47 MUTATION I~ESEARCH

t t YDROXYLAMI NE AS A MUTAGENIC AGENT FOR NEUROSPORA

CRA SSA
H. V. MALLI NG
Biolog 3' Division, Oak Ridge National Laboralor),, Oak l?idge, Tenn. ( U. S. d. )
(Received June 2ist, I000)
SUMMARY
The mutagenicity of hydroxylamine (HA) has been tested in 8 different adenine-
requiring mut ant s (ad-3B) of Neuro@ora crassa: 4 mut ant s of this tester set revert
after t reat ment with nitrous acid and ethyl methanesulfonate indicating that t hey
revert by a base-pair substitution, 2 mut ant s revert only after treatment with ICR-
17o indicating t hat they revert by a base-pair insertion or deletion, and 2 mut ant s
revert only spontaneously. HA induced reversions in 3 of the 4 mut ant s which revert
by base-pair substitution and in none of the others. The specificity of the action of
HA on the mut ant s 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
t hat 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 t hat 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 at t empt 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, adeni ne t hymi ne. I,;MS, ethvl mct hanesul fonat c. GC, guani ne cytosine.
HA, hydroxyl ami ne. HMC, hy~troxymethylcytos{ne. ICR-f7o, met hoxy 6-chloro 0-(3-et hyl -2-
chl oret hyl ]-ami nopropyl ami no)acri di ne dihydrochloride. NA, ni t rous acid. SP, spont aneous
reversion frequency in t he unt r eat ed series.
~Iulalion lees., 3 (I966) 47 476
CHEMICAL MUTAGENICITY IN N. crassa 471
less specific mut agens at t he mol ecul ar level by means of t est s for specific revert i bi l i t y.
Test s for t he i nduct i on of reversi ons in mut a nt s resul t i ng f r om known genet i c al t era-
t i ons is a much si mpl er met hod t o charact eri ze t he genet i c effects of mut agens t han
any known f or war d- mut at i on t echni que in Neurospora 6,19. The char act er i zat i on is
l i mi t ed b y t he ar r ay of genetic al t erat i ons in each mut a nt compri si ng a t est er set.
However , b y selecting mut a nt s r ever t i ng b y mos t common genet i c al t erat i ons, it is
possible t o obt ai n a first appr oxi mat i on of t he spect r um of genetic al t erat i ons pr oduced
b y a gi ven mut agen.
I n t hi s paper a t est er set of ad-3B mut a nt s consisting of 4 mut a nt s which r ever t
onl y by base- pai r subst i t ut i on, 2 mut a nt s which r ever t onl y by base- pai r i nsert i on or
deletion, and 2 mut a nt s which r ever t onl y spont aneousl y were each t r eat ed wi t h HA
and t hen screened t o det ect reversi ons t o wild t ype. The resul t s of such t est s show
cl earl y t hat in Neurospora HA produces reversi on onl y in t hose st rai ns which r ever t
by base- pai r subst i t ut i on. The specificity of t he act i on of HA on t he mut a nt s in t he
pr esent t est er set is consi st ent wi t h t he hypot hesi s t hat t he pr edomi nant class of
genet i c al t er at i on i nduced by HA in Neurospora is base- pai r t r ansi t i on f r om GC t o AT.
MATERIALS AND METHODS
(a) Strains
The mut a nt s wi t h t he prefix 2-17 were all i nduced by ni t rous acid in Neurospora
wi l d- t ype st rai n 74 A ( DE SERRES, BROCKMAN, BARNETT AND KOLMARK, in pr epar a-
tion). Mut ant No. 5-4-1 is of spont aneous origin (BROCKMAN, unpubl i shed). The mu-
t ant s have been i sol at ed b y t he di rect met hod 3.
(b) Preparation of the culture
I n all exper i ment s t he mut ageni c t r eat ment was carri ed out on suspensi ons of
conidia har vest ed f r om i 25- ml Er l enmeyer flasks cont ai ni ng 20 ml of gl ycerol com-
pl et e medi um 10 (IO m! glycerol per liter i nst ead of 20 ml ) +a de ni ne sul fat e (25 rag/l).
