JOURNAL OF BACTERIOLOGY, May 1971, p. 543-550

Copyright © 1971 American Society for MicrobiologyVol. 106, No. 2

Printed in U.S.A.

Effect of Gene Induction on the Rate ofMutagenesis by ICR-191 in Escherichia coli

ROBERT K. HERMAN AND NOMI B. DWORKIN

Department of Genetics and Cell Bi6logy, University of Minnesota, St. Paul, Minnesota 55101

Received for publication 16 February 1971

ICR-191, an acridine half-mustard known to cause frameshift mutations in bac-teria, was used to induce Lac- mutations revertible by ICR-191. The reversionrates of several of these mutations were stimulated approximately twofold by thepresence of lac inducer. The stimulatory effect of inducer was attributable to gene

induction rather than some other effect of inducer, since inducer did not stimulatereversion in a regulator constitutive strain. The stimulatory effect was not observedunless the gene to be reverted was induced during the period of exposure to ICR-191. The presence of a strong polar (nonsense) mutation on the operator side of a

frameshift mutation abolished the stimulatory effect of inducer on reversion of theframeshift mutation by ICR-191. (As expected, a nonpolar mutation on the oper-ator side of the frameshift mutation did not affect inducer-stimulated reversion.) Itwas concluded that some aspect of transcription or translation, or both, in theneighborhood of the ICR-191-induced mutation stimulated reversion by ICR-191.A recA mutation had no effect on reversion by ICR-191 in the presence or absenceof inducer. In one mutant, gene induction depressed reversion by ICR-191 aboutsevenfold. The difference between this exceptional strain and other mutants was

not attributable to different genetic backgrounds but seemed to be an inherent dif-ference in the original Lac- mutations.

When considering the molecular basis of muta-tion, it is natural to ask about the effect of geneactivity on the mutational process. A priori onemight expect the mutation rate in a gene to de-pend on whether the gene is induced or re-pressed. In addition to its relation to the mecha-nisms of mutation and gene induction, such aneffect, in the specific case of spontaneous muta-tion, would obviously be of some interest in thefield of evolutionary biology.

ICR- 191 [3-chloro-7-methoxy-9-(3-[chloro-ethyl] amino propylamino) acridine dihydrochlo-ride] is an acridine half-mustard known to bevery effective in causing frameshift mutations(additions and deletions of small numbers, notmultiples of three, of base pairs) in bacteria (2,3, 13, 14). Frameshift mutations induced byICR-191 are revertible by ICR-191. Many of therevertants are not true revertants but carry asecond frameshift mutation within the same cis-tron as the original mutation. Internally sup-pressed mutants of this type were first describedby Crick et al. (4). We have observed that ICR-191 is an effective mutagen under conditions inwhich cell growth and induction of the lactoseoperon take place normally. In this report, weshow that the activity of the structural genes of

the lactose operon affects the rate of reversion byICR-191 of certain Lac- mutations.

MATERIALS AND METHODS

Media. The minimal medium was M63 (16) invari-ably supplemented with 5 ,g of thiamine/ml and some-times supplemented with 0.25% Casamino Acids(CAA). Carbon-energy sources were glycerol (0.2%),glucose (0.5%), or lactose (0.5%). Isopropylthio-galactoside (IPTG) was used as inducer at 5 x 10-4 M.f,-Galactosidase was assayed by the method of Pardee,Jacob, and Monod (16). Complete agar was lac-tet (7).

Bacteria. The principal strains used are listed inTable 1. LH97, LH98, and LH99 were isolated afteracridine orange treatment of many Lac+ segregants ofLHII9 (see reference 8 for details). LH133, LH138,LH143, LH145, LH152, and LH161 were isolatedfrom their respective progenitors as Lac- segregants(red colonies) on lac-tet agar. After purification, eachwas tested for inability to segregate Lac+ recombinantson lactose-minimal agar, ability to give lac+ recombi-nants when mated with an F- carrying a different Lac-allele, and inability to give lac+ recombinants whenmated with an F- carrying the same Lac- allele.LH 173 was isolated by picking many progeny ofLH172 and testing inducibility for f3-galactosidase. Thedouble mutant strains LH141, LH147, LH157, LH159,and LH 175 were isolated by the following procedure.Heterozygous merodiploid progenitors (LH 140,LH 146, LH 156, LH 158, and LH 174, respectively) were

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HERMAN AND DWORKIN J. BACTERIOL.

