mutation rates in different organisms if the data are normalized for the amount of DNA (Table VI). Although a correlation with another possible structure (i.e. chromatin mass) has been postulated recently a, the data of ABRAH~,SISON et aI. require detailed analysis before their final confirmation. Irrom the data reported in the present paper it is possible to better define how the yeast S. pombe supports ABRAI~AMSONet al.'s data. A recent paper by NASIM has already reported a few data for S. pombeIL confirming the data obtained with the canavanine resistance mutational system used with Saccharo~,ces cerevisiae ~. In S. pombe, the content of DNA per cell (G2 stage) has been evaluated as o.o225 TABLE
VI
SPECIFIC LOCUS MUTATION RATE INDUCED
BY RADIATIONS a
Organism
Relative amount of D N A as compared with h u m a n ( ~ )
iVIutation frequency per locus per tad (B)
E. coli ~'. cerevisia~ N . crassa Drosophila melanogasler 3/Ius musculus H. vulgate Homo sapiens
4-5" lO 3 8.3 • IO -3
I . O . IO ~ i .6- io 9
2.2 i .9
1. 4 • i o - ~ 0.058
2. 7 • i o 9 1. 4 . I O s
1.9 2. 4
a From
ABRAHAMSON
Normalized value B / A ( . z o -~)
0.78
1. 7 . i o
7
2.2
3.45 i.o
i.o. IO -~ Average
2.9 2.2
et al. ~,
Inodified.
RADIATION MUTATION IN YEAST
173
9 LOPRIENO, ~X~., A m u t a t o r gene in the y e a s t Schizosaccharomyces pornbe, Genetics, Suppl. 73 (1973) 161-164. io LOPRIENO, ~7., A. ZkBBONDANDOLO, S. BARONCELLI, M. SIMILI AND R. ]~ARALE, Molecular n a t u r e of p o i n t m u t a t i o n s induced b y X - r a d i a t i o n s in S. pombe, Annual Report ~972. Programme Biology-Health Protection, 1973 , pp. 242-243. i i LOPRIENO, Iq., .A_. ABBONDANDOLO, S. BARONCELLI, ~1. SIMILI AND R. BARALE, Molecular n a t u r e of point m u t a t i o n s induced b y X - r a d i a t i o n s in S. pombe, Annual Report ~973. Programme Biology-Health Protection, 1974, pp. 276-277. 12 LOPRIENO, ~X~.,R. BARALE, C. ]~AUER, S. ]3ARONCELLI, G. BRONZETTI, .A,. CAMMELLINI, A. CINCI, G. CORm, C. LEPORINI, 1~. NIERI, M. NOZZOLINI AND CRISTINA SERRA, The use of different test s y s t e m s w i t h yeast for the evaluation of chemically induced ggne-conversions and genem u t a t i o n s , Mutation Res., 25 (1974) 197-217. 13 LURIA, S. ]~., AND •. DELBR~ICK, Mutations of bacteria from virus sensitivity to virus resistance, Genetics, 28 (1943) 491-511. 14 NASIM, A., Radiation-induced m u t a t i o n rate and D N A c o n t e n t in Schizosaccharomyces pombe, Mutation Res., 24 (1974) 211-212. 15 SCHUPBACH, *~/[.,The isolation a n d genetic classification of UV-sensitive m u t a n t s of Schizosaccharomyces pombe, Mutation Res., i i (1971) 361-371. 16 VqlTKIN, E. M., Ultraviolet-induced m u t a t i o n and D N A repair, Ann. Rev. Microbiol., 23 (1969) 487-514 .