The flasks were i nocul at ed and t hen i ncubat ed for I day at 3 o and t hen for 6- 9 days
at 25 . The conidia were har vest ed b y first shaki ng t he cul t ures wi t h glass beads
(4 mm di amet er) t o br eak up t he chai ns of coni di a; t hey were t hen suspended in saline
(o.9~o), filtered t hr ough a pl at i num st rai ner, washed twice by cent ri fugat i on, and
t hen r esuspended in saline. Ad-3 mut a nt s differ f r om each ot her wi t h respect t o
devel opment of t he purpl e pi gment in t he mycel i um and conidia when grown on
gl ycerol compl et e, but by addi ng 250 mg adeni ne sul fat e per liter purpl e pi gment
accumul at i on is essent i al l y el i mi nat ed. The densi t y of t he conidial suspensi ons were
measur ed on a col ori met er (Spectronic 20, Bausch and Lomb, Rochest er, New York)
at t he peak absor pt i ons of 750 m#.
(c) Treatments
All t r eat ment s were carri ed out wi t h conidial suspensi ons ( ~ 2 IoT/ml) in
Er l enmeyer flasks on a r ot a r y shaker in wat er bat h at 25 t o keep t he conidia in sus-
pensi on duri ng t he t r eat ment . 5 mi n before quenchi ng t he conidia were cent ri fuged
and t he s uper nat ant was decant ed. At t he t i me of quenchi ng af t er t r e a t me nt wi t h
ei t her NA, EMS, or ICR-I7O, t he conidia were resuspended in a sal t sol ut i on of
Fri es' mi ni mal 1 adj ust ed t o p H 8 wi t h NaOH. Thi s procedure was r epeat ed twice.
Mutation Res., 3 (1966) 470-476
472 t t . V. MAI A. I NG
The s a l t s ol ut i on of FRIES' mi n i ma l a d j u s t e d t o p H ha s been f ound t o s t o p t he r eac-
t i on b e t we e n cel l s a n d a l k y l a t i n g c o mp o u n d s a n d NA i mme d i a t e l y (MALLI~'C., un-
publ i s he d) .
(d) NA trealmenls
The c oni di a wer e s u s p e n d e d in 0. 05 M s o d i u m a c e t a t e buf f er at p H 4-5. One
v o l u me of f r e s hl y p r e p a r e d o. o2 M s o d i u m n i t r a t e s ol ut i on was a d d e d t o 3 vol ume s
of coni di a. The f i nal c o n c e n t r a t i o n was 0. oo 5 M NaNO, , , a n d t he t r e a t me n t was
q u e n c h e d as de s c r i be d a b o v e 4o mi n a f t e r t he s t a r t of t he t r e a t me n t .
(e) E MS treaOnent
The c oni di a wer e s u s p e n d e d i n a 0. 067 M p h o s p h a t e buf f er a t p H 7.0. The
t r e a t me n t was s t a r t e d b y a d d i n g e nough EMS t o b r i n g t he f i nal c o n c e n t r a t i o n up t o
o. I M; t he t r e a t me n t was q u e n c h e d 30o r ai n l a t e r .
(f ) I CR- z7o treatment
I CR- I 7 o is t he code n u mb e r a s s i gne d t o me t hoxy4) - c hl or o- 9- ( 3- [ e t hyl - 2-
c h l o r e t h y l i l - a mi n o p r o p y l a mi 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 I t . ,J. CREECH a n d co-
wor ke r s a t t he I n s t i t u t e f or Cancer Re s e a r c h, Ph i l a d e l p h i a . The c oni di a wer e sus-
p e n d e d in a o. o67 M p h o s p h a t e buf f er a t p H 7. o. The t r e a t me n t was s t a r t e d b y a d d i n g
I vol . of a f r e s hl y p r e p a r e d s ol ut i on of I CR- I 7O (25 rag/1 H20) t o 49 vol s. of t he
c oni di a s us pe ns i on whi c h ga ve a f i nal c o n c e n t r a t i o n of l O. 58/ ~M. The t r e a t me n t was
q u e n c h e d as de s c r i be d a b o v e 13o r ai n a f t e r s t a r t of t he t r e a t me n t . The t r e a t me n t
a n d ot he r ma n i p u l a t i o n s i nvol vi ng I CR- I 7O a nd c oni di a wer e p e r f o r me d u n d e r r e d
l i ght t o e l i mi n a t e t he p h o t o d y n a mi c ef f ect s a s s oc i a t e d wi t h t he a c r i di ne ringVa, ~a.
Pl a t e s wer e al so i n c u b a t e d in t he d a r k f or at l eas t 24 h t o al l ow suf f i ci ent t i me f or t he
c o n i d i a t o gi ve r i se t o s ma l l col oni es.
