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Chemicals which revert all commonly used Salmonella typhimurium tester strains
183
Mutation Research, 67 (1979) 183--187 © Elsevier/North-Holland Biomedical Press
Short Communication
CHEMICALS WHICH REVERT ALL COMMONLY USED Salmonella typbimurium TESTER STRAINS
R I C H A R D H. McKEE, JAMES G. T O M E T S K O and A N D R E W M. T O M E T S K O Litron Laboratories,Ltd., 1351 Mt. Hope Avenue Rochester, N Y 14620 (U.S.A.)
(Received 8 August 1978) (Revision received 16 January 1979) (Accepted 2 February 1979)
Previous uses of aryl azides to study biological systems have been based on their photosensitive properties. A variety of photosensitive derivatives have been synthesized for study in this laboratory and elsewhere [3]. Among these, 4-fluoro-3-nitrophenyl azide (FNPA) [4] has been shown to mediate photoinactivation of enzymes [10--12] and to photolytically inhibit some types o f amino acid transport in cultured mammalian cells [6]. Some classes of aryl azides can also photosensitize bacteria. In conjunction with investigations into the mechanism of bacterial photosensitivity, FNPA and dinitrophenyl azide (DNPA) were tested for mutagenicity in the Ames Salmonella typhimurium test system. The Ames test involves mutagenic testing of several strains of bacteria which contain mutations in genes for histidine biosynthesis [1]. Since the mutations carried by these strains include a missense and two different frameshift mutations, the test can be used to roughly classify the type of mutagenic event mediated by a particular test chemical. Although a variety of chemicals have been subjected to the Ames test [ 8], none have been described which induce reversions in all the tester strains. Chemicals which revert all the Ames strains could provide internal experimental controls not only to compare experiments within the same laboratory but also to normalize results from different laboratories. We now report results indicating that both FNPA and DNPA induce the reversion of all five of the c o m m o n l y used Salmonella tester strains (TA98, TA100, TA1535, TA1537 and TA1538).
Materials and methods The Salmonella typhimurium test strains used in these experiments were obtained from Dr. Bruce Ames (University of California, Berkeley). Mutagenesis testing with these strains was carried out according to the procedures described for plate-incorporation test studies [1]. The only modification of this procedure is that Vogel--Bonnet E buffer was added to the top agar in the same concentration as in the b o t t o m agar to maintain consistent buffering capacity
184 in all stages of the investigation. Each experimental point was plated in triplicate and reported as the average number of revertants per plate. The E. coli mutant, WP2s, was obtained from Dr. David Spencer (the Pennsylvania State University, University Park, PA). This m u t a n t is excisiondeficient and contains an ochre mutation in a gene for tryptophan biosynthesis which reverts primarily by ochre suppression [2,9]. Mutagenesis testing with this strain was carried o u t by a modification of the Ames' procedure: the top agar was supplemented with 1 pg/ml t r y p t o p h a n to allow limited growth of the bacteria in the presence of various mutagens. In control experiments with diagnostic mutagens, high levels of EMS- and MMS-induced reversion were observed with this plating technique. Preparation of FNPA was carried out as described by Fleet et al. [4]. DNPA was prepared by the m e t h o d of Yoshioka et al. [14]. The structures of these two chemicals are shown in Fig. 1. Both FNPA and DNPA are available from Litron Laboratories, Rochester, NY. Results and discussion In order to compensate for fluctuations within analytical data, a c o m m o n practice is to normalize the results of an u n k n o w n sample relative to the results obtained with a known substance. In this way, data obtained from different laboratories at different times and under slightly different experimental conditions can be readily compared. As in other analytical techniques, variations in results have been observed in mutagen screening experiments from different laboratories. However, normalization of the data for a more direct comparison is limited b y the absence of standard chemicals which will induce mutations in all c o m m o n l y used Salmonella typhimurium tester strains. FNPA and DNPA are two chemicals which induce mutation in all tester strains without metabolic activation, and, therefore, could be employed as standard mutagens. As shown in Table 1, both FNPA and DNPA revert all five of the tester strains in plate-incorporation tests without the addition of liver microsomes. This effect takes place in the dark where interactions with biological targets should be reversible [11,12]. Fig. 2 demonstrates that mutagenic induction is linear with all strains at FNPA concentrations varying from 100 to 400 pg per plate. A similar effect is observed with DNPA (Fig. 3): however high revertant yields are obtained at lower mutagen levels. FNPA yields 0.5 revertants per nanomole with TA100 whereas DNPA yields approximately 10 revertants per nanomole with TA98 and 5 with TA100. The tester strain pair T A 1 0 0 / T A 1 5 3 5 contain a missense mutation in c o m m o n and differ in that TA100 contains the
N:N~N
F FNPA
Fig. 1. S t r u c t u r e s o f F N P A a n d D N P A .
