Molec. gen. Genet. 152, 19-28 (1977) © by Springer-Verlag 1977
Bromouracil Mutagenesis in Escherichia coli Evidence for Involvement of Mismatch Repair Bj6rn Rydberg Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, 06520, USA
Summary. Bromouracil mutagenesis was studied in several strains of E. coli in combination with measurement of incorporation of bromouracil in DNA. For levels below 10% total replacement of bromouracil for thymine, mutagenesis was negligible compared with higher levels of incorporation. Such a nonlinear response occurred both when the bromouracil was evenly distributed over the genome and when a small proportion of the genome was highly substituted. Also, the mutation frequency could be drastically lowered by amino acid starvation following bromouracil incorporation. These observations suggest the involvement of repair phenomena. Studies of mutagenesis in recA- and uvrA mutants, as well as studies of prophage induction, did not support an "error prone" repair pathway of mutagenesis. On the other hand, uvrD- and uvrE- mutants, which are deficient in DNA mismatch repair, had much increased mutation frequencies compared with wild type cells. The mutagenic action of bromouracil showed specificity under the conditions used, as demonstrated by the inability of bromouracil to revert an ochre codon that was easily revertable by ultraviolet light irradiation. The results are consistent with a mechanism of bromouracil mutagenesis involving mispairing, but suggest that the final mutation frequencies depend on repair that removes mismatched bases.
Introduction The classical model of bromouracil (BrU) mutagenesis is that of mispairing during DNA replication (see Drake, 1970). The hypothesis is that BrU, which is normally incorporated in DNA in place of thymine, will occasionally pair with guanine instead of adenine. If this occurs during the actual incorporation of BrU, a G: C ~ A: T transition will result after segregation of the intermediate mismatch (G:BrU). On the other hand, if mispairing occurs during subsequent replica-
tion of DNA containing BrU (so-called "clean growth mutagenesis"), A : T -~ G: C transitions will result. Most experimental work seems to favor the first alternative, but the second alternative also has support (for a recent reference, see Ronen and Rahat, 1976). This model has been challenged by Pietrzykowska (1973), who reported that BrU mutagenesis in 2~phage apparently is affected by mutations uvrA, recA and lex in the host cell, and red in 2 phage, which control dark repair processes. She suggested a common mechanism for BrU and UV mutagenesis, involving a process dependent on recombinational repair. Pietrzykowska, Lewandowska and Shugar (1975) have also given evidence that repair processes modify the lethal effects of BrU incorporation. Witkin and Parisi (1974), on the other hand, showed that a tryptophan marker in E. eoli could be reverted by BrU equally effectively in mutants carrying recA, lex and uvrA mutations, which does not support the involvement of repair processes dependent on these gene functions. In the accompanying paper (Hutchinson and Stein, 1977) a similar conclusion is reached for mutagenesis in 2 phage. In the present work, evidence is given for the involvement, in connection with BrU mutagenesis in E. coli, of repair processes which are largely independent of recA and uvrA gene functions, but dependent on uvrD and uvrE functions. Nevers and Spatz (1975) have shown, by the use of a transfection assay with )v heteroduplex DNA, that uvrD- and uvrE mutants are defective in a process where mismatches are converted to homoduplexes (mismatch repair).
Materials and Methods Chemicals
5-bromouracil (BrU) was obtained from Sigma Chemical Co., and [methyl-3H] thymine, [methyl-14C] thymine and 5-[6-3H] bromouracil from New England Nuclear Corporation. The radiochemJcal
B. Rydberg: Bromouracil Mutagenesis in E. coli
20 purity of the labeled BrU (stored in 70% alcohol solution) was routinely checked by paper chromatography (Whatman No. 1 chromatography paper, descending in n-butanol:acetic acid:water 80:12:30 by volume). Only labeled BrU with a radiochemical purity better than 94% was used (the major breakdown product being labeled uracil). Ce//s The bacterial strains used are listed in Table 1. They are all derivatives of Escherichia coli K12. Bacteriophages T6 and 2 were standard laboratory wild type strains.
