INT . J . RADIAT . BIOL .,
1975,
VOL .
27,
NO .
5, 48 1-485
CORRESPONDENCE The induction of pyrimidine dimers in nuclear DNA after U.V.-irradiation during the synchronous cycle of Saccharomyces cerevisiae
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
R. CHANET, R . WATERS and E . MOUSTACCHI Fondation Curie-Institut du Radium, Universite de Paris-Sud, Batiment 110, 91405-ORSAY, France (Received 6 February 1975 ; accepted 21 March 1975)
1 . Introduction The lethal and genetic effects of radiation during the synchronous cell-cycle have been studied in various eucaryotic organisms (Terasima and Tolmach 1961, Kimball 1963, Davies 1965, Holliday 1965, Esposito 1968, Fabre 1973) . Considerable data have been accumulated regarding the fluctuation of such events after the U .V .-irradiation of synchronous cultures of Saccharomyces cerevisiae . These studies have involved wild-type cells (Chanet, Williamson and Moustacchi 1973), radiation-sensitive mutants (Chanet, Heude and Moustacchi 1974), and the influences of various post-irradiation treatments (Chanet and Heude 1974) . As regards survival, maximum U .V.-resistance occurs at the end of each cycle of DNA replication, that is, at the end of S phase and during the G2 period . Maximum U .V . sensitivity occurs just before the period of DNA synthesis or S phase, at the end of the G1 period (Chanet et al . 1973) . It has been shown that, in mammalian cells, the induction of pyr[ ]pyr by a specific U .V. incident dose varies during the cell cycle . 34 per cent more pyr[ ]pyr are induced in the S phase of Chinese hamster B-14 cells as compared to the amount induced in the G1 phase after incident doses of U .V . up to 10 4 ergs/mm 2 (Steward and Humphrey 1966) . Trosko, Kasschau, Covington and Chu (1966), also using Chinese hamster cells, found that there were 20 per cent more pyr[ ]pyr in the early S phase than in the G2 phase . Finally, differences in induction of pyr[ ]pyr of 50 per cent were reported by Watanabe and Harikawa (1974) following the irradiation of HeLa-H 3 cells with 200 ergs/mm2 of U.V. In this case the induction of pyr[ ]pyr closely follows changes in U .V . survival, in that the more pyr[ ]pyr induced, the lower the survival observed . In view of these results it was obviously necessary to determine whether fluctuations occur in the U .V . induction of pyr[ ]pyr during the synchronous cell-cycle of Saccharomyces cerevisiae, as any such changes would have to be considered when discussing variations in U .V.-induced lethal and genetic events . 2. Materials and methods 2 .1 . Strains, media and culture conditions his,) was used throughout this a+ d2 + study . The preparation of cells for synchronization was performed by the method of Williamson and Scopes (1962), and synchronized cultures were grown The diploid strain 10018 x N123 (a
a
482
Correspondence
at 1 x 10 7 cells/ml with aeration in a semi-defined medium at 25 ° C (Scopes and Williamson 1964) . Labelling was with 4 µCi/ml of (6-3H) uracil (28 Ci/mmole), and did not interfere with the synchrony obtained . Aliquots of synchronized cells were taken at 10-minute intervals for microscopic estimation of budding cells . The following cell classes have been distinguished : single cells without bud that are in G1, cells with a small bud that are in S phase, cells with a bud at about 2/3 of the size of the mother cell which are in G2 and the double cells as two cells in the Gl stage (Williamson 1965) .
