B.S. Rao

Non-equivalence of YEPD and synthetic complete media in yeast reversion studies

In yeast reversion studies, assay of the total number of cells is made by plating irradiated cells on agar plates containing yeast extract, peptone and dextrose (YEPD) medium. The number of revertants are scored by plating cells on synthetic complete (SC) medium deficient in the particular nutrient for which the reversion is tested. In this procedure equivalence for cell survival between the YEPD and the SC media is always assumed. However it is shown in this paper that this assumption is valid only up to dose levels where cell killing is not significant. At high doses, survivals on the two media differ significantly from each other for both high and low LET radiations. This difference influences the slope of the reversion frequency curve at high doses. Since the reversion frequency is expressed with reference to the number of survivors after a given radiation dose, it is essential to see that the same chance of survival is offered to the reverted and unreverted cells. Even though reversion is reported to vary linearly with dose, it is found that this linearity is restricted only to dose levels where cell killing is not significant. At higher doses, the reversion frequency varies in a very complex manner with dose for both high and low LET radiations. The complexity depends further on the reference medium chosen.

Liquid holding recovery in stationary and log phase cultures of diploid yeast exposed to gamma and alpha radiations

SummaryThe kinetics of liquid-holding recovery (LHR) in diploid yeast after gamma and alpha irradiation is studied. In case of stationary phase culture the rate and extent of LHR is found to be greater for gamma-ray-induced damage than for alpha-ray-induced damage. At 10% survival level, the half-time for recovery is 5.2 h for gamma-ray damage and 12 h for alpha-ray damage. Further, while the recovery factor for alpha damage reaches saturation at 5% survival level, that for gamma damage continues to increase as survival level decreases. Oxygen is required for the recovery process during LH after gamma irradiation. The cells can recover to the same extent from both oxygen-dependent and oxygen-independent components of damage. Log phase cells containing a high per cent of budding cells, however, exhibit negative liquid holding effect after gamma irradiation.

Dependence of the expression of the radiation-induced gene conversion to arginine independence in diploid yeast on the amino acid concentration: Effect on allelic mapping

The yield of radiation-induced gene conversion to arginine independence in diploid yeast depended on the concentration of the amino acid both in the plating medium and in the intracellular pool. By depletion of the level of arginine in the intracellular pool of amino acid or by provision of arginine at 0.4 mg/l of the plating medium the yield was varied by a factor as high as 20. This may be important in studies of the genetic mapping of alleles based on the slope of conversion frequency versus dose line.

Genetic control of repair of radiation damage produced under euoxic and anoxic conditions in diploid yeastSaccharomyces cerevisiae

SummaryDiploid wild type yeast strains 211, X2180 and the radiation sensitive mutantsrad2, 6, 9, 18, 50–55, and57 were exposed to cobalt-60 gamma radiation, in the presence and absence of oxygen, in order to identify the RAD loci involved in the repair of sublethal damage (SLD), recovery from potentially lethal damage (PLD) and oxygen enhancement ratio (OER). Response of wild type and mutants were compared in terms of survival curve parameters Dq, D10, D1, and D0. As compared to wild type the mutants showed increased sensitivity to radiation lethality, both under euoxic and hypoxic conditions, as judged by the reduction in Dq and D0 values. OER was reduced in therad2, 9, 18, 50, 51, and57 mutants indicating that these genes could be associated with the repair of gamma radiation damage produced under hypoxic condition.Shoulder (Dq) a measure of the ability of the cells to repair SLD, was reduced in therad6, 9, 18, 50, 53, and57 strains and was almost absent in therad51, 52, 54, and55 mutants. The ability to recover from PLD was equal to that of wild type strain in therad2, 6, 9, and18 strains, reduced in therad53, 55, and57 strains and was absent in therad50–52 and54 strains. In the mutants with liquid holding recovery ability, the extent of recovery from PLD produced under euoxic and hypoxic conditions was the same. These observations suggest that different groups of loci are involved in the control of different repair processes and that the expression of therad50–57 loci play a very important role in the repair of ionising radiation damage.On the basis of the liquid holding recovery data presented here and the observations made by others it is suggested that the unrepaired DSB constitute the PLD and that the repair of DSB involves recombination between homologous chromosomes.

