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Mutagenicity of fluorene derivatives: A proposed mechanism
Mutation Research, 63 (1979) 1--10 © Elsevier/North-Holland Biomedical Press
MUTAGENICITY OF F L U O R E N E DERIVATIVES: A PROPOSED MECHANISM DAVID E. LEVIN *, WILLIAM S. BARNES and ED KLEKOWSKI
Botany Department, University of Massachusetts, Amherst, MA 01003 (U.S.A.) (Received 3 May 1979) (Accepted 5 July 1979)
Summary Several derivatives of fluorene, a tricyclic, organic molecule, have been found to induce both frameshift mutations and base-pair substitutions in Salmonella typhimurium strains developed by Ames. Comparisons of the mutagenic p o t e n c y of these derivatives for several strains of Salmonella suggest the importance of a carbonyl group substituted at the carbon-9 position of mutagenic derivatives, with respect to mutagenic potency. In this study, we present a feasible mechanism for the interaction of mutagenic fluorene derivatives with deoxyribonucleic acid. This mechanism requires the interaction of the mutagenic molecule with carbon-8 of guanine and a second concurrent interaction with the C-4 amino group of an adjacent cytosine residue.
Several derivatives of the fluorene structure which carry electrophilic groups substituted at carbon-2 are mutagenic in the Ames test [2--4,8,9,20]. The current study presents data for several fluorene derivatives that have never been tested for mutagenicity. Since two (2,7
doublet b y extremely hisD3052 alternating derivatives
mutagenic fluorene derivatives. These mutagens have been found to be effective in reverting strains of S. typhimurium carrying the mutation which is very close to a DNA sequence composed of ---G--C-- residues. Structures of fluorene and several mutagenic are found in Table 1.
* Present address: Biochemistry Depaztment, Umversity of California, Berkeley, CA 9 4 7 2 0 (U.S.A.). To whom requests for reprints should be addressed.
TABLE
1
STRUCTURES
OF
FLUORENE
AND
SOME
/ O
\
FLUORE NE
X O
DIRECTLY
. ~ "
MUTAGENIC
DERIVATIVES.
rN C
,
2 7-DINIT ROF'LUORE NE
.
.
/
o
oc
2-NITROSOFLUORENE
\
2
27-DINITRO
c~
9 FLU¢~RENON 'r C ,2 \N \
/ O
2 - NIT ROFLUORE NE
. O
'' O
O
'O
2,4,7-T RINITRO -g -FLUORE NONE
MateriM~ and methods The four Salmonella typhimurium strains used were kindly provided by Dr. B.N. Ames. Each is m u t a n t in 1 of 3 histidine loci. Strain TA100, carrying the base substitution mutation hisG46 [4,5,20], responds to mutagens that cause single base-pair substitutions. Strain TA1537 contains the histidine frameshift mutation his3076 [4,5]. This mutation is an added G --C--
base pair resulting in --G--G--G--G--C-C--C--C-Reversion of this strain to histidine independence requires the deletion of the extra base pair (or the possible addition of two base pairs). Strains TA1538 and TA98 contain the histidine mutation his3052 [3,4,13]. This mutation is a - - 1 deletion which may be reverted by either of 2 mutational events: the insertion of a single base pair, or the deletion of 2 adjacent base pairs [13]. All 4 strains are "deep rough" (rfa) derivatives which have defective lipopolysaccharide coats and demonstrate increased permeability to m a n y chemicals. In addition, each strain carries a uvrB mutation, resulting in defective excision repair. Strains TA98 and TA100 also contain a resistance transfer factor (R factor) carried on the pKM101 plasmid for increased sensitivity to m a n y reactive chemical mutagens [20]. Mutation of the strains is detected by determining the reversion of His- to
His* on minimal medium. A 0.1-ml aliquot of a known concentration of the mutagen dissolved in dimethyl sulfoxide (DMSO) and 0.1 ml of an overnight bacterial culture were added to 2.0 ml of molten top agar containing a limiting supplement of histidine and excess biotin to allow growth. Positive controls included 9-aminoacridine (500 pg/ml) for TA1537, daunomycin (250 pg/ml) for TA98, methyl methanesulfonate (10%) for TA100 and 2-nitrofluorene (250 pg/ml) for TA1538. All chemicals were dissolved in DMSO except 9-aminoacridine, which was dissolved in 100% ethanol. A 20-pl aliquot was then spotted onto a minimal plate seeded with the appropriate tester strain. Each strain was routinely checked for retention of the rfa character with crystal violet. TA98 and TA100 were also checked for the presence of the ampicillin R factor residing on the pKM101 plasmid [5,20]. All chemicals were obtained from Aldrich Chemical Company except 2,4,7-trinitro-9-fluorenone which was supplied by Sigma Chemical Company. Molecular configuration was done with stick figure models (Minit molecular building system, Cochranes of Oxford Ltd). Results
Fig. 1 shows the mutagenic activity of 3 fluorene derivatives (2,7-dinitrofluorene; 2,7-dinitro-9-fluorenone; 2,4,7-trinitro-9-fluorenone) for Salmonella typhimurium strains TA98, TA100, TA1537 and TA1538. Strains TA98 and TA1538 displayed the greatest response to all tested derivatives, and acted similarly to each other with respect to mutagenic potencies of each compound. These results imply that frameshift mutation with reactive fluorene derivatives is more frequent than base-pair substitution. For all strains there was a marked decrease in revertant colonies per microgram in the range of 2.5--10.0 pg/plate for all compounds tested. Each experiment included 5 replicate plates per concentration of test chemical. Discussion J.A. Miller and E.C. Miller together with other investigators have examined the mutagenic, carcinogenic, electrophilic and nucleotide binding properties of many fluorene derivatives [8,10,14--16,18,21--23]. In a series of studies involving the reaction of fluorene derivatives with DNA or RNA bases in vitro, Kriek [15] showed that N-hydroxy-2-aminofluorene reacts with guanine bases of both DNA and RNA in vitro at pH lower than 6.0. Miller [21] subsequently demonstrated that guanine bases in vitro react readily at neutral pH with N-acetoxy-2-acetylaminofluorene. Kriek et al. [16] then found that N-acetoxyN-2-fluorenyl acetamide and guanine react readily at neutrality to yield a compound which they identified as 8-(N-2-fluorenyl-acetamido)guanosine. In a later study conducted by Kriek [14], he isolated guanosine-bound fluorene derivatives from nucleic acid hydrolysates following intraperitoneal injection of rats with C14-1abelled 2-aminofluorene, 2-acetylaminofluorene, or N-hydroxy2-acetylaminofluorene. These complexes were identified as N-(guanosin-8-yl)acetylaminofluorene and N-(guanosin-8-yl)aminofluorene.
