Abstract –We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8‐methoxypsoralen (8‐MOP) and ultraviolet A (UV‐A) light, on growth, colony forming ability and RNA synthesis in a repair‐proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8‐MOP and UV‐A repair‐deficient mutants were significantly more sensitive than the repair‐proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.
Abstract: We compared the photodynamic effects of thiopyronine (TP) and visible light, and 8‐methoxypsoralen (8‐MOP) and ultraviolet A (UV‐A) light, on growth, colony forming ability and RNA synthesis in a repair‐proficient Saccharomyces strain and three mutants deficient in DNA repair mechanisms (DNA repair assays). With 8‐MOP and UV‐A repair‐deficient mutants were significantly more sensitive than the repair‐proficient strain indicating that the system is sensitive for the detection of DNA damage. With TP and visible light, the photodynamic effects were comparable in the mutants and the control, indicating no DNA damage. These results support previous work showing that the main target of TP photosensitization in eukaryotes is not nuclear DNA.
In a specially bred series of Saccharomyces-strains of different ploidy (haploid to tetraploid) the content of DNA, RNA, folic acid, niacin, pantothenic acid, thiamine, and catalase has been determined. The cell content of these substances increased nearly proportional with increasing ploidy. Diploid strains, however, which were heterozygous for only the mating type alleles proved to have a remarkable higher cell content of DNA, catalase and undigested folic acid than diploid strains homozygous for the mating type alleles. Generally those strains, which are able to sporulate, proved to have a higher content of the last mentioned substances per dry weight than those which are able to mate. These latter results are an interesting biochemical parallele to the remarkable resistance of diploid aα-strains to ionizing radiations.
A method to prepare polyribosomes from yeasts by using the french-press is described. The highest yield of polyribosomes was derived from late log-phase cells. These polyribosomes, incubated in a cell-free system, were able to reinitiate protein synthesis, which was shown by inhibiting aminoacid incorporation by aurintricarboxylic acid, edeine and sodiumfluoride. We developed the translational system in order to look for the optimal ion-conditions of a DNA-dependent protein-synthesizing system. We found out that at the optimal MgCl2-concentration (6 mM) protein synthesis was strongly inhibited by Mangan ions which are required for transcription in yeast. If protein-synthesis was carried out with 2 mM and 3 mM MgCl2 maximal aminoacid incorporation was observed at 2 mM and 1.5 mM MnCl2.
Since Saccharomyces cells have only a very small uptake of thymine or thymidine, the uptake of labelled samples of these compounds cannot be used to measure the rate of DNA synthesis. Instead two other methods were used for both X-irradiated and unirradiated cells of a series of homozygous isogenic Saccharomyces strains of different ploidy: 1. Yeast cells were incubated with adenine-8-C 14 , the labelled DNA was isolated and completely separated from RNA, and the specific radioactivity of the DNA was determined. The reduction of the specific radioactivity after irradiation as a proportion of the specific radioactivity of unirradiated control cells gave the X-ray induced inhibition in the rate of DNA synthesis. 2. The uptake of thymine-2-C 14 -5′-monophosphate, which is the only thymine compound incorporated into the DNA of Saccharomyces in any appreciable amount, was measured after various incubation times for irradiated and control cells. The results of the two methods are compared and the possibilities for their application are discussed. The dose effect curve for the rate of DNA synthesis after X-irradiation shows two phases: a steep slope at low doses with a shallow slope at high doses. For low doses the proportional decrease with X-irradiation in the rate of DNA synthesis is of the same order of magnitude as that of the colony forming ability, and is considerably greater than that of other metabolic processes such as protein and RNS synthesis, respiration or fermentation. The dependence of this proportional decrease on degree of ploidy is also similar to that dependence shown by colony forming ability and is again different from the dependence shown by RNA or protein synthesis. The reduction in DNA synthesis is greater the greater the time of incubation after irradiation.
