Vol. 129, No. 2 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 698-701 Copyright ©D 1977 American Society for Microbiology
Ultraviolet Induction of Prophage Lambda During Inhibition of Deoxyribonucleic Acid Synthesis by Hydroxyurea B. K. LYDERSEN1 AND ERNEST C. POLLARD* Biophysics Laboratory, Department ofBiochemistry and Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 14 January 1976
Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) preand caused the cessation of DNA synthesis. It did not cause induction of X. Superinfection of an irradiated lysogen with Xind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased. The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection. The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of X prophage to occur. cursors
The precise character of the events that lead to the induction of prophage X by inducing agents such as ultraviolet irradiation (UV), ionizing radiation, mitomycin C, and nalidixic acid is not understood. It has been suggested that the disturbances in semiconservative deoxyribonucleic acid (DNA) replication caused by inducing agents result in the accumulation of DNA precursors, which leads to the inactivation of X repressors (4). Although most prophage-inducing treatments do inhibit semiconservative DNA synthesis to some degree, most have also been shown to result in breakdown of the DNA, suggesting that breakdown products may instead be the effector molecules leading to prophage induction (3, 7, 10). To provide some evidence in distinguishing between the two proposals, we have used hydroxyurea (HU) to block the formation of DNA precursors in lysogens treated with UV or gamma rays. If prophage X can be induced in the presence of HU, it would suggest that an accumulation of DNA precursors caused by the action of the inducing agent on the DNA is not an essential step in X induction. Rather, it would point to another effect that most inducing agents have in common, such as the breakdown of the DNA molecule. MATERIALS AND METHODS Strains and growth conditions. Escherichia coli W3350 and the lysogens W3350(Xt) and W3350(t857ind-) were obtained from A. Campbell through J. Cowlishaw (Rochester, Mich.). Phage preparations of Xt857ind- and Xvir were obtained I Present address: Department of Biophysics and Genetics, University of Colorado Medical School, Denver, CO
80220.
from heat induction of a lysogenic culture and infection of a nonlysogenic culture, respectively. Lysates were clarified of cell debris by two centrifugations in a tabletop centrifuge (Sorvall) after treatment with chloroform, and stored at 4°C for no more than a month. The growth medium was a minimal salts medium (6) containing 4.6 g of NaCl, 1.5 g of KCl, 1.1 g of NH4Cl, 0.2 g of MgCl2 * 6H2O, 30 mg of CaCl2 * 2H20, 350 mg of Na2SO4, 250 mg of KH2PO4, and 14.5 g of tris(hydroxymethyl)aminomethane (pH 7.5) per liter supplemented with 1.8 g of Casamino Acids and 5 g of maltose per liter. Liquid incubation was always at 3500 with aeration. Plaque assays were performed by pouring 12 ml of TMg agar at 45°C onto plates containing 0.3 ml of a dense culture of W3350 and 0.1 to 1.0 ml of sample; plates were incubated at 35°C for 14 to 18 h. TMg agar consisted of 5 g of KCl, 1.2 g of MgSO4, 10 g of tryptone (Difco), 7 g of agar (Difco), and 2.3 ml of 2 N NaOH per liter. All infections were carried out in the growth medium with MgSO4 added to 10-2 M. HU (K & K Laboratories, Plainview, N.Y.) was added to the growth medium at 1.0 M on the day it was used. Methods of induction. UV irradiation was performed with a germicidal lamp (GE 15T8) wrapped with tape to diminish the output to about 43 J/m2 per min. Cultures were irradiated in the growth medium in a petri dish (60 by 15 mm) in a volume of 8 ml under constant stirring. Subsequent operations were performed under yellow lights to avoid photoreactivation. Gamma irradiation was performed at ice temperature in a fiCo Gammacell 200 (Atomic Energy of Canada, Ltd.) at a rate of 7 krads/min. Mitomycin C (Sigma) and nalidixic acid (Sigma) treatments were at 5 and 35 g.tg/ml final concentrations, respectively.