TABLE 1. Bacterial strains

Genotype in lac region Otherrelevant

Strain no. Sex Chromosome F-merogenote phenotypic Derivationlicharacter-

I Z I Z iStiCSa

+ S+ U131+ ++ D6+ 23 ++ ++ 4+ D23+ L32+ L32+ L32+ D23+ D23+ U131+ U131+ U 131, L32+ U131+ U131+ 4+ 4+ D23+ 4, D23+ L32+ D6+ D6+ D63 G73 G73 L323 G73 D6+ 2+ 2+ L32+ D6+ L32+ L32+ D6+ D6+ D6+ L323 +3 ++ U131+ U 131, D233 +

+ +

+ +

+ L32+ +

+ D23

+ L32

U 131+

44

+ +

+ D6

+ L32

+ D6

+ +

+ 2

3

3

D6

L32

D23

Sm8 (7)Sms E. SignerSm9 Rec- B. LowSmR ICR-191 mutagenesis of LH99SmR LHl19 (8)SmR LH 119 (8)SmR LH 119 (8)SmA (9)SmR ICR-191 mutagenesis of LH99Sm ICR-191 mutagenesis of LH99SmR A327 x LH132

LH 1321Sm8 A327 x LH123

LH137SmR CA7049 x LH99

LH133 x LH139LH140

SMR A327 x LH139LH142

SmR A327 x LH105LH 144LH145 x LH123LH 146

SmRLys- ICR-191 mutagenesis of LH132SmR A327 x LH16

LH149SmRHis- ICR-191 mutagenesis of LH16

ICR-191 mutagenesis of LH98LH133 x LH155LH156LH152 x LH155LH 158

SmR A327 x LH97LH160

SmRLys+Rec- KL16-99 x LH148SmRHis+Rec- KL16-99 x LH153

LH152 x LH148LH166LH 166LH133 x LH153LH169LH169A327 x LH98LH 172LH138 x LH139LH174ICR-191 mutagenesis of LH173

Lys-Lys-L.ys-His-His-His-SmR

His-

a Phenotypic abbreviations are as follows: SmR and Sms, streptomycin resistance and sensitivity, respectively;Lys- and His-, lysine and histidine requirements, respectively; Rec-, deficient in recombination owing to recAlmutation.

isolated, after mating of parents, as Lac- (red colonieson lac-tet) strains capable of segregating Lac+ recom-binants when streaked on lactose-minimal agar. Manyindependent Lac+ segregants from each heterozygotewere cured by acridine orange treatment. The Z (struc-tural gene for f,-galactosidase) genotypes of the cured

strains were tested by crosses with appropriate homo-zygous F' donors on lactose-minimal agar. The I (regu-lator gene) genotypes of LH157, LH159, LH141, andLH 175 were identified by mating with LH 176 andtesting inducibility of Lac+His+ recombinants. LH164and LH 165 were identified as Rec- (recombination-

544

A327 ......CA7049 ...KL16-99 ...LH16 .....

LH97 .....

LH98 .....

LH99 .....

LH105 ....

LH 123 ....

LH 132 ....

LH 1321LH 133 ....

LH137 ....

LH 138 ....

LH139 ....

LH140 ....

LH141 ....

LH142 ....

LH143 ....

LH144 ....

LH145 ....

LH146 ....

LH147 ....

LH148 ....

LH149 ....

LH152 ....

LH 153 ....

LH 155 ....

LH 156 ....

LH 157 ....

LH 158 ....

LH159 ....

LH160 ....

LH161 ....

LH164 ....

LH165 ....

LH166 ....

LH167 ....

LH168 ....

LH169 ....

LH170 ....

LH171 ....

LH172 ....

LH 173 ....

LH174 ....

LH 175 ....

LH 176 ....

