Copyright 0 1982 by the Genetics Society of America

METHYL METHANESULFONATE MUTAGENESIS IN BACTERIOPHAGE T4l

JOHN W. DRAKE

Loborotory of Genetics, Notional Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

Manuscript received March 29, 1982 Revised copy accepted August 17,1982

ABSTRACT

MMS induces diverse rII mutations from a wild-type background in bacterio- phage T4. About 56% are base pair substitutions, about 30% are frameshift mutations, and the remainder is a miscellaneous set of rapidly reverting or leaky mutants of unknown composition; but deletions were not detected. MMS- induced forward mutation is sharply reduced by the mutations px and y, which also reduce ultraviolet, photodynamic and y-ray mutagenesis and increase killing by all of these agents. Thus, many of the mutations arise via the T4 WXY system. The induction of G:C + A:T transitions was detected even in a px or y background using sensitive reversion tests, and the few forward rII mutations that were induced from this background also behaved like transition mutations. Thus, some MMS-induced mutations arise independently of the WXY system, perhaps as a result of the (rather weak) ability of MMS to alkylate the O6 position of guanine.

HE in vitro induction of mutations in bacteriophages by alkylating agents T was first demonstrated by LOVELESS (1958, 1959), who observed the pro- duction of r mutations in bacteriophage T2 by EMS but not by MMS. BAUTZ and FREESE (1960) studied the mutagenicity of EES by scoring the induced reversion of T4rII mutations but observed only very small (and statistically insignificant) increases in r mutant and rII+ revertant frequencies after MMS treatments. STRAUSS (1961) also reported no r mutation induction by MMS. As a result, it has sometimes been assumed (KRIEG 1963b; DRAKE 1970) that MMS does not induce mutations in bacteriophage T4, even though it is mutagenic in many cellular systems (see LOVELESS 1966).

While studying the in vitro induction of r mutations in bacteriophage T4 by ultraviolet irradiation, DRAKE (1966) observed that the mutants arose as mottled plaques that usually contained a majority of wild-type particles; furthermore, these mottled plaques were difficult to detect unless special plating conditions were employed. Plating with very soft top agar, for instance, increased plaque sizes and very substantially increased the efficiency of scoring r and mottled (r/r+) plaques. The ability of MMS to induce r mutants became apparent using

' Abbreviations: ZAP = 2-aminopurine; A T = adenine:thymine base pair; BA = base analogues (ZAP or 5BU); 5BU = 5-bromouracil; EES = ethyl ethanesulfonate; EMS = ethyl methanesulfonate; G:C = guanine:cytosine (or, in the case of bacteriophage T4, guanine:S-hydroxymethylcytosine) base pair; HA = hydroxylamine; MMS = methyl methanesulfonate; PF = proflavin.

Genetics 102 639-651 December, 1962.

640 J. W. DRAKE

these plating conditions. Furthermore, some rrr mutants were readily reverted by MMS. These initial observations prompted a detailed examination of the process of MMS mutagenesis in bacteriophage T4, which has revealed an apparently dual mechanism. Preliminary reports of some of these results have already appeared (DRAKE 1973; GREEN and DRAKE 1974).

MATERIALS AND METHODS

Strains: Escherichin coli BB was the standard host for growing T4 stocks; it does not distinguish between wild-type T4 and rrr mutants. E. coli B was used for scoring r mutants. E. coli KB restricts the growth of rII mutants and is the differential host for scoring rll+ revertants. TZL, T4D and T4B were used as wild-type phage strains. The various rrr mutants have been described previously: rUV mutants by DRAKE (1963) and rSM mutants by DRAKE and MCGUIRE (1967a). The px mutant (DRAKE 1973) was extracted from T4Dx (HARM 1963,1964) and extensively backcrossed into T4B. It displays the same increased UV sensitivity, reduced rate of recombination and reduced burst size as the original T4Dx strain, but probably contains modifiers that increase plaque size and affect certain aspects of its behavior (M. A. CONKLING, personal communication). The y mutant (BOYLE and SYMONDS 1969; BOYLE 1969), which is largely epistatic to px and displays a very similar phenotype, was isolated in a T4D background and has also been backcrossed into T4B. The hm mutation was also extracted from T4Dx (DRAKE 1973). Its only established phenotype is enhancement of sponta- neous and UV-induced (and now MMS-induced) mutation rates.

