Hydroxylamine as a mutagenic agent for Nuerospora Crassa

47 °

MUTATION I~ESEARCH

t t Y D R O X Y L A M I N E AS A MUTAGENIC AGENT FOR N E U R O S P O R A CRA SSA

H. V. M A L L I N G Biolog3' Division, Oak Ridge National Laboralor),, Oak l?idge, Tenn. ( U . S . d . ) (Received J u n e 2ist, I000)

SUMMARY

The mutagenicity of hydroxylamine (HA) has been tested in 8 different adeninerequiring mutants (ad-3B) of Neuro@ora crassa: 4 mutants of this tester set revert after treatment with nitrous acid and ethyl methanesulfonate indicating that they revert by a base-pair substitution, 2 mutants revert only after treatment with ICR17o indicating that they revert by a base-pair insertion or deletion, and 2 mutants revert only spontaneously. HA induced reversions in 3 of the 4 mutants which revert by base-pair substitution and in none of the others. The specificity of the action of HA on the mutants in the present tester set is consistent with the hypothesis that the predominant class of genetic alterations induced by HA in Neuro@ora is base-pair transitions from guanine-cytosine to adenine thymine. Furthernlore, it was found that the reversion frequency after HA treatment increases in proportion to the square of the treatment time.

INTROI) UCTION

HA induces base-pair substitutions in phage and the type of the genetic alterations is preferentially 9 from GC to AT. The chemical reaction between HA and DNA has been analyzed by FREESE et al. 8 who found that HA reacts only with cytosine and HMC. Treatment of RNA with HA alters both uracil and cytosine; at pH 6.1 the reaction is much faster with cytosine than with uracil, whereas at higher pH the relationship is reversedlL HA has been shown to induce chromosonml damage in mammalian cells 1", but no previous attempt has been successful in identifying the genetic alteration(s) induced by HA in eukaryotes at the molecular level. The characterization of HA-induced mutations in Neurospora is particularly important because it may be as specific in eukaryotes as in phage in producing mutations by base-pair substitutions resulting from GC to AT transitions. Such specificity would make HA especially well-suited for the characterization of the genetic alterations induced by Abbrevi~tions: AT, adenine thymine. I,;MS, ethvl mcthanesulfonatc. GC, guanine cytosine. HA, hydroxylamine. HMC, hy~troxymethylcytos{ne. ICR-f7o, m e t h o x y 6-chloro 0-(3-ethyl-2chlorethyl]-aminopropylamino)acridine dihydrochloride. NA, nitrous acid. SP, spontaneous reversion frequency in the u n t r e a t e d series.

~Iulalion lees., 3 (I966) 47 ° 476

CHEMICAL MUTAGENICITY IN N . crassa

471

less specific mutagens at the molecular level by means of tests for specific revertibility. Tests for the induction of reversions in mutants resulting from known genetic alterations is a much simpler method to characterize the genetic effects of mutagens than any known forward-mutation technique in Neurospora 6,19. The characterization is limited b y the array of genetic alterations in each mutant comprising a tester set. However, b y selecting mutants reverting b y most common genetic alterations, it is possible to obtain a first approximation of the spectrum of genetic alterations produced b y a given mutagen. In this paper a tester set of ad-3B mutants consisting of 4 mutants which revert only by base-pair substitution, 2 mutants which revert only by base-pair insertion or deletion, and 2 mutants which revert only spontaneously were each treated with HA and then screened to detect reversions to wild type. The results of such tests show clearly that in Neurospora HA produces reversion only in those strains which revert by base-pair substitution. The specificity of the action of HA on the mutants in the present tester set is consistent with the hypothesis that the predominant class of genetic alteration induced by HA in Neurospora is base-pair transition from GC to AT. MATERIALS AND METHODS

(a) Strains The mutants with the prefix 2-17 were all induced by nitrous acid in Neurospora wild-type strain 74 A (DE SERRES, BROCKMAN, BARNETT AND KOLMARK, in preparation). Mutant No. 5-4-1 is of spontaneous origin (BROCKMAN, unpublished). The mutants have been isolated b y the direct method 3.

