Margarita Pesheva

The role of reactive oxygen species in the induction of Ty1 retrotransposition in Saccharomyces cerevisiae

Here we provide evidence for a dependence between the increased production of reactive oxygen species and the activation of Ty1 retrotransposition. We have found that the strong activator of Ty1 mobility, methylmethane sulphonate, can not induce Ty1 retrotransposition in cells with compromised mitochondrial oxidative phosphorylation (rho−; sco1Δ), which is the major source for production of reactive oxygen species (ROS) in Saccharomyces cerevisiae. The quantitative estimation of superoxide anions in living cells showed that rho+ cells exposed to methylmethane sulphonate increase Ty1 retrotransposition and superoxide levels. The increase of superoxide anions by the superoxide generator menadione is accompanied by induction of Ty1 mobility without any treatment with a DNA‐damaging agent. Higher frequencies of retrotransposition were found in rho+ and rho− cells treated with exogenously added hydrogen peroxide or in cells with disrupted YAP1 gene characterized by increased intracellular levels of hydrogen peroxide. These data indicate that increased levels of ROS may have an independent and key role in the induction of Ty1 retrotransposition. Copyright

Transposition of Saccharomyces cerevisiae Ty1 retrotransposon is activated by improper cryopreservation

Ty1 is a retrotransposon of the yeast Saccharomyces cerevisiae whose transposition at new locations in the host genome is activated by stress conditions, such as exposure to UV light, X-rays, nitrogen starvation. In this communication, we supply evidence that cooling for 2 h at +4 degrees C followed by freezing for 1 h at -10 degrees C and 16 h at -20 degrees C also increased Ty1 transposition. The mobility of Ty1 was induced by cooling at slow rates (3 degrees C/min) and the accumulation of trehalose inside cells or the cooling at high rates (100 degrees C/min) inhibited significantly the induction of the transposition. The freeze-induced Ty1 transposition did not occur in mitochondrial mutants (rho-) and in cells with disrupted SCO1 gene (Deltasco1 cells) evidencing that the Ty1 transposition induced by cooling depends on the mitochondrial oxidative phosphorylation. We also found that the freeze induced Ty1 transposition is associated with increased synthesis and accumulation of superoxide anions (O2-) into the cells. Accumulation of O2- and activation of Ty1 transposition were not observed after cooling of cells with compromised mitochondrial functions (rho-, Deltasco1), or in cells pretreated with O2- scavengers. It is concluded that (i) elevated levels of reactive oxygen species (ROS) have a key role in activation the transposition of Ty1 retrotransposon in yeast cells undergoing freezing and (ii) given the deleterious effect of increased ROS levels on cells, special precautions should be taken to avoid ROS production and accumulation during cryopreservation procedures.

The positive response of Ty1 retrotransposition test to carcinogens is due to increased levels of reactive oxygen species generated by the genotoxins

In previous laboratory and environmental studies, the Ty1 short-term test showed positive responses (i.e. induced mobility of the Ty1 retrotransposon) to carcinogenic genotoxins. Here, we provide evidence for a causal relationship between increased level of reactive oxygen species and induction the mobility of the Ty1 retrotransposon. Results obtained in concentration and time-dependent experiments after treatment, the tester cells with carcinogenic genotoxins [benzo(a)pyrene, benzo(a)anthracene, ethylmethanesulfonate, formamide], free bile acids (chenodeoxycholic, lithocholic acids) and metals (arsenic, hexavelant chromium, lead) showed a simultaneous increase in both cellular level of the superoxide anions and Ty1 retrotransposition rates. Treatment with the noncarcinogenic genotoxins [benzo(e)pyrene, benzo(b)anthracen, anthracene], conjugated bile acids (taurodeoxycholic, glycodeoxycholic acids) and metals (zinc, trivalent chromium) did not change significantly superoxide anions level and Ty1 retrotransposition rate. The induction by carcinogens of the Ty1 mobility seems to depend on the accumulation of superoxide anions, since the addition of the scavenger N-acetylcysteine resulted in loss of both increased amount of superoxide anions and induced Ty1 retrotransposition. Increased hydrogen peroxide levels are also involved in the induction of Ty1 retrotransposition rates in response to treatment with carcinogenic genotoxins, as evidenced by disruption of YAP1 gene in the tester cells. It is concluded that the carcinogen-induced high level of reactive oxygen species play a primary and key role in determination the selective response of Ty1 test to carcinogenic genotoxins.

