2.- mutagénesis

8/11/2019 2.- Mutagnesis

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47 MUTATION I~ESEARCH

t tYDROXYLAMINE AS A MUTAGENIC AGENT FOR NEUROSPORA

CRA SSA

H. V. MALLING

Biolog3 Division, Oak Ridge National Laboralor),, Oak l?idge,

Tenn.

(U.S.d .)

Received June 2ist,

I000

SUMMARY

The mutagenici ty of hydroxylamine (HA) has been tes ted 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 mut ant s 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 preferential ly 9 from GC to AT. The chemical reaction between H A 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

mammal ian cells 1 , but no previous a tt empt has been successful in identi fying 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 mutati ons by base-pair

subst itut ions resulting from GC to AT transi tions. Such specificity would make HA

especially well-suited for the characterization of the genetic alterations induced by

Abbrevi~ti ons: AT, adenine thy min e. I,;MS, ethvl m ctha nes ulfo natc . GC, guani ne cytosine.

HA, hydroxy lamine. HMC, hy~troxymethylcytos{ne. ICR-f7o, meth oxy 6-chloro 0- 3-ethyl-2-

chlorethyl]-aminopropylamino)acridine dihydrochloride. NA, nitrous acid. SP, spontaneous

reversion frequency in the untreated series.

~Iulalion lees., 3

I966) 47 476


2/7

C H E M IC A L M U T A G E N I C I T Y IN

N. crassa 471

l e ss s pe c i fi c m u t a g e n s a t t h e m o l e c u l a r l e v e l b y m e a n s o f t e s t s f o r s pe c i fi c r e v e r t i b i l i t y .

T e s t s f o r th e i n d u c t i o n o f r e v e r s io n s in m u t a n t s r e s u l ti n g f r o m k n o w n g e n e t ic a l t e r a -

t i on s i s a m u c h s i m p l e r m e t h o d t o c h a r a c t e r i z e t h e g e n e t i c e f fe c ts o f m u t a g e n s t h a n

a n y k n o w n f o r w a r d - m u t a t i o n t e c h n iq u e i n

Ne urosp ora 6,19.

T h e c h a r a c t e r i z a t i o n i s

l i m i t e d b y t h e a r r a y o f g e n e ti c a l t e r a t io n s i n e a c h m u t a n t c o m p r i s i n g a t e s t e r s e t .

H o w e v e r , b y s e l e c ti n g m u t a n t s r e v e r t i n g b y m o s t c o m m o n g e n e ti c a l te r a t i o n s, i t is

p o s si b le t o o b t a i n a f ir s t a p p r o x i m a t i o n o f t h e s p e c t r u m o f g e n e ti c a l t e r a t i o n s p r o d u c e d

b y a g i v e n m u t a g e n .

I n t h i s p a p e r a t e s t e r s e t o f

ad-3B

m u t a n t s c o n s is t in g o f 4 m u t a n t s w h i c h r e v e r t

o n l y b y b a s e - p a i r s u b s t i t u ti o n , 2 m u t a n t s w h i c h r e v e r t o n l y b y b a s e - p a i r i n s e r ti o n o r

d e l et io n , a n d 2 m u t a n t s w h i c h r e v e r t o n l y s p o n t a n e o u s l y w e r e e a c h t r e a t e d w i t h H A

a n d t h e n s c r e e n e d t o d e t e c t r e v e r s i o n s t o w i l d t y p e . T h e r e s u l t s o f s u c h t e s t s s h o w

c l e a r l y t h a t i n Neurospora H A p r o d u c e s r e v e r s i on o n l y in t h o s e s t r a in s w h i c h r e v e r t

b y b a s e - p a i r s u b s t i tu t i o n . T h e s p e c if i ci ty o f t h e a c t i o n o f H A o n t h e m u t a n t s i n th e

p r e s e n t t e s t e r s e t is c on s i s te n t w i t h t h e h y p o t h e s i s t h a t t h e p r e d o m i n a n t c la s s o f

g e n e t ic a l t e r a t i o n i n d u c e d b y H A i n

Neurospora

i s b a s e - p a i r t r a n s i t i o n f r o m G C t o A T .

