JOURNAL OF BACTERIOLOGY, Feb. 1978, p. 860-866 0021-9193/78/0133-0860$02.00/0 Copyright © 1978 American Society for Microbiology

Vol. 133, No. 2 Printed in U.S.A.

Near-UV Mutagenesis: Photoreactivation of 365-nm-Induced Mutational Lesions in Escherichia coli WP2s ROBERT B. WEBB

Division of Biological and Medical Research, Argonne National Laboratory, Argonne, Illinois 60439

Received for publication 16 August 1977

Reversion to tryptophan independence induced by 365-nm and 254-nm radiation was studied in Escherichia coli WP2s (B/r trp uvrA). Under aerobic conditions, the mutant frequency response was of the fluence-square or "twohit" type at both 365 and 254 nm when revertants were assayed on miniimal agar supplemented with 2% nutrient broth (SEM plates). In contrast, when mutants were assayed on miimal agar supplemented with tryptophan only, the revertant yield was reduced to very low values at 365 nm, whereas values substantially greater than with SEM plates were obtained at 254 nm. Premutational lesions induced by both 365-nm and 254-nm radiation were photoreactivated more than 10-fold when assayed on SEM plates, implicating pyrimidine dimers as premutational lesions at both wavelengths. The strong photoreactivation of 365-nm-induced mutagenesis contrasted strikingly with the complete absence of photoreactivation of 365-nm-induced lethality in this strain.

Early work clearly established that wavelengths of natural sunlight between 290 and 320 nm are mutagenic in Aspergillus crassa (14) and in liquid and frozen suspensions of Escherichia coli (2). However, efforts to induce mutations at wavelengths longer than 320 nm were either unsuccessful (14) or of doubtful validity (reviewed by Zelle and Hollaender [35]). More recent reviews on near-UV mutagenesis have been published by Eisenstark (11) and Webb

(27).

Induction of mutation to bacteriophage T5 resistance by wavelengths between 320 and 400 nm was unequivocally demonstrated by Kubitschek (15) in chemostat cultures of E. coli WP2s. A high mutation rate was obtained with low fluence rates from both a broad-spectrum nearUV source and an almost monochromatic 365nm source, both stringently filtered to remove wavelengths shorter than 330 nm. Mutagenesis at low fluence rates of visible light (wavelengths shorter than 400 nm removed by filtration) was reported by Webb and Malina (30, 31) in E. coli B/r. More recently, Speck and Rosenkranz (19) reported that visible light between 400 and 500 nm induced mutations of the base-substitution type to histidine independence in Salmonella typhimurium; frame-shift mutants were not produced. In addition, Witkin (34) has reported the induction of trp+ revertants in a tif derivative of E. coli B/r (tif mutants are induced for recA+ lexA+-dependent SOS repair by a short period of growth at 42°C) by illumination with broadspectrum fluorescent white light. The mutation

rate was much greater when the tif strain was incubated at 42°C for 60 min, suggesting the importance of the induced recA+ lexA+-dependent, error-prone repair in the near-UV and visible-light mutagenesis. The mutation rate was reduced by only 50% when wavelengths below 350 nm were removed by filtration. In this present study, photoreactivability of mutational lesions induced by 365-nm radiation was examined to assess the role of pyrimidine dimers in near-UV mutagenesis at high fluence rates in nongrowing cells. MATERIALS AND METHODS Bacterial strain. E. coli WP2s (B/r trp uvrA), obtained from E. M. Witkin, was used in this study. The mutation tested was tryptophan dependence (trp) to tryptophan independence (trp+). The tryptophan requirement is an ochre mutation that terminates a polypeptide in a cistron essential for tryptophan synthesis (3, 4). Irradiation procedures. Stationary-phase cells from nutrient agar plates incubated for 48 h at 37°C were suspended in M9 buffer (1) and were centrifuged and resuspended two times in M9 buffer to a final concentration of 1 x 10W to 5 x 10' cells per ml for 365-nm irradiation and 0.5 x 10' to 1 x 10" cells per ml for 254-nm irradiation. The upper value at each wavelength was the cell concentration that required a 10% correction in the fluence rate for shielding. Cell suspensions were irradiated in jacketed vessels made either of quartz for the 254-nm irradiation or of Pyrex for the 365-nm irradiation. The irradiation vessels had a 1-cm inside cross section and a capillary tube inserted at the bottom to provide for aeration and stirring of the cell suspension. Most irradiation at 254 860