Media: Standard L broth contains (per liter) 10 g Bacto tryptone, 5 g Bacto yeast extract, 1 g glucose and 5 g NaCl. It was used for all growth experiments. Standard D broth, used for plating dilutions only, contains (per liter) 2 g Bacto tryptone and 5 g NaC1. Nutrient agars were prepared from a standard mixture containing 10 parts (by weight) Bacto tryptone, 1 part Bacto yeast extract, 0.2 parts glucose, 5 parts NaCl and 10 parts Bacto agar. Bottom agar contains 26.2 g of this mixture per liter, standard top (soft) agar contains 17 g/liter, and super-soft top agar contains 10.3 g/liter.

MMS treatment: MMS (Eastman Organic Chemicals) was dissolved in 0.12 M NaP04, pH 7.0, immediately before use; 0.10 or 0.05 M MMS solutions were usually prepared. Phage stocks in L broth were diluted (typically about 10-fold, but medium carry-over effects were negligible in control experiments) to 10'"/ml in the same buffer. Both solutions were then brought to 44-45", and equal volumes were mixed to initiate treatment. Treatment was terminated by a twofold dilution into 1 M sodium thiosulfate; its carryover in this and the subsequent protocols affected neither r nor r+ genotype frequencies nor burst sizes. The pH of the treatment mixtures remained constant during the course of treatment. Controls were treated identically except for the absence of MMS in the phosphate buffer.

Treated and control suspensions either were plated immediately or were passaged through BB cells first. In the latter case, the suspensions in thiosulfate solution (containing the survivors from 2.5 X lo9 phage/ml) were mixed with an equal volume of L broth containing log-phase BB cells concentrated to 109/ml. (Both phage and cell suspensions were brought to 37' earlier.) After 10 min on a rotary shaker the complexes were diluted 50-fold into warm broth. After an additional 50 min lysis was completed by the addition of chloroform. Average burst sizes were usually in the range of 200-1000 for untreated controls and 40-400 for treated samples but were lower for phage strains carrying the mutations px or y.

Inactivation proceeded with nearly single-hit kinetics at rates similar to those observed by LOVELESS (1958,1959,1966) and BAUTZ and FREESE (1960). [An apparently much smaller rate reported by BALDY, STROM and BERNSTEIN (1971) is caused by a misprint: they actually used a final MMS concentration of 0.0832 M (H. BERNSTEIN, personal communication),]

Characterizing induced mutants: The induction of r mutants was followed by plating on 1-day- old plates with B cells and super-soft top agar. Plaques exhibiting mottling, increased size or a sharp edge were picked and streaked on B cells using sterile paper strips. The presence of r plaques among the several dozen to a few hundred plaques in the streak was taken as evidence that the original plaque contained r particles. From a few percent to as many as a third of the isolates failed to contain r mutants by this criterion. Induced r frequencies typically varied by less than twofold from day to day. Most of this variation appeared at the higher doses where plaque morphologies

MMS MUTAGENESIS IN PHAGE T4 641

are more diverse. Much of this rate variation could be suppressed by normalizing r frequencies to lethal hits rather than to mole-minutes. Comparisons of rates of inactivation and mutagenesis were based either on parallel or on severally repeated nonparallel determinations.

When further characterization was desired, isolated r plaques were picked from the streaks (restreaking where necessary) and spot tested on KB cells. Isolates that failed to grow were classified as rII, and those that grew with an r morphology were classified as rI. The remainder, which produced an r+ morphology on KB cells, consisted of an uncharacterized mixture of rIII and leaky or very rapidly reverting rll mutants not readily susceptible to further analysis. The tight rll mutants were then grown into stocks using BB cells and were subjected to base-analogue spot tests using the protocol of DRAKE (1966) with 2-aminopurine and 5-bromodeoxyuridine. (Spot tests were also performed on a few hundred rll mutants with various concentrations of MMS, but only one mutant responded unequivocally. Quantitative reversion tests were therefore performed with MMS on a selection of previously well-characterized rrl mutants.) In some instances the rrr mutants were also subjected to quantitative reversion analysis using base analogues, hydroxylamine and proflavin as described previously (DRAKE 1963).