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
2 .2 . U . V.-irradiation
At appropriate times in the cell-cycle, samples were taken, washed and quickly resuspended in saline at a concentration of 1 x 10' cells/ml for U .V.-irradiation at an incident dose of 6 x 10 3 ergs/mm 2. U .V. was supplied by a low pressure mercury lamp, most of the radiation being at 254 nm. The absorbed dose, equal to 90 per cent of the incident dose, as measured by the percentage transmission at a wave-length of 254 nm, indicated that differences in absorption at the different stages of the cell-cycle were negligible . 2 .3 . DNA extraction
This was begun immediately after U .V .-irradiation, and details of the isolation and purification by means of isopycnic CsC1 gradients and the subsequent profiles have been previously published (Waters and Moustacchi 1974 b). 2 .4 . Hydrolysis o f DNA, chromatography and scintillation counting
These were all carried out as previously described (Waters and Moustacchi 1974 a). 2 .5 . Estimation o f pyrimidine dimers
The profiles of chromatograms and induction of pyr[ ]pyr in the nuclear DNA of Saccharomyces cerevisiae have been previously described (Waters and Moustacchi 1974 a, Waters and Moustacchi 1975) . U .V . induced dimers were total c .p.m in pyr[ ]pyr The background radioactivity with estimated as : total c .p.m . i n pyrimidines an Rf the same as pyr[ ]pyr found in the non-irradiated sample has been substracted . Little variation in this background occurred with respect to cell cycle within any one experiment . Hence this could not account for differences in activity found in the pyr[ ]pyr regions . The data presented here are the results of three individual experiments . 3. Results and discussion The degree of synchrony obtained as measured by the percentage of budding cells, is presented in figure 1 (b) . The number of acid-precipitable c .p.m . found in the nuclear DNA at each time of sampling for irradiation verifies this synchronous condition (figure 1 (c)) . Figure 1 (a) shows the percentage induction of both thy[ ]thy and cyt[ ]thy dimers at specific stages in the cell-cycle after an incident U .V. dose of 6 x 10 3 ergs/mm2. Thy[ ]thy induction is seen to vary
48 3
Correspondence
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Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
B 80
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HOURS OF GROWTH
Figure 1 . Induction of pyrimidine dimers in nuclear DNA following a U .V.-dose of 6 x 10 3 ergs/mm 2 during the synchronous cycle of Saccharomyces cerevisiae . (a) Percentage of pyrimidine dimers as a function of time in the cell-cycle, three experiments are represented ( •, A, ∎) induction of thy[ ]thy ; (o, z~,, o) induction of cyt[ ]thy . (b) Budding curve . (c) Relative amount of acid-precipitable c .p .m . found in the nuclear DNA ; three experiments are represented (0, A, ∎) .
inversely with the percentage of budding cells ; that is the higher the percentage of budding cells in the population the smaller the percentage of thy[ ]thy induced . This variation is of the order of 30 per cent if one takes the greatest induction (0 . 85 per cent), when there are no buds, as being 100 per cent . Such a variation is not encountered as regards the induction of cyt[ ]thy dimers in the cell cycle . Very small differences can be seen to occur, but these do not fit a regular pattern . The fact that for one experiment less cyt[ ]thy appeared to be induced was due to a larger background in the non-irradiated sample for this region . However, this artefact does not alter the almost constant induction obtained . The addition of thy[ ]thy and cyt[ ]thy dimers, which are by far the greater part of pyr[ ]pyr induced in Saccharomyces cerevisiae, still results in a variation of at least 20 per cent . Waters and Moustacchi (1975) have shown that pyr[ ]pyr induction is the same in haploid and diploid cultures of yeast. Hence total amounts of RNA and protein do not appear to greatly influence pyr[ ]pyr induction . Although this observation does not exclude that, as suggested by Watanabe
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
484
Correspondence