(b) Preparation of the culture In all experiments the mutagenic treatment was carried out on suspensions of conidia harvested from i25-ml Erlenmeyer flasks containing 20 ml of glycerol complete medium 10 (IO m! glycerol per liter instead of 20 m l ) + a d e n i n e sulfate (25 rag/l). The flasks were inoculated and then incubated for I day at 3 °o and then for 6- 9 days at 25 °. The conidia were harvested b y first shaking the cultures with glass beads (4 m m diameter) to break up the chains of conidia; they were then suspended in saline (o.9~o), filtered through a platinum strainer, washed twice by centrifugation, and then resuspended in saline. Ad-3 mutants differ from each other with respect to development of the purple pigment in the mycelium and conidia when grown on glycerol complete, but by adding 250 mg adenine sulfate per liter purple pigment accumulation is essentially eliminated. The density of the conidial suspensions were measured on a colorimeter (Spectronic 20, Bausch and Lomb, Rochester, New York) at the peak absorptions of 750 m#.

(c) Treatments All treatments were carried out with conidial suspensions ( ~ 2 • IoT/ml) in Erlenmeyer flasks on a rotary shaker in waterbath at 25 ° to keep the conidia in suspension during the treatment. 5 min before quenching the conidia were centrifuged and the supernatant was decanted. At the time of quenching after treatment with either NA, EMS, or ICR-I7O, the conidia were resuspended in a salt solution of Fries' minimal 1 adjusted to p H 8 with NaOH. This procedure was repeated twice. Mutation Res., 3 (1966) 470-476

472

tt. V. MAIA.ING

The salt solution of FRIES' m i n i m a l a d j u s t e d to p H has been found to s t o p the reaction between cells a n d a l k y l a t i n g c o m p o u n d s a n d NA i m m e d i a t e l y (MALLI~'C., unpublished).

(d) N A trealmenls The conidia were s u s p e n d e d in 0.05 M sodium a c e t a t e buffer at p H 4-5. One volume of freshly p r e p a r e d o.o2 M sodium n i t r a t e solution was a d d e d to 3 volumes of conidia. The final c o n c e n t r a t i o n was 0.oo 5 M NaNO,,, a n d the t r e a t m e n t was q u e n c h e d as described a b o v e 4o min after the s t a r t of the t r e a t m e n t .

(e) E M S treaOnent The conidia were s u s p e n d e d in a 0.067 M p h o s p h a t e buffer at p H 7.0. The t r e a t m e n t was s t a r t e d b y a d d i n g enough EMS to b r i n g the final c o n c e n t r a t i o n up to o.I M ; the t r e a t m e n t was quenched 30o rain later.

(f) ICR-z7o treatment I C R - I 7 o is the code n u m b e r assigned to methoxy4)-chloro-9-(3-[ethyl-2c h l o r e t h y l i l - a m i n o p r o p y l a m i n o ) a c r i d i n e d i h y d r o c h l o r i d e b y It. ,J. CREECH a n d coworkers at the I n s t i t u t e for Cancer Research, Philadelphia. The conidia were susp e n d e d in a o.o67 M p h o s p h a t e buffer at p H 7.o. The t r e a t m e n t was s t a r t e d b y a d d i n g I vol. of a freshly p r e p a r e d solution of ICR-I7O (25 ° rag/1 H20) to 49 vols. of the conidia suspension which gave a final c o n c e n t r a t i o n of lO.58/~M. The t r e a t m e n t was quenched as described a b o v e 13o rain after s t a r t of the t r e a t m e n t . The t r e a t m e n t a n d other m a n i p u l a t i o n s involving ICR-I7O and conidia were p e r f o r m e d u n d e r red light to eliminate the p h o t o d y n a m i c effects associated with the acridine ringVa, ~a. P l a t e s were also i n c u b a t e d in the d a r k for at least 24 h to allow sufficient t i m e for the c o n i d i a to give rise to small colonies.

