Effects of a heterogeneous set of xenobiotics on RNA synthesis of yeast cells.

Previously the toxicity of 45 heterogeneous environmental chemicals on growth and membrane functions in Saccharomyces cerevisiae and Chinese hamster ovary (CHO) cells (Cascorbi et al., 1993) was examined. In this study the inhibitory effects of the same set of chemicals on yeast RNA synthesis rate, measuring [2-14C]uracil uptake during cell proliferation are presented. The sensitivity of this test system was three to six times higher than that of the proliferation rate assay. In addition, the range of the EC20 values were similar to the range from the mammalian CHO cell proliferation test. The relatively good correlation with the yeast cell growth rate (r = 0.78, P = 0.0001) suggests that yeast RNA synthesis is a simple and rapid method of detecting a wide range of toxic compounds without the disadvantage of the insensitivity of the growth rate assay.

Chromosomal damages by ethanol and acetaldehyde in Saccharomyces cerevisiae as studied by pulsed field gel electrophoresis.

We report on the effect of ethanol and acetaldehyde on yeast chromosomal DNA and on isolated DNA. Ethanol induced DNA single-strand breaks in repair deficient but not in repair proficient Saccharomyces cerevisiae. Acetaldehyde has a deleterious effect on chromosomal DNA in cells as well as on isolated DNA. The results presented support earlier data to show that ethanol is mutagenic via its first metabolite, acetaldehyde.

Thiopyronine- and 8-methoxypsoralen-sensitized photodynamic effect on cell growth, colony forming ability and RNA synthesis in Saccharomyces mutants deficient in DNA repair.

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.

Rapid estimation of chromosomal damage in yeast due to the effects of environmental chemicals using pulsed field gel electrophoresis.

We present a procedure to rapidly estimate the damage to yeast chromosomes by toxic chemicals. This procedure employs the following steps: incubation of yeast cells with the chemicals, DNA preparation in an agarose matrix, separation of chromosome-sized DNA molecules into reproducible band patterns by pulsed field gel electrophoresis, and quantification of the intensity of chromosomal bands by densitometry. Saccharomyces cerevisiae cells have been treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and cis-Platinum(II) diamminedichloride (cisPT), both of which are known to interact with DNA, and trichlorethylen (TCE), for which such an effect has not been shown in yeast. Treatment of cells with MNNG and cisPt led to an impairment of the intensity of the band pattern to an extent dependent on the concentration of the chemicals applied. For TCE a similar effect could not be discerned. This procedure will be useful as a screening test for the estimation of the biological hazards of toxic chemicals.

Thiopyronine-sensitized photodynamic effect on cell growth, RNA and DNA synthesis of Chinese hamster ovary cells.

After treatment of Chinese hamster ovary (CHO) cells with very low concentrations of thiopyronine (TP; 1 microgram/ml) and visible light, a delay in growth of cell cultures (prolongation of the lag phase] was observed. The lengthened lag phase, however, was followed by normal growth of the cells. The length of the lag period is dependent on the irradiation dose applied. A similar effect on DNA and RNA synthesis could be seen after photodynamic treatment with TP in CHO cells: the maxima of RNA and DNA synthesis occur later but are not significantly reduced after treatment with low concentrations of TP and irradiation with visible light. This result is further evidence that the photodynamic effect with TP does not involve attack on nuclear DNA in eukaryotic cells.

The effect of pentachlorophenol and its metabolite tetrachlorohydroquinone on RNA, protein, and ribosome synthesis in Saccharomyces cells.

The effect of pentachlorophenol (PCP) and its metabolite tetrachlorohydroquinone (TCH) were tested on growth, RNA, protein and ribosome syntheses, and ribosome content in yeast cells. Cells exposed to increasing concentrations of PCP show increasing inhibition to RNA and ribosome synthesis, and to cell growth. TCH causes a delay of the growth of the cell culture (prolongation of the lag phase) but does not cause inhibition. After treatment with TCH the maximum of the RNA synthesis was retarded, but subsequently reached nearly the same level as the untreated control cells. On ribosome synthesis and ribosome content, treatment with increasing concentrations of PCP, as well as of TCH, leads to a substantial decrease in ribosomal synthesis and, finally, total inhibition. Parallel to this, the content of free and membrane-bound ribosomes is diminished. PCP exhibits a stronger effect than TCH. The protein synthesis is only slightly reduced after treatment with PCP or TCH (with concentrations up to 20 micrograms/ml).

