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 adenine-

requiring 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 ICR-
17o 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 altera-
tions 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 charac-
terization 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-2-
chlorethyl]-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 altera-
tions 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 prepara-
tion). Mutant No. 5-4-1 is of spontaneous origin (BROCKMAN, unpublished). The mu-
tants 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 com-
plete 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 sus-
pension 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 reac-

tion 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., un-
published).

(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-2-
c 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 co-
workers at the I n s t i t u t e for Cancer Research, Philadelphia. The conidia were sus-
p 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 sub-
s 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 in-
s 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 popu-
lation :
Total population (surviving after the treatment)
(i)
Total population (untreated)+total population (surviving after treatment)
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 (2)
revertants in the treated population

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 muta-
tions. 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. How-
ever, 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 ICR-

Mutation Res., 3 (I966) 470-476

474 H . V . MALLING

TABLE I
THE 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 Survival percentage Reversions per ~o s survivors

No. NA EMS 1CR-I7o HA SP NA EMS ICR-zTO HA

2-17-8 75 77 86 54 I oa o 5 o
2-i7-23 8o 95 61 62 o. 3 o o o o
5-4-1 88 77 68 5° 0.2 o 5 1938 o
2-17-7 5° 69 7° 90 4 o 5 749 o
2-17-61 8o 68 62 60 5 183 198 o 45
2-17-155 93 99 72 62 I 79 377 8 20
2-17-18 61 71 61 64 3 182 IO 91 o
2-17-126 82 95 9° 68 4 136 77 146 8

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 base-
pair 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 rever-
sion 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 ob-
tained 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

~ 40-
60-
\
>

n~

~ 20-

~ 10 ----r-~
0 2 4 6
HOURS OF TREATMENT 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 Neuros-
pora 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-
/
°

50-

40-
0")

/
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 con-
M 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.

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Mutation Res., 3 (1966) 47/)-476