Photochernrr~ryand Phorohrology. Vol. 29. pp. 61 I 614.
8 P c r j m i o n Press Ltd.
M)3 1-8655 ’791030 I-MI Ilo2.00/0
1979. Printed in Great Britain
RESEARCH NOTE
MODIFICATION OF LETHAL INTERACTIONS BETWEEN NEAR-ULTRAVIOLET (365 nm) RADIATION AND DNA-DAMAGING AGENTS REX M. TYRRELL and IVANOSCA S. CORREIA Instituto de Biofisica, Centro de CiEncias da Saude, (Bloco G), Universidade Federal do Rio de Janeiro. Rio de Janeiro, Brazil (Received 23 May 1978: accepted 27 July 1978)
Abstract-The modification of the lethal interaction between near-UV (365 nm) radiation and a second DNA-damaging agent by incubation between treatments in either a minimal salts medium or complete growth medium has been studied in the wild-type bacterial strain Escherichia coli K12 A B 1157. The results indicate that the lethal interaction may be separated into at least two distinct processes whose evaluation may help in classifying DNA-damaging agents in terms of the repairability of the DNA lesions induced. An observation of changes when methyl methane sulphonate is given prior to the irradiation treatment indicates that this chemical irreversibly damages repair enzymes.
INTRODUCTION
The interaction of near-UV (ultraviolet) radiation with other physical agents (far-UV radiation, ionizing radiation and heat) in bacterial systems has recently been reviewed (see Tyrrell, 1978a). A strong lethal interaction has also been observed between radiation at several near-UV wavelengths and methyl methane sulphonate (MMS) (Correia and Tyrrell, 1978, accompanying paper). The precise mechanism of the interactions is not clear except that damage to DNA and interference with DNA repair by the two interacting agents appears to be involved. A further insight into the nature of the interactions may be obtained by allowing an incubation interval (in various media) between the two treatments. Far-UV induced sensitization to X-rays is rapidly lost by incubation in buffer alone (Okuda, 1973), whereas the interaction between near-UV and ionizing radiation disappears only when cells are incubated in complete medium in the interval between irradiations (Tyrrell, 1976a). In the latter case, a near-UV induced protection from the second treatment also develops. Smith and Martignoni (1976) observed a similar development of resistance during incubation of X-irradiated populations in complete growth medium before UV treatment. In the following study, the changes in the lethal action of various agents during post-near-UV incubation have been observed. The results indicate that the lethal interaction provoked by near-UV may be the result of at least two distinct cellular changes whose expression depends on the nature of the second agent.
Bacterial growth and irradiations were carried out as described in the accompanying paper (Correia and Tyrrell, 1979).The source for 254 nm radiation was a General Electric 15 W germicidal lamp (GI5 T8) and the dose rate (1 Wm-’) was measured by means of a Latarjet meter. Mild heating (52°C) was performed as previously described (Tyrrell, 1976b). Treatment with methyl methane sulphonate (0.1 M , 37°C) has been described in the accompanying paper (Correia and Tyrell. 1979). Incubation. Irradiated cells were either left in buffered minimal salts medium (M9) or centrifuged and resuspended in complete growth medium (M9 + 4.0mg// glucose + 2.5 mg/m/ Casamino acids + 10 pg/m/ thiamine) for incubation at 37’C. I n some cases chloramphenicol (50pg/m/. Sigma) was added to the incubation mix. Samples were taken at appropriate times, centrifuged and resuspended in M9 and treated with the second agent. In the experiments where MMS treatment was given first, cells were washed 3 times by centrifugation before incubation in the appropriate medium.
MATERIALS AND METHODS
Bacterial srroin. The repair proficient strain, Escherichia coli K12 AB 1157 was used in all experiments.