F'HfrHHfrF-F-F-F-F-F-F-F'F'F'F'F-F'F-F'F'F'F'F'F-F-

F'F'F-F-

FtF-

FtF-

F'F'F-

F-

F'F-

F-

F'F-

F-

F'FtFtF-

F'

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GENE INDUCTION AND ICR-191 MUTAGENESIS

deficient) by both their high sensitivity to ultravioletirradiation (Mineralight UVS-12) and their very poorability to form Lac+ recombinants when mated withLH161. LH167 and LH170 were derived by acridineorange curing of LH166 and LH169, respectively.LH168 and LH171 were isolated after acridine orangecuring of several Lac+ segregants of LH 166 andLH169, respectively. The genotypes of LH167, LH168,LH 170, and LH 171 were identified by mating withLH 133 and LH 152. Acridine orange treatments andcrosses were performed as previously described (8).

Isolation of mutants induced by ICR-191. ICR-191was generously supplied by H. J. Creech of the Insti-tute for Cancer Research, Philadelphia, Pa. Lac- mu-tants induced by ICR-191 were isolated as follows.LH99 (and LH98 for the isolation of LH155) wasgrown for about five doublings in glucose-minimalmedium containing 10 ug of ICR-191/ml. Afterwashing out the mutagen by centrifugation, the muta-genized culture was allowed to grow for roughly fourdoublings and was then shifted to lactose-minimal me-dium. After one doubling, the culture was subjected topenicillin selection (6), and samples were spread on lac-tet agar. Lac- mutants were picked. Only strains thatwere able to grow in glucose-minimal medium and thatshowed low spontaneous reversion but high ICR-191-induced reversion [as shown by the spot test of Amesand Whitfield (2)] were chosen for further study. Weshall call these mutants frameshift mutants, althoughwe have not characterized them beyond using ICR-191to induce them and noting their easy ICR-191 reverti-bility. The lysine requirement of LH 148 and the histi-dine requirements of LH153 and LH176 were intro-duced by an analogous ICR-191 treatment and peni-cillin selection.

RESULTSMapping of Lac- mutants induced by ICR-191.

Twenty-four independent Lac- mutants wereclassified into seven different segments of the lacregion by mating with six different F' strains(kindly provided by J. R. Beckwith) carrying thefollowing overlapping deletions: E65, 06, D40,F33, D34, and 015 (15). Mated mixtures wereplated on lactose-minimal medium to select forLac+ recombinants. The derived map positionsare indicated in Fig. 1. All strains were negativewith respect to d-galactosidase activity exceptthose carrying mutations A13, B17, and D38.These three mutants showed normal f,-galactosi-dase activities and presumably are mutant in thepermease gene, Y. The 24 mutants are said to beindependent by virtue of the fact that no twomembers of the same segment of the map wereisolated from the same mutagenized culture.

Rates of reversion of Lac+ induced by ICR-191in the presence and absence of inducer. The fol-lowing procedure was used to compare the ratesof reversion to Lac+ induced by ICR-191 in thepresence and absence of IPTG. ICR-191 wasadded to a mutant culture growing exponentiallyin minimal-glycerol-CAA medium at 37 C to

z yI U131 I 4 1 1

C27 1.3 A20 1.2 A21 IA B20 2.7 A29 1.8 A28 1.9 Al3 1.9D3 1.4 B14 1.4 CIl 1.5 D23 2.1 B12 2.2 85 1.8 B17 0.9E5 1.3 C32 1.2 C20 2.7 C8 2.0 D38 1.1

D5 1.2 D12 2.3D6 0.2 Eli 1.8

L32 2.1

FIG. 1. Map positions of Lac- mutations inducedby ICR-191 and revertible by ICR-191. To the right ofeach mutation name is given the ratio of reversion ratein the presence of IPTG to reversion rate in the ab-sence of IPTG. Mutations U131 and 4 are not frame-shift mutations but were employed in this study.