RESULTS Inhibition of DNA synthesis by HU. HU has been shown to reversibly block DNA synthesis 698
VOL. 129, 1977
PHAGE INDUCTION IN ABSENCE OF DNA SYNTHESIS
699
in both mammalian and bacterial cells by interTABLz 1. Effects of HU on X inductiona fering with the formation of deoxyribonucleoInfectious centers (% of cells forming Incubatide precursors of DNA by the enzyme ribonuplaques) in HU concn: tion in cleotide diphosphate reductase (2, 11, 13, 15). HU 0.06 HU at the concentration used for these experiOM (min) 00.03 M 0.1 M 0.2 M m ments has been shown to completely inhibit 0 0.9% DNA synthesis and cell division without a 60 2.2 1.5 1.1 1.1 2.0 great effect on the synthesis of RNA or protein 2.4 90 3.9 2.7 1.9 1.7 (12, 13). Figure 1 shows that 0.1 M HU causes a Samples of a log-phase W3350(A) culture were at least a 10-fold inhibition of [14C]thymidine incorporation under the experimental condi- centrifuged to remove free phage, resuspended, and tions we have used. diluted into prewarmed medium containing varying Effects of HU on A induction. The data in concentrations of HU, incubated at 35°C for the indiTable 1 indicate the effect of incubation of lyso- cated times, and diluted and plated for infectious genic cells in varying concentrations of HU. centers in the absence of HU. Data are recorded as percentage of cells at time zero forming plaques. The percentage of spontaneous infective centers Treatment separate samples with chloroform befor the control samples rises because the data fore platingofindicated that free phage did not conare recorded as percentage of cells at time zero. tribute significantly to the number of infectious cenAfter 90 min of incubation, the number of cells ters recorded. had increased fourfold. Cultures incubated in the presence of HU show some residual cell UV induces early steps in lytic development division for about 20 min, and a gradual loss of viability after 60 min (unpublished data). Since during incubation in HU. It is not possible to the number of infective centers is recorded as a determine whether induction of X by UV has percentage of cells at time zero, a very low level occurred in the presence of HU merely by dilutof induction at higher times and concentrations ing the cells out of HU (to allow DNA replicamay be indicated (5). However, this level of tion) and assaying for infective centers, because induction is not significant compared with the the induction could have begun before dilution. levels achieved by UV and other agents used, A probe must be introduced during the incubawhich ranged from 8 to 40% of the cell popula- tion in HU which will discriminate whether the induction process has begun or not. This can be tion. accomplished by supernfecting the irradiated lysogens with aX phage that codes for a repres50OOr sor that is not sensitive to irradiation induction (ind-). If the derepression of the prophage has not resulted in irreversible steps leading to lysis, the Ind- phenotype of the superinfecting c phage will be established and a plaque will not h. form when cells are diluted out of HU and plated for infective centers (14). This expression loooof the cI (repressor) gene of the infecting DNA c can occur in immune cells roughly 10 min after infection (8). HU should not greatly interfere 500with this process, since transcription and transA lation are hardly affected by the drug at the :t concentrations used (12, 13). On the other hand, ia if UV has caused irreversible early steps of prophage induction to begin, the Ind- phenotype of the superinfecting phage will not be established (14), and infective centers will form 50 IWWO0 IW upon subsequent dilution and plating. The ability of superinfecting Ind- phage to Minutes Incubation with Label the induction of prophage in irradiated prevent FIG. 1. Effect of HU on [4C]thymidine incorpora- cells depends on the elapsed time after irradiation. Strain W3350 growing on minimal medium tion. The solid circles in Fig. 2A show that, as with Casamino Acids was exposed to [14C]thymidine infection of irradiated cells (in the absence of at time zero (0). After 15 min, an aliquot was made 0.1 M HU (A), and samples were taken of both cul- HU) is delayed, more infective centers form upon subsequent dilution and plating. The tures for trichloroacetic acid-precipitable radioactivnumber of infective centers rises from the iniity. 0
0
/I
0
/0
0
a.
U)
0
O
-
700
J. BACTERIOL.
LYDERSEN AND POLLARD
--
I
I
I
B A
5s0o
0
0
o
0
0
U. C-
c
0
0
0-
2501 0-
0
0
..o
00
10
20
18
30 40 0 10 Minutes of Incubation
1
20
I 1
30
40
FIG. 2. Loss of repressor activity in irradiated lysogens. Cultures of W3350X at 1.3 x 108 cells per ml were treated with 75 s of UV exposure (A) or 13 krads of gamma irradiation (B), resulting in 40 and 8% induction and 0.1 and 7% cell survival, respectively. At the indicated time of incubation in the presence (0) or absence (@*) of0.1 MHU, 0.8-ml samples of the culture were added to 0.2 ml containing Xt857ind- at 7 x 109 phage per ml. After 5 min, at 35°C, 0.1 ml of A antiserum was added for 5 min, followed by chilling, dilution, and plating. Treatment of samples with chloroform showed that less than 1% of the plaques were due to unadsorbed phage. The number of infectious centers attained at 40 min corresponded to the number of induced cells, as indicated by the dashed line. The temperature during infection and subsequent incubations was kept below 35°C to avoid thermal inactivation of the heat-sensitive repressors coded for by phage Xt857ind-.
