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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=irab20 International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine ISSN: 0020-7616 (Print) (Online) Journal homepage: www.tandfonline.com/journals/irab19 Delayed Plating Recovery in Diploid Yeast of Different Sensitivities after X-ray and Alpha- particle Exposure E. Schneider & J. Kiefer To cite this article: E. Schneider & J. Kiefer (1976) Delayed Plating Recovery in Diploid Yeast of Different Sensitivities after X-ray and Alpha-particle Exposure, International Journal of Radiation Biology and Related Studies in Physics, Chemistry and Medicine, 29:1, 77-84, DOI: 10.1080/09553007614551581 To link to this article: https://doi.org/10.1080/09553007614551581 Published online: 03 Jul 2009. Submit your article to this journal Article views: 159 View related articles https://www.tandfonline.com/action/journalInformation?journalCode=irab20 https://www.tandfonline.com/journals/irab19?src=pdf https://www.tandfonline.com/action/showCitFormats?doi=10.1080/09553007614551581 https://doi.org/10.1080/09553007614551581 https://www.tandfonline.com/action/authorSubmission?journalCode=irab20&show=instructions&src=pdf https://www.tandfonline.com/action/authorSubmission?journalCode=irab20&show=instructions&src=pdf https://www.tandfonline.com/doi/mlt/10.1080/09553007614551581?src=pdf https://www.tandfonline.com/doi/mlt/10.1080/09553007614551581?src=pdf INT . J . RADIAT . BIOL ., 1976, VOL. 29, NO, 1, 77-84 Delayed plating recovery in diploid yeast of different sensitivities after X-ray and alpha-particle exposure E. SCHNEIDER and J. KIEFER Strahlenzentrum der Justus Liebig-Universitat, Giessen, Germany (Received 29 August 1975 ; accepted 28 November 1975) 1. Introduction Ionizing radiation is generally more effective in reducing colony forming ability if LET is increased . Since it is often assumed that this may be due to different reparability of the damage inflicted it is of interest to study LET- dependence in cells of different sensitivities and/or under conditions where recovery mechanisms are differently effective . Yeast cells offer the possibility to pursue both ways, because sensitive mutants are readily available and cell- survival may be increased by ` liquid holding' or ` delayed plating' (Patrick, Haynes and Uretz 1964) . This effect is assumed to reflect a specific recovery mechanism . We report here on experiments with three strains of diploid Saccharomyces cerevisiae which differed in U.V.- and/or X-ray sensitivity . They were exposed to 100 kV X-rays and 241Am-a-particles and plated immedia- tely or after a delay of several days . Colony-forming ability was followed over several decades, and the extent of delayed plating recovery assessed . 2 . Material and methods 2.1 . Cells and culture conditions The following diploid strains of S. cerevisiae were used : wild type, termed 211, originally received from Laskowski, Berlin, and the two radiation-sensitive mutants, S 2094 and S 2095, received from Averbeck (Averbeck 1970) . All strains have been maintained in our laboratory for several years . Strain S 2094 carries a mutation in the rad 2 gene and is presumably excision-negative (Game and Cox 1971, Resnick and Setlow 1972) . Strain 2095 is mutated in the rad 9 gene (Game and Mortimer 1974) . The cells were cultivated in a synthetic nutrient medium (modified Wickerham medium, Wickerham 1951) at 30°C under continuous bubbling with air. Strains 2094 and 2095 require isoleucin and valine which were added to the medium . Cells from the stationary phase (1 .3-1 .5 x 10 8 cells/ml) were taken for irradiation experiments . The percentage of budding cells was always smaller than 10 per cent for the strains 211 and S 2095 and smaller than 3 per cent for strain S 2094 . Generally the cells were placed on membrane filters with a pore diameter of 0 . 