Dissection of the functions of the Saccharomyces cerevisiae RAD6 postreplicative repair group in mutagenesis and UV sensitivity.

The RAD6 postreplicative repair group participates in various processes of DNA metabolism. To elucidate the contribution of RAD6 to starvation-associated mutagenesis, which occurs in nongrowing cells cultivated under selective conditions, we analyzed the phenotype of strains expressing various alleles of the RAD6 gene and single and multiple mutants of the RAD6, RAD5, RAD18, REV3, and MMS2 genes from the RAD6 repair group. Our results show that the RAD6 repair pathway is also active in starving cells and its contribution to starvation-associated mutagenesis is similar to that of spontaneous mutagenesis. Epistatic analysis based on both spontaneous and starvation-associated mutagenesis and UV sensitivity showed that the RAD6 repair group consists of distinct repair pathways of different relative importance requiring, besides the presence of Rad6, also either Rad18 or Rad5 or both. We postulate the existence of four pathways: (1) nonmutagenic Rad5/Rad6/Rad18, (2) mutagenic Rad5/Rad6 /Rev3, (3) mutagenic Rad6/Rad18/Rev3, and (4) Rad6/Rad18/Rad30. Furthermore, we show that the high mutation rate observed in rad6 mutants is caused by a mutator different from Rev3. From our data and data previously published, we suggest a role for Rad6 in DNA repair and mutagenesis and propose a model for the RAD6 postreplicative repair group.

Susceptibility of individual cells of Saccharomyces cerevisiae to the killer toxin K1.

The susceptibility of sensitive yeast to killer toxins is known to depend on various factors, such as the selected killer toxin, the exposed yeast strain, its growth phase and the state of culture under given experimental conditions. The aim of this paper was to find whether individual cells from one culture are equally susceptible to the impact of the killer toxin. For this purpose the rhodamine B assay in a modified form was used. In order to observe the fate of individual cell the method of fluorescence video microscopy with a digital picture analysis was applied. Four selected groups of specific cells (with no, small, medium, and large bud, respectively) were investigated. Different sensitivity of Saccharomyces cerevisiae cells to the killer toxin K1 was observed in these cell groups. The most susceptible appeared to be the cells which were in S-phase (cells with the small buds); the least susceptible were the M-phase cells with large buds. The enhanced susceptibility in S-phase results probably from coincidence in higher porosity of the cell wall, accumulation of surface receptors, and enlarged growth activity at the surface cell structures.

[Importance of the Genome Project]. / Význam genomových projektu.

Methodological and technological development of the molecular biology during the last decades of the twenties century has shifted our interest from the particular to synthetic problems. Studies has been undertaken both at the level of structure of the individual genetic information and on the level of its function and mutual relation within a single cell or in a multicellular organism. This crucial period culminated in the human genome analysis project, which is now celebrating its first major success. Parallel to it came the analysis of genomes of many other, though usually much thinner organisms.

Starvation-associated mutagenesis in yeast Saccharomyces cerevisiae is affected by Ras2/cAMP signaling pathway.

The number of revertants with restored ability to form colony increases in a time-dependent manner during long-term selective starvation of dense mutant microbial cultures. This is due to starvation-associated (also called adaptive) mutations that arise in a replication independent manner. Here we report that in Saccharomyces cerevisiae the frequency of starvation-associated reversions of mutant genes whose products are necessary for amino acids biosynthesis are influenced by Ras2/cAMP signaling pathway. This signaling pathway is a yeast general regulatory pathway involved in nutritional sensing, UV response, sporulation control and life span control and its changes are manifested in both, cell cycle and life cycle. Inactivation of the RAS2 gene causes an increase in number of starvation-associated revertants in comparison to an isogenic wild type strain and a strain with constitutively activated Ras2/cAMP signaling pathway. Therefore, we suggest that starvation-associated mutagenesis is different from spontaneous mutagenesis and is related to the cellular capacity to adopt distinct physiological states in response to environmental signals.

Development of papillae on colonies of two isopolyauxotrophic strains of Saccharomyces cerevisiae allelic in RAD6 during adenine starvation.

Papilla formation on colonies of two isopolyauxotrophic strains (ade2 his3 leu2 trp1 ura3) allelic in RAD6 was compared in order to find proper conditions for selecting mutants of Saccharomyces cerevisiae with altered starvation-induced mutability. The most promising for this purpose appeared to be culturing low numbers of colonies on suboptimal plates with a growth-limiting amount of adenine at 28 degrees C for 20 d. Inactivation of the RAD6 gene which suppresses the level of starvation-associated mutagenesis markedly enhanced papilla formation under these conditions. Formation of almost all papillae on 20-d-old colonies of BJC3 was caused by mutation. Most of the papillae (75%) were white Ade+ revertants. Three groups of these papillae were distinguished (Ade+, Ade+Rad6+ and Ade+Trp+). Both, Ade+Rad6+ and Ade+Trp+ double reversions were very probably caused by a suppressor mutation. The less frequent red papillae had the same auxotrophic markers and UV sensitivity as BJC3 but their outgrowth in liquid media was greater. It appears that creation of these papillae is caused by mutation affecting the cell response to growth limitation by low concentrations of adenine.