N:N~N
NO 2 DNPA
185
TABLE 1 I N D U C T I O N O F H I S T I D I N E R E V E R S I O N I N S A L M O N E L L A T Y ' P H I M U R I U M T E S T E R S T R A I N S BY FNPA AND DNPA. R e s u l t s l i s t e d are averages o f e x p e r i m e n t s p l a t e d in triplicate FNPA a
TA98
TA100
TA1535
TA1537
TA1538
0 0.1 1.0 10 100
34± 6 30± 5 30± 2 38± 5 235 ± 43
225* 20 2 2 3 ± 41 231 ± 6 243± 30 503 ± 100
35± 4 23 ± 3 24± 8 3 9 ± 12 306 ± 100
8±4 10±3 7 ±4 9±7 57 ± 6
11± 5 10± 1 11 ± 2 17± 2 1 2 9 ± 16
DNPA a
TA98
TAIO0
TA1535
TA1537
TA1538
0.0 0.1 1.0 10 100
27± 4 33± 4 83± 14 483± 59 1369 ± 268
221± 8 271± 19 305± 74 437± 78 1729 ± 514
24±4 27±4 29±3 57±6 70 ± 6
10± 4 10± 5 21± 6 99±17 7 1 5 ± 43
24± 5 23. 4 66± 1O 406± 58 1253 ± 186
(pg)
a C o n c e n t r a t i o n s are given as pg/Plate.
plasmid pKM101 which amplifies the activity of an inducible error-prone repair system in Salmonella, making carrier strains more responsive to a variety of mutagens [13]. The reversion of TA100 and TA1535 by both FNPA and DNPA implies a base-pair substitution mutagenic effect. As described by Ames [1], the TA98/TA1538 pair and TA1537 contain different frameshift mutations and respond to a somewhat different spectrum of mutagens. The reversion of both the TA98/TA1538 pair and the TA1537 strain by both mutagens indicates frameshift-inducing mutagenic activity.
1200.
TA 100 °/"
1000,
1
.1
t"
t. I t./°t
• 800. 0 Q.
•
TA 1535
V
t
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~ 600. 0
/"
Q ~ 400.
/"
I"
I"
/"
t °
v
. .......
.........o
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.,,.." TA 9 8 .o°°'°° ....."" . . " TA 1531
cJ
.A
TA 1537. ~ " D
200
1"
. ......
.~.~"
FNPA (pgrn/plate) Fig. 2. T h e f r e q u e n c y o f his r e v e r s i o n in Salmonella typhirnuriurn is p l o t t e d as a f u n c t i o n o f F N P A c o n centration.
186
1000. Q
38~T
/" • ,1" o,O J ,," , t * ..o*'*
TA 1538
800,
[] -~
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/
~6oo
"E
/
t"
/
/
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/.1".
.
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."
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....
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200
d..:" **11"
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A',i"
IL~'o ~ • ~.