(g) H A treatmenl Before the H A t r e a t m e n t , the conidia were s u s p e n d e d in 3 M NaCl a n d t h e n f u r t h e r d i l u t e d 5 times in the H A reaction m i x t u r e of STRACK, FREESE AND FREESE 17, giving a final H A c o n c e n t r a t i o n of i M. 5 min before the t r e a t m e n t was quenched, t h e conidia were centrifuged a n d d e c a n t e d , a n d at the quenching t i m e (i.e., 3oo rain a f t e r the s t a r t of the t r e a t m e n t ) the conidia were r e s u s p e n d e d in 3 M NaCl. This washing p r o c e d u r e was r e p e a t e d twice a n d then the conidia were suspended in t h e salt solution of Fries' m i n i m a l m e d i u m t a d j u s t e d to p H 8.

(h) Plating medium To e s t i m a t e the v i a b i l i t y of the t r e a t e d a n d u n t r e a t e d conidia, t h e y were p l a t e d in WESTERC.AARO'S m i n i m a l TM s u p p l e m e n t e d with sorbose (15 g/l), glucose (0. 5 g/l), fructose (0.5 g/l), casamino acid (200 rag/l), a v i t a m i n solution as in glycerol-complete (I ml/1), a n d adenine sulfate (25 rag/l). To e s t i m a t e the n u m b e r of r e v e r t a n t s the conidia were p l a t e d in the same subs t r a t e used for scoring survivors b u t s u p p l e m e n t e d with 0.2 rag/1 adenine sulfate ins t e a d of 25 rag/1 adenine sulfate. In the plates to d e t e r m i n e s u r v i v a l the d e n s i t y of the conidia was 5 IO conidia per ml s u b s t r a t e in a t o t a l v o l u m e of a b o u t IOO ml. F o r scoring of r e v e r t a n t s after

3/Iutation Res., 3 (I966) 47° 476

CHEMICAL MUTAGENICITY IN

N. crassa

473

NA, EMS, or ICR-I7O treatment, the conidia were plated to a density of IOe conidia per ml and 2 • lO5 conidia per ml, each ill a total volume of IOO ml. For scoring of the revertants after the HA treatment, the density of the conidia was 2 • lO 5 per ml in a total volume of 500 ml.

(i) Statistical test The test for significance is done according to BIRNBAUM(see ref. 2, p. 261). The number of revertants is considered as having a Poisson distribution. The probability is calculated by assuming that the following two ratios belong to the same population : Total population (surviving after the treatment) Total population (untreated)+total population (surviving after treatment)

(i)

Total number of revertants in the treated population Total number of revertants in the untreated p o p u l a t i o n + t o t a l number of revertants in the treated population

(2)

A probability lower than 5% indicates a significant difference between the number of reversions in the control and the treated series. RESULTS AND DISCUSSION

(a) Suppressors Identification of the genetic alteration in individual mutants by determining the specificity in the induction of reversions after treatment with different agents will be distorted by the occurrence of suppressor mutations along with reverse mutations. Revertants from 20 different mutants induced in the ad-3 loci (refs. 5, I I , and BARNETT, unpublished) have been analyzed for occurrence of extragenic suppressors, and none was found. We may therefore assume that suppressors occurring outside the ad-3A or ad-3B locus are rare or that they do not occur. The influence of the suppressors on the identification of the genetic alteration in individual ad-3 mutants will be discussed further in another paper (MALLING AND DE SERRES,in preparation, 1966 ). In addition a detailed analysis of induced reversions of ad-3B mutants is being made to provide further information on this point. However, since the revertants of the mutants in the present tester set appear early and are in the main part not accumulating the purple pigments usually done by ad-3 mutants, it seems likely that the frequency of extragenic suppressors in the present analysis is low.