6 tt for alpha- and X-irradiation which were kept on wet filter-paper to prevent drying and rotated during exposure . X-irradiations in suspension were also performed for comparison . Surviving cells were scored as macro- colonies after 4-5 days incubation on nutrient agar in plastic Petri dishes . For delayed plating the membrane filters with the cells were placed on 0 .9 per cent 78 Correspondence NaCl-containing agar for at least 4 days at 30°C and then transferred to nutrient agar . Delayed plating kinetics was followed in the same way for 6 days . 2 .2 . Irradiation and dosimetry The alpha-radiation source was an Americium-241 a-emitter foil of about 10 x 10 cm. The specific activity was 192 µC/cm 2 . Am-241 emits a-particles with an energy of 5 .5 MeV and has a half-life of 458 years . They-contamination (30 and 60 keV) is negligible, as shown by control experiments in which the cells were shielded against the a-particles by a thin plastic foil . The particle flux was determined by exposing nuclear emulsion film. The surface of the foil is protected by a thin coating which results in a reduction of the energy to about 4.5 MeV. The cells were exposed at a distance of 10 mm from the source. The maximum energy of a-particles hitting the cells is then reduced to 3 .4 MeV owing to the energy loss in air between the source and the object . The average energy is computed to be 2 .6 MeV, and we estimate a dose-rate of 3 .6 krad/min ± 10 per cent (Schneider 1974) . Ferrous sulphate dosimetry was also used, assuming a G-value of 4 . 8 ± 0 .5 (Bacq and Alexander 1961) . This method led to a value for the dose-rate of 4 .4 krad/min ± 10 per cent . A mean value of 4 krad/min was therefore taken for all dose determinations . X-irradiations were performed with a 100 kVp X-ray machine (Philips X-ray generator PW 1140) . At 100 kV voltage and 27 mA current a dose-rate of 80 krad/min ± 10 per cent at 10 cm focus distance for irradiations on membrane filters was determined . All experiments were repeated at least three times . Survival curves were fitted by eye, standard errors were estimated from single experiments . A DP-factor is defined as the ratio of doses resulting in the same surviving fraction under DP- and IP-conditions . 3. Results Delayed plating results with X-rays are shown in figure 1. There is no constant DP-factor ; with higher doses DP- and IP-survival curves become parallel. The main effect of delayed plating is therefore an increase in extra- polation number . Constancy of the DP-factor-as suggested by Patrick and Haynes (1964)-may be erroneously assumed if survival is not followed to low values. Control experiments showed that the relatively long time during which the cells were kept on membrane filters for a-irradiation did not influence survival . The results of the alpha-experiment are shown in figure 2 and are similar to the behaviour after X-rays . To eliminate the possible influence of the different irradiation conditions, we compared the DP kinetics after irradiation with high doses of X-rays when the cells are in suspension during irradiation with that when the cells are placed on membrane filters . No difference was found . The DP kinetics after a-irradiation are similar to that after X-irradiation (figure 3) . Comparison of the X-ray and the a-particle survival curves gives a r .b.e . value of 1 .3 ± 0 . 