tial level (which may indicate incomplete infection) at 0 and 8 min after UV to the number of infective centers obtained when no phage were added, or when A wild type was used for infection, as indicated by the dashed line. The open circles in Fig. 2 represent infective centers obtained when the cells were irradiated, incubated, and infected in the presence of 0.1 M HU. Equivalent results were obtained if cells were incubated in HU 30 min before irradiation. Figure 2B indicates that, for ionizing as well as for UV irradiation, the induction of X proceeds with approximately the same kinetics, and to the same magnitude, regardless of the presence of HU. Similar experiments with mitomycin C or nalidixic acid as inducing agents also showed that induction occurred in the presence of HU (B. K. Lydersen, Ph.D. thesis, Pennsylvania State University, University Park, 1974). Effect of temporary HU incubation on latent periods. The approximate 30-min extension of the latent period for induction by UVcompared with infection or heat induction of a temperature-sensitive lysogen appears to be due in part to the period required by the cell to inactivate the A repressor (14). If this process of repressor inactivation can proceed after irradiation in the presence of HU, as indicated in Fig. 2, then it would be predicted that the latent period for UV induction should not be ex-
tended very much even if the cells are incubated in HU during the period required for repressor inactivation and early transcription and translation. Figure 3 shows that a temporary incubation of irradiated cells in HU for 50 min immediately after irradiation extends the latent period only by about 10 min, and a temporary incubation of 100 min extends the latent period by about 60 min. If the same lysogenic cells were infected with Xvir or a temperaturesensitive lysogen was heat-induced and HU was present for the first 50 min, the latent periods were extended by 30 and 45 min, respectively (B. K. Lydersen, Ph.D. thesis, 1974). In the latter two cases it appears that some development takes place which is independent of A DNA synthesis (7). Also, it can be noted in Fig. 3 that a 50-min incubation in HU after irradiation does not significantly reduce the number of induced cells that give rise to infective centers, and that the number of phage particles liberated is not greatly reduced. The 10-min increase in latent period for cells incubated in HU for the first 50 min after UV further supports the conclusion that the early stages of prophage induction do occur in the presence of HU.
DISCUSSION We have inhibited the semiconservative synthesis of DNA by preventing the formation of DNA precursors with HU without inducing A prophage. HU treatment did not induce A pro-
VOL. 129, 1977
PHAGE INDUCTION IN ABSENCE OF DNA SYNTHESIS
701
of a disturbance in semiconservative DNA replication are not necessary for prophage induction. It is clear that lesions in the DNA are necessary for prophage induction (1). From the evidence discussed here, it is apparent that processing of these lesions into derepression of prophage is not always dependent on the results of the inhibition of DNA synthesis often caused by the lesions.
CD
0
a-
ACKNOWLEDGMENTS This work was supported by the U.S. Energy Research and Development Administration [E(11-1)-2362]. We thank Ralph Christensen for many useful discussions and for preparation of the lambda antiserum.
E z
Minutes of Incubation
FIG. 3. Effect ofHU on latent period of UV induction. A culture of W3350X was incubated in 0.1 M HU for 30 min before 200 ergslmm2 of UW irradiation. Immediately after UV irradiation, samples of the culture were diluted into the presence or absence (O) of 0.1 M HU and incubation at 35°C continued. At 50 min (A) and 100 min (0) after exposure to UV, HU was removed from samples by dilution, and liquid incubation was continued. At the times indicated on the abscissa, samples were chilled, diluted, and plated for plaque-forming ability. Essentially the same results are obtained if the pre-UV treatment with HU is omitted.
