Pencho Venkov

Activation of Ty transposition by mutagens.

The induction of Ty1 transposition by mutagens (MMS and 4NQO) in asynchronous cultures and cells blocked in G1 and G2/M suggested G1 dependence of activation of Ty1 element by DNA damage. Northern blot analysis revealed immediate five-fold increase in levels of Ty1 transcript after 20min incubation of cells with 1 microg/ml 4NQO and four-fold increase in Ty1 RNA after treatment the cells with 0.1% MMS. Western blot analysis showed no difference in TyA protein in treated and untreated with mutagen cells. Quantitative mutagenicity assay and Northern blot analysis demonstrated dependence of induction of Ty1 element by DNA-damaging agents on the function of RAD9 gene and independence on DUN1 gene.

Genotoxic effect of substituted phenoxyacetic acids.

Abstract. The potential toxic and mutagenic action of 2,4-dichlorophenoxyacetic acid has been studied in different test systems, and the obtained results range from increased chromosomal damage to no effect at all. We reexamined the effect of this herbicide by simultaneous using three tests based on yeast, transformed hematopoietic, and mouse bone marrow cells. The results obtained demonstrated that 2,4-dichlorophenoxyacetic acid has cytotoxic and mutagenic effects. The positive response of yeast and transformed hematopoietic cells was verified in kinetics and dose–response experiments. The analysis of metaphase chromosomes indicated a statistically proved induction of breaks, deletions, and exchanges after the intraperitoneal administration of 2,4-dichlorophenoxyacetic acid in mice. The study of phenoxyacetic acid and its differently chlorinated derivatives showed that cytotoxicity and mutagenicity are induced by chlorine atoms at position 2 and/or 4 in the benzene ring. The mutagenic effect was abolished by introduction of a third chlorine atom at position 5. Thus 2,4,5-trichlorophenoxyacetic acid was found to have very weak, if any mutagenic effect; however, the herbicide preserved its toxic effect.

Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae.

The srb1-1 mutation of Saccharomyces cerevisiae is an ochre allele which renders the yeast dependent on an osmotic stabilizer for growth and gives the cells the ability to lyse on transfer to hypotonic conditions. A DNA fragment which complements both of these phenotypic effects has been cloned. This clone contains a functional gene which is transcribed into a 2.3-kb polyadenylated mRNA molecule. Transformation of yeast strains carrying defined suppressible alleles demonstrated that the cloned fragment does not contain a nonsense suppressor. Integrative transformation and gene disruption experiments, when combined with classical genetic analysis, confirmed that the cloned fragment contained the wild-type SRB1 gene. The integrated marker was used to map SRB1 to chromosome XV by Southern hybridization and pulsed-field gel electrophoresis. A disruption mutant created by the insertion of a TRP1 marker into SRB1 displayed only the lysis ability phenotype and was not dependent on an osmotic stabilizer for growth. Lysis ability was acquired by growth in (or transfer to) an osmotically stabilized environment, but only under conditions which permitted budding. It is inferred that budding cells lyse with a higher probability and that weak points in the wall at the site of budding are involved in the process. The biotechnological potential of the cloned gene and the disruption mutant is discussed.

Enhanced cell permeability increases the sensitivity of a yeast test for mutagens

ts1 is a mutation which causes a general increase in permeability of Sacharomyces cerevisiae cells in an unspecific manner. The introduction of the ts1 mutation under homozygous conditions into the D7 diploid strain enhanced the sensitivity of the test system described by Zimmermann et al. (1975). The newly constructed strain D7ts1 responded with a four to six times higher frequency compared to the D7 strain for all genetic end-points induced with chemical mutagens (ethyl methanesulfonate, methyl methanesulfonate, hydroxyurea, benzpyrene). The increased sensitivity of D7ts1 is specific only for mutagens active in yeast, since treatment of D7ts1 cells with 5-bromouracil or 5-bromouridine, known to be non-mutagenic in yeast, did not result in the induction of any of the measured genetic alterations. Five out of 14 water samples taken from the environment induced recombinogenic events in D7ts1, whereas all 14 water samples were without effect in the D7 test system. We concluded that D7ts1 cells show a higher sensitivity in the detection of mutagenic or carcinogenic action because of their generally enhanced permeability due to the ts1 mutation.

Genetic analysis of an osmotic sensitive - Saccharomyces cerevisiae mutant

SummaryThe genetic analysis of VY1160 sorbitol dependent, osmotic sensitive yeast mutant led to the identification of three different nuclear recessive mutations. Two of them, designated sorb- and ts1 are closely linked to one another. The mutation sorb- determines the lysis, while the mutation tsl increases the ability for lysis of the sorbitol dependent cells. The third mutation ts2 segregates independently from the other two and confers the sensitivity of VY1160 mutant cells towards rifampicin.