M A T E R I A L S A N D M E T H O D S

a) Strains

T h e m u t a n t s w i t h t h e p r e f i x 2- 17 w e r e a ll i n d u c e d b y n i t r o u s a c i d i n Neurospora

w i l d - t y p e s t r a i n 7 4 A D E S E R R E S , B R O C K M A N , BARNETT AND KOLMARK i n p r e p a r a -

t i o n ) . M u t a n t N o . 5 - 4 - 1 i s o f s p o n t a n e o u s o r i g i n (B RO CK M AN , u n p u b l i s h e d ) . T h e m u -

t a n t s h a v e b e e n i s o l a te d b y t h e d i r ec t m e t h o d 3.

b) Prepa ration of the culture

I n a l l e x p e r i m e n t s t h e m u t a g e n i c t r e a t m e n t w a s c a r r i e d o u t on s u sp e n s io n s o f

c o n i di a h a r v e s t e d f r o m i 2 5 - m l E r l e n m e y e r f la s k s c o n t a i n in g 2 0 m l o f g l y c e ro l co m -

p l e t e m e d i u m 10 ( IO m g l y c e r o l p e r l i t e r i n s t e a d o f 2 0 m l ) + a d e n i n e s u l f a t e (2 5 r a g / l) .

T h e f la s k s w e r e i n o c u l a t e d a n d t h e n i n c u b a t e d f o r I d a y a t 3 o a n d t h e n f o r 6 - 9 d a y s

a t 2 5 . T h e c o n i d i a w e r e h a r v e s t e d b y f i rs t s h a k i n g t h e c u l t u r e s w i t h g l a s s b e a d s

(4 m m d i a m e t e r ) t o b r e a k u p t h e c h a i n s o f c o n i d ia ; t h e y w e r e t h e n s u s p e n d e d i n s a li n e

( o. 9~ o ), f i lt e r e d t h r o u g h a p l a t i n u m s t r a i n e r , w a s h e d t w i c e b y c e n t r i f u g a t i o n , a n d

t h e n r e s u s p e n d e d i n s a l i n e . Ad-3 m u t a n t s d i ff e r f r o m e a c h o t h e r w i t h r e s p e c t to

d e v e l o p m e n t o f t h e p u r p l e p i g m e n t i n t h e m y c e l i u m a n d c o n id i a w h e n g r o w n o n

g l y c e r o l c o m p l e t e , b u t b y a d d i n g 2 5 0 m g a d e n i n e s u l f a t e p e r l i t e r p u r p l e p i g m e n t

a c c u m u l a t i o n i s e s s e n t i a l l y e l i m i n a t e d . T h e d e n s i t y o f t h e c o n i d ia l s u s p e n s i o n s w e r e

m e a s u r e d o n a c o lo r i m e t e r (S p e c tr o n ic 2 0, B a u s c h a n d L o m b , R o c h e s t e r , N e w Y o r k )

a t t h e p e a k a b s o r p t i o n s o f 75 0 m # .

c) Treatments

A l l t r e a t m e n t s w e r e c a r r i e d o u t w i t h c o n i d i a l s u s p e n s i o n s ( ~ 2 I o T / m l ) i n

E r l e n m e y e r f l a s ks o n a r o t a r y s h a k e r i n w a t e r b a t h a t 25 t o k e e p t h e c o n i d i a i n s u s-

p e n s i on d u r i n g t h e t r e a t m e n t . 5 m i n b e f o r e q u e n c h i n g t h e c o n i d i a w e r e c e n t r if u g e d

a n d t h e s u p e r n a t a n t w a s d e c a n te d . A t th e t i m e o f q u e n c h i n g a f t e r t r e a t m e n t w i t h

e i t h e r N A , E M S , o r I C R - I 7 O , t h e c o n i d i a w e r e r e s u s p e n d e d i n a s a l t s o l u t i o n o f

F r i e s ' m i n i m a l 1 a d j u s t e d t o p H 8 w i t h N a O H . T h i s p r o c e d u r e w a s r e p e a t e d t w i ce .

Mutation Res.

