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Mutagenicity of actinomycin D in Neurospora crassa
187
Mutation Research, 3 3 ( 1 9 7 5 ) 1 8 7 - - 1 9 2 © Elsevier Scientific Publishing Company,
Amsterdam
- - P r i n t e d in T h e N e t h e r l a n d s
MUTAGENICITY OF ACTINOMYCIN D IN N E U R O S P O R A C R A S S A *
C.R. FISHER**,
H.V. MALLING t, F.J. DE SERRES t and SARA SNYDER tt
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.) (Received April 4th, 1975) (Revision received July 9th, 1975) (Accepted July 25th, 1975)
Summary Actinomycin D is known to bind to native DNA and is widely used as an antineoplastic agent and inhibitor of DNA-dependent R N A and protein synthesis. We report here the induction by actinomycin D of purple adeninerequiring mutants (ad-3) in wild-type Neurospora crassa. A significant increase in the frequency of ad-3 mutants was evident when the organism was grown vegetatively in the presence of actinomycin D; the mutation frequency was at least 3.6 per 106 survivors. The actinomycin D-induced ad-3 mutants were 29% ad-3A and 71% ad-3B. The ad-3B mutants were classed by complementation pattern as 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. The spectrum of complementation types of the actinomycin D-induced mutants most closely parallels that of mutants induced by ICR-170, known to induce base-pair insertions or deletions, or that of X rayinduced or spontaneous mutants. It is significantly different from spectra seen following treatment with nitrous acid or N-methyl-N'-nitro-N-nitrosoguanidine, agents known to induce mainly base-pair substitutions.
Introduction
Actinomycin D (Act D) is used clinically as an antineoplastic agent and in the laboratory as an inhibitor of DNA-dependent RNA and protein synthesis. It * Research was sponsored by the U.S. Atomic Energy 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 the Union Carbide Corporation. R e s u l t s w e r e r e p o r t e d in abstract form in Genetics, 64 (1970) $20. ** P r e s e n t address: R e s e a r c h and T e c h n i c a l S u p p o r t Division, Energy R e s e a r c h and D e v e l o p m e n t Administration, Post Office Box E, Oak Ridge, Tenn. 37830 (U.S.A.). t P r e s e n t address: Environmental Mutagenesis Branch, National Institute of Environmental Health Sciences, Post Office Box 12233, Research Triangle Park, N.C. 27709 (U.S.A.). 5"5" Oak Ridge Associated Universities Student Trainee from the University of Wyoming, Laramie.
188 is also of interest clinically because it has been reported to be one of the most p o t e n t teratogens known and to produce its effects at times during the developmental cycle not typical of other teratogens [ 17,18 ] ; furthermore, under certain conditions it is a carcinogen [6,7]. Genetically Act D has been observed to cause chromosome abnormalities [12,14] and give rise to nonnuclear genetic variants that persisted for several generations [10]. In other instances it has acted as an antimutagenic agent [13,15] and induced phenotypic reversions [8]. The mechanism of action of Act D was long assumed to be based upon its binding in the minor groove of the DNA helix, but more recent studies have shown that a different mechanism of Act D--DNA interaction exists. Studies now indicate that Act D intercalates between the DNA bases in a manner similar to the acridines [16]. There are consistencies within all of the data which show that there are at least two types of complexing of Act D with DNA, a weak and a strong binding type, and that some types of binding require double-stranded DNA with a suitable base sequence. The latter observation provides an explanation for n o n u n i f o r m [3H]Act D labeling of chromosomes [12] and offers a possible basis for nonrandom phenotypic effects observed previously [8]. We have investigated the ability of Act D to increase the frequency of purple adenine-requiring mutants (ad-3) in Neurospora crassa. Mutations in either of two genes (ad-3A or ad-SB), which code for sequential enzymes in the adenine biosynthetic pathway, result in the intraceltular accumulation of a purple pigment. Mutants at the ad-3A locus do not complement with one another, whereas ad-3B mutants may have nonpolarized or polarized complementation patterns or may be noncomplementing. Mutants recovered from these experiments were characterized by eomplementation testing. The complementation patterns obtained with the Act D-induced mutants were then compared with the eomplementation patterns