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.     

Mutagenesis of yeast by hydrazine: dependence upon post-treatment cell division.

Hydrazine was found to be mutagenic for yeast (Saccharomyces cerevisiae) at exposures (concentration × time) ranging over nearly three orders of magnitude. Little or no forward mutation from CAN1 to can1 was detectable upon immediate plating following treatment in neutral buffer suspension. Post-treatment cell division in yeast extract peptone dextrose complex growth medium was required for expression of induced mutation to canavanine resistance. Frequencies of induced mutation rose to levels approximately 10-fold higher than spontaneous levels for exposures between 0.1 and 12.0 min mol/l. Survival remained at 100%. For exposures greater than 80 min mol/l viability and mutation frequency began to decrease sharply. By contrast, single treatments of ethyl methanesulfonate, methyl methanesulfonate, N-methyl-N′-nitro-N-nitro-soguanidine, nitrous acid, hydroxylamine, and ultraviolet light were able to increase mutation frequency with this system upon immediate assay. Further growth-dependent increases in mutation frequency were not observed with HA and UV.Expression of HZ-induced mutation was detectable after treated cells had undergone less than one population doubling in YEPD. Such mutation expression could be blocked by the inhibitors cycloheximide and hydroxyurea, which block protein synthesis and DNA synthesis respectively. Results were similar to those obtained previously with Haemophilus influenzae and similarly suggest that, in this eukaryote, HZ-induced lesions lead to mutation by causing base mispairing at DNA replication rather than by means of an error-prone repair mechanism.     

A genetic study of x-ray sensitive mutants in yeast

A set of 64 mutants of Saccharomyces cerevisiae that confer sensitivity to X-ray inactivation were analyzed genetically to determine the number of genetic loci involved. The mode of interaction of various combinations of mutants was also determined. A minimum of 17 genes, when mutant, increase X-ray sensitivity of yeast, primarily by eliminating the resistance of budding haploid cells and by removing the shoulder on the survival curves of diploid cells. Eight mutant loci affect principally X-ray sensitivity while the remaining genes also control sensitivity to ultraviolet. Some of the genes when homozygous block sporulation or result in partial or complete sterility. Examination of the survival responses of multiple-mutant strains indicated a minimum of two pathways in the repair of X-ray damage. A number of the mutants have been mapped and these were found to be dispersed over the genome.     

Toward consistent and productive complex media for industrial fermentations: studies on yeast extract for a recombinant yeast fermentation process

Yeast extract (YE) is commonly used as a key component in the complex media for industrial fermentations. However, the lot-to-lot variation of this raw material frequently requires extensive "use testing" of many lots to identify only the few that support desired fermentation performance. Through extensive fermentation studies and chemical analyses, we have identified adenine and two metabolizable carbon sources, trehalose and lactate, as the principle components in YE that affect the production of a recombinant protein antigen by a yeast strain. Adenine is required for culture growth and the relationship between biomass and measured adenine can be expressed by a Michaelis-Menten model, while the slowly metabolized trehalose serves to maintain the energy supply to the continued antigen synthesis. The rapidly utilized lactate exerts an indirect positive effect by sparing some of the accumulated ethanol from being consumed for growth to being utilized in the product formation. The effects of these YE components are mutually dependent. Based on the database generated from 40 lots at laboratory scale, a relatively high level of carbon sources in YE (trehalose plus lactate, >9.5% w/w) and an intermediate level of adenine (0.14-0.24% w/w) appear to be the minimal requirement of a good lot for this recombinant yeast fermentation. Many poor lots were improved in lab fermenters by rational supplementation of trehalose, lactate, or adenine to compensate for their insufficiencies. At the large production scale, predictions based on adenine and trehalose/lactate contents in various YE lots used correlated reasonably well with culture growth and antigen yield, illustrating the feasibility of such a simple chemical/biochemical analysis as a rapid and reliable initial screening tool. Without incurring any compositional change to an established manufacturing medium, this study demonstrates an effective approach to achieve consistency in fermentations employing complex nutrients and to improve fermentation productivities supported by suboptimal lots of raw material.     

Dose-rate effects of gamma-ray-induced mutations in cultured mammalian cells

The dose-rate dependency of three radiobiological parameters, cell killing and mutations resistant to 6-thioguanine (6-TG^r) and to methotrexate (MTX^r), were studied in populations of mouse L5178Y cells exposed to gamma-rays. when the dose rate was reduced from 50 rad/min to 0.8 rad/min, the shape of the dose—response curves changed from sigmoidal to exponential for cell killing, from upward concave to linear in 6-TG^r mutations and remained linear in MTX^r mutations. A linear quadratic model appears capable of explaining the cell killing and 6-TG^r mutations but not the MTX^r mutations.The declining patterns of induced mutation frequencies of 6-TG^r and MTX^r with decreasing dose rate seem to be similar. The addition of DMSO resulted in protection of cells from cell killing, 6-TG^r and MTX^r mutations with acute exposure, but had little effect with chronic exposure. The reduction of mutation frequency of the 6-TG^r marker with chronic exposure was eliminated by holding cells in ice-cold condition during irradiation. These results suggest that there may be two components of induced mutation. One results primarily from repairable damage induced by the indirect action of radiation and shows a clear dose-rate dependency. The other is mainly from non-repairable damage by the direct action of radiation and is only slightly dose rate-dependent. Under chronic exposure conditions, the latter may predominate.     

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.     

酿酒酵母染色体设计与合成研究进展

随着合成生物学的蓬勃发展,基因组学的研究正在由读取基因组信息拓展到以编写基因组信息为主的合成基因组学时代。2009年,由Jef D. Boeke教授提出的人工合成酵母基因组计划(Sc2.0)旨在合成世界上首个真核生物基因组。在美、中、英、法、澳大利亚、新加坡等多国科学家的努力下,目前已经完成1/3的酵母染色体的人工合成。本文从合成基因组学领域的发展历程出发,介绍了Sc2.0计划中酿酒酵母(Saccharomyces cerevisiae)染色体设计与合成的最新进展,包括酿酒酵母9号染色体右臂、3号染色体、2号染色体、5号染色体、6号染色体、10号染色体和12号染色体的设计与合成过程,阐述了其各自的合成策略以及生物学意义,以期为合成基因组学的深入开展提供借鉴与参考。     

Host-mediated assay with yeast and rats using probenecid (Benemid®) to block the renal tubular excretion of cyclophosphamide metabolites

The genetic activity of cyclophosphamide (Cy) was tested in the host-mediated assay (injection of yeasts into the peritoneal cavity of rats) modified by the use of probenecid (Pro) (Benemid®) to block the renal tubular excretion of the genetically active metabolites. The genetic test system used was the induction of mitotic gene conversion in two unlinked loci of a diploid strain of Saccharomyces cerevisiae. By this method the genetic effect of Cy was doubled in comparison with the case of administering Cy alone. Compared with the animals which received only Pro, increases of conversion frequencies of 20 times in the ade2 locus and of 15 times in the trp5 locus were found.     

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.