phage. The accumulation of DNA precursors that may normally occur after a disturbance in DNA synthesis by inducing agents should not occur in the presence of HU, since an earlier step in DNA synthesis has already been blocked. The experiments reported in Fig. 2 and 3 show that the early steps in prophage induction take place in the presence of HU. Although the inhibition of DNA synthesis by HU may not have been absolute in these experiments, the similarity in kinetics and magnitude of induction in the presence or absence of HU leads us to conclude that the increased concentration of DNA precursors is not the cause of induction. The results presented here are in agreement with those of Monk and Gross (9), who showed the induction of X phage by UV in nonreplicating lysogens defective in initiation of DNA replication at high temperature. The fact that a high level of prophage induction occurs after the introduction of irradiated colI DNA into lysogens (indirect induction) without any detectable inhibition of host DNA replication (16) further supports the conclusion that the results
LITERATURE CITED 1. Borek, E., and A. Ryan. 1973. Lysogenic induction, p. 249-300. In J. Davidson and W. Cohn (ed.), Progress in nucleic acid research and molecular biology, vol. 13. Academic Press, Inc., New York. 2. Elford, H. 1972. Effect of hydroxyurea on ribonucleotide reductase. Biochem. Biophys. Res. Comm. 33:129-135. 3. Gudas, L., and A. Pardee. 1975. Model for regulation of Escherichia coli DNA repair functions. Proc. Natl. Acad. Sci. U.S.A. 72:2330-2334. 4. Hayes, W. 1968. The genetics of bacteria and their viruses, 2nd ed., p. 474-475. John Wiley & Sons, Inc., New York. 5. Heinemann, B. 1971. Prophage induction in lysogenic bacteria, p. 235-266. In A. Hollaender (ed.), Chemical mutagens, vol. 1. Plenum Press, New York. 6. Hertman, I., and S. Luria. 1967. Transduction studies on the role of a rec+ gene in the ultraviolet induction of prophage lambda. J. Mol. Biol. 23:117-133. 7. Joyner, A., L. Isaacs, H. Echols, and W. Sly. 1966. DNA replication and messenger-RNA production after induction of wild-type X bacteriophage and X mutants. J. Mol. Biol. 19:174-186. 8. Lieb, M. 1966. Studies of heat-inducible X mutants. II. Production of C, product by superinfectingA + in heatinducible lysogens. Virology 29:267-276. 9. Monk, M., and J. Gross. 1971. Induction of prophage X in a mutant of E. coli K12 defective in initiation of DNA replication at high temperatures. Mol. Gen. Genet. 110:299-306. 10. Monk, M., and J. Kinross. 1975. The kinetics of derepression of prophage X following ultraviolet irradiation of lysogenic cells. Mol. Gen. Genet. 137:263-268. 11. Pfieffer, S., and L. Tolmach. 1967. Inhibition of DNA synthesis in HeLa cells by hydroxyurea. Cancer Res. 27:124-129. 12. Rosencrantz, H., and J. Levy. 1965. A specific inhibitor of DNA synthesis. Biochim. Biophys. Acta 95:181183. 13. Sinha, N., and D. Snustad. 1972. Mechanism of inhibition of deoxyribonucleic acid synthesis in Escherichia coli by hydroxyurea. J. Bacteriol. 112:1331-1334. 14. Tomizawa, J., and T. Ogawa. 1967. Effect of ultraviolet irradiation on bacteriophage lambda immunity. J. Mol. Biol. 23:247-263. 15. Warner, H., and M. Hobbs. 1969. Effect of hydroxyurea on replication of bacteriophage T4 inEscherichia coli. J. Virol. 3:331-336.. 16. Wilkins, B., and S. Hollum. 1972. The kinetics of bacterial DNA synthesis during indirect induction of prophage X. Molec. Gen. Genet. 119:49-56.