(b) Genetic alteration induced by HA The mutagenicity of HA has been studied by determining whether there is any specificity in its action activity for inducing reversions with a tester set of 8 mutants (Table I). On the basis of present data, 4 of these strains appear to revert only by base-pair substitutions (revertible by NA and EMS), 2 strains appear to revert by a base-pair insertion and/or deletion (only revertible by ICR-I7O), and 2 strains revert only spontaneously. ICR-ITO is a monofunctional acridine mustard gas; forward mutations induced by ICR-I7O in Neurospora crassa seem mainly to be base-pair insertions or deletions 4. It is therefore likely to assume that mutants which revert after treatment with ICRMutation Res., 3 (I966) 470-476

474

H . V . MALLING

TABLE THE

I

SURVIVAL

PERCENTAGE

AND THE

REVERSION

FREQUENCIES

OF THE TESTER

SET AFTER

TRI~AT-

MENT WITH I C R - I 7 0 , N A , E M S AND H A

Mutant No.

Survival percentage NA EMS 1CR-I7o

HA

Reversions per ~o s survivors SP NA EMS ICR-zTO

HA

2-17-8 2-i7-23 5-4-1 2-17-7 2-17-61 2-17-155 2-17-18 2-17-126

75 8o 88 5° 8o 93 61 82

54 62 5° 90 60 62 64 68

I o. 3 0.2 4 5 I 3 4

o o o o 45 20 o 8

77 95 77 69 68 99 71 95

86 61 68 7° 62 72 61 9°

oa o o o 183 79 182 136

o o 5 5 198 377 IO 77

5 o 1938 749 o 8 91 146

a o m e a n s t h a t t h e r e v e r s i o n f r e q u e n c y is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h e s p o n t a n e o u s m u t a t i o n f r e q u e n c y a t t h e 5 % c o n f i d e n c e level.

17o and not after treatment with NA and EMS, which predominantly induces basepair substitutions, revert by a base-pair insertion or deletion. However, it was found (Table I) that mutants which revert by base-pair substitutions (2-17-155 , 2-17-18 , 2-17-126 ) also revert after treatment with ICR-I7O. This can be accounted for by the fact that ICR-I7O is a monofunctional mustard and therefore able to alkylate, and the result of an alkylation in the DNA is usually a base-pair substitution. The reversion frequencies after HA treatment are low compared with the reversion frequencies after treatment with NA and EMS at comparable survival frequencies. Strain 2-17-155 has been treated with HA at pH 6.2 for various lengths of time. A direct expression to show that HA has a mutagenic effect in Neurospora can be obtained by calculating the ratio (M/Mo) where (M) = the number of reversions per IO~ conidia after the HA treatment and (M0)=the number of spontaneous reversions per lO s conidia. In Fig. I it can be seen that we obtained 8 times as many mutants in the HA-treated series as in the control. We can therefore conclude that HA is 100 80-

i

°

°~ o

\

60-

~ 40> n~

~

0

2 4 6 HOURS OF TREATMENT

~

20-

10

----r-~ HOURS OF TREATMENT

Fig. I. T h e i n c r e a s e in fold of t h e n u m b e r of r e v e r s i o n s ( M ) s c o r e d a f t e r H A t r e a t m e n t o v e r t h e n u m b e r of s p o n t a n e o u s m u t a t i o n s (Mo) p l o t t e d a g a i n s t t h e t i m e of H A t r e a t m e n t . Fig. 2. T h e s u r v i v a l p e r c e n t a g e p l o t t e d a g a i n s t t h e t i m e of H A t r e a t m e n t .