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Fig. 1. A - - D . M u t a g e n i c i t y o f 3 f l u o r e n e derivatives for 4 strains o f S typhzmurzum. T h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t s ( 3 5 for T A 9 8 , 1 6 4 for T A 1 0 0 , 1 1 for T A 1 5 3 7 a n d 2 7 for T A 1 5 3 8 ) have b e e n s u b t r a c t e d f r o m t h e r e v e r t a n t values p l o t t e d against a m o u n t o f m u t a g e n ( 2 , 7 - D N F = 2 , 7 - d i n i t r o f l u o r e n e , 2 , 7 - D N F o n e ffi 2 , 7 - d i n i t r o - 9 - f l u o r e n o n e ; 2 , 4 , 7 - T N F o n e = 2 , 4 , 7 - t r i n i t r o - 9 - f l u o r e n o n e ) . Positive m u t a g e n e s i s c o n t r o l s w e r e i n c l u d e d in all e x p e r i m e n t s t o c o n f i r m t h e r e v e r s i o n p r o p e r t i e s o f e a c h strain. Each p o i n t is t h e m e a n o f 5 plates.
This evidence, combined with studies of the electrophilic nature of these and other fluorene derivatives allowed the Millers to elucidate the mechanism for covalent attachment of the C-2 nitrogen to C-8 of guanine. The general formula depicting this attachment appears in Fig. 2. The area has been reviewed by Heidelberger [ 10].
O m
X
.,N~
/J"" NH
(RNA) Fig. 2. G e n e r a l f o r m u l a depmting the covalent a t t a c h m e n t of electroPhilic f l u o r e n e d e r i v a t i v e s t o C-8 o f guamne.
In more recent studies, the mutagenic properties of many of these fluorene derivatives have been determined with the Ames test. All fluorene derivatives tested to date that have shown mutagenic activity for one or more strains also show a marked specificity for reversion of strains carrying the hisD3052 mutation. Isono and Yourno [13] have determined that the hisD3052 mutation of strain TA1538 (TA98 without the R factor plasmid pKM101) is a --1 deletion, probably the loss of a --G---C-pair from a sequence of --G--Cr-G---C--C--C-In light of this study, it was suggested [3] that mutations by reactive, electrophilic fluorene derivatives occur through frameshifts that follow covalent intercalation into the bacterial DNA. The result of such a mutation would be the introduction of a new base pair into the DNA sequence. The addition of one base pair to the defective gene would correct the offset reading frame of the original mutant. Assuming that the inserted base pair was positioned closely enough to the original base-pair deletion, reversion of the strain to histidine independence would occur [6]. The resultant protein produced by the double frameshift mutant (-- +) would be identical to the wild-type protein with exception of the region between the 2 frameshift mutations. Isono and Yourno [13] mapped histidine revertants induced by mutation with several c o m p o u n d s in strain T A 1 5 3 8 of S. typhimurium (which carries the hisD3052 frameshift mutation). They found that revertants induced by 2-nitrosofluorene invariably resulted from the deletion of a --G--C---C--G-d o u b l e t from the DNA sequence --C--G--C--G--C--G--C--G---G--C--G--C--G--C-G--C-which is close to the 3052 site. The deletion of this doublet produced a --3 deletion mutant with a correct reading frame. All revertants isolated that were
reduced by mutation with hycanthone and nitroquinoline-N-oxide were also of this nature. The prevailing mechanisms for intercalation-induced frameshift mutagenesis in bacteria can explain the insertion or deletion of single base pairs and the insertion of multiple base pairs [11,12,17,24]. None, however, allow for intercalation-induced deletion of multiple base pairs. Tsugita and associates [11,12, 24] have c o n d u c t e d studies with proflavine, a classic intercalating agent. They suggested that by stabilization of the DNA helix, the intercalated molecule can induce localized mispairing in sequences of DNA that contain some degree of repetition. This mispairing, if it occurs during DNA replication, or repair, can lead to the insertion of one or more base pairs in the repaired strand giving rise to a mutant heteroduplex heterozygote. They found that proflavine was capable of inducing single and multiple insertions in a DNA sequence, but was not able to delete more than one base pair. This observation is consistent with their proposed model for intercalation-induced frameshift mutations. In light of the fact that intercalating agents do not induce multiple deletions, together with the data presented by l.sono and Yourno [13], the model for covalent intercalation of mutagenic fluorene derivatives does n o t appear to be feasible. Therefore, we have considered another mechanism that is consistent both with the covalent attachment of the electrophilic molecule to C-8 of guanine and the deletion of 2 adjacent base pairs, either --G--C---C--G-or
--C--G--G--C-which hypothesizes the interaction of the reactive fluorene derivative with both adjacent bases. The proposed interaction involves the covalent attachment of C-8 of guanine to the C-2 nitrogen of the mutagen as described in the Millers' model, and requires a carbonyl group at the C-9 position o f the mutagen. The molecule would be positioned so that the C-9 carbonyl group of the fluorene derivative is free to undergo hydrogen bonding with the C-4 amino group of cytosine. Subsequent deletion of b o t h base pairs of a --1 deletion strain would result in the desired --3 deletion mutant. This interaction does not result in any unusual helix deformations. The structural formula and space filling model appear in Fig. 3. Reversion of the +1 frameshift mutant TA1537 by reactive fluorene derivatives is explicable in terms of a single covalent interaction with guanine in the absence of an adjacent cytosine. Deletion of a single base pair could occur in such a manner [25]. Reversion of the single base pair substitution mutation of strain T A 1 0 0 by reactive fluorene derivatives is entirely inexplicable b y means of the covalent intercalation model, and must be accounted for in some other manner. Because deletion of two adjacent base pairs by our mechanism requires the
o
..--o
7
H2C
O
O
I
H
A
Fig. 3. A, B. I n t e r a c t i o n o f a m u t a g e n i c fluorene derivative with adjacent G-C residues in D N A . Note the h y d r o g e n bonding w i t h C-4 a m i n o h y d r o g e n o f cytosine. The s t i c k f i g u r e m o d e l demonstrates that n o u n u s u a l stress is i n f l i c t e d u p o n the hehcal structure as a r e s u l t o f this interaction.
