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.     

Spontaneous mutability in yeast. I. Stability of lysine reversion rates to variation of adenine concentration

Novick and Szilard demonstrated that increasing the concentrations of adenine enhance the mutation rate of E. coli. We have found that the spontaneous mutation rate of the yeast Saccharomyces cerevisiae remains constant over a 200-fold range of adenine concentration.The system that we commonly use for measuring spontaneous mutation rate is reversion of the super-suppressible mutant lys1-1. In this system, growth of the yeast is limited by limiting the amount of lysine in the medium. A reversion to lysine independence will continue to grow. One of the other super-suppressible mutants in the test system is ade2-1, a mutant that causes accumulation of red pigment. By adjusting the concentration of adenine slightly above that of lysine, reversions of super-suppressors produce white colonies and reversions of the lys1-1 locus itself produce red colonies.     

Induction of dominant lethality by x-rays in radiosensitive strain of yeast

X-Ray-survival curves of haploid, diploid, triploid and tetraploid yeast strains homozygous for the X-ray-sensitive mutation rad52 (previously xs1 are presented. These curves suggest that strains carrying the rad52 mutation may be more susceptible than wild type to X-ray-induced dominant lethal damage. For the crosses (+ × +, rad52 × rad52, + × rad52, rad52 × +) in which only one parent was irradiated, the relationships between zygote survival and X-ray dose were similar except for rad52 × rad52. In this cross a considerably higher frequency of dominant lethal damage was observed. This observation indicates that the rad52 mutant lacks a repair system for X-ray damage and is consistent with the proposal that unrepaired chromosome damage is the event which leads to dominant lethality and reproductive cell death.     

Loss of hydrazine-induced mutability in wild-type and excision-repair-defective yeast during post-treatment inhibition ofcell division.

The fate of presumed premutational DNA lesions induced by hydrazine was studied under a variety of post-treatment conditions in wild-type and in excision repair-defective (rad2-1) strains of Saccharomyces cerevisiae. In all strains the full extent of hydrazine-induced, forward mutability from CAN1 to can1 (canavanine resistance) was dependent upon post-treatment cell division in mutagen-free synthetic or complex growth medium before plating on canavanine-containing selective agar and could be blocked by inhibitors of DNA synthesis (hydroxyurea) or protein synthesis (cycloheximide) contained in the growth medium. Following the growth-inhibitory period, cells were permitted to grow in fresh medium lacking inhibitors to determine the level of induced mutation remaining. Nearly all induced mutability was lost after a one-day growth inhibition, compared with mutagen-treated control samples subsequently grown twice in medium lacking inhibitor. In the wild type, half the induced mutability was lost after 3 h. The data suggest that premutational DNA lesions induced by hydrazine were removed, or possibly rendered non-mutagenic, by some error-free repair process that acted before mutation fixation by base mispairing during DNA replication. Since rad2-1 and RAD strains both exhibited loss of mutability, this process is not dependent upon the activity of an intact pyrimidine dimer excision-repair system.     

Effects of spermine on the detection of induced forward mutation at the Can1 locus in yeast: evidence for selection against canavanine-resistant mutants.

The effect of exogenous spermine tetrahydrochloride (0.5 mg/ml) on hydrazine- and nitrous acid-induced forward mutation to canavanine resistance (CAN1 leads to can1, normal to defective arginine permease) was examined in stationary-phase haploid Saccharomyces cerevisiae. Post-treatment cell division (specifically DNA replication) is required for hydrazine mutagenesis at this locus, whereas nitrous acid mutagenesis exhibits, in addition, a significant post-treatment-independent component. Spermine addition only during mutagenic treatments in buffer did not affect mutagen cytotoxicity, but did result in a slight yet consistent decrease in induced mutation frequencies. Addition of spermine to the yeast extract--peptone--dextrose (YEPD) post-treatment growth medium resulted in dramatic reductions of induced mutation frequencies, which could be alleviated by pregrowth in spermine-containing YEPD. Such a medium was found to cause an apparent temporary growth inhibition for almost 40 h, after which the growth rate of the culture increased rapidly. Cultures "recovering" from spermine inhibition were no longer inhibitable by spermine in fresh medium, suggesting an outgrowth of spontaneous and/or induced spermine-resistant derivatives. Genetic analysis of one isolate revealed a single dominant nuclear gene conferring resistance by some means other than defective spermine uptake. Growth of this mutant was only slightly inhibited by spermine (20% increase in doubling time), while mutation expression remained high. Results of competitive growth experiments indicated that spermine-containing YEPD exerted a selection pressure against canavanine-resistant cells, while YEPD by itself did not. The mechanism for this selection is not presently understood. With respect to replication-dependent induced mutation at CAN1, our initial observation of a strong apparent antimutagenic action of spermine was found to be best explained by this specific selection against can1 mutants. This underscores the need for caution in the interpretation of experiments designed to study physiological modification of mutagenic potential.     