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PHOTOREACTIVATION OF INDUCED MUTATIONAL LESIONS

and 365 nm was performed at 0°C (liquid) by circulating a mixture of ethanol and distilled water thermostatically controlled at -1°C through the vessel jacket. All procedures were carried out in a laboratory illuminated with yellow fluorescent bulbs (General Electric F40/GO) that transmit very little energy below 460 nm. Radiation sources. The source of far-UV radiation at 254 nm was a low-pressure mercury vapor lamp with an integral filter (G-275) (Penray Sc-il, Ultra-Violet Products) that provided more than 95% of the emitted radiation at 254 nm. The source of radiation at 365 nm was a 500-mm Bausch & Lomb monochromator (model 33-86-45-49) combined with a 2.5-kW mercury-xenon short-arc lamp (Hanovia 975C) in a Schoeffel LH52 housing and a predispersion prism (28). For 365 nm, stray light of wavelengths shorter than 335 nm was eliminated with a Corning 0-52 absorption filter ground to onehalf thickness (1% transmittance, 336 nm). Transmittance of this filter at 365 nm is 75% (28). Fluence rates at 254 nm were measured with a General Electric germicidal meter calibrated for the 254-nm wavelength. Fluence rates at 365 nm were measured with a calibrated YSI Kettering radiometer (model 65; Yellow Springs Instrument Co.). All calibrations were based on a National Bureau of Standards lamp. The procedures followed for photoreactivation (PR) were the same as those described previously (6, 29). The primary source of PR radiation was a projector with a 500-W quartz iodine lamp operated at 135 V. The beam was filtered by a 1-cm-light path liquid filter, containing 5% copper sulfate and 15% cobalt sulfate, and an Optics Technology LP400 absorption filter that transmitted wavelengths of approximately 370 to 440 nm (9). In some experiments, the 405-nm mercury line isolated by the Bausch & Lomb monochromator was used for PR. A PR fluence rate of about 75 W/m2 was measured from both sources with the YSI Kettering radiometer. Mutant assay. Generally, the method of Witkin (32) was used in measuring the number of revertants (reversion from tryptophan dependence [tip] to tryptophan independence [trp']) induced at the two wavelengths studied. Mutant yields were determined by spreading 5 x 10' to 4 x 107 cells on semienriched minimal (SEM) plates containing M9 buffer (1), 4 g of glucose per liter, 2% (vol/vol) nutrient broth (Difco), and 20 g of agar (Difco) per liter. In some experiments 1.5 or 3% nutrient broth supplement was used for the mutant assay. Survival was determined on the same plates used for mutant assays after appropriate dilution. Mutant and surviving colonies were counted after 4 days of incubation at 37°C. In some cases mutant and surviving cells were assayed on medium in the same manner as described above, except the nutrient broth supplement was replaced with a low concentration (0.15 pg/Ml) of L-tryptophan (TrpM plates). Spontaneous trp+ revertants were measured under all conditions employed. For most cultures of E. coli WP2s, fewer than one colony per plate appeared on M9 glucose plates in the absence of tryptophan or nutrient broth supplementation; these colonies arise from preexisting revertants in the population. In con-