obtained with mutants produced by mutagens known to induce primarily base-pair substitutions or base-pair insertions or deletions. Materials and Methods All experiments used the standard forward mutation system of De Serres and Kolmark [5] as modified by Brockman and De Serres [2] and wild-type N. crassa strains 74-OR8-1a and 74-OR23-1A or wild-type strains 74-OR223-25A and 74-OR223-23a, derived from a cross of 74-OR8-1a and 74-OR23-1A. Actinomycin D was a gift of Merck, Sharp and Dohme Research Laboratories. Single colony isolates were made from each strain and prepared as a silica gel stock [ 1] immediately prior to the start of the experiments. Vegetative growth was started by placing a few silica gel stock crystals on slants of Fries minimal medium [9] supplemented with 2% sucrose and incubating for 10 days at 25 ° C. Conidia harvested from the slants in sterile 0.9% saline solution, and 1 × 104 conidia were inoculated onto slants containing 10 ml of Fries minimal medium with 27o sucrose and either 0, 0.5 or 5 pg of Act D/ml. In each experiment slants were inoculated with the desired strain, and from this point on each culture (slant) was treated as an individual experiment. The cultures were incubated at 30°C for 10 days before the conidia were harvested in sterile 0.9%
189 saline solution. Conidia from each culture were inoculated into separate 12-liter flasks containing 10 liters of ad-3 forward m u t a t i o n medium to give a final conidial concentration of 1 × 106 conidia per flask. After the flasks were incubated in the dark with aeration for 7 days at 30°C, purple (ad-3) colonies were harvested and inoculated onto adenine-supplemented medium. Conidia from these cultures were plated to provide isolates h o m o k a r y o t i c for the induced ad-3 m u t a t i o n [5]. The h o m o k a r y o t i c mutants were then tested for their genotype and comnlementation pattern by inoculating them pairwise with each of nine standard tester strains [4]. This allowed mutants to be identified as ad-3A, non-complementing ad-3B, ad-3B with polarized complementation; ad-3B with nonpolarized complementation; or ad-3A, ad-3B. These were further classed as nonleaky or leaky, that is, showing residual growth in the absence of adenine supplementation. Results and Discussion In interpreting the results it must be noted that treatment was administered under vegetative growth conditions, not on isolated conidia. There was, therefore, the o p p o r t u n i t y for replication of the m u t a t e d genome before harvest and assay. Due to these circumstances we present the minimum number of mutants for each experiment; that is, the number of mutants clearly distinct as shown by their genotype and/or complementation pattern. For example, if five ad-3A colonies were obtained in a single experiment only one mutational event was scored. The ad-3B mutants were scored in the same manner unless complementation tests showed them to be the product of separate mutational events. The m u t a t i o n frequency reported is therefore u n d o u b t e d l y much lower than the actual m u t a t i o n frequency. Mutation frequencies are shown in Table I. Act D (5 pg/ml) increased the frequency at least 10 times: the average following treatment was 3.6 per 106 survivors. There is no statistical difference in sensitivity between the two mating types. The overall average m u t a t i o n frequency for mating type A was 3.7 per
TABLE I F R E Q U E N C Y O F A C T I N O M Y C I N D - I N D U C E D ad-3 M U T A T I O N S Neurospora strains
Act D (/~g/ml)
Number of experiments
74-OR23-1A
0 0.5 5.0
10 10 10
74-ORS-la
0 0.5 5.0
74-OR223-23a 74-OR223-25A
N u m b e r of survivors X 106
Number of mutants recovered
Minimum number of mutants/106
8.5 7.1 6.7
2 4 20
0.2 0.6 3.0
10 10 10
9.3 8.9 8.7
3 7 20
0.3 0.8 2.3
0 5.0
15 14
10.8 5.6
1 30
0.1 5.4
0 5.0
15 13
13.0 6.2
1 28
0.1 4.5
190
T A B L E II GENOTYPES
AND
COMPLEMENTATION
TYPES
ACTINOMYCIN D - I N D U C E D
AMONG
ad-3
MUTANTS
Neurospora strain
Number of mutants of each eomplementation type
Number of mutants
ad-3 A
ad-3 B NP
74-OR23-1A 74-ORS-1a 74-OR223-23a 74-OR223-25A
P
NC
26 27 30 28
9
4
3
9
7
1
9 5
4 5
2 5
10 10 15 13
111
32
20
11
48
I n o n e e x p e r i m e n t b o t h a l e a k y and a n o n l e a k y , n o n c o m p l e m e n t i n g a d - 3 B m u t a n t w e r e r e c o v e r e d . N P , n o n p o l a r i z e d c o m p l e m e n t i n g ; P, p o l a r i z e d c o m p l e m e n t i n g ; NC, n o n c o m p l e m e n t i n g .
