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.

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.

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(-); sco1Delta), 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.

Ty1 transposition induced by carcinogens in Saccharomyces cerevisiae yeast depends on mitochondrial function.

The transposition of the Ty mobile genetic element of Saccharomyces cerevisiae is induced by carcinogens. While the molecular background of spontaneous Ty1 transposition is well understood, the detailed mechanism of carcinogen induced Ty1 transposition is not clear. We found that mitochondrial functions participate in the Ty induced transposition induced by carcinogens. Contrary to the parental rho(+) cells rho(-) mutants (spontaneous or induced by ethidium bromide) do not increase the rate of Ty1 transposition upon treatment with carcinogens. Preliminary results strongly suggest that the absence of oxidative phosphorylation in rho(-) mutants is the reason for the inhibited Ty transposition. The lack of carcinogen induced Ty1 transposition in rho(-) cells is not specific for a particular carcinogen and represents a general feature of different carcinogenic substances inducing rho(-). It is concluded that carcinogen induced Ty1 transposition depends on the functional state of mitochondria and cannot take place in cells with compromised mitochondrial function (rho(-)).

Enhanced secretion of heterologous proteins in Kluyveromyces lactis by overexpression of the GDP-mannose pyrophosphorylase, KlPsa1p.

GDP-mannose is the mannosyl donor for the glycosylation reactions and is synthesized by GDP-mannose pyrophosphorylase from GTP and d-mannose-1-phosphate; in Saccharomyces cerevisiae this enzyme is encoded by the PSA1/VIG9/SRB1 gene. We isolated the Kluyveromyces lactis KlPSA1 gene by complementing the osmotic growth defects of S. cerevisiae srb1/psa1 mutants. KlPsa1p displayed a high degree of similarity with other GDP-mannose pyrophosphorylases and was demonstrated to be the functional homologue of S. cerevisiae Psa1p. Phenotypic analysis of a K. lactis strain overexpressing the KlPSA1 gene revealed changes in the cell wall assembly. Increasing the KlPSA1 copy number restored the defects in O-glycosylation, but not those in N-glycosylation, that occur in K. lactis cells depleted for the hexokinase Rag5p. Overexpression of GDP-mannose pyrophosphorylase also enhanced heterologous protein secretion in K. lactis as assayed by using the recombinant human serum albumin and the glucoamylase from Arxula adeninivorans.

The Ty1 transposition assay: a new short-term test for detection of carcinogens.

An assay based on induction by carcinogens of Ty1 transposition in Saccharomyces cerevisiae is proposed. A tester strain was developed that contains a marked Ty1 element, which allows following the transposition in the genome as a whole and a mutation, which increases cellular permeability. Hypersensitivity to chemical agents, higher cell wall porosity and transformability with plasmid DNA evidenced an enhanced cellular permeability of the tester cells. The increased permeability resulted in higher sensitivity to carcinogens. The treatment with different laboratory carcinogens induced Ty1 transposition rates in the tester strain by a factor of 10 to 20, compared to the controls. The induction is not stress-generated by the cytotoxicity of carcinogens, since treatment with NaN3 at concentrations killing 50% of the cells did not increase the transposition rate. The increase of Ty1 transposition in tester cells is specific for active carcinogens and a positive response with procarcinogens was obtained only in presence of S9 mix. The Ty1 transposition test responded positively to a number of Ames-test or DEL-test negative carcinogens. The positive response of Ty1 test was statistically significant and verified in kinetics and concentration-dependent experiments. It is concluded that the Ty1 transposition test can be used, in addition to the Ames assay, as a short-term test for detection of carcinogens.

Characterization of a Saccharomyces cerevisiae thermosensitive lytic mutant leads to the identification of a new allele of the NUD1 gene.

To improve our understanding of the factors involved in the osmotic stability of yeast cells, a search for novel conditional Saccharomyces cerevisiae cell lysis mutants was performed. Ten temperature-sensitive (ts) mutant strains of S. cerevisiae were isolated that lyse at the restrictive temperature on hypotonic, but not on osmotically supported medium. The ten mutants fell into four complementation groups: ts1 to ts4. To clone the wild-type gene corresponding to the ts4 mutation, a strategy aimed at complementing the thermosensitive phenotype-using low-copy and high-copy DNA libraries--was followed, but only two extragenic suppressors were identified. Another approach, in which classic genetic methods were combined with the use of yeast artificial chromosomes and traditional cloning procedures, allowed the identification of the NUD1 gene--which codes for a component of the spindle-pole body-as the wild-type gene corresponding to the ts4 mutation. Cloning and sequencing of the defective allele from the chromosome of the mutant cells resulted in the identification of a point mutation that produces a single amino acid change in the protein: a Gly-to-Glu change at position 585 (the nud1-G585E allele). Further analysis revealed that cells carrying this allele show a thermosensitive growth defect. At the restrictive temperature, the cells arrest with large buds, elongated spindles, and duplicated nuclei. In addition, with longer incubation times they are unable to maintain cellular integrity and lyse. Our results have allowed the identification of the first single amino acid mutation in NUD1, and suggest a link between cell cycle progression and cellular integrity.

KlSEC53 is an essential Kluyveromyces lactis gene and is homologous with the SEC53 gene of Saccharomyces cerevisiae.

Phosphomannomutase (PMM) is a key enzyme, which catalyses one of the first steps in the glycosylation pathway, the conversion of D-mannose-6-phosphate to D-mannose-1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for the glycosylation reactions in eukaryotic cells. In the yeast Saccharomyces cerevisiae PMM is encoded by the gene SEC53 (ScSEC53) and the deficiency of PMM activity leads to severe defects in both protein glycosylation and secretion. We report here on the isolation of the Kluyveromyces lactis SEC53 (KlSEC53) gene from a genomic library by virtue of its ability to complement a Saccharomyces cerevisiae sec53 mutation. The sequenced DNA fragment contained an open reading frame of 765 bp, coding for a predicted polypeptide, KlSec53p, of 254 amino acids. The KlSec53p displays a high degree of homology with phosphomannomutases from other yeast species, protozoans, plants and humans. Our results have demonstrated that KlSEC53 is the functional homologue of the ScSEC53 gene. Like ScSEC53, the KlSEC53 gene is essential for K. lactis cell viability. Phenotypic analysis of a K. lactis strain overexpressing the KlSEC53 gene revealed defects expected for impaired cell wall integrity.

Dimerization of Rhizobium meliloti NifH protein in Saccharomyces cerevisiae cells requires simultaneous expression of NifM protein.

Compared to free living diazotrophs, the nitrogenase system of symbiotic microorganisms, like Rhizobium (Synorhizobium) meliloti, was poorly studied. The aim of our research was to investigate whether (by analogy with Klebsiella pneumoniae) the NifM product is required and sufficient to obtain active R. meliloti Fe-protein. We cloned nifH gene of R. meliloti and nifM gene of K. pneumoniae in suitable yeast vectors. When introduced into Saccharomyces cerevisiae cells, both genes were effectively expressed to proteins similar to the native products in its immunoreactivity and apparent molecular mass. The association of R. meliloti NifH protein into dimer structure required co-expression of NifM that also conferred stability of NifH polypeptide. However, the NifH protein synthesized in yeast did not show enzyme activity, suggesting that the NifM of K. pneumoniae is incapable of activating the NifH protein of R. meliloti.