and Harikawa (1974), conformational changes in DNA or varying amounts of protecting substances such as specific proteins may be responsible for the variation of pyr[ ]pyr in the cell cycle . One possible explanation for the much greater variation in thy[ ]thy induction (30 per cent) as compared to cyt[ ]thy induction may be found in the fact that cyt[ ]thy induction, although shown by Waters and Moustacchi (1975) to be approximately linear up to an incident dose of 4 x 10 3 ergs/mm2 becomes biphasic and tends to form a plateau at higher doses of 6 x 10 3 ergs/mm2 (Setlow and Carrier 1966, Unrau, Wheatcroft, Cox and Olive 1973) . Setlow and Carrier (1966) suggested this was the result of U .V.-splitting as well as forming cyt[ ]thy at higher doses. On the contrary, as found by these authors and ourselves (Waters and Moustacchi 1974), thy[ ]thy induction increases linearly up to doses considerably higher than 6 x 10 6 ergs/mm 2. Finally the pyr[ ]pyr induced in the first synchronous cycle (figure 1 (a)) is slightly greater at each stage compared with the second and third cycles . This may be the result of two factors . Firstly the cells are in a starved condition before synchronization and hence are in a physiological state different from that encountered in normal growth . Secondly the degree of synchronization is more precise in the first cycle, a degeneration occurring in the second and third cycles . The use of a zonal rotor to separate cells grown under normal physiological conditions should resolve this factor . The implications of these results on the variation in U .V. -sensitivity seen in the cell-cycle may now be discussed . Previous experiments have indicated that the U .V.-sensitivity of a RAD strain also changes in accord with the percentage of budding cells in the cycle . That is, the more budding cells there are, the higher the U .V. resistance observed (Chanet et al. 1973) . Hence one must consider could the variation in pyr[ ]pyr induction possibly account for the variation in sensitivity observed? A variation of 50 per cent in the lethality produced on the irradiation of a synchronized RAD strain of Saccharomyces cerevisiae (Chanet et al . 1973) could not be explained by a 20 per cent fluctuation in pyr[ ]pyr content alone . However, we have used a high U .V . dose of 6 x 10 3 ergs/mm 2 in our experiments, this by no means being related to the previous physiological experiments. The lower doses used by Watanabe and Harikawa (1974) gave a greater fluctuation (50 per cent) than at the higher doses used by both Trosko et al . (1966) and Humphrey and Steward (1966) who reported fluctuations of 20 per cent and 34 per cent, respectively . Admittedly, they used different cell-lines, which makes any comparison speculative . Whether on the basis of such data the fluctuation is greater or not at low U .V. doses may be questioned . Techniques enabling a better resolution of pyr[ ]pyr induction in yeast will resolve this problem . To date at least three repair pathways would appear to be responsible for the repair of U .V. damage in yeast (Game and Cox 1973) . When one regards the cyclic fluctuations of a double mutant rec5 rad1_3 (Chanet et al . 1974) which is extremely U .V.-sensitive and hence deficient in some of the major repair pathways, it is seen to be of the order of 30 per cent . The fluctuation in pyr[ ]pyr induction after high doses could possibly account for these variations in the viability of rec 5rad1_3 seen for lower doses . Thus differences in the amounts of pyr[ ]pyr induced during the cell-cycle may be more important : (i) for low doses when a strain is deficient in the major repair pathways of U .V. damage ; (ii) for high doses, when the major repair pathways are saturated .
Correspondence
485
ACKNOWLEDGMENTS
The authors wish to thank Madame R . Guilbaud and Mademoiselle A . Boulet for technical assistance . One of us (R .W .) is indebted to EMBO for supplying a long term fellowship . This investigation was supported in part by the Commissariat a 1'Energie Atomique (Saclay, France) and Euratom (contract n° 126-74-7-BIOF) .
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
REFERENCES CHANET, R ., and HEUDE, M ., 1974, Molec . gen . Genet ., 131, 21 . CHANET, R ., HEUDE, M ., and MOUSTACCHI, E ., 1974, Molec . gen . Genet., 132, 23 . CHANET, R ., WILLIAMSON, D . H ., and MOUSTACCHi, E ., 1973, Biochim . biophys . Acta, 324, 290 . DAVIES, D . R ., 1965, Mutation Res ., 2, 477 . ESPOSITO, R . E., 1968, Genetics, 59, 191 . FABRE, F., 1973, Radiat . Res ., 56, 528 . GAME, J . C ., and Cox, B . S ., 1973, Mutation Res., 20, 35 . HOLLIDAY, R ., 1965, Genet. Res . (Camb .), 6,104. KIMBALL, R . F ., 1963, Repair from Genetic Radiation Damage, edited by F. H . Sobels (London : Pergamon), p . 167 . SCOPES, A . W., and WILLIAMSON, D . H ., 1964, Expl Cell Res ., 35, 361 . SETLOW, R . B ., and CARRIER, W . L ., 1966, Y. molec. Biol ., 17, 237 . STEWARD, D . L ., and HUMPHREY, R . M ., 1966, Nature, Lond ., 212, 298 . TERASIMA, T ., and TOLMACH, L . J ., 1961, Nature, Lond ., 190, 1210 . TROSKO, J . E ., KASSCHAU, M ., COVINGTON, L ., and CHU, E . H . Y ., 1966, Radiat . Res ., 27, 535 . UNRAU, P ., WHEATCROFT, R ., Cox, B . S ., and OLIVE, T ., 1973, Biochim . biophys . Acta, 312, 626 . WATANABE, M ., and HARIKAWA, M ., 1974, Biochem . biophys . Res . Commun., 58,185 . WATERS, R ., and MOUSTACCHI, E ., 1974 a, Biochim . biophys. Acta, 353, 407 ; 1974 b, Ibid., 366, 241 ; 1975, Y . Bact ., 121, 901 . WILLIAMSON, D . H., and SCOPES, A . W ., 1962, Nature, Lond., 193, 256 . WILLIAMSON, D . H ., 1965, `J. cell Biol ., 25, 517 .
1975,
VOL .
27,
NO .