Media and Growth Conditions What will be referred to as " M 9 " buffer contains : 19 mM NH4C1; 42raM NazHPO4; 22raM KHzPO4; l m M MgSO4; 0.1mM CaC12 and 0.1 gg/ml thiamine (B0. K medium is made up by adding 5 ml 20% glucose and 10 ml 10% casamino acids (decolorized
Table 1. Bacterial strains Strain AB2497
Genetic markers F - thr-1, leu-6, proA2,
Source P. Howard-Flanders
his-4, argE3, lacY1, galK2, ara-14, xyl-5, mtl-1, thi-1, tsx-33, strA31, sup-37, thyA ~, drm-" AB2487
as AB2497, also recA13 P. Howard-Flanders
AB2500
as AB2497, also uvrA6
P. Howard-Flanders TRIM-selection b of W3101 (obtained from P. Howard-Flanders)
B82
F - galT22, thyA-, drm-"
B84
F
galT22, thyA-, dra- a, recA
TRIM-selectionb of W3101 recA (obtained from P. Howard-Flanders)
CR34
F
leu-6, thr-l, lacY1, thi-1, tonA21, supE44, thyA- drm- ~
Laboratory strain
KMBL1854
serAlO1, metA90, pheA1, A. R6rsch hisA323,proC32, trp-lO1, malB78, lacZ46, bio-2, thyA301, drm-301
KMBL1851
as KMBL1854, also uvrDlO1
A. R6rsch
KMBLI853
as KMBL1854, also uvrE502
A. R6rsch
B82 (2) B84 (2) AB2497 (2) AB2487 (2)
2-1ysogen of B82 2-1ysogen of B84 2-1ysogen of AB2497 2-1ysogen of AB2487
" Tentative designation obtained by testing the strain for growth inhibition from extraneous supplied thymidine (Lomax and Greenberg, 1968) b TRIM-selections were done according to Stacey and Simson (1965)
and vitamin-free) per 100 ml M9 buffer. The general liquid growth medium used was K medium supplemented with 0.1 mM thymine and, when growing strains KMBL1851-4, also supplemented with 50 gg/ml DL-tryptophan and 1 gg/ml biotin. Luria broth contains: 10 g/l (Difco) Bacto tryptone; 5 g/1 (Difco) Bacto yeast extract; 0.5 g/1 NaC1 and 0.12 g/1 NaOH. Luria plates contain Luria broth ÷ 1.85% (Difco) Bacto agar. Lambda (2) plates contain: 5 g/1 (Difco) Bacto tryptone; 8 g/1 (Difco) Bacto peptone; 1 g/1 NaC1, and 15 g/1 (Difco) Bacto agar. Rif-plates were made by adding 100 mg rifampicin (Sigma Chemical Co.), dissolved in 2 ml methanol, to one liter Luria agar just before pouring plates (Miller, 1972). Soft agar is 0.6% (Difco) Bacto agar with 0.8% NaC1 and 0.1 mM thymine. Medium 56 agar contains: 2.64 g/1 KH2PO4; 4.34 g/1 Na2HPO4; 1 g/1 (NH4)2SO4; 100 mg/l MgSO4.7HzO ; 5 mg/1 Ca(NO3)z; 0.25 mg/l FeSO4.7H20, and 1.5% (Difco) Bacto agar, SEMhis- plates were made from medium 56 agar with the addition of 2 g/1 glucose, 83 ~tg/ml DL-threonine, 83 gg/ml DL-leucine, 167 gg/ml L-proline, 147 ~tg/ml L-arginine, 0.17 ~tg/ml thiamine, 12.6 gg/ml thymine, and 1% Luria broth. SEMthr- plates had the same composition except that threonine was omitted and 87 ~tg]ml DL-histidine was added. SEMmet plates were made from medium 56 agar with the addition of 2 g/1 glucose, 167 gg/ml L-proline, 87 gg/ml DLhistidine, 42/~g/ml L-phenylalanine, 83 gg/ml DL-serine, 40 gg/ml DL-tryptophan, 1 gg/ml biotin, 12.6 gg/ml thymine and 1% Luria broth. In some experiments, the thymine in these plates was replaced by various thymine-BrU mixtures at a total concentration of 0.1 raM. Cells were grown in liquid medium with aeration either in a roller drum or in a water bath shaker at 35-37°C with cell doubling times (dependent on the strain) of 33-50 rain during exponential growth. Media were generally changed by centrifugation (5K, 5 10 min, room temp.) and resuspension of the pellet. When so noted, membrane filters (0.45 gm pore size) were used for quick washes and changes of media. Manipulations with cells containing BrU were in yellow light or with aluminum foil-covered tubes. Incubations were in the dark.
