Analysis of induced mutations in Aspergillus nidulans I. UV- and HNO2-induced mutations

145

MUTATION RESEARCH

ANALYSIS OF INDUCED MUTATIONS IN ASPERGILLUS N I D U L A N S I. UV- AND HNO2-INDUCED MUTATIONS C L A R A C A L V O R 1 AND G 1 O R G I O M O R P U R ( ; O Centro Intef~aziotzale di Chimica Microbiologica, lstit~.tto .fuperiove di Sanitg~, leoma (Italy) ( R e c e i v e d J u l y 28th, 1965)

SUMMARY

A method is described that permits tile selective detection of forward and back mutations for a single cistron in Aspergillus nid,dans. The method also permits an easy phenotypic distinction among true back mutants and suppressor mutations. The rate of spontaneous, UV- and HNO,-induced retromutations has been studied in 15 UV- and 14 HNO~-induced mutants. Most of the UV-induced mutants can be interpreted as due to a base substitution while HNO2-induced mutants must be due to a different mechanism. Two mutants of this group present a rate of spontaneous retromutation 2o-4o times higher than the rate of forward mutation in the whole cistron. The spontaneous rate of retromutation is not modified by treatment with five reference mutagens.

I NTRODUCTION

Oil the basis of extensive analysis of induced and spontaneous mutations in phages (for a review see ref. 5) the nature of the mutation process at the molecular level has been defined and the mechanism of action of a large number of mutagenic agents has been inferred. In phages, according to the models of FREESE,mutations can be due to one of these primary events: (I) substitution of bases; this type is further subdivided in (a) transition (purine-purine or pyrimidine-pyrimidine substitution), and (b) transversion namely purine-pyrimidine substitution; (2) addition or deletion of bases; (3) larger rearrangement or deletions. Most of the commonest mutagens produce mutations of Types I and 3- Mutations of Type 2 are apparently produced by acriflavine and by spontaneous mutational process 5,9,1,,. These results have been largely achieved by analysing the pattern of retromutation with different mutagenic agents. Very few data are at present available on the nature of induced mutation at molecular level in higher organisms with a chromosomal structure. Some data on mutagen specificity in Neurospora and yeast have been presented by DE SERRESa and by I~AKAR, ZIMMERMANAND WAGNER8 but nothing comparable with the work on phages was yet been done. The obvious problems are: (I) if the same general mechA b b r e v i a t i o n : P F P , p - f l u o r o p h e n y l a l a n i n e . H N - 2 , Methyl-bis(fl-chloroethyl)amine.

2VIutation Res., 3 (1966) 145 151

I40

C. CALVORI, G. MORPURGO

anism apply to the process of mutation in chromosomal and non-chromosomal organisms; (2) to test if the same mutagen produces mutations of the same nature in higher organisms and in phages. For such a study the necessary requirement is a system which permits the selection of forward and reverse mutations. Mutants for resistance to p-fluorophenylalanine (PFP) in A@ergilhts ~ziduIa~s met the necessary requirement. All the pfp-r mutants belong to the same cistron ~0 and back mutants can be easily selected on a medium supplemented with aminotyrosine and phenylanthranilic acid. pfp-r mutants are leaky requiring tyrosine~°; addition of aminotyrosine and phenylanthranilic acid into the medium enhance this requirement and completely prevent their growth. A more difficult problem is presented by the choice of the mutagens. In Aspergillus and probably in most of higher organisms (except yeast) most of the more specific nmtagens do not work: we unsuccessfully tried hydroxylamine, methoxyamine, N-methyl hydroxylamine, 2-aminopurine and 5-aminoacridine. Choice is therefore necessarily limited to the classical mutagens. In the present paper the specificity of the mutations obtained with UV light and with HNO,, has been investigated. MATERIALS AND METHODS

Media

A minimal medium, which has already been described, has been used~. Strains

Two strains of A. nidulans have been used: strain 18 s u - z ad-2o ribo-~ p r o - z ad- 2 o bi- I pyro - 4 and strain 35 paba- z an - z y meth- r hie-2 nic- 8 S - z 2. The symbols of PONTECORVO AND KAFER 11 for mutant alleles were adopted. All HNO,, mutants were from strain I8 and all the UV-induced mutants were from strain 35. Plating procedure

Conidia were taken by fresh cultures and suspended ill water except otherwise stated (see mutagenic treatment). Conidia were spread on the surface of the selective medium at a density of 2O.lO~ 3o' lO6 per dish. Selectio~2 of forward mutation