3 1 9 6 6 ) 4 7 0 - 4 7 6


3/7

47

t t . V . M A I A . I N G

T h e s a l t s o l u t i o n o f F R I ES m i n i m a l a d j u s t e d t o p H h a s b e e n f o u n d t o s t o p t h e re a c -

t i o n b e t w e e n c e l ls 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 N A i m m e d i a t e l y (M ALLI~ C ., u n -

p u b l i s h e d ) .

d ) N A t r e a l m e n l s

T h e c o n i d i a w e re s u s p e n d e d i n 0 .0 5 M s o d i u m a c e t a t e b u f f e r a t p H 4 -5 . O n e

v o l u m e o f f r e s h l y p r e p a r e d o . o 2 M s o d i u m n i t r a t e s o l u t i o n w a s a d d e d t o 3 v o l u m e s

o f c o n i d i a . T h e f in a l c o n c e n t r a t i o n w a s 0 . o o 5 M N a N O , , , a n d t h e t r e a t m e n t w a s

q u e n c h e d a s d e s c r i b e d a b o v e 4 o m i n a f t e r t h e s t a r t o f t h e t r e a t m e n t .

e ) E M S tr ea O ne nt

T h e c o n i d i a w e r e s u s p e n d e d i n a 0 .0 6 7 M p h o s p h a t e b u f f e r a t p H 7 .0 . T h e

t r e a t m e n t w a s s t a r t e d b y a d d i n g e n o u g h E M S t o b r i n g th e f in a l c o n c e n t r a t i o n u p t o

o . I M ; t h e t r e a t m e n t w a s q u e n c h e d 3 0 o r a in l a t e r .

f ) I C R - z 7 o t r e a t m e n t

I C R - I 7 o i s t h e c o d e n u m b e r a s s i g n e d t o m e t h o x y 4 ) - c h l o r o - 9 - ( 3 - [ e t h y l - 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 I t . ,J. C R E E CH a n d c o -

w o r k e r s a t t h e I n s t i t u t e f o r 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 . T h e c o n i d i a w e re s us -

p e n d e d i n a o .o 6 7

M

p h o s p h a t e b u f f e r a t p H 7 .o . T h e t r e a t m e n t w a s s t a r t e d b y a d d i n g

I v o l . o f a f r e s h l y p r e p a r e d s o l u t i o n o f I C R - I 7 O (2 5 r ag /1 H 2 0 ) t o 4 9 v o l s. o f t h e

c o n i d i a s u s p e n s i o n w h i c h g a v e a f i n a l c o n c e n t r a t i o n o f lO . 5 8 / ~ M . T h e t r e a t m e n t w a s

q u e n c h e d a s d e s c r ib e d a b o v e 1 3o ra in a f t e r s t a r t o f t h e t r e a t m e n t . T h e t r e a t m e n t

a n d o t h e r m a n i p u l a t i o n s i n v o l v i n g I C R - I7 O a n d c o n i d i a w e r e p e r f o r m e d u n d e r r e d

l i g h t t o e l i m i n a t e t h e p h o t o d y n a m i c e f f ec t s a s s o c i a t e d w i t h t h e a c r i d i n e ring Va, ~a.

P l a t e s w e r e a l s o i n c u b a t e d i n t h e d a r k f o r a t l e a s t 2 4 h t o a l l o w s u ff i c ie n t t i m e f o r t h e

c o n i d i a t o g i v e r i se t o s m a l l c o lo n i e s .

g ) H A t r e a t m e n l

B e f o re t h e H A t r e a t m e n t , t h e c o n i d i a w e r e s u s p e n d e d in 3 M N a C l a n d t h e n

f u r t h e r d i l u t e d 5 t i m e s i n t h e H A r e a c t i o n m i x t u r e o f ST RA CK , F R E ES E AND F R E ES E

17

g i v i n g a f in a l H A c o n c e n t r a t i o n o f i M . 5 m i n b e f o r e th e t r e a t m e n t w a s q u e n c h e d ,

t h e c o n i d i a w e r e ce n t r i f u g e d a n d d e c a n t e d , a n d a t t h e q u e n c h i n g t i m e

i.e.,

3 o o r a i n

a f t e r t h e s t a r t o f t h e t r e a t m e n t ) t h e c o n i d i a w e re r e s u s p e n d e d i n 3 M N a C l . T h i s

w a s h i n g p r o c e d u r e w a s r e p e a t e d t w i c e a n d t h e n t h e c o n i d i a w e r e s u s p e n d e d i n t h e

s a l t s o l u t i o n o f F r i e s m i n i m a l m e d i u m t a d j u s t e d t o p H 8.