Comparison of Sensitivity of Rad Mutants of Diploid Yeast to Heat and Gamma Radiation: Cellular Target for Heat Inactivation

Wild type and radiation-sensitive mutants rad 53, 54 and 55 of the diploid yeast Saccharomyces cerevisiae, in stationary and log phase were exposed to gamma radiation and hyperthermia (51 degrees C) in order to compare their sensitivity to these agents. The wild type diploid strain exposed to gamma rays showed a sigmoidal survival curve both in stationary and log phase cultures. Log phase cells were significantly more resistant than stationary phase cells. When compared to wild type, the gamma radiation response of the mutants indicated that the mutations in these RAD loci render the cells sensitive in stationary phase and very sensitive in log phase. The response of mutants to hyperthermia was similar to that of wild type cells in both the phases. The log phase cells of both wild type and mutants wee gamma radiation response of the mutants indicated that the mutations in these RAD loci render the cells sensitive in stationary phase and very sensitive in log phase. The response of mutants to hyperthermia was similar to that of wild type cells in both the phases. The log phase cells of both wild type and mutants wee gamma radiation response of the mutants indicated that the mutations in these RAD loci render the cells sensitive in stationary phase and very sensitive in log phase. The response of mutants to hyperthermia was similar to that of wild type cells in both the phases. The log phase cells of both wild type and mutants were more sensitive to heat than stationary phase cells. These results suggest that the RAD loci are not involved in the repair of hyperthermic damage. Since it is known that the products of the RAD genes are involved in the repair of DNA damage, the wild type response of these rad mutants to hyperthermia indicates that the DNA may not be the principal target for hyperthermic killing. Furthermore, the enhanced thermal sensitivity of log phase cells, containing higher amounts of active enzymes and sensitive membrane, strongly suggests that proteins and/or membranes could be the primary targets for thermal inactivation.

Hyperthermic inactivation of diploid yeast and the interaction of damage caused by hyperthermia and ionizing radiation.

Inactivation of diploid yeast by hyperthermia has been studied. DO and Dq decrease with temperature for euoxic and anoxic conditions. The Arrhenius plot shows a break at 52 degrees C; the inactivation energies above and below this temperature are 153 and 94kcal/mol respectively. Hyperthermia (20 min at 51 degrees C) also potentiates the lethal action of gamma rays in diploid yeast cells under both euoxic and anoxic conditions. The interaction between hyperthermic and radiation damage appears to be largely at the sublethal level. The euoxic cells, the hyperthermic potentiation decreases with increasing time between the application of hyperthermia and radiation, being completely lost after 24 hours. However, in the anoxic cells there was no decrease in the hyperthermic potentiation with increasing time interval. These results suggest that yeast cells are capable of repairing hyperthermic sublethal damage, but require oxygen to do so. Thus there is a similarity in the process of repair of sublethal damage caused by ionizing radiation on the one hand and hyperthermia on the other.

Genetic control of budding-cell resistance in the diploid yeast Saccharomyces cerevisiae exposed to γ-radiation

The gamma-radiation response of stationary and budding cells of wild-type diploid strains (RAD) and radiation-sensitive strains rad2, 6, 9, 18, 50-55, 57 and rec4 was studied. As compared with the wild-type strains, mutants generally showed enhanced sensitivity in both stages of the cell cycle. Budding-cell resistance was totally absent from rad50-55 strains. Mutants rad6, 9 and 18 showed some degree of budding-cell resistance. The response of rad2 and rec4 strains was identical with that of the corresponding wild-type strains. These results suggest that the pathway dependent upon the expression of RAD50-55 loci functions more efficiently in budding cells compared with the pathway dependent on RAD2 and RAD6, 9 and 18 loci. Recombination between sister chromatids appears to play an important role in budding-cell resistance, and this process is under the control of the RAD52 repair pathway. The relationship between the repair pathways associated with budding-cell resistance and post-irradiation cellular recovery (LHR) is discussed.