Mutants for p-fluorophenylalanine resistance (pfp-r) were selected on minimal agarized medium in which the concentration of the drug was I mM. On such a medium mutants are scored as well sporulating colonies on a light brownish background. Selection of revertants

Revertants can be easily selected (Fig. I) on minimal agarized medium to which L-3-aminotyrosine and phenylanthranilic acid were added to a final concentration of o.oi and o.o47% (w/v) respectively. On this medium wild-type colonies can grow while pfp-r mutants are totally incapable of growth. The pH of the medium must be carefully adjusted to 4.5 or 6.5 otherwise the medium can be toxic also for wild-type conidia. The method was developed in collaboration with Drs. SERMONTI, P E T R E L L I AND RICCI.

2YIutation Res., 3 (1966) 1 4 5 - 1 5 l

A N A L Y S I S OF I N D U C E D M U T A T I O N S IN

A. nidulans. I.

147

Fig. 1. B a c k m u t a n t s of s t r a i n 16N s e l e c t e d on A I F m e d i u m . D i s h e s w e r e s e e d e d w i t h a p p r o x . 2" IO~ c o n i d i a .

3I~ttage~zic treatment In the present work treatment with HNO,,, UV-rays, X-rays, diepoxybutane and the nitrogen mustard methyl-bis(fi-chloroethyl)amine have been used. HNO~a. 9.67 ml of a suspension of conidia in acetate buffer at pH 4.5 were mixed with <>.33 ml of I M NaNO2 and incubated 5 min at 37 °. The reaction was stopped by a 5-fold dilution of the conidial suspension with phosphate buffer (pH 7.1). UV-rm's. Conidia were treated directly on the surface of the selective agar medium. A germicidal Hanovia lamp was used as source of UV-rays. X-rm;s. Conidia were treated suspended in water--depth of the liquid was about 1.6 mm. The apparatus used was a (;ilardoni Neoderma Be model (7° kV, 5 mA). ]'he dose rate in the air at the locus of the cells was approx. 125oo R/rain. Methyl-bis(l']-chloroeth3~l)ami~,e (H~N'-2) was used at a concentration of 6.5 mM in an aqueous solution of 3.I5}/o NaHCOa (w/v). Treatment of the conidia was interrupted by diluting the suspension witb a decontaminating solution containing glycine. After the treatment and before plating it is necessary to centrifuge and wash the conidia to avoid changes of the pH of the medium. Diepo&vbutane. Conidia suspended in water were treated 5 rain with 0.o2 mM diepoxybutane. After the treatment it is necessary to centrifuge and resuspend conidia for 4 h on complete liquid medium before plating on I.-3-aminotyrosine+phenylanthranilic acid ( A + F ) medium otherwise this is lethal to diepoxybutane-treated conidia. ..VIutation Res.,

3 (1966) 145-1.51

148

C.

CALVORI,

(;.

MORPUI{GO

RESULTS

HN()2 a n d UV-ravs were used to induce forward m u t a n t s . T r e a t m e n t with different m u t a g e n s of wild-type a n d m u t a n t conidia produces identical survival and selective et~ect can be ruled out. i n Table I the m u t a t i o n frequency and the survival with s t a n d a r d t r e a t m e n t "1".\ 1~1.1~

1

FRKQUFNCY (IF SPONTANI'OUS AND INDUCI£D ]b./p y MUTANTS

Strain

Trealm
Spontaneous, lnitotic Spontancous,

18 35

meiotic

[" V-Ffl \'S

Methyl bis(/3-chloroethyl)aminc l)icpoxybutane

lereque~wy

Sz~r~'iz,al

( ~ ±o9

(.:: ±o ')

(%)