Mutation Res., 3 (1966) 4 7 0 - 4 7 6

CHEMICAL MUTAGENICITY IN ]~. c r a s s a

475

mutagenic in Neurospora. The survival was not lower than 42% even for the longest treatment (Fig. 2). It was found that HA induces reversions in 3 of the 4 strains which revert b y base-pair substitutions (Table I). The failure of certain base-pair substitution mutants to revert with HA can be accounted for if we assume that it induces predominantly transitions from GC-AT in Neurospora as it does in phage. If that is the case, then we would expect that certain base-pair substitution mutants exist which cannot be reverted b y HA.

(c) The kinetics of induction of reversions The HA-induced reversion frequencies are not proportional to time in Neurospora but follow a second-order kinetics (Fig. 3). However, HA-induced forward and reverse mutation in phage follow first-order kinetics 8,9. This inconsistency could result from several mechanisms: ( z ) that an induced reversion in Neurospora must express 60-

/ °

50o

400")

m 30_o 20-

]0-

/ 15

£

35

4~

~"

SQUARE OF HOURSOF TREATMENT (t a)

Fig. 3. K i n e t i c s of t h e i n d u c t i o n s of r e v e r s i o n s b y H A i n t h e b a s e - p a i r s u b s t i t u t i o n m u t a n t 2- x 7-15.5. The f r e q u e n c y of t h e r e v e r s i o n s per lO 8 s u r v i v o r s are p l o t t e d a g a i n s t t h e s q u a r e of t h e t i m e of HA treatment.

itself in a conidium, which has an average of 2 nuclei, and the expression depends on the inactivation of one of the 2 nuclei, (2) that the HA treatment will promote a faster penetration of HA into the cell, or most probably (3) that the reaction of HA with cytosine occurs in two steps (for review, see SCHUSTER AND WITTMAN15) and that the reaction rate of the 2 steps are more nearly equal in Neurospora than in phage. The reaction rate of cytosine with HA depends on p H ; increasing pH gives decreasing rates of reaction 14. Experiments to investigate the relationship between the mechanism of HA mutagenesis in virus and Neurospora b y studying the effect of pH on the mutation rates are now in progress.

(d) Forward mutations induced by HA Experiments have been carried out to obtain more detailed information on the genetic effects of HA in Neurospora by treatment of a genetically marked balanced dikaryon 7. The forward-mutation frequency after the 6-h treatment under the conM u t a t i o n Res., 3 (1966) 4 7 0 - 4 7 6

476

n.v.

MALLING

d i t i o n s d e s c r i b e d h e r e g a v e 600 m u t a n t s p e r IO ~ s u r v i v i n g c o n i d i a . M u t a t i o n s o b t a i n e d in t h i s t e s t s y s t e m are n o w in t h e p r o c e s s of b e i n g a n a l y z e d w i t h r e g a r d t o g e n o t y p e (single o r m u l t i l o c u s m u t a t i o n s ) , allelic c o m p l e m e n t a t i o n ( p e r c e n t a g e of c o m p l e m e n t i n g m u t a n t s as well as t h e t y p e s of c o m p l e m e n t a t i o n p a t t e r n s ) , a n d specific r e v e r t i b i l i t y a f t e r t r e a t m e n t w i t h N A , E M S , I C R - I 7 O or o t h e r a g e n t s a n d will b e r e p o r t e d elsewhere. ACKNOWLEDGEMENT I w i s h t o a c k n o w l e d g e g r a t e f u l l y Dr. F. J. DE SERRES' v a l u a b l e s u g g e s t i o n s a n d c o o p e r a t i o n , t h e a i d of Dr. MARVIN I{ASTENBAUM in t h e s t a t i s t i c a l a n a l y s i s , a n d Dr. H . J . CREECH a n d c o - w o r k e r s of t h e I n s t i t u t e for C a n c e r R e s e a r c h , P h i l a d e l p h i a , for t h e i r g i f t of I C R - I 7 O . T h i s r e s e a r c h w a s s p o n s o r e d j o i n t l y b y t h e N a t i o n a l I n s t i t u t e s of H e a l t h a n d b y t h e U . S . A t o m i c E n e r g y C o m m i s s i o n u n d e r c o n t r a c t w i t h t h e Union Carbide Corporation. REFERENCES I BEADLE, G. W. AND E. L. TATUM, Neurospora, I1. Methods of producing and detecting muta-