h ) P l a t in g m e d i u m

T o e s t i m a t e t h e v i a b i l i t y o f t h e t r e a t e d a n d u n t r e a t e d c o n id i a , t h e y w e r e p l a t e d

i n W E S TE R C.A A R O S m i n i m a l T s u p p l e m e n t e d w i t h s o r b o s e (1 5 g / l) , g l u c o s e (0. 5 g / l) ,

f r u c t o s e ( 0 . 5 g / l ) , c a s a m i n o a c i d ( 2 0 0 r a g / l ) , a v i t a m i n s o l u t i o n a s i n g l y c e r o l - c o m p l e t e

( I m l / 1 ) , a n d a d e n i n e s u l f a t e ( 2 5 r a g / l ) .

T o e s t i m a t e t h e n u m b e r o f r e v e r t a n t s t h e c o n i d i a w er e p l a t e d in t h e s a m e s u b -

s t r a t e u s e d f o r sc o r i n g s u r v i v o r s b u t s u p p l e m e n t e d w i t h 0 .2 r a g /1 a d e n i n e s u l f a te i n -

s t e a d o f 25 r ag / 1 a d e n i n e s u l f a t e .

I n t h e p l a t e s t o d e t e r m i n e s u r v i v a l t h e d e n s i t y o f t h e c o n i d i a w a s 5 IO c o n i d i a

p e r m l s u b s t r a t e i n a t o t a l v o l u m e o f a b o u t IOO m l . F o r s c o r in g o f r e v e r t a n t s a f t e r

3/Iutation Res.,

3 (I966) 47 476


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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 to tal volume of IOO ml. For scoring of the

rever tants 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 untre ated)+ total population surviving after treatment)

Total number of revertants in the treated population

Total number of revertants in the untreated popu latio n+to tal number of 2)

revertants in the treated population

A probabi lity lower than 5 indicates a significant difference between the

number of reversions in the control and the treated series.

RESULTS NDDISCUSSION

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, II, 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

n 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 acti vity 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 must ard gas; f orward mutations induced

by ICR-I7O in

Neurospora crassa

seem main ly 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


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474 H.V. MALLING

TABLE I

T H E S UR V I V A L P E R C E N T A G E A N D T H E R E V E R S I O N F R E Q U E N C I E S O F T H E T E S T E R S E T A F T E R T R I ~A T

MENT WITH ICR-I70, NA, EMS AND HA

Reversions per ~o s survivors

ut ant Survival percentage

No. NA EM S 1CR-I7o HA SP NA EM S ICR-zTO HA

2-1 7-8 75 77 86 54 I oa o 5 o

2- i7 -2 3 8o 95 61 62 o. 3 o o o o

5-4-1 88 77 68 5 0.2 o 5 1938 o

2-1 7-7 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

2-1 7-1 8 61 71 61 64 3 182 IO 91 o

2-1 7-1 26 82 95 9 68 4 136 77 146 8

a o m eans tha t t he r ever s ion f r equen cy i s no t s ign i f i can t ly d i f f e r en t f rom the spon tane ous muta -

t ion f r equ ency a t t he 5 conf idence l eve l .

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 mut an ts which revert by base-pair subst itut ions 2-17-155 , 2-17-18

2-17-126 ) also revert after t reat ment with ICR-I7O. This can be accounted for by t he

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 t reat ment are low compared with the rever-

sion frequencies after t reatment 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 treatm ent and M0)= the numb er of spontaneous reversions

per lO s conidia. In Fig. I it can be seen that we obtained 8 times as many mutant s

in the HA-treated series as in the control. We can therefore conclude that HA is

0 2

4

6

HOURS OF TREATMENT

100

80

60

~ 40-

>

n~

~ 20-

~

~ 10 r ~

HOURS OF TREATMENT

Fig. I . The increase in fold of the n umb er of revers ions

M)

s c o r ed 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 o f 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 o f H A t r e a t m e n t .

F ig . 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 o f H A t r e a t m e n t .

Mutation Res.,

3 (1966) 470-476


6/7

CHEMICAL MUTAGENICITY IN ]~.

crassa 475

m u t a g e n i c i n

Neurospora.