RESULTS

Inactivation: T4B, T4D, T2L and both v and rII mutants of T4B exhibited identical inactivation rates, which were reproducible to about +20% from day to day. Typical single-hit inactivation rate constants are given in Table 1. The hm mutant also exhibited the wild-type inactivation rate, but both px and y were more sensitive. As also observed by BALDY, STROM and BERNSTEIN (1971) for x, px exhibited an irregular bi- or multi-phasic (concave) inactivation curve below survivals of about 1%.

Induction of r mutants: Mutant frequencies were determined as a function of MMS dose using the super-soft top agar method. Some typical results are shown in Figure 1 and Table 1. Induced mutants almost always appeared in the form of mottled plaques containing both r and wild-type particles, with the latter usually in considerable excess. At the low to moderate doses (up to about eight lethal hits, corresponding to survivals greater than about 3 x lop4), the induction of r mutants was approximately linear. At higher doses mutant scoring becomes unreliable for two reasons: the increasingly great variability in plaque size makes the detection of r mutants difficult (especially among the smaller plaques), decreasing apparent mutant frequencies; whereas multiplicity or cross-reactivation on the plate may increase apparent mutant frequencies.

As shown in Table 1, the rate of induction of r mutants is strongly influenced by the T4 genotype. The mutations px and y sharply reduce MMS mutagenesis, whereas hm nearly doubles it. The mutation rate is indistinguishable, however, in T4B, T4D, T2L and T4Bv. When rI mutants were assayed by plating on BB cells, the r+ -+ r mutation rate was the same in both wild-type and rII backgrounds, about 4 x

Properties of MMS-induced r mutants: In the initial experiments, all r mutants appearing in treated and untreated samples were picked and screened for the subsets of rII mutants (Table 2). The rII/r ratios (column 5) reflected both MMS treatment and phage genotype. Although these ratios are only crude indices to mutagen specificity, and many of the data subsets of column 5 are too small to provide comfortable levels of statistical confidence, differences between MMS- induced and spontaneous mutants are clearly revealed. It has previously been noted in this laboratory that transition mutagens such as hydroxylamine and

per lethal hit.

642 1. W. DRAKE

TABLE 1

Rotes of MMS-induced inactivation and mutagenesis

Inactivation rate (hits Mutation rate per survivor Phage genotype per mole-minute) (r per lo4 lethal hits)

T4B, T4D, T2L, T4Bv 9 6 T4Bpx 23 -1.5 T4By 18 -0.2 T4Bhm 9 11

Lethal hits were calculated as in Figure 1. Mutation rates are only approximate for values below about 2 because of the very small factors of increase (if any) over the spontaneous background.

base analogues induce relatively low rII/r ratios, whereas spontaneous muta- genesis and the frameshift mutagen proflavin induce much higher ratios. The basis of this difference is not understood, but the low rII/r ratio (33%) with MMS suggests that many of the induced mutations consist of base pair substi- tutions. The high ratio (73%) for the spontaneous mutations suggests a prepon- derance of frameshift mutations or deletions. The values in the hm background simply reflect previous observations suggesting that hm specifically enhances base pair substitution mutagenesis (DRAKE 1973). The profile in the px back- ground is similar to that in the wild type.

The rII mutants from these experiments were then subjected to base analogue reversion spot tests (columns 6 and 7 of Table 2). MMS clearly induced transition-revertible mutations in all three genetic backgrounds, confirming the implications of the rII/r ratios. MMS mutagenesis generates a somewhat higher proportion of base pair substitution mutations in the hm background than in the wild-type background. The results with px confirm that the small MMS- induced increase in r mutant frequency seen in this background is real, since the treated and untreated samples were qualitatively different [O% vs. ca. 57% (uncorrected) or ca. 100% (corrected) BA(+) mutants].