(g) HA treatmenl
Bef or e t he HA t r e a t me n t , t he c oni di a wer e 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 t i me s i n t he HA r e a c t i o n mi x t u r e of STRACK, FREESE AND FREESE 17,
gi vi ng a f i nal HA c o n c e n t r a t i o n of i M. 5 mi n bef or e t he t r e a t me n t was que nc he d,
t h e c oni di a wer e c e nt r i f uge d a n d d e c a n t e d , a n d at t he que nc hi ng t i me (i.e., 3oo r ai n
a f t e r t he s t a r t of t he t r e a t me n t ) t he c oni di a wer e r e s u s p e n d e d i n 3 M NaCl . Thi s
wa s hi ng p r o c e d u r e was r e p e a t e d t wi ce a n d t he n t he c oni di a wer e s u s p e n d e d in t h e
s a l t s ol ut i on of Fr i e s ' mi n i ma l me d i u m t a d j u s t e d t o p H 8.
(h) Plating medium
To e s t i ma t e t he v i a b i l i t y of t he t r e a t e d a n d u n t r e a t e d coni di a, t h e y wer e p l a t e d
in WESTERC.AARO'S mi n i ma l TM s u p p l e me n t e d wi t h s or bos e (15 g/ l ) , gl ucose (0. 5 g/ l ) ,
f r uc t os e (0.5 g/ l ) , c a s a mi n o a c i d (200 r ag/ l ) , a v i t a mi n s ol ut i on as in g l y c e r o l - c o mp l e t e
( I ml/1), a n d a de ni ne s ul f a t e (25 r ag/ l ) .
To e s t i ma t e t he n u mb e r of r e v e r t a n t s t he c oni di a wer e p l a t e d i n t he s a me s ub-
s t r a t e us e d for s cor i ng s u r v i v o r s b u t s u p p l e me n t e d wi t h 0.2 rag/ 1 a de ni ne s ul f a t e i n-
s t e a d of 25 rag/1 a de ni ne s ul f at e.
I n t he p l a t e s t o d e t e r mi n e s u r v i v a l t he d e n s i t y of t he c oni di a was 5 IO c oni di a
pe r ml s u b s t r a t e i n a t o t a l v o l u me of a b o u t IOO ml . Fo r s cor i ng of r e v e r t a n t s a f t e r
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 IO e conidia
per ml and 2 lO 5 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 t hat the following two ratios belong to the same popu-
lation :
Total population (surviving after t he treatment)
(i)
Total population (unt reat ed)+t ot al population (surviving after treatment)
Total number of revertants in the t reat ed population
Total number of revert ant s in t he unt reat ed popul at i on+t ot al number of (2)
revert ant s in the t reat ed population
A probability lower than 5% indicates a significant difference between the
number of reversions in the control and the treated series.
RESULTSANDDISCUSSION
(a) Suppressors
Identification of the genetic alteration in individual mut ant s 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 t hat suppressors occurring outside
the ad-3A or ad-3B locus are rare or t hat t hey do not occur.
The influence of the suppressors on the identification of the genetic alteration
in individual ad-3 mut ant s 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 mut ant s is being made to provide further information on this point. How-
ever, since the revertants of the mut ant s 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 t hat 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 mut ant s
(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 t hat mutants which revert after treatment with ICR-
Mutation Res., 3 (I966) 470-476
474 H. V. MALLING
TABLE I
THE SURVI VAL PERCENTAGE AND THE REVERSI ON FREQUENCI ES OF THE TESTER SET AFTER TRI ~AT-
MENT WITH I CR- I 70, NA, EMS AND HA
Reversions per ~o s survivors Mut ant Survival percentage
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- i 7- 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 me a ns t ha t t he r ever s i on f r e que nc y is not s i gni f i cant l y di f f er ent f r om t he s pont a ne ous mut a -
t i on f r e que nc y at t he 5% conf i dence 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) t hat 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 t hat 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 t hat we obtained 8 times as many mut ant s
in the HA-treated series as in the control. We can therefore conclude t hat HA is
i
0 2 4 6
HOURS OF TREATMENT
100 -
80-
60-
~ 40-
>
n~
~ 20-
~ o
\
~ 10 - - - - r - ~
HOURS OF TREATMENT
Fi g. I. The i ncr ease in fol d of t he numbe r of r ever si ons ( M) scor ed af t er HA t r e a t me nt over t he
numbe r of s pont a ne ous mut a t i ons (Mo) pl ot t e d a ga i ns t t he t i me of HA t r e a t me nt .
Fi g. 2. The s ur vi val pe r c e nt a ge pl ot t e d a ga i ns t t he t i me of HA t r e a t me nt .