T h e s u r v i v a l w a s n o t l ow e r t h a n 4 2 % e v e n f o r t h e l o n g e s t

t r e a t m e n t ( F ig . 2). I t w a s f o u n d t h a t H A i n d u c e s r e v e r s i o n s in 3 o f t h e 4 s t r a i n s

w h i c h r e v e r t b y b a s e - p a i r s u b s t i t u t i o n s ( T a b l e I ) . T h e f a i l u r e o f c e r t a i n 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 s t o r e v e r t w i t h H A c a n b e a c c o u n t e d f o r i f w e a s s u m e t h a t i t

i n d u c es p r e d o m i n a n t l y t r a n s i t io n s f r o m G C - A T in Neurospora a s i t d o e s i n p h a g e .

I f t h a t i s t h e c a s e, t h e n w e w o u l d e x p e c t t h a t c e r t a i n b a s e - p a i r s u b s t i tu t i o n m u t a n t s

e x i s t w h i c h c a n n o t b e r e v e r t e d b y H A .

c) The kinetics of induction of reversions

T h e H A - i n d u c e d r e v e r s io n f r e q ue n c i es a r e n o t p r o p o r t i o n a l t o t i m e i n

Neuros-

pora b u t f o l l ow a s e c o n d - o r d e r k in e t i c s (F ig . 3). H o w e v e r , H A - i n d u c e d f o r w a r d a n d

r e v e r s e m u t a t i o n i n p h a g e f o ll o w f i r s t - o rd e r k i n e t i c s 8,9. T h i s i n c o n s i s t e n c y c o u l d r e s u l t

f r o m s e v e r a l m e c h a n i s m s : ( z ) t h a t a n i n d uc e d r e v e r s i o n in Neurospora m u s t e x p r e s s

60-

50-

40-

0 )

m 30-

_o 20-

]0-

/

15 35 4~ ~

SQUARE OF HOURS OF TREATMENT t a)

Fig. 3. Kinet ics of the indu cti ons of reversions by HAi n the base- pair subst itu tio n mut an t 2- x 7-15.5.

The f requency of the revers ions per lO 8 survivors are plot ted against the square of the t ime of

HA t rea tment .

i t s e lf in a c o n i d i u m , w h i c h h a s a n a v e r a g e o f 2 n u c le i , a n d t h e e x p r e s s i o n d e p e n d s o n

t h e i n a c t i v a t i o n o f o n e o f t h e 2 nu c l ei , 2 ) t h a t t h e H A t r e a t m e n t w ill p r o m o t e a

f a s t e r p e n e t r a t i o n o f H A i n t o t h e c ell, o r m o s t p r o b a b l y

3 )

t h a t t h e r e a c t io n o f H A

w i t h c y t o s i n e o c c u r s i n t w o s t e p s ( f o r r e v i e w , s e e S C H U ST E R A ND W I TT M A N 15 ) a n d

t h a t t h e r e a c t i o n r a t e o f t h e 2 s t e ps a r e m o r e n e a r l y e q u a l i n

Neurospora

t h a n i n p h a g e .

T h e r e a c t i o n r a t e o f c y t o s i n e w i t h H A d e p e n d s o n p H ; i n c re a s i n g p H g i v e s

d e c r e a s i n g r a t e s o f r e a c t i o n 14. E x p e r i m e n t s t o i n v e s t i g a t e t h e r e l a t i o n s h i p b e t w e e n

t h e m e c h a n i s m o f H A m u t a g e n e s i s i n v i r u s a n d

Neurospora

b y s t u d y i n g t h e e f fe c t o f

p H o n th e m u t a t i o n r a t e s a r e n o w in p ro g r es s .

d) Forward mutations induced by H A

E x p e r i m e n t s h a v e b e e n c a r r i e d o u t t o o b t a i n m o r e d e t a i l e d i n f o r m a t i o n o n t h e

g e n e t i c e f f e c ts o f H A i n

Neurospora

b y t r e a t m e n t o f a g e n e ti c a ll y m a r k e d b a l a n c e d

d i k a r y o n 7. T h e f o r w a r d - m u t a t i o n f r e q u e n c y a f t e r t h e 6 - h t r e a t m e n t u n d e r t h e c o n -

Mutat ion Res.