presence of a carbonyl group at the C-9 position of the mutagen, the absence of the carbonyl group would allow only a single interaction resulting in the induction of only single base-pair deletions. Table 2 presents the relative mutagenic potencies of several fluorene derivatives for 2 frameshift strains; TA1538 (a --1 deletion mutant) and TA1537 (a +1 insertion mutant). Note that the addition of a C-9 carbonyl group to 2,7-dinitrofluorene (giving, 2,7-dinitro9-fluorenone) results in a considerable increase in reversion of TA1538 and a corresponding decrease in reversion of TA1537, precisely what our mechanism would predict. Consider the effect of a compound that induces only single deletions; this compound would not effectively revert the --1 strain (TA1538), but would be quite effective in reversion of the +1 strain (TA1537). A compound that induces double deletions would preferentially revert the --1 strain as described above, but would "skip over" the reversion point of the +1 strain, therefore demonstrating a lower mutagenic potential for TA1537 than its structural cousin which lacks the C-9 carbonyl group. It is apparent that the presence of a carbonyl group at the C-9 position of
TABLE 2 RELATIVE REVERSION FREQUENCIES OF 3 FLUORENE M U T A N T A N D A --1 D E L E T I O N M U T A N T a,b
DERIVATIVES
F O R A +1 I N S E R T I O N
Strain
2,7-DNF c
2,7-DNFone d
2,4,7-TNFone e
T A 1 5 3 8 (--1) T A 1 5 3 7 (+1)
2931 289
5650 77
8068 214
a All values correspond to 1 n m of mutagen. b All values were taken from linear portions of dose--response curves from which spontaneous revertants have been subtracted. c 2 , 7 - D N F = 2,7-dinitrofluorene. d 2 , 7 - D N F o n e = 2,7-dinitro-9-fluorenone. e 2,4,7-TNFone = 2,4,7-trinitro*9-fluorenone.
reactive fluorene derivatives induces different results in a +1 insertion strain and a --1 deletion strain of Salmonella typhimurium. The unusually high reversion rates observed in strains that carry the hisD3052 mutation m a y be attributed to the presence of a run of alternating G--C residues near the original deletion. This run represents the mutational h o t spot of this gene and is particularly susceptible to mutation by these c o m p o u n d s [13]. Because fluorene and 9-fluorenone {fluorene with a C-9 carbonyl group) are not mutagenic for any of the 4 tester strains used [Levin, unpublished], the presence of an electrophilic side group appears to be a prerequisite for mutagenesis. There appear to be other factors affecting mutagenesis b y this group, as reversion of both --1 strains was detected with derivatives that do not carry the C-9 carbonyl. It is conceivable that derivatives that do not carry the carbonyl group are metabolized by the bacteria to derivatives carrying a C-9 carbonyl. This would account for the observed mutagenicity of such c o m p o u n d s for TA1538 and TA98. Because these c o m p o u n d s are activated b y bacterial nitroreductases [19], the enzymes might preferentially activate one c o m p o u n d to a greater extent than another. This, however, should n o t affect the relative mutagenicities among strains {assuming that the active enzymes are identifical in compared strains). Although the mechanism proposed in the present study appears to be feasible, other interactions are possible and it would not be appropriate to assume that mutation by a group of c o m p o u n d s occurs b y one mechanism alone. Clearly, full elucidation of the mechanisms b y which these c o m p o u n d s induce mutation will require further study.
Note added in p r o o f Studies conducted by Weinstein and Grunberger [26] have resulted in a model for base displacement by N-acetoxy-2-acetylaminofluorene in which the modified quanine residue is shifted o u t of the double helix, while the fixed carcinogen is inserted. This would result in a stacking interaction between the fluorene residue and one of the next adjacent bases. However, a recent study [27] stated that although N-acetoxy-2-acetylaminofluorene and 7-fluoro-N-2acetylaminofluorene were capable o f inducing such a stacking interaction, 7-iodo-N-2-acetylaminofluorene was t o o bulky for insertion. Based upon this
evidence, it would seem unlikely that molecules of such bulk as those in the present study would be capable of insertion into the helix. Acknowledgements This work was supported by grants to Edward J. Klekowski Jr. from the U.S. National Science Foundation and from the Office of Water Resources Research, U.S. Department of the Interior under the Water Resources Research Act of 1964, as amended. This work was also supported in part by the University of Massachusetts Experiment Station, project 445, grant to R.E. Levin. This study constitutes the senior honors thesis of D.E. Levin who wishes to express his gratitude to R.E. Levin, A.I. Stern and C.B. Thorne for invaluable discussion, and to R.R. Holmes for stick figure models. Special thanks to B.N. Ames for critical review and discussion. References 1 The Aldrich Catalog-Handbook of Orgamc and Blochemmals (1977--1978) 730. 2 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . 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Miller, C o m p a r a t i v e e l e c t r o p h f l i c i t y , m u t a genicity, DNA repair induction activity and carcinogenimty of some N- and O-acyl derivatives of N-hydroxy-2-aminofluorene, Cancer Res., 37 (1977) 1461--1467. 9 D u r s t o n , W . E . , a n d B.N. A m e s , A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s m U r i n e : S t u d i e s w i t h t h e c a r c i n o g e n 2 - a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 1 0 H e i d e l b e r g e r , C., C h e m i c a l c a r c i n o g e n e s i s , A n n u . R e v . B l o c h e m . , 4 4 ( 1 9 7 5 ) 7 9 - - 1 2 1 . 