tions concerned with nutritional requirement, Am. J. Bota.ny, 32 11945) 678-686. 2 BIRNBAUM A., Statistical methods for Poisson processes and exponential populations, ./-. Am. Statist. Assoc., 49 (1954) 254. 3 BROCKMAN, H. E. AND F. j. DE SERRES, Induction of ad-3 mutants of Neurospora crassa by 2-aminopurine, Genetics, 48 (1963) 597-6o4. 4 BROCKMAN H. E. AND W. GOBEN, Mutagenicity of a monofunctional alkylating agent derivative of acridine in Neurospora, Science, I47 11965) 75o-751. 5 I)F SERRES F. J., Studies with purple adenine mutants in Neurospora crassa, 11 t. Reversion of X ray-induced mutants, Genetics, 43 (1958) 187-2°6. 6 Dv SERRES F. j. AND H. G. KOLMARK, A direct method for determination of forward-mutation rates in Ne~rospora crassa, Nature, 182 (I958) 1249-125o. 7 DE SERRES F. J. AND R. S. OSTFRBIND, Estimation of the relative frequencies of X rayinduced viable and recessive lethal mutations in the ad-3 region of Neurospora cvassa, (;e~zetics, 47 11962) 793-7968 t?REESE, E., g. BAUTZ AND E. B. FREESE, The chemical and mutagenic specificity of hydroxylamine, Prec. Natl. Acad. Sci. (U.S.), 47 11961) 845. 9 FREESE, E., E. BAUTZ-FREESEAND E. BAUTZ, Hydroxylauline as a mutagenic an(! inactivating agent, J. 3foi. Biol., 3 11961) i33 -143. t o HOROWITZ, N. }t., Methionine synthesis in Neurospora: The isolation of cystathionine, J. Biol. Chem., I7I 11947) 255 264. 11 tkOLMARK, (). AND N. H GILES, Comparative studies on nlonoepoxides as inducers of reverse mutation in Neurospora, Genetics, 4° 11955) 89o-9o2. i2 NAKA[, S. AND T. SACKI, Induction of mutation by photodynanlie action m Eseherichia cell, Genetical Res., 5 (1964) 158. 13 RITCHIF;, O. A., Mutagenicity with light and proflavine in phage T 4, Genel. Res., 5 (I964) 168. 14 SCHUSTER, H., The reaction of tobacco mosaic virus ribonucleic acid with hydroxylamine, J. J}Iol. Biol., 3 (I961) 447-455. 15 SCHUSTVR,H. aND G.-G. WlTTMA.~N, The inactivating and nlutagenic action of hydroxylamine on tobacco mosaic virus ribonucleic acid, Virology, 19 (I963) 421-43 o. i 0 SOMERS, C. AND T. C. HSU, Chromosome damage induced by hydroxylamine in mammalian cells, Prec. Na~l. Acad. Sci. (U.S.), 48 (I962~ 937. I 7 STRACK, H . B., t . ]:~. FREESE AND g . FREESE, Comparison of mutation and inactivation rates induced in bacteriophage and transforming DNA by various nlutagens, 2afutation Res., i 11964) iO 2 I .

18 XYESTERGAARD,M. AND H. K. MITCHELl., Neurospora, V. A synthetic medium favoring sexual reproduction, Am. J. Botany, 34 (1947) 573-578. I9 WOOD\VARD, V. ~V., I. R. DE Zt';EUW AND A. M. SRB, The separation and isolation of particular biochemical mutants of Neurospora by differential germination of conidia, followed by filtration and selective plating, Proc. Natl. Acad. Sci (U.S.), 4° (1954) 192-2oo. Mutation Res., 3 (1966) 47/)-476