5, 48 1-485
CORRESPONDENCE The induction of pyrimidine dimers in nuclear DNA after U.V.-irradiation during the synchronous cycle of Saccharomyces cerevisiae
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
R. CHANET, R . WATERS and E . MOUSTACCHI Fondation Curie-Institut du Radium, Universite de Paris-Sud, Batiment 110, 91405-ORSAY, France (Received 6 February 1975 ; accepted 21 March 1975)
1 . Introduction The lethal and genetic effects of radiation during the synchronous cell-cycle have been studied in various eucaryotic organisms (Terasima and Tolmach 1961, Kimball 1963, Davies 1965, Holliday 1965, Esposito 1968, Fabre 1973) . Considerable data have been accumulated regarding the fluctuation of such events after the U .V .-irradiation of synchronous cultures of Saccharomyces cerevisiae . These studies have involved wild-type cells (Chanet, Williamson and Moustacchi 1973), radiation-sensitive mutants (Chanet, Heude and Moustacchi 1974), and the influences of various post-irradiation treatments (Chanet and Heude 1974) . As regards survival, maximum U .V.-resistance occurs at the end of each cycle of DNA replication, that is, at the end of S phase and during the G2 period . Maximum U .V . sensitivity occurs just before the period of DNA synthesis or S phase, at the end of the G1 period (Chanet et al . 1973) . It has been shown that, in mammalian cells, the induction of pyr[ ]pyr by a specific U .V. incident dose varies during the cell cycle . 34 per cent more pyr[ ]pyr are induced in the S phase of Chinese hamster B-14 cells as compared to the amount induced in the G1 phase after incident doses of U .V . up to 10 4 ergs/mm 2 (Steward and Humphrey 1966) . Trosko, Kasschau, Covington and Chu (1966), also using Chinese hamster cells, found that there were 20 per cent more pyr[ ]pyr in the early S phase than in the G2 phase . Finally, differences in induction of pyr[ ]pyr of 50 per cent were reported by Watanabe and Harikawa (1974) following the irradiation of HeLa-H 3 cells with 200 ergs/mm2 of U.V. In this case the induction of pyr[ ]pyr closely follows changes in U .V . survival, in that the more pyr[ ]pyr induced, the lower the survival observed . In view of these results it was obviously necessary to determine whether fluctuations occur in the U .V . induction of pyr[ ]pyr during the synchronous cell-cycle of Saccharomyces cerevisiae, as any such changes would have to be considered when discussing variations in U .V.-induced lethal and genetic events . 2. Materials and methods 2 .1 . Strains, media and culture conditions his,) was used throughout this a+ d2 + study . The preparation of cells for synchronization was performed by the method of Williamson and Scopes (1962), and synchronized cultures were grown The diploid strain 10018 x N123 (a
a
482
Correspondence
at 1 x 10 7 cells/ml with aeration in a semi-defined medium at 25 ° C (Scopes and Williamson 1964) . Labelling was with 4 µCi/ml of (6-3H) uracil (28 Ci/mmole), and did not interfere with the synchrony obtained . Aliquots of synchronized cells were taken at 10-minute intervals for microscopic estimation of budding cells . The following cell classes have been distinguished : single cells without bud that are in G1, cells with a small bud that are in S phase, cells with a bud at about 2/3 of the size of the mother cell which are in G2 and the double cells as two cells in the Gl stage (Williamson 1965) .