Mutation Res., 3 (1966) 470- 476
CHEMICAL MUTAGENICITY IN ]~. crassa 475
mut ageni c in Neurospora. The sur vi val was not lower t han 42% even for t he longest
t r eat ment (Fig. 2). I t was found t hat HA induces reversi ons in 3 of t he 4 st rai ns
whi ch r ever t b y base- pai r subst i t ut i ons (Table I). The failure of cert ai n base- pai r
subst i t ut i on mut a nt s t o r ever t wi t h HA can be account ed for if we assume t ha t it
induces pr edomi nant l y t ransi t i ons from GC- AT in Neurospora as it does in phage.
I f t ha t is t he case, t hen we woul d expect t hat cert ai n base- pai r subst i t ut i on mut a nt s
exi st whi ch cannot be r ever t ed b y HA.
(c) The kinetics of induction of reversions
The HA- i nduced reversi on frequencies are not pr opor t i onal t o t i me in Neuros-
pora but follow a second-order ki net i cs (Fig. 3). However , HA- i nduced f or war d and
reverse mut at i on in phage follow fi rst -order kinetics 8,9. Thi s i nconsi st ency could resul t
f r om several mechani sms: ( z ) t hat an i nduced reversi on in Neurospora mus t express
60-
50-
o
40-
0" )
m 30-
_o 20-
]0-
/

/
15 35 4~ ~ "
SQUAREOFHOURSOFTREATMENT(t a)
Fig. 3. Ki net i cs of t he i nduct i ons of r ever si ons by HAi n t he base- pai r s ubs t i t ut i on mut a nt 2- x 7-15.5.
The f r equency of t he r ever si ons per lO 8 sur vi vor s are pl ot t ed agai ns t t he squar e of t he t i me of
HA t r eat ment .
itself in a coni di um, which has an aver age of 2 nuclei, and t he expressi on depends on
t he i nact i vat i on of one of t he 2 nuclei, (2) t hat t he HA t r eat ment will pr omot e a
f ast er penet r at i on of HA i nt o t he cell, or most pr obabl y (3) t hat t he react i on of HA
wi t h cyt osi ne occurs in t wo st eps (for review, see SCHUSTER AND WITTMAN15) and
t ha t t he react i on r at e of t he 2 st eps are more near l y equal in Neurospora t han in phage.
The react i on r at e of cyt osi ne wi t h HA depends on p H; i ncreasi ng pH gives
decreasi ng r at es of react i on 14. Exper i ment s t o i nvest i gat e t he rel at i onshi p bet ween
t he mechani sm of HA mut agenesi s in vi rus and Neurospora b y st udyi ng t he effect of
pH on t he mut at i on r at es are now in progress.
(d) Forward mutations induced by HA
Exper i ment s have been carri ed out t o obt ai n mor e det ai l ed i nf or mat i on on t he
genet i c effects of HA in Neurospora by t r eat ment of a genet i cal l y mar ked bal anced
di kar yon 7. The f or war d- mut at i on f r equency af t er t he 6-h t r eat ment under t he con-
Mut at i on Res., 3 (1966) 470- 476
476 n. v. MALLING
di t i ons de s c r i be d her e gave 600 mut a nt s per IO ~ s ur vi vi ng coni di a. Mut a t i ons obt a i ne d
in t hi s t es t s ys t e m are now in t he pr oces s of bei ng a na l yz e d wi t h r egar d t o ge not ype
(si ngl e or mul t i l ocus mut a t i ons ) , allelic c ompl e me nt a t i on ( per cent age of c ompl e me nt -
i ng mut a nt s as wel l as t he t ype s of c ompl e me nt a t i on pat t er ns ) , a nd speci fi c r ever t i -
bi l i t y af t er t r e a t me nt wi t h NA, EMS, I CR- I 7O or ot he r agent s a nd wi l l be r e por t e d
el sewher e.
ACKNOWLEDGEMENT
I wi sh t o a c knowl e dge gr at ef ul l y Dr. F. J . DE SERRES' val uabl e s ugges t i ons a nd
cooper at i on, t he ai d of Dr. MARVIN I{ASTENBAUM in t he s t at i s t i cal anal ysi s, a nd
Dr. H. J. CREECH a nd co- wor ker s of t he I ns t i t ut e for Cancer Res ear ch, Phi l adel phi a,
for t hei r gi f t of I CR- I 7O. Thi s r es ear ch was s pons or e d j oi nt l y by t he Na t i ona l I ns t i -
t ut e s of He a l t h a nd by t he U. S. At omi c Ene r gy Commi s s i on unde r c ont r a c t wi t h t he
Uni on Car bi de Cor por at i on.
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