861

trast, when the cells were assayed on 2% SEM plates, approximately 15 colonies appeared per plate over the range of 9.3 x 104 to 3.7 x 10' cells per plate (see Fig. 1), regardless of the number of unirradiated cells present. Other results indicate that this constancy extends from 1 x 104 to 4 x 108 cells per plate. If spontaneous revertants arise as a function of the total number of cell divisions that occur on the plate, this result is to be expected, since the limiting amount of added tryptophan (or tryptophan in the nutrient broth supplement) provides for approximately the same total number of cell divisions regardless of the number of cells initially plated. The 0.15-pLg/ml tryptophan supplementation in the TrpM plates resulted in approximately 10 spontaneous trp' colonies per plate over a similar range of cells plated (data not shown). With strain WP2s, altering the amount of tryptophan or nutrient broth supplementation of the minimal medium produced a corresponding change in the number of spontaneous revertants that appeared per plate. For example, 1% nutrient broth resulted in eight colonies per plate, and 4% nutrient broth resulted in 30 colonies per plate. In contrast, the yield of radiation-induced revertants remained approximately constant over the concentration range of 0.1 to 1.0 pg of tryptophan per ml and 1 to 10% (vol/vol) nutrient broth (data not shown). The levels of tryptophan (0.15 pg/ml) and nutrient broth supplementation (2%) were chosen to provide the maximum expression of radiation-induced mutants, a relatively low level of spontaneous "plate" mutants, and colonies large enough for easy recognition on the total count plates. Induced mutant frequencies were calculated by a modification of a method suggested by Kubitschek (16). When the number of preexisting mutants in the unirradiated population is significant (>1 per plate), induced mutant frequency M can be obtained with the

following expression: (ma- amp.S) -(mb- bmp,,S) M-Na-Nb

(1)

where ma is the number of mutant colonies that occur when Na viable cells are plated, mb is the number of mutant colonies that occur when Nb viable cells are plated (a is the dilution factor giving Na viable cells; b is the dilution factor giving Nb viable cells; since b can be expressed in terms of a, no generality is lost in setting a = 1; good results with strain WP2s were obtained for ratios of a:b of between 0.5 and 0.1), mpa is the number of preexisting mutants in a population of Na untreated cells, and S is the surviving fraction of the total population after treatment. The value of mpa is obtained by plating Na untreated cells in the absence of tryptophan or nutrient broth supplementation. Since preexisting mutants are killed at approximately the same rate as are nonmutants, the number present after irradiation or other treatment is given by mp,S. The total mutant colonies ma after plating Na viable cells are comprised of induced mutants mia, spontaneous plate mutants m.,, and preexisting mutants ampaS; thus, ma = mi. + ms. + amp,S. Similarly, the total mutant colonies mb after plating Nb viable cells are comprised of induced mutants mib, spontaneous

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WEBB

plate mutants m,b, and preexisting mutants bmp,,S; therefore, mb = mib + m.b bmp,S. As the number of spontaneous plate mutants is independent of the number of cells plated over several orders of magnitude (Fig. 1), it is evident that mi. = m..b. Also, since the number of induced mutants is proportional to the number of viable cells plated up to 4 x 107 per plate (Fig. 1), it follows that m,ib bmi.. In addition, by definition, Nb = bNW. Therefore, by substitution, the following relationship for mutation frequency can be obtained: M= [(amin + m,,, + amiAS) - amp,,S] -[(bmia + m. + bmS) - bm,S]

by the mutagen decreases (or increases) the number of spontaneous plate mutants that develop, the method described by equations 1 and 2 will give accurate results, but the method of Green and Muriel (12) will not reveal a change in spontaneous plate mutants, since the plate mutants are measured with untreated cells.

=

Na - bNa By simplification, setting a = 1, Mia- bmi M

mi(1

-

b)

mi.