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
2 .2 . U . V.-irradiation
At appropriate times in the cell-cycle, samples were taken, washed and quickly resuspended in saline at a concentration of 1 x 10' cells/ml for U .V.-irradiation at an incident dose of 6 x 10 3 ergs/mm 2. U .V. was supplied by a low pressure mercury lamp, most of the radiation being at 254 nm. The absorbed dose, equal to 90 per cent of the incident dose, as measured by the percentage transmission at a wave-length of 254 nm, indicated that differences in absorption at the different stages of the cell-cycle were negligible . 2 .3 . DNA extraction
This was begun immediately after U .V .-irradiation, and details of the isolation and purification by means of isopycnic CsC1 gradients and the subsequent profiles have been previously published (Waters and Moustacchi 1974 b). 2 .4 . Hydrolysis o f DNA, chromatography and scintillation counting
These were all carried out as previously described (Waters and Moustacchi 1974 a). 2 .5 . Estimation o f pyrimidine dimers
The profiles of chromatograms and induction of pyr[ ]pyr in the nuclear DNA of Saccharomyces cerevisiae have been previously described (Waters and Moustacchi 1974 a, Waters and Moustacchi 1975) . U .V . induced dimers were total c .p.m in pyr[ ]pyr The background radioactivity with estimated as : total c .p.m . i n pyrimidines an Rf the same as pyr[ ]pyr found in the non-irradiated sample has been substracted . Little variation in this background occurred with respect to cell cycle within any one experiment . Hence this could not account for differences in activity found in the pyr[ ]pyr regions . The data presented here are the results of three individual experiments . 3. Results and discussion The degree of synchrony obtained as measured by the percentage of budding cells, is presented in figure 1 (b) . The number of acid-precipitable c .p.m . found in the nuclear DNA at each time of sampling for irradiation verifies this synchronous condition (figure 1 (c)) . Figure 1 (a) shows the percentage induction of both thy[ ]thy and cyt[ ]thy dimers at specific stages in the cell-cycle after an incident U .V. dose of 6 x 10 3 ergs/mm2. Thy[ ]thy induction is seen to vary
48 3
Correspondence
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Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
B 80
Q
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D ~
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20
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0
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HOURS OF GROWTH
Figure 1 . Induction of pyrimidine dimers in nuclear DNA following a U .V.-dose of 6 x 10 3 ergs/mm 2 during the synchronous cycle of Saccharomyces cerevisiae . (a) Percentage of pyrimidine dimers as a function of time in the cell-cycle, three experiments are represented ( •, A, ∎) induction of thy[ ]thy ; (o, z~,, o) induction of cyt[ ]thy . (b) Budding curve . (c) Relative amount of acid-precipitable c .p .m . found in the nuclear DNA ; three experiments are represented (0, A, ∎) .
inversely with the percentage of budding cells ; that is the higher the percentage of budding cells in the population the smaller the percentage of thy[ ]thy induced . This variation is of the order of 30 per cent if one takes the greatest induction (0 . 85 per cent), when there are no buds, as being 100 per cent . Such a variation is not encountered as regards the induction of cyt[ ]thy dimers in the cell cycle . Very small differences can be seen to occur, but these do not fit a regular pattern . The fact that for one experiment less cyt[ ]thy appeared to be induced was due to a larger background in the non-irradiated sample for this region . However, this artefact does not alter the almost constant induction obtained . The addition of thy[ ]thy and cyt[ ]thy dimers, which are by far the greater part of pyr[ ]pyr induced in Saccharomyces cerevisiae, still results in a variation of at least 20 per cent . Waters and Moustacchi (1975) have shown that pyr[ ]pyr induction is the same in haploid and diploid cultures of yeast. Hence total amounts of RNA and protein do not appear to greatly influence pyr[ ]pyr induction . Although this observation does not exclude that, as suggested by Watanabe
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
484
Correspondence