11 I m a d a , M., M. I n o u y c , M. E d a a n d A . T s u g l t a , F r a m e s h i f t m u t a t i o n m t h e l y s o z y m e g e n e o f b a c t e r i o phage T4: Demonstration of the msertmn of four bases and the preferential occurrence of base addit m n m a c r i d i n e m u t a g e n e s l s , J . Mol. B m l . , 5 4 ( 1 9 7 0 ) 1 9 9 - - 2 1 7 . 1 2 I n o u y e , M., E. A k a b o s h l , A. T s u g i t a , G . S t r e l s m g e r a n d Y. O k a d a , A f r a m e s h i f t m u t a t i o n r e s u l t i n g m t h e d e l e t i o n o f t w o b a s e p a i r s m t h e l y s o z y m e g e n e o f b a c t e r i o p h a g e T 4 , J . Mol. Biol., 3 0 ( 1 9 6 7 ) 3 9 - 47. 1 3 I s o n o , K . , a n d J. Y o u m o , C h e m i c a l c a r c i n o g e n s as f r a m e s h i f t m u t a g e n s ' S a l m o n e l l a D N A s e q u e n c e sensitive t o m u t a g e n e s l s b y p o l y c y c l i c c a r c i n o g e n s , P r o c . N a t l . A c a d . ScL ( U . S . A . ) , 71 ( 1 9 7 4 ) 1 6 1 2 - 1617. 1 4 K r m k , E., B i n d i n g o f 2 - a c e t y l a m i n o f l u o r e n e a n d N - h y d r o x y - 2 - a c e t y l a m m o f l u o r e n e t o r a t - l i v e r n u c l e i c a m d s m vivo, C h e m . - B i o l . I n t e r a c t . , 1 ( 1 9 6 9 / 1 9 7 0 ) 3 - - 1 7 . 1 5 K n e k , E., O n t h e i n t e r a c t i o n o f N - 2 - f l u o r e n y l h y d r o x y l a m i n e w i t h n u c l e i c a c i d s in v i t r o , B i o c h e m . Biophys. Res. Commun.° 20 (1965) 793--799. 1 6 K r l e k , E., J . A . Miller, U. J u h l a n d E.C. Miller, 8 - ( N - 2 - F l u o r e n y l a c e t a m i d o ) q u a n o s t n e , a n a r y l a m l d a t l o n r e a c t i o n p r o d u c t o f g u a n o s i n e a n d t h e c a r c i n o g e n N - a c e t o x y - N - 2 - f l u o r e n y l a c e t a m i d e in n e u t r a l s o l u t m n , B i o c h e m i s t r y , 6 (1967,) 1 7 7 - - 1 8 2 . 1 7 L e r m a n , L . S . , T h e s t r u c t u r e o f t h e D N A - a c n d m e c o m p l e x , P r o c . N a t l . A c a d . ScL ( U . S . A . ) , 4 9 ( 1 9 6 3 ) 94--102. 1 8 Maher0 V . M . , E . C . Miller, J . A . Miller a n d W. S z y b a i s k l , M u t a t i o n s a n d d e c r e a s e s m d e n s i t y o f t r a n s forming DNA produced by derivatives of the carcinogens 2-acetylammofluorene and N-methyl-
10
4-anunoazobenzene, Mol. Pharmacol., 4 (1968) 411--426. 1 9 M c C a n n , J., 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 as m u t a g e n s In t h e S a l n e l l 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 , D i s c u s s i o n , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 3 ( 1 9 7 6 ) 9 5 0 - - 9 5 4 . 2 0 M c C a n n , J., E. S p i n g a r n , J. K o b a r 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 as m u t a g e n s : B a c t e r i a l t e s t e r s t r a i n s w i t h R f a c t o r p l a s m l d s , P r o c . N a t l . A c a d . Sci ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 . 21 Miller, E.C., N u c l e i c a c i d g u a n i n e : R e a c t i o n w i t h t h e c a r c i n o g e n N - a c e t o x y - 2 - a c e t y l a m m o f l u o r e n e , Science, 153 (1966) 1125--1127. 2 2 Miller, E . C . , a n d J . A . Miller, B i o c h e m i s t r y o f c a r c i n o g e n e s i s , A n n u . R e v . B l o c h e m . , 2 8 ( 1 9 5 9 ) 2 9 1 - 320. 2 3 Miller, J . A . , C a r c i n o g e n e s i s b y c h e m i c a l s " A n o v e r w e w , G . H . A . C l o w e s M e m o r i a l L e c t u r e , C a n c e r Res., 30 (1970) 559--576. 2 4 O c a d a , Y., S. A m a g a s e a n d A . T s u g i t a , F r a m e s h l f t m u t a t i o n m t h e l y s o z y m e g e n e o f b a c t e r i o p h a g e T 4 : D e m o n s t r a t i o n o f t h e i n s e r t i o n o f b y e b a s e s , a n d a s u m m a r y o f in vlvo c o d o n s a n d l y s o z y m e a c t i v i t i e s , J. Mol. Biol., 5 4 ( 1 9 7 0 ) 2 1 9 - - 2 4 6 . 2 5 Y o u r n o , J . , E x t e r n a l l y s u p p r e s s i b l e +1 " g l y c i n e " f r a m e s h i f t : Possible q u a d r u p l e t i s o m e r s f o r g l y c i n e a n d p r o l i n e , N a t u r e N e w Biol., 2 3 9 ( 1 9 7 2 ) 2 1 9 - - 2 2 1 . 2 6 L e v i n e , A . F . , L.M. F i n k , I.B. W e i n s t e i n a n d D. G r u n b e r g e r , E f f e c t o f N - 2 - a c e t y l a m i n o f l u o r e n e m o d i f i cation on the conformation of nucleic acids, Cancer Res., 34 (1974) 319--327. 2 7 S a n t e l l a , R . M . , R . P . P . F u e h s a n d D. G r u n b e r g e r , M u t a g e n i e i t y o f 7 - i o d o a n d 7 - f l u o r o d e n v a h v e s o f N - h y c l r o x y - a n d N - a c e t o x y - N - 2 - a c e t y l a m i n o f l u o r e n e in t h e Salmonella t y p h i m u r i u m a s s a y , M u t a t i o n Res., 67 (1979) 85--87.
MUTAGENICITY OF F L U O R E N E DERIVATIVES: A PROPOSED MECHANISM DAVID E. LEVIN *, WILLIAM S. BARNES and ED KLEKOWSKI
Botany Department, University of Massachusetts, Amherst, MA 01003 (U.S.A.) (Received 3 May 1979) (Accepted 5 July 1979)
Summary Several derivatives of fluorene, a tricyclic, organic molecule, have been found to induce both frameshift mutations and base-pair substitutions in Salmonella typhimurium strains developed by Ames. Comparisons of the mutagenic p o t e n c y of these derivatives for several strains of Salmonella suggest the importance of a carbonyl group substituted at the carbon-9 position of mutagenic derivatives, with respect to mutagenic potency. In this study, we present a feasible mechanism for the interaction of mutagenic fluorene derivatives with deoxyribonucleic acid. This mechanism requires the interaction of the mutagenic molecule with carbon-8 of guanine and a second concurrent interaction with the C-4 amino group of an adjacent cytosine residue.