Mutagenesis and Scoring of Mutants BrU mutagenesis in liquid culture was performed by changing over exponentially growing cells in supplemented K medium to the same medium containing a defined BrU-thymine molar ratio, allowing growth at 37°C for a defined period of time, and then washing and resuspending the cells in M9 buffer containing 0.1 mM thymine. The number of viable cells after BrU uptake was generally close to that of a control culture grown in thymine. Auxotrophic revertants were scored by plating dilutions of the mutagenized cells on appropriate SEM plates and incubating the plates for two days. For scoring rifampicin resistant mutants, mutagenized cells were resuspended in supplemented K medium at 107 cells/ml (5-10 ml) and incubated overnight to allow 6-9 generations posttreatment growth. The number of rifampicin-resistant mutants per ml in an overnight culture was found by spreading appropriate dilutions on rif-plates (not more than one week old), and counting the resulting colonies after 18-24 h incubation at 37°C. When T6-resistant mutants were scored, the mutagenized cells were diluted to 107 cells/ml, grown to about 0.3 x 109 cells/ml, and then diluted again to 107 cells/ml, to allow a total of 11 14 generations posttreatment growth (necessary for segregation and phenotypic expression). The number of T6-resistant mutants per ml in an overnight culture was found by plating dilutions of the cells in 2.5 ml soft agar, containing about 109 plaque forming units of T6 phage, onto Luria plates. After hardening, a second layer of soft agar with the same amount of phage (but no cells) was poured
B. Rydberg: Bromouracil Mutagenesis in E. coli on top of the first layer. T6-resistant mutants were then counted after 18-24 h incubation at 37°C. The frequency of mutants in a culture was calculated as the number of mutants per ml (estimated as described above) divided by the total number of viable cells/ml, usually found by spreading dilutions on Luria plates. Control experiments were done to test for eventual selection against rifampicin-resistant or T6-resistant mutants. After mutagenesis and phenotypic expression, the frequency of rifampicinresistant mutants in exponentially growing cultures (in supplemented K medium) declined by a factor of 2 per five generations (strains AB2497, AB2487, AB2500, B82, B84) or three generations (strains KMBL1851-4), while the frequency of T6-resistant mutants (strains B82, B84) declined less than a factor of 2 in ten generations. During stationary phase in supplemented K medium (at saturation), the frequency of mutants did not change significantly in 20 h at 37°C. Amino acid starvation for 30 min at 37°C (protocol as in legend to Fig. 4) did not change the subsequent growth rate of rifampicin resistant cells relative to wild type cells. From these data it is estimated that within the context of each experiment, the maximal possible variation caused by selection after phenotypic expression is less than a factor of 2. BrU mutagenesis on plates was performed by spreading out about 2 x 107 unmutagenized cells from an overnight culture on SEM-plates containing various BrU-thymine ratios. Mutagenesis was thus obtained, in this case, during the growth that occurred on the plates before exhaustion of methionine (SEMmet- plates) or threonine (SEMthr- plates). Revertants were scored as colonies on a limited lawn of cells after two days incubation. When the total number of colonies on such a plate exceeded several thousand, the colonies on several small areas of the plates were counted with a stereomicroscope and the total number was then estimated. UV-mutagenesis was performed by irradiating shallow layers of cells at less than l0 s cells/ml in M9 buffer (supplemented with 0.1 mM thymine) with a low pressure Mercury germicidal lamp (15 W) giving predominantly 254 nm light. The dose rate was l J/ m2/s.
21 The BrU-thymine ratio of the labeled DNA was calculated by comparing the 3H/1'C counting rates on the paper slip and the growth medium of known BrU-thymine ratio. Calibration for different counting efficiences in the two systems was achieved by using [methyl-3H] thymine and [methyl-14C] thymine as labeled constituents under identical conditions and assuming the molar ratio of 3H/14C in this case to be identical in the DNA and in the medium. (Also, under identical conditions, 3H or ~4C label alone was used to get information of spillovers between the 3H and 1¢C channels in the scintillation counter for the two systems.) A possible source of systematic error in this procedure is the incorporation of 3H-uracil into DNA after conversion to 3H-cytosine (McCarthy and Britten, 1962). It was found, however, that a two-fold increase in concentration of unlabeled uracil had no effect on 3H-uptake into DNA for the strains tested, suggesting that this was of minor importance. Omitting nonradioactive uracil, however, resulted in increased 3H-uptake by the cells despite RNA hydrolysis.