1, calculated from the Do-values of the respective survival curves . There is no change in extrapolation number between X-rays and a-particles . Figures 4 and 5 show the DP results of the mutant S 2094. The DP effect is expressed in the same way ; the amount of the DP repair is even slightly 10 0 10 -2 10 -3 10 -4 10-5 10-6 Figure 1 . Delayed plating recovery after X-irradiation on membrane filters, strain 211 IP = immediate plating, DP =delayed plating, IP : Do=18 krad, DP-factor (10 per cent)= 2 .3 . 10 0 10"1 50 Correspondence 100 X - ray dose 150 d - dose 40 60 120 160 200 krad ~~o\ ¢\0 211 z N I\ N~~DP \ 1P . I\ 200 10 -4 M\ Figure 2. Delayed plating recovery after a-particle irradiation strain krad, DP-factor (10 per cent)= 2-4 . 25o krad 79 211, IP : Do =14 80 Correspondence larger both after X-rays and after a-particles especially in the low dose range (DP-factor at 10 per cent survival about 2 .6) . The DP-results of the strain S 2095 are shown in figures 6 and 7 . This strain has a two-phasic survival curve, but at least for the first part of the survival curve (X-rays, figure 6) a constant dose-modifying DP-factor of about 2 .6 . In the case of a-irradiation (figure 7), the effect is also dose-modifying but reduced (DMF =1 .9) . 10 -2 C 0 u 10 -3 rnc 10' 5 Figure 3 . Delayed plating kinetics of strain 211 after X-irradiation in suspension (I), a-irradiation (II) and X-irradiation (III) on membrane filters . 100 101 16-4 X-ray doss 50 100 150 200 krad \- S 20940 x -\o .\ DP x ?\. DP IP X\_ o\ Figure 4. Delayed plating recovery after X-irradiation, strain S 2094, DP-factor (10 per cent) =2-5, IP : Do = 17-5 krad . °-I 0 o . ~- III r 1 1 1 1 1 1 2 3 4 5 6 (days) delayed plating time 10 0 C 0 ro 10-2 rn c N 10-3 Figure 5 . Delayed plating recovery after «-irradiation, strain per cent) =2-7, IP : Da =11 . 5 krad . 25 50 Correspondence 81 oc -dose 75 100 125 150 175 krad J S 2094, DP-factor (10 Figure 6. Delayed plating recovery after X-irradiation, strain S 2095, DP-factor (10 per cent)= 2-6, Do-values (see the table) . 4. Discussion 4.1 . Relative biological efficiency of alpha particles X-ray and alpha-particle survival curves have essentially the same general shape with all strains studied in our experiments . This behaviour is different from that of mammalian cells, where increasing LET results in a progressive loss of the shoulder (Barendsen, Walter, Fowler and Bewley 1963, Todd 1967) . Since it has been suggested that repair-deficient mutants of yeast may resemble mammalian systems in this respect (Raju, Gnapurani, Martins, Madhvanath, R.B . F 82 Correspondence Figure 7 . Delayed plating recovery after a-irradiation, strain S 2095, DP-factor (10 per cent= 1 .9, Do -values (see the table). Howard, Lyman and Mortimer 1971), one has to conclude from our results that this obviously depends on the nature of the mutation . More detailed studies would be helpful to elucidate the nature of the LET-effect . Unfortunately, virtually nothing is known about the molecular mechanisms of repair phenomena in yeast after exposure to ionizing radiation . It would be interesting to see whether high LET action may be related to the ability to repair double-strand breaks, which was demonstrated in yeast very recently (Resnick and Martin 1974) . As X-ray and alpha-survival curves have the same extrapolation numbers, r.b.e. values can be calculated from the slopes in the exponential region . The relevant data are summarized in table 1 . The behaviour of strain 2095 deserves further discussion . Compared with the wild type it is more sensitive to X-rays by a factor of 3, as judged from survival curve slopes in the initial region . The broken character of the survival curve in the sensitive strain cannot simply be attributed to population heterogeneity, since the terminal parts do not extrapolate to the same ordinate value in the IP- and DP-experiments . The cause of this particular shape cannot be understood at present; it should be noted, however, that the break occurs at the same dose-level . In contrast to strain 211 and 2094, the r .b .e. values are different in strain 2095 for immediate and delayed plating ; they are higher for the latter treatment, which suggests that delayed plating recovery after alpha-exposure is less efficient in this strain . This is particularly obvious for low survival levels . Again, without any knowledge of the mechanism that leads to the higher sensitivity, no explanation can be offered . 