106 survivors, and for mating type a, 3.5 per 106 survivors. The distribution of mutants by genotype and complementation type is shown in Table II. 29% of the mutants were ad-3A and 71% were ad-3B. There were no ad-3A, ad-3B double mutants. Of the ad-3B mutants, the complementation types were: 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. There was a marked absence of leaky mutants -- only 5 in the 111 mutants recovered. It should be evident that if a larger percentage of complementing ad-3B mutants had been induced it would have been possible to distinguish many individual mutants in this group by their complementation pattern. There are, however, low percentages of complementing ad-3B mutants in all of our studies with Act D. We are left, therefore, with primarily two classes of mutants, ad-3A and noncomplementing ad-3B. This decreases the number of mutations definable as independent events by our criteria and undoubtedly makes our mutant frequencies lower than the true value. We have
TABLE III SPECTRUM OF COMPLEMENTATION WITH VARIOUS MUTAGENS
Mutant origin a
Act D ICR-170 X ray SP MNNG NA
T Y P E S A M O N G ad-3B M U T A N T S I N N E U R O S P O R A
INDUCED
Percentage of each complementation type NP
P
NC
25 9 10 9 81 60
14 14 15 29 1 12
61 77 75 62 18 28
a ICR-170, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]amino propylamino)acridine dihydrochloride; M N N G , N - m e t h y l - N ~ - n i t r o - N - n i t r o s o g u a n i d i n e ; N A = n i t r o u s acidi S P = s p o n t a n e o u s . NP = nonpolarized complementing, P = polarized complementing, NC = noncomplementing.
191
not attempted to further subdivide these groups by additional genetic tests. The cumulative results for different mutagens show that the type of mutational alteration induced is reflected in the frequency of leakiness and the spectra of ad-3B complementation types recovered. A comparison of the present data with those obtained with mutants from other mutagenic origins (Table III) indicates that Act D produces a spectrum most closely resembling that of mutants induced by ICR-170, a mutagen known to induce primarily base-pair additions and deletions, or that of X ray-induced or spontaneous mutants. The spectrum is very different from that of nitrous acid and N-methylN'-nitro-N-nitrosoguanidine, agents known to induce primarily base-pair substitutions. The observed characteristics of the mutants are in agreement with those expected if Act D intercalates into DNA in the manner of ~the acridines; however, other types of mutational processes may also be occurring, as indicated by the nonpolarized complementing ad-3B mutants obtained. It is of interest that in the complementation testing the majority of mutants classed here as nonpolarized complementing did not show vigorous complementation. Their growth was very sparse, and they were normally heavily pigmented when complementing with other ad-3B mutants. Their complementation with ad-3A mutants was normal. No further testing of these atypical ad-3B complementing mutants has been undertaken at this time.
Acknowledgements The authors extend their appreciation to the excellent technical staff of the Fungal Genetics Group of the Biology Division, ORNL.