Mutagenic effect of freezing on nuclear DNA of Saccharomyces cerevisiae.

Although fragmentation of DNA has been observed in cells undergoing freezing procedures, a mutagenic effect of sub‐zero temperature treatment has not been proved by induction and isolation of mutants in nuclear DNA (nDNA). In this communication we supply evidence for mutagenicity of freezing on nDNA of Saccharomyces cerevisiae cells. In the absence of cryoprotectors, cooling for 2 h at +4°C and freezing for 1 h at −10°C and 16 h at −20°C, with a cooling rate of 3°C/min, resulted in induction of frame‐shift and reverse mutations in microsatellite and coding regions of nDNA. The sub‐zero temperature exposure also has a strong recombinogenic effect, evidenced by induction of gene‐conversion and crossing‐over events. Freezing induces mutations and enhances recombination with a frequency equal to or higher than that of methylmethanesulphonate at comparable survival rates. The signals for the appearance of nDNA lesions induced by freezing are detected and transduced by the DNA damage pathway. Extracellular cryoprotectors did not prevent the mutagenic effect of freezing, while accumulation of trehalose inside cells reduced nDNA cryodamage. Freezing of cells is accompanied by generation of high ROS levels, and the oxidative stress raised during the freeze–thaw process is the most likely reason for the DNA damaging effect. Experiments with mitochondrial rho– mutants or scavengers of ROS indicated that mutagenic and recombinogenic effects of sub‐zero temperatures can be decreased but not eliminated by reduction of ROS level. The complete protection against cryodamage in nDNA required simultaneous usage of intracellular cryoprotector and ROS scavenger during the freeze–thaw process. Copyright

The response of Ty1 test to genotoxins

The Ty1 assay is a short-term test for detection of genotoxins based on induction of the transposition of a gene-engineered Ty1 retrotransposon in Saccharomyces cerevisiae cells. Here, we provide evidence that the Ty1 test responds positively in concentration-dependent manner to the carcinogenic genotoxins benz(a)anthracene, benzo(a)pyrene, chenodeoxycholic and taurodeoxycholic free bile acids and to environmental soil samples polluted with carcinogenic substances. The Ty1 test gives negative results with the noncarcinogenic mutagens benz(b)anthracene, benzo(e)pyrene, lithocholic and taurodeoxycholic conjugated bile acids and to soil samples not polluted with carcinogens. Presence or absence of genotoxins in soil samples was evidenced by chemical analysis. Several explanations for the sensitive differential test’s response to genotoxins are proposed and discussed. It is concluded that the Ty1 test can complement existing assays in laboratory and environmental studies showing high sensitivity to a wider spectrum of carcinogenic genotoxins.

New Cell-Based Assay Indicates Dependence of Antioxidant Biological Activity on the Origin of Reactive Oxygen Species

The mobility of the Ty1 transposon in Saccharomyces cerevisiae was found to vary proportionally with the level of ROS generated in cells, which provides the possibility to determine antioxidant activity by changes in a cellular process instead of using chemical reactions. The study of propolis, royal jelly, and honey with the newly developed Ty1antiROS test reveals an inverse exponential dependence of antioxidant activity on increased concentrations. This dependence can be transformed to proportional by changing the source of ROS: instead of cell-produced to applied as hydrogen peroxide. The different test responses are not due to excess of added hydrogen peroxide, as evidenced by the exponential dependence found by usage of yap1Δ tester cells accumulating cell-generated ROS. Results indicate that the activity of antioxidants to oxidative radicals depends on the origin of ROS, and this activity is elevated for cell-generated ROS compared to ROS added as reagents in the assay.