JOURNAL OF BACTERIOLOGY, Feb. 1977, p. 698-701 Copyright ©D 1977 American Society for Microbiology
Ultraviolet Induction of Prophage Lambda During Inhibition of Deoxyribonucleic Acid Synthesis by Hydroxyurea B. K. LYDERSEN1 AND ERNEST C. POLLARD* Biophysics Laboratory, Department ofBiochemistry and Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 14 January 1976
Hydroxyurea inhibited synthesis of certain deoxyribonucleic acid (DNA) preand caused the cessation of DNA synthesis. It did not cause induction of X. Superinfection of an irradiated lysogen with Xind- could prevent induction, but the percentage of cells protected decreased as the time between irradiation and superinfection increased. The presence of hydroxyurea did not increase the time during which cells could be rescued by superinfection. The accumulation of DNA precursors after ultraviolet or ionizing radiation was not necessary for the induction of X prophage to occur. cursors
The precise character of the events that lead to the induction of prophage X by inducing agents such as ultraviolet irradiation (UV), ionizing radiation, mitomycin C, and nalidixic acid is not understood. It has been suggested that the disturbances in semiconservative deoxyribonucleic acid (DNA) replication caused by inducing agents result in the accumulation of DNA precursors, which leads to the inactivation of X repressors (4). Although most prophage-inducing treatments do inhibit semiconservative DNA synthesis to some degree, most have also been shown to result in breakdown of the DNA, suggesting that breakdown products may instead be the effector molecules leading to prophage induction (3, 7, 10). To provide some evidence in distinguishing between the two proposals, we have used hydroxyurea (HU) to block the formation of DNA precursors in lysogens treated with UV or gamma rays. If prophage X can be induced in the presence of HU, it would suggest that an accumulation of DNA precursors caused by the action of the inducing agent on the DNA is not an essential step in X induction. Rather, it would point to another effect that most inducing agents have in common, such as the breakdown of the DNA molecule. MATERIALS AND METHODS Strains and growth conditions. Escherichia coli W3350 and the lysogens W3350(Xt) and W3350(t857ind-) were obtained from A. Campbell through J. Cowlishaw (Rochester, Mich.). Phage preparations of Xt857ind- and Xvir were obtained I Present address: Department of Biophysics and Genetics, University of Colorado Medical School, Denver, CO
80220.
from heat induction of a lysogenic culture and infection of a nonlysogenic culture, respectively. Lysates were clarified of cell debris by two centrifugations in a tabletop centrifuge (Sorvall) after treatment with chloroform, and stored at 4°C for no more than a month. The growth medium was a minimal salts medium (6) containing 4.6 g of NaCl, 1.5 g of KCl, 1.1 g of NH4Cl, 0.2 g of MgCl2 * 6H2O, 30 mg of CaCl2 * 2H20, 350 mg of Na2SO4, 250 mg of KH2PO4, and 14.5 g of tris(hydroxymethyl)aminomethane (pH 7.5) per liter supplemented with 1.8 g of Casamino Acids and 5 g of maltose per liter. Liquid incubation was always at 3500 with aeration. Plaque assays were performed by pouring 12 ml of TMg agar at 45°C onto plates containing 0.3 ml of a dense culture of W3350 and 0.1 to 1.0 ml of sample; plates were incubated at 35°C for 14 to 18 h. TMg agar consisted of 5 g of KCl, 1.2 g of MgSO4, 10 g of tryptone (Difco), 7 g of agar (Difco), and 2.3 ml of 2 N NaOH per liter. All infections were carried out in the growth medium with MgSO4 added to 10-2 M. HU (K & K Laboratories, Plainview, N.Y.) was added to the growth medium at 1.0 M on the day it was used. Methods of induction. UV irradiation was performed with a germicidal lamp (GE 15T8) wrapped with tape to diminish the output to about 43 J/m2 per min. Cultures were irradiated in the growth medium in a petri dish (60 by 15 mm) in a volume of 8 ml under constant stirring. Subsequent operations were performed under yellow lights to avoid photoreactivation. Gamma irradiation was performed at ice temperature in a fiCo Gammacell 200 (Atomic Energy of Canada, Ltd.) at a rate of 7 krads/min. Mitomycin C (Sigma) and nalidixic acid (Sigma) treatments were at 5 and 35 g.tg/ml final concentrations, respectively.