The involvement of the RAD6 gene in starvation-induced reverse mutation in Saccharomyces cerevisiae.

The accumulation of Ade+ revertants during adenine starvation and Trp+ revertants during tryptophan starvation in haploid polyauxotrophic strains of Saccharomyces cerevisiae occurs in a time-dependent manner. Accumulation of revertants is enhanced in Rad6- strains, suggesting that starvation-induced reversion is influenced by some of the RAD6 gene functions. The higher frequency of adaptive reversions in Rad6- strains is somewhat influenced by, but does not totally depend on, the genetic background. Therefore, the RAD6 gene product is involved in maintaining a low level not only of spontaneous mutation but also of starvation-induced reversion. The starvation-induced Ade+ and Trp+ reversions both appear to be adaptive. The analysis of growth characteristics and the genotype of revertants shows a difference between early and late-appearing revertants. These results support the hypothesis that the adaptivity of starvation-induced reversion is based on the selective fixation of random mutations, and particularly on transcription-enhanced repair and/or mutagenesis processes.

Accumulation of Ade(+) reversions in isoauxotrophic strains ofSaccharomyces cerevisioe allelic inRAD6 during adenine starvation.

A comparative method based on an analysis of accumulation of starvation-induced Ade(+) reversions and cell death during adenine starvation was developed and exploited for estimating the role ofRAD6 in the starvation-induced reversions. It was shown that inactivation ofRAD6 function inSaccharomyces cerevisioe markedly enhances the accumulation of Ade(+) reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells.

Accumulation of Ade+ reversions in isoauxotrophic stains of Saccharomyces cerevisiae allelic in RAD6 during adenine starvation.

A comparative method based on an analysis of accumulation of starvation-induced Ade+ reversions and cell death during adenine starvation was developed and exploited for estimating the role of RAD6 in the starvation-induced reversions. It was shown that inactivation of RAD6 function in Saccharomyces cerevisiae markedly enhances the accumulation of Ade+ reversions, and therefore it is likely that this gene is taking part in maintaining the low level of starvation-induced mutations in yeast cells.

Uracilless death and papillae formation in rad6-1 polyauxotrophic strains of Saccharomyces cerevisiae.

Both uracilless death and papillae formation during uracil starvation are markedly more extensive in rad6-1 than in RAD6 strains. Osmotic stabilization with 1 mol/L glucitol improves the growth of rad6-1 polyauxotrophic strains in supplemented minimal medium and partially suppresses both the uracilless death and cannibalistic growth of papillae on colonies.

Formation and reversion of Schizosaccharomyces pombe cells with apical protoplast protuberances.

Lytic enzymes from the hepatopancreas of Helix pomatia do not induce a uniform digestion of the cell wall of Schizosaccharomyces pombe over the entire cell surface. Perforations are formed in growth zones through which a protoplast can locally protrude. Conditions were found under which the frequency of formation of apical protoplast protuberances is higher than 90% cells with such protuberances can reverse to normally multiplying cells.

Electrofusion of oriented Schizosaccharomyces pombe cells through apical protoplast-protuberances.

The electrofusion of oriented Schizosaccharomyces pombe cells through apical protoplast-protuberances was demonstrated. The protuberances arose after an exposure of early-exponential phase cells to digestive enzymes from hepatopancreas of Helix pomatia. The orientation of cylindric cells within pearl chains was produced by the application of inhomogenous alternating electric fields.

Expression of hepatitis B virus large envelope protein in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for hepatitis B large envelope protein was cloned under the lac promoter in bacterial vector pUC-8 and under the ADH1 promoter in yeast expression shuttle vector pVT103-U, and expression of HBsAg in bacteria and yeast was determined. The strongest expression of large envelope protein was obtained after transformation of the protease-deficient yeast strain BJ1991. The recombinant large envelope protein did not form complex 22-nm particles and was not secreted into medium.

The use of a killer factor in the selection of hybrid yeast strains.

A new selection method of yeast cell hybrids, based on the application of yeast killer factors, has been developed in the model system of induced protoplast fusion of Saccharomyces cerevisiae superkiller strain T158C (alpha, his-) and different sensitive strains. The method is comparable in its efficiency to the currently used selection techniques based on the auxotrophy or respiration deficiency of the parental cells. Selection by means of a killer factor, when compared to other methods, has the following advantages: (1) Sensitivity to a killer factor is a current natural property of most of the yeast strains. (2) Killer strains of different species of yeast are available and the killer character can be easily transmitted from strain to strain. (3) Hybrid cells produce a killer factor (K+) and are resistant to it (R+). Both these properties are easily and independently testable. (4) Hybrid cells can be cured of the "killer character" by growth at elevated temperatures. Therefore the killer character can also serve as a temporary marker for the purposes of selection of hybrid cells and can be eliminated when no longer desired. On an example of interspecific fusion of strain T158C of S. cerevisiae and strain P9 of S. uvarum the procedure has been shown to be advantageous particularly for the construction of new industrial yeast strains because mutagenesis of the original industrial strain is not required.