2.~2 2O

18 > 35

42

IS

IO I4

35 18 ~8 35 IS 18

X-F~/VS HNt) 2

~\'z~mber observed

0.20 o.5o

IOO loo

0.07

100

172 118

(~ 24 ~5 9 6

77 t"7 I ~'5 75 55

38 2]4

33 78 77.5 22.5 38.5

is shown. I n the same table data are reported also on the m u t a g e m c action and survival following t r e a t m e n t with X-rays, HN-2 and d i e p o x y b u t a n e which were used in some experiments. 15 U\7 a n d 14 HNO2 pJp-r induced m u t a n t s were examined for the occurrence of spontaneous, UV- and HNO.2-induced revertants. Conidia from pfp-r n m t a n t s to be scored for r e v e r t a n t s were t a k e n from colonies grown on m e d i u m s u p p l e m e n t e d with P F P , on which r e v e r t a n t s c a n n o t grow. In such a way it is possible to avoid completely formation of large clones of r e v e r t a n t s which on n o n - s u p p l e m e n t e d m e d i u m arc at a selective over the m u t a n t s . In Tables I I and II1 data are given on the reversion of UV- a n d HNO..,-induced m u t a n t s . In the tables n u m b e r s refer directly to true back m u t a n t s . Suppressor m u t a tions could be quite easily b u t laboriously distinguished by crossing the re~ertant with a wild-type strain a n d selecting pfp-r segregants, or by e x a m i n i n g diploids of the general structure

p.lP rx

I

s~t-p¢p-r,

PIP ry

which, on the a s s u m p t i o n t h a t the suppressor is recessive, should be ~fp-r. t%rtun a t e l y suppressors can also be distinguished p h e n o t y p i c a l l y being capable of growth either on media s u p p l e m e n t e d with P F P or with Aq F. In all the cases we have tested the three methods have exactly the same value a n d therefore the suppressors can be distinguished b y their p h e n o t y p e (Fig. 2). l:rom Tables II a n d I I I it can be seen t h a t the level of spontaneous r e t r o n m t a tions is generally less t h a t I . I 0 s both in UV- a n d HNO2-induced m u t a t i o n s . The r e t r o m u t a t i o n of UV-induced m u t a t i o n s is generally inducible b y UV itself, b u t not b y HNO2. The r e t r o m u t a t i o n of HNOo.-induced m u t a t i o n s is in most of cases not inducible b y either UV or HNO.2, at least in the limits of our experiments.

31z#afioJ~ Rcs., 3 (t960) 145 15t

ANALYSIS

OF

INDUCEI)

MUTATIONS

IN

A. nidulans. I.

149

F i g . 2. D i s t i n c t i o n a n l o n g b a c k m u t a n t s an¢l s u p p r e s s o r m u t a t i o n s . A, m i n i m a l m e n t e d w i t h P F P ; B, m i n i m a l m e d i u m s u p p l e m e n t e d w i t h A F F ; 1, w i l d t y p e ; p r e s s o r o f p l p - r ; 4, b a c k m u t a n t . TABLE [~r

mediunl supple2, pfp-r; 3, s u p -

11

1NDUCEI) MUTATIONS

Spontaneous back mutants Frequency N u m b e r tested

U V - i n d u c e d back mutants Frequency N u m b e r tested

HNO=-induced back mutants Frequency ,Number tested

(~ ±o')

(×±oU

(×zo')*

(>±o')**

(×1o')*

(
-

21 21 25

it.8 -.--

6 5 11

---

7 8 8

4UV

"

17

--

5UV 817V 9UV loWr IIUV I2UV I3UV I4UV 16UV I7UV I8UV

--

13 27 23 23 19 IO 4 I9 17 27 16

Code

No.

liT\: 2U\: 3UV

------

?*** 7.4 2. 4 I ?*** ?*** I. 5 5 1.9 6.6

5 2 IO 13 13 5 I I 6 6 IO 5

8 ?*** 0.25 --?*** ?*** -.---

7 8 8 8 8 8 8 8 8 8 8

* Freqnency on the surviving fraction. ** S u r v i v i n g f r a c t i o n . T r e a t m e n t s b y U V a n d H N O 2 g i v e 34°'¢, a n d 77.5°.o s u r v i v a l r e s p e c t i v e l y . *** T h e n u m b e r o f s u p p r e s s o r s d o e s n o t p e r m i t t h e m e a s u r e m e n t of the frequency of retromutation.

Among the HNO2-induced mutations two of them exhibit a remarkable pattern of retromutation. The spontaneous level is extremely high, about 2o-4 o times the level of spontaneous forward mutation in the whole pfp cistron. We have tried experimentally to raise the spontaneous level in the strain I6N by treatment with UV-light, HNO~, X-rays, HN-2 and diepoxybutane. The data are shown in Table IV from which it can be observed that the spontaneous frequency of retromutation is not increased bv any of the treatments. This anomalous high frequency of retromutation could be explained by the presence of a mutator gene. To test this hypothesis three back mutants from I6N 3/Iutation Res., 3 ( 1 9 6 6 ) I 4 5 - I 5 t

I50

C. CALVORI, G. MORPURGO

T A B L E 111 H.N~*O2-INDUCED M U T A N T S

Code No.