The nature of MMS-induced rII mutations was then examined in much greater detail in a second set of mutants subjected to quantitative base analogue, hydroxylamine, and proflavin reversion tests. As shown in the last column of Table 3, about 56% of the induced mutants were reverted by base analogues, indicating ability to revert by a transition (but not excluding revertibility by transversions). Of these mutants, some 40% (0.41/1.03) were also reverted by hydroxylamine, indicating ability to revert along the specific pathway G:C + AT. The majority of the remaining mutants contained proflavin-revertible frameshift mutations, but, although the spontaneous frameshift mutations were mainly (12/18) occupants of the two major rII mutational hot spots, only a minority (4/12) of the MMS-sample mutants occupied these sites, and most of these (two or three of the four) were expected from the spontaneous background. The mutants reverting spontaneously but induced to revert neither by base analogues nor by proflavin are of unknown composition. The “other” mutants, of equally unknown composition, are not susceptible to reversion analysis in the first place because they are too leaky, too rapidly reverting spontaneously

MMS MUTAGENESIS IN PHAGE T4 643

I 1 1 I

2 4 6 8 Phage Lethal Hits

FIGURE 1.-MMS-induced T4r mutant frequencies as a function of phage lethal hits. Lethal hits (h) were calculated from the relationship S = exp(-h), where S = surviving fraction (0.05 M MMS). Standard deviations were estimated from the square roots of the mutant counts and were about 20% in the case of the 0-dose values.

or, in rare cases, nonreverting. The frequency of nonreverting mutations (e.g., deletions) was not detectably increased by MMS treatments.

Reversion of rII mutants by MMS: Numerous reversion tests were performed using well-characterized rrr mutants in various genetic backgrounds, with and without a single cycle of growth (“passaged”) in permissive (BB) cells after treatment to permit mutation expression before selection of revertants. Typical results with mutants in wild-type and hm backgrounds appear in Table 4. “G:C”

644 J . W. DRAKE

TABLE 2

Preliminary classification of spontaneous and MMS-induced mutants

Approxi- Average mate % factor of Approxi- Among rJJ MMS-in-

Genetic back- increase in mate I, duced ground MMS r mutants rll/r rlJ BA(+) BA(-) BA(+) rll

Wild-type - 16/22 73 2 14 + 7.4 51/132 33 15 36 40

hm - 6/19 32 1 5 + 10.7 103/267 39 53 50 55

- 9/16 56 0 9 PX + 3.2 14/37 29 a 6 106

The MMS-induced mutants derived from several experiments and doses and the average factor of increase in r mutant frequency was calculated as [sum of (number of r mutants at each particular dose) X (factor of increase over spontaneous background at that dose)] divided by (total number of r mutants at all doses). Thus, the percentage of rII mutants among total r mutants, which in the case of MMS treatments are corrected for spontaneous backgrounds, are approximate.

BA(+) [or BA(-)] indicates reversion [or not] in spot tests using ZAP or 5BU. The approximate percentage of MMS-induced rII mutants that were BA(+) was calculated by determining the absolute frequencies of BA(+) and BA(-) mutants at each MMS dose, subtracting the respective BA(+) and BA(-) spontaneous frequencies, determining the individual percent BA(+) at each MMS dose, and averaging these percentages each weighted against the number of rII mutants a t that MMS dose. Although the numbers of spontaneous mutants are small, their characteristics are similar to those of spontaneous mutants from a larger parallel study of ultraviolet mutagenesis, where the corresponding division into the BA(+) and BA(-) categories were 2 and 28 in the wild- type and 15 and 41 in the hm backgrounds.

mutants (revertible by hydroxylamine and, therefore, capable of reversion by G:C + A:T transitions) all responded to MMS. (rUV7+ revertants exhibit two distinct phenotypes when plated on B cells: semi-r and wild-type. Both types of revertants were approximately equally promoted by MMS.) The hm mutation, which nearly doubles the MMS-induced forward ( r+ + r ) mutation rate (Table I), had no consistent effect on the reversion of “G:C” mutants. It had no significant effect on the spontaneous or induced revertant frequencies of rSM94 and rUV13. It did, however, increase the spontaneous revertant frequency of rUV7 by approximately fivefold and the after-passage MMS-induced revertant frequency about twofold.