61 1
RESULTS AND DISCUSSION
When cell populations are irradiated at 365 nm (106Jm-’) and then immediately exposed to mild heat (52”C),the lethal action of the mild heat is strongly amplified (see Fig. 1 and Tyrrell, 1976b). A smaller interaction occurs between near-UV and immediate far-UV (Fig. 2; Webb et al., 1978; Tyrrell, 1978b) or MMS (Fig. 3) treatment in this repair proficient K12 strain. If the cells are incubated in M9 buffer between the two treatments, the near-UV: mild heat interaction is reduced slightly (Fig. I), there is an intermediate loss in the near-UV: MMS interaction (Fig. 3) and the near-UV: far-UV interaction disappears completely (Fig. 2). Previous studies (Tyrrell, 1976a) have shown that the strong lethal interaction between near-UV and ionizing radjation hardly changes during incubation in buffer. Detailed kinetic studies with all the agents tested (results not shown) indicate that
REX M. TYRRELL and IVANOSCA S . CORREIA
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z
2
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I
I
I
5
10
15
20
2
MMS INCUBATION TIME [min)
10-3
30
90
60
HEATING TIME [ 5 2 ” ) MIN
Figure I. The modification of the lethal interaction between near-UV (106Jm-2 at 365 nm)* radiation and mild-heat (52°C) by incubation for 2 h between the radiation and heat-treatments in either M9 (0) or complete growth medium (A). Curves for populations that were treated only with mild heat ( 0 )or treated with mild heat immediately after irradiation with 106Jm-’ at 365 nm (H) are also shown. Curves redrawn from Tyrrell, 1978a. by permission Plenum Publishing. the change in response during incubation in buffer is essentially complete within 60 min. Incubation in complete growth medium following irradiation treatment leads to a complete loss in the I00
2
0
L 10-1 a
a
LL
W
z
> >
5 10-2 In
10-3
I
I
I
I
I
I
20
40
60
80
100
120
UV DOSE(254nm) Jm-*
Figure 2. The modification of the lethal interaction between near-UV (106Jm-* at 365 nm) radiation and far-UV (254 nm) radiation by incubation for 2 h between the radiation treatments in either M9 (0) or complete growth medium (A). Curves for populations that were treated only with far-UV (0)or treated with far-UV immediately after irradiation with 106Jm-’ at 365nm (H) are also shown. *The survival of irradiated populations (106Jm-’ at 365 nm. surviving fraction approximately 0.7) has been normalized to 1.0 in Figs 1, 2 and 3.
Figure 3. The modification of the lethal interaction between near-UV (106JnV2 at 365 nm) radiation and MMS (0.1 M . 37°C) by incubation for 90 min between the two treatments in either M9 (0) or complete growth medium (A). Curves for populations that were treated only or treated with MMS immediately after with MMS (0) irradiation with 106Jm-* at 365nm (H) are also shown. near-UV induced sensitization in all cases (Figs. 1, 2 and 3). The loss occurs within 60min (results not shown) and then a resistance to the second agent develops. A summary of the results for incubation in both media is shown in Table 1. The development of antagonism following near-UV treatment has been previously discussed (Tyrrell, 1978a), is not well understood and will not be further considered here. It appears that at least two changes leading to the loss of the interaction occur during the post-near-UV incubation, at least one of which does not occur in buffer. The effect which is lost in buffer appears to be totally responsible for the 365 nm: 254 nm interaction (Fig. 2), partially responsible for the 365 nm: MMS interaction (Fig. 3) and only very slightly responsible for the 365 nm: ionizing radiation/heat interactions. Considerable evidence suggests that repair systems are inhibited by near-UV radiation (for review of near-UV effects, see Webb, 1977). The buffer-reversible effects may be related to this repair inhibition as originally proposed by Tyrrell and Webb (1973). However, recent studies (Tyrrell, 1979) have indicated that near-UV induced lesions in phage DNA interfere with host-mediated repair systems. Thus, we must now be open to the possibility that the buffer-reversible 365 nm: 254 nm interaction does not reflect 365 nm-induced damage to the repair systems themselves but rather a modification of the efficiency of repair by damage induced in the DNA. The changes that occur in buffer would then represent repair of this type of damage. The reversal of the lethal interaction during incubation in complete medium between near-UV and ionizing radiation is probably due to a reversal of the 365 nm-inhibition of rec-controlled single-strand break-rejoining (Tyrrell, 1974). A similar mechanism may also be ‘responsible for the loss of interaction