X-ray induced translocations in spermatogonia. I. Dose and fractionation responses in mice

The frequency of recirprocal translocations, inducedm by X-irradiation of mouse spermatogonial cells and observed at diakinesis-metaphase I in primary spermatocytes, was measured over a dose range of 0–1200 R. The resulting dose-response curve gave a best fit to the model Y = bD+CD² over the range of 0–500 R. Above 600 R, howeverm, the yeild of translocations decreased with increasing dose, leadiong to a “humped” dose-response curve over the whole dose range studied, as has been observed by several worker previously.The significance of the nonlinear dose-response curve over the lower dose range is discussed in terms of the known fractionation and dose-rate effects for reciprocal translocations induced in spermatogonia.A dose of 800 R was split into two 400-R fractions separated by 8 weeks, or one of 1200 R into three equal parts, each separated by an 8-week interval. The resulting yield of translocations was the same as the sum of two, or three, separate 400-dose doses, but was much higher than a single dose of 800 R or 1200 R.It is suggested that these results, namely the shape of the dose-response curve and the “reverse” fractionation effect, can be explained in terms of resistant and sensitive stem-cell populations, but that any one cell can be in either population, depending upon the stage of the cell cycle in which it is at the time of irradiation.     

Simplified luciferase assay of NAD+ applied to microsamples from liver,kidney and pancreatic islets

A new single-step procedure for the bioluminescence assay of NAD⁺, permitting measurements on the pmol level (10^?12 mol) is described. Acid extracts of NAD⁺ were prepared in different tissues. The acidification destroys reduced pyridine nucleotides and most enzymes which are present in the tissue sample. After neutralization the extract is added to a light-yielding solution, and the luminescence is measured with a photomultiplier. The maximal height of the signal is measured by means of a digital voltmeter. The light yielder is bacterial luciferase with appropriate additives and supplemented with malate and malate dehydrogenase.The modified light-yielding solution provides for continuous formation of NADH resulting in a durable level of light emission. The cycle involved was shown not to operate with NADP⁺. The slow fading of the emission permits simplification of the measuring procedure. Rapid injection in front of the phototube can thus be omitted and replaced by ordinary mixing before the reaction cell is positioned for the measurement. Furthermore, the instrumentation required is less elaborate than in photokinetic assay, since it is not necessary to record and integrate the time course of the emission. To test the applicability of the method, analyses of pmol amounts were performed in the islets of Langerhans and in tissue samples of much smaller size than fine needle biopsies.     

Radiation-induced human chromosome aberrations. II. Human in vitro irradiation compared to in vitro and in vivo irradiation of marmoset leukocytes

Whole blood cultures from humans and from the New World primate, Saguinus fuscicollis, were irradiated with various doses of 250 kV X-rays. The resulting centric ring plus dicentric aberration yields were fitted to the three models, Y = a+bD, Y = a+bD+cD², and Y = a+cD², by least squares regression. In both instances the best fit was to the model Y = a+bD+cD², with coefficients of the one- and two-track components for human and marmoset being: b = (0.78 ± 0.09)·10⁻³, c = (5.92 ± 0.31)·10⁻⁶, and b = (1.11 ± 0.36)·⁻³, c = (7.7 ± 1.7)·10⁻⁶, respectively.     

Mutagenic effectiveness of 14.1 MeV neutrons and 200 kV X-rays at the dumpy complex locus of Drosophila melanogaster.

The effectiveness of 14.1 MeV neutrons relative to 200 kV X-rays for the induction of the various kinds of dumpy mutation in mature sperm of Drosophila melanogaster was investigated. The estimated RBE values are: 0.52 for all complete mutations; 0.64 for the (olv, ov) types; 0.33 for the (ol, lv, o, v, c) types; 0.33 for all fractional mutations. These data lend support to the thesis that (1) complete dumpy mutations of the olv and ov types are more frequently associated with chromosomal aberrations than those of the ol, lv, o, v and c types, and (2) fractional mutations and complete mutations of the (ol, lv, o, v, c) types are most probably point mutational events.     

Chromosomal rearrangements induced in mouse spermatogonia by 14.5-MeV neutrons

Inbred CBA male mice were irradiated with 14.5-MeV neutrons. Three acute doses, 75, 150 and 250 rad, and one chronic dose, 250 rad, were given. The percentages of affected spermatocytes as counted from reciprocal translocations which had been induced in spermatogonia were 0.7, 0.8 and 1.6 respectively for the acute series and 2.2 after chronic exposure. The data could be fitted to a linear or concave curvilinear regression line. There seemed to be a slight increase of damage with dose, even if the percentages were generally lower than those reported earlier for fast neutrons with energies around 1 MeV. The existence of dose-rate effects is discussed, and the conclusion drawn so far is that there seems to be no such effect either for 1-MeV fast neutrons or 14.5-MeV high energy neutrons. The term “reversed dose-rate effect”, as used earlier, relates to another phenomenon. The difference between the point estimates for the chronic and acute 250 rad series is not significant. The effectiveness of neutrons with energies around 14 MeV versus neutrons with energies around 1 MeV is discussed.     