give a final concentration of 10 ,g/ml. The cul-ture was immediately split into two subcultures.Inducer was added to one of the subcultures, andboth were then incubated with shaking. All ma-nipulations and incubations with ICR-191 werecarried out in dim light. After a 1- or 2-hr in-cubation with mutagen, each culture was washedtwice by centrifugation and plated (after suitabledilutions) on lactose-minimal and complete agarto measure the ratio of Lac+ cells to total viablecells. Background frequencies were approxi-mately 10-7 Lac+/total or less. Lac+ mutantfrequencies obtained after a I-hr exposure tomutagen ranged from about 10-6 to 10-1Lac+/total for the 24 strains listed in Fig. 1.The ratio of reversion rate in the presence of

inducer to reversion rate in the absence of in-ducer is given in Fig. I for each strain. Eachnumber represents an average of at least twoindependent measurements. The best indicationof the reproducibility of these measurements isprovided by the data obtained with L32, themutant we have studied most extensively. Theratio given in Fig. I for this strain is the samplemean obtained from 12 independent experiments;the sample standard deviation for these 12 meas-urements was equal to 16% of the sample mean(estimated standard deviation of the mean was5% of the mean). The ratios given in Fig. I formost of the other mutants were obtained fromonly two experiments per mutant. Several of themutants gave a roughly twofold greater reversionrate in the presence of inducer. One mutant, D6,gave about a sevenfold reduction in reversionrate in the presence of inducer. The reversion ofthe remaining mutants by ICR-191 was stimu-lated only slightly, if at all, by inducer. PossiblyIPTG would exert a greater effect on the latterstrains if a lower concentration of ICR-191 wereused. This possibility has not been tested. It maybe significant that within the Z gene the mutantsappear to fall roughly into two classes; thosemapping in the thrde segments on the Y side are

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stimulated by inducer to revert somewhat morethan those mapping in the three segments on theoperator side of Z (aside from D6, which seemsto be an exception to the general pattern). Threeof the mutations have been used in subsequentstudies reported below: L32, D23, and D6.

Dependence of reversion rates on concentrationof ICR-191. Reversion frequencies obtained withstrain LH132 after a 2-hr exposure to four dif-ferent concentrations of ICR-191 (in minimal-glycerol-CAA medium) in the presence and ab-sence of inducer are plotted in Fig. 2. The con-centration dependencies are roughly parallel,with inducer increasing the mutation rate overthe full range of concentrations of mutagen.Strain LH 16 behaved similarly except that in-ducer decreased the reversion rate over the samerange of concentrations. Typical frequencies ofreversion of LH16 after a 1-hr exposure to 10 ugof ICR-191/ml (in minimal-glycerol-CAA me-dium) were 1.5 x 1-' and 1 x 10-i in the pres-ence and absence of inducer, respectively.

Kinetics of mutagenesis by ICR-191. LH132was mixed with 10 ytg of ICR-191/ml in min-imal-glycerol-CAA medium with and without in-ducer. Samples were removed after 20, 40, and60 min, washed twice by centrifugation, resus-pended in the minimal-glycerol-CAA medium(omitting ICR-191 and IPTG), and assayed forLac+ mutants and total viable cells. As can beseen in Fig. 3, the mutant frequency increasedroughly linearly with time of exposure to mu-tagen both in the presence and in the absence ofinducer. After the 60-min treatment, washedsamples were returned to 37 C (in the same me-dium but lacking the mutagen and inducer), andsamples were removed to determine Lac+ mu-

,o/TOTAL

o1-5

0 5 10 15

CONCENTRATION OF ICR-191 (.US/ml)FIG. 2. Concentration dependence of reversion of

LH132 to Lac+ by ICR-191.

tants per milliliter and total cells per milliliter.Both cultures maintained their exponentialgrowth throughout the course of the experiment(doubling time: 44 min, not shown in the figure),but the number of Lac+ mutants per milliliterdid not begin to increase until at least 2 hr afterthe mutagenesis was initiated. This delay is quiteexpected and can be attributed to the lag in seg-regation of the mutant genes. It is clear that thepresence of inducer during mutagenesis has vir-tually no effect on the kinetics of this segrega-tion. The same can be said for LH 16, which dis-played the same kinetics of mutagenesis.Does inducer affect mutagenesis by altering the