Na-bNa Na(1-b) Na

Therefore, equation 1 is a valid expression of the induced mutant frequency. When the number of preexisting mutants is small (51 colony per plate), as is the case in most of the experiments described here, the number can be considered zero, and equation 1 can be simplified as follows:

(2)

m,-mb

M

Na -Nb If the spontaneous plate mutants are not subtracted from the initial colony counts or if they are treated as though they were preexisting mutants, calculated mutant frequencies in the irradiated samples will be much larger than the actual frequencies. A different method for dealing with plate and preexisting mutants has been presented by Green and Muriel (12). The two methods give essentially the same results if the number of spontaneous plate mutants is not altered by the experimental treatment. However, if treatment

'to1'1 ' 460

RESULTS The number of tip+ revertants as a function of the number of 365-nm-irradiated cells plated on SEM plates is shown in Fig. 1. Below 4 x 107 viable cells per plate, the number of induced revertant colonies was strictly proportional to the number of viable cells plated, provided the spontaneous plate revertants were subtracted. The number of revertant colonies remained constant from 5 x 107 to 2 x 10" cells per plate. A similar result was obtained with 254-nm radiation, except the proportionality of revertants to the number of cells plated extended to approximately 10O cells per plate (data not shown). The induction of reversion to tryptophan independence (tip

-.

tip+)

in E. coli WP2s

by

monochromatic wavelengths at 254 and 365 nm is shown in Fig. 2. This strain is approximately 7 x 10 times more sensitive to 254-nm than to 365-nm radiation (22, 28). The survival-curve shape of E. coli WP2s at 254 nm typically is an exponential with a small shoulder. At 365 nm, in contrast, the survival curve of this strain has a large shoulder (22, 28). When assayed on SEM plates, the mutational response at both wavelengths followed approximately a fluence-square relationship that can be interpreted as a "twohit" response. Two-hit relationships are typical of mutagenesis by far-UV radiation (3, 5, 10, 34).

~~~I

w

140_

IL

120 _/

so

40-+~~~~~~~~~~~~~~~

~~~~~~~UnirmAdOMe

I

20-

9g

x

le,ix

5x!07 NUMBES

iXola6

I5x10

2xIO0&?IO2

OF VIABLE CELLS PLATED

FIG. 1. Number of spontaneous trp+ revertants per plate (0) is plotted as a function of the number of cells of E. coli WP2s plated. In addition, the number of 365-nm-induced trp+ revertants that occur per plate (0) as a function of the number of viable cells of E. coli WP2s plated is shown. The 365-nm fluence was 1.5 x 10i Jim2. All cells were plated on SEMplates (2% nutrient broth). Surviving fraction of the 365-nm-irradiated cells was 0.4.

VOL. 133, 1978

When the nutrient agar-grown stationaryphase cells of E. coli WP2s were irradiated at 365 nm and assayed on the TrpM plates instead of 2% nutrient broth (SEM plates), the revertant frequency was much lower, approaching the limit of detection (Fig. 2). Cell survival at 365 nm also was decreased when the irradiated cells were plated on TrpM plates; however, the effect was smaller than for 365-nm mutagenesis. In contrast, when this population was irradiated at 254 nm, the revertant frequency obtained when the irradiated cells were assayed on TrpM plates was substantially greater than when assayed on SEM plates (Fig. 2). In addition, with this uvrA strain, survival after 254-nm irradiation also was greater when the cells were plated on TrpM plates instead of SEM plates. The effects of PR on lethality and mutagenesis after 365-nm irradiation at 00C are depicted for E. coli WP2s in Fig. 3 and 4. The absence of PR of lethality after aerobic near-UV irradiation supports results previously obtained with another uvrA strain, E. coli K-12 AB1886 (29). The absence of change in survival of cells inactivated at 365 nm and 00C upon exposure to PR irradiation at 25°C (Fig. 3 and 4) or at 00C (Fig. 4) suggests that concomitant PR is not a significant factor in survival when the cells are inactivated by near-UV radiation at 00C. In contrast to the absence of PR effects on cell survival, the revertant frequency was reduced more than 10-fold by exposing the 365nm-irradiated suspension to PR (370 to 440 nm or 405 nm) radiation at 250C for 30 min (Fig. 3 SEM