Neither the “A:T” mutants (revertible by 2AP but not by hydroxylamine and, therefore, capable of reversion by A:T -+ G:C but not by G:C -+ A:T transitions) nor the frameshift mutants (revertible by proflavin) were markedly reverted by MMS, subject to the reservations outlined below. In two cases, “A:T” mutants were also tested in hm backgrounds, still without notable revertibility. Two of the mutants, rSMlO and rUV200, are capable of reverting by transversions from A:T base pairs (RIPLEY 1975), and their nonresponsiveness to MMS suggests that this pathway is not readily induced. Two other mutants (rSM1OAM and rUV200AM, derived from ochre mutants) contain amber mutations known from their suppressibility patterns and other results to contain G:C base pairs capable

645 MMS MUTAGENESIS IN PHAGE T4

TABLE 3

Characteristics of spontoneous and MMS-induced rII mutants

Spontaneous

Pcr 10:’ Category No. survivors

Total r mutants 53 0.72 Total rll mutants 36 0.49

Reversion patterns of rII mutants

BA(+)HA(+) BA(+)HA(-)

BA(-) PF(-) PF(+)

Other

3 0.041 4 0.054

4 0.054 7 0.095

18 0.24

Major hot spots 12 0.16

Net (MMS-in- duced) fre-

MMS-treated quencies

Per 10’ Per 10” survi- survi-

No. vors vors W

104 6.9 6.2 35 2.3 1.8

10 0.66 0.62 34 7 0.46 0.41 22 12 0.80 0.55 30 3 0.20 0.14 8

6 3 0.20 0.10

100 4 0.27 0.10 6

-

~~

The MMS-induced mutants described above were harvested after exposure of wild-type T4B to 0.05 M MMS for 18 min. BA(+) = reverted by ZAP and/or 5BU in quantitative tests; HA(+) = reverted by hydroxylamine; PF(+) = reverted by proflavin. “Other” mutants consist of mixtures of nonreverting, leaky and very rapidly reverting mutants not susceptible to base analogue reversion tests. “Major hot spots” consist of proflavin-revertible mutants mapping at the r i i 7 and ri31 sites (in the expected ratio of about 21).

of reverting by transversions but not by transitions (RIPLEY 1975). Their lack of response to MMS emphasizes the general trend exhibited by the data of Table 4: only rII mutants capable of reverting by G:C + A:T transitions gave detectable responses to MMS.

Both the “A:T” and frameshift mutants exhibited very small but fairly uniform decreases in revertant frequency (average 16%) after MMS treatment with direct plating and small increases (average 65%) in revertant frequencies after passaging. We do not understand the reason for the small decreases in revertant frequencies observed upon direct plating, but identical decreases were observed when artificial mixtures of an rrr mutant and wild type (total r+ frequency adjusted to well above the rrr mutant itself) were treated similarly. Thus, the decreases are interpreted as selection effects, despite the lack of any systematic differences in the MMS sensitivities of the wild-type and several rII mutants when plated separately on permissive cells. If the small increases after passaging are normalized to the decreases before passaging [e.g., for rSM10, (8 X 34)/(14 x 6)], then, for both the “A:T” and frameshift mutants, the average MMS-induced increase is X.l-fold, and again there is no significant hm effect.