Banding of DNA in CsCI Gradients Approximately 5 x 107 cells were suspended in 0.5 ml TE (0.05 M Tris; 0.05 M EDTA, pH 8.0) containing 200 ~tg/ml newly dissolved lysozyme and incubated for 15 minutes at 37°C. Sodium sarcosyl was then added to give 0.5%. After complete lysis, 1.5 ml TE and 2 ml chloroform:isoamyl alcohol (24: 1) was added and the mixture vortexed at maximum speed for one min to deproteinize and shear the DNA. After centrifugation (5K, 10 rain), 1.70 ml of the upper phase was withdrawn and mixed with 2.25 g CsC1. When dissolved, the solution was transferred to 5 ml cellulose nitrate centrifuge tubes, overlaid with mineral oil, and centrifuged at 30 K, 20°C, for 43 h in a Beckman SW50.1 rotor. The tubes were punctured at the bottom and 0.075 ml fractions were collected on paper slips, which, after drying, were washed three times in ice-cold 5% TCA, twice in ethyl alcohol, and twice in acetone. Radioactivity was then measured as described above for paper slips.
Estimation of BrU Uptake into DNA In experiments in which BrU-uptake was measured, [methyl14C] thymine was added at 0.04 gCi/ml and 5-[6-3H] bromouracil at 1 gCi/ml. Uracil at 50 gg/ml was also added to the medium in order to minimize uptake of [6-3H] uracil, which is a radiochemical breakdown product of 5-[6-3H] bromouracil. Higher uracil concentration was avoided because of an inhibitatory effect on thymine uptake (Budman and Pardee, 1967). In most instances, the cells were prelabeled with a4C-thymine for several generations to ensure that, at the end of the BrU uptake period, all thymine in DNA was uniformly labeled with t'C, and all BrU was labeled with 3H. The radioactivity in cellular DNA was estimated by the following procedure. About 10s cells in 0.2 ml M9 buffer (supplemented with 0.1 mM thymine) was added to paper slips (Whatman #17, cut in 1 x3 cm pieces). After 30 min to lh, the still wet paper slips were placed in separate test tubes containing 0.5 M NaOH and incubated for one hour at 37°C for hydrolysis of RNA (Watson and Yamazaki, 1973). The NaOH was then replaced by ice-cold 5% TCA. After allowing to stand for 10-20 rain, the slips were washed on a Buchner funnel three times with ice-cold 5% TCA, twice with ethyl alcohol and twice with acetone. When completely dry, the radioactivity of the slips was measured in a liquid scintillation counter, using 4 ml toluene-based counting fluid (14.4 g PPO and 0.18 g POPOP in 3 liters toluene). The radioactivity in the growth media was measured by mixing 0.4 ml of a 1/20 dilution in distilled water with 3.8 ml scintillation mixture (18 g Permablend (Packard Instruments) in 2 liters toluene + 1 liter Triton X-100).
Prophage Induction Total number of plaque forming units (pfu) in a treated, or untreated, suspension of lysogenic cells was estimated by plating out appropriate dilutions in 2.5 ml soft agar onto 2 plates, together with an indicator strain (CR34). The number of free phage was measured by briefly shaking the suspension with a drop of chloroform, spinning down the cells (5K, 10 min), and plating suitable dilutions of the supernatant onto ). plates.