References 1 B r o c k m a n , H.E. a n d F.J. De Serres, Viability of N e u r o s p o r a c o n i d i a f r o m s t o c k c u l t u r e s on silica gel, N e u r o s p o r a N e w s l e t t e r , 1 ( 1 9 6 2 ) 8. 2 B r o c k m a n , H . E . a n d F.J. De Serres, I n d u c t i o n of ad-3 m u t a n t s of N e u r o s p o r a crassa b y 2 - a m i n o p u r i n e , G e n e t i c s , 48 ( 1 9 6 3 ) 5 9 7 - - 6 0 4 . 3 B r o c k m a n , H . E . , F.J. De Serres a n d W.E. B a r n e t t , A n a l y s i s o f ad-3 m u t a n t s i n d u c e d b y n i t r o u s acid in a h e t e r o k a r y o n of N e u r o s p o r a crassa, M u t a t i o n Res., 7 ( 1 9 6 9 ) 3 0 7 - - 3 1 4 . 4 De Serres, F.J., H.E. B r o c k m a n , W.E. B a r n e t t a n d H . G . K ~ l m a r k , M u t a g e n s p e c i f i c i t y in N e u r o s p o r a crassa, M u t a t i o n Res., 12 ( 1 9 7 1 ) 1 2 9 - - 1 4 2 . 5 De Serres, F.J. a n d H . G . K ¢ l m a r k , A d i r e c t m e t h o d for d e t e r m i n a t i o n o f f o r w a r d - m u t a t i o n r a t e s in N e u r o s p o r a crassa, N a t u r e , 1 8 2 ( 1 9 5 8 ) 1 2 4 9 - - 1 2 5 0 . 6 Di Paolo, J.A. a n d J. Elis, E f f e c t o f a c t i n o m y c i n D a n d u r e t h a n o n successive litters a n d g e n e r a t i o n s o f mice, Teratology, 3 (1970) 53--58. 7 Di Paolo, J . A . a n d P. K o t i n , T e r a t o g e n e s i s - o n c o g e n e s i s : a s t u d y of possible r e l a t i o n s h i p s , A r c h . P a t h o l . , 81 ( 1 9 6 6 ) 3 - - 2 3 . 8 Fisher, C.R., A c t i n o m y c i n D - i n d u c e d p h e n o t y p i c r e v e r s i o n in N e u r o s p o r a crassa, G e n e t i c s , 57 ( 1 9 6 7 ) 189--194. 9 H o r o w i t z , N.H. a n d G.W. Beadle, A m i c r o b i o l o g i c a l m e t h o d f o r t h e d e t e r m i n a t i o n o f c h o l i n e b y u s e o f a m u t a n t o f N e u r o s p o r a , J. B i o l . C h e m . , 1 5 0 ( 1 9 4 3 ) 3 2 5 - - 3 3 3 . 10 K a l i n i n a , L . V . , H e r e d i t a r y c h a n g e s in t h e a m o e b a c a u s e d b y a c t i n o m y c i n D, T s i t o l o g i y a , 10 ( 1 9 6 8 ) 1588--1597. 11 Mailing, H . V . a n d F.J. De Series, C h e m i c a l l y i n d u c e d p o i n t m u t a t i o n s a n d c h r o m o s o m e d e l e t i o n s in t h e ad-3 r e g i o n of a t w o - c o m p o n e n t h e t e r o k a r y o n o f N e u r o s p o r a crassa, Oral p r e s e n t a t i o n a t Proc. X t h Int. C o n g r . Microbiol., M e x i c o C i t y , 1 9 7 0 . 12 Miles, C.P., L a b e l i n g a n d o t h e r e f f e c t s o f a c t i n o m y c i n D o n h u m a n c h r o m o s o m e s , Proc. Nail. A c a d . Sci. ( U S A ) , 6 5 ( 1 9 7 0 ) 5 8 5 - - 5 9 2 .
192
13 M u k h e r j e e , R . , A c t i n o m y c i n D e f f e c t s o n t h e f r e q u e n c y o f r a d i a t i o n - i n d u c e d m u t a t i o n s in D r o s o p h i l a , G e n e t i c s , 51 ( 1 9 6 5 ) 3 6 3 - - 3 6 7 . 1 4 O s t e r t a g , W. a n d W. K e r s t e n , T h e a c t i o n o f p r o f l a v i n a n d a c t i n o m y c i n D in c a u s i n g c h r o m a t i d b r e a k a g e in h u m a n cells, E x p . Cell Res., 3 9 ( 1 9 6 5 ) 2 9 6 - - 3 0 1 . 1 5 Pugiisi, P.P., A n t i m u t a g e n i c a c t i v i t y o f a c t i n o m y c i n D a n d b a s i c f u c h s i n e , Mol. G e n . Genet.~ 1 0 3 (1968) 248--252. 16 S o b e l l , H . M . , T h e s t e r e o c h e m i s t r y o f A c t i n o m y c i n b i n d i n g t o D N A , C a n c e r C h e m o t h e r . R e p . , 5 8 (1974) 101--116. 17 T u c h m a n n - D u p l e s s i s , H. a n d L. M e r c i e r - P a r o t , T h e t e r a t o g e n i c a c t i o n o f t h e a n t i b i o t i c a c t i n o m y c i n D, Ciba Found. Syrup. on Congenital Malformations, (1960) 115--133. 1 8 W i l s o n , J . G . , E m b r y o l o g i c a l c o n s i d e r a t i o n s in t e r a t o l o g y , A n n . N.Y. A c a d . Sci., 1 2 3 ( 1 9 6 5 ) 2 1 9 - - 2 2 7 .