Several derivatives of the fluorene structure which carry electrophilic groups substituted at carbon-2 are mutagenic in the Ames test [2--4,8,9,20]. The current study presents data for several fluorene derivatives that have never been tested for mutagenicity. Since two (2,7
doublet b y extremely hisD3052 alternating derivatives
mutagenic fluorene derivatives. These mutagens have been found to be effective in reverting strains of S. typhimurium carrying the mutation which is very close to a DNA sequence composed of ---G--C-- residues. Structures of fluorene and several mutagenic are found in Table 1.
* Present address: Biochemistry Depaztment, Umversity of California, Berkeley, CA 9 4 7 2 0 (U.S.A.). To whom requests for reprints should be addressed.
TABLE
1
STRUCTURES
OF
FLUORENE
AND
SOME
/ O
\
FLUORE NE
X O
DIRECTLY
. ~ "
MUTAGENIC
DERIVATIVES.
rN C
,
2 7-DINIT ROF'LUORE NE
.
.
/
o
oc
2-NITROSOFLUORENE
\
2
27-DINITRO
c~
9 FLU¢~RENON 'r C ,2 \N \
/ O
2 - NIT ROFLUORE NE
. O
'' O
O
'O
2,4,7-T RINITRO -g -FLUORE NONE
MateriM~ and methods The four Salmonella typhimurium strains used were kindly provided by Dr. B.N. Ames. Each is m u t a n t in 1 of 3 histidine loci. Strain TA100, carrying the base substitution mutation hisG46 [4,5,20], responds to mutagens that cause single base-pair substitutions. Strain TA1537 contains the histidine frameshift mutation his3076 [4,5]. This mutation is an added G --C--
base pair resulting in --G--G--G--G--C-C--C--C-Reversion of this strain to histidine independence requires the deletion of the extra base pair (or the possible addition of two base pairs). Strains TA1538 and TA98 contain the histidine mutation his3052 [3,4,13]. This mutation is a - - 1 deletion which may be reverted by either of 2 mutational events: the insertion of a single base pair, or the deletion of 2 adjacent base pairs [13]. All 4 strains are "deep rough" (rfa) derivatives which have defective lipopolysaccharide coats and demonstrate increased permeability to m a n y chemicals. In addition, each strain carries a uvrB mutation, resulting in defective excision repair. Strains TA98 and TA100 also contain a resistance transfer factor (R factor) carried on the pKM101 plasmid for increased sensitivity to m a n y reactive chemical mutagens [20]. Mutation of the strains is detected by determining the reversion of His- to
His* on minimal medium. A 0.1-ml aliquot of a known concentration of the mutagen dissolved in dimethyl sulfoxide (DMSO) and 0.1 ml of an overnight bacterial culture were added to 2.0 ml of molten top agar containing a limiting supplement of histidine and excess biotin to allow growth. Positive controls included 9-aminoacridine (500 pg/ml) for TA1537, daunomycin (250 pg/ml) for TA98, methyl methanesulfonate (10%) for TA100 and 2-nitrofluorene (250 pg/ml) for TA1538. All chemicals were dissolved in DMSO except 9-aminoacridine, which was dissolved in 100% ethanol. A 20-pl aliquot was then spotted onto a minimal plate seeded with the appropriate tester strain. Each strain was routinely checked for retention of the rfa character with crystal violet. TA98 and TA100 were also checked for the presence of the ampicillin R factor residing on the pKM101 plasmid [5,20]. All chemicals were obtained from Aldrich Chemical Company except 2,4,7-trinitro-9-fluorenone which was supplied by Sigma Chemical Company. Molecular configuration was done with stick figure models (Minit molecular building system, Cochranes of Oxford Ltd). Results
Fig. 1 shows the mutagenic activity of 3 fluorene derivatives (2,7-dinitrofluorene; 2,7-dinitro-9-fluorenone; 2,4,7-trinitro-9-fluorenone) for Salmonella typhimurium strains TA98, TA100, TA1537 and TA1538. Strains TA98 and TA1538 displayed the greatest response to all tested derivatives, and acted similarly to each other with respect to mutagenic potencies of each compound. These results imply that frameshift mutation with reactive fluorene derivatives is more frequent than base-pair substitution. For all strains there was a marked decrease in revertant colonies per microgram in the range of 2.5--10.0 pg/plate for all compounds tested. Each experiment included 5 replicate plates per concentration of test chemical. Discussion J.A. Miller and E.C. Miller together with other investigators have examined the mutagenic, carcinogenic, electrophilic and nucleotide binding properties of many fluorene derivatives [8,10,14--16,18,21--23]. In a series of studies involving the reaction of fluorene derivatives with DNA or RNA bases in vitro, Kriek [15] showed that N-hydroxy-2-aminofluorene reacts with guanine bases of both DNA and RNA in vitro at pH lower than 6.0. Miller [21] subsequently demonstrated that guanine bases in vitro react readily at neutral pH with N-acetoxy-2-acetylaminofluorene. Kriek et al. [16] then found that N-acetoxyN-2-fluorenyl acetamide and guanine react readily at neutrality to yield a compound which they identified as 8-(N-2-fluorenyl-acetamido)guanosine. In a later study conducted by Kriek [14], he isolated guanosine-bound fluorene derivatives from nucleic acid hydrolysates following intraperitoneal injection of rats with C14-1abelled 2-aminofluorene, 2-acetylaminofluorene, or N-hydroxy2-acetylaminofluorene. These complexes were identified as N-(guanosin-8-yl)acetylaminofluorene and N-(guanosin-8-yl)aminofluorene.
)~¢
A
2 4 " TNFon~
0
J
' ]
/
/
2,7 bNFone
© cJ
2~ ~00
~_
//
,,,,,,, b L
5t
CL 1 5 0
C 0
0
0 ~ C CJ
25
L
8 fr
02
i
I
I
2
~. /u ,
I
I
6 N'lut~qcr
30¢, 0 0 x.