Results Nonlinear Dependence o f Mutagenesis on B r U Incorporation W i l d t y p e E. coli cells w e r e g r o w n in l i q u i d c u l t u r e f o r t h r e e g e n e r a t i o n s in t h e p r e s e n c e o f v a r i o u s B r U t h y m i n e m i x t u r e s . A s s h o w n in F i g u r e 1, p a n e l s A a n d B, v e r y little m u t a g e n e s i s is i n d i c a t e d w i t h 1 0 % B r U - s u b s t i t u t i o n o r less in D N A , w h i l e levels a b o v e about 10% appear highly mutagenic. I n t h e e x p e r i m e n t s s h o w n in p a n e l s C a n d D , m u t a g e n e s i s w a s p e r f o r m e d b y g r o w i n g t h e cells f o r
B. Rydberg: Bromouracil Mutagenesis in E. coli
22 A I
I
I
B I
I
i
I
--~i
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r
r
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1120 i
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800
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_o
Bromouracil Mutagenesis in Escherichia coli Evidence for Involvement of Mismatch Repair Bj6rn Rydberg Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, 06520, USA
Summary. Bromouracil mutagenesis was studied in several strains of E. coli in combination with measurement of incorporation of bromouracil in DNA. For levels below 10% total replacement of bromouracil for thymine, mutagenesis was negligible compared with higher levels of incorporation. Such a nonlinear response occurred both when the bromouracil was evenly distributed over the genome and when a small proportion of the genome was highly substituted. Also, the mutation frequency could be drastically lowered by amino acid starvation following bromouracil incorporation. These observations suggest the involvement of repair phenomena. Studies of mutagenesis in recA- and uvrA mutants, as well as studies of prophage induction, did not support an "error prone" repair pathway of mutagenesis. On the other hand, uvrD- and uvrE- mutants, which are deficient in DNA mismatch repair, had much increased mutation frequencies compared with wild type cells. The mutagenic action of bromouracil showed specificity under the conditions used, as demonstrated by the inability of bromouracil to revert an ochre codon that was easily revertable by ultraviolet light irradiation. The results are consistent with a mechanism of bromouracil mutagenesis involving mispairing, but suggest that the final mutation frequencies depend on repair that removes mismatched bases.
Introduction The classical model of bromouracil (BrU) mutagenesis is that of mispairing during DNA replication (see Drake, 1970). The hypothesis is that BrU, which is normally incorporated in DNA in place of thymine, will occasionally pair with guanine instead of adenine. If this occurs during the actual incorporation of BrU, a G: C ~ A: T transition will result after segregation of the intermediate mismatch (G:BrU). On the other hand, if mispairing occurs during subsequent replica-
tion of DNA containing BrU (so-called "clean growth mutagenesis"), A : T -~ G: C transitions will result. Most experimental work seems to favor the first alternative, but the second alternative also has support (for a recent reference, see Ronen and Rahat, 1976). This model has been challenged by Pietrzykowska (1973), who reported that BrU mutagenesis in 2~phage apparently is affected by mutations uvrA, recA and lex in the host cell, and red in 2 phage, which control dark repair processes. She suggested a common mechanism for BrU and UV mutagenesis, involving a process dependent on recombinational repair. Pietrzykowska, Lewandowska and Shugar (1975) have also given evidence that repair processes modify the lethal effects of BrU incorporation. Witkin and Parisi (1974), on the other hand, showed that a tryptophan marker in E. eoli could be reverted by BrU equally effectively in mutants carrying recA, lex and uvrA mutations, which does not support the involvement of repair processes dependent on these gene functions. In the accompanying paper (Hutchinson and Stein, 1977) a similar conclusion is reached for mutagenesis in 2 phage. In the present work, evidence is given for the involvement, in connection with BrU mutagenesis in E. coli, of repair processes which are largely independent of recA and uvrA gene functions, but dependent on uvrD and uvrE functions. Nevers and Spatz (1975) have shown, by the use of a transfection assay with )v heteroduplex DNA, that uvrD- and uvrE mutants are defective in a process where mismatches are converted to homoduplexes (mismatch repair).
Materials and Methods Chemicals
5-bromouracil (BrU) was obtained from Sigma Chemical Co., and [methyl-3H] thymine, [methyl-14C] thymine and 5-[6-3H] bromouracil from New England Nuclear Corporation. The radiochemJcal
B. Rydberg: Bromouracil Mutagenesis in E. coli
20 purity of the labeled BrU (stored in 70% alcohol solution) was routinely checked by paper chromatography (Whatman No. 1 chromatography paper, descending in n-butanol:acetic acid:water 80:12:30 by volume). Only labeled BrU with a radiochemical purity better than 94% was used (the major breakdown product being labeled uracil). Ce//s The bacterial strains used are listed in Table 1. They are all derivatives of Escherichia coli K12. Bacteriophages T6 and 2 were standard laboratory wild type strains.