Mutation Research, 3 3 ( 1 9 7 5 ) 1 8 7 - - 1 9 2 © Elsevier Scientific Publishing Company,
Amsterdam
- - P r i n t e d in T h e N e t h e r l a n d s
MUTAGENICITY OF ACTINOMYCIN D IN N E U R O S P O R A C R A S S A *
C.R. FISHER**,
H.V. MALLING t, F.J. DE SERRES t and SARA SNYDER tt
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 (U.S.A.) (Received April 4th, 1975) (Revision received July 9th, 1975) (Accepted July 25th, 1975)
Summary Actinomycin D is known to bind to native DNA and is widely used as an antineoplastic agent and inhibitor of DNA-dependent R N A and protein synthesis. We report here the induction by actinomycin D of purple adeninerequiring mutants (ad-3) in wild-type Neurospora crassa. A significant increase in the frequency of ad-3 mutants was evident when the organism was grown vegetatively in the presence of actinomycin D; the mutation frequency was at least 3.6 per 106 survivors. The actinomycin D-induced ad-3 mutants were 29% ad-3A and 71% ad-3B. The ad-3B mutants were classed by complementation pattern as 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. The spectrum of complementation types of the actinomycin D-induced mutants most closely parallels that of mutants induced by ICR-170, known to induce base-pair insertions or deletions, or that of X rayinduced or spontaneous mutants. It is significantly different from spectra seen following treatment with nitrous acid or N-methyl-N'-nitro-N-nitrosoguanidine, agents known to induce mainly base-pair substitutions.
Introduction
Actinomycin D (Act D) is used clinically as an antineoplastic agent and in the laboratory as an inhibitor of DNA-dependent RNA and protein synthesis. It * Research was sponsored by the U.S. Atomic Energy 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 the Union Carbide Corporation. R e s u l t s w e r e r e p o r t e d in abstract form in Genetics, 64 (1970) $20. ** P r e s e n t address: R e s e a r c h and T e c h n i c a l S u p p o r t Division, Energy R e s e a r c h and D e v e l o p m e n t Administration, Post Office Box E, Oak Ridge, Tenn. 37830 (U.S.A.). t P r e s e n t address: Environmental Mutagenesis Branch, National Institute of Environmental Health Sciences, Post Office Box 12233, Research Triangle Park, N.C. 27709 (U.S.A.). 5"5" Oak Ridge Associated Universities Student Trainee from the University of Wyoming, Laramie.