effective concentration of ICR-191 in the cell?The double mutant LH148 was isolated to an-swer this question. The ICR-191-induced muta-tion giving rise to the lysine requirement hasbeen mapped by blendor experiments and mapsclose to min 55 of the E. coli linkage group (un-published experiments), which is far away fromthe lac region (18). A 1-hr exposure to 10 Mg ofmutagen/ml gave the same frequency of Lys+revertants/total cells in the presence and absenceof inducer (1.3 x 10-5 as compared with a back-ground frequency of less than 10-7), whereasinducer stimulated the reversion to Lac+ by afactor of 2.3, which is characteristic of L32. Anal-ogous results were obtained with LH 153: in-ducer decreased the reversion of D6 to Lac+ inthe usual manner but had no significant effect onreversion to His+. We also noted that the rever-sion to Lys+ by LH 148 and the reversion to His+by LH 153 showed dependencies on concentrationof ICR-191 that were similar to those for rever-sion to Lac+; in particular, concentrations higherthan 10 gg/ml gave higher reversion rates. We

-j4

I

LAC+/L

TIME (MIN)

FIG. 3. Kinetics of reversion of LH132 to Lac+ byICR-191 and kinetics of segregation of revertant al-leles. The ordinate on the left applies to the samplestaken from cultures containing mutagen (first 60 min).The ordinate on the right applies to samples takenafter mutagen was removed.

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GENE INDUCTION AND ICR-191 MUTAGENESIS

therefore conclude that I PTG does not affectICR-191 mutagenesis by altering the effectiveconcentration of ICR-191 in the cell.

Is ICR-191 mutagenesis influenced by geneinduction or by the inducer molecule per se?Strain LH157, which carries a regulator constitu-tive mutation (I- phenotype) and L32, was iso-lated to answer this question. Clearly, if it is theinduction of the Z gene and not the inducer mol-ecule per se which is critical, then inducer willnot stimulate mutagenesis by ICR-191 in thisstrain. Indeed, the frequencies of reversion ofLH157 in the presence and absence of inducerwere identical (2.24 x 10-5 and 2.29 x 10-5,respectively, after a 1-hr exposure to 10 ug ofICR-191/ml). We conclude that some aspect ofgene expression and not the inducer molecule it-self stimulates the rate of ICR-induced reversionof the L32 mutation. The same conclusion holdsfor the D6 mutation since LH159 (which is I-)gave the same frequency of reversion to Lac+ inthe presence and absence of inducer (about 10-5Lac+/total after a 1-hr exposure to 10 gg ofICR-191 /ml).Does gene induction before or after exposure to

mutagen modify mutant yield? In the experimentsso far described, inducer was present in the me-dium coincidentally with mutagen. We next askwhether inducer affects the rate of mutagenesis ifit is present either before or after (but not dur-ing) exposure of cells to ICR-191. The answer isthat for 30-min treatments with ICR-191 (10ug/ml), neither induction (for 60 min) immedi-ately prior to exposure to mutagen nor induction(for 60 min) immediately after exposure to mu-tagen had any significent stimulatory effect onthe rate of reversion of LH 132 or any significantinhibitory effect on the rate of reversion of LH 16.We conclude that, in order for gene induction tomodify the mutant yield, it must occur withinminutes at most of the time of exposure of cellsto mutagen. This indicates that inducer is notexerting its effect by selection for (in the case ofL32) or against (in the case of D6) Lac+ mu-tants. This conclusion was substantiated by re-construction experiments in which revertants ofL32 and D6 (five reverted by ICR-191 in thepresence and five in the absence of IPTG foreach parental strain) were purified, mixed indi-vidually with their Lac- parental strain at a ratioof 1:100 (Lac+ to Lac-, respectively), andtreated for I hr with 10 Mg of ICR-191/ml. Therecovery of Lac+ colonies was not affected byIPTG.

Reversion by ICR-191 of frameshift mutationsin strains bearing a base substitution mutation(polar or nonpolar) between the frameshift muta-tion and the lac operator. The simplest picture we

can draw from the foregoing experiments is asfollows: transcription or translation [or both,since they are intimately associated (1, 10, 11)]occurring in the neighborhood of the frameshiftmutation during or shortly after binding of mu-tagen to deoxyribonucleic acid (DNA), modifies(by some unknown mechanism since the mecha-nism of action of ICR-191 is unclear) the yieldof mutants. This picture can be tested further bymaking use of certain nonsense mutations.