TA 1537 ~.,4.~ . ~ ' ~ . ...- •
, ......
o~.~
r
y
~,
5
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,~ 153s
-r2
,.
2'0
D N P A ( p c j m / p l a te) Fig. 3. The frequency of his reversion in Salmonella typhimurium is plotted as a function of D N P A concentration.
Sodium azide is used in the preparation of both FNPA and DNPA and has been shown to be highly mutagenic with TA100 and TA1535 [8]. Since we can detect sodium azide induced reversion of TA100 and TA1535 at dose levels as low as 0.5 pg per plate, we were concerned with the possibility that the induced reversion of TA100 and TA1535 could be due to the presence of contaminating sodium azide. To rule out this possibility, 25 mg of FNPA was dissolved in ether. The ether layer was passed through a glass wool column, concentrated to dryness, and the residue was redissolved in ether. This process was carried out 5 times. A total of 16.6 mg of material was recovered after the final evaporation. This material was dissolved in dimethyl sulfoxide (1.0 ml), and 25++1 aliquots were tested for mutagenic activity with TA1535. This material was significantly mutagenic (542 + 1 3 1 revertants per plate); approximately a 10-fold enhancement over spontaneous levels (49 + 8). Conversely, when an equal a m o u n t sodium azide was treated in the same way, the final product showed no mutagenic activity (44 + 6 revertants per plate with 100-pl aliquots tested). Consequently, we are confident that the observed basepair substitution mutagenic activity is associated with FNPA rather than a contaminant. To further characterize the mutagenic activity of FNPA and DNPA, both chemicals were tested against an E. coli nonsense m u t a n t , WP2s. WP2s is highly revertable by ochre suppression induced by UV and alkylating agents [5]. Neither FNPA nor DNPA significantly enhances the reversion of WP2s above background levels at drug concentrations ranging from 1 to 400 pg per plate, although some growth inhibition was observed at the highest doses in the DNPA experiments. This result suggests that the types of base-pair substitution events induced by FNPA and DNPA m a y be restricted to specific types or sites. Kleinhofs and Smith [7] have shown that, whereas TA1535 and TA100 are reverted by sodium azide, a variety of other tester alleles including frameshift,
187
ochres, ambers or other missense mutants are not reverted. Consistent with these findings, we observed no enhancement of WP2s reversion in the presence of 40 pg sodium azide per plate. The results with FNPA and DNPA suggest that the base-pair substitution mutagenic events induced by these agents involve sites affected by sodium azide, whereas the frameshift mutagenic activity may be a more general effect. References 1 A m e s , B.W., J . M . M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e H a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 Bridges, B.A., R.E. Dennis and R.J. Munson, Differential induction and repair of ultra-violet damage l e a d i n g t o t r u e r e v e r s i o n s a n d e x t e r n a l s u p p r e s s o r m u t a t i o n s o f a n o c h r e c o d o n i n E s c h e r i c h i a coil B / R W P 2 , G e n e t i c s , 57 ( 1 9 6 7 ) 8 9 7 - - 9 0 8 . 3 D a f f i e r , F., an.