188 is also of interest clinically because it has been reported to be one of the most p o t e n t teratogens known and to produce its effects at times during the developmental cycle not typical of other teratogens [ 17,18 ] ; furthermore, under certain conditions it is a carcinogen [6,7]. Genetically Act D has been observed to cause chromosome abnormalities [12,14] and give rise to nonnuclear genetic variants that persisted for several generations [10]. In other instances it has acted as an antimutagenic agent [13,15] and induced phenotypic reversions [8]. The mechanism of action of Act D was long assumed to be based upon its binding in the minor groove of the DNA helix, but more recent studies have shown that a different mechanism of Act D--DNA interaction exists. Studies now indicate that Act D intercalates between the DNA bases in a manner similar to the acridines [16]. There are consistencies within all of the data which show that there are at least two types of complexing of Act D with DNA, a weak and a strong binding type, and that some types of binding require double-stranded DNA with a suitable base sequence. The latter observation provides an explanation for n o n u n i f o r m [3H]Act D labeling of chromosomes [12] and offers a possible basis for nonrandom phenotypic effects observed previously [8]. We have investigated the ability of Act D to increase the frequency of purple adenine-requiring mutants (ad-3) in Neurospora crassa. Mutations in either of two genes (ad-3A or ad-SB), which code for sequential enzymes in the adenine biosynthetic pathway, result in the intraceltular accumulation of a purple pigment. Mutants at the ad-3A locus do not complement with one another, whereas ad-3B mutants may have nonpolarized or polarized complementation patterns or may be noncomplementing. Mutants recovered from these experiments were characterized by eomplementation testing. The complementation patterns obtained with the Act D-induced mutants were then compared with the eomplementation patterns obtained with mutants produced by mutagens known to induce primarily base-pair substitutions or base-pair insertions or deletions. Materials and Methods All experiments used the standard forward mutation system of De Serres and Kolmark [5] as modified by Brockman and De Serres [2] and wild-type N. crassa strains 74-OR8-1a and 74-OR23-1A or wild-type strains 74-OR223-25A and 74-OR223-23a, derived from a cross of 74-OR8-1a and 74-OR23-1A. Actinomycin D was a gift of Merck, Sharp and Dohme Research Laboratories. Single colony isolates were made from each strain and prepared as a silica gel stock [ 1] immediately prior to the start of the experiments. Vegetative growth was started by placing a few silica gel stock crystals on slants of Fries minimal medium [9] supplemented with 2% sucrose and incubating for 10 days at 25 ° C. Conidia harvested from the slants in sterile 0.9% saline solution, and 1 × 104 conidia were inoculated onto slants containing 10 ml of Fries minimal medium with 27o sucrose and either 0, 0.5 or 5 pg of Act D/ml. In each experiment slants were inoculated with the desired strain, and from this point on each culture (slant) was treated as an individual experiment. The cultures were incubated at 30°C for 10 days before the conidia were harvested in sterile 0.9%
189 saline solution. Conidia from each culture were inoculated into separate 12-liter flasks containing 10 liters of ad-3 forward m u t a t i o n medium to give a final conidial concentration of 1 × 106 conidia per flask. After the flasks were incubated in the dark with aeration for 7 days at 30°C, purple (ad-3) colonies were harvested and inoculated onto adenine-supplemented medium. Conidia from these cultures were plated to provide isolates h o m o k a r y o t i c for the induced ad-3 m u t a t i o n [5]. The h o m o k a r y o t i c mutants were then tested for their genotype and comnlementation pattern by inoculating them pairwise with each of nine standard tester strains [4]. This allowed mutants to be identified as ad-3A, non-complementing ad-3B, ad-3B with polarized complementation; ad-3B with nonpolarized complementation; or ad-3A, ad-3B. These were further classed as nonleaky or leaky, that is, showing residual growth in the absence of adenine supplementation. Results and Discussion In interpreting the results it must be noted that treatment was administered under vegetative growth conditions, not on isolated conidia. There was, therefore, the o p p o r t u n i t y for replication of the m u t a t e d genome before harvest and assay. Due to these circumstances we present the minimum number of mutants for each experiment; that is, the number of mutants clearly distinct as shown by their genotype and/or complementation pattern. For example, if five ad-3A colonies were obtained in a single experiment only one mutational event was scored. The ad-3B mutants were scored in the same manner unless complementation tests showed them to be the product of separate mutational events. The m u t a t i o n frequency reported is therefore u n d o u b t e d l y much lower than the actual m u t a t i o n frequency. Mutation frequencies are shown in Table I. Act D (5 pg/ml) increased the frequency at least 10 times: the average following treatment was 3.6 per 106 survivors. There is no statistical difference in sensitivity between the two mating types. The overall average m u t a t i o n frequency for mating type A was 3.7 per
TABLE I F R E Q U E N C Y O F A C T I N O M Y C I N D - I N D U C E D ad-3 M U T A T I O N S Neurospora strains
Act D (/~g/ml)
Number of experiments
74-OR23-1A
0 0.5 5.0
10 10 10
74-ORS-la
0 0.5 5.0
74-OR223-23a 74-OR223-25A
N u m b e r of survivors X 106
Number of mutants recovered
Minimum number of mutants/106
8.5 7.1 6.7
2 4 20
0.2 0.6 3.0
10 10 10
9.3 8.9 8.7
3 7 20
0.3 0.8 2.3
0 5.0
15 14
10.8 5.6
1 30
0.1 5.4
0 5.0
15 13
13.0 6.2
1 28
0.1 4.5
190
T A B L E II GENOTYPES
AND
COMPLEMENTATION
TYPES
ACTINOMYCIN D - I N D U C E D
AMONG
ad-3
MUTANTS
Neurospora strain
Number of mutants of each eomplementation type
Number of mutants
ad-3 A
ad-3 B NP
74-OR23-1A 74-ORS-1a 74-OR223-23a 74-OR223-25A
P
NC
26 27 30 28
9
4
3
9
7
1
9 5
4 5
2 5
10 10 15 13
111
32
20
11
48
I n o n e e x p e r i m e n t b o t h a l e a k y and a n o n l e a k y , n o n c o m p l e m e n t i n g a d - 3 B m u t a n t w e r e r e c o v e r e d . N P , n o n p o l a r i z e d c o m p l e m e n t i n g ; P, p o l a r i z e d c o m p l e m e n t i n g ; NC, n o n c o m p l e m e n t i n g .