Research Note
613
Table 1. Change in lethal interactions observed as a result of incubation of bacterial populations between two DNAdamaging treatments ~
~~
Near-UV: heat
Near-UV :* ionizing radiation
Near-UV: Far-UV
Near-UV: MMS
MMS: Near-UV
I ar ge
large
intermediate
intermediate
large
slight loss
slight loss
complete
intermediate
slight
loss
loss
loss
complete
complete
intermediate
loss
loss
loss
slight protection
slight protection
-~ ~
Initial sensitization (no incubation) Change in sensitization observed after incubation in M9 Change in sensitization observed after incubation in complete growth medium
complete loss large protection
complete loss large protection
*Tyrrell ( 1 976a) seen during incubation between near-UV and heat (Fig. 1) and to some extent between near-UV and MMS (Fig. 3) since both agents lead to damage that is susceptible to rec-controlled repair (Bridges et al., 1969; Correia and Tyrrell, 1978). However this type of repair inhibition does not appear to be critical for 254 nm damage (Fig. 2). This result may be explained by assuming that DNA lesions induced by various agents may vary considerably with respect to how long they can remain in the DNA in the absence of effective repair, and still eventually be repaired. Damage induced by 254 nm radiation can probably remain for relatively long periods whereas ionizing radiation damage must be repaired rapidly or the cell will die (Tyrrell, 1976a).
The changes in response of MMS-treated cells to subsequent treatment with 365 nm radiation, during incubation, are shown in Fig. 4. Neither incubation in M9 or complete medium can completely reverse the interaction between the two agents. Furthermore, if chloramphenicol (a protein synthesis inhibitor) is present during incubation in complete medium, no reversal occurs and there is some additional sensitization to the subsequent radiation treatment. Chloramphenicol only slightly modifies the changes that occur during post-near-UV irradiation incubation prior to MMS-treatment during incubation (results not shown). This appears to be good supporting evidence (see accompanying paper) that MMS irreversibly damages repair enzymes so that repair systems only regain their functional activity after the cells are incubated under conditions which allow de nouo protein synthesis. An alternative explanation of the chloramphenicol effect is that MMS, but not 365 nm radiation damage, signals the induction of an inducible repair system . These studies, although admittedly complex to interpret, suggest that DNA-damaging agents may be classified according to the time that they remain repairable in the absence of effective repair and that near-UV radiation may be a useful probe in determining this parameter. Both ionizing radiation and heat may lead to irreversible DNA lesions if DNA repair cannot function immediately or soon after their in10-3 duction. Damage induced by 254 nm radiation L. T appears to be susceptible to repair for long periods 60 120 I80 while MMS could induce a mixture of the two types INCUBATION TIME(min 1 of damage. In addition, certain agents may induce Figure 4. Modification of the sensitivity of cells to radi- damages which lead to reversible (365 nm) or irreversation at 365 nm (106Jm- z, during incubation in either M9 ible inhibition of repair. Further studies are clearly or complete growth medium following MMS treatment needed to substantiate these suggestions. (0.1 M , 3 7 T , 5 min). Key: MMS only, incubate in growth medium with (+)or without )1. 50 pg/m( chlorampheni- Acknowledgemetits -The work was supported by the folcol; MMS only, incubate in M9 (0);MMS, incubate in lowing Brazilian granting agencies: CNPq (National growth medium, irradiate at 365 nm (A);MMS, incubate Research Council); CNEN (National Nuclear Energy in M9. irradiate at 365 nm (A);MMS, irradiate at 365 nm, Council), CEPCjUFRJ (University Council for Post-Grathen incubate in growth medium ( 0 ) ;MMS, irradiate at duate Studies) and FINEP/FNDCT-375/CT (Study and 365nm, then incubate in M9 ( 0 ) ; MMS, incubate in Project Grants/National Fund for Scientific Development). growth medium with 50 pg/m/ chloramphenicol, irradiate Part of the work appears in an M.S. thesis of the University of S l o Paulo by one of us (I.S.C.). at 365nm (V).