Effect of acrylonitrile on the transcription of specific genes in Saccharomyces cerevisiae.

We have studied the effect of acrylonitrile on the transcription of specific genes of Saccharomyces cerevisiae. The results presented demonstrate that ACN disturbs the coordinated response of ribosomal protein genes and causes a dramatic induction of the LEU2 gene, which might be due to metabolites of ACN.

Isolation of yeast ribosomal proteins L3 and L2 for immunological studies.

We report on a rapid method for the isolation and purification of the yeast ribosomal proteins L3 and L2 using a simple instrumentation. Preparative dodecyl sulfate polyacrylamide gel electrophoresis was applied to the separation of cytoplasmatic ribosomal proteins of the large subunit from the yeast Saccharomyces cerevisiae. The polypeptides were removed from gel slices by electrophoretic elution. Subsequent analytical electrophoresis showed groups of proteins in all but two fractions. The latter were further analysed by a two-dimensional gel electrophoresis system which disclosed the purity of two polypeptides. They were identified as L3 and L2. Their molecular masses were 51.5 and 44 kDa as estimated from the gels. A possible application to the isolation of other yeast ribosomal proteins is discussed. An antiserum against the polypeptide L3 was raised in a rabbit. Applying an enzyme-linked immunosorbent assay (ELISA) we were able to determine the relative antibody concentration. Its specificity was demonstrated by immunoblotting.

Investigation of a test system for the rapid differentiation of nuclear and cytoplasmic damage in eucaryotes.

The effect of various agents which cause cell inactivation on the growth curves and RNA synthesis rates of yeast cells has been studied. On the basis of these investigations it was concluded that such studies can be used as a rapid test system for drawing preliminary conclusions as to whether a particular agent primarily damages the DNA of the cell nucleus or cytoplasmic structures.

Thiopyronine and 8-methoxypsoralen sensitized photodynamic effect on DNA synthesis in yeast.

The synthesis of DNA in growing yeast cells was investigated after photodynamic treatment of the cells with thiopyronine (TP) and visible light or with 8-methoxypsoralen (8-MOP) and UVA light. DNA synthesis was inhibited after photodynamic treatment with 8-MOP but not after photodynamic treatment with TP. This result is further evidence that the photodynamic effect with TP does not attack nuclear DNA in eucaryotic cells.

The effect of trichloroethylene and acrylonitrile on RNA and ribosome synthesis and ribosome content in Saccharomyces cells.

The effects of trichloroethylene (TCE) and acrylonitrile (ACN) on growth, RNA synthesis, ribosome synthesis, and ribosome content were tested in yeast cells. TCE causes a delay of the growth of a cell culture (prolongation of the lag phase), but does not cause inhibition. Cells exposed to increasing concentrations of ACN show increasing damage, so that, at a certain point of the growth curve, cell division stops altogether. Similar results were obtained when RNA synthesis was investigated: After treatment with TCE, the maximum RNA synthesis of the cell culture was retarded, but subsequently reached the same level as the untreated control cells. In the presence of ACN, however, the rate of RNA synthesis was lowered with increasing ACN concentrations. The same effect was observed upon investigation of ribosome synthesis: Whereas TCE produces only a slight effect, treatment with increasing concentrations of ACN leads to a substantial decrease in ribosome synthesis, and finally to total inhibition. Parallel to this, the content of free and membrane-bound ribosomes is diminished. Obviously, the decrease in ribosome content is caused not only by an inhibition of ribosome synthesis, but also by a degradation of existing ribosomes, as well as by induction of a ribosome-associated RNase.