RESULTS Inhibition of DNA synthesis by HU. HU has been shown to reversibly block DNA synthesis 698
VOL. 129, 1977
PHAGE INDUCTION IN ABSENCE OF DNA SYNTHESIS
699
in both mammalian and bacterial cells by interTABLz 1. Effects of HU on X inductiona fering with the formation of deoxyribonucleoInfectious centers (% of cells forming Incubatide precursors of DNA by the enzyme ribonuplaques) in HU concn: tion in cleotide diphosphate reductase (2, 11, 13, 15). HU 0.06 HU at the concentration used for these experiOM (min) 00.03 M 0.1 M 0.2 M m ments has been shown to completely inhibit 0 0.9% DNA synthesis and cell division without a 60 2.2 1.5 1.1 1.1 2.0 great effect on the synthesis of RNA or protein 2.4 90 3.9 2.7 1.9 1.7 (12, 13). Figure 1 shows that 0.1 M HU causes a Samples of a log-phase W3350(A) culture were at least a 10-fold inhibition of [14C]thymidine incorporation under the experimental condi- centrifuged to remove free phage, resuspended, and tions we have used. diluted into prewarmed medium containing varying Effects of HU on A induction. The data in concentrations of HU, incubated at 35°C for the indiTable 1 indicate the effect of incubation of lyso- cated times, and diluted and plated for infectious genic cells in varying concentrations of HU. centers in the absence of HU. Data are recorded as percentage of cells at time zero forming plaques. The percentage of spontaneous infective centers Treatment separate samples with chloroform befor the control samples rises because the data fore platingofindicated that free phage did not conare recorded as percentage of cells at time zero. tribute significantly to the number of infectious cenAfter 90 min of incubation, the number of cells ters recorded. had increased fourfold. Cultures incubated in the presence of HU show some residual cell UV induces early steps in lytic development division for about 20 min, and a gradual loss of viability after 60 min (unpublished data). Since during incubation in HU. It is not possible to the number of infective centers is recorded as a determine whether induction of X by UV has percentage of cells at time zero, a very low level occurred in the presence of HU merely by dilutof induction at higher times and concentrations ing the cells out of HU (to allow DNA replicamay be indicated (5). However, this level of tion) and assaying for infective centers, because induction is not significant compared with the the induction could have begun before dilution. levels achieved by UV and other agents used, A probe must be introduced during the incubawhich ranged from 8 to 40% of the cell popula- tion in HU which will discriminate whether the induction process has begun or not. This can be tion. accomplished by supernfecting the irradiated lysogens with aX phage that codes for a repres50OOr sor that is not sensitive to irradiation induction (ind-). If the derepression of the prophage has not resulted in irreversible steps leading to lysis, the Ind- phenotype of the superinfecting c phage will be established and a plaque will not h. form when cells are diluted out of HU and plated for infective centers (14). This expression loooof the cI (repressor) gene of the infecting DNA c can occur in immune cells roughly 10 min after infection (8). HU should not greatly interfere 500with this process, since transcription and transA lation are hardly affected by the drug at the :t concentrations used (12, 13). On the other hand, ia if UV has caused irreversible early steps of prophage induction to begin, the Ind- phenotype of the superinfecting phage will not be established (14), and infective centers will form 50 IWWO0 IW upon subsequent dilution and plating. The ability of superinfecting Ind- phage to Minutes Incubation with Label the induction of prophage in irradiated prevent FIG. 1. Effect of HU on [4C]thymidine incorpora- cells depends on the elapsed time after irradiation. Strain W3350 growing on minimal medium tion. The solid circles in Fig. 2A show that, as with Casamino Acids was exposed to [14C]thymidine infection of irradiated cells (in the absence of at time zero (0). After 15 min, an aliquot was made 0.1 M HU (A), and samples were taken of both cul- HU) is delayed, more infective centers form upon subsequent dilution and plating. The tures for trichloroacetic acid-precipitable radioactivnumber of infective centers rises from the iniity. 0
0
/I
0
/0
0
a.
U)
0
O
-
700
J. BACTERIOL.
LYDERSEN AND POLLARD
--
I
I
I
B A
5s0o
0
0
o
0
0
U. C-
c
0
0
0-
2501 0-
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..o
00
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30 40 0 10 Minutes of Incubation
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30
40
FIG. 2. Loss of repressor activity in irradiated lysogens. Cultures of W3350X at 1.3 x 108 cells per ml were treated with 75 s of UV exposure (A) or 13 krads of gamma irradiation (B), resulting in 40 and 8% induction and 0.1 and 7% cell survival, respectively. At the indicated time of incubation in the presence (0) or absence (@*) of0.1 MHU, 0.8-ml samples of the culture were added to 0.2 ml containing Xt857ind- at 7 x 109 phage per ml. After 5 min, at 35°C, 0.1 ml of A antiserum was added for 5 min, followed by chilling, dilution, and plating. Treatment of samples with chloroform showed that less than 1% of the plaques were due to unadsorbed phage. The number of infectious centers attained at 40 min corresponded to the number of induced cells, as indicated by the dashed line. The temperature during infection and subsequent incubations was kept below 35°C to avoid thermal inactivation of the heat-sensitive repressors coded for by phage Xt857ind-.