Y 25C ~D ID
o
FL 20,9 Ck © ~
~0
0
0
0 C cJ L (1) O~
',CJ
i
i 4
~q
C
c
1'
3
4
I 5
~ j taqr'r
Fig. 1. A - - D . M u t a g e n i c i t y o f 3 f l u o r e n e derivatives for 4 strains o f S typhzmurzum. T h e n u m b e r o f s p o n t a n e o u s r e v e r t a n t s ( 3 5 for T A 9 8 , 1 6 4 for T A 1 0 0 , 1 1 for T A 1 5 3 7 a n d 2 7 for T A 1 5 3 8 ) have b e e n s u b t r a c t e d f r o m t h e r e v e r t a n t values p l o t t e d against a m o u n t o f m u t a g e n ( 2 , 7 - D N F = 2 , 7 - d i n i t r o f l u o r e n e , 2 , 7 - D N F o n e ffi 2 , 7 - d i n i t r o - 9 - f l u o r e n o n e ; 2 , 4 , 7 - T N F o n e = 2 , 4 , 7 - t r i n i t r o - 9 - f l u o r e n o n e ) . Positive m u t a g e n e s i s c o n t r o l s w e r e i n c l u d e d in all e x p e r i m e n t s t o c o n f i r m t h e r e v e r s i o n p r o p e r t i e s o f e a c h strain. Each p o i n t is t h e m e a n o f 5 plates.
This evidence, combined with studies of the electrophilic nature of these and other fluorene derivatives allowed the Millers to elucidate the mechanism for covalent attachment of the C-2 nitrogen to C-8 of guanine. The general formula depicting this attachment appears in Fig. 2. The area has been reviewed by Heidelberger [ 10].
O m
X
.,N~
/J"" NH
(RNA) Fig. 2. G e n e r a l f o r m u l a depmting the covalent a t t a c h m e n t of electroPhilic f l u o r e n e d e r i v a t i v e s t o C-8 o f guamne.
In more recent studies, the mutagenic properties of many of these fluorene derivatives have been determined with the Ames test. All fluorene derivatives tested to date that have shown mutagenic activity for one or more strains also show a marked specificity for reversion of strains carrying the hisD3052 mutation. Isono and Yourno [13] have determined that the hisD3052 mutation of strain TA1538 (TA98 without the R factor plasmid pKM101) is a --1 deletion, probably the loss of a --G---C-pair from a sequence of --G--Cr-G---C--C--C-In light of this study, it was suggested [3] that mutations by reactive, electrophilic fluorene derivatives occur through frameshifts that follow covalent intercalation into the bacterial DNA. The result of such a mutation would be the introduction of a new base pair into the DNA sequence. The addition of one base pair to the defective gene would correct the offset reading frame of the original mutant. Assuming that the inserted base pair was positioned closely enough to the original base-pair deletion, reversion of the strain to histidine independence would occur [6]. The resultant protein produced by the double frameshift mutant (-- +) would be identical to the wild-type protein with exception of the region between the 2 frameshift mutations. Isono and Yourno [13] mapped histidine revertants induced by mutation with several c o m p o u n d s in strain T A 1 5 3 8 of S. typhimurium (which carries the hisD3052 frameshift mutation). They found that revertants induced by 2-nitrosofluorene invariably resulted from the deletion of a --G--C---C--G-d o u b l e t from the DNA sequence --C--G--C--G--C--G--C--G---G--C--G--C--G--C-G--C-which is close to the 3052 site. The deletion of this doublet produced a --3 deletion mutant with a correct reading frame. All revertants isolated that were
reduced by mutation with hycanthone and nitroquinoline-N-oxide were also of this nature. The prevailing mechanisms for intercalation-induced frameshift mutagenesis in bacteria can explain the insertion or deletion of single base pairs and the insertion of multiple base pairs [11,12,17,24]. None, however, allow for intercalation-induced deletion of multiple base pairs. Tsugita and associates [11,12, 24] have c o n d u c t e d studies with proflavine, a classic intercalating agent. They suggested that by stabilization of the DNA helix, the intercalated molecule can induce localized mispairing in sequences of DNA that contain some degree of repetition. This mispairing, if it occurs during DNA replication, or repair, can lead to the insertion of one or more base pairs in the repaired strand giving rise to a mutant heteroduplex heterozygote. They found that proflavine was capable of inducing single and multiple insertions in a DNA sequence, but was not able to delete more than one base pair. This observation is consistent with their proposed model for intercalation-induced frameshift mutations. In light of the fact that intercalating agents do not induce multiple deletions, together with the data presented by l.sono and Yourno [13], the model for covalent intercalation of mutagenic fluorene derivatives does n o t appear to be feasible. Therefore, we have considered another mechanism that is consistent both with the covalent attachment of the electrophilic molecule to C-8 of guanine and the deletion of 2 adjacent base pairs, either --G--C---C--G-or
--C--G--G--C-which hypothesizes the interaction of the reactive fluorene derivative with both adjacent bases. The proposed interaction involves the covalent attachment of C-8 of guanine to the C-2 nitrogen of the mutagen as described in the Millers' model, and requires a carbonyl group at the C-9 position o f the mutagen. The molecule would be positioned so that the C-9 carbonyl group of the fluorene derivative is free to undergo hydrogen bonding with the C-4 amino group of cytosine. Subsequent deletion of b o t h base pairs of a --1 deletion strain would result in the desired --3 deletion mutant. This interaction does not result in any unusual helix deformations. The structural formula and space filling model appear in Fig. 3. Reversion of the +1 frameshift mutant TA1537 by reactive fluorene derivatives is explicable in terms of a single covalent interaction with guanine in the absence of an adjacent cytosine. Deletion of a single base pair could occur in such a manner [25]. Reversion of the single base pair substitution mutation of strain T A 1 0 0 by reactive fluorene derivatives is entirely inexplicable b y means of the covalent intercalation model, and must be accounted for in some other manner. Because deletion of two adjacent base pairs by our mechanism requires the
o
..--o
7
H2C
O
O
I
H
A
Fig. 3. A, B. I n t e r a c t i o n o f a m u t a g e n i c fluorene derivative with adjacent G-C residues in D N A . Note the h y d r o g e n bonding w i t h C-4 a m i n o h y d r o g e n o f cytosine. The s t i c k f i g u r e m o d e l demonstrates that n o u n u s u a l stress is i n f l i c t e d u p o n the hehcal structure as a r e s u l t o f this interaction.