Since the mutations px and y reduced the rate of MMS mutagenesis in the forward direction (Table I), their effects were also tested upon MMS-induced reversion. The results appear in Table 5 but require careful interpretation. First and most simply, the small revertant frequency increases in treated “A:T” and

646 J. W. DRAKE

TABLE 4

MMS-induced reversion of T4rII mutants

Mutant Type No. of tests

rSM94

rSM94-hm r UV7

rUV7-hm rUVZ3

rUV13-hm rUV343

rSMl0 rSMlOAM rUV30 rUV183 rUV183-hm rUV199 rUV199-hm rUV200 r UV200AM

r UV28 r UV58 rUV113

G:C 4 2“ 2”

2“ 2“

zn 2“

G:C 4

G:C 4

G:C 2

A:T 2 A:T 2 A:T 2 A:T 4

3 A:T 4

3 A:T 2 A:T 2

FS 2 FS 5 FS 2

Revertants per 10“ survivors

Unpassaged Passaged

Control MMS __ 60 330 30 420 30 470 50 60 50 110

200 280 80 480 40 1200 GO 970 20 160

30 10 20 20 30 30 60 40

340 300 40 30

180 200 3 4 4 2

50 40 20 10 40 30

Control MMS

30 1000 9 170

10 160 30 GBO 40 350

190 860 GO 1800 30 590

9 540

___________

30 680

6 8 10 20

90 100 520 590 40 GO

280 510 2 4 5 8

40 ao

80 110 20 40 70 120

The mutants were treated with 0.05 M MMS for 3.5 min, and survivals ranged from 8 to 30%. Revertant frequencies, which are averages of the indicated numbers of tests, frequently varied by as much as twofold in repeated measurements. “G:C” mutants are revertible by hydroxylamine. “A:T” mutants are revertible by 2-aminopurine but not by hydroxylamine. “FS” (frameshift) mutants are revertible by proflavin.

These tests were run in a different decade, a different laboratory, and by a different individual from the other tests listed in this table; incubations were for 10 min. and survivals averaged 26%.

frameshift mutants after passaging were not affected by px or y, regardless of how the results were viewed. The average increase in revertant frequency was 19% greater in the px or y background compared with the wild-type background, and this increase was insignificant, whereas this 19% value dropped to 10% (again insignificant) when the normalizing procedure described in the preceding paragraph was applied.

In the case of the “G:C” mutants there was a apparent px/y-mediated decrease in MMS-induced reversion. Assuming a linear dose-response relation- ship, however, this was merely (and exactly) the result of the smaller dose applied to the px and y strains. The average reduction in Net Revertants was 61% unpassaged and 57% passaged. However, the relative dose to the px and y strains compared with the wild type was (0.025 M) (3.0 min) compared with (0.05 M) (3.5 min), a dose reduction of 4/7 = 57%. Assuming linear kinetics,

MMS MUTAGENESIS IN PHAGE T4 647

TABLE 5

Effects of px and y on MMS-induced reversion of T4rII mutants

Mutant

rSM94

rUV7 rSM94-y

rUV7-px rUV7-y rUV13 rUV13-px rUV13-y

rUV183 T UV183-PX rUV199 rUV199-y

rUV58

rUV113 rUV58-y

rUV113-y

Revertants per 10’ survivors

Unpassaged Passaged No. of

Type tests Control MMS Net Control MMS Net

G:C 3 4

G:C 4 4 4

G:C 3 4 3

A:T 1 2

A:T 1 2

FS 2 3

FS 2 3

130 305 175 202 191 -11 77 101 24

113 116 3 40 55 15

134 682 548 150 491 341 313 654 341

67 35 496 340 35 32

246 149

18 8 55 37 61 40 96 74

55 741 686 98 235 137 64 667 603 97 304 207 55 370 315 49 1148 1099 69 602 533

131 812 681

71 90 460 591

58 50 242 245

28 40 46 113 75 159 68 123

Mutants in wild-type backgrounds were treated with 0.05 M MMS for 3.5 min, and mutants in px or y backgrounds were treated with 0.025 M MMS for 3.0 min, thus producing equivalent survivals (ranging from 15 to 35%). “Net” indicates average MMS-induced revertant frequencies less the control values; values have not been rounded off to the one or two significant figures given in previous tables in order to display the calculations more explicitly.

therefore, neither px nor y reduced MMS mutagenicity at “G:C” base pairs on a mole-minute basis.