Thiopyronine-sensitized photodynamic effect on RNA synthesis in Saccharomyces cells in vivo.

The effect of photodynamic treatment with thiopyronine and visible light on RNA metabolism in yeast cells was investigated at different times during logarithmic growth. The results show that RNA synthesis in the nucleus of the cells is not directly inhibited photodynamically. In the endoplasmic reticulum of photodynamically treated cells one finds mRNA in about the same relative amounts and quality as in untreated control cells, but the binding of polysomes on membranes in the cytoplasm as the first step of protein synthesis is inhibited for a long time after treatment as well as the synthesis of ribosomes.

Thiopyronine sensitized photodynamic effect on ribosome synthesis and ribosome content in Saccharomyces cells.

The content and the synthesis of membrane bound and free ribosomes in growing yeast cells was investigated after photodynamic treatment of the cells with thiopyronine and visible light. It was shown that the synthesis of ribosomes is inhibited after photodynamic treatment and that as a consequence, the content of ribosomes in the cell is diminished.

[The content of membrane-bound and free ribosomes in growing saccharomyces cells at different temperatures (author's transl)]. / Uber den Gehalt an membrangebundenen und freien Ribosomen in Saccharomyceszellen bei verschiedenen Wachstums-Temperaturen.

The distribution of membrane-bound and free ribosomes was investigated at different temperatures in growing yeast cells. During duplication phases of the cell a changing pattern of the amount of membrane-bound and free ribosomes can be found at all temperatures investigated: at points where there is a maximum of membrane-bound ribosomes, one can always find a minimum of free ribosomes. The ribosomal content is significantly higher at 30 degrees C than it is at 20 degrees C, and higher at 20 degrees C than at 40 degrees C, whereas the duplication time of the cells is only slightly longer at 40 degrees C, but extremely longer at 20 degrees C than at 30 degrees C.

Protein synthesis on membrane-bound and free ribosomes in growing yeast.

1. A method for the study of membrane-binding of ribosomes and the rate of synthesis of protein on free and bound ribosomes has been examined critically. 'Brij 58' has been found to be more suitable for detaching ribosomes from membranes than 'Triton X-100' or sodium deoxycholate since it does not alter the structure or biological activity of the ribosomes. Triton and deoxycholate remove amino acid- and ribonucleotide-containing material from ribosomes, thus decreasing their activity during protein synthesis in a cell free system. 2. The rate of protein synthesis on membrane-bound and free ribosomes, the total amount of ribosomes, and the percentage distribution of free and membrane-bound ribosomes were determined in a culture of synchronously growing yeast cells. The results show that in all probability protein synthesis in yeast cells occurs mainly on membrane-bound ribosomes. Free ribosomes are considered here to be a ribosome pool which allows the cell to increase its rate of protein synthesis rapidly. Membrane-bound and free ribosomes are viewed as different functional states, one being a state of protein synthesis and the other a latent state.

Distribution of membrane-bound and free ribosomes in growing yeast.

1. The distribution of membrane-bound and free ribosomes was investigated in stationary as well as in growing yeast cells. the relative amount of free ribosomes varies with the growth phase of the cell culture. During the duplication phases of the cell, relative maxima of free ribosomes can be found. However, the absolute amount of free ribosomes is fairly constant during the growth of the cells. 2. Membrane-bound ribosomes show lower polypeptide synthesis activity in a cell-free, poly (U)-dependent system than free ribosomes. 3. There is no difference in the distribution pattern of free and membrane-bound ribosomes in growing yeast cells of different ploidy. 4. A turnover between free and membrane-bound ribosomes is suggested to be in agreement with the hypothesis of Branes and Pogo ((1975) Eur. J. Biochem. 54, 317-328).

[Initiation in a polyribosome-dependent protein-synthesizing cell-free system from Saccharomyces (author's transl)]. / Initiation in einem Polyribosomen-abhängigen Protein-synthetisierenden zellfreien System aus Saccharomyces

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.