3 1966) 470- 476


7/7

476 n . v . MALLING

di t ions desc r i bed he re gave 600 mutan t s pe r IO ~ su rv iv ing con id ia. Muta t ions ob ta ined

in th i s t e s t sys t em a re now in the p rocess o f be ing ana lyzed wi th rega rd to geno type

(s ing le or mul t i l ocus m uta t ions ) , a ll el ic compl ement a t io n (pe rc en tage o f compl ement -

ing m utan t s a s we l l a s t he types o f compl ement a t io n pa t t e rns ) , and spec i fi c r eve r t i -

b i l i t y a f t e r t r ea tment wi th NA, EMS, ICR-I7O or o the r agen t s and wil l be repo r t ed

e l sewhere .

ACKNOWLEDGEMENT

I wish to acknowle dge g ra t e fu l ly Dr . F .

J

DE SERRES' va luabl e sugg est i ons an d

cooperat ion, the aid of Dr. MARVIN I{ASTENBAUM in the stat ist ical analysis, and

Dr . H. J . CREECH and co-work e rs o f t he Ins t i t u t e fo r Cance r Resea rch , Ph i l ad e lph ia ,

fo r t he i r g i ft o f ICR-I7O. Th i s r e se a rch was sponso red jo in t ly by the N a t iona l Ins t i -

t u t e s o f He 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 ss i o n u n d e r c o n t r a c t w i t h t h e

Un i o n C a r b i d e C o r p o r a t i o n .

REFERENCES

I BEADLE

G. W. AND E. L. TATUM, Neurospora, I1. Methods of producing and detectin g muta-

tions concerned with nutritional requirement, Am. J. Bota.ny, 32 11945) 678-686.

2 BIRNBAUM A., St atistical met hod s for Poisson processes and expon entia l population s, ./-. Am.

Statist. Assoc., 49 (1954) 254.

3 BROCKMAN H. E. AND F. j . DE SERRES, Induc tio n of ad-3 mutants of Neurospora crassa by

2-aminopurine, Genetics, 48 (1963) 597-6o4.

4 BROCKMAN H. E. AND W. GOBEN, Mutagenic ity of a monofun ctiona l alkyla ting agent deriv-

ative 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) 187-26.

6 Dv SERRES F. j . AND H. G. KOLMARK A direct method for determination of forward-muta-

tion rates in Ne~rospora crassa, Nature, 182 (I958) 1249-125o.

7 DE SERRES F. J. AND R. S. OSTFRBIND, Es tima tion of the rela tive frequenc ies of X ray-

induced viable and recessive lethal mu tation s in the ad-3 region of Neurospora cvassa, ;e~zetics,

47 11962) 793-796-

8 t?REESE, E., g. BAUTZ AND E. B. FREESE, The chemical and mut age nic specifi city of hydr oxy l-

amine, Prec. Natl. Acad. Sci. U. S. ), 47 11961) 845.

9 FREESE, E., E. BAUTZ-FREESE AND E. BAUTZ, Hyd rox yla uli ne as a mut agenic an( ina cti vat ing

agent, J. 3foi. Biol., 3 11961) i33 -143.

t o HOROWITZ, N. }t., Met hio nine syn thes is 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, Ind ucti on of mut ati on by pho tody nanl ie action m Eseherichia cell,

Genetical Res., 5 (1964) 158.

13 RITCHIF;, O.

A.

Mutag enicit y with li ght an d prof lavine in phage T 4, Genel. Res., 5 (I964) 168.

14 SCHUSTER, H., The rea ction of tobac co mosaic virus ribonucleic acid wit h hydr oxy lami ne,

J. J}Iol. Biol., 3 (I961) 447-455.

15 SCHUSTVR, H. aND G.-G. WlTTMA.~N, The inact ivati ng a nd nlut agen ic action of hy dro xy lam ine

on tobacco mosaic virus ribonucleic acid, Virology, 19 (I963) 421-43 o.

i0 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 trans forming DNA by various nlutagens, 2afutation Res., i 11964)

iO 2I.

18 XYESTERGAARD,M. AND H. K. MITCHELl., Neurospora, V. A synthetic med ium 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 filtra-

tion a nd selective plating, Proc. Natl. Acad. Sci U. S. ), 4 (1954) 192-2oo.

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

2.- mutagénesis

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