Mannan structure analysis of the fragile Saccharomyces cerevisiae mutant VY1160

The fragile Saccharomyces cerevisiae mutant VY1160 has a cell-wall mannan with a lower molecular weight and a higher nitrogen content than the parental S288C strain. More mannobiose but less mannotriose and mannotetraose were found in O-glycosidically-bound oligosaccharides in the mutant. Acetolysis analysis of the alkali-stable mannan also showed a reduction of mannotetraose and an increase of the mannobiose fractions. Methylation analysis of total mannan, or side chains recovered after acetolysis, showed that in the mutant the amount of alpha(1-3)-linked mannose units prevailed over alpha(1-2)-linked residues.

Degradation of ribosomal precursor and polyadenylic acid-containing ribonucleic acids in Saccharomyces cerevisiae caused by actinomycin D

Abstract The synthesis of RNA in the osmotic-sensitive yeast mutant VY 1160 is inhibited by actinomycin D at concentrations which are without effect on the parental wild-type strain. Protein synthesis is not affected initially even at doses which block RNA synthesis completely. Lower concentrations of actinomycin D inhibit preferentially the synthesis of rRNA as compared to that of poly(A)+-RNA. Unspecific degradation of prelabeled precursors of rRNAs (pre-rRNA) and poly(A)+-RNA is caused by actinomycin D at doses which block new RNA synthesis. This degradation could not be correlated with changes in the nuclease activity of yeast cell lysates. It appears that the post-transcriptional degradation of pre-rRNA and poly(A)+-RNA caused by actinomycin D is not connected with the action of the drug on transcription.

The action of snake venom phosphodiesterase on liver ribosomal ribonucleic acids

Abstract The stepwise hydrolysis of rat-liver ribosomal RNAs (rRNA) with snake venom phosphodiesterase (EC 3.1.4.1) has been studied. The following results have been obtained: 1. 1. Hydrolysis of poly (U), poly (A) and equimolar mixtures of them proceeds to completion and yields pU and pA as the only major products of the reaction. Secondary structure in poly (U) · poly (A) has no detectable influence on the rate and the extent of hydrolysis. The substrate (Ap)nCp is resistant to the action of this enzyme, while (Ap)nC is rapidly degraded to give pA and pC. 2. 2. As shown by agar-gel electrophoresis, the liver rRNA studied is constituted of homogeneous 28-S and 18-S RNA molecules. They display secondary structure transitions typical for native rRNAs. The preparations studied are virtually free from endonucleolytic contaminants. 3. 3. Hydrolysis of rRNAs with venom phosphodiesterase goes to completion and yields pG, pC, pA and pU as the only major products. Analysis by determination of the nucleosides originating from chain terminals during hydrolysis, shows that the endonuclease contaminants in this system may account for no more than 1 to 2 internal breaks per RNA molecule. 4. 4. The molar ratio of the mononucleotides liberated on stepwise hydrolysis of liver rRNAs with venom phosphodiesterase varies with progress of enzymatic degradation. The product obtained at early stages of enzyme action has a higher content of pA and pU and a lower content of pG and pC. The same correlation is observed with 18-S rRNA and partly with 28-S rRNA. It is suggested that in rRNA the segment near the 3′-end of the chain contains more pA and pU and less pG and pC as compared with the segment near the 5′-end of the chain. 5. 5. Labeling in vivo of free uridine 5′-phosphates and cytidine 5′-phosphates with [6-14C]orotic acid shows with both nucleotides a maximum incorporation at 40 min. Ribosomal RNAs labeled for 90 min in vivo with [6-14C]orotic acid were analysed by partial degradation with venom phosphodiesterase and determination of the radioactivity of uridylic acid residues. The labeling of 18-S RNA is higher than of 28-S RNA. Enzymatic liberation of labeled uridylic acid from 18-S and 28-S RNA is non-uniform. With both 18-S and 28-S RNA, uridylic acid liberated at the initial stages of enzyme action is more highly labeled than uridylic acid from the remaining RNA fragments.

Toyocamycin inhibition of ribosomal ribonucleic acid processing in an osmotic-sensitive adenosine-utilizing Saccharomyces cerevisiae mutant

An adenosine-utilizing mutant of Saccharomyces cerevisiae (SY 15 ado) is isolated after remutagenesis of an osmotic-sensitive strain, auxotrophic for adenine, with ethyl methanesulfonate. It is shown that the SY15ado mutant can be used to achieve experimental conditions under which cell growth and RNA Synthesis are directly dependent on exogenous adenosine. After starvation for adenosine, toyocamycin is incorporated into pre-rRNA chains of SY15ado cells replacing adenosine residues. The extent of this replacement depends on the concentration of added toyocamycin. Lower doses slow down processing of pre-rRNA into mature rRNA with an accumulation of 27 S and 20 S pre-rRNA. At higher concentrations toyocamycin blocks the last steps of pre-rRNA processing i.e. the conversions 27 S pre-rRNA leads to 25 S rRNA and 20 S pre-rRNA leads to 18 S rRNA. It appears that the main site of toyocamycin action is at the last steps of ribosome formation, while transcription and the early stages of pre-RNA processing are less affected.

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