plates

zI

0

P>

*t

I

1-

FLUENCE,

lo-

36wnm,

Jm-2s

Vs

FIG. 3. Lethal (O, *) and mutagenic (0, 0) effects of 365-nm radiation at 0°C are shown for stationaryphase cells of E. coli WP2s in the presence (U, 0) and absence (0, 0) of maximal PR. The fluence rates were 1,100 W/m2 for 365-nm irradiation and 75 W/m2 for PR irradiation (370 to 440 nm). Maximal PR was obtained after a 30-min irradiation (370 to 440 nm) at 250C.

z > >

00-

_ and 0C 10 256C

n

gzc

14-

~> WrpM plates plates

2 1-

863

PHOTOREACTIVATION OF INDUCED MUTATIONAL LESIONS

i2

trpM

U.

SEM

a:

plates

0

Fr r

C

A

!

I

0

S

10

>

FLUENCE, 254nm, Jm-

FLUENCE, 365nm, Jm2xlO06

FIG. 2. Lethality (0, *) and mutagenesis (0, O) for stationary-phase E. coli WP2s (uvrA) irradiated at 254 nm at 25'C and at 365 nm at 0°C. Revertants were assayed on SEM plates (2% nutrient broth) (0) or on TrpMplates (0). Fluence rates were 0.65 W/m2 at 254 nm and 1,100 W/m2 at 365 nm.

20 15 PR TIME, min

25

30

35

FIG. 4. Effect of PR at 405 nm under nitrogen anoxia on lethality (0, A) and revertant frequency (0, A) in stationary-phase E. coli WP2s is shown after a single fluence (2.15 x 1(0 J/m2) of 365-nm radiation given at 0°C in air. The effect of PR is shown at 0°C (0, 0) and 25'C (A, A). The fluence rates were 1,000 W/m2 for 365-nm irradiation and 75 W/m2 for PR irradiation (405 nm).

and 4). This result indicates that PR of mutational lesions induced at 365 nm is similar to the results obtained at 254 nm with this strain by Witkin (32). The rate of PR of 365-nm-produced mutational lesions (T1/2 = 7 min) at a