Spontaneous back mutants Frequen O, Number tested

U V-induced back mutants Frequency Number tested "

HNO2-induced back matanls F~equencv Number tested

(x±o~)

(x~o~)

(x±o~)* (xio')**

(×±o')*

(:~o:)**

- - -

-

°.15

81 94 16 33 4° 33

~3 2t 5 .5 29 18

O.17

23

2N 3N 4N 6N 9N IoN

o. 15

[IN

3 .8 iz 5 i~ ii 0

0.27 0.83 O. I 2

-- o.17 0.27

8

--

I 2N

54.4

4

39

5

41 '4

5

l tin

9 1 .O

i6

75

7

48"5

0

17 N

17

I~

-

8N 19N

.38 1o

9 6

-

20 34

2

5

12

22

2oN 24N

- "

0.72

(5

2.{52

14 29 7

* F r e q u e n c y o f t h e s u r v i v i n g fraction. ** S u r v i v i n g fraction. T r e a t m e n t s b y_ I'k: a n d [tN().a give o~8 ° o a n d -8 °, o s u r v i v a l r e s p e c t i \ e h. . / TABI,E

IV

FREQUENCY ('

IO 7) OK R E T R O M U T \ T I O N OF I().N \VITH VARIOUS T R E A T M E N T

Trealme~zt

Survival ( % )

Conlrol*

Treated

UV-ravs HN(),a X-rays 1)iepoxybutane Meth yl-bis(/~-chloroeth yl) aln ine

38 78 33 38.5 22. 5

9i 53 5{~ 53 63

75 4I 00 51 41

* l q g u r e refers t o five s e p a r a t e e x p e r i m e n t s . T h e h i g h e s t vahle could bc t e n t a t i v e l y e x p l a i n e d as a clonal incidence. were

examined

mutants

to determine

were found among

5"uppressors. H N 0 2 cerning the suppressibility 2 are suppressible. Among dicated

and UV-induced bv external

the UV-induced

by an asterisk

detection

the frequency

of forward

mutation

-cersHs h / ~ - r . N o

1 8 . IO 6 p l a t e d c o n i d i a .

mutants

mutations

suppressors.

13 a r e s u p p r e s s i b l e

in t h e t a b l e ) t h e n u m b e r

of the back nmtants

are profoundly

Among

different con-

the HNO,~ nmtants a n d in t h r e e of t h e m

of s u p p r e s s o r s

is s o h i g h t h a t

only (inthe

is i m p o s s i b l e .

DISCUSSION From

t h e s e d a t a i t is e v i d e n t

that

the spectrum

of retronmtation

is d i f f e r e n t

in t h e H N O , 2 - a n d U V - i n d u c e d m u t a n t s . A c c o r d i n g t o F R E E S E 5 a n d H O W A R D AND TESSMAN 6 i n p h a g e s , H N O 2 - i n d u c e d mutants are of transitional type; HNO2 can induce the two possible transitions and t h e r e f o r e t h e r e v e r s i o n o f t h e m u t a t i o n s t h a t i t h a s i n d u c e d . F r o m o u r d a t a it is possible to see that retromutation is n o t i n c r e a s e d b y H N O , a , a n d i n t h e m a j o r i t y o f c a s e s n e i t h e r b y U V . I t is p r o b a b l e t h a t i n A s p e r g i l l u s , H N O , ~ a c t s b y a d i f f e r e n t m e c h anism producing non-transitional mutants. Also in Salmonella DEMEREC 2 found that among the mutations produced by NaNO,a more than 6o°~ were long or short deletions.