tial level (which may indicate incomplete infection) at 0 and 8 min after UV to the number of infective centers obtained when no phage were added, or when A wild type was used for infection, as indicated by the dashed line. The open circles in Fig. 2 represent infective centers obtained when the cells were irradiated, incubated, and infected in the presence of 0.1 M HU. Equivalent results were obtained if cells were incubated in HU 30 min before irradiation. Figure 2B indicates that, for ionizing as well as for UV irradiation, the induction of X proceeds with approximately the same kinetics, and to the same magnitude, regardless of the presence of HU. Similar experiments with mitomycin C or nalidixic acid as inducing agents also showed that induction occurred in the presence of HU (B. K. Lydersen, Ph.D. thesis, Pennsylvania State University, University Park, 1974). Effect of temporary HU incubation on latent periods. The approximate 30-min extension of the latent period for induction by UVcompared with infection or heat induction of a temperature-sensitive lysogen appears to be due in part to the period required by the cell to inactivate the A repressor (14). If this process of repressor inactivation can proceed after irradiation in the presence of HU, as indicated in Fig. 2, then it would be predicted that the latent period for UV induction should not be ex-
tended very much even if the cells are incubated in HU during the period required for repressor inactivation and early transcription and translation. Figure 3 shows that a temporary incubation of irradiated cells in HU for 50 min immediately after irradiation extends the latent period only by about 10 min, and a temporary incubation of 100 min extends the latent period by about 60 min. If the same lysogenic cells were infected with Xvir or a temperaturesensitive lysogen was heat-induced and HU was present for the first 50 min, the latent periods were extended by 30 and 45 min, respectively (B. K. Lydersen, Ph.D. thesis, 1974). In the latter two cases it appears that some development takes place which is independent of A DNA synthesis (7). Also, it can be noted in Fig. 3 that a 50-min incubation in HU after irradiation does not significantly reduce the number of induced cells that give rise to infective centers, and that the number of phage particles liberated is not greatly reduced. The 10-min increase in latent period for cells incubated in HU for the first 50 min after UV further supports the conclusion that the early stages of prophage induction do occur in the presence of HU.
DISCUSSION We have inhibited the semiconservative synthesis of DNA by preventing the formation of DNA precursors with HU without inducing A prophage. HU treatment did not induce A pro-
VOL. 129, 1977
PHAGE INDUCTION IN ABSENCE OF DNA SYNTHESIS
701
of a disturbance in semiconservative DNA replication are not necessary for prophage induction. It is clear that lesions in the DNA are necessary for prophage induction (1). From the evidence discussed here, it is apparent that processing of these lesions into derepression of prophage is not always dependent on the results of the inhibition of DNA synthesis often caused by the lesions.
CD
0
a-
ACKNOWLEDGMENTS This work was supported by the U.S. Energy Research and Development Administration [E(11-1)-2362]. We thank Ralph Christensen for many useful discussions and for preparation of the lambda antiserum.
E z
Minutes of Incubation
FIG. 3. Effect ofHU on latent period of UV induction. A culture of W3350X was incubated in 0.1 M HU for 30 min before 200 ergslmm2 of UW irradiation. Immediately after UV irradiation, samples of the culture were diluted into the presence or absence (O) of 0.1 M HU and incubation at 35°C continued. At 50 min (A) and 100 min (0) after exposure to UV, HU was removed from samples by dilution, and liquid incubation was continued. At the times indicated on the abscissa, samples were chilled, diluted, and plated for plaque-forming ability. Essentially the same results are obtained if the pre-UV treatment with HU is omitted.
phage. The accumulation of DNA precursors that may normally occur after a disturbance in DNA synthesis by inducing agents should not occur in the presence of HU, since an earlier step in DNA synthesis has already been blocked. The experiments reported in Fig. 2 and 3 show that the early steps in prophage induction take place in the presence of HU. Although the inhibition of DNA synthesis by HU may not have been absolute in these experiments, the similarity in kinetics and magnitude of induction in the presence or absence of HU leads us to conclude that the increased concentration of DNA precursors is not the cause of induction. The results presented here are in agreement with those of Monk and Gross (9), who showed the induction of X phage by UV in nonreplicating lysogens defective in initiation of DNA replication at high temperature. The fact that a high level of prophage induction occurs after the introduction of irradiated colI DNA into lysogens (indirect induction) without any detectable inhibition of host DNA replication (16) further supports the conclusion that the results
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