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WEBB

J. BACTERIOL.

per 2.5 x 109 daltons of DNA per J per m2 in pot' cells (24) and at 7.5 x 10' per 2.5 x 109 daltons of DNA per J per m2 for a pot strain (R. D. Ley, personal communication). The number ofdimers per F37 (fluence yielding a surviving fraction of 0.37) in E. coli WP2s was 75 at 254 nm and 66 at 365 nm for aerobic conditions. As the PR sector (specific for pyrimidine dimers [18]) for lethality in this strain at 254 mm is approximately 0.75 (PR ratio of 4:1), pyrimidine dimers appear to be the major lethal lesion at this far-UV wavelength (data not shown). In contrast, whereas the number of dimers induced at 365 nm at 00C for a survival value of 0.37 under aerobic conditions was almost as great (66 dimers) as at 254 nm (75 dimers), no PR for lethality after 365-nm inactivation was detectable (Fig. 3 and 4) (29). The strong photoreactivability of mutational lesions at 250C under all conditions reported here clearly implicates pyrimidine dimers in trp+ reversion induction (Fig. 3 and 4) for aerobic irradiation at 254 and 365 nm. However, evidence has been presented that oxygen-dependent DNA lesions (nondimer) account for most of the lethal effect of 365-nm radiation (24). A mutation-induction response of the fluencesquare or two-hit type may occur when these or some other oxygen-dependent 365-nm lesions interact with the pyrimidine dimers. MutageneDISCUSSION sis at 365 nm was 4.5 times more efficient than Under aerobic conditions, pyrimidine dimers at 254 nm as a function of lethality (Fig. 5). are induced by 365-nm radiation at 5.5 x 10' Similarly, mutagenesis at 365 nm was 2.5 times dimers per 2.5 x l09 daltons of DNA per J per more efficient than at 254 nm as a function of m2 in uvrA, phr, and wild-type strains (22), and pyrimidine dimers per cell at the two wavesingle-strand breaks are induced at 3.0 x 10-' lengths (Fig. 2) (22). The greater efficiency of mutagenesis at 365 nm supports the occurrence of DNA lesions that are not formed at 254 nm. The data suggest the interaction of nondimer oxygen-dependent lesions with photoreactivable lesions (dimers) in 365-nm mutagenesis. Although oxygen-dependent single-strand breaks are strong candidates for the additional lesions (24), other DNA lesions also are produced by 365-nm radiation. A pyrimidine adduct (not a cyclobutane dimer) is produced in purified DNA from Haemophilus influenzae (8), and pyrimidine glycols are induced at 365 nm (13; P. A. Cerutti, personal communication). The high efficiency of the production of dimers and single-strand breaks relative to the absorbance of purine and pyrimidine bases at 10-2 10-, 365 nm, together with the complete oxygen deSURVIVING FRACTION FIG. 5. Relationship between mutagenesis and le- pendence of single-strand break formation (24), thality in stationary-phase cells of E. coli WP2s ir- suggests that the chromophore for these DNA radiated at 365 nm (0°C) (-, U) or at 254 nm (250C) lesions is not a component of DNA. Further(0, 0). Cels were assayed for lethality and mutagen- more, dimers and single-strand breaks were deesis on both SEM (0, 0) and TrpM plates (Or, I. tected only at very low levels in purified DNA Data are replotted from Fig. 2 and 3. of H. influenzae; a pyrimidine adduct not of the

PR fluence rate above saturation (9) was considerably less than the rate of PR for survival previously reported (TI/2 = 2.2 min) under similar conditions at 254 nm. Partial destruction of the PR enzyme by 365-nm radiation (23, 26) may account for the reduced PR rate for mutagenesis at 365 nm. The absence of PR of mutagenesis at 00C is consistent with enzymatic PR, as very little PR occurred with this strain at 0°C after 254-nm inactivation (9). The PR of the premutational lesions implicates cyclobutylpyrimidine dimers as the major mutational lesions induced at high 365-nm fluence rates. The relationship between revertant frequency (equation 1 or 2) and surviving fractionis shown in Fig. 5. The mutant frequency as a function of cell inactivation was much greater at 365 nm than at 254 nm when revertants were assayed on SEM plates. If 0.37 is taken as the surviving fraction that represents an average of one lethal event per cell, based on the Poisson distribution, revertant frequency can be expressed as revertants per lethal event. At 254 nm, 1.4 x 10-6 revertants were induced per lethal event; at 365 nm, 6.4 x 10-6 revertants were induced per lethal event. Therefore, the rate of mutagenesis relative to the inactivation rate was 4.6 times greater at 365 nm than at 254 nm when SEM plates were used for the assay.