Mulation Res., 3 ([960) I45 I51

ANALYSIS OF INDUCED MUTATIONS IN

A. nidulans. I.

151

According to HOWAR3 AnD TESSMAN 7 UV-rays produce a majority of transitional m u t a n t s of t y p e C - 7 T and s o m e which are possibly due to transversion. F r o m our data one can deduce that in m o s t cases U V does not act by inducing unidirectional transition, because the m u t a t i o n s can be reverted by the same UV-rays. Probably m u t a t i o n s can be attributed to bidirectional transition or transversion, but a test with more reference m u t a g e n s is necessary to get more precise information. As shown in Table I I I two m u t a t i o n s induced by HNO2 revert spontaneously with a frequency that is about 20 4 ° times higher than the frequency of forward m u t a t i o n in the whole pfp-cistron. Cases in which the rate of retromutation was exceptionally high were found also by BENZER1 but have not yet been satisfactory explained. \Ve tried to deduce the nature of the mutation by trying to increase the spontaneous rate of reversion by t r e a t m e n t with different mutagens: we were however i n a n e either to modify the spontaneous rate of mutation, or to induce suppressor mutations. These data permit the rejection as highly improbable of the idea that the m u t a t i o n could be due to a transition or a transversion. An explanation based on addition or deletion of a base is obviously unlikely as it cannot explain such a high rate of retromutation, especially during mitosis. Moreover we were not able ~o found in back m u t a n t s of I 6 N any evidence of internal suppression. Further work is therefore necessary to decide if this type of m u t a t i o n can be in s o m e w a y explained on the basis of simple molecular models. ACKNO'WLEDGEMENTS

This research has been partially supported by a grant from Consiglio Nazionale delle Ricerche (research group directed by Prof. GIORGIOTECCE). Thanks are due to Mr. G. CONTI for able technical assistance. Thanks are also due to Simes S.p.A. for a generous gift of HN-2. I~EFI;~RENCES I BENZl~R, S., M u t a t i o n s . In \ ¥ . SHULL, 2~d Conf. on Genetics, J o s i a h M a e y Jr. F o u n d a t i o n , October I6-19, I96O, P r i n c e t o n , N . J . , T h e U n i v e r s i t y of M i c h i g a n Press, 19(o2, p. i41. 2 DEMER1~C, M., M u t a t i o n s . I n W. SHt'LL, and Conf. on Genetics, J o s i a h M a c y Jr. F o u n d a t i o n , O c t o b e r i 6 - i 9 , 196o, P r i n c e t o n , N.J., T h e U n i v e r s i t y of Michigan Press, 1962, p. ~19. 3 DE SERRES, F. J., M u t a g e n e s i s a n d c h r o m o s o m e structure. J. Cellular Comp. Pt(vsiol., 64, Suppl. r (1904) 33-42. 4 FRATEI.LO, B., G. MORPURGO AND G. SERMONTI, I n d u c e d s o m a t i c s e g r e g a t i o n in Aspergillus nidulans. Genetics, 45 (19(m) 785 8oo. 5 FREESE, I=.., Molecular m e c h a n i s m of m u t a t i o n s . Molecular (;e~etics, P a r t t, A c a d e m i c Press, N e w York, I9(~3, p. 2o7-269. (0 HOWARD, B. D. AND 1. TESSMAN, Identification of the altered bases in n l u t a t e d single s t r a n d e d I)NA. 1. In vitro m u t a g e n e s i s b y h y d r o x y l a m i n e , e t h y l m e t h a n e s u l f o n a t e and n i t r o u s acid. J. Molec. Biol., 9 (1964) 352-3637 HOWARD, B. D. AND I. TESS.XIAN, Identification of the altered bases in m u t a t e d single s t r a n d e d D N A . 111. M u t a g e n e s i s b y u l t r a v i o l e t light. J. Molec. Biol., 9 (t9(04) 372--375 . 8 IkAKAR, S. N., F. ZIMMERMAN AND R. P. WAGNER, R e v e r s i o n b e h a v i o n r of i s o l e u c i n e - v a l i n e m u t a n t s of yeast. Mutation Res., I (1964) 381-38(0. 9 NIAGNI, (;., T h e origin of s p o n t a n e o u s m u t a t i o n d u r i n g meiosis. Proc. Natl. Acad. Sci. (U..','.), 5 ° (1964) 975-98o. IO MORPURGO, G., A n e w m e t h o d of e s t i m a t i n g forward m u t a t i o n in fungi: R e s i s t a n c e to 8-azag u a n i n e a n d p - f l u o r o p h e n y l a l a n i n e . Sci. Repls. Ist. Sup. Sanit£, 2 (i962) 9 - I 2 . I I PONTECORVO, G. AND 1?,. tX~.FER, Genetic a n a l y s i s b a s e d on m i t o t i c r e c o m b i n a t i o n . Advan. Genel., 9 (I958) 71 lO412 STRIGINI, P . , O n t h e m e c h a n i s m of s p o n t a n e o u s reversion a n d genetic r e c o m b i n a t i o n in bacteriophage T 4. Genetics, 52 (1965) 759-77(0.

~ViutationRes., 3 (1960) 145- 151