4.2 . Delayed plating It has been shown previously that survival curves from wild-type diploid yeast retain shoulders even with high-LET radiation (Mortimer, Brustad and Correspondence 83 m a y ,p +7" l O C +I +I CV M O N +I +I N 'i' O M LI +I O' 1~ bD p 71 e O N +I +I (71 Ln N ~ v> .n +I +I T ~ O O +I +I 00 00 h N A m N O N +I +1 Ln 00 M O L +I +I ~Ln N N Ln +I +I Ieq ,- . .x A on Q ¢, a~ e I- .r? i+ +1 +I M M .n +I 'n .n .-1 'I MI M 00 _ 2S O 00 00O O +I +I t- OO .-++I +I M MO N+I +I y 00 +I +I 00 00 CO +I +I .n .n O(V +I +I O •n N'Og cm a4 .~ v) V] - rn oN ON O N 84 Correspondence Cormack 1965), and that DP-recovery can also be shown under these conditions (Lyman, Haynes and Tobias 1963, Lyman and Haynes 1967) . The present communication confirms these data and extends them to two sensitive strains . Obviously, yeast behaves differently from mammalian cells (Barendsen 1970) . One reason may be the different size of sensitive targets in the cells, so that the low-LET delta-rays may have higher relative importance in yeast than in mammalian systems . Haploid yeast, which is more sensitive to X-rays than diploid, has an exponen- tial survival curve and does not show DP recovery after exposure to ionizing irradiation (Patrick et al . 1964) . It appears from the present study that the abilities to accumulate sub-lethal damage and to show delayed plating recovery are interrelated . REFERENCES AVERBECK, D., 1970, Thesis, Freie Universitat Berlin, Germany . BACQ, Z. M ., and ALEXANDER, P., 1961, Fundamentals of Radiobiology (Oxford : Pergamon Press) . BARENDSEN, G . W., 1964, Int . Y. Radiat . Biol., 8, 453 ; 1970, Proc. 2nd Symposium on Microdosimetry, edited by H. G . Ebert, p. 83 . BARENDSEN, G. W., WALTER, H. M. D ., FOWLER, J . F ., and BEWLEY, D. K., 1963, Radiat . Res ., 18, 106 . BILUSHI, V., and KOROGODIN, V. I ., 1961, Dokl. Akad. Nauk. SSSR, 138, 1208 . GAME, J . C ., and Cox, B. S., 1971, Mutation Res ., 12, 328 . GAME, J . C ., and MORTIMER, R. K., 1964, Mutation Res ., 24, 281 . HAYNES, R . H., 1964, Physical Processes in Radiation Biology (London, New York : Academic Press), p. 51 . HAYNES, R. H., PATRICK, M. H ., and BAPTIST, J . E ., 1964, Radiat . Res., 22, 194 . KOROGODIN, V. I ., BILUSHI, V., MARKOVA, L . I ., and SHEKHTMAN, Y.L ., 1963, Radio- biologiya, 3, 39 . LYMAN, J. T., and HAYNES, R . H., 1967, Radiat . Res ., Suppl ., 7, 222 . LYMAN, J . T ., HAYNES, R. H., and TOBIAS, C . A., 1963, Radiat . Res ., 19, 237 . MORTIMER, R., BRUSTAD, T., and CORMACK, D . V ., 1965, Radiat . Res., 26, 465 . PATRICK, M. H ., and HAYNES, R . H., 1964, Radiat. Res ., 23, 564 . PATRICK, M. H ., HAYNES, R. H., and URETZ, R . B., 1964, Radiat . Res ., 21,144 . RAJU,M.R., GNAPURANI,M., MARTINS, B. I ., MADHVANATH, U., HOWARD, J ., LYMAN, J . T., and MORTIMER, R . K., 1971, Radiat. Res ., 47, 635 . RESNICK, M. A., and MARTIN, P., 1974, 5th Int. Congr. Radiat . Biol ., Seattle, U.S .A ., Abstract B-13-6 . RESNICK, M. A., and SETLOW, J . K., 1972, Y. Bacteriol., 109, 979 . SCHNEIDER, E., 1974, Thesis, D26, Justus Liebig-Universitat, Giellen, Germany . TODD, P., 1967, Radiat . Res., Suppl ., 7, 196 . WICKERHAM, L . J., 1951, U.S. Dep . agric. Technol . Bull ., No . 1029 . page 1 page 2 page 3 page 4 page 5 page 6 page 7 page 8