Y1'-
1 L-
614
REX M. TYRRELL and IVANOWA S. CORREIA REFERENCES
Bridges, B. A., M. J. Ashwood-Smith and R. J. Munson (1969) J . Gen. Microhiol. 58, 115-124. Correia. 1. S. and R. M. Tyrrell (1979) Photochem. Photohiol. 29, 521-526. Okuda. A. (1973) PhfJtoChem. Photohiol. 18, 335-337. Smith. K. C. and K. D. Martignoni (1976) Photochum. Phorohiol. 24, 514-525. Tyrrell. R. M. (1974) Int. J . Radiut. Biol. 25. 373-390. Tyrrell. R. M. (1976a) Pholochem. Photohiol. 23, 13-20. Tyrrell. R . M . (l976b) Phofochem. Phorohiol. 24, 345-351. Tyrrell. R. M. (1978a) In Phorochemicul und Photobiological Reviews (Edited by K. C. Smith). Vol. 3. pp. 35-113. Plenum, New York. Tyrrell. R. M. (1978b) Muruf. Res. 52, 25-35. Tyrrell. R . M. (1979) Photochum. Photohiol. in press). Tyrrell, R. M. and R. B. Webb (1973) Murat. Res. 19, 361-364. Webb. R. B. (1977) In Photochemical cmd Photohioloqical Rerieus (Edited by K. C. Smith). Vol. 2, pp. 169-261. Plenum, New York. Webb. R. B.. M.S. Brown and R. M. Tyrrell (1978) Roditrt. Res. 74, 298-311.
8 P c r j m i o n Press Ltd.
M)3 1-8655 ’791030 I-MI Ilo2.00/0
1979. Printed in Great Britain
RESEARCH NOTE
MODIFICATION OF LETHAL INTERACTIONS BETWEEN NEAR-ULTRAVIOLET (365 nm) RADIATION AND DNA-DAMAGING AGENTS REX M. TYRRELL and IVANOSCA S. CORREIA Instituto de Biofisica, Centro de CiEncias da Saude, (Bloco G), Universidade Federal do Rio de Janeiro. Rio de Janeiro, Brazil (Received 23 May 1978: accepted 27 July 1978)
Abstract-The modification of the lethal interaction between near-UV (365 nm) radiation and a second DNA-damaging agent by incubation between treatments in either a minimal salts medium or complete growth medium has been studied in the wild-type bacterial strain Escherichia coli K12 A B 1157. The results indicate that the lethal interaction may be separated into at least two distinct processes whose evaluation may help in classifying DNA-damaging agents in terms of the repairability of the DNA lesions induced. An observation of changes when methyl methane sulphonate is given prior to the irradiation treatment indicates that this chemical irreversibly damages repair enzymes.
INTRODUCTION
The interaction of near-UV (ultraviolet) radiation with other physical agents (far-UV radiation, ionizing radiation and heat) in bacterial systems has recently been reviewed (see Tyrrell, 1978a). A strong lethal interaction has also been observed between radiation at several near-UV wavelengths and methyl methane sulphonate (MMS) (Correia and Tyrrell, 1978, accompanying paper). The precise mechanism of the interactions is not clear except that damage to DNA and interference with DNA repair by the two interacting agents appears to be involved. A further insight into the nature of the interactions may be obtained by allowing an incubation interval (in various media) between the two treatments. Far-UV induced sensitization to X-rays is rapidly lost by incubation in buffer alone (Okuda, 1973), whereas the interaction between near-UV and ionizing radiation disappears only when cells are incubated in complete medium in the interval between irradiations (Tyrrell, 1976a). In the latter case, a near-UV induced protection from the second treatment also develops. Smith and Martignoni (1976) observed a similar development of resistance during incubation of X-irradiated populations in complete growth medium before UV treatment. In the following study, the changes in the lethal action of various agents during post-near-UV incubation have been observed. The results indicate that the lethal interaction provoked by near-UV may be the result of at least two distinct cellular changes whose expression depends on the nature of the second agent.