IOu

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PHOTOREACTIVATION OF INDUCED MUTATIONAL LESIONS

cyclobutane type accounted for most of the photoproducts detected in the purified DNA (8). This result suggests that chromophores accounting for the production of dimers and singlestrand breaks at 365 nm in intact cells may be removed during purification of DNA. Recently, Tuveson and Satterthwaite (21) reported that mutations at the adenine locus in Neurospora crassa were not induced by nearUV radiation under conditions that readily produced mutations at 254 nm. In addition, Cabrera-Juarez and Espinosa-Lara (7) failed to observe significant near-UV mutagenesis under some cultural conditions with H. influenzae. The failure to observe near-UV-induced mutagenesis may be the result of a greater susceptibility of the error-prone recA+ lexA+-dependent repair system than of other components of repair to near-UV damage (23, 25) by the broad-spectrum source used. The greatly decreased mutant frequency after plating 365-nm-irradiated cells on TrpM plates instead of SEM plates (Fig. 2) is consistent with selective damage to repair systems or selective recovery on semienriched minimal medium of repair systems involved in mutagenesis. NearUV radiation was shown by Swenson and Setlow (20) to inhibit induced enzyme formation. If the error-prone inducible function (SOS repair) reported for far-UV mutagenesis (17, 33, 34) is inhibited at 365 nm, mutagenesis should require conditions that allow recovery of this function, a requirement that may be provided by SEM assay medium. ACKNOWLEDGMENTS I thank H. E. Kubitschek, B. S. Hass, and R. C. Bockrath for helpful discussions. This work was supported by the U.S. Energy Research and Development Administration. LITERATURE CITED 1. Anderson, E. H. 1946. Growth requirements of virusresistant mutants in Escherichia coli strain "B." Proc.

Natl. Acad. Sci. U.S.A. 32:120-128. 2. Ashwood-Smith, M. J., J. Copeland, and J. Wilcockson. 1967. Sunlight and frozen bacteria. Nature (London) 214:33-35. 3. Bridges, B. A. 1969. Mechanisms of radiation mutagenesis in cellular and subcellular systems. Annu. Rev. Nucl. Sci. 19:139-178. 4. Bridges, B. A., R. E. Dennis, and R. J. Munson. 1967. Differential induction and repair of ultraviolet damage leading to true reversions and external suppressor mutations of an ochre codon in Escherichia coli B/r WP2. Genetics 57:897-908. 5. Bridges, B. A., J. Law, and R. J. Munson. 1968. Mutagenesis in Escherichia coli. II. Evidence for a common pathway for mutagenesis by ultraviolet light, ionizing radiation, and thymine deprivation. Mol. Gen. Genet. 103:266-273. 6. Brown, M. S., and R. B. Webb. 1972. Photoreactivation of 365 nm inactivation of Escherichia coli. Mutat. Res.