presence of a carbonyl group at the C-9 position of the mutagen, the absence of the carbonyl group would allow only a single interaction resulting in the induction of only single base-pair deletions. Table 2 presents the relative mutagenic potencies of several fluorene derivatives for 2 frameshift strains; TA1538 (a --1 deletion mutant) and TA1537 (a +1 insertion mutant). Note that the addition of a C-9 carbonyl group to 2,7-dinitrofluorene (giving, 2,7-dinitro9-fluorenone) results in a considerable increase in reversion of TA1538 and a corresponding decrease in reversion of TA1537, precisely what our mechanism would predict. Consider the effect of a compound that induces only single deletions; this compound would not effectively revert the --1 strain (TA1538), but would be quite effective in reversion of the +1 strain (TA1537). A compound that induces double deletions would preferentially revert the --1 strain as described above, but would "skip over" the reversion point of the +1 strain, therefore demonstrating a lower mutagenic potential for TA1537 than its structural cousin which lacks the C-9 carbonyl group. It is apparent that the presence of a carbonyl group at the C-9 position of
TABLE 2 RELATIVE REVERSION FREQUENCIES OF 3 FLUORENE M U T A N T A N D A --1 D E L E T I O N M U T A N T a,b
DERIVATIVES
F O R A +1 I N S E R T I O N
Strain
2,7-DNF c
2,7-DNFone d
2,4,7-TNFone e
T A 1 5 3 8 (--1) T A 1 5 3 7 (+1)
2931 289
5650 77
8068 214
a All values correspond to 1 n m of mutagen. b All values were taken from linear portions of dose--response curves from which spontaneous revertants have been subtracted. c 2 , 7 - D N F = 2,7-dinitrofluorene. d 2 , 7 - D N F o n e = 2,7-dinitro-9-fluorenone. e 2,4,7-TNFone = 2,4,7-trinitro*9-fluorenone.
reactive fluorene derivatives induces different results in a +1 insertion strain and a --1 deletion strain of Salmonella typhimurium. The unusually high reversion rates observed in strains that carry the hisD3052 mutation m a y be attributed to the presence of a run of alternating G--C residues near the original deletion. This run represents the mutational h o t spot of this gene and is particularly susceptible to mutation by these c o m p o u n d s [13]. Because fluorene and 9-fluorenone {fluorene with a C-9 carbonyl group) are not mutagenic for any of the 4 tester strains used [Levin, unpublished], the presence of an electrophilic side group appears to be a prerequisite for mutagenesis. There appear to be other factors affecting mutagenesis b y this group, as reversion of both --1 strains was detected with derivatives that do not carry the C-9 carbonyl. It is conceivable that derivatives that do not carry the carbonyl group are metabolized by the bacteria to derivatives carrying a C-9 carbonyl. This would account for the observed mutagenicity of such c o m p o u n d s for TA1538 and TA98. Because these c o m p o u n d s are activated b y bacterial nitroreductases [19], the enzymes might preferentially activate one c o m p o u n d to a greater extent than another. This, however, should n o t affect the relative mutagenicities among strains {assuming that the active enzymes are identifical in compared strains). Although the mechanism proposed in the present study appears to be feasible, other interactions are possible and it would not be appropriate to assume that mutation by a group of c o m p o u n d s occurs b y one mechanism alone. Clearly, full elucidation of the mechanisms b y which these c o m p o u n d s induce mutation will require further study.
Note added in p r o o f Studies conducted by Weinstein and Grunberger [26] have resulted in a model for base displacement by N-acetoxy-2-acetylaminofluorene in which the modified quanine residue is shifted o u t of the double helix, while the fixed carcinogen is inserted. This would result in a stacking interaction between the fluorene residue and one of the next adjacent bases. However, a recent study [27] stated that although N-acetoxy-2-acetylaminofluorene and 7-fluoro-N-2acetylaminofluorene were capable o f inducing such a stacking interaction, 7-iodo-N-2-acetylaminofluorene was t o o bulky for insertion. Based upon this
evidence, it would seem unlikely that molecules of such bulk as those in the present study would be capable of insertion into the helix. Acknowledgements This work was supported by grants to Edward J. Klekowski Jr. from the U.S. National Science Foundation and from the Office of Water Resources Research, U.S. Department of the Interior under the Water Resources Research Act of 1964, as amended. This work was also supported in part by the University of Massachusetts Experiment Station, project 445, grant to R.E. Levin. This study constitutes the senior honors thesis of D.E. Levin who wishes to express his gratitude to R.E. Levin, A.I. Stern and C.B. Thorne for invaluable discussion, and to R.R. Holmes for stick figure models. Special thanks to B.N. Ames for critical review and discussion. References 1 The Aldrich Catalog-Handbook of Orgamc and Blochemmals (1977--1978) 730. 2 A m e s , B.N., W.E. D u r s t o n , E. Y a m a s a k i a n d F . D . L e e , C a r c i n o g e n s a r c m u t a g e n s : A s i m p l e t e s t syst e m c o m b i n i n g h v e r h o m o g e n a t e s f o r a c t i v a t i o n a n d b a c t e r i a f o r d e t e c t i o n , P r o c . N a t l . A c a d . ScL (U.S.A.), 7 0 ( 1 9 7 3 ) 2 2 8 1 - - 2 2 8 5 . 3 A m e s , B.N., E . G . G u r n e y , J . A . Miller a n d H. B a r t s c h , C a r c i n o g e n s as i r a m e s h i f t m u t a g e n s : m e t a b o h t e s a n d d e r i v a t i v e s o f 2 - a c e t y l a m m o f l u o r e n e a n d o t h e r a r o m a t i c a m i n e c a r c i n o g e n s , P r o c . N a t l . A c a d . Sci. (U.S.A.), 69 (1972) 3128--3132. 