Nonsense mutations lead to the termination oftranslation at the nonsense codon. In addition,nonsense mutations located near the operatorend of the Z gene exert a strong polar effect onthe expression of the Y and A genes, and lead tomarkedly reduced levels of lac operon messengerribonucleic acid (21). It is of interest, then, to seewhat effect the presence of a strong polar muta-tion located on the operator side of a frameshiftmutation has on the rate of reversion of theframeshift by ICR-191 in the presence and ab-sence of inducer. The double mutant strainsLH141 (U131, L32) and LH175 (U131, D23)were isolated for this purpose. The U131 muta-tion is an amber mutation and is strongly polar(15); see Fig. I for map position. The doublemutants are not reverted by ICR-191 since theycarry U131, which is a base-substitution muta-tion and is not revertible by ICR-191; therefore,to follow reversion of the frameshift mutation insuch a double mutant we must insert U131+ intothe genome after mutagenesis. This can be ac-complished very efficiently by mating the muta-genized culture (after removal of the mutagen)with a homozygous F-lac donor carrying U131+and also carrying the appropriate frameshiftmutation so that Lac+ colonies will not beformed unless the recipient's frameshift mutationhas been reverted by mutagen.The design of this set of experiments was as

follows. The double mutant was grown in min-imal-glycerol-CAA medium to about 108cells/ml. The culture was split into two parts.ICR-191 was added (10 or 15 gg/ml) to onepart, which was immediately split again into twoparts, to one of which was added IPTG. Thethree resulting cultures (one with mutagen andinducer, one with mutagen alone, and the thirdwith neither mutagen nor inducer) were incu-bated with shaking at 37 C for 60 min (90 min inone experiment), washed two times by centrifu-gation, and resuspended in minimal-glycerol-CAA medium without further supplements.After incubation for 20 min, samples were takenfrom each culture to assay the concentration oftotal viable cells, and each culture was matedwith about 108 F' donors/ml for 60 min, afterwhich samples were plated on lactose-minimal

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medium. The results of one experiment for eachstrain are presented in Table 2. All results havebeen confirmed by repeating the experiments.(Perhaps it is worth noting that the spontaneousbackground given in Table 2 would be more inline with our previously mentioned figure of 10-'it the data were given in terms of Lac+ coloniesper total viable cells, donors and recipients,present at the time of sampling.)

In control experiments, LH139, which carriesU131 and no frameshift mutation, was used inplace of the double mutants to measure the effi-ciency at which U131+ was inserted by the F-lacdonors. The results (see last entry in Table 2 forone experiment) showed that the efficiency ofinsertion is about 20% and is unaffected by thepresence of inducer in the recipient's mediumprior to the mating. This very high frequency ofrecombination within the Z gene is attributableto the fact that after the F-lac is transmitted tothe recipient it is permitted many chances for a

"mitotic recombinational event" during the re-

sidual growth of the heterozygous merodiploidon the lactose-minimal plate.

The results with LH141 and LH175 (Table 2)show that the strong polar mutation virtuallyabolished the stimulatory effect of inducer on theICR-191-induced reversion of L32 and D23.Conversely, as expected, when a nonpolar muta-tion was located on the operator side of D23 (instrain LH147), inducer gave a greater than two-fold stimulation of reversion of D23, which isusual for this mutation. These results are con-

sistent both with our picture of the effect of geneinduction on mutagenesis by ICR-191 and withcurrent ideas about polarity. In particular, theresults rule out a model in which the reversion of

L32 or D23 is affected solely by the state of theoperator (viz., bound to repressor or not):LH141 and LH175 presumably are capable ofnormal inducer-repressor-operator interactions(revertants are inducible). We conclude thatsome aspect of normal transcription or transla-tion, or both, in the neighborhood of the frame-shift mutation influences the back-mutation rate,and that this particular aspect of gene expression(which we expect to be closely associated withthe Z gene because of its effect on mutation)does not function normally on the operator-distalside of a strong polar mutation.