TY ELEMENTS OF THE YEAST SACCHAROMYCES CEREVISIAE

ABSTRACT Transposable elements in the yeast Saccharomyces cerevisiae are a heterogeneous group of mobile genetic elements. Ty elements transpose via an intermediate RNA-molecule using the same replication and integration mechanisms as the metazoan retroviruses. Ty1 is a retrotransposons with life cycle very similar to that of oncogenes. This similarity provides an attractive model for virus-host interaction in a genetically tractable eukaryote. Like oncogenes, responds to carcinogens with an increased formation of Ty1 mRNA. There are many articles, concerning the structure, transposition cycle and regulation, as well as post-transcriptional regulation of transposition by Saccharomyces cerevisiae Ty retrotransposons. In this review, we will observe recent advances in our understanding of structure, transposition and regulation of Ty retrotransposition in Saccharomyces cerevisiae.

The Ty1 Retrotransposition Short-Term Test for Selective Detection of Carcinogenic Genotoxins

Long-term tests are bioassays for carcinogenicity using animals (mainly rats or mice) as testers. Targets of the carcinogenic action are either organs or the whole body of the tester animal studied by dissection and histochemical analysis. These bioassays are not suitable for the study of a large number of substances because they are time consuming, expensive, require special facilities and qualified personal. A full long-term assay is completed in an average period of two years which stimulated the development of medium-term bioassays (Ito et al. 1998). These assays determine formation of neoplasms in various tissues of rats or mice within a period of 12-18 months. However, the existence of thousands genotoxins with potential carcinogenic effect requires the application of much faster assays and short-term tests have been developed and widely used. According to the timeframe required to obtain results in an assay, the transgenic mouse systems are intermediate between long-term and short-term tests. Although designated as “short-term bioassays” (Tennant et al. 1995) results from these tests are obtained in 4-6 months. The modified genes of transgenic animals cause them to respond rapidly to carcinogens (Cannon et al. 1997). Transgenic mouse systems are statistically reliable in vivo assays and the positive results obtained in the tests are highly predictive of rodent carcinogenicity.

TRANSPOSITION OF SACCHAROMYCES CEREVISIAE Ty1 RETROTRANSPOSON DEPENDS ON THE FUNCTION OF MITOCHONDRIA

ABSTRACT Ty1 is a mobile genetic element (transposon) which is transposed spontaneously with a frequency rate of about 1×10−7/element/generation. It was found recently that carcinogens can induce Ty1 transposition to about 1×10−6/element/generation. While molecular background of the spontaneous Ty1 transposition is studied in details, the mechanism of carcinogen induced Ty1 transposition is not known. We found that mitochondrial functions participate in Ty1 transposition. The carcinogen induced Ty1 transposition can not take place in cells with dysfunctional mitochondria (rho− mutants) or cells which fulfill their energy requirement on glycolysis. Contrary to this, the rate of spontaneous Ty1 transposition in mitochondrial mutants is increased. The obtained results suggest the existence of different mechanisms for spontaneous and carcinogen induced transposition of the Ty1 retrotransposon.

CONSTRUCTION OF HYBRID YEAST STRAINS WITH INCREASED FLOCCULATION AND FERMENTATION ACTIVITY FOR RED SPARKING WINE MANUFACTURE

ABSTRACT Polyploid hybrid strains of yeast Saccharomyces cerevisiae were constructed in order to improve their fermentation and flocculation activity for manufacturing of red sparking wine. It was observed that the flocculation ability of the new hybrids depended on the expression of the FLO1 gene. The constructed hybrids DM1, DM2, DM3 and DM4 showed better fermentation activity in laboratory and semi-industrials trials compared to their parental strains. DM1 proved to be suitable for industrial application.