4 A m e s , B . N , F . D . Lee a n d W . E . D u r s t o n , A n i m p r o v e d b a c t e r m l s y s t e m f o r t h e d e t e c t i o n a n d classific a t i o n o f m u t a g e n s a n d c a r c i n o g e n s , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 0 ( 1 9 7 3 ) 7 8 2 - - 7 8 6 . 5 A m e s , B.N., J . M c C a n n a n d E. Y a m a s a k l , 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 l l 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 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 . 6 A m e s , B . N . , a n d H~I. Whitfleld~ J r . , F r a m e s h l f t m u t a g e n e s i s m S a l m o n e l l a , C o l d S p r i n g H a r b o r S y m p . Q u a n t . Biol., 31 ( 1 9 6 6 ) 2 2 1 - - 2 2 5 . 7 H u t z i n g e r , O., a n d W.D. J a m l e s o n , Mass s p e c t r o p h o t o m e t r i c i d e n t i f i c a t i o n a n d I s o l a t i o n o f i n d o l e s as polymtrofluorene complexes, Anal. Blochem., 35 (1970) 351--358. 8 B a r t s c h , H., C. Malavellle, H . F . S t m h , E.C. Miller a n d J . A . Miller, C o m p a r a t i v e e l e c t r o p h f l i c i t y , m u t a genicity, DNA repair induction activity and carcinogenimty of some N- and O-acyl derivatives of N-hydroxy-2-aminofluorene, Cancer Res., 37 (1977) 1461--1467. 9 D u r s t o n , W . E . , a n d B.N. A m e s , A s i m p l e m e t h o d f o r t h e d e t e c t i o n o f m u t a g e n s m U r i n e : S t u d i e s w i t h t h e c a r c i n o g e n 2 - a c e t y l a m i n o f l u o r e n e , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 71 ( 1 9 7 4 ) 7 3 7 - - 7 4 1 . 1 0 H e i d e l b e r g e r , C., C h e m i c a l c a r c i n o g e n e s i s , A n n u . R e v . B l o c h e m . , 4 4 ( 1 9 7 5 ) 7 9 - - 1 2 1 . 11 I m a d a , M., M. I n o u y c , M. E d a a n d A . T s u g l t a , F r a m e s h i f t m u t a t i o n m t h e l y s o z y m e g e n e o f b a c t e r i o phage T4: Demonstration of the msertmn of four bases and the preferential occurrence of base addit m n m a c r i d i n e m u t a g e n e s l s , J . Mol. B m l . , 5 4 ( 1 9 7 0 ) 1 9 9 - - 2 1 7 . 1 2 I n o u y e , M., E. A k a b o s h l , A. T s u g i t a , G . S t r e l s m g e r a n d Y. O k a d a , A f r a m e s h i f t m u t a t i o n r e s u l t i n g m t h e d e l e t i o n o f t w o b a s e p a i r s m t h e l y s o z y m e g e n e o f b a c t e r i o p h a g e T 4 , J . Mol. Biol., 3 0 ( 1 9 6 7 ) 3 9 - 47. 1 3 I s o n o , K . , a n d J. Y o u m o , C h e m i c a l c a r c i n o g e n s as f r a m e s h i f t m u t a g e n s ' S a l m o n e l l a D N A s e q u e n c e sensitive t o m u t a g e n e s l s b y p o l y c y c l i c c a r c i n o g e n s , P r o c . N a t l . A c a d . ScL ( U . S . 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S . , T h e s t r u c t u r e o f t h e D N A - a c n d m e c o m p l e x , P r o c . N a t l . A c a d . ScL ( U . S . A . ) , 4 9 ( 1 9 6 3 ) 94--102. 1 8 Maher0 V . M . , E . C . Miller, J . A . Miller a n d W. S z y b a i s k l , M u t a t i o n s a n d d e c r e a s e s m d e n s i t y o f t r a n s forming DNA produced by derivatives of the carcinogens 2-acetylammofluorene and N-methyl-
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4-anunoazobenzene, Mol. Pharmacol., 4 (1968) 411--426. 1 9 M c C a n n , J., 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 as m u t a g e n s In t h e S a l n e l l 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 , D i s c u s s i o n , P r o c . N a t l . A c a d . Sci. ( U . S . A . ) , 7 3 ( 1 9 7 6 ) 9 5 0 - - 9 5 4 . 2 0 M c C a n n , J., E. S p i n g a r n , J. K o b a r 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 as m u t a g e n s : B a c t e r i a l t e s t e r s t r a i n s w i t h R f a c t o r p l a s m l d s , P r o c . N a t l . A c a d . Sci ( U . S . A . ) , 7 2 ( 1 9 7 5 ) 9 7 9 - - 9 8 3 . 21 Miller, E.C., N u c l e i c a c i d g u a n i n e : R e a c t i o n w i t h t h e c a r c i n o g e n N - a c e t o x y - 2 - a c e t y l a m m o f l u o r e n e , Science, 153 (1966) 1125--1127. 2 2 Miller, E . C . , a n d J . A . Miller, B i o c h e m i s t r y o f c a r c i n o g e n e s i s , A n n u . R e v . B l o c h e m . , 2 8 ( 1 9 5 9 ) 2 9 1 - 320. 2 3 Miller, J . A . , C a r c i n o g e n e s i s b y c h e m i c a l s " A n o v e r w e w , G . H . A . C l o w e s M e m o r i a l L e c t u r e , C a n c e r Res., 30 (1970) 559--576. 2 4 O c a d a , Y., S. A m a g a s e a n d A . T s u g i t a , F r a m e s h l f t m u t a t i o n m t h e l y s o z y m e g e n e o f b a c t e r i o p h a g e T 4 : D e m o n s t r a t i o n o f t h e i n s e r t i o n o f b y e b a s e s , a n d a s u m m a r y o f in vlvo c o d o n s a n d l y s o z y m e a c t i v i t i e s , J. Mol. Biol., 5 4 ( 1 9 7 0 ) 2 1 9 - - 2 4 6 . 2 5 Y o u r n o , J . , E x t e r n a l l y s u p p r e s s i b l e +1 " g l y c i n e " f r a m e s h i f t : Possible q u a d r u p l e t i s o m e r s f o r g l y c i n e a n d p r o l i n e , N a t u r e N e w Biol., 2 3 9 ( 1 9 7 2 ) 2 1 9 - - 2 2 1 . 2 6 L e v i n e , A . F . , L.M. F i n k , I.B. W e i n s t e i n a n d D. G r u n b e r g e r , E f f e c t o f N - 2 - a c e t y l a m i n o f l u o r e n e m o d i f i cation on the conformation of nucleic acids, Cancer Res., 34 (1974) 319--327. 2 7 S a n t e l l a , R . M . , R . P . P . F u e h s a n d D. G r u n b e r g e r , M u t a g e n i e i t y o f 7 - i o d o a n d 7 - f l u o r o d e n v a h v e s o f N - h y c l r o x y - a n d N - a c e t o x y - N - 2 - a c e t y l a m i n o f l u o r e n e in t h e Salmonella t y p h i m u r i u m a s s a y , M u t a t i o n Res., 67 (1979) 85--87.