Effect of recA on reversion by ICR-191. Onemodel for the mechanism of action of acridinesin mutagenesis involves a recombination step(12). Certain bacterial mutants, designated Rec-,are defective both in the ability to recombinelinked genetic markers and in the ability to re-

cover from damage to DNA induced by ultravi-olet light. Therefore, it is obviously of interest toinvestigate the effect of rec mutations on ICR-191-induced mutagenesis. We found, usingstrains LH164 and LH165, that recA had no sig-nificant effect on reversion of L32 or D6 byICR-191, either in the presence or in the absenceof inducer. The recA marker did have a markedeffect on the ability of LH 164 and LH 165 toform colonies on complete agar after exposure tomutagen. After exposure to 10 ,ug of ICR-191/ml for 60 min, about 90% of the cells ofthese two strains did not form colonies on com-plete agar. (The same treatment had essentiallyno effect on the viability of LH132 and LH16.)The ratios of Lac+ revertants to total viable cellswere approximately the same as in recA+ strains,however. For example, in a typical experiment

TABLE 2. Effect ofstrong polar mutation on reversion offrameshift mutation in the same gene

Mutagenized strain Donor" ICR-191 treatment Inducer" Lac+ coloniesc

LH141 (U131, L32) LH133 (L32/F-L32) 60 min, lO g/ml + 4.9 x 10-660 min, 10 Ag/ml - 4.7 x 10-6None - 0.5 x 10-6

LH175 (U131, D23) LH138 (D23/F-D23) 90 min, 15 1sg/ml + 15.8 x 10-690 min, 15 jug/ml - 13.5 x 10-6None - 0.3 x 10-6

LH147 (4, D23) LH138 (D23/F-D23) 60 min, 10 gg/ml + 2.28 x 10-660 min, 10 tg/ml - 1.02 x 10-6None - 0.12 x 10-6

LH139 (U131) LH133 (L32/F-L32) 60 min, 10tlg/ml + 0.1960 Inin, 10 gg/ml - 0.18

a Postmutagenic donor of wild-type allele of base-substitution mutation.Presence (+) or absence (-) of inducer during mutagenesis.

c Lac+ colonies per viable recipient present at beginning of mating.

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(10 ug of ICR-191/ml for I hr in minimal-glyc-erol-CAA medium) with LH164, the ratios ofLac+ revertants to total viable cells in the pres-ence and absence of inducer were 1.28 x 10-4and 0.67 x 10-4, respectively.

On the difference between D6 and L32. As al-ready noted, the effect of gene induction on thereversion of D6 is peculiar compared with theother frameshift mutants we have isolated, inthat the rate of reversion of LH 16 and its deriva-tives which carry D6 is markedly reduced bygene expression. We have entertained the possi-bility that the aberrant behavior of D6 is due toa secondary mutation, perhaps affecting someaspect of the mechanism of ICR-191 mutagen-esis-maybe DNA repair-or some aspect ofgene expression. To test this possibility, we haveput L32 into the genetic background of LH148,which previously carried D6, and, conversely, wehave put D6 into the genetic background ofLH153, which previously carried L32. This ex-change of lac regions was accomplished by firstconstructing two heterozygous merodiploids:LH166 and LH169. From the former strain, weisolated two F- segregants: LH167 (D6) andLH168 (L32). Two F- segregants from LH169were also isolated: LH170 (D6) and LH171(L32). (See the Materials and Methods for detailsof the derivations of these strains.) It is clearthat except for the genetic material contained inthe F' factor, LH167 and LH168 have identicalgenetic backgrounds. The same statement can bemade for LH170 and LH171. Moreover, the F'factor carried by LH166 and LH169 is derivedfrom the F-lac of A327, which carries no morethan 1 min of the Escherichia coli linkage mapsince it carries neither proB nor proC (unpub-lished experiments). The results of reversionstudies are that inducer stimulates reversion ofLH168 and LH171 approximately twofold anddecreases reversion of LH167 and LH170 ap-proximately sevenfold. These results make it veryunlikely that the aberrant behavior of D6 com-pared with L32 is due to different genetic back-grounds. The most straightforward interpretationis that the nature of the D6 mutation itself isresponsible for its unique reversion property.