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15:348-352. 7. Cabrera-Juarez, E., and M. Espinosa-Lara. 1974. Lethal and mutagenic action of black light (325 to 400 nm) on Haenophilus influenzae in the presence of air. J. Bacteriol. 117:960-964. 8. Cabrera-Juarez, E., and J. K. Setlow. 1977. Formation of a thymine photoproduct in transforming DNA by near ultraviolet irradiation. Biochim. Biophys. Acta 475:315-322. 9. Davies, D. G. G., S. A. Tyler, and R. B. Webb. 1970. A sequential repair model of photoreactivation in bacteria. Photochem. Phytobiol. 11:371-386. 10. Doudney, C. 0. 1975. The lesion hypothesis for UVinduced mutation in relation to recovery of capacity for DNA replication, p. 389-392. In P. Hanawalt and R. B. Setlow (ed.), Molecular mechanisms for the repair of DNA, part A. Plenum Press, New York. 11. Eisenstark, A. 1971. Mutagenic and lethal effects of visible and near-ultraviolet light on bacterial cells, p. 167-198. In E. W. Caspari (ed.), Advances in genetics, vol. 1w. Academic Press Inc., New York. 12. Green, M. H. L, and W. J. Muriel. 1976. Mutagen testing using trp+ reversion in Escherichia coli. Mutat. Res. 38:3-32. 13. Hariharan, P. V., and P. A. Cerutti. 1977. Formation of products of the 5,6-dihydroxydihydrothymine type by ultraviolet light in HeLa cells. Biochemistry 16:2791-2795. 14. Hollaender, A., and C. W. Emmons. 1946. Induced mutations and speciation in fungi. Cold Spring Harbor Symp. Quant. Biol. 11:78-84. 15. Kubitschek, H. E. 1967. Mutagenesis by near-visible light. Science 155:1545-1546. 16. Kubitschek, H. E. 1970. Introduction to research with continuous cultures. Prentice-Hall, Inc., Englewood Cliffs, N.J. 17. Radman, M. 1974. Phenomenology of an inducible mutagenic DNA repair pathway in Escherichia coli: SOS repair hypothesis, p. 128-142. In L. Prokash, F. Sherman, M. Miller, C. Lawrence, and H. W. Tabor (ed.), Molecular and environmental aspects of mutagenesis. Charles C Thomas Publisher, Springfield, Ill. 18. Setlow, R. B. 1966. Cyclobutane-type pyrimidine dimers in polynucleotides. Science 153:379-386. 19. Speck, W. T., and H. S. Roseitkranz. 1975. Base substitution mutations induced in Salmonella strains by visible light. Photochem. Photobiol. 21:369-371. 20. Swenson, P. A., and R. B. Setlow. 1970. Inhibition of the induced formation of tryptophanase in Escherichia coli by near-ultraviolet radiation. J. Bacteriol. 102:815-819. 21. Tuveson, R. W., and M. A. Satterthwaite. 1976. Comparison of ultraviolet and blacklight for the induction of nutritional independence at two loci in Neurospora crassa. Mutat. Res. 36:165-170. 22. Tyrrell, R. M. 1973. Induction of pyrimidine dimers in bacterial DNA by 365 nm radiation. Photochem. Photobiol. 17:69-73. 23. Tyrrell, R. M. 1976. RecA+-dependent synergism between 365 nm and ionizing radiation in log-phase Escherichia coli: a model for oxygen-dependent near-UV inactivation by disruption of DNA repair. Photochem. Photobiol. 23:13-20. 24. Tyrrell, R. M., R. D. Ley, and R. B. Webb. 1974. Induction of single-strand breaks (alkali-labile bonds) in bacterial and phage DNA by near-UV (365 nm) radiation. Photochem. Photobiol. 20:395-398. 25. Tyrrell, R. M., and R. B. Webb. 1973. Reduced dimer excision following near ultraviolet (365 nm) radiation. Mutat. Res. 19:361-364. 26. Tyrrell, R. M., R. B. Webb, and M. S. Brown. 1973. Destruction of photo-reactivating enzyme by 365 nm radiation. Photochem. Photobiol. 18:249-254.

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27. Webb, R. B. 1977. Lethal and mutagenic effects of nearultraviolet radiation, p. 169-261. In K. C. Smith (ed.), Photochemical and photobiological reviews, vol. 2. Plenum Press, New York. 28. Webb, R. B., and M. S. Brown. 1976. Sensitivity of strains of Escherichia coli differing in repair capability to far UV, near UV and visible radiations. Photochem. Photobiol. 24:425432. 29. Webb, R. B., M. S. Brown, and R. M. Tyrrell. 1976. Lethal effects of pyrimidine dimers induced at 365 nm in strains of E. coli differing in repair capability. Mutat. Res. 37:163-172. 30. Webb, R. B., and M. M. Malina. 1967. Mutagenesis in Escherichia coli by visible light. Science 156:1104-1105. 31. Webb, R. B., and M. M. Malina 1970. Mutagenic effects of near ultraviolet and visible radiant energy on contin-

cultures of Escherichia coli. Photochem. Photobiol. 12:457-468. Witkin, E. M. 1969. Ultraviolet induced mutation and DNA repair. Annu. Rev. Microbiol. 23:487-514. Witkin, W. M. 1975. Elevated mutability of polA and uvrA poLA derivatives of Escherichia coli B/r at sublethal doses of ultraviolet light: evidence for an inducible error-prone repair system ("SOS repair") and its anomalous expression in these strains. Genetics 79:199-213. Witkin, E. M. 1976. Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40:869-907. Zelle, M. R., and A. Holaender. 1955. Effects of radiation on bacteria, p. 365-440. In A. Hollaender (ed.), Radiation biology, vol. 2. McGraw Hill, New York. uous

32. 33.

34. 35.