Media and Growth Conditions What will be referred to as " M 9 " buffer contains : 19 mM NH4C1; 42raM NazHPO4; 22raM KHzPO4; l m M MgSO4; 0.1mM CaC12 and 0.1 gg/ml thiamine (B0. K medium is made up by adding 5 ml 20% glucose and 10 ml 10% casamino acids (decolorized
Table 1. Bacterial strains Strain AB2497
Genetic markers F - thr-1, leu-6, proA2,
Source P. Howard-Flanders
his-4, argE3, lacY1, galK2, ara-14, xyl-5, mtl-1, thi-1, tsx-33, strA31, sup-37, thyA ~, drm-" AB2487
as AB2497, also recA13 P. Howard-Flanders
AB2500
as AB2497, also uvrA6
P. Howard-Flanders TRIM-selection b of W3101 (obtained from P. Howard-Flanders)
B82
F - galT22, thyA-, drm-"
B84
F
galT22, thyA-, dra- a, recA
TRIM-selectionb of W3101 recA (obtained from P. Howard-Flanders)
CR34
F
leu-6, thr-l, lacY1, thi-1, tonA21, supE44, thyA- drm- ~
Laboratory strain
KMBL1854
serAlO1, metA90, pheA1, A. R6rsch hisA323,proC32, trp-lO1, malB78, lacZ46, bio-2, thyA301, drm-301
KMBL1851
as KMBL1854, also uvrDlO1
A. R6rsch
KMBLI853
as KMBL1854, also uvrE502
A. R6rsch
B82 (2) B84 (2) AB2497 (2) AB2487 (2)
2-1ysogen of B82 2-1ysogen of B84 2-1ysogen of AB2497 2-1ysogen of AB2487
" Tentative designation obtained by testing the strain for growth inhibition from extraneous supplied thymidine (Lomax and Greenberg, 1968) b TRIM-selections were done according to Stacey and Simson (1965)
and vitamin-free) per 100 ml M9 buffer. The general liquid growth medium used was K medium supplemented with 0.1 mM thymine and, when growing strains KMBL1851-4, also supplemented with 50 gg/ml DL-tryptophan and 1 gg/ml biotin. Luria broth contains: 10 g/l (Difco) Bacto tryptone; 5 g/1 (Difco) Bacto yeast extract; 0.5 g/1 NaC1 and 0.12 g/1 NaOH. Luria plates contain Luria broth ÷ 1.85% (Difco) Bacto agar. Lambda (2) plates contain: 5 g/1 (Difco) Bacto tryptone; 8 g/1 (Difco) Bacto peptone; 1 g/1 NaC1, and 15 g/1 (Difco) Bacto agar. Rif-plates were made by adding 100 mg rifampicin (Sigma Chemical Co.), dissolved in 2 ml methanol, to one liter Luria agar just before pouring plates (Miller, 1972). Soft agar is 0.6% (Difco) Bacto agar with 0.8% NaC1 and 0.1 mM thymine. Medium 56 agar contains: 2.64 g/1 KH2PO4; 4.34 g/1 Na2HPO4; 1 g/1 (NH4)2SO4; 100 mg/l MgSO4.7HzO ; 5 mg/1 Ca(NO3)z; 0.25 mg/l FeSO4.7H20, and 1.5% (Difco) Bacto agar, SEMhis- plates were made from medium 56 agar with the addition of 2 g/1 glucose, 83 ~tg/ml DL-threonine, 83 gg/ml DL-leucine, 167 gg/ml L-proline, 147 ~tg/ml L-arginine, 0.17 ~tg/ml thiamine, 12.6 gg/ml thymine, and 1% Luria broth. SEMthr- plates had the same composition except that threonine was omitted and 87 ~tg]ml DL-histidine was added. SEMmet plates were made from medium 56 agar with the addition of 2 g/1 glucose, 167 gg/ml L-proline, 87 gg/ml DLhistidine, 42/~g/ml L-phenylalanine, 83 gg/ml DL-serine, 40 gg/ml DL-tryptophan, 1 gg/ml biotin, 12.6 gg/ml thymine and 1% Luria broth. In some experiments, the thymine in these plates was replaced by various thymine-BrU mixtures at a total concentration of 0.1 raM. Cells were grown in liquid medium with aeration either in a roller drum or in a water bath shaker at 35-37°C with cell doubling times (dependent on the strain) of 33-50 rain during exponential growth. Media were generally changed by centrifugation (5K, 5 10 min, room temp.) and resuspension of the pellet. When so noted, membrane filters (0.45 gm pore size) were used for quick washes and changes of media. Manipulations with cells containing BrU were in yellow light or with aluminum foil-covered tubes. Incubations were in the dark.