~ A.M. T o m e t s k o , A p p l i c a t i o n s o f l i g h t sensitive c h e m i c a l s t o p r o b e b i o l o g i c a l p r o cesses, in: B. W i n s t e i n (Ed.), T h e C h e m i s t r y a n d B i o c h e m i s t r y o f A m i n o a c i d s , P e p t i d e s a n d P r o teins, Vol. 5, M a r c e l D e k k e r , N e w Y o r k , 1 9 7 9 , p p . 3 1 - - 9 3 . 4 F l e e t , G . W . J . , J . R . K n o w l e s a n d 1t.1t. P o r t e r , T h e a n t i b o d y b i n d i n g site, L a b e l l i n g o f a s p e c i f i c a n t i b o d y a g a i n s t t h e p h o t o - p r e c u r s o r o f a n a r y l n i t r e n e , B i o c h e m . J., 1 2 8 ( 1 9 7 2 ) 4 9 9 - - 5 0 8 . 5 G r e e n , M . H . L . , a n d W.J. Muriel, M u t a g e n t e s t i n g u s i n g T R P + r e v e r s i o n o f E s c h e r i c h i a coli, M u t a t i o n Res., 3 8 ( 1 9 7 6 ) 3 - - 3 2 . 6 H a r e , J . D . , G . V . M a r i n e t t i , A.I. Meisler a n d A.M. T o m e t s k o . D i f f e r e n t i a l i n a c t i v a t i o n o f t h e " L " a n d " L y +'' a m i n o a c i d t r a n s p o r t s y s t e m s b y s u l f h y d r y l r e a g e n t a n d a p h o t o a f f i n i t y p r o b e , B i o c h i m . Biophys. Acta, 443 (1976) 485--493. 7 K l e i n h o f s , A., a n d J . A . S m i t h , E f f e c t o f e x c i s i o n r e p a i r o n a z i d e - i n d u c e d m u t a g e n e s i s , M u t a t i o n R e s . , 41 ( 1 9 7 5 ) 2 3 3 - - 2 4 0 . 8 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l i a / m i c r o s o m e t e s t : A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 5 1 3 5 - 5139. 9 O s b o r n e , M., a n d S. P e r s o n , C h a r a c t e r i z a t i o n o f r e v e r t a n t s o f E. coli W U 3 6 - 1 0 a n d W P 2 u s i n g a m b e r mutants and an ochre mutant of bacteriophage T4, Mutation Res., 4 (1967) 504--507. 1 0 S e n i o r , A . E . , a n d A.M. T o m e t s k o , A c t i v a t i o n a n d p h o t o - i n a c t i v a t i o n o f F 1 b y a 4 - f i u o r o - 3 - n i t r o p h e n y l a z i d e , in: E. Q u a g l i a r e l l o , S. P a p p a , F. P a l m i e r i , E.C. S l a t e r a n d N. S i H p r a n d i ( E d s . ) , S y m p o s i u m o n E l e c t r o n T r a n s f e r C h a i n s a n d O x i d a t i v e P h o s p h o r y l a t i o n , Elsevier, A m s t e r d a m , 1 9 7 6 , pp. 155--160. 11 T o m e t s k o , A . M . , a n d J. T u r u l a , E v a l u a t i n g t h e s t a b i l i t y a n d r e a c t i v i t y o f a l i g h t - s e n s i t i v e p r o b e b y enzyme analysis, Photochem. Photobiol., 23 (1976) 579--585. 1 2 T o m e t s k o , A . M . , a n d J. T u r u l a , I n a c t i v a t i o n o f t r y p s i n a n d c h y m o t r y p s i n w i t h a p h o t o s e n s i t i v e p r o b e , I n t . J. P e p t i d e P r o t e i n R e s . , 8 ( 1 9 7 6 ) 3 3 1 - - 3 3 6 . 13 Walker, G.C., Isolation and characterization of mutants of the plasmid pKM101 deficient in their a b i l i t y t o e n h a n c e m u t a g e n e s i s a n d r e p a i r , J. B a c t e r i o l . , 1 3 3 ( 1 9 7 8 ) 1 2 0 3 - - 1 2 1 1 . 1 4 Y o s h i o k a , M., J. L i f t e r , C.L. H e w , C . A . C o n v e r s e , M . Y . K . A r m s t r o n g , W.H. K o n i g s b e r g a n d F . F . Richards, Studies on the combining region of protein 460, a mouse ~A immunoglobulin which binds several h a p t e n s . B i n d i n g a n d r e a c t i v i t y o f t w o t y p e s o f p h o t o a f f i n i t y l a b e l i i n g r e a g e n t s , B i o c h e m i s t r y , 12 (1973) 4679--4685.