106 survivors, and for mating type a, 3.5 per 106 survivors. The distribution of mutants by genotype and complementation type is shown in Table II. 29% of the mutants were ad-3A and 71% were ad-3B. There were no ad-3A, ad-3B double mutants. Of the ad-3B mutants, the complementation types were: 25% nonpolarized complementing; 14% polarized complementing; and 61% noncomplementing. There was a marked absence of leaky mutants -- only 5 in the 111 mutants recovered. It should be evident that if a larger percentage of complementing ad-3B mutants had been induced it would have been possible to distinguish many individual mutants in this group by their complementation pattern. There are, however, low percentages of complementing ad-3B mutants in all of our studies with Act D. We are left, therefore, with primarily two classes of mutants, ad-3A and noncomplementing ad-3B. This decreases the number of mutations definable as independent events by our criteria and undoubtedly makes our mutant frequencies lower than the true value. We have
TABLE III SPECTRUM OF COMPLEMENTATION WITH VARIOUS MUTAGENS
Mutant origin a
Act D ICR-170 X ray SP MNNG NA
T Y P E S A M O N G ad-3B M U T A N T S I N N E U R O S P O R A
INDUCED
Percentage of each complementation type NP
P
NC
25 9 10 9 81 60
14 14 15 29 1 12
61 77 75 62 18 28
a ICR-170, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]amino propylamino)acridine dihydrochloride; M N N G , N - m e t h y l - N ~ - n i t r o - N - n i t r o s o g u a n i d i n e ; N A = n i t r o u s acidi S P = s p o n t a n e o u s . NP = nonpolarized complementing, P = polarized complementing, NC = noncomplementing.
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not attempted to further subdivide these groups by additional genetic tests. The cumulative results for different mutagens show that the type of mutational alteration induced is reflected in the frequency of leakiness and the spectra of ad-3B complementation types recovered. A comparison of the present data with those obtained with mutants from other mutagenic origins (Table III) indicates that Act D produces a spectrum most closely resembling that of mutants induced by ICR-170, a mutagen known to induce primarily base-pair additions and deletions, or that of X ray-induced or spontaneous mutants. The spectrum is very different from that of nitrous acid and N-methylN'-nitro-N-nitrosoguanidine, agents known to induce primarily base-pair substitutions. The observed characteristics of the mutants are in agreement with those expected if Act D intercalates into DNA in the manner of ~the acridines; however, other types of mutational processes may also be occurring, as indicated by the nonpolarized complementing ad-3B mutants obtained. It is of interest that in the complementation testing the majority of mutants classed here as nonpolarized complementing did not show vigorous complementation. Their growth was very sparse, and they were normally heavily pigmented when complementing with other ad-3B mutants. Their complementation with ad-3A mutants was normal. No further testing of these atypical ad-3B complementing mutants has been undertaken at this time.
Acknowledgements The authors extend their appreciation to the excellent technical staff of the Fungal Genetics Group of the Biology Division, ORNL.
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