Mutagenesis and Scoring of Mutants BrU mutagenesis in liquid culture was performed by changing over exponentially growing cells in supplemented K medium to the same medium containing a defined BrU-thymine molar ratio, allowing growth at 37°C for a defined period of time, and then washing and resuspending the cells in M9 buffer containing 0.1 mM thymine. The number of viable cells after BrU uptake was generally close to that of a control culture grown in thymine. Auxotrophic revertants were scored by plating dilutions of the mutagenized cells on appropriate SEM plates and incubating the plates for two days. For scoring rifampicin resistant mutants, mutagenized cells were resuspended in supplemented K medium at 107 cells/ml (5-10 ml) and incubated overnight to allow 6-9 generations posttreatment growth. The number of rifampicin-resistant mutants per ml in an overnight culture was found by spreading appropriate dilutions on rif-plates (not more than one week old), and counting the resulting colonies after 18-24 h incubation at 37°C. When T6-resistant mutants were scored, the mutagenized cells were diluted to 107 cells/ml, grown to about 0.3 x 109 cells/ml, and then diluted again to 107 cells/ml, to allow a total of 11 14 generations posttreatment growth (necessary for segregation and phenotypic expression). The number of T6-resistant mutants per ml in an overnight culture was found by plating dilutions of the cells in 2.5 ml soft agar, containing about 109 plaque forming units of T6 phage, onto Luria plates. After hardening, a second layer of soft agar with the same amount of phage (but no cells) was poured
B. Rydberg: Bromouracil Mutagenesis in E. coli on top of the first layer. T6-resistant mutants were then counted after 18-24 h incubation at 37°C. The frequency of mutants in a culture was calculated as the number of mutants per ml (estimated as described above) divided by the total number of viable cells/ml, usually found by spreading dilutions on Luria plates. Control experiments were done to test for eventual selection against rifampicin-resistant or T6-resistant mutants. After mutagenesis and phenotypic expression, the frequency of rifampicinresistant mutants in exponentially growing cultures (in supplemented K medium) declined by a factor of 2 per five generations (strains AB2497, AB2487, AB2500, B82, B84) or three generations (strains KMBL1851-4), while the frequency of T6-resistant mutants (strains B82, B84) declined less than a factor of 2 in ten generations. During stationary phase in supplemented K medium (at saturation), the frequency of mutants did not change significantly in 20 h at 37°C. Amino acid starvation for 30 min at 37°C (protocol as in legend to Fig. 4) did not change the subsequent growth rate of rifampicin resistant cells relative to wild type cells. From these data it is estimated that within the context of each experiment, the maximal possible variation caused by selection after phenotypic expression is less than a factor of 2. BrU mutagenesis on plates was performed by spreading out about 2 x 107 unmutagenized cells from an overnight culture on SEM-plates containing various BrU-thymine ratios. Mutagenesis was thus obtained, in this case, during the growth that occurred on the plates before exhaustion of methionine (SEMmet- plates) or threonine (SEMthr- plates). Revertants were scored as colonies on a limited lawn of cells after two days incubation. When the total number of colonies on such a plate exceeded several thousand, the colonies on several small areas of the plates were counted with a stereomicroscope and the total number was then estimated. UV-mutagenesis was performed by irradiating shallow layers of cells at less than l0 s cells/ml in M9 buffer (supplemented with 0.1 mM thymine) with a low pressure Mercury germicidal lamp (15 W) giving predominantly 254 nm light. The dose rate was l J/ m2/s.
21 The BrU-thymine ratio of the labeled DNA was calculated by comparing the 3H/1'C counting rates on the paper slip and the growth medium of known BrU-thymine ratio. Calibration for different counting efficiences in the two systems was achieved by using [methyl-3H] thymine and [methyl-14C] thymine as labeled constituents under identical conditions and assuming the molar ratio of 3H/14C in this case to be identical in the DNA and in the medium. (Also, under identical conditions, 3H or ~4C label alone was used to get information of spillovers between the 3H and 1¢C channels in the scintillation counter for the two systems.) A possible source of systematic error in this procedure is the incorporation of 3H-uracil into DNA after conversion to 3H-cytosine (McCarthy and Britten, 1962). It was found, however, that a two-fold increase in concentration of unlabeled uracil had no effect on 3H-uptake into DNA for the strains tested, suggesting that this was of minor importance. Omitting nonradioactive uracil, however, resulted in increased 3H-uptake by the cells despite RNA hydrolysis.