DISCUSSIONThe experiments reported in this paper show

that the activity of a gene can modify the rate ofICR-191-induced mutation within that gene. Theeffect of gene expression, however, was notunique: gene induction stimulated ICR-191-in-duced reversion of several Lac- mutations (orig-inally induced by ICR-191) approximately two-fold but inhibited reversion of another Lac-mutation approximately sevenfold. The differ-

ence between these two responses was not attrib-utable to different genetic backgrounds butseemed to be an inherent difference in the orig-inal Lac- mutations. Each type of response wasspecific for the lac operon and depended on geneexpression, not merely on the presence of an in-ducer molecule. Moreover, in each case, themodification of mutant yield by gene expressionrequired that the gene be active during (or atleast within minutes of) the period of exposure ofcells to ICR-191. The simplest hypothesis toaccount for the two distinctly different effects ofgene induction on reversion would be that thereversion even in one case requires a deletion andin the other case requires an addition, and thatthese two reversion events are modified in dif-ferent ways by gene expression. An obvious testof this idea would be to identify each of ourframeshift mutations as to whether it is an addi-tion or deletion. This we have not done.We have shown that the presence of a strong

polar (nonsense) mutation on the operator sideof L32 or D23 abolished the stimulatory effect ofgene induction on reversion of the latter muta-tions by ICR-191. We have concluded that someaspect of transcription or translation or both inthe neighborhood of L32 or D23 influences theirrate of reversion by ICR-191, and that this par-ticular aspect of gene expression does not func-tion normally throughout the operator-distal por-tion of the gene in which the strong polar muta-tion resides. This conclusion might also explainwhy gene induction stimulates reversion of mostmutations located in the front (operator-proxi-mal) three segments of the Z gene in Fig. I sig-nificantly less than the mutations located nearerthe Y gene. Frameshift mutations located in thefront three segments are generally strongly polar(14), possibly because of the generation of non-sense codons. Gene induction might then have areduced effect in stimulating reversion of thesemutations because of reduced activity of the Zgene on the operator-distal side of the frameshiftmutations (or the nonsense codons generated bythe frameshift mutations).An understanding of the mechanism of action

of gene expression on ICR-191 mutagenesis ob-viously depends on a better understanding thanwe now possess of the molecular basis of muta-genesis by ICR-191. The recombination of ho-mologous DNA segments is probably not in-volved in the mutagenic action of ICR-191, be-cause we found that the recA1 mutation had noeffect on the reversion of our Lac- mutations byICR-191. This result is in marked contrast tothat reported for mutagenesis by ultraviolet irra-diation, in which the recA+ function is claimedto be essential for the formation of mutants (20).

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HERMAN AND DWORKIN

Our result with the recAI mutants also appearsto contradict a proposed mechanism for the ac-tion of acridines involving recombination (12). Itis, of course, not clear that the mutagenic actionof ICR-191 is basically the same as that of acri-dines in bacteriophages. It is perhaps worthnoting in this regard that the complex concentra-tion dependence of ICR-191 mutagenesis in E.coli (Fig. 2) is rather similar to the concentrationdependence of proflavine mutagenesis of intracel-lular bacteriophage T4 (5).

Streisinger et al. (17) have proposed a mecha-nism for the production of frameshift mutationsinvolving mispairing errors occurring during therepair of single-strand interruptions in a regionof repeating bases or repeating base doublets in a

double-stranded DNA molecule. They have sug-gested that acridines act to stabilize improbablemispairing configurations, thereby making therepair of mispaired regions more likely. Thismodel generates deletions and additions byslightly different mechanisms (nuclease action isrequired in the formation of deletions but notadditions), thereby making it possible for geneexpression to affect the formation of these twoclasses of frameshift mutations in different ways.An obvious line of pursuit of the idea that geneexpression influences ICR- 191 mutagenesis byaffecting DNA repair is to employ various mu-

tants defective in DNA repair.Finally, it should be of interest to note that the

length of the segregation lag observed after ICR-191 mutagenesis (Fig. 3) should depend in a

simple way on the position of the reverted genein the sequence of gene replication, as pointedout by Vielmetter, Messer, and Schutte for mu-

tations induced in growing cultures by shortpulses of nitrosoguanidine (19). This propositionis presently under investigation.

ACKNOWLEDGMENTS

We thank Chio Tan for capable technical assistance, J. R.Beckwith, B. Low, and E. Signer for bacterial strains, and H.J. Creech for ICR-191.

This work was supported by Public Health Service grantAM1 1321 from the National Institute of Arthritis and Meta-bolic Diseases.

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