Bacterial growth and irradiations were carried out as described in the accompanying paper (Correia and Tyrrell, 1979).The source for 254 nm radiation was a General Electric 15 W germicidal lamp (GI5 T8) and the dose rate (1 Wm-’) was measured by means of a Latarjet meter. Mild heating (52°C) was performed as previously described (Tyrrell, 1976b). Treatment with methyl methane sulphonate (0.1 M , 37°C) has been described in the accompanying paper (Correia and Tyrell. 1979). Incubation. Irradiated cells were either left in buffered minimal salts medium (M9) or centrifuged and resuspended in complete growth medium (M9 + 4.0mg// glucose + 2.5 mg/m/ Casamino acids + 10 pg/m/ thiamine) for incubation at 37’C. I n some cases chloramphenicol (50pg/m/. Sigma) was added to the incubation mix. Samples were taken at appropriate times, centrifuged and resuspended in M9 and treated with the second agent. In the experiments where MMS treatment was given first, cells were washed 3 times by centrifugation before incubation in the appropriate medium.
MATERIALS AND METHODS
Bacterial srroin. The repair proficient strain, Escherichia coli K12 AB 1157 was used in all experiments.
61 1
RESULTS AND DISCUSSION
When cell populations are irradiated at 365 nm (106Jm-’) and then immediately exposed to mild heat (52”C),the lethal action of the mild heat is strongly amplified (see Fig. 1 and Tyrrell, 1976b). A smaller interaction occurs between near-UV and immediate far-UV (Fig. 2; Webb et al., 1978; Tyrrell, 1978b) or MMS (Fig. 3) treatment in this repair proficient K12 strain. If the cells are incubated in M9 buffer between the two treatments, the near-UV: mild heat interaction is reduced slightly (Fig. I), there is an intermediate loss in the near-UV: MMS interaction (Fig. 3) and the near-UV: far-UV interaction disappears completely (Fig. 2). Previous studies (Tyrrell, 1976a) have shown that the strong lethal interaction between near-UV and ionizing radjation hardly changes during incubation in buffer. Detailed kinetic studies with all the agents tested (results not shown) indicate that
REX M. TYRRELL and IVANOSCA S . CORREIA
612 I00
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7
P IV
a LL a
”
10-2
z
2
>
l Y
l3 n I
I
I
I
5
10
15
20
2
MMS INCUBATION TIME [min)
10-3
30
90
60
HEATING TIME [ 5 2 ” ) MIN
Figure I. The modification of the lethal interaction between near-UV (106Jm-2 at 365 nm)* radiation and mild-heat (52°C) by incubation for 2 h between the radiation and heat-treatments in either M9 (0) or complete growth medium (A). Curves for populations that were treated only with mild heat ( 0 )or treated with mild heat immediately after irradiation with 106Jm-’ at 365 nm (H) are also shown. Curves redrawn from Tyrrell, 1978a. by permission Plenum Publishing. the change in response during incubation in buffer is essentially complete within 60 min. Incubation in complete growth medium following irradiation treatment leads to a complete loss in the I00
2
0
L 10-1 a
a
LL
W
z
> >
5 10-2 In
10-3
I
I
I
I
I
I
20
40
60
80
100
120
UV DOSE(254nm) Jm-*
Figure 2. The modification of the lethal interaction between near-UV (106Jm-* at 365 nm) radiation and far-UV (254 nm) radiation by incubation for 2 h between the radiation treatments in either M9 (0) or complete growth medium (A). Curves for populations that were treated only with far-UV (0)or treated with far-UV immediately after irradiation with 106Jm-’ at 365nm (H) are also shown. *The survival of irradiated populations (106Jm-’ at 365 nm. surviving fraction approximately 0.7) has been normalized to 1.0 in Figs 1, 2 and 3.