Mutation Research, 67 (1979) 183--187 © Elsevier/North-Holland Biomedical Press
Short Communication
CHEMICALS WHICH REVERT ALL COMMONLY USED Salmonella typbimurium TESTER STRAINS
R I C H A R D H. McKEE, JAMES G. T O M E T S K O and A N D R E W M. T O M E T S K O Litron Laboratories,Ltd., 1351 Mt. Hope Avenue Rochester, N Y 14620 (U.S.A.)
(Received 8 August 1978) (Revision received 16 January 1979) (Accepted 2 February 1979)
Previous uses of aryl azides to study biological systems have been based on their photosensitive properties. A variety of photosensitive derivatives have been synthesized for study in this laboratory and elsewhere [3]. Among these, 4-fluoro-3-nitrophenyl azide (FNPA) [4] has been shown to mediate photoinactivation of enzymes [10--12] and to photolytically inhibit some types o f amino acid transport in cultured mammalian cells [6]. Some classes of aryl azides can also photosensitize bacteria. In conjunction with investigations into the mechanism of bacterial photosensitivity, FNPA and dinitrophenyl azide (DNPA) were tested for mutagenicity in the Ames Salmonella typhimurium test system. The Ames test involves mutagenic testing of several strains of bacteria which contain mutations in genes for histidine biosynthesis [1]. Since the mutations carried by these strains include a missense and two different frameshift mutations, the test can be used to roughly classify the type of mutagenic event mediated by a particular test chemical. Although a variety of chemicals have been subjected to the Ames test [ 8], none have been described which induce reversions in all the tester strains. Chemicals which revert all the Ames strains could provide internal experimental controls not only to compare experiments within the same laboratory but also to normalize results from different laboratories. We now report results indicating that both FNPA and DNPA induce the reversion of all five of the c o m m o n l y used Salmonella tester strains (TA98, TA100, TA1535, TA1537 and TA1538).
Materials and methods The Salmonella typhimurium test strains used in these experiments were obtained from Dr. Bruce Ames (University of California, Berkeley). Mutagenesis testing with these strains was carried out according to the procedures described for plate-incorporation test studies [1]. The only modification of this procedure is that Vogel--Bonnet E buffer was added to the top agar in the same concentration as in the b o t t o m agar to maintain consistent buffering capacity
184 in all stages of the investigation. Each experimental point was plated in triplicate and reported as the average number of revertants per plate. The E. coli mutant, WP2s, was obtained from Dr. David Spencer (the Pennsylvania State University, University Park, PA). This m u t a n t is excisiondeficient and contains an ochre mutation in a gene for tryptophan biosynthesis which reverts primarily by ochre suppression [2,9]. Mutagenesis testing with this strain was carried o u t by a modification of the Ames' procedure: the top agar was supplemented with 1 pg/ml t r y p t o p h a n to allow limited growth of the bacteria in the presence of various mutagens. In control experiments with diagnostic mutagens, high levels of EMS- and MMS-induced reversion were observed with this plating technique. Preparation of FNPA was carried out as described by Fleet et al. [4]. DNPA was prepared by the m e t h o d of Yoshioka et al. [14]. The structures of these two chemicals are shown in Fig. 1. Both FNPA and DNPA are available from Litron Laboratories, Rochester, NY. Results and discussion In order to compensate for fluctuations within analytical data, a c o m m o n practice is to normalize the results of an u n k n o w n sample relative to the results obtained with a known substance. In this way, data obtained from different laboratories at different times and under slightly different experimental conditions can be readily compared. As in other analytical techniques, variations in results have been observed in mutagen screening experiments from different laboratories. However, normalization of the data for a more direct comparison is limited b y the absence of standard chemicals which will induce mutations in all c o m m o n l y used Salmonella typhimurium tester strains. FNPA and DNPA are two chemicals which induce mutation in all tester strains without metabolic activation, and, therefore, could be employed as standard mutagens. As shown in Table 1, both FNPA and DNPA revert all five of the tester strains in plate-incorporation tests without the addition of liver microsomes. This effect takes place in the dark where interactions with biological targets should be reversible [11,12]. Fig. 2 demonstrates that mutagenic induction is linear with all strains at FNPA concentrations varying from 100 to 400 pg per plate. A similar effect is observed with DNPA (Fig. 3): however high revertant yields are obtained at lower mutagen levels. FNPA yields 0.5 revertants per nanomole with TA100 whereas DNPA yields approximately 10 revertants per nanomole with TA98 and 5 with TA100. The tester strain pair T A 1 0 0 / T A 1 5 3 5 contain a missense mutation in c o m m o n and differ in that TA100 contains the
N:N~N
F FNPA
Fig. 1. S t r u c t u r e s o f F N P A a n d D N P A .