Banding of DNA in CsCI Gradients Approximately 5 x 107 cells were suspended in 0.5 ml TE (0.05 M Tris; 0.05 M EDTA, pH 8.0) containing 200 ~tg/ml newly dissolved lysozyme and incubated for 15 minutes at 37°C. Sodium sarcosyl was then added to give 0.5%. After complete lysis, 1.5 ml TE and 2 ml chloroform:isoamyl alcohol (24: 1) was added and the mixture vortexed at maximum speed for one min to deproteinize and shear the DNA. After centrifugation (5K, 10 rain), 1.70 ml of the upper phase was withdrawn and mixed with 2.25 g CsC1. When dissolved, the solution was transferred to 5 ml cellulose nitrate centrifuge tubes, overlaid with mineral oil, and centrifuged at 30 K, 20°C, for 43 h in a Beckman SW50.1 rotor. The tubes were punctured at the bottom and 0.075 ml fractions were collected on paper slips, which, after drying, were washed three times in ice-cold 5% TCA, twice in ethyl alcohol, and twice in acetone. Radioactivity was then measured as described above for paper slips.
Estimation of BrU Uptake into DNA In experiments in which BrU-uptake was measured, [methyl14C] thymine was added at 0.04 gCi/ml and 5-[6-3H] bromouracil at 1 gCi/ml. Uracil at 50 gg/ml was also added to the medium in order to minimize uptake of [6-3H] uracil, which is a radiochemical breakdown product of 5-[6-3H] bromouracil. Higher uracil concentration was avoided because of an inhibitatory effect on thymine uptake (Budman and Pardee, 1967). In most instances, the cells were prelabeled with a4C-thymine for several generations to ensure that, at the end of the BrU uptake period, all thymine in DNA was uniformly labeled with t'C, and all BrU was labeled with 3H. The radioactivity in cellular DNA was estimated by the following procedure. About 10s cells in 0.2 ml M9 buffer (supplemented with 0.1 mM thymine) was added to paper slips (Whatman #17, cut in 1 x3 cm pieces). After 30 min to lh, the still wet paper slips were placed in separate test tubes containing 0.5 M NaOH and incubated for one hour at 37°C for hydrolysis of RNA (Watson and Yamazaki, 1973). The NaOH was then replaced by ice-cold 5% TCA. After allowing to stand for 10-20 rain, the slips were washed on a Buchner funnel three times with ice-cold 5% TCA, twice with ethyl alcohol and twice with acetone. When completely dry, the radioactivity of the slips was measured in a liquid scintillation counter, using 4 ml toluene-based counting fluid (14.4 g PPO and 0.18 g POPOP in 3 liters toluene). The radioactivity in the growth media was measured by mixing 0.4 ml of a 1/20 dilution in distilled water with 3.8 ml scintillation mixture (18 g Permablend (Packard Instruments) in 2 liters toluene + 1 liter Triton X-100).
Prophage Induction Total number of plaque forming units (pfu) in a treated, or untreated, suspension of lysogenic cells was estimated by plating out appropriate dilutions in 2.5 ml soft agar onto 2 plates, together with an indicator strain (CR34). The number of free phage was measured by briefly shaking the suspension with a drop of chloroform, spinning down the cells (5K, 10 min), and plating suitable dilutions of the supernatant onto ). plates.
Results Nonlinear Dependence o f Mutagenesis on B r U Incorporation W i l d t y p e E. coli cells w e r e g r o w n in l i q u i d c u l t u r e f o r t h r e e g e n e r a t i o n s in t h e p r e s e n c e o f v a r i o u s B r U t h y m i n e m i x t u r e s . A s s h o w n in F i g u r e 1, p a n e l s A a n d B, v e r y little m u t a g e n e s i s is i n d i c a t e d w i t h 1 0 % B r U - s u b s t i t u t i o n o r less in D N A , w h i l e levels a b o v e about 10% appear highly mutagenic. I n t h e e x p e r i m e n t s s h o w n in p a n e l s C a n d D , m u t a g e n e s i s w a s p e r f o r m e d b y g r o w i n g t h e cells f o r
B. Rydberg: Bromouracil Mutagenesis in E. coli
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