Figure 3. The modification of the lethal interaction between near-UV (106JnV2 at 365 nm) radiation and MMS (0.1 M . 37°C) by incubation for 90 min between the two treatments in either M9 (0) or complete growth medium (A). Curves for populations that were treated only or treated with MMS immediately after with MMS (0) irradiation with 106Jm-* at 365nm (H) are also shown. near-UV induced sensitization in all cases (Figs. 1, 2 and 3). The loss occurs within 60min (results not shown) and then a resistance to the second agent develops. A summary of the results for incubation in both media is shown in Table 1. The development of antagonism following near-UV treatment has been previously discussed (Tyrrell, 1978a), is not well understood and will not be further considered here. It appears that at least two changes leading to the loss of the interaction occur during the post-near-UV incubation, at least one of which does not occur in buffer. The effect which is lost in buffer appears to be totally responsible for the 365 nm: 254 nm interaction (Fig. 2), partially responsible for the 365 nm: MMS interaction (Fig. 3) and only very slightly responsible for the 365 nm: ionizing radiation/heat interactions. Considerable evidence suggests that repair systems are inhibited by near-UV radiation (for review of near-UV effects, see Webb, 1977). The buffer-reversible effects may be related to this repair inhibition as originally proposed by Tyrrell and Webb (1973). However, recent studies (Tyrrell, 1979) have indicated that near-UV induced lesions in phage DNA interfere with host-mediated repair systems. Thus, we must now be open to the possibility that the buffer-reversible 365 nm: 254 nm interaction does not reflect 365 nm-induced damage to the repair systems themselves but rather a modification of the efficiency of repair by damage induced in the DNA. The changes that occur in buffer would then represent repair of this type of damage. The reversal of the lethal interaction during incubation in complete medium between near-UV and ionizing radiation is probably due to a reversal of the 365 nm-inhibition of rec-controlled single-strand break-rejoining (Tyrrell, 1974). A similar mechanism may also be ‘responsible for the loss of interaction
Research Note
613
Table 1. Change in lethal interactions observed as a result of incubation of bacterial populations between two DNAdamaging treatments ~
~~
Near-UV: heat
Near-UV :* ionizing radiation
Near-UV: Far-UV
Near-UV: MMS
MMS: Near-UV
I ar ge
large
intermediate
intermediate
large
slight loss
slight loss
complete
intermediate
slight
loss
loss
loss
complete
complete
intermediate
loss
loss
loss
slight protection
slight protection
-~ ~
Initial sensitization (no incubation) Change in sensitization observed after incubation in M9 Change in sensitization observed after incubation in complete growth medium
complete loss large protection
complete loss large protection
*Tyrrell ( 1 976a) seen during incubation between near-UV and heat (Fig. 1) and to some extent between near-UV and MMS (Fig. 3) since both agents lead to damage that is susceptible to rec-controlled repair (Bridges et al., 1969; Correia and Tyrrell, 1978). However this type of repair inhibition does not appear to be critical for 254 nm damage (Fig. 2). This result may be explained by assuming that DNA lesions induced by various agents may vary considerably with respect to how long they can remain in the DNA in the absence of effective repair, and still eventually be repaired. Damage induced by 254 nm radiation can probably remain for relatively long periods whereas ionizing radiation damage must be repaired rapidly or the cell will die (Tyrrell, 1976a).