N:N~N
NO 2 DNPA
185
TABLE 1 I N D U C T I O N O F H I S T I D I N E R E V E R S I O N I N S A L M O N E L L A T Y ' P H I M U R I U M T E S T E R S T R A I N S BY FNPA AND DNPA. R e s u l t s l i s t e d are averages o f e x p e r i m e n t s p l a t e d in triplicate FNPA a
TA98
TA100
TA1535
TA1537
TA1538
0 0.1 1.0 10 100
34± 6 30± 5 30± 2 38± 5 235 ± 43
225* 20 2 2 3 ± 41 231 ± 6 243± 30 503 ± 100
35± 4 23 ± 3 24± 8 3 9 ± 12 306 ± 100
8±4 10±3 7 ±4 9±7 57 ± 6
11± 5 10± 1 11 ± 2 17± 2 1 2 9 ± 16
DNPA a
TA98
TAIO0
TA1535
TA1537
TA1538
0.0 0.1 1.0 10 100
27± 4 33± 4 83± 14 483± 59 1369 ± 268
221± 8 271± 19 305± 74 437± 78 1729 ± 514
24±4 27±4 29±3 57±6 70 ± 6
10± 4 10± 5 21± 6 99±17 7 1 5 ± 43
24± 5 23. 4 66± 1O 406± 58 1253 ± 186
(pg)
a C o n c e n t r a t i o n s are given as pg/Plate.
plasmid pKM101 which amplifies the activity of an inducible error-prone repair system in Salmonella, making carrier strains more responsive to a variety of mutagens [13]. The reversion of TA100 and TA1535 by both FNPA and DNPA implies a base-pair substitution mutagenic effect. As described by Ames [1], the TA98/TA1538 pair and TA1537 contain different frameshift mutations and respond to a somewhat different spectrum of mutagens. The reversion of both the TA98/TA1538 pair and the TA1537 strain by both mutagens indicates frameshift-inducing mutagenic activity.
1200.
TA 100 °/"
1000,
1
.1
t"
t. I t./°t
• 800. 0 Q.
•
TA 1535
V
t
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~ 600. 0
/"
Q ~ 400.
/"
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I"
/"
t °
v
. .......
.........o
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cJ
.A
TA 1537. ~ " D
200
1"
. ......
.~.~"
FNPA (pgrn/plate) Fig. 2. T h e f r e q u e n c y o f his r e v e r s i o n in Salmonella typhirnuriurn is p l o t t e d as a f u n c t i o n o f F N P A c o n centration.
186
1000. Q
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800,
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/
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/
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.
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....
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TA 1537 ~.,4.~ . ~ ' ~ . ...- •
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y
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,~ 153s
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D N P A ( p c j m / p l a te) Fig. 3. The frequency of his reversion in Salmonella typhimurium is plotted as a function of D N P A concentration.