DISCUSSION

MMS does, after all, induce mutations in bacteriophage T4. The induced r mutants are unlikely to have arisen from selection artifacts, since the types of mutants produced differ from the spontaneous background in the decreased frequency of rrr mutants relative to total r mutants, the increased frequency of rII mutants revertible by base analogues, and the absence of the two major rII frameshift hot spots. The marked allele specificity revealed in the mapping and reversion tests also indicates that selection artifacts are not important, at least in the case of the strongly responding “G:C” mutants. It seems very likely that the previous nondetection of r mutants (LOVELESS 1959; BAUTZ and FREESE 1960; STRAUSS 1961) was simply caused by the unavailability of a suitable detection system. (LOVELESS’S use of T2 instead of T4 is unlikely to have been of significance in this respect, since we readily detected mutagenesis in T2 using the improved detection system.) Similarly, the failure to detect induced rever- sion of rrr mutants was probably the result of simply not testing suitable “G:C” mutants.

648 J. W. DRAKE

The more detailed results of this study will be discussed in the context of two mechanisms that seem likely to mediate MMS mutagenesis. The first depends upon the x and y genes, components of the WXY system; this mechanism is most easily observed in the induction of forward mutations. The second is independent of the WXY system and depends upon the induction of base tautomerism and consequent mispairing; this mechanism is most easily ob- served in the induction of reverse mutations.

An intact WXY system is required in T4 for the induction of r mutations by ultraviolet (GREEN and DRAKE 1974), photodynamic (DRAKE and MCGUIRE 1967b) and y-ray (CONKLING, GRUNAU and DRAKE 1976) irradiation, although a require- ment for w + function has been demonstrated only in the case of ultraviolet mutagenesis (M. A. CONKLING, unpublished results). Besides exhibiting reduced mutagenesis, x and y mutants are more easily killed. The WXY system functions in postreplication repair (MAYNARD-SMITH and SYMONDS 1973; M. A. CONKLING, unpublished results), as well as in mutagenesis, and resembles, in phenotype if perhaps not in mechanism, the recA-controlled system of the host (WITKIN 1976). However, at least with respect to ultraviolet irradiation, the T4 response is independent of the functional state of the host system (GREEN and DRAKE 1974), even when that system is fully induced (M. A. CONKLING, unpublished results).

Most MMS-induced forward mutation requires an intact WXY system; the exception, discussed below, involves the few mutants induced in a px back- ground. MMS-induced rII mutations are clearly diverse. Slightly more than half can revert by transitions and, of these, approximately % revert by A:T -+ G:C transitions and ?h by G:C -+ A:T transitions. Among the mutants not reverted by base analogues, most (12/15) were reverted by proflavin and, therefore, contained frameshift mutations; however, MMS did not promote frameshift mutagenesis at either of the two major rII frameshift mutational hot spots, nor has any other mutagen examined to date (e.g., BENZER 1961). It is possible that transversions were also induced but were not detected in the available reversion tests. Transversion-induced rII mutants would fall either into the transition-revertible class (when allowed by codon degeneracy or amino acid acceptability) or into the small class reverted neither by base analogues nor by proflavin. (This latter class could also contain frameshift and/or transi- tion mutations that happened not to respond to the corresponding diagnostic mutagens.) Deletions were not detectably induced by MMS.

In contrast to the bulk of the forward mutations, MMS-induced reversion of “G:C” mutants occurs independently of the WXY system: it was neither reduced in px or y backgrounds nor consistently enhanced by hm. This process also presumably accounts for the handful of transition-revertible rII mutations induced in a px background (Table 2).

A number of mutagens act independently of the mutagenic propensities of postreplication repair systems, probably by altering bases so as to cause them to mispair efficiently. A good example is EMS in T4 (R. R. GREEN, unpublished results) and in E. coli (KONDO et al. 1970). Comparisons between MMS and EMS are instructive at both the genetical and the chemical levels. EMS induces