The changes in response of MMS-treated cells to subsequent treatment with 365 nm radiation, during incubation, are shown in Fig. 4. Neither incubation in M9 or complete medium can completely reverse the interaction between the two agents. Furthermore, if chloramphenicol (a protein synthesis inhibitor) is present during incubation in complete medium, no reversal occurs and there is some additional sensitization to the subsequent radiation treatment. Chloramphenicol only slightly modifies the changes that occur during post-near-UV irradiation incubation prior to MMS-treatment during incubation (results not shown). This appears to be good supporting evidence (see accompanying paper) that MMS irreversibly damages repair enzymes so that repair systems only regain their functional activity after the cells are incubated under conditions which allow de nouo protein synthesis. An alternative explanation of the chloramphenicol effect is that MMS, but not 365 nm radiation damage, signals the induction of an inducible repair system . These studies, although admittedly complex to interpret, suggest that DNA-damaging agents may be classified according to the time that they remain repairable in the absence of effective repair and that near-UV radiation may be a useful probe in determining this parameter. Both ionizing radiation and heat may lead to irreversible DNA lesions if DNA repair cannot function immediately or soon after their in10-3 duction. Damage induced by 254 nm radiation L. T appears to be susceptible to repair for long periods 60 120 I80 while MMS could induce a mixture of the two types INCUBATION TIME(min 1 of damage. In addition, certain agents may induce Figure 4. Modification of the sensitivity of cells to radi- damages which lead to reversible (365 nm) or irreversation at 365 nm (106Jm- z, during incubation in either M9 ible inhibition of repair. Further studies are clearly or complete growth medium following MMS treatment needed to substantiate these suggestions. (0.1 M , 3 7 T , 5 min). Key: MMS only, incubate in growth medium with (+)or without )1. 50 pg/m( chlorampheni- Acknowledgemetits -The work was supported by the folcol; MMS only, incubate in M9 (0);MMS, incubate in lowing Brazilian granting agencies: CNPq (National growth medium, irradiate at 365 nm (A);MMS, incubate Research Council); CNEN (National Nuclear Energy in M9. irradiate at 365 nm (A);MMS, irradiate at 365 nm, Council), CEPCjUFRJ (University Council for Post-Grathen incubate in growth medium ( 0 ) ;MMS, irradiate at duate Studies) and FINEP/FNDCT-375/CT (Study and 365nm, then incubate in M9 ( 0 ) ; MMS, incubate in Project Grants/National Fund for Scientific Development). growth medium with 50 pg/m/ chloramphenicol, irradiate Part of the work appears in an M.S. thesis of the University of S l o Paulo by one of us (I.S.C.). at 365nm (V).
Y1'-
1 L-
614
REX M. TYRRELL and IVANOWA S. CORREIA REFERENCES
Bridges, B. A., M. J. Ashwood-Smith and R. J. Munson (1969) J . Gen. Microhiol. 58, 115-124. Correia. 1. S. and R. M. Tyrrell (1979) Photochem. Photohiol. 29, 521-526. Okuda. A. (1973) PhfJtoChem. Photohiol. 18, 335-337. Smith. K. C. and K. D. Martignoni (1976) Photochum. Phorohiol. 24, 514-525. Tyrrell. R. M. (1974) Int. J . Radiut. Biol. 25. 373-390. Tyrrell. R. M. (1976a) Pholochem. Photohiol. 23, 13-20. Tyrrell. R . M . (l976b) Phofochem. Phorohiol. 24, 345-351. Tyrrell. R. M. (1978a) In Phorochemicul und Photobiological Reviews (Edited by K. C. Smith). Vol. 3. pp. 35-113. Plenum, New York. Tyrrell. R. M. (1978b) Muruf. Res. 52, 25-35. Tyrrell. R . M. (1979) Photochum. Photohiol. in press). Tyrrell, R. M. and R. B. Webb (1973) Murat. Res. 19, 361-364. Webb. R. B. (1977) In Photochemical cmd Photohioloqical Rerieus (Edited by K. C. Smith). Vol. 2, pp. 169-261. Plenum, New York. Webb. R. B.. M.S. Brown and R. M. Tyrrell (1978) Roditrt. Res. 74, 298-311.