Sodium azide is used in the preparation of both FNPA and DNPA and has been shown to be highly mutagenic with TA100 and TA1535 [8]. Since we can detect sodium azide induced reversion of TA100 and TA1535 at dose levels as low as 0.5 pg per plate, we were concerned with the possibility that the induced reversion of TA100 and TA1535 could be due to the presence of contaminating sodium azide. To rule out this possibility, 25 mg of FNPA was dissolved in ether. The ether layer was passed through a glass wool column, concentrated to dryness, and the residue was redissolved in ether. This process was carried out 5 times. A total of 16.6 mg of material was recovered after the final evaporation. This material was dissolved in dimethyl sulfoxide (1.0 ml), and 25++1 aliquots were tested for mutagenic activity with TA1535. This material was significantly mutagenic (542 + 1 3 1 revertants per plate); approximately a 10-fold enhancement over spontaneous levels (49 + 8). Conversely, when an equal a m o u n t sodium azide was treated in the same way, the final product showed no mutagenic activity (44 + 6 revertants per plate with 100-pl aliquots tested). Consequently, we are confident that the observed basepair substitution mutagenic activity is associated with FNPA rather than a contaminant. To further characterize the mutagenic activity of FNPA and DNPA, both chemicals were tested against an E. coli nonsense m u t a n t , WP2s. WP2s is highly revertable by ochre suppression induced by UV and alkylating agents [5]. Neither FNPA nor DNPA significantly enhances the reversion of WP2s above background levels at drug concentrations ranging from 1 to 400 pg per plate, although some growth inhibition was observed at the highest doses in the DNPA experiments. This result suggests that the types of base-pair substitution events induced by FNPA and DNPA m a y be restricted to specific types or sites. Kleinhofs and Smith [7] have shown that, whereas TA1535 and TA100 are reverted by sodium azide, a variety of other tester alleles including frameshift,
187
ochres, ambers or other missense mutants are not reverted. Consistent with these findings, we observed no enhancement of WP2s reversion in the presence of 40 pg sodium azide per plate. The results with FNPA and DNPA suggest that the base-pair substitution mutagenic events induced by these agents involve sites affected by sodium azide, whereas the frameshift mutagenic activity may be a more general effect. References 1 A m e s , B.W., J . M . M c C a n n a n d E. Y a m a s a k i , M e t h o d s f o r d e t e c t i n g c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e H a / m a m m a l i a n - m i c r o s o m e m u t a g e n i c i t y t e s t , M u t a t i o n R e s . , 31 ( 1 9 7 5 ) 3 4 7 - - 3 6 4 . 2 Bridges, B.A., R.E. Dennis and R.J. Munson, Differential induction and repair of ultra-violet damage l e a d i n g t o t r u e r e v e r s i o n s a n d e x t e r n a l s u p p r e s s o r m u t a t i o n s o f a n o c h r e c o d o n i n E s c h e r i c h i a coil B / R W P 2 , G e n e t i c s , 57 ( 1 9 6 7 ) 8 9 7 - - 9 0 8 . 3 D a f f i e r , F., an.~ A.M. T o m e t s k o , A p p l i c a t i o n s o f l i g h t sensitive c h e m i c a l s t o p r o b e b i o l o g i c a l p r o cesses, in: B. W i n s t e i n (Ed.), T h e C h e m i s t r y a n d B i o c h e m i s t r y o f A m i n o a c i d s , P e p t i d e s a n d P r o teins, Vol. 5, M a r c e l D e k k e r , N e w Y o r k , 1 9 7 9 , p p . 