MMS MUTAGENESIS IN PHAGE T4 649

transitions in both directions but favors G:C + A:T (KRIEG 1963a, b; U. RAY, L. BARTENSTEIN and J. W. DRAKE, unpublished results with EMS that amply confirm the earlier reports). EMS alkylates the O6 position of guanine and, to a lesser extent, the O4 position of thymine, in both cases promoting “G:T” mispairs (MEHTA and LUDLUM 1976, 1978; SAFFHILL and ABBOTT 1978; ABBOTT and SAFFHILL 1977, 1979); but guanine O6 is more frequently alkylated, favoring G:C + A:T transitions as observed. MMS, on the other hand, induces G:C + A:T transitions to a modest extent and A:T + G:C transitions hardly at all (discussed further below). MMS is also much less efficient than EMS in the alkylation of guanine at the O6 compared with the N7 position and does not detectably alkylate at thymine O4 (LAWLEY and SHAH 1972). Thus, although other expla- nations are possible, the WXY-independent mechanism of MMS mutagenesis is most simply explained by guanine O6 alkylation and subsequent “G:T” mis- pairing .

There remain two poorly explained aspects of MMS mutagenesis in phage T4: the patterns of immediate-vs.-delayed reversion of “G:C” mutants and the contrast between the very weakly induced reversion of “A:T” and frameshift rrr mutations and the readily induced forward mutation of the same pathways.

It is sometimes possible to identify the target base of a reverting base pair according to its pattern of immediate or delayed expression (LEVISOHN 1967; BALTZ, BINGHAM and DRAKE 1976; BINGHAM et al. 1976). If MMS converts a guanine to a mispairing residue in a T4 gene whose function is required prior to DNA replication, then the revertant will be expressed immediately (“unpas- saged”) if the guanine occupies the transcribed strand; while expression will be delayed (“passaged”) if the guanine occupies the complementary strand. From previous tests (BALTZ, BINGHAM and DRAKE 1976; P. R. TEMPEST and L. S. RIPLEY, unpublished results), the rSM94 guanine residue is deduced to occupy the transcribed strand, and the rUV7 and rUV13 guanines to occupy the comple- mentary strand. rUV7 consistently exhibited the expected delayed MMS rever- sion response, but rUV13 and rSM94 exhibited patterns that were somewhat variable but clearly similar despite their opposite purine:pyrimidine orienta- tions. The rUV23 and rSM94 results, therefore, would have to be attributed to experimental error (including, for instance, leakiness) or would require some combination of ad hoc explanations.

MMS clearly induces frameshift mutations in the forward direction: the absence of the major spontaneous hot spots precludes selection artifacts. MMS probably induces A:T + G:C transitions in the forward direction, as indicated by the substantial fraction of rrr mutations revertible by HA. In contrast, MMS was unable to generate unequivocal reversion responses of frameshift or “A:T” mutations. (Consistently small reversion responses were observed in passaged stocks, whereas revertant frequencies were consistently but weakly reduced in MMS-treated, unpassaged stocks. It is unclear whether these responses are caused by mutagenesis.) A similar inconsistency between forward and reverse mutational patterns has been observed with ultraviolet mutagenesis (J. W. DRAKE and M. A. CONKLING, unpublished results). One possible explanation is that few “A:T” or potential frameshift sites are capable of responding to MMS;

650 J. W. DRAKE

although detected in forward-mutation screens, such sites might happen not to be represented in the mutations chosen for reversion tests.

It should be noted that by no means all T4 DNA repair genes have been examined for effects upon MMS mutagenesis. Candidates for interesting effects include mms, which is sensitive to MMS lethality (EBISUZAKI, DEWEY and BEHME 1975), and uvs79, which is sensitive to ultraviolet (CUPIDO, SCHREIJ-VISSER and VAN DER REE 1982).

This study was initiated while the author was a National Institute of Health Special Fellow in the Department of Molecular Biology at the University of Edinburgh in 1971-72. It was continued in the Department of Microbiology at the University of Illinois at Urbana, where many of the experiments were carried out with the skilled assistance of DAVID W. RIPLEY and RONALD R. GREEN and with the support of grant VS-5L from the American Cancer Society, grant GB30604 from the National Science Foundation and Public Health Service grant A104886 from the National Institute of Allergy and Infectious Diseases. I am indebted to LYNN S. RIPLEY and BARRY W. GLICKMAN for their incisive comments on various drafts of this report.

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