Post-irradiation modification of radiation-induced heteroallelic reversion in diploid yeast: Effect of nutrient broth

The effect of post-irradiation growth in complete rich medium on the expression of the reversion to arginine-independence induced by gamma and alpha radiation in a heteroallelic diploid yeast strain (Saccharomyces cerevisiae BZ34) has been studied. During the post-irradiation treatment the reversion frequency increased, reached a peak at about 90 min and decreased thereafter reaching a constant value for treatment periods exceeding 6 h. As determined by the increase in number of budding cells, extensive DNA synthesis took place in cells incubated only in the nutrient medium and not in the omission medium. Hence the observed increase in the reversion frequency is explained on the basis that post-irradiation DNA synthesis is necessary for the expression of gene conversion. The decrease in the reversion frequency for continued treatment with yeast extract, peptone, dextrose (YEPD) is related to the fact that only one daughter of the post-irradiation first cell division is a revertant. The broth effect was not lost when the irradiated cells were first incubated for 90 min in arginine-less medium and then transferred to the broth. Similarly, the broth effect persisted even at doses high enough to induce considerable division delay. These results suggest that the radiation-induced pre-conversional lesions are not susceptible to repair by alternative pathways.

Modification of High LET Radiation-induced Damage and Its Repair in Yeast by Hypoxia

The lethal response of a diploid yeast strain BZ34 to densely ionizing radiations from the reaction 10B(n, alpha)7 Li was studied. The values for relative biological effectiveness (r.b.e.) and oxygen enhancement ratio (o.e.r.) for this radiation compare favourably with the data obtained with charged particles on the same strain of yeast. Recovery from potentially lethal damage was also studied by post-irradiation holding under non-nutrient conditions. In order to understand the role of oxygen in the recovery process, the investigation covered the following treatment regimens: (a) aerobic irradiation and aerobic holding (A-A), (b) aerobic irradiation and hypoxic holding (A-H), (c) hypoxic irradiation and hypoxic holding (H-H) and (d) hypoxic irradiation and aerobic holding (H-A). It has been found that the presence of oxygen is essential for recovery from the damage induced by both gamma rays and high linear energy transfer (LET) radiations. The extent of recovery was larger for gamma-induced damage than for damage induced by high LET radiation (alpha + 7Li) for the A-A condition. In the H-H condition, while only a slight recovery was seen for gamma-induced damage, it was totally absent for high LET damage. For the modality A-H, it was found that there is not recovery from the sparsely ionising gamma radiation-induced damage. The implications of these results for the treatment of malignant tumours by radiotherapy are briefly discussed.

Induction of gene conversion in yeast cells continuously cultured at high radiation background.

The induction of genetic damage was investigated by culturing diploid yeastSaccharomyces cerevisiae D7 cells continuously at radiation levels ranging from 0.383 µSv/h to 1.275 mSv/h by selecting appropriate concentrations of tritiated water in the growth medium. These radiation levels correspond to 3–10000 times the natural background. Parameters such as growth kinetics, gene conversion frequency at background radiation and after a challenging dose of acute gamma-radiation or alkylating agentN-methyl-N′-nitro-N-nitrosoguanidine (MNNG) were assessed. The gene conversion frequency in most of the assays was in the range of 5–10 convertants per 106 cells, as in the case of controls. However, a number of the cultures showed conversion frequencies above 20 per 106 viable cells. This stochastic phenomenon occurred more frequently in cells which were incubated at higher radiation levels and for longer durations. This suggests that radiation is responsible for the phenomenon. When subculturing continued beyond 900 h, gene conversion frequencies reverted back to normal values in all cultures in spite of elevated background radiation levels, thus suggesting an adaptive response. The generation time of the cells was 78 min in all cultures irrespective of the radiation level. The response of the cells cultured at elevated background radiation levels to subsequent challenging treatment with gamma-radiation or MNNG was identical to that of the control cultures. Our results suggest that in eukaryotic yeast, low-level radiation may induce an adaptive response to chronic radiation, whereas no such response could be detected when the cells were challenged with acute high-dose exposure or with MNNG.