3 1 - - 9 3 . 4 F l e e t , G . W . J . , J . R . K n o w l e s a n d 1t.1t. P o r t e r , T h e a n t i b o d y b i n d i n g site, L a b e l l i n g o f a s p e c i f i c a n t i b o d y a g a i n s t t h e p h o t o - p r e c u r s o r o f a n a r y l n i t r e n e , B i o c h e m . J., 1 2 8 ( 1 9 7 2 ) 4 9 9 - - 5 0 8 . 5 G r e e n , M . H . L . , a n d W.J. Muriel, M u t a g e n t e s t i n g u s i n g T R P + r e v e r s i o n o f E s c h e r i c h i a coli, M u t a t i o n Res., 3 8 ( 1 9 7 6 ) 3 - - 3 2 . 6 H a r e , J . D . , G . V . M a r i n e t t i , A.I. Meisler a n d A.M. T o m e t s k o . D i f f e r e n t i a l i n a c t i v a t i o n o f t h e " L " a n d " L y +'' a m i n o a c i d t r a n s p o r t s y s t e m s b y s u l f h y d r y l r e a g e n t a n d a p h o t o a f f i n i t y p r o b e , B i o c h i m . Biophys. Acta, 443 (1976) 485--493. 7 K l e i n h o f s , A., a n d J . A . S m i t h , E f f e c t o f e x c i s i o n r e p a i r o n a z i d e - i n d u c e d m u t a g e n e s i s , M u t a t i o n R e s . , 41 ( 1 9 7 5 ) 2 3 3 - - 2 4 0 . 8 M c C a n n , J., E. C h o i , E. Y a m a s a k i a n d B.N. A m e s , D e t e c t i o n o f c a r c i n o g e n s a n d m u t a g e n s w i t h t h e S a l m o n e l i a / m i c r o s o m e t e s t : A s s a y o f 3 0 0 c h e m i c a l s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 5 1 3 5 - 5139. 9 O s b o r n e , M., a n d S. P e r s o n , C h a r a c t e r i z a t i o n o f r e v e r t a n t s o f E. coli W U 3 6 - 1 0 a n d W P 2 u s i n g a m b e r mutants and an ochre mutant of bacteriophage T4, Mutation Res., 4 (1967) 504--507. 1 0 S e n i o r , A . E . , a n d A.M. T o m e t s k o , A c t i v a t i o n a n d p h o t o - i n a c t i v a t i o n o f F 1 b y a 4 - f i u o r o - 3 - n i t r o p h e n y l a z i d e , in: E. Q u a g l i a r e l l o , S. P a p p a , F. P a l m i e r i , E.C. S l a t e r a n d N. S i H p r a n d i ( E d s . ) , S y m p o s i u m o n E l e c t r o n T r a n s f e r C h a i n s a n d O x i d a t i v e P h o s p h o r y l a t i o n , Elsevier, A m s t e r d a m , 1 9 7 6 , pp. 155--160. 11 T o m e t s k o , A . M . , a n d J. T u r u l a , E v a l u a t i n g t h e s t a b i l i t y a n d r e a c t i v i t y o f a l i g h t - s e n s i t i v e p r o b e b y enzyme analysis, Photochem. Photobiol., 23 (1976) 579--585. 1 2 T o m e t s k o , A . M . , a n d J. T u r u l a , I n a c t i v a t i o n o f t r y p s i n a n d c h y m o t r y p s i n w i t h a p h o t o s e n s i t i v e p r o b e , I n t . J. P e p t i d e P r o t e i n R e s . , 8 ( 1 9 7 6 ) 3 3 1 - - 3 3 6 . 13 Walker, G.C., Isolation and characterization of mutants of the plasmid pKM101 deficient in their a b i l i t y t o e n h a n c e m u t a g e n e s i s a n d r e p a i r , J. B a c t e r i o l . , 1 3 3 ( 1 9 7 8 ) 1 2 0 3 - - 1 2 1 1 . 1 4 Y o s h i o k a , M., J. L i f t e r , C.L. H e w , C . A . C o n v e r s e , M . Y . K . A r m s t r o n g , W.H. K o n i g s b e r g a n d F . F . Richards, Studies on the combining region of protein 460, a mouse ~A immunoglobulin which binds several h a p t e n s . B i n d i n g a n d r e a c t i v i t y o f t w o t y p e s o f p h o t o a f f i n i t y l a b e l i i n g r e a g e n t s , B i o c h e m i s t r y , 12 (1973) 4679--4685.