The regulation of human exposure to mutagens amidst scientific controversy

Human peripherial lymphocytes were irradiated with different doses of 15.0-MeV neutrons. The frequency of different aberration types was determined and the dose-response relation was calculated. The data were fitted by least-squared regression analysis to different models. The dicetric, dicentric+centric ring, and different acentric data gave the best fit to the linear quadratic model. The RBE of 15.0-MeV neutrons versus 220 kV X-rays decreased significantly with increasing dose.     

Physical mapping and cloning of RAD56

Here we report the physical mapping of the rad56-1 mutation to the NAT3 gene, which encodes the catalytic subunit of the NatB N-terminal acetyltransferase in Saccharomyces cerevisiae. Mutation of RAD56 causes sensitivity to X-rays, methyl methanesulfonate, zeocin, camptothecin and hydroxyurea, but not to UV light, suggesting that N-terminal acetylation of specific DNA repair proteins is important for efficient DNA repair.     

Ethidium-bromide-induced loss and retention of cytoplasmic drug resistance factors in yeast

By using a strain of Saccharomyces cerevisiae harboring three cytoplasmic resistance factors to oligomycin, erythromycin and chloramphenicol, the effect of ethidium bromide (EB) on the loss and retention of the resistance factors was examined. Comparison was also made of the petite mutation with the loss of each resistance factor.Although resistance to each drug was specific and separately determined, EB induced the segregation of the erythromycin-resistance factor from the chloramphenicol-resistance factor with a very low frequency, and segregation of both from the oligomycin-resistance factor at a higher rate. There was a close correlation between the rate of the petite mutation and that of the loss of any resistance factro in the whole cell population treated with EB.We interpret these findings as indicating that the drug-resistance factors studied are linked together in the mitochondrial genome.     

The genetic control of dark recombination in yeast

The post-UV effects of dark holding and photoreactivating light on survival and intragenic recombination in UV-sensitive strains of yeast have been studied. The results indicate that two liquid holding dark repair pathways exist in this organism: the excision pathway which does not lead to intragenic recombination (“silent” repair) and a dark recombination pathway which does lead to intragenic recombination. A third pathway also exists which is plating-dependent and leads to intragenic recombination via a postreplication pathway.     

Pathways of ultraviolet mutability in Saccharomyces Cerevisiae. IV. The relation between canavanine toxicity and ultraviolet mutability to canavanine resistance.

Seven umr mutants of Saccharomyces cerevisiae which had reduced capacity for ultraviolet light (UV)-induced forward mutation from CAN1 to can1 were tested for sensitivity to L-canavanine relative to one wild-type UMR strain and one slightly UV-sensitive but phenotypically umr⁺ strain (mutant 306). Relative UV mutation resistance was estimated by dividing the UV fluence needed to yeild a particular induced mutation frequency by that needed to reach the same frequency in the genotypic wild-type strain. The umr5 and umr6 strains were especially sensitive to canavanine growth inhibition, while umr1 was no more sensitive than either wild type; umr2, umr3, umr4, a umr7, and α umr7 were equally sensitive to an intermediate degree. Incubation at 30°C of wildtype cells plated on canavanine-selective agar for increasingly longer times before UV irradiation resulted in decreasing UV mutation frequencies (reduced to 50% in 1.6 h). All umr strains tested in this way lost UV mutability faster than wild type, including mutant 306, umr1 (not sensitive to growth inhibition), and umr6 (very sensitive to growth inhibition). Cells were grown to stationary phase in YEDP growth medium and assayed for arginine and tryptophan transport into the cell. The umr6 strain, which had weak UV mutation resistance but high sensitivity to canavanine growth inhibition, transported arginine and tryptophan at essentially wild-type levels. The umr1 strain, however, which had moderate UV mutation resistance and normal canavanine toxicity, transported both amino acids at rates tenfold higher than wild type. The data suggest that increased canavanine toxicity does not necessarily lead to defective mutability at CAN1, and that mutational deficiency cannot result solely from increased canavanine toxicity. Although exposure to canavanine was shown to block mutation fixation and/or expression, it is suggested that the degree of growth inhibition is not strictly correlated with the degree of mutation resistance.     

Suppressor studies on ilvI mutants of Saccharomyces cerevisiae

In order to answer the question whether with the X-ray induction of an achromatic lesion (a gap) an increase of the chromatid length is involved (e.g. by a localized failure of spiralization), the length ratios of 26 straight and 22 flexed metaphase chromosomes of Vicia faba were measured. (The length ratio is defined as the quotient between the length of the chromatid with a gap and that of its sister-chromatid without a gap.) Our results agree with the hypothesis that the induction of a gap does not increase the length of the chromatid. Remarkably in each of the 22 flexed chromosomes the gap was located at the “outer” chromatid. The bearing of our results on the nature of X-ray-induced achromatic lesions is discussed.