and Harikawa (1974), conformational changes in DNA or varying amounts of protecting substances such as specific proteins may be responsible for the variation of pyr[ ]pyr in the cell cycle . One possible explanation for the much greater variation in thy[ ]thy induction (30 per cent) as compared to cyt[ ]thy induction may be found in the fact that cyt[ ]thy induction, although shown by Waters and Moustacchi (1975) to be approximately linear up to an incident dose of 4 x 10 3 ergs/mm2 becomes biphasic and tends to form a plateau at higher doses of 6 x 10 3 ergs/mm2 (Setlow and Carrier 1966, Unrau, Wheatcroft, Cox and Olive 1973) . Setlow and Carrier (1966) suggested this was the result of U .V.-splitting as well as forming cyt[ ]thy at higher doses. On the contrary, as found by these authors and ourselves (Waters and Moustacchi 1974), thy[ ]thy induction increases linearly up to doses considerably higher than 6 x 10 6 ergs/mm 2. Finally the pyr[ ]pyr induced in the first synchronous cycle (figure 1 (a)) is slightly greater at each stage compared with the second and third cycles . This may be the result of two factors . Firstly the cells are in a starved condition before synchronization and hence are in a physiological state different from that encountered in normal growth . Secondly the degree of synchronization is more precise in the first cycle, a degeneration occurring in the second and third cycles . The use of a zonal rotor to separate cells grown under normal physiological conditions should resolve this factor . The implications of these results on the variation in U .V. -sensitivity seen in the cell-cycle may now be discussed . Previous experiments have indicated that the U .V.-sensitivity of a RAD strain also changes in accord with the percentage of budding cells in the cycle . That is, the more budding cells there are, the higher the U .V. resistance observed (Chanet et al. 1973) . Hence one must consider could the variation in pyr[ ]pyr induction possibly account for the variation in sensitivity observed? A variation of 50 per cent in the lethality produced on the irradiation of a synchronized RAD strain of Saccharomyces cerevisiae (Chanet et al . 1973) could not be explained by a 20 per cent fluctuation in pyr[ ]pyr content alone . However, we have used a high U .V . dose of 6 x 10 3 ergs/mm 2 in our experiments, this by no means being related to the previous physiological experiments. The lower doses used by Watanabe and Harikawa (1974) gave a greater fluctuation (50 per cent) than at the higher doses used by both Trosko et al . (1966) and Humphrey and Steward (1966) who reported fluctuations of 20 per cent and 34 per cent, respectively . Admittedly, they used different cell-lines, which makes any comparison speculative . Whether on the basis of such data the fluctuation is greater or not at low U .V. doses may be questioned . Techniques enabling a better resolution of pyr[ ]pyr induction in yeast will resolve this problem . To date at least three repair pathways would appear to be responsible for the repair of U .V. damage in yeast (Game and Cox 1973) . When one regards the cyclic fluctuations of a double mutant rec5 rad1_3 (Chanet et al . 1974) which is extremely U .V.-sensitive and hence deficient in some of the major repair pathways, it is seen to be of the order of 30 per cent . The fluctuation in pyr[ ]pyr induction after high doses could possibly account for these variations in the viability of rec 5rad1_3 seen for lower doses . Thus differences in the amounts of pyr[ ]pyr induced during the cell-cycle may be more important : (i) for low doses when a strain is deficient in the major repair pathways of U .V. damage ; (ii) for high doses, when the major repair pathways are saturated .
Correspondence
485
ACKNOWLEDGMENTS
The authors wish to thank Madame R . Guilbaud and Mademoiselle A . Boulet for technical assistance . One of us (R .W .) is indebted to EMBO for supplying a long term fellowship . This investigation was supported in part by the Commissariat a 1'Energie Atomique (Saclay, France) and Euratom (contract n° 126-74-7-BIOF) .
Int J Radiat Biol Downloaded from informahealthcare.com by University of Toronto on 12/29/14 For personal use only.
REFERENCES CHANET, R ., and HEUDE, M ., 1974, Molec . gen . Genet ., 131, 21 . CHANET, R ., HEUDE, M ., and MOUSTACCHI, E ., 1974, Molec . gen . Genet., 132, 23 . CHANET, R ., WILLIAMSON, D . H ., and MOUSTACCHi, E ., 1973, Biochim . biophys . Acta, 324, 290 . DAVIES, D . R ., 1965, Mutation Res ., 2, 477 . ESPOSITO, R . E., 1968, Genetics, 59, 191 . FABRE, F., 1973, Radiat . Res ., 56, 528 . GAME, J . C ., and Cox, B . S ., 1973, Mutation Res., 20, 35 . HOLLIDAY, R ., 1965, Genet. Res . (Camb .), 6,104. KIMBALL, R . F ., 1963, Repair from Genetic Radiation Damage, edited by F. H . Sobels (London : Pergamon), p . 167 . SCOPES, A . W., and WILLIAMSON, D . H ., 1964, Expl Cell Res ., 35, 361 . SETLOW, R . B ., and CARRIER, W . L ., 1966, Y. molec. Biol ., 17, 237 . STEWARD, D . L ., and HUMPHREY, R . M ., 1966, Nature, Lond ., 212, 298 . TERASIMA, T ., and TOLMACH, L . J ., 1961, Nature, Lond ., 190, 1210 . TROSKO, J . E ., KASSCHAU, M ., COVINGTON, L ., and CHU, E . H . Y ., 1966, Radiat . Res ., 27, 535 . UNRAU, P ., WHEATCROFT, R ., Cox, B . S ., and OLIVE, T ., 1973, Biochim . biophys . Acta, 312, 626 . WATANABE, M ., and HARIKAWA, M ., 1974, Biochem . biophys . Res . Commun., 58,185 . WATERS, R ., and MOUSTACCHI, E ., 1974 a, Biochim . biophys. Acta, 353, 407 ; 1974 b, Ibid., 366, 241 ; 1975, Y . Bact ., 121, 901 . WILLIAMSON, D . H., and SCOPES, A . W ., 1962, Nature, Lond., 193, 256 . WILLIAMSON, D . H ., 1965, `J. cell Biol ., 25, 517 .
